1841846791

THE
SCIENTIFIC
BASIS OF
UROLOGY
THIRD EDITION
Edited by
Anthony R. Mundy
John M. Fitzpatrick
David E. Neal
Nicholas J. R. George
The Scientific Basis of Urology

Third Edition
Edited by
Anthony R. Mundy, MS, FRCP, FRCS

Professor of Urology, Institute of Urology and Nephrology,
University College Hospital, London, UK
John M. Fitzpatrick, MCh, FRCSI, FRC(Urol), FRCSGlas, FRCS

Professor of Surgery, Surgical Professorial Unit,
University College Dublin, Ireland
David E. Neal, FMed Sci, MS, FRCS

Professor of Surgery, Department of Surgery,
School of Surgical and Reproductive Science,
University of Cambridge, UK
Nicholas J. R. George, MD, FRCS

Consultant Urologist, Withington Hospital,
and Senior Lecturer, Department of Urology,
University Hospitals of South Manchester, UK
First published in 1999 by Isis Medical Media, Ltd, Oxford, UK

This edition published in 2010 by Informa Healthcare, Telephone House, 69-77 Paul Street, London EC2A 4LQ, UK.
Simultaneously published in the USA by Informa Healthcare, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017, USA.
# 2010 Informa UK Ltd, except as otherwise indicated.
No claim to original U.S. Government works.
Reprinted material is quoted with permission. Although every effort has been made to ensure that all owners of copyright material
have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions
brought to our attention.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise, unless with the prior written permission of the publisher or in
accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited
copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP, UK, or the Copyright Clearance
Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA (http://www.copyright.com/ or telephone 978-750-8400).
Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without
intent to infringe.
This book contains information from reputable sources and although reasonable efforts have been made to publish accurate
information, the publisher makes no warranties (either express or implied) as to the accuracy or fitness for a particular purpose of
the information or advice contained herein. The publisher wishes to make it clear that any views or opinions expressed in this book by
individual authors or contributors are their personal views and opinions and do not necessarily reflect the views/opinions of the
publisher. Any information or guidance contained in this book is intended for use solely by medical professionals strictly as a
supplement to the medical professionals own judgement, knowledge of the patients medical history, relevant manufacturers
instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice
on dosages, procedures, or diagnoses should be independently verified. This book does not indicate whether a particular treatment is
appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her
own professional judgements, so as appropriately to advise and treat patients. Save for death or personal injury caused by the
publishers negligence and to the fullest extent otherwise permitted by law, neither the publisher nor any person engaged or employed
by the publisher shall be responsible or liable for any loss, injury or damage caused to any person or property arising in any way from
the use of this book.
A CIP record for this book is available from the British Library.
ISBN-13: 978-1-84184-679-8
Orders may be sent to: Informa Healthcare, Sheepen Place, Colchester, Essex CO3 3LP, UK
Telephone: +44 (0)20 7017 5540
Email: [email protected]
Website: http://informahealthcarebooks.com/
For corporate sales please contact: [email protected]
For foreign rights please contact: [email protected]
For reprint permissions please contact: [email protected]
Typeset by MPS Limited, A Macmillan Company
Printed and bound in the United Kingdom
Preface
It is 10 years since the first edition, and 5 years since the second edition of this
book. We are pleased that there is demand for a third edition. Once again, we
have included some new chapters, but more importantly, the whole book has
been revised to reflect a changing understanding of the scientific basis of urology
over the last few years. When the editorssenior gentlemen all!completed
their training in urology and became consultants, there was a tacit assumption
that our knowledge base would last us through for the rest of our careers. Now,
instead of reckoning on a 25-year life expectancy for our knowledge base, it is
probably nearer 2.5 years. One considerable benefit from editing this book is that
the four of us have managed to keep up to date, and we hope that other readers
will benefit in the same way. We all believe that a sound scientific basis is
essential for a good clinical practice and therefore hope that this book will be of
interest to all urologists.
Tony Mundy
Contents
Preface . . . . iii
Contributors . . . . vii
1. Introduction to Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Haley L. Bennett and Hing Leung
2. The Cell and Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
David E. Neal
3. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Neville Woolf
4. Immunology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Anne C. Cunningham, Graeme OBoyle, and John A. Kirby
5. The Nature of Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
George B. Haycock
6. Principles of Radiological Imaging of the Urinary Tract . . . . . . . . . . . . . . . . . 83
Uday Patel and Miles Walkden
7. Upper Urinary Tract Obstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Neil G. Docherty and John M. Fitzpatrick
8. Interactive Obstructive Uropathy: Observations and Conclusions
from Studies on Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9. Urinary Tract Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
10. The Scientific Basis of Urinary Stone Formation
William G. Robertson
........................
162
11. Pathophysiology and Management of Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Iain M. J. Mackenzie
12. Acute Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Rona Smith and John R. Bradley
13. Chronic Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Sanjay Ojha and John R. Bradley
14. Structure and Function of the Lower Urinary Tract
Anthony R. Mundy
.....................
15. Physiological Properties of the Lower Urinary Tract

Christopher H. Fry
....................
221
244
16. Scientific Basis of Urodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

Michael Craggs and Sarah Knight
17. Scientific Basis of Male Hypogonadism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Thang S. Han and Pierre-Marc G. Bouloux
vi
Contents
18. Male Sexual Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Giulio Garaffa, Suks Minhas, and David J. Ralph
19. The Prostate and Benign Prostatic Hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . 316
Mark Feneley, Anthony R. Mundy, and Mark Emberton
20. Genetic and Biological Alterations in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Vincent J. Gnanapragasam
21. General Biology of Cancer and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
David E. Neal
22. Tumor Suppressor Genes and Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
David E. Neal
23. The Molecular Genetics and Pathology of Renal Cell Carcinoma . . . . . . 360
Maxine G. B. Tran, Tim OBrien, and Patrick H. Maxwell
24. Transitional Cell Carcinoma of the Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
T. R. Leyshon Griffiths and Nick Mayer
25. Prostate Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Freddie C. Hamdy and Craig N. Robson
26. Testis Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Danish Mazhar and Michael Williams
27. Radiotherapy: Scientific Principles and Practical Application
in Urogical Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Angela Swampillai, Rachel Lewis, Mary McCormack, and Heather Payne
28. Principles of Systemic Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
Christina Thirlwell, John Bridgewater, and Judith Cave
29. Embryology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
David F. M. Thomas
30. Urological and Biochemical Aspects of Transplantation Biology . . . . . . . 454
David Talbot and Naeem Soomro
31. Tissue Transfer in Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Daniela E. Andrich and Anthony R. Mundy
32. Energy Sources in Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
Andy Symes and Ken M. Anson
33. Instrumentation in Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Rashmi Singh and Ken M. Anson
34. Minimally Invasive Technologies in the Treatment of Renal
and Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
Hashim U. Ahmed, Caroline Moore, Manit Arya, and Mark Emberton
35. Stratified Risk Assessment for Urological Surgery . . . . . . . . . . . . . . . . . . . . . . 523
Thiru Gunendran, Nicholas A. Wisely, and Nicholas J. R. George
36. Screening in Urology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542
37. Evidence-Based Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Kieran J. OFlynn
Index . . . . 575
Contributors
Hashim U. Ahmed Department of Urology, Division of Surgical and

Interventional Sciences, University College London, London, U.K.
Daniela E. Andrich Institute of Urology and Nephrology, University College
Hospital, London, U.K.
Ken M. Anson
Department of Urology, St. Georges Hospital, London, U.K.
Manit Arya Department of Urology, Division of Surgical and Interventional

Sciences, University College London, London, U.K.
Haley L. Bennett
Beatson Institute for Cancer Research, Glasgow, U.K.
Pierre-Marc G. Bouloux Department of Endocrinology, Royal Free and

University College Hospital Medical School, Royal Free Hospital, London, U.K.
John R. Bradley Cambridge University Hospitals NHS Foundation Trust,
Cambridge, U.K.
John Bridgewater University College London Cancer Institute, University
College London Medical School, London, U.K.
Judith Cave
Southampton General Hospital, Southampton, U.K.
Michael Craggs London Spinal Cord Injuries Centre, Royal National

Orthopaedic Hospital, Stanmore, U.K.
Anne C. Cunningham
Sunderland, U.K.
Faculty of Applied Sciences, University of Sunderland,
Neil G. Docherty The Conway Institute, University College Dublin, Ireland

Mark Emberton Department of Urology, Division of Surgical and Interventional
Sciences, University College London, London, U.K.
Mark Feneley Institute of Urology and Nephrology, University College
John M. Fitzpatrick
Dublin, Ireland
Mater Misericordiae Hospital and University College
Christopher H. Fry
Guildford, U.K.
Postgraduate Medical School, University of Surrey,
Giulio Garaffa
London, U.K.
Department of Urology, University College London Hospitals,
Nicholas J. R. George Department of Urology, Withington Hospital, University

Hospitals of South Manchester, Manchester, U.K.
Vincent J. Gnanapragasam Uro-oncology Group, Department of Oncology,
Hutchinson MRC Research Centre, University of Cambridge, Cambridge, U.K.
T. R. Leyshon Griffiths Urology Group, Department of Cancer Studies and
Molecular Medicine, University of Leicester, Leicester, U.K.
viii
Contributors
Thiru Gunendran Department of Urology, University Hospital of South

Manchester, Manchester, U.K.
Freddie C. Hamdy
Oxford, U.K.
Nuffield Department of Surgery, University of Oxford,
Thang S. Han Department of Endocrinology, Royal Free and University College Hospital Medical School, Royal Free Hospital, London, U.K.
George B. Haycock
London, U.K.
Academic Department of Paediatrics, Guys Hospital,
John A. Kirby Institute of Cellular Medicine, Newcastle University,

Newcastle upon Tyne, U.K.
Sarah Knight London Spinal Cord Injuries Centre, Royal National Orthopaedic
Hospital, Stanmore, U.K.
Hing Leung
Rachel Lewis Department of Oncology, University College Hospitals, London,

U.K.
Iain M. J. Mackenzie Department of Anaesthesia and Critical Care, University
Hospital Birmingham NHS Foundation Trust, Edgbaston, Birmingham, U.K.
Patrick H. Maxwell
London, U.K.
Nick Mayer
Division of Medicine, University College Hospital,
University Hospitals of Leicester, Leicester, U.K.
Danish Mazhar Department of Medical Oncology, Addenbrookes Hospital,

Cambridge, U.K.
Mary McCormack
London, U.K.
Suks Minhas
London, U.K.
Department of Oncology, University College Hospitals,
Caroline Moore Department of Urology, Division of Surgical and

Interventional Sciences, University College London, London, U.K.
Anthony R. Mundy Institute of Urology and Nephrology, University College
David E. Neal Department of Oncology, University of Cambridge,
Addenbrookes Hospital, Cambridge, U.K.
Graeme OBoyle Institute of Cellular Medicine, Newcastle University,
Tim OBrien Department of Urology, Guys and St Thomas Hospital, London,
U.K.
Kieran J. OFlynn Department of Urology, Salford Royal Foundation Trust,
Salford, Manchester, U.K.
Sanjay Ojha Cambridge University Hospitals NHS Foundation Trust,
Cambridge, U.K.
Uday Patel
St Georges Hospital and Medical School, London, U.K.
Heather Payne
London, U.K.
Contributors
David J. Ralph
London, U.K.
William G. Robertson Physiology Department, Royal Free and University

College Medical School, London, U.K.
Craig N. Robson Northern Institute for Cancer Research, The Medical School,
University of Newcastle, Newcastle, U.K.
Rashmi Singh
London, U.K.
Department of Urology, Kingston and St. Georges Hospital,
Rona Smith Cambridge University Hospitals NHS Foundation Trust,

Cambridge, U.K.
Naeem Soomro Department of Urology, Freeman Hospital,
Angela Swampillai
London, U.K.
Andy Symes
Department of Urology, St Georges Hospital, London, U.K.
David Talbot Department of Hepatobiliary and Transplant Surgery,

Freeman Hospital, Newcastle upon Tyne, U.K.
Christina Thirlwell University College London Cancer Institute, University
College London Medical School, London, U.K.
David F. M. Thomas Department of Paediatric Urology, St Jamess University
Hospital, Leeds, U.K.
Maxine G. B. Tran
Cambridge, U.K.
Miles Walkden
Department of Urology, Addenbrookes Hospital,
University College Hospital, London, U.K.
Michael Williams
Cambridge, U.K.
Department of Clinical Oncology, Addenbrookes Hospital,
Nicholas A. Wisely Department of Anaesthesia, University Hospital of South

Manchester, Manchester, U.K.
Neville Woolf
London, U.K.
Medical School Administration, University College London,
ix
1
Introduction to Cell Biology
Haley L. Bennett and Hing Leung
INTRODUCTION
Cell biology is a discipline that is no longer solely the
domain of the bench-bound scientist. Mainstream
awareness of the concepts that this discipline entails
is increasing, and while many a patient may not know
what DNA stands for, they will be well aware of the
impact of genetics. As the field of cell biology has
expanded and diversified, so have its translational
applications within the clinic. Cell biologists are identifying novel key drug targets and gaining a more
thorough understanding of the cellular action of currently available therapies. In turn, medical professionals can design therapeutic regimes specifically
targeted to the needs of the individual patient,
thus narrowing the gap between the bench and the
bedside.
How then is cell biology important to urologists?
Like in any branch of medicine, a keen knowledge of
the biology of the cell allows for an appreciation of the
molecular basis of pathologies and the resulting cellular dysfunction, and how this dysfunction can manifest at the level of the tissue and/or organ. Within the
field of urology, many recent developments have
stemmed from better understanding of the molecular
and cellular processes in disease, including urological
oncology and andrology.
The aim of this chapter is to present a thorough
overview of the main aspects of cell biology as they
relate to cellular behavior. The reader should gain an
appreciation of structural and molecular components
of the cell and how these components interact to
govern cellular growth, proliferation, differentiation,
and other key biological behaviors. Finally, using
cancer as an example, this chapter will evaluate the
mechanisms whereby normal cellular behaviors can
be manipulated or subverted, resulting in a pathological state.
THE MOLECULES OF LIFE

The cell is the fundamental unit of all organisms. In
essence, it is a fluid-filled sac enclosed by a lipid
membrane, yet this sac possesses the ingredients and
machinery to drive its own replication, react to its
external environment, and control its energy expenditure. To gain a full understanding of cell biology and
the mechanisms that underlie cellular behavior, we

must first be familiar with the molecules that make up
the cell. The cell is 90% water, and the remaining 10%
comprises 50% protein, 15% DNA and RNA, 15%
carbohydrate, and 10% lipid. The structure and function of these classes of biomolecules will be discussed
in the following section.
Proteins
Proteins are macromolecules comprising a chain of
amino acids (Fig. 1A). There are 20 different amino
acids that can be stringed together in any order by
peptide bonds to make up an almost infinite number
of proteins. Usually, proteins are of 100 to 1000 amino
acids in length. The primary structure of a protein
refers to its amino acid sequence. Once assembled,
small sections of a protein will fold into a secondary
structure stabilized by hydrogen bonding between
neighboring or proximal amino acids. These secondary structures include a-helices, b-sheets, and turns,
and a protein may contain many regions with different secondary structures that stabilize each other. The
overall three-dimensional or tertiary structure of a
protein in its entirety is determined by the intramolecular bonds between local and distant amino acids.
These bonds are weaker than those maintaining secondary structure, and thus the tertiary structure can
be manipulated or altered in certain conditions. This
propensity for flexibility has allowed proteins to
assume enumerable functional roles in the cell, as
will be discussed later. Finally, a single protein, or
monomer, can assemble with other monomers to
form multimeric structures, and in many cases,
multimeric assembly is required for protein function.
This quaternary structure is defined by the number
and relative order of protein monomers within a
multimeric complex.
The relative abundance of proteins in a cell
compared with that of other biomolecules suggests
the necessity of this class in all aspects of cellular
function. In fact, most cellular tasks are executed by
proteins. Some functional classes of proteins include
structural components (cytoskeletal, matrix), enzymatic components (catalytic), control of gene expression
(transcription factors), and intercellular communication (growth factors, hormones). Proteins are classified
Bennett and Leung
into families on the basis of structural homology. As a

proteins structure governs its function, generally these
classifications translate into functional divisions.
Deoxyribonucleic Acid
Deoxyribonucleic acid (DNA) is a polymer of single
nucleotides linked by phosphodiester bonds. Nucleotides consist of a five-carbon sugar with a phosphate
group attached to carbon 5 and a purine or pyrimidine
base attached to carbon 2 (Fig. 1B). There are four
nucleotide basesthymine, cytosine, adenine, and
guanineand the nucleotide itself is often referred
to as the base subunit that it contains. RNA, or
ribonucleic acid, also comprises these bases except

for thymine, which is replaced by uracil. Nucleotides
are arranged as two complimentary antiparallel
strands in a double helix and the order in which
they are linked is referred to as its primary structure.
In the cell nucleus, DNA helices are tightly coiled into
protein-associated chromosomes, whereas RNA exists
predominantly as single-stranded polymers and are
found both in the nucleus and cytoplasm.
Sections of DNA contain genes, which are
regions of the molecule that code for a protein. The
process whereby genes are manufactured into proteins is known as gene expression, and is described in
detail in chapter 2. Briefly, gene expression is initiated
Figure 1 Chemical structures of some key classes of

biomolecules: (A) amino acid, (B) phosphoglyceride,
(C) nucleotide, (D) monosaccharide, and (E) ATP.
Chapter 1: Introduction to Cell Biology
when protein-coding regions of DNA are copied into

messenger RNA by RNA polymerases, an event
known as transcription. Messenger RNA then guides
the manufacture of protein by specifying the sequence
of amino acids to be joined to make up the protein.
This process, known as translation, takes place on
ribosomes on rough endoplasmic reticulum.
Lipids
Fatty acids are a vital energy resource and can be
modified to generate numerous types of lipids with a
variety of functions. Fatty acids comprise a hydrocarbon chain attached to a carboxyl group. The length of
the hydrocarbon chain can vary, and the longer the
chain, the lower the solubility in water. Saturated fatty
acids contain no double bonds between carbon atoms
in the hydrocarbon chain, whereas those that do are
referred to as unsaturated. Fatty acids used for energy
are stored in the cell in the form of triacylglycerols.
Phospholipids, another fatty acid derivative, are
the building blocks of the plasma membrane, a waterimpermeable phospholipid bilayer that surrounds the
cell and protects its contents from the external environment. The membrane itself is composed of two
layers of phospholipid molecules that are arranged
with their hydrophobic fatty acyl tails buried within
the bilayer, forming a hydrophobic core, while the
hydrophilic polar ends are in contact with the cell
interior (cytosol) or exterior (exoplasmic). Phosphoglycerides are the most abundant phospholipid found
in membranes, followed by sphingolipids and cholesterol (Fig. 1C). Proteins are also found embedded
within or attached to the membrane and play vital
roles in cell-cell and cell-matrix communication as
well as facilitating the transport of molecules in and
out of the cell. All membrane components can move
laterally along the plane of the membrane, allowing
both stability and fluidity.
Carbohydrates
Sugars, or monosaccharides, are the smallest unit of
carbohydrates (Fig. 1D). Monosaccharides comprise
up to seven carbon atoms bound to an equal ratio of
water molecules, the most common being hexoses and
pentoses. Monosaccharides polymerize into disaccharides, such as sucrose and lactose, or can form
short or long chains known as oligosaccharides or
polysaccharides, respectively. These chains are held
together by glycosidic bonds. Oligosaccharide chains
can also be covalently linked to proteins and lipids.
Glucose, a hexose sugar, is the main fuel source used
in animal cells and can be stored in the form of
glycogen, a multibranched polymer.
Adenosine Triphosphate
The small molecule adenosine triphosphate (ATP) is
utilized by all cells in the body as a means to transfer
energy harvested from the breakdown of glucose, a
process known as respiration (Fig. 1E). The conversion
of ATP, which contains three phosphate groups

attached to adenosine, to ADP involves the transfer
of a phosphate group to a substrate protein. This
releases energy required to catalyze energetically
unfavorable reactions within the cell. This process
mediates a multitude of cellular events such as the
synthesis of new macromolecules, the transport of
molecules against their concentration or electrochemical
gradient, cellular movement, or signal transduction.
HOW CELLS RESPOND TO THEIR

ENVIRONMENT
Every eukaryotic cell shares a number of key features
a plasma membrane enclosing its contents, a cytoskeleton that maintains shape and structure, a nucleus
that contains the genetic material, and organelles that
each have a unique function that contributes to
normal cellular activity. The key architectural components of the cell are discussed in greater detail in
chapter 2.
The main aim for any cell is to generate the
biomolecules required to maintain a homeostatic environment and to perform its required function at the
level of the tissue or organ. The requirement of the cell
may be to differentiate, to proliferate, or to undergo
programmed cell death, to name some examples. Such
cellular behaviors are directed or modified by external
cues, detected by molecules that sense alterations to
the cells environment. How these cues are detected
and how they drive changes to the cell by utilizing or
synthesizing the biomolecules, discussed in the previous section, is the focus of the remainder of this
chapter.
Detection of Changes in the Extracellular

Environment
Because of the relative impermeability of the plasma
membrane, specific transmembrane proteins are
embedded within it that can detect conditions outside
the cell. These integral proteins can respond to chemical signals by the binding of ligand to their extracellular domain. These receptors have exquisite sensitivity
for specific ligands, which can be soluble (e.g., growth
factors, cytokines, neurotransmitters) or insoluble
(e.g., collagen, fibronectin, and other components of
the extracellular matrix). Binding of their cognate
ligand alters the conformation of the receptor and
drives the formation of a multiprotein signaling complex. This complex, via specific protein-protein interactions and enzymatic activation, initiates a cascade of
signaling events. Thus, the message that originated
at the cell surface by ligand-receptor binding is passed
from protein to protein by a series of enzymatic
reactions and eventuates in a particular cellular
event, such as gene transcription or ion flux. This
process is known as signal transduction.
Other transmembrane proteins function to regulate the flow of molecules between the cells exterior
and the cytosol. Although gases and lipophilic molecules, such as steroids, can passively diffuse through
Bennett and Leung
Figure 2 Detection of changes in the extracellular environment. Receptors bind ligands that are present in the extracellular space,
diffuse the plasma membrane, or enter the cell by transmembrane pores. These signals initiate a cellular response such as gene
transcription, which in turn can lead to protein production.
the membrane, most ions, proteins, and small molecules, such as glucose and amino acids, require assistance to gain entry or exit. Transmembrane protein
complexes form hydrophilic pores or channels that
facilitate flow of molecules either down their electrochemical or concentration gradient, or alternatively
against the gradient, at the expense of ATP. In this
manner, cells maintain fine control over their membrane potential by the regulated transport of ions and
over their energy status by monitoring the flow of
nutrients. Receptors for lipophilic ligands that can
diffuse the membrane, such as steroids, reside in the
cytoplasm rather than being presented on the cell
surface. Steroid receptors are activated upon binding
of their ligand in a manner homologous to transmembrane receptors. Upon activation, these receptors enter
the nucleus and assume their role as transcription
factors (Fig. 2).
Mechanisms of Signal Transduction

Protein-Protein Interactions
The cascade of signaling events initiated by an extracellular cue is facilitated by a number of types of relay
molecules that are recruited to form the multimeric
signaling complex (Fig. 3). These molecules are divided
into three main classesadapter, docking, and catalytic

proteins. Adapters act to bridge proteins of various
classes to the activated receptor but do not possess
enzymatic activity (e.g., Shc and the p85 subunit of
PI3-kinase). Similarly, docking proteins do not contain
catalytic domains and rather provide a scaffold on which
proteins of various classes can assemble and maintain
proximity (e.g., insulin receptor substrates 14). Finally,
catalytic proteins contain an enzymatic activity, such
as kinase, lipase, or phosphatase. However, within
the sea of molecules available, how does a protein
recognize its specific binding partner? Proteins contain various functional modules, unique sequences of
amino acids that fold into a specific conformation that
links two proteins together. By directing particular
protein-protein interactions, these modules determine
subsequent signal specificity. A brief outline of some of
the well-characterized functional modules is given in
the following paragraph.
Proteins containing Src-homology (SH)2 domains
or phosphotyrosine-binding (PTB) domains bind to
specific phosphorylated tyrosine residues on other proteins. Examples include the adapters Shc and the catalytic protein tyrosine kinase Src. SH3 domains recognize
sequences on target proteins that are rich in the amino
acid proline. Src, for example, also contains SH3
domains. In addition to protein-protein interactions,
Figure 3 Mechanisms of signal transduction by receptor tyrosine kinases. Growth factor binding to the extracellular domain of the
receptor induces autophosphorylation of tyrosine residues within the kinase domain. These residues provide binding sites for SH2
domaincontaining proteins, allowing for recruitment of effector proteins to the activated receptor. Interactions between effector proteins
are maintained by specific functional domains and may be transient or constitutive. Proteins with catalytic activity then propagate the
membrane-initiated signal to other downstream cellular effectors.
other protein modules allow for recognition of certain

lipids, DNA sequences, or other molecules. Examples
include the pleckstrin homology domain, which recognizes specific membrane phospholipids and thus plays
a key role in determining intracellular localization (e.g.,
Akt) and DNA-binding domains (DBD), which allow
for transcription factors to recognize particular regions
on DNA and thus regulate transcription.
Posttranslational Modifications
For a cell to propagate a signal initiated by an extracellular cue to the subcellular target, for example, to the
nucleus to drive the transcription of certain genes, a
series of chemical reactions must occur. These reactions
commonly result in the posttranslational modification
of proteins within the signal cascadethat is, a chemical alteration or addition made to a protein that changes
its function or activity without altering its primary
amino acid sequence. These modifications allow a
protein (the activator) to talk to the next protein in
the sequence (the effector) and pass on the message
downstream. There are several types of posttranslational modifications, some of which are discussed below.
Phosphorylation. Phosphorylation of proteins

refers to the transfer of a phosphate group, commonly
from ATP, by a class of enzymatic proteins called
kinases to a substrate. This supplies the substrate with
high potential energy. Phosphorylation can both activate and inhibit a protein substrate, depending on its
energy status. If the protein exists predominantly in a
conformation that does not favor activation, then phosphorylation can drive the protein into a high-energy
state. On the other hand, energy may be required to
switch off a protein, and in this case phosphorylation
is needed to induce an energy-unfavorable inhibitory
conformation.
Specific amino acid residues within a protein act
as acceptors of phosphate groups. Phosphorylation
occurs at tyrosine, serine, or threonine residues, and
the kinases that catalyze this transfer are highly specific. Kinases can have a transmembrane localization [e.g.,
receptor tyrosine kinases, which include the epidermal
growth factor receptor (EGFR) family] or can be found
intracellularly [Src family kinases, mitogen-activated
protein (MAP) kinases, Akt]. The target proteins of
kinases include both catalytic proteins such as other
kinases, or noncatalytic proteins such as adapters or
Bennett and Leung
scaffolding proteins that form the skeleton of multiprotein signaling complexes. Another class of enzymes
known as phosphatases can remove the phosphate
group from a particular residue of a protein, thereby
altering its activity. The antagonistic action of kinases
and phosphatases thus tightly regulates both the
duration and magnitude of signal transduction by
phosphorylation (Fig. 4A).
Ubiquitylation. Another key posttranslational
modification is ubiquitylation, a process whereby a
small molecular weight protein called ubiquitin is
covalently attached to lysine residues on a target protein
(Fig. 4B). This is achieved by the sequential action of
ubiquitin-activating (E1), ubiquitin-conjugating (E2),
and ubiquitin-ligating (E3) enzymes. The mechanism
of ubiquitylation is analogous to phosphorylation in
that it can be activated by cell surface ligands, is
reversible, and can determine protein-protein interactions. However, unlike phosphorylation, multiple
types of ubiquitylation can occur, rendering it a more
complex system. Ubiquitin can be ligated as a single
molecule (monoubiquitylation) or as a polymeric chain
(polyubiquitylation). The biological effects of different

forms of ubiquitylation are diverse, but the best characterized is endocytosis. In this process, transmembrane proteins tagged with ubiquitin are internalized
by the cell into a vesicle known as endosome. The type
of ubiquitylation then determines whether the protein
is recycled back to the membrane, or whether it proceeds to the lysosome where it is degraded into its
constituent amino acids. Ubiquitylation can also play a
role in the nucleus, for example, by decreasing the
stability of transcription factors, and in DNA repair
pathways.
SUMOylation, acetylation, and other posttranslational modifications. SUMO (small ubiquitin-related
modifier) is a small molecular weight protein that is
structurally related to ubiquitin, and indeed many
similarities exist between the two in terms of ligation
and lysine attachment. The role of SUMOylation
appears to be substrate specific; however, it has been
implicated as a negative regulator of transcription,
potentially by promoting the interaction of transcription factors with corepressors. Cross talk with another
Figure 4 Proteins are subject to posttranslational modifications. (A) Phosphorylation is the process whereby a phosphate group is transferred
from ATP to a substrate. This process is catalyzed by protein kinases and reversed by protein
phosphatases. (B) Ubiquitylation describes the
ligation of one or several ubiquitin molecules to
a substrate by a series of reactions catalyzed by
E1, E2, and E3 enzymes. The outcomes of
ubiquitylation (mono- or polyubiquitylation) are
diverse and include endocytosis or proteosomal
degradation of the substrate protein.
mechanism of posttranslation modification, acetylation, may compete with SUMO for binding, and
many have antagonistic effects. Acetylation occurs
when an acetyl group (CH3CO) is covalently attached
to lysine and this process is catalyzed by a group of
enzymes known as acetyl transferases. Acetylation
protects proteins from rapid digestion by intracellular
proteases, although other functions for this modification are being revealed.
In addition, other protein modifications exist
that not only add to the diversity of protein function
but also play key roles in controlling their degradation
or intracellular localization. These include methylation, glycosylation, biotinylation, and ribosylation.
Posttranslational modifications can be overlapping,
antagonistic, or cooperative, and both the functional
outcomes and mechanisms underlying their individual
and combined regulation are an emerging field within
cell biology.
Key Signaling Cascades
Although there is an enormous variety of extracellular

stimuli that elicit a cellular response, they commonly
share the same signaling pathways within the cell.
Examples of frequently utilized signaling cascades
include the MAP kinase, PI3-kinase/Akt, IKK/NFkB, and JAK/STAT pathways. The cascades activated
by certain ligands are cell-type specific, although there
may be common preferences. For example, one MAP
kinase cascade, the Ras/Erk (extracellular-regulated
kinase) pathway, is activated by epidermal growth
factor (EGF) in many cell types. In the most basic
model, a number of known proteins are recruited and
activated in a particular sequence, which, for simplification, occurs in this order: EGFR ? Grb2 ? Sos ?
Ras ? Raf ? Mek ? Erk1/2 ? transcription factors
? gene expression. Of course, in reality the system is
much more complex; the signal is not propagated in
such a linear fashion and rarely is only one cascade
activated by one stimulus. Rather, signal cascades
cross talk can be synergistic or antagonistic and, at
any given time, can be activated by a variety of stimuli.
Given that many signaling pathways utilize the
same proteins, activated in the same sequence, how
does the cell generate specific outcomes to different
extracellular cues? This specificity is often achieved by
the regulation of the duration and magnitude of the
signal. For example, the aforementioned kinases
Erk1/2 are activated by a variety of extracellular
signals, such as growth factors, lipid hormones, and
insoluble matrix components. However, the length of
time that Erk1/2 remain activated after stimulation is
a vital determinant in the cellular response, as is the
strength, or magnitude, of activation. In addition, the
shuttling of proteins such as Erk1/2 to different
locations around the cell determines its access to
substrates; thus, intracellular trafficking is another
mechanism that is utilized to govern signal specificity.
In these ways, although many signaling pathways
may intersect at particular effector proteins like
Erk1/2, the cellular response can be as diverse as
proliferation, differentiation, or migration.
Signal Termination
Once a cell has received a cue from its environment

and has utilized the cellular machinery to elicit a
behavioral response, it must then deactivate the signaling cascade. Termination of the signal is crucial for
cellular homeostasis and disruption to this process is a
key hallmark of oncogenesis. Numerous mechanisms
exist that lead to signal termination. Initially, the
deactivation of key enzymes that facilitate phosphorylation or other posttranslational modifications that
positively mediate signaling are countered by the
action of negative mediators, such as phosphatases.
In addition, the levels of proteins themselves are
regulated, and there are many examples of activated
proteins that become targeted for degradation by
ubiquitylation, such as EGFR. Ubiquitin moieties
attached to a targeted protein are recognized by
adapters that deliver them to proteosomes, resulting
in their degradation by hydrolysis. This latter mechanism is also used to turn over long-lived proteins that
are no longer required for normal cellular function, by
lysosomal degradation.
CELLULAR BEHAVIORS
The previous section highlighted the ways in which
individual cells can respond to the extracellular environment and the mechanisms that facilitate signal
propagation through a cell. This section will describe
the aspects of cellular behavior that can be affected by
various signals and how this can impact on tissue
homeostasis (Fig. 5).
Proliferation
Many mitogenic signals, such as hormones and
growth factors, can stimulate cell replication. Some
cell types, such as epithelia, are constantly being
renewed and thus are highly responsive to mitogenic
cues. These cues can be blood-borne (endocrine), be
produced by neighboring or nearby cells (paracrine),
or be secreted by an individual cell for self-stimulation
(autocrine). For a cell to divide, it must make the
machinery required to replicate its DNA, breakdown
the nuclear envelope, segregate the new chromosomes, reform the nuclear membrane, and divide the
cytoplasm. This is an enormous task and numerous
checkpoints exist along the way to ensure the cell can
commit to the next stage of division.
The sequence of events that underlies cell division is called the cell cycle. Cells that are actively
moving through the cell cycle are known as proliferating cells. Cells that are nondividing are referred to as
quiescent and are maintained in G0 phase. The start of
the cycle occurs at G1 phase, a rest period that prepares
the cells for a period of DNA synthesis, known as the
S phase. This is followed by G2, which leads to mitosis
(M phase) whereby cells undergo active division. The
cell cycle and the proteins that control its progression,
namely the cyclins and the cyclin-dependent kinases,
are discussed in detail in chapter 2.
Bennett and Leung
Figure 5 Summary of cellular behaviors elicited by

changes in the extracellular environment. All contribute
to the maintenance of tissue homeostasis and are
subject to deregulation in pathologies such as cancer.
It is vital that proliferation be tightly regulated to

maintain tissue homeostasis. Indeed, tumor cells subvert the normal cell cycle regulatory checkpoints to
obtain a capacity for uncontrolled proliferation. This
ability can be achieved by a number of mechanisms. A
frequent event in many cancers in the deregulation of
growth factor signaling, either by amplification or
mutation of surface receptors (e.g., EGFR) or positive
effector proteins (e.g., Ras), or loss of negative regulators (e.g., PTEN) that lie within the signaling transduction cascade. This leads to the upregulation of
proproliferative genes, pushing cells through the cell
cycle. If this increase in proliferation outweighs cell
death, then hyperplasia or tumor growth will result.
Cell Growth
Cell growth refers to an increase in physical size of an
individual cell, as opposed to the larger tissue mass
that results from proliferation. This process, known as
hypertrophy, reflects an increase in metabolism of an
individual cell in the condition of high nutrient availability and the absence of environmental cellular
stressors. In this event a cell is free to drive de novo
RNA and protein synthesis, and these macromolecules constitute the increased cellular bulk. As this is
an anabolic event, it is crucial that the cell has at its
disposal not only sufficient energy but also the building blocks required for synthesis.
A cell ascertains its nutrient and energy availability (glucose, amino acids, oxygen, and ATP) by
various intracellular sensors, which initiate signal
cascades that culminate at a key protein known as
mammalian target of rapamycin (mTOR). A multitude
of signaling pathways feed in to the mTOR pathway,
and thus this master regulator integrates many inputs
informing the cellular environment. For example, the
binding of insulin to the transmembrane insulin

receptor signals via the Akt pathway to mTOR, reflecting an abundance of glucose for energy production.
Conversely, low ATP levels are detected by AMP
kinase, an enzyme that is activated by AMP and
negatively regulates mTOR activity. Free amino
acids activate mTOR signaling via the class III PI3kinase, hVsp43; however, the precise mechanisms
underlying this activation are still to be elucidated.
The level of mTOR activity is determined by the
relative strength of these various inputs. Downstream
targets of mTOR include S6 kinase, which activates
ribosomal protein S6, and 4E-BP1, an inhibitor of capdependent translation. These proteins in turn directly
regulate the synthesis and translation of mRNA to
create new protein.
Environmental stressors negatively impact on
mTOR signaling and act to halt protein synthesis
and conserve energy. Such stressors include starvation, hypoxia, DNA damage, oxidative or osmotic
stress, and heat shock. In certain conditions such as
starvation, cells can increase available nutrients by the
degradation of existing proteins, membrane components, or entire organelles. Free amino acids, carbohydrates, and lipids can be generated for reuse by
proteolytic or lysosomal degradation. Once the levels
of available biomolecules are sufficient, then mTOR
signaling can be switched back on and protein synthesis reinitiated. In this way, cells can maintain a
degree of autonomy from the extracellular environment when under stress to allow for greater chance of
survival. This capacity for adaptation when under
metabolic or environmental stress is a key feature of
cancer cells, and the mTOR pathway is frequently
hyperactivated in cancer. Given its role as a key
integrator of numerous cascades, mutation or overexpression of upstream regulators renders this protein
vulnerable to deregulation.
Cell Death
Cell death is an event vital for normal embryonic and
neonatal development as well as for proper maintenance of adult tissues. In development, certain cells
are genetically programmed to die to permit the
formation of glandular and ductal lumen and this
same program is utilized in adult tissue for morphogenetic remodeling in response to injury or cellular
stressors. Programmed cell death, otherwise known as
apoptosis or type I cell death, is defined by a cascade
of molecular events that results in a characteristic
morphological appearancecellular shrinkage, the
breakdown of organelles, the condensation of DNA
into chromatin, and the leakage of cytosol into the
extracellular space (blebbing). Two mechanisms
referred to as the extrinsic and intrinsic pathways
lead to apoptotic cell death. The extrinsic pathway is
initiated by the activation of transmembrane receptors
of the tumor necrosis factor receptor (TNFR) superfamily by soluble ligands such as Fas or TRAIL,
whereas the intrinsic pathway is activated by cellular
stressors such as ionizing radiation or chemotherapeutic drugs. These pathways utilize intracellular
proteins from the Bcl-2 family to permeabilize the
mitochondrial membrane, releasing cytochrome C
into the cytosol and activating a family of proteolytic
enzymes, known as caspases, to degrade intracellular substrates. Alternatively, these pathways can
promote cell death by a caspase-independent mechanism by the permeabilization of lysosomes and the
subsequent generalized degradation of cytosolic proteins and organelles by the lysosomal proteases cathepsins. Dead cells are then cleared from tissue by
macrophages.
Type II, or autophagic, cell death is a process
whereby a cell eats itself. Damaged or long-lived
proteins, cytoplasm, or entire organelles are engulfed
by vacuoles called autophagosomes, which fuse with
lysosomes wherein their contents are degraded. A cell
can eventually die via the digestion of its entire
contents and be cleared from tissue by macrophages,
and thus autophagy is used as a mechanism to maintain tissue homeostasis and remodeling alongside
apoptosis. Intriguingly, autophagy is also used as a
survival mechanism to generate nutrients from preexisting proteins and organelles in periods of starvation
or stress. However, the point at which nutrient generation ends and death by self-digestion begins is a
complex story that is only starting to be elucidated.
Many key molecules utilized during autophagy overlap with apoptotic pathways, such as p53 and Bcl-2. It
was suggested that the two processes are mutually
exclusive and inhibitory; however, evidence suggests
that they can occur simultaneously, adding further
complexity to the mechanisms that underlie cell death.
Finally, type III, or necrotic, cell death describes
the process when cells are killed by damage that is
beyond repair and usually affects large numbers of
cell within a tissue. It is characterized by cellular
swelling, irreversible plasma membrane and organelle
damage, and random DNA cleavage. Cellular contents are extruded into the extracellular space, and
this, unlike during apoptosis, provokes an inflammatory response. Like apoptosis and autophagy, necrosis
is controlled by molecular signaling cascades that
overlap, and indeed, in certain cases, this form of
cell death can be recruited upon failure of apoptotic
or autophagic induction. Given the extensive cross
talk between the death cascades, it has been difficult
to describe specific necrotic effectors; however, the
kinase RIP1 as well as reactive oxygen species (ROS)
and calcium have been implicated in this process.
As previously mentioned, the evasion of cell
death by cancer cells is a key factor for tumorigenesis.
Many oncogenes have been implicated in such evasion by their disruption of apoptotic signaling and/or
promotion of survival, for example, overexpression of
antiapoptotic proteins Bcl-2 or Bcl-XL, the increased
Akt signaling due to PTEN deletion, or the loss of the
autophagy regulatory gene Beclin-1. Prosurvival signaling is essential for cancer cells to endure the lack of
proper vascularization within early tumors and the
onslaught of chemotherapeutic drugs, or to form
secondary tumors within new microenvironments.
Differentiation
The process of differentiation entails the expression of
particular genes within a precursor cell that drives
its functional specialization. This process underlies
embryonic development and is also vital for the
homeostatic functioning and maintenance of adult
tissues via the differentiation of stem cells. Stem cells
themselves can divide to form other stem cells, or can
be committed to a pathway of differentiationfrom a
restricted potential stem cell, to a progenitor cell and
finally to a terminally differentiated cell. This process
is usually determined by the presence of extracellular
factors that drive expression of certain genes. Once
differentiated, most cells are committed to serve that
function and are unable to dedifferentiate.
Numerous signaling pathways are implicated in
driving differentiation, or alternatively in maintaining
the stem cell population. Soluble extracellular factors
such as transforming growth factor (TGF) a and b,
stem cell factor (SCF)/kit or certain cytokines have all
been shown to activate differentiation in particular
settings. Similarly, specific transcription factors have
been implicated in differentiation including b-catenin
via the Wnt pathway, Hedgehog, and Notch.
As with other cell behaviors discussed in this
chapter, the process of differentiation can be used by
cancer cells to promote tumorigenesis. Genes that are
switched off in a terminally differentiated cell can be
reexpressed because of oncogenic activity and this can
alter the differentiation status of that cell. A key
histological marker of aggressive disease is the degree
of dedifferentiation within tumor tissue. Altered differentiation also underlies the ability of tumor cells to
form metastases. During development, mesenchymal
cells migrate to their respective positions within tissues where many then undergo terminal epithelial
differentiation. Tumor cells can reverse this process
by reexpressing mesenchymal genes and allowing
10
Bennett and Leung
epithelial cells to obtain a migratory phenotype. It is a

hotly debated issue within the cancer field, however,
as to whether de- or transdifferentiated cells actually
represent the altered state of preexisting cells that
were terminally differentiated, or whether these populations are derived from a stem cell or progenitor that
had acquired oncogenic characteristics. Evidence suggests cancer stem cells may exist in certain tumor types
and, if definitively proven, has far-reaching implications on the development of future cancer therapies.
Migration
The capacity for cells to actively move around the body
or locally within tissues is necessary for development,
wound healing, and the regular tissue maintenance.
Migration through tissue is facilitated by a number of
mechanisms involving the de-adhesion of a cell to its
neighbors (in the case of cells originating within a
given tissue), the remodeling of the actin cytoskeleton
to push the cell forward, coupled with the release of
extracellular proteases that degrade the surrounding
extracellular matrix. Migration can be stimulated by a
number of cytokines and guidance factors, which
can either act as chemoattractive/repulsive agents or
alternatively to promote the disassembly of cell-cell or
cell-matrix interactions.
Cells that are structurally bound within a tissue
must first disassemble their cell-cell contacts to
become motile. Between neighboring cells, a number
of junctions exist that facilitate adhesion, communication, and structural support. Adherens junctions are
supported by the calcium-regulated binding of the
extracellular domain of cadherin molecules of adjacent cells. The expression pattern of cadherins is
highly specific for different cell types, for example,
epithelial cells express E-cadherin and vascular endothelial cells express VE-cadherin. Cadherin molecules
are bound by their cytoplasmic domain to b-catenin
and via interactions with a-catenin and other actinbinding molecules; the complex is bridged to the actin
cytoskeleton. Tight junctions are portions of plasma
membrane between adjacent cells that appear to be
fused together at points, providing an impermeable
barrier to small hydrophilic molecules and ions. Tight
junctions also prevent the diffusion of membrane-bound
proteins between the apical and basolateral surface; thus
they maintain functional polarity. Gap junctions are small
pores between adjacent cells that allow for diffusion of
small molecules and ions. Finally, desmosomes comprise
specific cadherins known as desmoglein and desmocollin that are bound to intermediate filaments via the
cytosolic plakoglobin and desmoplakins, providing
mechanical strength. Likewise, intermediate filament
bundles secure adhesion to the extracellular matrix
through structures called hemidesmosomes.
Once detached from its neighbors, a cell

migrates by the formation of transient sites of interaction with the extracellular matrix, called focal adhesions. The focal adhesion complex comprises a
multitude of different proteins such as Fak, Src, and
paxillin, and acts to bridge extracellular matrixbound
integrins to the actin cytoskeleton. Focal adhesions are
linked to stress fibers composed of actin and myosin,
which in turn provide the mechanical contractility to
promote cell migration. The coordinated activity of
proteins of the Rho family of GTPases act at the
leading edge of the migrating cell to reorganize actin
filaments, generating protrusive finger-like (filopodia)
and sheet-like (lamellipodia) structures, while also
stimulating contraction at the rear of the cell by
regulating the assembly of acto-myosin filaments. In
this manner, the signals generated at focal adhesions
promote the directional migration of the cell. Migrating cells can also secrete proteases such as matrix
metalloproteases (MMPs) to degrade the local matrix
and provide space into which the cell can move.
Understanding the processes that regulate cell
migration is vital as it is the acquisition of this behavior that underlies invasive, and therefore lethal, forms
of cancer. Various oncogenes have been shown to
increase motility by the alteration of actin dynamics.
In addition, the disassembly of cell-cell junctions by
the deregulation or loss of E-cadherin and the switch
to its mesenchymal counterpart is frequently observed
in epithelial cancers. Similarly, changes in integrin
expression can promote migration and survival of
metastatic cells in unfamiliar microenvironments.
Cancer therapeutics that specifically undermine invasive potential are thus a key mechanism to combat
metastatic disease.
SUMMARY
This chapter has presented the basic concepts of cell
biologythe ways in which cells use biomolecules to
maintain structure, sense their environment, and react
accordingly. The molecular mechanisms underlying
the main cellular behaviorsproliferation, growth,
death, differentiation, and migrationwill be discussed in greater detail in the next chapter.
SUGGESTED READING
1. Lodish H, Baltimore D, Berk A, et al. Molecular Cell Biology.
5th ed. New York: Scientific American Books, 2004.
2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell
2000; 100(1):5770.
3. Seet BT, Dikic I, Zhou MM, et al. Reading protein modifications with interaction domains. Nat Rev Mol Cell Biol
2006; 7(7):473483.
2
The Cell and Cell Division
David E. Neal
Department of Oncology, University of Cambridge, Addenbrookes Hospital, Cambridge, U.K.
STRUCTURE OF THE CELL

Cell Membrane
The cell membrane confines the contents of the cell. It
consists of a continuous bilayer of phospholipid with
the polar hydrophilic ends forming the outer and
inner layers and the hydrophobic tails forming the
central core of the bilayer. The hydrophilic heads on
the two sides of the cell membrane are of different
composition, those on the outside often being modified by glycosylation: a process that involves the
addition of various sugar residues. Embedded in
this lipid bilayer are proteins whose function can be
classified as follows:
l
Signal transduction. These proteins mediate the

action of external ligands such as growth factors
and neurotransmitters). These receptor proteins
cross the cell membrane and may have intrinsic
enzyme functions (such as the tyrosine kinase
activity of the receptor for epidermal growth
factor) or may be linked to other proteins such
as G proteins (e.g., the muscarinic acetylcholine
receptor) (Fig. 1).
Cell-cell or cell-matrix contact. Specialized junctions
between cells and between cells and the intercellular matrix occur, involving transmembrane proteins such as integrins and cadherins that interact
on the outside with molecules such as fibronectin,
and on the inside with molecules such as catenin,
talin, and vinculin that act as intermediate links to
the cell skeleton, which is made of actin fibers.
These proteins form specialized cellular contacts,
such as desmosomes and tight junctions.
Carrier proteins. These are involved in the transport
of small molecules and ions across the cell membrane (as in glucose, the Na1/K1 pump or the
multidrug resistance pump). These proteins may
be energy dependent or passiveif they are
active, they usually utilize adenosine triphosphate
(ATP) as a source of energy. These carrier proteins
often transport other molecules at the same time in
the opposite direction (Fig. 2).
Channel proteins. These are involved in the transport of ions across the cell membrane. These
function in a passive way and effectively are
hydrophilic pores; they function more efficiently
than carrier proteins and can carry ions more than

1000-fold faster. They are selective for certain ions
and may be closed or open. The stimuli for opening these channels may be electrical current,
ligand binding (as with acetylcholine binding to
nicotinic receptors), or mechanical deformation.
Intracellular organelles. These are also bounded by
membranes and include rough and smooth endoplasmic reticulum (ER), Golgi apparatus, and
nuclear membranes. These intracellular membranes compartmentalize the cell into functionally
distinct units (Fig. 3) and are extensive, having a
surface area 10- to 25-fold greater than the cell
membrane itself. Intracellular proteins are synthesized on free cytosolic ribosomes and remain in
the cytoplasm, but proteins for export begin their
life by being synthesized on ribosomes that have
special signaling molecules that dock with receptors on the ER, Golgi apparatus, lysosomes, etc.,
which mediate transport to their final destination.
There are specialized areas of the cell membrane. These
include the structure of the synapse or neuromuscular junction, which are dealt with elsewhere in
the book. In addition, cells have small vesicles that
can be involved in the storage of neurotransmitters. Calveolae are small vesicles formed by budding off the cell membrane. Clathrin-coated pits
are other specialized areas of the cell membrane
that are responsible for internalization of activated
ligand-bound receptors, thereby reducing the
intensity of the associated signaling cascade. In
such areas of the cell, adapter proteins are found,
which may act as signal transduction molecules.
Endoplasmic Reticulum
The ER is convoluted and may occupy up to 10% of the
cell volume; it plays a central role in protein and lipid
biosynthesis and is the site for synthesis of proteins
and lipids that are destined to be incorporated in other
cell organelles such as mitochondria (Fig. 4).
Ribosomes are found free in the cytosol or
attached to the ER (rough ER) when making a protein
destined for export. In the latter circumstance, a
signaling molecule targets the ribosome with its
mRNA to the ER, into which it secretes its protein
(Fig. 5).
12
Neal
Smooth ER is abundant in some cells. It is

involved in the storage of calcium ions and therefore
has a role in the contraction of smooth muscle cells,
which is brought about by calcium release from these
structures as well as transmembrane influx. Together
with the Golgi apparatus, it is involved in the synthesis of lipoproteins. In liver and some other cells, it
contains the cytochrome p450 enzymes, which detoxify
lipid-soluble drugs by converting them into watersoluble forms.
Golgi Apparatus
This system of flattened sacs, which lie in continuity
with the ER, is involved in sorting, packaging, and
modifying macromolecules for secretion or for delivery
to other intracellular organelles (Fig. 6). It consists of a
stack of four to six flattened cisternae with an entry
(cis) and exit (trans) surface. Glycosylation of proteins
such as mucin takes place in the Golgi apparatus. Other
proteins are sorted for differential transport to certain
organelles; for instance, acid hydrolase is transported
from here to the lysosomes, and if the cell synthesizes
hormones or neurotransmitters, these are excreted by
small, smooth vesicles budding off from the Golgi
apparatus before transport to the cell membrane,
where exocytosis takes place.
Mitochondria
Figure 1 A schematic drawing of a G-protein-linked receptor.

Receptors that bind protein ligands have a large, extracellular,
ligand-binding domain formed by the part of the polypeptide
chain shown in light green. Receptors for small ligands such as
adrenaline (epinephrine) have small extracellular domains, and
the ligand-binding site is usually deep within the plane of the
membrane, formed by amino acids from several of the transmembrane segments. The parts of the intracellular domains that
are mainly responsible for binding to trimeric G proteins are
shown in orange, while those that become phosphorylated during
receptor desensitization (discussed later) are shown in red.
These are the powerhouse of the cell and are responsible for the production of most of the high-energy
phosphate intermediates, such as ATP. They are present in virtually all cells and mediate oxidative respiration, in which pyruvate is converted to carbon
dioxide and water with the production of about
30 molecules of ATP. Mitochondria consist of an
outer membrane and a convoluted inner membrane
containing the inner space or matrix, which is packed
with hundreds of enzymes. Acetyl coenzyme A is the
central intermediate produced by fatty acid oxidation
and glycolysis (Fig. 7), and the citric acid cycle takes
place in the mitochondria (Fig. 8). In the mitochondria,
Figure 2 Three types of carrier-mediated transport.

The schematic diagram shows carrier proteins functioning as uniports, symports and antiports.
Chapter 2: The Cell and Cell Division
13
Figure 3 The major intracellular compartments of an

animal cell. The cytosol, endoplasmic reticulum, Golgi
apparatus, nucleus, mitochondrion, endosome, lysosome, and peroxisome are distinct compartments
isolated from the rest of the cell by at least one
selectively permeable membrane.
Figure 4 The intracellular compartments of the eukaryotic cell

involved in the biosynthetic-secretory and endocytic pathways.
Each compartment encloses a space that is topologically equivalent to the outside of the cell, and they all communicate with one
another by means of transport vesicles. In the biosyntheticsecretory pathway, protein molecules are transported from the
ER to the plasma membrane or (via late endosomes) to lyosomes. In the endocytic pathway, molecules are ingested in
vesicles derived from the plasma membrane and delivered to
early endosomes and then (via late endosomes) to lyosomes.
Many endocytosed molecules are retrieved from early endosomes and returned to the cell surface for reuse; similarly,
some molecules are retrieved from the late endosome and
returned to the Golgi apparatus, and some are retrieved from
the Golgi apparatus and returned to the ER. All of these retrieval
pathways are shown with arrows. Abbreviation: ER, endoplasmic
reticulum.
Figure 5 Free and membrane-bound ribosomes. A common

pool of ribosomes is used to synthesize both the proteins that
stay in the cytosol and those that are transported into the ER. It is
the ER signal peptide on a newly formed polypeptide chain that
directs the engaged ribosome to the ER membrane. The mRNA
molecule may remain permanently bound to the ER as part of a
polyribosome, while the ribosomes that move along it are
recycled; at the end of each round of protein synthesis, the
ribosomal subunits are released and rejoin the common pool in
the cytosol. Abbreviation: ER, endoplasmic reticulum.
high-energy electrons from NADH and FADH2 are

passed to oxygen during oxidative phosphorylation
by the respiratory chain, which is situated on the inner
membrane of the mitochondria (Fig. 9). There are
14
Neal
It is thought that ATP is formed by a process of

chemiosmotic coupling. The passage of electrons
down each of the three parts of the respiratory chain
results in H1 being pumped into the intermembrane
space of the mitochondria to set up an electrochemical
proton gradient. The subsequent backflow of H1 into
the matrix provides the energy to drive the enzyme
ATP synthase. Mitochondria are also actively
involved in the induction of caspases during the
process of apoptosis (Fig. 11).
Mitochondrial DNA
Figure 6 The major intracellular compartments of an animal

cell. The cytosol, endoplasmic reticulum, Golgi apparatus, nucleus, mitochondrion, endosome, lysosome and peroxisome are
distinct compartments isolated from the rest of the cell by at least
one selectively permeable membrane.
three large enzyme groups embedded in the inner

membrane (Fig. 10).
1.
2.
3.
The NADH dehydrogenase complex, which contains a flavin and ubiquinone

The cytochrome b-c1 complex, which contains
three hemes and iron-sulfur protein
The cytochrome oxidase complex, which contains
two cytochromes and copper
Mitochondria and chloroplasts contain the DNA that

encodes the crucial structural and functional proteins,
which permit the mitochondria to divide during cell
division. Mitochondrial DNA utilizes biochemically
distinct pathways from those of nuclear DNA. For
instance, protein synthesis taking place in the cytoplasm but directed by nuclear DNA is blocked by
cyclohexidine, whereas protein synthesis directed by
mitochondrial DNA is blocked by chloramphenicol,
erythromycin, and tetracycline. Mitochondria are not
synthesized de novo, but arise from division of the
organelle, which occurs during mitosis and is driven
by mitochondrial DNA. Mitochondrial DNA is a
circular structure like that of bacteria; in mammalian
cells, it contains around 16.5 kbp. It synthesizes
2 ribosomal RNAs, 22 transfer RNAs (tRNAs), and
13 peptides. Unlike nuclear DNA, most of the nucleotides are direct coding sequences with little or no
space left for regulatory codons (Table 1). Analysis
of the genetic code shows that the codon sequence is
relaxed, allowing many tRNA molecules to recognize
Figure 7 The fatty acid oxidation cycle. The cycle is

catalyzed by a series of four enzymes in the mitochondrial matrix. Each turn of the cycle shortens the
fatty acid chain by two carbons (red), as indicated,
and generates one molecule of acetyl CoA and one
molecule each of NADH and FADH2. The NADH is
freely soluble in the matrix. The FADH2, in contrast,
remains tightly bound to the enzyme fatty acyl-CoA
dehydrogenase; its two electrons will be rapidly
transferred to the respiratory chain in the mitochondrial inner membrane, regenerating FAD.
15
Figure 8 The citric acid cycle. The intermediates are

shown as their free acids, although the carboxyl
groups are actually ionized. Each of the indicated
steps is catalyzed by a different enzyme located in
the mitochondrial matrix. The two carbons from acetyl
CoA that enter this turn of the cycle (shadowed in red)
will be converted to CO2 in subsequent turns of the
cycle: it is the two carbons shadowed in blue that are
converted to CO2 in this cycle. Three molecules of
NADH are formed. The GTP molecule produced can
be converted to ATP by the exchange reaction
GTP ADP ? GDP ATP. The molecule of
FADH2 formed remains protein-bound as part of the
succinate dehydrogenase complex in the mitochondrial inner membrane; this complex feeds the
electrons acquired by FADH2 directly to ubiquinone
(see below). Abbreviations: ATP, adenosine triphosphate; GTP, guanosine triphosphate.
Figure 9 A summary of mitochondrial energy

metabolism. Pyruvate and fatty acids enter the
mitochondrion, are broken down to acetyl CoA,
and are then metabolized by the citric acid cycle,
which produces NADH (and FADH2, which is not
shown). In the process of oxidative phosphorylation, high-energy electrons from NADH (and
FADH2) are then passed to oxygen by means of
the respiratory chain in the inner membrane,
producing adenosine triphosphate by a chemiosmotic mechanism. NADH generated by glycolysis
in the cytosol also passes electrons to the respiratory chain (not shown). Since NADH cannot
pass across the mitochondrial inner membrane,
the electron transfer from cytosolic NADH must
be accomplished indirectly by means of one of
several shuttle systems that transport another
reduced compound into the mitochondrion; after
being oxidized, this compound is returned to the
cytosol, where it is reduced by NADH again.
16
Neal
Figure 10 The path of electrons through the three

respiratory enzyme complexes. The size and shape of
each complex is shown, as determined from images of
two-dimensional crystals (crystalline sheets) viewed in
the electron microscope at various tilt angles. During the
transfer of two electrons from NADH to oxygen (red
lines), ubiquinone and cytochrome c serve as carriers
between the complexes.
Figure 11 Factors in apoptosis. This diagram shows

that DNA-damaging agents result in induction of
apoptosis through release of cytochrome c from
mitochondria.
Table 1 Differences Between Nuclear and Mitochondrial Coding

Codon
Standard
Mitochondrial
UGA
AUA
AGA/AGG
STOP
Ile (isoleucine)
Arg (arginine)
Trp (tryptophan)
Met (methionine)
STOP
any one of four nucleotides in the third or wobble

position. In addition, in man, 3 of the 64 codons have
different meanings from the standard codons.
Because mitochondrial inheritance is cytoplasmic, one would expect mitochondria to be inherited from the maternal side because the sperm has
little cytoplasm. It is thought that both mitochondria
and chloroplasts evolved from endosymbiotic bacteria
more than a billion years ago. However, over time,
some mitochondrial genes have been transferred to
nuclear DNA; for instance, lipoproteins in the mitochondrial membranes are synthesized from nuclear
genes and modified in the cells Golgi apparatus.
THE NUCLEUS
This is the most striking organelle in the cell. It is
separated from the rest of the cell by the nuclear
membrane, which consists of a lipid bilayer fenestrated
by multiple nuclear pores. Inside the nucleus is the

nucleolus, which is the factory that produces ribosomes. All chromosomal DNA is held in the nucleus,
but it is associated with an equal mass of histone
proteins. The dense nuceolus is the site for the assembly of ribosomes.
Nuclear pores (Fig. 12) are highly specialized
structures, which mediate the transfer of macromolecules across the nuclear membrane. Many proteins
(e.g., the androgen receptor) contain a nuclear localization signal (NLS), which comprise short sequences
of highly basic amino acids such as lysine. This NLS
binds to importin-a and is actively transported into
the nucleus. This whole process is driven by a concentration gradient of RAN-GTP.
Arrangement of DNA
Each DNA molecule in the nucleus is packaged as a
chromosome, which, in essence, is an enormously long
molecule arranged as two strands of a double helix.
There are 23 pairs of chromosomes (22 autosomes and
1 sex chromosome: X or Y), which contain about 6 "
109 nucleotide pairs. Each DNA molecule not only has
to be able to code for many different proteins (Fig. 13),
but also has to be able to replicate itself reliably during
mitosis and to be able to repair itself when damaged
17
Figure 12 (A) Translocation of nuclear hormone receptors from the cytoplasm into the nucleus using the importin alpha (yellow box) and
importin beta (orange box) components. This process is under the control of RanGTP, which causes the dissociation of importin beta
from the process. (B) Structure of the nuclear pore through which this process takes place.
Figure 13 DNA double helix. In a DNA molecule, two

antiparallel strands that are complementary in their
nucleotide sequence are paired in a right-handed double helix with about 10 nucleotide pairs per helical turn.
A schematic representation (top) and a space-filling
model (bottom) are illustrated here.
by chemicals or radiation. At the ends of each chromosome are the telomeres and at the center is the centromere, which serves to anchor the chromosome via the
kinetochore to the mitotic spindle during cell division.
Not all DNA in the chromosomes is used to produce

mRNA; indeed, most of it is arranged into regulatory
elements (noncoding sequences) found in introns.
The function of much of the DNA sequence structure is
18
Neal
Figure 14 Nucleosome-free regions in 30-nm fibers. A

schematic section of chromatin illustrating the interruption
of its regular nucleosomal structure by short regions
where the chromosomal DNA is unusually vulnerable to
digestion by DNase I. At each of these nucleasehypersensitive sites, a nucleosome appears to have
been excluded from the DNA by one or more sequencespecific DNA-binding proteins.
not understood. It used to be known as junk DNA,

but some of these sequences encode small segments of
RNA, known as short hairpin (because of its shape)
RNA, which can act to regulate the half-life of other
messenger RNA, and hence protein synthesis. Totally
unknown until a few years ago, this system is now
known to be an important means of regulating cell
function in most organisms.
Histone Proteins
These proteins allow formal packaging of DNA in the
nucleus; without them, DNA in mammalian cells
would not be arranged in a regular way, and cell
division and gene transcription would not be possible.
They are small proteins with a large amount of dibasic
amino acids, such as arginine and lysine, which bind
to DNA because of their positive charges. There are of
five types: the H1 histones and the nucleosomal
histones (H2A, H2B, H3, and H4), which are highly
evolutionarily conserved. The nucleosomal histones
are responsible for the coiling of DNA into nucleosomes (Fig. 14), which are essential to the accommodation of DNA in the nucleus. It is thought that
nucleosome histones are preferentially bound to
areas of DNA that are adenine/thymine (AT) rich;
these proteins can be prevented from binding by the
attachment of inhibitory proteins. Nucleosomes are
themselves packed together even more tightly by H1
histone proteins.
Figure 15 shows how DNA within chromosomes
is ordered. DNA that is actively being transcribed into
mRNA is unfolded, but is at its most condensed
during mitosis, when individual chromosomes can
be recognized by their specific banded structure.
Histone acetylation and deacetylation (mediated by
histone actetylases, deacetylases, and their inhibitors)
are crucial to the process of transcription, as acetylation allows unwinding of the chromatin and access to
general and specific transcription factors (Fig. 16).
DNA Replication
Replication of DNA requires that it is unwound at socalled replication forks, each strand acting as a template for synthesis. DNA polymerase a is used on the
lagging strand and DNA polymerase d on the leading
strand (Fig. 17). Both sections are synthesized in the 50
to the 30 direction (on the new strand), and the lagging
strand is initially synthesized as short segments called
Okazaki segments. In man, each molecule of DNA

(the full length of the chromosome) is so long that
several replication forks are required; these are often
clustered together in areas of DNA that are transcriptionally active. For instance, in women whose second
X chromosome is condensed as heterochromatin (Barr
body), the active X chromosome replicates throughout
each S phase, which lasts for about eight hours,
whereas the heterochromatin replicates only late in
the S phase. During mitosis, a huge amount of histone
protein has to be synthesized (histone protein forms
an equal mass to DNA), and in man there are
20 replicated sets of histone protein genes arranged
as tandem repeats on several chromosomes, each set
of which contains all five histone proteins.
DNA Replication Errors

Errors can arise during DNA replication owing to
insertion of the wrong base or the insertion of a run
of microsatellite repeats (replication errors). These are
normally repaired by nucleotide excision enzymes or
replication error repair enzymes, respectively. Errors
in DNA repair are a common feature of most human
cancer.
Telomeres and Telomerase

Because of its structure and the way that it synthesizes
nucleotides, DNA polymerase cannot replicate the
very ends of chromosomes, which are modified into
special regions called telomeres. In many species,
these consist of tandem repeats rich in G (in man,
GGGTTA). This region is replicated by a special
enzyme called telomerase, which recognizes the
GGGTTA sequence. Because there is no complementary DNA strand to replicate, telomerase uses an RNA
template, which is structurally part of the telomerase,
as a temporary extension for replication of the other
strand. After several rounds of extension, one strand
is longer and therefore can be used in turn as a
template for replication of the second strand by
DNA polymerase. It is thought that the telomeres
shorten after each round of cell division and that
this may limit the lifespan of a cell (senescence).
Some malignant tumors are known to have high levels
of telomerase, a feature that may mean that the cell
can divide without the lengths of the telomeres being
a limiting factor.
19
Figure 15 Model of chromatin packing. This schematic

drawing shows some of the many orders of chromatin
packing postulated to give rise to the highly condensed
mitotic chromosome.
This rather simplistic view has been replaced by

one in which capping of the telomeres is important.
It is true that during aging of the cell, the telomeres
become shorter, but while the telomeres are capped,
cell division can carry on. If the capping process
fails, senescence and apoptosis can occur, but, more
importantly, fusion of chromosomes can occur. This
process is thought to be important in carcinogenesis
(Fig. 18).
CELL DIVISION AND SENESCENCE

Senescence
Normal human cells cannot go on dividing forever in
tissue culture; eventually, they senesce and will not
divide further in response to mitogens. This process is
accompanied by loss of telomerase, failure to phos-
phorylate the retinoblastoma protein in response to

mitogens, and high levels of p21 (WAF1) and p16,
which are cyclin-dependent kinase inhibitors that
profoundly inhibit cell division.
Cell Division
Cell division (Fig. 19) involves duplication of DNA,
mitosis (nuclear division), and cytokinesis (division of
the cytoplasm). Following replication of DNA, the
centrosome divides to form the mitotic spindle, and
the chromosomes condense and align in the center of
the cell, where they are pulled apart by the mitotic
spindle.
Mitosis: the M Phase
Prophase. The chromatin condenses into chromosomes that have duplicated and hence are formed
20
Neal
Figure 16 Conventional view of how histone acetylases interact with DNA. This cartoon shows how the acetylation of histone residues
opens up the DNA by release of histone proteins. A more dynamic process is the current view.
Figure 17 A mammalian replication fork. The mammalian replication fork is important in several
respects. First, it makes use of two DNA polymerases, one for the leading strand and one for the
lagging strand. It seems likely that the leading-strand
polymerase is designed to keep a tight hold on the
DNA, whereas that on the lagging strand must be
able to release the template and then rebind each
time that a new Okazaki fragment is synthesized.
Second, the mammalian DNA primase is a subunit of
the lagging-strand DNA polymerase, while that of
bacteria is associated with the DNA helicase.
of sister chromatids held together by the centromere.

The centrosome divides to form the mitotic spindle.
The nucleolus disperses.
Prometaphase. The nuclear membrane disrupts.
The kinetochores begin to form. These are specialized
proteins attached to the centromere to which the
microtubule proteins of the spindle pole bind. Patients
with scleroderma form autoantibodies to kinetochore
protein.
Metaphase. The chromosomes are aligned at the
metaphase plate at the center of the cell.
Anaphase. The paired kinetochores suddenly

separate, allowing the chromosomes to separate and
to be pulled toward each spindle pole in a matter of
minutes. Contraction of the microtubules pulls the
chromosomes toward the spindle pole.
Telophase. The nuclear membrane begins to
reform and the microtubules disappear. The nucleolus
reappears.
Cytokinesis. The cytoplasm separates at a specialized contractile ring situated at the center of the
cell, actively cutting the cytoplasm into two parts.
21
Figure 18 Telomeres. Telomeres are normally

capped. Even shortened telomeres can be associated with normal cell cyclingprovided they are
capped. If the capping is lost, cells either undergo
apoptosis or may be prone to chromosome fusion
and malignancy.
THE CELL CYCLE

In the adult human, cells are continuously being lost
by the process of planned, programmed cell death
(apoptosis). Some cells do not divide at all, although
they can be destroyed (neurons and skeletal muscle
fibers; however, recent work on stem cells suggest that
these continue to exist in brain and muscle into adult
life): some divide slowly, and others have to divide
rapidly (such as cells in the bone marrow and lining of
the gut). During cell division, DNA is replicated and
reproduction of intracellular organelles takes place.
Replication of DNA takes place during a specific part
of mitosis known as the S phase. G2 is the period of
rest before the prophase part of the M phase starts.
The G1 phase occupies the period between the completion of the previous mitosis and the S phase; some
mature cells, however, enter a specialized period of
rest, G0, which can last for months or years.
The proportion of dividing cells can be measured by a number of different tests. Administration
of radiolabelled thymidine or bromodeoxyuridine
can allow labeling of cells in the S phase to be
demonstrated by the use of photographic plates or
monoclonal antibodies, respectively. Other antibodies
can be used to measure the Ki67 antigen (Ki67 or
MIB1) or proliferating cell nuclear antigen, which are
expressed during particular parts of the cell cycle;
these are useful in measuring cell proliferation within
tumors. The amount of DNA within a population of
cells can be estimated by the use of fluorescent stains
such as ethidium bromide measured in a fluorescenceactivated cell sorter.
The Control of Cell Division

Figure 19 The four successive phases of a standard eukaryotic
cell cycle. During interphase, the cell grows continuously; during
M phase, it divides. DNA replication is confined to the part
of Interphase known as S phase. G1 phase is the gap between
M phase and S phase; G2 is the gap between S phase and
M phase.
This process is tightly controlled at certain critical

points of the cell cycle, which are known as cell
cycle checkpoints, at which certain brakes can be
applied to stop the process if conditions are unfavorable (Fig. 20). Checkpoints are found at the G1/S
22
Neal
Figure 20 Checkpoints and inputs of regulatory

information to the cell-cycle control system. Feedback from downstream processes and signals from
the environment can prevent the control system
from passing through certain specific checkpoints.
The most prominent checkpoints are where the
control system activate the triggers shown in yellow
boxes.
transition (the start checkpoint); another is found at

G2/M. Most studies have concentrated on the G2/M
transition (the mitosis checkpoint). This cell cycle
control mechanism is based on two series of proteins:
the cyclin-dependent protein kinases (CDKs), which
phosphorylate a series of downstream proteins on
serine and threonine residues, and the cyclins, which
along with other regulatory proteins bind to CDKs to
alter their activity. Different cyclins are synthesized
during different parts of the cell cycle (mitotic cyclins
and G1 cyclins) (Fig. 21). In mammalian cells, there are
at least six types of cyclin (A, B, C, D, E, and F). Entry
into mitosis is stimulated by activation of a CDK by
mitotic cyclin; in amphibians, this is known as maturation promotion factor (MPF) and consists of a cyclin
and a cyclin-dependent kinase called cdc2another is
known as cdc4. Repetitive synthesis and degradation
of cyclins is associated with the cell cycle. Activation
of MPF drives mitosis, and degradation of cyclin then
allows the cell to enter the S phase. As we shall see
later, there is a set of proteins that can inhibit CDK
known as cyclin-dependent kinase inhibitors (or
inhibitors of cyclin-dependent kinasesINKs). The
activity of the CDK can therefore be abruptly switched

on and off during different parts of the cell cycle.
In part, this process is controlled by the synthesis
and degradation of the cyclins, but it is also affected
by phosphorylation of the CDK on two sites (Fig. 22),
which is controlled by two phosphatases known as
Wee1 and MO15. Removal of one of the phosphate
residues by means of another phosphataseCdc25
is then needed for activation of MPF. It is thought that
the MPF autocatalytically activates itself, resulting in a
steady rise in MPF levels during the cell cycle until the
critical point when an explosive increase in activity
takes place and drives the cell irretrievably into the M
phase. Cdc2 is associated with the G1/S, and the G2/M
transitions and cdc4 and cdc6 are associated with
start, but are bound to different cyclins (cyclin B at
mitosis and cyclin E and A at start at G1/S). The Kip/cip
family of cyclin-dependent kinases, which include p21,
p27 and p57, are capable of binding to and inhibiting
most cyclin/CDK complexes. The expression of these
CDK inhibitors is often dependent on upstream events
that are activated by physiological signals, such as
DNA damage, serum deprivation, or contact inhibition.
23
In contrast, the INK4 family of CDK inhibitors, including p15, p16, p18, and p19, bind to and inactivate
D-type cyclins.
Growth Factors and the Control of the Cell Cycle
Figure 21 The core of the cell cycle control system. CDK is

thought to associate successively with different cyclins to trigger
downstream processes of the cycle. CDK activity is terminated
by cyclin degradation. Abbreviation: CDK, cyclin-dependent protein kinase.
In mammalian cells, many peptide growth factors are

potent inducers of cell division. These include plateletderived growth factor; epidermal growth factor (EGF);
insulin and insulin-like growth factors I and II; acidic
and basic fibroblast growth factors (a-FGF and b-FGF);
transforming growth factor a (TGF-a), which binds to
the EGF receptor); and transforming growth factor b
(TGF-b), which is structurally unrelated to TGF-a and
is generally inhibitory. Many of these growth factors
also act as proto-oncogenes.
These growth factors act over very short distances and may act on the cell that produces them
(autocrine action) or on neighboring cell types (paracrine action). Many are found in serum, and this is one
reason why serum is required to maintain cells in
culture. High-affinity receptors for these growth factors are found on the cell membrane, and, in culture,
cells compete for growth factors. As well as requiring
growth factors for cell division, many cells require
signals produced by intercellular contact and adherence (anchorage) mediated by means of adhesion
molecules before they divide (anchorage-dependent
growth).
Many growth factor receptors contain an endogenous tyrosine kinase, which is activated by ligand
binding (Fig. 23). These receptors interact with protooncogenes, such as G proteins (such as ras), because
tyrosine phosphorylation of the receptors facilitates
binding of intermediate adapter proteins with socalled SH2 domains, which then link with other
proteins (SOS, GRB2) that activate the ras pathway.
Phosphorylation of downstream protein by growth
factor receptors activates several early-response
genes, which are stimulated within a few minutes of
adding growth factor. In contrast, delayed-response
gene activation requires protein synthesis. Many
early-response genes activated by growth factor receptors control gene transcription; they include myc, jun,
and fos, which are known to be crucial for gene
transcription. In particular, myc is thought to be
closely linked to activation of cell division.
Figure 22 Genesis of MPF activity. Cdc2 becomes associated with cyclin as the level of cyclin gradually increases; this enables Cdc2
to be phosphorylated by an activating kinase on an activating site, as well as by Wee1 kinase on Cdc2s catalytic site. The latter
phosphorylation inhibits Cdc2 activity until this phosphate group is removed by the Cdc25 phosphatase. Active maturation promotion
factor (MPF) is thought to stimulate its own activation by activating Cdc25 and inhibiting Wee1, either directly or indirectly.
24
Neal
Figure 23 Typical signaling pathway for stimulation of cell

proliferation by a growth factor. This greatly simplified diagram
shows some of the major steps. It omits many of the intermediate
steps in the relay system.
to engulf and digest it. How this recognition process is

mediated is unclear. The macrophages do not secrete
inflammatory cytokines and chemokines; therefore,
inflammation does not occur: apoptosis is quite different from necrosis.
Several genes are involved in this process. For
instance, in thymic cells damaged by radiation, p53 is
upregulated, an effect that increases the level of p21
(an inhibitor of cyclin-dependent kinase). This, in
turn, slows down cell division, placing the cell in G1
arrest and allowing DNA repair to take place. It is
thought that p21 is not directly concerned with the
onset of apoptosis. In some cell types, such DNA
damage initiates apoptosis, which is also associated
with the upregulation of p53. There are a number of
proteins known to promote apoptosis, including the
bax family. Bax binds to and is inhibited by Bcl2.
Homodimers of bax, which is upregulated by p53,
bring on apoptosis. The Bcl2 protein binds and inactivates bax homodimers, thereby preventing apoptosis.
Some types of apoptosis are not activated through
p53. It is thought that the end point of apoptosis is the
activation of a set of ICE-like cysteine proteases.
Production of activated caspases is crucial to this
process and may occur as a consequence of DNA
damage. Detection of DNA damage and determination of the outcome, whether repair, apoptosis or
carcinogenesis, is the responsibility of several genes,
including BRCA1, BRCA2, ATM (ataxia telangiectasia), and p53. It is a highly complex process, which as
noted above is frequently disrupted in cancer.
Aberrant expression of p53 and upregulation of
Bcl2 will decrease the rate of apoptosis (although
apoptosis is often higher in tumors with mutated
p53 and upregulated Bcl2, because other mechanisms
are activated). The point is that, in many tumors, DNA
damage and the presence of mutated tumor suppressors and oncogenes will stimulate cell division, which
will be in excess of the rate of apoptosis, so continued
deregulated proliferation will probably lead to more
DNA mutations occurring with each cell cycle.
APOPTOSIS OR PROGRAMMED CELL DEATH
STRUCTURE OF DNA AND RNA
In many tissues, such as those of the hematopoietic

system and the lining of the gut wall, proliferation and
cell division are balanced by a process of planned cell
death, the control of which is every bit as important as
that involved in cell division. Programmed cell death
also occurs in tumors, but the control of it is disrupted.
Apoptosis is a carefully orchestrated event in
which the cell is programmed to die. The nucleus
becomes shrunken and pyknotic, and the cytoplasm
shrinks. The nucleus is sometimes extruded and may
be engulfed by neighboring cells. No inflammatory
reaction is excited by the process. This process
is characterized by disintegration of the nuclear
envelope and marked condensation of DNA into
chromatin. On electrophoresis, the DNA assumes a
characteristic banding pattern, implying that it
has been cut by endonuclease enzymes. Nearby
macrophages recognize the apoptotic cell and begin
Nucleic acids form the essential basis of DNA and RNA.

These are made of the following three compounds:
Bases: Nitrogenous ring compounds called
purines (adenine and guanine) or pyrimidines
[cytosine, thymine (DNA), or uracil (RNA)].
2. Pentose sugars: Two typesribose (b-D-ribose:
RNA) and deoxyribose (b- D -2 deoxyribose:
DNA).
3. Phosphate: The phosphate bond is attached to the
50 C hydroxyl group, and in a nucleic acid, it
bonds to the 30 C position of the adjacent sugar
residue.
The nucleoside is defined as a base plus a sugar
(adenosine, guanosine, cytidine, thymidine, or uridine).
The nucleotide is defined as a base plus a sugar plus a
phosphate (Fig. 24). In addition to being the building
blocks of DNA and RNA, nucleotides are used.
1.
25
Figure 24 DNA synthesis. The addition of a deoxyribonucleotide to the 30 end of a polynucleotide chain is the
fundamental reaction by which DNA is synthesized. As
shown, base-pairing between this incoming deoxyribonucleotide and an existing strand of DNA (template strand) guides
the formation of a new strand of DNA with a complementary
nucleotide sequence.
As high-energy intermediates (such as ATP or

guanosine triphosphateGTP)
In combination with other structures to form
enzymes (coenzyme A)
As signaling molecules (such as cyclic adenosine
monophosphateAMP)
RNA forms a single chain, whereas DNA forms

a double helix held together by hydrogen bonding
between bases on opposing strands; the individual
nucleotides are held together by phosphate linkages
between sugar residues (Fig. 24), the individual bases
being suspended from the other end of the sugar
residue. The sequence of bases forms the basis for all
genetic information and is organized as a series of
triplets, which code for individual amino acids. The
hydrogen bonding between bases on opposing
strands is not random, and on opposite sides of
DNA, the following bases are always matched as

complementary pairs (or Watson-Crick base pairs):
l
l
G with C
A with T (or A with U in RNA)
The triplet code for DNA and the corresponding

amino acids are shown in Tables 2 and 3. A section of
DNA can be read in a number of different reading
frames, depending on which particular nucleotide is
the start of the reading frame.
The double helix formed by DNA occupies 3.4 nm
for one complete turn, which contains 10 nucleotide
pairs per turn and has a major and a minor groove. A
single strand of DNA acting as a template will induce
the formation of a second complementary strand if the
appropriate nucleoside triphosphates and DNA polymerase are present in solution. Errors introduced by
such complementary replication will induce a point
26
Neal
Table 2 The Genetic Code

1st position
(50 end)
Table 4 The Size of Some Human Genes
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
Sea
Sea
Sea
Sea
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
Tyr
Tyr
STOP
STOP
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
Cys
Cys
STOP
Trp
Arg
Arg
Arg
Arg
Sea
Sea
Arg
Arg
Gly
Gly
Gly
Gly
3rd position
(30 end)
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
Table 3 Amino Acids and Their Symbols

Letter
Symbols
Amino acid
Codons
Ala
Alanine
C
D
E
F
G
Cys
Asp
Glu
Phe
Gly
Cysteine
Aspartic acid
Glutamic acid
Phenylalanine
Glycine
H
I
K
L
His
Ile
Lys
Leu
Histidine
Isoleucine
Lysine
Leucine
M
N
P
Met
Asn
Pro
Methionine
Asparginine
Proline
Q
R
Gln
Arg
Glutamine
Arginine
Sea
Serine
Thr
Threonine
Val
Valine
W
Y
Trp
Tyr
Tryptophan
Tryosine
GCA, GCC, GCG,

GCU
UGC, UGU
GAC, GAU
GAA, GAU
UUC, UUU
GGA, GGC, GGG,
GGU
CAC, CAU
AUA, AUC, AUU
AAA, AAG
UUA, UUG, CUA,
CUC, CUG,
CUU
AUG
AAC, AAU
CCA, CCC, CCG,
CCU
CAA, CAG
AGA, AGG, CGA,
CGC, CGG,
CGU
AGC, AGU, UCA,
UCC, UCG,
UCU
ACA, ACC, ACG,
ACU
GUA, GUC, GUG,
GUU
UGG
UAC, UAU
mutation where a single inappropriate base is

inserted.
BASIC GENETIC MECHANISMS

Genes
Certain sections of DNA are arranged into genes,
which are defined as lengths of DNA that produce
specific mRNA and protein, although this definition is
Protein
Gene
size (in kb)
MRNA
size (in kb)
Number
of introns
b-globulin
Insulin
Protein kinase C
Albumin
Catalase
LDL receptor
Factor VIII
Thyroglobulin
Dystrophin
1.5
1.7
11
25
34
45
186
300
>2000
0.6
0.4
1.4
2.1
1.6
5.5
9
8.7
17
2
2
7
14
12
17
25
36
>50
not quite correct because, in some organisms, several

species of RNA may be produced from one gene, and
spliced variants of mRNA in higher organisms are
commonly tissue specific (such as splice variants for
FGF receptors and p15, which is an alternative spliced
variant of p16). The size of genes varies a great deal
depending partly on the size of the protein to be
produced (Table 4). Even within a gene sequence,
although all of it is transcribed as primary RNA, not
all sections are exported as mature RNA (Fig. 25). The
gene is arranged into exons, which are exported from
the nucleus as mature mRNA, and introns, which are
excised from the primary transcript mRNA (Fig. 25)
by means of RNA splicing and degraded in the
nucleus.
Each gene produces messenger RNA (mRNA),
and most genes (except those encoding ribosomes and
tRNA) eventually produce proteins, some of which
control the synthesis and modification of other compounds, such as nucleic acids, lipids, and carbohydrates. Less than 1% of DNA is transcribed into
mature mRNA. Proteins have myriad functions, ranging from structural proteins, such as actins, right
through to those that precisely control the rate of
gene transcription.
mRNA Synthesis
After the opening up of the chromatin structure that
occurs as a result of acetylation of histones, the next
step is the synthesis of primary or heterogeneous
mRNA (hnRNA), which is initiated by RNA polymerase type II, a large multiunit enzyme (Fig. 26). This
enzyme binds to the promoter unit of the gene that is
to be transcribed in conjunction with several other
proteins called transcription factors.
The promoter region contains the site at which
transcription factors bind and that is rich in TATA
sequences (the so-called TATA box); it is situated
about 25 nucleotides upstream of the start site of the
gene. An AUG codon (methionine) always represents
the start of each gene. RNA polymerase opens up the
double-stranded helix, and RNA is synthesized by the
polymerase, moving from the 30 to the 50 direction of
DNA (i.e., the RNA is extended from the 50 to the 30
direction) (Fig. 26). This elongation continues until the
polymerase reaches a STOP signal (UAA; UAG).
27
Figure 25 The organization of genes on a typical vertebrate chromosome. Proteins that bind to the DNA in
regulatory regions determine whether a gene is transcribed; although often located on the 50 side of a gene,
as shown here, regulatory regions can also be located in
introns, in exons, or on the 30 side of a gene. Intron
sequences are removed from primary RNA transcripts to
produce messenger RNA (mRNA) molecules. The figure
given here for the number of genes per chromosome is a
minimal estimate.
Figure 26 The synthesis of an RNA molecule by RNA

polymerase. The enzyme binds to the promoter sequence
on the DNA and begins its synthesis at a start site within the
promoter. It completes its synthesis at a STOP (termination)
signal, whereupon both the polymerase and its completed
RNA chain are released. During RNA chain elongation,
polymerization rates average about 30 nucleotides per
second at 378C. Therefore, an RNA chain of 5000 nucleotides takes about three minutes to complete.
28
Neal
Thirty nucleotides are added per second, so a chain of

5000 nucleotides will take about three minutes to
make. Each strand of the double helix could, in theory,
be used to copy into RNA, but, in any one section of
DNA, only one strand is used, although, in any one
chromosome, different strands are used in different
parts.
There are three types of RNA polymerase (types
I, II, and III). Type I RNA polymerase makes large
ribosomal RNA and type III makes tRNA and the
small 5S ribosomal RNA. Type II RNA polymerase is
responsible for synthesis of the other types of RNA.
Both ends of mRNA are modified. The 50 end is
capped by a methylated G nucleotide, whereas to
the 30 end a polyadenylated (poly-A) tail is added. The
50 cap plays an important part in protein synthesis and
protects the mRNA from degradation. The poly-A tail
aids the export of mRNA, it may stabilize mRNA in
the cytoplasm, and it serves a recognition signal for
the ribosome. The primary RNA transcript from the
gene is known as hnRNA, most of which is rapidly
destroyed by removal and splicing of the remaining
mRNA. The presence of the poly-A tail allows purification of the mRNA from other types of RNA, a fact
that is useful in purifying mRNA for studies.
Introns and Exons: mRNA Processing

Early evidence for the presence of introns was the
finding that mature RNA is relatively short and
when, in experiments, it was annealed to DNA containing the gene of interest, it was found that the DNA
formed several large loops, only short sections of DNA
being adherent to mRNA. Primary mRNA transcribed
from the loops of DNA not bound to the mature

mRNA does not form part of mature mRNA and is
excised as introns. hnRNA, which is the primary
transcript of the gene, is much longer than the mature
mRNA; although it has the 50 cap and poly-A tail, it
also contains several introns. The hnRNA in the nucleus is coated with proteins and small nuclear ribonucleoproteins (snRNPs), which are somewhat similar to
ribosomes, but much smaller (250 kDa compared with
4500 kDa); they are crucial to the excision of introns.
Patients with systemic lupus erythematosas form autoantibodies against one or more snRNPs. As pointed out
above (Table 4), introns form much of the largest part
of hnRNA and of the genome itself. Introns can accumulate several mutations without necessarily affecting
protein function (i.e., they comprise the so-called junk
DNA). However, point mutations near the exon/intron
junction can have disastrous effects on protein function
if they result in introns not being removed (as in
thalassemia) and in the formation of longer sections
of mRNA and therefore of new types of protein.
Spliceosomes are formed by aggregation of several
snRNPs, ATP, and other proteins onto the exon/intron
junctionthey remove the intron as a lariat or
noose-like structure (Fig. 27). Most genes contain several introns. After excision of introns, the exons are
spliced together to form mature mRNA, which is then
exported from the nucleus via the nuclear pores.
In the formation of some proteins, differential
RNA splicing is the norm, producing different forms
of RNA and hence different proteins. This is achieved
by differential binding of distinct repressors or activators to the primary RNA transcript, which can alter
the splice site and therefore alter the exact structure of
Figure 27 The RNA splicing mechanism. RNA

splicing is catalyzed by a spliceosome formed
from the assembly of U1, U2, U5 and U4/U6
snRNPs (green circles) plus other components
(not shown). After assembly of the spliceosome,
the reaction occurs in two steps: in step 1, the
branch-point A nucleotide in the intron sequence,
which is located close to the 30 splice site, attacks
the 50 splice site and cleaves it; the cut 50 end of
the intron sequence thereby becomes covalently
linked to this A nucleotide, forming the branched
nucleotide. In step 2, the 30 -OH end of the first
exon sequence, which was created in the first
step, adds to the beginning of the second exon
sequence, cleaving the RNA molecule at 30 splice
site; the two exon sequences are thereby joined
to each other and the intron sequence is released
as a lariat. The spliceosome complex sediments
at 60S, indicating that it is nearly as large as a
ribosome. These splicing reactions occur in the
nucleus and generate mRNa molecules from
primary RNA transcripts (mRNA precursor
molecules).
the final mature mRNA. This occurs with the fibroblast growth factor receptors, which have several
different spliced variants and different affinities for
various members of the FGF family.
Control of Transcription
Following histone acetylation, various proteins bind to
the promoter regions of genes to act as promoters and
repressors. These can be classified into the following
types:
l
Helix-turn-helix proteins. These solely comprise

amino acids and have a structure that facilitates
binding into the major groove of DNAtheir
binding may block transcription or, conversely,
may force bending of the DNA molecule facilitating transcription. The homeo-domain proteins are
a type of helix-turn-helix protein involved in
sequential embryonic development. Each of these
homeo-domain proteins contains an identical section of 60 amino acids.
Zinc finger proteins. Some transcription factors are
rich in histidine and cysteine residues, which can
bind zinc, thereby bending the protein into a
finger-like shape. Steroid hormone receptors also
contain several zinc fingers and are thought to
function as transcription factors.
Proteins with a leucine zipper motif. Certain a protein
chains can form Y-shaped dimmers, which can
attach to DNA; the two chains are held together by
interactions between hydrophobic regions that are
rich in leucine.
Helix-loop-helix proteins. These form structures similar to leucine zippers.
These protein regulators of gene transcription

can turn genes on or off depending on how they link
with other transcription factors and RNA polymerase.
It is also clear that many gene-regulatory proteins act
at a distance upstream or downstream of the gene itself
(Fig. 28); in some instances this can be a considerable
distance from the promoter. These gene-regulatory proteins can have their function changed by increased de
novo synthesis when needed, by ligand binding, by
phosphorylation, or by combination with a second agent.
29
In addition to these specific transcription factors,

general transcription factors are also required. Several
proto-oncogenes are transcription factors. Fos and jun
combine when phosphorylated to form the AP-1 protein, which functions as a transcription factor. Several
growth factors interact through ras to activate MAP
kinase (mitogen-activated kinase). MAP kinase phosphorylates a protein called Elk-1, which itself activates
the transcription of fos.
Modification of DNA Can Alter Transcription

DNA that is tightly packaged around nucleosomes
or the type that is packaged into heterochromatin is
not easily transcribed. Unwinding of chromatin
is controlled by acetylation of histone residues (deacetylation producing chromatin condensation). Condensation of the second X chromosome in women may
affect the maternal or paternal copy at random, but
small zones of neighboring cells in tissues will have
the same X chromosome inactivated as a mosaic
pattern. Another method of inactivating genes is
heavy methylation of cytosine bases, which can result
in inactivation of either the maternal or the paternal
copies of particular genes. For instance, only the
paternal insulin-like growth factor II gene is active;
the maternal copy is inactivated or imprinted
throughout the whole body. Recent data have suggested that some tumor suppressor genes can be
inactivatednot through mutations or deletions, as
in the case of retinoblastoma or p53but through
heavy methylation [such as the MTS (multitumor
suppressor) gene, which encodes the p16 protein].
Posttranscriptional Modifications in mRNA

Stability
Another method of controlling how much protein
is produced by mRNA is to alter the stability of
mRNA, which is normally a nascent molecule with a
half-life of around 30 minutes to 10 hours. Some
mRNAs have lengths of AU-rich noncoding sequences at the 30 end, which stimulate the removal of the
poly-A tail, making the mRNA more stable. Some
Figure 28 Integration at a promoter. Multiple sets of

gene regulatory proteins can work together to influence a promoter, as they do in the eve stripe 2 module.
It is not yet understood in detail how the integration of
multiple inputs is achieved.
30
Neal
steroid hormones also increase the stability of certain

mRNA molecules. Modification of the mRNA molecule can be mediated by proteins or, in some instances, can be mediated by RNA sequences that have
intrinsic enzymic activity (i.e., they are RNA-ases).
Posttranslational modifications in peptide chains can
also lead to increased protein stability and increased
duration of action.
Short Hairpin RNA

Some years ago it was found in plants and some
primitive organisms that short sequences of RNA
forming a hairpin shape could regulate the half-life
of mRNA. Such short regulatory sequences are in fact
used in cell biology experiments to knock down the
function of certain genes. It was then found that the
production of such short hairpin RNA (shRNA)
sequences is an important part of the regulation of
mammalian gene function. The expression of such
shRNA sequences is now being actively studied in
human health and disease.
Ribosomal RNA
During cell division, a large number of new ribosomes
are required, and because the amplification process
involved in protein synthesis (mRNA being translated
into many protein molecules) is not available, most
cells contain tandemly arranged multiple copies of
ribosomal genes (around 200 per cell) on five pairs of
different chromosomes. A tandem repeat is formed
when duplicated DNA segments are joined head to
tail. The initial ribosomal RNA (rRNA) (Fig. 29) transcript is 13,000 nucleotides long and is cut into three to
form the 28S rRNA (5000 nucleotides), the 18S rRNA
(2000 nucleotides), and the 5.8S rRNA (160 nucleotides)
components of the ribosome. The 5S component of the
large ribosomal subunit is transcribed separately by
RNA polymerase III, and there are 2000 copies of the 5S
rRNA genes arranged as a single cluster.
Packaging of ribosomal RNA occurs in the nucleolus, in which we find several large loops of DNA
containing the tandem repeats for rRNA, which are
known as nucleolar organizer regions. Here rRNA is
transcribed by RNA polymerase I.
PROTEIN SYNTHESIS
When the mature mRNA reaches the cytoplasm after
passing through the nuclear pores, it becomes
attached to ribosomes, which catalyze the production
of a peptide chain. Each amino acid is brought to the
ribosome by its own specific small molecule of RNA,
the tRNA (Fig. 30), which has an anticodon at its
base corresponding to the codon for that particular
amino acid. Specific enzymes couple specific amino
acids to their particular tRNA molecule after the
amino acid is activated by the attachment of AMP,
after which it is known as adenylated amino acid. The
Figure 29 Eukaryotic ribosomes. Ribosomal components are

commonly designated by their S values, which indicate their
rate of sedimentation in an ultracentrifuge. Despite the differences in the number and size of their rRNA and protein components, both types of ribosomes have nearly the same structure,
and they function in very similar ways. Although the 18S and 28S
rRNAs of the eukaryotic ribosome contain many extra nucleotides not present in their bacterial counterparts, these nucleotides are present as multiple insertions that are thought to
protrude as loops and leave the basic structure of each rRNA
largely unchanged.
tRNA connected to its amino acid is referred to as an

aminoacyl tRNA because of the bond between the
amino acid and the tRNA. These mechanisms ensure
that the right amino acid is brought by the correct
tRNA to its specified position within the peptide
chain. The genetic code is degenerate because most
amino acids are coded for by more than one triplet, so
that there is more than one tRNA for each amino acid,
and a single tRNA can bind with more than one
codon. Many amino acids require that the codon is
accurate only in its first two positions (Table 2) and
will tolerate wobble in the third position. Each
incoming amino acid is attached to the carboxy end
of the growing peptide chain.
31
Figure 30 The cloverleaf structure of tRNA. This is

a view of a molecule after it has been partially
unfolded. There are many different tRNa molecules,
including at least one for each kind of amino acid.
Although they differ in nucleotide sequence, they all
have the three stem loops shown plus an amino acidaccepting arm. The particular tRNA molecule shown
binds phenylalanine and is therefore denoted tRNAPhe. In all tRNa molecules, the amino acid is
attached to the A residue of a CCA sequence at the
30 end of the molecule. Complementary base-pairings
are shown by red bars. Abbreviation: tRNa, transfer
RNA.
Each ribosome consists of two major parts: the

60S and 40S subunits. The large 60S subunit comprises
three separate parts: a 28S rRNA, a 5.5S rRNA, and a
5S rRNA (Fig. 31). These two subunits dock separately
(the small subunit attaches first) on the mRNA at the
AUG start codon (Fig. 31) and dock separately when
the STOP codon (UAG) is reached (Fig. 32). Identification of the initiation site is important because, in
principle, the mRNA can be read in any one of the
reading frames, depending on the exact start site.
Initiation factors assemble with the methionine
tRNA at the start site before translation occurs.
Usually, there are several ribosomes attached to
each mRNA molecule, so that at any one time there
are several peptide chains in various stages of completion of synthesis. About 50% of the weight of the
ribosome is RNA; the remainder comprises several
types of proteins. Initially, it was thought that these
proteins carried out the catalytic reactions of the RNA,
but it is now clear that the RNA itself has these

properties, and the function of the proteins is to
modify these enzymatic functions. The ribosome has
three binding sites for RNA; one for mRNA, one for
incoming tRNA (A site), and one for outgoing tRNA
and peptide chain (P site). On binding of an incoming
tRNA, the aminoacyl-RNA bond is lysed, and the
peptide bond is formed between the new amino acid
and the peptide chain. The peptidyl-RNA in the A site
is then moved to the P site by means of an energydependent reaction (GTP driven), freeing the A site
for a new amino acid.
Obviously, recognition of the correct amino acid
attached to the correct tRNA molecule is important.
Firstly, the attachment of the amino acid to the tRNA
is specific, and if an incorrect amino acid is bound to
the aminoacyl synthetase enzyme, it is removed by
hydrolysis. Secondly, the tRNA is attached to the
mSteRNA as a complex with elongation factors,
32
Neal
Figure 31 The initiation phase of protein synthesis in eukaryotes.
Figure 32 The final phase of protein synthesis. The binding of

release factor to a STOP codon terminates translation. The
completed polypeptide is released, and the ribosome dissociates
into its two separate subunits.
which prevent the amino acid from immediately

undergoing attachment to the peptide chain; and
there is a short delay, which allows an incorrect
tRNA to exit from the codon. It should be noted that
the control of the levels of protein formation is
achieved by the following three factors:
1.
2.
Controlling over gene transcription

Controlling the speed of mRNA degradation
3.
33
Controlling posttranslational peptide and altering

protein stability
For instance, upregulation of p53 in epidermal

cells following exposure to sunlight is achieved by
increasing the stability of protein that has already
been made.
3
Inflammation
Neville Woolf
Medical School Administration, University College London, London, U.K.
INTRODUCTION
Leukocyte Activation
Inflammation is a reaction to injury in living tissue

resulting in the accumulation of phagocytic cells and
components of circulating plasma in the injured area.
The inflammatory response is mediated via two biological pathways: the first involves changes in the
caliber and permeability of the vessels constituting
the microcirculation; the second is a complex series of
operations leadingtoactivationof leukocytes. Basically, the inflammatory reaction is a protective one
elicited by tissue injury or by the entry into a host of
nonself elements such as pathogenic microorganisms. Inflammationacts to digest the components of
dead tissue and to destroy and isolate the nonself
elements just alluded to. It should not be forgotten,
however, that the same functions which protect can
also have devastating consequences, as, for example,
when the inflammatory process becomes inappropriately activated in a systemic context, as in septic
shock.
This is a complex set of sequential processes, which

include
l
adhesion of leukocytes to microvascular endothelium in affected areas,

migration of leukocytes through gaps between
endothelial cells and across the basement membranes to reach the extravascular compartment,
attachment of phagocytes to infecting microorganisms, to dead or injured cells or to foreign material
at the site of injury,
engulfment (phagocytosis) by the leukocytes of
microorganisms or cell/tissue debris,
microbial killing or digestion of cellular debris by
phagocytes, and
effects on surrounding cells/tissues of chemical
mediators, either secreted or otherwise released
from leukocytes.
CAUSES OF INFLAMMATION
Changes in the Microcirculation
The causes of inflammation are as follows:
Injury is followed by an increase in caliber of arterioles, capillaries and venules at the site of its application.
Arterioles dilate as a result of a chemically mediated
relaxation of smooth muscle; capillaries and venules
do so passively as a result of the increased flow
mediated by arteriolar dilatation. This increase in
local blood flow leads to two of the classic signs of
acute inflammation: redness and heat.
Inflammation is characterized also by an
increase in permeability of the microvessels, thus
allowing more water and electrolyte, derived from
plasma, to escape into the interstitial tissue, as well as
the escape of high-molecular-weight proteins, such as
fibrinogen, not normally found in extravascular interstitial fluid. This process, termed exudation, causes
another of the classic signs of inflammation, swelling,
which may be localized, or may be the accumulation
of edema fluid within serosa-lined spaces, such as the
pericardial sac or peritoneal cavity.
l
l
l
l
l
Mechanical trauma such as cutting or crushing

Injury caused by living organisms
Chemical injury (exogenous or endogenous)
Excess ultraviolet or X-radiation
Injury due to extremes of heat (burns) or cold
(frostbite)
Ischemia sufficiently severe to cause death or
underperfused tissues
Injury caused by excessive or inappropriate operation of the immune system
THE FORMATION OF THE INFLAMMATORY

EXUDATE
Inflammatory edema and the formation of exudates
are the expression of a net increase in the volume of
water and electrolyte moving from the vascular to
the extravascular compartment in the injured area,
Chapter 3: Inflammation
35
Table 1 Patterns of Increased Vascular Permeability in Acute Inflammation

Immediate transient
Delayed persistent
Immediate persistent
Injury
Onset
Peak time
Mild
Almost immediately
5 min
Moderate
Up to 24 hr
424 hr
Severe
Within a few minutes
1560 min in experimental
circumstances
Site of leakage
Postcapillary venules
Duration
Mechanism
15 min
Histamine-induced
interendothelial gap formation
Mild type 1 hypersensitivity
Capillaries (relatively early peaking);

venules plus capillaries in later
peaking
Hours or days
Gap formation plus mild endothelial
cell injury
Sunburn
Example
this being associated with the escape also of largemolecular-weight proteins (such as fibrinogen) that
are normally held within the vascular compartment.
This process can be looked at in two ways. First,
we need to consider the forces that determine the
normal relationship between intra- and extravascular
fluid and, second, the anatomical routes via which
transfer of fluid and protein occurs.
Water, Solute, and Protein Leakage

Transport of water and solute across the microvascular
endothelium shows many of the characteristics of
ultrafiltration. Here, physical forces control the movement of fluid and electrolytes. The two forces normally
acting (in opposition to each other) in this context are
as follows:
l
Intravascular hydrostatic pressure, which tends to

push fluid out into the extravascular compartment
Plasma oncotic pressure exerted by proteins, most
notably albumin
An increase in the first force and a decrease in the

second greatly increase fluid loss from the vascular
compartment, a situation that is by no means confined
to the context of inflammation. Normally, the result of
interaction between these opposing forces is a small
net outflow of fluid from the microcirculation. This
excess fluid then drains from the extravascular compartment via the lymphatics. The importance of this
last step is dramatically illustrated in patients with
lymphoedema.
Chemical analysis of inflammatory exudates
shows a protein concentration and pattern that is
simply not attainable by the mechanisms mentioned
above and for which other explanations must be
canvassed. Such an explanation is readily found in
the many data indicating that injury is followed by an
increase in the permeability of microvessels. The
patterns of exudate formation that we see in mammalian inflammation are the functional expressions of the
severity and duration of injury and of the effects that
such injury has on microvascular endothelium.
In mild injury, such as that caused by histamine,
serotonin, or bradykinin, Majno and Palade proposed
that plasma leakage was due to the formation of gaps
Microvessels of all types

Severe damage to endothelial
cells and pericytes
Serious burns, major
mechanical trauma
between the endothelial cells of postcapillary venules.

Recent studies have confirmed this and show that
these gaps are coterminous with the sites of plasma
leakage.
Patterns of increased vascular permeability are
usually characterized in terms of two variables:
the time between the injury and the first recordable
changes in microvascular permeability, and the duration of the increase in such permeability. These patterns are described briefly in Table 1.
Neutrophil and Leukocyte Actions and

Activation in Acute Inflammation
One of the most important components in defense
against nonself elements is phagocytosis, first
described by the Nobel laureate Elie Metchnikoff. If
there is a lack of phagocytic cells or if those cells
cannot seek out, engulf and destroy infectious organisms, the life of the affected individual will be endangered by increased susceptibility to infection.
The pivotal cell in the early phases of any
inflammatory reaction is the neutrophil, which is
present in relatively high concentrations in the
blood, rapidly replaceable from bone marrow precursors and able to move more quickly than other
leukocytes.
The two sets of lysosomal granules, that have
led to their being called by some granulocytes contain
enzymes such as muramidase (lysozyme), proteases,
cationic proteins which act as reinforcing signals,
peroxidase, lactoferrin (an iron-binding protein
that inhibits bacterial multiplication) and alkaline
phosphatase.
The operations necessary for effective neutrophil
function have been listed on page 34. Few are more
intriguing than the first of these: the adhesion between
neutrophils and the microvascular endothelium, without which emigration of phagocytes from the blood
into areas of tissue injury cannot occur. Failure to
achieve a bond between neutrophils and endothelium
occurs in some rare inherited syndromes and is associated with repeated episodes of bacterial infection.
Adhesion between leukocytes and endothelium
is a two-stage process. In the first of these, the neutrophils move to the periphery of the column of
36
Woolf
flowing blood, adhere momentarily to the endothelial

surface and then roll along that surface. In the second
phase, the cells bind to the endothelial surface and
flatten out before migrating through interendothelial
cell gaps.
These two phases are mediated by two distinct
sets of ligands and receptors. In the rolling phase, the
microvascular endothelial cells in an injured area
express molecules known as selectins because of
their structural resemblance to lectins, a set of proteins, widely distributed in nature, that can distinguish with exquisite sensitivity between different
complex sugars. Two types of selectin are known.
One, E-selectin, is synthesized and expressed only
after injury. This occurs about 30 minutes after the
injury and usually reaches its peak in two to four
hours. E-selectin binds to a complex sugar on the
surface of white cells known as sialy Lewis X. Its
expression is triggered by several signals, including
the important cytokines interleukin-1 (IL-1) and tumor
necrosis factor (TNF)-a.
The second, P-selectin, is synthesized constitutively and is stored in organelles, the Weibel-Palade
bodies, within the endothelial cytoplasm. Following
stimulation by appropriate triggers, the P-selectin is
translocated to the endothelial surface, where it binds
to another leukocyte surface sugar, lacto-N-fucopentaose III.
In the second phase of leukocyte adhesion, the
endothelial components in the ligand-receptor interaction belong to a set of proteins known as the
immunoglobulin gene superfamily. These include
heavy and light chains of immunoglobulin, the a
and b chains of the T-lymphocyte receptor, major
histocompatibility peptides of both classes, b2-microglobulin, CD4 and CD8 molecules on T lymphocytes
and adhesion molecules on endothelium. The lastnamed comprise intercellular adhesion molecule
(ICAM) 1 and 2, vascular cell adhesion molecule
(VCAM)-1, and platelet-endothelial cellular adhesion
molecule (PECAM)-1. ICAM 1 and 2 play a significant
role in the binding of leukocytes to the endothelial
surface. PECAM-1 contributes to the egress of leukocytes from the microvessels, and treatment with antibodies raised against PECAM inhibits leukocyte
emigration, despite adhesion being perfectly normal.
ICAM 1 and 2 are expressed constitutively on endothelium, but at very low levels. Release of the cytokines IL-1 and TNF-a upregulates IC expression,
which reaches its peak about 24 hours after injury.
The leukocyte components belong to a family
of two-chained transmembrane proteins known as
integrins, which are expressed in many cell types.
Some integrins are cell specific; this is the case with
leukocytes, which express LFA-1, Mac1 and p150,95.
These have a common b chain (CD18), and it is
absence of this chain that underlies leukocyte adhesion syndromes.
Adhesion of leukocytes is followed by emigration from the small vessels, a process taking two to
nine minutes. Leukocyte pseudopodia are inserted
into interendothelial cell gaps, and the cells then
move across the vessel wall into the surrounding
tissues. As already mentioned, PECAM-1 plays an

important part in this process, as does C-X-C chemokine known as IL-8.
Movement of leukocytes out of the microvasculature and through the extravascular compartment
uses the same mechanisms that are involved in both
phagocytosis and intracellular granule movement.
They involve the plasma membrane of activated leukocytes and the peripheral zone of the leukocyte
cytoplasm.
As with any cell capable of movement, leukocytes move as a result of interaction between the
contrac-tile proteins actin and myosin. Stimulation of
leukocytes by chemical signals results in an increasing
proportion of the intracellular actin becoming
arranged in a filamentous form, a process which is
controlled by the expression of various proteins that
can be stimulated or inhibited by calcium fluxes
within the cells.
Phagocytes are stimulated not merely to move in
the course of inflammatory reactions but also to move
in a directed and purposive manner toward areas of
tissue injury or of invasion by microorganisms. This
is termed chemotaxis, which, as its name implies, is
controlled by a series of chemical messengers. For
such a system to work, there must be adequate
l
generation of signals that attract leukocytes to the

site of injury,
reception of these signals by protein or glycoprotein molecules on the surfaces of leukocytes, and
transduction of the received signals so that the
target leukocytes translate the former into action.
These signals, the chemical mediators of inflammation, are discussed in a later section of this chapter.
Reception and transduction of chemotactic signals is likely to start with the binding of the signal to a
transmembrane receptor protein on the surface of the
phagocyte. This is known to be true in the case of
both the 5a component of the complement system and
the formylated peptides released from bacteria in the
course of bacterial protein synthesis. Occupation of a
ligand-binding site on a surface receptor produces
conformational changes that activate a G protein
lying just beneath the plasma membrane and connected to the latter by a farnesyl bond. This in turn
activates phospholipase, which cleaves phosphoinositol diphosphate (PIP2). Cleavage of this molecule
releases diacyglycerol and inositol triphosphate. The
former activates protein kinase C, and the resulting
protein cascade causes degranulation and secretion of
granule contents; the latter produces calcium fluxes
within the cell, which mediate chemotactic movement, and also releases arachidonic acid from cell
membranes, with synthesis of prostaglandins and
leukotrienes.
Cyclic nucleotides also play an important role in
initiating leukocyte movement. cAMP and cGMP act
in an opposing manner and may constitute a control
system for regulating chemotactic movement and
degranulation. cGMP enhances chemotactic movement, the release of histamine and leukotrienes from
mast cells, the release of lymphokines from activated
T lymphocytes and the release of granule contents

from neutrophils. In this context, the main effect of the
cyclic nucleotides is probably exerted on the microtubules. cGMP promotes assembly of microtubules
from tubulin, and cAMP has the opposite effect.
Thus, drugs (such as levamisole) that raise the intracellular content of cGMP enhance the movement of
phagocytes toward the source of chemoattractant substances and reverse the depression in chemotaxis that
may follow a number of viral infections.
PHAGOCYTOSIS
Phagocytosis is the biological raison detre of neutrophils. Before it can occur, the neutrophils traveling up
their chemotactic gradient must recognize the particles, living or dead, which are to be engulfed and
attach to them by means of ligand-receptor interactions. It has long been known that bacteria coated with
immunoglobulin or dead tissue particles that have
been exposed to fresh serum are more readily phagocytosed than those that have not. This coating of
particles with protein is called opsonization.
Opsonins are either of the following:
l
Immunoglobulins of the IgG class. For successful

attachment of phagocytes to take place, the Fc
fragment of the immunoglobulin must be intact.
The C3b component of complement. This is of
great biological value in defense against infection
since complement activation by the alternate pathway does not require interaction between bacterial
antigens and specific antibody.
The apparent restriction of opsonization to these

two protein classes indicates that the phagocyte plasma membrane is the site of receptors both for the Fc
fragment of immunoglobulin and the C3b component
of complement.
For engulfment of bacteria, nonliving foreign
particles, or cell and tissue debris, finger-like pseudopodia project from the plasma membrane of the
phagocyte and fuse on the far side of the object to
be engulfed, thus enclosing it within a vesicle formed
of plasma membrane (the phagosome). The phagosome then buds off from the inner surface of the
plasma membrane and migrates into the cytoplasm
of the neutrophil or macrophage. The boundaries of
the phagosome are formed of inverted plasma membrane, the inner lining of the phagosome being composed of the outer layer of the phagocyte plasma
membrane.
Phagocytosis is an energy-dependent process
that is inhibited by anything that interferes with
ATP production. It is likely that the same mechanisms
that power phagocyte movement are involved in
phagocytosis and substances that inhibit chemotactic
movement also inhibit phagocytosis.
Lysosomal Fusion and Degranulation

Phagosome formation is followed by movement of the
lysosomes and fusion of the phagosome/lysosome
37
membranes, this being associated with disappearance

of the intralysosomal granules. The process is
extremely rapid, and, again, as is the case with
phagocytosis, is inhibited by any chemical substance
that interferes with phagocyte movement.
BACTERIAL KILLING
The principal method of bacterial killing is oxygendependent. Phagocytosis is associated with a burst of
oxidative activity known as the respiratory burst. This
results in a stepwise reduction of molecular oxygen,
ending in the formation of hydrogen peroxide, as
follows:
l
The respiratory burst is associated with a 2- to

20-fold increase in oxygen consumption, compared with a resting phagocyte, and there is a
significant increase in glucose metabolism via the
hexose monophosphate shunt.
The reduction of molecular oxygen is catalyzed by
a nonheme protein oxidase believed to be localized in the phagocyte plasma membrane. The
presence of this oxidase is crucial for this type of
bacterial killing; its absence leads to the congenital
disorder known as chronic granulomatous disease
of childhood.
The hydrogen donor for oxygen reduction is either
reduced nicotinamide adenine dinucleotide
(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The presence of one
of these is an essential link in the chain because
hydrogen peroxide formation requires regeneration of NAD to NADP. The logical correlate of
this is a requirement for adequate amounts of
intracellular glucose-6-phosphate dehydrogenase
if bacterial killing is to proceed normally.
The reduction of molecular oxygen involves the
gain of only one electron, resulting in the formation of an oxygen free radical, the superoxide
anion (O2"). About 90% of the molecular oxygen
consumed in the respiratory burst is converted
into superoxide anion.
The reaction of two superoxide anions in the
presence of water results in the formation of
hydrogen peroxide and molecular oxygen. This
is termed a dismutation reaction and is catalyzed
by the enzyme superoxide dismutase. Other highly reactive oxygen species formed in association
with phagocyte activation include the highly reactive hydroxyl radical (OH), singlet oxygen and
hypochlorous acid.
It is now believed that the most potent oxygendependent bactericidal activity in phagocytes is associated with hydrogen peroxide. This molecule, by
itself, has significant bactericidal activity, but this is
very markedly potentiated by a reaction occurring
between hydrogen peroxide and intracytoplasmic
halide ions that is catalyzed by the lysosomal enzyme,
myeloperoxidase. The bactericidal effect is probably
mediated by halogenation or oxidation of cell-surface
components.
38
Woolf
Other Mechanisms of Bacterial Killing
DEFECTS IN NEUTROPHIL FUNCTION
While oxygen-dependent killing is pivotal in defense

against infection, it is not the only bactericidal mechanism. Some are related to the character of lysosomes
and include the following:
These defects may be quantitative or qualitative.

Even if all the operations described above are
carried out normally, successful neutrophil defense
against microorganisms requires an adequate number
of these cells. Thus, neutropaenia from any cause
confers a major increment of risk of infection. This
situation can occur in any form of marrow failure such
as may be caused by the following:
Low pH within the lysosome (3.54.0). This in

itself may be bactericidal or bacteriostatic. In addition, the acid milieu promotes the production of
hydrogen perioxide.
Lysosomes containing lysozyme (muramidase), a
lowmolecular weight cationic protein that attacks
the mucopeptide cell walls of some bacteria.
Lactoferrin, an iron-binding protein found within
lysosomes that inhibits the growth of several
microorganisms.
Lysosomal cationic proteins and hydrolases.
l
l
Drugs, chemotherapeutic agents and toxins

Bone marrow infiltration by large numbers of
tumor cells
Bone marrow fibrosis
Qualitative defects, of which there are many, can

be considered most conveniently in relation to the
specific functions which are defective. This is set out
in Table 2.
Table 2 Defects in Neutrophil Function

Function affected
Disorder
Characteristics
Migration and
chemotaxis
Lazy leukocyte syndrome
Defective response to chemotactic signals; nature of defect not yet

elucidated.
Typically affects fair-skinned, red-haired females; patients suffer from
recurrent cold staphylococcal abscesses.
Poor leukocyte movement reversed by insulin and glucose.
Rare autosomal recessive syndrome occurring in humans, cattle, mink,
and certain strains of mouse; characterized by partial albinism, giant
lysosomal granules in neutrophils and enhanced susceptibility to
infection; believed to be related to failure of assembly of microtubules
from tubulin; can be reversed (ex vivo) by agents that increase
intracellular cGMP.
Because of absence of the CD18 b subunit of the integrins expressed on
neutrophil surface; affects neutrophils only since other leukocytes
express and adhesion molecule binding to Vascular cell adhesion
molecule on endoethelium.
Can occur after steroids or phenylbutazone
Jobs syndrome
Poorly controlled diabetes mellitus
Chediak-Higashi syndrome
Leukocyte adhesion deficiency

syndrome
Disorders of
phagocytosis
Drug-related inhibition of leukocyte

movement
Deficiencies in release of
chemotactic signals
Opsonin deficiencies
Failure of engulfment
Failures in lysosomal fusion
Disorders of
bacterial killing
Chronic granulomatous diseases

of childhood
Affects mainly the complement system.

Deficiencies in complement or IgG; some patients with sickle-cell disease
show opsonin deficiency believed to be due to failure to activate the
alternate pathway of complement activation.
Can occur in hyperosmolar states; may also be caused by drugs such as
morphine analogues.
May be caused by drugs (steroids, colchicine, certain antimalarial
agents).
A rare disorder of childhood, sometimes X-linked and sometimes
occurring as an autosomal recessive disorder; characterized by
recurrent bacterial infections involving skin, bones, lungs and lymph
nodes; lymph nodes draining infected areas are often enlarged and
show sinusoids packed with mononuclear phagocytes and focal
granuloma-like aggregates of mononuclear cells.
The functional defect is failure of the respiratory burst (see page 37) and
thus failure of oxygen-dependent killing.
This reflects a defect in the cytochrome b-245 oxidase system that has
two subunits. The X-linked syndrome is due to a mutation in the gene
encoding the larger subunit, and in most cases, no gene product at all
is produced.
About one-third of patients with CGD have the autosomal recessive form,
and here the defect arises as the result of a mutation in the gene
encoding the smaller subunit or in the cytosolic components of the
nicotinamide adenine dinucleotide phosphate oxidase system.
CHEMICAL MEDIATORS OF ACUTE

INFLAMMATION
The process involved in acute inflammation, especially those controlling the activation of neutrophils, are
under the control of a very large number of chemical
signals. These mediators can be broadly classified
under the rubrics exogenous and endogenous.
Only one group of exogenous mediators is recognizedthe formylated peptides. These are low
molecular weight compounds produced by microorganisms in the course of protein synthesis, and all
have methionine as the N-terminal residue. The most
powerfully acting of these peptides is formylated
methionine-leucyl-phenylalanine (FMet-Leu-Phe), for
which neutrophils possess a surface receptor. FMetLeu-Phe can also cause the expression of some adhesion molecules by microvascular endothelium.
39
great biological importance in the defense against

infection.
Whichever pathway is involved, activation of
complement yields chemical species that
l
coat microorganisms, thus functioning as opsonins;

lead to lysis of cell membranes via the formation
of a membrane-attack complex; and
influence both the vascular and cellular components of the inflammatory reaction.
Protein Mediators: Cytokines and Chemokines
Activation of cells involved in inflammation and in

the immune response is associated with the release of
many lowmolecular weight proteinsthe cytokines
and chemokines. Only a few of the most important
will be mentioned here.
Endogenous Mediators
Tumor Necrosis Factor a
The endogenous mediators of inflammation are

grouped as shown in Table 3.
While it is impossible in a chapter of this length
to comment in any detail on these multiple signaling
systems, a few points are worth making.
This molecule derives its name from the fact that the
serum of animals given bacterial endotoxin causes
necrosis when injected into tumors. The active principle of such serum is the cytokine TNF-a. It is synthesized and secreted by activated macrophages, mast
cells and T lymphocytes. At low concentrations, TNFa upregulates the inflammatory response. It induces
expression of adhesion molecules, thus promoting
adhesion and migration of leukocytes from the microvessels to the extravascular compartment. It enhances
the killing of intracellular organisms such as Mycobacterium tuberculosis and Leishmania and acts as a positive feedback loop for the production of a variety of
cytokines.
At high concentrations (such as we may see in
endotoxin shock), TNF-a acts systemically. In this
context, it acts as a pyrogen, activates the clotting
system, stimulates production of acute-phase proteins
by the liver, inhibits myocardial contractility by stimulating nitric oxide production and causes inappropriate vasodilatation by the same mechanism.
Complement
The complement system consists of about 20 different

proteins, functionally interlinked to play a significant
part in immune responses and inflammation. Activation of complement may be achieved in two ways. In
the first, the classical pathway, the union of antibody and antigen leads to the binding of a protein
known as C1q. This step initiates the whole complement activation cascade. An alternate pathway
exists in which no antibody/antigen union is
required, and which can be triggered in many ways,
including the presence of many strains of bacteria.
The existence of the alternate pathway is clearly of
Table 3 Endogenous Mediators of Inflammation.
Interleukin-1
Source
Mediators
Activated plasma
protein cascades
The complement system

The kinin system
The intrinsic blood-clotting pathway
The fibrinolytic system
Amines and proteins

stored within cells
and released on
demand
Histamine
5-hydroxytryptamine
Lysosomal enzymes and other
proteins
Newly synthesized in
cells and released
from them on
demand
Lipids
Prostaglandins
Leukotrienes
Platelet-activating factor
The principle sources of IL-1 are monocytes and

macrophages, but other cell types, such as endothelial
cells and certain epithelial cells, may also produce this
cytokine. IL-1 is a potent regulator of both local
inflammatory events and many systemic ones. Its
synthesis is stimulated by various microbial products,
most notably bacterial endotoxin, and also by various
cytokines. Locally, it upregulates adhesion molecule
expression by endothelial cells, thus promoting adhesion of both leukocytes and platelets. Systemically,
IL-1 produces fever and promotes the release of
prostaglandins, glucocorticoids and acute-phase proteins. While it shares some of the functions of TNF-a,
it lacks certain others, such as the ability to produce
hemorrhagic necrosis in tumors.
This commonality of some functions between
two molecules as different from each other as IL-1
Proteins
Cytokines such as interleukin-1
and tumor necrosis factor a and
a and b chemokines
40
Woolf
and TNF-a is not without interest. Many genes that

are switched on in inflammatory diseases have their
expression induced by IL-1 and TNF-a and, in some
cases, TNF-neutralizing antibodies have had a beneficial effect in certain inflammatory disorders, such as
rheumatoid arthritis. The link between these two
cytokines appears to be that, after binding to their
individual cell-surface receptors, both TNF and IL-1
activate a cytoplasmic form of the transcription factor
NF-kB. DNA-binding motifs for NF-kB are found in
the promoters and enhancers of more than 50 genes
that are known to be activated in inflammation.
Chemokines
Chemokines are lowmolecular weight proteins (811

kDa) that are important in initiating and sustaining
inflammatory reactions. Some preferentially attract
neutrophils; others attract monocytes. They are divided
into two families: a and b.
The a family is encoded by genes located on
chromosome 4. They are characterized by an amino
acid sequence in which two terminal cysteines are
separated by some other amino acid, thus giving the
sequence Cys-X-Cys. Included in this group are IL-8,
b-thromboglobulin, gro-a, gro-b, and gro-g, and platelet factor 4.
The main source for the a chemokines is the
monocyte, though some are produced also by T lymphocytes and endothelial cells. IL-8 mediates the rapid
accumulation of neutrophils in inflamed tissues by
inducing the expression of neutrophil-binding adhesion molecules on the surface of endothelial cells in the
microvessels in injured areas. The chemokine gro acts
also as a neutrophil chemoattractant as well as promoting the release of lysosomal enzymes. b thromboglobulin and platelet factor 4 are released from activated
platelets and stimulate fibroblasts.
b chemokines are encoded by genes located on
chromosome 17. They are potent attractants for memory T cells and monocytes.
Monocytes and Macrophages in Acute

Inflammation
The cellular component of acute inflammation
includes another type of phagocyte, the mononuclear
phagocyte, which exists in two forms. An intermediate form known as the monocyte circulates in the
blood; after migration into the tissues, it differentiates
into the macrophage.
Monocytes have a half-life of about 222 hours,
three times as long as that of the neutrophil. They are
derived from bone marrow, and bone marrow
destruction leads to a failure of injury to elicit a
mononuclear cell response. Maturation from monocyte to macrophage within the tissues is accompanied
by considerable structural and functional increase in
the phagocytic, lysosomal and secretory apparatus, so
that
l
l
the cell becomes larger,

the plasma membrane becomes more convoluted,
l
l
lysosomes increase in number, and

both the Golgi apparatus (secretion) and the endoplasmic reticulum (protein synthesis) become
more prominent.
Like the neutrophil, the macrophage possesses

surface receptors for the Fc component of immunoglobulin as well as for complement components.
Again, like the neutrophil, the macrophage depends
on anaerobic glycolysis for its energy needs during
phagocytosis and has a respiratory burst after engulfment. Macrophages have certain advantages over
neutrophils in that they
l
l
have a longer life span,

can synthesize and membranes and intracellular
enzymes expended during phagocytosis (as the
neutrophil cannot),
can ingest particles far larger than those that can
be engulfed by neutrophils, and
can undergo mitotic division.
Many pathogenic microorganisms are engulfed

within macrophages. Some of these parasitize the
macrophages and multiply within phagosomes. Such
organisms include Listeria, Brucella, Salmonella, Mycobacteria, Chlamydia, Rickettsia, Leishmania, Toxoplasma,
Trypanosoma, and Legionella. This symbiotic relationship is destroyed as a result of what is effectively a
dialogue between macrophages and T lymphocytes.
Macrophage activation via this route leads to destruction of the guest organisms.
Chemical Signals That Influence

Macrophage Function
Factors chemotactic for macrophages include those
already mentioned in relation to neutrophils. In addition, however, there is an important group of chemical
signals that trigger a wide range of macrophage functions other than chemotaxis. These are the lymphokines, secreted by T lymphocytes activated as a
consequence of encountering specific antigens. T lymphocytes synthesize and secrete many products. Noteworthy in the context of macrophage activation is
interferon-c; this is the most powerful enhancer of the
ability of macrophages to kill intracellular parasites.
It should not be forgotten that macrophages also
play a dominant role in the natural history of both
healing and chronic inflammation. Their importance
in relation to both of these processes rests on the fact
that these cells have the power to
l
l
l
l
phagocytose living and nonliving foreign material

(including tissue debris, old or abnormal red
blood cells, immune complexes and modified
lipoprotein),
kill many microorganisms,
synthesize and release tissue-damaging products,
synthesize and release mediators of acute inflammation and fever,
present antigens to both T and B lymphocytes and
in this way initiate immune responses,
l
become activated by signals released from activated lymphocytes,

release mediators that are chemoattractant for
cells involved in repair and new blood vessel
formation,
41
synthesize and release growth factors that are

important in healing, and
regulate their own activities to a certain extent
through the operation of autocrine loops.
4
Immunology
Anne C. Cunningham
Faculty of Applied Sciences, University of Sunderland, Sunderland, U.K.
Graeme OBoyle and John A. Kirby

Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, U.K.
INTRODUCTION
The immune system plays a key role in maintaining
health and preventing disease. It has the capacity to
destroy a very wide range of pathogens to prevent
infection. In addition, it plays a key role in the
recognition and destruction of transformed body
cells and the prevention of cancer. To do that, it has
to sense danger and respond by unleashing an
effective and flexible arsenal to fight disease-causing
organisms and cancerous cells. The immune system is
not a discrete organ but a whole body system. To be
effective, it must have the ability to respond to anything dangerous anywhere in the body. In reality,
the immune system faces outward toward our
barriers with the environment (musosal surfaces,
skin) and is a highly dynamic, well-organized system.
This chapter will discuss the structure, function,
and regulation of the immune system. It will explain
the role of inflammation in the activation of immune
effector mechanisms capable of destroying intracellular and extracellular pathogens. A well-functioning
immune system represents a balance between making
effective responses against dangerous agents while
ignoring harmless things such as normal body components, food, and commensal organisms. This is a
very exciting area of research in immunology, and it is
clear that regulatory mechanisms exist to moderate
the destructive capacity of immune responses. This
harmful potential of immune responses are well demonstrated by the damage associated with immunopathologies seen in diseases such as glomerulonephritis,
rheumatoid arthritis, and the extreme vigor of acute
allograft rejection. The morbidity associated with
genetic or induced immunodeficiency is also indicative of the importance of effective immune responses.
The chapter concludes with a specific discussion of
immunological examples drawn from the fields of
clinical transplantation and cancer immunobiology.
STRUCTURE AND ORGANIZATION

There are two systems of immunity: the innate (or
natural) responses that are phylogenetically older and
present in all multicellular organisms and the

acquired (or adaptive) responses, which are only
present in vertebrates (with a jaw, including fish,
reptiles, birds, and mammals) and evolved approximately 400 million years ago. The key difference
between these responses is how they recognize danger. Innate receptors are not specific but recognize
unique microbial structures found on pathogens. They
are commonly referred to as pattern recognition
receptors and they recognize pathogen associated
molecular patterns (PAMPs), generally carbohydrates and lipids (1). These receptors can be most
simply described as being biased to the enemy. Pathogens generally have a short cell cycle (minutes or
hours) and can evolve rapidly to avoid recognition
and destruction. However, we also possess adaptive
receptors, which are produced by combining a diverse
range of antigen receptor genes randomly (2). Each
individual, therefore, also has many different rearranged antigen receptors and they can be most simply
described as nonbiased. They are only expressed by T
and B lymphocytes and generally recognize proteins.
Innate responses sense danger and coordinate
the most effective adaptive immune responses for a
particular pathogen. They lead to the activation of
immune response genes and the activation of a specialized group of white blood cells, the antigen-presenting
cells (APC). Once recognition and activation has taken
place, then appropriate effector mechanisms remove
the offending pathogen from the body. This does not
happen everywhere in the body but is coordinated into
different compartments. All white blood (immune)
cells are produced in the bone marrow. This is the
primary lymphoid organ. In addition, T lymphocytes mature in the thymus (where they undergo
receptor rearrangement and a very important selection
process). Innate recognition takes place in tissues
(inductive sites), and inflammatory responses are generated. The purpose of inflammation is to focus on
responses where they are needed. It results in an
increase in immune cell/mediator recruitment to the
site of infection. Ultimately, it is followed by the repair
process. Figure 1 illustrates the principle behind
Chapter 4: Immunology
43
Figure 1 Relationship of innate and adaptive immune system.
inflammation; when danger is sensed, signals are

released that lead to the recruitment of immune cells
and mediators from the blood into tissue.
Adaptive immune responses take place in lymph
nodes (secondary lymphoid organs). These unique
structures are essentially antigen capture devices.
Following inflammation, specialized APC in tissue
(dendritic cells) drain to the regional lymph nodes
where they activate T lymphocytes. Armies of effector
lymphocytes are generated, which then home back to
the inductive site to destroy pathogens/cancerous
cells. Blood contains many immune cells traveling
around the body; however, the really interesting
action is taking place in the tissue and lymph nodes.
INNATE IMMUNITY
Nonspecific barriers to infection are the first obstacles
a pathogen must overcome to achieve successful invasion of its host. These barriers are not acquired, do not
change, and allow the specific immune system time to
mount a response. The innate system of immune
defense is, therefore, fast-acting but relatively nonspecific and nonadaptive. However, it is increasingly
clear that the type of innate response influences the
quality and quantity of the subsequent specific
immune response. At the simplest level, the innate
immune system consists of physical and biochemical
barriers such as the skin, mucus, acid in the stomach,
and lysozyme in many secretions, which all help to
prevent the entry of microorganisms into the body. In
addition, a battery of complex mechanisms designed
to eliminate microbes that have penetrated normal
body tissues also exists. More recently, the receptors
by which danger is sensed by the innate immune

system have been described.
l
l
l
l
C-type lectin receptors (CLRs)

Toll-like receptors (TLRs)
Nod-like receptors (NLRs)
Retinoid acidinducible gene-1 (Rig-1)-like receptors (RLRs)
Danger signals can be derived from pathogens,

or from damaged tissue (endogenous signals), which
enable the function of recognition and activation to
take place. Each family of sensors has a distinct
structure, which facilitates recognition and ultimately
activation of immune responses. Recent evidence
indicates that mutations in innate sensors can contribute to disease (3).
C-type Lectin Receptors

The structure of CLRs enables them to recognize
repeating sugars found on yeasts, bacteria, and fungal
cell walls. They consist of a collagen stalk, a neck
region, and multiple carbohydrate recognition
domains. They have multivalent binding domains,
which increase the affinity and avidity of these molecules. This also explains why innate responses are
broadly reactive rather than specific. These receptors
exist as soluble molecules such as the mannan-binding
lectin (MBL) and surfactant proteins A and D, or as
receptors such as the mannose receptors expressed by
macrophages. MBL is a very interesting molecule in
that it can not only sense danger by its carbohydrate
recognition domains but also activate the complement
system (see later) (4).
44
Cunningham et al.
Figure 2 Features of the toll-like receptor signaling pathway.
Toll-like Receptors
TLRs represent a highly conserved gene family, which
are very important in transducing signals via the transcription factor NFkb and activating immune response
genes. Each TLR is a type 1 membrane protein, and
ligand recognition takes place via an extracellular leucine-rich domain complex. Signaling occurs via the
intracellular tail, which activates nuclear transcription
factors (NFkb) and leads to the transcription of immune
response genes (Fig. 2). There are 10 TLRs in humans,
and they recognize a range of distinct cell wall and
nucleic acid structures found in microbes and a range of
endogenous molecules (Table 1).
In general, TLR1, 2, and 6 sense gram-positive

bacteria and fungi, TLR4 senses gram-negative bacteria, TLR5 senses flagellum, and TLR3, TLR7, and
TLR8 sense viruses (5).
Nod-like Receptors
The NLR family consists of 22 cytoplasmic proteins,
which are primarily expressed by leukocytes but can
also be present in many other cell types, including
epithelial cells. These receptors generally recognize
specific bacterial molecules, although viral nucleic
acids can also stimulate some of these receptors. Following activation, the NLRs and TLRs can cooperate
Table 1 Ligand Specificity of Human TLRs

TLR
1
2
3
4
5
6
7
8
9
10
11
Ligand
(heterodimer with TLR2) triacylated bacterial lipopeptides, mycobacterial lipomannans
Lipoproteins, peptidoglycan, zymosan, liposaribomannan, staphylococcal enterotoxin B, HMGB-1, biglycan
Double-stranded RNA, poly(I:C)
LPS, heat shock proteins, taxol (mouse), RSV fusion protein, pneumolysin, fibronectin, lipotechoic acid, oligosaccharides from
heparan sulfate and hyaluronic acid, b-defensin 2 (mouse), HMGB-1, saturated fatty acids (lauric acid) biglycan (with TLR2)
Flagellin
(heterodimer with TLR2) mycoplasmal lipopeptide, MALP-2, diacetylated lipoproteins, zymosan
Imidazoquinolines (anti viral compounds) single-stranded RNA (mouse)
Small synthetic antiviral compounds, single-stranded RNA (human)
Unmethylated CpG DNA
Pseudogene in mouse
Not present in humans, uropathogenic bacteria in mouse
Abbreviation: TLR, toll-like receptor.
through some common signaling systems leading to

proinflammatory cell activation. However, activation of
some NLRs also results in production of a macromolecular inflammasome, which activates the proapoptotic
enzyme caspase 1, which also generates active proinflammatory cytokines, including IL-1b and IL-18 (6).
Rig-1-like Receptors
Rig-1-like intracellular receptors primarily sense infection by some viruses, including influenza, by recognition of a characteristic 5 0 -triphosphate moiety
associated with single-strand RNA sequences. Activation of these receptors leads to the production of the
antiviral cytokine, interferon-b (IFN-b) (7).
To summarize, these innate sensors recognize
danger in nature and often exploit differences
between mammalian cells and pathogens (e.g., repeating sugars or distinct nucleic acid structures) and
coordinate inflammatory responses by activation of
distinct immune response genes (8).
Complement Activation
The complement system consists of up to 20 serum

proteins that are involved in three interrelated
enzyme cascades termed the classical, the alternative, and the lectin pathways. The end result of
these cascades is the generation of a membrane
attack complex, which forms a potentially lethal
membrane pore and a series of proinflammatory
mediators, which recruit and activate other effector
cells including neutrophils, macrophages, and mast
cells. The alternative pathway may be activated directly, but nonspecifically, by a range of bacterial or yeast
cell wall components, such as lipopolysaccharide
(Fig. 3). The lectin pathway is initiated by MBL,
which recognizes repeating mannose residues in bacterial cell walls. Once bound, this associates with a
family of MBL-associated serine proteases, which
activate serum C4 in a way similar to the classical
pathway (see later), resulting in bacterial cell lysis and
the generation of inflammatory mediators (C3a, C4a,
and C5a).
Natural Killer Cells
Natural killer (NK) cells are frequently termed large

granular lymphocytes on the basis of their morphology.
The key feature of NK cells is their ability to kill virally
infected or transformed body cells. For example,
patients with defective NK cells frequently suffer severe
infection by viruses such as cytomegalovirus. In many
ways, they are related to cytotoxic T cells. However,
their recognition of targets and control of killing are
clearly different. NK cells possess receptors that can
recognize class I major histocompatibility (MHC) antigens (see section on Cancer Immunotherapy), which
deliver a strong inhibitory signal to the NK cell, preventing the lysis of healthy cells (9). Many tumors have
defective genes or defective genetic regulation, which
causes a lowering of the expression of MHC antigens.
This reduces the classical immunogenicity of these cells
but appears to enhance their susceptibility to NK cell-
45
mediated cytolysis. Furthermore, infected or transformed cells express abnormal proteins, which can
then be recognized by NK-activating receptors, and
stimulate the delivery of a lethal hit.
Phagocytic Leukocytes
Phagocytes engulf and digest microorganisms and

particles that are harmful to the body. Cells in this
category can be divided into polymorphonuclear and
mononuclear, but are all derived from common stem
cells within the bone marrow. Neutrophils, the most
common white blood cell, are generally the first
cellular components to respond to tissue injury.
They migrate from the blood across the endothelium
and into the tissues within seconds of the recognition
of a chemotactic stimulus. These stimuli may be
chemicals specifically produced by bacteria, factors
produced by damaged body cells, or peptides produced during the activation of complement.
Other phagocytic cells include tissue macrophages, such as mesangial cells in the kidney, Kupffer
cells in the liver, alveolar macrophages in the lung,
histiocytes in connective tissue, and microglial cells in
the nervous system. Macrophages are highly phagocytic and contain numerous lysosomes and endocytic
vesicles. They possess an enhanced antimicrobicidal
capacity following activation. Phagocytes are also
capable of secreting soluble agents able nonspecifically to damage cells and pathogens within the microenvironment. The most active of these factors are oxygen
radicals, which are produced during the respiratory
burst that follows cell activation.
Dendritic Cells
A specific class of mononuclear phagocytic cells forms

a link between the innate and adaptive immune
systems (10). These dendritic cells are named for the
network of dendrite-like processes, which penetrate
tissues to capture and phagocytose extracellular material. In normal situations, this phagocytosis is an
immunologically silent process, which may contribute
to the maintenance of immunological tolerance. However, following activation as a consequence of the
innate system sensing danger by recognition of
PAMPs, these cells acquire expression of a series of
costimulatory molecules, which are required for
induction of an adaptive immune response and also
respond to chemokines by migration to regional
lymph nodes. Crucially, dendritic cells break down
proteins acquired by phagocytosis into short peptides,
which can be presented in the context of MHC antigens to the antigen receptor on T lymphocytes.
Chemokines
The ability of cells to migrate is a highly regulated and

specific biology. Leukocytes act to defend against
infection and maintain balance. It is insufficient for
leukocytes to merely react subsequent to gross tissue
damage during infection; mechanisms exist to institute active surveillance of organs and tissues. The
chemokine family of chemotactic cytokines is small
46
Cunningham et al.
Figure 3 Complement activation.
47
Figure 4 Stages of chemokine-stimulated leukocyte transendothelial migration.
(810 kDa); cytokines have a crucial role in leukocyte

trafficking (11).
Leukocytes travel through the blood at high
speed, and to penetrate a tissue, the multistep process
of transendothelial migration must occur (Fig. 4).
Shear forces in the blood causes leukocytes to roll
along the surface of the vasculature because of weak
adhesion between selectins on the leukocyte and
endothelial surface. Chemokine receptors found on
the cell membrane of the leukocyte may then encounter their chemokine ligand, causing activation of the
adhesion process. Integrins on the leukocyte are activated within milliseconds, firmly tethering the leukocyte to the vessel wall. The leukocyte then migrates
across the vessel surface toward a docking structure
on the endothelial cell. Once leaving the high-pressure
blood stream, chemokines then direct the leukocyte to
migrate within the tissue along a chemokine gradient.
Over 45 different chemokines have been
described, and they bind to over 22 different receptors. Chemokines can be subdivided on the basis of
their functional role. CXCL12 is an example of a
homeostatic chemokine; it is widely expressed and is
important in immune surveillance by nave lymphocytes. During disease or infection, inflammatory chemokines, such as CCL2, are produced and these direct
activated leukocytes from the blood into the tissue.
Inflammatory chemokines are typically stored within
the endothelial cells lining blood vessels and are
rapidly released upon endothelial stress or activation
by cytokines. The chemokine system is thought to
consist of a complex network with a large degree of

cross talk and redundancy. This is because individual
chemokines can bind a number of chemokine receptors expressed on a range of leukocytes. In turn, each
receptor may bind a range of chemokine. For example,
the chemokine CCL7 is able to activate four different
chemokine receptors, and up to 11 different chemokines bind the chemokine receptor CCR1, which is
expressed by lymphocytes and mononuclear phagocytes. Because of this redundancy, targeting chemokines in human disease has proven difficult, although
animal models have suggested a number of possible
approaches, such as blocking groups of receptors at
once to limit inflammation. Chemokine nomenclature
is based around their structure and the position of
highly conserved cysteine residues. C-chemokines
have a single cysteine, CC-class chemokine have two
conserved cysteines, CXC chemokines have two conserved cysteines with an intervening amino acid, and
CX3C chemokine have three amino acids between
their conserved cysteines.
Aside from their role in leukocyte migration,
chemokines are also able to direct some immune effector mechanisms. Some chemokines, particularly those
that act to attract polymorphonuclear cells, are able to
cause respiratory burst and degranulation, while also
influencing the movement of these cells. Chemokines
play a role in the activation of lymphocytes by APC,
and can influence the polarization of the immune
response toward antibody- or cell-based responses.
Chemokines are also involved in the metastasis of
48
Cunningham et al.
cancer cells. Cancers are characterized by a distinct

metastatic pattern involving the regional lymph nodes,
and tissues such as bone marrow, lung, and liver. For
example, it has been demonstrated that the chemokine
receptor CCR7 is highly expressed in many human
cancer cells, malignant tumors, and metastases. Its
ligand CCL21 is expressed in organs representing the
first destinations of cancer metastasis.
ADAPTIVE IMMUNITY
The adaptive immune system has evolved to provide
a versatile defense mechanism against infectious
pathogens. The innate immune system is able to
hold pathogens at bay for a time, but infectious microorganisms rapidly evolve to bypass these defenses.
Each process of adaptive immunity is dependent on
the function of lymphocytes and is characterized by
an escalating response with a high degree of specificity. Furthermore, adaptive responses are characterized
by immunological memory, which enables a more
vigorous reaction after secondary exposure to a specific agent.
Resting lymphocytes are small mononuclear
cells with little cytoplasm. However, following exposure to an antigen, a small proportion of antigenspecific cells expand rapidly and begin to divide.
This process of clonal expansion is an essential feature
of adaptive immunity. Lymphocytes may be divided
functionally into T and B cells.
T cells and Cellular Immunity

The T-cell precursor is generated within the bone
marrow but migrates to the thymus before maturing
and developing the ability to recognize foreign antigen.
Each newly formed T cell expresses numerous copies
of an identical T-cell antigen receptor (TCR). However,
the receptor on each different T cell varies in sequence
and antigen specificity. This seemingly random variability enables at least a few cells within the total T-cell
population to respond to antigens on any given pathogen. Following antigen encounter, these few specific
cells divide to generate a sufficient number of responsive cells to mount a useful immune response. Estimates show that a single antigen-specific T cell can
proliferate to generate a clone of over 1000 identical
cells. This mechanism is termed clonal selection.
In recent years, the process by which maturing T
cells generate their enormous receptor diversity has
been determined. The TCR is a heterodimer and, for
more than 90% of the cells, consists of an a and a b
chain. Each chain is composed of a variable region, a
constant region, a transmembrane region, and a cytoplasmic tail. The initial genomic sequence of DNA
encoding each chain contains a large number of possible variable sequences followed by multiple diversity and joining sequences. Each mature chain is
produced after a genetic rearrangement process that
involves extensive deletion of genomic DNA and the
resplicing of single, variable, joining, and diversity
regions. It has been estimated that this process can
yield a total of 1017 different ab T-cell receptors.
A proportion of T cells, particularly those found

close to the epithelial cells of the mucosa (including
the genitourinary tract), express an alternative form of
the TCR. This consists of a g and d chain heterodimer.
The function of these gd T cells is not clear. They
appear to be less variable than ab T cells, and are
thought to represent an older, more primitive lineage.
Receptors expressed by each T cell are generated
by a random process, and it is therefore likely that
some newly formed cells will recognize antigens produced by healthy, or self, body cells. The capacity to
discriminate between self and nonself, and to prevent a
response to self, is vital for normal immune function.
This important requirement is fulfilled by the thymus,
which is essential for the generation of T-cell selftolerance. Equally essential to normal immune function
is the thymus other functionto provide a microenvironment suitable for T-cell maturation.
Thymic Tolerance
T-cell function is regulated by the affinity of the
antigen receptor for a given ligand. The T cell will
be activated by interaction with an antigen only if the
affinity is sufficiently high. Essentially, the thymus
selects newly formed cells that show no more than a
low affinity for all self-antigens but deletes any cellbearing receptors that recognize self-antigens with a
dangerously high affinity (12). It has been estimated
that only 3% of newly formed T cells survive this
selection process and escape the thymus to join the
recirculating pool.
T-Cell Recirculation
T cells that have not encountered their specific antigen, and are therefore naive, migrate from the circulation across specialized high endothelial venules to
secondary lymphoid sites, such as the spleen, lymph
nodes, and Peyers patches in the genitourinary tract.
These sites provide the ideal environment for contact
with foreign antigens, and enable antigen-specific
clonal expansion and the generation of effector/memory subsets. If a naive lymphocyte is not activated
after 10 to 20 hours, it recirculates from the lymph
node, through the lymphatic system and back into the
blood at the thoracic duct. However, during an
immune response, the blood flow through a lymph
node rises dramatically and increases the number of
lymphocytes within the node. Nonspecific resting
cells leave the node followed, after three to four
days, by a large number of activated antigen-specific
T cells. These cells recirculate to the site of primary
inflammation and to local lymphoid tissues. Finally,
after a week or so, small, long-lived memory cells
begin to leave the lymph node to join the recirculating
pool. It has been estimated for adults that almost 50%
of recirculating T cells are memory cells. These memory cells migrate differently from naive T cells and
recirculate through the peripheral tissue where they
first encountered their specific antigen (such as the
skin, genitourinary, gastrointestinal, or respiratory
system).
49
Figure 5 Crystal structures of (A) class I and (B) class II major histocompatibility antigens.
Major Histocompatibility Antigens

Human cells can express two classes of MHC antigen.
The class I antigens are expressed constitutively on
most cells, whereas the class II antigens are generally
induced by stimulation with proinflammatory cytokines such as IFNg or tumor necrosis factor-a (TNFa).
Class II MHC antigens are generally only expressed
constitutively by specialized cells of the immune
system that are adapted for antigen presentation
(such as dendritic cells). MHC molecules are essentially peptide receptors. They are specialized glycoproteins that bind a diverse array of peptides and
present them to T cells.
Resolution of the structure of the MHC antigens
(Fig. 5) has been one of the most significant advances
in immunology during the past few years (13). Class I
and class II MHC antigens have a similar threedimensional structure but a different subunit structure. Class I MHC consists of a transmembrane achain that has three domains and a noncovalently
attached b2-microglobulin. Class II MHC is a heterodimer of two transmembranous glycoproteins. A
remarkable feature of these molecules is the prominent groove on their membrane-distal surface, which
is always occupied by short peptide sequences. Class I

MHC molecules typically contain nine amino acid
peptides, whereas class II molecules can accommodate much larger peptides of up to 25 amino acid
residues.
The genes encoding MHC antigens are extremely
polymorphic within the human population. This polymorphism is generally restricted to the region of the
groove and allows different forms of the MHC molecule to bind different families of short peptides. Table 2
shows the number of polymorphisms for the human
class I (HLA-A, HLA-B, and HLA-C) and class II
(HLA-DR, HLA-DP, and HLA-DQ) MHC alleles.
T-Cell Antigens
The TCR can recognize only short peptide epitopes
bound in the groove of MHC antigens. Evidence
suggests that the T-cell receptor interacts simultaneously with the MHC molecule and the peptide. In
general, peptides from intracellular proteins are loaded into class I antigens, and peptides from phagocytosed extracellular proteins are loaded into class II
antigens. However, a cross-priming pathway in
specialized APC such as dendritic cells can allow
50
Cunningham et al.
Table 2 Polymorphism of Human MHC Antigens

MHC
Class I
Class II
Name
Number of allelic variants
HLA-A
697
HLA-B
1109
HLA-C
381
HLA-DR
693
HLA-DP
129
HLA-DQ
158
Data from the Anthony Nolan Trust, 2008.

Abbreviation: MHC, major histocompatibility.
extracellular proteins to yield peptides, which are

loaded into class I MHC antigens.
T-cell self-tolerance is vital, given the inability of
MHC molecules to discriminate between loading peptides from normal self-proteins and peptides from, for
example, a pathogenic organism. The lymphocyte is
activated only when the T-cell receptor binds to an
MHC-peptide complex with an affinity greater than a
triggering level. Thus, thymic deletion of all T cells
that recognize MHC-self-peptide complexes with an
affinity above this level will produce cells that can
reach the required affinity only by interaction with
complexes formed with nonself peptides.
Helper or CD4 T Cells

Approximately 60% of human peripheral T cells
express the CD4 antigen on their cell surface. This
antigen binds to a domain on the class II MHC antigen
during interaction between the T-cell receptor and the
class II MHC-peptide complex. Formation of this
complex plays an important role in T-cell activation
and has an additional implication for the immune
response. As class II MHC antigens primarily form
complexes with peptides derived from extracellular
proteins, CD4 T cells are activated, albeit indirectly, by
extracellular antigens (Fig. 6).
It is now clear that CD4 T cells are responsible
for the regulation of most phases of the adaptive
immune response. These cells are generally not directly cytotoxic, but produce a range of soluble mediators,
or cytokines, able to affect other cells in the local
environment. For this reason, they are often termed
helper cells. Recent studies of individual clones of
activated helper cells have shown that they can be
divided functionally into several subpopulations.
These are termed Th1, Th2, Th17, and immunoregulatory T cells (Treg) cell types and are distinguished
from each other by the range of cytokines they produce or by their biological functions (14).
The Th2 subpopulation is generated in the presence of IL-4 and produces a range of cytokines that are
involved in antibody responses mediated by B cells
and in mast-cell proliferation, eosinophilia and granuloma formation. These cytokines include IL-3, IL-4,
IL-5, and IL-10. Significantly, IL-4 stimulates B cells to
produce the IgE class of antibody, and these antibodies are involved in the release of histamine by
degranulating mast cells.
The Th1, Th17, and Treg subpopulations may
share a common precursor and are increasingly
thought to be closely related, with a possibility for
cytokine-induced transformation between these phenotypes (Fig. 7).
The Th1 lymphocyte subpopulation appears to
direct delayed-type hypersensitivity reactions by the
production of cytokines such as IFNg and TNFb.
These factors enhance the local recruitment and activation of phagocytes and stimulate the division of
antigen-specific lymphocytes, including the CD8 cytotoxic cells described in the next section.
The Th17 phenotype was only named in 2005,
but is already recognized as playing a major role
during the early stages of a number of inflammatory
disease processes. These cells produce IL-17, which
induces many body cells to secrete a range of chemokines, which recruit and activate innate immune cells,
leading to further inflammatory damage.
Treg can reduce T cellmediated immune processes. While TGFb and IL-10 are known to modulate
T-cell activity and Th1 cell differentiation, the precise
mechanism through which Treg function is not clear.
An important subset of Treg can be identified by the
transcription factor foxp3, which appears to be a
master switch for regulatory function. Study of immunoregulation is one of the most exciting areas of
Figure 6 The presentation of antigen to CD4 T cells.
51
Figure 7 The differentiation of CD4+ helper T

cells.
immunological research and holds great promise for

the understanding and treatment of immunopathologies, and even cancer.
Cytotoxic or CD8 T Cells

Approximately 40% of peripheral T cells express the
CD8 antigen. This molecule binds to a domain on the
class I MHC antigen during interaction between the
T-cell receptor and the class I MHCpeptide complex.
The formation of this complex plays an important role
in the activation of CD8 T cells. As class I MHC
antigens primarily form complexes with peptides
derived from intracellular proteins, it follows that
CD8 T cells are activated by intracellular antigens
(Fig. 8). However, the cross-priming pathway in dendritic cells can allow presentation of, for example,
viral peptides from the extracellular milieu to CD8 T
cells, which can then recognize other cells infected
with the same virus.
The CD8 lymphocyte subpopulation fulfills two
roles. Its primary role is its involvement in the process
of antigen-specific target cell lysis followed by cytokine secretion. Following activation, the CD8 T cell
differentiates from a resting, or precursor, state to
form a cytotoxic effector cell. This cell efficiently
kills target cells that express the specific class I

MHCpeptide complex. The direct lytic process
involves lymphocyte degranulation and the secretion
of agents including perforin, which forms pores in the
target cell membrane, and granzymes, which induce
the fragmentation of DNA within the target cell by a
process termed apoptosis.
The ability of CD8 cytotoxic cells to lyse target
cells containing nonself proteins is consistent with
their involvement in the prevention of the spread of
viruses by killing virally infected cells. Virally infected
cells are recognized by the cytotoxic lymphocyte
because nonself viral peptides are expressed in the
peptide-binding groove of their class I MHC antigens.
A similar mechanism may allow cytotoxic cells to kill
class I MHC antigenexpressing tumor cells that produce novel tumor-associated antigens (see section on
Cancer Immunotherapy).
B Cells and Humoral Immunity

In humans, B cells constitute approximately 10% of
the circulating lymphocytes and are produced within
the bone marrow. Resting B cells are morphologically
similar to T cells despite being functionally distinct
from them. B cells use a membrane-bound form of
Figure 8 The presentation of antigen to CD8+ T cells.
52
Cunningham et al.
immunoglobulin to recognize antigen. The receptor

can also be secreted as an immunoglobulin molecule
or antibody following B-cell activation. Like T cells, B
cells also have a clonal specificity for antigen, with
each activated cell producing soluble immunoglobulins with a single specificity. B cells also express class
II MHC antigens constitutively, and can therefore
present foreign peptide to CD4 T cells, which can
then help further in B cell maturation and potentiate
immunoglobulin production.
Immunoglobulins
The immunoglobulins (Igs), or antibodies, are a group
of five glycoproteins that are divided into five classes,
termed IgM, IgG, IgA, IgD, and IgE. These classes
differ from each other in structure and molecular
weight, but their functions are broadly similar. One
portion of the molecule, the variable region, binds to a
specific site on an antigen, whereas the constant
region may interact with, and regulate the function
of, additional components of the immune system,
such as complement, phagocytes, or cytotoxic cells.
Unlike the T-cell receptor, immunoglobulins are not
restricted to peptide binding and are able to bind
efficiently to carbohydrates, nucleic acids, proteins,
and a range of chemical and biochemical compounds.
Approximately 10% of the total immunoglobulin
pool consists of IgM, which is a large, pentameric
structure normally restricted to the blood. After primary infection, IgM is the first immunoglobulin produced by antigen-specific B cells. It has a low affinity
for antigen, but the multivalency confers a relatively
high avidity of overall binding. IgM is able to activate
complement efficiently. Activated helper T cells, particularly of the Th2 phenotype, produce cytokines that
are able to stimulate B cells to class-switch from
production of IgM to IgG. This smaller immunoglobulin makes up about 75% of the immunoglobulin pool,
can diffuse more rapidly than IgM and, in addition to
the activation of complement, can bind to a range of
cellular components of the innate immune system.
IgA makes up about 15% of the total immunoglobulin pool and is generally restricted to mucosal
sites. It is present in colostrum, saliva, and tears.
Nearly all the IgD is associated with antigen recognition on the surface of B cells, whereas IgE is found on
the surface of mast cells.
overall immunoglobulin response tends to become

more specific as the immunoglobulins produced by
the mutant cells possess an increased affinity for their
antigen. This process is termed affinity maturation.
Classical Pathway of Complement Activation

The classical pathway of complement is activated by
the interaction between subunit q of the complement
C1 complex and the constant region of IgM or IgG
molecules bound to their specific antigen. The activated C1 complex initiates the classical pathway of
complement activation, in which classical C3 and C5
convertase are produced (Fig. 9). Beyond this point,
both the alternative and classical pathways are identical and cause cell damage in a similar way.
Opsonization
Many human leukocytes express one or more of three
classes of Fc receptor for domains on the constant
region of IgG molecules. These cells include mononuclear phagocytes, neutrophils, eosinophils, and NK
cells. These Fc receptors enable the leukocytes to
recognize antibody-coated antigens by a process
known as opsonization. This greatly enhances the
efficiency of phagocytosis and increases the specificity
of cellular elements of the innate immune response.
Antibody-Dependent, Cell-Mediated
Cytotoxicity
Many NK cells express a relatively low-affinity Fc
receptor for IgG. This receptor enables these cells to
bind to IgG-coated targets and triggers a process that
results in target cell lysis. As the receptor has a low
IgG affinity, it binds aggregated IgG more readily than
monomeric immunoglobulin in the plasma. This
appears to prevent the inappropriate activation of
NK cells in the blood.
ADHESION MOLECULES
A series of specialized adhesion and signaling molecules is involved in the migration of leukocytes across
vascular endothelium and into body tissues and also
in the regulation of T-cell activation.
Immunoglobulin Diversity
The diversity of immunoglobulin specificities is generated by genetic splicing in a manner analogous to
that of the rearrangement of the genes encoding the
TCR. In the case of human immunoglobulins, it has
been estimated that this process can produce up to
1011 different immunoglobulin molecules. However,
unlike T cells, B cells supplement this process by
rearranging immunoglobulin genes after antigen recognition by somatic hypermutation. The mutated B
cells, which, coincidentally, have a higher affinity for
the antigen than the original clone, survive. The
Ligands and Receptors

Three main families of molecules are involved in
binding and supporting the migration of leukocytes.
These are the selectins and members of the integrin
and immunoglobulin superfamilies. The involvement
of selectins is mainly restricted to an initial lowaffinity interaction typified by leukocytes rolling
across the vascular endothelium. Members of the
other two families are of importance during leukocyte
stimulation, tight adhesion to endothelium, and
extravasation into tissues (15).
53
Figure 9 The classical pathway of complement activation.
Selectins
The three members of the selectin family are designated L-selectin, E-selectin, and P-selectin after the lymphocyte, endothelial cell, and platelet on which they
were respectively identified. These molecules contain
three structural regions and a cytoplasmic tail. The
N-terminal domain is closely related to calciumdependent lectins, whereas the central domain shares
characteristics with an epidermal growth factor
sequence. The third domain contains a number of
repeating sequences homologous to a sequence
found in proteins that regulate the activity of complement. Each selectin is thought to possess the ability to
bind to a number of carbohydrate ligands.
Integrins
Integrins are a superfamily of transmembrane glycoproteins consisting of noncovalently linked a and,
generally smaller, b subunits. They are widely
expressed by cells of the body and are usually
grouped by virtue of their common b chains into
eight subfamilies. At least 14 discrete a subunits
have been identified, and it is clear that a given a
chain may associate with more than one b chain
(Table 3). The extracellular portion of the a chain

contains three or four sites that bind divalent cations
essential for integrin function. Both chains generally
have short cytoplasmic regions able to interact with
the cytoskeleton, and these may be phosphorylated.
Phosphorylation is often associated with cell activation and can enhance the affinity of adhesion to the
ligand (inside-out signaling).
Integrins play a key role in both cell-cell and cellmatrix adhesion. They bind to components of the
extracellular matrix, such as collagen, fibronectin,
laminin, and vitronectin. Some integrins have affinity
for specific peptide domains, such as the well-characterized arginine-glycineaspartic acid sequence, which
is present in a number of extracellular matrix components. Three subfamilies of integrins are of particular
importance for leukocyte adhesion. These include a
member of the b1 family of very late antigens
(VLA)-4 (a4b1), b2 integrins termed the leukocyte
cell adhesion molecules (LCAM; aLb2, aMb2, and
aXb2), and b7 integrins (a4b7 and aeb7).
The b1 integrin VLA-4 is expressed by many
mononuclear leukocytes and binds to the immunoglobulin superfamily member vascular cell adhesion
molecule (VCAM)-1 in addition to fibronectin. The b2
54
Cunningham et al.
Table 3 The Integrin Family

Integrin
b subunit
Other names
VLA-1
VLA-2
VLA-3
VLA-4
VLA-5
VLA-6
b1a7
b1a8
b1aV
LFA-1
Mac-1
p150, 95
CD41a
b3aV
b1
b1
b1
b1
b1
b1
CD29
VLAb
gpIIa
b2
CD18
b3
CD61
or
gpIIIa
b4a6
b5aV
b6aV
LPAM-1
CD103
b8aV
b4
b5
b6
b7
bX, bS
Bp
b8
a subunit
Other common
name
Ligands
a1
a2
a3
a4
a5
a6
a7
a8
aV
aL
aM
aX
aIIb
aV
CD49a
CD49b
CD29c
CD49d
CD49e
CD49f
Laminin (collagen)
Collagen (laminin)
Fibronectin, laminin, collagen
VCAM-1, fibronectin
Fibronectin
Laminin
Laminin
CD51
CD11a
CD11b
CD11c
CD41
CD51
Fibronectin
ICAM-1, ICAM-2, ICAM-3 ICAM-1, C3bi,
fibrinogen C3bi
a6
aV
aV
a4
ae
aV
CD49f
CD51
CD51
CD49d
CD51
Fibrinogen, fibronectin, vitronectin, Von

Willebrand factor
As above 1-thrombospondin
Laminin
Vitronectin, fibronectin
Fibronectin; activation of latent TGFb
VCAM-1, MadCam1, fibronectin
E-cadherin
Activation of latent TGFb by dendritic
cells
Abbreviations: VLA, very late antigen; LFA, lymphocyte functionassociated antigen; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion
molecule.
subfamily is restricted to cells of the leukocyte lineage

and includes lymphocyte functionassociated antigen1 (LFA-1) and Mac-1, which both serve to anchor
leukocytes to cells that express intercellular adhesion
molecule (ICAM)-1. The b7 family is expressed by
mucosal lymphocytes. The a4b7 integrin has recently
been demonstrated to be the gut-homing receptor,
enabling gut-trophic lymphocytes to enter the Peyers
patches. The aEb7 (CD103) integrin is expressed by
nearly all lamina propria lymphocytes in the gut, and
by many intraepithelial lymphocytes in mucosal surfaces, including the gut, airways, and the urogenital
system.
Immunoglobulin Superfamily Members

ICAM-1 is a large transmembrane glycoprotein bearing five domains containing the folded b sheet characteristic of immunoglobulins. The molecule is
expressed constitutively by endothelial cells but is
significantly upregulated by stimulation with IL-1,
TNFa, and IFNg. The cytokines TNFa and IFNg also
induce and upregulate the expression of ICAM-1 on a
range of parenchymal cells, including fibroblasts and
epithelial cells in the skin, lung, and liver. This process
enhances the adhesion of b2 integrinexpressing
leukocytes.
Structural studies have shown that VCAM-1 contains seven immunoglobulin domains. The first and
fourth of these regions are homologous and function
during lymphocyte adhesion. VCAM-1 is constitutively
expressed on endothelial cells at a low level, but
expression is upregulated within 12 hours and then
maintained at high levels by stimulation with the
cytokines IL-1 and TNFa. Immunocytochemical studies
have also demonstrated the presence of VCAM-1 on a

variety of nonvascular cells, including renal epithelial
cells, neural cells, and the synovial cells of inflamed
joints.
Costimulation
Ligation of a T-cell receptor with its specific MHCpeptide ligand is insufficient to activate the T cell. This
observation has generated the two-signal hypothesis
for lymphocyte activation. Signal one is defined as
interaction with specific MHC and peptide, and signal
two is a nonspecific costimulatory signal.
Studies have indicated that the TCR has only a
very modest affinity for its specific MHC-peptide
ligand. This has been estimated as between 1 # 10$5
M and 5 # 10$5 M, which is considerably lower than
the value for a typical IgG molecule, which is of the
order of 1 # 10$9 M. This affinity is too low to allow
stable conjugates to form between T-cell receptors and
the 210 to 340 specific MHC-peptide ligands required
for lymphocyte activation. The multiplicity of antigenindependent adhesion molecule interactions plays a
vital role in stabilizing the T-cell and antigen-presenting
cell complex sufficiently to allow T-cell receptor signal
transduction to take place. This adhesion is rapidly
increased following T-cell receptor ligation by an
increase in the affinity of LFA-1 (inside-out signaling).
Appropriate ligation of the lymphocyte adhesion
molecules LFA-1 and VLA-4 is known to generate
costimulatory signals able to augment lymphocyte
activation. Furthermore, monoclonal antibodies specific
for LFA-1 have been shown to stimulate the proliferation of resting lymphocytes. The best characterized
costimulatory molecule is the T-cell-surface molecule
CD28, which interacts with members of the B7 ligand

family (CD80, CD86) on antigen-presenting cells, leading to the generation of a signal important for the
activation and proliferation of antigen-specific T cells
(16). Expression of the B7 family of ligands is restricted
to a small group of specialized mononuclear cells,
including dendritic cells and B cells. Other costimulatory molecules contribute to the response, including
CD40 ligand (CD40L), which binds to CD40 on antigen-presenting cells. This leads to upregulation of B7
and therefore further T-cell stimulation. Antigen presentation in the absence of satisfactory costimulatory
signal transduction may even produce stable lymphocyte hyporeactivity. Indeed, the therapeutic elimination
of CD28 signaling by the blockade of B7 molecules with
the soluble receptor-like construct CTLA4-Ig has
resulted in partial cardiac allograft tolerance in a
murine model.
EXAMPLES OF CLINICAL IMMUNOLOGY

Acute Allograft Rejection
The extreme vigor of allograft rejection can be
explained by the process termed alloreactivity.
Although mature T cells have been selected in the
thymus for specific tolerance of all potential self-MHC
antigen-self-peptide complexes, the cells have not
been selected for tolerance of the subtly different
MHC moleculepeptide complexes expressed on the
surface of donor cells. It has been estimated that up to
2% of all recipient T cells may respond to the allogeneic MHC molecules expressed by graft tissues. Furthermore, as this situation reflects an artificial crossreaction, a significant proportion of the responsive
lymphocytes are present as memory cells, which
facilitate the rapid initiation of the rejection response.
It is not clinically feasible to match the organ
donor and recipient for identical MHC antigens, given
the enormous polymorphism of MHC alleles (Table 2).
Consequently, it has been necessary to develop drugs
to suppress the immune response. One of the most
successful immunosuppressive drugs is cyclosporin
A, a calcineurin inhibitor, which revolutionized organ
transplantation in the 1980s. This cyclic peptide blocks
the production of IL-2 during T-cell activation. The
function of this cytokine is best illustrated by its
original name, which was T-cell growth factor.
Without IL-2, the graft-specific lymphocytes are
unable to divide and cannot initiate the rejection
response.
Cancer Immunotherapy
The prospects for successful cancer immunotherapy
depend on the ability of T cells to recognize tumor
antigens. During the 1950s, some chemically induced
tumors were shown to express specific, nonself
antigens that allowed sensitized animals to reject
transplanted tumor cells. More recent studies have
shown that T cells are able to recognize tumor-derived
peptides complexed in the groove of MHC molecules.
55
A range of tumor-specific antigens have been

detected, including the MAGE family (MAGE-1),
which is found in 37% of human melanomas, a
smaller proportion of other tumors, and no normal
tissues except the immunologically privileged testis.
Peptides from MAGE-1 associate with the class I
molecules HLA-A1 and HLA-Cw16 and can be recognized by cytotoxic lymphocytes. The related molecule
MAGE-3 is associated with a greater proportion of
cancers and yields immunogenic peptides that complex with HLA-A1 and HLA-A2. Studies have demonstrated that up to 35% of transitional bladder
cancers express MAGE antigens, with the proportion
increasing to 61% of invasive tumors. Further tumorassociated proteins have been identified, including
BAGE, and GAGE-1 and GAGE-2, which also yield
peptides that can be recognized by cytotoxic T cells
and are expressed by 10% to 20% of bladder cancers.
In addition to the identification of proteins expressed
by a range of cancer cells, tumors also express unique
peptide epitopes derived from mutated genes. Recent
work has identified such mutations in murine cancers,
which yield peptides recognizable by both CD8 CTL
and CD4 helper T cells.
It is, however, becoming increasingly clear that
many cancer cells evolve a capacity to evade a local
immune response (17). Not only do some cells express
reduced levels of MHC molecules, the primary T-cell
receptor ligand, but some also acquire a capacity to
actively kill specific effector T cells by the Fas
counterattack. In this system, the cancer cell develops expression of Fas-ligand, which can engage the
Fas receptor on local T cells, thereby initiating a
proapoptotic cycle, which results in T-cell death.
Another potential example of an immune evasion
strategy is loss of the expression of E-cadherin by
many epithelial cancer cells. It is now known that
this molecule is an effective adhesion receptor for
intraepithelial T cells, which are thought to be responsible for normal immune surveillance of mucosal
surfaces. It is possible that the acquisition of these
successful phenotypic attributes (from the cancers
point of view!) is a reflection of microevolution within
the developing cancer, leading to selection of phenotypic variants that can evade an otherwise cancerclearing immune response.
BCG Treatment of Bladder Cancer

The treatment of superficial bladder cancer by intravesical administration of BCG is almost a unique
example of successful cancer immunotherapy. The
mechanism by which BCG can cause bladder cancer
regression is not clear (18). However, it is known that
BCG is an effective agent for the activation of dendritic cells in vitro, presumably by a mechanism involving
ligation of multiple PAMPs, including the TLRs. Furthermore, model systems have shown that both activated T cells and NK cells are potentially involved in
the clearance of bladder cancer cells. A challenge
remaining in this area is the precise definition of
optimal ways to induce immunological clearance of
56
Cunningham et al.
bladder cancer cells without resorting to the administration of viable, and often harmful, BCG bacteria.
Indeed, current research is focusing on methods to
induce an intravesical immune response by administration of defined subfractions of BCG by ligation of
an optimal repertoire of PAMPs.
REFERENCES
1. Janeway CA Jr. Presidential address to the American
association of immunologists. The road less traveled by:
the role of innate immunity in the adaptive immune
response. J Immunol 1998; 161:539544.
2. Cooper MD, Alder MN. The evolution of adaptive immune
systems. Cell 2006; 124:815822.
3. McDermott MF, Tschopp J. From inflammasomes to fevers,
crystals and hypertension: how basic research explains
inflammatory diseases. Trends Mol Med 2007; 13:381388.
4. Holmskov UL. Collectins and collectin receptors in innate
immunity. APMIS Suppl 2000; 100:159.
5. Mitchell JA, Paul-Clark MJ, Clarke GW, et al. Critical role
of toll-like receptors and nucleotide oligomerisation
domain in the regulation of health and disease. J Endocrinol 2007; 193:323330.
6. Delbridge LM, ORiordan MX. Innate recognition of intracellular bacteria. Curr Opin Immunol 2007; 19:1016.
7. Saito T, Gale M Jr. Principles of intracellular viral recognition. Curr Opin Immunol 2007; 19:1723.
8. Creagh EM, ONeill LA. TLRs, NLRs and RLRs: a trinity of

pathogen sensors that co-operate in innate immunity.
Trends Immunol 2006; 27:352357.
9. Bottino C, Castriconi R, Moretta L, et al. Cellular ligands of
activating NK receptors. Trends Immunol. 2005; 26:221226.
10. Watts C, Zaru R, Prescott AR, et al. Proximal effects of tolllike receptor activation in dendritic cells. Curr Opin Immunol 2007; 19:7378.
11. Jin T, Xu X, Hererld D. Chemotaxis, chemokine receptors
and human disease. Cytokine 2008; 44:18.
12. Naeher D, Daniels MA, Hausmann B, et al. A constant
affinity threshold for T cell tolerance. J Exp Med 2007;
204:25532559.
13. Bjorkman PJ. Finding the groove. Nat Immunol 2006;
7:787789.
14. Chen Z, OShea JJ. Th17 cellsa new fate for differentiating
helper T cells. Immunol Res 2008; 41:87102.
15. Rose DM, Alon R, Ginsberg MH. Integrin modulation and
signaling in leukocyte adhesion and migration. Immunol
Rev 2007; 218:126134.
16. Bour-Jordan H, Bluestone JA. Regulating the regulators:
costimulatory signals control the homeostasis and function
of regulatory T cells. Immunol Rev 2009; 229:4166.
17. Pettit SJ, Seymour K, OFlaherty E, et al. Immune selection
in neoplasia: towards a microevolutionary model of cancer
development. Br J Cancer 2000; 82:19001906.
18. Brandau S, Suttmann H. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success
story with room for improvement. Biomed Pharmacother
2007; 61:299305.
5
The Nature of Renal Function
George B. Haycock
Academic Department of Paediatrics, Guys Hospital, London, U.K.
HOMEOSTASIS
Homeostasis is the regulation of the volume and
composition of the intracellular fluid (ECF), the internal environment that bathes the cells. The kidney is
the preeminent organ of homeostasis, although the
lungs, gut, and skin also contribute to it. One component of homeostasis is excretion of products of metabolism: failure of excretion leads to accumulation of
these products with progressive pollution of the ECF.
A second component is conservation of ECF solutes
(mainly nutrients) that are too valuable to be allowed
to escape into the urine. The third component is
constant adjustment of the urinary excretion rate of
water and inorganic solutes to maintain their concentrations in the ECF within the normal range, in the
face of unpredictably changing input from the diet
and from other body water compartments. This last
might be called homeostasis proper. To achieve it, the
kidney must
1.
2.
detect small changes in ECF volume, or in the

concentration of any of its many constituents,
before such changes become serious; and
respond by altering the volume or composition of
the urine so as to correct the tendency to abnormality.
This is achieved by negative feedback: a rise in

the ECF concentration of, say, potassium leads to an
increase in its excretion rate with a consequent return
of the ECF concentration to normal, while a fall in its
concentration leads to a decrease in excretion rate,
defending the ECF against hypokalemia. The kidneys
capacity to alter the volume of urine and the concentration of its constituent solutes in urine is very large.
An adult can vary daily urine volume between a
minimum of about 500 mL and a maximum of more
than 20 L, while the urinary concentration and excretion rate of the major ECF cation, sodium, can vary by
at least three orders of magnitude.
The Concept of External Balance

Homeostasis requires (in the nongrowing organism)
that net input of any substance into the ECF be exactly
equal to net output from it. In health, body composition remains constant because output, mainly via the
kidney, can be varied continuously in response to

changes in input. The net external balance (NEB) for
any substance is the algebraic difference between
input and output over any specified period of time;
that is,
NEB input ! output
Balance may be positive (input > output), negative

(output > input), or zero (input output). Normally,
NEB for all nonmetabolized substances is zero, except
in growing individuals, in whom it is continuously
positive. Since the main determinant of input is food
intake, an obvious survival advantage enjoyed by an
animal with a large reserve of renal function is the
ability to eat a varied diet. As renal function is lost,
dietary freedom becomes constrained. In advanced
renal failure, survival is possible only on a rigorously
controlled diet, reversing the normal physiological
state of affairs: homeostasis is achieved by adjusting
intake to match a relatively constant urinary output.
Growth and Renal Function

The size of the kidneys, and therefore their functional
capacity, increases with growth of the body as a
whole. Between the age of two years and maturity,
glomerular filtration rate (GFR) and renal plasma flow
in healthy humans are approximately proportional to
body surface area (BSA) at about 120 mL/min/1.73 m2
(range 90150) and 600 mL/min/1.73 m2 (range 450
750), respectively. The association between renal function and BSA is empirical, and probably does not
reflect any underlying physiological principle. Some
have argued that it should be standardized to ECF
volume or body weight: the latter undoubtedly gives a
better fit than BSA in newborn infants (Fig. 1) (1).
GFR in healthy, term, newborn infants is about
30 mL/min/1.73 m2, rising to 50 mL/min/1.73 m2 at
1 month, 80 mL/min/1.73 m2 at 6 months, and 100 mL/
min/1.73 m2 at one year, and attaining the adult value by
18 months to two years.
Whatever standardizing factor is applied, renal
function is low at birth and during the first few
months of extrauterine life, even corrected for body
size. Despite this, normal infants thrive and show no
clinical or biochemical evidence of failure of excretion
58
Haycock
Figure 1 GFR corrected for body surface area

(interrupted line) and weight (continuous line) in
newborn infants. Within the range studied, GFR
factored by surface area increases threefold while
GFR factored by weight less than doubles. Abbreviation: GFR, glomerular filtration rate. Source:
From Ref. 1.
or homeostasis. This can be explained by the fact that

the infant is growing very rapidly at this stage,
increasing body weight by up to 5% per week. This
results in a strongly positive NEB for most nutrients,
particularly since they are supplied by the babys
normal diet (human milk) in quantities that provide
only just enough energy, protein, and minerals to
support normal growth and activity. The residue
remaining for excretion is, therefore, very small:
growth provides the infant with a third kidney
(2). A major advantage to the infant of constraining
glomerular filtration at a low level is conservation of
energy by the mother-infant dyad. Approximately
99% of filtered sodium is absorbed, with its attendant
anions, by the renal tubule. This is an active process
(see below) and accounts for almost all of the energy
expenditure of the kidney. The additional energy cost
of reabsorbing the sodium filtered at an adult level
of glomerular filtration would be significant, given the
marginal sufficiency provided by human milk,
amounting to about 3% of basal metabolic rate (3,4).
The negative aspect of the low GFR of the newborn is
its lack of reserve. If growth is interrupted for any
reason (such as intercurrent infection), or if an unphysiological diet containing too much protein (such as
unmodified cows milk) is given, the solute load
presented for excretion can overwhelm the kidney,
leading to a form of acute renal insufficiency with
resulting metabolic imbalance. This principle is illustrated by the syndrome of late metabolic acidosis of
prematurity (5).
The Concept of Renal Clearance

It follows from the above that, in a steady state, the
composition of the urine is determined by dietary and
metabolic input to the ECF, and not by renal function:
individuals with a GFR of 100 and 10 mL/min,
respectively, will produce identical urine if they are

eating identical diets. Since the function of the kidneys
is to regulate the ECF, one way of quantifying renal
function is to measure the rate of processing of ECF
(plasma). For any substance (x) present in both plasma
and urine, it is simple to calculate the volume of
plasma containing the quantity of x excreted in the
urine in a specified period of time. This volume is the
renal clearance of x, the volume of plasma (not urine)
cleared of x per unit of time. Renal clearance is given
by the formula
Cx
Ux % V
Px
where C, U, and P represent clearance, urine, and

plasma concentrations, respectively, and V is the urine
flow rate. C is expressed in the same units as those
chosen to express V (e.g., mL/min, L/day).
In the special case of a substance that is freely
filtered by the glomerulus, such that its concentrations
in plasma water and glomerular filtrate are the same,
and which is not secreted or reabsorbed by the tubule
or metabolized by the kidney, the filtration rate of x
(Fx) must equal its excretion rate (Ex). The renal
clearance of such a substance is equal to the GFR.
No endogenous substance has been described that
fulfills these criteria exactly in humans. Inulin, a
starch-like polymer of fructose, does meet them, and
is therefore an ideal marker for measurement of GFR.
The chelating agents EDTA and DTPA, which can be
labeled with radioisotopes for easy assay in plasma
and urine, and the radio contrast chemical, sodium
iothalamate, are excellent alternatives. Creatinine is an
ideal marker for GFR in dog; unfortunately, in
humans, it is secreted by the tubule to a small extent
when renal function is normal but to a considerably
greater extent when renal function is reduced. Despite
Chapter 5: The Nature of Renal Function
this limitation, a carefully performed creatinine clearance test is an adequate measure of GFR for most
clinical purposes (6).
Any substance whose clearance is greater than
GFR must be secreted by the tubule. Conversely, any
freely filtered substance with a clearance less than
GFR must be reabsorbed by the tubule. Dividing Cx by
GFR gives the fractional excretion of x (FEx). If creatinine clearance (Ccr) is taken as GFR,

Ux % Pcr
% 100
3
FEx %
Ucr % Px
Note that it is not necessary to measure V to calculate
FEx: simultaneously obtained blood and urine samples are sufficient.
A substance that is completely cleared by the
kidney, that is, its concentration in renal venous
plasma is zero, has a clearance equal to renal plasma
flow (RPF). The organic anions paraaminohippurate
(PAH) and orthoaminohippurate (hippuran) are
almost ideal markers for measurement of RPF in
most circumstances. An exception is in the newborn,
in whom renal extraction of PAH is incomplete (7).
Dividing GFR by RPF gives the filtration fraction (FF),
the proportion of renal arterial plasma removed from
the circulation as glomerular filtrate.
FF
GFR Cinulin U=Pinulin Uinulin % PPAH
CPAH
U=PPAH
UPAH % Pinulin
RPF
Typical normal values after infancy for GFR, RPF, and

FF are 120 & 30 mL/min/1.73 m2, 600 & 150 mL/min/
1.73 m2, and 0.2, respectively.
59
tubule is further divided into the early (S1) and late

(S2) proximal convoluted tubule, the proximal straight
tubule (pars recta, S3), and the loop of Henle. About
90% of the human nephron population (superficial
cortical nephrons) have short loops of Henle that
descend only into the outer medulla before bending
and returning toward the surface of the cortex. The
remaining 10% have long loops of Henle that descend
deep into the inner medulla and bend at or near the
papillary tip. These are the tubules that arise from
those glomeruli situated in the deepest layer of the
cortex, the juxtamedullary nephrons: they form an
important part of the mechanism for urinary concentration and dilution. The parts of the loop that extend
into the inner medulla are thin-walled and are called
the thin descending and thin ascending limbs. The
cortical part of the ascending limb is relatively thickwalled and is referred to as the thick ascending limb
(TAL). The TAL of each nephron passes through the
vascular pole of its own glomerulus where it is
intimately related to the afferent and efferent arterioles in a structure called the juxtaglomerular apparatus
(JGA). The JGA marks the functional division between
the proximal and distal tubules.
The distal tubule is also divided into distal
convoluted tubule, connecting tubule, cortical collecting duct (CCD), and medullary collecting duct. The
different segments of the nephron are identifiable not
only by their anatomical relationships but also by the
ultrastructural and histochemical characteristics of
their epithelia. Details may be found in relevant
anatomical and pathological studies (810).
The Function of the Glomerulus

FUNCTIONAL SEGMENTATION OF THE
NEPHRON
Each nephron consists of a glomerulus and a tubule
(Fig. 2). The tubule is divided into two major segments: the proximal and distal tubules. The proximal
The glomerulus is a network of specialized capillaries

that act as a size- and electrical chargeselective ultrafilter, retaining large molecules such as proteins within the lumen while allowing the passage of small
molecules (water, electrolytes, and other crystalloid
Figure 2 Schematic diagram of two nephrons, one of

which is a juxtamedullary nephron (long loop of Henle)
and one a superficial cortical nephron (short loop of
Henle). The figure is not drawn to scalethe loop of
Henle of the juxtamedullary nephron is actually much
longer in proportion to the rest of the nephron than
represented here.
60
Haycock
solutes) into Bowmans space. The concentration of

these solutes in glomerular filtrate is almost identical
with that in plasma, with a small correction for the
Gibbs-Donnan effect (because of the fact that negatively charged proteins are at much higher concentration within the capillary lumen than in glomerular
filtrate). Ultrafiltration is a passive process, and the
concentrations of small solutes in glomerular filtrate
depend entirely on their concentrations in plasma and
whether they are bound to plasma albumin. Glomerular filtrate is, therefore, the precursor of urine, the
final composition of which is extensively modified by
tubular reabsorption and secretion. Some important
waste products of protein metabolism are entirely
(e.g., urea) or mainly (e.g., creatinine) excreted by
glomerular filtration. Although the excretion rate of
these compounds is maintained at reduced levels of
GFR, this is at the cost of sustained elevation of their
plasma concentrations.
The Function of the Proximal Tubule
been reabsorbed at entry into the distal tubule. The

major function of the loop is concentration and dilution of urine: reabsorption in this segment is, therefore, no longer isotonic.
Concentration and dilution of urine are both
powered by the same energy-dependent process:
absorption of salt without water in the TAL. The
fluid entering the distal tubule is hypotonic with
respect to plasma, irrespective of whether the final
urine is concentrated or dilute at the time. Salt reabsorption in the TAL also generates hypertonicity in the
interstitium surrounding the TAL. This hypertonicity
is amplified by countercurrent exchange and multiplication in the loops of Henle and vasa recta, leading to
the establishment of an osmotic gradient in the medulla with the highest osmolality at the papillary tip. In
the presence of antidiuretic hormone (ADH), the
collecting duct becomes permeable to water, which
is osmotically extracted from the lumen by the hypertonic medulla, leading to the formation of concentrated
urine.
Pars Convoluta and Pars Recta
The Function of the Distal Tubule
Two-thirds of the volume of the glomerular filtrate is

reabsorbed in the proximal tubule. The nature of the
reabsorptive process can be summarized as follows:
The distal tubule adjusts the composition of the final

urine by selectively reabsorbing or secreting individual solutes according to the need of the moment. For
example, reabsorption of Na from the tubular fluid is
virtually complete in Na depletion, while in Na repletion exactly sufficient Na escapes reabsorption to
balance dietary input. Many substances (such as sodium, chloride, calcium, and phosphate) are normally
filtered in amounts greatly exceeding dietary input,
and the excretion rate is regulated by varying the
amount reabsorbed. Other important substances,
notably hydrogen ion and potassium, generally enter
the distal tubule in very small amounts, and the rate of
excretion depends mainly on varying the rate of active
secretion into the tubular fluid. The rate of water
excretion is determined by ADH, which increases
the water permeability of the collecting duct and
thus regulates the rate of osmotic reabsorption of
water into the hypertonic medullary and papillary
interstitium.
1.
2.
3.
4.
It is isotonic.
Some solutes (bicarbonate, glucose and amino
acids) are preferentially reabsorbed in the initial
segments of the proximal tubule, leading to their
almost complete removal from the tubular fluid
and a rise in the concentration of chloride (Cl!).
It is energy and oxygen dependent.
Some solutes (such as inulin, creatinine, and urea)
are reabsorbed little or not at all, and are therefore
concentrated about threefold with respect to plasma by the end of the proximal tubule.
Organic anions such as PAH and penicillin are

actively secreted in this segment. The fluid delivered
into the loop of Henle is therefore the following:
1.
2.
3.
4.
Isotonic with plasma.

Chloride-rich and bicarbonate-poor.
At a pH lower than that of plasma (more acid).
More concentrated than plasma with respect to
substances excreted solely or principally by glomerular filtration. The ratio of the tubular fluid
inulin concentration to the plasma inulin concentration (TF:Pinulin) at any point along the tubule is
a measure of the volume of filtrate that has been
absorbed at that point. For example, if TF:Pinulin is
10, 90% of filtered water has been reabsorbed at
that point. Similarly, the urine-to-plasma inulin
ratio (U:Pinulin) is a measure of the amount of
filtered water that is reabsorbed between glomerular filtrate and the final urine (as is, approximately, U:Pcreatinine).
Loop of Henle
Net reabsorption continues in the loop so that about

85% of filtered sodium and 70% of filtered water have
GLOMERULAR FILTRATION
GFR is determined by the interaction of the physical
forces driving filtration and the permeability of the
filtration barrier (the glomerular capillary wall) to
water and small solutes. The composition of the
filtrate in health is that of a protein-free ultrafiltrate
of plasma, because of the almost complete impermeability of the glomerular capillaries to protein.
The Glomerular Capillary Wall

Structure
The glomerular capillary wall consists of three layers:

a lining endothelium composed of thin, fenestrated
polygonal squames, a basement membrane, and an
outer epithelium consisting of cells (podocytes) each
of which possesses a central body from which radiate

fern-like foot processes (pedicels) that interdigitate
with those of adjacent cells. In section, the transected
foot processes appear as islands of cytoplasm intimately attached to the basement membrane and connected to one another by a fine membrane. The view
afforded by scanning electron microscopy shows these
islands to be sections through alternating processes
from adjacent cells (11). The basement membrane is a
hydrated proteoglycan gel with a thickness of about
in the adult and rather less in the infant.
3200 A
Ultrastructural examination reveals a central relatively electron dense lamina densa, sandwiched between
a lamina rara interna and a lamina rara externa. The
thin squames of the endothelium contain numerous
diameter. This differentiates them
fenestrae of 700 A
from the endothelium of capillaries in other tissues.
All three layers take up cationic stains, indicating that
they are negatively charged.
Location of the Filtration Barrier
All three layers of the glomerular capillary wall probably contribute to the retention of macromolecules in
the capillary lumen. Ultrastructural studies using
macromolecular tracers of different sizes and electrical
charge suggest that very large molecules are
completely excluded from the basement membrane,
while smaller ones penetrate it to varying degrees
(12). Small quantities of molecules of approximately
the size of albumin appear to traverse the basement
membrane but are retained at the filtration slits
between the epithelial foot processes (13). The electrical charge, as well as the size, of molecules is an
important determinant of their ability to cross the
capillary wall into Bowmans space (14). Proteinuric
states are associated with loss of negative charge from
one or more layers of the capillary wall. The passage
of large amounts of protein from the plasma into the
glomerular filtrate is prevented partly by the physical
structure of the proteoglycan mesh of the basement
membrane and partly by its electronegativity. The
small amount of albumin that crosses the basement
membrane is further contained by the polyanionic
coat surrounding the epithelial cells and extending
to the filtration slits between them. The relative importance of charge and size selectivity is still controversial. The filtration slits between foot processes are
bridged by a fine membrane, the slit diaphragm,
which consists mainly of a specialized membrane
protein, nephrin (15). Nephrin molecules from adjacent foot processes interdigitate to form a zipper-like
structure, which is thought to be the limiting part of
the filtration complex for albumin. Mutations in the
gene for nephrin cause a severe form of recessively
inherited congenital nephrotic syndrome (CNS),
known as CNS of the Finnish type (16). Other specialized proteins expressed in the foot processes are also
essential to the proper formation of the filtration
barrier, and mutations in the genes that code for
them cause other, even rarer, forms of CNS. The role
of these proteins, if any, in the pathogenesis of
acquired forms of nephrotic syndrome is unknown.
61
Dynamics of Glomerular Filtration

Starling Forces
Glomerular filtration is driven by the balance between

the hydrostatic pressure gradient between capillary
and Bowmans space, generated by myocardial contraction, and the oncotic pressure (colloid osmotic
pressure) gradient generated by the protein concentration gradient between the same two compartments.
Hydrostatic pressure favors, and oncotic pressure
opposes, filtration. These forces are referred to as
Starling forces. Starling forces govern the movement
of fluid between the intravascular and extravascular
compartments of the extracellular fluid throughout the
body as a whole, glomerular filtration being a special
example of their application. The net ultrafiltration
pressure (PUF) is, therefore, the algebraic sum of the
hydrostatic (P) and oncotic (p) pressure gradients
between capillary (C) and Bowmans space (BS).
PUF PC ! PBS ! #$C ! $BS
Since pBS is insignificant and s (the reflection coefficient for albumin) has a value of unity,
PUF PC ! PBS ! $C
P must be higher at the arterial than at the venous end

of the glomerular capillary plexus, or blood would not
flow. The pressure drop is small, however, since the
glomerular capillaries are low-resistance vessels
between two high-resistance components (the afferent
and efferent arterioles). As plasma passes through the
glomerulus, filtration occurs and the plasma proteins
are progressively concentrated toward the venous end
by removal of water. The force opposing filtration (pC)
therefore increases. The combination of falling PC and
rising pC causes PUF to diminish toward the venous
end of the capillary. These pressures have been directly measured in several species. At least in the MunichWistar rat and the squirrel monkey, PUF at the venous
end of the circuit is zero (17) This means that filtration
actually stops before the efferent arteriole is reached, a
condition referred to as filtration equilibrium. A normalized plot of filtration pressure gradients can therefore be drawn (Fig. 3). The mean ultrafiltration
pressure is shown as the integrated area between the
curves for P and p. For technical reasons, it is not
possible in filtration equilibrium to measure at what
point along the notional distance between afferent and
efferent arteriole equilibrium is achieved. If all other
variables are held constant, and plasma flow is
increased or decreased, this point will move to the
right or the left, respectively: there is an infinite family
of curves yielding pressure equilibrium at the efferent
arteriole. Under these conditions, filtration fraction is
constant, and
GFR ' GPF
That is, GFR is plasma flow dependent (18). It is not

known whether filtration equilibrium is the normal
condition in humans.
62
Haycock
Figure 3 Hydrostatic (P) and oncotic (P) pressure profiles along an idealized glomerular capillary. DP and DP refer, respectively, to the
hydrostatic and oncotic pressure gradients
between the capillary lumen and Bowmans
space. As plasma traverses the capillary bed
from afferent to efferent arteriole, removal of fluid
by filtration causes the plasma protein concentration to rise, with a consequent increase in DP. In
filtration equilibrium (unbroken lines), DP rises to
equal DP, and filtration stops before the efferent
arteriole is reached. An infinite number of lines
(e.g., upper interrupted line) can be drawn that
yield filtration equilibrium: these cannot be experimentally distinguished. As flow increases, the point
of intersection of DP and DP moves to the right,
eventually producing filtration disequilibrium (lower
interrupted line). In filtration disequilibrium, measurements of afferent and efferent arteriolar pressures allow a unique curve for DP to be plotted,
from which Kf can be calculated (see text). The
shaded area represents P UF . Source: From
Ref. 20.
The Ultrafiltration Coefficient
At any given PUF, filtration rate is inversely proportional to the resistance offered by the filtering membrane. The reciprocal of this resistance is the
ultrafiltration coefficient (Kf). Kf is the product of the
surface area of the membrane (S) and its hydraulic
permeability or conductivity (k), the latter being
expressed as rate of flow (Q) per unit S per unit PUF.
k Q % S!1 % P!1
UF
Although S can be estimated morphometrically, it is

difficult or impossible to measure effective S, the area
participating in filtration at any given time. In the
absence of a reliable value for S, k cannot be calculated. The composite term Kf is, therefore, preferred for
most purposes. Thus,
GFR PUF % k % S PUF % Kf
In filtration equilibrium, Kf is not limiting to GFR and

cannot be calculated from experimental measurements. When filtration disequilibrium is induced by
massive volume expansion, Kf becomes rate limiting,
and a unique value can be calculated for it from
measurements of hydrostatic and oncotic pressures
in the afferent and efferent arterioles and in Bowmans
space, and single nephron GFR. Identical values for
single nephron Kf of 0.08 nL/sec/mmHg have been
found experimentally in both rat and dog, despite the
fact that filtration equilibrium probably does not
obtain in the latter species (19,20). Depending on the
value taken for S, this yields an estimate for k in the
range 25 to 50 nL/sec/mmHg/cm2. This is 10 to 100
times higher than that found in capillaries from other

tissues, enabling filtrate to be formed at a very high
rate despite quite low values for PUF (<10 mmHg). It
is evident from equation (9) that GFR may change as a
result of changes in factors affecting one or more of
the components of PUF (changes of glomerular plasma
flow, and changes of plasma albumin concentration or
urinary tract obstruction), changes in S (reduction of
nephron numbers), or changes in k (diffuse glomerular disease). More than one of these may be involved
in progressive renal disease.
Regulation of Glomerular Filtration
Factors affecting PUF. As discussed above, GFR is

plasma flow dependent if
1.
2.
3.
filtration equilibrium exists,

DP ! Dp at the afferent arteriole does not change,
and
Kf is constant.
Under physiological conditions, these requirements are probably met, at least approximately.
Plasma flow is determined by arterial pressure and
renal vascular resistance, which resides mostly in the
afferent and efferent glomerular arterioles. Angiotensin II (AII) and norepinephrine (noradrenaline, NE)
constrict both afferent and efferent arterioles; these
hormones are released in ECF volume contraction and
other conditions in which arterial filling, and therefore
renal perfusion pressure is reduced. Efferent arteriolar
constriction raises PC, and therefore PUF, more than
it reduces glomerular blood flow, leading to maintenance of GFR by an increase in FF. Conversely,
vasodilators such as prostaglandins E 2 and I 2
(prostacyclin) and bradykinin, despite a lowering of

arterial blood pressure, cause renal plasma flow to rise
because of dilatation of both afferent and efferent
arterioles. Here again, GFR remains relatively constant with an associated fall in FF. It is likely that
both vasodilator and vasoconstrictor hormones also
affect Kf (see below). Obstruction of the flow of urine
causes reduction or abolition of glomerular filtration
because of a rise in PBS.
Factors affecting Kf. The mesangial cells that
support the glomerular capillaries are contractile
(21). Their contraction causes constriction of the
capillaries, with a resulting fall in S and therefore Kf.
AII, NE, and prostaglandins all alter Kf to the degree
necessary to offset their effects on PUF; in other words,
the glomerular hemodynamic response to vasoactive
substances is nicely balanced by changes in Kf, maintaining GFR approximately constant. Both AII and
ADH cause cultured mesangial cells to contract, and
this may be a physiologically important effect in vivo.
Autoregulation. In common with other vital
organs such as the brain, the kidneys can regulate
their own blood flow, even in an artificial perfusion
system where systemic humoral and nervous controlling factors cannot operate. This is because of differential constriction of the afferent and efferent
arterioles. Analysis of the determinants of GFR in
this model system suggests that the damping effect
on GFR is mediated in part by autoregulation of
plasma flow and partly by compensatory changes in
PC, producing parallel changes in PUF. As mentioned
in the previous section, a role for mesangial cell
contraction in autoregulation is likely, probably by
altering Kf; locally produced AII may be involved in
this process. Autoregulation fails at extremes of blood
pressure. In severe hypertension, the exposure of the
kidney to excessive pressure overwhelms the resistance of the afferent arterioles, causing the phenomenon of pressure natriuresis. When blood pressure falls
below the autoregulatory range, RPF and GFR decline
and prerenal failure develops.
PROXIMAL TUBULAR FUNCTION

About two-thirds of filtered salt and water is reabsorbed in the proximal tubule. Other solutes including
glucose, amino acids, bicarbonate, and lowmolecular
weight proteins are almost completely reclaimed in
this tubule segment, as is about 90% of filtered inorganic phosphate. Epithelial transport of many of these
is linked to sodium transport and dependent on it for
its energy supply. Exceptions include some classes of
amino acid, which are transported without sodium by
a complicated series of co- and countertransporters.
Salt and Water Reabsorption in the Proximal

Tubule
Sodium Reabsorption
The proximal tubular epithelial cells are arranged in

a single layer lining the cylindrical basement
membrane. They exhibit polarity: the membranes on
63
Figure 4 Schematic view of a proximal tubular cell. The apical

(brush border) membrane (left) is depicted as a wavy line. The
basolateral membrane (right and lining the intercellular space) is
separated from the apical membrane by the tight junction (dark
squares). S represents any substance that enters the cell
across the apical membrane by secondary active transport via a
sodium-S cotransporter. The enzyme Na, K-ATPase (upper
right) maintains a steep gradient for sodium across the apical
membrane by constantly extruding sodium across the basolateral membrane.
opposite sides of the cells have different characteristics. The part of the cell membrane that faces the
tubular lumen is called the apical membrane; the part
that faces the basement membrane and the intercellular space is the basolateral membrane. Adjacent cells are
attached to one another at the tight junction, which
separates the apical from the basolateral membranes
(Fig. 4).
The enzyme sodium, potassium adenosine triphosphatase (Na, K-ATPase, also known as the
sodium pump) is located in the basolateral membrane.
Powered by energy released by the hydrolysis of ATP,
it transports sodium out of the cell interior and
potassium into it, with a stoichiometry of 3 Na:
2 K. This process is called primary active transport.
A consequence of the action of Na, K-ATPase is a
very low intracellular sodium concentration, establishing a steep concentration gradient between the
tubular fluid and the cell interior that favors sodium
entry across the apical membrane. This membrane is
impermeable to sodium, but contains proteins of
several different types that act as specialized sodium
channels. Each of these not only allows sodium entry
but also transports another solute into or out of the
cell. This is called secondary active transport, since the
energy driving it is provided indirectly by Na, KATPase at a site remote from the transport process
itself. Inhibition of Na, K-ATPase by ouabain blocks
sodium reabsorption in all nephron segments (22).
One of these proteins is NHE3, a member of a
family of transporters called Na, H-exchangers (23).
64
Haycock
Ion exchangers or antiporters are so called because

they transport two species of ions, in this case sodium
and hydrogen ions (protons), in opposite directions.
Sodium entry, therefore, leads to alkalinization of the
cell interior and acidification of the tubular fluid. By
an indirect process described later in this chapter, this
effectively causes reabsorption of bicarbonate in an
amount chemically equivalent to the amount of sodium entering via NHE3. This takes place early in the
proximal tubule, and because bicarbonate, in common
with other solutes described below, is reabsorbed in
preference to chloride (24), the concentration of chloride in the tubular fluid increases to a level substantially higher than that in the peritubular fluid.
Chloride, therefore, moves down its concentration
gradient from tubular fluid to peritubular space, partly by simple diffusion across the tight junction (which
is not really chemically tight) and partly via chloride
channels in the apical and basolateral membranes.
Diffusion across the tight junction, bypassing the cell
interior, is referred to as reabsorption via the paracellular shunt pathway. Because chloride is an anion,
its movement from the luminal to the peritubular
aspects of the tubule generates an electrical voltage,
lumen positive, across the epithelium that acts as a
further driving force for sodium reabsorption (25). At
least some of this also occurs through the paracellular
shunt pathway.
Other apical membrane proteins transport sodium and another solute (the cotransportate) in the
same direction, and are called cotransporters. For
example, the sodium-glucose cotransporter is activated when one sodium ion and one glucose molecule
attach to specific receptors on the protein on the
luminal side of the membrane. The molecule then
either rotates in the membrane or alters its configuration in such a way that the sodium and glucose are
transferred to the cytoplasmic side of the apical membrane, where they are released. Other cotransporters
include a sodium phosphate transporter and a whole
set of specific transporters for different amino acids.
The general mode of operation of secondary active
transport in the apical membrane of the proximal
tubule is schematized in Figure 4. Both the antiporter
and the cotransporters are potentially bidirectional. It
is only the maintenance of the steep concentration
gradient favoring sodium entry, resulting from sodium removal by Na, K-ATPase, that causes them to
transport protons out of the cell and the various
cotransportates to the cell interior. The antiporter
and several of the apical membrane cotransporter
proteins have now been sequenced and the corresponding genes mapped and cloned.
Water Reabsorption in the Proximal Tubule
Water is reabsorbed by osmosis, diffusing down the

osmotic gradient established by solute reabsorption.
The epithelium is freely permeable to water so that
osmotic equilibration is virtually instantaneous, and
the osmolality of the tubular fluid at all points in the
proximal tubule is not measurably different from that
of plasma. An unknown, but probably substantial,
proportion of water reabsorption takes place through

the paracellular shunt pathway and provides yet
another mechanism promoting sodium reabsorption.
Active transport of sodium across the basolateral
membrane into the lateral intercellular spaces leads
to a small, transient osmotic water gradient favoring
water reabsorption across the tight junction. The
resulting water flux causes salt to be transported
passively by a process known as convection or solvent
drag, given that the tight junction is permeable to
sodium and chloride. The relative importance of
transcellular and paracellular reabsorption of sodium
and water is uncertain.
Effect of Physical Forces on Proximal Tubular Reabsorption
Water and solute reabsorbed from the tubular lumen

is returned to the blood in the peritubular capillary
plexus, which is perfused by blood draining from
glomerular efferent arterioles. The peritubular
capillaries are therefore in series with and downstream from the glomerulus. The rate of reabsorption
of tubular fluid is governed by the Starling forces
across the peritubular capillary wall (26). The importance of the peritubular oncotic pressure in this process has been demonstrated both by micropuncture in
the intact animal and by studies in the isolated,
perfused rabbit proximal tubule (27,28). The effect of
changes in peritubular hydrostatic pressure is less
clear. Since the protein concentration of postglomerular plasma is proportional to the filtration fraction
[eq. (4)], it follows that changes in glomerular function
may induce secondary changes in proximal tubular
reabsorption rate; this may be important in
maintenance of glomerulotubular balance (GTB) (see
below).
Proximal Tubular Reabsorption of Other Solutes

Glucose
Glucose is reabsorbed by cotransport with sodium,

chiefly in the early proximal tubule. Glucose is not
merely acting as an energy source for sodium transport, since the nonmetabolized glucose analogue
a-methyl-D-glucoside can be substituted for it, but its
transport is dependent on sodium reabsorption. At
least two sodium-glucose cotransporters have been
identified in mammalian proximal tubule; they have
been named SGLT-1 and SGLT-2, respectively. SGLT-1
binds one sodium ion and one glucose molecule, while
SGLT-2 has a stoichiometry of 2Na:1 glucose. SGLT-2
is probably the more important of the two (29). It is
saturable, and when the filtered load of glucose
exceeds its maximal transporting capacity (Tm), the
excess is excreted in the urine. The relationship
between glucose filtration, reabsorption and excretion
is shown in Figure 5. Glycosuria occurs in hyperglycemia (as in diabetes mellitus) because the filtered load of
glucose exceeds the Tm of the transport system. Glycosuria because of abnormalities of the tubular glucose
transport system (renal glycosuria) occurs when the
renal threshold for glucose is less than the normal
65
Figure 5 Curves of filtration, excretion, and reabsorption of three solutes reabsorbed actively from
glomerular filtrate, plotted against the plasma concentration of the solute. Values plotted on the
abscissa (x-axis) are plasma concentrations
(mmol/L); those on the ordinate (y-axis) are the
quantities filtered, excreted, and reabsorbed
(mmol/100 mL glomerular filtration rate).
blood glucose concentration and is because of mutations in the gene for SGLT-2 (30).
Phosphate
The majority of filtered phosphate is reabsorbed with

sodium in the early proximal tubule. Studies of phosphate excretion at different plasma phosphate concentrations show that the ion is handled in a manner
similar to glucose, that is, by a saturable transport
system with a Tm and a threshold; hence, a similar set
of titration curves can be drawn (Fig. 5). In contrast to
glucose, some degree of phosphaturia is normally
present, since dietary phosphate in excess of requirement must be excreted: in the adult (nongrowing)
subject, dietary intake and urinary excretion are
equal (NEB 0). Thus, the normal plasma phosphate
concentration is just above the threshold value, and is
indeed defined by it, whereas, in health, the plasma
glucose concentration is always below the renal
threshold. The mechanism of proximal tubular phosphate reabsorption is discussed in more detail later in
this chapter.
Amino Acids
Amino acids are reabsorbed in the same general

manner as glucose and phosphate. At least five transport proteins exist, some of which transport sodium
and one of the major subclasses of amino acids with
high specificity, while others operate as exchangers,
transporting one group of amino acids out of the cell
in exchange for entry of another (31). The normal
plasma concentrations of all the amino acids are
below threshold in health, so that normal urine contains virtually no amino acids of any kind. Several
inherited defects of proximal tubular amino acid
transport systems have been described. By far, the

commonest is cystinuria. This is caused by mutations
in an apical membrane system that operates as an
amino acid exchanger. It transports cystine and the
dibasic amino acids arginine, lysine, and ornithine
into the cell in exchange for exit of neutral amino
acids. This is another form of secondary active transport. The driving force for exchange is the steep cellto-lumen gradient for neutral amino acids generated
by sodium-coupled entry of the latter, also across the
apical membrane. The exchanger is a heterodimer of
two components, labeled rBAT and bo,AT, respectively (32). The mutations that cause cystinuria affect
rBAT, and numerous loss of function mutations have
so far been identified in different families (33). The
only clinical significance of cystinuria is the tendency
to form cystine stones in the urinary tract: affected
individuals are otherwise healthy.
Proteins
Several lowmolecular weight proteins are present in

normal plasma in measurable concentrations. Examples
are a-1 microglobulin, b-2 microglobulin, and retinolbinding protein (RBP). Because they are smaller than
the size barrier of the glomerular ultrafilter, significant
amounts are present in glomerular filtrate and enter the
proximal tubule, where they are reabsorbed by pinocytosis into the tubular epithelial cells where they are
digested to their component amino acids and returned
to the body protein pool. They are therefore virtually
absent from normal urine. Proximal tubular dysfunction, however, leads to a urinary leak of these proteins
and a characteristic pattern of lowmolecular weight
proteinuria known as tubular proteinuria. Measurement
of urinary excretion of b-2 microglobulin and RBP,
66
Haycock
usually expressed as fractional excretion rates, is a

useful means of distinguishing between proteinuria of
glomerular and nonglomerular (tubulo-interstitial)
origin (34).
Only very small amounts of albumin and other
large plasma proteins normally pass the glomerular
filter. These are essentially completely reabsorbed by a
mechanism similar to that for lowmolecular weight
proteins. At low filtered loads, the system behaves as a
high-affinity, low-capacity transporter with virtually
complete clearance of albumin from the tubular fluid,
while at high filtered loads this mechanism becomes
saturated and a high-capacity, low-affinity mode operates, so that reabsorption continues to rise with increasing load, but about one-third of the filtered albumin
escapes reabsorption and appears in the urine (35). In
such conditions of glomerular proteinuria, the urinary
albumin excretion rate is always considerably less than
the amount filtered and catabolized. This probably
explains why proteinuria of apparently trivial amounts
(<10 g/day) may cause hypoalbuminaemia in some
cases of the nephrotic syndrome.
Proximal Tubular Secretion of Organic Anions

Many organic substances, anionic at physiological pH,
are actively secreted by the proximal tubule. These
include endogenous substances such as hippurate and
exogenous substances such as penicillin, probenecid,
and derivatives of hippuric acid such as orthoaminohippurate (hippuran) and paraaminohippurate
(PAH). The renal clearance of these compounds
exceeds GFR, and in the case of hippuran and PAH
extraction is almost complete. The secretory site is the
proximal convoluted tubule, especially the middle
and late segments. The small amount of PAH that
escapes secretion is accounted for by blood perfusing
the deepest (juxtamedullary) nephrons, in which the
postglomerular blood flows directly into the medullary vasa recta system without passing through the
cortical peritubular capillary plexus, bypassing the
secretory site for PAH. In the adult, only about 10%
of nephrons are of this type, so the underestimation of
renal plasma flow by PAH clearance is small. In the
newborn infant, in contrast, the superficial cortical
nephrons (the last to be formed) function little or not
at all, and the juxtamedullary nephrons provide the
lions share of renal function. Clearance of PAH and
similar substances is, therefore, an unreliable measure
of renal plasma flow in babies and newborn animals.
PAH secretion is saturable and Tm-limited; below the
renal threshold, excretion increases in parallel with
rising plasma concentration, while above this value,
there is no further rise in excretion. A similar, but
separate, transport system exists for the secretion of
organic cations.
GLOMERULOTUBULAR BALANCE
Delivery of fluid to the distal tubule is, by definition,
the difference between GFR and proximal tubular
reabsorption rate. Since the rate of distal fluid delivery
is an important determinant of distal tubular function,

and therefore of homeostasis, it is important that
glomerular and proximal tubular function are coupled
together. This coupling is known as GTB: changes in
GFR are partially offset by parallel and proportional
changes in proximal tubular reabsorption (36). GTB
has been shown experimentally to exist: When GFR is
manipulated by altering renal perfusion pressure,
changes in proximal tubular reabsorption take place
as the theory predicts. At least three mechanisms are
involved.
Peritubular Physical Forces

The protein concentration in postglomerular plasma is
determined by arterial plasma protein concentration
and filtration fraction. If GFR increases without a
parallel increase in plasma flow, filtration fraction
rises and peritubular capillary oncotic pressure is
increased. This alters the balance of Starling forces in
the peritubular environment in a direction that favors
reabsorption. Conversely, a fall in GFR and filtration
fraction leads to a reduction in the pressure gradient
for reabsorption (37). The oncotic pressure of the
peritubular capillary plasma has been shown to be a
powerful, and possibly rate-limiting, factor in proximal tubular reabsorption (27).
Filtration of Preferentially Reabsorbed

Substances
Sodium is reabsorbed in the early proximal tubule
with bicarbonate, phosphate, amino acids, lactate and
citrate in preference to chloride; glucose is also
absorbed by cotransport. The more these substances
are presented to the tubule, the more sodium (and
therefore water) will be reabsorbed. At any plasma
concentration, a change in GFR will produce a parallel
and proportionate change in the rate of filtration of
these substances, inducing a change in proximal tubular reabsorption rate in the same direction. This effect
will be amplified by the secondary effect on downstream, passive sodium reabsorption described in a
previous section. Thus, any random fluctuation in
GFR will cause secondary changes in both active
proximal tubular sodium reabsorption and in the
peritubular environment, reinforcing one another in
the preservation of GTB.
Tubuloglomerular Feedback
The ascending limb of the loop of Henle passes
through the vascular pole of its own glomerulus,
where it is intimately associated with the afferent
and efferent arterioles in the JGA. There is strong
evidence that delivery of some solute, probably chloride, is sensed at the JGA, where it provides the
afferent stimulus for regulation of GFR by negative
feedback (38). In experiments where the loop of
Henle was perfused in both orthograde and retrograde directions with solutions of various NaCl concentrations and at various rates, a clear inhibitory
effect was seen on RPF and GFR as concentration (or

flow rate) was increased (39). The effect is probably
mediated at least in part by local production and
conversion of angiotensin, producing glomerular
vasoconstriction (especially of the efferent arteriole)
and increasing vascular resistance. TGF may provide
an emergency defense against drastic salt and water
depletion in tubular injury. If proximal tubular NaCl
reabsorption were severely impaired by anoxia or the
action of a nephrotoxic drug, the resulting flood of
sodium-rich urine would lead rapidly to fatal dehydration and electrolyte depletion unless GFR were to
fall by some means. The oliguria of acute renal failure
may be seen, in this light, as a life-preserving
response rather than an undesirable feature of the
syndrome. Recognition that the TAL of the loop of
Henle is the most vulnerable part of the nephron to
ischemic injury (40) is entirely consistent with this
view, since the TAL is included in the anatomical
feedback loop consisting of glomerulusproximal
tubuleloop of HenleJGA.
SODIUM EXCRETION AND EXTRACELLULAR

FLUID VOLUME
Sodium and Extracellular Fluid Volume
The major osmotically active solute in the ECF is
sodium chloride: 90% of the sodium in the body,
and almost all the chloride, is located in the extracellular compartment. Since the tonicity of body fluids is
held within very narrow limits by regulation of water
intake and excretion, it follows that a gain or loss of
total body NaCl leads to expansion or contraction of
ECF volume. Conversely, ECF volume is the major
determinant of sodium excretion rate. In subjects on a
salt-restricted diet, a tiny increase in ECF volume is
followed by an immediate natriuresis (41). ECF volume expansion beyond a certain threshold volume is a
stimulus to sodium excretion, the magnitude of the
natriuresis being proportionate to the amount by
which this volume is exceeded. Conversely, sodiumfree urine is produced when ECF volume is below this
threshold, while sodium appears in the urine as soon
as this volume is even slightly exceeded. The relationship between ECF volume and sodium excretion is
shown in Figure 6. It is evident that the stimulus
producing the increase in sodium excretion cannot
be plasma sodium concentration, which does not
change, but ECF volume. How changes in this volume
are sensed and whether it is the total ECF volume or a
particular component or function of it that is important have been the subject of much investigation.
Control of Sodium Excretion

The control system regulating sodium excretion and,
therefore, ECF volume is in three parts: an afferent
(sensory) component, which detects a signal indicating the need to excrete or conserve sodium; an effector
organ (the kidney); and a means of transmitting the
67
Figure 6 The relationship between ECF volume, total body

sodium and urinary sodium excretion rate, assuming that a
gain or loss of 1 L of ECF corresponds to a gain or loss of 150
mmol sodium, and that the ECF sodium concentration does not
change. Negative values for sodium excretion indicate the magnitude of the sodium deficit that must be made up before sodium
appears in the urine, which is actually sodium free at points to the
left of the vertical zero line. The solid oblique line describes the
relationship as observed in recumbent subjects; the interrupted
line shows the relationship in the upright (quiet standing) position. The line is displaced upward, indicating sodium retention,
but the slope of the relationship is unchanged. Abbreviation:
ECF, extracellular fluid. Source: From Ref. 41.
response to the afferent stimulus to the kidney, the

efferent (messenger) component.
The Afferent Stimulus
Both acute (saline loading) and chronic (high dietary

sodium intake) volume expansion are natriuretic stimuli. Maneuvres that expand intrathoracic blood volume, such as head-out water immersion, are
natriuretic even though total ECF volume becomes
contracted as natriuresis continues (42). The magnitude of the response is correlated with left atrial
volume, and left atrial stretch receptors probably
mediate the effect. Stimulation of carotid sinus baroreceptors by underfilling of the arterial tree, as by
hypotension or vasodilatation, is antinatriuretic;
observations in subjects with arteriovenous fistulae
indicate that underfilling of the high-pressure (arterial) compartment overrides the effect of overfilling of
the low-pressure (venous) compartment, so that the
net response is antinatriuretic (43). Volume or sodium
receptors have been postulated in the liver or portal
venous system, following the observation that saline
infused into the portal vein or ingested orally is more
natriuretic than the same amount infused into a
peripheral vein. The presence of baroreceptors in the
interstitial space is suggested by the differential natriuretic effects of saline infusions of different oncotic
pressures; the fluid that expands intravascular volume
the least and the interstitial compartment the most
68
Haycock
(isotonic saline) is much more natriuretic than an

equivalent volume of plasma or hyperoncotic albumin
(44). Receptors in the pulmonary interstitium may
subserve this function.
Changes in the perfusion pressure perceived by
the kidney itself, in consequence of changes in arterial
blood pressure, lead to changes in urinary sodium
excretion both by intrarenal mechanisms and by
affecting the rate of renin secretion. It is likely that
most or all of these receptors play a part in determining the natriuretic status of the kidney, perhaps via a
(hypothetical) integrating mechanism located in the
hypothalamus or elsewhere. In general, reduction in
effective arterial volume, that is, underfilling of the
arterial tree in relation to its holding capacity, seems
to engender a sodium-retaining response sufficient to
override conflicting or interfering stimuli from elsewhere. Examples include congestive cardiac failure,
the nephrotic syndrome, and some cases of cirrhosis
of the liver.
Renal Effector Responses
Theoretically, the renal sodium excretion rate could be

altered either by changing GFR without an accompanying change in tubular reabsorption, or by the
reverse. In fact, changes in tubular reabsorption are
undoubtedly responsible for physiological regulation
of this function. Patients with chronic renal failure are
able to remain in external sodium balance on a fixed
sodium intake despite progressively falling GFR; an
adjustment of tubular reabsorption must have taken
place. It has been shown in many experimental models that the natriuretic response to volume expansion
is not abolished if GFR is prevented from increasing,
or even artificially decreased. Furthermore, experimental manipulations, which produce large increases
in GFR are not necessarily natriuretic. For example, in
dogs, protein feeding, dexamethasone administration,
dopamine infusion, and saline infusion all increase
GFR by up to 30%: of these, only saline loading is
consistently natriuretic (45).
If sodium excretion is modulated by changes in
tubular reabsorption, which part of the tubule is
mainly responsible? Experimental studies favor the
distal nephron as the important effector. Natriuresis
can be dissociated not only from GFR (see above) but
also from absolute and fractional proximal tubular
sodium reabsorption (46). Humoral factors known to
influence sodium reabsorption rate [such as aldosterone and atrial natriuretic peptide (ANP)] act mainly
on distal nephron segments. In rats, saline loading
causes marked changes in sodium reabsorption in the
collecting duct, resulting in apparent sodium secretion
in response to extreme volume expansion. Newborn
infants, in whom fractional proximal tubular sodium
reabsorption is lower than that in adults, maintain
sodium balance perfectly well, presumably by distal
tubular compensation. Furthermore, it appears logical
that the most distal part of the nephron is responsible
for the final adjustment of the sodium excretion rate
since, by definition, further downstream adjustments
are not possible.
It should be emphasized that the response to

saline volume expansion is normally a reduction in
sodium reabsorption in both proximal and distal
nephron segments, and that the dissociation between
these mentioned above was produced by unphysiological experimental manipulations. The fact that the
distal parts of the nephron can maintain a degree of
sodium homeostasis in the absence of parallel
responses in the proximal tubule probably reflects
the extreme importance of control of ECF volume; if
proximal tubular reabsorption is impaired or abnormal, external balance for sodium can still be maintained. However, the cost of maintaining sodium
balance in these circumstances may be secondary
disturbances of potassium and hydrogen ion excretion, as in Bartters syndrome.
Intrarenal Control of Sodium Excretion
Sodium excretion is correlated with renal artery pressure. ECF volume is an important determinant of
renal artery pressure, with resulting effects on intrarenal haemodynamics. The main resistance vessels in
the renal microcirculation are the afferent and efferent
glomerular arterioles. The latter are interposed
between the glomerular and peritubular capillary
plexuses. The hydrostatic pressure in the peritubular
capillaries, therefore, varies in proportion to renal
artery pressure and in inverse proportion to renal
arteriolar resistance. Volume expansion directly
increases perfusion pressure and, partly by inhibition
of the rennin-angiotensin-aldosterone system (RAAS),
reduces arteriolar resistance: volume contraction has
the reverse effects. Thus, volume expansion leads to
an increase, and volume contraction to a decrease, in
peritubular capillary pressure. The effect of angiotensin II on the renal circulation is complex. It causes both
preglomerular (afferent arteriole) and postglomerular
(efferent arteriole) vasoconstriction, but the postglomerular effect predominates, largely because of the
production of vasodilator prostaglandins in the afferent arteriole that offset the vasoconstrictor action of
angiotensin II. Conditions in which the RAAS is
stimulated, such as volume contraction, therefore
increase glomerular capillary pressure, leading to an
increase in filtration fraction and postglomerular
plasma oncotic pressure. Suppression of the RAAS
(volume expansion) has the opposite effect. In sum,
volume expansion causes an increase in hydrostatic
pressure and a decrease in oncotic pressure in the
peritubular capillaries. This reduces the Starling
pressure gradient for fluid reabsorption from the
peritubular interstitium and hence from the proximal
tubule, favoring natriuresis. Volume contraction
initiates the opposite sequence of events and is therefore antinatriuretic. These processes are schematized
in Figure 7 (47).
Although the above discussion has focused on
the proximal tubule, the rate of fluid absorption in the
distal nephron is also influenced by Starling forces,
since the cortical part of the distal tubule is supplied
by the same peritubular capillary network that supplies the proximal tubule, and the medullary part by
69
Figure 7 Schematic diagram of the forces

responsible for filtration of fluid from the glomerular capillaries and reabsorption of fluids into
the peritubular capillaries. The forces are
expressed in mmHg and are considered representative of those found in normal humans.
Source: From Ref. 47.
capillaries that receive part of their blood supply from

the efferent arterioles of juxtamedullary (deep cortical)
nephrons. It has sometimes been argued that physical
forces alone are sufficient to explain the regulation of
renal sodium excretion, but this hypothesis is rendered untenable by other observations, notably by the
fact that saline expansion is natriuretic even if renal
perfusion pressure is artificially prevented from rising
or even reduced. It follows that extrarenal factors are
also involved.
The Efferent Limb in the Regulation of Sodium Excretion
The fact that sodium excretion is influenced by

changes in the apparent volume of ECF subcompartments remote from the kidney indicates not only that
volume sensors exist at those sites, but also that there
must be some means of altering renal sodium excretion in response to the signals they receive. This
message (increase or decrease sodium excretion)
may be transmitted from sensor to effector by a
number of efferent pathways.
The Renal Nerves
The renal nerve supply contains efferent postganglionic

fibers located in the splanchnic nerves: stimulation of
these is antinatriuretic while denervation is natriuretic.
a-Adrenergic endings have been identified in close
approximation to proximal and distal tubules as well
as blood vessels; a direct effect on epithelial transport
seems likely. Denervation of one kidney causes natriuresis in the ipsilateral and antinatriuresis in the contralateral kidney, suggesting a role also for the afferent
fibers in the control of sodium excretion, perhaps by
integrating the responses of the two kidneys via a
renorenal reflex (48).
Aldosterone
The mineralocorticoid aldosterone is an important

regulator of tubular sodium handling. Released in
response to activation of the renin-angiotensin system,
which in turn is stimulated by volume contraction, it
acts on the distal convoluted tubule and collecting duct
to stimulate sodium reabsorption, where it also promotes secretion of K and H (see below). Although
other factors undoubtedly interact with aldosterone
and can even override it in the mineralocorticoid escape
phenomenon, its importance in sodium homeostasis is
illustrated by the finding that adrenalectomized animals, on a fixed replacement dose of mineralocorticoid, achieve sodium balance only at the cost of greatly
exaggerated changes in ECF volume, body weight,
blood pressure, and potassium concentration (49).
Catecholamines
Catecholamines affect sodium excretion, noradrenaline (norepinephrine) being antinatriuretic and dopamine natriuretic; both a-adrenergic and dopaminergic
receptors have been identified in the kidney. It has
been suggested that dopamine is an important mediator of sodium excretion, particularly in chronic renal
failure; however, it is likely that the major effects of
these amines are mediated via their action as locally
released neurotransmitters, rather than as circulating
hormones proper. The fact that L-dopa, a precursor of
dopamine, is natriuretic when infused into the renal
artery further supports the view that local synthesis
accounts for the origin of most or all of the dopamine
present in the kidney. This does not exclude an
important role for the amine as a second messenger,
that is, a locally acting vasoactive and natriuretic
factor, the activity of which may be increased by
other, circulating, substances.
70
Haycock
Prostaglandins
The vasodilator prostaglandins PGE2 and PGI2 (prostacyclin) are synthesized within the kidney and are
natriuretic. Inhibitors of prostaglandin synthase such
as indomethacin and ibuprofen cause RPF and GFR to
fall and the fractional proximal tubular sodium reabsorption to increase. These effects are partly offset by
volume expansion and greatly exaggerated by volume
contraction; the combination of volume contraction
and a nonsteroidal antiinflammatory agent may lead
to acute renal failure. Angiotensin II stimulates renal
release of prostaglandins: The main physiological role
of renal prostaglandins may be to maintain glomerular plasma flow and filtration rate in volume-contracted states, when AII levels are high.
The Kallikrein-Kinin System
The kinins, bradykinin and kallidin, are another class

of vasodilator and natriuretic substances produced
locally within the kidney. They are formed by the
action of the proteinase kallikrein, produced in the
distal tubule, on a circulating precursor. The reninangiotensin-aldosterone system, prostaglandins, and
kinins all interact in complex ways. AII and aldosterone both stimulate kallikrein production, while
kinins promote prostaglandin synthesis. Angiotensin-converting enzyme (ACE) not only converts AI
to AII but also inactivates bradykinin; it is also known
as kininase II. ACE therefore causes vasoconstriction
and antinatriuresis by both AII production and bradykinin degradation; ACE inhibition has the opposite
effects, accounting for the fact that ACE inhibitors
lower blood pressure even when the RAAS is not
activated. Catecholamines, the RAAS, prostaglandins,
and kinins can be regarded as components of a
complex intrarenal paracrine system that normally
act synchronously to regulate and stabilize renal
blood flow and GFR in response to changes in extrarenal influences such as ECF volume and circulating
hormones.
acids 130), vessel dilator hormone (3167) and

kaliuretic hormone (7998). ANP is a systemic and
renal vasodilator, and also reduces pulmonary vascular resistance. It acts directly on arteriolar smooth
muscle by a pathway not involving prostaglandins
or nitric oxide. Its actions on the renal vasculature
include dilatation of the arcuate arteries and afferent
arterioles, and antagonism of mesangial cell contraction induced by both AII and vasopressin. These
effects combine to cause an increase in GFR and a
reduction in fractional proximal tubular sodium reabsorption, probably because of altered physical (Starling) forces. It also has direct effects on the distal renal
tubule, decreasing sodium reabsorption by inhibiting
both its entry into the tubular cells through sodium
channels in the apical membrane and its exit from the
cell across the basolateral membrane by reducing Na1,
K1-ATPase activity. At the cellular level, the actions of
ANP are mediated by cyclic guanosine monophosphate (c-GMP) and c-GMP-dependent protein kinase
(52). Other actions of ANP include suppression of
renin release, inhibition of aldosterone synthesis, and
antagonism of AII-mediated vasoconstriction. Natriuretic peptides are the subject of much ongoing
research, and are beginning to be used experimentally
as therapeutic agents. Further details are available
from recent reviews (5355).
There is a compelling logic in atrial distension
being an important natriuretic stimulus, since in most
circumstances atrial stretch is a sensitive indicator of
circulating volume. The right atrium is the main
source of ANP, suggesting that the observed pulmonary effects of the hormone may be physiologically
important. In pathological states such as congestive
heart failure, atrial distension may become dissociated
from ECF and circulatory volume. It is theorized that
ANP release is beneficial in heart failure both by
reducing afterload and by mitigating the salt and
water retention characteristic of that condition. ANP
is involved, and may be preeminent, in the natriuresis
of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The major actions of ANP are
schematized in Figure 8.
Natriuretic Peptides
Volume expansion causes natriuresis in dogs even

when GFR is artificially constrained and supramaximal doses of mineralocorticoid and vasopressin are
given (50). The design of the experiments in which
this was demonstrated excluded all feasible explanations for the natriuresis except the action of a natriuretic hormone. In the 1980s, a new class of peptide
hormones was identified (51). The first of these was
ANP, so called because it is synthesized in the cardiac
atria and released in response to atrial stretch. Related
peptides are produced elsewhere in the body, including the brain, probably the anterior hypothalamus.
Human ANP is composed of 28 amino acids, including a 17amino acid ring formed by a disulfide bridge.
It is formed by cleavage of a 126amino acid prohormone. At least three other fragments of the same
prohormone have hormonal activity and are known,
respectively, as long-acting natriuretic peptide (amino
SODIUM REABSORPTION BEYOND THE

PROXIMAL TUBULE
About one-third of filtered sodium is reabsorbed
distal to the proximal tubule. As in the proximal
tubule, active extrusion of sodium by Na, K-ATPase
across the basolateral membrane accounts for the exit
step of sodium from the cell interior, and also generates a steep concentration gradient across the apical
membrane. Entry from tubular fluid to cell interior
takes place by secondary active transport down this
gradient via a family of Na-K-Cl! cotransporters,
coded for by genes collectively named SLC12, at least
three of which are expressed in different segments of
the distal nephron, and an amiloride-sensitive sodium
channel. In contrast to events in the proximal tubule,
where reabsorption is isotonic, salt and water reabsorption are dissociated in more distal segments.
71
Figure 8 Schematic outline of the major actions of atrial natriuretic peptide.
The Loop of Henle

About 25% of filtered sodium is reabsorbed in the
loop of Henle, all of it in the TAL. Entry into the cell
here is effected by the transporter NKCC2 (the corresponding gene is SLC12A2; see above), which as its
name suggests transports 1 Na, 1 K, and 2Cl!. Since
the number of positively and negatively charged ions
transported is equal, transport is electroneutral.
Ammonium (NH4), if present in the tubular fluid,
can substitute for K. NKCC2 is inhibited by bumetanide and furosemide (frusemide), a fact that
accounts for the diuretic action of these drugs. The
gene is located on chromosome 15q, and mutations of
it cause one form of Bartters syndrome of hypokalemia, alkalosis, hypercalciuria and hyper-reninaemia
with normal blood pressure, (56) as had been predicted from the close similarity between Bartters
syndrome and the effect of chronic administration of
loop diuretics. The very large amount of sodium
reabsorbed by this transport system, and its relatively
distal location, accounts for the great pharmacological
power of the loop diuretics. The activity of NKCC2
causes large amounts of potassium and chloride, as
well as sodium, to enter the cell from the tubular fluid.
The absorbed potassium is recycled back into the
tubule lumen via another apical membrane transporter, ROMK, and the chloride leaves the cell across the
basolateral membrane via a chloride channel, ClC-Kb.

Mutations in both ROMK and ClC-Kb can also cause
forms of Bartters syndrome (57,58) since NKCC2 can
operate efficiently only if the absorbed potassium and
chloride are prevented from accumulating within the
cell. The relationship between the various transporters
in the TAL cell is shown in Figure 9.
The Distal Convoluted Tubule

About 5% to 8% of filtered sodium is reabsorbed in the
DCT. Apical sodium entry in this segment takes place
via an electroneutral, thiazide-sensitive Na -Cl !
cotransporter (TSC). The gene is SLC12A3, located at
16q1213. Mutations of this gene cause Gitelmans
syndrome, a variant of Bartters syndrome distinguished from the classical forms by milder clinical
course, hypocalciuria and hypomagnesemia (59). As
would be expected, inhibition of the TSC by thiazide
diuretics mimics the features of Gitelmans syndrome
precisely. Because much less sodium is reabsorbed by
the TSC than by NKCC2, thiazides are much weaker
diuretics than loop diuretics. However, if a loop
diuretic [such as furosemide (frusemide)] and a thiazide (such as metolazone) are used together, the two
effects reinforce one another and massive diuresis
results.
72
Haycock
coded for by three separate genes. The gene for

a-ENaC is on chromosome 12, while those for b- and
g-ENaC are close together on 16p. Two distinct hereditary diseases are caused by mutations in ENaC genes.
Liddles syndrome, a form of dominantly inherited
hypertension with hypokalemia and suppression of
renin and aldosterone, was first shown to be because
of mutations in the b-ENaC gene that cause the
channel to be constitutively open even in the absence
of mineralocorticoid (60). The consequence is overabsorption of sodium, volume expansion, and
increased potassium excretion. It was subsequently
shown that the syndrome could also be caused by
mutations in the gene for g-ENaC. The rare disorder
pseudohypoaldosteronism type 1 (PHA1) is physiologically the mirror image of Liddles syndrome with
salt wasting, hyperkalemia, and acidosis despite high
levels of renin and aldosterone. It is because of loss of
function mutations in the genes for ENaC (61). The
inheritance of PHA1 is recessive in some pedigrees
and dominant in others. Some families with PHA1
have mutations in the a-ENaC gene; others in that for
b-ENaC. The three subunits of ENaC have considerable sequence homology and structural similarity,
suggesting descent from a common ancestor gene.
Figure 9 Membrane transporters in the thick ascending limb
(TAL) of the loop of Henle. Orientation as in Figure 4. Sodium,
potassium, and chloride enter the cell via NKCC-2 (upper left).
Sodium is then returned to the peritubular fluid by the action of
Na, K-ATPase (upper right); chloride is returned to the peritubular fluid by ClC-Kb (lower right) and potassium is recycled to
the tubular lumen via the potassium channel ROMK (lower left).
Mutations in any of these three transport proteins (NKCC-2,
ROMK, or ClC-Kb) cause Bartters syndrome (see text).
The Collecting Duct

Only 3% to 5% of filtered sodium enters the CCD
in most circumstances. However, the filtered load
of sodium is so large that this amounts to 750 to
1250 mmol/day, several times the normal daily intake.
Regulation of sodium reabsorption in this terminal
nephron segment is therefore crucial to homeostasis,
since no further adjustment of urine composition can
occur at a more distal site. Sodium enters the cell in
this segment via a highly sodium-selective, lowconductance, amiloride-sensitive sodium channel
located in the apical membrane of the principal cells,
commonly abbreviated to ENaC (epithelial sodium
channel). Opening of ENaC is under the control of
aldosterone, and therefore volume contraction promotes, and volume expansion inhibits, sodium reabsorption through it. Entry of sodium across the apical
membrane, without an accompanying anion, creates a
lumen-negative voltage across the epithelium, which
promotes the movement of H and K in the opposite
direction. Blockade of this channel with amiloride
therefore leads to H and K retention, as does
aldosterone deficiency. The channel consists of three
subunits, designated a-, b-, and g-ENaC, respectively,
RENAL CONTROL OF POTASSIUM AND

HYDROGEN ION SECRETION
Potassium Excretion
On a normal diet and at normal levels of renal
function, the rate of potassium filtration exceeds that
of potassium excretion 5- to 10-fold. At first sight, this
might suggest that excretion is controlled by varying
the rate of tubular reabsorption of filtered potassium,
as is the case for sodium. In fact, almost all the filtered
potassium is reabsorbed in the proximal tubule; potassium excretion depends on distal tubular secretion of
the ion.
Proximal Tubular Potassium Reabsorption
Potassium is reabsorbed isotonically throughout the

length of the proximal convoluted tubule, a process
apparently continued in the proximal straight tubule.
As described above, it is absorbed by NKCC2 in the
TAL but recycled to the lumen via ROMK; the loop of
Henle probably makes little or no net contribution to
potassium reabsorption or secretion. The fluid issuing
from the loop into the early distal convoluted tubule
contains relatively little potassium (515% of the filtered load); this fraction remains constant while urinary potassium excretion varies from minimum to
maximum, a 200-fold range. It is therefore logically
necessary that changes in potassium excretion are
effected at a more distal site.
Potassium Transport in the Distal Tubule and Collecting Duct
Potassium is secreted in the distal tubule in potassium

repletion and reabsorbed in potassium depletion; the
latter case is unusual. The major site of potassium
73
Table 1 The Effects of Various Conditions on the Factors Determining Renal Potassium Excretion
Condition
Volume expansion
Volume contraction
K loading
K depletion
Acute metabolic acidosis
Chronic metabolic acidosis
Metabolic alkalosis
Mineralocorticoid deficiency
Mineralocorticoid excess
Proximal tubulopathies
Loop diuretics, thiazides
K-sparing diuretics
D Distal sodium delivery
D Aldosterone
D Intracellular (K)
DUKV
DPK
:
;
;
:
:
:
;
;
:
;
:
:
:
:
:
;
;
:
?;
?:
?:
?;
:
;
;
:
:
;
:
:
:
;
:
;
:
;
;
:
;
;
;
:
Notes: ; Direction of change in UkV produced to restore Pk to normal. ? shows the direction of change indicated by the relevant arrows is probable but not certain.
Data from references cited in the text.
Abbreviations: UkV, urinary potassium excretion rate; Pk, plasma potassium concentration.
secretion is the late distal convoluted tubule and CCD,

corresponding to the location of ENaC (see the section
on sodium reabsorption in the collecting duct). There
are two major cell types in the CCD: principal cells
and intercalated cells. Potassium secretion is a function of the formerreabsorption, of the latter. The
driving force for potassium secretion is the lumennegative voltage resulting from sodium reabsorption.
Although perhaps 90% of this potential difference is
effaced by secondary, passive chloride reabsorption,
the remaining 10% or so is associated with countermovement of potassium and H. Electrophysiological
studies indicate that epithelial potassium transport
is a complex process involving several component
steps.
Intracellular potassium concentration is an
important modulator of potassium secretion into the
tubular lumen. Like all cells, distal tubular cells transport sodium out and potassium in across both basolateral and apical membranes. The apical (luminal)
membrane of principal cells, however, allows potassium to leave the cell down its electrochemical gradient
by two mechanisms: one is the potassium channel
ROMK and the other a potassium chloride symporter
(cotransporter). A rise in the peritubular (ECF) potassium concentration stimulates Na, K-ATPase in the
basolateral membrane, thus increasing potassium
entry. The net force driving potassium secretion is
therefore made up of two components: a concentration
gradient, resulting mainly from intracellular potassium
concentration, and an electrical potential gradient,
resulting from sodium reabsorption, as described
above. The electrical driving force is proportional to
the rate of sodium reabsorption. This is determined by
two main factors. The first is the rate of sodium entry
into the CCD: anything that increases distal sodium
delivery, such as inhibition of its reabsorption in more
proximal segments, stimulates sodium entry into principal cells and hence potassium secretion. The second
is aldosterone, which increases both sodium entry
across the apical membrane via ENaC and sodiumpotassium exchange across the basolateral membrane
by upregulating Na, K-ATPase (62,63).
The interaction between distal sodium delivery
and aldosterone is crucially important in the normal
control of potassium excretion in response to changes

in ECF volume. Volume expansion increases distal
sodium delivery by inhibition of reabsorption at more
proximal sites, and suppresses activity of the RAAS
and therefore aldosterone secretion. The resulting
effects on distal sodium reabsorption, and thus on
potassium secretion, are mutually opposed and cancel
one another. Volume contraction produces opposite
effects (reduced distal Na delivery, increased aldosterone secretion). Therefore, physiological changes in
sodium excretion rate because of changes in ECF
volume do not cause inappropriate secondary effects
on potassium excretion. Conditions in which the
normal relationship between distal sodium delivery
and aldosterone release are disrupted, however, cause
predictable disturbances of potassium balance: some
examples are given in Table 1.
Acute and chronic changes in acid-base status
exert important effects on potassium balance. Acute
metabolic alkalosis enhances, and metabolic acidosis
inhibits, tubular potassium secretion, leading to hypokalemia and hyperkalemia, respectively. This is probably secondary to changes in intracellular potassium
concentration: alkalosis promotes potassium entry
into cells, thus increasing the cell-to-tubular lumen
potassium gradient, while acidosis has the opposite
effect. The rate of secretion of H may have a further
effect on potassium secretion: in acidosis, increased
H flux attenuates the potential difference between
cell and tubular fluid, reducing the driving force for
potassium extrusion. Chronic metabolic acidosis, in
contrast to acute, is accompanied by increased potassium secretion and eventual potassium depletion.
This is probably because of increased distal delivery
of sodium secondary to hyperchloraemia: bicarbonate
deficiency inhibits proximal sodium reabsorption by
mechanisms discussed in an earlier section. The fact
that chronic respiratory acidosis, in which bicarbonate
levels are elevated, is not accompanied by potassium
wasting lends support to this view. Diuretics that
inhibit sodium reabsorption at sites proximal to the
CCT cause urinary potassium wasting because of the
simultaneous induction of increased distal sodium
delivery and hyperaldosteronism secondary to volume contraction. These include loop diuretics
74
Haycock
such as mercurials, furosemide (frusemide), and ethacrynic acid, as well as thiazides, carbonic anhydrase
inhibitors and osmotic diuretics. Diuretics such as
spironolactone, triamterene, and amiloride, which
work by inhibiting sodium reabsorption at the potassium-secreting site, cause potassium retention and
hyperkalemia.
Hydrogen Ion Excretion

Volatile and Nonvolatile Acids
The pH of the ECF is regulated within a very narrow

range (7.40 & 0.04) despite a number of perturbing
influences. By far, the largest of these is the continuous generation of carbonic acid as the end product of
carbohydrate metabolism. Because carbonic acid can
be dehydrated to CO2 and water by the enzyme
carbonic anhydrase (CA) in the lungs, and the resulting CO2 excreted in expired air, it is known as volatile
acid (VA). Regulation of CO2 excretion, therefore,
depends on pulmonary function, and its retention or
excessive loss result in respiratory acidosis and alkalosis, respectively. In addition, a much smaller
amount of nonvolatile acid (NVA), acid that cannot
be dehydrated to anhydrides in the lung, is generated
from other metabolic pathways. The principal components of NVA are as follows:
1.
2.
3.
Sulfuric acid, from combustion of sulfur-containing amino acids

Phosphoric acid, mainly from dietary organic
phosphate
Organic acids (OA), such as lactic and aceto-acetic
acids, from incomplete combustion of carbohydrate and fat, respectively
Organic acids usually form only a small component of NVA but may become important in disease
states (diabetic ketoacidosis, lactic acidosis and hereditary organic acidemias). The amount of sulfuric acid
produced depends on the intake of animal protein:
strict vegetarians (vegans), who eat no animal products at all, generate predominantly alkaline products
of metabolism and therefore excrete alkaline urine.
Since, by definition, NVA cannot be excreted via the
lungs it must be excreted by the kidney if the pH of
the ECF is not to become progressively, and eventually fatally, lowered. The disproportion between the
production rates of VA and NVA is enormous. An
adult human on a mixed Western diet produces about
14,000 mmol of carbonic acid daily but only about 50
to 100 mmol of NVA. This fact is dramatically illustrated by the difference in the rate of development of
acidosis following total obstruction of the trachea, as
opposed to total obstruction of the urinary tract.
Intracellular Fluid Buffers
A quantity of acid added to plasma or whole blood

lowers the pH far less than it would if added to a similar
volume of water. This is because of the presence of
powerful buffer systems in both plasma and red cells.
The most important of the ECF buffers is the carbonic

acidbicarbonate buffer system.
pH 6:1 log
HCO3 ! *
H2 CO3 *
10
Addition of H depletes (HCO!

3 ) by titrating it to
H2CO3 with a consequent fall in pH. The role of the
kidney is to regenerate HCO3! (buffer base) by excreting H, thus back-titrating or recharging the buffer
system. Addition of 1 mol of HCO3 (as NaHCO3) is
equivalent to the excretion of 1 mmol of H; loss of
1 mmol of HCO3! (as in diarrhea or bicarbonaturia)
causes metabolic acidosis quantitatively equivalent to
the addition of 1 mol of H (e.g., as hydrochloric acid)
to the ECF.
Reabsorption of Filtered Bicarbonate
The bicarbonate concentration in glomerular filtrate is

the same as that in plasma. At normal GFR (180 L/day)
and normal plasma bicarbonate (26 mmol/L) this represents a potential daily loss of more than 4500 mmol
of bicarbonate, equivalent to adding 4500 mmol of
acid to the ECF. All, or very nearly all, of this filtered
bicarbonate must be reabsorbed to prevent severe
acidosis. This takes place almost entirely in the proximal tubule(64), according to the scheme shown in
Figure 10. Note (a) that bicarbonate reabsorption is
dependent on H secretion, (b) that it is accompanied
by reabsorption of an equivalent amount of sodium,
and (c) that the HCO3! ion restored to the ECF is
synthesized in the proximal tubular cell, and is not the
same one that is titrated by secreted H in the tubular
lumen. The process is dependent on intracellular CA
to provide a source of H and HCO3! ions by hydration of CO2, and intraluminal CA (derived from the
brush border of the apical cell membrane) to remove
the H2CO3 formed there by dehydration to CO2;
the high diffusibility of CO2 leads to its rapid movement down its concentration into the cell, completing
the cycle and preventing the intraluminal reaction
from coming to equilibrium and stopping.
Titration curves similar to those for glucose and
phosphate (Fig. 5) can be plotted for bicarbonate and a
Tm and threshold value identified. An abnormally low
threshold defines proximal renal tubular acidosis
(RTA), in which urinary acidification capacity is normal but only when the plasma bicarbonate has fallen
below the (low) threshold value, that is, at the cost of
sustained metabolic acidosis (Fig. 11). Proximal RTA
is most commonly seen as part of a generalized
disorder of proximal tubular function (the Fanconi
syndrome), but has been described as an isolated
tubular defect in at least two forms (65,66).
Distal Tubular Hydrogen Ion Secretion
Proximal reabsorption of filtered HCO3! does not

contribute to the excretion of ingested or endogenously formed NVA and is therefore irrelevant to external
H balance: it merely prevents acidosis because of
renal HCO3! wasting. To achieve acid balance, H
must be secreted into the urine in a quantity exactly
75
Figure 10 Proximal tubular reabsorption of filtered

bicarbonate. The process is dependent on carbonic
anhydrase present in the apical membrane of the proximal but not the distal tubule.
pump hydrogen ions uphill from high pH [low

(H)] to low pH [high (H)]. This takes place in the
distal nephron, particularly the collecting duct. The
reaction whereby H is generated for secretion is the
same as that operating in the proximal tubule: intracellular synthesis and ionization of H2CO3 from CO2
and water. The process of secretion is, however,
different utilizing an active, energy-dependent H
transporter (proton pump). The tubular H pump
can sustain a maximum pH gradient of about 103
(ECF pH 7.4, urine pH 4.4). At pH 4.4, 1 L of water
contains only about 0.4 mmol H: at a daily urine flow
rate of 2 to 3 L, this is clearly inadequate to achieve H
balance. The ability to do so depends on the presence
of urinary buffers.
Urinary Buffers
Figure 11 Urine pH plotted against plasma bicarbonate concentration in normal subjects and patients with proximal and
distal RTA. In distal RTA, much commoner of the two kinds, urine
pH does not fall below that of plasma, even in severe metabolic
acidosis. In proximal RTA, normal urine acidification is achieved
but only at abnormally low plasma bicarbonate concentrations.
The curve of the relationship in proximal RTA shows the same
sigmoid form as in normal subjects but is shifted to the left to
varying degrees depending on the severity of the condition,
passing through the shaded area. Abbreviation: RTA, renal
tubular acidosis. Source: From Ref. 65.
equivalent to dietary and metabolic input of NVA,

typically 50 to 100 mmol/day for an adult (depending
on diet). This requires that the pH of the urine be
lowered below that of ECF; that is, the tubule must
The principal buffer in normal urine is inorganic

phosphate. The buffering reaction is as follows:
H Na2 HPO4 $ Na NaH2 PO4
11
The pK of this reaction is 6.8, leading to the following

version of the Henderson-Hasselbalch equation:
pH 6:8 log
NaHPO4 ! *
Na2 HPO4 *
12
The pK of 6.8 is well suited to the normal ECF pH of

7.4, since maximal buffering will take place within one
pH unit of 7.4, that is, well within the capacity of the
H pump.
The other important means of accommodating a
large amount of secreted H within the available
range of urinary pH is by the formation of ammonium
(NH4). This is formed in the urine from secreted H
76
Haycock
and ammonia (NH3), the latter being synthesized from

glutamine by the tubular cells. Thus,
NH3 H NaCl NH4 Cl Na
13
The Na ion is reabsorbed in exchange for the secreted

H and restored to the ECF as NaHCO3. The reactions
between secreted H and urinary phosphate and
ammonia are illustrated in Figure 12. Unlike urinary
phosphate excretion, ammonia synthesis can be upregulated in response to increased need to secrete H;
this is a very important adaptive response to chronic
acidosis. The classical (distal) type of RTA is because of
inability to lower urinary pH significantly below that
of plasma; an acquired form of distal RTA may be seen
in obstructive uropathy (67).
Relationship Between Hydrogen Ion Secretion and Sodium Ion

Reabsorption
As discussed above, H secretion requires the reabsorption of an equivalent amount of sodium. Conversely, failure of H secretion is associated with
impairment of Na reabsorption: Patients with proximal and distal RTA have proximal and distal Na
wasting, respectively. Control of H secretion is fundamentally dependent on changes in Na reabsorption at different nephron sites (68).
DIVALENT CATIONS, PHOSPHATE, AND

VITAMIN D
Renal Handling of Calcium
Calcium is filtered in amounts greatly exceeding its
rate of dietary intake and therefore of excretion; it is
reabsorbed in all the actively transporting segments of
the tubule by a variety of mechanisms. Reabsorption
occurring in the distal tubule is responsive to changes
in calcium intake and to other factors known to
influence calcium excretion; that in more proximal
segments is not.
Glomerular Filtration
Calcium is present in plasma in three forms: protein

bound, complexed to anions (principally phosphate),
and ionized (Ca2+). The last two together form ultrafilterable calcium (CaUF). The concentration of calcium
in glomerular filtrate is the same as that of plasma
CaUF, approximately 1.58 mmol/L (6.3 mg/dL).
Proximal Tubule
Calcium is reabsorbed approximately isotonically in

the proximal tubule: A slight rise in tubular fluid
concentration occurs in the initial (S1) segment, with
little or no change in the remainder of the proximal
tubule. Reabsorption is partly passive and dependent
on sodium transport, but an active transport system
also exists in the basolateral membrane probably
involving sodium-calcium countertransport; that is,
calcium is extruded from the cell as sodium enters.
The interdependence of proximal sodium and calcium
reabsorption is very close: factors such as changes in
ECF volume that affect sodium absorption produce
proportionate and parallel changes in calcium absorption. As with sodium, about 60% of filtered calcium is
reabsorbed in the proximal tubule (69).
Loop of Henle
Figure 12 Distal tubular hydrogen ion secretion (urinary acidification). (A) Titration of urinary phosphate buffer. (B) Buffering by
secreted ammonia. See text for details.
Calcium reabsorption continues in parallel with that

of sodium in the TAL; factors affecting the delivery of
sodium out of the loop produce proportionate
changes in calcium delivery. The reabsorption of
both is markedly inhibited by furosemide (frusemide)
(70). As previously explained, the NKCC2 transporter
is electroneutral, but back-leak of potassium into the
tubular fluid takes place through potassium channels
(ROMK) in the apical membrane. This recycling of
potassium creates a lumen-positive voltage across the

epithelium, which drives other positively charged
ions, including calcium, into the peritubular space
via the paracellular shunt pathway, accounting for
calcium reabsorption in this part of the tubule.
Distal Tubule
Calcium ions are actively reabsorbed in the distal

convoluted tubule and collecting duct. Transport in
this segment varies independently of sodium reabsorption: It is probable that homeostatic changes in
calcium excretion are effected here. Factors increasing
calcium reabsorption include parathyroid hormone,
hypocalcemia (probably by PTH-independent mechanisms as well as by promoting PTH release), metabolic
alkalosis, and hyperphosphatemia. Hypoparathyroidism, hypercalcemia, chronic metabolic acidosis, and
phosphate depletion are inhibitory (i.e., they increase
calcium excretion). Vitamin D has been reported to
have both enhancing and inhibiting effects on calcium
excretion: a physiologically important role for vitamin D
in renal calcium excretion has not been demonstrated
in man. The apparent discrepancies among these
reports may be because of species differences. Calcium
enters distal tubular cells across the apical membrane
through nifedipine-sensitive calcium channels. The
driving force appears to be the electrical polarization
of the membrane, which is strongly influenced by
intracellular chloride concentration. Measures that
decrease intracellular chloride (such as thiazide diuretics) cause hyperpolarization of the membrane and
enhance calcium uptake, thus decreasing its excretion.
This explains why thiazides decrease calcium excretion
while increasing sodium excretion (71).
77
response to changes in factors influencing the latter,

but to a lesser extent.
Loop of Henle
Eighty percent of the magnesium delivered into the

loop is reabsorbed there; this segment, therefore,
accounts for quantitatively the most important fraction of magnesium reabsorption. To what extent active
and passive (voltage-dependent) transport is responsible is not known. Furosemide (frusemide) strongly
inhibits magnesium reabsorption, suggesting that the
same mechanism that accounts for calcium reabsorption in the loop is involved.
Distal Tubule
Only a small proportion (<5%) of filtered magnesium

is reabsorbed in the distal tubule and collecting duct.
The mechanism responsible has not been identified.
Although it is tempting to dismiss distal tubular
reabsorption as unimportant because the amount
involved is small, this is misleading because 5% of
the filtered load represents a large quantity of magnesium in relation to dietary intake, and the final regulation of magnesium excretion is presumably effected
at the most distal site capable of magnesium reabsorption, probably the distal convoluted tubule.
Renal Handling of Phosphate
Magnesium is processed by the kidney in a manner

similar, but not identical, to calcium. The major difference is that the proximal tubule is proportionately less
important and Henles loop more important in the
case of magnesium. Moreover, unlike calcium, overall
renal magnesium handling is of the Tm-threshold type
(Fig. 5).
Inorganic phosphate (P) is filtered and reabsorbed by a

Tm-limited transport system (Fig. 5). Although FEP can
be calculated by equation (3), marked variability in TmP
secondary to changes in GFR makes FEP an imprecise
way of describing tubular P reabsorption. The quotient
TmP/GFR defines the theoretical P threshold, that is,
the intercept on the x-axis of Figure 5 obtained by
extending to the left the horizontal segment of the P
excretion curve and ignoring the splay. It is this quantity that is altered by changes in circulating PTH and
other modulators of tubular P transport. TmP/GFR can
be calculated from the equation (72)

TmP
UP % Pcreatinine
14
PP !
GFR
Ucreatinine
Glomerular Filtration
Segmental Tubular Phosphate Reabsorption
Like calcium, magnesium is present in plasma in

protein-bound, complexed, and ionized (Mg 2+ )
forms. MgUF is about 80% of total plasma magnesium,
a rather higher fraction than for calcium.
The bulk of filtered P is reabsorbed in the proximal

tubule, where it is responsive to influences such as
changes in ECF volume, which alter sodium reabsorption. Entry into the cell is via sodium phosphate
cotransporters (NPT) in the apical membrane. Two
families of such transporters have now been characterized in renal cortex from several species, including
man. Designated as type I and type II transporters,
respectively, both are present in proximal tubular
apical membranes. Type II transporters are thought
to be the main physiologically important regulators of
P reabsorption. NPT IIa is a protein of 635 amino
acids, probably has eight transmembrane-spanning
domains and has sites for protein phosphorylation
that probably regulate transporter activity via protein
Renal Handling of Magnesium
Proximal Tubule
Magnesium is absorbed less avidly in the proximal

tubule than sodium and calcium, so that its concentration rises along the length of the segment to a value
about 1.6 times that in glomerular filtrate. This probably reflects a lower epithelial permeability to magnesium than to calcium, the forces driving reabsorption
of the two being similar. Proximal magnesium reabsorption changes in parallel with that of sodium in
78
Haycock
kinase C. It transports with a stoichiometry of 3Na:1P.

The gene is located on chromosome 5 (that for the type I
transporter is on chromosome 6).
PTH and Phosphate Reabsorption
PTH inhibits P reabsorption in both proximal and

distal segments. Changes in proximal P reabsorption
are paralleled by changes in sodium reabsorption, as
would be expected from the nature of the transport
process, whereas in the distal tubule, PTH has no
effect on sodium transport. PTH binds to proximal
tubular cell receptors that activate the adenylate
cyclase and protein kinase C pathways. Acute changes
in transporter activity are mediated by activation of
protein kinase C, which reduces activity of the sodium
phosphate cotransporter (NPT IIa). Chronic changes
probably involve alterations in gene transcription and
translation. PTH release is controlled directly and
indirectly by phosphate intake. Increasing P intake
leads to transient hyperphosphatemia, which in turn
causes hypocalcemia. Hypocalcemia stimulates PTH
release both directly and by altering vitamin D metabolism (see below). The increased level of PTH inhibits
P reabsorption, leading to phosphaturia that compensates for the increase in dietary P.
Phosphatonin, Phex, Dmp1, Klotho, and Phosphate Reabsorption
The activity of NPT IIa is inhibited by not only PTH,

but also a recently described protein of the fibroblast
growth factor family, FGF23(73), produced in osteocytes. FGF23 is also secreted by some tumors that
cause tumor-induced osteomalacia, a paraneoplastic syndrome leading to renal phosphate wasting, hypophosphatemia, and rickets/osteomalacia (74). FGF23 is
inactivated by a yet unidentified peptidase that
cleaves it into two inactive fragments. Mutations in
FGF23 that render it resistant to enzymatic cleavage
but do not inhibit its activity cause a rare, autosomal
dominant, form of hypophosphatemic rickets (75).
Two other proteins are also expressed in osteocytes,
and are believed to be involved in the regulation of
expression of the gene for FGF23. The gene for the first
of these to be described is called PHEX (a phosphateregulating gene with homologies to genes for endopeptidases on the X chromosome), and is mutated in
patients with the relatively common X-linked hypophosphatemic rickets (76). The second is called DMP-1
(dentin matrix protein-1) and the gene for it is mutated in the very rare autosomal recessive hypophosphatemic rickets (77). The phenotypes of all these forms of
hereditary hypophosphatemia are similar, and are
caused by inhibition of NPT IIa either by the uncontrolled release of FGF23 or by failure of its enzymatic
degradation, leading to inhibition of P reabsorption by
NPT IIa, phosphaturia, and hypophosphatemic rickets. Yet another gene, rather quaintly called klotho in
honor of the Greek goddess who spins the thread of
life, is mutated in transgenic mice with a premature
aging syndrome. Further studies have shown that
klotho is expressed in the renal tubule and is an
essential coreceptor for FGF23. Klotho-deficient mice
and FGF-deficient mice develop identical phenotypes,

indicating that klotho expression in the renal tubules
is necessary for the renal effect of FGF23. These
interrelationships are reviewed at greater length by
Kurosu and Kuro-o (78).
Renal Metabolism of Vitamin D

The active end product of vitamin D metabolism is the
hormone 1,25-dihydroxycholecalciferol (1,25-DHCC,
1,25-dihydroxyvitamin D3). It is produced from vitamin D by 25-hydroxylation, which takes place in the
liver, and subsequent 1a-hydroxylation, which takes
place in the mitochondria of the proximal tubular
cells. In conditions of 1,25-DHCC repletion, 1ahydroxylation is suppressed and the inactive metabolite 24,25-DHCC is produced instead (79). The kidney
is thus an endocrine organ producing a hormone
under negative feedback control: According to need,
conversion of 25-HCC oscillates between 1a- and 24hydroxylation. Apart from the circulating concentration of 1,25-DHCC, other factors promoting 1ahydroxylation include, in descending order of
power, hypocalcemia, hypophosphatemia, and PTH.
As well as regulating the production of 1,25-DHCC,
the kidney is one of its target organs, along with
intestine and bone. Localization of the hormone to
the nuclei of distal tubular cells occurs (80) and
presumably mediates its renal action, which is to
enhance tubular calcium reabsorption, reinforcing
the elevation of ECF Ca and P concentrations mediated by its other major actions: stimulation of intestinal
Ca and P transport and mobilization of bone mineral.
Failure of renal 1a-hydroxylation of vitamin D is one
of the three main processes that result in bone disease
because of chronic renal failure, the other two being
secondary hyperparathyroidism and chronic metabolic
acidosis.
CONCENTRATION AND DILUTION OF URINE

Osmolality and Tonicity of Body Fluids
In health, the osmolality of body fluids is controlled
within narrow limits (291 & 4 mosmol/kg H2O) by
regulation of water intake and excretion. Osmolality is
a physical property of solutions that depends on the
total molar concentration of all solutes present, in
other words the number of solute particles dissolved
in 1 kg of solvent (water), and is measured in the
laboratory by various techniques such as freezing
point depression. Tonicity refers to effective osmolality, that is, that part of the total osmolality of the fluid
in question (e.g., ECF) that is contributed by solutes to
which the cell membrane is impermeable. Normally
sodium and its attendant anions make up nearly all of
the tonicity of the ECF, urea and glucose being relatively permeant, although in insulin deficiency (type 1
diabetes mellitus) glucose is impermeant and does
contribute to tonicity. Some exogenous solutes can
contribute to tonicity (alcohol, mannitol) in special
circumstances, but these are not present in the body
fluids in normal conditions. In the absence of exogenous, osmotically active substances, osmolality of
plasma can be approximated by the formula
Osmolality 2 % PNa PUrea PGlucose
15
where the plasma concentrations of sodium, urea, and

glucose are all expressed in mmol/L. If the osmolality
as measured in the laboratory is significantly greater
than the result of equation (15), this is evidence for the
presence of an abnormal osmolyte (most commonly
alcohol in the comatose or confused adult patient).
Changes in ECF tonicity are sensed by osmoreceptors located in the anterior hypothalamus. ECF
hypertonicity produces thirst, leading to increased
water intake (providing the subject has access to
water), and release of ADH, leading to a reduction
in water excretion: hypotonicity has the opposite
effects. Mammals in the wild state drink in response
to thirst: they therefore oscillate between isotonicity,
the point at which thirst disappears, and marginal
hypertonicity, which stimulates further water intake.
They rarely, if ever, need to excrete dilute urine but
may need to excrete highly concentrated urine if water
is not available in response to the thirst stimulus.
Civilized man, in contrast, frequently drinks in excess
of biological need for social and cultural reasons and
because of the availability of flavored (and otherwise
adulterated) drinks. The ability to excrete urine more
dilute than ECF is therefore essential to the avoidance
of dilutional hypotonicity.
79
Countercurrent Multiplication and Exchange

The processes of countercurrent multiplication and
exchange convert the hyperosmolality of the interstitium surrounding the TAL, and resulting from NaCl
reabsorption, into an axial concentration gradient
maximal at the papillary tip. The elements of the
countercurrent system are the loops of Henle (multipliers) and the vasa recta (exchangers), the collecting
duct being the site of final osmotic water reabsorption.
The mechanism was described in principle many
years ago, but a more detailed understanding of the
process required methods of investigation not available until much later. A general description of the
medullary countercurrent system is beyond the scope
of this chapter: the reader is referred to an authoritative symposium published in 1983 (82), and to a lucid
account of the properties of such systems in biology
(83). A recurring difficulty has been to account for
concentration of solute in the inner medullary and
papillary interstitium (where the tonicity is highest) in
the apparent absence of active solute reabsorption in
the thin (inner medullary) segment of the ascending
limb (Fig. 2). This probably depends on passive recycling of urea through facilitated diffusion in the
medulla. Various models have been constructed to
account for the observed hypertonicity in the inner
medulla without the need to postulate active solute
reabsorption in the thin ascending limb, but none has
been unequivocally validated to date.
The Antidiuretic Hormone

The Diluting Segment
As previously discussed, proximal tubular fluid reabsorption is isotonic. The dissociation between water
and solute reabsorption necessary for the production
of urine with osmolality different from that of ECF
takes place in more distal parts of the nephron.
Micropuncture studies showed that the fluid in the
early distal convoluted tubule is hypotonic to plasma
(about 100 mosmol/kg H2O) irrespective of whether
the final urine was concentrated or dilute at the time
(81). Solute is therefore actively reabsorbed without
water in this segment. This is now known to be via the
Na-K-2Cl! transporter NKCC2 (see above). The
TAL and early distal convoluted tubule therefore
comprise the obligate diluting segment. Further dilution occurs in the early distal convoluted tubule via
the thiazide-sensitive NaCl! cotransporter TSC.
When maximally dilute urine is being produced, the
late distal tubule and collecting duct also becomes
water impermeable in the absence of ADH, and continuing solute (NaCl) reabsorption in the late distal
convoluted tubule and the collecting duct leads to the
elaboration of urine of osmolality about 40 mosmol/
kg H 2 O. The production of concentrated urine
depends on the establishment of interstitial hypertonicity in the medulla, a process that ingeniously
utilizes the same transporting process that is responsible for dilution, that is, active electrolyte reabsorption in the TAL.
The human ADH is the cyclic nonapeptide arginine

vasopressin (AVP):
Cys ! Tyr ! Phe ! Glu ! Asp ! Cys ! Pro ! Arg ! Gly ! NH2
A ring structure is formed by a disulfide bridge
between the two cysteine residues. AVP is synthesized in cells whose bodies lie in the supraoptic and
paraventricular nuclei of the anterior hypothalamus,
and is released from their axonal endings in the
posterior pituitary. It is released in response to both
osmoreceptor stimulation and carotid sinus and aortic
arch baroreceptor stimulation (84). The former is
responsible for maintaining tonicity of body fluids in
the normal range, while the latter causes renal water
retention in the face of underfilling of the high-pressure
compartment of the circulation because of, for example,
volume depletion, vasodilatation or left ventricular
failure. Baroreceptor-mediated AVP release is not suppressible by hypotonicity, and so hyponatremia is
commonly seen in conditions associated with it. A
much larger proportional volume change is needed
than osmolar change to initiate an AVP response
(Fig. 13). An increase of only 1% to 2% in plasma
osmolality suffices to elicit a maximally antidiuretic
plasma level (5 pg/mL), while a reduction in blood
volume or pressure of about 7% to 8% is needed before
a significant AVP response is seen (85). However, very
high AVP levels are produced when hypovolemia or
hypotension become more severe than this.
80
Haycock
an autosomal recessive condition and affects both

sexes equally (89).
Free Water Clearance and Reabsorption

The urinary solute excretion rate is determined by
dietary and metabolic factors, but is usually in the
region of 500 to 1500 mosmol/day for an adult on an
average diet. Taking a notional value of 580 mosmol/
day, and assuming a plasma osmolality of 290 mosmol/kg H2O, it is evident that 2 L of isotonic urine
would suffice to excrete this amount of solute. Any
additional water excretion will render the urine hypotonic to plasma: such additional water excretion is
called free water clearance, CH2O. Osmolar clearance
(COSM) can be calculated by the following equation:
COSM
Figure 13 The antidiuretic response to isovolemic decrease in

plasma osmolality (straight line) and isotonic depletion of plasma
volume (curved line). The negative value of the exponent of the
equation relating PAVP to volume arises from the fact that the
volume change (Dvol) is in a negative direction. Source: From
Ref. 85.
Antidiuretic Hormone Receptors
AVP binds to specific receptors (V2 receptors) on the

collecting duct cells, causing activation of adenylate
cyclase and conversion of ATP to cyclic AMP. This
initiates a chain of intracellular events that culminate
in the insertion of water channels in the apical membrane of the cell, rendering it permeable to water. The
gene for the V2 receptor is on the long arm of the X
chromosome: loss of function mutations in it is
responsible for the disease X-linked diabetes insipidus
(86). As recently as 2005 a new syndrome was
described, termed nephrogenic syndrome of inappropriate antidiuresis (87), shown to be because of gain of
function mutations in the same gene. The clinical
features are similar to those seen more commonly in
the syndrome of inappropriate secretion of ADH: the
production of inappropriately concentrated urine in
the presence of hyponatremia and normovolemia, but
with low, rather than high, ADH levels. The condition
has since been described in neonates (88).
Aquaporins
The water channels referred to in the previous paragraph belong to a family of proteins known as aquaporins, of which different members are expressed in
different cell types. The human collecting duct aquaporin is aquaporin 2, and the gene that codes for it is
located on chromosome 12 in the region 12q13. Mutations in the gene for aquaporin 2 cause a form of
diabetes insipidus clinically indistinguishable from
that caused by mutations in the V2 receptor gene
except that, as would be expected, it is inherited as
UOSM % V
POSM
16
where V is urine flow rate, OSM is osmolality, and U

and P refer to urine and plasma, respectively. When
urine is isotonic with plasma, U OSM POSM (by
definition), and therefore COSM V. When urine is
hypotonic to plasma, COSM < V, and
CH2 O V ! COSM
17
When urine is hypertonic, COSM > V, and therefore the

calculated value for CH2O is negative. Reversing the
sign, negative free water clearance becomes free water
reabsorption, usually designated TcH2O.
TcH2 O COSM ! V
CH2O and TcH2O define the limiting
water excretion, and therefore for
ingestion. At a solute excretion rate
day, COSM is 2 L a day. Rearranging
V
COSM % POSM
UOSM
18
values for renal
tolerated water
of 580 mosmol/
equation (15),
19
and taking values of 40 and 1200 mosmol/kg H2O as

minimal and maximal U OSM, respectively, yields limiting values for V of about 15 and 0.5 L daily. Corresponding maximum values for CH2O and TcH2O are
12.5 and 1.52 L/day. Thus, at any given solute excretion rate, the defense against overhydration and dilution (CH2O) is much more effective than that against
dehydration and concentration (TcH2O), a conclusion
borne out by clinical experience. This huge capacity to
protect the body fluids from dilution may be an
evolutionary inheritance from our fishy and amphibian ancestors, to whom (in fresh water) osmotic dilution is a constant threat.
REFERENCES
1. Coulthard MG, Hey EN. Weight as the best standard for
glomerular filtration in the newborn. Arch Dis Child 1984;
59:373375.
2. McCance RA. The maintenance of stability in the newly
born. 1. Chemical exchange. Arch Dis Child 1959; 34:
361370.

3. Dugdale AE. Evolution and infant feeding. Lancet 1986;
1:14411442.
4. Manz F, Kalhoff H, Remer T. Renal acid excretion in early
infancy. Pediatr Nephrol 1997; 11:231243.
5. Kildeberg P. Disturbances of hydrogen ion balance occurring in premature infants. II. Late metabolic acidosis. Acta
Paediatr 1964; 53:517526.
6. Arant BS Jr, Edelmann CM Jr, Spitzer A. The congruence of
creatinine and inulin clearances in children: use of the
technicon autoanalyzer. J Pediatr 1972; 81:559561.
7. Calcagno PL, Rubin MI. Renal extraction of para-aminohippurate in infants and children. J Clin Invest 1963;
42:16321639.
8. Tisher CC, Bulger RE, Trump BF. Human renal ultrastructure. I. Proximal tubule of healthy individuals. Lab Invest
1966; 15:13571394.
9. Bulger RE, Tisher CC, Myers CH, et al. Human renal ultrastructure. II. The thin limb of Henles loop and the interstitium in healthy individuals. Lab Invest 1967; 16:124141.
10. Tisher CC, Bulger RE, Trump BF. Human renal ultrastructure. 3. The distal tubule in healthy individuals. Lab Invest
1968; 18:655668.
11. Arakawa M. A scanning electron microscopic study of the
human glomerulus. Am J Pathol 1970; 64:457466.
12. Kanwar YS, Farquar MG. Anionic sites in the glomerular
basement membrane. In vivo and in vitro localization in
the laminae rarae by cationic probes. J Cell Biol 1979;
81:137153.
13. Venkatachalam MA, Karnovsky MJ, Fahimi HD, et al. An
ultrastructural study of glomerular permeability using
catalase and peroxidase as tracer proteins. JExp Med
1970; 132:11531167.
14. Brenner BM, Hostetter TH, Humes HD. Glomerular permselectivity: barrier function based on discrimination of
molecular size and charge. Am J Physiol 1978; 234:
F455F460.
15. Ruotsalainen V, Ljungberg P, Wartiovaara J, et al. Nephrin
is specifically located at the slit diaphragm of glomerular
podocytes. Proc Natl Acad Sci U S A 1999; 96:79627967.
16. Tryggvason K, Ruotsalainen V, Wartiovaara J. Discovery of
the congenital nephrotic syndrome gene discloses the
structure of the mysterious molecular sieve of the kidney.
Int J Dev Biol 1999; 43:445451.
17. Maddox DA, Deen WM, Brenner BM. Dynamics of glomerular filtration. VI. Studies in the primate. Kidney Int
1974; 5:271278.
18. Brenner BM, Troy JL, Daugherty TM, et al. Dynamics of
glomerular ultrafiltration in the rat. II. Plasma flow dependence of GFR. Am J Physiol 1972; 223:11841190.
19. Deen WM, Troy JL, Robertson CR, et al. Dynamics of
glomerular filtration in the rat. IV. Determination of the
ultrafiltration coefficient. J Clin Invest 1973; 52:15001508.
20. Navar LG, Bell PD, White RW, et al. Evaluation of the
single nephron glomerular filtration coefficient in the dog.
Kidney Int 1977; 12:137149.
21. Ausiello DA, Kreisberg JJ, Roy C, et al. Contraction of
cultured rat glomerular mesangial cells after stimulation
with angiotensin II and arginine vasopressin. J Clin Invest
1980; 65:754760.
22. Burg MB, Orloff J. Electrical potential difference across
proximal convoluted tubules. Am J Physiol 1970; 219:
17141716.
23. Brant SR, Yun CH, Donowitz M, et al. Cloning, tissue
distribution, and functional analysis of the human Na/
H exchanger isoform, NHE3. Am J Physiol 1995; 269:
C198C206.
24. Burg MB, Green N. Bicarbonate transport by isolated
perfused rabbit proximal convoluted tubules. Am J Physiol
1977; 233:F307F314.
81
25. Kokko J. Proximal tubule potential difference. Dependence

on glucose, HCO3 and amino acids. J Clin Invest 1973;
52:13621367.
26. Martino JA, Earley LE. Relationship between intrarenal
hydrostatic pressure and hemodynamically induced
changes in sodium excretion. Circ Res 1968; 23:371386.
27. Spitzer A, Windhager E. Effect of peritubular oncotic
pressure changes on proximal tubular fluid reabsorption.
Am J Physiol 1970; 218:11881193.
28. Berry CA, Cogan MG. Influence of peritubular protein on
solute reabsorption in the rabbit proximal tubule. J Clin
Invest 1981; 68:506516.
29. Mackenzie B, Loo DD, Panayotova-Heiermann M, et al.
Biophysical characteristics of the pig kidney Na+/glucose
cotransporter SGLT2 reveal a common mechanism for
SGLT1 and SGLT2. J Biol Chem 1996; 271:3267832683.
30. Wright EM, Martin MG, Turk E. Familial glucosegalactose
malabsorption and hereditary renal glycosuria. In: Scriver
CR, Beaudet AL, Sly WS, et al., eds. The Metabolic and
Molecular Bases of Inherited Disease. 8th ed. New York:
McGraw-Hill, 2001:48914908.
31. Silbernagl S. The renal handling of amino acids and
oligopeptides. Physiol Rev 1988; 68:9111007.
32. Palacn M, Estevez R, Bertran J, et al. Molecular biology of
mammalian plasma membrane amino acid transporters.
Physiol Rev 1998; 78:9691054.
33. Palacn M, Goodyer P, Nunes V, et al. Cystinuria. In:
Scriver CR, Beaudet AL, Sly WS, et al., eds. The Metabolic
and Molecular Bases of Inherited Disease. 8th ed. New
York: McGraw-Hill, 2001:49094932.
34. Waldmann TA, Strober W, Mogielnicki BP. The renal
handling of low molecular weight proteins. II. Disorders
of serum protein catabolism in patients with tubular proteinuria, the nephrotic syndrome, or uremia. J Clin Invest
1972; 51:21622174.
35. Park CH, Maack T. Albumin absorption and catabolism by
isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 1984; 73:767777.
36. de Wardener HF. The control of sodium excretion. In: Orloff
J, Berliner RW, eds. Handbook of Physiology. Washington,
DC: American Physiological Society, 1973:677720.
37. Ichikawa I, Hoyer JR, Seiler MW, et al. Mechanism of
glomerulotubular balance in the setting of heterogeneous
glomerular injury. Preservation of a close functional linkage between individual nephrons and surrounding microvasculature. J Clin Invest 1982; 69:185198.
38. Wright FS, Briggs P. Feedback regulation of glomerular
filtration rate. Am J Physiol 1977; 233:F1F7.
39. Schnermann J, Ploth DW, Hermle M. Activation of tubuloglomerular feedback by chloride transport. Pflugers Arch
Eur J Physiol 1976; 362:229240.
40. Brezis M, Rosen S, Silva P, et al. Selective vulnerability of
the medullary thick ascending limb to anoxia in the isolated perfused rat kidney. J Clin Invest 1984; 73:182190.
41. Strauss MB, Lamdin E, Smith WP, et al. Surfeit and deficit
of sodium: a kinetic concept of sodium excretion. Arch
Intern Med 1958; 102:527536.
42. Epstein M. Cardiovascular and renal effects of head-out
water immersion in man. Circ Res 1976; 39:619628.
43. Epstein FH, Post RS, McDowell M. Effects of an arteriovenous fistula on renal hemodynamics and electrolyte excretion. J Clin Invest 1953; 32:233241.
44. Martino JA, Earley LE. Demonstration of the role of
physical factors as determinants of natriuretic response to
volume expansion. J Clin Invest 1967; 46:19631978.
45. Lindheimer MD, Lalone RC, Levinsky NG. Evidence that
an acute increase in glomerular filtration rate has little
effect on sodium excretion in the dog unless extracellular
volume is expanded. J Clin Invest 1967; 46:256265.
82
Haycock
46. Howards SS, Davis BB, Knox FB, et al. Depression of fractional sodium reabsorption by the proximal tubule of the dog
without sodium diuresis. J Clin Invest 1968; 47:15611572.
47. Arendshorst WJ, Navar LG. Renal circulation and glomerular hemodynamics. In: Schrier RW, Gottschalk CW, eds.
Diseases of the Kidney. Boston: Little, Brown & Co., 1993:75.
48. Moss NG. Renal function and renal afferent and efferent
nerve activity. Am J Physiol 1982; 243:F425F433.
49. Young DB, Guyton AC. Steady state aldosterone dose
response relationships. Circ Res 1977; 40:138142.
50. de Wardener HE, Mills IH, Clapham WF, et al. Studies on
the efferent mechanism of the sodium diuresis which
follows the administration of intravenous saline in the
dog. Clin Sci 1961; 21:249258.
51. Flynn TG, Davies PL. The biochemistry and molecular
biology of atrial natriuretic factor. Biochem J 1985;
232:313321.
52. Brenner BM, Ballermann BJ, Gunning ME, et al. Diverse
biological actions of atrial natriuretic peptide. Physiol Rev
1990; 70:665699.
53. McGrath MF, de Bold ML, de Bold AJ. The endocrine
function of the heart. Trends Endocrinol Metab 2005;
16:469477.
54. de Bold AJ. Cardiac natriuretic peptides: gaining further
insights into structurefunction relationships. J Am Coll
Cardiol 2009; 54:10331034.
55. Rubattu S, Sciarretta S, Valenti V, et al. Natriuretic peptides: an update on bioactivity, potential therapeutic use,
and implication in cardiovascular diseases. Am J Hypertens 2008; 21:733741.
56. Simon DB, Karet FE, Hamdan JM, et al. Bartters syndrome, hypokalemic alkalosis with hypercalciuria, is
caused by mutations in the NaK2Cl transporter
NKCC2. Nat Genet 1996; 13:183188.
57. Simon DB, Karet FE, Rodriguez-Soriano J, et al. Genetic
heterogeneity of Bartters syndrome revealed by mutations
in the K channel, ROMK. Nat Genet 1996; 14:152156.
58. Simon DB, Bindra RS, Mansfield TA, et al. Mutations in the
chloride channel gene, CLCNKB, cause Bartters syndrome
type III. Nat Genet 1997; 17:171178.
59. Simon DB, Nelson-Williams C, Johnson Bia M. Gitelmans
variant of Bartters syndrome, inherited hypokalaemic
alkalosis, is caused by mutations in the thiazide-sensitive
Na-Cl cotransporter. Nat Genet 1996; 12:2430.
60. Shimkets RA, Warnock DG, Bositis CM, et al. Liddles
syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel.
Cell 1994; 79:407414.
61. Chang SS, Grunder S, Hanukoglu A, et al. Mutations in
subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 1996; 12:248253.
62. Rossier BC, Palmer LG. Mechanism of aldosterone action
on sodium and potassium transport. In: Seldin DW, Giebisch G, eds. The Kidney, Physiology and Pathophysiology. New York: Raven Press, 1992:13731409.
63. Feldman D, Funder JW, Edelman IS. Subcellular mechanisms in the action of adrenal steroids. Am J Med 1972;
53:545560.
64. Gottschalk CW, Lassiter WE, Mylle M. Localisation of
urine adicification in the mammalian kidney. Am J Physiol
1960; 198:581585.
65. Rodriguez Soriano J, Boichis H, Stark H, et al. Proximal
renal tubular acidosis. A defect in bicarbonate reabsorption
with normal urinary acidification. Pediatr Res 1967;
1:8198.
66. Brenes LG, Brenes JN, Hernandez MM. Familial proximal
renal tubular acidosis: a distinct clinical entity. Am J Med
1977; 63:244252.
67. Hutcheon RA, Kaplan BS, Drummond KS. Distal renal

tubular acidosis in children with chronic hydronephrosis.
J Pediatr 1976; 89:372376.
68. Schwartz WB, Cohen JJ. The nature of the renal response to
chronic disorders of acidbase equilibrium. Am J Med
1978; 64:417428.
69. Lassiter WE, Gottschalk CW, Myolle M. Micropuncture
study of renal tubular reabsorption of calcium in normal
rodents. Am J Physiol 1963; 205:771775.
70. Burg MB, Stoner L, Cardinal J, et al. Furosemide effect on
isolated perfused tubules. Am J Physiol 1973; 225:119124.
71. Gesek FA, Friedman PA. Mechanism of calcium transport
stimulated by chlorothiazide in mouse distal convoluted
tubule cells. J Clin Invest 1992; 90:429438.
72. Brodehl J, Krause A, Hoyer PF. Assessment of maximal
tubular phosphate reabsorption: comparison of direct measurement with the nomogram of Bijvoet. Pediatr Nephrol
1988; 2:183189.
73. Yu X, White KE. FGF23 and disorders of phosphate
homeostasis. Cytokine Growth Factor Rev 2005; 16:
221232.
74. Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced
osteomalacia. Proc Natl Acad Sci U S A 2001; 98:65006505.
75. White KE, Evans WE, ORiordan JLH, et al. Autosomal
dominant hypophosphataemic rickets is associated with
mutations in FGF23. Nat Genet 2000; 26:345348.
76. Beck L, Soumounou Y, Martel J, et al. Pex/PEX tissue
distribution and evidence for a deletion in the 30 region
of the Pex gene in X-linked hypophosphatemic mice. J Clin
Invest 1997; 99:12001209.
77. Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes
rickets and osteomalacia and identifies a role for osteocytes
in mineral metabolism. Nat Genet 2006; 38:13101315.
78. Kurosu H, Kuro-o M. The Klotho gene family as a regulator of endocrine fibroblast growth factors. Mol Cell Endocrinol 2009; 299:7278.
79. DeLuca HF, Schnoes HK. Metabolism and actions of vitamin D. Annu Rev Biochem 1976; 45:631666.
80. Stumpf WE, Sar M, Reid FA, et al. Target cells for 1, 25dihydroxyvitamin D. Science 1979; 206:11881190.
81. Gottschalk CW, Mylle M. Micropuncture study of the
mammalian urinary concentrating mechanism: evidence
for the countercurrent hypothesis. Am J Physiol 1959;
196:927936.
82. Stephenson JL, Kriz W, Jamison RL, et al. Symposium on
the renal concentrating mechanism. Federation Proc 1983;
42:23772405.
83. Scholander PF. The wonderful net. Sci Am 1957; 196:96107.
84. Schrier RW, Bichet DG. Osmotic and nonosmotic control of
vasopressin release and the pathogenesis of impaired
water excretion in adrenal, thyroid and edematous disorders. J Lab Clin Med 1981; 98:115.
85. Dunn FL, Brennan TJ, Nelson AE, et al. The role of blood
osmolality and volume in regulating vasopressin secretion
in the rat. J Clin Invest 1973; 52:32123219.
86. Knoers N, van den Ouweland A, Dreesen J, et al. Nephrogenic diabetes insipidus: identification of the genetic
defect. Pediatr Nephrol 1993; 7:685688.
87. Feldman BJ, Rosenthal SM, Vargas GA, et al. Nephrogenic
syndrome of inappropriate antidiuresis. N Engl J Med
2005; 352:18841890.
88. Marcialis MA, Faa V, Fanos V, et al. Neonatal onset of
nephrogenic syndrome of inappropriate antidiuresis.
Pediatr Nephrol 2008; 23:22672271.
89. van Lieburg AF, Verdijk MA, Knoers VV, et al. Patients
with autosomal nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene.
Am J Hum Genet 1994; 55:648652.
6
Principles of Radiological Imaging of the Urinary Tract
Uday Patel
St Georges Hospital and Medical School, London, U.K.
Miles Walkden
University College Hospital, London, U.K.
INTRODUCTION
This chapter provides an overview of the main modalities used in urological imaging and the physics
behind them (except nuclear medicine which is covered elsewhere). The mention of the word physics
may concern many, but this is after all a book about
basic sciences. As with most things in medicine, basic
concepts allow the reader a deeper understanding.
The history of the various techniques, which includes
contributions from urologists in the development of
iodinated contrast media (CM), is also discussed.
Until fairly recently, radiology was only really
involved in producing anatomical information. Functional information, such as perfusion or filtration
rates, was only measurable with nuclear scintigraphy.
Even then the spatial resolution (how readily two
adjacent structures can be seen as separate) was
poor. Improvements in technology such as multislice
computed tomography (CT) scanning and faster MRI
sequences have not only allowed improved spatial
and contrast resolution but also started to open the
door to functional imaging by increasing temporal
resolution (how quickly a scan can be obtained).
This means that physiological information can be
obtained about a tissue. This is particularly so with
prostate imaging, where the possibility of diagnosing,
grading, and staging cancer with MRI is becoming a
distinct possibility. This could allow more focused
prostate biopsy in the future.
CREATING AN IMAGE
To make an image, a body tissueenergy interaction
must be created, precisely located and measured. The
different radiological modalities, X ray, MRI, and
ultrasound, simply utilize different energies to create
that body tissueenergy interaction. The ability to see a
structure within the patient rest on three main factors.
1.
2.
Spatial resolution: The ability to identify two adjacent structures as separate entities.
Contrast resolution: The ability to identify adjacent
tissues as texturally distinct, depending on
3.
differences in the transmission of whatever

energy source is being used.
Signal to noise ratio (SNR): This is the amount of
useful energy coming from the patient that can be
used to make the image, compared with the
degree of useless energy (quantum mottle and
scatter) that contains no meaningful information
and merely degrades the image.
Further intrinsic and extrinsic parameters also

affect an image. Intrinsic parameters cannot be
changed and are factors inherent to the tissue being
imaged such as tissue density. Extrinsic parameters
are those that can be manipulated or changed such as
the current or voltage used in plain film radiography.
It is by manipulating these extrinsic factors that an
image is created.
PLAIN AND CONTRAST RADIOGRAPHY

Plain Radiography
Plain radiography as well as CT uses the X ray to
create an image. X rays are part of the electromagnetic
spectrum with a very short wavelength of around
10 10 m. They have large amounts of energy, which
when absorbed in body tissues can cause ionization
and tissue damage. X rays are therefore a type of
ionizing radiation, whereas other energies used in
radiology such as radiowaves in MRI have insufficient
energy for ionization.
In the late 19th century, many people were
experimenting with cathode tubes. It was however
Wilhelm Rontgen, a German physics professor, who
first wrote about X rays in a paper titled On a new
kind of Rays, a preliminary communication, in
December 1895 (1). The discovery was a serendipitous
finding while he was experimenting with electricity.
His cathode tube made a nearby barium platinocyanide screen fluoresce. Intrigued, he named his newly
discovered rays X rays, as this is the scientific
denomination for an unknown quantity. He concentrated his research on this ray and went to produce the
first human radiograph when he X-rayed his wifes
84
Patel and Walkden
hand. On seeing the plate, she is said to have

exclaimed I have seen my death; but its medical
potential was immediately obvious. Indeed, by May
1896 the first dedicated radiology journal was published (Archives of Clinical Skiagraphy described as
illustrating applications of the new photography to
Medicine and Surgery and the predecessor of the
present British Journal of Radiology) and Ro ntgen
became the first Nobel laureate of Physics in 1901.
Yet, he died nearly bankrupt of cancer of the intestines in 1923. His death was not felt to be radiation
induced, but soon after the more harmful effects of Xradiation became known, and are discussed below.
The medical evaluation of the plain radiograph was
undertaken in many countries, and even now, after
over a century of clinical use and although increasingly challenged, it remains the workhorse modality
in radiology.
Urology was one of the first areas to exploit the
use of X rays for the assessment of the renal tract.
James Adams, a Glasgow surgeon, was the first to
x-ray a renal stone in 1896. He later removed the
stone as proof. The first complete retrograde pyelogram was produced by Voelcher and von Lichtenberg
in 1906 (2).
X-Ray Production
An X-ray tube is shown in Figure 1. It consists of a
negative electrode (cathode), a heating filament, and a
positive electrode (anode), all made from tungsten
and housed within a vacuum tube. The tungsten
filament is heated to incandescence and emits electrons by thermionic emission. These negatively
charged electrons are repelled by the negative cathode
and attracted toward the positive anode at speeds of
up to 1000 m/sec. On impact with the positive anode,
they suddenly decelerate, and the lost kinetic energy
is converted into X rays (1%) and heat (99%).
Once directed at the human body, X rays are
differentially attenuated by body tissues, proportionate to that tissues atomic number, density, and thickness. The attenuation occurs as either absorption or
scatter. Scatter introduces noise and degrades the
image while the emergent beam carries information
on the thickness and composition of the intervening
body tissue. This information is translated into a
visible image when the X ray falls on a detector. In the
case of analogue plain film, this is a photographic
plate made of silver haloid. X rays convert the silver
ions into stable, dense, and visible silver atoms.
Five basic radiographic densities can be differentiated on plain filmair, fat, soft tissue, bone, and
contrast agents. Density is the main determining factor. Air only minimally attenuates and therefore
appears black. In comparison, calcium-containing
structures are substantially attenuating, and therefore
a renal stone will appear white. How well the stone
can be seen depends on its calcium content (or rather
its density): the more the calcium, the denser it is, and
hence whiter and more apparent. Thick structures
also attenuate more than thin structures of the same
Figure 1 (A) A typical X-ray tube in cross section. Abbreviations: C, cathode; F, filament within the cathode; A, rotating
anode; T, target on the anode where the electrons hit; E, electron
beam. Body tissues attenuate the beam by variable amounts,
depending on tissue density, tissue thickness, and atomic number (as illustrated by the bar chartlungs being less dense allow
more X rays to pass through). The emergent X-ray beam
carrying this (mainly density related) information is then detected
by a TFT screen and converted into a digital radiograph. The
radiograph (B) Bilateral staghorn calculi (white arrows), seen as
white (or relatively unexposed) areas as they are dense.
Chapter 6: Principles of Radiological Imaging of the Urinary Tract
composition, which is why you can clearly see the

bulky psoas muscle, as opposed to the thinner anterior
abdominal muscles.
An anatomical structure will only be seen on a
plain radiograph if it is outlined by tissue of a substantially different density. The kidneys (soft-tissue
density) can only be seen as separate from the remaining retroperitoneal structures (also of soft-tissue density) because they are separated by the perinephric fat.
Structures within the kidney cannot be differentiated
as their X-ray attenuation is similar. The collecting
system becomes visible only once filled by the much
denser iodine-containing CM.
Table 1 (with details of film interpretation in
Tables 2 and 3) shows the current uses of the plain
film in urology, but a further deficiency of the plain
film is the superimposition of structures on one another. Plain film tomography can partly overcome this, as
it allows a slice of the patient to be obtained. The
X-ray tube and film are simultaneously moved around
a pivot point centered in the plane of interest. The
structures above and below the focal plane are blurred
out by motion of the tube and film, whereas structures
within the focal plane are seen clearly (Fig. 2). This
technique is used during excretion urography to
obtain better detail of the renal collecting system.
Fluoroscopic equipment is based on the same
principles of X-ray imaging except that the X rays fall
onto a fluoroscopy screen. The light from this is
amplified electronically by an image intensifier and
displayed on a monitor. This allows real-time radiographic visualization of moving structures. Fluoroscopic images are of lower radiating dose to the
85
Table 3 How to Interpret an IVU

1. Check the name date and side-marker on the film.
2. Control filmcheck all the same points as for a KUB.
3. Immediate film.
a. This should show bilateral symmetrical nephrograms.
b. Assess the renal outline for any masses or scarring.
4. 5- and 10-min films of the kidney.
a. Assess the calyceal anatomy.
b. Ensure that all calyceals have been identified.
c. Look for any filling defects.
5. Full-length release film.
a. Ensure both ureters have been well opacified.
b. Assess the bladder; looking for any filling defects.
c. Postmicturition film.
d. Assess adequate bladder emptying and also drainage of
the upper tracts.
Table 1 Current Uses of Plain Films in Urology

KUB
Good for following known radio-opaque stones during treatment
or as a screening tool for diagnosis of stones. However, in this
respect it has probably been largely replaced by CT KUB.
IVU
IVUs were still used. However, with the introduction of multislice
CT urography is starting to replace the IVU as it can assess not
only the urothelium, but all the components of the renal tract as
well as other intra abdominal organs.
Table 2 How to Interpret a KUB

1. Check the name, date, and side-markers on the film.
2. Look for calcification. Inspiratory and expiratory films may help
as a renal stone should move on respiration.
i. Kidneysensure both are present and of a normal size
and outline.
ii. Uretersnot normally seen on plain film but their course
should be followed looking particularly at the PUJ, Pelvic
brim and VUJ.
iii. Bladdercan normally just be made out in the pelvis.
3. Assess the soft tissues.
i. Look for the psoas shadows, their absence may indicate
renal pathology.
ii. Inspect other organs, such as the liver and spleen.
iii. Asses the bowel to make sure there is no obvious bowel
obstruction.
4. Assess the bones for any evidence of metastasis.
Figure 2 The principles of linear tomography. (A) The plane of

interest is set for the kidneys (K). Structures outside the plane of
interest such as the bowel (B) and the vertebral body (V) are
blurred as their position on the film moves whereas structures in
the plane of interest are projected in the same position and
therefore appear sharp. (B) A tomogram through the kidneys
during an IVU series. The plane of interest is set at 10 cm, which
is the average depth for most kidneys. The calyceal system can be
clearly seen whereas overlying structures have been blurred out.
86
Patel and Walkden
patient; however, spatial resolution is poorer compared with the plain film.
within the file, so that the image can never be separated

from this information by mistake.
Digital Radiography and PACS
Contrast Media
The plain radiograph is being increasingly replaced by

digital radiography (DR), where the image is acquired
electronically. A thin-film transistor (TFT) screen
replaces the photographic plate and converts the X
ray into an electronic signal to create an image,
composed of a matrix of individual cells or pixels
(picture elements). Each pixel has an assigned value
that is related to the intensity of X rays that falls onto
it. Pixel size determines the spatial resolution
smaller pixels have better spatial resolution. The spatial resolution of digital images is currently poorer
than conventional radiographs. Plain film is able to
resolve 10 line pairs/mm, compared with the DRs 5.5
line pairs/mm. So, fine structure such as renal calcification is more readily apparent on conventional radiographs. Digital films do, however, have the advantage
that they have superior contrast resolution, employing
a wider grayscale, which can be further manipulated
to the observers satisfaction. This partly compensates
for its modest spatial resolution. But most importantly, DR, being electronic, is easier to store and transmit,
allowing PACS (picture archiving and communication
systems) to be used.
PACS is revolutionizing radiology departments,
with film-less departments that can rapidly transmit
images through electronic links. Multimodality
images can be instantly recalled from memory banks
and displayed on a single monitor with image incorporation for surgical planning. Departments are
becoming digital at a rapid pace as unit costs decline
and the necessary cultural changes take place, such
that radiologists and clinicians feel comfortable using
monitors alone to view films. The promise is not
necessarily cost saving, but promises more efficient
patient managementlost films have become a thing
of the past, and a complete imaging history is instantly available at any point inside or even outside the
hospital, day or night.
Before the 1980s, it was impossible for anyone
other than the manufacturer of a specific imaging
machine to decode their images. With the increasing
use of digital images and the need to be able to view
these images around the hospital and indeed around
the country/ world, it was realized that a standard
format for viewing, storing, and transmitting medical
images was required. In 1983, the American College of
Radiologists (ACR) and the National Electrical Manufacturers Association (NEMA) formed a joint committee and in 1985 produced the ACR-NEMA
standard. This was revised in 1993 and renamed
Digital Imaging and Communications in Medicine
(DICOM) (3). This standard is now updated yearly.
DICOM enables the integration of scanners, servers,
workstations, printers, and network hardware from
multiple manufacturers into a PAC system. DICOM
differs from other data formats in that it groups information into data sets. This means that a file of a KUB X
ray, for example, actually contains the patient ID
One way to alter and improve the contrast resolution

of a tissue is to use radio-opaque CM to outline it. CM
can either be positive, such that it has a higher
attenuation density than the surrounding tissues
(and appearing white on the image), or it can be
negative with a lower attenuation density (e.g., air)
that absorbs less radiation and so appears black on the
final image. Positive-contrast agents are substances
with high atomic numbers. The three main categories
in use in radiology currently are iodine-based, gadolinium-containing, and barium-based. Barium-based
preparations are used only for the alimentary tract
and are thus not readily encountered in urology, and
gadolinium agents are used in magnetic resonance
imaging (MRI).
Currently, the commonest intravenous contrast
agent used is iodine (atomic weight 127)-containing
compounds. The idea of using iodine-based compounds as an intravenous contrast agent came from
the discovery that the urinary bladder could be visualized on radiographs of patients who had taken large
doses of sodium iodine for the treatment of syphilis
(4). Unfortunately, the collecting system was only
poorly seen and the sodium iodine was found to be
too toxic for clinical radiological use. In 1925, two
Germans, Bruz and Rath, managed to produce less
toxic iodinecontaining compounds based on the pyridine ring that were initially used to treat coccal
infections. In 1928 an American urology intern,
Moses Swick, working with some of these drugs
realized that they may be of use in visualizing the
renal tract and the intravenous urogram was born (5).
All soft tissues in the body enhance with iodinated CM, but none as much as the kidneys. This is
because they are the principle source of excretion
(99%) with a half-life of two hours. Renal excretion
is by free glomerular filtration without tubular reabsorption: this means that CT contrastenhanced perfusion studies can be used to calculate GFR.
Adverse Effects of Contrast Media

and Current Recommendations
A limitation of current CM agents is their toxicity with
two broad types of adverse reactionanaphylactoid
and chemotoxic. The exact mechanism of either reaction is unclear and they can occur either early (within
one hour of administration) or be delayed (one hour to
one week following administration).
Anaphylactoid reactions occur unpredictably
and independently of the dose or concentration of
the agent. They are believed at least in part to be
because of the ionic makeup of the CM, as many of the
earlier ionic agents caused severe anaphylactoid reactions. With the introduction of nonionic CM, these
reactions have become less frequent. Chemotoxic
effects are related to the total dose, molecular toxicity,
and physiological characteristics (such as osmolality)
87
Table 4 Prevention of Adverse Reactions Because of CM

1.
2.
In all CM studies, the dose of CM

should be kept as low as possible.
Nonrenal adverse reactions.
3.
Renal adverse reactions
a. Identify patients at increased risk of reaction.

i. Previous moderate/severe acute reaction
ii. Asthma
iii. Allergy requiring medical treatment
b. If increased risk detected then do the following:
i. Reassess the clinical indications, if still necessary.
ii. Use nonionic CM under careful monitoring.
iii. Keep the patient in the department for 30 min post administration.
iv. Premedication prophylaxis such as 12-hr 30-mg prednisolone and 2-hr pre-CM
administration is no longer recommended.
a. Identify patients with an increased risk of developing CM-induced nephropathy.
i. Raised serum creatinine particularly secondary to diabetic nephropathy.
ii. Dehydration.
iii. Congestive heart failure.
iv. Age >70.
v. Concurrent administration of nephrotoxic drugs such as NSAIDs.
vi. Non-insulin-dependent patient with diabetes taking metformin. Metformin is
excreted unchanged in the urine. In the presence of renal failure such as can be
induced by CM, metformin may accumulate in sufficient amounts to cause lactic
acidosis.
b. If increased risk then do the following:
i. If taking metformin, stop a time of administration of CM, check creatinine at 48 hr
and if unchanged then restart metformin.
ii. Stop nephrotoxic drugs 24 hr prior to CM administration.
iii. Give IV hydration 6 hr prior to administration and for up to 24 hr after the study
iv. Use low or iso-osmolar CM
Special situations are as follows:

Pregnancy and lactation: No teratogenic effects of CM have been described. The free iodine does, however, have the potential to depress fetal/neonatal
thyroid function. If CM is given during pregnancy, the neonatal thyroid function should be checked within the first week. Such tiny amounts of iodine enter
the breast milk that there is no contraindication to breast feeding.
Thyroid disease: As CM contains small amounts of free iodine, it may cause thyrotoxic crisis in patients with Graves disease. These patients can still
receive CM but should be closely followed. The free iodine can also interfere with diagnostic and therapeutic nuclear medicine procedure for up to two
months.
Phaeochromocytoma: Crises can be precipitated by CM. Patients with known phaeochromocytoma should have A and B blockade prior to the
administration of CM.
Extracted from ESUR guidelines version 6.
of each agent. These are the ones thought to be

responsible for nephrotoxicity. As well as being nonionic, modern CM is of low or iso-osmolality compared with plasma, and of lower chemotoxicity.
CM nephropathy is a diagnosis of exclusion,
defined as an increase in serum creatinine by more
than 25% or 44 nm/L occurring within three days of
the use of IV CM with no alternative explanation (6).
There are no characteristic or diagnostic, biochemical
or histological abnormalities. It is the third highest
cause of hospital-acquired acute renal failure (ARF)
accounting for 12% of the cases (7). The patients with
the highest risk for developing contrast-induced ARF
are those with preexisting renal impairment or risk
factors for renal impairment (e.g., diabetes mellitus,
etc.) (8,9). The degree of renal impairment present also
determines the severity of CM nephropathy. At least
two risk scores for the prediction of CM-induced
nephropathy have been developed (10,11) but these
are not widely used, and in most radiology departments a creatinine cutoff of 200 ng/mL is used, above
which intravenous CM is not used unless deemed
clinically justified.
A number of preventative measures can be
taken to reduce CM nephrotoxicity. These include
intravenous hydration (which is probably the only

intervention that has some evidence to support
it), vasodilators, and antioxidants such as Nacetylecysteine and ascorbic acid (12). Currently, the
European Society of Urogenital Radiology (ESUR)
guidelines do not recommend any pharmacological
manipulation for routine use in prevention of CMinduced nephropathy (13) (Table 4).
The Hazards of Ionizing Radiation

Soon after Rontgens discovery of the X ray, their
harmful effects became increasingly evident. In 1896,
the first case of radiation dermatitis and hair loss were
reported (14), followed soon after by cases of radiation-induced malignancy. Their frequency was sufficiently alarming for the Rontgen Society (formed in
1896) to set up a committee in 1898 to investigate
radiation-induced side effects, followed in 1915 by a
code of practice document with recommendations for
radiation protection.
Ionization of tissues by absorption of radiation
energy is the cause, and the most vulnerable molecules are proteins and deoxyribonucleic acid (DNA).
Damage occurs either directly by rupture of the
88
Patel and Walkden

Table 5 Effective Radiation Dose for Patients Undergoing
Radiological Investigations
Chest X ray
Lumbar spine X ray
IVU
Bone scan
CT
Head
Abdomen
(Renal stone protocol)
(CT urogram)
Renal scan
DTPA
Mag 3
Barium enema
Effective
dose
(mSv)
Approximate
natural-background
radiation
0.02
1.3
2.5
4.0
3 days
7 mo
14 mo
1.8 yr
2.3
10
(4.7)
(1015)
1 yr
4.5 yr
1.0
1.0
7.0
6 mo
6 mo
3.2 yr
Average effective dose in the United Kingdom: 2.2 (87% natural sources;
13% from artificial sources of which 11% is medical sources).
Figure 3 The damaging ionizing action of X rays has cellular

and genetic consequences (see text for further explanation of
stochastic and nonstochastic effects).
covalent bond or indirectly by free-radical production.

Indirect free-radical production is the most commonly
occurring reaction (Fig. 3). Terminologically, there are
two broad groups of ionizing hazards: stochastic
effects and deterministic effects (nonstochastic).
Stochastic effects means they arise by chance and
have no threshold dose. The risk of an effect occurring
increases with the dose, but the severity of the effect
does not. Stochastic effects result in cell transformation and cancer induction. Deterministic effects have a
value below which they will not occur, that is, there is
a threshold dose, and include effects such as erythema
and cataract formation. Once the threshold dose is
exceeded, the likelihood of that effect increases rapidly up to a level, beyond which it becomes inevitable.
Deterministic effects are rate dependent because the
body is able to repair the damage (except in the eye,
where the dose is cumulative and particular care
should be taken to protect it from radiation).
Doses in diagnostic radiology on the whole do
not reach deterministic threshold levels, except in
interventional radiology (IR) and cumulative CT
doses on the lens of the eye. Stochastic effects, however, are a risk with all X-ray imaging, no matter how
small the dose. Typical doses in urological imaging
are given in Table 5. It should be remembered that
normal background radiation also has a risk of inducing a cancer. The quoted risk of inducing a cancer is
1:15,000/mSv. In the United Kingdom, it is estimated
that diagnostic X ray causes approximately 700 cancers a year (16). Normal background radiation for a
person in this country is approximately 2.2 mSv/yr
(15), unless you happen to live in Cornwall where
radon gas increases this total to approximately 6 mSv/
yr. Imaging accounts for approximately 14% (17) of
this background radiation but is increasing. One of the
main contributors to this increase is CT, which currently accounts for 10% of all radiological investigations and 40% of all radiation (18). We, therefore, need
to keep the radiation dose as low as possible. Radiating radiological investigations should always have a
clinical justification and, whenever possible, a substitute safer alternative should be chosen (e.g., ultrasound or MRI).
CROSS-SECTIONAL IMAGING
Plain and contrast radiography is limited to a twodimensional (2D) format with no depth perception.
However, if the source of the body tissue/energy
interaction can be precisely located in the three perpendicular planes, this digitized information can be
used to build a slice-by-slice sectional image. Crosssectional imaging has made possible the noninvasive
imaging of those internal tissues and organs beyond
resolution by conventional radiography. CT, ultrasound, and MRI are all examples. CT uses X-radiation
while ultrasound and MRI exploit novel energies and
tissue/energy interactions.
Computed Tomography
CT was first developed by Sir Godfrey Hounsfield in
1973, working at the Atkinson Morley Hospital in
London (19). At the time he was working with the
music company EMI, who had many other nonmusic
interests as well and who had recently signed the
Beatles. Some of the substantial revenue that the
Beatles generated for EMI made possible the investment in the development of the CT scanner. It has
89
Figure 4 Principles of CT. The X-ray source

does a 3608 rotation around the patient to obtain
a slice. This slice is divided up into a matrix made
up of voxels. Each voxel is a cuboid with x, y, and
z values. The z-value (or slice thickness was
traditionally larger than the x and y values and
thus reformats in planes other than the one of
acquisition, that is, axial resulted in a loss of
resolution). The sum attenuating value from the
voxel is then taken and converted into a linear
density scale (as Hounsfield units), which are
displayed as pixels in shades of gray on the final
image. Multislice machines simply acquire multiple
slices per rotation, allowing the slice thickness to
be significantly reduced and an isotropic voxel
obtained, which allows accurate reformatting of
the image in any plane.
therefore been said by some that the development of

the CT scan was at least partly down to the success of
the Beatles.
CT also uses X rays, and the fundamental physics of X ray/tissue interaction, as described earlier,
still apply. For CT, the X-ray beam is generated as a
precise pencil-thin beam, which is detected by a bank
of detectors positioned diametrically opposite to the
source. As the beam rotates around the body, the
detectors convert the transmitted beam intensity into
a cross-sectional image of between 0.6 and 10 mm
thick. The image itself is composed of numerous
picture elements (pixels) (Fig. 4). As structures are

no longer superimposed on one another, a far superior
contrast resolution is possible compared with the
plain radiograph, with at least a 10-fold improvement.
Each pixel is assigned a grayscale value according to
the sum attenuating power of the tissue in its corresponding volume (or voxel). The sum attenuating
power is known as the CT or Hounsfield unit, and
Figure 5 gives some typical values for normal body
tissue. By convention, water was assigned a neutral
density at zero and all other values are measured
relative to water. Air is 1000, fat 100, and bone
90
Patel and Walkden
Figure 5 (A) A scale showing the CT (or Hounsfield) numbers of various body tissues. Water is conventionally graded as zero, air as
1000, renal stones as >200 and soft tissue such as kidney as +40 to +60 units unenhanced and +40 to +150 enhanced. The figure also
demonstrates the concept of windowing to allow better contrast resolution. For soft-tissue windows the window level is set at +200. The
window width 200 units either side, that is, 0 to +400. Any structure lying within this range will be seen as a shade of gray. Any structure
with a value higher or lower than the window width will appear white or black, respectively, and will not be separable from other structures
outside the window width. The windows are set according to the structure being scrutinized. For the majority of urological imaging, softtissue windows are employed. (B) A coronal image of the abdomen on soft-tissue windows (white arrow shows the window level has
been set at 70 and the window width at 400). The RCC in the left lower pole is well seen. (C) The same coronal image on bony windows
(white arrow shows the window level at 300 and the window width at 1800); now good bony detail is seen but the left RCC is less well
seen.
above 200. The kidneys are in the soft-tissue range of

40 to 60, rising to around 150 units after intravenous contrast. Renal calculi are near the bone range
and are readily identified. Structures that have a high
positive value appear as white, for example, bone/
renal calculi, and structures with a high negative
value appear as black, for example, air.
The Hounsfield value is converted to a grayscale
value for display. There are a wide range of Hounsfield
values ranging from 1000 to 3000. The human eye is
only able to distinguish approximately 60 shades of

gray. It is, therefore, necessary to focus in on levels of
interest (Fig. 5) by windowing. A window level is set
depending on the structure of interest; for example, for
the kidneys as they lie in the soft-tissue range, the level
is set at 20. A window width of approximately 200
Hounsfield units is then set on either side of this value.
Any structure with a value inside the window width
will be assigned a shade of gray, and it will be possible
to differentiate it. Any structure with a value greater
91
than 200 will be white and any structure less than

200 will appear black.
Spiral and Multidetector CT

The first CT scans obtained data one slice at a time, in
between which the table had to be moved. This
resulted in long scan times, misregistration between
slices, and movement artifact. Helical scanning was
then developed in which the patient is moved through
the scanner at a constant speed while the X-ray tube
continuously rotates around the patient. A continuous
volume of data was thus obtained. Scan times were
shorter and reconstruction of overlapping slices could
be undertaken to improve visualization of small
lesions. However, spiral CT only obtained one slice
per rotation and the calculated voxels were still large
(or cuboid) with the length of the Z-axis (i.e., the sliced
thickness) still considerably larger than the X and Y
axes (Fig. 4). This meant that image quality was good
in the plane of acquisition, that is, the axial plane, but
poor when attempts were made to reformat the data
into other planes or three-dimensional (3D) volume
images.
The advent of multidetector CT (the acquisition
of multiple slices during each spiral rotation) changed
this. Now instead of a single slice, 64 or even 128 slices
can be obtained with one rotation of the gantry. Scan
times have now plummeted with thinner slices possible, so that isotropic voxels (cubes rather than
cuboids) could be obtained (Fig. 4). Isotropic voxels
allow for more accurate, true 3D imaging: no matter
how the data set is projected, there is no significant
loss of resolution. Diagnostic quality images can be
obtained in any plane. Table 6 lists the current uses of
CT in urology.
Multiplanar Reformat and 3D Volume Rendering

As data are acquired as a volume with near isotopic
voxels, it is possible to postprocess this information to
create images in different planes and in 3D. These
images do not contain any more information than the
axial data, but they offer a more rapid appreciation of
spatial information and anatomical relationships. This
can be particularly useful in surgical planning (Fig. 6).
Multiplanar Reformats
The sagittal and coronal planes are the most commonly used and easiest to orientate. In urology the coronal
Table 6 Current Uses of CT in Urology
1.
CT KUB
2.
CT of kidneys
3.
4.
CT angiography
CT urography
Gold standard for the investigation of

renal colic. Sensitivities and
specificities approach 100%. Also has
the advantage of detecting other
pathology in up to a third of cases.
For evaluation of renal masses, renal
infections, etc.
For suspected renal artery stenosis.
For the investigation of upper tract
causes of hematuria.
Figure 6 (A) Maximum-intensity projection (MIP) image of the

kidneys. Note that high-density contrast in the collecting systems
is well seen as is the stone in the lower pole of the left kidney
(black arrow), but the low attenuation renal parenchyma and
surrounding soft tissue is not well seen. (B) A 3D volumerendered image of the same kidneys as in A. This was obtained
prior to PCNL to allow delineation of the stone (white arrow) and
its relationship to the collecting system (dashed arrow), which is
filled with contrast. The 3D anatomical information provided by
modern imaging has the potential to aid presurgical planning and
navigation.
plane is excellent for visualizing the kidneys, ureters

and bladder in a single view. Oblique reformats can
also be made and are useful for viewing the hepatic
and pancreatic regions. Curved reformats allow tortuous tubular structures such as blood vessels to be seen
in one view.
Maximum- and Minimum-Intensity Projections
In maximum-intensity projection viewing, rays are

traced from the viewer through the object to the
display, and only the relative maximum value
detected along each ray path is retained to create the
image. This means that high attenuation structures
such as bone and contrast are well seen and lower
attenuation structures are not well visualized. This
method is utilized in vascular imaging. Minimumintensity projection involves detecting the minimum
value along each ray path.
Volume Rendering
Rendering is the process of mapping a 3D data set

onto a 2D screen for display. In volume rendering the
CT numbers that make up the image are assigned to
be either visible or invisible and can be displayed with
varying colors and varying opacity levels (20). 3D
volumerendered images have proved particularly
92
Patel and Walkden
useful in preoperative planning for partial nephrectomy where tumor relationship to the collecting system
and renal vasculature can be seen (21).
It is from using prospective rendering techniques that so-called virtual endoscopy images can be
created, which are starting to be used for virtual
cystography (22).
Multiphase CT Imaging
With the fast imaging capability of multidetector CT,
the different phases in the handling of the contrast by
the kidneys can be identified and different pathologies
can be evaluated (Fig. 7).
1.
2.
3.
4.
5.
6.
Noncontrast: This is used for detecting stones and

also is a baseline for enhancement of renal masses
(Table 7).
Arterial phase: This occurs at about 15 seconds post
injection. This allows high-quality multiplane
images of the renal arteries to be obtained.
Cortical phase: This occurs 30 to 40 seconds post
injection. Contrast filters into the proximal convoluted tubule. It permits corticomedullary differentiation. Scars are best seen and it helps differentiate
renal pseudotumor (normal renal tissue) from
pathology. This phase has, however, been shown
to hinder mass detection in some cases and a
nephrographic phase tends to be preferred.
Nephrographic phase: This phase occurs 90 to 120
seconds post injection. Contrast enters the collecting tubules. A homogenous nephrogram is
obtained in which corticomedullay differentiation
is lost. The detection of true renal masses is easiest
in this phase.
Excretory phase: Occurs typically 10 minutes following injection. Contrast enters the pelvicalyceal
system (PC), ureters, and bladder. This is the CT
equivalent of the IVU. This allowed detection of
filling defects within the collecting system, such as
TCC, blood clot, stone, and vascular impression.
As these data are obtained as a volume, it can be
processed to give virtual endoscopic views.
Functional imaging: A modern 64-slice CT scanner
can obtain multiple data acquisition some two or
three times per second. Its ability to acquire data
at such speed allows contrast-enhancing agents to
be used as tracer substances, similar to many of
the dynamic nuclear medicine investigations. CT
number versus time curves can be constructed,
which give information on parameters such as
perfusion and also GFR.
If all these phases were obtained in a single scan,

the radiation dose would be unacceptably high. It is
therefore necessary to tailor the phases obtained to the
clinical question being asked. Accurate clinical information facilitates focused imaging.
Ultrasound
Ultrasound employs sound waves that occur at a
frequency above the upper limit of human hearing,
Figure 7 (A) Unenhanced image of the kidney shows an

exophytic renal mass. It has HU above 15 and is therefore not
a cyst. (B) Nephrographic phase obtained 100 seconds post
injection of CM. The mass is seen to enhance compared with the
precontrast image but not as much as the surrounding renal
parenchyma. This is typical for a renal cell cancer. (C) Excretory
phase coronal image shows contrast outlining the pelvicalyceal
system. This is the CT equivalent of the IVU.
typically above 20 kHz, which are inaudible to the

human ear. Ultrasound was first developed toward
the end of the war in the form of SONAR (sound
navigation and ranging) to detect submarines. Medical ultrasound started to be used in the 1940s as a
therapeutic modality rather than a diagnostic one,

Table 7 How to Interpret a Low-Dose CT KUB
Table 8 Current Uses of Ultrasound in Urology
1. Look at the kidneysassess size, cortical thickness and any

evidence of stones or hydronephrosis.
2. Follow the course of the ureterfollow antegradely from the
kidneys to the pelvic brim, then retrogradely from the VUJ up
to the pelvic brim. Assess for any stones, thickening or
stranding. Three common areas for stones to be found are at
the PUJ, pelvic brim and VUJ.
3. The bladderassess for stones and masses.
4. Assess the other organs, particularly paying attention to the
appendix, pancreas, gallbladder, sigmoid colon, and in
females the uterus and ovaries.
Renal
Differentiating a stone from a phlebolith: Phleboliths have comet tail of

soft tissue (the tail sign), tend to be more rounded, and have a central
lucency. Calculi demonstrate a rim sign (a rim of surrounding soft tissue).
Signs of obstruction: The signs include renal enlargement, calyceal
dilatation, and perinephric stranding. There is a relationship between
the signs of obstruction and the duration of the pain. If the patient is
imaged within two hours of pain commencing, many of these signs of
obstruction will not be present.
Source: From Refs. 23 and 24.
utilizing its heating and tissue disrupting effects.

William Fry first used ultrasound to destroy parts of
the basal ganglia in patients with Parkinsons disease
(25). It was not until the 1950s that reproducible
diagnostic ultrasound pictures were obtained by
Holmes and Howry in America (26). One of the
earliest produced was of a normal human neck and
was obtained by submerging a volunteer in water
(contained in a disused B29gun turret!) and scanning
through 3608. Medical ultrasound can be used for both
therapeutic (250 kHz to 2000 kHz) and diagnostic (3
20 MHz) purposes, the difference being the frequency
of the sound wave and thus the amount of energy that
is deposited in the tissue.
Diagnostic Ultrasound
This provides the cornerstone of urological imaging

from which much important information on the renal
tract can be obtained and of such importance that
many urologists have begun to use the technique in
their clinics. Diagnostic ultrasound is the safest,
cheapest, and most versatile of the cross-sectional
modalities and Table 8 lists its current uses in urology.
The sound waves are nonionizing and so far no
serious tissue consequences have been proven. It
does, however, cause some minor tissue heating but
normal tissue perfusion dissipates this. The ultrasound probe or transducer is both a transmitter and
a receiver and continuously switches from one to the
other. Within the probe is a piezoelectric disk. When a
current is applied to the disk, it expands and once the
current is reversed, the disk contracts emitting a
sound wave proportional to the applied current. In
its receiver mode, the disk is compressed by the
retuning echo, producing a current proportional to
the pressure.
Sound waves in the diagnostic range (320 MHz)
will transmit through the body, but are altered in many
ways by the intervening tissues and tissue-tissue
Bladder
Scrotum
Prostate
Penis
93
Hematuria
Loin pain
Suspected hydronephrosis
Renal impairment
Renal calculi
Bladder outflow obstruction
Hematuria
Pelvic pain
Lower urinary tract symptoms
Bladder emptying
Testicular masses
Testicular pain
Suspected torsion
Infertility
Lower urinary tract symptoms
Prostate size estimation
Suspected prostate cancer
Peyronies disease
Erectile dysfunction
interfaces. Of the numerous alterations, only three are

commonly exploited for imagingthe absorption of
sound by tissue, the reflection of sound at tissue-tissue
interfaces, and the apparent change in frequency of
sound at moving interfaces (the Doppler effect). The
first two are used to build the familiar grayscale
ultrasound image, and the last allows vascular sonography or duplex/color Doppler.
Grayscale Ultrasound
The amount of sound absorbed by a given tissue is
calculated by analysis of the reflected echo and this is
used to assign a value on a grayscale. The factors
that influence absorption and reflection are given in
Figure 8. The weaker the reflection, the more gray
or darker is the pixel value. Varying shades of grays
from different points in an organ are used to build an
image that can be viewed on a television monitor. As
water is a good sound transmitter and poor reflector, a
renal cyst is seen as black; while fat, a good reflector, is
seen as white. Bone and calcium are highly sound
attenuating and reflective, and demonstrate a sharp
interface with a shadow beyond (Fig. 9). Unlike the
other cross-sectional modalities, this is a real-time,
interactive method and ideally suited for biopsy and
percutaneous intervention of soft tissues.
The Doppler Effect

Christian Doppler described the change in perceived
frequency of sound emitted by a moving source in
1843, and an everyday example is the crescendo/
decrescendo sound of the moving train whistle, as
heard by the observer on the platform. Medical ultrasound is also subject to this Doppler shift effect, and
the frequency change can be recalculated to give the
velocity of the moving interfacethe equation for this
Doppler effect is given in Figure 10. The common
94
Patel and Walkden
Figure 8 Principles of medical ultrasound. Spatial

localization is calculated from the time delay before
sound is received back. Sound from deeper structures
takes longer. The composition of a structure is calculated by the percentage of sound waves that are
reflected back. A renal calculus reflects more sound
waves than a renal cyst. The stone, therefore,
appears white (hyperechoic) on ultrasound whereas
a cyst appears black (hypoechoic).
moving interface in human tissues is the red blood cell

and blood flow and tissue hemodynamics can be
studied. Calculated velocities can be presented quantitatively as a continuous, time-framed trace of true
velocity (the Doppler or spectral waveform) or
semiquantitatively/qualitatively as a color Doppler
image.
Spectral or Duplex Doppler

Spectral Doppler represents the full spectrum of
recorded velocities in a given time period. It can be
used to diagnose and to grade arterial stenosis using
the velocity gradient across a narrowing. This is of
some value in the assessment of renal artery stenosis.
As well as velocity information, the shape of a spectral
waveform encodes further qualitative or semiquantitative data about tissue character. The peripheral
resistance (PR) or stiffness of the tissue bed alters
the waveform. Increased PR, as seen immediately
after acute ureteric obstruction, restricts flow during
diastole with reduced diastolic velocities. Waveform
changes can be quantified by calculating so-called
waveform indices, for example, the resistive index
(RI) (Fig. 10). RI analysis is of some value in evaluation
of renal dilatation. However, the diagnostic specificity
of RI is modest, as adaptive physiological responses
after obstruction counteract the elevated PR. Conversely, tumors have a low RI and elevated diastolic
flowsbecause of intratumoral arteriovenous shunts
and the lack of a complete smooth muscle wall in
tumoral neovesselsand are seen on color Doppler as
areas of hypervascularity (Fig. 11). This improves the
diagnostic specificity in tumor diagnosis.
Color Doppler
With color Doppler, the peak velocity in a given time
frame is assigned a value on a color scale, and this is
superimposed on the grayscale image. Conventionally,
red represents flow toward the probe and blue away.

Conveniently, renal arterial flow is usually toward and
venous flow away from the probe, and so the gray
and color Doppler findings are easily assimilated by
the observer. Sonographic evaluation of the morphology of soft tissues as well as its vascularity can be
carried out simultaneously, adding a qualitative or
functional element to the grayscale ultrasound image.
Most disease processes have some alteration of the
blood flow, either increased or decreased, and color
Doppler assists diagnostic evaluation. Tumors, such as
renal cell carcinoma or angiomyolipoma, are seen as
hypervascular because of the increased vessel density, as well as the higher mean velocity within tumor
microvessels because of the reduced PR in the tumor.
Ultrasound Contrast Media

As with radiographic CM, certain injected agents can
be used to enhance the ultrasound signal/noise.
Essentially, these agents are stabilized microbubbles
(210 m range), which are sonovisible (27). Only intravascular agents are available at the moment with no
soft-tissue distribution, but they are useful for vascular studies as they improve the strength of the Doppler effect and allow more detailed vascular analysis.
They can also, under some circumstances, return
signal of a specific frequency (or harmonic) that can
be individually isolated and analyzed. Such harmonic
imaging is currently being evaluated, and with the
development of more refined agents with a tissue
distribution, harmonic imaging of soft-tissue abnormalities may expand the diagnostic range of grayscale
ultrasound.
Many of the recent developments in medical
ultrasound are to do with improved transducer design
and postprocessing. This has improved the quality of
the ultrasound, but some disease processes remain
frustratingly beyond current visibility. Prostate cancer
is a germane examplealthough the prostate gland is
95
exploits the difference in stiffness of tissues. A

tumor is normally significantly stiffer than the
background soft tissue. When a mechanical compression such as an ultrasound wave is applied, the tumor
deforms less than the surrounding tissue and this can
be detected. Sonoelasticity has been explored in the
prostate gland but has not yet proven useful (28,29).
Therapeutic Ultrasound
Figure 9 (A) Ultrasound image of a renal stone (being measured with callipers) showing it to be hyperechoic because of the
increased reflection of sound waves (thick arrow). It also casts a
hypoechoic shadow (thin arrow) as no sound waves can penetrate through it. Not all renal stones show this characteristic
shadowing, for example, small stones may not cast a shadow.
(B) A small hyperechoic lesion is seen in the lower ureter (white
arrow) but it does not cast an acoustic shadow. Is it a stone?
(C) With the color Doppler switched on, the lesion demonstrates
twinkle artifact (white arrow), suggesting that it represents a
stone. Ureteric jets can also be seen in the bladder (dashed
arrow), suggesting that the ureter is not obstructed.
ideally suited for ultrasound examination, as with a

high frequency transrectal probe, it is very close to the
probe surface, with few intervening tissues to degrade
the sound; yet many biopsy-proven prostate cancers
are not visible. This highlights the limitation of current
grayscale ultrasound. Even the most modern probes
poorly resolve early or small-volume disease in the
prostate gland or elsewhere. To overcome this limitation, other fundamental properties of tissue-sound
interaction are being investigated for sonography.
These include the imaging of harmonic frequencies
(as above) and tissue sonoelasticity. Elastography
After over 30 years of constant use, there has not been

even one reported case of harm attributable to the use
of diagnostic ultrasound, although there are theoretical risks because of local heating or microcavitation in
tissues. Theraputic ultrasound exploits these tissueheating effects. High-intensity focused ultrasound
(HIFU) is being explored as a minimally invasive
treatment for solid cancers. This uses low frequency
sound waves and focuses them in a small target area.
The high concentration of energy at the focus point
causes a dramatic rise in temperature to >558C in a
matter of seconds. This causes coagulation necrosis of
the targeted tissue. The volume of destructed tissue is
small1 to 3 mm in width and 5 to 20 mm in height
(30,31). The probe, therefore, has to be moved sequentially to allow a larger treatment area. The tissue
outside the targeted area is not heated to cytotoxic
levels; therefore, HIFU provides a trackless method of
delivering ablative energy. HIFU has been used in
urology to treat renal (32) and testicular cancers (33),
but it is in the treatment of prostate cancer that it has
gained most acceptance (3436). It can treat those who
are not candidates for standard radical therapy, as
well as a salvage treatment in patients who have local
recurrence following external beam radiotherapy (37).
A future promising area is the use of ultrasound
beams to destabilize injected microbubbles to release
therapeutic agents precisely within the organ of
disease (38).
MAGNETIC RESONANCE IMAGING

The nuclear magnetic resonance (NMR) phenomenon
was first described in 1937 by Professor Rabi of
Columbia University in the United States (39). However, it is only in the last 25 to 30 years that the
medical applications of NMR have been realized.
The physics of NMR is often viewed as complex.
However, the essence of the technique may be understood by reviewing extracts from quantum mechanical
and classical models.
In brief, the steps in the production of an image
are as follows:
l
The patient is placed in a powerful magnetic field.

(Typically in most hospitals this is a 1.5-Tesla
magnet. This is some 30,000 times stronger than
the earths magnetic field.)
This magnetic field aligns hydrogen nuclei within
the body, creating a net magnetization.
Radiowaves alter the alignment of the net magnetization.
96
Patel and Walkden
Figure 10 Principles of Doppler ultrasound. The Doppler effect is used to calculate the velocity of a moving substance, such as blood.
The velocity can be presented as a continuous Doppler or spectral waveform or can be superimposed on the grayscale ultrasound image
as a color Doppler imageas shown on the right with intrarenal arteries clearly demonstrated. The left-hand image is a power Doppler
image showing the fine details of the penile vasculature. The right-hand image is a montage showing a color and spectral Doppler
imagea waveform has been measured with an RI of 0.63, indicating normal PR in this nonobstructed kidney.
After the radiowave is switched off, the net magnetization realigns to its original position and in
doing so emits a weak radio signal.
The frequencies contained within the radio signal
can be manipulated and thereby encode spatial
information.
The signal generates a current within a coil of wire
and this current is converted into an image.
To understand this process in more depth, a

number of fundamental principles need to be appreciated (Fig. 12).
Useful MR Nuclei
Figure 11 (A) Ultrasound of a patient with histologically proven

seminoma of the left testicle shows the homogenously hypoechoic
tumor that displays increased vascularity on color Doppler. (B) The
patients normal right testicle with its normal vascular pattern.
A nucleus of an atom contains protons and neutrons

that are normally spinning. Nuclei, which have an
odd number of constituents, have a net charge and,
because they are spinning by the law of electromagnetic induction, must generate a small magnetic field.
These nuclei are the MR active nuclei and include
hydrogen. The hydrogen nucleus (consisting of a
single proton only) is the one used in clinical MRI
because of its natural abundance within the body.
Each hydrogen nucleus is in essence an extremely
weak bar magnet. Luckily, the human body is not
normally magnetized as these mini magnets are
randomly aligned and the magnetic fields cancel
each other out.
97
Figure 12 Principles of MRI. (A) Protons have a net spin

that makes them act like mini bar magnets. When placed
in a strong magnet field, they develop a special type of
spin called precession and they align to give an NMV.
(B) Under the influence of an externally applied radiowave, the NMV flips into the transverse plane. Once the
radiowave is switched off the NMV decays in the transverse plane and recovers in the longitudinal plane. This
happens in a spiral motion giving rise to the MR signal.
(C) T1 is the time for the longitudinal magnetization to
recover to 63% of its maximum value. T2 is the time for the
MR signal to fall to 37% of its maximum value.
The Effect of an External Magnet

When the body is placed in a strong external magnetic
field (B0), two things happen.
1.
2.
Hydrogen nuclei within the body now adopt one

of two distinct energy levels (high or low). Individual hydrogen nuclei constantly flip between
these two levels. But at any given time, there are
more nuclei in the low-energy state. Summed
together, these additional low-energy hydrogen
nuclei create a small net magnetic field parallel
to the direction of the external magnetic field. It is
the existence of this net magnetic vector (NMV),
which makes MR possible.
The hydrogen nuclei begin to precess as well as
continue to spin. The motion of precession can be
described by analogy to a spinning top. As a
spinning top spins on its own axis, it begins to
tilt. As it tilts the top of the highest point of the
spinning top starts moving in a circular orbit
(precesses) while the spinning top continues to
spin on its own axis. Continuing with the top
analogy, the speed of precession depends on the

strength of gravity, and a top would precess far
slower on the moon than on Earth. For hydrogen
nuclei, the speed of precession depends on, and is
in fact proportional to, the strength of the external
magnetic field B0.
Resonance
All materials have a natural frequency at which they
find it easiest to vibrate, which is termed as resonant
frequency. If a periodical force is applied at the same
frequency as an objects natural frequency, the object
will tend to absorb the applied energy. As this energy
builds, the amplitude of its oscillations will continue
to increase. The object will then either change its
dynamic properties, which then alter its resonant
frequency, or it loses the energy in some form. If it
does not lose the energy, then the object can break
apart. An example of this occurred in World War II
when soldiers marching in step across a bridge caused
it to collapse as they were marching at the resonant
98
Patel and Walkden
frequency of the bridge. To this day soldiers still break

step when crossing bridges.
It so happens that the resonant frequency of
hydrogen nuclei lies in the radiowave bandwidth.
Application of Radiofrequency Radiation (Pulse)

Why then do resonance and nuclear magnetism
come together to give the term nuclear magnetic
resonance? Recall that in an external magnetic field
a NMV is set up by the alignment of the hydrogen
nuclei and that this NMV lies parallel to the external
magnetic field. In this equilibrium state this magnetism cannot be detected. However, when the body is
exposed to radiowaves at the resonant frequency of the
hydrogen nuclei, energy can be transferred from the
radiowaves to the hydrogen nuclei.
This has two effects on the nuclei.
1.
2.
More nuclei gain enough energy to enter the highenergy state, so that the total number of excess
nuclei in the low-energy state at any given time is
reduced. This has the result of decreasing the
NMV. If the radiofrequency pulse is applied for
a sufficient time, then the NMV in the direction of
the external magnetic field can be made to reduce
to zero, and this is termed a 908 radiofrequency
pulse.
Another very useful property of a radiofrequency
pulse is that it reforms the NMV in the plane
perpendicular to the external magnetic field. It is
because of this transverse magnetic vector that
NMR signal can be detected.
When the radiowave is stopped, the hydrogen nuclei start to lose energy and more and more
nuclei once again enter the low-energy state until
equilibrium is reached. Furthermore, the transverse magnetic vector begins to precess. As it
precesses it generates an oscillating magnetic
field, which induces an alternating current in the
receiver coil. By application of a series of magnetic
field gradients, the current frequencies can be
varied in a controlled fashion to allow spatial
encoding. This multifrequency current is then
amplified and mathematically converted to an
onscreen MR image.
The contrast in every MR image is dependent on

three factors.
(1) The rate at which the hydrogen nuclei return from
the high- to low-energy state (T1)
(2) How quickly the transverse magnetization disperses (T2)
(3) The density of protons within a particular area (r)
The greater the proton density, the higher the
signal from a tissue and the brighter the MR image.
The contribution of T1 and T2 to an image can be
altered by manipulation of the imaging program
(radiowaves and magnetic field gradients applied to
the body to generate an image) and is completely
under user control. For proton density to predominate, the T1 and T2 effects must be diminished.
Different body tissues may have different T1, T2

times and also vary in proton density, thus generating
a contrast between them. If two tissues differ in T2
times, then producing a T2-weighted image will show
a difference in contrast between these two tissues and
this can sometimes be useful in differentiating tumor
from normal tissue (i.e., prostate carcinomalow T2
signal intensity; from normal peripheral zonehigh
T 2 signal intensity). If T1-weighted images were
acquired from the peripheral zone of the prostate
with tumor, then the tumor may be masked as the
T1 times of both tissues are similar. The multiparameter-dependent contrast of an MRI is a distinct advantage over CT where contrast is simply related to
density of material.
In general, T1 images show fluid as low signal
(dark) and fat as high signal (bright). They are generally good for anatomy. They can also allow the detection of blood (at certain stages in its breakdown) such
as that caused by prostate biopsy, which appears as
high signal. T2 images show fluid as high signal
(bright) and tend to be more useful for distinguishing
between normal and pathological tissues (Fig. 13).
Heavily T2-weighted images are used to craft MR
urographic studies (Fig. 14).
A typical MRI examination consists of a selection
of images of different contrasts (T1, T2, etc.) acquired
in differing anatomical planes (saggital, coronal, axial,
etc.). Exactly which planes and contrasts are used
depends on the information that is required about
the subject tissue (Fig. 15). Table 9 shows the current
uses of MRI in urology.
Improving Signal to Noise Ratio

Improving the SNR has been a major goal in MRI. It
can be achieved in many ways, but often these result
in either increased scan time or loss of spatial resolution, and achieving the optimum image is a trade-off
between SNR, scan time, and spatial resolution.
A simple (but rather expensive) way of getting
more signal is to use a stronger magnet to generate a
larger external magnetic field. Increasing the magnet
strength from 1.5 to 3 Tesla should improve the SNR
by a factor of 2, and 3-Tesla MRI machines are now
commercially available. This extra signal can be
traded, if required, for faster imaging or higher spatial
resolutions.
Another method of improving SNR is to place
the coil used to pick up the signal as close as possible
to the tissue generating the signal. In urology, using
an endorectal coil has been shown to improve spatial
and contrast resolution for prostate imaging. Initially,
there was little evidence to suggest that this dramatically improved staging accuracy, but now increasingly
improved sensitivity, specificity and accuracy are
being reported (40).
Contrast Media
MRI-specific CM are also available. These are paramagnetic or supaparamagnetic metal ions that affect
99
Figure 14 MR urography. A heavily T2-weighted image shows

fluid as high signal as seen here in an MR urography. A hydronephrotic left kidney is seen (thin solid arrow) caused by an
upper ureteric stone (dashed arrow), which appears as a signal
void. The bladder is well seen (thick solid arrow). The patient is
also pregnant and the fetus can be seen in the uterus (multiple
white arrows). The ability to assess the kidneys in pregnant
women is a major advantage of MR urography.
Figure 13 Magnetic resonance images of the bladder and

pelvic anatomy. (A) T1 image. Note that the urine is of low
signal. (B) T2 image, where fluid (urine) is always of high signal.
the MR signal properties of surrounding tissues. Like

other radiological CM they can be used to improve the
SNR. Two main types existnonspecific extracellular
agents and organ-specific agents. The organ-specific
contrast agents are mainly iron oxide based, used for
liver imaging, and therefore are not that relevant to
urological imaging. They are, however, currently
undergoing investigation for detection of lymph
node metastases (41), which could have a significant
effect on the accurate staging of pelvic tumors.
Gadolinium chelates are the most widely used
extracellular, nonspecific contrast agents. They are
strongly paramagnetic and shorten both T1 and T2

times of the hydrogen nuclei. Gadolinium chelates in
general clinical use short-lived intravascular agents
that leak into the extravascular spaces but remain
extracellular. They are excreted by the kidneys by
glomerular filtration without tubular secretion or
reabsorption. Gadolinium-based CM can cause similar
adverse reactions to iodinated CM, but much less
frequently. They are quoted to occur in approximately
1% of all patients and are normally mild and transient.
The prevalence of anaphylactoid reactions has been
reported as between 1/100,000 and 1/500,000 (42).
Contrast-induced nephropathy is not a clinical concern, but recently a link between certain gadoliniumbased agents, renal failure, and nephrogenic fibrosing
dermopathy has been established (43). The ESUR has
published guidelines suggesting that gadolinium contrast agents should not be used in patients with a GFR
<30 mL/min (44).
Dynamic ContrastEnhanced MRI

Cancers excite neovascularity and abnormal perfusion
compared with normal tissue. DCE MRI is able to
detect such aberrant perfusion by obtaining a rapid
series of images over a short period, following a bolus
administration of gadolinium. Signal intensitytime
100
Patel and Walkden
Figure 15 MRI series from a man with histological proven prostate cancer. (A) The peripheral zone normally displays high T2 signal
(dashed arrow). The low signal in the left peripheral zone (PZ, white arrow) is typical for tumor. (B) An image from a dynamic contrastenhanced series shows the tumor to enhance early (black arrow) compared with the normal PZ (dashed arrow). (C, D) Signal intensity
curves drawn from four regions of interest. 1 on the tumor nodule shows the tumor to have a characteristic type I curve with early
enhancement and washout compared with 2, which represents normal PZ. 3 is placed in the central gland, and 4, on the femoral artery.
(E) An ADC map shows the tumor to have restricted diffusion seen as low signal (solid arrow) compared with the normal PZ which has
normal diffusion seen as high signal (dashed arrow). If a lesion demonstrates all these findings, then a confident diagnosis of cancer can
be made.
Table 9 Current Use of MRI in Urology

Prostate imaging
Renal imaging
MR urography
Penile imaging
Prostate cancer staging is probably the

most common use of MRI in urology.
With standard T1 and T2 sequences
alone sensitivity and specificity are
approximately 80% and 60%,
respectively. The use of the newer
sequences such as HMRS and diffusionweighted images may increase this.
Faster sequences have allowed improved
imaging of the kidneys and MRI is often
used in renal mass evaluation its
improved contrast resolution being a
distinct advantage over CT.
Particularly in pregnant women where
radiation is an issue.
To stage penile cancer. It is also useful in
the assessment of corporal fibrosis and
priapism assessment.
curves can be created (Fig. 15), and these data can be

assessed qualitatively or quantitatively (45). Qualitative assessment is based on the shape of the curve or
descriptors, such as time to maximum enhancement,
gradient of curve, or maximum signal intensity, and is
relatively easy to obtain. Quantitative information
involves creating concentration-time curves from
pharmacokinetic models and requires special software
programs (46). Currently, it is not known if the
quantitative data provide more accurate information.
It has been shown that prostate cancer in the peripheral zone demonstrates early nodular enhancement,
before normal parenchyma and early washout (47).
This pattern of enhancement, while highly suggestive
of tumor, is not pathognomonic with other processes,
most notably prostatitis, having a similar curve. Nevertheless, DCE is felt to improve specificity and is
currently undergoing clinical evaluation.
Hydrogen Magnetic Resonance Spectroscopy

Hydrogen magnetic resonance spectroscopy (HMRS)
provides metabolic information about the tissue being
investigated by displaying the relative concentrations
of chemical metabolites within a voxel. Cancers can
have a different metabolic makeup compared with
their normal surrounding tissue, because of the
enhancement of the phospholipid cell membrane
turnover associated with tumor cell proliferation,
increased cellularity, and growth. For example, prostate cancer has a decreased level of citrate and an
increased level of choline when compared with normal prostatic tissue (48) and RCC has been shown to
demonstrate a decrease in free cholesterol and unsaturated fatty acids compared with normal renal parenchyma (49). HMRS has been shown to improve
prostate cancer localization in the peripheral zone
and provides an indication of tumor grade (50). It
may also be particularly useful in detecting cancer in
the previously treated prostate gland (51,52). HMRS
will benefit from the use of 3T magnets as it will allow
smaller voxels to be obtained, reducing volume averaging and improving the separation of the metabolite
peaks (53).
Diffusion-Weighted Imaging
Diffusion is the process by which particles intermingle
as the result of spontaneous movement caused by
thermal agitation. MRI can be used to examine the
microscopic diffusion of water molecules within tissues. Unlike free water, tissue water is limited in its
movement by cellular barriers. An increase in the
restriction of water diffusion has been seen in pathological tissues such as tumor and is thought to be
related in part to increased cellular density. Diffusionweighted images are able to detect and quantify this
change in diffusion. Areas of restricted diffusion
appear of increased signal (bright) on diffusionweighted images and are correspondingly dark on
quantitative diffusion maps (Fig. 15).
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ACKNOWLEDGMENT
20.
We would like to thank Dr Shonit Punwani for his

help with the physics of MRI.
21.
REFERENCES
22.
1. Mould RF. Invited review: Rontgen and the discovery of

X-rays. Br J Radiol 1995; 68(815):11451176.
2. Voeleker F, von Lichtenburg A. Pyelography (Rontgenographie des Nierenbeckens nach kullargolfullung). Munchener Med Wochenschr 1906; 53:105106.
3. Digital Imaging and Communication in Medicine. Strategic
document version 4.0. June, 2005. Available at: http://
medical.nema.org.
4. Osbourne ED, Sutherland CG, Scholl AJ, Jr., et al. Roentgenology of the urinary track during excretion of sodium
iodide. J Am Med Assoc 1923; 80:368373.
5. Swick M. Darstellung der Niete und Harnwege in Roentgenbild durch intravenous Einbringung eines neuen
23.
24.
25.
101
kontraststoffes:des uroselectans. Klin Wochenschr 1923;

8:20872089.
Morcos SK, Thomsen HS, Webb JA. Contrast-mediainduced nephrotoxicity: a consensus report. Contrast
media safety committee, European Society of Urogenital
Radiology (ESUR). Eur Radiol 1999; 9(8):16021613.
Nash K, Hafeez A, Hou S. Hospital-acquired renal insufficiency. Am J Kidney Dis 2002; 39(5):930936.
Parfrey PS, Griffiths SM, Barrett BJ, et al. Contrast materialinduced renal failure in patients with diabetes mellitus,
renal insufficiency, or both. A prospective controlled
study. N Engl J Med 1989; 320(3):143149.
Rudnick MR, Goldfarb S, Wexler L, et al. Nephrotoxicity
of ionic and nonionic contrast media in 1196 patients:
a randomized trial. The Iohexol Cooperative Study. Kidney
Int 1995; 47(1):254261.
Bartholomew BA, Harjai KJ, Dukkipati S, et al. Impact of
nephropathy after percutaneous coronary intervention
and a method for risk stratification. Am J Cardiol 2004;
93(12):15151519.
Mehran R, Aymong ED, Nikolsky E, et al. A simple risk
score for prediction of contrast-induced nephropathy
after percutaneous coronary intervention: development
and initial validation. J Am Coll Cardiol 2004; 44(7):
13931399.
Pannu N, Wiebe N, Tonelli M Prophylaxis strategies for
contrast-induced nephropathy. JAMA 2006; 295(23):
27652779.
Thomsen HS. European Society of Urogenital Radiology
(ESUR) guidelines on the safe use of iodinated contrast
media. Eur J Radiol 2006; 60(3):307313.
Cipollaro VA. Radiation dermatitis today. J Eur Acad
Dermatol Venereol 2001; 15(4):300301.
The Royal College of Radiologists. Making the Best Use of
Clinical Radiology Services: Referral Guidelines. London:
The Royal College of Radiologists, 2007.
Berrington de Gonzalez A, Darby S. Risk of cancer from
diagnostic X-rays: estimates for the UK and 14 other
countries. Lancet 2004; 363(9406):345351.
United Nations Scientific Committee on the Effects of
Atomic Radiation. Sources and effects of ionising radiation. New York: United Nations, 2000.
Shrimpton P, Jones DG, Hillier MC, et al. Survey of CT
practice in the UK. Part 2: dosimetric aspects. NRPB Report
R249. Oxfordshire: National Radiological Protection Board,
1991.
Ambrose J, Hounsfield G. Computerized transverse axial
tomography. Br J Radiol 1973; 46(542):148149.
Cody DD. AAPM/RSNA physics tutorial for residents:
topics in CT. Image processing in CT. Radiographics
2002; 22(5):12551268.
Coll DM, Herts BR, Davros WJ, et al. Preoperative use of
3D volume rendering to demonstrate renal tumors and
renal anatomy. Radiographics 2000; 20(2):431438.
Browne RF, Murphy SM, Grainger R, et al. CT cystography
and virtual cystoscopy in the assessment of new and
recurrent bladder neoplasms. Eur J Radiol 2005; 53(1):
147153.
Boridy IC, Nikolaidis P, Kawashima A, et al. Ureterolithiasis: value of the tail sign in differentiating phleboliths from
ureteral calculi at nonenhanced helical CT. Radiology 1999;
211(3):619621.
Varanelli MJ, Coll DM, Levine JA, et al. Relationship
between duration of pain and secondary signs of obstruction of the urinary tract on unenhanced helical CT. AJR
Am J Roentgenol 2001; 177(2):325330.
Fry WJ, Mosberg WH Jr., Barnard JW, et al. Production of
focal destructive lesions in the central nervous system with
ultrasound. J Neurosurg 1954; 11(5):471478.
102
Patel and Walkden
26. Holmes JH, Howry DH, Posakony GJ, et al. The ultrasonic
visualization of soft tissue structures in the human body.
Trans Am Clin Climatol Assoc 1954; 66:208225.
27. Cosgrove D. Ultrasound contrast agents: an overview. Eur
J Radiol 2006; 60(3):324330.
28. Linden RA, Halpern EJ. Advances in transrectal ultrasound imaging of the prostate. Semin Ultrasound CT MR
2007; 28(4):249257.
29. Pallwein L, Aigner F, Faschingbauer R, et al. Prostate
cancer diagnosis: value of real-time elastography. Abdom
Imaging 2008; 33(6):729735.
30. Watkin NA, ter Haar GR, Rivens I. The intensity dependence
of the site of maximal energy deposition in focused ultrasound surgery. Ultrasound Med Biol 1996; 22(4):483491.
31. Chapelon JY, Margonari J, Theille`re Y, et al. Effects of highenergy focused ultrasound on kidney tissue in the rat and
the dog. Eur Urol 1992; 22(2):147152.
32. Wu F, Wang ZB, Chen WZ, et al. Preliminary experience
using high intensity focused ultrasound for the treatment
of patients with advanced stage renal malignancy. J Urol
2003; 170(6 pt 1):22372240.
33. Kratzik C, Schatzl G, Lackner J, et al. Transcutaneous highintensity focused ultrasonography can cure testicular cancer in solitary testis. Urology 2006; 67(6):12691273.
34. Blana A, Murat FJ, Walter B, et al. First analysis of the longterm results with transrectal HIFU in patients with localised prostate cancer. Eur Urol 2008; 53(6):11941201.
35. Muto S, Yoshii T, Saito K, et al. Focal therapy with highintensity-focused ultrasound in the treatment of localized
prostate cancer. Jpn J Clin Oncol 2008; 38(3):192199.
36. Illing RO, Leslie TA, Kennedy JE, et al. Visually directed
high-intensity focused ultrasound for organ-confined prostate cancer: a proposed standard for the conduct of therapy. BJU Int 2006; 98(6):11871192.
37. Gelet A, Chapelon JY, Poissonnier L, et al. Local recurrence
of prostate cancer after external beam radiotherapy: early
experience of salvage therapy using high-intensity focused
ultrasonography. Urology 2004; 63(4):625629.
38. Postema M, Gilja OH. Ultrasound-directed drug delivery.
Curr Pharm Biotechnol 2007; 8(6):355361.
39. Rabi II, Zacharias JR, Millman S, et al. A new method of
measuring nuclear magnetic moment. Phys Rev 1938; 53:318.
40. Futterer JJ. MR imaging in local staging of prostate cancer.
Eur J Radiol 2007; 63(3):328334.
41. Harisinghani MG, Barentsz J, Hahn PF, et al. Noninvasive
detection of clinically occult lymph-node metastases in
prostate cancer. N Engl J Med 2003; 348(25):24912499.
42. Shellock FG, Kanal E. Safety of magnetic resonance imaging
contrast agents. J Magn Reson Imaging 1999; 10(3):477484.
43. Grobner T. Gadoliniuma specific trigger for the development of nephrogenic fibrosing dermopathy and nephrogenic systemic fibrosis? Nephrol Dial Transplant 2006; 21:
11041108.
44. Thomsen HS. ESUR guideline: gadolinium-based contrast

media and nephrogenic systemic fibrosis. Eur Radiol 2007;
17(10):26922696.
45. Alonzi R, Padhani AR, Allen C. Dynamic contrast
enhanced MRI in prostate cancer. Eur J Radiol 2007;
63(3):335350.
46. Tofts PS, Brix G, Buckley DL, et al. Estimating kinetic
parameters from dynamic contrast-enhanced T(1)-weighted
MRI of a diffusable tracer: standardized quantities and
symbols. J Magn Reson Imaging 1999; 10(3):223232.
47. Turnbull LW, Buckley DL, Turnbull LS, et al. Differentiation of prostatic carcinoma and benign prostatic hyperplasia: correlation between dynamic Gd-DTPA-enhanced MR
imaging and histopathology. J Magn Reson Imaging 1999;
9(2):311316.
48. Heerschap A, Jager GJ, van der Graaf M, et al. In vivo
proton MR spectroscopy reveals altered metabolite content
in malignant prostate tissue. Anticancer Res 1997;
17(3A):14551460.
49. Katz-Brull R, Rofsky NM, Morrin MM, et al. Decreases in
free cholesterol and fatty acid unsaturation in renal cell
carcinoma demonstrated by breath-hold magnetic resonance spectroscopy. Am J Physiol Renal Physiol 2005;
288(4):F637F641.
50. Coakley FV, Qayyum A, Kurhanewicz J. Magnetic resonance imaging and spectroscopic imaging of prostate
cancer. J Urol 2003; 170(6 pt 2):S69S75; discussion
S75S76.
51. Parivar F, Hricak H, Shinohara K, et al. Detection of locally
recurrent prostate cancer after cryosurgery: evaluation
by transrectal ultrasound, magnetic resonance imaging,
and three-dimensional proton magnetic resonance
spectroscopy. Urology 1996; 48(4):594599.
52. Pucar D, Shukla-Dave A, Hricak H, et al. Prostate cancer:
correlation of MR imaging and MR spectroscopy with
pathologic findings after radiation therapy-initial experience. Radiology 2005; 236(2):545553.
53. Futterer JJ, Scheenen TW, Huisman HJ, et al. Initial experience of 3 tesla endorectal coil magnetic resonance imaging
and 1H-spectroscopic imaging of the prostate. Invest
Radiol 2004; 39(11):671680.
FURTHER READING
Westbrook C, Kaut Roth C, Talbot J. MRI in Practice. 3rd ed.
Oxford: Blackwell Publishing, 2005.
Allisy-Roberts P, Williams J. Farrs Physics for Medical Imaging.
2nd ed. London: WB Saunders, 2008.
Silverman SG, Cohan R. CT Urography: An Atlas. Philadelphia:
Lippincott Williams & Wilkins, 2007.
7
Upper Urinary Tract Obstruction
Neil G. Docherty
The Conway Institute, University College Dublin, Ireland
John M. Fitzpatrick
Mater Misericordiae Hospital and University College Dublin, Ireland
ETIOLOGY
Upper urinary tract obstruction can result from a
variety of both congenital and acquired conditions,
impeding urinary flow from the renal pelvis to the
bladder. Implicit in this statement is the fact that the
structure affected is either the kidney (renal pelvis) or
the ureter. Albeit that bilateral hydronephrosis resulting from bladder outlet obstruction shares many
features of more proximal obstruction, we will restrict
our discussion in this chapter to obstruction of the
upper urinary tract.
The nature of ureteric obstruction (UO) can be
further delineated according to whether the obstruction is intraluminal or extrinsic. The degree of obstruction can also vary from partial, to progressively
occluding to acutely presenting complete obstruction.
Commonly, UO is unilateral (UUO) and may involve
obstruction of proximal or distal segments.
A major concern following obstruction is its effect
on the proximal kidney, an effect which will depend on
the site (renal pelvis, proximal or distal ureter), degree
(partial, complete) and duration (acute or chronic). The
combination of these considerations will determine the
incidence of obstructive nephropathy in the affected
kidney and in cases of UUO in individuals with a
solitary kidney, the severity of acute renal failure
encountered. A summary of the causes of upper urinary
tract obstruction is found in Table 1.
INCIDENCE
Upper urinary tract obstruction is a frequently
encountered diagnosis in urology. However, because
of the fact its etiology is varied, reliable cumulative
incidence rates do not exist.
Starting with neonatal obstruction, the criteria
for diagnosis requires that hydronephrosis is present,
with the fetal kidney producing isoosmolar urine
within the first trimester of pregnancy. Antenatally
hydronephrosis id detectable via ultrasonography and
its incidence is reported to be around 1% (1).
The major cause of upper urinary tract obstruction in adult urology is intrinsic, intraluminal nephro-
lithiasis and urolithiasis. Calcium oxalate and calcium

phosphate stone disease predominates, accounting for
80% of cases, with hypercalciuria a major risk factor,
underpinned by both genetic and lifestyle factors. The
lifetime incidence for men is reported at 12% versus 6%
in women (2).
CLINICAL PRESENTATION
Presentation of upper urinary tract obstruction is not
homogeneous, reflecting differences in etiology and
degree of obstruction.
Antenatal obstruction presents during routine
imaging. In postnatal (inc. adult) obstruction, clinical
presentation depends on the whether the obstruction is
unilateral or bilateral, acute or chronic and partial or
complete. Colicky flank pain is characteristic of acute
UUO, with the presence of fever suggestive of infection.
DIAGNOSIS AND TREATMENT

Management of Neonatal Obstruction
Neonatal hydronephrosis secondary to obstruction
frequently has its origin in antenatal uretero-pelvic
junction (UPJ) obstruction or uretero-vesicular junction (UVJ) obstruction, (primary megaureter) which
can be visualized on antenatal ultrasound.
Intrauterine management of obstruction is limited
to close observation of the obstruction to decide the
course of action in the neonate. While evidence of
hydronephrosis per se does not exclude the possibility
of meaningful renal function postnatally, evidence of
renal hypoplasty and dysplasty predicts a poor outcome in the neonate, and reflects obstructive injury of
first-trimester origin.
Postnatal ultrasound revealing dilatation should
lead to the ordering of a voiding cystourethrogram
(VCUG) and/or diuretic renography. Sustained deterioration of split renal function indicates pyeloplastic
surgical intervention in cases of UPJ obstruction. In
cases of megaureter, if adequate ureteral drainage is
observed a conservative approach of antibiotic theapy
throughout the first year of life is indicated. However
104
Docherty and Fitzpatrick
Table 1 Causes of Congenital and Acquired Upper Tract Obstruction

Congenital
Intraluminal
Extraluminal
Acquired
Intraluminal
PUJ obstruction
Ureterocoele (ectopic, orthotopic)
Calcus
Ureteric actresia
Bladder diverticulum
Stricture
Ureteric valve
Vascular (retrocaval ureter, retroiliac

ureter, lower pole renal vessels,
persistent umbilical artery)
Urothelial tumor
Ureteric folds
Congenital stricture
Vesicoureteric reflux
Primary megaureter
VUJ obstruction
Extraluminal
Malignancy (pelvic: prostate, colorectal, ovarian,
uterine, cervical; petroperitoneal: lymphoma,
sarcoma, mesothelioma, metastases)
Gastrointestinal (pancreatitis, appendicitis,
diverticulitis, Crohns disease)
Vascular (abdominal aortic aneurysm, lliac
artery aneurysm)
Blood clot
Pregnancy
Sloughed papilla
Gynaecological (fibrosis, endometriosis)
Benign Polyp
Retrperitoneal fibrosis
Foreign body (stent)
Fungal ball
The major causes of upper urinary tract obstruction are presented and subdivided, firstly, according to cause (congenital vs. acquired) and, secondly,
according to site (intraluminal vs. extraluminal).
recurrent UTIs and/or renal dysfunction indicate

surgical straightening and tapering of the ureter to
restore urinary flow.
Adult Obstruction
Further to history and physical examination, imaging
techniques are required to make a conclusive diagnosis. These may include intravenous or retrograde
pyelography and kidney-ureter-bladder CT scanning.
If urosepsis is suspected, the presence of infection can
be confirmed via culture and quantification of white
cell counts and erythrocyte sedimentation rates. With
urosepsis, percutaneous nephrostomy is indicated
immediately with provision of parenteral gram-negative targeted antibiotic therapy.
In the absence of the requirement for nephrostomy, ultrasound may assist in the diagnosis of the
grade of associated hydronephrosis. Immediate symptomatic relief is generally provided using oral diclofenac (50 mg) t.i.d.
An examination of the renal effects may be established using serum and urine biochemistry, with isotope renography also useful to assess split renal
function, though rarely used in first-line diagnostics.
Once a diagnosis of obstruction has been established and the presence or absence of sepsis determined,
a decision on management of obstruction can be made.
Nephrolithiasis
In the case of calculi contained within the renal pelvis,

extracorporal shock wave lithotripsy (ESWL) is preferred for small to medium sized stones (<2cm), with
some indication that its effectiveness is more limited
in lower pole nephrolithiasis due to gravitational
effects. Percutaneous nephrolithotomy (PNL) is preferred for larger or more numerous stones such as
large pelvic staghorn calculi. Open surgery for renal
calculi is rapidly disappearing but its use continues in
cases where open pyelolithotomy or nephrectomy is
the indicated course of action (3).
Urolithiasis
Obstruction of the ureter may require surgery if the

cause is extrinsic or the result of ureteric stricturing.
When ureteric calculi are the cause, the clinical decision

is based on whether spontaneous expulsion of the stone
is possible. The criteria for determining this are the size
and position of the calculus with small distally located
calculi most amenable to spontaneous expulsion. Duration of symptoms and degree of hydronephrosis have
also been shown to be predictive of spontaneous passage, possibly related to the effect of these parameters
on ureteric peristalsis. Promising pharmacological promotion of spontaneous passage of distally located
calculi has been demonstrated using the combination
of an a-adrenergic blocker with a corticosteroid (e.g.,
prednisolone + tamsulosin) (4,5). The beneficial effect of
medical expulsive therapy in distally located calculi is
likely to be a result of the preferential expression of
a1d-adrenergic receptors and adrenergic transmission
in the distal versus proximal ureteric muscularis (6).
When spontaneous passage is discounted, ESWL
and ureteroscopy constitute the major therapeutic
modalities.
THE EFFECT OF URETERIC OBSTRUCTION ON

URETERIC FUNCTION
Normal Pyelourteric Peristalsis
The peristaltic propulsion of a urine bolus from the
renal pelvis to the bladder depends on coordinated
contractions of the renal pelvis and ureter. In times of
low urine output, renal pelvic contractions outnumber
ureteric contraction. During diuresis however, contractility in the renal pelvis is increased to a point
where it instigates myogenic transfer of action potentials to the ureteric muscularis, leading to coordinated
pelvic/ureteric contractions, which have the combined effect of propelling urine to the bladder. Despite
existence of evidence of both sympathetic and parasympathetic input and receptor expression in the
renal pelvis and proximal ureter, central integrative
control of ureteric peristalsis remains poorly understood. It is currently believed that the primary oscillator of ureteric peristalsis is renal pelvic urine
volume and its effect on pyeloureteric pacemaker
activity (7).
As mentioned above, peristalsis in the distal
ureter is more dependent on adrenergic control of
Chapter 7: Upper Urinary Tract Obstruction
peristalsis making medically expulsive therapy an

exciting emerging field in the treatment of distally
located calculi.
Pacemaker coordination of pyelourteric peristalsis has been suggested to depend primarily on the
activity of atypical smooth muscle cells and interstitial
cells of Cajal-like cells which predominate in the renal
pelvis, have a low depolarization threshold and act to
drive depolarization (and peristalsis) in naturally
more refractory typical smooth muscle cells (8).
The tonic effect of urothelium-derived factors on
contractility remains an interesting area. Diclofenac is
effective in preventing obstruction-associated colic
and is a nonspecific inhibitor of prostaglandin producing cycloxygenase enzymes, which are localized to
the urothelium. No consensus has been reached on the
effect of individual prostaglandin species on ureteric
contractility (7).
Acute Changes in Peristaltic Activity Following Obstruction
The frequency and amplitude of pelvic and ureteric

contraction changes dramatically in response to
obstruction. Graded elongation of the human and
sheep intrarenal and extrarenal pelvis and distal ureter in vitro demonstrates that stretch increases both
the basal and active tension (amplitude) in all segments but only increases spontaneous frequency of
contraction in the pelvic segments (9).
Lennon et al. (1993) examined the effect of both
complete and partial UUO of one month in duration on
pelvic and ureteric contractility in vivo in dogs (10).
In the case of complete obstruction baseline pelvic
pressure was 1.9 mmHg, with a frequency of 9.2/min
and an amplitude of 2.5 mmHg. Ureteric baseline
pressure was 2.1 mmHg with a frequency of single
spike contractions of 8.9/min and an amplitude of
36.2 mmHg. Following one month of obstruction both
the pelvis and ureter were aperistaltic with raised
baseline pressures of 15 mmHg and 16.3 mmHg,
respectively. This aperistaltic effect is likely related
to changes in the length/active tension relationship of
the ureteric and pelvic muscularis caused by sustained urinary pooling.
A different picture was observed following one
month of partial obstruction. Both pelvis and ureter
remained contractile with a 50% and 66% reduction in
contraction frequency, respectively. This was due to a
change in contraction type to a multiphasic response
with increased refractory periods between spikes. Interestingly a fivefold increase in pelvic amplitudes and a
twofold increase in ureteric amplitudes were observed.
Urosepsis has also been shown to perhaps play a
role in decreased contraction frequency in vitro. Urothelial application of live gram-negative species
has been shown to reversibly inhibit ureteric contractility (11).
Postobstructive Ureteric Function
In the same key paper by Lennon et al. (2003)

described above, measurements of ureteric function
were made eight weeks after relief of both complete
105
and partial obstruction in dogs (10). No statisitical

difference versus original baseline measures was
observed in ureteric contractile frequency or amplitude in the pelvis or ureter from animals with complete obstruction although baseline pressure remained
twofold higher in the ureter. Baseline ureteric pressure was also increased in the partial obstruction
group, however these animals retained twofold higher
amplitude rates in both the pelvis and ureter
and continued to show a mixed single/multphasic
waveform.
OBSTRUCTIVE NEPHROPATHY
Obstructive Nephropathy Following UUO
in Animal Models
The above description of the changes in ureteric
function in obstructive injury has important implications for the upstream kidney.
To explain the pathophysiology of obstructive
nephropathy, it is best to examine the findings of
acute UUO in adult animals, which approximates
most accurately to acute and complete stone mediated
obstruction of the ureter in the adult human. While
doing so, it is imperative to keep in mind that the
results of such studies may have important differences
to obstruction in divergent clinical settings (e.g., partial obstruction, bilateral affectation, neonatal vs.
adult).
Overview of Macroscopic and Microscopic

Changes in Acute, Complete UUO
With complete obstruction comes associated urinary
pooling which, in combination with changes in ureteric contractility, gives rise to retrograde pressure
transfer to the proximal kidney and hydronephrosis.
These events set in motion the major gross morphological and microanatomical changes observed in
renal structure. With increasing time post obstruction,
the kidney becomes swollen because of pooling of
urine proximal to the site of obstruction. As the renal
pelvis expands, the renal parenchyma becomes affected.
Flattening of the renal papilla and back filling of the
collecting system occurs. This leads to compression of
the renal cortex causing the characteristic cortical
thinning observed in obstruction. When obstruction
is sustained, tubular swelling extends to the more
proximal portions of the nephron where flattening of
the tubular epithelium can be observed. These
mechanical changes are associated with renal tubular
cell stress, which is associated with increased oxidative stress (12), the induction of cell death by apoptosis, and the initiation of both proinflammatory and
profibrotic signaling (13) (Fig. 1). The result of sustained UUO is therefore progressive tubular atrophy
accompanied by the development of tubulointerstitial
fibrosis.
Following UUO there is relative preservation of
renal microvascular structure and glomerulosclerosis
106
Figure 1 Mechanical stretch as a primer of obstructive nephropathy. Changes in both the frequency and amplitude of pyeloureteral
contractions combined with sustained urinary pooling proximal to the site of obstruction contribute to mechanical stretch injury of the
renal tubular epithelium. Apoptotic, inflammatory and haemodynamic changes thereafter give rise to the characteristic features of acute
obstructive renal injury which can progress to fibrotic deterioration of the kidney.
is only a feature of the late stages of long-term

chronically sustained obstruction.
The contralateral kidney undergoes adaptive
changes in renal blood flow, glomerular filtration
rate (GFR) and single nephron function and with
sustained obstruction undergoes compensatory
hypertrophy (14,15).
LINK BETWEEN CHANGES IN RENAL PELVIC

PRESSURE AND BLOOD FLOW POST UUO
A well-defined phasic response to UUO is known to
occur. In the first phase (090 minutes) there is an
increase in intrapelvic pressure from values of 6 to 7 to
50 to 70 mmHg (16). Initial elevations in ipsilateral
renal pelvic pressure induce a reno-renal reflex characterized by substance P release from renal sensory
neurons that leads to decreased efferent sympathetic
output (17,18). This neural adaptation underpins the
contralateral diuresis observed in UUO. In chronic
UUO in rats, artificial elevation of ipsilateral intrapelvic pressure fails to induce a further reno-renal
reflex (18).
Accompanying these phasic pressure changes
are nitric oxide/prostaglandin mediated decreases in
afferent arteriolar resistance, which in turn raise glomerular capillary pressure to the extent that glomerular filtration rate (GFR) is maintained at around 80% of
normal values despite large increases in intratubular
pressure (19).
In the second phase, occurring approximately
between 90 minutes and four hours after obstruction,
elevated intratubular pressure is sustained but renal

blood flow decreases secondary to afferent and efferent arteriolar vasoconstriction in response to vasoconstrictory substances including thromboxane A2
and endothelin (20,21). GFR is thus reduced to
approximately 20% of control levels (22). Phasic
changes in glomerular haemodynamics in acute
UUO are presented in Figure 2.
After the initial 24 hours of obstruction, collecting system pressure is reduced, but remains around
50% elevated versus normal levels (23). The continued
pooling of urine in the renal pelvis is likely to contribute to the sustained dilatation of particularly the distal
tubular segments (24).
As obstruction persists, adaptive dilatation of the
efferent renal lymphatics occurs secondary to
increased venous pressure (25). This limits the urinary
pooling effect, helps to maintain glomerular filtration
by reducing intratubular pressure and ultimately limits renal damage.
Ureteric compliance also increases with sustained
obstruction and the retrograde peristaltic effect is lessened as the ureter takes on a baggy appearance.
MECHANOSENSATION IN RENAL
TUBULAR CELLS
As outlined in the above section, phasic changes in
renal pelvic pressure/ureteric contraction and urinary
pooling constitute a significant stress stimulus to the
proximal kidney. The mechanism of mechanosensation
and the subsequent responses invoked provide a link
107
Figure 2 Changes in glomerular arteriolar

tone in UUO. Early after complete UUO,
vasodilatory mediators such as nitric oxide
and prostaglandin E2 cause afferent arteriolar
dilatation which acts to maintain renal blood
and glomerular ultrafiltration coefficient in
spite of rises in intratubular hydrostatic pressure. As obstruction proceeds and an inflammatory response is established, increased
release of vasoconstrictory substances (e.g.,
thromboxane A2and endothelin-1) lead to
constriction of both afferent and efferent arteriolae, leading to reduced renal blood flow and
a decrease in GFR.
between the biophysical stress stimuli in UUO and the

apparition of renal injury. Mechanical deformation of
cultured renal tubular epithelial cells elicits a wide
variety of changes in cell signaling and gene expression
which coordinates a set of responses which in many
aspects recapitulate early tubular cell responses in
UUO in vivo (26). For example, in vitro cyclic mechanical stretch of tubular epithelial cells leads to increases
in transforming growth factor b-1 (TGF-b1) expression,
which compliments similar finding in postobstructed
renal tissue in man (27,28).
Sensing of Stretch
There are a number of mechanisms by which epithelia
can sense mechanical perturbations. Firstly, epithelial
adhesion to the basement membrane is mediated via
the association of heterodimeric proteins called integrins to specific arginine-glycine-aspartic acid motifs
on extracellular matrix (ECM) proteins such as fibronectin (29). Intracellularly the integrin heteromdimers
are linked to the actin cytoskeleton at focal adhesions,
sites at which the initating components of various cell
signaling pathways are condensed. Mechanical
stretching of cells or tissues leads to mechanosensation via this ECM-integrin-cytoskeleton complex (30).
Such responses are dependent on elevations in intracellular calcium derived both from internal stores and
from influx from the extracellular compartment (31).
Transient Receptor Potential Cationic Channel-1
(TRPC-1) has been identified as the putative stretchactivated calcium channel in man and is expressed in
the renal tubular epithelium (32,33).
Additionally, the apical membrane of tubular
cells has the capacity for mechanosensation. It is
known that the renal epithelium has apically located
nonmotile cilia with TRPC-1 channels residing in their
base (34). Deformation of these cilia by pressure transfer leads to activation of inward calcium currents.
Microvilli of the proximal tubular brush border are
also known to be mechanically sensitive and respond
adaptively to changes in GFR, and proximal tubular flow
rate by altering the activity of the sodium-hydrogen

exchanger (35).
EFFECT OF MECHANICAL INSULT ON

TUBULAR CELLS
The set of responses induced in cells mechanically
stressed cells are pivotal in generating the pathophysiological changes observed in obstructive
nephropathy.
The Early Epithelial Stress Response

The epithelial stress response leads to oxidative stress
in affected cells. An increase in superoxide radical
prodiction and a reduction in hydrogen peroxide
metabolizing catalase enzyme expression has been
observed both in rodent models of obstruction and
during in vitro mechanical stretch (36). Such changes
destabilize the mitochondria and promote the release
of cytochrome C, which subsequently activates the
cysteine aspartate specific protease (Caspase) mediated
pathway of apoptosis. Release of cytochrome C following obstruction is also promoted by a decrease in the
expression of the mitochondrial transition pore guardian Bcl-2 (37). The production of tumor necrosis factor a
(TNF-a) and TGF-b1, Fas and Fas ligand also provide a
means for the autocrine/paracrine induction of apoptosis early after obstruction (13).
Establishment of Renal Inflammation in UUO

However, the induction of tubular cell apoptosis
following UUO in rats only attains pathological significance after all the early initial pressure and
haemodynamic changes are complete (>1 day).
This coincides with the establishment of a profound
inflammatory infiltrate in the kidney. Early activation of the renal vascular endothelium occurs characterized by increases in adhesion molecule expression
(e.g., VCAM-1 and ICAM-1) (38). The infiltrate is
108
Figure 3 Fibrosis in sustained UUO in the rat. Panel A shows mature fibrillar collagen (green) in tubulointerstitial areas of the rat kidney
10 days post UUO. Panel B demonstrates the presence of a smooth muscle actin positive interstitial cells in the same animals, reflecting
the proliferation of a matrix producing myofibroblast population.
initially composed of suppressor T-lymphocytes and

macrophages, the latter possibly reflecting the fact
that mechanically stretched cells(39) secrete large
amounts of monocyte chemotactic protein following
obstruction (40,41). Activated macrophages are a rich
source of proapoptotic and profibrotic cytokines such
as TNF-a and TGF-b1, leading to an exacerbation of
renal injury (42).
Development of Renal Fibrosis

Sustained obstruction compounded by a progressive
renal hypoxia leads to the continued production of the
various growth factors and cytokines (33). In particular, TGF-b1 and its associated signaling pathways are
crucial in the development of tubulointerstitial fibrosis
(TIF) in the affected kidney.
Mature fibrillar collagen is laid down in the interstitium, particulary in the medullary interstitium. In
addition, there is a net increase both in matrix degrading
metalloproteinase expression and the expression of
corresponding matrix metalloproteinase inhibitors the
balance of which coordinates pathological remodeling.
The fibrotic phenomenon is associated with the
increased presence of fiber producing interstitial a
smooth muscle actin positive myofibroblasts (Fig. 3).
INTRAOBSTRUCTIVE TUBULAR DYSFUNCTION

Associated with the development of renal injury as
described above, and in addition to changes in renal
haemodynamics and GFR, are a number of specific
defects in tubular function that are characteristic of
obstructive nephropathy. Despite the negligible GFR
in the obstructed kidney, a significant amount of urine
continues to pool, indicating that major changes in the
systems coordinating renal reabsorption of filtered

electrolytes and water is impaired in UUO.
Sodium and Potassium Handling

The opposing active transport of sodium and potassium at the basolateral membrane of the proximal
tubule, the ascending limb of the loop of Henle and
distal tubule is crucial to the fine tuning of renal
control of extracellular fluid volume. Sodium reabsorption is coupled to luminal secretion of potassium
and linked to osmotic movement of water back to the
blood.
Aside from the energetic limitations on active
transport imposed by progressive hypoxia. Decreased
expression of apical sodium antiporters and catalytic
units of the basolateral Na-K-ATPase has been
described in UUO (43). Loss of sodium reabsorption
interrupts the maintenance of the corticomedullary
gradient and inhibits normal function of the renal
countercurrent multiplier. In UUO, a reduced GFR
also contributes to reductions in potassium excretion,
however in the presence of a competent contralateral
renal function, hyperkalemia does not ensue (44).
Evidence in a rat model of UUO shows that there
is also a lesser but significant reduction in sodium
transporter expression in the contralateral kidney,
which contributes to early natriuresis (45).
It is interesting to note that these changes in
sodium handling occur in spite of the fact that activation of the intrarenal renin angiotensin system (RAS)
occurs in the obstructed kidney. Key amongst the
variety of pressor effects of RAS activation are stimulation of renal sodium reabsorption, both via increases in
aldosterone secretion and directly via induction of
sodium transporter subunits. However, in UUO,
these RAS system effects seem limited and more is
109
Figure 4 Defective AVP-collecting duct axis in UUO. The figure illustrates the defect in transmission of the AVP signal in the collecting
duct during/after obstructive injury. The failure of AVP to stimulate cAMP induced PKA activation prevents adaptive AQP2 membrane
translocation and CREB mediated AQP2 upregulation. The net result is a failure to appropriately reabsorb water from the filtrate leading
to dilution of the urine (AVP-arginine vasopressin, AQP2-aquaporin 2, cAMP-cyclic adenosine monophosphate, PKA-protein kinase A
CREB-cyclic adenosine monophosphate responsive element binding protein).
known regardingits role in pathophysiological changes

including apoptosis, inflammation and fibrosis (46).
Distal Tubular Acidification and Collecting Duct

Function
The addition of hydrogen ions to the filtrate is dependent on activity of the apical sodium coupled antiport,
particulary at the level of the distal tubule. As both the
expression of the antiport and general sodium reabsorptive mechanisms are impaired in the Obstructed
kidney, an associated alkalization of urine is observed
during and following release of obstruction (47).
The collecting duct normally receives hypotonic
filtrate from the distal tubule and acts to vary the
concentration of the final urine by modifying free
water reabsorption. This system is tightly controlled
by changes in baroreceptor and osmoreceptor firing in
response to changes in blood pressure and osmololality.
A decreased circulatory volume or increased plasma
osmolality excites magnocellular and parvocellular
neurosecretory cells of the hypothalamus to release
arginine vasopressin (AVP) into the circulation from
their axon terminals in the neuro-hypophysis. Interaction of AVP with V2 receptors in the collecting duct
leads to cyclic adenosine-monophosphate (cAMP) generation, protein kinase A activation and the stimulation
of both the expression and apical membrane localization
of aquaporin-2 (AQP2) water channels.
Experimental studies in rats have demonstrated

impairement of this axis in the collecting duct of
kidneys with obstruction (Fig. 4) (48).
A further impairement in collecting duct function
in UUO centers on the failure to correctly reabsorb
urea from the filtrate. Reabsorption of urea is reliant on
appropriate expression of A and B type urea transporters. Expression of these transporters is downregulated in the obstructed kidney (49). Impaired urea
reabsorption has an impact on interstitial osmolality,
as urea accounts for 50% of interstitial osmolality. The
net result is to collapse the corticomedullary gradient
required for correct functioning of the countercurrent
multiplier.
The above description of changes in renal sodium, urea, and water handling explain how sustained
urinary pooling can occur during obstruction in spite
of such marked reductions in GFR and increases in
lymphatic drainage of urine.
POSTOBSTRUCTIVE RENAL FUNCTION

Predicting Functional Recovery
Degree and duration of obstruction are clearly parameters that can affect recoverability of ipsilateral renal
function in UUO, both of which are factors that are
difficult to control for outwith the experimental field.
Recuperation of GFR post obstruction has been noted
110
to bear an inverse relationship to the length of prior

obstruction in rats (50).
In an attempt to increase the ability to predict the
outcome of obstruction in the clinical setting, a recent
prospective study was made of 91 adult patients with
UUO and a normally functioning contralateral kidney
(51). This demonstrated that on multivariate analysis,
only preoperative renographic clearance and perfusion of the obstructed kidney predicted recoverability
of function. Kidneys with a renographic GFR of less
than 10 mL/min/1.73 m2 were subsequently found to
be irreversibly damaged.
Postobstructive Tubular Function

The postobstructed kidney initially produces hypotonic urine. However, this is generally insignificant in
individuals with intact contralateral function and only
becomes clinically relevant in cases of BUO or solitary
kidney UUO.
The recovery of a normal urinary osmolality and
pH has been shown to take up to one month to stabilize
in rats subjected to a reversible UUO of 24 hours
duration reflecting the time required to reestablish
correct epithelial polarity and vectorial transport (52).
Recovery of renal tubular structure concomitant
with reversal has been observed in adult mice following the release of UUO of 10 days duration (53).
Effect of Nephrogenic Stage in Neonatal

Obstruction
Completion of nephrogenesis is delayed postnatally in
the rat, it serves as a good model to compare recovery
following transient complete neonatal obstruction at
different time points versus temporary and complete
obstruction in adult animals. Study of animals three
months subsequent to unilateral obstruction during the
early postnatal phase (days 15, 10% of nephrons
formed) revealed a relatively attenuated injury versus
the results obtained in animals obstructed at two weeks
of age when nephron number has peaked (54). Such
studies may offer important insights into debate regarding the best time to operate on the obstructed neonate.
Evidence of Long-Term Renal Injury in UUO

Despite acute recovery of renal function in terms of
the measurable GFR and renal blood flow, recent
studies in rats indicate that complete obstruction of
three days duration causes a progressive histological
renal injury following relief (55). The authors in this
study equate this progression to the establishment of
an irreversible fibrotic lesion in the kidney.
EMERGING THERAPIES IN OBSTRUCTIVE

NEPHROPATHY
The model of UUO in rodents is extensively used by
those with a broader interest in the prevention of renal
injury both in terms of the acute tubular apoptosis and
chronic fibrotic progression. Administration and/or
neutralization of a number of growth factors and
cytokines show a beneficial effect in reducing renal

injury intraobstructively (13). Chief among the growth
factors which have been shown to have a positive
effect intraobstructively in vivo are the administration
of bone-morphogenic protein-7 (BMP-7), an epithelial
growth factor downregulated in renal injury, epidermal growth factor (EGF) therapy, which provides an
antiapoptotic proproliferatory stimulus to the renal
epithelium, and hepatocyte growth factor (HGF)
administration, which has been shown to work chiefly
through the inhibition of TGF-b1 signaling (5658).
Separately, neutralizing anti-TGF-b1 antibody has
been demonstrated to ameliorate tubular apoptosis
following UUO in rats (59).
While the results of these studies are both interesting and useful, they are limited by two factors.
Firstly, many of the demonstrations of efficacy are
based on prophylactic deployment at the initiation of
obstruction, a scenario irreplicable clinically and secondly, the use of recombinant proteins may in many
cases prove economically unviable.
A number of studies have centered on the effectiveness of established pharmaceutical agents on injury in UUO. Angiotensin type 1 receptor antagonists
and angiotensin converting enzyme inhibitors are
particularly attractive agents given the involvement
of the RAS in all aspects of renal injury following
obstruction (13). These have been shown to particularly
effective in preventing the glomerular vasoconstriction
in UUO. Enalapril and Losartan have also been shown
to prevent fibrosis following UUO in rats (60). This
effect has been related to preservation of renal nitric
oxide synthesis (61). In the same article, provision of
the nitric oxide substrate L-arginine had a similar
protective effect.
One recent study used the generic, tested and
inexpensive anti-inflammatory sulfasalazine as an
anti-inflammatory strategy implemented three days
after induction of UUO in rats (62). Treated animals
showed much reduced indices of renal injury in terms
of inflammation and fibrosis. Results with this
delayed approach to therapy have been replicated in
the case of HGF in adult obstruction in rats and EGF
in neonatal obstruction in mice (63,64).
The HMG-CoA reductase inhibitors simvastatin,
fluvastatin and pravastatin have also shown promising antioxidative, anti-inflammatory and antifibrotic
effects in UUO in rodents (6567).
However, particular clinical relevance should
be attached to studies in which therapies have been
initiated in the postobstructive phase. A good example is a study in which provision of the nitric oxide
synthase substrate L-arginine ad libitum to rats after
the relief of a three-day UUO, significantly reduced
tubular apoptosis, macrophage infiltration and interstitial fibrosis in the absence of any beneficial effect
on GFR and renal plasma flow (68).
CONCLUSIONS
Upper urinary tract obstruction can occur secondary to
a diverse range of aetiologies. Primary considerations
are the type of obstruction, its location and the

relevant medical and surgical approaches required
to relieve obstruction. Obstructive nephropathy as a
consequence of upper urinary tract obstruction as a
pathology of particular importance in urology. This
chapter has placed an emphasis on describing how
upper urinary tract obstruction translates into
changes in ureteric function and renal damage via
the key primary stimulus of retrograde pressure
transfer and mechanical stretch. The subsequent
changes in renal haemodynamics and establishment
of renal inflammation and fibrosis lead us to the
characteristic features of obstructive nephropathy.
The wealth of information in the literature regarding
the pathophysiology of obstructive nephropathy and
its treatment, combined with advances in the relief of
obstruction, (particularly medical expulsive therapy)
hold promise for the future minimization of obstruction induced renal injury.
REFERENCES
1. Shi Y, Pedersen M, Li C, et al. Early release of neonatal
ureteral obstruction preserves renal function. Am J Physiol
2004; 286(6):F1087F1099.
2. Coe FL, Evan A, Worcester E. Kidney stone disease. J Clin
Invest 2005; 115(10):25982608.
3. Galvin DJ, Pearle MS. The contemporary management of
renal and ureteric calculi. BJU Int 2006; 98(6):12831288.
4. Dellabella M, Milanese G, Muzzonigro G. Medicalexpulsive therapy for distal ureterolithiasis: randomized
prospective study on role of corticosteroids used in combination with tamsulosin-simplified treatment regimen and
health-related quality of life. Urology 2005; 66(4):712715.
5. Nakada SY. Tamsulosin: ureteric motility. BJU Int 2008;
101(9):10611062.
6. Itoh Y, Kojima Y, Yasui T, et al. Examination of alpha
1 adrenoceptor subtypes in the human ureter. Int J Urol
2007; 14(8):749753.
7. Lang RJ, Davidson ME, Exintaris B. Pyeloureteral motility
and ureteral peristalsis: essential role of sensory nerves
and endogenous prostaglandins. Exp Physiol 2002;
87(2):129146.
8. Lang RJ, Tonta MA, Zoltkowski BZ, et al. Pyeloureteric
peristalsis: role of a typical smooth muscle cells and
interstitial cells of Cajal-like cells as pacemakers. J Physiol
2006; 576(pt 3):695705.
9. Thulesius O, Angelo-Khattar M, Sabha M. The effect of
ureteral distension on peristalsis. Studies on human and
sheep ureters. Urol Res 1989; 17(6):385388.
10. Lennon GM, Ryan PC, Fitzpatrick JM. Recovery of ureteric
motility following complete and partial ureteric obstruction. Br J Urol 1993; 72(5 pt 2):702707.
11. Lennon GM, Ryan PC, Fitzpatrick JM. The ureter in vitro:
normal motility and response to urinary pathogens. Br J
Urol 1993; 72(3):284290.
12. Kawada N, Moriyama T, Ando A, et al. Increased oxidative
stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int 1999; 56(3):10041013.
13. Docherty NG, OSullivan OE, Healy DA, et al. Evidence
that inhibition of tubular cell apoptosis protects against
renal damage and development of fibrosis following ureteric obstruction. Am J Physiol 2006; 290(1):F4F13.
14. Paulson DF, Fraley EE. Compensatory renal growth after
unilateral ureteral obstruction. Kidney Int 1973; 4(1):2227.
111
15. Boberg U, Wahlberg J, Persson AE. Tubuloglomerular

feedback response in the contralateral kidney after
24-hour unilateral ureteral obstruction. Ups J Med Sci
1985; 90(2):193199.
16. Michaelson G. Percutaneous puncture of the renal pelvis,
intrapelvic pressure and the concentrating capacity of the
kidney in hydronephrosis. Acta Med Scand Suppl 1974;
559:126.
17. Kopp UC, Cicha MZ. PGE2 increases substance P release
from renal pelvic sensory nerves via activation of N-type
calcium channels. Am J Physiol 1999; 276(5 pt 2):
R1241R1248.
18. Ma MC, Huang HS, Chen CF. Impaired renal sensory
responses after unilateral ureteral obstruction in the rat.
J Am Soc Nephrol 2002; 13(4):10081016.
19. Gaudio KM, Siegel NJ, Hayslett JP, et al. Renal perfusion
and intratubular pressure during ureteral occlusion in the
rat. Am J Physiol 1980; 238(3):F205F209.
20. Whinnery MA, Shaw JO, Beck N. Thromboxane B2 and
prostaglandin E2 in the rat kidney with unilateral ureteral
obstruction. Am J Physiol 1982; 242(3):F220F225.
21. Hegarty NJ, Young LS, ONeill AJ, et al. Endothelin in
unilateral ureteral obstruction: vascular and cellular
effects. J Urol 2003; 169(2):740744.
22. Yarger WE, Griffith LD. Intrarenal hemodynamics following chronic unilateral ureteral obstruction in the dog. Am J
Physiol 1974; 227(4):816826.
23. Hsu CH, Kurtz TW, Rosenzweig J, et al. Intrarenal hemodynamics and ureteral pressure during ureteral obstruction. Invest Urol 1977; 14(6):442445.
24. Kinn AC, Bohman SO. Renal structural and functional
changes after unilateral ureteral obstruction in rabbits.
Scand J Urol Nephrol 1983; 17(2):223234.
25. Naber KG, Madsen PO. Renal function during acute total
ureteral occlusion and the role of the lymphatics: an
experimental study in dogs. J Urol 1973; 109(3):330338.
26. Quinlan MR, Docherty NG, Watson RW, et al. Exploring
mechanisms involved in renal tubular sensing of mechanical stretch following ureteric obstruction. Am J Physiol
Renal Physiol 2008; 295(1):F1F11.
27. Kaneto H, Ohtani H, Fukuzaki A, et al. Increased expression of TGF-beta1 but not of its receptors contributes to
human obstructive nephropathy. Kidney Int 1999; 56(6):
21372146.
28. Sato M, Muragaki Y, Saika S, et al. Targeted disruption of
TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest 2003; 112(10):14861494.
29. Pytela R, Pierschbacher MD, Ruoslahti E. Identification
and isolation of a 140 kd cell surface glycoprotein with
properties expected of a fibronectin receptor. Cell 1985;
40(1):191198.
30. Matthews BD, Overby DR, Mannix R, et al. Cellular
adaptation to mechanical stress: role of integrins, Rho,
cytoskeletal tension and mechanosensitive ion channels.
J Cell Sci 2006; 119(pt 3):508518.
31. Glogauer M, Arora P, Chou D, et al. The role of actinbinding protein 280 in integrin-dependent mechanoprotection. J Biol Chem 1998; 273(3):16891698.
32. Goel M, Sinkins WG, Zuo CD, et al. Identification and
localization of TRPC channels in the rat kidney. Am J
Physiol 2006; 290(5):F1241F1252.
33. Maroto R, Raso A, Wood TG, et al. TRPC1 forms the
stretch-activated cation channel in vertebrate cells. Nat
Cell Biol 2005; 7(2):179185.
34. Raychowdhury MK, McLaughlin M, Ramos AJ, et al.
Characterization of single channel currents from primary
cilia of renal epithelial cells. J Biol Chem 2005; 280(41):
3471834722.
112
35. Du Z, Yan Q, Duan Y, et al. Axial flow modulates proximal

tubule NHE3 and H-ATPase activities by changing microvillus bending moments. Am J Physiol 2006; 290(2):F289F296.
36. Sunami R, Sugiyama H, Wang DH, et al. Acatalasemia
sensitizes renal tubular epithelial cells to apoptosis and
exacerbates renal fibrosis after unilateral ureteral obstruction. Am J Physiol 2004; 286(6):F1030F1038.
37. Zhang G, Oldroyd SD, Huang LH, et al. Role of apoptosis
and Bcl-2/Bax in the development of tubulointerstitial
fibrosis during experimental obstructive nephropathy.
Exp Nephrol 2001; 9(2):7180.
38. Ricardo SD, Levinson ME, DeJoseph MR, et al. Expression
of adhesion molecules in rat renal cortex during experimental hydronephrosis. Kidney Int 1996; 50(6):20022010.
39. Higgins DF, Kimura K, Bernhardt WM, et al. Hypoxia
promotes fibrogenesis in vivo via HIF-1 stimulation of
epithelial-to-mesenchymal transition. J Clin Invest 2007;
117(12):38103820.
40. Crisman JM, Richards LL, Valach DP, et al. Chemokine
expression in the obstructed kidney. Exp Nephrol 2001;
9(4):241248.
41. Schreiner GF, Harris KP, Purkerson ML, et al. Immunological aspects of acute ureteral obstruction: immune cell
infiltrate in the kidney. Kidney Int 1988; 34(4):487493.
42. Diamond JR, Kees-Folts D, Ding G, et al. Macrophages,
monocyte chemoattractant peptide-1, and TGF-beta 1 in
experimental hydronephrosis. Am J Physiol 1994; 266(6 pt 2):
F926F933.
43. Kim SW, Lee J, Jung K, et al. Diminished expression of
sodium transporters in the ureteral obstructed kidney in
rats. Nephron Exp Nephrol 2004; 96(3):e67e76.
44. Buerkert J, Martin D, Head M. Effect of acute ureteral
obstruction on terminal collecting duct function in the
weanling rat. Am J Physiol 1979; 236(3):F260F267.
45. Li C, Wang W, Kwon TH, et al. Altered expression of major
renal Na transporters in rats with unilateral ureteral
46. Chevalier RL, Thornhill BA, Wolstenholme JT. Renal cellular response to ureteral obstruction: role of maturation and
angiotensin II. Am J Physiol 1999; 277(1 pt 2):F41F47.
47. Ribeiro C, Suki WN. Acidification in the medullary collecting duct following ureteral obstruction. Kidney Int 1986;
29(6):11671171.
48. Frokiaer J, Christensen BM, Marples D, et al. Downregulation of aquaporin-2 parallels changes in renal water
excretion in unilateral ureteral obstruction. Am J Physiol
1997; 273(2 pt 2):F213F223.
49. Li C, Klein JD, Wang W, et al. Altered expression of urea
transporters in response to ureteral obstruction. Am J
Physiol 2004; 286(6):F1154F1162.
50. Harris RH, Yarger WE. Renal function after release of
unilateral ureteral obstruction in rats. Am J Physiol 1974;
227(4):806815.
51. Khalaf IM, Shokeir AA, El-Gyoushi FI, et al. Recoverability
of renal function after treatment of adult patients with
unilateral obstructive uropathy and normal contralateral
kidney: a prospective study. Urology 2004; 64(4):664668.
52. Valles P, Merlo V, Beron W, et al. Recovery of distal
nephron enzyme activity after release of unilateral ureteral
obstruction. J Urol 1999; 161(2):641648.
53. Cochrane AL, Kett MM, Samuel CS, et al. Renal structural
and functional repair in a mouse model of reversal of
ureteral obstruction. J Am Soc Nephrol 2005; 16(12):
36233630.
54. Chevalier RL, Thornhill BA, Chang AY, et al. Recovery
from release of ureteral obstruction in the rat: relationship
to nephrogenesis. Kidney Int 2002; 61(6):20332043.
55. Ito K, Chen J, El Chaar M, et al. Renal damage progresses
despite improvement of renal function after relief of unilateral ureteral obstruction in adult rats. Am J Physiol 2004;
287(6):F1283F1293.
56. Mizuno S, Matsumoto K, Nakamura T. Hepatocyte
growth factor suppresses interstitial fibrosis in a mouse
model of obstructive nephropathy. Kidney Int 2001;
59(4):13041314.
57. Hruska KA, Guo G, Wozniak M, et al. Osteogenic protein-1
prevents renal fibrogenesis associated with ureteral
58. Kennedy WA 2nd, Buttyan R, Garcia-Montes E, et al.
Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction. Urology 1997; 49(6):
973980.
59. Miyajima A, Chen J, Lawrence C, et al. Antibody to transforming growth factor-beta ameliorates tubular apoptosis
in unilateral ureteral obstruction. Kidney Int 2000; 58(6):
23012313.
60. Jones EA, Shahed A, Shoskes DA. Modulation of apoptotic
and inflammatory genes by bioflavonoids and angiotensin
II inhibition in ureteral obstruction. Urology 2000;
56(2):346351.
61. Morrissey JJ, Ishidoya S, McCracken R, et al. Nitric oxide
generation ameliorates the tubulointerstitial fibrosis of
obstructive nephropathy. J Am Soc Nephrol 1996;
7(10):22022212.
62. Demirbilek S, Emre MH, Aydin EN, et al. Sulfasalazine
reduces inflammatory renal injury in unilateral ureteral
obstruction. Pediatr Nephrol 2007; 22(6):804812.
63. Yang J, Liu Y. Delayed administration of hepatocyte growth
factor reduces renal fibrosis in obstructive nephropathy. Am
J Physiol 2003; 284(2):F349F357.
64. Chevalier RL, Goyal S, Thornhill BA. EGF improves recovery following relief of unilateral ureteral obstruction in the
neonatal rat. J Urol 1999; 162(4):15321536.
65. Vieira JM Jr, Mantovani E, Rodrigues LT, et al. Simvastatin
attenuates renal inflammation, tubular transdifferentiation
and interstitial fibrosis in rats with unilateral ureteral
obstruction. Nephrol Dial Transplant 2005; 20(8):15821591.
66. Mizuguchi Y, Miyajima A, Kosaka T, et al. Atorvastatin
ameliorates renal tissue damage in unilateral ureteral
obstruction. J Urol 2004; 172(6 pt 1):24562459.
67. Moriyama T, Kawada N, Nagatoya K, et al. Fluvastatin
suppresses oxidative stress and fibrosis in the interstitium
of mouse kidneys with unilateral ureteral obstruction.
Kidney Int 2001; 59(6):20952103.
68. Ito K, Chen J, Seshan SV, et al. Dietary arginine supplementation attenuates renal damage after relief of unilateral
ureteral obstruction in rats. Kidney Int 2005; 68(2):515528.
8
Interactive Obstructive Uropathy: Observations and
Conclusions from Studies on Humans
Department of Urology, Withington Hospital, University Hospitals of South Manchester,
Manchester, U.K.
INTRODUCTION
It has been known for many years that dysfunctional
abnormalities of the lower urinary tract may affect the
performance of the upper urinary tract in several
respects. Typical examples of such bladder dysfunction include those associated with benign prostatic
hypertrophy and the changes that are observed after
neurologic damage to the spinal cordthe neuropathic bladder. It is recognized that the renal damage
caused by such interaction between the lower and
upper tract may be severe, silent, and progressive,
leading to terminal renal failure if the abnormalities
are not recognized and corrected.
In this chapter, the basis of our physiologic
understanding of the mechanisms involved in the
interactive urinary tract dysfunctional states will be
explored. Animal studies of such abnormalities have
been few, partially because of the difficulties with
complex animal experimentation and partly because
natural models of lower/upper tract dysfunction do
not exist. This account therefore deals with human
subjects found to have a particular form of bladder
dysfunction (high-pressure chronic retention) that is
particularly appropriate to demonstrate the physiologic changes that occur synchronously within the
lower and upper tract. Naturally, all subjects gave
informed consent for the procedures, which, being
undertaken without any form of anesthesia, benefited additionally from the ability of the patient to
speak and comment throughout on the test procedures. Before I describe the observations and discuss
the conclusions of these studies, it is pertinent to
review the historical perspective relating to interactive dysfunction; such an appreciation explains previous misunderstandings and lays a more secure
foundation for a rational understanding of the interactive disorder.
HISTORICAL PERSPECTIVE
In the 1840s dissections by Guthrie in Britain and
Civiale in France identified a number of abnormalities
in the region of the bladder neck (Fig. 1). Clearly
visible in many cases were large lateral lobes of the

prostate with a bladder showing trabeculation and
sacculation. However, in a number of cases, there was
equivalent detrusor hypertrophy, trabeculation, etc.,
but little to be seen in the way of prostatic hypertrophy, a thickened median bar at the bladder neck being
the only possible source of obstruction (Fig. 1B). A
short while later, again in Paris at the Necker hospital,
Professor Guyon described a third type of dysfunctional bladder that was essentially thin-walled yet still
associated with typical prostatic symptoms. He
called this entity prostatisme vesicale. At the time,
the relationship between these three apparently discrete forms of bladder dysfunction was far from clear,
and the situation was not assisted by the very high
prevalence of lower urinary tract stones and infection
(urgency, frequency, and incontinence) in both
middle-aged and older men.
A hypothesis was however formed, which in the
next 70 to 100 years became known as the three-stage
theory of prostatism (Fig. 2). In this theory, the
bladder first becomes trabeculated and hypertrophied
because of outflow tract obstruction, usually as a
result of an easily palpable and enlarged prostate.
As obstruction develops, sacculation and diverticulum formation take place, and with increased pressure the ureters and subsequently upper tracts dilate,
leading to hydronephrosis and eventual renal failure.
In the third phase of the classically described course of
events, the bladder becomes decompensatedflaccid,
large, and overdistended. Overflow incontinence was
said to occur.
The three-stage theory of prostatism was widely
accepted in the latter part of the 19th century and first
half of the 20th century, and typical accounts are to be
found in urologic texts published as late as the 1980s.
Examination of the theory, however, revealed inconsistencies, which were impossible to explain on a
rational basis. In particular, it was not clear why a
detrusor muscle, which in stage two had developed
hypertrophy, trabeculation, and sacculation, should
suddenly give way into a thin-walled atonic bag
associated with overflow incontinence and upper
tract hydronephrosis.
114
George
Figure 1 Drawings of original dissections by early investigators.
Figure 2 The classically described three-stage theory

of prostatism.
In fact, an explanation of this dilemma had

been offered by some careful observational studies
undertaken after World War II. In 1948, Badenoch
studied 26 patients with bladder neck obstruction
(Fig. 1B) whose mean age was 43, 16 of the patients
being under 50 years. He noted no prostatic enlargement in these cases but made the highly significant
observation that 25 of the 26 cases had diverticulum
formation and bilateral hydroureter with hydronephrosis in the majority. In 1951, Wallace complemented this study. He looked at the association
between lateral lobe enlargement, median lobe
enlargement, and normal and dilated upper tracts.
He found that 89% of patients with large prostatic
lateral lobes had no upper tract dilatation, but 72%
of patients with bladder neck hypertrophy alone
demonstrated clear hydronephrosis with hydroureter.
These studies showed clearly that while upper
tract dilatation was indeed related to obstruction, this
relatively rarely involved the expected prostatic
hypertrophy but was much more strongly correlated
with pure bladder neck obstruction. These and other
studies eventually led to the conclusion that the
unifying three-stage theory of prostatism was unlikely to be correct, and that individual or discrete
disorders of the lower urinary tract (i.e., bladder
neck obstruction or primary atonic/thin-walled bladder) offered a more plausible explanation for the
observed clinical symptom complexes. The particular
group of patients with upper tract dilatation associated with bladder neck hypertrophy originally
described and clarified by Badenoch were subsequently investigated in greater detail, and the advent
of sophisticated urodynamic measuring equipment
enabled precise recordings of this dysfunctional
statehigh-pressure chronic retentionto be made
for the first time. Simultaneous advances in uroradiology and nephrostomy placement, in particular,
finally made possible for sophisticated simultaneous
urodynamic and radiologic studies of both upper
and lower urinary tracts. This chapter, therefore,
describes and illustrates the physiologic interactive
changes in both lower and upper tract, using records
obtained by the author and colleagues from such
studies on patients with uncomplicated (sterile
urine) high-pressure chronic retention.
Chapter 8: Interactive Obstructive Uropathy
115
Figure 3 Volumes and events during the micturition cycle.
THE MICTURITION CYCLE

It is important to consider the bladder in terms of the
micturition cycle. Traditionally, the terms obstruction and blockage have been associated with
difficulty in passing urine, and the idea of blockage
has somehow become extended to the upper tracts
rather as in the three-stage theory of prostatism.
However, a moments consideration of the micturition cycle (Fig. 3) shows that the bladder spends very
little time indeed on micturition and that the vast
percentage of its functional existence is spent in
storage mode. A normal 70-kg adult will micturate
four times in 24 hours passing approximately 1500
mL of urine. Assuming that each micturition takes
approximately 1 minute to complete, it is clear that
the bladder is contracting for only 0.3% of 24 hours;
more emphatically, the bladder is in storage phase
for 23 hours and 56 minutes of the day. Even in a case
severe enough to cause the patient to spend five
minutes micturating every hour, the bladder still
spends 92% of its time in the filling phase.
These simple calculations show that if there is to
be any effect of the lower tract on the upper tract, any
abnormality thought to be responsible must act chiefly
during the filling phase. An abnormality during the
micturition phasehowever severewould not have
sufficient time to act and lead to permanent change in
the ureters or renal pelvis. Hence, the state of the
bladder during filling is of critical importance to a
concept of interactive urinary tract dysfunction, and in
this respect the mechanical and physical properties of
the bladder wall (bladder compliance) require
detailed consideration.
Figure 4 Endoscopic appearance in high-pressure chronic

retention.
THE BLADDER WALL IN HIGH-PRESSURE

RETENTION
The bladder wall in patients with high-pressure
chronic retention is characteristically thick with massive detrusor hypertrophy. Endoscopically, bars of
trabecular muscle stand out, often described as a
cathedral roof appearance (Fig. 4). Histologic examination and analysis of sections taken from such trabecular bars (Fig. 5A, B) show degeneration of the
Figure 5 Histologic appearances of detrusor muscle in highpressure chronic retention. Muscle hypertrophy (A) may degenerate (B) with collagen infiltration (green stain).
116
George
ureter, as seen in Figure 7, with a wire probe emerging

from the ureteric orifice.
EFFECT OF BLADDER WALL HYPERTROPHY
Figure 6 Ureteric passage through thickened detrusor wall.

Normal (right ), loss of peristalsis (center ), and increasing wall
thickness after micturition (left ).
As might be expected, the increase in detrusor muscle

mass together with the collagen infiltration illustrated
above fundamentally alters the urodynamic characteristics of the bladder as a storage organ. Normal
bladders with normal compliance exhibit little or no
intrinsic pressure rise during filling to capacity. By
contrast, in high-pressure chronic retention, intrinsic
detrusor pressure (related to abnormal sensation as
discussed below) remains within the bladder after
micturition has been completed, and accurate recording of this end void pressure is critical with regard
to the analysis of associated renal dysfunction. It is not
known at the present time (and it is extremely difficult
to determine by experimental means) whether the
upper tract dilatation develops as a result of the
bladder wall hypertrophy illustrated in Figure 6 or
as a result of the intrinsic detrusor postvoid pressure
remaining within the bladder after micturition. This is
perhaps a slightly academic point, as regardless of
whether pressure precedes hypertrophy or vice versa,
the end resultprogressive upper tract dilatation
remains the same.
MEASUREMENT OF END VOID PRESSURE
Figure 7 Whole mount bladder from case of high-pressure

retention. Note the wire in the ureteric orifice (arrow ).
smooth muscle bundles into collagen, as illustrated

with the Masson trichrome stain. It is not difficult to
imagine that the course of the ureter to the vesicoureteric junction (VUJ) through the wall of such a bladder
may well become obstructed. The normal coapting
process of ureteric peristalsis may be lost (Fig. 6, top);
indeed, obstruction at the VUJ may become worse as
the bladder empties (Fig. 6, bottom left). The situation
is well illustrated by occasional whole mounts of such
bladders (Fig. 7). Massive trabeculation and hypertrophy may well cause intramural obstruction to the
As noted above, the accurate recording of end void

pressure is an important measurement, which correlates with renal dysfunction. Figure 8 illustrates such
a measurement taking place. The typical patient
with high-pressure retention (painless protuberant
bladder) micturates in an entirely normal fashion in
private and without any Valsalva maneuver (artificial
pushing). Following the void, the patient lies on the
table, and under local anesthetic a small needle is
placed suprapubically through the abdominal and
bladder wall. Fluid rises up the tube as illustrated,
clearly demonstrating the end void intrinsic detrusor
pressure remaining within the bladderthis can easily be measured with reference to the pubic bone, as
is standard practice, and is responsible for the
high pressure name applied to this form of chronic
retention.
This simple procedure is important because it is
undertaken during the patients natural state without
stimulation of urethral or sphincteric sensory receptors. On occasion, investigators have attempted to
obtain such measures following conventional urodynamic investigation of the bladder, during which the
organ, after drainage of the residual, has almost
invariably been filled at supranormal rates. Such
abnormal filling rates significantly disrupt the functional performance of high-pressure bladders, and
false recordings are obtained (see below). The measurement of end void pressure as described above is
the simplest and the best method of obtaining the
critical pressure that relates to renal dysfunction.
117
Figure 8 Direct measurement by suprapubic puncture of intrinsic detrusor pressure after void.
SUMMARY OF URODYNAMIC DATA RELATING

TO BLADDERS WITH HIGH-PRESSURE
RETENTION
Figure 9A illustrates in cartoon format the important
concept of the pressure cycle that occurs within the
bladder of patients with high-pressure chronic retention.
Following micturition (EVP, end void point), the
pressure within the bladder rises as filling takes
place from the upper tracts. Eventually, the end fill
pressure (EFP) is reached, at which point the patient is
experiencing the normal desire to void. A detrusor
contraction takes place (over and above the intrinsic
pressure within the bladder), and urine is expelled in
the usual fashion. At the end of micturition, the
pressure returns once again to the EVP, thus completing the pressure loop.
It can thus be seen that in the state of highpressure chronic retention the bladder fills and empties in cyclical fashion at an abnormally raised pressure throughout the 24 hours. Micturition may occur
with apparent normality, but at no time are the upper
tracts able freely to empty into the bladder storage
organ. Thus, it might be predicted that within the
upper tracts a similar cycle might be observed
reduced pressure when urine was able to drain easily
into the bladder but rising pressure as the bladder
filled and passage of urine distally through the vesicoureteric junction became more difficult. This
hypothesis is tested by the experiment described
below.
Patients with high-pressure retention never drain
their bladder below the EVP unless urine is removed
artificially. Evidence from cases observed at intervals
(years) before initiation of treatment suggest that the
EVP gradually rises with time, and as the range of
pressure within the bladder cyclesfill/void/fill/void,
etc., slowly elevates it becomes progressively more

difficult for the ureter to empty into the storage organ.
This change is initially observed radiologically as a
snakes head at the lower end followed by a gradually filling hydroureter to a full hydroureteronephrosis
(Fig. 12A) when the intrinsic detrusor pressure at EVP
becomes significantly elevated. If a catheter is placed in
the bladder and urine drained (Fig. 9A, dashed line),
pressure will drop toward zero, and, of course, the
suprapubic mass will disappear. As already noted,
subsequent measurements taken under these circumstances are highly misleading and do not give useful
prognostic information regarding renal function.
Fill-Void Observations
The broad categories of filling phase abnormality that
may be seen in the human bladder are noted in
Figure 9B. Patients with normal (normal compliance)
inflow phases have a pressure rise to the physiologic
bladder capacity (approximately 500 mL) of less than
5 cm water. Cases of poor compliance are those in
which the bladder pressure rises to a greater degree
during filling. This mechanical process can be easily
understood when the bladder wall has been replaced
by fibrous tissue such as occurs in interstitial cystitis
or tuberculosis, or after radiation therapy. By contrast, in high-pressure retention, the reduced compliance during filling is related to a pathophysiologic
abnormality of smooth muscledetrusor hypertrophy and associated collagen formation, as discussed
earlier. In any one case the proportion of compliance
loss because of muscle hypertrophy (ameliorated to a
degree by stress relaxation) or collagen (essentially
unable to relax) cannot be known with certainty;
however, there is little doubt that those bladders
118
George
Figure 9 (A) Pressure-volume relationship in high-pressure

chronic retention. (B) Types of abnormal bladder compliance.
Abbreviations: EFP, end fill pressure; EVP, end void pressure;
RU, residual urine.
with a higher proportion of collagen replacement are

more injurious to the upper tract and renal function,
as is seen in the significant therapeutic challenge
offered by the pediatric valve bladder.
By definition, storage organs with abnormal
compliance will be variably sensitive to filling rates,
whether by ureter or urodynamic catheter. Figure 10
panels A and B are from an ambulatory (natural fill)
study on a 61-year-old man with relatively early
HPCR visualized radiologically as retention with
snakes head lower ureters but minimal hydronephrosis. By comparison, a medium-fill (60 mL/min)
videocystometric inflow study is shown from a
patient with a similar condition (Fig. 10C). In the latter
the familiar smooth curve (to 45 cm H2O) of poor
compliance is seen, whereas under conditions of ureteric filling (Fig. 10A) such traces are in reality shown
to be made up of a series of contractions gradually
rising from EVP 9 cm H2O to maximum 46 cm H2O
prior to the void. The patient remains completely
unaware of filling until, in this case, a sensory threshold
of about 35 cm H2O is breached. The filling rate is thus

responsible for the appearance of the urodynamic
trace.
The reason for the development of the highpressure characteristic remains unknown. Undoubtedly, this detrusor reaction to obstruction may occur
with any form of distal urethral lesion, such as dense
phimosis, urethral stricture or, in children, urethral
valves (the final common pathway). Such pathologic
or embryologic abnormalies (or iatrogenic damage
such as catheter damage to the urethra) carry a
significant risk of silent and irreversible renal damage.
By contrast, obstruction that involves the prostate
rarely seems to lead to the high-pressure filling type
of change (as noted in the historical series). The
typical prostate (lateral lobe) obstruction characterized by high-pressure/low-flow voiding seldom
develops filling phase abnormalities other than phasic
overactive waves against a background of near-normal
compliance (Fig. 9B). This may perhaps be related to
the irritation caused by prostatic obstruction, such as
detrusor overactivity protecting against the development of painless retention.
Abnormal sensation appears to be the critical
factor in the development of this condition. Urethral
sensation, as judged during catheterization, remains
normal to touch, thermal stimuli, and pain as classically described and illustrated. Likewise, sensation
from periurethral and external urethral sphincter
regions appears normal as a catheter passes into the
bladder. The sensation of bladder filling, however, is
lost until some point toward the end of filling when
gradually the patient becomes aware of the
impending need to void. At the EFP a perfectly
synergic voiding episode takes place with detrusor
contraction and synchronous urethral relaxation, no
Valsalva maneuver being required (Fig. 10B). Subsequently, all sensation is lost as the pressure settles
back to the EVP prior to the start of a new cycle.
Whether this loss relates to receptor failure, pelvic
nerve dysfunction, or spinal long tract disorder
remains unknown at the present time.
RADIOLOGIC ASSESSMENT OF HIGHPRESSURE BLADDERS

Reflux cystograms (Fig. 11) show, as might be
expected, a trabeculated, sacculated picture entirely
consistent with the cystoscopic appearance. It might
also be expected that reflux would not be seen when
one considers the oblique path of the ureter through
the massively hypertrophied bladder wall (Fig. 7).
Naturally, for the purpose of these human studies
into interactive dysfunction, all patients received cystograms to exclude the possibility of reflux, which
would be expected to lead to unrelated and entirely
different changes within the upper tracts. In the
patients studied, urine was always sterile and reflux
never present.
Intravenous urography demonstrates the typical
picture of well-preserved renal parenchyma with general distension of the collecting system from the
119
Figure 10 HPCR. (A) Ambulatory (natural fill) record

of one cycle lasting 1 hour and 40minutes taken from
filling phase. (B) Ambulatory record from above
expanded to illustrate voiding characteristics. (C) Conventional medium fill (60mL/mn) urodynamics in HPCR
after bladder residual drained.
120
George
calyces to the vesicoureteric junction (Fig. 12). Figure

12A particularly well illustrates the typical snakes
head appearance of high-pressure retention at the
lower end of the left uretera near-certain sign of
raised intravesical pressure and a warning, if not
otherwise suspected, that renal function might be at
risk. Such appearances allow a relatively easy target
for a skilled nephrostomist, and the placement of
such a tube at last allows the synchronous measurement of upper and lower urinary tract pressures to be
contemplated.
INTERACTIVE PRESSURE RECORDINGS IN

PATIENTS WITH HIGH-PRESSURE CHRONIC
RETENTION AND HYDROURETER/
HYDRONEPHROSIS
Figure 11 Reflex cystogram in high-pressure retention, demonstrating competent vesicoureteric junction.
Figure 12 (A, B) Radiologic aspects of high-pressure retention.
Following careful screening to exclude default conditions such as prostate cancer or urinary tract infection, consenting patients underwent the investigation
protocol illustrated in Figure 13. A nephrostomy tube
was placed in the renal pelvis (via the renal substance
to avoid leakage) and connected to standard Whitaker
121
Figure 13 Experimental protocol for bladder and

renal pressure measurement.
pump/measurement apparatus. A suprapubic pressure line was inserted, and finally a urethral catheter
allowed bladder volume to be increased or decreased
at will. The patient was made warm and comfortable
on the investigation table, as the studies took an
extended period of time. Patients rarely experienced
any discomfort during the tests and commonly fell
asleep during measurements.
(Fig. 15), the bladder was filled at a rate of 60 mL/min

and the effect on bladder and pelvic pressure noted.
As expected from the pressurevolume characteristic
of high-pressure bladders, the bladder pressure rose
rapidly, but at the same time pelvic pressure was also
seen to rise, albeit much more slowly. When 100 mL
of water was removed from the bladder (Fig. 16),
States of Hydration
Three differing states of hydration were identified.
Baseline hydration determined that the patient was
comfortableenough water had been drunk to satisfy
thirst. Water drinking determined that patients were
asked to drink 1 to 1.5 L of water fairly rapidly, a load
well in excess of what they would normally ingest.
A third level of fluid loading was attained by administration of furosemide (frusemide) 0.5 mg/kg IV.
These three forms of hydration were used to frontend load the urinary tractthat is present differing
diuretic loads to the drainage system of the renal
mass.
In addition to fluid loads presented to the kidney, it was possible to vary the rate with which the
fluid passed through the vesicoureteric junction. This
was performed by filling and emptying the bladder
through the urethral catheter, the resultant pressure
being recorded by the suprapubic pressure line. As
will be evident from Figure 9, such artificial filling and
emptying of the bladder results in marked swings of
intrinsic detrusor pressure, which may be expected to
affect vesicoureteric transport.
Figure 14 Baseline recordings of renal pelvic (Pp) and bladder

(Pb) pressures.
Experimental Observations
The results of these experiments are illustrated in
Figures 14 to 20. During prolonged baseline recordings (Fig. 14), in which the patient was resting comfortably and simply hydrated, there was no
identifiable correlation or connection between bladder and pelvic pressure measurements. Occasional
pelvic pressure waves were seen, as were occasional
detrusor contractions (unstable waves), but these bore
no temporal relationship to each other. Subsequently
Figure 15 Subsequent pressure variations.
122
George
Figure 16 Effect of bladder pressure variation.
Figure 17 Further pressure variations with water loading and

an additional 90 mL in the bladder.
bladder pressure dropped rapidly, as again would be

expected, but pelvic pressure also dropped following
the slow initial rise, demonstrating that the artificial
cycling of bladder pressure (see above) had
induced a form of artificial cycling of the upper
tract pressure.
The cycling linkage continued but was more

precisely interlinked when oral water loading
increased the perfusion pressure at the proximal end
of the urinary tract (Fig. 17). Natural diuresis and
subsequent bladder drainage produced sharp swings
in bladder and pelvic pressure measurements,
although, as might be expected, bladder pressure
swings were more acute than those seen in the renal
pelvis.
Subsequently, under maximal front-end loading caused by furosemide IV, bladder pressure
cycling via the urethral catheter led to similar sharp
swings of pressure being recorded from the renal
pelvis. Occasionally, small reductions in pelvic pressure were observed that were not related to any
variation of bladder volume; these were thought to
represent periods of ureteric creep, during which
smooth muscle of the upper urinary tract dilated
marginally, thus reducing pressure and accommodating a greater volume. This is the mechanism thought
experimentally to account for the development of
hydroureter and hydronephrosis over the long term
(Fig. 19).
The interactive pressure experiments are summarized in Figure 20. During baseline periods, bladder and renal pressures are dissociated, and variation
of bladder pressure in particular has no effect on
upper tract distension. However, prolonged increase
of bladder pressure (unlikely to be tolerated by the
patient) or increased fluid loading/diuretic therapy (a
reasonably common occurrence) might well prime
the upper tracts, and thereafter pressures within the
pelvis may mimic precisely pressures within the bladder. It is very important to appreciate that this synchronous pressure state occurs in the absence of the
vesicoureteric reflux. The pressure measured within
the pelvis is not a reflection of bladder pressure
passing backward up the ureter; it is a pressure
required to be exerted by the upper tract if profusion
through the vesicoureteric junction is to occur.
It is proposed therefore that these experiments
give some insight into the development of hydronephrosis in the simple, uncomplicated (uninfected)
Figure 18 Coordinated pressure movements

within bladder and renal pelvis. Drainage volumes
not shown. C: Possible time of ureteric creep..
123
Figure 19 Schematic diagram of ureteric creepgradual

dilatation under stress.
Figure 21 Urographic changes in early high-pressure retention.
Figure 20 Summary of interactive pressure-volume effects.
case of high-pressure retention. It is now possible to

consider what might be the consequences of such
changes for renal function. By the same experimental
model, it is possible to examine both renographic and
biochemical aspects of renal function, and the technique and results of these studies will be briefly
described.
EXCRETORY FUNCTION MEASURED BY

GAMMA CAMERA RENOGRAPHY
It has already been noted that the lack of coapting
peristaltic waves would be likely fundamentally to
affect drainage function from the upper urinary tract.

The ureter, previously divided into segments,
becomes an open column of fluid as hydronephrosis
supervenes, and such an open column is capable of
exerting forces very dissimilar to those acting in the
normal state. It might be hypothesized, for example,
that such an open hydroureter would, under certain
critical circumstances, exert pressures on the vesicoureteric junction in the vertical position that
would not be present in the horizontal position.
Clearly, in this respect, the pressure on the other
side of the vesicoureteric junction (i.e., within the
bladder) would be critical, but there might be defined
circumstances where significant differences in drainage from the upper tracts would be seen when the
patient was in the erect rather than the horizontal
position.
Such patients can be identified, and one such is
illustrated in Figures 21 to 23. The IVP of this 41-yearold man is not, at first glance, overtly hydronephrotic,
but an open hydroureter can be seen on both sides,
most marked on the left, and the typical snakes head
at the lower end of the ureter typical of high-pressure
124
George
position. This simple study in a young man with early

high-pressure retention demonstrates conclusively
that drainage through the vesicoureteric junction can
be affected by postural conditions; loss of the normal
coapting mechanism might well be responsible for the
observations.
GRAVITATIONAL THEORY OF DRAINAGE
Figure 22 Erect and supine renographic images.
This gravitational theory of the open hydroureter may

be tested with the human high-pressure retention
model (Fig. 24). In these studies, men with sterile
high-pressure retention have a suprapubic line and
catheter inserted under local anesthetic. Thus, the
volume and hence pressure within the bladder may
be varied at will while the patient is seated or lying in
front of a gamma camera.
The results of various studies are depicted in
Figures 25 to 28. In the first experiment, a man underwent 123I gamma camera renography in the sitting
position. He was comfortably hydrated but had been
asked not to void prior to the examination. It was thus
envisaged that his bladder pressure would be relatively advanced up the pressurevolume curve illustrated in Figure 9A. Injection of the isotope was given,
and the curves elaborated are shown. At 25 minutes,
120 mL of urine was withdrawn through the suprapubic catheter, and the effect on renal clearance of the
isotope observed. The acute reduction in intravesical
volume and thus pressure allowed vesicoureteric
transport to take place, urine from the previously
Figure 23 Erect and supine renographic curves.
retention can be seen. The relatively small but highpressure bladder fails to opacify. 123I gamma camera
renography was performed in erect and supine positions under identical circumstances a few days apart.
Figure 22 shows accumulated frames between 10 and
15 minutes. The panel showing the erect study clearly
demonstrates upper tract drainage, particularly from
the left, and bladder filling, while the panel relating to
the supine study shows near-total stasis within the
renal pelvis and calyces and no image whatever of the
bladder. Graphical analysis (Fig. 23) shows equally
good uptake for either test but little excretion in the
supine position as compared with near-normal excretion (renal area of interest demonstrated) in the erect
Figure 24 Experimental protocol for drainage experiments.
Figure 25 Effect of bladder drainage.
obstructed kidney passing rapidly onward and down

the hydroureter. In Figure 26, the same experiment is
repeated, but the intravesical pressure is recorded on
the same time base as that for the renogram curves.
Under these conditions, little isotope escaped the renal
substance until the reduction of the bladder volume
once again caused a sharp drop of bladder pressure,
following which rapid reduction in isotope counts
from the kidney took place. These experiments demonstrate an important hydrodynamic aspect of highpressure retention. A vertical line drawn at the
moment isotope counts begin to reduce (urine leaving
the kidney) bisects the intravesical pressure recording
125
at approximately 25 cm of water. Thus, when the

intravesical pressure is above this level, little or no
drainage occurs from the upper tract. As the pressure
falls through 25 cm, rapid drainage starts to commence, suggesting that the figure of 25 cm is particularly important for the maintenance of upper tract
function.
The robust and resilient nature of the lower
tract changes are illustrated in Figure 27. In these
experiments, bladder pressure was recorded as
before during gamma camera renography. However,
to stimulate to the maximum the upper tractand
thus, if possible, to force perfusion into the bladder
an injection of furosemide was given 15 minutes after
the isotope injection. As can be seen, despite this
front-end loading, the renographic counts do not
diminish and the renographic curves do not deflect
until, once again, bladder pressure has been significantly reduced by removing fluid from within.
Lower tract intrinsic pressure and dynamics appear
to dominate renal function, and such an effect may
still be seen in relatively advanced obstructive renal
failure (Fig. 28). In this case, relatively poor uptake
during gamma camera renography signifies relatively high serum creatinine, but, on draining the bladder, there is still an effect seen in the erect position
that is not identified if the renography is carried out
supine.
In conclusion, therefore, it is possible to
advance a hypothesis on high-pressure chronic retention to account for the variation seen in upper and
lower tract pressures. It is postulated that an open
hydroureter (approximate length in the adult, 25 cm)
(Fig. 29) may maintain profusion through the vesicoureteric junction as long as the cyclic intrinsic
detrusor pressure remains under 25 cm for the
Figure 26 Bladder pressure and renal drainage

compared.
126
George
Figure 27 Lack of furosemide (frusemide) effect

until bladder drained. See text for details.
Figure 28 Bladder effect in advanced renal failure.
majority of the cycle. If the intrinsic detrusor pressure rises above 25 cm for the majority of the filling
phase, ureteric stasis should supervene and
obstructed renal failure would follow. This hypothesis was explored in the original report describing
high-pressure retention where a graph of intrinsic
detrusor pressure against serum creatinine (Fig. 30)
appeared to show that above 25 cm of water there
was indeed a steep rise in creatinine, as would be
expected from the experimental evidence. Although
there were few patients with significantly raised
creatinine levels in this series, other results from
workers have tended to support the importance of
the 25- to 30-cm water pressure range with respect to
maintenance of renal profusion and function.
Figure 29 Hypothesis for gravitational theory in open hydroureter.
APPLICATION TO RECONSTRUCTIVE
SURGERY
The urodynamic observations on HPCR patients may
assist those considering orthotopic or neobladder
techniques for patients requiring radical pelvic surgery. Naturally, bladder walls constructed from other
127
Figure 30 Intrinsic detrusor pressure related to

serum creatinine.
Figure 31 Volume excretion before and after

relief of obstruction. Day 0: prior to relief of
obstruction by bladder catheterization.
than the original detrusor muscle will be neurologically independent of the patient micturition reflex, so
sensory/motor and compliance abnormalities might
be anticipated following such surgery.
Filling phase pressure within the reconstructed
storage organ will clearly be critical for successful
outcome with regard to incontinence, enuresis, and
upper tract function. Physiologic wall characteristics,
detubularization techniques, and other refinements of
design should enable low-pressure storage, ideally
less than 25 cm of water but certainly no greater
than 35 cm of water for the majority of the micturition cycle. Failure to attain these objectives will
inevitably lead to complications as predicted by the
high-pressure model.
BIOCHEMICAL STUDIES BEFORE AND AFTER

THE RELIEF OF OBSTRUCTION
This subject has been extensively studied in the literature and in our own department during the last few
years by Jones and coworkers. An understanding of
the abnormalities of tubular function that may occur
under such circumstances is important for the practicing urologist, who not infrequently has to manage a
patient with postobstructive diuresis on the urology
ward. Absolute volumes (Fig. 31) and electrolyte
excretion (Fig. 32) are maximal within 24 hours and
usually stabilize by 14 days. Synchronous studies of
glomerular filtration rate by various mechanisms
(Fig. 33) illustrate that most of the early improvement
is related to tubular recovery, although a late glomerular recovery phase can be identified. Precisely similar
urodynamic, renographic, and biochemical changes
may be seen in children as well as adultsinfants
and children with urethral valves have a near-identical
syndrome to high-pressure retention (the valve bladder), and in those cases that are not associated with
reflux, similar patterns of obstructive renal failure take
place. Unfortunately, the destructive nature of the
severe obstruction on the detrusor muscle frequently
leads to marked collagen infiltration and fibrotic
damage, resulting in very poor compliance. By contrast, in the adult, long-term studies both from our
own unit and others suggest that the majority of
patients recover well, both as regards bladder and
renal function.
128
George
Figure 32 Sodium excretion before and after

release of obstruction. Day 0: as in Figure 31.
Figure 33 Summary of glomerular and tubular

changes in obstructive uropathy before and after
the release of obstruction.
The observations recorded above have, of

course, been made in a particular group of patients
studied with a particular form of bladder dysfunction.
It might be argued that patients with neurogenic
bladder dysfunctionacknowledged to be chiefly at

risk because of obstructive renal problemscould not
be linked urodynamically or therapeutically with the
high-pressure retention group. There is no doubt that
neurogenic bladders may demonstrate a profile of

much more aggressive hyperreflexic contractions
than that seen in high-pressure retention cases
(although, in the latter, moderate overactivity may
be seen on ambulatory studies), and it is possible
that the hyperreflexic contractions themselves may
be responsible for the obstructive renal failure. Nevertheless, there are enough signs in common (clinical,
radiologic, renographic, and biochemical) to consider
that the conditions may be associated at least in part;
therapeutic and reconstructive lessons learned in one
group may well be applicable to the other.
CLINICAL ADDENDUM
The classical high-pressure presentation of a tense,
painless bladder, late onset enuresis, hypertension,
and progressive renal impairment associated with
bilateral hydroureteronephrosis is comparatively
uncommon, occurring in 10% or less of patients
attending lower urinary tract clinics. Patients
whose urine remains sterile do not complain of prostatic symptoms such as hesitancy or poor stream and
are often astonished when the distended bladder is
pointed out to them.
Unfortunately, the combination of suboptimal
diagnostic skills and the many reasons for abnormal
renal function in the elderly male have led to overdiagnosis of the condition with subsequent illogical
treatment of a postobstructive diuresis, which is usually no more than resolving acute on chronic (low
pressure) retention or residual urine associated with
underactive detrusor function. By association, the real
threat posed by a marked diuresis in a genuine case
may pass unnoticed until postural hypotension supervenes. Informed history taking, accurate fluid chart
records, appropriate radiology and familiarity with
renal function in the elderly can help reduce these
errors of clinical judgment.
FURTHER READING
1. Badenoch AW. Congenital obstruction of the bladder neck.
Ann R Coll Surg Engl 1949; 4:295307.
129
2. George NJR, OReilly PH, Barnard RJ, et al. High-pressure

chronic retention. BMJ 1983; 286:17801783.
3. George NJR, OReilly PH, Barnard RJ, et al. Practical
management of patients with dilated upper tracts and
chronic retention of urine. Br J Urol 1984; 56:912.
4. George NJR, Feneley RC, Roberts JBM. Identification of the
poor risk patient with prostatism and detrusor failure.
Br J Urol 1986; 58:290295.
5. Ghose RR. Prolonged recovery of renal function after
prostatectomy for prostatic outflow obstruction. BMJ
1990; 300:13761377.
6. Holden D, George NJR, Rickards D, et al. Renal pelvic
pressures in human chronic obstructive uropathy. Br J Urol
1984; 56:565570.
7. Jones DA, George NJR, OReilly PH, et al. Reversible
hypertension associated with unrecognised high-pressure
chronic retention of urine. Lancet 1987; 11:10521054.
8. Jones DA, George NJR, OReilly PH. Post-obstructive renal
function. Semin Urol 1987; 5:176190.
9. Jones DA, George NJR, OReilly PH, et al. The biphasic
nature of renal functional recovery following relief of
chronic obstructive uropathy. Br J Urol 1988; 61:192197.
10. Jones DA, Holden D, George NJR. Mechanism of upper
tract dilatation in patients with thick walled bladders,
chronic retention of urine and associated hydroureteronephrosis. J Urol 1988; 140:326329.
11. Jones DA, Lupton EW, George NJR. Effect of bladder
filling on upper tract urodynamics in man. Br J Urol
1990; 65:492496.
12. Jones DA, Atherton JC, OReilly PH, et al. Assessment of
the nephron segments involved in post-obstructive diuresis in man, using lithium clearance. Br J Urol 1989; 64:
559563.
13. Jones DA, Gilpin SA, Holden D, et al. Relationship
between bladder morphology and long-term outcome of
treatment in patients with high pressure chronic retention
of urine. Br J Urol 1991; 67:265285.
14. Sacks SH, Aparicio SAJR, Bevan A, et al. Late renal failure
due to prostatic outflow obstruction: a preventable disease.
BMJ 1989; 298:156159.
15. Styles RA, Neal DE, Ramsden PD. Chronic retention of
urine. The relationship between upper tract dilatation and
bladder pressure. Br J Urol 1986; 58:647651.
16. Styles RA, Neal De, Griffiths CJ, et al. Long term monitoring of bladder pressure in chronic retention of urine: the
relationship between detrusor activity and upper tract
dilatation. J Urol 1988; 140:330334.
17. Wallace DM. The bladder neck in urinary obstruction. Proc
R Soc Med 1951; 44:434437.
9
Urinary Tract Infection
Manchester, U.K.
INTRODUCTION
The infectious process encompasses a highly complex
series of events that surround the relationship
between the defending host and the offending parasite. Virulence factors available to the microorganism
will be combated by a wide range of specific and
nonspecific defense mechanisms, and the result of this
encounter, the microbiological battleground, will
determine whether infectious disease is established.
Conventionally, accounts of infection of the urinary tract concentrate on the response of the urothelium to bacterial invasion. However, a continuing and
perhaps increasing tendency to open surgery in certain groups of patients determines that a basic understanding of the broader concepts of the infectious
process is likely to be advantageous for the practicing
urological surgeon. Therefore, in this account of urinary tract infection (UTI), before dealing with specific
issues relating to organisms and the urothelium, a
general description will be made of the host-parasite
relationship as it applies to the urogenital system both
in health and disease. Some important fundamental
definitions are noted in Table 1. Such general microbiological points may be considered under the following headings.
1.
2.
3.
4.
Colonizing microorganisms in health

General defense mechanisms
General modifying factors
Properties of commensal organisms
Colonizing Microorganisms in Health

Table 2 lists common colonizing microorganisms by
site in healthy humans. The widespread presence of
staphylococci and streptococci on the skin and
Table 1 Essential Definitions of Basic Microbiological Terms
Term
Definition
Pathogenicity
Opportunistic infection
Ability to cause disease

Weakened defenses predispose to
infection, often by nonpathogens
Degree of pathogenicity
Virulence
surrounding the lower genitourinary tract will be

appreciated, as will the occurrence of Candida and
lactobacilli within vaginal flora. The colon contains
enormous numbers of bacteriaup to 1011 organisms
per gram. The majority of these organisms are obligate
anaerobes, although aerobic and facultative anaerobic
organisms, such as enterobacteriaceae and Enterococcus spp., are present in significant numbers (approximately 108/g of colonic contents), these species being
the most common source of uropathogens.
The normally balanced environment of the
bowel flora is significantly affected by antimicrobial
agents. Antibiotic therapy results in normally sensitive Escherichia coli strains as well as anaerobic species
being replaced by more resistant strains and organisms such as Pseudomonas aeruginosa (1). Cessation of
broad-spectrum therapy allows recolonization by resident flora, but slower-growing anaerobic organisms
may initially be displaced by faster-growing enterobacteriaceae. Hence, injudicious broad-spectrum
Table 2 Organisms by Site in Health (Normal Flora): Commensal Organisms That Exist in Symbiotic Relationship with the Host
Protecting Against Uropathogens
Skin
Staphylococci (Staphylococcus aureus and Staphylococcus
epidermidis)
Corynebacterium spp.
Candida spp.
Lower genitourinary tract
Staphylococci
Streptococci
Anaerobic cocci
Corynebacterium spp.
Lactobacilli (vagina)
Large intestine
Anaerobes
Bacteroides spp.
Clostridium spp.
Fusobacterium spp.
Aerobes/facultative anaerobes
Escherichia coli
Klebsiella spp.
Streptococci Enterococci (Streptococcus faecalis)
Yeasts
Chapter 9: Urinary Tract Infection
131
Table 3 General Nonspecific (Constitute) Host Defense Mechanisms

Surface
Compromised
Subsurface
Commensal flora
Mechanical integrity
Acidity
Antibiotics
Surgery
Cannulae
Lysozyme
Lactoferrin
Acute-phase
response
Secretions
Lysozyme
Lactoferrin
IgA
Flow
Peristalsis
Irritation
Compromised
Cellular
Compromised
Drugs
Corticosteroids
Immunosuppression
Infections
Phagocytosis
Polymorphonuclear
Mononuclear
Alcholism
Advanced cancer
Renal disease
Liver disease
HIV
Inflammatory
response
Surgery
Obstruction
Complement
Fibronectin
These are conveniently described in terms of the degree to which the organism penetrate the surface. General factors that may compromise these defenses
are noted.
therapy may increase the size of the colonic reservoir

from which uropathogens are normally drawn (2); as
noted above, these organisms usually account for only
0.1% of total colonic flora. The presence of a large
volume of potential uropathogens may clearly be an
ascending threat to the lower urinary tract, particularly in the presence of any abnormality such as outflow
tract obstruction or congenital anomalies.
General Defense Mechanisms

Nonspecific host defense mechanisms are outlined in
Table 3. General resistance to invasion may be
described in terms of events at the surface of the
host, events at a deeper level, and mechanisms that
depend on cellular function.
The role of commensal flora is further explored
below. The mechanical integrity of skin and mucous
membranes may clearly be breached in a number of
ways. The breakdown of lipids into fatty acids
(approximate pH 56) by skin flora constitutes a
mildly hostile environment for pathogens. Lysozyme,
found in every mucosal secretion, splits the muramic
acid linkage in cell walls of gram-positive organisms
in particular. The iron-binding properties of lactoferrin disrupt the normal metabolism of the microorganism. IgA secretion may prevent attachment of
organisms to host cells.
Penetration of the initial line of defense leads to
more substantial but nonspecific reactions such as the
acute-phase response and the inflammatory response.
Humoral and cellular components, such as protease
inhibitors and adherence proteins, are delivered to the
site, and the classical inflammatory reaction supervenes. Activation of the complement cascade by the
alternative pathway may lead to bacterial lysis as well
as enhancement of phagocytosis. The antiadherence
properties of the glycoprotein fibronectin may prevent
attachment of pathogenic organisms. Increased
phagocytosis by various cells, including neutrophil
polymorphs, mononuclear phagocytes, and naturalkiller cells, is stimulated by a complex series of events,
which may vary according to the nature of the microbiological challenge.
General Modifying Factors

A number of generalized factors may affect the standard host defense mechanisms. Patients at the
extremes of life are vulnerable to infections. Postmenopausal hormonal changes in the lower urinary tract
are of particular interest to urologists.
Poor nutrition or overt malnutrition, particularly
in the elderly, may weaken defenses in a number of
ways relating to protein synthesis and vitamin deficiency. Generalized disorders, such as diabetes mellitus, alcoholism, and renal failure, may markedly
increase susceptibility to disease, as may overwhelming infection and certain types of drug therapy as well
as the general debility related to advanced malignancy.
Pathological conditions, which further expose the
individual to risk of infection, such as stone disease
or obstruction, will be considered below.
Properties of Commensal Organisms

Commensal organisms have an important role to play
in the protection of the host by resisting the growth of
more pathogenic organisms. The mechanisms by
which they attain this objective are listed in Table 4.
Competition for a limited supply of nutrients acts to
restrict the growth of pathogens, while the ability of
the commensal organisms to occupy certain cell surface receptors (tropism) limits the adherence possibilities for the invader. Bacterial products known as
bacteriocins may be toxic to other organisms, often
of the same species. As noted above, fatty acid production by sebum and lipids results in a hostile
microenvironment. Low-level but continued stimulation
of the immune system, as well as the stimulation of
cross-reacting natural antibodies (such antibodies
Table 4 Mechanisms of Protection by Commensal Flora
Competition for nutrients (interference)
Competition for receptors (tropism)
Bacteriocin production
Fatty acid production
Stimulation of immune system
Stimulation of natural antibodies
132
George
are raised against organisms, which the host has not

encountered because of antigenic cross-reaction with
organisms that have been experienced), further
enhances resistance to pathogenic bacteria. Apart
from antibiotic therapy, the normal commensal flora
may be significantly affected by general factors such
as diet, hygienic habits, and underlying disease.
The described general mechanisms are, under
the normal circumstances of health, remarkably effective at excluding pathogenic invasion. For a more
complete account of the highly complex processes
involved, the reader is referred to standard bacteriological texts. The remainder of this chapter will
address, first, specific microbiological aspects of UTI
in humans and, second, a clinical account of the more
important forms of inflammatory disease.
HOST DEFENSE MECHANISMS: LOWER

URINARY TRACT
Apart from the broad concepts outlined above, the
lower urinary tract has a number of specific defense
mechanisms, which allow it to counter the threat posed
by the reservoir of potential pathogens, which is located
chiefly in the lower bowel and on the perineal skin.
Naturally, the anatomy of the male and in particular the
length of the urethra determine that ascending infection
is extremely uncommon when compared with the
female. However, the lower urogenital tract, while
clearly offering a bacteriological threat to the female
urothelium, does offer some defense mechanisms
against ascending infection by pathogenic bacteria.
Commensal Organisms
An outline of the advantages of commensal flora has
been described above. During the reproductive years,
circulating estrogens affect the vaginal epithelium,
which stores increased amounts of glycogen within
the cells (Fig. 1). The glycogen is metabolized by
Lactobacillus acidophilus into lactic acid, and the resultant drop in pH produces an unfavorable microenvironment for the majority of pathogenic bacteria
attempting to ascend into the bladder (see also discussion of adhesion theory). Lactobacilli and other
gram-positive rods are collectively known as Doderleins
bacilli, and disruption of this vaginal flora by vaginitis
or other infection leads to a rise in pH and loss of the
natural defense barrier. Lactobacilli are one of the
main causes of milk going sour and indeed are the
active organisms in live yoghurt, which for this
reason is frequently advocated by health magazines
as a topical application that can prevent recurrent
lower UTI without the need for antibiotic therapy.
Naturally, such protection is not available either
before or after the menopause, one fact that possibly
explains the increased incidence of ascending lower
UTI in elderly women, whose introital skin is oftenthin
and atrophic because of lack of circulating estrogens.
Urine
Urine, normally a good culture medium, may under
some circumstances be inhibitory or even bactericidal
against some uropathogens. Low urinary pH levels in
particular, as well as raised blood urea and high
osmolarity, are inhibitory for some organisms (3).
Genital Skin
Although strictly unrelated to the urothelium, penetration of the natural barrier afforded by genital skin can
have serious consequences for the patient (Fig. 2A, B).
Any perforation of penile skin may be followed by
infection, but this is particularly the case when the
patient is diabetic; such patients are commonly
exposed to increased risk during self-administration
of vasoactive substances for erectile dysfunction.
Urine Flow
Urine flow from the kidney by ureteric peristalsis and
from the bladder by periodic detrusor contraction
constitutes the main defense mechanism of the urinary tract against ascending infection (4). Loss of
competence at the vesicoureteric junction for any
reason may lead under certain circumstances to significant renal damage; additionally, reflux prevents
efficient bladder emptying, thus compromising the
flushing mechanism of micturition, which is similarly
impeded by any form of lower urinary tract obstruction.
Bladder Surface Mucin
Figure 1 Vaginal epithelial cells stuffed with glycogen. Produced

in response to estrogens, the glycogen is broken down by Lactobacillus acidophillus, thus increasing local vaginal acidity.
In a series of experiments on rabbit bladders, Parsons

et al. (57) proposed that a bladder surface mucin
layer consisting of a glycosaminoglycan (GAG) was
secreted by transitional cells and acted as an antiadherence factor by inhibiting bacterial attachment
to bladder mucosa, thereby facilitating removal of
bacteria by the voiding process. These workers demonstrated that removal of the GAG layer by acid
133
Figure 3 Effect of bladder surface mucin. Binding of 14Clabelled Escherichia coli to normal bladder mucosa and acidtreated mucosa. Acid treatment removes the mucin layer, and
bacterial adhesion to the bladder mucosa increases significantly.
Twenty-four hours after acid treatment, the mucin layer has
recovered sufficiently to prevent epithelial attachment (rabbit
bladder). Source: From Ref. 8.
protective barrier for the urothelium, thus preventing

bacterial adherence to the uroepithelial cells. Subsequently, the bacteria trapped in the GAG layer are
expelled by urination (57).
Figure 2 The importance of skin epithelial integrity. (A) Large

blister on forearmno infection within perfect subblister culture
medium because of integrity of ultrathin residual epithelial covering. (B) Puncture site of intracavernosal therapy in diabetic
despite full precautions, serious widespread inflammation has
taken place.
markedly increased adhesion to the urothelium (Fig. 3)

and, furthermore, that instillation of heparin (a synthetic GAG) into bladders previously denuded of their
GAG layer by acid resulted in restoration of the antimucosal adherence properties. Pretreatment of the bacteria with heparin had no effect on adherence, and the
authors concluded that the surface mucin provided a
Tamm-Horsfall Protein
Tamm-Horsfall protein is secreted by the cells of the
ascending loop of Henle and is the most common
mucoprotein of renal origin in urine. Also known as
uromucoid, Tamm-Horsfall protein was originally
noted to react with influenza virus (9), but subsequently, it was established that the protein was capable of binding strongly to E. coli expressing type 1
mannose-sensitive fimbriae (10), probably because of
mannose-containing side chains within the mucoprotein. After entrapment, it is proposed that the uromucoid/coliform complex is mechanically cleared from
the urinary tract by urination. Recent studies confirm
that in these circumstances the mannose-binding
properties of type I pili are critically related to the
FimH tip adhesin (see below) (11).
134
George
Mucosal Shedding
Attachment to uroepithelial cells by pathogenic bacteria
offers both defense and attack possibilities for host and
invading organism alike. Exfoliation rates for bladder
mucosal cells under normal conditions of sterile urine
are approximately four weeks (12); following infection,
greatly increased rates are observed within hours of
inoculation, a process apparently linked to FimH adhesion and the apoptotic pathway involving caspases and
epithelial cell DNA fragmentation (13,14).
Exfoliation of cells with adherent bacteria (Fig. 4)
by bulk flow of urine is likely to be of benefit to the
host, although the consequent exposure of less mature
epithelial cells may provide an opportunity for colonization and invasion by other uropathogenic organisms. It has been suggested that such sequestration of
organisms within deeper layers of the bladder wall
might explain problems related to persistent infection
in some cases of cystitis (17).
Local Immune Response

The role of immunity in the defense of the urinary tract
remains poorly understood. Although considerable
humoral and cellular response may be observed in

upper UTI, serum production of urinary antibody in
bladder infection is characteristically difficult to detect,
perhaps reflecting the relative superficiality of the
infection.
VIRULENCE MECHANISMS
Virulence mechanisms are those properties of the
parasite that enable it first to colonize and then
flourish within the host. Such mechanisms may be
directed against external agents administered to eradicate the organism or against the host itself, including
natural host defenses that have developed over time
to counter the invading microorganism (Table 5).
Virulence Against External Agents:

Antimicrobial Resistance
Before World War II there were few agents with
reliable antimicrobial properties, and the virulence
capability of organisms was almost entirely directed
against the natural properties of the host. After the
discovery and clinical usage of penicillin, organisms
learnt rapidly to develop systems for evasion of the
toxic effects of antibiotics, and the consequent emergence of resistant strains today constitutes a significant threat, particularly for certain groups of the
population such as hospital inpatients. Staphylococcus
aureus, once almost entirely penicillin sensitive, is now
approximately 90% resistant to the agent. Neisseria
gonorrheae remained penicillin sensitive for many
years, but its acquisition of b-lactamase activity has
resulted in high levels of penicillin resistance. Hospital-based doctors will be well aware of the extremely
serious threat posed by methicillin-resistant S. aureus
(MRSA) infections, which may spread rapidly
throughout whole wards and effectively be untreatable. These examples underscore the extreme flexibility and adaptability of the invading microorganism,
which, through evolutionary processes, has managed
to match and subsequently often overcome the best
efforts of scientific antibiotic endeavor.
Types of Resistance
Figure 4 Mucosal shedding of epithelial cells in response to

Escherichia coli infection. (A) Scanning electron micrograph
of superficial epithelial cells of uninfected mouse bladder.
(B) Following infection with type 1 pilated E. coli, superficial
cells exfoliate, exposing deeper less mature cells. Scale bars 50
mm. (C) Magnified view of exfoliated mouse epithelial cell showing
vesical surface coated with E. coli while the recently separated
inferior surface is devoid of organisms ( 440). Source: From
Refs. 15 and 16.
Organisms may acquire antimicrobial resistance by

two basic mechanisms. Intrinsic resistance means that
the organism may naturally resist antibiotics by the
production of natural enzymes, such as b-lactamases;
or because the usual antibiotic target within the cell is
lacking; or by the presence of an impenetrable cell
wall. Acquired resistance develops as a result of the
evolution of new or altered genetic material, which
may occur through either mutations or gene transfer.
Table 5 General Classification of Virulence Mechanisms
Against external agents
Against the host
Antimicrobial resistance
Toxin production
General mechanisms
Adherence
mechanisms
135
Table 6 Target Sites of Common Antimicrobial Drugs

Drug
Target/mechanism
b-Lactams (penicillin, cephalosporin)

Aminoglycosides
Erythromycin
Tetracyclines
Trimethoprim
Fluoroquinolones (ciprofloxacin)
Cell wall: disruption of peptidoglycan X-linkage

Ribosomal interference: misreading mRNA
Translocation interference
RNA-binding interference
Nucleic acid interference: dihydrofolate reductase inhibition
Inhibit DNA gyrase
It is convenient to consider the action of the antibiotic as it relates to bacterial cell structurecell wall, cytoplasmic membrane,
ribosomal function, and nucleic acid synthesis.
Such mutations and transfers result in a number of

well-described mechanisms by which the organism
may evade the action of the antibiotic.
Enzyme inactivation. b-Lactamase production is
the most important resistance mechanism against the
penicillins and cepha losporins. The enzyme hydrolyzes the b-lactam bond in the antibiotic structure,
rendering it inactive. b-Lactamase production is commonly found in S. aureus, N. gonorrhea, and enterobacteria.
Altered permeability. Various alterations in receptor activity and transport mechanisms may prevent
access of the antibiotic to the microorganism. Aminoglycosides and tetracyclines are actively taken up into
organisms, and resistance may occur either by inactivation of the transport mechanism or by development
of additional systems, allowing increased expulsion of
the drug from the cell.
Alteration of binding site. Antibiotics may bind to
specific targets within the cell. Genetic variation may
alter or delete the antibiotic target, thus leading to
resistance to the drug. The site of action of the more
commonly used antibiotics is summarized in Table 6.
The complex nature of antimicrobial resistance determines that a detailed account is beyond the scope of this
text; for a comprehensive overview, the reader is referred
to definitive studies of resistance mechanisms (18).
VIRULENCE AGAINST THE HOST ITSELF

Toxin Production
Toxins are proteins that are able to harm the cells or
tissues of the host. Many organisms elaborate toxins
that may either assist with the process of local invasion or be themselves responsible for the characteristics of disease. Important examples of such virulent
toxins are those that produce the clinical manifestations of diphtheria, tetanus, and botulism.
A number of organisms, including E. coli, exhibit
hemolysin activity, which may damage erythrocytes
in a variety of ways, including phospholipase C
activity (cell-membrane damage) and osmotic lysis.
E. coli strains commonly associated with UTI (01, 02,
04, 06, 07, 016, 018, and 075) frequently elaborate
hemolysins, and such strains may be disproportionately promoted from the colonic reservoir (19,20).
Cytotoxic necrotizing factor 1 is another toxin elaborated by pathogenic coliforms to facilitate colonization
of the host (21). Despite the strong association of such
strains with the ability to cause ascending UTI, the
precise mechanism of enhanced virulence remains

unknown.
General Mechanisms
Apart from toxin production, there are a number of
general mechanisms that facilitate the organisms
attempt to enter and multiply within the host.
1.
2.
3.
4.
Penetration
Antihumoral activity
Evasion of phagocytosis
Competition for nutrients
Penetration
In general, organisms are unable to penetrate the intact

epithelium of the host unless prior damage (Fig. 2B) has
taken place. However, certain parasites may do so, the
best example in the urinary tract being the fork-tailed
cercariae of Schistosoma haematobium, which are able to
penetrate unbroken skin for a few hours after being
shed by the intermediate snail host (Fig. 5A).
Antihumoral Activity
As noted above, antibodies such as secretory IgA may

be active against organisms before mucosal invasion
occurs. Organisms such as N. gonorrheae and Proteus
mirabilis elaborate anti-IgA proteases that inactivate
this defense mechanism. A further antihumoral virulence factor is exhibited by certain isolates of N.
gonorrheae that are able to resist the lytic effects of
complement. Such strains are thus able to multiply
and enter the host bloodstream, whereas those isolates
that lack this virulence factor are unable to invade and
so remain localized on the surface of the genital tract
(22). Resistance to the bactericidal effect of serum/
complement has been linked to the integrity of the
lipolysaccharide cell wall (O-antigen). Additionally,
loss of capsular polysaccharide (K antigen) has been
associated with lack of resistance to compliment mediated lysis (23).
Evasion of Phagocytosis
A number of bacteria have developed mechanisms for

resisting phagocytic attack by the host. Variation in
surface antigens and the properties of some polysaccharide capsules may constitute a successful defense
mechanism by preventing interaction between the
phagocytic cell and the invading organism (22).
136
George
Table 7 Recognized Adhesion Mechanisms
Afimbrial adhesions
Adherence pedestals
Fimbriae
fully understood at that time. General observations of

bacteria and algae adhering to inert plastic or rocks in
streams were brought together with other observations
of various bacteria adhering in vivo to a number of
epithelial surfaces. It is now possible to distinguish the
individual mechanisms by which adhesion takes place in
each of these specialized circumstances (Table 7).
Although fimbriae are of overriding importance in UTI,
it is helpful briefly to describe and contrast other methods of adhesion. Adherence is defined as the initial
interaction of a microorganism with the host. An adhesin
is a microbiological molecule that leads to bacterial
adhesion to cells or tissues. Adhesins predominantly
react with specific receptors on the host cell surface,
although nonspecific adhesion (as by surface charge)
may occur (26).
Types of Adhesins
Afimbrial Adhesions
Figure 5 Schistosoma haematobium. (A) The fork-tailed cercariae have a free-swimming existence of 24 to 28 hours. Intact
skin or mucosa may be penetrated, the fork-tailed structure being
lost in the process, allowing the organism (now known as a
schistosomulum) to reach the subcutaneous tissues. (B) The
spike-like projection at the end of the egg identifies the organism,
in contrast to the projections of Schist. mansoni, which are
located in the equatorial region. The eggs sequester within the
wall of the urinary tract and are shed to the exterior in the urine.
Typical egg dimensions of S. haematobium: 112/170 40/70 mm.
Source: Courtesy of Dr Allan Curry, UHSM.
Afimbrial adhesins consist of polymers, polysaccharides, lipoteichoic acid, and other proteins associated
with the cell wall of the organism. Collectively, these
are known as a glycocalix, and together they serve
to attach the organism to the target cell. The classical
studies into glycocalix formation involved Streptococcus mutans, an organism that colonizes human teeth,
leading to decay. Enzymatic activity at the cell surface
degrades sucrose, providing fructose for ongoing
nutrition, but it also polymerizes glucose into polysaccharide chains used to construct the glycocalix. Thus,
the organism attaches itself to its target, at the same
time protecting itself against attack and concentrating
its nutrients by wrapping itself within the glycocalix
also known as a biofilm (27).
Competition for Nutrients
Helicobacter pylori
To be successful, any invading microorganism will

require an adequate supply of nutrients. In particular,
free iron is required for metabolism and multiplication of E. coli, iron uptake being facilitated by the
siderophore aerobactin and enterobactin (21). The
ability to produce these agents thus confirms an
advantage on strains invading the urinary tract, and
an association has been observed between aerobactin
production and the expression of P fimbriae (see
below) in patients with symptomatic UTI (24).
Helicobacter pylori provides a typical example of an

organism that attaches itself via small cellular projections
known as adherence pedestals. This organism, with its
unmistakable flagella (approximately three times the size
of fimbriae; see below), attaches itself to cells within the
gastric epithelium, leading to ulceration and perhaps
gastric carcinoma (Fig. 6A). Presumably, the flagella
drive the organism downward into the mucosa, following which adhesion to the target cells takes place
(Fig. 6B).
Adherence Mechanisms
Fimbriae
The adherence of a cell to another structure is an

extremely important biological characteristic that can
be observed widely throughout nature. Early reports
of this phenomenon (25) were slightly misleading, as
the particular mechanisms of attachment were not
Fimbriae (also known as pili) are very important

virulence structures that mediate attachment to host
tissues and are particularly important in the pathogenesis of E. coli UTI. A typical organism may have
100 to 500 appendages, each approximately 5 to 10 nm
Figure 6 Helicobacter pylori. (A) The flagella of the organism

are easily identified (significantly larger than fimbriae). (B) Flagella used to propel organism into gastric mucosa where adhesins provide attachment. Source: Courtesy of Dr Alan Curry,
UHSM.
in diameter and 2 mm in length (Fig. 7). Isolates of

E. coli may produce a number of antigenically distinct
fimbriae, although some strains produce no fimbriae
at all.
Supramolecular Adhesins Associated with

Pathogenic Strains of Escherichia coli
Early distinction between types of fimbriae was made
by the ability of mannose, respectively, to inhibit
(mannose sensitive) or not to inhibit (mannose resistant) the agglutination of guinea pig erythrocytes. For
some years, it had been known that type I pili were
specifically bound to Tamm-Horsfall protein (10) and
foreign bodies such as catheters and prosthetic
implants (28). More recently, a variety of techniques,
including hemagglutination, electron microscopy, Xray crystallography, and molecular methodologies,
have enabled a more precise identification of a number of specific adhesion organelles that are recognized
as critically important virulence factors for uropathological E. coli (UPEC) (Table 8).
137
Figure 7 Escherichia coli, showing fimbriae. Source: Courtesy

of Dr Pauline Handley, University of Manchester.
Type I Fimbriae
These structures are the most common adhesion organelles found on pathogenic coliforms isolated from the
urinary tract (32). They are of variable length (12 mm)
and approximately 7 nm thick, being of helical construction (Fig. 8). The FimH terminal adhesin is a twodomain protein approximately 3 nm in width to which
mannose-containing glycoprotein receptors can attach
(33,34). Uroplakin (UP1a) is a complex glycoprotein that
covers the internal surface of the bladder in a form of
membrane or plaque (35). This and other related compounds (UP1b, UPII and UPIII) are the primary receptors (docking sites) for the type I pilus of pathogenic
bacteria (34) (Table 8). The defensive role of TammHorsfall protein, which preferentially binds type I pili,
has been mentioned above (11).
Initiation of the Infective Process

Traditionally, pathogenic coliforms were considered
to exert their influence by epithelial adherence and
138
George
Table 8 Adhesin and Receptor Characteristics Identified in Pathogenic Coliform Infections
Organelle
Guinea pig
hemaglutination
characteristic
Type I
fimbriae
Inhibited by
mannose
(mannose
sensitive)
Type P
fimbriae
Not inhibited by
mannose
(mannose
resistant)
S
F1C
Fimbriae
Dr adhesins
Adhesin
Host receptors
Host cells
Clinical disease
Fim H
Uroplakin 1a
(mannosylated glyco
protein), Tamm-Horsfall
protein (uromucoid),
collagen types I, IV,
laminin, fibronectin
Globotriaosylceramide
Globotetraceramide
(globoside)
Globopentaceramide
(Forssmann antigen)
a-sialyl-2,3-b-galactoside
Plasminogen
lactosylceramide
Type IV collagen *5b,
integrin
Bladder:kidney: buccal
epithelial cells,
erythrocytes, neutrophils,
foreign bodies
(i.e., catheters)
Cystitis
Sepsis
PapG
Epitope
Class
Sfas
SfaA/FocH
Various
I
II
III
Kidney epithelial cells

Human P
blood group antigen
Bladder/kidney
Epithelial cells
Erythrocytes
Bladder/kidney
Epithelial cells
Not in human
Pyronephritis
Cystitis
Ascending UTI
Sepsis
Meningitis
Cystitis
Diarrhea
Sepsis
The description of the organelle, the characteristic of the tip adhesin and specific host receptor docking port on particular host cells are tabulated. The
critical chemical structure of the PapG receptor has been emphasized by bold underlined text. For further details concerning the site of the disaccharide
Gal-Gal core receptor, see Ref. 29. Globo A is similar but not identical to the Forsmann antigen and has been investigated as part of comparative receptor
studies.
Source: From Refs. 15, 21, 30, and 31.
subsequent metabolic disruption as a result of factors

expressed from the epithelial locus. Recent work
suggests that under certain circumstances coliform
organisms may become opportunistic intracellular
pathogens. Using human bladder epithelial cells, Martinez and coworkers observed cellular invasion by
organisms expressing type I pili, but not by those with
type G pili (36). The process required FimH-activated
submembranous actin organization, phosphoinositide
3-kinase activation and tyrosine phosphorylation
(Fig. 9A). Such internalization might allow (assuming
no shedding) the organisms to replicate in the relative
shelter (vacuole) of the uroepithelial cell, thus propagating the infective process. Similar observations have
been made relating to bacterial survival within host
macrophages (37). Recent studies (38) have shown the
sophistication of such intracellular bacterial communities (IBCs) wherein UPEC are able to adjust virulence
systems including iron acquisition mechanisms and
various adhesins that may be up- or downregulated
presumably to give the organism a survival advantage
on release (Fig. 9B). Klebsiella pneumoniae may also form
IBCs though expression of type I pili is minimal as
compared with UPEC (39) (see below).
Schwan and coworkers varied growth conditions

and found that fewer type I pili were expressed in a
low-pH/high-osmolarity environment (40). Other
cues include temperature change, oxygen tension,
and nutrient availability. Transcription of type I fimbrial genes is controlled by a promoter located on an
invertible element; during UTI, the orientation of the
element (on) that allows the expression of type I pili
was maximal at 24 hours, suggesting that the switch
(phase variation) was a significant virulence mechanism in the early stages of an infection (41). Expression of organelles is also significantly affected by other
adhesins that may be present on the bacterium. PapB,
a regulator of type P pilus expression, may modulate
type I expression, increasing the off phase noted
above (42). Such interpilus variations may enable the
organism to adapt and change targets; for example,
such a mechanism would be advantageous for a PapP
pathogenic organism trying to reach the upper tracts
via the epithelial barrier of the bladder.
Further studies have explored the complex relationship between type I pilus expression and motility
as related to flagellum expression. The presence of
type I pili significantly reduced flagella expression
and motility while loss of type I surprisingly did not
result in increased motility (43).
Expression of Type I Pili

Expression of adhesins can be markedly affected by a
wide variety of factors, including environmental cues,
adhesin cross-talk and the adherence environment
in which the organism is placed; coliforms in liquid
culture (no adherence required) may fail to develop
any adhesion organelles at all.
Type P Fimbriae
P fimbriae are particularly important in disease of the
upper urinary tract, and their clinical significance is
discussed below. It has already been noted that some
pathogenic organisms may variably express both type I
Figure 8 Model of the type I pilus. (A) side view; (B) top view.
External diameter approximately 70 A. FimH tip adhesin; rod
predominantly FimA subunits. Source: Courtesy of Ref. 33.
139
Figure 9 (A) Possible mechanism of type I pilus-mediated

invasion of bladder epithelial cells. FimH adhesion to cell surface
triggers envelopment followed by activation of a cascade involving protein tyrosine kinases, PI-3 kinases and Cdc 42. The
transduction cascade facilitates the internalization of the organism within membrane-bound vacuoles that protect it from host
defense mechanisms. The organisms replicate within the intracellular environment, effectively converting the epithelial cell into
a bacterial factory. (B) Finally, the organisms burst out and are
released from the surface of the epithelial cell. Note smaller
rupture at base of cell with emerging rod-like organisms. Bar
5 mm. Source: Courtesy of Refs. 15, 17, and 36.
and P pili (44), a factor that may complicate attempts to

identify virulence factors by in vitro bacterial studies.
Structure and Ultrastructure of P Fimbriae

Advances in molecular biology have enabled the
structure of P fimbriae to be elucidated (Fig. 10). The
Pap pili (pili associated with pyelonephritis) essentially
consist of four proteins, PapA, PapE, PapF and PapG,

constructed and assembled on a platform of PapC
protein, the fiber being 7 nm wide. PapG is chiefly
responsible for binding to the receptor, while protein
PapA constitutes the bulk of the stem of the pilus,
140
George
Figure 10 Pilus assembly and structure. The Pap pilus is

constructed and assembled on a platform of PapC protein. The
adhesion characteristics are chiefly determined by the terminal
PapG protein, which must be some distance from the platform
before it is able to function (", no adhesion; , able to adhere).
The PapA subunits are formed in an helical fashion into the stem
of the pilus, the entire structure being approximately
1000 subunits long. Source: From Ref. 45.
approximately 1000 subunits being arranged in a helical fashion between the surface of the organism and the
active tip proteins (21). Experimental observations suggest that the adhesion characteristics are maintained by
the GFE tip proteins even in the absence of the helical
structure of PapA stem subunits. It has been suggested
that the reason for the seemingly dispensable fiber
structure is that this length places the adhesin outside
the lipopolysaccharide cell surface structure of E. coli
(see below), thus maintaining the integrity of the
individual virulence mechanisms (45).
Recent advances in the understanding of the
receptor structure for the G tip protein are leading to
significant advances in knowledge concerning the
mechanisms of UTI. Table 8 annotates the globoseries
glycolipid isoreceptor types, each of which contains
the disaccharide a-GAL-1-4-b-GAL (known colloquially as GAL-GAL), and their association with disease.
Different positioning of the disaccharide within the
molecule in each of the isoreceptors determines the
adherence capability of the G tip proteins. Organisms
with class I tip proteins do not adhere and do not
cause disease in man because of the absence of the
globo isoreceptor. Those with class II are strongly
associated with pyelonephritis, while class III adhesins are commonly found in patients with cystitis (29).
Hence, the structure of the globo isoreceptor determines the outcome between invading organism and
hostpatients with infections from coliforms expressing class II adhesins are unlikely to suffer from cystitis, globoside being the major isoreceptor in the
human kidney (46). Similarly, the association of class
III adhesion with cystitis suggests a predominance of
Forssman receptors on the urothelium of the lower
urinary tract. Further studies of receptor expression

by means of differential blood group analysis (30)
have demonstrated that minor differences in receptor
core structure profoundly affect disease patterns, and
emphasize the importance of precise fitness if bacteria are to persist and multiply within the lower
urinary tract (31). However, the complexity of the
relationship between PapG adhesins and their receptors on target cells has recently been reemphasized.
Most E. coli express a single fimbrial type at any one
time while loss may promote another, a form of
compensatory virulence. The multiple renal putative
targets for Pap pili are mentioned below; this and the
variable surface adhesion expression of experimental
coliforms make specific conclusions as to the etiology
of infectious disease problematical (47,48). Nevertheless, despite the complexity of adhesin/receptor interactions, the importance played by nonspecific
mechanisms, such as electrostatic and hydrophobic
attractive forces, should not be overlooked (46).
Historical Relevance of Fimbrinated Status

Long before the mechanisms of adherence were fully
understood, observational studies had indicated a
connection between the macroscopically observed
bacterial adherence and severity of UTI. This phenomenon was first reported in The Lancet in 1976 by
Catharina Svanborg Eden and colleagues from the
Department of Immunology in Goteborg, Sweden, a
department that has continued at the forefront of
adherence research. Uroepithelial cells from freshly
voided morning urine samples were added to bacterial cells, and the mixture was incubated during rotation for 60 minutes. Adherent bacteria (Fig. 11) were
counted under direct light microscopy. The results
(Fig. 12) demonstrated convincingly that pyelonephritic strains were more adherent than strains from
other disorders of the lower urinary tract (49). This
effect was negated when the organisms were incubated with antibodies against the strain tested. Two years
later the same laboratory described hairlike appendages (Fig. 7), which might have been responsible for
the effect (50). These observations were extended by
Fowler and Stamey, who studied adherence to vaginal
cells from controls and from women susceptible to
lower UTI (Fig. 13). Significant adherence was demonstrated, suggesting the possibility that general cellular characteristics might be involved in the
adherence process (51). This concept was taken further by Schaeffer and coworkers, who confirmed the
findings on vaginal cells and then showed that the
same phenomenon could be observed on buccal cells
(Fig. 14). These observations clearly indicated that
there might be a widespread alteration in the surface
characteristics of mucosal epithelial cells in particular
susceptible individuals (52).
Previously, Sellwood and coworkers at the Veterinary Agricultural Research Council had noted that
E. coli strains expressing K-88 surface antigen caused
neonatal diarrhea when administered to some piglets,
but not others. This phenomenon was observed to be
because of K-88 adherence to intestinal cell brush
141
Figure 12 Escherichia coli adhering to 40 healthy, human,

urinary tract epithelial cells. Organisms from patients with ABU
adhered minimally when compared with those from cases of
pyelonephritis. Source: From Ref. 49.
Figure 11 Examples of bacterial adherence. (A) Adherence of
Escherichia coli to epithelial cell. (B) Adhesion of colonies to the
surface of a urinary catheter. Such adhesion is thought to be
mediated by type 1 fimbriae. Source: From Ref. 28.
borders in piglets that developed diarrhea, while

those that remained well did not show this phenomenon (53,54). Subsequently, it was found that adhesive and nonadhesive piglets inherited these
intestinal cellular characteristics in a simple Mendelian manner, and these findings have since been
acknowledged as the first report of a genetic basis
for resistance to enteric disease (55). Schaeffer, quoting
this work, noted that it might explain in part why
some patients resistance to infectious disease could
correlate with their blood groups (56).
Subsequent ability to identify P-fimbriated varieties of E. coli allowed further investigation into the
mechanisms of UTI. Kallenius et al. (57) examined
97 children with UTI and compared them with
82 healthy controls. P-fimbriated forms were found
in 33/35 (91%) of urinary strains causing acute pyelonephritis, but in only 19% of patients with cystitis and
14% of cases with asymptomatic bacteriuria (ABU). By
contrast, only 7% of fecal isolates from healthy controls carried such fimbriae. Further evidence of the
importance of P fimbriation was observed by Johnson
et al. (58), who investigated host conditions that might
be associated with increased frequency of P fimbriae
or other virulence factors. These studies clearly
showed that, while P-fimbriated forms remained relatively common in the presence of anatomical urinary
tract abnormality or after instrumentation, the fimbriated form was absolutely essential if infection was to
occur when none of these predisposing abnormalities
were present. In summary, coliform strains with a
variety of characteristics are capable of causing upper
UTI in the presence of (i.e., with the help of) obstruction or other abnormalities; however, for infection to
supervene in a completely normal upper urinary tract,
the presence of the P-fimbriated form of E. coli is
almost essential. Nevertheless, other adhesins have
been mapped to other docking molecules at various
sites within the human kidney (Fig. 15), underscoring
the complexity of ascending UPEC infection in the
urinary tract.
Recently, in an elegant experiment, Wullt and
coworkers examined the ability of P fimbriae to
142
George
Figure 13 In vitro adherence of Escherichia coli to vaginal

epithelial cells in patients susceptible to recurrent UTI compared
with controls. Source: From Ref. 51.
induce inflammation in the human urinary tract, and

at the same time they sought to satisfy Kochs molecular postulates linking P pilus expression to the host
inflammatory response. Kochs postulates state that,
from diseased tissue, isolates should be recoverable,
be reproducible in culture and, on reintroduction to a
susceptible host, be the cause of further recognizable
disease. At molecular level, the same postulates can be
examined with respect to various factors such as the
putative virulence mechanisms of the coliform bacillus.
It has already been noted that P fimbriation is
associated with pyelonephritis in approximately 90%
of cases, but in 20% of patients with ABU (57,60,61).
Nevertheless, other adhesinstype I, type S, and Dr
adhesinsmay also be isolated from patients with
upper tract infection, and the question arises as to
which of these characteristics is the preeminent cause
of disease. However, other lines of evidence (the
ability to enter the bloodstream, to enhance experimental infection and to promote a cytokine response)
suggest strongly that P fimbriae are indeed the significant independent virulence factors. To resolve this
problem, 17 human volunteers received intravesical
inoculation of either a nonfimbriated ABU coliform
strain or Pap transformants of the same strain, the
former acting as control to negate the possibility of
nonspecific adhesin formation linked to DNA sequences carried by the ABU strain. The result (Fig. 16)
demonstrated that the presence of the Pap transformants invariably caused higher neutrophil and cytokine
responses; furthermore, loss of Pap expression was
linked to a reduction in background inflammatory
levels. Control strains did not cause a significant
host response. Taken as a whole, the results provided
convincing evidence that P fimbriation per se converted a low- into a high-virulence strain, suggesting
in turn that Kochs molecular postulates were indeed
fulfilled (62).
Figure 14 In vitro adherence of Escherichia coli to vaginal cells (left panel ) and buccal cells (right panel ) from patients with recurrent
UTIs as compared with controls. Source: From Ref. 52.
143
Figure 15 Binding sites of various Escherichia coli adhesins in the human kidney. The complexity of variable adhesin expression on
uropathogenic E. coli and the possible relationship to pyelonephritis can be appreciated. Adhesins in red. Source: Courtesy of Ref. 59.
Type S Pili
Less well defined than the preceding types of pili, S
types nevertheless share a similar structure of Sfa A
subunits, with the Sfa S subunit localized to the tip and
interacting with sialic acid receptors on renal vascular
and epithelial cells. The fimbriae may facilitate bacterial
dissemination, and the type has been associated with
pathogenic strains that are associated with sepsis and
meningitis as well as ascending UTI (63). FIC pili are
homologous with type S and may be present on
approximately 14% of pathogenic coliforms.
Dr Adhesin Family
The family includes both pilus-like adhesin Dr and
nonfimbrial adhesion molecules. Their function
remains open to question, but they can be isolated in
a high proportion of children with UTI and one-third
of pregnant women with pyelonephritis. They may be
responsible for long-term bacterial persistence within
the upper tract (64).
Clinical Aspects of Adhesion Theory

The ability to adhere and thus to persist within the
host is an advantage to the microorganism in a number of circumstances; it helps the pathogen to remain
within its source (reservoir), survive during the journey to the target tissue, and establish and replicate on
arrival within the lower urinary tract. Receptors for P

fimbriae have been identified within the colon, and
this attachment mechanism has been proposed to
explain persistence of pathogenic organisms within
the reservoir (65). Such strains expressing Pap established themselves more rapidly in children susceptible
to UTI than organisms without the adhesin (61). Recent
studies of cystitis in young schoolgirls (66), chosen (by
age) to exclude the effect of sexual intercourse,
observed that the clones of organisms responsible for
the infection were P fimbriated, whereas those forms
were not usually found on the perineumwhere
clones exhibiting type I fimbriae and other virulence
factors predominated. These observations, when summarized, indicate that both type I and type P fimbriae
are commonly found in the fecal reservoir, as well as
the periurethral zone, and may variously facilitate the
initial move toward the lower urinary tract. Clearly,
phase variation, cross-talk, and an altered microenvironment (the effect of L. acidophyllus is noted above)
may significantly alter fimbrial expression (Fig. 17),
both to evade host defenses and maintain optimal
pathogenicity en route to the target cells. Needless to
mention, this ability rapidly to vary expression makes
the study of virulence factors particularly difficult for
microbiological researchers. Hence, it is not entirely
clear which virulence factors are responsible for colonization and development of clinical disease in the
lower urinary tract. Certainly, the variability mentioned above means that it is difficult at this time to
144
George
Figure 17 Microenvironment and pathogenicity. Effect of

altered pH in human urine on pilus expression. With a sodium
phosphate buffer, the expression of FimB almost doubles at pH
7.0, whereas FimE levels remain constant throughout. Source:
From Ref. 40.
Figure 16 Neutrophil, IL-6 and IL-8 responses to intravesical

inoculation with ABU Escherichia coli 83972 nonfimbriated strain
(gray bars) and the same E. coli 83972 strain transformed with
recombinant plasmids encoding class II or class III P fimbriae
(black bars). Responses identified in urine; see text for details.
Abbreviation: Precol, precolonization. Source: From Ref. 62.
predict infection on the basis of adhesin analysis of

introital or periurethral organisms (66).
Finally, it should be noted that the identification
of certain forms of an organism associated with particular tissues does not per se prove a relationship
with clinical diseasehence the need to test Kochs
postulates. In particular, type I fimbriae are found in a
majority of enterobacteria, on both nonpathogenic as
well as pathogenic E. coli. In the experiments of Wullt
and coworkers mentioned above, it was acknowledged that the inflammatory responses induced in
urine were not observed systemically, where all
patients remained well (62). This, however, was an
experiment on the bladder of humans, and not on the
acknowledged target of Pap pili, the urothelium of the
renal pelvis and calyces, a fact that might explain the

observed lack of systemic response. Both type I and
Pap III/Forsmann antigen mechanisms have been
implicated in cystitis, but the interaction remains
obscure, as do the various forms of inflammatory
responses initiated during infection (37,67). Transmission of the organism and subsequent establishment of
clinical disease are clearly highly complex microbiological processes that remain minimally understood at
the present time.
Therapeutic Implications
The observations noted above might assist treatment
of affected individuals in a number of ways. Variation
in adhesion potential might identify at-risk groups.
Fimbriae would appear to be a rational target for
antimicrobial therapy and vaccine development. In
the veterinary piglets experiment referred to above,
effective vaccines based initially on the K-88 antigen
were developed and successfully prevented neonatal
diarrhea in the susceptible groups. Immunization
with purified P fimbriae has been attempted in a
number of animal models, with variable success. In
a primate setting, vaccination with a FimH adhesin
chaperone complex protected three of four treated
monkeys while all control animals developed cystitis,
suggesting that such techniques might have application in humans with chronic lower UTI (68). As might
be expected, phase variation and other types of antigen variability might be expected to cause problems in
this type of therapeutic approach.
Virulence Factors in Other Uropathogens

Not surprisingly, studies of other species involved in
UTI have demonstrated the presence of adherence
mechanisms. P. mirabilis and Klebsiella spp. have been
found to express fimbriae in animal experiments (69,70),
although the fimbrial expression of type I pili is considerably less than that of UPEC, which might explain why
Klebsiella is a less prevalent agent for UTI than the
former organism (39). Staphylococcus saprophyticus is
known to be able to adhere avidly to uroepithelial
cells, probably by nonspecific adherence mechanisms.
Summary of Virulence Factors in Urinary

Tract Infections
It is appropriate to bring together those factors that are
thought to be important with respect to the virulence
of gram-negative bacteria in general and E. coli in
particular (Fig. 18 and Table 9).
The antigenic structure of the bacterial surface is
classically described in terms of three classes of antigens. O-Antigens represent the polysaccharide side
chains of the lipopolysaccharide structure found in all
gram-negative bacteria. The polysaccharide is
anchored to the outer membrane by lipid A (Fig. 18),
Figure 18 Schematic diagram of cell-wall gram-negative bacteria and associated structures. Abbreviations: CM, cytoplasmic
membrane; OM, outer membrane. Note differential size of flagella and fimbriae. Lipid A (see text) is on the innermost aspect of
the lipopolysaccharide O antigen, next to the outer membrane
(see also Fig. 28).
Table 9 Virulence Factors Associated with Escherichia coli

Specific O serotypes
K capsular antigen
Adherence mechanisms
Resistance to serum bactericidal activity
Hemolysin production
Aerobactin production
Colicin V
145
the agent thought to be responsible for endotoxic

shock, as described below. O-antigens are heat stable
and classically certain serogroups (01, 02, 04, 06, 07,
016, 018, and 075) responsible for urinary infections,
such strains being responsible for up to 80% of cases
of pyelonephritis. Modern theory suggests that the
O-antigen is not itself specifically responsible for
pathogenicity; rather, the identified serogroups represent clones of organisms with a selection or panel of
various virulence properties that enable successful
colonization of the urinary tract. Other serogroups
and hence other combinations of virulence factors
may enable successful colonization of other areas
such as the gastrointestinal tract. K capsular antigen
is partially heat stable and may, on occasion, partially
obscure the O-antigen (Fig. 18). K capsular polysaccharide antigen has been strongly associated with
pyelonephritis for many years, both in adults (71)
and in children (72). Some 70% of strains from children with pyelonephritis were associated with K1, K2,
K3, K12, and K13 antigens, of which K1 is acknowledged to be the most frequently associated strain with
pyelonephritic disease. Interestingly, K1 strains have
also been associated with 80% of E. coli strains causing
neonatal meningitis (72).
Adherence mechanisms have been fully
described above, and the relationship between type
1 fimbriae and P fimbriae and their ability to cause
UTI have been noted. Typically, E. coli may be killed
by serum bactericidal activity relating to both the
classical and alternative pathway of complement.
Cell wall lipopolysaccharide (O-antigen) is thought
to provide a degree of resistance against complementmediated digestion, and, recently, Leying and associates have suggested that the K1 polysaccharide
antigen may also confer significant levels of serum
resistance on the organism (23). The advantages conferred on those organisms expressing hemolysin and
aerobactin have been noted above. Colicin V is another virulence factor that has selectively been found to
be present in isolates from urine, but not from isolates
in the fecal reservoir. Colicin V is assumed to interfere
with host defense mechanisms (19,20).
ORGANISMS RESPONSIBLE FOR URINARY

TRACT INFECTION
The great majority of UTIs are caused by single
bacterial species; among these, E. coli is by far the
most common, accounting for approximately 85% of
general or community-based infections at the present
time. Previous studies (Table 10) have emphasized the
differences between general practice and hospital
practice, where E. coli accounts for only approximately
50% of the isolates. Proteus, Klebsiella, enterococci, and
Pseudomonas are all more frequently isolated from
hospital patients, especially if an in-dwelling catheter
is present (73,74). Remaining organisms include Enterobacter, Citrobacter, and Serratia; fungal infections are
rarely encountered outside hospital practice. These
classical studies, though dated, serve to emphasize
the differing bacteriological environments between
146
George
Table 10 Organisms Causing Urinary Tract Infection in Community and Hospital Practice
General practice
Escherichia coli
Proteus mirabilis
Staphylococci
Streptococcus faecalis
Klebsiella spp.
Pseudomonas
Remainder
Hospital practice
1976
1971
1971
1978
72
9
6
3.3
2.7
7.0
78.5
9.2
5.1
2.3
2.3
2.6
55.4
11.4
3.3
4.0
16.8
2.7
6.4
50.7
10.6
2.7
4.3
21.6
2.8
7.3
Although the proportions of organisms have remained relatively similar, it is accepted that in the 1990s approximately 80% to 85% of
community UTI was related to E. coli. The comparable figure for hospital-based infections has remained steady at approximately 50%.
Source: Data from Refs. 73 and 74.
hospital and community that has only partially been

affected by the publicity surrounding the superbug
threats of MRSA and Clostridium difficile.
The presence of abnormalities in the urinary
tract, as may be found in congenital abnormality or
obstruction, often leads to infection by noncoliform
organisms, such as Proteus and Klebsiella, and, under
these circumstances, mixed infections are also frequently encountered. Naturally, obstruction related
to stone formation is often associated with P. mirabilis.
S. saprophyticus has been identified, particularly in the
United States of America, as a cause of acute cystitis in
young, sexually active females (75). Anaerobic organisms are rarely pathogens in the urinary tract. Maskell
et al. supported the concept that slow-growing CO2dependent, gram-positive bacteria might be responsible
for the urethral syndrome (76), although these suggestions that fastidious organisms could be responsible for the symptom complex were strongly denied by
other workers (77). Apart from lactobacilli, Gardinerella
vaginalis and Ureaplasma urealyticum are not infrequently
isolated, but their role in infection of the lower urinary
tract remains unproven.
Routes of Infection
Organisms may enter the urinary tract via the ascending route, the hematogenous route, or the lymphatic
route.
Ascending Route
The difference in the incidence of lower UTI between

men and women strongly suggests that the ascending
urethral route is the most common pathway for infection; in the female, organisms have been isolated from
the bladder after both urethral massage (78) and
sexual intercourse (79). One insertion of a urinary
catheter into the bladder has been observed to result
in lower UTI in 1% to 2% of patients (80). It is widely
agreed that the presence of a urethral catheter for
more than 36/48 hours almost invariably results in
bladder bacteriuria. Recent studies suggest that spermicidal agents encourage the colonization of the
introital region with uropathogenic bacteria (81). The
relationship between the introital flora and lower UTI

is further considered below.
The question of ascent into the upper tract from
the bladder in the absence of reflux appears problematical. Presumably, various virulence factors must aid
progression through the vesicoureteric junction; perhaps mucosal edema caused by local inflammation
disrupts the valvular mechanisms and allows passage
of organisms, which then successfully colonize the
upper tracts by means of the appropriate fimbriated
structures. Diuresis and loss of the usual coapting
ureteral mechanisms have also been suggested as a
mechanism whereby organisms may gain entry into
the upper tract.
Hematogenous Route
Blood-borne infection of the kidney is a welldescribed, though uncommon, mechanism of renal

infection in individuals who are otherwise normal.
Staphylococcal spread from dental abscesses may
occur, and these and other organisms may originate
from sites such as diseased heart valves. Hematogenous spread of Candida albicans has been observed in
experimental circumstances, although this fungus is
not usually observed unless a chronic in-dwelling
catheter is present. Interestingly, if one ureter is tied
and bacteria are introduced into the bladder during
experimentation on animals, infection supervenes in
the nonobstructed kidney, but not on the hydronephrotic side (82). This convincingly demonstrates the
overriding importance of the ascending route and
the relative resistance to hematological spread, even
in the presence of severe obstructionconditions
under which it is acknowledged that hematogenous
infection should easily supervene.
Lymphatic Route
Theoretically, infection could spread via lymphatics

into various parts of the urinary tract, but, in practice,
there is little clinical evidence for such a mode of
infection. Occasional experimental reports have failed
to convince that the lymphatic route is other than of
academic interest.
CLINICAL ASPECTS OF URINARY TRACT

INFECTION
The Significance of Significance
Historically, there have always been difficulties distinguishing between true bacteriuria, defined as actual
residence of bacteria within the urinary tract, and
contamination, defined as the adventitious entry of
bacteria into the urine during the collection of the
specimen. This problem was most notably tackled in
the mid-1950s by Edward H. Kass from Boston, who
performed a number of studies on women with various disorders of the lower urinary tract in both the
normal and pregnant state. In his most important
study, the urine of female patients attending as outpatients was studied, specimens being obtained by
catheterization and cultured promptly thereafter (83).
He found (Fig. 19) that the patients could broadly be
divided into two groups. In the first group were
patients with bacterial counts between 0 and
100,000/mL of urine; of these, only approximately
15% had a past history of UTI, instrumentation, or
catheterization of the urinary tract. He noted that the
bacteria obtained in this group were usually the
common saprophytes of the urinary tract, and his
contention that these were contaminated specimens
was further supported by the fact that second samples
obtained from the same group usually demonstrated
dissimilar organisms and counts.
By contrast, in the second group with more than
100,000 bacteria/mL, 55% had a past history of UTIs,
and repeat sampling in these patients revealed similarly high counts, both specimens yielding commonly
accepted pathogens of the urinary tract. These simple
yet ground-breaking observations define the level of
bacterial significance widely used to this day105
organisms or colony-forming units (CFU) per milliliter
from midstream urine.
For a quarter of a century, the scientific distinction between patients with contaminants and patients
with true infection was broadly welcomed. Gradually,
however, it became clear that there were a significant
147
number of women with dysuria and frequent urination whose midstream urines did not contain significant bacteriuria, and to these women the label acute
urethral syndrome was applied. In an important
study, Stamm et al. investigated 59 women with
acute urethral syndrome, from whom bladder urine
was obtained either by suprapubic aspiration or clean
urethral catheterization. Forty-two patients had
abnormal pyuria, and 37 of these were infected with
coliforms, S. Saprophyticus, or Chlamydia trachomatis.
Patients without pyuria had little demonstrable infection. Stamm et al. concluded that the classic Kass criteria
of 105 CFU/mL was an insensitive diagnostic criterion
when applied to symptomatic lower UTI in this group
of relatively young, sexually active women (84).
This and other similar studies (85) provoked a
flurry of editorial comment (86,87). Doubt was cast
over the suggestion that 102 CFU/mL organisms
could reliably discriminate between patients with
infected and uninfected lower urinary tracts. Additionally, it was noted that many midstream urine
cultures were mixedignoring the conventional wisdom that true pathogens are usually found in pure
culture. Nevertheless, it was acknowledged that
most of Stamms bacteriuric patients (102, 105) had
pyuria, suggesting that this was indeed a true infection
(60). Stamm himself made further comment and
reviewed the situation in 1984 (87). He pointed out
that the essence of Kasss original work (often forgotten)
concerned patients with pyelonephritis, not women
with acute frequency/dysuria lower tract symptoms.
He made a plea for closer communication between
clinicians and laboratory so as to obtain better information from the more flexible approach to quantitative
bacteriological sampling. There is little doubt that urologists should be aware that no significant growth
may mean different things according to definitions in
different bacteriological laboratories. It is the responsibility of each clinician to determine whether such a
report refers to 105 CFU/mL or 102 CFU/mLas usual,
optimum results result only from close cooperation
between clinical and laboratory service.
Figure 19 Bacterial counts in urine of various

population groups. Kass noted that most of the
patients with low counts did not have a history of
UTI, and in these cases reculture frequently demonstrated different organisms. However, patients
with >105 organisms frequently had pure cultures
associated with significant clinical infections
(pyelonephritis). Source: From Ref. 83.
148
George
The Introital Question

Another major microbiological debate concerns the
means whereby pathogenic organisms pass from the
(presumed) fecal reservoir to the lower urinary tract.
In particular, the importance of organisms that colonize the vaginal introitus and periurethral region has
been discussed at great length, and the debate continues to the present day.
There seems little doubt that E. colipathogenic
or otherwisemay obtain a foothold in the introital
area, and, in this respect, it has been noted above that
mannose-sensitive type 1 pili adhere powerfully to
vaginal epithelial cells (52), vaginal mucus (88) and
uromucoid (10). It has also been emphasized that type
1 fimbriae are to be found on nonpathogenic as well as
pathogenic enterobacteria.
Three essential questions may be asked.
1.
2.
3.
Do women at risk of UTIs carry pathogenic organisms in the introital and periurethral area?
If so, are these organisms responsible for the
symptomatic bladder or pyelonephritic infection?
In such cases, what is the state of the introital and
periurethral area between overt symptomatic clinical infections?
A number of workers support the concept that

abnormal periurethral flora are to be found in women
with recurrent infections. Hinmans group (89) studied 43 patients and found that the flora contained a
higher percentage of pathogenic microorganisms than
that of female subjects without urological disease. In a
general practice study from London, Gru neberg
observed that the infecting strain of E. coli was isolated
from rectal, vaginal, and periurethral flora in nearly
all cases. Furthermore, he noted that chemotherapy
eradicated the organism from the urine but not necessarily from the introital/urethral area (90). Stamey
studied cultures from 20 premenopausal controls
compared with cultures from nine women with recurrent urinary infections (91). He observed that not only
was introital colonization significantly higher in the
patients, but also enterobacteria persist after the infectious episode, and he postulated that the introital
mucosa in women with infection was biologically different from the same area in women who never suffer
from lower UTI. This postulate was supported some
years later by Pfau and Sacks (92), who had previously
shown that the predominant bacterial flora of the
introital and periurethral area consisted of lactobacilli
and staphylococci, gram-negative bacteria being infrequent and transitory. These workers found E. coli to be
the predominant microorganism recovered from 68% of
introital, 60% of vaginal, and 42% of urethral cultures.
Despite these apparently conclusive results by
American researchers, a number of British workers
failed to confirm the findings (9396). Nevertheless,
although an absolute association between periurethral
flora and lower UTI could not be demonstrated, it was
acknowledged that the presence of E. coli in the
introital area might constitute a permissive factor
for the subsequent development of overt infection
(93). Similarly, OGrady et al. could find no difference
in the carriage rate between normal women and
women with symptoms suggestive of UTI, although,
again, these workers acknowledged that introital bacteria were more commonly recovered in patients
when symptomatic (34%) than when symptom free
(19%)(94). In a further development, Brumfitt and
coworkers observed that women with recurrent urinary infections were susceptible to perineal and periurethral colonization with gram-negative bacteria, but
they noted that the infection need not be with the
colonizing enterobacteria (95). Kunin, in an editorial
comment, attempted to reconcile these positions (92)
by suggesting that most workers could agree that
infections were indeed preceded by colonization of
the periurethral area with gram-negative bacteria, but
he considered that the evidence for colonization of this
area between infections was less convincing. It may be
argued that the presence or absence of organisms is
not so critical as the ability of any organisms that may
be presentthat is, the virulence mechanism carried
on those organismsto ascend and invade the lower
urinary tract. In summary, colonization is important,
but the critical factor relates to the presence or absence
of essential virulence mechanisms.
Urinary Tract Infection: Variation by Age and Sex

It is appropriate at this point to review the changes that
may occur in the urinary tract of either sex as a result of
invasion by microorganisms. Table 11 records the prevalence of bacteriuria by age in either sex as determined
by studies in the literature. It is immediately apparent
that bacteriuria is more common in females at all times
of life, with the single exception of babies under three
months old, among whom boys are more than twice as
likely to have clinical infection than girls.
Table 11 Prevalence of Bacteriuria by Age (%)

Age (years)
<1=12
Male
0.075
Female
Reference
0.077
73
<3=12
<5
Circumcised, 0.07
0.5
Noncircumcised, 0.77
0.3
4.5
73
77
Young
men
0.03
<0.1
1.2
7882
1.3
83
47
82, 71, 85
35
80, 81
The identified groups of patients are fully discussed in the text.
Nonpregnant Pregnant
females
females
Pregnant females
(previous
bacteriuria)
School
age
6570
>80
23
>20
20
86
>20
86
149
Figure 20 Urinary tract infection in infants under one

year. The incidence of infection between sexes is
approximately similar under one month, but thereafter
girls have more infections than boys unless the boys
have not been circumcised. Circumcised; uncircumcised. Source: From Ref. 97.
Infants
In the first month of life, there is a virtually identical

incidence of UTI in girls and boys (97). Interestingly,
in infants under three months old, the infection rate in
males depends on whether circumcision has been
performed. For those who have had the operation,
the incidence of infection is significantly less than that
of girls of the same age. For uncircumcised infants,
however, the risk is considerably greater, and it is
clear that the presence of the foreskin is linked to an
increased incidence of lower UTI. These observations
were made in a remarkable study of 422,328 children
born to members of the U.S. services over a 10-year
period. Precision military record keeping allowed the
result of circumcision to be accurately documented
(Fig. 20). A surprising 80% of the male population
underwent circumcision, but the 20% who did not
suffered 70% of the male UTIs (97). The authors later
proved their point by noting that a subsequent decline
in the circumcision rate was associated with an
increased incidence of male infant infection (98).
Preschool Children
Urinary tract infection is important in children of this

age group, as most pediatricians consider renal development to be most at risk at this time. Both symptomatic
and asymptomatic infections are more common in girls,
although the line between the two is difficult to draw
careful history taking in children with asymptomatic
bacteriuria often reveals symptoms strongly suggestive
of UTI (99). From the first year onward, infections
become increasingly uncommon in male infants; indeed,
the presence of such an infection may indicate significant disease or other abnormality of the urinary tract.
Infections in girls may be troublesome and, as noted
above, permanent damage relating to the reflux of
infected urine may occur by mechanisms such as those

described by Ransley (100). It is difficult to avoid the
conclusion that bacteriuria is a very important finding in
this group of children (101).
Bacteriuria in Schoolchildren
As can be seen from Table 11, the problem of bacteriuria in schoolchildren relates almost entirely to girls.
There is an impressive body of evidence concerning
the nature and outcome of such infections. In Charlottesville, Virginia, an area with a stable local population, Kunin prospectively studied the characteristics
and natural history of UTI in schoolgirls between 1959
and 1968. It was observed that bacteriuria was common in schoolgirls and symptoms were often absent;
recurrence frequently occurred (102). Approximately
one-third of the girls had symptoms of the infection at
the time of detection. In the United Kingdom, Meadow et al. reported broadly similar findings in Birmingham schoolchildren. Infection in schoolboys was
essentially undetectable, but 1% of girls had significant ABU (103). In another important prospective
study, the Cardiff/Oxford bacteriuria study group
followed 208 girls from 5 to 12 years of age who had
been identified as suffering from bacteriuria. The girls
were followed for four years, and the authors noted
that treatment had little effect on the emergence of
symptoms, the clearance of vesicoureteric reflux, renal
growth, or the progression of renal scars. These observations seemed to suggest that renal damage had
occurred before five years of age, as noted above (104).
Effect of bacteriuria on subsequent pregnancy. Both
Kunins group and the Oxford/Cardiff group continued to follow their young women as they grew up and
eventually became pregnant. These irreplaceable studies have emphasized the importance of vigilance with
respect to young bacteriuric schoolgirls.
150
George
Gillenwater reported the U.S. results in 1979. Sixty

schoolgirls with bacteriuria and 38 matched controls
had been followed for periods ranging up to 18 years.
Renal scars and/or caliectasis were observed to occur
only in the bacteriuric group, but renal function and
blood pressure were not affected. The study group had
10 times as many bacteriuric episodes as did controls,
and infections were particularly common during subsequent pregnancy. Most interestingly, seven children of
the bacteriuric mothers, but none of the controls themselves, showed UTIs (105).
The Oxford/Cardiff group studied 52 pregnancies in 34 women who had been found to have
bacteriuria in childhood. At the first antenatal visit,
the prevalence of bacteriuria in the study group was
significantly greater (35%) than that in the control
group (5%). During pregnancy, pyelonephritis developed in 10% of the study group and in 4% of controls.
The data suggested that previously bacteriuric women
known to have renal scars were at increased risk of
hypertension and preeclampsia of pregnancy, findings that have not been universally accepted. No
comment was made about the children resulting
from these pregnancies and their susceptibility or
otherwise to UTI (106).
In summary, therefore, these observations show
that ABU in schoolgirls persists over long periods.
Subsequently, such young women are at greater risk
of infection during pregnancy, and if renal damage
has occurred in earlier life, the pregnancy may perhaps be complicated by hypertension or preeclampsia.
The children of such women appear to inherit an
ongoing susceptibility to bacteriuria and infection.
pregnant women who were bacteriuric as schoolgirls

carry a significantly greater risk of UTI during
pregnancy (104,105).
Urinary Tract Infection in Young Adult Men
As previously emphasized, UTI in otherwise healthy

adult men is very uncommon. Presumably, the large
difference in prevalence between men and women is
related to the length of the urethra and the difficulties
that face uropathogens attempting to reach the urethral meatus from the fecal reservoir. The antibacterial
nature of prostatic fluid is noted below. Presumably,
most infections that occur arise because of sexual
intercourse with an infected female partner, or, in
the case of homosexuality, direct contamination from
the fecal reservoir.
Older Patients
The bias in favor of female patients that operates

throughout most of life begins to reverse in old age.
Presumably because of prostatic obstruction, residual
urine, and other problems in the lower urinary tract
of older men, the incidence of bacteriuria rises steeply
to significant levels, particularly after the age of 70
(Table 11). It has been observed that the place of
residence has an important influence on the presence
or otherwise of bacteriuria. Older men living at home
have a lesser incidence of bacteriuria than those living
in nursing homes, where the prevalence in both sexes
is approximately 20%. Figures for those resident in
hospital inpatient facilities are even higher (110).
Uncomplicated Cystitis in Females
Young, Nonpregnant Females
It is generally accepted that the prevalence of bacteriuria in this group is approximately 1% per decade. To
investigate the assumption that these cases were
largely intercourse-related, Kunins group compared
the prevalence of significant bacteriuria in nuns and
married women. As expected, they found that celibacy
was associated with a lower frequency of infection, but
young nuns still had a higher frequency of urinary
infection than young males. He commented later that it
was not clear from this study whether there was a
subpopulation of women who were inherently susceptible to urinary infection that was not the result of
sexual intercourse (107).
Bacteriuria During Pregnancy
There is general agreement that 4% to 7% of pregnant

women have bacteriuria (108,109), and of these 20% to
40% will develop symptomatic infection later in the
pregnancy, usually in the third trimester. Bacteriuria
in the lower urinary tract more commonly leads to
pyelonephritis in pregnant than nonpregnant women,
presumably because of the various changes that occur
in the upper tracts as a result of the pregnancy. The
prevalence of bacteriuria during pregnancy rises with
parity, sexual activity, age, diabetes mellitus, and
sickle-cell trait. It has already been mentioned that
Approximately 20% of women experience an episode

of simple cystitis during their lifetime. Most of these
settle rapidly, but 2% to 3% suffer from repeat infections. In a Danish study, nonpregnant women 16 to
65 years of age were referred to the medical outpatient
clinic, where a placebo study of patients with acute
symptomatic lower UTI was undertaken. Fifty-three
female patients were given placebo and were followed
for longer than 12 months following the initial infection;
43 of these (81%) spontaneously cleared their urine
within 5 months (111). Unfortunately, nearly half
these patients became reinfected within a year, and
similar observations were found in the antibiotic-treated
group. The author concluded that host defense and
eradication mechanisms could be very effective, but to
keep recurrence of bacteriuria to a minimum, it would
be necessary to recheck urine samples for at least six
months after the initial elimination of bacteriuria.
Typically, uncomplicated lower UTI is caused by
E. coli in 80% of domicillary cases. S. saprophyticus may
be implicated in up to 5% of cases, this organism being
particularly noted in the literature from North America.
The causes of the acute urethral syndrome, as reported
by Stamm et al., have already been noted (84). It seems
reasonable that a pure growth of organism at concentrations between 102 and 105 accompanied by pyuria,
should be accepted as a case of true cystitis. The case of
bladder urine with mixed organisms and equivocal
pyuria is much more debatable; these may perhaps be

better described as equivocal cystitis, in contradistinction to the true urethral syndrome described below in
which the symptoms of urethral irritation are accompanied neither by organisms nor by pyuriathe female
equivalent of prostatodynia.
The Urethral Syndrome
A critical definition of the female urethral syndrome

refers to the symptoms of frequency and dysuria in
the absence of bacteriuria and pyuria in both initial
[voided bladder 1 (VB1)] and midstream (VB2) urine
when analysis is made on several separate occasions.
This definition assumes that such female patients have
been thoroughly screened to exclude vesical motor
dysfunction (bladder overactivity) as well as systemic
and local (such as trauma, tumor and irradiation
injury) disease (112).
A number of workers have stressed the importance of pyuria in patients with such urethral symptomatology. OGrady et al. (113) proposed that pyuria
was significant even when tests failed to identify
bacteriuria. In their studies, if cultures were performed over extended periods, bacteria were eventually identified; thus, this patient group was designated
as being between infections (113). C. trachomatis was
identified in 10 of 16 patients with pyuria, but in only
one of 16 patients without white cells in the urine (57).
The controversy surrounding more fastidious carbon
dioxidedependent organismschiefly lactobacilli
has also been noted (76). In summary, although considerable effort may be required, the presence of
pyuria may often give a clue as to the true bacteriological cause of the frequency dysuria syndrome.
Those who have neither bacteriuria nor pyuria constitute the essential core of those who are said to suffer
from the true urethral syndrome.
Pyelonephritis
The clinical, radiographic, and therapeutic aspects of

acute and chronic pyelonephritis are beyond the remit
of this work. The important virulence factors that
enable colonization of the upper tract have been
noted, as have those factors in childhood that later
predispose to pyelonephritis of pregnancy. It is generally agreed that uncomplicated pyelonephritis in
adultsas opposed to infants under five yearsrarely leads to permanent and progressive renal damage
with scarring. Renal function usually remains stable.
Stones and Infection

Most urinary infections are caused by E. coli, but a
substantial minorityaround 10%may be because
of P. mirabilis, which is also found in the normal fecal
flora. Most patients with simple Proteus infections do
not form stones, but stone formation is a risk, this
being particularly high when stones are already present. Urease-producing bacteria, of which Proteus is the
most notable genus, may split urea into ammonia and
carbon dioxide, with alkalization of the urine and
151
precipitation of crystals of magnesium, ammonia,

and calcium phosphate, triple phosphate, leading
to stone formation. Such struvite stones may grow
rapidly in infected urine, and a vicious circle ensues
whereby the organisms themselves are trapped within
the stone safe from the action of antibiotics and the
natural host defense mechanisms. Urine cultures at
these times may show pyuria but, misleadingly, no
bacterial growth. Hence, removal of the stone is an
essential part of the treatment of patients with Proteus
and other urea-splitting UTIs.
Infections of the Male Genital Tract

Prostatitis
Until relatively recently, considerable confusion surrounded the symptom complex of men thought to
have prostatitis. In no small part, this was related to
problems of terminologyvarious authorities were
describing aspects of prostatitis in the literature but
calling these symptom complexes by different names
such as pelvic floor tension myalgia. As a result, no
one was sure exactly who was investigating which
group of patients.
This situation was clarified by Drach et al., who
suggested a classification in a letter to the Journal of
Urology (114), which was accepted by most workers
in the field. Subsequently, this suggestion was in large
part taken up by an NIH consensus classification in
1998 as illustrated in Table 12.
These four conditions have many symptoms in
common, but they are also distinguished by specific
clinical and microbiological features. Successful treatment in each group depends on meticulous attention to
diagnostic detail, without which failure is inevitable.
Organisms responsible for acute bacterial and
chronic bacterial prostatitis are generally similar and
resemble those organisms responsible for lower UTI.
The majority are grown in pure culture; most often,
E. coli is isolated, while institutionalized patients may
harbor more virulent organisms such as Pseudomonas
or Streptococcus faecalis. It has been suggested that the
(rare) episodes of cystitis that occur in young men are
all secondary to infection of the prostatic ducts.
Etiological factors thought to be important in
acute or chronic bacterial prostatitis include ascending
urethral infection and reflux of infected urine into
ejaculatory and prostatic ducts. Blacklock (115) noted
Table 12 The Classification of Drach et al. and Subsequent

Adjustments by the NIH Consensus Committee (1998)
Drach et al.
Clinical descriptor
1
2
3
Acute bacterial prostatis

Chronic bacaterial prostatis
Nonbacterial prostatitis
Inflammatorya
Prostatodyniab noninflammatory
Asymptomatic inflammatory
4
a
NIH Consensus
I
II
III
IIIA
IIIB
IV
WBC semen > 106/mL, WBC EPS > 5p hpf, WBC VB3 > 10p hpf.
Drach defined as literally prostatic pain without evidence of inflammation.
b
152
George
that patients with chronic bacterial prostatitis frequently had the same pathogens as identified in
vaginal cultures of their sexual partners. Kirby et al.
injected carbon particles into the bladders of men
about to undergo transurethral resection and found
the particles within the prostate on later histological
examination, thus proving that significant intraprostatic reflux had taken place (116).
evident, diagnostic and analytical care is required to

make an exact diagnosis by Drach/NIH criteria. The
schema illustrated (Fig. 21) are still necessary to isolate
conventional bacteriological organisms, but modern
point of care tests based on molecular techniques have
largely replaced standard culture methodologies for
C. trachomatis, as described in chapter 36. The prevalence of this organism is discussed below.
Localization of Infection
Examination of Prostatic Fluid
It is evident that with a unified genitourinary tract

emerging at the urethral meatus, specimens obtained
there may relate to urethral, prostatic, or bladder
infections. To overcome this difficulty, specific techniques have been developed for localization of infection
(117). A possible scheme for carrying out such studies
is illustrated in Fig. 21. The localization tests are not
difficult, but they do require attention to detail and a
state of preparednessfailure to attend to such detail
usually results in negative findings and a disappointed tertiary referral.
Following arrival in the clinic, the patient passes
a VB1 specimen (10 mLvoided bladder 1). This is a
washout specimen and relates to urethral disease.
After passage of a further 100 to 200 mL, the patient
collects a standard specimen of midstream urine
(VB2). Following this, the prostate is massaged in
the usual fashion, and prostatic secretion is collected
for analysis, as described below. Subsequently, the
next voided 10 mL is collected for further examination
(VB3), and, within this specimen, organisms of prostatic origin may be cultured. Hence, by comparison of
specimens obtained from urethral urine, bladder
urine, and postmassage urine, it is generally possible
to localize the source of the patients infection. As is
Examination of material obtained after prostatic massage is an important step in the diagnosis of genital
infection. In general, cases of prostatic inflammation
are found to have more than 15 white cells per highpowered field when such microscopy is performed; as
noted above, it is important to check that urethral and
bladder specimens do not have similar levels of
pyuria. A number of biochemical examinations may
be made of expressed prostatic fluid (Fig. 22). The pH
of the fluid, normally around 6.5, becomes alkaline as
a result of decreased levels of citric acid (119). Zinc
levels also reduce significantly (118), this element
having previously been known as prostatic antibacterial factor because of its potent bactericidal action on
most bacteria capable of causing UTI. It is not clear,
however, whether these changes are the cause or the
result of bacterial infection of the prostate gland.
Nonbacterial Prostatitis
The cause of nonbacterial prostatitis is essentially

unknown. Meticulous investigations may reveal
pyuria, but no positive cultures will be obtained by
the selective methodology described above. It is not
clear whether the symptom complexwhich is
Figure 21 Possible investigation schedule for female (A) and male (B) patients with possible urogenital infection. The tests are time
intensive and require meticulous planningsee text for details.
153
Figure 22 Differences in expressed prostatic secretion composition (EPS) between normal samples and men with prostatitis. Circles:
median values; boxes: central 50% of samples. Error bars 10th/90th percentile. All differences significant (P < 0.001). Infection causes a
rise in pH but a decrease in citrate and zinc concentration. See text for details. Source: From Ref. 118.
Figure 23 The spectrum of microbiological activity in expressed prostatic secretion. Anticipated levels of both organisms and white
cells in the various clinical groups as classified by Drach and NIH criteria (see text). The major clinical problems relate to patients with
nonbacterial prostatitis and prostatodynia.
similar to chronic bacterial prostatitisis related to

infectious disease by an unidentified pathogen or is a
noninfectious inflammatory process (Fig. 23). A number of causes of nonbacterial prostatitis have been
discussed in the literature, including previous antibiotic therapy, viral infection (herpes), U. urealyticum,
chemical inflammatory processes, and autoimmune
disease. The role of C. trachomatis is controversial.
154
George
Mardh et al. studied 53 patients with nonbacterial

prostatitis and found only one positive chlamydial
isolate (120), and most other investigators have been
similarly unsuccessful in identifying this organism in
prostatic fluid or even in prostatic aspirates obtained
by direct transrectal ultrasound-guided needle puncture of the gland (121). Nevertheless, identification of
this agent as the causative organism of epididymitis in
younger men (Figs. 24 and 25) raises questions as to
how the infection reaches the epididymis. In this
study, a tender, swollen epididymis was associated
with chlamydial infection in 19 of 23 patients (83%) 15
to 25 years of age, and these findings were consolidated by the observation that 9 of 12 consorts were also
positive for this infection (122). Presumably, these
observations are reconciled by assuming that the
organism may pass from urethra to epididymis, but

the biological environment within the prostate ducts
themselves is hostile to this agent. Further defined
studies using the new diagnostic techniques noted
below should resolve this question.
Prevalence and Diagnois of Chlamydia trachomatis
Infection rates with C. trachomatis have markedly

increased in recent years. Younger sexually active
males (1829 years) and females (1624 years) are, as
expected, the main source of the infection (see Fig.10
of chapter 36, page 554), 2% to 6% of the population
under the age of 25 being infected by the organism
(123,124). The alarming increase in prevalence has
prompted the U.K. Department of Health to institute
a National Chlamydia Screening Programme (NCSP)
within the United Kingdom as described in chapter 36.
In part, the increase in prevalence might be because of
better health awareness, increased sporadic screening,
or more sensitive diagnostic tests (125). The new rapid
point of care tests in particular are expected to make a
significant impact on both immediate treatment and
contact tracing (126).
Antibiotic Penetration in Prostatitis
Figure 24 Age distribution of patients with epididymitis according to recovered organism. negative for Chlamydia trachomatis, positive for C. trachomatis. Source: From Ref. 97.
The specific physicochemical characteristics of the

prostate and prostatic fluid govern the penetration
of antibiotics into the organ. To penetrate the lipid
membrane of the prostatic epithelium, a drug must be
lipid soluble; furthermore, the acid-base characteristic
of the drug will determine the concentration of the
antibiotic in the fluid. Experiments on normal dog
prostates (secretion pH 6.4) demonstrate that only
bases are able to penetrate successfully into prostatic
fluid. However, as noted above, during infection of
the human prostate, pH becomes more alkaline and
the dissociation gradient may reverse. Nevertheless,
fat-soluble bases, such as erythromycin and trimethoprim, are effective in acute or chronic bacterial
prostatitis despite experimental evidence, which suggests that concentration of the drug should deteriorate as the pH of the expressed prostatic fluid
becomes more alkaline.
Tuberculosis
Figure 25 Tuberculous epididymitis removed from 38-year-old

white patient; histology revealed florid tuberculous changes
within the descending genital tract.
Tuberculosis remains an important disease worldwide

with an estimated 8 to 10 million new cases per
annum. The incidence of the disease in its pulmonary
form is increasing steadily, particularly in certain
ethnic groups and geographical locations. A national
survey in England and Wales in 1998 identified an
annual rate of 10.9 per 100,000 population, representing increases of 11% and 21%, respectively, over
similar surveys undertaken in 1993 and 1988 (127).
However, these figures mask a more complex
picture; there has been relatively little change in most
regions, but a 71% increase in London and other
major urban areas with significant ethnic population.
The per 100,000 rate among the whole population
155
Table 13 Annual Number of Patients with Tuberculosis and Rate of Disease in England and Wales by Ethnic Group
Annual number cases/rate per 100,000
Ethnic group
1988
1993
1998
White
Indian subcontinent
Black African
Black Caribbean
Chinese
2504/5.36
1784/132
77/64.4
137/29.4
48/36.2
2267/4.78
2101/128
355/151
104/21.6
41/30.7
2108/4.38
2141/121
743/210
125/26.4
103/77.3
Note the relatively stable data in whites and patients from the Indian subcontinent but marked increases within the Chinese and Black African groups,
especially those recently arrived from their native countries.
(4.4) was many times lower than that of immigrants

from the Indian subcontinent (121), while significant
increases were recorded for groups of black Africans
(210) and Chinese (77.3) origin (Table 13). As
expected, recent immigrants had a higher incidence
of the disease than those who had arrived more than
five years previously (127), and the demographic
profile of the disease shows wide variation between
ethnic groups (Fig. 26). Nonpulmonary disease in
developed countries presents chiefly as lymph
adenopthy or genitourinary disease (128), the latter
accounting for approximately 25% of cases surveyed

in North America and the United Kingdom. Paradoxically, in these populations, genitourinary tuberculosis
is less common in ethnic minority groups (approximately 5%) than in white Europeans (27%), although
these observations might be related to age differences
at diagnosis (129). Approximately 3% of all adults with
pulmonary or nonpulmonary disease are coinfected
with HIV (130).
Clinical Features
Descriptions of the classical presentation of genitourinary tuberculosis are to be found in clinical textbooks;
some, however, are worthy of note to urologists.
Disseminated disease may affect patients with renal
or other types of organ transplantation, a situation
further complicated by therapeutic immunosuppression (131). Prophylactic chemotherapy (isoniazid) has
been demonstrated to be of benefit in such high-risk
renal transplant situations. In one study, 6 of
27 patients without prophylaxis developed disease
while no treated patients became infected (132).
Length of treatment prophylaxis is probably best
linked to the necessary duration of immunosuppression. Rarely, patients with chronic renal failure may
develop genitourinary tuberculosis and hypercalcaemia (133). Usually, raised calcitriol levels are more
commonly found in patients with disseminated but
nongenitourinary disease, probably because of active
synthesis of vitamin D by activated macrophages
within granulomas.
Laboratory Diagnosis
Figure 26 Age and sex distribution of proportion of patients with

tuberculosis, comparing (top) ethnic white population with (bottom) black African patients. Note very different age distribution
and rate per 100,000 population. : male; : female; : male
rate; : female rate. Source: From Ref. 127.
The great majority of cases are infected by the human

tubercle bacillus, Mycobacterium tuberculosis. M. bovis is
now rarely (1%) a cause of disease although it may be
isolated in reactivated dormant disease or that associated with HIV infection. Bacillus Calmette-Guerin
vaccine strain-associated lesions are now regularly
encountered by laboratories in those centers where
intravesical immunotherapy of superficial bladder
cancer is widely practiced.
Classically, the diagnosis of genitourinary tuberculosis was made by culture on solid LowensteinJensen median, the process taking up to six weeks.
In cases with a high index of clinical suspicion,
modern liquid culture systems may deliver a diagnosis
156
George
in half this time. In recent years, polymerase chain

reaction (PCR) amplification techniques have been
investigated for the diagnosis of both pulmonary and
genitourinary tuberculosis and have been found to
deliver good sensitivity and specificity (135,136).
Other workers have attempted to use the technique to
diagnose active pulmonary tuberculosis in HIV patients
by means of urine analysis (134). It was concluded that
PCR diagnostics may provide much faster confirmation
of disease (2448 hours), although the intermittent
excretory nature of M. tuberculosis may lead to falsenegative results, and urine concentration techniques
may be required to reduce this error (135).
To an extent, this diagnostic divide is a good
example of the difference between an efficacy trial
(what works in perfect conditions) and an effectiveness trial (what works in the real world) (136). It is
well known that techniques that function satisfactorily
with dedicated researchers and refined techniques
perform poorly when applied to mass populations
and routine laboratories. PCR diagnostics are suspect
at organism concentrations of 103 CFU/mL (roughly
the lower detectable limit for light microscopy), and
for most situations a high index of clinical suspicion,
effective communication, and modern culture systems
leads to optimal outcomes in the search for M. tuberculosis in the genitourinary tract.
Bacteraemia and Septic Shock

Septic shock is a relatively common and extremely
serious complication of infection, usually of gramnegative origin. In urological practice, the complication is most often encountered in the management of
stones, usually complex in nature and located in the
upper tract. Septicemic shock, however, may occur
after apparently simple and uncomplicated instrumentation of the urinary tract.
Figure 27 illustrates organisms isolated from
blood cultures, and the surgical procedures involved
are taken from a typically busy stone service. The
endotoxin, which may be encased within the stone
(137), may not always respond to appropriate antibiotic prophylaxis (138). The toxin lies between the
outer membrane and the core oligosaccharide that
makes up the O-serotype antigen common to gramnegative bacteria (Fig. 28). Known as lipid A, this
lipopolysaccharide may trigger release of large
amounts of cytokines, such as tumor necrosis factor
and the interleukins, which participate in the classically described cascade illustrated in Fig. 29. A full
description of the sepsis syndrome is beyond the
scope of this text, but urologists will be aware of the
urgent need to maintain adequate tissue profusion by
volume replacement at the same time as instituting
appropriate antimicrobial therapy as judged by
repeated blood cultures if necessary.
Papilloma Viruses and Cancer

Human papilloma viruses (HPV) are a group of DNAcontaining viruses numbering over 100 types, which
stimulate rapid cell division. External genital warts
Figure 27 Organisms (A) recovered from blood cultures following procedures (B) carried out in a busy interventional stone
center. Unfortunately, septic shock occurs in a significant number of these cases.
are most frequently caused by HPV types 6 and 11

(Fig. 30). Other HPV types (mainly 16 and 18, less
often 31 and 33) are frequently present in the anogenital region and have been associated with the development of high-grade, premalignant cervical lesions
[cervical intraepithelial neoplasia (CIN) III] as well as
anogenital cancer, principally of the anus and vulva.
Types 16 and 18 are thought to cause over 70% of
squamous cell cancers while type 18 may be responsible in 50% adenocarcinomas from the endocervix.
DNA sequences from such HPVs are detectable in
the majority of cervical and anal tumors; PCR studies
of apparently normal cervical smears showed
increased levels of sequences from high-risk virus
types, and subsequent colposcopy confirmed the
Figure 28 Detailed structure of the O polysaccharide antigen

associated with Escherichia coli. Lipid adjacent to the outer
membrane (lipid A) is thought to be responsible for the manifestations of endotoxic shock.
157
Figure 30 Exophytic papilloma virus, usually caused by type 6

or type 11 infection.
to be important. Early reports suggested high efficacy in

women not previously exposed to virus (preintercourse) although later studies were less clear-cut, generating considerable controversy (140,141).
The vaccines (Gardasil, MSD, Cervarix GSK) naturally are not active against existing HPV infection or
established cervical neoplasia; Gardasil alone, however,
is active against the genital wart types 6 and 11. The
cost, marketing, and ethicswhere vaccination has to
be given to girls not yet exposed to virus (sexually
inactive, target 1213 years)continue to generate comment on both sides of the Atlantic (142,143).
Early surveys involving PCR assays for HPV
DNA in squamous cell carcinoma of the penis have
been reported. About half of patients with Basaloid
and warty squamous cell carcinomas were found to be
associated with HPV 16 and 18. The role of vaccines in
such disease has yet to be determined (144).
Localization Studies in the Urinary Tract
Figure 29 The classical cascade described in endotoxic septicemic shock. Abbreviations: ARDS, adult respiratory distress
syndrome; DIC, disseminated intravascular coagulation;
MODS, multiple organ dysfunction syndromee.
presence of underlying high-grade CIN III (139). Not

all women infected with high-risk HPV types develop
cancer, and the reason for this discrepancy remains
unclear at the present time.
Recombinant vaccines have been developed
against the higher risk type 16 and 18 viruses based
on L1 protein shell particles; however, at least
15 oncogenic types have been identified, which lead
to neoplastic transformation under certain circumstances, and hence routine cervical screening will continue
It is pertinent to summarize the various localization

studies that may be employed to identify the site of
infection of any particular organism. When efficiently
and accurately performed, such studies can be of great
help to the urologist attempting to identify the source
of organisms within the urinary tract.
Studies on prostatic fluid described by Meares
and Stamey (117) have already been noted. Stamey has
also refined localization studies designed to determine
the source of bacteriuria emanating from the upper
urinary tract. Essentially, this test involves ureteric
catheterization of the appropriate unit in question
after meticulous steps to eradicate bacteria from the
bladder that would otherwise contaminate the ureteric
sample. Such techniques have been equally useful when
attempting to determine the source of cells with abnormal cytological features. Earlier methods for differentiating kidney from bladder infections (Fairley bladder
washout test) have fallen into disuse, although such
studies can, on occasion, be clinically helpful.
158
George
Summary of Definitions
Adherence
Adhesin
Bacteriuria
Contamination
Fitness
GAL-GAL
Pathogenicity
Phase variation
Tropism
Virulence
Initial interaction of microorganism with host

Microbiological molecule that leads
to adhesion to cells or tissues
The residence of bacteria within
the urinary tract
Adventitious entry of bacteria during urine collection
The ability of bacteria to establish
and maintain a population in a
specific ecological habit
Essential core of disaccharide
receptor for P fimbriae
The ability to cause disease
Variation of virulence antigens,
often to protect organism
The restriction of commercials and
pathogens to certain host tissues
and cell types
The degree of pathogenicity
REFERENCES
1. Lincoln K, Lidin-Janson G, Winberg J. Resistant urinary
infections resulting from changes in resistance pattern of
faecal flora induced by sulphonamide and hospital environment. BMJ 1970; 3:305309.
2. Finegold SM, Mathisen GE, George WL. Changes in
human intestinal flora related to administration of antimicrobial agents. In: Hentges DJ, ed. Human Intestinal
Microflora in Health and Disease. New York: Academic
Press, 1983:355446.
3. Kaye D. Antibacterial activity of human urine. J Clin
Invest 1968; 47:23742390.
4. Cox CE, Hinman F, Jr. Experiments with induced bacteriuria, vesical emptying and bacterial growth on a mechanism of bladder defence to infection. J Urol 1961; 86:
739748.
5. Parsons CL, Greenspan C, Mulholland SG. The primary
antibacterial defence mechanism of the bladder. Invest
Urol 1975; 13:7276.
6. Parsons LC, Shrom SH, Hanno PM, et al. Bladder surface
mucinexamination of possible mechanisms for its antibacterial effect. Invest Urol 1978; 60:196200.
7. Parsons CL, Mulholland SG, Anwar H. Antibacterial activity
of bladder surface mucin duplication by exogenous glycosaminoglycan (heparin). Infect Immun 1979; 24:552557.
8. Parsons CL, Greenspan C, Moore SW, et al. Role of surface
mucin in primary antibacterial defense of bladder. Urology
1977; 9:4852.
9. Tamm I, Horsfall FL. A mucoprotein derived from human
urine which reacts with influenza, mumps and Newcastle
disease viruses. J Exp Med 1952; 95:7197.
10. Orskov I, Ferencz A, Orskov F. TammHorsfall protein or
uro-mucoid is the normal urinary slime that traps type I
fimbriated Escherichia coli. Lancet 1980; 1:887.
11. Pak J, Pu Y, Xhang ZT, et al. TammHorsfall protein binds
to type I fimbriated E. coli and prevents E. coli from
binding to uroplakin 1a and 1b receptors. J Biol Chem
2001; 276:99249930.
12. Jost SP. Cell cycle of normal bladder urothelium in
developing and adult mice. Virchows Arch B Cell Pathol
Incl Mol Pathol 1989; 57:2736.
13. Mulvey MA, Lopez-Boado YS, Wilson CL, et al. Induction

and evasion of host defences by type I pilated uropathological E. coli. Science 1998; 282:14941497.
14. Klump DJ, Weiser AC, Sangupta S, et al. Uropathogenic
E. coli potentiates type I pilus induced apoptosis by suppressing NF-kappaB. Infect Immun 2001; 69:66896695.
15. Mulvey MA. Adhesion and entry of uropathogenic
Escherichia coli. Cell Microbiol 2002; 4:257271.
16. Gunther NW, Lockatelle V, Johnson DE, et al. In vivo
dynamics of type I fimbria regulation in uropathogenic
Escherichia coli during experimental urinary tract infection. Infect Immun 2001; 69:28382853.
17. Mulvey MA, Schilling JD, Hultgren SJ. Establishment of a
persistent E. coli reservoir during the acute phase of
bladder infection. Infect Immun 2001; 69:45724579.
18. Mayer KH, Opal SM, Mederios AA. Mechanisms of
antibiotic resistance. In: Mandell GL, Douglas, Benett JE,
et al., eds. Principles and Practice of Infectious Diseases.
Edinburgh: Churchill Livingstone, 1995:212224.
19. Cooke EM, Ewins SP. Properties of strains of Escherichia
coli isolated from a variety of sources. J Med Microbiol
1975; 8:107111.
20. Minshew BH, Jorgenson J, Swanstrum M. Some characteristics of E. coli strains isolated from extra-intestinal infections of humans. J Infect Dis 1978; 137:648654.
21. Johnson JR. Virulence factors in E. coli urinary tract
infection. Clin Microbiol Rev 1991; 4:80128.
22. Mayer TF. Pathogenic neisseriaea model of bacterial
virulence and genetic flexibility. Int J Microbiol 1990;
274:135154.
23. Leying H, Suerbaum S, Kroll H P, et al. The capsular
polysaccharide is a major determinant of serum resistance
in K-1 positive blood culture isolates of Escherichia coli.
Infect Immun 1990; 58:222227.
24. Jacobson SH, Hammarlind M, Lidefeldt KJ, et al. Incidence of aerobactin-positive Escherichia coli strains in
patients with symptomatic urinary tract infection. Eur J
Clin Microbiol Infect Dis 1988; 7:630634.
25. Costerton JW, Geesey GG, Cheng K J. How bacteria stick.
Sci Am 1978; 238:8695.
26. van Oss CJ, Good RJ, Chaudhury MK. The role of Van de
Waals forces and hydrogen bonds in hydrophobic interactions between biopolymers and low energy surfaces.
J Colloid Interface Sci 1986; 3:378384.
27. Gibbons RJ, van-Houte J. Bacterial adherence in oral
microbial ecology. Ann Rev Microbiol 1975; 29:1941.
28. Mobley HLT, Chippendale GR, Tenney JH, et al. Expression of type I fimbriae may be required for persistence of
Escherichia coli in the catheterised urinary tract. J Clin
Microbiol 1987; 25:22532257.
29. Roberts JA. Tropism in bacterial infections: urinary tract
infections. J Urol 1996; 156:15521559.
30. Senior D, Baker N, Cedergren B, et al. Globo-Aa new
receptor specificity for attaching Escherichia coli. FEBS Lett
1988; 237:123127.
31. Lindstedt R, Larson G, Falk P, et al. The receptor repertoire defines the host range for attaching Escherichia coli
strains that recognise Globo-A. Infect Immun 1991;
59:10861092.
32. Langermann S, Palaszynski S, Barnhart M, et al. Prevention of mucosal E. coli infection by FimH adhesin based
systemic vaccination. Science 1997; 276:607611.
33. Choudhury D, Thompson A, Stojanoff V, et al. Xray structure of the FimCFimH chaperoneadhesin complex from uropathogenic E. coli. Science 1999; 285:
10611066.
34. Zhou G, Mo WJ, Sebbel P, et al. Uroplakin is the urothelial
receptor for uropathogenic E. coli: evidence from in vitro
FimH binding. J Cell Sci 2001; 114:40954103.

35. Sunn TT, Zhao H, Provet J, et al. Formation of asymmetric
unit membrane during urothelial differentiation. Mol Biol
Rep 1996; 23:311.
36. Martinez TS, Mulvey MA, Schilling JD. Type I pilus
mediated bacterial invasion of bladder epithelial cells.
EMBO J 2000; 19:28032812.
37. Baorto DM, Gao Z, Malaviya R, et al. Survival of FimH
expressing enterobacteria in macrophages relies on glycolipid traffic. Nature 1997; 389:636639.
38. Berry RE, Klumpp DJ, Schaeffer AJ. Urothelial cultures
support intracellular bacterial community formation by
uropathogenic E. coli. Infect Immun 2009; 77:27622772.
39. Rosen DA, Pinkner JS, Jones JM, et al. Utilization of an
intracellular bacterial community pathway in Klebsiella
pneumoniae UTI and the effects of FimK on type I pilus
expression. Infect Immun 2008; 76:33373345.
40. Schwan WR, Lee JL, Lenard FA, et al. Osmolarity and pH
growth conditions regulate fim gene transcription and
type I pilus expression in uropathogenic E. coli. Infect
Immun 2002; 70:13911402.
41. Gunther WW, Synder JA, Lockatell V, et al. Assessment of
virulence of uropathogenic E. coli type I fimbrial mutants
in which the invertible element is phase locked on or off.
Infect Immun 2002; 70:33443354.
42. Xia Y, Gally D, Forsman-Sembk, et al. Regulatory cross
talk between adhesin operant in E. coli: inhibition of
fimbriae expression by the PapB protein. EMBO J 2000;
19:14501457.
43. Lane MC, Simms AN, Mobley HLT. Complex interplay
between Type I Fimbrial Expression and Flagellum mediated motility of uropathogenic E. coli. J Bacteriol 2007;
189:55235533.
44. Eisenstein BI. Phase variation of type I fimbriae in Escherichia coli is under transcriptional control. Science 1981; 214:
337339.
45. Lindberg F, Lund B, Johansson L, et al. Localisation of the
receptor-binding protein adhesin at the tip of the bacterial
pilus. Nature 1987; 328:8487.
46. Roberts JA, Kaack MB, Baskin G, et al. Epitotes of the
P-fimbrial adhesin of E. coli cause different urinary tract
infections. J Urol 1997; 158:16101613.
47. Lane MC, Mobley HLT. Role of P-fimbrial-mediated
adherence in pyelonephritis and persistence of uropathogenic E. coli (UPEC) in mammalian kidney. Kidney Int
2007; 72:1925.
48. Holden NJ, Gally DL. Switches, cross-talk and memory in
E. coli adherence. J Med Microbiol 2004; 53:585593.
49. Svanborg, Eden C, Hanson LC5, Jodal U, et al. Variable
adherence to normal human urinary tract epithelial cells
of Escherichia coli strains associated with various forms of
urinary tract infection. Lancet 1976; 2:490492.
50. Eden CS, Hansson HA. E. coli pili as possible mediators of
attachment to human urinary tract epithelial cells. Infect
Immun 1978; 21:229237.
51. Fowler JE, Stamey TA. Studies of introital colonisation in
women with recurrent urinary tract infectionsrole of
bacterial adherence. J Urol 1977; 177:472476.
52. Schaeffer AJ, Jones JM, Dunn JK. Association of in vitro
Escherichia coli adherence to vaginal and buccal epithelial
cells with susceptibility of women to recurrent urinary
tract infections. N Engl J Med 1981; 304:10621066.
53. Sellwood R, Gibbons RA, Jones GW, et al. A possible basis
for the breeding of pigs relatively resistant to neonatal
diarrhoea. Vet Rec 1974; 95:574575.
54. Sellwood R, Gibbons RA, Jones GW, et al. Adhesion of
enteropathogenic Escherichia coli to pig intestinal brush
borders: the existence of two pig phenotypes. J Med
Microbiol 1975; 8:405411.
159
55. Rutter JM, Burrows MR, Sellwood R, et al. A genetic basis

for resistance to enteric disease caused by E. coli. Nature
1975; 257:135136.
56. Buckwalter JA, Naifeh GS, Auer JE. Rheumatic fever and
the blood groups. BMJ 1962; 2:10231027.
57. Kallenius G, Mollby R, Svenson SB, et al. Occurrence of Pfimbriated Escherichia coli in urinary tract infections. Lancet 1981; 2:13691372.
58. Johnson JR, Roberts PL, Stamm WE. P-fimbriae and other
virulence factors in Escherichia coli urosepsis: association
with patients characteristics. J Infect Dis 1987; 156:
225228.
59. Virkola R, Westerlund B, Holthofer H, et al. Binding
characteristics of E. Coli adhesins in human urinary
bladder. Infect Immun 1988; 56:26152622.
60. Leffler H, Svanborg-Eden C. Glycolipid receptors for
uropathogenic E. coli on human erythrocytes and uroepithelial cells. Infect Immun 1981; 34:920929.
61. Plos K, Carter T, Hull S, et al. Intestinal carriage of P
fimbriated E. coli and the susceptibility to urinary
tract infection in young children. J Infect Dis 1995; 171:
625631.
62. Wullt B, Bergsten G, Connell H, et al. P-fimbriae trigger
mucosal responses to E. coli in the human urinary tract.
Cell Microbiol 2001; 3:255264.
63. Hacker J, Kestler H, Hoschntzky H, et al. Cloning and
characterisation of the S fimbrial adhesin II complex of
E. coli 018 K1 meningitis isolate. Infect Immun 1993; 61:
544550.
64. Nowicki B, Selvarangan R, Nowick S. Family of E. coli Dr
adhesins: delay-accelerating factor receptor recognition
and invasiveness. J Infect Dis 2001; 183:S24S27.
65. Wold A, Thorssen M, Hull S, Svenborg-Eden C. Attachment of E. coli via mannose or Gal-Gal containing receptors to human colonic epithelial cells. Infect Immun 1988;
56:25312537.
66. Schlager TA, Whittam TS, Hendley JO, et al. Comparison
of expression of virulence factors by Escherichia coli causing cystitis and E. coli colonising the peri-urethra of
healthy girls. J Infect Dis 1995; 172:772778.
67. Hedlund M, Svensson M, Wilsson A, et al. Role of the
ceramide-signalling pathway in cytokine responses to P
fimbriated E. coli. J Exp Med 1996; 183:10371044.
68. Langermann S, Mollby R, Burlein JE, et al. Vaccination
with Fim H adhesin protects cynomolgus monkey from
colonisation and infection by uropathogenic E. coli.
J Infect Dis 2000; 181:774778.
69. Silverblatt FS. Hostparasite interaction in the rat renal
pelvis: a possible role of pili in the pathogenesis of
pyelonephritis. J Exp Med 1974; 140:16961699.
70. Fader RC, Davis CP. Effect of pilation on Klebsiella pneumoniae infection in rat bladders. Infect Immun 1980;
30:554561.
71. Glynn AA, Brumfitt W, Howard CJ. K antigens of Escherichia coli and renal involvement in urinary tract infections.
Lancet 1971; 1:514516.
72. Kaijser B, Hanson LA, Jodal U, et al. Frequency of E. coli K
antigens in urinary tract infections in children. Lancet
1977; 2:663664.
73. Crump J, Pead L, Maskell R. Urinary infections in general
practice. Lancet 1976; 1:1184
74. Gruneberg RN. Antibiotic sensitivities of urinary pathogens, 19711978. J Clin Pathol 1980; 33:853856.
75. Jordan PA, Iravani A, Richard GA. Urinary tract infection
caused by Staphylococcus saprophyticus. J Infect Dis 1980;
142:510515.
76. Maskell R, Pead L, Allen J. The puzzle of urethral
syndrome: a possible answer? Lancet 1979; 1:10581059.
160
George
77. Brumfitt W, Hamilton-Miller JMT, Ludlam H, et al. Lactobacilli do not cause frequency and dysuria syndrome.
Lancet 1981; 2:393394.
78. Bran JL, Levison ME, Kaye D. Entrance of bacteria into the
female urinary bladder. N Engl J Med 1972; 286:626629.
79. Buckley RM, McGuckin M, MacGregor RR. Urine bacterial counts following sexual intercourse. N Engl J Med 1978;
298:321324.
80. Hinman F, Jr. Mechanisms for the entry of bacteria and
the establishment of urinary infection in female children. J
Urol 1966; 96:546550.
81. Hooton TM, Hillier S, Johnson C. Escherichia coli bacteriuria and contraceptive method. JAMA 1991; 265:6469.
82. Vivaldi E, Cotran R, Zangwill DP. Ascending infection as
a mechanism in pathogenesis of experimental nonobstructive pyelonephritis. Proc Soc Exp Biol Med 1959;
102:242247.
83. Kass EH. Bacteriuria and the diagnosis of infections of the
urinary tract. AMA Arch Intern Med 1957; 100:709714.
84. Stamm WE, Wagner KF, Amsell R, et al. Causes of the
acute urethral syndrome in women. N Engl J Med 1980;
303:409415.
85. Stamm WE, Counts GW, Running KR, et al. Diagnosis of
coliform infection in acutely dysuric women. N Engl J
Med 1982; 307:463468.
86. Editorial. Can Kasstigation beat the truth out of the
urethral syndrome? Lancet 1982; 2:694695.
87. Stamm WE. Quantitative urine cultures revisited. Editorial.
Eur J Clin Microbiol 1984; 3:279281.
88. Venegas MF, Navas EL, Gaffney RA, et al. Binding of type
I pilated Escherichia coli to vaginal mucous. Infect Immun
1995; 73:416421.
89. Cox CE, Lacy SS, Hinman F, Jr. The urethra and its
relationship to urinary tract infection. II. The urethral
flora of the female with recurrent urinary infection. J
Urol 1968; 99:632638.
90. Gruneberg RN. Relationship of infecting urinary organism to the faecal flora in patients with symptomatic
urinary infection. Lancet 1969; 2:766768.
91. Stamey TA, Sexton CC. The role of vaginal colonisation
with enterobacteriaceae in recurrent urinary infections.
J Urol 1975; 113:214217.
92. Pfau A, Sacks T. The bacterial flora of the vaginal vestibule, urethra and vagina in premenopausal women with
recurrent urinary tract infections. J Urol 1981; 126:630634.
93. Marsh FP, Murray M, Panchamia P. The relationship
between bacterial cultures of the vaginal introitus and
urinary infection. Br J Urol 1972; 44:368375.
94. OGrady FW, Richards B, McSherry MA, et al. Introital
enterobacteria, urinary infection and the urethral syndrome. Lancet 1970; 2:12081210.
95. Brumfitt W, Grogan RA, Hamilton-Miller JMT. Periurethral enterobacterial carriage preceding urinary infection.
Lancet 1987; 1:824826.
96. Cattell WR, McSherry MA, Northeast A, et al. Periurethral
enterobacterial carriage in the pathogenesis of recurrent
urinary infection. BMJ 1974; 4:136139.
97. Wiswell TE, Roscelli JD. Corroborative evidence for the
decreased incidence of urinary tract infections in circumcised male infants. Paediatrics 1986; 78:9699.
98. Wiswell TE, Enzenauer RW, Holton ME, et al. Declining
frequency of circumcision: implications for changes in the
absolute incidence and male to female sex ratio of urinary
tract infections in early infancy. Paediatrics 1987; 79:
338342.
99. Feld L, Greenfield S, Ogra P. Urinary tract infections in
infants and children. Paediatr Rev 1989; 11:7177.
100. Ransley PG, Risdon RA. The pathogenesis of reflux
nephropathy. Contrib Nephrol 1979; 16:9098.
101. Siegel S, Siegel B, Sokoloff B. Urinary infection in infants

and preschool children. Am J Dis Child 1980; 134:369372.
102. Kunin CM. A ten year study of bacteriuria in schoolgirls:
final report of bacteriologic, urologic, and epidemiologic
findings. J Infect Dis 1970; 122:382393.
103. Meadow RS, White RHR, Johnston NM. Prevalence of
symptomless urinary tract disease in Birmingham schoolchildren. I. Pyuria and bacteriuria. BMJ 1969; 3:8184.
104. Cardiff/Oxford Bacteriuria Study Group. Sequelae of
covert bacteriuria in schoolgirls. A four-year follow-up
study. Lancet 1978; 1:889894.
105. Gillenwater JY, Harrison RB, Kunin CM. Natural history
of bacteriuria in schoolgirls. N Engl J Med 1979; 301:396
399.
106. Sacks SH, Verrier Jones K, Roberts R, et al. The effect of
symptomless bacteriuria in childhood on subsequent
pregnancy. Lancet 1987; 2:991994.
107. Kunin CM. Sexual intercourse and urinary infections. N
Engl J Med 1978; 298:336337.
108. Norden CW, Kass EH. Bacteriuria of pregnancy: a critical
appraisal. Annu Rev Med 1968; 19:431437.
109. Kass EH. Bacteriuria and pyelonephritis of pregnancy.
AMA Arch Intern Med 1960; 105:194198.
110. Brocklehurst JC, Dillane JB, Griffiths L. The prevalence
and symptomatology of urinary infection in an aged
population. Gerontol Clin 1968; 10:242253.
111. Mabeck CE. Treatment of uncomplicated urinary tract
infection in non-pregnant women. Postgrad Med J 1972;
48:6975.
112. George NJR. Urethral syndromeclinical features. In:
George NJR, Gosling JA, eds. Sensory Disorders of the
Bladder and Urethra. New York: Springer-Verlag,
1986:91102.
113. OGrady FW, Charlton CAC, Fry IK, et al. Natural history
of intractable cystitis in women referred to a special
clinic. In: Brumfitt W, Ascher AW, eds. Urinary Tract
Infection. Oxford: Oxford University Press, 1973:8191.
114. Drach GW, Fair WR, Meares EM, Stamey TA. Classification of benign diseases associated with prostatic pain:
prostatitis or prostatodynia? J Urol 1978; 120:226.
115. Blacklock NJ. Anatomical factors in prostatitis. Br J Urol
1974; 46:4750.
116. Kirby RS, Lowe D, Bultitude MI. Intraprostatic urinary
reflux: an aetiological factor in abacterial prostatitis. Br J
Urol 1982; 54:729731.
117. Meares EM, Jr, Stamey TA. Bacteriologic localisation
patterns in bacterial prostatitis and urethritis. Invest
Urol 1968; 5:492518.
118. Kavanagh JP, Darby C, Costello CB. Differences in
expressed prostatic secretion composition (EPS) between
normal samples and from men with prostatitis. Int J
Androl 1982; 5:487496.
119. Blacklock NJ, Beavis JP. Response of prostatic fluid pH in
inflammation. Br J Urol 1974; 46:537542.
120. Mardh PH, Ripa KT, Colleen S, et al. Role of Chlamydia
trachomatis in non-acute prostatitis. Br J Vener Dis 1978;
54:330334.
121. Doble A, Thomas BJ, Walker MM. The role of Chlamydia
trachomatis in chronic abacterial prostatitis: a study using
ultrasound guided biopsy. J Urol 1989; 141:332335.
122. Grant JBF, Costello CB, Sequeira PJL, et al. The role of
Chlamydia trachomatis in epididymitis. Br J Urol 1987;
60:355359.
123. Anderson B, Olesen F, Moller JK, et al. Population based
strategies for outreach screening of urogenital Chlamydia
trachomatis infections: a randomised controlled trial.
J Infect Dis 2002; 185:252258.
124. Fenton KA, Korovessis C, Johnson AM, et al. Sexual
behaviour in Britain: reported sexually transmitted infections
125.
126.
127.
128.
129.
130.
131.
132.
133.
and prevalent genital Chlamydia trachomatis infection. Lancet

2001; 358:18511854.
Macleod J, Salisbury C, Low N, et al. Coverage and
uptake of systemic postal screening for genital Chlamydia
trachomatis and prevalence of infection in the United
Kingdom general population: cross-sectional study. BMJ
2005; 230:940943.
Mahilum-Tapay L, Laitila V, Wawrzyniak JJ, et al. New
point of care Chlamydia rapid testbridging the gap
between diagnosis and treatment: performance evaluation study. BMJ 2007; 335:11901194.
Rose AMC, Watson JM, Graham C, et al. Tuberculosis at
the end of the 20th century in England and Wales: the
results of a national survey in 1998. Thorax 2001; 56:
173179.
Garcia-Rodriguez JA, Garcia-Sanchez JE, Munoz Bellido
JL, et al. Genitourinary TB in Spain: a review of 81 cases.
Clin Infect Dis 1994; 18:557561.
Grange JM, Yates MD, Ormerod LP, et al. Factors determining ethnic differences in the incidence of bacteriologically confirmed genitourinary tuberculosis in south east
Engl J Infect 1995; 30:3740.
Churchill D, Hannan M, Miller R, et al. HIV associated
culture proved tuberculosis has increased in north central
London from 19901996. Sex Transm Infect 2002; 76:4345.
Gwoeltje KF, Matthew A, Rothstein M, et al. Tuberculosis
infection and anergy in haemodialysis patients. Am J
Kidney Dis 1998; 31:848852.
Higgins RM, Cahn AP, Porter D, et al. Microbacterial
infections after renal transplantation. Q J Med 1991;
78:145153.
Paces R, de la Torre M, Alcazar F, et al. Genitourinary
tuberculosis as the cause of unexplained hypercalcaemia
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
161
in a patient with pre-end stage renal failure. Nephrol Dial

Transplant 1998; 13:488490.
Romano L, Sanquinetti, Posteraro B, et al. Early detection
of negative BACTEC MCIT 960 cultures by PCR-reverse
cross blot hybridization assay. J Clin Microbiol 2002;
40:34993501.
Van Vollenhoven P, Heynes CF, de Beer PM, et al. PCR in
the diagnosis of urinary tract tuberculosis. Urol Res 1996;
24:107111.
When to act on evidence? Editorial. BMJ 2002; 325:7371.
Mc Aleer IM, Kaplan GW, Bradley JS, et al. Endotoxin
content in renal calculi. J Urol 2003; 169:18131814.
Mariappen P, Smith G, Mousssa SA, et al. One week of
ciprofloxacin before percutaneous nephrolithotomy significantly reduces upper tract infection and urosepsis: a
prospective controlled study. BJU Int 2006; 98:10751079.
Cuziack J, Szarewski A, Terry G. Human papilloma virus
testing in primary cervical screening. Lancet 1995; 345:
15331536.
The Future II study group: quadrivalent vaccine against
human papilloma virus to prevent high-grade cervical
lesions. N Engl J Med 2007; 356:19151927.
Haug C. Human papilloma virus vaccinationreason for
caution. N Engl J Med 2008; 359:861862.
Editorial. Cheaper HPV vaccines needed. Lancet 2008;
371:1638.
Rothman SM, Rothman DJ. Marketing HPV vaccine.
JAMA 2009; 302:781786.
Miralies-Guri C, Bruni L, Cubilla A. HPV prevalence and
type destribution in penile carcinoma. J Clin Pathol 2009;
62:870878.
10
The Scientific Basis of Urinary Stone Formation
William G. Robertson
Physiology Department, Royal Free and University College Medical School, London, U.K.
INTRODUCTION
Urolithiasis is a disorder that has cut across all historical, geographical, demographic and social boundaries. From the days of the predynastic Egyptians
until the present time, kidney stones have perplexed
patients and physicians alike and, although during
that time the methods for removing stones have
advanced from the crudely barbaric to the highly
sophisticated, the problem of how successfully to
prevent their recurrence in a given patient continues
to challenge urologists and nephrologists.
If patients are not provided with proper preventative management, the risk of recurrence is traditionally high40% within 3 years rising to 74% at 10 years
and to 98% at 25 years in the days when open surgery
and transurethral basket or loop extraction were the
main techniques for removing stones (1). In the era of
extracorporeal shock wave lithotripsy (ESWL) and
percutaneous nephrolithotomy (PCNL), the recurrence rate became even higher (Fig. 1), a fact which
is not surprising since both techniques, particularly
ESWL, often leave particles behind in the kidney that
provide ideal nuclei for further stone formation (2).
However, with the recent advent of flexible ureterorenoscopy (FURS), a procedure which promises to be
more efficient with respect to the removal of stones
and their fragments, it may be that recurrence rates
may decrease to those experienced in the days of open
surgery. Unfortunately, the relative success of ESWL,
PCNL and FURS in the disintegration and removal of
stones has lulled many into the belief that the problem
can be managed solely by these means, a trend that
has been increased by opportune cost-cutting by many
Health Authorities, for although these minimally
invasive techniques may be the procedures of choice
for the removal of stones, they do not treat the
underlying cause(s) of stone formation. Without biochemical screening and appropriate dietary and/or
medical management, the patient will generally return
for further stone removal in the future.
Not only is the failure to provide proper prophylactic treatment for the patient bad clinical management, in the long term it is economically more
expensive (3). Financial analysis has shown that the
projected cost of treating stone patients solely by
removing their stones by minimally invasive technologies every time they form them is considerably more
expensive than removing their initial stones and then
screening the patients thoroughly to identify their risk
factors to provide them with appropriate prophylactic
management (4).
During the six millennia since the formation of
the earliest recorded stones, the pattern of urolithiasis
has changed in many respects, particularly within the
past century. In Western countries before 1900, for
example, stones occurred commonly in children, particularly boys, and were formed mainly in the bladder. These stones usually consisted of ammonium
urate and/or calcium oxalate and were caused by
poor nutrition. Although this form of the disorder is
still found today in rural areas within the endemic
stone belt stretching from Jordan, through Iraq, Iran
and the Indian subcontinent to the furthest extremities
of South-East Asia, it is rapidly disappearing with
improving standards of nutrition, as it did in most
developed countries about 100 years ago (5).
As the incidence of bladder stones in children
has decreased, however, the prevalence of upper
urinary tract stones in adults has increased. Within
this general increase in stone occurrence, there have
been peaks and troughs in incidence that coincide
with periods of economic prosperity and recession,
respectively (Fig. 2). Kidney stones occur more frequently in the more industrially developed nations
and are less common in those countries whose economies are more dependent on agriculture. Overall, the
incidence of upper tract stone disease increases in
parallel with the level of affluence, presumably
through the effect of the latter on diet and lifestyle (6).
Other changes in the pattern of stone formation
have also been noted during this time. Although
stones generally occur more frequently in men than
in women (male: female ratio about 2.5:1), recent
studies have shown that, within the past 25 years,
there has been a progressive decrease in the age at
onset of stone formation in both males (Fig. 3A) and
females (Fig. 3B), particularly females (7). Within the
population of stone formers as a whole, the male:
female ratio among patients who formed their first
stone before the age of 20 is now 1.5:1 (cf. 3.0:1 in
1975). In those patients currently aged under 20, the
Chapter 10: The Scientific Basis of Urinary Stone Formation
163
Figure 1 The percentage of patients with new stones

or with new residual stones within three years of the
(A) open surgery, (B) percutaneous nephrolithotomy
(PCNL), (C) extracorporeal shock wave lithotripsy
using a Dornier HM3 lithotripter [ESWL (HM3)], or
(D) extracorporeal shock wave lithotripsy using 2nd
or 3rd generation lithotripters [ESWL (2,3)].
Figure 2 The number of stone cases per 1000

patients attending hospital in Norway and in the
United Kingdom during the past 100 years. Oil
Crisis refers to the Middle East Oil crisis in the
early 1970s. Abbreviation: WW, World War.
Figure 3 The decrease in age at onset of

stone formation in males and females in the
UK during the past 25 years.
164
Robertson
ratio has fallen to 1.15:1 (cf 1.6:1 in 1975). The main

changes have taken place in the teenage years and
may be attributed to alterations in diet and lifestyle in
that group during the past 33 years. The combination
of an earlier age at onset and an increasing life
expectancy means that more young first-time stone
formers will have a longer span of life over which they
are likely to experience recurrence of the disorder.
This will add to the burden on urological resources
required to manage the stone problem in the future.
STONE COMPOSITION
The vast majority (7580%) of urinary calculi consist
predominantly of calcium oxalate (Fig. 4), either on its
own or mixed with calcium phosphate or, increasingly
more frequently, mixed with uric acid (8). In about
90% of these cases, there is no obvious metabolic cause
for their stones (idiopathic stone formers). In the
remainder, stones form secondarily to some disorder
of calcium metabolism, oxalate metabolism or acidbase balance (see section on calcium stone disease).
Infection stones composed of magnesium
ammonium phosphate, usually in conjunction with
calcium phosphate, constitute between 4% and 12% of
all calculi. They are caused by urinary tract infections
involving a urea-splitting organism and occur more
commonly in women than in men (9). They may also
occur secondarily to the formation of most types of
sterile stone. The relative incidence of infection stones
has decreased over the past 25 years in most Western
countries, presumably as a result of better clinical
diagnosis and earlier treatment of urinary tract infections.
Uric acid calculi constitute between 4% and 15%
of all stones and generally depend on the relative
consumption of animal and vegetable protein in the
population concerned. Over the past two decades,
there has been an increase in the occurrence of uric
acidcontaining stones in many Western countries

which has been attributed to the increase in the
prevalence of metabolic syndrome, a disorder associated with obesity, hypertension and type 2 diabetes
(10) (see later). Pure uric acid stones are rare in
developing countries and are most common in the oilrich states of the Arabian Gulf (Fig. 4) and other
affluent populations, or in countries where there is a
cheap local source of animal protein (8). Most uric
acid stones are idiopathic in origin; a small number
form secondarily to some disorder of purine metabolism or to some condition in which there is a high
tissue turnover.
In all stone series, 1% to 3% of stones consist of
one of a range of rare constituents derived either
from some hereditary or congenital inborn error
of metabolism, such as cystinuria, xanthinuria or
2,8-dihydroxyadeninuria, or from a prescribed drug
or one of its metabolites that is relatively insoluble in
urine, such as silica, sulfonamides (such as sulfadiazine), indinavir or triamterene.
All stones contain a small percentage by weight
of mucoproteinaceous matrix. A few consist almost
entirely of mucoprotein (matrix calculi) and usually
result from inflammation of the urinary tract in
patients whose urine is not sufficiently supersaturated
with calcium salts to mineralize the organic matrix
(11). Apart from these rare entities, the role of the
organic matrix in the formation of stones is unclear.
By analogy with organized mineralized tissues such
as bones and teeth, where the matrix plays a major
role not only in the laying down of the mineral phase
but also in defining the structure and architecture of
the completed tissue, some researchers believe that
the matrix of stones plays an equally important role in
their initiation and growth (1214). Others, however,
maintain that it is present merely as an adventitious
inclusion, as a consequence of the calcium-binding
properties of certain glycoproteins in the matrix
material (8).
Figure 4 The percentage of urinary stones

classified according to the predominant mineral in the United Kingdom (UK), United
States of America (USA), and Kingdom of
Saudi Arabia (KSA). Abbreviations: CaOx,
calcium oxalate; CaP, calcium phosphate.
THEORIES OF STONE FORMATION

The essential features of a complete theory of stone
formation are that it should account for the formation
and retention within the urinary system of some
critical nucleus, which then enlarges by the processes
of crystal growth and agglomeration until it produces
the clinical symptoms associated with the disorder,
namely, renal colic, dysuria and hematuria. To understand the current theories on how stones begin to
form, it is necessary to appreciate some simple chemical principles involved in the stone-forming process.
CHEMICAL PRINCIPLES OF URINARY

STONE FORMATION
Supersaturation
The overriding factor that is common to all types of
stone is the relative insolubility of their respective
mineral component(s) in urine. When a substance is
allowed to dissolve in water, dissolution proceeds
until the rate of return of the dissolved material to
the solid phase equals the rate at which the solid
phase goes into solution. The concentration of the
substance in solution at this equilibrium point is
known as the substances solubility under the conditions of temperature and ionic strength concerned.
Examples of the solubilities in water at 378C and at
pH 6 of various salts and acids present in urine are
shown in Table 1. Clearly, the substances that turn up
in kidney stones have much lower solubilities than
those that do not. For a sparingly soluble substance, it
is usual to express this equilibrium value as a product
of the concentrations (or more correctly activities) of
the constituent ions rather than as the absolute solubility of the substance in mass per unit volume. This
equilibrium activity product is known as the thermodynamic solubility product of the substance concerned and is a constant at a given temperature.
In the absence of crystals of a particular substance, it is possible to add the ionic constituents of
that substance to water and reach an activity product
in the ensuing solution that is considerably above the
solubility product of the substance without crystal
nucleation occurring (8). Such a solution is said to be
in a state of metastable supersaturation, and it may
survive in this state without de novo crystals forming
Table 1 Solubilities of Various Possible Urinary Salts and
Acids in Water at 378C and pH 6
Salt
Solubility (g/l)
Calcium oxalate
Calcium phosphate
Uric acid
Cystine
Magnesium ammonium
phosphate
0.0071
0.08
0.08
0.17
0.36
Calcium sulfate
Calcium citrate
Magnesium sulfate
Calcium chloride
2.1
2.2
293
560
Properties
All occur in kidney

stones.
Never occur in
kidney stones.
165
for several hours. There is, however, an upper limit to

this region of metastability, known as the formation
product of the substance. This is not a true thermodynamic constant, like the solubility product, but
covers a band of supersaturation values within
which de novo crystal nucleation may take place
within a relatively short interval of time. Nucleation
is dependent on the time of incubation and on the
concentration of heterogeneous nuclei (that is, particles consisting of materials other than the substance
under consideration) in the system. The upper limit of
this band of formation products, beyond which the
solution becomes completely unstable, corresponds to
the level at which homogeneous nucleation takes
place (8).
From these general considerations of solubility,
it is evident that urine may fall into one of three zones
of relative supersaturation with respect to each of the
stone-forming minerals (Fig. 5). Urines lying below
the solubility product of a given mineral are said to be
undersaturated with respect to that mineral; in this
region, no new crystals can form and any existing
crystals will dissolve. There is no risk of forming
stones in undersaturated urine. Urines lying between
the solubility product and the formation product band
are in a state of metastable supersaturation; in this
region, urines can exist for quite long time periods
without new crystals forming, but any existing crystals of the mineral concerned would be expected to
grow [subject to the presence of possible inhibitors of
crystallization (see below)]. Urines lying above the
formation product band are said to be labile or
unstable. Since urine is never a pure solution but
usually contains particles of cell debris, polymerized
glycoproteins, or even crystals of another salt, it is
extremely unlikely that the point of homogeneous
nucleation will ever be attained, since these particles
will most likely have caused spontaneous heterogeneous nucleation to take place long before reaching
this level of supersaturation (8).
No simple method exists for measuring supersaturation directly in urine, and this has to be
obtained indirectly by computational techniques. To
calculate the relative supersaturation of urine with
respect to every stone-forming salt and acid requires
the measurement of the concentrations of 15 different
urinary constituents (15,16). These are fed into a
computer that calculates the concentrations of all the
soluble complexes formed between these constituents,
the ionic strength of the urine and the activity coefficients of all the ions in solution. From this information, the activity products of each of the stone-forming
substances are calculated and compared with the
relevant solubility products. The ratio of activity
product to solubility product is termed the relative
supersaturation of urine with respect to the substance
concerned. Values of 1 indicate that the urine is at the
solubility product of the substance; values below 1
indicate that the urine is undersaturated with respect
to the substance, and values greater than 1 indicate
that the urine is supersaturated with that substance
(16).
166
Robertson
Figure 5 Diagram showing the three main

regions of relative supersaturation of urine
with respect to a sparingly soluble mineral
and the crystallization processes that may
occur in each region.
Whether or not supersaturation is the only factor

responsible for the formation of stones is still open to
debate. Broadly, there are two main schools of thought
regarding the initiation of stones. Proponents of the
free-particle model of urolithiasis believe that
stones are initiated when urine becomes so excessively
supersaturated with one of the salts or acids that turn
up in kidney stones that crystals of that constituent
spontaneously precipitate in urine (17,18). If this happens frequently and if the crystals grow or aggregate
sufficiently within the transit time of urine through
the kidney, the risk increases that one of these particles will become trapped at some narrow point in the
urinary tract and form the focus around which a stone
may form (19). Alternatively, blockage may occur
through a log-jamming mechanism in a urinary
stream overcrowded with crystals (Fig. 6) (8).
An alternative theory claims that the formation
of a critical particle by such a free-particle mechanism
cannot take place within the transit time of urine
through the kidney and that the only way that a
stone can be initiated is through chemical fixation
of a crystal or aggregate of crystals to the renal
epithelial lining (Fig. 6). In this fixed-particle theory, formation is postulated to occur either through
damage to the cell walls (caused either by the crystals
themselves or by viruses or bacteria) and/or through
the participation of some gluing material present in
the urine of stone formers (but absent from the urine
of nonstone formers) that causes crystals to adhere to
these sites and there act as foci for stone formation
(2026). Recently, a variant of the fixed-particle model
has been proposed which postulates that stone formation is initiated by so-called Randalls plaques,
subepithelial flecks of calcium phosphate that are
more frequently found in the renal papillae of stone
formers than in those of individuals who do not have
a history of urolithiasis (27,28). The plaques, originally
described by Randall in 1937 (29), are considered to
Figure 6 Diagrammatic representation of how stones are initiated according to the free-particle and fixed-particle models
of stone formation.
form as a result of high supersaturation of tubular

fluid with respect to calcium phosphate in the
descending limb of the Loop of Henle (27,28). If
these plaques erupt through the papillary epithelium,
they may be exposed to urine supersaturated with
respect to calcium oxalate and/or other stone-forming
salts and acids in the renal calyces, thereby leading to
the nucleation of a stone at the tip of renal papilla
concerned. All the above theories require urine to be
supersaturated to some degree with respect to the
stone-forming salt or acid concerned, sufficient to
cause crystals to be formed by spontaneous or heterogeneous nucleation.
Processes of Crystallization
167
Nucleation, as described above, is the first step in the

process of crystallization. This causes a fall in the
supersaturation of urine as new crystals are formed
but does not usually reduce the level of supersaturation immediately down to the solubility product.
There is then a period during which the initially
formed nuclei grow in the remaining metastable
urine, and in the moving stream of urine, the growing
crystals may impact with each other and aggregate to
form larger particles. This process is called agglomeration and is the stage in the crystallization process
during which the particle size increases most rapidly
(18,19,30). In the free-particle theory, crystal agglomeration is essential to create a particle that is large
enough to be retained in the collecting tubule before
the urine is expelled through the ducts of Bellini
(18,19,31,32). Crystal growth per se is normally too
slow a process to allow this to happen within the
transit time of urine through the renal tubule.
Agglomeration may be less important in the
fixed-particle model, since the initial particle that
causes the stone to form round it is considered, by
definition, to be glued to the renal tubular epithelial
wall or formed round an exposed Randalls plaque.
Once the initial entrapment (free-particle model) or
fixation step (fixed-particle model) has taken place,
both theories require the immobilized particle to
increase in size through further crystal growth and
agglomeration until it becomes a stone that causes
problems for the patient (1720,29,31,32).
proteins and glycoproteins (5557) and the polymerized form of Tamm-Horsfall protein (uromucoid)
(5860).
Unquestionably, urine does possess a certain
ability to modify the rate of crystal growth and/or
agglomeration of calcium oxalate crystals and may
also contain factors that influence the binding of these
crystals to renal epithelial cells. Currently, it would
seem, however, that the crystallization-modifying
activity is unlikely to be attributable to one single
magic factor X but is probably due to the net effect of
all the above promoters and inhibitors (and probably
others not yet identified). None of the above factors
appears to dominate the kinetics of crystal nucleation,
growth, aggregation and binding to cells, and none
has yet been accepted as being uniquely different,
either quantitatively or qualitatively, between stone
formers and normal subjects. Therefore, the assertion
that a deficiency in one particular inhibitor or an
excess of one particular promoter is the cause of
stone formation is open to question (61).
In the final analysis, stone formation is probably
due to an abnormal combination of factors that affect
both the thermodynamic driving force (supersaturation) and the kinetic (rate-controlling) processes
involved in the crystallization of the various stoneforming minerals. For some types of stone formation
(cystine, xanthine, 2,8-dihydroxyadenine, uric acid
and probably magnesium ammonium phosphate),
the thermodynamic factors appear to predominate;
in others (calcium-containing stones), both sets of
factors may be involved (8).
Modifiers of Crystallization
Crystalluria
One factor that may affect the kinetics of the processes

involved in both theories is the presence or absence in
urine of so-called modifiers of crystallization (8).
These are claimed to be of particular importance in
the formation of calcium-containing stones. Indeed, no
specific, naturally occurring modifier has been
reported to have any effect on the crystallization of
cystine, uric acid or magnesium ammonium phosphate. One group of crystallization modifiers is
claimed to retard the rate of growth and/or aggregation of crystals of calcium salts and/or the binding of
calcium-containing crystals to cell walls. These are
known as inhibitors of crystallization and include
magnesium (33), citrate (34), pyro-phosphate (35,36),
adenosine diphosphate (37), adenosine triphosphate
(37), at least two phosphopeptides (38), various glycosaminoglycans (39,40), nonpolymerised TammHorsfall protein (also known as uromodulin) (41,42),
nephrocalcin (4346), calgranulin (47), various plasma
proteins (12), osteopontin (also known as uropontin)
(48,49), a-1-microglobulin (13), b-2-microglobulin (50),
urinary prothrombin fragment 1 (51,52) and inter-atrypsin inhibitor (bikunin light chain) (53,54).
The second group of modifiers is claimed to
stimulate one or more of the processes involved in
the crystallization of calcium salts. These are known as
promoters of stone formation and include matrix
substance A (13), various uncharacterized urinary
Whatever the detailed mechanism of stone initiation,

an individuals propensity to produce crystals of one
of the stone-forming constituents is an important
prerequisite for stone formation, either because the
crystals lead directly to the initiation of stones (as
postulated in the free-particle model) or because their
production is a signal that urine is so supersaturated
with respect to that constituent that heterogeneous
nucleation and crystal growth are likely to occur at
one of the critical sites postulated in the fixed-particle
model. Crystalluria occurs more frequently and more
abundantly in the urine of stone formers than in that
of controls, and, in patients with calcium stone disease, the crystals are larger and more agglomerated
(1819). The severity of the disorder, as defined by the
stone episode rate in a given patient is proportional to
the percentage of large crystals and aggregates in the
patients urine (Fig. 7). It would appear that particle
size is a vital factor in the stone-forming process.
CYSTINE STONE FORMATION

Cystinuria is a congenital disorder characterized by a
defect in the renal tubular reabsorption of cystine,
lysine, ornithine and arginine, but the lesion also
affects the intestine (62,63). As a consequence, the
urinary excretion of these amino acids is greatly
168
Robertson
Figure 7 The relationship between the severity of stone formation (as defined by the stone episode rate) in recurrent calcium
oxalate stone formers and the proportion of large calcium oxalate
crystals and aggregates in their freshly voided urine: ., untreated
patients; o, patients on orthophosphate supplements.
increased. Lysine, ornithine and arginine are all fairly

soluble in urine, but cystine is relatively insoluble and,
as a result, these patients tend to have cystine crystalluria and to form cystine stones (64,65).
The sole risk factor for cystine stone formation is
the supersaturation of urine with respect to cystine, as
defined by the urinary concentration of cystine and
urine pH (Fig. 8). Within the normal pH range of urine
(57), the supersaturation is defined by the concentration of cystine. In normal urine, this is generally low
(10100 mmol/l), at which concentrations urine is well
undersaturated with respect to cystine; therefore, cystine crystalluria is never found in normal urine. In
heterozygous cystinurics, whose urinary cystine concentrations are in the range 20 to 1000 mmol/l, the
supersaturation values are higher but still below the
solubility of cystine in urine. As a rule, these patients
do not have cystine crystalluria in fresh urine and do
not form cystine stones (66). Homozygous cystinurics,
however, have much higher urinary concentrations of
cystine (14004200 mmol/l), above the solubility of
Figure 9 A risk factor model of cystine stone formation.
Figure 8 The relative supersaturation (RS) of urine with respect

to cystine in homozygous cystinurics, heterozygous cystinurics
and normal subjects. The data are plotted on a log scale (right
axis), where the solubility product (SP) has a value of 1, and on a
normalized log scale (left axis) such that the SP has a value of 0
and the upper limit of the formation product band (FP) has a
value of 1.
cystine in saline at 378 (1250 mmol/l), and so their

urines are considerably supersaturated with respect to
cystine (Fig. 8). These patients often pass large cystine
crystals in their urine and form cystine stones (64,65).
This form of stone formation is therefore readily
explained in terms of excessive excretion ? excessive
supersaturation ? abnormal crystalluria ? stones.
The complete risk factor model of cystine stone formation is outlined in Figure 9.
Prevention of cystine stones requires the supersaturation of urine to be substantially reduced to
prevent continued crystalluria (Fig. 10). Although
cystine is more soluble in alkaline urine than under
acidic or neutral conditions, alkali therapy is not
always practicable, as large doses of sodium bicarbonate are required to maintain urinary pH above 7.6 (67).
Less alkali may be required if the patient is also put on
a high fluid intake (>3 l/day) (68). Alternatively,
reduction in cystine concentration may be achieved
169
Figure 10 The effect of various treatments on the

relative supersaturation (RS) of urine with respect to
cystine in cystinuric stone formers. The data are
plotted on a log scale (right axis), where the solubility
product (SP) has a value of 1, and on a normalized
log scale (left axis) such that the SP has a value of 0
and the upper limit of the formation product band
(FP) has a value of 1.
xanthine and silica) simply on the basis of their

insolubility in urine. These stones form purely from
the excessive excretion in urine of the particular
substance concerned, either as a result of some metabolic defect or from the administration of a drug that
is itself insoluble (or one of its metabolites is insoluble)
in urine. Urine becomes excessively supersaturated
with the substance concerned, resulting in abnormal
crystalluria and stone formation (8).
URIC ACID STONE FORMATION
Figure 11 The structures of cystine, cysteine and various

analogues which form S-S complexes with cysteine that are
soluble in urine.
by administration of 2 to 4 g/day of D-penicillamine,

which forms an S-S link with the cysteine subunits
of cystine to form a disulfide that is more soluble
than cystine (Fig. 11) (69). However, this form of
therapy has a high risk of side-effects, including
rashes, neutropenia, thrombo penia, nephrotic
syndrome and fever. Severe proteinuria is experienced in about 10% to 15% of patients (65). A number
of analogues, such as N-acetyl-D-penicillamine and amercaptopropionylglycine, have been tried with some
degree of success as less toxic alternatives (7072). If
the above forms of therapy are pursued tenaciously,
the supersaturation of urine with respect to cystine
can be reduced (Fig. 10) and with it the risk of stone
recurrence (65,73,74).
It is also possible to explain the formation of
the other rare stones (such as 2,8-dihydroxyadenine,
Uric acid is the end-product of purine metabolism and

is normally excreted in urine in the range 2 to
5 mmol/day (75,76). Patients with urinary excretions
above this range are prone to form uric acid stones,
but hyperuricosuria per se is not the sole cause or
even the most important factor in uric acid lithiasis
(77). Dehydration is as important as hyperuricosuria,
and a low urine pH is more critical than both of these
factors (78,79). The main causes of hyperuricosuria are
listed in Table 2. The most recent addition to this list is
fructose, which has been shown to increase the urinary excretion of uric acid following the breakdown of
fructose by fructokinase (80).
Table 2 Causes of Hyperuricosuria

Primary gout
Increased purine intake
Increased fructose intake
Glycogen storage diseaseglucose-6-phosphatase deficiency
Increased phosphoribosyl pyrophosphate synthetase activity
Hypoxanthineguanine phosphoribosyltransferase deficiency
(Lesch-Nyhan syndrome)
Neoplastic disease
Secondary polycythaemia
Anaemia and haemoglobinopathy
Psoriasis
Cystinuria
170
Robertson
Figure 12 The relative supersaturation (RS) of urine

with respect to uric acid in idiopathic uric acid stone
formers, patients with an ileostomy and uric acid
stones and normal subjects. The data are plotted on
a log scale (right axis), where the solubility product
(SP) has a value of 1, and on a normalized log scale
(left axis) such that the SP has a value of 0 and the
upper limit of the formation product band (FP) has a
value of 1.
As with the formation of rare stones, uric acid

stone formation can be completely explained in terms
of excessive supersaturation of urine with respect to
uric acid (8). Chemically, the two factors that determine the risk of uric acid crystalluria are the concentration of uric acid and the pH of urine. The pKa of
uric acid is 5.46, so that any urine with a pH of less
than this figure will contain more of the relatively
insoluble, undissociated form of the acid. Uric acid
crystalluria becomes increasingly more likely as urine
pH drops below 5.3 (81). Normal urine, on the other
hand, generally lies in the metastable or undersaturated region with respect to uric acid (8) with little or no
risk of forming uric acid crystals (Fig. 12).
The causes of an acid urine are listed in Table 3.
These include a high acid-ash diet containing large
amounts of meat, fish and poultry (82). As this type of
diet is also high in purines, these patients usually have
hyperuricosuria in conjunction with their acid urine,
and so the risk of uric acid stone formation is compounded (8). In the idiopathic uric acid stone formers,
the tendency to pass an acid urine has been attributed
to a low rate of ammonia synthesis;(78,83) ammonia is
normally synthesized in the kidney to buffer hydrogen ions. Impaired renal ammoniagenesis leading to
the passage of a more acidic urine than expected from
the potential renal acid load, is probably the defect
which leads to the formation of uric acidcontaining
stones in patients with metabolic syndrome,
through the effects of insulin-resistance on both
Table 3 Possible Causes of Acid Urine
Dehydration
Oliguria
Diarrhoea
Permanent ileostomy
Colostomy
Low rate of renal production of ammonia
Increased titratable acidity
High acid ash diet (as in a high intake of animal protein)
Metabolic syndrome
phosphate-dependent and phosphate-independent

ammonia production in the renal tubules (84,85).
Patients with a permanent ileostomy also pass urine
that is highly supersaturated with respect to uric acid
(86). As a result their urine is highly concentrated and
very acidic as a result of losing water and bicarbonate
through their ileostomy. The risk factor model of uric
acid stone formation is shown in Figure 13.
Treatment is designed to lower the supersaturation of urine with respect to uric acid (Fig. 14). This
can be achieved relatively cheaply by increasing urinary pH to above 6.2 (but not higher than 6.5 to avoid
formation of calcium phosphate stones) with oral
sodium bicarbonate or potassium citrate and maintaining the urine volume above 2.5 l/day with a high
fluid intake (78,79,87). Allopurinol, which inhibits the
metabolic production of uric acid from xanthine by
blocking the enzyme xanthine oxidase, may be necessary for patients who cannot tolerate alkali (88,89).
Allopurinol has few side-effects, but care has to be
taken with patients with enzyme defects that lead to a
very high production of uric acid (such as LeschNyhan syndrome or neoplastic disease), since allopurinol administration may result in xanthinuria and
xanthine stones (90,91).
INFECTED STONE FORMATION

Urinary tract infections involving urea-splitting
organisms often lead to the formation of stones that
consist of calcium phosphate often mixed with magnesium ammonium phosphate (92). Chemically, the
process involves the breakdown of urea by the
enzyme urease to form ammonium, bicarbonate and
hydroxyl ions (Fig. 15) with an accompanying marked
alkalinization of urine (pH > 7.2). Under these conditions, the supersaturation of urine with respect to
both calcium phosphate and magnesium ammonium
phosphate reaches values that cause spontaneous
precipitation of these salts and massive crystalluria
171
Figure 13 A risk factor model for uric acid stone formation.
Figure 14 The effect of various treatments on the

relative supersaturation (RS) of urine with respect to
uric acid in uric acid stone formers. The data are
plotted on a log scale (right axis), where the solubility product (SP) has a value of 1, and on a normalized log scale (left axis) such that the SP has a
value of 0 and the upper limit of the formation
product band (FP) has a value of 1.
Figure 15 The breakdown of urea by urease to form ammonium ions and alkali.
(Fig. 16) (92,93). In addition, two of the known inhibitors of calcium phosphate crystal growth, pyrophosphate and citrate, are often low in the urines of
patients with urinary tract infections, and this may

allow the formation of larger than normal crystals and
aggregates of calcium phosphate, thereby increasing
the risk of stone formation (8). A list of the organisms
causing urinary tract infection and their ability to
produce urease is contained in Table 4. The risk
factor model of infected stone formation is shown in
Figure 17.
Treatment of infected stone patients usually
involves the combined efforts of the surgeon and the
physician. The first important stage is to remove the
stone completely and, if possible, to correct any anatomical obstruction that might have caused the underlying infection. Medical management is necessary
172
Robertson
Table 4 The Relative Prevalence of Organisms Causing Urinary Tract Infection and Their Ability to Produce Urease
Organisms
causing urinary
tract infection
Escherichia
Proteus
Klebsiella
Streptococcus
Staphylococcus
Pseudomonas
Ureaplasma urealyticum
Patients with infection (%)
Ability to produce urease
In hospital
At home
Frequently
Occasionally
5
16
9
7
5
3
1
0
5
2
0
3
0
0
0
1
0
0
1
0
1
1
0
1
0
0
1
0
Figure 16 The relative supersaturation (RS) of urine with respect to calcium phosphate (CaP) (left panel) and magnesium ammonium
phosphate (MAP) (right panel) in infected stone formers and normal subjects. The data are plotted on a log scale (right axis of each
panel), where the solubility product (SP) has a value of 1, and on a normalized log scale (left axis of each panel) such that the SP has a
value of 0 and the upper limit of the formation product band (FP) has a value of 1.
Figure 17 A risk factor model of infected stone formation.
173
Figure 18 The effect of various treatments on the relative supersaturation (RS) of urine with respect to calcium phosphate (CaP) (left
panel) and magnesium ammonium phosphate (MAP) (right panel) in infected stone formers. The data are plotted on a log scale (right
axis of each panel), where the solubility product (SP) has a value of 1, and on a normalized log scale (left axis of each panel) such that
the SP has a value of 0 and the upper limit of the formation product band (FP) has a value of 1. Abbreviation: AHA, acetohydroxamic
acid.
before and after stone removal to sterilize the urine

(94). This may not always be easy to achieve since the
infecting organism may be resistant to several antibiotics (95,96). In this situation, urease-inhibiting
drugs, such as acetohydroxamic acid or one of its
analogues, may be helpful (9799), although there
may be side-effects (Fig. 18) (97). A high fluid intake
is necessary in all patients, and some acidification may
be required to lower the urine pH to around 6.
Drinking cranberry juice may be beneficial in this
respect (100). It should be noted that cranberry juice
is the only fruit juice known to acidify urine; all other
fruit juices including those which are acidic to the
taste, alkalinise urine.
CALCIUM STONE DISEASE

Stones consisting of calcium oxalate, often mixed with
calcium phosphate, are the most common types of
calculi in most series (8). Pure calcium phosphate
stones are quite rare, suggesting that calcium oxalate
is the main problem behind the formation of calciumcontaining stones. The reason for this is the very
poor solubility of calcium oxalate in aqueous media
(Table 1). Whereas urine can often be undersaturated
with respect to all other constituents of urinary calculi,
particularly in nonstone formers, in the case of calcium oxalate urine is always supersaturated to some
degree with the salt, even in normal subjects (Fig. 19).
This means, firstly, that urine is relatively close to the
formation product of calcium oxalate in most individuals and, secondly, that once calcium oxalate deposits
form and become lodged in the kidney, they can never
dissolve spontaneously. Stones containing calcium
oxalate can be disintegrated by ESWL and other

methods and the fragments (probably) passed, but
they cannot be easily redissolved, even with aggressive irrigation techniques.
The vast majority of calcium stone formers are
said to be idiopathic, since they exhibit no obvious
metabolic cause for their stones. About 10% to 12% of
patients are found to have a metabolic cause, such as
primary hyperparathyroidism, distal renal tubular acidosis or primary or enteric hyperoxaluria (8). The
complete list of conditions and lifestyle practices that
lead to secondary calcium stones is contained in Table 5.
Unlike most other types of stone formation, calcium lithiasis does not appear to be attributable to a single
urinary abnormality or even to a simple combination of
abnormalities. It is a truly multifactorial disorder in
which various combinations of (often) small increases
or decreases in certain urinary factors may compound
the risk of forming stones (8). These include a low urine
volume (<1.3 l/day) (101,102), a raised urinary pH
(>6.3) (8,103). hypercalciuria (>6 mmol/day) (104108),
mild hyperoxaluria (>0.45 mmol/day) (29,109,110),
hypocitraturia (<2 mmol/day) (111113), hyperuricosuria (>4.5 mmol/day) (82) and a low urinary magnesium excretion (<3.2 mmol/day) (114). All of these
factors have been found to be different between stone
formers and normals, although in each case there is a
considerable overlap between the data in the two groups
(115,116). All the abnormalities described are known to
increase the supersaturation of urine with respect to
calcium oxalate and/or calcium phosphate, although
they are not all equally important in this respect. In
addition to their ability to influence supersaturation,
citrate and magnesium are mild inhibitors of crystallization of calcium salts (33,34), and uric acid is a mild
174
Robertson
Figure 19 The relative supersaturation (RS) of urine with respect to calcium oxalate (CaOx) (left panel) and calcium phosphate (CaP)
(right panel) in recurrent idiopathic calcium stone formers (RSF) and normal subjects. The RSF are divided into those with pure CaOx
stones (.) and those with mixed CaOx/CaP stones (~). The data are plotted on a log scale (right axis of each panel), where the
solubility product (SP) has a value of 1, and on a normalized log scale (left axis of each panel) such that the SP has a value of 0 and the
upper limit of the formation product band (FP) has a value of 1.
Table 5 List of Medical Conditions and Lifestyle Practices That

May Lead to Secondary Calcium Stone Formation
Primary hyperparathyroidism
Distal renal tubular acidosis (type I)
Hereditary hyperoxaluria
Enteric hyperoxaluria
Pancreatitis
Medullary sponge kidney
Cushings disease and steroid treatment
Sarcoidosis
Immobilization
Milk-alkali syndrome
Vitamin D intoxication
Bariatric surgery
Betel nut chewing
Laxative abuse
Antacid abuse (with calcium carbonatecontaining preparations)
Stress
Tropical holidays
Strenuous exercise (without sufficient rehydration)
promoter of calcium stone formation (117,118). Risk

factor analysis has shown that, taking all their properties
into account, the overall order of importance of the
above seven factors in determining the risk of forming
calcium-containing stones is as follows: low urine volume > mild hyperoxaluria > raised urinary pH >
hypercalciuria > hypocitraturia > hyperuricosuria >
hypomagnesiuria (116).
The risk factor model of calcium stone formation
makes use of these seven measurements to generate a
number of algorithms that represent an estimate (PSF)
of the overall biochemical risk of forming various types
of calcium-containing stones (115,116). The model has
recently been extended to include uric acid-containing
stones so that predictions can be made from an analysis

of two 24-hour urines on the likelihood of forming all
combinations of stone types involving uric acid, calcium oxalate and calcium phosphate (116). The values of
PSF, which are calculated on a probability scale from
0 to 1, are higher in stone formers than in nonstone
formers. In recurrent stone formers, the values are
generally over 0.5, whereas in normal subjects they
are usually under 0.5. This function therefore provides
quite a good discriminator between the two groups
(Fig. 20). Furthermore, within the group of recurrent
stone formers, the severity of the disorder (as defined
by the stone recurrence rate of the patient) is related to
the patients average PSF value. This method of combining risk factors is a useful means of assessing the
risk of forming stones in a given individual and for
following the efficacy of the particular form of prophylaxis prescribed for the patient. A summary of the
urinary risk factors involved in all types of stone
formation is given in Table 6.
Epidemiological Factors in the Formation

of Urinary Stones
There are three groups of epidemiological factors that
have been found to be of importance in the formation
of urinary stonesdemographic, environmental and
pathophysiological. Each of these groups contains
a number of categories. These are summarized in
Table 7. Each has been shown to increase the risk of
stone formation through its effect on the balance
between supersaturation, inhibitors and promoters
of crystallization in urine (8). For calcium stone formation, the most common form of the disorder, the
175
Figure 20 The overall relative probability of forming calcium-containing stones (PSF) in normal children and adults and in single episode
(SSF) and recurrent episode (RSF) idiopathic calcium stone formers. Also shown are the PSF values in various groups of patients at risk
of forming secondary calcium stones: hyperparathyroid, patients with primary hyperparathyroidism; dRTA, distal renal tubular acidosis;
enteric, patients with enteric hyperoxaluria; hereditary, patients with hereditary hyperoxaluria.
Table 6 Summary of the Urinary Risk Factors for the Various Types of Stone Formation and Their Effects on the Parameters of
Crystallization
Stone type
Urinary risk factor
Chemical effect
Rare stones
: Cystine, : xanthine,
: 2,8-dihydroxyadenine,
: silica, : indinavir,
: triamterene, etc.
: Supersaturation with respect to corresponding stone constituent
Cystine stones
Cystine
: Cystine supersaturation
Uric acid stones
; pH
: Uric acid
; Volume
: Uric acid supersaturation
Infection stones
: pH
: Ammonium ions
: Mucosubstances
: Magnesium ammonium phosphate and : calcium phosphate supersaturation

: Agglomeration of crystals
Calcium stones
;
:
:
:
;
;
;
:
:
Volume
>
>
>
>
Oxalate
>
=
Calcium
pH
>
>
>
>
Citrate
>
;
o
Magnesium
Macromolecular inhibitors
o
Uric acid
Macromolecular promoters
: Calcium oxalate and/or calcium phosphate supersaturation
; Crystallization-inhibitory activity
: Crystallization-promotive activity
main epidemiological factors are age (8), gender (8),

season (119,120), climate (121,122), stress (123,124),
occupation (122,125,126), affluence (127129), diet
(including fluid intake) (8,130134), genetic/metabolic
(135137) and lifestyle factors (8).
The role of diet, in particular, has been studied in

detail, and it appears to explain much of the changing
pattern of stone incidence over the past 100 years
(8,132,138), As the composition of the diet becomes
richer in a given population (owing to an increased
176
Robertson
Table 7 The Epidemiological Factors Involved in Uric Acid and Calcium Stone Formation and Their Effects on Urinary Risk Factors
Epidemiological factor
Urinary risk factors
Age and sex
: Calcium, : oxalate, : uric acid, : pH, ; volume, ; citrate, ; magnesium,

; macromolecular inhibitors, : promoters
; Volume, : calcium, : oxalate, ; pH
: Calcium, : oxalate, : uric acid, ; magnesium
; Volume, ; pH
: Calcium, : oxalate, : uric acid, ; citrate, ; pH
Climate and season

Stress
Low fluid intake or strenuous exercise
Affluence and diet
Metabolic and lifestyle disorders
Gout
Glycogen storage disease
Lesch-Nyhan syndrome
Neoplastic disease
Ileostomy
Metabolic syndrome
Primary hyperparathyroidism
Distal renal tubular acidosis
Hereditary hyperoxaluria
Enteric hyperoxaluria
Cushings disease
Bariatric surgery
Topiramate
Sarcoidosis
Immobilization
Betel-nut chewing
Antacid abuse
Tropical holidays
: Uric acid
: Uric acid
: Uric acid
: Uric acid
;; Volume, ;; pH
: Uric acid, ;; pH
:: Calcium, : pH
: pH, : calcium, ; citrate
:: oxalate
: Oxalate, ; pH, ; citrate, ; magnesium
: Calcium
: Calcium, : pH
:: Calcium
: Calcium, : pH
: Oxalate
: Calcium, : pH, ; citrate
:: Calcium
: Calcium, :: pH (from urinary tract infection)
: Calcium, : pH
:: Calcium, : pH
; Volume, : calcium
consumption of protein, particularly animal protein, fat,

refined sugars and salt (the so-called bad Western
diet), the incidence of stones increases (132,138)
(Fig. 21). This often follows periods of economic expansion (Fig. 2). During periods of recession, on the other
hand, the incidence of stones has been noted to decrease

in parallel with a return to a more healthy form of diet
containing more fiber and fewer energy-rich foods
(8,138). The risk factor model for calcium stone formation,
incorporating all of these factors, is shown in Figure 22.
Figure 21 The effects of the bad Western diet on the risk of forming calcium oxalate and uric acid stones.
177
Figure 22 A risk factor model for calcium stone formation.
Prevention of Stone Recurrence

The main aim in the prevention of stone recurrence is
to decrease the likelihood of crystals forming in the
urinary tract by reducing the supersaturation of urine
with respect to the particular constituent(s) that occur
in the patients stones (139141). A summary of the
available dietary and medical treatments for the various types of urinary calculi is shown in Table 8.
Although most of these are effective in reducing the

risk of stone recurrence, the main problem in the longterm management of stone patients is compliance
(142). Generally, stone formers feel well for most of
the time, except when they are experiencing an attack
of renal colic. It is often difficult, therefore, to maintain
their cooperation and motivation to adhere to their
preventative treatment over a long period after their
Table 8 Medical Methods for Prevention of Urinary Stone Disease

Stone type
Treatment
2,8-Dihydroxyadenine
Silica
Xanthine
Very high fluid intake (>3 l/day) allopurinol (300 mg/day)

Discontinue magnesium trisilicate antacids
Hereditary form: high fluid intake oral alkali (urine pH >7.4)
Iatrogenic form: withdraw allopurinol
Very high fluid intake (>3 l/day) oral alkali (urine pH >7.5) or D-penicillamine (24 g/day) or
a-mercaptopropionylglycine
High fluid intake (>2.5 l/day) oral alkali (urine pH >6.2) or allopurinol (300 mg/day) or reduce
purine intake
High fluid intake antibiotics oral acid (pH < 6.2)
Cystine
Uric acid
Infected
Calcium
Idiopathic
Hyperparathyroid
Hereditary hyperoxaluric
Enteric hyperoxaluric
Renal tubular acidotic
Corticosteroid induced
Sarcoidosis
Betel nut chewing
Immobilization
Iatrogenic
Antacid abuse
Tropical holidays
High fluid intake dietary advice or thiazide diuretics (bendrofluazide 10 mg/day) or

magnesium supplements (500 mg mg/day) or potassium citrate (20 mequiv t.d.s.)
Parathyroidectomy or, if contraindicated, high fluids oral acid
High fluid intake (>3 l/day) pyridoxine (300 mg/day)
High fluid intake low oxalate/high calcium diet or potassium citrate
High fluid intake thiazides or potassium citrate
Treat as for idiopathic
Discontinue corticosteroids: treat as for idiopathic
High fluid intake
Discontinue alkali and moderate calcium intake high fluid intake
Discontinue high vitamin D intake high fluid intake
Discontinue practice
High fluid intake; remobilize as far as possible; treat any urinary tract infection with antibiotics
Discontinue drug concerned as far as possible and replace with alternative therapy high fluid
intake
Discontinue calcium-containing antacids; change to proton pump inhibitors
Protect skin from UV radiation increase fluid intake (not alcohol) while on holiday
178
Robertson
stone episode. If they do not have a recurrence of their

problem within a few months of their episode, most
stone formers will regress to their original abnormal
pattern of urine biochemistry within three to six
months and will eventually produce another stone
(143). Once they have had several episodes of renal
colic, it is usually easier to motivate them on a more
continuous basis. It is important to review the patient
regularly as an outpatient and to repeat the 24-hour
urine analysis, preferably annually but at least biennially, to ensure that they are adhering to the prophylaxis prescribed and to check that their biochemical
risk of stones remains low (116,142).
REFERENCES
1. Williams RE. Long-term survey of 538 patients with
upper urinary tract stone. B J Urol 1963; 35:416437.
2. Pearl MS, Clayman RV. Outcomes and selection of surgical therapies of stones in the kidney and ureter. In: Coe
FL, Favus MJ, Pak CYC, et al., eds. Kidney Stones
Medical and Surgical Management. Philadelphia:
Lippincott-Raven, 1996:709755.
3. Robertson WG. The economic case for the biochemical
screening of stone patients. In: Rodgers AL, Hibbert BE,
Hess B, et al., eds. Urolithiasis 2000. Cape Town: University
of Cape Town, 2000:403405.
4. Parks JH, Coe FL. The financial effects of kidney stone
prevention. Kidney Int 1996; 50:17061712.
5. Andersen DA. Environmental factors in the aetiology of
urolithiasis. In: Cifuentes Delatte L, Rapado A, Hodgkinson
A, eds. Urinary Calculi. Basel: Karger, 1973:130144.
6. Robertson WG, Peacock M, Heyburn PJ, et al. The risk of
calcium stone-formation in relation to affluence and dietary animal protein. In: Brockis JG, Finlayson B, eds.
Urinary Calculus. Littleton, MA: PSG Publishing,
1981:312.
7. Robertson WG, Whitfield HN, Unwin RJ, et al. Possible
causes of the changing pattern of the age of onset of
urinary stone disease in the UK. In: Rodgers AL, Hibbert
BE, Hess B, et al., eds. Urolithiasis 2000. Cape Town:
University of Cape Town, 2000:366368.
8. Robertson WG. Urinary tract calculi. In: Nordin BEC,
Need AG, Morris HA, eds. Metabolic Bone and Stone
Disease. 3rd ed. Edinburgh: Churchill Livingstone,
1993:249311.
9. Griffith DP, Osborne CA. Infection (urease) stones. Miner
Electrolyte Metab 1987; 13:278285.
10. Maalouf NM, Cameron MA, Moe OW, et al. Low urine
pH: a novel feature of the metabolic syndrome. Clin J Am
Soc Nephrol 2007; 2:883888.
11. Wickham JEA. Matrix and the infective renal calculus. Br J
Urol 1976; 47:727732.
12. Boyce WH, King JS. Present concepts concerning the
origin of matrix and stones. Ann N Y Acad Sci 1963;
104:563578.
13. Morse RM, Resnick MI. A new approach to the study of
urinary macromolecules as a participant in calcium oxalate crystallization. J Urol 1988; 139:869873.
14. Morse RM, Resnick MI. A study of the incorporation of
urinary macromolecules onto crystals of different mineral
compositions. J Urol 1989; 141:641644.
15. Robertson WG. Measurement of ionized calcium in biological fluids. Clin Chim Acta 1969; 24:149157.
16. Werness P, Brown C, Smith LH, et al. EQUIL2: a BASIC
computer program for the calculation of urinary saturation. J Urol 1985; 134:12421244.
17. Vermeulen CW, Ellis JE, Hsu TC. Experimental observations on the pathogenesis of urinary calculi. J Urol 1966;
95:681690.
18. Robertson WG, Peacock M, Nordin BEC. Calcium crystalluria in recurrent renal stone-formers. Lancet 1969;
2:2124.
19. Kok DJ, Papapoulos SE, Bijvoet OLM. Crystal agglomeration is a major element in calcium oxalate urinary stoneformation. Kidney Int 1990; 37:5156.
20. Finlayson B, Reid F. The expectation of free and fixed
particles in urinary stone disease. Invest Urol 1978;
15:442448.
21. Kumar S, Sigmon D, Miller T, et al. A new model of
nephrolithiasis involving tubular dysfunction/injury.
J Urol 1991; 146:13841389.
22. Bigelow MW, Wiessner JH, Kleinman JG, et al. Calcium
oxalate crystal attachment to cultured kidney epithelial
cell lines. J Urol 1998; 160:15281532.
23. Verkoelen CF, van der Boom BG, Houtsmuller AB, et al.
Increased calcium oxalate monohydrate crystal binding to
injured tubular epithelial cells in culture. Am J Physiol
1998; 274:F958F965.
24. Scheid C, Koul H, Hill WA, et al. Oxalate toxicity in LLCPK1 cells: role of free radicals. Kidney Int 1996; 49:
413419.
25. Lieske JC, Leonard R, Swift HS, et al. Adhesion of calcium
oxalate monohydrate crystals to anionic sites of renal
epithelial cells. Am J Physiol 1996; 270:F192F199.
26. Koul H, Menon M, Koul S, et al. Effect of oxalate on
calcium oxalate crystal adherence to renal epithelial cells
in culture. In: Rodgers AL, Hibbert BE, Hess B, et al, eds.
Urolithiasis 2000. Cape Town: University of Cape Town,
2000:267269.
27. Matlaga BR, Williams JC, Kim SC, et al. Endoscopic
evidence of calculus attachment to Randalls plaque.
J Urol 2006; 175:17201724.
28. Matlaga BR, Coe FL, Evan AP, et al. The role of Randalls
plaques in the pathogenesis of calcium stones. J Urol 2007;
177:3138.
29. Robertson WG, Peacock M, Nordin BEC. Calcium oxalate
crystalluria and urine saturation in recurrent renal stoneformers. Clin Sci 1971; 40:365374.
30. Randall A. The origin and growth of renal calculi. Ann
Surg 1937; 105:10091027.
31. Kok DJ, Khan SR. Calcium oxalate nephrolithiasis, a free
or fixed particle disease? J Urol 1994; 46:847854.
32. Robertson WG. Kidney models of calcium oxalate stoneformation. Nephron Physiol 2004; 98:2130.
33. Borden TA, Lyon ES. The effects of magnesium and pH
on experimental calcium oxalate stone disease. Invest
Urol 1969; 6:412422.
34. Meyer JL, Smith LH. Growth of calcium oxalate crystals.
II. Inhibition by natural urinary crystal growth inhibitors.
Invest Urol 1975; 13:3639.
35. Fleisch H, Bisaz S. Isolation from urine of pyrophosphate,
a calcification inhibitor. Am J Physiol 1962; 203:671675.
36. Fleisch H, Bisaz S. The inhibitory effect of pyrophosphate
on calcium oxalate precipitation and its relation to urolithiasis. Experientia 1964; 20:276277.
37. Meyer JL, McCall JT, Smith LH. Inhibition of calcium
phosphate crystallization by nucleoside phosphates.
Calcif Tissue Res 1974; 15:289293.
38. Howard JE, Thomas WC, Barker LM, et al. The recognition and isolation from urine and serum of a peptide
inhibitor to calcification. Johns Hopkins Med J 1967;
120:119136.
39. Robertson WG, Peacock M, Nordin BEC. Inhibitors of the
growth and aggregation of calcium oxalate crystals in
vitro. Clin Chim Acta 1973; 43:3137.

40. Ryall RL, Harnett RM, Marshall VR. The effect of urine,
pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate
crystals in vitro. Clin Chim Acta 1981; 112:349356.
41. Robertson WG, Scurr DS, Bridge CM. Factors influencing
the crystallization of calcium oxalate in urine a critique.
J Cryst Growth 1981; 53:182194.
42. Worcester EM, Nakagawa Y, Coe FL. Glycoprotein calcium oxalate crystal growth inhibitor in urine. Miner
Electrolyte Metab 1987; 13:267272.
43. Worcester EM, Nakagawa Y, Wabner CL, et al. Crystal
adsorption and growth slowing by nephrocalcin, albumin
and Tamm-Horsfall protein. Am J Physiol 1988; 255:
F1197F1205.
44. Nakagawa Y, Ahmed MA, Hall SL, et al. Isolation from
human calcium oxalate stones of nephrocalcin, a
glycoprotein inhibitor of calcium oxalate crystal growth.
Evidence that nephrocalcin from patients with calcium
oxalate nephrolithiasis is deficient in gammacarboxyglutamic acid. J Clin Invest 1987; 79:17821787.
45. Hess B, Nakagawa Y, Coe FL. Inhibition of calcium
oxalate monohydrate crystal aggregation by urine proteins. Am J Physiol 1989; 257:F99F106.
46. Coe FL, Parks JH. Defenses of an unstable compromise:
crystallization inhibitors and the kidneys role in mineral
regulation. Kidney Int 1990; 38:625631.
47. Pillay SN, Asplin JR, Coe FL. Evidence that calgranulin is
produced by kidney cells and is an inhibitor of calcium
oxalate crystallization. Am J Physiol 1998; 275:
F255F261.
48. Shiraga H, Min W, Van Dusen WJ, et al. Inhibition of
calcium oxalate crystal growth in vitro by uropontin:
another member of the aspartic acid-rich protein superfamily. Proc Natl Acad Sci USA 1992; 89:426430.
49. Tsuji H, Tohru U, Hirotsugu U, et al. Urinary concentration of osteopontin and association with urinary
supersaturation and crystal formation. Int J Urol 2007;
14:630634.
50. Dussol B, Geider S, Lilova A, et al. Analysis of the soluble
matrix of five morphologically different kidney stones.
Urol Res 1995; 23:4551.
51. Stapleton AMF, Dawson CJ, Grover PK, et al. Further
evidence linking urolithiasis and blood coagulation:
urinary prothrombin fragment 1 is present in stone
matrix. Kidney Int 1996; 49:880888.
52. Grover PK, Ryall RL. Inhibition of calcium oxalate crystal
growth and aggregation by prothrombin and its fragments in vitro: relationship between protein structure
and inhibitory activity. Eur J Biochem 1999; 263:5056.
53. Dawson CJ, Grover PK, Ryall RL. Inter-alpha-inhibitor in
urine and calcium oxalate urinary crystals. Br J Urol 1998;
81:2026.
54. Evan AP, Bledsoe S, Worcester EM, et al. Renal interalpha-trypsin inhibitor heavy chain 3 increases in calcium
oxalate stone-forming patients. Kidney Int 2007; 72:
15031511.
55. Spector AR, Gray A, Prien EL. Kidney stone matrix.
Differences in acidic protein composition. Invest Urol
1976; 13:387389.
56. Lian JB, Prien EL, Glimcher MJ, et al. The presence of
protein-bound c-carboxyglutamic acid in calcium-containing renal calculi. J Clin Invest 1977; 59:11511157.
57. Jones WT, Resnick MI. The characterization of soluble
matrix proteins in selected human renal calculi using twodimensional polyacrylamide gel electrophoresis. J Urol
1990; 144:10101014.
58. Rose GA, Sulaiman S. Tamm-Horsfall mucoproteins promote calcium oxalate crystal formation in urine: quantitative studies. J Urol 1982; 127:177179.
179
59. Scurr DS, Robertson WG. Modifiers of calcium oxalate

crystallization found in urine. III. Studies on the role of
Tamm-Horsfall mucoprotein and of ionic strength. J Urol
1986; 136:505507.
60. Grover PK, Ryall RL, Marshall VR. Does Tamm-Horsfall
mucoprotein inhibit or promote calcium oxalate crystallization in human urine? Clin Chim Acta 1990; 190:223238.
61. Robertson WG. Take-home message on the epidemiology and basic science of stone-formation. In: Rodgers AL,
Hibbert BE, Hess B, et al., eds. Urolithiasis 2000. Cape
Town: University of Cape Town, 2000:841849.
62. Dent CE, Senior B, Walshe JM. The pathogenesis of
cystinuria. II. Polarographic studies of the metabolism of
sulphur-containing amino acids. J Clin Invest 1954;
33:12161226.
63. Milne MD, Asatoor AM, Edwards KDG, et al. The intestinal absorption defect in cystinuria. Gut 1961; 2:323337.
64. Ettinger B, Kolb FO. Factors involved in crystal formation
in cystinuria. In vivo and in vitro crystallization dynamics
and a simple colorimetric assay for cystine. J Urol 1971;
106:106110.
65. Dahlberg PJ, Van den Berg CJ, Kurtz SB, et al. Clinical
features and management of cystinuria. Mayo Clin Proc
1977; 52:533542.
66. Crawhall JC, Purkiss P, Watts RWE, et al. The excretion of
amino acids by cystinuric patients and their relatives.
Ann Hum Genet 1969; 33:149169.
67. Dent CE, Friedman M, Green H, et al. Treatment of
cystinuria. Br Med J 1965; 1:403407.
68. Dent CE, Senior B. Studies on the treatment of cystinuria.
Br J Urol 1955; 27:317332.
69. Crawhall JC, Scowen EF, Watts RWE. Effect of penicillamine on cystinuria. Br Med J 1963; 1:588590.
70. Stokes GS, Potts JT, Lotz M, et al. New agent in the
treatment of cystinuria: N-acetyl-D-penicillamine. BMJ
1968; 1:284288.
71. Remien A, Kallistratos G, Burchardt P. Treatment of
cystinuria with thiola (a-mercapto-propionyl glycine).
Eur Urol 1975; 1:227228.
72. Pak CYC, Fuller C, Sakhaee K, et al. Management of
cystine nephrolithiasis with mercaptopropionylglycine. J
Urol 1986; 136:10031008.
73. Frimpter GW. Medical management of cystinuria. Am J
Med Sci 1968; 255:348357.
74. Koide Y, Kinoshita K, Takemoto M, et al. Conservative
treatment of cystine calculi: effect of oral alphamercaptopropionylglycine on cystine stone dissolution
and on prevention of stone recurrence. J Urol 1982;
128:513516.
75. Gutman AB, YFC TF. Uric acid nephrolithiasis. Am J Med
1968; 45:657779.
76. Seegmiller JE, Grayzel AI, Laster L, et al. Uric acid
production in gout. J Clin Invest 1961; 40:13041314.
77. Atsmon A, De Vries A, Frank M. Uric Acid Lithiasis.
Amsterdam: Elsevier, 1963.
78. Metcalfe-Gibson A, MacCallum FM, Morrison RBI, et al.
Urinary excretion of hydrogen ion in patients with uric
acid calculi. Clin Sci 1965; 28:325345.
79. Kursh ED, Resnick MI. Dissolution of uric acid calculi
with system alkalinization. J Urol 1984; 132:286287.
80. Johnson RJ, Segaj MS, Sautin Y, et al. Potential role of
sugar (fructose) in the epidemic of hypertension, obesity
and the metabolic syndrome, diabetes, kidney damage
and cardiovascular disease. Am J Clin Nutr 2007; 86:
899906.
81. Cifuentes Delatte L, Rapado A, Abehsera A, et al. Uric
acid lithiasis and gout. In: Cifuentes Delatte L, Rapado A,
Hodgkinson A, eds. Urinary Calculi. Basel: Karger,
1973:115118.
180
Robertson
82. Coe FL, Moran E, Kavalich AG. The contribution of

dietary purine over-consumption to hyperuricosuria on
calcium oxalate stone-formers. J Chron Dis 1976; 29:
793800.
83. Henneman PH, Wallach S, Dempsey EF. The metabolic
defect responsible for uric acid stone-formation. J Clin
Invest 1962; 41:537542.
84. Maalouf NM, Sakhaee K, Parks JH, et al. Kidney Int 2004;
65:14221425.
85. Robertson WG, Nair D, Lang C, et al. The role of
Metabolic Syndrome in the formation of uric-acidcontaining stones. Urol Res 2010 (in press).
86. Bambach CP, Robertson WG, Peacock M, et al. Effect of
intestinal surgery on the risk of urinary stone formation.
Gut 1981; 22:257263.
87. Pak CYC, Sakhaee K, Fuller C. Successful management of
uric acid nephrolithiasis with potassium citrate. Kidney
Int 1986; 30:422428.
88. Rundles WR, Wyngaarden JB, Hitchings GH, et al. Effect
of xanthine oxidase inhibitor on thiopurine metabolism,
hyperuricaemia and gout. Trans Assoc Am Physicians
1963; 76:126140.
89. Godfrey RG, Rankin TJ. Uric acid renal lithiasis: management by allopurinol. J Urol 1969; 101:643647.
90. Greene ML, Fujimoto WY, Seegmiller JE. Urinary xanthine stones a rare complication of allopurinol therapy.
N Engl J Med 1969; 280:426427.
91. Band PR, Silverberg DS, Henderson JF, et al. Xanthine
nephropathy in a patient with lymphosarcoma treated
with allopurinol. N Engl J Med 1970; 283:354357.
92. Griffith DP, Musher DM, Itin C. Urease. The primary
cause of infection-induced urinary stones. Invest Urol
1976; 13:346350.
93. Robertson WG, Peacock M, Nordin BEC. Activity products in stone-forming and non-stone-forming urine. Clin
Sci 1968; 32:579594.
94. Lerner SP, Gleeson MJ, Griffith DP. Infection stones.
J Urol 1989; 141:753758.
95. Cox CE. Urinary tract infection and renal lithiasis. Urol
Clin N Am 1974; 1:279297.
96. Chinn RH, Maskell R, Mead JE, et al. Renal stone and
urinary infection: a study of antibiotic treatment. BMJ
1976; 2:14111413.
97. Griffith DP, Gibson JR, Clinton CW, et al. Acetohydroxamic
acid: clinical studies of a urease inhibitor in patients with
staghorn renal calculi. J Urol 1978; 119:915.
98. Martelli A, Buli P, Cortecchia V. Urease inhibitor therapy
in infected renal stones. Eur Urol 1981; 7:291293.
99. Griffith DP, Gleeson MJ, Lee H, et al. Randomized,
double-blind trial of Lithostat (acetohydroxamic acid) in
the palliative treatment of infection-induced urinary calculi. Euro Urol 1991; 20:243247.
100. Kinney AB, Blount M. Effect of cranberry juice on urinary
pH. Nurs Res 1979; 28:287290.
101. Pak CYC, Sakhaee K, Crowther C, et al. Evidence justifying a high fluid intake in treatment of nephrolithiasis.
Ann Intern Med 1980; 93:3639.
102. Jaeger P, Portmann L, Jacquet AF, et al. Drinking water
for stone-formers: is the calcium content relevant? Eur
Urol 1984; 10:5354.
103. Marshall RW, Cochran M, Robertson WG, et al. The
relation between urine saturation and stone composition
in patients with calcium-containing renal stones. Clin Sci
1972; 43:433441.
104. Albright F, Henneman P, Benedict PH, et al. Idiopathic
hypercalciuria. Proc R Soc Med 1953; 46:10771081.
105. Hodgkinson A, Pyrah LN. The urinary excretion of calcium and inorganic phosphate in 344 patients with calcium
stone of renal origin. Br J Surg 1958; 46:1018.
106. Pak CYC, Kaplan RA, Bone H, et al. A simple test for the
diagnosis of absorptive, resorptive and renal hypercalciurias. N Engl J Med 1975; 292:497500.
107. Coe FL, Favus MJ. Idiopathic hypercalciuria in calcium
nephrolithiasis. Disease-a-Month 1980; 26:136.
108. Halabe A, Sutton RAL. Primary hyperparathyroidism and
idiopathic hypercalciuria. Miner Electrolyte Metab 1987;
13:235241.
109. Robertson WG, Peacock M. The cause of idiopathic calcium stone disease: hypercalciuria or hyper oxaluria?
Nephron 1980; 26:105110.
110. Rose GA. Current trends in urolithiasis research. In: Rous
SN, ed. Stone Disease: Diagnosis and Management.
Orlando, FL: Grune & Stratton, 1987:383416.
111. Rudman D, Kutner MH, Redd SC, et al. Hypocitraturia
in calcium nephrolithiasis. J Clin Endocrinol Metab 1982;
55(6):10521057.
112. Nicar MJ, Skurla C, Sakhaee K, et al. Low urinary citrate
excretion in nephrolithiasis. Urology 1983; 21:814.
113. Hosking DH, Wilson JW, Liedtke RR, et al. Urinary citrate
excretion in normal persons and patients with idiopathic
calcium urolithiasis. J Lab Clin Med 1985; 106:682689.
114. Tiselius H G, Almgard LE, Larsson L, et al. A biochemical
basis for grouping of patients with urolithiasis. Eur Urol
1978; 4:241249.
115. Robertson WG, Peacock M, Heyburn PJ, et al. Risk factors
in calcium stone disease of the urinary tract. Br J Urol
1978; 50:449454.
116. Robertson WG. A risk factor model of stone-formation.
Front Biosci 2003; 8:13301338.
117. Coe FL, Kavalich AG. Hypercalciuria and hyperuricosuria in patients with calcium nephrolithiasis. N Engl J Med
1974; 291:13441350.
118. Grover PK, Ryall RL. Urate and calcium oxalate stones:
from repute to rhetoric to reality. Miner Electrolyte Metab
1994; 20:361370.
119. Prince CL, Scardino PL, Wolan CT. The effect of temperature, humidity and dehydration on the formation of renal
calculi. J Urol 1956; 75:209215.
120. Robertson WG, Peacock M, Marshall RW, et al. Seasonal
variations in the composition of urine in relation to calcium stone-formation. Clin Sci Mol Med 1975; 49:597602.
121. Pierce LW, Bloom B. Observations on urolithiasis among
American troops in a desert area. J Urol 1945; 54:466470.
122. Blacklock NJ. The pattern of urolithiasis in the Royal
Navy. In: Hodgkinson A, Nordin BEC, eds. Proceedings
of the Renal Stone Research Symposium. London:
Churchill, 1969:3347.
123. Brundig P, Berg W, Schneider HJ. Stress und Harnsteinbildungsrisiko. I. Einfluss von Stress auf lithogene Harnsubstanz. Urol Int 1981; 36:199207.
124. Diniz DHMP, Schor N, Blay SL. Stressful life events and
painful recurrent colic of renal lithiasis. J Urol 2006;
176:24832487.
125. Larsen JF, Phillip J. Studies on the incidence of urolithiasis. Urol Int 1962; 13:5354.
126. Clark JY. Renal calculi in army aviators. Aviat Space
Environ Med 1990; 61:744747.
127. Robertson WG, Peacock M, Hodgkinson A. Dietary
changes and the incidence of urinary calculi in the UK
between 1958 and 1976. J Chron Dis 1979; 32:469476.
128. Robertson WG, Peacock M, Baker M, et al. Studies on the
prevalence and epidemiology of urinary stone disease in
men in Leeds. Br J Urol 1983; 55:595598.
129. Power C, Barker DJP, Blacklock NJ. Incidence of renal
stones in 18 British towns. Br J Urol 1987; 59:105110.
130. Robertson WG, Peacock M, Heyburn PJ, et al. Should
recurrent calcium oxalate stone-formers become vegetarians? Br J Urol 1979; 51:427443.

131. Robertson WG, Peacock M, Marshall DH. Prevalence of
urinary stone disease in vegetarians. Eur Urol 1982;
8:334339.
132. Robertson WG. Diet and calcium stones. Miner Electrolyte
Metab 1987; 13:228234.
133. Iguchi M, Umekawa IT, Ishikawa T, et al. Dietary intake
and habits of Japanese renal stone patients. J Urol 1990;
143:10931095.
134. Curhan GC, Willett WC, Rimm EB, et al. A prospective
study of dietary calcium and other nutrients and the risk
of symptomatic kidney stones. N Engl J Med 1993;
328:833838.
135. Resnick MI, Pridgen DB, Goodman HO. Genetic predisposition to formation of calcium oxalate renal calculi. N
Engl J Med 1968; 278:13131318.
136. Coe FL, Parks JH, Moore ES. Familial idiopathic hypercalciuria. N Engl J Med 1979; 300:337340.
137. Aladjem M, Modan M, Lusky A, et al. Idiopathic hypercalciuria: a familial generalized renal hyper excretory
state. Kidney Int 1983; 24:549554.
181
138. Blacklock NJ. Epidemiology of renal lithiasis. In: Wickham JEA, ed. Urinary Calculous Disease. Edinburgh:
Churchill Livingstone, 1979:2139.
139. Coe FL, Parks JH, Asplin JR. The pathogenesis and
treatment of kidney stones medical progress. N Engl J
Med 1992; 327:11421152.
140. Fine JK, Pak CYC, Preminger GM. Effect of medical
management and residual fragments on recurrent stone
formation following shock wave lithotripsy. J Urol 1995;
153:2733.
141. Pak CYC. Kidney stones. Lancet 1998; 351:17971801.
142. Robertson WG. Is it possible to motivate patients
with recurrent stones to adhere to their treatment
regimen?In: Rodgers AL, Hibbert BE, Hess B, et al., eds.
Urolithiasis 2000. Cape Town: University of Cape Town,
2000:624627.
143. Norman RW, Bath SS, Robertson WG, et al. When should
patients with symptomatic stone disease be evaluated
metabolically? J Urol 1984; 132:11371139.
11
Pathophysiology and Management of Shock
Iain M. J. Mackenzie
Department of Anaesthesia and Critical Care, University Hospital Birmingham NHS
Foundation Trust, Edgbaston, Birmingham, U.K.
The manifestation of a rude unhinging of the machinery of life.

Dr Samuel D. Gross
A momentary pause in the act of death.
Dr R. Adams Cowley
WHAT IS SHOCK?
The war of the Spanish Succession was eventually
concluded by the treaty of Utrecht on the April 11,
1713, after two grueling battles on the Franco-Belgian
border, which left staggering numbers of dead and
wounded in their wake: about 37,000 at the Battle of
Malplaquet in 1709 and a further 23,000 at the Battle
of Denain in 1712. Even compared with earlier battles
of the campaign, such as the Battles of Blenheim (1704)
and Ramillies (1706), these casualty figures were
alarmingly high. Besides the change in military tactics
that had occurred since the Thirty Years War almost a
half century earlier, the other major contributor was
the replacement of the infantrymans pike for the
flintlock musket. Tending to the wounded in Louis
XIVs army was the young military surgeon Henri Le
Dran (16851770), who was later appointed as a surgeon at La Charite in Paris (1724), and eventually
became the Chief Surgeon to the French army. Drawing on his extensive experience, Le Dran published in
1737 a monograph on the management of gun-shot
wounds (1) in which he undoubtedly described both
hemorrhagic and septic shock, although contrary to
some accounts he never used the word choc himself. Six years later, in 1743, the English translation of
Le Drans now famous treatise was published in
London, and it is in the translation that the first use
of the word shock is made as a mistranslation of the
French secousse.
Although subsequent military conflicts, such as
the Crimean Wara (18531856), the American Civil
Warb (18611865), and the Spanish-American Warc
(1898), contributed a number of theories to the etiology
a
Shock identified as a distinct entity from haemorrhage.

George Crile.
c
Shock identified with infection.
of wound shock, a proper understanding of this

complex phenomenon required hemodynamic measurements that only became available during the course
of the 20th century (2).
In clinical terms shock describes a constellation
of signs and symptoms that reflect widespread organ
dysfunction caused by cellular metabolic failure. This
may arise from an intracellular failure of substrate
utilization, but is often driven, or at least accompanied
by, a failure of substrate delivery, either locally or
systemically (Fig. 1). More formally the definition
of shock varies depending on the clinical context
(Tables 1 and 2). By the time that the clinical features
attract attention, hypotension, relative or absolute, is
a common finding, accompanied by one or more
features of organ dysfunction such as confusion or
oliguriawith the conspicuousness of the clinical
presentation arising from the interplay between
severity and duration. Both the severity and duration
of the initiating insult determine the extent and
reversibility of cellular/organ dysfunction, exacerbated by stress signals from other sites.
The number of patients admitted to an intensive
care unit (ICU) with shock has risen steadily since
1997 (Fig. 2), although the overall proportion of
patients admitted in shock has remained much the
same. As the Intensive Care National Audit and
Research Centre (ICNARC) only collects data from
Englandd, one can extrapolate this figure on a population basis to estimate an incidence of over 31,300
cases per year for the United Kingdom as a whole. Of
note, the proportion of patients admitted in shock
with an underlying genitourinary problem has almost
doubled in the same period, now accounting for just
under 7% of all cases of shock admitted to ICU (Fig. 3).
The proportion by pathophysiological cause is hard to
By the 2001 census 94% of the UK population lived in England.
Chapter 11: Pathophysiology and Management of Shock
183
Nevertheless, it is likely that these three forms of

shock account for up to 75% or 80% of cases. The
hospital mortality of these patients increases with the
severity of shock and the number of failed organs, but
overall varies from around 33% in patients with septic
shock (8), 41.6% in patients with cardiogenic shock (9),
and up to 54% in patients with hemorrhagic shock (10).
HEMODYNAMIC PATHOPHYSIOLOGY
Traditionally, shock has been divided into four etiological categories, namely hypovolemic, cardiogenic,
obstructive, and distributive (11), with the later addition of endocrine shock as a fifth category. This
classification implies a single underlying hemodynamic problem in each category, which is almost invariably
not the case. The schema shown in Figure 1 illustrates
the cascade of dependencies, with tissue substrate
utilization requiring adequate peripheral delivery,
which itself requires an adequate central supply.
Central Supply
Figure 1 Schematic overview of the pathophysiological cascade of dependencies leading to shock. The top half of the
diagram illustrates the cascade of dependencies, where tissue
substrate utilization depends on the regional (distal) delivery of
substrate via arterioles and capillaries, which in turn is dependent
on the central supply of this substrate from the heart and major
arteries. The bottom half of the diagram illustrates the interplay
between the severity and duration of metabolic failure that then
result in a spectrum of abnormalities from an adaptive response
at one end of the spectrum to death at the other.
determine, but in a consecutive series of patients

admitted to ICU with shock, 53% had septic shock,
28% cardiogenic shock, and 19% hypovolemic shock
(7). However, this data is from a small series and was
performed in a surgical ICU, explaining the absence
of the many other, nonsurgical, causes of shock.
The central supply of substrate is generated by the

cardiac output, which is the product of stroke volume
and heart rate.
Cardiac output CO stroke volume SV
# heart rate HR
Stroke Volume
There are three factors that determine the stroke

volume: preload, contractility, and afterload (Fig. 4).
The factors that influence each of these is discussed
below.
Preload. The maximum force that a strip of
cardiac muscle can exert is an intrinsic property of
its precontraction length and determined by the
degree of overlap between the thick (myosin-bearing)
and thin (actin-bearing) myofilaments (Fig. 5). The
three-dimensional corollary of this two-dimensional
Table 1 Formal Definitions of Septic and Cardiogenic Shock

Septica
Cardiogenicb
SBP < 80 mmHg
or
;MAP $ 30%
Intravascular volume
SBP < 90 mmHg

or
;SBP > 40 mmHg
or
MAP < 60 mmHg
and
Adequate volume resuscitation
Other criteria 1
and
Other organ dysfunctionc
Other criteria 2
and
Sepsisa
Blood pressure
and
LVEDP > 18 mmHg
or
RVEDP > 10 mmHg
and
CI < 1.8 L/min1/m2 without support
or
CI < 2.2 L/min1/m2 with support
Data taken from Ref. 3

c
Abbreviations: CI, cardiac index; LEVDP, left ventricular end-diastolic pressure; MAP, mean arterial pressure; REVDP, right ventricular end-diastolic
pressure; SBP, systolic blood pressure.
b
184
Mackenzie
Table 2 Classification of Hypovolemic Shock
Volume loss (%)

Pulse (beats/min)
Blood pressure
Respiratory rate (breaths/min)
Urine output (mL/hr)
Neurology
Class I
Class II
Class III
Class IV
<15%
<100
Normal
1420
>30
Normal
1530%
100120
Decreased
2130
2030
Anxious
3140%
121140
Decreased
3140
519
Confused
>40%
>140
Decreased
>35
<5
Obtunded
Figure 2 Annual incidence of shock. Annual

admissions to ICUs in England participating in
the ICNARC Case Mix Programme divided by
(i) admission type (elective versus emergency) and (ii) the presence or absence or shock.
Shock in this instance is defined as following:
1. Cardiovascular failure (SBP & 90 mmHg
or MAP & 70 mmHg for at least one hour
despite adequate fluid resuscitation, or
the use of vasopressors)
2. Failure of at least one other organ system
by the PROWESS criteria (data taken
from Ref. 6)
Data courtesy of Case Mix Programme Database, ICNARC (Intensive Care National Audit
and Research Centre), Tavistock House,
Tavistock Square, London WC1H 9HR.
phenomenon is that stroke volume is determined by

the precontraction ventricular volume, which is itself
determined by the rate and duration of ventricular
filling. Ventricular filling has three phases: early, mid-,
and end diastolic (13).
Figure 3 Annual proportion of shock cases arising from a

genitourinary condition. Data courtesy of Case Mix Programme
Database, ICNARC (Intensive Care National Audit and Research
Centre), Tavistock House, Tavistock Square, London WC1H
9HR.
Figure 4 Conceptual analogy for the relationship between

preload, contractility, and afterload in determining stroke volume.
Using the analogy of an arrow being fired into a target, it
becomes intuitively obvious that the depth of penetration (stroke
volume) is determined by the draw of the bowstring [(A) preload],
the stiffness of the bow [(B) contractility], and the resistance of
the target [(C) afterload].
185
Figure 5 Relationship between precontraction

sarcomere length and tension generated on
contraction. Source: Adapted from Ref. 12.
Early diastolic ventricular filling is augmented

by the elastic recoil of the ventricle that springs back
to its resting volume at the beginning of diastole,
sucking blood into the ventricle as it does so.
Mid-diastolic ventricular filling, which under
normal circumstances is the principle determinant of
the end-diastolic ventricular volume, occurs at a rate
determined by (i) the veno-ventricular pressure gradient, more commonly referred to as the filling pressure, and (ii) ventricular stiffness (Fig. 6). It is worth
emphasizing three points that are the source of common misconceptions. First, preload is defined by
the ventricular end-diastolic volume, and not, as is
widely believed, simply the filling pressure (often
measured clinically as the central venous pressure
[CVP]). This is illustrated in Figure 6 as a normal
stroke volume arising from the combination of either a
low filling pressure and a compliant ventricle (panel
A), or a high filling pressure and a stiff ventricle
(panel D). Second, filling pressure arises from the
combined effects of circulating volume (also referred
to as blood volume or intravascular volume) and
venous tone, not just circulating volume alone. This
is illustrated in Figure 7 where it can be seen that a
normal circulating volume can generate a high (Panel A),
normal (Panel C), or low (Panel E) filling pressure
depending on whether the venous tone is high, normal,
or low, respectively. Conversely, a normal filling pressure can arise with a low (Panel B), normal (Panel C), or
high (Panel D) circulating volume if the venous tone is
high, normal, or low, respectively. Third, body weight
cannot be used to estimate circulating volume in
patients who may have leaky capillaries and interstitial edema (Fig. 8). Stiffness, properly termed
elastance, describes the relationship between pressure
(y-axis) and volume (x-axis), and for a simple elastic
body, for example, a rubber balloon, plots an upward
curve (Fig. 9). Passive ventricular stiffness is determined by the viscoelastic properties and mass of the
ventricle, and is increased in conditions that result in
either myocardial fibrosis, or ventricular wall thickening, displacing the elastance curve upward (Fig. 9A).
Unlike a rubber balloon, however, the ventricle is

encased in a pericardial sac that is not only considerably stiffer, but whose limited volume has to be
shared with the other cardiac chambers. Therefore,
at low/normal diastolic volumes ventricular elastance
is determined by the ventricle alone (Fig. 10A, B, C),
but at a critical diastolic volume pericardial elastance
becomes superimposed on the ventricular elastance
and, being much greater, becomes dominant
(Fig. 10D). Although pericardial elastance itself can
increase even further as a result of pericardial fibrosis
or calcification, the impact of the pericardiums
increased stiffness becomes much more significant if
the critical diastolic volume is reduced. This may
occur if any of the other cardiac chambers are
enlarged, or if there is fluid within the pericardial
sac (Fig. 10B). The second difference from a simple
elastic body is that the ventricle is composed of
cardiac muscle. Total ventricular elastance is therefore
composed of a passive component, described above,
and an active component that is determined by
the extent of ventricular relaxation in diastole. In
molecular terms relaxation is a process in which the
myofibrillar myosin heads disengage from the actin
filaments as a result of energy-dependent calcium
sequestration into the sarcoplasmic reticulum. Being
energy-dependent, relaxation is impaired in ischemic
myocardium.
Finally, ventricular volume is given a presystolic
boost by atrial contraction. This effect is lost in
patients with atrial fibrillation or flutter, but this is of
little consequence in patients with well-preserved ventricular function. However, in patients with significant
impairment of left ventricular function, the loss of the
atrial boost is much more noticeable, and may precipitate or exacerbate symptoms of heart failure such as
exercise intolerance, dyspnea, and fluid retention.
In the absence of changes in contractility, or
afterload, increases in preload result in an increase
in the force generated by myofibril contraction (Fig. 5)
and stroke volume (Fig. 10) up to the point of optimal
preload. Thereafter, further increases in preload cause
186
Mackenzie
Figure 6 Effect of filling pressure and ventricular elastance (stiffness) on end-diastolic ventricular volume. Preload is defined by the
ventricular end-diastolic volume (LVEDV) and may result in a normal stroke volume (A and D) with a low filling pressure and a compliant
ventricle (A), or a high filling pressure and a stiff ventricle (D). A low filling pressure and stiff ventricle (B) results in a low LVEDV and a
low stroke volume, while a high filling pressure and compliant ventricle (C) results in a high LVEDV and a high stroke volume.
a decrease in both the force generated by myofibril

contraction (Fig. 5) and stroke volume (Fig. 11). Factors affecting preload are shown in Table 3.
Contractility. Changes in myocardial contractility account for changes in stroke volume under similar
conditions of preload and afterload (Fig. 11) and may
be effected physiologically, pharmacologically, or
pathologically (Table 2). In practical terms, the commonest causes of depressed contractility in patients
with shock are myocardial ischemia or infarction, or
sepsis. Clinically contractility is optimized by correcting any relevant electrolyte disturbances, and avoid-
ing or minimizing the use of pharmacological agents

that reduce contractility (negative inotropes). Finally,
contractility can be directly augmented by drugs that
increase the effect of myocyte cytosolic calcium on
troponin, either by increasing the myocyte calcium
content, or by sensitizing the tropinin to the effects of
calcium (Table 4).
Afterload. Constant blood flow through vital
organs is achieved by maintaining a constant perfusion pressure by the regulation of afferent and
efferent vasomotor tone. Increases in mean arterial
pressure (MAP) are regulated by afferent arteriolar
187
Figure 7 Effect of intravascular volume and venous tone on filling pressure. Filling pressure arises from the combined effects of
circulating volume and venous tone. Thus, a normal circulating volume can generate a high (A), normal (C), or low (E) filling pressure
depending on whether the venous tone is high, normal, or low, respectively. Conversely, a normal filling pressure can arise with a low
(B), normal (C), or high (D) circulating volume if the venous tone is high, normal, or low, respectively.
Figure 8 Body weight and circulating volume. In the

absence of capillary leak and third space losses body
weight provides a reasonable estimate of intravascular volume (A and B). However, in the context of systemic inflammation (C), which is invariably associated with capillary leak,
body weight and intravascular volume are not related.
188
Mackenzie
Figure 9 Increases in ventricular elastance (stiffness). An

increase in ventricular elastance as a consequence of either
increased ventricular muscle mass (pressure overload of the
ventricle, e.g., hypertension or stenosis of the ventricular exit
valve) or ventricular fibrosis (ischemic heart disease) causes the
elastance curve to be displaced upward (A). A reduction in the
ventricular volume at which pericardial elastance becomes dominant (B) arises when the available pericardial volume is reduced,
either by the overdistension of any of the other cardiac chambers, or by the presence of pericardial fluid (pericardial effusion
or hemopericardium) resulting in pericardial tamponade.
vasoconstriction, while decreases in MAP are accommodated initially by afferent arteriolar vasodilatation.
However, when arteriolar vasodilatation is maximal,
perfusion pressure is maintained by efferent vasoconstriction, which inevitably reduces flow. This means
that the perfusion of these vital organs is compro-
Figure 11 Effect of changes in contractility on the relationship

between end-diastolic volume (preload) and stroke volume. The
green curve represents the normal relationship between stroke
volume and end-diastolic volume. The red curve shows the effect of
an increase in contractility, while the blue curve shows the effect of
a decrease in contractility. At the same end-diastolic volume, an
increase in contractility results in an increase in stroke volume,
while a decrease in contractility has the opposite effect.
mised by reductions in MAP. If the amount of cardiac

work per unit time (cardiac power) is held constant
then the MAP is proportional to systemic vascular
resistance [SVR, eq. (2)], which is a measure of cardiac
Figure 10 Ventricular elastance curves. In each of the panels the cartoon above the graph represents the ventricle (brown) surrounded
by the pericardium (blue). In panels A, B, and C ventricular volume can be seen to trace a smooth upward curve in relation to ventricular
pressure, and as the ventricular volume increases progressively more of the pericardial slack is taken up. At a critical volume,
represented by panel D, the pericardial slack is completely taken up by the expanding ventricle. Ventricular elastance is now principally
determined by the pericardium, and as the latter is considerably stiffer than the ventricle, the gradient of the pressure/volume curve (the
elastance) becomes markedly steeper.
189
Table 3 Factors Affecting Preload
Physiological
Pharmacological
Pathological
Increased preload
Decreased preload
. Increased intravascular volume

Renal retention of salt and water
(: ADH, : angiotensin II,
: cortisol, ; ANP)
: Fluid intake (thirst)
. Increased venous tone
: Sympathetic drive
. Intravenous fluids
. Decreased intravascular volume

Renal diuresis (; ADH, ; angiotensin II, ; cortisol, : ANP)
; Fluid intake
. Decreased venous tone
: Parasympathetic drive
. Fluid retention
Renal failure
Cardiac failure
Liver failure

Diuretics
Diuretics
Opiates
Hemorrhage
Inappropriate renal diuresis (polyuric ATN, diabetes insipidus,
diabetes mellitus, hypocortisolism)
Fluid loss from GI tract (vomiting, diarrhea) or impaired fluid
reabsorption from GI tract (ileus)
Increased insensible losses (fever, burns)
Capillary leak (systemic inflammation, anaphylaxis)
. Increased intrathoracic pressure
Tension pneumothorax
Positive pressure ventilation
Continuous positive airway pressure (CPAP)
. Reduced diastolic ventricular compliance
Pericardial effusion
Ventricular hypertrophy or infiltration
Ventricular dilatation (Bernheim effect)
Table 4 Factors Affecting Cardiac Contractility

Increased contractility
Physiological
Pharmacological
Pathological
Cardiac sympathetic nerve stimulation (sympathetic

nerves ? noradrenaline ? cardiac b1-adrenoreceptors)
Adrenomedullary drive (adrenaline release ? cardiac
a1-adrenoreceptors)
Hypercalcemia
: Myocyte calcium load
: cAMP synthesis
* b1-adrenergic receptor agonists (adrenaline,
dopamine, dobutamine, dopexamine, noradrenaline)
* Glucagon receptor
; cAMP degradation
Phosphodiesterase inhibitors (milrinone, amrinone)
: Na/Ca2 exchange
* Cardiac glycosides (digoxin)
: Extracellular calcium concentration (calcium)
: Myocyte calcium sensitivity (levosimendan)
Hyperthyroidism (early)
Decreased contractility
l
b-Blockers
Calcium channel blockers (verapamil,
nicardipine)
Amiodarone
Volatile anesthetic agents
l
l
l
l
l
l
l
l
l
l
Ischemia/hypoxia
Infarction
Sepsis
Electrolyte disturbance
Hypocalcemia
Hyperkalemia
Hypokalemia
Hypothermia
Acidemia
Cardiomyopathy
Myocarditis
190
Mackenzie
Table 5 Factors Affecting Afterload
Physiological
Pharmacological
Pathological
Increased LV afterload
Decreased LV afterload
. Arteriolar vasoconstriction
Cold
Pain
Fear
. Arteriolar vasoconstrictors
a-Adrenergic agonists
(noradrenaline, metaraminol)
b-Adrenergic antagonists
. Arteriolar vasodilatation
Exercise
Hypercapnia
Pyrexia
. Arteriolar vasodilators
a-Adrenergic antagonists (phentolamine, phenoxybenzamine,
doxasozin)
b-Adrenergic agonists (dobutamine, dopexamine, dopamine)
: Smooth muscle cGMP (nitrates)
Smooth muscle Ca2 channel blockers (nifedipine, verapamil)
; Angiotensin II (angiotensin converting enzyme inhibitors,
angiotensin II receptor antagonists)
Smooth muscle relaxant (hydralazine, prostacyclin)
. Arteriolar vasodilatation
Sepsis
Anaphylaxis
. Systemic AV shunts
. Loss of sympathetic tone
Thoracic epidural or high spinal
Cervical or thoracic spinal cord damage
.
.
.
.
:Sympathetic drive (hypovolemia)

18 or 28 hypertension (LV)
Aortic valve stenosis
Aortic coarctation
Increased RV afterload
Physiological
Pharmacological
Pathological
Decreased RV afterload
. Low partial pressure of inspired gas

(high altitude, hypoxic gas mixture,
upper airway obstruction)
. Very low (pneumothorax) or very high
lung volumes
. Pulmonary AV shunts
. Inhaled nitric oxide

. Pulmonary AV shunts
. Acute or chronic pulmonary

thromboembolism
. Pulmonary hypertension
. Pulmonary valve stenosis
. Mitral valve stenosis
afterload, and the MAP is inversely proportional to

the cardiac output [eq. (3)].
SVR / MAP
1
/ cardiac output
SVR
What these equations tell us is that under conditions of constant cardiac power output, a reduction
in afterload allows the cardiac output to rise, whereas
an increase in afterload (MAP) causes the cardiac
output to fall. In this latter situation, achieving the
minimum MAP required to maintain vital organ
perfusion may only be possible with an increase in
afterload that then reduces total blood flow to less
than that required by the vital organs. Taken together,
this tells us that the perfusion of vital organs requires
the central supply of both pressure and flow.
Clinically, cardiac power output is only ever
fixed when a patient is operating at the upper limit
of their hearts capacity. This occurs when there is an
imbalance between the demand being placed on the
heart, and the hearts capacity, which may entail an
increase in demand, a decrease in capacity, or both.
Factors affecting afterload are presented in Table 5.
Heart Rate
By virtue of the relationship between cardiac output

and heart rate defined above, cardiac output declines
linearly as the heart rate falls, providing that the
stroke volume remains constant. Under normal circumstances the decline in cardiac output is mitigated
by a protective reflex mediated by sympathetic drive
that increases the stroke volume. This mechanism is
limited by a maximum stroke volume, beyond which
further reductions in heart rate result in a steep
decline in cardiac output. Where the falling heart
rate is being driven by parasympathetic (vagal)
drive, or in patients who cannot mount an adequate
sympathetic response (sympathetic outflow blocked
by a thoracic epidural or high spinal block, a adrenergic blockade, Table 6), this protective reflex is
severely compromised and the cardiac output
declines more precipitously. As the cardiac output
falls below the critical perfusion thresholds for brain
and heart, the patient starts to lose consciousness and
cardiac function is further compromised by ischemia.
Commonly, this sequence of events occurs over a
minute or two and, in the absence of intervention,
culminates in cardiorespiratory arrest. While some
patients will be found to be asystolic, others develop
a degenerative rhythm under these conditions such as
191
Table 6 Factors Affecting Cardiac Output

Increased cardiac output
l
l
l
l
Optimized preload
Increased contractility
Decreased afterload
Increased heart rate (within normal range)
Decreased cardiac output

l
l
l
l
Suboptimal or supraoptimal preload

Decreased contractility
Increased afterload
Bradycardia
* Reflex (pneumoperitoneum, mesenteric traction, pain, nausea, inferior MI)
* Opiates
* Volatile anesthetic agents
; Sympathetic drive
; Sinus node automaticity
Atrioventricular conduction defect
Tachycardia
ventricular fibrillation or torsades de pointes. In rapidly progressive bradycardia this sequence of events
can be aborted providing (i) appropriate monitoring
is in place to provide both visual and auditory
warning of the impending disaster, (ii) intravenous
access is immediately available, and (iii) appropriate
rescue therapy, such as atropine (0.51 mg) or ephedrine (612 mg), is administered quickly enough. In
urological practice, where spinal anesthesia is commonly employed in the frail and elderly undergoing
transurethral procedures, rapidly progressive bradycardia is a trap for the unwary that may spring
immediately following the completion of surgery. At
this time, the combination of (i) sympathetic blockade
from a high spinal anesthetic, (ii) hypovolemia from
preoperative fasting, vasodilatation, and intraoperative blood loss, and (iii) nausea secondary to hypotension, all synergize to produce vagal drive and
hypotension precipitated by removing the patients
legs from the lithotomy stirrups. Not uncommonly,
the anesthetist will already have removed the monitoring equipment in anticipation of transferring the
patient into the recovery area, and with the distraction of theater staff preparing for the next case, the
patients loss of consciousness may not be immediately appreciated.
Although it may seem obvious that an increase
in heart rate would de facto result in an increase in
cardiac output, this is not, in fact, the case. This is
explained by the observation that as the heart rate
increases there is progressively less time during diastole for the left ventricle to fill with blood, resulting in
a paradoxical decrease in stroke volume. Under most
circumstances, however, increases in heart rate are
driven by stimuli, which also increase the stroke
volume, for example, endogenous or exogenous catecholamines. In the absence of an extracardiac stimulus for an increased heart rate, a symptomatic
tachycardia is usually driven by an atrial or ventricular dysrhythmia. The urgency of intervention is determined by the degree of circulatory failure and, where
the patient is symptomatically hypotensive, or
shocked, requires immediate recognition and synchronized DC cardioversion.
ASSOCIATED PATHOPHYSIOLOGY
Although shock is primarily defined by the characteristic hemodynamic disturbance that leads to the failure of substrate delivery, the shocked state is
inextricably linked to associated abnormalities in
other organ systems that this failure of substrate
delivery provokes.
Hypotension and Organ Ischemia

The loss or reduction of normal organ perfusion will
have consequences that will depend not only on the
extent and duration of hypoperfusion, but also on the
presence of coexisting abnormalities, such as ischemic
heart disease, peripheral vascular disease, or hypertension. In susceptible patients, therefore, shock can
precipitate ischemia or infarction of the heart, gut,
kidneys, liver, or brain. Myocardial ischemia or infarction has immediate hemodynamic consequences,
either by reducing myocardial contractility or by
precipitating abnormalities of rhythm or conduction,
thus complicating the hemodynamic picture. The consequences of gut, kidney, liver, or brain damage may
be less immediately damaging, but are no less important in terms of their impact on morbidity and mortality. Gastrointestinal ischemia can lead to bacterial
translocation, discussed below, as well as precipitating gastric or duodenal stress ulceration and GI hemorrhage, ileus, or even GI perforation. Hepatic
ischemia is rather more subtle in its manifestations,
which may range from an elevation of hepatic
enzymes and hypoabuminemia, which are very
common, to a failure of hepatic function with prolongation of the prothrombin time, worsening lactic
acidemia and intractable hypoglycemia, which are less
common. Renal function is almost always disturbed in
patients with shock, although oliguria may not be
present if in fact it is a failure of renal concentrating
ability, which is responsible for, or contributing to, the
hypovolemia. Although renal function is readily supported by hemofiltration, renal failure remains an
independent risk factor for mortality, the reason for
which remains unclear. Finally, neurological
192
Mackenzie
Figure 12 Positive feedback cycle connecting

shock, cellular dysfunction, and inflammation.
disturbances are commonly seen in the acute phase of

shock, but persisting brain failure in the form of
delirium is commonly seen in patients during the
recovery phase of critical illness.
Systemic Inflammation
Although a systemic inflammatory response may
result in shock, for example, as a result of sepsis,
anaphylaxis, or pancreatitis, shock can itself provoke
a systemic inflammatory response by exposing the
tissues to an ischemic insult. For example, pure hemorrhagic shock has been shown to cause significantly
increased plasma concentrations of the inflammatory
cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor a (TNFa)(14), and in patients with trauma
particular cytokine profiles have been associated with
outcome (15). Disseminated activation of the innate
immune system has a number of consequences. Within the circulation endothelial cell activation leads to
the expression of cell surface receptors, promoting the
margination and vascular emigration of activated
leukocytes. Activationt of the vascular endothelium
leads to a profound relaxation of the interendothelial
cell junctions, resulting in fluid extravasation, and
increased endothelial expression of tissue factor
resulting in intravascular activation of the coagulation
cascade and the deposition of microthrombi in the
capillaries. A number of inflammatory mediators,
which include TNFa and nitric oxide, have also been

implicated in the loss of smooth muscle and myocardial contractility. Thus, the hypovolemia precipitated
by the capillary leak is then compounded by both
arteriolar and venous vasodilatation as well as by
reduced myocardial contractility, creating a positive
feedback loop for hemodynamic deterioration. Within
the splanchnic circulation, ischemia and circulating
inflammatory mediators also impair the barrier function of the intestinal epithelium, allowing the ingress
of bacteria and bacterial toxins such as endotoxin.
These agents are themselves potent activators of the
innate immune system and there is a growing body of
circumstantial and animal data to suggest that this
process of bacterial translocation plays a role in
driving the pathophysiology of multiple organ failure
(16) (Fig. 12).
In septic patients with an elevated cardiac output but evidence of tissue hypoxia (lactic acidosis) and
organ dysfunction, it was originally believed that the
problem was caused by regional ischemia, with flow
being diverted away from the tissues by arteriovenous
shunts or intracapillary microthrombi. While this has
certainly been seen to occur in some tissue beds (17),
elevated tissue oxygen tensions have been documented in both septic animals (18) and man (19).
These observations, and others, have led to the identification of a phenomenon referred to as cytopathic
dysoxia (20), a condition in which cellular dysfunction
arises from a failure of substrate utilization rather than

delivery. Although more typically associated with
primary systemic inflammation, especially sepsis,
rather than shock-induced secondary systemic
inflammation, it is likely to be a feature of both
conditions, and may represent a cellular stress
response in which mitochondrial function is downregulated rather than actually driving the observed
abnormalities in cellular function. This is discussed
further below.
MANAGEMENT OF SHOCK
The range of clinical conditions associated with the
presence of cellular metabolic failure (i.e., shock) can
vary from cardiac arrest, at one end of the spectrum,
to subtle biochemical abnormalities in a patient who
otherwise gives no cause for concern, at the other end
of the spectrum. However, the same logical and
methodical approach to the assessment and management for all these patients ensures that the former
are treated effectively, and shock in the latter is not
overlooked.
The assessment begins with an evaluation of
respiratory function and, in particular, the quality of
the airway and breathing. Four broad situations
can usually be recognized by (i) respiratory arrest,
(ii) agonal respiration, (iii) abnormal breathing, and
(iv) normal breathing. Tachypnea is an early and
useful sign of clinical deterioration (21) and, in
patients with shock, may reflect acidosis (tissue hypoperfusion, renal dysfunction) or increased carbon
dioxide production associated with the hypermetabolism of sepsis. More recently, it has been shown that
sepsis (22) induces early contractile failure in the
diaphragm, a fact that may explain ventilatory failure
in patients with an extrapulmonary source of sepsis.
In adults, respiratory arrest is almost always secondary to cardiac arrest, the management of which is
beyond the scope of this chapter. Agonal respiration
is usually easily recognized as intermittent stertorous
and labored respiration accompanied by marked
impairment of neurological function. Assessment
using the Glasgow scale is likely to reveal a patient
whose eyes open to pain (2) or not at all (1), who is
able to make incomprehensible sounds (2), and who
either localizes (5) or withdraws from a painful stimulus. This peri-arrest situation requires immediate
attention, with urgent tracheal intubation and
mechanical ventilation by members of the ICU team.
In the meantime, having established vascular access
and initiated fluid resuscitation, further examination
of the patient is likely to provide clues as to the
underlying problem. Warm peripheries with a brisk
capillary refill are typical of systemic inflammation,
and in some patients gentle pressure on the nail-bed
elicits the capillary pulsation of Quinckes sign. Otherwise, cool peripheries with a prolonged capillary
would be expected. Most patients in shock are likely
to have a tachycardia up to 150/min, with a bounding
pulse being typical of systemic inflammation, and a
weak thready pulse indicating other types of shock.
193
Pulse rates higher than this may simply reflect profound hemodynamic disturbance, but may in fact
indicate a tachydysrhythmia. If this is confirmed on
electrocardiography, and the rhythm is causing symptomatic hypotension, consideration should be given to
immediate DC cardioversion. If time allows, blood gas
analysis may reveal hypokalemia, which ought to be
corrected before, or as soon as possible after, cardioversion. Bradycardia or a normal pulse rate would be
highly unusual in most cases of shock, unless the
bradycardia itself was driving the hemodynamic condition, for example, in patients with significant parasympathetic drive. Finally, in most cases of overt
shock one would expect hypotension. Serious hypotension (systolic blood pressure <70 mmHg) that does
not appear to be responding to fluid therapy deserves
a little pharmacological support. In tachycardic
patients increments of 1 mg of metaraminol, a potent
a-agonist, are usually very effective in preventing any
further deteriorations in the blood pressure, amd may
be accompanied by a modest fall in the heart rate. In
bradycardic patients incremental doses of 3 to 6 mg of
ephedrine may be effective in increasing both the
heart rate and blood pressure, as would small doses
(0.51 mL) of adrenaline 1:10,000. In the seriously
compromised patient additional hemodynamic monitoring is required to clarify the hemodynamics and
guide therapy.
Assessment and Optimization of Preload

Clinical assessment of the jugular venous pressure can
only serve as a very rough guide to right ventricular
preload, which is best measured by the placement of a
central venous catheter. Even then, the CVP is itself a
relatively poor indicator of left ventricular preload
and makes the following assumptions:
1.
2.
3.
4.
5.
6.
7.
Left ventricular end-diastolic pressure (LVEDP) /

Left ventricular end-diastolic volume (assumes
constant and normal LV compliance)
Left atrial pressure (LAP) / LVEDP (assumes
normal mitral valve)
Pulmonary artery pressure (PAP) / LAP (assumes
constant and normal pulmonary resistance)
Right ventricular end-diastolic volume (RVEDV)
/ PAP (assumes normal pulmonary valve)
Right ventricular end-diastolic pressure (RVEDP)
/ RVEDV (assumes constant and normal RV
compliance)
Right atrial pressure (RAP) / RVEDP (assumes
normal tricuspid valve)
CVP / RAP
However, providing ventricular compliance and

pulmonary resistance remain constant, changes in
CVP will reflect changes in ventricular preload within
an individual. Between individuals there is too much
variation in these factors to detect any relationship
even between pulmonary artery occlusion pressure
(PAOP) and LVEDV (Fig. 13). Nevertheless, it is worth
noting that it is almost impossible for a correctly
measured CVP to underestimate ventricular preload,
194
Mackenzie
Figure 13 Relationship of the left ventricular end-diastolic index (LVEDVI) to the PAOP. Data from 12 normal subjects (left). Data from
61 patients with acute respiratory distress syndrome (ARDS) (right). Source: From Refs. 23 and 24.
and therefore an abnormally low CVP almost certainly

reflects functional hypovolemia.
The pulmonary artery catheter (PAC) eliminates
some of the assumptions inherent in the CVP and
allows direct measurement of the PAP, which is very
helpful in patients with right venticular dysfunction or
raised PAPs (pulmonary thromboembolism or pulmonary hypertension). In unselected patients use of the
PAC has not been shown in either of two large prospective trials to be associated with any benefit (25,26),
and its use is falling out of favor. In place of the PAC
some centers are now using other techniques for hemodynamic assessment such as esophageal Doppler or
pulse-contour analysis. These techniques are not only
less invasive, but also provide continuous measurement of stroke volume, cardiac output, and SVR. This is
particularly helpful in allowing dynamic assessment of
the patients response to fluid loading. Where there is
doubt of the patients preload status, this is an effective
technique and involves challenging the patients with
repeated boluses of fluid (sufficient to cause an elevation of CVP or PAOP of >3 mmHg) until there is no
further improvement in cardiac output.
Transthoracic echocardiography is a rapid, noninvasive, and useful method of assessing the hemodynamics, if somewhat underutilized. Echocardiography
not only provides information about contractility and
preload, but also eliminates valve failure, pericardial
effusion/tamponade, and pulmonary embolism as
causes for shock.
There is currently no evidence to favor isotonic
crystalloids over colloids although large volume fluid
resuscitation with 0.9% sodium chloride risks hyperchloremic acidosis and may be associated with a
number of other adverse effects. In cases of hemorrhage these fluids must be accompanied by packed
red blood cells. Current recommendations (27) are to
give fresh frozen plasma (1015 mL/kg) if the prothrombin time is $ 1.5 times normal, with cryopreci-
pitate if the plasma fibrinogen concentration falls

below 1 g/L and platelet concentrate to maintain the
count above 50,000 cells/mm3. Overt blood loss, such
as hemorrhage from an open wound, is impossible to
overlook, but equally significant blood loss can occur
covertly, such as that associated with pelvic and long
bone fractures, gastrointestinal bleeding, or even following transurethral resection of the prostate. In the
young or fit patient significant hypovolemia can be
obscured by highly effective reflex mechanisms that
are capable of maintaining an almost normal blood
pressure and heart rate. In these patients hemodynamic decompensation is a late and precipitous event,
and there may be other signs of covert shock such
as metabolic acidosise or elevated plasma lactate concentration. Thus, in patients with septic shock, for
example, the addition of a mixed venous saturation
greater than 70% as a therapeutic end point for
resuscitation was associated with a 16% reduction of
hospital mortality (28). Although some patients
respond to volume resuscitation alone, most will
also require pharmacological support of either contractility, or afterload, or both.
Management of Contractility and Afterload

The effect of vasopressor and inotropic agents is most
commonly determined by their activity at endogenous
receptors of the adrenergic (a and b) and dopaminergic families, although there are agents that work in
other ways (Table 4). This knowledge combined with
an understanding of the effects of receptor activation
on preload, contractility, and afterload allows clinicians to estimate which agent, or combination of
agents, is most likely to produce the desired effects
(Table 7). However, the actual effects produced in
e
Indicated by a fall in pH or an elevated base deficit.
195
Table 7 Agents Used to Manipulate Preload, Contractility, and Afterload
Adrenaline
Dobutamine
Dopamine (<5 mg/kg/min)
Dopamine (510 mg/kg/min)
Dopamine (>10 mg/kg/min)
Dopexamine
Nitrates
Isoprenaline
Milrinone
Noradrenaline
Phentolamine
Preload
CO
SVR
MAP
HR
SBF
)
*
)
))
)
)
NC
NC
))
*
)
)
)
))
))
:
;
:
:
:
;
;
;
;
:
;
:
:
(:)
(:)
(:)
:
:
:
:
;
:
;
:
:
?
;
:
?
?
?
: or ;
:
Abbreviations: CO, cardiac output (a clinical surrogate for contractility but is strongly influenced by afterload); SVR, systemic vascular resistance (a good
reflection of afterload); MAP, mean arterial pressure; HR, heart rate; SBF, splanchnic blood flow; ?, unknown or unpredictable; NC, no change.
vivo may differ from those expected because of the

effects of adrenoreceptor up- and downregulation
during critical illness (29), the effect of polymorphisms
in the adrenoreceptor genes (30), and alterations to
adrenoreceptor activationf caused by free radicals (31).
In practice, therefore, the desired effect may require a
therapeutic trial of more than one agent, or the use of a
combination of agents. Unfortunately, the quality of
evidence on which to base the choice, or combination,
of agents, is generally low (32) and largely based on
animal data, small studies in patients, and expert
opinion. Currently, either norepinephrine or dopamine is recommended as first-line agents (33) for the
treatment of septic shock. For norepinephrine this
represents the culmination of two decades of rehabilitation, having originally been believed to increase
blood pressure at the expense of tissue (especially
splanchnic) perfusion. Norepinephrine increases the
blood pressure more rapidly and reliably than
dopamine (34) and improves renal function, but only
produces a modest rise in cardiac output. Its effects on
the liver and gastrointestinal mucosa are unpredictable. Dopamine, on the other hand, despite increasing
splanchnic blood flow at low doses does produce
increased gut oxygen consumption or improvements
in hepatic function. Moreover discomfort is growing
about a range of negative effects attributable to
dopamine, including the reduction of gut motility,
hypoprolactinemia-mediated immunosuppression,
reduced anabolism related to a fall in the production
of growth hormone and dehydroepiandrosterone, and
impaired thyroid function (35). In a recent observational study involving 198 centers in 24 European
countries, the use of dopamine in the subset of
patients with sepsis (n = 1058) was associated with
an increased risk of hospital death (36) (relative risk
1.19, 95% confidence interval 1.041.36). Epinephrine
is now rarely used as a single agent, if at all, as it
causes a fall in splanchnic perfusion and, in some
patients, a lactic acidosis.
See also: Inactivation of catecholamines by superoxide gives new

insights on the pathogenesis of septic shock. Proc Natl Acad Sci U S A.
2000 Aug 15;97(17):9753-8.
CELLULAR RESPONSES TO SHOCK: THE

MOLECULAR BASIS OF MULTIPLE ORGAN
FAILURE
Adaptation to adverse conditions is the foundation of
evolution and has been the driving force behind the
cumulative complexity and sophistication of metazoan organisms. This applies not only to the acquisition
and inheritance of traits that enhance survival in the
face of long-term changes in conditions, such as
changes in the availability of food sources, but also
to the acquisition of defensive mechanisms against
unfavorable conditions arising in the short term. In
other words, one of evolutions end points is the
acquisition of adaptability itself. Within the confines
of the normal variation of environmental conditions, this adaptability has resulted in mechanisms
that maintain homeostasis at every level of the organism, including mechanisms to deal with tissue injury
and attack from other organisms, large or small.
Clinicians are most familiar with these stress
responses at the level of entire organs and organ
systems, and indeed this has formed the basis of this
chapter so far. However, stress responses also exist at
a cellular level, and indeed are quite specific to the
various cellular components.
Cytosolic Response to Stress

The existence of an organized response to cellular
stress was uncovered entirely by accident. In the early
1960s Ferrucio Ritossa was trying to identify the type
of nucleic acid synthesized in the chromosomal puffs
of salivary gland cells of Drosophila when a colleague
inadvertently increased the temperature of the incubator. The following morning Ritossa was struck by
the different puffing pattern of the chromosomes and
had the foresight to wonder whether this was connected to the change in temperature (37). Not surprisingly this change in gene expression was
subsequently referred to as the heat shock
response, and the protein products of these genes,
identified in 1974 (38), became known as the heat
shock proteins (Hsp). It has since emerged that Hsp
196
Mackenzie
are among the most phylogenetically conserved proteins identified, having been found in all three kingdoms of life, g and that their expression can be
induced by a host of stressors besides heat, including
oxidant stress, ischemia/reperfusion, hypoxia, and
sepsis (39). Even under basal conditions Hsp are
normal constituents of the cells housekeeping
armamentarium, where they act as molecular chaperones. In this role they are responsible for ensuring the
successful attainment and maintenance of correct
protein tertiary (folding) and quaternary (subunit
assembly) structure, transmembranous protein transport for secretion or entry into cellular organelles, and
finally the identification and disposal of denatured or
abnormal proteins. By convention, the Hsp in vertebrates are categorized into families on the basis of
molecular size, for example, Hsp70, Hsp90 or Hsp110,
although the discovery that previously identified proteins have a role in the stress response, for example,
heme oxygenase (Hsp32), ubiquitin, or inhibitor of kB
(ikBa), means that the convention is not water tight.
Increased gene expression is mediated by a small
family of transcription factors with a helix-turn-helix
DNA-binding domain called the heat shock factors
(HSF), of which three have been identified in man,
HSF1, HSF2 and HSF4.h HSF1 is a 57 kD protein
coded on the long arm of chromosome 8 that is
constitutively present in a monomeric form in the
cytosol, where it is sequestered by forming complexes
with the chaperone Hsp90. An excess of chaperone
demand over supply distracts the HSF1-anchoring
Hsp90, liberating the naked HSF1 monomers to
homotrimerize, whereupon DNA-binding activity is
acquired. The HSF1 trimers then migrate to the nucleus where they bind recognition sequences, known as
heat shock elements (HSE), in the promoter regions
of the Hsp. Although DNA binding is an essential
step in gene regulation, binding alone is not sufficient
for regulatory activity that requires modification of
HSF-1 by phosphorylation, sumoylation, or the activity
of coregulatory factors such as DAXX (40). A
genome-wide search of functional HSE in human
cells (HeLa) under conditions of heat shock identified
46 and 26 genes that were up- and downregulated,
respectively, by HSF1 (41). Upregulated genes included those coding for Hsp, for example, Hsp70 and
Hsp90, thus redressing the imbalance between the
supply and demand for molecular chaperones, as
well as genes involved in oxidant defense, immune
function, gut epithelial permeability, and antiapoptotic proteins. Downregulated genes include those
that code for IL-1b, TNFa, and genes induced by
FOS. Mice lacking a functional HSF-1 allele are less
likely to survive to birth, with survivors showing a
number of phenotypic abnormalities including
growth-retardation, female infertility, and a significantly reduced survival following endotoxin challenge (42).
Archaea, Bacteria and Eukaria.

Hsf-3 appears to be uniquely avian.
Endoplasmic Reticulum Stress Responses

While ATP might be the currency of life and DNA the
secret of life, i proteins are the fabric of life. Proteins
that are destined for secretion (hormones, cytokines,
neurotransmitters) or expression on the cell surface
(receptors, adhesion molecules) are guided through
aqueous pores by the Sec61 complex into the lumen of
the endoplasmic reticulum (ER) as they are translated
by cytosolic ribosomes. In contrast to any other cellular compartment, the ER maintains an oxidizing environment necessary for the formation of disulfide
bonds and glycosylation, processes vital to the stabilization of a functional tertiary conformation following
protein folding. The correct execution of these posttranslational events are mediated and monitored by a
repertoire of molecular chaperones that are able to
detect misfolded or aggregated proteins, which are
then exported to the cytosol for proteasomal degradation. When the capacity for ER processing cannot meet
demand, for example, as a consequence of increased
protein synthesis or degradation of the ERs processing capacity (hypoxia, hypoglycemia, oxidative
stress), the ER chaperone Grp78 (BiP) is distracted
from the lumenal aspect of the ER membrane where it
anchors the N-terminal transmembranous portion of
three molecules that are then freed to generate the
unfolded protein response (UPR) (43).
The first of these, IRE1 (inositol-requiring transmembrane kinase and endonuclease), migrates in the
plane of the membrane to form oligomers that transactivate each other by phosphorylation. Phosphorylated IRE1 is then able to initiate two separate pathways.
On the one hand, its carboxy terminus acquires endonuclease activity, which removes a 26-nucleotide
intron from native XBP-1 mRNA. This generates a
frameshift that translates a 371 amino acid protein
from the edited mRNA instead of a 267 amino acid
protein from the native mRNA. Unlike the product of
the native mRNA, the translation product of the
edited XBP-1 mRNA is a transcription factor of the
basic leucine zipper family (bZIP) responsible for the
upregulation of a number of genes that code for ER
chaperones (44). On the other, the phosphorylated
IRE1 forms a scaffold for the attachment of TRAF2
(TNF receptor-associated factor 2), which can then
bind and activate the ASK1/JNK kinase cascade.
This pathway not only cross talks with the cytosolic
version of the UPR by upregulating the transcription
of HSF-1 but also promotes insulin resistance and
gluconeogenesis by phosphorylating IRS1 (insulin
receptor substrate 1) (45) at serine 307 and activating
glucose-6-phosphatase, respectively. Finally, this pathway also promotes autophagy, although intermediaries in this process remain to be identified (46).
The second, ATF6 (activating transcription factor
6), is a bZIP transcription factor that on release of its
BiP anchor migrates to the Golgi where the N-terminal
intramembranous domain is cleaved by the S1P and
S2P serine proteases. This frees the carboxy terminus
i
See Watson J. DNA. The Secret of Life. London: Random House;

2003. Page 53.
moiety to translocate to the nucleus and engage in

transcriptional regulation, where it induces increased
expression of genes coding for ER chaperones.
The third, PERK (PKR-like ER kinase), is a
serine/threonine kinase that, like IRE1, oligomerizes
on release of its BiP anchor and autoactivates by
transphosphorylation. Its substrate is the eukaryotic
translation initiation factor 2a (eIF2a), which in its
naked form binds eIF2B to exchange GDP for GTP to
become active. Activated eIF2a can then load tRNAMet on to the 40S ribosome to initiate protein translation. Phosphorylation of eIF2a on serine 51 prevents it
from interacting with eIF2B, thus shutting down general protein translation. However, protected translation from ATF4 (activating transcription factor 4) is
enhanced following phosphorylation of eIF2a,
because of the presence of an alternative open reading
frame 50 upstream in the ATF4 mRNA. As with both
XBP-1 and ATF6, ATF4 increases the production of
molecular chaperones and antioxidant systems, as
well as being important in gluconeogenesis (47).
Nuclear Responses to Stress

The physiological circumstances that induce the cytosolic and ER UPR increase the generation of DNA
damage such as nucleotide depurination or deamination, oxidative damage, single- and double-strand
breaks, and the generation of nonsense mRNA.
DNA damage is recognized by, and activates, a family
of phosphoinositide-3-kinase-related protein kinases,
including ataxia telangiectasia and Rad-3 related,
ataxia telangiectasia mutated, DNA-dependent protein kinase, and SMG1, that not only effect DNA
repair,j which is important in its own right, but also
all converge on the activation of the transcription
factor p53. Although more commonly viewed as a
tumor suppressor because of its key role in precipitating cell cycle arrest, p53 also exerts pressure on other
cellular processes where, for example, it suppresses
translation,k increases antioxidant defenses (48), and
enhances anaerobic metabolism by activation of AMPdependent protein kinase (49) (AMPK). In persistently
unfavorable circumstances p53 enhances autophagy, a
highly conserved mechanism for autocannibalizing
and recycling old or damaged organelles (see
below). This mechanism is invoked under these circumstances in a last-ditch bid to source cellular
substrate for continued cell function, and is driven
by p53 both through the downregulation of mTOR
and the upregulation of damage-regulated autophagy
modulator (50). Finally, where the situation is deemed
irrecoverable, p53 invokes a dignified apoptotic cell
death both through transrepression of BCL-2 (an
inhibitor of apoptosis) as well as upregulation of
PUMA, BAX, BIDD, and PIDD. However, in situations
where the genetic damage is extensive, overwhelming
activation of the enzyme poly-ADP ribose polymer-
ase-1 leads to the rapid cellular depletion of both

NAD and ATP, culminating in cellular necrosis (51).
Energy Depletion, Hypoxic Stress, and the

Mitochondrial Responses to Stress
Oxygen was entirely absent from the Earths early
atmosphere but was produced as a by-product of the
photosynthetic activity of marine cyanobacteria.
Although there is evidence that cyanobacteria (and
eucaria) were present at least 2700 million years ago
(MYA) (52), oxygen did not begin to accumulate for a
further 250 million years and only reached present
day concentrations a further 1850 to 1900 million years
later (550600 MYA).l The oxygenation of earths
atmosphere appears to have been permissive for
metazoan life-forms, and within a geologically short
time resulted in the appearance of a multitude of these
organisms in the fossil record recognized as the
Cambrian explosion, which started 540 MYA.
Today, the vast majority of metazoan organisms rely
on oxygen and oxidative phosphorylation to generate
their cellular energy. But in the absence of any means
of storing this component of the energy supply chain
(unlike other substrates), metazoan cells had to develop mechanisms to cope with temporary interruptions
to its supply.
A fall in the supply of oxygen is detected in three
ways. First, the rate at which the mitochondrial
F1F0ATPase (complex V) can generate ATP falls,
and in the absence of attenuated consumption this
immediately results in a fall in the intracellular ratio of
ATP to AMP, activating the cytosolic AMPK. Second,
as the supply of oxygen continues to fall, mitochondrial generation of reactive oxygen species (ROS)
increases (53). Third, the fall in oxygen tension induces the expression of the transcription factor HIF-1
(hypoxia-induced factor-1).
AMPK is a heterotrimeric protein kinase composed of a catalytic a subunit (a1 or a2) and two
regulatory subunits (b1 or b2 and g1, g2 or g3) that is
only susceptible to activation (phosphorylation) when
AMP is bound to the g subunit. In addition, AMP
allosterically activates AMPK and renders the activated kinase less susceptible to the action of deactivating
phosphatases. The action of adenylate kinase in maintaining the ADP:ATP ratio in equilibrium by converting ADP into ATP and AMP acts as a gearing ratio
between ATP and AMP. This, together with AMPs
activating influence, makes AMPK activation exquisitely sensitive to energy deprivation. Not surprisingly, therefore, AMPK activity aims to redress the
balance between energy consumption and energy
production. Energy consumption is reduced in a
number of ways. First, phosphorylation of tuberous
sclerosis complex 2 promotes the conversion of GTP to
l
j
Nucleotide excision repair (NER), base excision repair (BER) and

nonsense mutation.
k
Via the TSC2 ? mTOR ? S6 kinase and 4EBP1 pathways.
197
For further information on this fascinating subject see: (1) Knoll AH.
Life on a Young Planet. Princeton: Princeton University Press, 2003,
and (2) Lane N. Oxygen. The Molecule That Made the World. Oxford:
Oxford University Press, 2002.
198
Mackenzie
GDP on RHEB, inactivating mTOR and shutting down

protein translation. Second, cell division is arrested
both directly, through activation of a cyclin-dependent kinase inhibitor, and indirectly by upregulation
of signaling through the family of forkhead transcription factors (54). Energy synthesis, on the other hand,
is enhanced through both glycolysis and b-oxidation.
Cellular glucose uptake is enhanced by the translocation of GLUT1 and GLUT4 to the plasma membrane,
while glycolytic flux is promoted by the inhibition of
glycogen synthase. Insulin signaling is inhibited by
phosphorylation of IRS-1 at Ser794 and oxidative
phosphorylation is downregulated by AMPK-mediated increased transcription of pyruvate dehydrogenase
kinase 4, an enzyme that inactivates the pyruvate
dehydrogenase complex, thus preventing the entry
of pyruvate into the Krebs cycle. b-Oxidation is
enhanced by AMPK-mediated inhibition of acetylcoA carboxylase. This prevents the synthesis
of malonyl-CoA, removing the latters allosteric
inhibition of long-chain acyl-CoA transport into mitochondria by carnitine palmitoyltransferase-1.
HIF-1 is another phylogenetically conserved
molecule. This heterodimer is made up of a constitutively expressed HIF-1b subunit and an HIF-1a subunit that is hydroxylated in the presence of oxygen,
ascorbate, Fe(II), and a-ketoglutarate. The stable HIF1ab heterodimer is therefore only formed under hypoxic conditions when it translocates into the nucleus
to regulate the expression of a range of glycolytic
enzymes, including lactate dehydrogenase, that promote anaerobic glycolysis. HIF-1 also increases the
transcription of genes involved in angiogenesis, erythropoeisis, and the maintenance of blood pressure
(endothelin 1, tyrosine hydroxylase). Another important role for HIF-1 appears to involve the suppression
of mitochondrial function during hypoxia. Under
hypoxic conditions there is a paradoxical increase in
the mitochondrial generation of ROS from complex III
of the electron transport chain (55), which can then
lead to mitochondrial depolarization, the release of
cytochrome C from the inner mitochondrial membrane, and triggering of apoptosis. To prevent this,
HIF-1 shuts down oxidative phosphorylation by activating pyruvate dehydrogenase kinase 1, which in
turn phosphorylates and inactivates the pyruvate
dehydrogenase complex, thus preventing pyruvate
from entering the Krebs cycle. Furthermore, HIF-1
appears to reduce mitochondrial biogenesis, mitochondrial respiration, and the generation of ROS by
inhibiting MYC-regulated activation of PPAR-g coactivator 1b (56) (PGC-1b), as well as promoting the
dismantling (autophagy) of mitochondria to provide
raw materials (57).
Autophagy, Apoptosis, and Necrosis

In the normal course of cellular activity intracellular
components become damaged or redundant. These
components are then biochemically tagged and
then incorporated into double-membrane vesicles,
which then fuse with lysosomes. The contents of
these autophagosomes are then digested, and the
metabolic building blocks reexported to the cytoplasm for recycling. As described above, this highly
selective process, known as autophagy, may
become nonselective when cell survival is at risk
(58). Ultimately, under circumstances where the supply of essential substrates continues to fall short of the
requirement for cellular survival, a decision has to be
made to either struggle on, and risk a necrotic cell
death, or to abandon the fight gracefully, and activate
a highly ordered form of cell death referred to as
apoptosis. The apoptotic pathway involves two distinct phases. The first phase, commitment, is believed
to be integrated by endogenous signals arriving at the
mitochondria that provide information on cellular
well-being and culminates in an irreversible
increase in mitochondrial membrane permeability.
The molecular details of this decision phase remain
to be elucidated in detail. In contrast the second
phase, execution, is more clearly understood and
involves the activation of a cascade of enzymes,
known as caspases, that culminate in the orderly
dismantling of the cellular contents, including the
nucleus, which are then packaged into vesicles for
phagocytosis by neighboring cells (59). In marked
contrast, cellular necrosis occurs when the integrity
of the cell membrane can no longer be maintained.
Membrane rupture results in the release of intracellular content and provokes a local inflammatory reaction. This increases the metabolic demands of the
local environment, which, in the context of a local
shortage of substrate supply, may then jeopardize the
survival of neighboring cells.
Multiple Organ Dysfunction

The cellular outcomes from the pathways described
above are limited, varying along the continuum
between normal function and a threshold of dysfunction that then triggers a commitment to cellular senescence, apoptosis, or necrosis (Fig. 14). As our
understanding of the cellular mechanisms of stress
improves, it is becoming apparent that the pathways
identified may vary between different species, different tissues types, or indeed between different experimental situations between transformed or native cells
in vitro, or whole tissues in vivo. Furthermore, the
ultimate outcome for any given cell will depend on
the integration of a complex interplay between multiple pathways, some of which undoubtedly remain to
be described, as well as the influence of particular
genotypes. However, common end points clearly
emerge from the stress pathways described above,
which include the minimization of free-radical damage (enhanced antioxidant defenses, reduced oxidative phosphorylation), enhanced anaerobic glycolysis,
the recycling of endogenous substrate by autophagy,
and the conservation of energy by reduced protein
translation. Ultimately, where survival appears
impossible, energy is expended in an apoptotic cell
death in an altruistic effort to minimize damage to
neighboring cells (Fig. 15). At the organ level, where
each constituent cell is subjected to particular conditions of substrate supply and extracellular signals, the
199
Figure 14 The spectrum of cellular end points.
Figure 15 Molecular pathways mediating multiple organ dysfunction.
outcome can be very heterogeneous, with the function

of the whole organ reflecting the sum of all the
different outcomes for each constituent cell. Significant organ dysfunction (Table 8), sufficient to contribute to a patients death, can be present with only
modest cellular evidence of apoptosis (60). Survival
in this condition for days or weeks is an entirely
artificial situation made possible by modern medicine,
and the cellular stress response, akin to hibernation, in
the context of modern intensive care and organ support, establishes a vicious cycle (Fig. 12). While there
has been strong evolutionary pressure to develop
mechanisms to cope with, and survive, short periods
of acute severe illness, this is not the case for prolonged critical illness (Fig. 16). An excellent example
of this phenomenon is stress hyperglycemia. This
particular response to stress is highly phylogenetically
conserved and is seen not only in mammals, but even
in fish (61), crustacea (62), and insects (63). This trait
must, therefore, confer a survival benefit. In man,
however, stress hyperglycemia has been associated
with a poorer outcome in patients following stroke
(64), acute myocardial infarction (65), and admission
200
Mackenzie
Table 8 Clinical Manifestations of Cell Stress and Cell Death

Clinical manifestations of cell stress (
Nervous system
Respiratory
system
Heart
Kidneys
Liver
Gut
Skeletal muscle
Immune system
Bone marrow
Connective tissue
Metabolism
cell senescence)
Impaired arousal (stupor, coma)

Impaired content (confusion, delirium)
Motor and/or sensory neuropathy
Failure to synthesize surfactant
Failure to repopulate alveoli with type I pneumocytes
Failure of gas exchange (hypoxia and hypercapnia)
Contractile failure of respiratory muscles
Persistent pulmonary inflammation
Depressed contractility
Rhythm disturbances
Impaired handling of water and solutes
Impaired synthetic function
Impaired drug and metabolite clearance
Impaired gastric acid buffering/protection leading to gastric
erosions
Impaired enterocyte production leading to delayed healing
Impairment of gut barrier function leading to bacterial
translocation
Impaired motility (ileus and constipation)
Atrophy
Impaired contractility
Failure to clear infection
Clinical manifestations of cell death

Irreversible neurological deficits
Raised plasma concentration of cardiac

enzymes
ECG changes
Anuric renal failure
Raised plasma concentration of hepatic
enzymes
Rhabdomyolysis
Lymphocytopenia
Neutropenia
Anemia
Neutropenia
Thrombocytopenia
Poor and slow wound healing
Insulin resistance
Impaired glucose tolerance
Figure 16 Time course of acute severe illness in man. The red zone represents the course of acute severe illnesses that are, and
always have been, fatal. The green zone represents the course of acute severe illnesses that men, in some cases, have been able to
survive. The responses to these conditions have therefore been under evolutionary selective pressure. The gray zone represents the
course of acute severe illness that, in the absence of medical developments introduced in the past 50 years, would have been fatal. This
zone represents a biological condition for which there has been no evolutionary precedent.
to intensive care (66). Furthermore, tight control of

blood glucose with intravenous insulin has been
shown to reduce morbidity and mortality in critically
ill adults in both a historical cohort comparison (67)
and two large prospective randomized trials (68,69).

For this reason teleological and phylogenetic arguments that the stress response must serve some
purpose cannot be applied in the context of patients
whose acute severe illness and multiple organ failure

have been artificially prolonged by modern intensive care.
20.
21.
REFERENCES
1. Le Dran H. Traite Ou Reflexions Tirees De La Pratique
` Feux. Paris: Charles Osmont, 1737.
Sur Les Playes Darmes A
2. Mackenzie IMJ. The haemodynamics of human septic
shock. Anaesthesia 2001; 56:130144.
3. Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/
ESICM/ACCP/ATS/SIS International Sepsis Definitions
Conference. Crit Care Med 2003; 31(4):12501256.
4. Reynolds HR, Hochman JS. Cardiogenic shock: current
concepts and improving outcomes. Circulation 2008; 117
(5):686697.
5. Ferreira FL, Bota DP, Bross A, et al. Serial evaluation of the
SOFA score to predict outcome in critically ill patients.
JAMA 2001; 286(14):17541758.
6. Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and
safety of recombinant human activated protein C for
severe sepsis. N Engl J Med 2001; 344:699709.
7. Caille V, Chiche JD, Nciri N, et al. Histocompatibility
leukocyte antigen-D related expression is specifically
altered and predicts mortality in septic shock but not in
other causes of shock. Shock 2004; 22(6):521526.
8. Sprung CL, Annane D, Keh D, et al. Hydrocortisone
therapy for patients with septic shock. N Engl J Med
2008; 358(2):111124.
9. Zeymer U, Vogt A, Zahn R, et al. Predictors of in-hospital
mortality in 1333 patients with acute myocardial infarction
complicated by cardiogenic shock treated with primary
percutaneous coronary intervention (PCI): results of the
primary PCI registry of the Arbeitsgemeinschaft Leitende
Kardiologische Krankenhausarzte (ALKK). Eur Heart J
2004; 25(4):322328.
10. Heckbert SR, Vedder NB, Hoffman W, et al. Outcome after
hemorrhagic shock in trauma patients. J Trauma 1998;
45(3):545549.
11. Weil MH, Shubin H. Proposed reclassification of shock
states with special reference to distributive defects. Adv
Exp Med Biol 1971; 23(0):1323.
12. Sonnenblick EH, Skelton CL. Reconsideration of the ultrastructural basis of cardiac length-tension relations. Circ Res
1974; 35:517526.
13. Gilbert JC, Glantz SA. Determinants of left ventricular
filling and of the diastolic pressure-volume relation. Circ
Res 1989; 64(5):827852.
14. Kentner R, Rollwagen FM, Prueckner S, et al. Effects of
mild hypothermia on survival and serum cytokines
in uncontrolled hemorrhagic shock in rats. Shock 2002;
17(6):521526.
15. Lausevic Z, Lausevic M, Trbojevic-Stankovic J, et al.
Predicting multiple organ failure in patients with severe
trauma. Can J Surg 2008; 51(2):97102.
16. Clark JA, Coopersmith CM. Intestinal crosstalk: a new
paradigm for understanding the gut as the motor of
critical illness. Shock 2007; 28(4):384393.
17. Spronk PE, Zandstra DF, Ince C. Bench-to-bedside review:
sepsis is a disease of the microcirculation. Crit Care 2004;
8(6):462468.
18. VanderMeer TJ, Wang H, Fink MP. Endotoxemia causes
ileal mucosal acidosis in the absence of mucosal hypoxia in
a normodynamic porcine model of septic shock. Crit Care
Med 1995; 23(7):12171226.
19. Boekstegers P, Weidenhofer S, Pilz G, et al. Peripheral
oxygen availability within skeletal muscle in sepsis and
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
201
septic shock: comparison to limited infection and cardiogenic shock. Infection 1991; 19(5):317323.
Fink MP. Bench-to-bedside review: cytopathic hypoxia.
Crit Care Dec 2002; 6(6):491499.
Fieselmann JF, Hendryx MS, Helms CM, et al. Respiratory
rate predicts cardiopulmonary arrest for internal medicine
inpatients. J Gen Intern Med 1993; 8(7):354360.
Lanone S, Taille C, Boczkowski J, et al. Diaphragmatic
fatigue during sepsis and septic shock. Intensive Care Med
Dec 2005; 31(12):16111617.
Kumar A, Anel R, Bunnell E, et al. Pulmonary artery
occlusion pressure and central venous pressure fail to
predict ventricular filling volume, cardiac performance,
or the response to volume infusion in normal subjects.
Crit Care Med 2004; 32(3):691699.
Raper R, Sibbald WJ. Misled by the wedge? The SwanGanz catheter and left-ventricular pre-load. Chest 1986;
89(3):427434.
Harvey S, Harrison DA, Singer M, et al. Assessment of the
clinical effectiveness of pulmonary artery catheters in management of patients in intensive care (PAC-Man): a randomised controlled trial. Lancet 2005; 366(9484):472477.
Wheeler AP, Bernard GR, Thompson BT, et al. Pulmonaryartery versus central venous catheter to guide treatment of
acute lung injury. N Engl J Med 2006; 354(21):22132224.
Spahn DR, Cerny V, Coats TJ, et al. Management of
bleeding following major trauma: a European guideline.
Crit Care 2007; 11(1):R17.
Rivers E, Nguyen B, Havstad S, et al. Early goal-directed
therapy in the treatment of severe sepsis and septic shock.
N Engl J Med 2001; 345(19):13681377.
Bucher M, Kees F, Taeger K, et al. Cytokines downregulate alpha1-adrenergic receptor expression during
endotoxemia. Crit Care Med 2003; 31(2):566571.
Schaak S, Mialet-Perez J, Flordellis C, et al. Genetic variation of human adrenergic receptors: from molecular and
functional properties to clinical and pharmacogenetic
implications. Curr Top Med Chem 2007; 7(2):217231.
Takakura K, Taniguchi T, Muramatsu I, et al. Modification
of alpha1-adrenoceptors by peroxynitrite as a possible
mechanism of systemic hypotension in sepsis. Crit Care
Med 2002; 30(4):894899.
Mullner M, Urbanek B, Havel C, et al. Vasopressors for
shock. Cochrane Database Syst Rev 2004(3):CD003709.
Beale RJ, Hollenberg SM, Vincent JL, et al. Vasopressor and
inotropic support in septic shock: an evidence-based
review. Crit Care Med 2004; 32(11 suppl):S455S465.
Martin C, Papazian L, Perrin G, et al. Norepinephrine or
dopamine for the treatment of hyperdynamic septic shock?
Chest 1993; 103(6):18261831.
Debaveye YA, Van den Berghe GH. Is there still a place for
dopamine in the modern intensive care unit? Anesth Analg
2004; 98(2):461468.
Sakr Y, Reinhart K, Vincent JL, et al. Does dopamine
administration in shock influence outcome? Results of
the Sepsis Occurrence in Acutely Ill Patients (SOAP)
Study. Crit Care Med 2006; 34(3):589597.
Ritossa F. A new puffing pattern induced by temperature
shock and DNP in Drosophila. Experientia 1962; 18
(12):571573.
Tissieres A, Mitchell HK, Tracy UM. Protein synthesis in
salivary glands of Drosophila melanogaster: relation to
chromosome puffs. J Mol Biol 1974; 84(3):389398.
Ryan AJ, Flanagan SW, Moseley PL, et al. Acute heat stress
protects rats against endotoxin shock. J Appl Physiol 1992;
73(4):15171522.
Boellmann F, Guettouche T, Guo Y, et al. DAXX interacts
with heat shock factor 1 during stress activation and
202
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
Mackenzie
enhances its transcriptional activity. Proc Natl Acad Sci U S A
2004; 101(12):41004105.
Page TJ, Sikder D, Yang L, et al. Genome-wide analysis of
human HSF1 signaling reveals a transcriptional program
linked to cellular adaptation and survival. Mol Biosyst
2006; 2(12):627639.
Xiao X, Zuo X, Davis AA, et al. HSF1 is required for extraembryonic development, postnatal growth and protection
during inflammatory responses in mice. EMBO J 1999;
18(21):59435952.
Marciniak SJ, Ron D. Endoplasmic reticulum stress signaling in disease. Physiol Rev 2006; 86(4):11331149.
Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a
subset of endoplasmic reticulum resident chaperone
genes in the unfolded protein response. Mol Cell Biol
2003; 23(21):74487459.
Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum
stress links obesity, insulin action, and type 2 diabetes.
Science 2004; 306(5695):457461.
Ogata M, Hino S, Saito A, et al. Autophagy is activated for
cell survival after endoplasmic reticulum stress. Mol Cell
Biol 2006; 26(24):92209231.
Scheuner D, Song B, McEwen E, et al. Translational control
is required for the unfolded protein response and in vivo
glucose homeostasis. Mol Cell 2001; 7(6):11651176.
Bensaad K, Tsuruta A, Selak MA, et al. TIGAR, a p53inducible regulator of glycolysis and apoptosis. Cell 2006;
126(1):107120.
Feng Z, Hu W, de Stanchina E, et al. The regulation of
AMPK beta1, TSC2, and PTEN expression by p53: stress,
cell and tissue specificity, and the role of these gene
products in modulating the IGF-1-AKT-mTOR pathways.
Cancer Res 2007; 67(7):30433053.
Crighton D, Wilkinson S, OPrey J, et al. DRAM, a p53induced modulator of autophagy, is critical for apoptosis.
Cell 2006; 126(1):121134.
Virag L, Szabo C. The therapeutic potential of poly(ADPribose) polymerase inhibitors. Pharmacol Rev 2002; 54(3):
375429.
Brocks JJ, Logan GA, Buick R, et al. Archean molecular
fossils and the early rise of eukaryotes. Science 1999;
285(5430):10331036.
Clanton TL. Hypoxia-induced reactive oxygen species
formation in skeletal muscle. J Appl Physiol 2007; 102(6):
23792388.
Greer EL, Oskoui PR, Banko MR, et al. The energy sensor
AMP-activated protein kinase directly regulates the
mammalian FOXO3 transcription factor. J Biol Chem
2007; 282(41):3010730119.
Guzy RD, Schumacker PT. Oxygen sensing by mitochondria at complex III: the paradox of increased reactive
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
oxygen species during hypoxia. Exp Physiol 2006;

91(5):807819.
Zhang H, Gao P, Fukuda R, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient
renal cell carcinoma by repression of C-MYC activity.
Cancer Cell 2007; 11(5):407420.
Zhang H, Bosch-Marce M, Shimoda LA, et al. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic
response to hypoxia. J Biol Chem 2008; 283(16):
1089210903.
Kundu M, Thompson CB. Autophagy: basic principles
and relevance to disease. Annu Rev Pathol 2008; 3:
427455.
Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007;
87(1):99163.
Hotchkiss RS, Swanson PE, Freeman BD, et al. Apoptotic
cell death in patients with sepsis, shock, and multiple
organ dysfunction. Crit Care Med 1999; 27(7):12301251.
Suski CD, Cooke SJ, Danylchuk AJ, et al. Physiological
disturbance and recovery dynamics of bonefish (Albula
vulpes), a tropical marine fish, in response to variable
exercise and exposure to air. Comp Biochem Physiol A
Mol Integr Physiol 2007; 148(3):664673.
Telford M. The effects of stress on blood sugar composition
of the lobster, Homarus americanus. Can J Zool 1968; 46
(5):819826.
Schilder RJ, Marden JH. Metabolic syndrome and obesity
in an insect. Proc Natl Acad Sci U S A 2006; 103(49):
1880518809.
Capes SE, Hunt D, Malmberg K, et al. Stress hyperglycaemia and prognosis of stroke in nondiabetic and
diabetic patients: a systematic overview. Stroke 2001; 32:
24262432.
Capes SE, Hunt D, Malmberg K, et al. Stress hyperglycaemia and increased risk of death after myocardial infarction
in patients with and without diabetes: a systematic
overview. Lancet 2000; 355(9206):773778.
Krinsley JS. Association between hyperglycaemia and
increased hospital mortality in a heterogeneous population
of critically ill patients. Mayo Clin Proc 2003; 78:
14711478.
Krinsley JS. Effect of an intensive glucose management
protocol on the mortality of critically ill adult patients.
Mayo Clin Proc 2004; 79(8):9921000.
Van den Berghe G, Wouters P, Weekers F, et al. Intensive
insulin therapy in critically ill patients. N Engl J Med 2001;
345(19):13591367.
Van den Berghe G, Wilmer A, Hermans G, et al. Intensive
insulin therapy in the medical ICU. N Engl J Med 2006; 354
(5):449461.
12
Acute Renal Failure
Rona Smith and John R. Bradley
Cambridge University Hospitals NHS Foundation Trust, Cambridge, U.K.
INTRODUCTION
PRESENTATION
Until recently more than 30 definitions for acute renal

failure have been used in the literature. The majority
mention a rapid deterioration in renal function, with
the accumulation of nitrogenous wastes in the body,
often associated with oliguria or anuria. However,
there was no agreed definition. The huge variation
not only caused confusion but also made comparisons
between studies very difficult. As a consequence, a
group of experts, the acute dialysis quality initiative
(ADQI), developed the RIFLE criteria as a consensus
definition for acute kidney injury (AKI), an alternative
term now commonly used and which henceforth will
be used in this chapter (1). The RIFLE criteria divides
renal dysfunction into five categoriesrisk, injury,
failure, loss, and end stage kidney disease (ESKD)
on the basis of glomerular filtration rate (GFR) and
urine output criteria, which are illustrated in Figure 1.
AKI is common, although the incidence depends
on the definition used. In a United Kingdombased
community study, the incidence of severe AKI (creatinine >500 mmol/L) was 172 cases/million adults/yr,
with 22 cases/million adults/yr requiring dialysis (2).
AKI complicates 5% to 20% of hospital admissions
and approximately 30% of admissions to the intensive
care unit. Rates of survival are dependent on cause.
However, mortality is high (4080%) in patients with
multiple organ failure, with death being likely if AKI
is associated with failure of more than three other
organ systems. In patients acquiring AKI in the community, mortality is much lower (1030%) (3).
The potential causes of AKI can be divided into
three categories: prerenal failure (approximately 55%
of cases) due to inadequate perfusion of blood through
the kidney, often termed physiological prerenal failure,
which resolves when the underlying hypovolaemia is
corrected, and pathological prerenal failure due to an
interruption of blood flow to the kidney when there is
an anatomical cause for disturbed renal blood flow,
such as an aortic dissection; renal failure as a consequence of intrinsic injury to the kidney (about 40% of
cases); and postrenal failure (less than 5% of AKI cases)
due to the obstruction of urine outflow (Fig. 2). However, in 50% of cases of hospital acquired AKI, the
cause is multifactorial, with hypotension, sepsis, and
nephrotoxic drugs commonly being involved (4).
Presenting symptoms are usually dominated by those

of the underlying etiology, especially in cases of
prerenal AKI. Often symptoms are those of hypovolaemia, namely thirst, dizziness, decreased urine output, and postural hypotension. Conversely, patients
may present in a state of fluid overload, with peripheral edema and shortness of breath. Symptoms of
uremia are nonspecific, but include anorexia, nausea,
vomiting, lethargy, weakness, myoclonic jerks, seizures, confusion, and coma.
INVESTIGATIONS
On any sick patient, a number of investigations are
performed. It may only be at this point that a diagnosis
of AKI is made, when elevated serum urea and creatinine levels are identified on blood tests. Once the
diagnosis is made, further tests are required to determine the severity and urgency of any treatment
required, such as dialysis, and also the etiology of the
renal failure. The key tests required are listed below.
Blood Tests
Biochemical tests identify high urea and creatinine
levels. Other features of AKI include high serum
potassium and phosphate levels due to reduced
renal excretion, and typically a low calcium level. A
high serum calcium level would make one suspicious
of multiple myeloma, which would be confirmed by
performing serum protein electrophoresis, and the
presence of a monoclonal immunoglobulin band.
Elevated creatinine kinase (CK) levels are seen in
rhabdomyolysis. Serum levels of any potential nephrotoxins (vancomycin, gentamicin, cyclosporine, and
tacrolimus) should be determined.
Hematological investigations include a full
blood count and coagulation screen. Anemia is a
feature of chronic renal failure due to a number of
factors, but predominantly decreased erythropoietin
levels. Thrombocytopaenia is suggestive of sepsis or
systemic lupus erythematosus (SLE) or thrombotic
thrombocytopaenic purpura (TTP). A blood film
may be useful if hemolysis is suspected as in hemolytic uremic syndrome (HUS). Clotting may be
deranged if severe sepsis or liver disease is present
204
Smith and Bradley
Urine output
(UO) criteria
GFR criteria
Risk
Injury
Failure
Loss
ESKD
UO < 0.5mL/kg/hr !
6 hr
UO < 0.5mL/kg/hr !
12 hr
UO < 0.3mL/kg/hr !
24 hr or anuria !
12 hr
Persistent AKI complete loss of kidney function
>4 wk
End-stage kidney disease (>3 mo)
Increased creatinine ! 1.5

or GFR decrease >25%
Increased creatinine ! 2
or GFR decrease >50%
Increase creatinine ! 3 or
GFR decrease >75%
Figure 1 RIFLE criteria for acute renal dysfunction.
Prerenal
Volume depletion
Decreased cardiac
output
Systemic vasodilatation
Afferent arteriolar
constriction
Efferent arteriolar
constriction
Intrinsic
Acute tubular injury
Glomerulonephritis
Tubulointerstitial
nephritis
Acute vascular
nephropathy
Postrenal
Ureteric obstruction
Bladder neck
obstruction
GI losses (diarrhoea and vomiting)

Renal losses (diuretics)
Hemorrhage
Pulmonary embolus
Heart failure/myocardial infarction/
severe valvular disease
Abdominal compartment
syndrome
Sepsis/anaphylaxis
Hypercalcaemia
Drugscalcineurin inhibitors,
NSAIDS, radio contrast agents
Hepatorenal syndrome
ACE inhibitors or angiotensin
receptor blockers
Ischaemic
Cytotoxicheme pigment
(rhabdomyolosis), crystals,
drugs, for example, gentamicin
Antiglomerular basement membrane
disease
ANCA associated, for example,
Wegners granulomatosis, MPA
Immune complex GNlupus, post
infective, cryoglobulinaemia
Drugs, for example, NSAIDS,
penicillin
Infection
Systemic diseasefor example,
sarcoid
Renal artery obstruction
Renal vein obstruction
Microangiopathy
Atheroembolic disease
Intrinisic (stones), extrinsic
(retroperitoneal fibrosis, lymph
nodes)
Prostate carcinoma, benign
prostatic hypertrophy, bladder
tumours, hemorrhage/clot
Figure 2 Causes of acute renal failure.
but must be performed if a biopsy is contemplated.

Elevated erythrocyte sedimentation rate (ESR) is seen
in vasculitis and multiple myeloma.
Serological investigations are performed if glomerular pathology is suspected. Key tests are antinuclear antibody (ANA) and double-stranded DNA
levels, which are positive in SLE; antineutrophil
cytoplasmic antibodies (ANCA), which are divided
into proteinase PR3 positive in Wegners granulomatosis; and myeloperoxidase (MPO) positive; and antiGBM (glomerular basement membrane) antibody is
found in Goodpastures disease. Complement levels,
namely C3 and C4, should be measured and cryoglobulins tested in patients with unexplained glomerular
disease. Serum complement levels tend to be low in
immune-mediated renal conditions because of consumption within the kidney. Cryoglobulins are immunoglobulin proteins that precipitate in the cold and can
be deposited in the kidney where they are associated
with immune inflammation. They may be idiopathic or
associated with autoimmune disease, hematological
disease, or infection, including hepatitis C.
Urine Testing
Dipstick testing identifies hematuria and proteinuria,
which are suggestive of glomerular pathology as a
cause of intrinsic renal failure. In cases of prerenal failure,
ATN, and obstruction, the urine should be bland. Leukocytes and nitrites may also be identified and if that is
the case a urine culture should be performed.
Microscopy looks for red cells or white cells, or
cellular casts, in which red or white cells adhere to
proteins excreted by tubules. Red cells are seen in
bleeding from anywhere in the renal tract, but red cell
casts usually indicate glomerular bleeding. White cell
casts imply tubular inflammation.
Osmolality and urinary sodium concentrations
can help distinguish prerenal failure from established
ATN. Prerenal failure is characterised by a urine sodium <20 mmol/L and an urine/plasma urea ratio >8,
because the tubules are working normally and reabsorbing salt and water maximally. Once ischemic damage occurs to the tubules and ATN is established, the
tubules can no longer reabsorb sodium or concentrate
the urine and so the urine sodium concentration is
typically high at >40 mmol/L and the urine/plasma
urea ratio low at <3. Sometimes this information is
presented as a fractional excretion of sodium (FeNa),
which is calculated using the following formula.
FeNa (urine Na/plasma Na)/
(urine creatinine/plasma creatinine)
A FeNa <1% is suggestive of prerenal AKI,
while a FeNa >1% indicates tubular damage and
ATN (5). However, in the following situations, the
FeNa may be <1%, but the cause of renal impairment
is not prerenalurinary tract obstruction, hepatorenal
syndrome, renal allograft rejection, contrast induced
ATN, rhabdomyolysis, and acute glomerulonephritis.
Imaging
Chest radiography is performed on a routine basis to
look for evidence of pulmonary edema and fluid
Chapter 12: Acute Renal Failure
overload. It is also important to look for pulmonary

infiltrates, which can be seen in pulmonary renal
syndromes, such as Wegners granulomatosis and
Goodpastures syndrome.
Renal ultrasound looks at the size, shape, and
texture of the kidneys. In AKI, kidneys are often
normal sized or larger than normal and may show
increased echogenicity. However, in chronic renal
failure, the kidneys are not only smaller than
expected, but also show increased echogenicity, probably because of fibrosis. Ultrasound can also identify
signs of hydronephrosis, such as a dilated pelvicalyceal system, although it must be remembered that a
normal ultrasound does not completely exclude
obstruction.
Renal Biopsy
Renal biopsies are an invasive procedure and should
therefore be reserved for evaluation of cases of AKI
when the cause is uncertain. They are particularly
useful when a glomerular pathology is suspected not
only for confirming the diagnosis but also for guiding
treatment decisions and indicating prognosis.
MANAGEMENT OF ACUTE KIDNEY INJURY

While investigation into the cause of AKI progresses,
early medical intervention to reverse any potential
underlying cause, such as hypovolaemia, and prevent
further iatrogenic injury, for example, by the administration of nephrotoxic drugs, is key. Attention must be
focused on optimal fluid balance, whether that is
aggressive rehydration with intravenous fluids if dry
or attempts at inducing a diuresis with various agents
if the converse is true. If lower urinary tract obstruction is suspected, a urinary catheter should be
inserted. Its placement will also help with accurate
fluid balance measurement, but it is a potential source
of infection, and urinary catheter placement is not
obligatory to manage fluid balance closely.
Patients with nonoliguric AKI have decreased
mortality rates, and improved renal recovery rates (6).
Therefore, in the past diuretics were often used in an
attempt to convert oliguric AKI into nonoliguric AKI.
However, randomised double-blind controlled trials
have failed to show any benefit (7), and now diuretics
are only used in fluid overloaded patients to promote
a diuresis in an attempt to avoid the need for dialysis.
Furosemide is the most commonly used agent. Very
high doses, up to 250 mg intravenously may be
required to have an effect when renal function is
poor, but carry a risk of ototoxicity.
There is also often confusion regarding the role
of low-dose dopamine, generally considered to be a
dose less than 2.5 mg/kg/min, in AKI. Dopamine is a
potent vasodilator acting via DA1 receptors, and also
inhibits active sodium transport in the proximal
tubule leading to a naturesis and diuresis. At moderate doses it has b-adrenergic effects increasing cardiac
output and renal blood flow and perfusion. A metaanalysis by Kellum (8) and a randomised controlled
205
trial by Bellomo et al. (9), have both shown that lowdose dopamine may increase urine output, but has no
beneficial effect in terms of decreased mortality or the
need for renal replacement therapy (RRT). Documented side effects include depression of respiratory
drive, cardiac arrhythmias, and tissue necrosis and
digital gangrene (10). Therefore, at present, the use of
dopamine in AKI, whether it be prophylactically or
therapeutically, is not advised.
COMPLICATIONS OF ACUTE KIDNEY INJURY

Hyperkalemia
Hyperkalaemia is a medical emergency. In AKI serum
potassium levels rise for several reasons including
decreased GFR, reduced tubular secretion, a catabolic
state and a metabolic acidosis. It is particularly
marked in situations where endogenous potassium
production is high, such as in rhabdomyolysis or
tumor lysis syndrome. Often the patient will not
have any specific symptoms, but the ECG may show
typical changes including tented T waves, broadening
of the QRS complex, flattening of P waves and prolongation of the PR interval. If the K+ >7 mmol, then
treat immediately. Most people would advocate treating a K+ level greater than 6.5 mmol if ECG changes
are present. Initially 10 mL 10% calcium gluconate
should be given intravenously to stabilize the myocardium. It does not lower the potassium level. This is
achieved by administering 50 mL 50% dextrose with
10 units of actrapid insulin intravenously over 20 to
30 minutes to drive potassium intracellularly. 5 mg
nebulized salbutamol can be given as an adjunct to
drive potassium into cells, as can intravenous sodium
bicarbonate. Calcium resonium, 15g orally, four times
a day, reduces absorption of dietary potassium from
the gut, but works over many hours. Drugs that raise
serum potassium levels, such as ACE inhibitors,
angiotensin receptor blockers and potassium sparing
diuretics must be stopped. Ultimately, if hyperkalaemia proves refractory to the above treatments, then
dialysis will be required.
Fluid Overload
Patients with AKI may present with fluid overload
because of salt and water retention and a decrease in
GFR. It also commonly occurs after oliguric/anuric
patients are given intravenous fluids by medical staff
in an attempt to improve urine output. Clinically,
patients complain of feeling short of breath, and on
physical examination signs of fluid overload include a
raised jugular venous pressure, peripheral edema, a
gallop rhythm, and bibasal crepitations on auscultation of the chest.
Bleeding Tendency
Patients with severe renal failure have an increased
likelihood of bleeding, which may occur even though
their platelet counts and clotting are normal. This is
206
Smith and Bradley
thought to occur because platelets do not adhere

normally to the endothelium and stop bleeding.
Experimental work in rats has suggested that nitric
oxide is the mediator of bleeding tendency in uremia.
Rats that have extensive surgical ablation of renal
tissue develop renal failure because of progressive
glomerulosclerosis. Like uremic humans, these rats
have a prolonged bleeding time. Administration of
L-NMMA, a specific inhibitor of nitric oxide normalized the bleeding time in uremic rats. This effect was
completely reversed by giving the animals the NO
precursor L-arginine (11).
Infection
Infections develop in 30% to 70% of patients with
ATN, probably because of a combination of factors
including impaired defences due to uremia, excessive
use of antibiotics and multiple invasive procedures.
DIALYSIS
Indications for urgent dialysis or RRT are as follows:
1.
2.
3.
4.
Persistent hyperkalaemia (K+ >7 mmol).

Fluid overload refractory to diuretic therapy.
Acidosis (pH <7.1, or bicarbonate <12 mmol).
Symptomatic uremia (tremor, cognitive impairment,
fits, coma or pericarditis). This usually occurs when
urea >45 mmol.
Three principle methods for RRT exist, namely

intermittent hemodialysis, peritoneal dialysis and
continuous venovenous haemofiltration (CVVH).
Each method has its advantages and disadvantages.
Intermittent hemodialysis is probably the most
widely available technique, and it is very efficient
at removing volume from the intravascular compartment. However, its use is often complicated by
hypotension, and it is in this situation that CVVH

may be more appropriate. Peritoneal dialysis can be
used in the acute setting, but this is certainly more
unusual, and it is more difficult to remove large fluid
volumes.
ACUTE TUBULAR NECROSIS

Pathogenesis of Acute Tubular Necrosis
The commonest form of intrinsic AKI is acute tubular
necrosis (ATN) secondary to ischemic and, or nephrotoxic injury. It is often the result of multiple insults,
rather than a single event, and multiple mechanisms
contribute to the final outcome of reduced urine
output. The key processes involved are illustrated in
Figure 3. The clinical course of ATN can be divided
into three phases: initiation, maintenance and recovery. Figure 4 illustrates the key features of each phase.
Research into the pathogenesis of ATN aims to
develop strategies to prevent it developing initially,
and the secondly to discover interventions that might
accelerate recovery.
ATN is characterised pathologically by varying
degrees of tubular cell damage and cell death. In
biopsy specimens regenerating cells are often found
together with freshly damaged cells, suggesting multiple cycles of injury and repair. The histological
sequence of events is as follows:
l
l
l
Patchy loss of tubular epithelial cells, leading to gaps

and exposure of a denuded basement membrane
Loss of proximal tubular brush border
Patchy necrosis of tubules most commonly in the
outer medulla
Tubular dilatation and formation of intraluminal
casts
Cellular regeneration
Figure 3 Key process involved in acute tubular necrosis.
207
Figure 4 The clinical course of acute tubular

necrosis.
In almost all experimental models of ATN and in

clinical experience of patients with AKI, volume loading with saline and the establishment of natriuresis,
will minimize or even prevent ATN developing. Conversely, volume depletion increases the risk of ATN.
Therefore, alterations in renal haemodynamics and
tubular function are two key factors in the pathogenesis of ATN.
Alterations in Renal Hemodynamics
The kidneys (less than 0.5% of total body mass) have

one of the highest blood flow rates per unit tissue
mass in the body, receiving approximately 20% of
cardiac output. This is mainly to filter the blood,
with only a small percentage being required for their
metabolism. If cardiac output, or effective intravascular volume decreases, compensatory mechanisms
such as afferent arteriolar dilatation (as a result of
myogenic responses and prostaglandins) and efferent
arteriolar constriction (via angiotensin II) attempt to
maintain renal perfusion. However, if prolonged,
decompensation occurs. Excessive stimulation of the
renin angiotensin system and the sympathetic nervous system, leads to profound renal vasoconstriction
and tubular hypoxia and damage. Drugs that interfere
with these compensatory mechanisms, such as
NSAIDS inhibiting prostaglandin synthesis, calcineurin inhibitors (e.g., cyclosporine and tacrolimus),
which are potent vasoconstrictors, and ACE inhibitors
accelerate the process.
Blood flow in the outer cortex is approximately
six fold greater than the outer medulla and twenty
fold greater than the inner medulla. The limited blood
flow in the medulla prevents wash out of the gradients of solute formed by the loops of Henle, which
are important for normal urine concentration. Therefore, even under normal conditions the medulla is
relatively hypoxic, with a partial pressure of oxygen
being 10 to 20 mmHg, compared with 50 mmHg in the
cortex. So, tubules in the medulla are particularly
vulnerable to ischemic damage when renal perfusion
is reduced (12). Although reduction in renal blood
flow is thought to be the dominant cause of ATN, it

cannot be the sole factor, as restoration of adequate
blood flow by volume expansion or vasodilatation
does not correct GFR.
Mediators such as endothelin-1, adenosine and
angiotensin II, all potent vasoconstrictors and
reduced levels of vasodilators such as nitric oxide
and prostaglandins are thought to lead to prolonged
medullary vasoconstriction and hypoxic damage to
tubules. It is not understood why this occurs. During
periods of ischemia and reperfusion, the ET-1 gene is
upregulated (13) and elevated levels of endothelin-1
have been demonstrated in experimental models of
AKI, and endothelin-1 receptor antagonists shown to
improve renal function and renal histological features of ischemic injury (14,15). Adenosine is thought
to play a role in contrast nephropathy. Pretreatment
with adenosine receptor antagonists, such as theophylline, prior to the administration of contrast have
been shown to limit the reduction in GFR, but have
no effect in established ATN (16). The role of angiotensin II is somewhat unclear. Expansion of the
juxtaglomerular apparatus and an increase in plasma rennin levels have been shown in experimental
cases of ATN, but inhibition of angiotensin II, rather
than ameliorate AKI has been shown to precipitate
renal failure in patients with reduced blood volume.
A deficiency of vasodilators, such as endotheliumderived nitric oxide and prostaglandins may play a
role in the initiation of ATN, but no evidence exists
that administration of either molecule alters the course
of established ATN. As a consequence of the initial
severe vasospasm occurring in an attempt to maintain
renal perfusion, focal and segmental necrosis of the
media in resistance arterioles is found. Endothelial
cells swell, and platelets and leukocytes adhere to sites
of endothelial cell injury, obstructing the lumens of
capillaries. Activated adherent neutrophils inactivate
endothelium-derived nitric oxide, promoting further
vasoconstriction and renal hypoperfusion and further
leukocyte adhesion, probably via up-regulation of
adhesion molecules, such as ICAM-1 (Intercellular
adhesion molecule-1). Administration of monocloncal
antibodies to ICAM-1, protected rats against AKI (18),
208
Smith and Bradley
and ICAM-1 deficient mice are protected against

ischemic renal injury (18).
There is a rationale for giving vasodilators in
ATN. However, nitric oxide donors such as nitroprusside, do not improve the course of ischemic ATN (19).
The lack of effect may not be due to a failure in
vasodilatation, but due to the paradoxical toxic effect
of nitric oxide on the proximal tubules, by the generation of oxygen free radicals. The cell swelling seen
after ischemic damage in ATN, not only obstructs the
tubular lumens, but together with interstitial edema
obstructs the ascending vessels impairing venous
return from the medulla, further exacerbating tubular
ischemia.
Alterations in Tubular Function
Tubular obstruction due to the exfoliation of mainly

viable rather than necrotic tubular cells into the
lumen, contributes to reduction in GFR. It also leaves
behind an exposed basement membrane, and the loss
of tight junctions between the proximal tubule cells
results in tubular back leak of various substances in
the ultrafiltrate. The sloughed cells in the lumen
exacerbate this by increasing intratubular pressure.
Tubular obstruction is especially relevant in certain causes of AKI. In myeloma, haemoglobulinuria,
myoglobinuria and crystalluria, casts contribute to
tubular obstruction in addition to denuded tubular
cells. In these situations, establishing a diuresis and
alkalinizing the urine are strategies to minimize the
risk of tubular obstruction.
Activation of tubuloglomerular feedback also
contributes to the reduction in GFR. As well as losing
their tight junctions, proximal tubule cells lose their
polarity, by the relocation of the Na+/K+ adenosine
triphosphate (ATP)ase from the basolateral to the
apical membrane. This impairs sodium and water
transport, and consequently the loss of chloride reabsorption in the thick ascending limb of the loop of
Henle activates the feedback mechanism at the macula
densa because of increased chloride delivery. This
leads to afferent arteriolar constriction via the adenosine A1 receptor (A1AR) in the glomerulus and a
decrease in the GFR. Therefore, it may be expected
that knockout of the A1AR may limit the decline in
GFR. However, paradoxically the reverse is true (20).
Activation of A1AR is actually protective, probably by
limiting delivery of ions and solutes to the damaged
cells of the proximal tubule, thereby reducing the
demand for ATP dependent reabsorption. An alternative explanation may be that adenosine has differential effects in the cortex and medulla. In the cortical
circulation it is a potent vasoconstrictor via A1 receptors, but in the medulla it is a vasodilator acting via A2
receptors.
Oxygen deprivation, as occurs with renal hypoperfusion, leads to the catabolism of ATP to adenosine
diphosphate (ADP) and adenosine monophosphate
(AMP). Prolonged ischemia results in the breakdown
of adenine nucleotides to adenosine and hypoxanthine. Consequently, ATP dependent transport mechanisms are inhibited. One effect is a rise in
intracellular free calcium, as ATP is required to

pump calcium out of cells or sequester it in the
endoplasmic reticulum. High calcium levels activate
proteases and phospholipases, cause mitochondrial
damage, generate oxygen free radicals and disrupt
the cell cytoskeleton. Calcium channel blockers have
been show to limit ischemic renal injury in animal
models, although the exact mechanism is unclear (21).
Possible effects include membrane stabilization of
tubular epithelial cells, or antagonism of calmodulin,
or improved renal haemodynamics in addition to
preventing calcium overloading of cells. Calcium
channel blockers have been shown to ameliorate
ATN in humans. The best evidence comes from the
transplant population, where administration of calcium channel blockers to recipients reduces the prevalence of ATN (22,23). In addition, there is some
evidence that administration of calcium channel
blocking agents prior to radiocontrast materials offers
some protection to their nephrotoxicity (21).
Recovery from Acute Tubular Necrosis

Once renal perfusion has been reestablished, the
majority of individuals with ATN recover most of
their renal function. For recovery to occur, tubular
cell polarity and the integrity of tight junctions need to
be restored in viable cells. In addition, dead cells must
be removed by apoptosis, and lost tubular epithelial
cells must regenerate. Following reperfusion, there is
marked up-regulation of a number of genes involved
in cell proliferation, including insulin-like growth
factor 1 (IGF-1) (24), fibroblast growth factor (FGF),
hepatocyte growth factor (HGF) (25) and epidermal
growth factor (EGF) (26). In animal models of AKI,
administration of these various growth factors accelerated renal recovery. However, when IGF-1 was
given to human subjects with AKI, no benefit was
demonstrated. In addition to the above factors, heat
shock proteins (HSP) are likely to play a key role in
tubular regeneration. Both HSP-70 and HSP-25 have
been shown to be over expressed in renal tubular cells
following ischemia and reperfusion in animal models
(27,28). Their role in humans is yet to be determined.
CONCLUSIONS
Treatment of patients with AKI is still largely supportive. Of course strategies to prevent the development of
AKI, such as volume expansion prior to administration of radiocontrast materials is important, but most
research has focused on interventions to accelerate
regeneration and recovery of the tubular epithelium
following an episode of ATN. Much of this work is
still very experimental, and although successes have
been seen in animal models, translating such work
into human subjects remains difficult. It is likely that
as the cause of most cases of ATN is multifactorial,
and the development of ATN requires a number of
pathological processes, that any single drug therapy
will be the cure, and multiple agents will be required
to promote a rapid recovery.
REFERENCES
1. Bellomo R, Kellum JA, Ronco C. Defining and classifying
acute renal failure: from advocacy to consensus and validation of the RIFLE criteria. Intensive Care Med 2007; 33
(3):409413.
2. Lameire N Van Biesen W, Vanholder R. Acute renal
failure. Lancet 2005; 365(9457):417430.
3. Chertow GM, Christionsen CL, Cleary PD, et al. Prognostic
stratification in critically ill patients with AKI requiring
dialysis. Arch Intern Med 1995; 155(14):15051511.
4. Kalra PA. Early management and prevention of acute renal
failure. EDTNA ERCA J. 2002(suppl 2):3438, 42.
5. Miller TR, Anderson RJ, Linas SL, et al. Urinary diagnostic
indices in acute renal failure: a prospective failure. Ann
Intern Med 1978; 89:4750.
6. Anderson RJ, Linas SL, Berns AS, et al. Nonoliguric acute
renal failure. N Engl J Med 1977; 296:11341138.
7. Shilliday I, Quinn K, Allison M. Loop diuretics in the
management of acute renal failure: a prospective, doubleblind, placebo-controlled, randomized study. Nephrol Dial
Transplant 1997; 12(12):25922596.
8. Kellum JA, Decker J. Use of dopamine in acute renal failure: a
meta-analysis. Crit Care Med 2001; 29(8):15261531.
9. Bellomo R, Chapman M, Finfer S, et al. Low-dose dopamine in patients with early renal dysfunction: a placebocontrolled randomised trial. Lancet 2000; 356:21392143.
10. Debaveye YA, Van de Berghe GH. Is there still a place for
dopamine in the modern intensive care unit? Anesth Analg
2004; 98:461468.
11. Remuzzi G, Perico N, Zoja C, et al. Role of endotheliumderived nitric oxide in the bleeding tendency of uremia.
J Clin Invest 1990; 86(5):17681771.
12. Heyman SN, Fuchs S, Brezis M. The role of medullary
ischemia in acute renal failure. New Horiz 1995; 3(5):
597607.
13. Wilhelm SM, Simonson MS, Robinson AV, et al. Endothelin up-regulation and localization following renal ischemia
and reperfusion. Kidney Int 1999; 55:10111018.
14. Gellai M, Jugus M, Fletcher T, et al. Nonpeptide endothelin
receptor antagonists. V. Prevention and reversal of acute
renal failure in the rat by SB 209670. J Pharmacol Exp Ther
1995; 275:200206.
15. Gellai M, Jugus M, Fletcher T, et al. Reversal of postischemic acute renal failure with a selective endothelinA receptor antagonist in the rat. J Clin Invest 1994; 93:900906.
209
16. Ix JH, McCulloch CE, Chertow GM. Theophylline for the

prevention of radiocontrast nephropathy: a meta-analysis.
Nephrol Dial Transplant 2004; 19(11):27472753.
17. Kelly KJ, Williams WW Jr., Colvin RB, et al. Antibody to
intercellular adhesion molecule 1 protects the kidney
against ischemic injury. Proc Natl Acad Sci USA 1994; 91:
812816.
18. Kelly KJ, Williams JJ, Colvin RB, et al. Intracellular adhesion molecule-I deficient mice are protected against renal
ischemia. J Clin Invest 1996; 97:10561063.
19. Lopez-Neblina F, Toledo-Pereyra LH, Mirmiran R, et al.
Time dependence of Na-nitroprusside administration in
the prevention of neutrophil infiltration in the rat ischaemic kidney. Transplantation 1996; 61(2):179183.
20. Lee HT, Xu H, Nasr SH, et al. A1 adenosine receptor
knockout mice exhibit increased renal injury following
ischemia and reperfusion. Am J Physiol Renal Physiol
2004; 286:F298F306.
21. Michael U, Lee SM. The role of calcium antagonists in
nephrotoxic models of renal failure. In: Epstein M, Loutzenheimer R, eds. Calcium Antagonists and the Kidney.
Philadelphia: Hanley and Belfus, 1990:187201.
22. Neumayer HH, Kunzendorf U, Schreiber M. Protective
effects of calcium antagonists in human renal transplantation. Kidney Int 1992; 41:S87S93.
23. Alcaraz A, Oppenheimer F, Talbot-Wright R, et al. Effect of
diltiazem in the prevention of acute tubular necrosis, acute
rejection, and cyclosporine levels. Transplant Proc 1991;
23(5):23832384.
24. Miller SB, Martin DR, Kissane J, et al. Insulin-like growth
factor I accelerates recovery from ischaemic acute tubular
necrosis in the rat. Proc Natl Acad Sci 1992; 89(24):
1187611880.
25. Miller SB, Martin DR, Kissane J, et al. Hepatocyte growth
factor accelerates recovery from acute ischaemic renal injury
in rats. Am J Physiol Renal Physiol 1994; 266:F129F134.
26. Humes HD, Cieslinski DA, Coimbra TM, et al. Epidermal
growth factor enhances renal tubule cell regeneration and
repair and accelerates the recovery of renal function in
postischemic acute renal failure. J Clin Invest 1989; 84:
17571761.
27. Aufricht C. Heat-shock protein 70: molecular supertool?
Pediatr Nephrol 2005; 20:707713.
28. Kelly KJ. Heat shock (stress response) proteins and renal
ischemia/reperfusion injury. Contrib Nephrol 2005; 148:
86106.
13
Chronic Renal Failure
Sanjay Ojha and John R. Bradley
Cambridge University Hospitals NHS Foundation Trust, Cambridge, U.K.
DEFINITION
The kidneys perform a number of roles, which include
regulation of water and inorganic ion balance, removal of metabolic waste products from the blood, and
secretion of certain hormones. Chronic renal failure is
a state in which there has been irreversible loss of
these functions, sufficient to cause an impact on an
individuals health. Given that there is a significant
amount of renal reserve, this only occurs when
more than 50% of renal excretory capacity has been
lost; for example, removal of a single kidney from a
healthy person (e.g., living kidney donors) does not
cause any long-term sequelae.
Traditionally, serum creatinine has been used as
a marker of renal function. This has always posed two
problems. First, creatinine is affected by a number of
variables apart from glomerular filtration rate (GFR),
such as age, gender, race and body mass. Second,
serum creatinine tends not to rise outside the normal
range until around 50% of GFR has been lost. As a
consequence certain groups of patients with renal
diseasethe frail and the elderlytend to be detected
late.
Estimated GFR (eGFR) is now being used to
provide a more accurate assessment of kidney function. This utilizes the four-variable modification of
diet in renal disease (MDRD) equation, which takes
into account age, gender, and race as well as serum
creatinine, to give a measure of renal function.
The U.S. Kidney Disease Outcomes Quality Initiative (K/DOQI) has introduced the term chronic
kidney disease (CKD) to replace chronic renal failure.
This classifies renal impairment into five stages on the
basis of eGFR (Table 1) (1).
All stages are associated with hypertension as
well as an increased risk of cardiovascular disease.
From stage 3 onward complications of renal failure
such as renal anemia and renal bone disease start
to develop. Stage 4 tends to be the point at which
uremic symptoms occur. Stage 5 should prompt
consideration of initiating renal replacement therapy
(RRT), unless a conservative management plan has
been agreed.
The chief advantage to this classification is that it
allows early identification of those patients who are
at risk of progressive renal impairment (i.e., stages
1 and 2). In addition, it provides a standardized nomenclature worldwide for severity of chronic renal disease.
EPIDEMIOLOGY
CKD is a common condition. Extrapolating data from
the third National Health and Nutrition Examination
Survey (NHANES III) in the United States indicates
a prevalence of 11% in the adult population (2).
However, only a small proportion of these patients
require RRT. The majority of patients with CKD fall
into stages 1 to 3 and will not progress to more severe
renal failure. They often require no specific interventions, apart from addressing cardiovascular risk
factors.
Accurate data for the incidence and prevalence
of end-stage renal failure (ESRF) exist, thanks to the
establishment of various national renal registries
(Table 2) (35). In 2006, 113 patients per million
population were accepted onto dialysis programs in
the United Kingdom. This is approximately double
the number compared with 20 years ago, and
although this increase is in part because of improved
provision of RRT, there has undoubtedly been a real
increase in the incidence of ESRF. A number of
elements have contributed to this. Diabetes and obesity, both of which predispose to CKD, are now far
commoner. In addition, there is a larger elderly population to care for, and it is well recognized that the
incidence of ESRF rises exponentially with age. The
median age of patients starting dialysis in the United
Kingdom was 65 years in 2006, with the highest
acceptance rate in the 75- to 79-year age group (3).
CKD is also commoner in ethnic populations.
Afro-Caribbeans and Asians are two to four times
more likely to start RRT than the white (Caucasian)
population (4). To an extent this is explained by the
predisposition of these groups to develop type 2
diabetes. Furthermore, hypertension is particularly
common among people of Afro-Caribbean background. However, this is not the full story. Other
genetic and environmental factors contribute to the
tendency of these populations to develop ESRF. For
example, conditions such as lupus nephritis and focal
segmental glomerulosclerosis are also overrepresented
in the ethnic communities. In addition to the greater
Chapter 13: Chronic Renal Failure

Table 1 Staging of CKD
CKD
stage
GFR (mL/
min/1.73 m2)
>90
6089
3
4
5
3059
1529
<15
Table 3 Common Causes of ESRF in the United Kingdom

Description
Cause of ESRF
Normal renal function but other

evidence of kidney damagea
Mild reduction in renal function, with
other evidence of kidney damagea
Moderately reduced GFR
Severely reduced GFR
End-stage renal failure or on dialysis
Diabetes
Glomerulonephritis
Pyelonephritis/interstitial nephritis
Polycystic kidney disease
Hypertension
Renovascular disease
Etiology uncertaina
Others
a
Evidence of kidney damage is suggested by abnormal urinalysis for
blood and/or protein, structural abnormalities (e.g., abnormal renal
imaging), or known inherited renal disease (e.g., ADPKD).
Abbreviations: CKD, chronic kidney disease; GFR, glomerular filtration
rate.
Table 2 Incidence and Prevalence of End-Stage Renal Failure

United
Kingdom
(2006)
Incidence (pmp/yr)
Prevalence (pmp)
211
113
725
United States
of America
(2006)
347
1569
Australia
(2006)
115
778
frequency of renal diseases in these two populations,

it has been noted that there is a faster decline in GFR
in Afro-Caribbeans and Asians with nephropathy
compared with Caucasians. This may partly explain
the overrepresentation of these groups on dialysis
programs.
There is a disparity between the genders with a
male to female ratio of 1.5:1 in both the U.S. and the
U.K. dialysis cohorts (3,4). This may be related to the
fact that diseases such as atherosclerosis and hypertension are commoner in men. In addition, there is
evidence that many renal diseases (including diabetic
nephropathy, ADPKD, IgA nephropathy, and membranous nephropathy) progress more quickly in males
than females (6).
There continues to be a steady increase in the
number of patients initiating dialysis at a rate of 5% to
7% per year, and it seems likely that a steady state will
not be reached for at least another 10 years.
ETIOLOGY
There are a number of causes for renal failure. Their
frequency will vary according to the population that is
evaluated. For example, in the United States, diabetes
is responsible for 45% of all new patients commencing
dialysis (4). In contrast, glomerulonephritis and
hypertension together account for over 90% of new
cases of ESRF in sub-Saharan Africa (7). This geographical discrepancy largely reflects differences in
diet, lifestyle, and environmental factors that exist
between Western and developing countries.
Table 3 shows the common diseases that result in
ESRF in the U.K. population. In most cases, there is a
chronic underlying process. However, 16% of patients
who have severe acute renal failure do not recover
renal function and remain on long-term dialysis (8).
Percentage of
all patients
22.2
10.4
7.2
6.7
5.4
6.8
26.2
15.3
a
In many cases the cause is presumed to be glomerulonephritis, but this
is not biopsy proven.
Abbreviation: ESRF, end-stage renal failure.
The underlying diagnosis changes depending on

the age group that is considered. In children, congenital urinary system anomalies and reflux nephropathy
constitute the commonest causes of ESRF. In the
elderly, renal vascular disease and hypertension are
more prevalent. In the U.K. dialysis population, renal
vascular disease is five times more common in those
aged over 65 compared with patients under 65.
The commonest presentation of CKD is the finding of
an elevated urea and creatinine on blood tests in an
otherwise asymptomatic individual. It is well recognized that the symptoms of renal failure are nonspecific and occur lateusually when 80% to 90% of renal
function has been lost. Consequently, it is now recommended that renal function should be measured at
least annually in adult patients who are at high risk of
silent development of CKD (Table 4) (9).
Table 4 Indications for Annual Measurement of Renal Function

Diseases associated with high risk of silent development of CKD
l
Hypertension
l
Diabetes
l
Heart failure
l
Atherosclerotic disease (coronary/cerebral/peripheral vascular)
Diseases associated with high risk of obstructive nephropathy
l
Urinary stone disease
l
Urinary diversion surgery
l
Known or suspected bladder outflow obstruction
l
Neurogenic bladder
Diseases requiring long-term treatment with potentially nephrotoxic drugs
l
ACE inhibitors and angiotensin receptor blockers
l
Nonsteroidal anti-inflammatory drugs
l
Lithium
l
Calcineurin inhibitors
Multisystem conditions that may involve the kidney
l
Systemic lupus erythematosus
l
Vasculitis
l
Rheumatoid arthritis
Previously diagnosed CKD
Abbreviation: CKD, chronic kidney disease.
212
Ojha and Bradley
Identifying these patients early is important as it

allows measures to be instituted to preserve kidney
function, as well as ensuring that the complications of
renal failure can be prevented or ameliorated in a
timely fashion.
Despite this targeted screening process, many
patients with CKD are detected late. Twenty-three
percent of patients who commence dialysis will have
seen a nephrologist for the first time less than 90 days
prior to starting RRT (3).
The typical early symptoms of renal failure are
lethargy, malaise, fatigue, and dyspnea, which are
predominantly a consequence of renal anemia. Later
symptoms include pruritus, anorexia, nausea and
vomiting, headache, difficulty in concentrating, unintentional weight loss, peripheral edema, and restless
legs.
It is less common now for ESRF to present as a
uremic emergency, but in these situations, symptoms
of encephalopathy will often be present. Patients will
have a reduced conscious level and evidence of neuromuscular irritability such as asterixis. In addition,
they have an increased bleeding tendency; there
may be evidence of skin bruising or gastrointestinal
bleeding. Uremic pericarditis (characterized by a pericardial friction rub) or pulmonary edema can also
occur. The presence of any of these features is an
indication to commence dialysis urgently.
CLINICAL APPROACH TO CKD

When faced by an individual with impaired renal
function, there are four key aspects to be considered
in the initial assessment (Table 5):
Is the Renal Failure Acute or Chronic?

Determining chronicity is vital as it provides prognostic information and alters patient management. The
best way of distinguishing between acute and chronic
renal failure is to find out what the patients renal
function has been in the past. Looking through old
notes and laboratory records or phoning the patients
Table 5 Clinical Approach to Chronic Kidney Disease
Acute vs. chronic
Underlying diagnosis
Reversible factors
Complications
Historical values for renal function

Ultrasound of kidneys
History and examination
Ultrasound
Immunology
Renal biopsy
Hypovolemia
Nephrotoxins
Obstruction
Urinary tract infection
Fluid overload
Hypertension
Hyperkalemia
Acidosis
Anemia
Calcium/phosphate imbalance
Renal osteodystrophy
GP are simple tasks, which can yield important

information.
In the absence of old blood results, ultrasonography of the kidneys may be helpful. The finding of
two shrunken kidneys with increased echogenicity
can allow a diagnosis of chronic renal failure to be
made with confidence.
What Is the Underlying Diagnosis?

It is vital to make a diagnosis where possible as some
causes of CKD are treatable. In addition, if the condition is inherited there are implications for family
members.
The diagnosis may sometimes be obvious from
the history and exam; a patient with longstanding
diabetes and evidence of retinopathy is almost certain
to have diabetic nephropathy. Older gentlemen with
longstanding urinary symptoms and a palpable
bladder on abdominal examination are likely to have
obstructive uropathy because of prostatic pathology.
An ultrasound scan is a mandatory investigation
in all patients with CKD. It not only looks for evidence
of obstruction, but also allows diagnoses such as
ADPKD to become apparent. Furthermore, in patients
with evidence of atherosclerotic disease, the finding
of asymmetric kidneys on ultrasound should raise
the possibility of renovascular disease, which can be
further investigated with either magnetic resonance
angiogram or formal invasive angiography.
Urine dipstick is the other obligatory test; the
presence of blood and protein on urinalysis should
raise the possibility of a chronic glomerulonephritis.
In these circumstances, it would be reasonable to
check immunological blood tests to assess for an
underlying autoimmune condition.
If the initial assessment fails to elucidate the
etiology, consideration should be given to performing
a renal biopsy. This should not be undertaken, however, if the kidneys are small. A biopsy is unlikely to
provide useful information and the risk of bleeding is
significant in this situation.
Are There Any Reversible Factors

That Can Be Addressed?
Even when renal failure is confirmed to be chronic,
there are a number of factors that can cause an acute
decline in GFR (termed acute-on-chronic renal failure). Hypovolemia, use of nephrotoxins, urinary tract
sepsis, and urinary tract obstruction should all be
actively looked for and excluded.
Have Any Complications

of Renal Failure Arisen?
Questioning should be directed at determining whether there are symptoms of uremia or fluid overload.
The examination should also focus on these, looking
for evidence of hypertension, peripheral or pulmonary edema, and excoriations, which would suggest
pruritus.
A number of blood tests assessing for electrolyte

disturbances, acidosis, anemia, and hyperparathyroidism should be checked.
The complications of renal failure will be dealt
with in more detail later in the chapter.
PATHOPHYSIOLOGY
This section will deal with the mechanisms that lead
to the development and progression of CKD. It will
then address the interventions that may be undertaken to prevent deterioration of renal function in
patients with established CKD.
213
response ensues with release of proinflammatory and

profibrotic cytokines, as well as growth factors (such
as PDGF). This leads to mesangial cell proliferation
and extracellular matrix deposition. Combined with
the platelet aggregation and fibrin deposition that
also occur following endothelial injury, the end point
is glomerulosclerosis.
In addition to this process, glomerular hypertension with consequent endothelial and epithelial cell
stress leads to proteinuria, which also plays a pathogenic role in progression of kidney disease (see
below).
Tubulointerstitial Scarring
Mechanisms of Progression of Renal Disease
The first step in the development of renal failure is an
insult, which results in loss of functioning nephrons.
This may be acute, for example, hemodynamic compromise leading to renal ischemia, or chronic such as
in the case of a persistent glomerulonephritis. Not all
insults lead to progressive kidney disease; for example, patients with renal impairment secondary to urinary tract obstruction can maintain stable renal
function in the long-term once the obstruction has
been relieved.
However, once a threshold has been passed
usually when 60% to 70% of renal function has been
lost, CKD tends to become progressive. This is the
consequence of pathogenic processes, which lead to
glomerulosclerosis and tubulointerstitial fibrosis.
Glomerulosclerosis
Glomerular scarring can arise because of ongoing
damage from the primary disease process (glomerulonephritis and diabetes being the main culprits here).
However, it also occurs because of maladaptive
responses, which attempt to compensate for the loss
of functioning renal mass. The evidence for this comes
from an animal model in which rats underwent subtotal nephrectomy (10). In the remnant kidney, hemodynamic changes were noted, specifically preferential
vasodilatation of afferent glomerular arterioles, together with increases in glomerular transcapillary hydraulic
pressure and filtration fraction. This functional
changeso-called hyperfiltrationis an attempt by
the remaining nephrons to maintain GFR.
Unfortunately, hyperfiltration contributes to the
progressive destruction of remaining glomeruli; over
a period of weeks, these rats developed structural
lesions within the remaining kidney tissue, including
mesangial expansion and focal segmental glomerulosclerosis. This was associated with progressive hypertension, proteinuria, and renal insufficiency, eventually
leading to the death of the animals.
The mechanism by which hyperfiltration causes
renal scarring is thought to relate to increased glomerular capillary pressure, which in turn causes
stretch of these capillaries and initiates endothelial
cell damage. As a result of this, an inflammatory
The extent of tubular and interstitial damage seen

within a kidney often correlates better with longterm renal prognosis than the degree of glomerular
injury.
As with glomerulosclerosis, tubulointerstitial
inflammation and fibrosis may arise as a consequence
of a primary disease process (Table 6).
However, it can also be a secondary effect of
glomerular pathology and this is thought to be largely mediated by proteinuria. There are a number of
mechanisms that have been implicated. Tubular cell
injury can occur either because of the direct toxic
effects of filtered proteins, or as a consequence of
complement activation within the tubules (11).
This in turn leads to release of proinflammatory
cytokines with the development of an inflammatory
infiltrate within the interstitium. Resolution of this is
by fibrosis.
Increased deposition of extracellular matrix in
the interstitium is driven by a number of profibrotic
factors within the filtrate, which have the effect of
activating renal fibroblasts, as well as increasing
their number by causing tubular cells to change
phenotype.
The outcome of tubulointerstitial damage is
chronic ischemia within this part of the kidney.
This arises because of loss of peritubular capillaries,
causing impaired blood flow to the interstitium. In
addition, there is reduced oxygen diffusion because
of the interstitial fibrosis. As a consequence of the
hypoxia, further tubular damage occurs, and so a
Table 6 Primary Causes of Tubulointerstitial Nephritis

Drugs
NSAIDs
Calcineurin inhibitors
Lithium
Cystic diseases
ADPKD
Medullary cystic disease
Infection
Pyelonephritis
Heavy metals
Lead
Cadmium
Metabolic
Hyperuricemia
Hyperoxaluria
Chronic hypokalemia
Obstructive
Obstructive uropathy
Reflux nephropathy
Sickle cell nephropathy
Immunological
Sjogrens
Sarcoidosis
214
Ojha and Bradley
Table 7 Risk Factors for Progression of Chronic Kidney

Disease
Modifiable
Nonmodifiable
Hypertension
Proteinuria
Hyperglycemia
Dyslipidemia
Obesity
Smoking
Age
Gender
Race
Genetics
Underlying renal disease
vicious cycle of self-perpetuating renal damage

emerges (12).
Slowing Progression of Renal Disease

The majority of patients with CKD will not progress to
a point where they require RRT. Death from cardiovascular disease is a far more likely outcome. However, a significant proportion of patients do progress
to ESRF and there are a number of factors that
predispose to this (Table 7).
The strategy for slowing progression of renal
disease entails three approaches.
1.
2.
3.
Diagnosis and treatment of the underlying disease:

Not all diseases are treatable; polycystic kidney
disease tends to have a relentless progression.
However, many causes of chronic renal failure
are amenable to treatment, which is why a diagnosis should always be sought. For example,
relieving urinary tract obstruction is often a
straightforward procedure that can yield satisfying results. More complex cases such as glomerulonephritis and vasculitis may entail interventions
such as immunosuppression, which can address
the underlying pathogenic process and preserve
renal function.
Avoidance of nephrotoxins and other insults to the
kidney: Kidneys that are already damaged are
more vulnerable to new insults, which can cause
further, sometimes irreversible, decline in GFR.
This can be avoided through simple measures;
patients should be told to steer clear of NSAIDs.
Radiocontrast and aminoglycoside antibiotics
should be used with caution. Urinary tract infections and episodes of hypovolemia need to be
treated promptly.
Modulating factors that promote progression: The
importance of this cannot be overstated, for all
of these influences are recognized as risk factors
for cardiovascular disease. Hence controlling
them will provide a dual health benefit. These
will be dealt with in detail.
Hypertension
Given the kidneys central role in the control of blood
pressure, it is not surprising that hypertension is a
finding in most patients with CKD. It arises because
of salt and water retention with subsequent expansion
of the extracellular space. Furthermore, the renin-
angiotensin-aldosterone system (RAAS) is upregulated in renal disease. This contributes to salt and
water retention through increased production of
aldosterone, as well as promoting arteriolar vasoconstriction and sympathetic nervous system activation
via the effects of angiotensin II.
Systemic hypertension promotes renal damage,
as it exacerbates glomerular capillary hypertension
and thus induces increased glomerular filtration and
proteinuria.
There is a clear association between elevated
blood pressure and a faster rate of progression of
CKD, although this relationship is modulated by the
amount of proteinuria that is also present. The MDRD
trial demonstrated that a lower blood pressure target
(125/75 vs. 140/90) in patients with more than 1 g
proteinuria per day was associated with a slower
decline in GFR (13). A more recent meta-analysis
showed that in nondiabetic patients with CKD, the
lowest risk of progression was in those who achieved
a systolic blood pressure between 110 and 129 (14).
Current guidelines state that all patients with
CKD should have a target blood pressure of less than
130/80 (15). In cases where there is significant proteinuria (>1 g/day) tighter blood pressure control is
desirable with a target of less than 125/75.
Treatment of hypertension needs to closely
involve the patient; restricting dietary sodium load
is a vital aspect of management, as is ensuring
compliance with any medications that are prescribed.
It often requires three or more agents to achieve the
appropriate blood pressure target. The choice of
antihypertensive medication is determined by several variables. In general, ACE inhibitors and angiotensin II receptor blockers (ARBs) are first line as they
have antiproteinuric as well as antihypertensive
effects (see below). Furthermore, in patients with
concomitant diabetes or cardiovascular disease,
they are recognized to reduce morbidity and mortality. However, this has to be tempered by the fact that
they often exacerbate hyperkalemia, which is a familiar complication of CKD. Their use can also be
limited by a decline in renal function that is seen in
some cases soon after these drugs are started. The
commonest situation in which this is observed is
silent renovascular disease. In this setting, angiotensin II plays a vital role in maintaining renal perfusion
pressure by preferentially vasoconstricting the efferent arteriole. When this is blocked the adverse effect
on renal hemodynamics causes an acute drop in GFR.
It is imperative that all patients commenced on an
ACE inhibitor or ARB have their renal function
checked within seven days. If there is a fall in eGFR
of >20%, these drugs should be stopped, and consideration given regarding investigation for renovascular disease (9).
Diuretics are useful second-line antihypertensives, especially in the context of fluid overload.
However, in more advanced CKD (stages 4 and 5)
thiazide diuretics tend to be ineffective. Calcium
channel blockers, a-blockers, b-blockers, and centrally
acting antihypertensive drugs can all be used
thereafter.
Proteinuria
Proteinuria is a powerful risk factor for the progression
of renal disease, via mechanisms that have been
explained above. It is widely accepted that in proteinuric renal disease, the aim should be to achieve less
than 1 g/day proteinuria. The rationale for this comes
from the observation that the greater the degree of
proteinuria, the more rapidly patients will progress to
ESRF. A seminal study from 1996 looked at renal
survival in patients with a variety of proteinuric kidney
disease; in the group that had <1 g/day proteinuria,
less than 10% had progressed to ESRF after two years.
Conversely, nearly 25% of the group with >3 g/day
proteinuria had reached this end point by 18 months
(16). A number of other studies have confirmed this
relationship between proteinuria and poorer renal outcomes (9).
Indeed, on the basis of the strength of this link,
reduction in proteinuria is now accepted as a good
surrogate marker for assessing whether an intervention has a role in slowing kidney damage.
A number of antiproteinuric interventions in
CKD have been evaluated. The largest body of evidence exists for blockade of the RAAS. Overactivation
of this system leads to increased production of
angiotensin II, which drives renal efferent arteriolar
vasoconstriction. This causes increased glomerular
capillary pressure and hence hyperfiltration and
proteinuria.
In both diabetic and nondiabetic patients with
renal impairment, ACE inhibitors and ARBs have
been shown to reduce protein leak and progression
of nephropathy (1721). Each of these drugs targets a
different step in the RAAS pathway, and there are
theoretical reasons why individually they cannot provide complete blockade of this system. For example,
angiotensin I can be converted to angiotensin II by
enzymes that are not affected by ACE inhibitors
(e.g., chymase). Conversely, ARBs cause elevated
angiotensin II levels. Normally, angiotensin II inhibits
renin secretion via a negative feedback mechanism
mediated by the angiotensin receptor (AT1). When
this is blocked, the subsequent rise in renin and
angiotensin II levels can antagonize the therapeutic
effects of the ARB.
With this rationale in mind, there has been a
focus on using dual blockade therapy, especially in
those patients who have persistent proteinuria of
more than 1 g/day despite monotherapy. The COOPERATE study assessed the efficacy of combined ACE
inhibitor (trandolapril) and ARB (losartan) treatment
in nondiabetic patients with chronic proteinuric
nephropathy (22). Patients who received dual therapy
had a 50% reduction in their relative risk of doubling
their serum creatinine or developing ESRF compared
with patients on monotherapy.
In type 2 diabetic patients, the CALM study has
also provided evidence that combination treatment
with ACE inhibitor (lisinopril) and ARB (candesartan)
produces better reductions in blood pressure and
microalbuminuria compared with using these drugs
in isolation (23).
215
The main concern with using these drugs together is the risk of severe hyperkalemia. Given that nondihydropyridine calcium channel blockers (ditiazem
and verapamil) have also been shown to reduce
proteinuria and progression of renal disease, a safer
option is to add in one of these medicines to achieve
satisfactory control of proteinuria.
Rather more controversial than RAAS blockade,
is the role for dietary protein restriction as an antiproteinuric intervention. Analysis of the MDRD study
showed that in those patients with severe renal
impairment (GFR 1324 mL/min), reduction in dietary protein intake by 0.2 g/kg/day slowed decline in
GFR by 1.15 mL/min/yr (13).
A meta-analysis of data from 1413 nondiabetic
patients confirmed that dietary protein restriction
effectively slows the progression of renal disease
(24). Nonetheless, many physicians will avoid this
strategy because of concerns that it may result in
malnutrition. It is recognized that a significant proportion of patients with CKD have an inadequate
calorie intake and this could be made worse if dietary
restrictions are imposed.
Hyperglycemia
Diabetes is the commonest cause of ESRF in the
Western world. Therefore, it is imperative that this
disease is optimally managed to reduce the incidence
of renal complications. Data from the Diabetes Control
and Complications Trial (DCCT) showed that tight
glycemic control in type 1 diabetics prevented the
development of microalbuminuria by 34%, and
reduced progression of microalbuminuria to frank
diabetic nephropathy by 56% (25). In type 2 diabetics,
the United Kingdom Prospective Diabetes Study
(UKPDS) showed that maintaining HbA1C levels at
7% as compared with 7.9% also reduced the development of microalbuminuria (26). What remains to be
seen, however, is whether attaining tighter glycemic
control has any impact on the rate of progression of
established diabetic renal disease.
Obesity
There is a growing body of evidence linking obesity to
the development of CKD. One study has shown that
patients with a BMI of over 30 are more likely to
develop proteinuria and renal insufficiency following
nephrectomy compared with nonobese patients (27).
The pathophysiology underlying this is not clear.
Many patients with obesity will have associated
hypertension, impaired glucose tolerance, and dyslipidemia. This is known as the metabolic syndrome
and is associated with hyperfiltration.
It is also postulated that deposition of excessive
adipose tissue in the renal viscera can cause compression of the loop of Henle, resulting in sluggish tubular
flow. Consequently, there is increased sodium reabsorption and so reduced delivery of sodium to the macula
densa. The outcome is vasodilatation of the afferent
arteriole and hence glomerular hyperfiltration (28).
216
Ojha and Bradley
Smoking
Salt and Water
Smoking should be discouraged in all patients with

CKD, not only to preserve renal function, but also to
reduce the risk of cardiovascular disease and cancer. It
is well recognized that smoking increases the risk of
ESRF, though the precise mechanisms for this remain
unclear (2931) It is postulated that cigarette smoke
causes renal injury through adverse effects on renal
hemodynamics. More recently, it had been shown that
nicotine promotes mesangial cell proliferation and
production of extracellular matrix. This could accelerate glomerulosclerosis (32).
In healthy individuals the kidneys are able to maintain sodium and water homeostasis despite wide
variations in the intake of these substances. Salt intake
may be up to 20 g/day in a Western diet, while water
intake may fluctuate hugely on a day-to-day basis.
Nonetheless, renal mechanisms ensure that serum
sodium is tightly regulated between 135 and 145
mmol/L and serum osmolality is kept between 280
and 300 mOsm.
Dyslipidemia
Sodium balance is managed because of the ability of

the tubules to vary the fractional excretion of filtered
sodium from less than 1% normally, up to 30% in
advanced CKD. However, once GFR falls to <10 mL/
min, the kidneys can no longer cope with sodium
loads. The consequence of this is salt and water
retention, which manifests itself as hypertension and
tissue edema.
To prevent this, patients should be advised to
limit their salt intake to <4 g/day. When hypertension
and fluid overload are present, diuretics should be
used to promote natriuresis. Loop diuretics are the
most effective, but their action can be potentiated by
addition of a thiazide diuretic. Caution needs to be
taken when using these drugs as they can precipitate
dehydration and hypotension, which can worsen
renal failure.
Dyslipidemia is common in patients with CKD. Both

experimental and clinical studies show that it is
associated with faster progression of renal disease.
Hyperlipidemia can cause both increased glomerular
permeability, which can progress to glomerulosclerosis, as well as tubulointerstitial injury (33). In the
MDRD study a low serum HDL was an independent
risk factor for more rapid decline in GFR (13).
However, it remains unclear as to whether lipidlowering therapy is beneficial in retarding progression
of CKD. A meta-analysis of 13 small trials suggests
that treatment of hyperlipidemia does preserve GFR
and reduce proteinuria in patients with renal disease
(34). Further evidence is awaited though. The Study of
Heart and Renal Protection (SHARP) trial is currently
ongoing and aims to give a definitive answer as to
whether lipid-lowering drugs improve outcomes in
patients with CKD.
HMG CoA reductase inhibitors do carry an
increased risk of side effects in patients with CKD,
notably myositis and rhabdomyolysis. Hence, current
recommendations are that they should only be used to
address cardiovascular risk, rather than as a therapy
to protect renal function.
CONSEQUENCES OF ADVANCED CKD

Having considered the ways in which GFR can be
preserved, we now need to reflect on the consequences of advanced CKD and how to manage the complications that arise when there is insufficient renal
function to maintain health.
Table 8 shows the functions that the kidneys
perform. The effect of loss of each of these functions
will be considered individually.
Table 8 Functions of the Kidney
Regulation of sodium and water balance
Regulation of inorganic ion balance
Regulation of acid-base balance
Removal of metabolic waste products
Secretion of hormones
l
Renin
l
Erythropoietin
l
1,25-Dihydroxycholecalciferol
Gluconeogenesis
Sodium
Water
The kidneys control water balance through their
capacity to produce very dilute or concentrated
urine. This is mediated by variations in water reabsorption from the collecting duct from <1% (for a
water-loaded individual) to >24% (for a dehydrated
person).
In renal failure, the kidneys lose the ability to
produce concentrated urine. This is because of a
number of mechanisms.
l
Hyperfiltration leads to an increased solute load in

each nephron, thus promoting a diuresis.
Tubulointerstitial scarring diminishes the high
interstitial osmolality that drives water reabsorption from the collecting ducts.
Tubular damage causes the collecting ducts to
become resistant to antidiuretic hormone.
As a consequence patients tend to develop polyuria, nocturia, and then thirst. They are prone to
dehydration if they cannot maintain satisfactory oral
fluid intake.
Hyperkalemia
Extracellular potassium ion concentration needs to be
tightly regulated; otherwise, life-threatening cardiac
dysrhythmias occur. Potassium excretion by the kidneys is predominantly controlled by the effect of

Table 9 Causes of Hyperkalemia in Chronic Kidney Disease
Table 11 Effects of Uremic Toxins
High potassium intake

Hyporeninemic hypoaldosteronism
l
Diabetes
l
Interstitial nephritis
Medications
l
ACE inhibitors/angiotensin II receptor blockers
l
Aldosterone antagonists (spironolactone)
l
NSAIDs
l
b-Blockers
Acidosis
Chronic inflammation
Accelerated atherogenesis
Sensorimotor peripheral neuropathy
Autonomic neuropathy
Defective immune system
Impaired platelet function
aldosterone, which increases potassium secretion by

the principal cells of the distal tubules.
Hyperkalemia does not tend to be a problem
until GFR falls to <10 mL/min as the kidneys retain
the ability to excrete potassium normally till this stage.
There are some exceptions to this rule: diseases in
which there is reduced production of aldosterone (e.
g., diabetes) or cases where the distal tubule becomes
less responsive to aldosterone (obstructive uropathy,
chronic pyelonephritis) can cause hyperkalemia
despite a much higher GFR.
In CKD a number of other factors can promote
hyperkalemia (Table 9). The management of hyperkalemia relies mainly on restriction of dietary intake
of potassium. In a normal diet an individual will
consume around 100 mmol potassium each day. A
low-potassium diet will contain half this amount.
Other measures that should be taken are correction of acidosis and stopping medications that promote hyperkalemia. If the serum potassium remains
elevated despite these measures, it is an indication to
start dialysis.
Acidosis
The kidneys regulate acid-base balance through excretion of hydrogen ions, as well as reabsorption of
bicarbonate. Acidosis only occurs once GFR has fallen
to <20 mL/min.
This has multiple detrimental consequences on
health (Table 10). The respiratory system attempts to
compensate for metabolic acidosis through hyperventilation. Consequently, patients describe symptoms of
breathlessness and exhaustion. Acidosis also exacerbates hyperkalemia by stimulating movement of
potassium ions from the intracellular to the extracellular compartment, in exchange for hydrogen ions.
Bone attempts to buffer the acidosis, but this
leads to calcium loss from bone as well as impaired
mineralization, hence contributing to renal osteodystrophy. Finally, acidosis increases skeletal muscle
Table 10 Consequences of Acidosis
Hyperventilation
Hyperkalemia
Exacerbation of renal bone disease
Skeletal muscle breakdown
Reduced cardiac output
217
catabolism, contributing to loss of body mass and

general weakness.
To avoid these complications, acidosis should be
treated with oral sodium bicarbonate once the serum
bicarbonate concentration is <20 mmol/L. Unfortunately, this therapy contributes to salt and water
retention and can worsen hypertension.
Uremia
One of the principal functions of the kidneys is the
excretion of organic waste products. These include
breakdown products of protein metabolism, such
as urea and creatinine, as well as metabolites of
various hormones. Although urea and creatinine
are used as measures of renal function, they are
not themselves harmful (35). However, when their
levels are elevated it reflects the accumulation of a
number of small (<500 Da) and middle (5005000 Da)
molecules, which are normally excreted by healthy
kidneys. These molecules are termed uremic toxins
and they exert several deleterious effects on the body
(Table 11).
Over 90 such compounds have been recognized,
which include free water-soluble solutes such as guanidines, protein-bound solutes such as phenol, and
middle molecules including b2 microglobulin, complement factor D, and multiple cytokines (36). Many
other uremic toxins remain unidentified.
These molecules are responsible for many of the
symptoms that are observed in advanced CKD,
including anorexia, nausea and vomiting, the features
of neuropathy and encephalopathy, and pericarditis.
If any of these symptoms develop, dialysis should be
commenced.
Anemia
Anemia is a common complication of CKD and can
occur earlyonce GFR has fallen to <50 mL/min.
Typically, it is normochromic and normocytic. There
are a number of factors that contribute to its development; the primary mechanism is reduced renal production of the peptide hormone erythropoietin (EPO).
This hormone is secreted by peritubular cells in the
interstitium, in response to a reduction in the partial
pressure of oxygen in the kidneys. It stimulates erythroid lines within the bone marrow to proliferate and
mature, hence increasing production of red blood
cells.
Other explanations for anemia include reduced
intake of iron and folic acid because of anorexia,
218
Ojha and Bradley
impaired intestinal absorption of iron as a consequence of uremia, gastrointestinal blood loss

because of bleeding tendency, and shortened red cell
survival.
Anemia has a significant impact on the quality of
life in patients with CKD. They suffer from fatigue,
reduced exercise tolerance, slowing of cognitive function, and reduced libido. In patients with ESRF, there
is also evidence that anemia contributes to increased
morbidity and mortality. This is thought to be secondary to increased left ventricular hypertrophy.
Correction of anemia is therefore a priority in the
management of CKD patients. Iron stores must
be assessed first. If they are deficient, they can be
restored through the use of oral or intravenous iron.
Once patients are iron replete, recombinant EPO can
be administered.
There has been much debate over what hemoglobin target to aim for. Until recently, it was mistakenly thought that a normal hemoglobin target would
improve clinical outcomes. However, the publication
of two trialsCREATE (cardiovascular risk reduction
by early anemia treatment with epoetin b) (37) and
CHOIR (correction of hemoglobin and outcomes in
renal insufficiency) (38)has exploded this myth. In
these trials, patients with a GFR of between 15 and
50 mL/min were found to derive no benefit from
achieving a higher (normal) hemoglobin target.
Indeed, the CHOIR study found that patients with
CKD-related anemia were at higher risk of death from
cardiovascular events when the target hemoglobin
level was 13.5 g/dL rather than 11.3 g/dL.
Consequently, current guidelines state that all
patients with CKD should aim to achieve target
hemoglobin between 10.5 g/dL and 12.5 g/dL.
Renal Bone Disease

Renal bone disease is a complex entity that starts to
develop early in the course of progressive renal disease. It is characterized by a spectrum of metabolic
bone disorders, all of which have the effect of reducing bone strength and hence predisposing to fractures.
The main pathologies are as follows:
l
Osteitis fibrosa cystica (high turnover bone disease): This occurs because of secondary hyperparathyroidism (SHPT) with increased osteoblast
and osteoclast activity, leading to rapid bone
formation and resorption. The outcome is weakened bone structure.
Adynamic bone disease (low turnover bone disease): This is characterized by reduced bone
formation and resorption. Parathyroid hormone
(PTH) levels are suppressed in this case, either
as a consequence of excessive use of vitamin D
analogues, or because the patient has undergone
parathyroidectomy.
Osteomalacia: This normally arises as a result of
low 1,25-dihydroxycholecalciferol levels, as well
as metabolic acidosis. Both lead to impaired bone
mineralization. Osteomalacia commonly coexists
with adynamic bone disease.
Mixed renal osteodystrophy: This has features of

osteitis fibrosa, adynamic bone disease, and osteomalacia.
The interplay between a number of factors is

involved in the evolution of renal osteodystrophy. The
two main mechanisms are SHPT and 1,25-dihydroxycholecalciferol deficiency.
Secondary Hyperparathyroidism
SHPT is driven by three processes. Hyperphosphatemia arises in renal failure because of the reduction in
filtered phosphate load that occurs once GFR falls
below 40 mL/min. It promotes SHPT through a direct
effect on the parathyroid glands, as well as by precipitating a fall in serum calcium levels.
Loss of functioning renal mass leads to reduced
1a-hydroxylase activity. This enzyme is produced by
renal tubular cells and is required for the production
of active vitamin D (1,25-dihydroxycholecalciferol).
CKD thus results in low 1,25-dihydroxycholecalciferol
levels, which then stimulate release of PTH.
Both the mechanisms mentioned above cause
hypocalcemia. This is detected by the calcium-sensing
receptor on the parathyroid glands and acts as the
strongest driver for PTH release.
The effects of elevated PTH are diverse, with an
overall aim of attempting to maintain calcium-phosphate homeostasis. PTH increases urinary excretion of
phosphate as well as stimulating 1a-hydroxylase in an
effort to increase active vitamin D levels. It also tries to
correct hypocalcemia through increased resorption of
calcium from bone. This effect is responsible for
causing high turnover bone disease.
Prolonged hyperparathyroidism can have other
detrimental consequences. These include left ventricular hypertrophy and cardiac fibrosis, as well as EPOresistant anemia.
1,25-Dihydroxycholecalciferol Deficiency
Active vitamin D plays a number of roles in bone
metabolism. Low levels prevent mineralization of
bone and hence predispose to osteomalacia, which is
characterized by bone fragility and proximal muscle
weakness.
It is well recognized that the abnormal mineral
metabolism that occurs in CKD can also have extraskeletal effects; disturbances of calcium and phosphate homeostasis lead to their deposition in soft
tissues. Blood vessels are a prime target for the ensuing calcification and this leads to arterial stiffening as
well as accelerated atherosclerosis. Calcification of
heart valves (usually the mitral valve annulus or
aortic valve leaflets) plays a role in the development
of left ventricular dysfunction. Both of these factors
contribute to cardiovascular morbidity and mortality
in patients with CKD.
Treatment of renal bone disease requires a multipronged approach. Control of hyperphosphatemia is
the primary objective. There is evidence showing that
hyperphosphatemia is an independent risk factor for
all-cause and cardiovascular mortality in hemodialysis patients (39). This is in part because it promotes
vascular calcification. Current guidelines suggest
phosphate targets of 0.9 to 1.5 mmol/L in stages 3
and 4 CKD, and 1.1 to 1.8 mmol/L in stage 5 CKD.
Achieving this requires a combination of dietary
phosphate restriction, as well as the use of oral phosphate binders, which are taken with meals.
There are a number of phosphate binders currently available, which fall into two categories: calcium-based binders (calcium acetate and calcium
carbonate) and non-calcium-based binders (sevelamer
hydrochloride and lanthanum carbonate). Use of the
calcium-based preparations is limited by the fact that
patients with CKD should not take in more than 2 g of
elemental calcium per day, as this may again promote
vascular calcification.
Correction of SHPT is the other key target in the
treatment of renal osteodystrophy. A certain degree of
SHPT is desirable to maintain bone health, so a careful
balancing act needs to be achieved. For patients with
ESRF, PTH levels should ideally be maintained
between 150 and 300 pg/mL. This is based on the
observation that these levels are associated with normal bone turnover as monitored by bone histology.
Therapy for SHPT involves the use of active
forms of vitamin D. These can cause hypercalcemia,
in which case second-line agents such as vitamin D
analogues (e.g., paracalcitol) or calcimimetic agents
(cinacalcet) should be prescribed. In cases of refractory hyperparathyroidism, surgical removal of the parathyroid glands may be needed.
Cardiovascular Complications of CKD

The commonest cause of death in patients with CKD
is cardiovascular disease. Individuals at each stage of
CKD are more likely to die from a cardiovascular
event than progress to ESRF. The outlook is worst
for those patients who require dialysis; they have an
annual mortality rate of 10% to 15% and cardiovascular disease accounts for 55% of all deaths in ESRF (40).
The explanation given for this is that they have
accelerated atherogenesis. It is certain that these
patients have a preponderance of the so-called classical
risk factors for atherosclerosis including hypertension,
dyslipidemia, glucose intolerance, and hyperinsulinemia. As described above these factors also promote
accelerated loss of GFR. Hence, close attention needs to
be paid to addressing these problems.
Controversy remains as to how effective statins
are in reducing cardiovascular risk in CKD. No randomized controlled studies thus far have shown them
to be of benefit in dialysis patients.
This is probably attributable to a number of
pathogenetic factors, which are not modifiable by
statins. The uremic milieu promotes a state of inflammation and oxidative stress that encourages the formation of atherosclerotic plaques. Furthermore,
hyperhomocysteinemia is a common finding in ESRF,
and this is also believed to accelerate atherogenesis.
It is not only atherosclerosis that causes cardiovascular mortality. Left ventricular hypertrophy and
219
dysfunction, which predispose to sudden cardiac

death are highly prevalent in CKD. These arise as a
consequence of hypertension, volume overload, anemia, and loss of arterial elasticity because of vascular
calcification.
The combination of all these factors creates a
significant challenge for physicians who care for the
CKD population.
RENAL REPLACEMENT THERAPY

Commencement of RRT should be considered once
patients have an eGFR of less than 15 mL/min. This is
not a precise cut-off and the decision regarding initiation of dialysis has to take into account the patients
overall well-being. Some individuals do not feel
unwell despite this low level of renal function, and
are reluctant to start a treatment that will involve
major disruption to their lives. Conversely, if symptoms of uremia develop, RRT should not be delayed.
There are three modalities of RRT available: renal
transplantation, hemodialysis, and peritoneal dialysis.
For those patients fit enough to withstand the operation and subsequent immunosuppression, the transplant option is best. It provides better quality of life as
well as increased longevity compared with dialysis.
The majority of patients who commence RRT
will start out on dialysis though. Ideally, these
patients should have been under the care of a nephrologist well in advance so that they can be prepared
physically (in terms of dialysis access and vaccination
against hepatitis B) as well as psychologically for the
rigors of dialysis.
Not all patients are suitable for dialysis; the frail
elderly patient with multiple comorbidities may not
tolerate this treatment. In cases such as this, good
conservative care aimed at managing the complications and relieving the symptoms of ESRF is likely to
be a kinder option.
REFERENCES
1. National Kidney Foundation. Kidney disease outcome
quality initiative. Am J Kidney Dis 2002; 39(suppl 1):
S1S266.
2. Coresh J, Astor BC, Greene T, et al. Prevalence of chronic
kidney disease and decreased kidney function in the adult
US population: Third National Health and Nutrition
Examination Survey. Am J Kidney Dis 2003; 41:112.
3. The UK Renal Registry. The Tenth Annual Report. December
2007.
4. US Renal Data System: USRDS Annual Data Report. 2007.
5. ANZDATA Registry. The 30th Annual Report. 2007.
6. Locatelli F, Del Vecchio L. Natural history and factors
affecting the progression of chronic renal failure. In:
El Nahas AM, Anderson S, Harris KPG, eds. Mechanisms
and Management of Progressive Renal Failure. London:
Oxford University Press, 2000:2079.
7. Naicker S. End-stage renal diseases in sub-Saharan and
South Africa. Kidney Int 2003; 63:S119S122.
8. Bhandari S, Turney JH. Survivors of acute renal failure
who do not recover renal function. Q J Med 1996; 89:
415421.
220
Ojha and Bradley
9. Joint Specialty Committee on Renal Medicine of the Royal

College of Physicians and the Renal Association, and the
Royal College of General Practitioners. Chronic Kidney
Disease in Adults: UK Guidelines for Identification, Management and Referral. London: Royal College of Physicians, 2006.
10. Hostetter TH, Olson JH, Rennke HG, et al. Hyperfiltration
in remnant nephrons: a potentially adverse response to
renal ablation. Am J Physiol 1981; 241:F85F93.
11. Hong SI, Couser WG. Chronic progression of tubulointerstitial damage in proteinuric renal disease is mediated by
complement activation: a therapeutic role for complement
inhibitors? J Am Soc Nephrol 2003; 14(7 suppl 2):S186S191.
12. Nangaku M. Chronic hypoxia and tubulointerstitial injury:
a final common pathway to end-stage renal failure. J Am
Soc Nephrol 2006; 17:1725.
13. Klahr S, Levey AS, Beck GJ, et al. The effects of dietary
protein restriction and blood-pressure control on the progression of chronic renal disease. N Engl J Med 1994; 330:
877884.
14. Jafar TH, Stark PC, Schmid CH, et al. Progression of
chronic kidney disease: the role of blood pressure control,
proteinuria, and angiotensin-converting enzyme inhibition: a patient-level meta-analysis. Ann Intern Med 2003;
139:244252.
15. Li PK, Weening JJ, Dirks JA, et al. A report with consensus
statements of the International Society of Nephrology 2004
consensus workshop on prevention of progression of renal
disease, Hong Kong, June 29, 2004. Kidney Int Suppl 2005;
94:S2S7.
16. Locatelli F, Marcelli D, Comelli M, et al. Proteinuria and
blood pressure as causal components of progression to
end-stage renal failure. Nephrol Dial Transplant 1996;
11:461467.
17. Lewis EJ, Hunsicker LG, Bain RP, et al.; For the Collaborative Study Group. The effect of angiotensin-converting
enzyme inhibition on diabetic nephropathy. N Engl J
Med 1993; 329:14561462.
18. The GISEN group. Randomized placebo-controlled trial of
effect of ramipril on decline in glomerular filtration rate
and risk of terminal renal failure in proteinuric non-diabetic nephropathy. Lancet 1997; 349:18571863.
19. Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective
effect of the angiotensin-receptor antagonist irbesartan in
patients with nephropathy due to type 2 diabetes. N Engl J
Med 2001; 345:851860.
20. Brenner BM, Cooper ME, de Zeeuw D, et al. For the
RENAAL study investigators. Effects of losartan on renal
and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 2001; 345:861869.
21. Parving HH, Lehnert H, Brochner-Mortensen J, et al. The
effect of irbesartan on the development of diabetic
nephropathy in patients with type 2 diabetes. N Engl J
Med 2001; 345:870878.
22. Nakao N, Yoshimura A, Morita H, et al. Combination
treatment of angiotensin-II receptor blocker and angiotensin-converting-enzyme inhibitor in non-diabetic renal disease (COOPERATE): a randomized controlled trial. Lancet
2003; 361:117124.
23. Morgensen CE, Neldam S, Tikkanen I, et al. Randomised
controlled trial of dual blockade of reninangiotensin system in patients with hypertension, microalbuminuria and
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
non-insulin dependent diabetes: the candesartan and lisinopril microalbuminuria (CALM) study. BMJ 2000; 321:
14401444.
Pedrini MT, Levey AS, Lau J, et al. The effect of dietary
protein restriction on the progression of diabetic and
nondiabetic renal diseases: a meta-analysis. Ann Intern
Med 1996; 124(7):627632.
The effect of intensive treatment of diabetes on the development and progression of long-term complications in
insulin-dependent diabetes mellitus. The Diabetes Control
and Complications Trial Research Group. N Engl J Med
1993; 329:977986.
Intensive blood-glucose control with sulphonylureas or
insulin compared with conventional treatment and risk of
complications in patients with type 2 diabetes (UKPDS 33).
UK Prospective Diabetes Study (UKPDS) Group. Lancet
1998; 352:837853.
Praga M, Hernandez E, Herrero JC, et al. Influence of
obesity on the appearance of proteinuria and renal insufficiency after unilateral nephrectomy. Kidney Int 2000;
58:21112118.
Hall JE. Mechanisms of obesity-associated cardiovascular
and renal disease. Am J Med Sci 2002; 324:127137.
Gonzalez E, Gutierrez E, Morales E, et al. Factors influencing the progression of renal damage in patients with
unilateral renal agenesis and remnant kidney. Kidney Int
2005; 68(1):263270.
Orth SR, Stockmann A, Conradt C, et al. Smoking as a risk
factor for end-stage renal failure in men with primary renal
disease. Kidney Int 1998; 54:926931.
Schiffl H, Lang SM, Fischer R. Stopping smoking slows
accelerated progression of renal failure in primary renal
disease. J Nephrol 2002; 15:270274.
Jaimes EA, Tian RX, Raij L. Nicotine: the link between
cigarette smoking and the progression of renal injury? Am
J Physiol Heart Circ Physiol 2007; 292:7682.
Moorhead JF, Chan MK, El-Nahas M, Varghese Z. Lipid
nephrotoxicity in chronic progressive glomerular and
tubulo-interstitial disease. Lancet 1982; 2(8311):13091311.
Fried LF, Orchard TJ, Kasiske BL. Effect of lipid reduction
on the progression of renal disease: a meta-analysis. Kidney Int 2001; 59:260269.
Johnson WJ, Hagge WW, Wagoner RD, et al. Effects of urea
loading in patients with far-advanced renal failure. Mayo
Clin Proc 1972; 47:2129.
Vanholder R, De Smet R, Glorieux G, et al. Review on
uremic toxins: classification, concentration, and interindividual variability. Kidney Int 2003; 63:19341943.
Drueke TB, Locatelli F, Clyne N, et al. Normalization of
hemoglobin level in patients with chronic kidney disease
and anaemia. N Eng J Med 2006; 355:20712084.
Singh AK, Szczech L, Tang KL, et al. Correction of anemia
with epoetin alfa in chronic kidney disease. N Eng J Med
2006; 355:20852098.
Block GA, Hulbert-Shearon TE, Levi NW, et al. Association
of serum phosphorus and calcium
phosphate product
with mortality risk in chronic haemodialysis patients: a
national study. Am J Kidney Dis 1998; 31:607617.
Baber U, Toto RD, de Lemos JA. Statins and cardiovascular
risk reduction in patients with chronic kidney disease and
end-stage renal failure. Am Heart J 2007; 153:471477.
14
Structure and Function of the Lower Urinary Tract
Anthony R. Mundy
Institute of Urology and Nephrology, University College Hospital, London, U.K.
INTRODUCTION
Before starting, the reader should be aware of certain
problems in discussing the subject.
First of all, a great deal is known (relatively
speaking) about the structure of the bladder, urethra
and pelvic floor, but as one works back proximally
through the innervation of the lower urinary tract to
the spinal cord and up to the brain, and as one turns
more from structure to function, so knowledge of the
subject becomes exponentially less.
Secondly, much of the published research on the
lower urinary tract has been done on animals other
than humans and there are considerable species differences that make interpretation of such work very
difficult.
Thirdly, in the same vein, many of the experimental studies have been done after neuronal ablation
or otherwise in circumstances that are very far from
physiological. Extrapolation from ablative pathophysiology in experimental animals to normal physiology
in humans is also problematic.
Fourthly, many experimental studies have been
based on the identification of receptors for neurotransmitters, or on the demonstration by radioimmunoassay of the presence of neurotransmitters
themselves or have attempted to infer the presence
of a physiologically significant mechanism from the
presence of one component of a presumed reflex
arc. Two examples will show the fallacy of such an
extrapolation. Firstly, one of the most significant medical advances in recent years has been in the development of b-adrenergic receptor-active drugs for the
treatment of bronchospasm, but there is no significant
b sympathetic innervation to the human lung. Receptors are present that may be therapeutically manipulated, but they have no apparent physiological
significance. Secondly, and more obviously, receptors can be identified in human platelets but no one
imagines that platelets have an innervation.
Finally, it should be appreciated that just
because a reflex mechanism exists does not mean
that that mechanism is active, let alone important in
normal circumstances. Thus, a reflex may be present
or elicitable or evident in disease, but it does not
necessarily mean that it is active or important in
health.
These various points should be borne in mind

when reading about studies of the structure and
function of the lower urinary tract. Equally, it is
hoped that the reader will forgive the author for the
speculation that will creep in to provide a reasonably
smooth narrative when substance is lacking.
The discussion begins distally in the bladder and
urethra and then moves proximally, considering
structure first and function second but integrating
both as far as possible.
THE STRUCTURE OF THE BLADDER

AND URETHRA
The bladder and urethra have three layers to which is
added, in the male, the additional component of the
prostate. The prostate is considered in detail in chapter 19 and is not considered further here. These three
layers are, from the outside in, the adventitial layer,
the muscular layer and the epithelial layer.
The Adventitial Layer

The bladder and proximal urethra lie in the pelvis,
covered over the dome of the bladder with peritoneum. Deep to the peritoneum the bladder is held in
place by an adventitial layer consisting of two fascial
layersthe first on the anterior and lateral aspect of
the bladder and the second on the dome and posterior
aspect of the bladderthe two fusing together
anteriorly and laterally to form what Uhlenhuth (1)
described as the superior hypogastric wing (Fig. 1),
which runs laterally to the external iliac vessels and
anteriorly onto the anterior abdominal wall, ensheathing the medial umbilical ligaments and the urachus.
The same two layers form the anterior layer of the
sheath around the ureters and the superior and inferior vesical vessels posterolaterally in what Uhlenhuth
described as the inferior hypogastric wing (Fig. 2).
Posteriorly, the sheet of fascia that covers the dome
and posterolateral aspect of the bladder sweeps back
onto the posterolateral pelvic side wall and to
ensheath the rectum as Uhlenhuths presacral fascia
(Fig. 3). These fascial layers and the neurovascular
structures that they ensheath hold the bladder in
place, as does the urethra, and the prostate in males,
222
Mundy
Figure 1 Diagram of the distribution of pelvic

fascia to show the superior hypogastric wing.
with which the bladder is continuous. The urethra and

prostate have their attachments too, notably the endopelvic fascia, which tethers them to the pelvic side
wall and the periurethral component of the levator ani
muscle in males (it is vestigial in females) and the
anterior vaginal wall in females, to which the female
urethra is intimately related (Fig. 4).
There are other supporting structures that are
thought to be important in lower urinary tract function, particularly in the female.
First and foremost, in the female, are the
pubourethral ligaments, which sling the full length
of the urethra but particularly the proximal urethra
from the inferior pubic area (Fig. 5). These have been
particularly studied by Zacharin (5), and his findings
have since been confirmed by others, and all of these
authors have sought somehow to link a deficiency in
these ligaments to the genesis of stress incontinence.
The homologous structures in the male are the
puboprostatic ligaments, which have become of particular interest since the development by Walsh (6) of
his technique of radical retropubic prostatectomy.
One supporting structure identified in the anatomical and more traditional urological literature, the
existence of which is now disputed, is the so-called
urogenital diaphragm. Described as the layer on
which the prostate sits, (7) it has proved elusive to
others (8) and probably does not exist as a separate
entity.
THE MUSCULAR LAYER

The smooth muscle of the bladder, which accounts for
the most component of the bladder wall, is called the
detrusor. There have been various attempts to identify
specific bundles of muscle within the bladder wall, (9)
largely to substantiate a hypothetical mechanism to

account for the opening of the bladder neck at the
initiation of voiding, but there is no good evidence
that such layering exists (10). The bladder should be
considered as a single homogeneous layer of relatively
large muscle bundles in a relatively small amount of
connective tissue. These muscle bundles have no
particular orientation (Fig. 6). As one approaches the
bladder base from above down, there is the triangular
region of the trigone between the two ureters and the
internal urinary meatus, where there is a flimsy additional superficial layer of smooth muscle quite separate from the underlying smooth muscle, which is
typical detrusor. This additional trigonal layer is
derived from the ureters and shows several distinct
characteristics. The muscle fibers form bundles that
are much smaller with a higher connective tissue
component and a fairly dense adrenergic innervation
(11) (Fig. 7). The trigonal muscle was once thought
also to have a role in opening the bladder neck, but
this too has not been substantiated. As one approaches
closer to the bladder neck, the disposition and orientation of the detrusor change uniformly around it. The
muscle bundles get smaller, there is a relatively greater amount of connective tissue, and the orientation of
the muscle bundles becomes more uniform, to loop
obliquely around the bladder neck (Fig. 8).
Below the bladder neck in both sexes, the muscle
bundles continue to be relatively small in size and
interspersed with a relatively larger connective tissue
component and they continue to show an oblique
looping arrangement around the urethra. The vascular
component of the wall increases by comparison with
the bladder, particularly in females (12). In the male,
the prostate forms an additional component at this
level, and at the upper part, where the prostate and
Chapter 14: Structure and Function of the Lower Urinary Tract
223
Figure 2 (A) Diagram of the pelvic fascia to

show the inferior hypogastric wing. (B) Crosssection of the inferior hypogastric wing to show
its structure, origin, disposition and contents.
the bladder neck merge, there is the preprostatic

sphincter in which adrenergically innervated smooth
muscle bundles form a distinct sphincter around the
urethra to prevent retrograde ejaculation (Fig. 9) (11).
It should be emphasized that this adrenergically innervated smooth muscle componentthe preprostatic
sphincterwhich is present in males but not in

females, is a genital sphincter and not the bladder
neck urinary sphincter mechanism in the strict sense,
which is presumably the same in both sexes.
The preprostatic sphincter and the trigone are the
only areas to show a distinct adrenergic innervation;
224
Mundy
Figure 3 Diagram of the pelvic fascia to show

the distribution of the presacral fascia. Source:
From Ref. 2.
otherwise the only adrenergic neurons in the bladder

are those that supply the blood vessels (11). This is not
to say that there are not adrenergic receptors present;
indeed, there are a receptors in the bladder base and
b receptors elsewhere in the bladder dome, (13) but
there is no evidence that there is a functionally important innervation to these receptors in the normal
individual. Further down the urethra at the apex of
the prostate in males and in the mid-urethra in
females, there is the urethral sphincter mechanism,
sometimes called the distal sphincter mechanism to
distinguish it from the proximal sphincter mechanism,
which is the other name for the bladder neck. Whereas
there is a readily identifiable sphincter mechanism in
the sphincter-active area of the urethra in both
sexes, there is no anatomically identifiable sphincter
mechanism at the bladder neck in either sex (using the
term bladder neck in its strictest senseas distinct
from the preprostatic sphincter). The urethral sphincter mechanism has three components (Fig. 10). The
innermost is the urethral smooth muscle and the
middle layer is the striated muscle component within
the urethral wall. These two components within the
urethral wall itself are separated from the third component, which is the periurethral component of the
levator ani, or pubouretheral sling, which is slung
around the urethra in much the same way as the
puborectal sling is disposed round the rectum (4).
These two sling components, the pubourethral and
the puborectal slings, are distinct from and below the
diaphragmatic layer of the levator ani (Fig. 11) (14).
They both form substantial components of their
respective sphincter mechanisms and they share several common features.
THE URETHRAL SPHINCTER MECHANISM

Of the three layers of the urethral sphincter mechanism, two are relatively quickly dealt with. The outermost layerthe pubourethral slingis the levator ani
component most closely related to the urethra; (4) it is
typical striated muscle; it has typical striated muscle
innervation; it is relatively insignificant in the female
compared with the male because of the presence of the
vagina; and it is activated along with the rest of the
levator ani under stress conditions, acting specifically on the urethra at such times to augment urethral
occlusion pressure.
The urethral smooth muscle is dealt with relatively quickly for an entirely different reason: we know
very little about its function. The innervation is more
complicated than the detrusor with both excitatory
and inhibitory innervation. Small ganglia, smaller
than in and around the bladder, are present, and
both acetylcholine and noradrenaline (norepinephrine) mediate contraction. The sympathetic innervation involves a distinct subtype of the a1 adrenoceptor
in the male, particularly in the prostatic urethra, which
has particular implications for the treatment of bladder outflow obstruction due to benign prostatic hyperplasia as described in chapter 19.
When the striated muscle of the urethral sphincter
mechanism is paralyzed, the urethral smooth muscle
225
Figure 5 The orientation of the pubourethral ligaments. Abbreviations: AAW, anterior abdominal wall; PB, pubic bone; BN,
bladder neck; BW, bladder wall; AVW, anterior vaginal wall; ISM,
inner smooth muscle; OSM, outer striated muscle; IUM, internal
urinary meatus; EUM, external urinary meatus; SPUL, superior
pubourethral ligament; SPPUL, subpubic pubourethral ligament;
IPML, inferior pubomeatal ligament. Source: From Ref. 4.
Figure 4 (A) The relationship of the urethra to the anterior

vaginal wall. (B) Cross-section to show the integration of the
urethra, below the bladder neck within the anterior vaginal wall.
Source: From Refs. 2 and 3.
continues to produce a high-pressure zone, which suggests that it is tonically active (14)whereas the bladder
smooth muscle is phasically activeand recent experimental studies suggest that this tonic smooth muscle
contraction may be relaxed by nitric oxide (1517).
Indeed, there are both ganglia and nerves innervating
the urethral smooth muscle, which contain nitric oxide
synthase. The current opinion is that this nitric oxiderelated relaxant mechanism in this area of the urethra
and bladder neck is responsible for the opening of
the bladder neck and the relaxation of the urethra
that occurs when the bladder contracts at the onset of
voiding (17).
It appears that it is the striated muscle component within the urethral wall that is the most important component for continence. It is sometimes called
the intrinsic rhabdosphincter (11). This striated muscle
is unusual in a number of ways (11). It is orientated
predominantly anteriorly in both the vertical and
horizontal planes and is relatively deficient posteriorly, giving it, overall, a signet ring distribution (Fig. 12).
This is clearly seen microscopically in both sexes
(Fig. 13). The reason for this is not clear, but it is
common experience that the easiest way of stopping
Figure 6 Low-power microscopy of the detrusor stained with

Massons trichrome to show the orientation of the smooth muscle
bundles. Source: From Ref. 3.
water flowing through a hosepipe is to kink it rather

than to squeeze it circumferentially. It may be that the
distribution of the fibers of the intrinsic striated muscle of the urethra produces a kinking rather than a
circumferential compression when it contracts, for this
sort of reason, but this is entirely conjectural. This
intrinsic striated muscle sphincter is composed of
relatively very small muscle fibers when compared
with typical striated muscle; the fibers themselves are
disposed in small muscle bundles within a very much
greater connective tissue component than is seen in
typical striated muscle (Fig. 14); the muscle fibers
themselves are stuffed with mitochondria; there are
226
Mundy
Figure 7 (A) Low-power microscopy of the detrusor stained

with Massons trichrome to show the distribution of the smooth
muscle bundles in relation to the trigone superficially, compared
with the detrusor proper, more deeply. (B) Immunofluorescent
microscopy study to show the presence of adrenergic nerves in
the trigone. Source: From Ref. 3.
no muscle spindles; and there are several distinct

histochemical staining characteristics of these muscle
fibers, most notably a uniform staining with acidstable myosin adenosine triphosphatase (ATPase)
(Fig. 15) (18). By contrast, the typical striated muscle
of the pubourethral sling that is immediately adjacent
to the intrinsic rhabdosphincter shows larger muscle
fibers in larger muscle bundles, with little connective
tissue, fewer mitochondria, muscle spindles present
and a mixed pattern of staining for acid-stable myosin
ATPase (Fig. 16). Furthermore, whereas the nerve
supply to the levator ani originates in typical cell
bodies in the motor cell nuclei of the anterior horn
of the sacral spinal cord, the cell bodies of the fibers
that innervate the intrinsic rhabdosphincter appear to
arise in a nucleus more medially situated in the sacral
anterior horn, as do the fibers innervating the anal
sphincter mechanism, and this spinal cord nucleus is
known as spinal nucleus X, sometimes called Onufs
nucleus (Fig. 17) (19). The fibers from Onufs nucleus
to the intrinsic rhabdosphincter run out of the anterior
Figure 8 (A) Low-power microscopy of the detrusor stained

with Massons trichrome to show the distribution of the smooth
muscle bundles at the bladder neck. (B) Immunofluorescent
microscopy to show the presence of adrenergic nerve fibers in
the bladder neck. Source: From Ref. 3.
primary rami and the nerve fibers run initially with the
nervi erigentes, which are the preganglionic parasympathetic neurons. They run with these fibers through
the pelvic plexuses and down with the postganglionic
parasympathetic neurons into the urethra (8).
Whether this is the sole innervation of the intrinsic rhabdosphincter or whether there is a separate
component arising from the pudendal nerve is not
clear, (20) but after pudendal neurectomy or pudendal
nerve blockade, the urethral sphincter mechanism is
intact so there is clearly a source other than the
pudendal nerve.
These characteristics of the intrinsic rhabdosphincter are very unusual but not unique; the intrinsic
laryngeal muscles are very similar in structure and in
the nature of their innervation.
The Epithelial Layer

The urothelial layer of the bladder is a multilayered
transitional epithelium that is continuous with the
ureters above and the prostatic urethra below.
Under the light microscope, the urothelium is similar
227
Figure 11 Illustration of the various components of levator ani

showing separate sling and diaphragmatic components. Abbreviations: A, pubourethral sling; B, puborectal sling; C, levator
diaphragm.
Figure 9 A diagram to show the preprostatic sphincter. Abbreviations: CZ, central zone; PZ, peripheral zone; S, preprostatic
sphincter; V, verumontanum.
Figure 12 Low-power microscopy of a transverse section of the

male sphincter-active urethra to show the signet ring distribution
of the intrinsic rhabdosphincter (anterior to the right of the figure).
Figure 10 Diagrammatic representation of the components of

the urethral sphincter mechanism. Abbreviations: D, detrusor;
T, trigone; PSM-BN, proximal sphincter mechanism bladder neck;
DSM, distal sphincter mechanism; USM, urethral smooth muscle;
IR, intrinsic rhabdosphincter; PUM, periurethral musculature.
in the bladder and the prostatic urethra, but there are

differences on scanning electron microscopy that
become more marked the further one proceeds
down the urethra toward the bulbar segment where
it changes to a columnar epithelium and changes
again toward the meatus to a squamous epithelium.
The urothelium has numerous so-called tight junctions (chap. 1), which make it impermeable to fluids
and solutes. This relative impermeability should not
Figure 13 Low-power microscopy of a transverse section of the

female sphincter-active urethra to show the signet ring distribution of the intrinsic rhabdosphincter (anterior to the top of the
figure).
228
Mundy
Figure 17 Diagram to show the separate origin of the innervation of the intrinsic rhabdosphincter from Onufs nucleus as
distinct from the site of origin of typical striated muscle from
anterior horn cells. Abbreviations: A, a motor neuron group;
B, Onufs nucleus.
Figure 14 Low-power microscopy of the intrinsic rhabdosphincter to show the relative distribution of muscle bundles and
connective tissue. Source: From Ref. 3.
Figure 15 Low-power histochemistry of the intrinsic rhabdosphincter to show the distribution of acid-stable myosin ATPase.
be taken to mean that the urothelium is inert. Indeed,

various drugs can be absorbed from the bladder and
instilled intravesically. In addition, the urothelium has
a sensory role. Various sensory nerves have been
recognized in the epithelium and subepithelium,
which have been presumed to act as touch receptors
and chemoreceptors. There are also cells in the epithelium sensitive to hydrostatic pressure, which appear
to have an important role in continence and voiding.
Deep to the basal lamina of the epithelial layer,
in the lamina propria, is a thin layer of muscle cells,
formerly called interstitial cells and now more commonly known as myofibroblasts (21). Although only a
thin layer, these cells are closely connected to each
other by numerous gap junctions facilitating electrical
coupling (22). They are also closely connected to the
overlying epithelium by numerous fine nerves, some
of which are related to the cells, receptors and nerves
mentioned in the last paragraph. Together these three
componentsepithelium, myofibroblasts and the
interconnecting network of nervesare thought to
act as a sort of unitary stretch receptor responsible
for sensing bladder filling (23,24). This is discussed
further below.
THE INNERVATION OF THE BLADDER

AND URETHRA
Figure 16 Low-power histochemistry of the typical striated

muscle, in this case the pubourethral sling, to show the distribution of acid-stable myosin ATPase. Source: From Ref. 3.
Four separate neuronal pathways to the lower urinary

tract have been alluded to (Fig. 18). The principal one
is derived from cell bodies in the intermediolateral
column of the second, third and fourth sacral segments of the spinal cord. These are preganglionic
parasympathetic fibers that run out of the anterior
primary rami of S2, S3, and S4 and then separate out
from the somatic component, which runs to the sacral
plexus, to run as the nervi erigentes to the pelvic
plexuses. These preganglionic parasympathetic neurons end by synapsing in ganglia on the cell bodies of
the postganglionic parasympathetic nerves, which
then run to the bladder and to the urethra (and
more proximally to the rectum and the genital structures). In humans, 50% of the ganglia of the pelvic
plexus are in the adventitial tissue around the base
and posterolateral aspects of the bladder and 50% are
229
Figure 18 Diagram to show the four components of innervation

of the lower urinary tract. , sympathetic; , parasympathetic;
, somatic innervation of intrinsic rhabdosphincter; , pudendal nerve. Source: From Ref. 3.
within the bladder wall itself (11). For this reason it is,
strictly speaking, technically impossible to denervate
the bladder because the 50% within the bladder wall
itself will still remain and there will therefore still be
reflex activity, even if this is not physiologically
significant. A more semantically correct term would
therefore be decentralization rather than denervation when discussing the stripping of the nerves
from around the outside of the bladder.
The somatic nerve fibers that arise from Onufs
nucleus and that travel with the otherwise autonomic
parasympathetic nerve fibers of the nervi erigentes
and pass ultimately to the intrinsic rhabdosphincter
have already been mentioned.
The third component is the sympathetic nerve
component that arises from the intermediatolateral
column of the 10th, 11th and 12th thoracic and the
1st and 2nd lumbar segments of the spinal cord. These
preganglionic sympathetic fibers and their postganglionic sympathetic derivatives travel as the hypogastric
nerves, which innervate the trigone, the blood vessels
of the bladder and the smooth muscle of the prostate
in males, including the preprostatic sphincter. They
also have postganglionic branches that end in the
parasympathetic ganglia, (11) where they exert an
inhibitory effect that is described in detail below.
Finally, there is the pudendal nerve component,
also arising from S2, S3, and S4, in this instance from
typical anterior horn motor neuron cell bodies, which
innervates the urethra and the pelvic floor musculature and provides afferent innervation to the urethra.
Figure 19 (A) Cadaveric dissection of the right half of the pelvis

to show the pelvic plexus and its origins and distribution on that
side, and its anatomical relationships. (B) Diagram of the dissection identifying the structures demonstrated. Source: From Ref. 25.
By comparison with typical anatomical nerves

such as the obturator nerve, the femoral nerve and
even the sciatic nerve, the total mass of the autonomic
nerves in the pelvis is large. The autonomic nerves are
not as discrete and easily identifiable as the somatic
nerves referred to, disposed as they are as a sheet
within the fascial layers that invest the rectum, the
genital structures and the lower urinary tract, but if
the nerve fibers are dissected out and considered as a
whole, their volume is considerable (Fig. 19). It is this
sheer mass that protects pelvic autonomic function
from extensive damage during pelvic surgery such as
hysterectomy or rectal resection, but they are nonetheless vulnerable to traction injuries during such procedures (26), as of course they are in females during
childbirth.
FUNCTIONAL ASPECTS OF THE INNERVATION

OF THE BLADDER AND URETHRA
Cholinergic nerves cannot be stained directly for
microscopical studyonly indirectly by staining for
acetylcholinesterase, the enzyme that breaks down
acetylcholine at the neuromuscular junction. Staining
for acetylcholinesterase shows a dense presumptive
cholinergic innervation of the detrusor smooth
230
Mundy
Figure 22 Diagram of a nerve ending and neuromuscular

junction to show the release of fast neurotransmitters into the
neuromuscular junction. Source: From Ref. 29.
Figure 20 Low-power microscopy of the muscle wall after
staining with acetylcholinesterase to show the distribution of
presumptive cholinergic nerves in the bladder wall. Source:
From Ref. 3.
muscle (Fig. 20) with a nerve to muscle cell ratio of

about 1:1, and a less dense cholinergic innervation of
the urethral smooth muscle (11). Under the electron
microscope, the terminal branches of the parasympathetic neurons within the bladder wall are seen to
contain varicosities (Fig. 21). These varicosities are
there because of the presence of numerous vesicles
within the terminal neuron at that point and these
vesicles contain neurotransmitter substances. Alongside the varicosity is the specialized area of the
smooth muscle cell membrane that constitutes the
receptor site, although this is not very specialized by
comparison with the receptor sitethe neuromuscular junctionin striated muscle. Adjacent smooth
muscle cells are connected by so-called regions of
close approach, like gap junctions in effect, which
could, at least theoretically, allow electrotonic spread
of activity from one smooth muscle cell to the next and
Figure 21 Electron microscopy of a terminal neuron within the

detrusor smooth muscle layer to show a varicosity containing small
clear vesicles containing acetylcholine. Source: From Ref. 27.
which thereby confer so-called cable properties on

the smooth muscle fibers. This would supplement the
spreading neuronal stimulus as a sort of parallel
pathway, and help to ensure a uniform simultaneous
contraction of all the bladder smooth muscle cells at
the time of voiding.
Vesicles are of two main types (28): small clear
vesicles and larger vesicles with a dense core. The
small clear vesicles are thought to contain so-called
fast neurotransmitter substances, which are
released directly into the area between the neuron
and the adjacent neuron across a synapse, or the
adjacent smooth muscle cell across a neuromuscular
junction (Fig. 22) to open ligand-gated ion channels on
the receptor site that will initiate an action potential
so-called electromechanical coupling. Outside the
central nervous system, the commonest neurotransmitter to be found in these small clear vesicles is
acetylcholine and the commonest receptor is the nicotinic acetylcholine receptor, although acetylcholine
does not exclusively act as a fast neurotransmitter to
open ligand-gated ion channels in this way, nor is the
nicotinic receptor the only type of acetylcholine receptor (see below). In the central nervous system,
g-aminobutyric acid (GABA) is also found in small
clear vesicles (28).
Large, dense-cored vesicles contain so-called
slow neurotransmitters (30), which are called slow
because they are released nonspecifically from around
the area of the varicosity (Fig. 23) rather than specifically into the receptor site; and because they generally
act by binding to G proteinlinked receptors (chap. 1)
and initiating smooth muscle contraction through second messengers systems. This is so-called pharmacomechanical coupling of the neuronal stimulus to
smooth muscle contraction, as distinct from the electromechanical coupling initiated by the binding of
ligand-gated ion channels by fast neurotransmitters.
A single nerve impulse will empty about half the
vesicles in a varicosity, each of which will release about
5000 molecules of acetylcholine as a quantum. These
vesicles empty their contents in response to a rise in
cytosolic calcium within the varicosity as a result of the
opening of membrane calcium channels induced by
the neuronal electrical impulse (voltage-gated calcium
231
Figure 23 Diagram of a nerve ending and neuromuscular

junction to show the release of slow neurotransmitters from
around the neuromuscular junction. Source: From Ref. 29.
Figure 25 A frequency-response curve before and after Tetrodotoxin showing a very small residual contraction that is not
nerve mediated and due to direct smooth muscle stimulation.
channels), causing an influx of extracellular calcium

(30).
There are other ways of demonstrating the primacy of parasympathetic cholinergic activity in the
excitatory innervation of the bladder other than by
showing the density of staining of acetylcholinesterase
on light microscopy and the vesicles on electron
microscopy. The most convincing way is by the physiological study of strips of detrusor smooth muscle in
an organ bath (Fig. 24). The organ bath keeps the
muscle strip at the right temperature, sufficiently
oxygenated and in the right fluid medium to keep it
viable sufficiently long for adequate study. The excitatory nerve supply to the muscle fibers within the
muscle strip is stimulated by using an electrical
impulse. If the strength and amplitude are optimized
and the frequency of the electrical stimulus is then
varied, a frequency-response curve is produced

between about 0.5 Hz and 20 Hz, above which the
response rate plateaus until the electrical impulse is of
sufficiently high frequency to cause damage (Fig. 25).
This response can be abolished by the application of
tetrodotoxin, which is a sodium channel blocker
which, therefore, blocks nerve conduction (32).
(Tetrodotoxin is extracted from the liver of the puffer
fishcalled fugu in Japanand will be known as
such to James Bond aficionados.) If a frequencyresponse curve is plotted after the application of
tetrodotoxin, there will be a very small response at
higher frequencies that is not nerve mediated but due
to direct stimulation of the smooth muscle cell membrane itself. If the frequency-response curve is performed after the application of atropine at a sufficient
dose to give complete cholinergic blockade, then the
Figure 24 Diagrammatic representation of an organ bath

experiment. Source: From Ref. 31.
232
Mundy
It has indeed been shown to do so (32), but it does not

appear to be a component of normal human voiding.
It may, however, be a cause of abnormal detrusor
function in detrusor instability.
OTHER NEUROTRANSMITTERS
Figure 26 A frequency-response curve before and after atropine in a human showing a very small residual response. In this
case it is a graphical representation of the percentage response.
only response left will be the same as that after

tetrodotoxin (Fig. 26). In other words, in the normal
human specimen (stressing both adjectives), there is
no excitatory component left after atropine blockade
and there is no atropine-resistant component to
excitatory neurotransmission. Normal excitatory neurotransmission in the human bladder is therefore
exclusively muscarinic cholinergic (3335). In other
mammals, it is a completely different story. In some
small mammals, there may be as much as 30% of the
frequency-response curve that is atropine resistant.
This is thought to be due to the presence of an
alternative excitatory neurotransmitter that, in most
animal species showing this type of excitatory neurotransmission, is thought to be adenosine triphosphate
(ATP) (30). This is thought to give rise to the type of
bladder contraction responsible for the excretion of a
small amount of urine for the purposes of territorial
marking. In those animals that exhibit territorial marking, it seems likely that a normal voiding detrusor
contraction is cholinergic in origin, whereas the smallvolume squirt of urine excreted for territorial marking is under so-called purinergic (ATP-mediated)
innervation.
This is not to say that ATP cannot be made to
cause contraction of human detrusor smooth muscle.
To qualify as a neurotransmitter, a potential candidate

must satisfy certain stringent criteria. In the last
20 years or so, numerous compounds have been identified as potential neurotransmitters, many of which
fail to satisfy these criteria but nonetheless appear to be
involved in the process of neurotransmission, either as
neurotransmitters or as neuromodulators (30).
A further observation is that many of these
compounds seem to be related to nerves that show
the characteristics of cholinergic or adrenergic neurons. In other words, the putative neurotransmitter or
neuromodulator appears to be colocalized with a
known classical transmitter or with another putative neurotransmitter (26).
Several of these putative neurotransmitters are
peptides, which have been grouped together as neuropeptides. This group includes substance P (36,37)
vasoactive intestinal polypeptide (38,39) and neuropeptide Y, to name but a few. Substance P is thought
to be involved in afferent neurotransmission (36).
Vasoactive intestinal polypeptide relaxes detrusor
smooth muscle (39) and has been shown in some
studies to be coreleased with acetylcholine. Similarly,
neuropeptide Y has been colocalized in adrenergic
neurons. This concept of colocalization, corelease
and cotransmission is interesting as it provides a
means of modifying the effects of neural activity either
temporally or spatially. Thus, the same nerves could
produce two transmitters with different effects, both
being simultaneously released but with one predominating in one area and the other in the other area. For
example, an excitatory and an inhibitory transmitter,
if coreleased, might cause bladder contraction if the
former predominated in the bladder and relaxation of
the bladder neck if the latter predominated at that site.
This is indeed what is thought to happen with acetylcholine and nitric oxide, colocated in and coreleased
from the parasympathetic nerves that supply this area
(17,40). This type of neuromodulatory activity could
have other effects, which would less dramatically act
to fine tune the actions of the lower urinary tract.
RECEPTORS
The acetylcholine released from the varicosities at the
ends of parasympathetic nerves has a variety of different actions depending on exactly where it is released
and particularly on the nature of the receptors present.
As a general rule, the same applies to all transmitters
and all nerves. Many transmitters are associated with
several different types of receptors and each type of
receptor has several subtypes, each with different
effects. When acetylcholine is released from preganglionic nerve fibers in the parasympathetic ganglia of the
pelvic plexus and in the bladder wall its principal
targets are the receptors on the cell bodies of the

postganglionic nerves. These receptors are nicotinic in
type, which are ligand-gated ion channels.
At the neuromuscular junctions in the detrusor
smooth muscle itself, the most important excitatory
receptors are muscarinicthe other main type of
cholinergic receptorand specifically of the M3 subtype (41,42). These are not the only muscarinic receptors presentthere are muscarinic receptors on the
nerve terminals from which the acetylcholine is
released which can enhance (M1 subtype) or suppress
(M4 subtype) transmitter release depending on the
intensity of the nerve impulse (43) There are also M2
receptors on the bladder smooth muscle cells themselves as a separate population of muscarinic receptors whose function is not clear. M2 receptors have
various actions, both as ligand-gated ion channels and
as G proteinlinked receptors; M3 receptors are G
proteinlinked receptors.
Muscarinic receptors are not restricted to the
ganglia and the neuromuscular junctions; they are
also found in the urothelim and in the suburothelial
lamina propria, in relation to myofibroblasts, where
have a role in bladder sensation and probably also in
normal motor function. Acetylcholine is not the only
transmitter released by parasympathetic nerves and
muscarinic receptors are not the only receptors to be
found in the bladder and urethra. Nitric oxide is
released by parasympathetic nerves, particularly in
the bladder neck and urethra (17,40); there are
purinergic receptors of various different types (41),
and there are of course a few sympathetic nerves with
various associated types and subtypes of receptors,
mainly associatedoutside the prostatewith vasomotor function.
Because ATP was first identified in experimental
animals as the factor responsible for atropine resistance in detrusor contraction most attention until
recently was concentrated on its contractile properties.
ATP acts through two types of purinergic receptor: the
P2X receptor (with seven subtypes), which is an ion
channel, and the P2Y receptor (with eight subtypes),
which is a G proteinlinked receptor (41). The contractile response to ATP is mediated through the P2X
receptor subtypes; the sensory role of ATP is mediated
by P2Y receptor subtypes.
Mechanisms of Contraction and Relaxation

of the Bladder and Urethral Smooth Muscle
in Health and Disease
This is described in detail in the next chapter.
THE AFFERENT INNERVATION

OF THE BLADDER
So far, we have mainly concentrated on the efferent
innervation of the bladder. The afferent innervation is
equally important, but is less well understood. Afferent
impulses arise from the nerve plexus in the lamina
propria associated with the myofibroblasts immediately
deep to the urothelium, from within the muscle layer
itself, and, presumably, from the adventitia. Afferent
233
nerve endings have been identified, but their nature is

poorly understood (44). Substance P and ATP are
thought to be sensory neurotransmitters and there is
good evidence to support this view, but again the
mechanism is not well understood. Other presumptive
sensory nerves have been shown to contain calcitonin
gene-related peptide (CGRP) and other transmitters,
including neurokinin A (45).
The emerging explanation for the sensation of
bladder filling centers on the layer of myofibroblasts in
the lamina propria, referred to above as forming a sort
of unitary stretch receptor with the epithelium and
the interconnecting network of nerves between the two
(2124). Thus, although there are stretch receptors (in
the traditional physiological sense) throughout the
bladder wall, current opinion is that the bulk of afferent activity originates in this functional syncitium
forming the unitary stretch receptor; the receptors
involved in this system are many and varied but
principally muscarinic and purinergic; and that
onward transmission of the afferent impulse in the
pelvic and other afferent nerves is largely purinergic.
Importantly it is now thought likely that the
activity in these receptors in the lamina propria
might be a better explanation for bladder overactivity
states and for the clinical response to anticholinergic
medication in such states than the activity in the
receptors in the detrusor layer (46,47).
The majority of the afferents from the bladder
run in the pelvic nerves (48). Only a small, albeit
important, percent run in the sympathetic and somatic
nerves. Pelvic nerve afferents consist of myelinated
A-d and unmyelinated C axons. The A-d myelinated
axons arise from tension receptors in the smooth
muscle of the bladder and respond to increasing
muscle wall tension during bladder filling. These are
the afferent neurons responsible for eliciting the socalled micturition reflex in normal healthy individuals. Most of the C-fiber unmyelinated axons arise from
receptors in the suburothelial layer, although some
nociceptors sensitive to overdistension may also
be found in the muscle and adventitia. Some of the
suburothelial receptors respond to stretching of the
mucosa and thus act as bladder volume sensors.
The remainder are insensitive to normal bladder distension and are referred to as silent afferents (49).
These afferents are thought to become mechanosensitive when irritated or inflamed, thereby lowering the
threshold of bladder contraction in such situations. It
is thought that this may be due to the release of
neurokinin A, which then acts on its own specialist
receptor binding sites and sensory nerve endings in
the suburothelial layer (50).
What is clear, from the work of Nathan (51), is that
there are three types of sensation arising from the
bladder (Fig. 27). The first and most general sensation
of bladder filling arises from receptors throughout the
bladder. The afferent fibers run with the parasympathetic nerves back to the sacral cord, where there is some
local synapsing on preganglionic parasympathetic cell
bodies in the intermediolateral column of the sacral
cord, but the majority of afferent neurons run in the
ascending tracts of the spinal cord and up to the pons,
where they synapse with preganglionic neurons in the
234
Mundy
Figure 27 The nervous pathways concerned

with the three different types of sensation from
the lower urinary tract. , from the bladder; ,
from the trigone; , from the urethra and pelvic
floor. Source: From Ref. 31.
nucleus locus coeruleus in the rostral pons. Other fibers

pass up to the cerebral cortex to give rise to sensory
awareness. This type of afferent stimulus gives rise to a
volume-related awareness of bladder filling that is
easily suppressed.
Less easily suppressed is the next type of bladder sensation, which is stimulated by definite fullness
of the bladder. This is a stimulus that arises in the
trigonal area and the afferent impulses are transmitted
in neurons that run with the sympathetic nerves up to
the thoracolumbar cord. Once again, there are local
relays in the cord, but most fibers run up in the
ascending tracts to the pons and to the cerebral cortex
to give awareness.
Finally, there is the feeling of severe urgency and
the sense that voiding is imminent. This sensation
cannot be overlooked. It arises in the urethra and the
afferent fibers run with the pudendal nerve, again
giving rise to local relays in the sacral cord, but with
most fibers running in the ascending tracts to the pons
and to the cerebral cortex.
Within the spinal cord, the ascending and
descending fibers all run in an equatorial plane
through the spinal canal, as also shown by Nathan
(Fig. 28) (52). The medial tracts are visceral efferent,
the intermediate tracts are somatic efferent to the
intrinsic rhabdosphincter and the pelvic floor, and
the most lateral fibers lying at the periphery of the
cord between the corticospinal and the spinothal amic
tracts are visceral afferent. The location of these tracts
in relation to the urinary tract was found by postmortem studies of patients who had undergone the
neurosurgical procedure of percutaneous cordotomy
during life for relief for severe visceral pain, usually
malignant in origin.
Figure 28 The orientation within the spinal cord of the pathways related to lower urinary tract function. Abbreviations:
1, autonomic efferent; 2, somatic efferent; 3, afferent. Source:
From Ref. 31.
The nature of the neurotransmitters in both the

afferent and efferent pathways of the spinal cord (53)
is still largely unknown (54). Glutamic acid is thought
to be the principal excitatory transmitter of both the
ascending and descending limbs of the micurition
reflex pathway as well as in the reflex pathway
controlling sphincter function at both the spinal and
supraspinal levels. Likewise, GABA and the opioid
peptides (enkephalins) are the most important inhibitory modulators. It is clear that several other substances may exert significant modulatory influences,
including supraspinal cholinergic and dopaminergic
influences at the supraspinal level and adrenergic
influences at the spinal level. All of these transmitters
have variable effects according to their site of action
within the neuraxis and the type of receptor activated.
235
of the sphincter mechanismoccur and are coordinated. If there is such a thing as a micturition center,
then this is it (55).
This view of a unitary pontine micturition
center has been revised in recent years largely by
the work of Blok and Holstege (56). They have shown
experimentally that micturition and continence are
independently organized in the brain, and this has
since been confirmed by PET scanning in humans (55).
Their experimental studies in the cat have shown that
the pontine micturition center is exactly where Barrington said it was in the medial part of the dorsolateral pons (55,56) (Fig. 30). This they call the M-region.
Neurons from the M-region project downward to the
CEREBRAL CONTROL OF VOIDING

Within the brain, there are five areas that are concerned with continence and voiding (Fig. 29). The first
has already been alluded to the nucleus locus coeruleus in the rostral pons where afferent nerves synapse
on the cell bodies of efferent nerve fibers. There are
two connections of importance at this site. The first is
the synapse, by which means an adequate afferent
impulse gives rise to an efferent impulse that will
generate a detrusor contraction that is sufficient in
amplitude and duration to cause complete bladder
emptying. The second connection that occurs at this
site coordinates this contraction with relaxation of the
bladder outflow to given unobstructed voiding. The
details of these two mechanisms and their coordination are unclear; it is, however, quite clear that this is
the site where both these actionsgeneration of a
normal voiding contraction and reciprocal relaxation
Figure 29 Five areas of the brain concerned with lower urinary

tract function. Abbreviations: 1, medial aspect of frontal lobe;
2, septal and preoptic nuclei; 3, hypothalamus; 4, nucleus locus
coeruleus (Barringtons pontine micturition center); 5, paracentral
lobule. Source: From Ref. 31.
Figure 30 Schematic diagram of the spinal and supraspinal

structures and their pathways involved in micturition. Pathways
are indicated on one side only. Abbreviations: IC, inferior cellulus;
S2, second sacral segment.
236
Mundy
sacral intermediolateral gray column. There are also

projections to the sacral intermediomedial cell column, which is also known as the dorsal gray commissure (DGC). Thus, impulses from the M-region
simultaneously excite the preganglionic parasympathetic neurons in the sacral intermediolateral gray
column to cause bladder contraction and excite
GABA-ergic interneurons in the DGC in the same
area of the spinal cord, which inhibit the urethral
sphincter motor neurons and thereby cause relaxation
of the sphincter. The presence of the M-region has
been shown in exactly the same area in humans by
PET.
It has also been shown that afferent impulses do
not project directly to the pontine micturition center
(M-region) but to a relay center in the mesencephalic
periaqueductal gray matter (PAG). This is an important relay center between the pontine micturition
center and a number of higher centers in the brain,
which seems to act as an interface between the afferent
and efferent components of bladder control and specifically in registering the degree of bladder filling
both with the pontine micturition center and with the
higher centers responsible for conscious awareness
(57).
Ventral and lateral to the pontine micturition
center of M-region is another group of neurons
responsible for the storage of urine during continence.
This is known as the pontine storage center (PSC) or
L-region (Fig. 31). Neurons project from the L-region
to the urethral sphincter motor neurons in Onufs
nucleus, reducing tonic urethral sphincter contraction.
The presence of this center in humans has also been
shown on PET scanning but with less certainty than
the M-region.
Clearly, as this is largely an autonomic event,
parasympathetically mediated, the hypothalamus is
important, and this is the second of the five centers.
The third is the paracentral lobule of the cerebral
cortex, which is responsible for the control of the
pelvic floor musculature. The importance of this center becomes manifest in spastic conditions such as
congenital cerebral palsy, in which failure of relaxation can cause quite severe voiding difficulties. The
normal physiological role of other related areas of the
brain, such as the basal ganglia, is not clear, although
diseases that affect these areas, such as Parkinsons
disease and multiple system atrophy, have obvious
adverse effects.
At the junction of the diencephalon and telencephalon are the septal and preoptic nuclei where the
associated acts of both micturition and defecation
(and, for that matter, coition) are coordinated (58). In
some animals, such as the cat, these associated acts of
voiding are quite elaborate, but in the human being
they are restricted to fixation of the diaphragm and
anterior abdominal wall musculature.
Finally, there is the area in the medial aspect of
the frontal lobespecifically the right inferior frontal
gyrus (57)that, although described initially as an
inhibitory area (59), seems to facilitate or inhibit
voiding according to circumstances. Thus, it would
appear, this area of the frontal lobe can facilitate the
Figure 31 Schematic diagram of the spinal and supraspinal

structure and their pathways involved in bladder control problems.
nucleus locus coeruleus to cause voiding when the

bladder is only partially full and afferent activity is
therefore subthreshold. This is the way in which the
bladder can be emptied before going to bed or before
undertaking a long journey to avoid, perhaps, the
need to void during the night or the journey. Equally,
if afferent activity has reached a threshold level but
there is a good program on the television, then this
area of the frontal lobe can suppress the need to void
until a more appropriate time.
The details of our understanding of the spinal
cord mechanisms involved in voiding are sparsein
the brain, our knowledge of the mechanisms involved
in continence and voiding is scant indeed.
237
THE NORMAL BLADDER

FILLING-VOIDING CYCLE
So far, a summary has been given of our anatomical
and physiological knowledge peripherally in the bladder and urethra themselves and then centrally within
the nervous system. It has been seen that the most
important reflex arcs are mediated through the nucleus
locus coeruleus in the rostral pons; that excitatory
neurotransmission in the normal human is exclusively
cholinergic, and specifically muscarinic in origin; that
relaxation of the bladder neck and urethral smooth
muscle is probably mediated through the action of
nitric oxide; and that the intrinsic rhabdosphincter is
the most important component of the sphincter mechanism for continence and must be reciprocally relaxed
through a coordinating mechanism in the rostral pons
for normal voiding to occur. However, these issues and
the other matters discussed do not explain all that we
need to know about normal continence and voiding.
It is apparent during the normal filling and
voiding cycle of a cystometrogram, with synchronous
measurement of pressure within the sphincter-active
urethra (Fig. 32), that bladder pressure stays almost
completely unchanged throughout filling to a normal
capacity despite an increasing afferent stimulus.
Indeed, in health, the bladder is quiescent for over
99.5% of the day. There is, however, a small but
definite rise in intraurethral pressure that is volume
related. Then, just before or synchronous with the
onset of voiding, there is a drop in intraurethral
pressure, matched by a cessation of the electromyographic activity of the intrinsic rhabdosphincter, after
which or synchronous with which detrusor pressure
starts to rise (14). This rise in pressure is then sustained until the bladder is empty, by which time
detrusor pressure has returned to normal and urethral
resistance has risen back to normal. The cycle then
starts over again.
WHAT KEEPS THE BLADDER PRESSURE

LOW DURING FILLING?
This ability of the bladder to keep its pressure almost
unchanging irrespective of bladder volume and afferent stimulation is known as compliance. The bladder is
highly compliant: it shows very little change in pressure for a substantial change in volume. A steady rise
in pressure during filling, which is sometimes seen
when the bladder wall is stiff because of disease, is
known as low compliance or poor compliance.
The exact nature of normal bladder compliance
is not clear, but it can be observed to be present in the
bladder post-mortem, at least up to a certain filling
volume (60). This has been explained on the basis of
the physical characteristics of the protein fibers that
constitute the cellular and connective tissue structures
of the bladder wall. They can be imagined as being
coiled in the collapsed bladder and filling simply
uncoils them, at least until 100 to 200 mL of filling
has occurred (Fig. 33). Detrusor smooth muscle cells
have a striking ability to change their length without
Figure 32 Synchronous urethral and bladder pressure studies

to show the urethra pressurized during bladder filling and pressure fall before the onset of detrusor contraction. Source: From
Ref. 31.
238
Mundy
Figure 35 The gating mechanism showing that, with threshold afferent and preganglionic efferent activity, efferent activity is
transmitted to postganglionic neurons.
Figure 33 The contribution of passive properties of the bladder

wall to normal bladder compliance. Source: From Ref. 31.
any change in tension. They may lengthen as much as

fourfold during bladder filling, in a linear relationship
with increasing bladder radius.
Our understanding of what happens over and
beyond this elastic component and a certain viscoelastic property of the bladder is due to the work of
de Groat and his coworkers, who have studied this
mechanism in great detail in the cat (61). In a series of
elegant experiments, de Groat has shown that there is a
gating mechanism in the parasympathetic ganglia of
the pelvic plexuses, which means that subthreshold
activity in the preganglionic neurons is not transmitted
to postganglionic efferent neurons (Fig. 34). There is
also an inhibitory interneuron mechanism within the
spinal cord that helps to keep afferent impulses from
being transmitted onward until they reach a critical
level. At low levels of afferent activity, the inhibitory
interneurons prevent the transmission of impulses

from the afferent nerves to the preganglionic efferent
nerves. As afferent activity builds up, the inhibitory
interneurons are progressively inhibited and impulses
start to appear in the preganglionic efferent neurons,
but the gating mechanism prevents these from being
transmitted to the postganglionic efferent neurons
until preganglionic efferent activity has reached a
critical threshold level. When this level is reached,
a barrage of impulses is then transmitted down the
postganglionic efferent neuron to the bladder, which
therefore contracts (Fig. 35).
In addition, there are the inhibitory effects of the
sympathetic neurons that have postganglionic
branches ending on parasympathetic ganglion cells,
as mentioned earlier in the chapter. The afferent fibers
are presumably those that arise in the trigone, conveying fullness of the bladder and running up to the
thoracolumbar cord, where the local relay gives rise to
efferent sympathetic activity that tends to inhibit
neurotransmission across the parasympathetic ganglia
of the pelvic plexuses, thereby enhancing de Groats
gating mechanism (Fig. 36).
WHAT CAUSES THE RISE IN URETHRAL

PRESSURE DURING BLADDER FILLING?
This appears to be due to local reflex activity within
the sacral cord by which afferent impulses from the
bladder cause a local reflex rise in efferent activity to
the urethral smooth muscle. It is thought to be spinal
because it persists after complete spinal cord transection above the level of the sacral segments. Other than
that, the mechanism is unclear.
WHAT CAUSES THE FALL IN URETHRAL

PRESSURE AT THE ONSET OF VOIDING?
Figure 34 The gating mechanism by which, with minor
degrees of activity in the afferent and preganglionic efferent
nerves, there is no transmission to postganglionic efferent
nerves.
The mechanism of this is not clear either, but it has

regularly been observed that intraurethral pressure
drops to a degree that indicates that both the smooth
and the striated components of the urethral sphincter
mechanism are relaxed (14). Cessation of rhabdosphincter electromyogram activity synchronous with
239
Figure 36 The action of sympathetic fibers. , on

parasympathetic ganglia. Source: From Ref. 31.
the fall in urethral pressure has also been observed to

support this conclusion. This possibly explains why
the most urgent sense of a very full bladder is more a
urethral than a bladder sensation, transmitted through
the pudendal nerve. It seems, in fact, that the urethral
sphincter mechanism is reflexly opening, and only
voluntary contraction of the pelvic floor and suppression of the micturition reflex can stop it occurring. It
seems, therefore, that at this point there has been
threshold activation of the efferent cell bodies in the
nucleus locus coeruleus and only positive intervention
by higher centers to suppress the process can prevent
the urethra from relaxing and stop reflex detrusor
contraction from following.
Figure 37 Diagrammatic representation of the theory of reciprocal innervation.
HOW DOES THE BLADDER NECK OPEN?

There have been several theories to account for this. It
used to be thought that there was a reciprocal innervation of the bladder by the parasympathetic system
and of the bladder neck by the sympathetic system
and that when the one caused contraction the other
caused relaxation (Fig. 37) (62), but that was discounted when the role of the sympathetic system in
the lower urinary tract was effectively ruled out, as
discussed above. With the recent discovery of the role
of nitric oxide, however, this reciprocal innervation
theory has been resuscitated, although not in so
many words. Nitric oxide may be cotransmitted
with acetylcholine and it may be that the acetylcholine

component causes contraction of the detrusor smooth
muscle, whereas at the bladder neck and in the
urethra there is a dominant nitric oxide effect released
from the same type of neurons, which causes bladder
neck and urethral relaxation. This does not explain the
relaxation of the intrinsic rhabdosphincter, but if one
presupposes that relaxation of the rhabdosphincter is
a necessary precondition for detrusor contraction to
occur, then bladder neck and urethral smooth muscle
relaxation at the onset of detrusor contraction is all
that is left to explain.
240
Mundy
Figure 38 Diagrammatic representation of Lapides theory of

bladder neck opening.
Another explanation popularized by Lapides

(63) (Fig. 38) was that the bladder neck and the
urethral musculature were continuous and fixed like
a system of guy ropes at the urogenital diaphragm.
Contraction therefore caused shortening, which therefore caused opening of the bladder neck. This theory
was never widely held and was eventually discounted
by the demonstration that the urogenital diaphragm
did not exist.
The next theory to explain bladder neck opening
was Hutchs base plate theory (Fig. 39) (64). This
was based on the importance of the trigonal musculature and of certain bands of detrusor smooth muscle
that acted to trip open the base plate of the bladder
neck, but the presence of such layers and bands has
never been demonstrated convincingly by anybody
else and so this theory, too, has been discounted.
There is the simple hydrokinetic observation that
there is only actually one way out of the bladder and
that is through the bladder neck, so that when the
detrusor contracts, the force is likely to be transmitted
in that direction (Fig. 40). It may well be that this
simplistic point of view is at least in part correct,
perhaps in conjunction with the reciprocal relaxation
Figure 40 A simple hydrokinetic explanation of bladder neck

opening.
theory modified to incorporate the action of nitric

oxide rather than the sympathetic nervous system.
In addition, it should be noted that it has been
observed in spinal cord-injured patients fitted with a
sacral anterior root stimulator that the bladder neck
can be made to open and close separately from events
in the main body of the detrusor, indicating that there
may be a separate innervation to the bladder neck
(Giles Brindley, personal communication). Clearly, as
it can only be demonstrated in this way and then only
under certain circumstances and in some patients, this
innervation cannot readily be dissected out from
innervation of the bladder as a whole, but again it
would tend to support the reciprocal innervation
theory, with the corelease of nitric oxide in the
sphincter-active area to explain opening coincident
with cholinergic-mediated detrusor contraction.
VESICOURETHRAL REFLEXES
As many as 12 micturition reflexes have been
described, largely following the descriptions by
Barrington earlier this century (55) and by Kuru more
Figure 39 (A, B) Diagrammatic representation of Hutchs theory of bladder neck opening.

Figures refer to hypothetical muscle bundles
identified by Hutch. Source: From Ref. 9.
241
amplitude and duration and the second to coordinate

this with reciprocal relaxation of the intrinsic rhabdosphincter.
In addition, there is a reflex facilitation that occurs
during a detrusor contraction that is mediated by afferents in the pudendal nerve. The mechanism for this is
unclear, but Brindley has noted that after pudendal
blockade, the force of detrusor contraction is considerably reduced (66): hence, the afferent pathway can be
identified as pudendal but the rest of the reflex is
unclear.
This is not to say that other reflexes do not exist
and particularly that other reflexes do not exist in
disease, but these are the only ones that can be
positively identified as being important in health.
SUMMARY
Figure 41 Diagram to show the bladder reflexes described in

this chapter. , reflex 1; , reflex 2; , reflex 3; , reflex 4.
recently (65). Some of these are only active in experimental animals subject to various neural ablation procedures and at least one of them is the genital reflex of
closing off the bladder neck to ensure antegrade ejaculation. Some have been observed in animals but not in
humans. So far in this chapter, reference has been made
to four reflexes (Fig. 41). The first is the afferent impulse
routed up to the pons to cause the parasympathetic
efferent contraction of the bladder of sufficient amplitude and duration to give complete bladder emptying;
and the second is that which causes reciprocal relaxation of the intrinsic rhabdosphincter to allow unobstructed voiding. The third is the local spinal reflex
increase in urethral pressure during bladder filling.
The fourth reflex is the one that causes sympathetic
inhibition of parasympathetic ganglionic transmission
in the pelvic plexuses with more advanced degrees of
bladder filling.
There is, therefore, one local sacral reflex causing a
rise in urethral pressure during filling, and one thoracolumbar reflex causing sympathetic inhibition of
ganglionic transmission in the parasympathetic innervation to the bladder, thereby supporting de Groats
gating mechanism that keeps the bladder quiescent
during filling. Then, there are the two pontine reflexes,
the one to cause a bladder contraction of adequate
It will be apparent to the reader that there is a great

deal to be learnt about the structure and even more
about the function of the lower urinary tract, particularly about the spinal and supraspinal mechanisms
that control it.
The fundamental features are that during bladder
filling, intravesical pressure changes very little despite
the bladder filling by 400 mL or more, and this compliance is achieved by a gating mechanism that prevents afferent neuronal activity being transmitted to
postganglionic efferent activity until that afferent activity reaches a critical level. Critical afferent activity,
probably originating in the myofibroblasts of the lamina
propria of the bladder, is transmitted by ascending
spinal pathways that synapse on cell bodies in the
nucleus locus coeruleus in the rostral pons in addition
to providing conscious awareness of bladder filling.
When afferent activity to the nucleus locus coeruleus,
subject to suprapontine facilitation or inhibition,
reaches a threshold level, efferent activity is initiated,
mediated through the pelvic parasympathetic nerves
and muscarinic receptors. This causes a detrusor contraction of sufficient amplitude and duration to give
complete bladder emptying and a synchronous coordinated reciprocal relaxation of the urethral sphincter
mechanism to allow unobstructed voiding until the
bladder is empty. It seems clear that excitatory neurotransmission in the normal human detrusor is exclusively cholinergic, and it is becoming increasingly clear
that reciprocal relaxation of the urethral sphincter is a
prerequisite for detrusor contraction, and that the synchronous relaxation of the bladder neck and urethral
smooth muscle is probably mediated by nitric oxide,
which is cotransmitted with acetylcholine from postganglionic parasympathetic neurons. Unfortunately,
although this fundamental mechanism appears reasonably clear, a lot of the details are lacking at present.
REFERENCES
1. Uhlenhuth E, Hunter DT, Loechel WE. Problems in the
Anatomy of the Pelvis. Philadelphia: Lippincott, 1953.
2. Mundy AR. Urodynamic and Reconstructive Surgery of
the Lower Urinary Tract. Edinburgh: Churchill Livingstone, 1993.
242
Mundy
3. Gosling JA, Dixon JS, Humpherson JR. The Functional

Anatomy of the Urinary Tract. Edinburgh: Churchill Livingstone, 1983.
4. Chilton CP. The urethra. In: Webster G, Kirby R, King L,
et al., eds. Reconstructive Urology. Cambridge: Blackwell
Scientific, 1993:5973.
5. Zacharin RF. The anatomical supports of the female urethra. Obstet Gynaecol 1968; 32:754759.
6. Walsh PC, Partin AW. Anatomic radical retropubic prostatectomy. In: Wein AJ, Kavoussi LR, Novick AC, et al.,
eds. CampbellWalsh Urology. 9th ed. Philadelphia:
Saunders, 2007:29562978.
7. Redman JF. Anatomy of the genitourinary system. In:
Gillenwater JR, Grayhack JT, Howards SS, et al., eds.
Adult and Pediatric Urology. 3rd ed. Philadelphia:
Mosby, 1996:361.
8. Kaye KW, Milne N, Creed K, et al. The urogenital diaphragm external urethral sphincter and radical prostatectomy. Aust N Z J Surg 1997; 67:4044.
9. Hutch JA. Anatomy and Physiology of the Bladder, Trigone and Urethra. London: Butterworths, 1972.
10. Gosling J. The structure of the bladder and urethra in
relation to function. Urol Clin North Am 1979; 6:3138.
11. Dixon J, Gosling J. Structure and innervation in the human.
In: Torrens M, Morrison FB, eds. The Physiology of the
Lower Urinary Tract. London: Springer Verlag, 1987:322.
12. Raz S, Caine M, Zeigler M. The vascular component in the
production of intraurethral pressure. J Urol 1972; 108:9396.
13. Sundin T, Dahlstrom A, Norlen L, et al. The sympathetic
innervation and adrenoreceptor function of the human
lower urinary tract in the normal state and after parasympathetic denervation. Invest Urol 1977; 14:322328.
14. Tanagho EA. The anatomy and physiology of micturition.
Clin Obstet Gynaecol 1978; 5:326.
15. James MJ. Relaxation of the human detrusor. University of
Nottingham, MD Thesis, 1993.
16. Bridgewater M, MacNeil HF, Brading AF. Regulation of
tone in pig urethral smooth muscle. J Urol 1993; 150:223228.
17. de Groat WC, Fraser MO, Yoshiyama M, et al. Neural
control of the urethra. Scand J Urol Nephrol Suppl 2001;
207:3543.
18. Gosling JA, Dixon JS, Critchely HOD, et al. A comparative
study of the human external sphincter and periurethral
levator ani muscles. Br J Urol 1981; 53:3541.
19. Schroder HD. Onufs nucleus X: a morphological study of
a human spinal nucleus. Anat Embryol (Berl) 1981;
162:443453.
20. Zrara P, Carrier S, Kour NW, et al. The detailed neuroanatomy of the human striated urethral sphincter. Br J Urol
1994; 74:182187.
21. Wiseman OJ, Fowler CJ, Landon DN. The role of the human
bladder lamina propria myofibroblast. BJU Int 2003; 91:8993.
22. Sui GP, Rothery S, Dupont E, et al. Gap junctions and
connexin expression in human suburothelial interstitial
cells. BJU Int 2002; 90(1):118129.
23. Wu C, Sui GP, Fry CH. Purinergic regulation of guinea pig
suburothelial myofibroblasts. J Physiol 2004; 559:231243.
24. Fowler CJ, Griffiths D, de Groat WC. The neural control of
micturition. Nat Rev Neurosci 2008; 9(6):453466.
25. Kirkham APS, Mundy AR, Heald RJ, et al. Cadaveric
dissection for the rectal surgeon. Ann R Coll Surg Engl
2001; 83:8995.
26. Mundy AR. An anatomical explanation for bladder dysfunction following rectal and uterine surgery. Br J Urol
1982; 54:501504.
27. Gosling JA, Dixon JS. Anatomy of the bladder, urethra and
pelvic floor. In: Mundy AR, Stephenson TP, Wein AJ, eds.
UrodynamicsPrinciples, Practice and Application. Edinburgh: Churchill Livingstone, 1984.
28. Burnstock G. Autonomic innervation and transmission. Br

Med Bull 1979; 35:255262.
29. Goodman SR. Medical Cell Biology. Philadelphia: JB Lippincott, 1994.
30. Burnstock G. The changing face of autonomic transmission. Acta Physiol Scand 1986; 126:6791.
31. Mundy AR, Thomas PJ. Clinical physiology of the bladder,
urethra and pelvic floor. In: Mundy AR, Stephenson TP,
Wein AJ, eds. UrodynamicsPrinciples, Practice and Application. 2nd ed. Edinburgh: Churchill Livingstone, 1994.
32. Brading AF. Physiology of the urinary tract smooth muscle. In: Webster G, Kirby R, King L, et al., eds. Reconstructive Urology. Cambridge: Blackwell Scientific, 1993:1526.
33. Brindley GS, Craggs MD. The effect of atropine in the
urinary bladder of the baboon and of man. J Physiol 1975;
255:55P.
34. Kinder RB, Mundy AR. Atropine blockade of nervemediated stimulation of the human detrusor. Br J Urol
1985; 57:418421.
35. Sibley GA. An experimental model of detrusor instability
in the obstructed pig. Br J Urol 1985; 57:292298.
36. Alm P, Alumets J, Brodin E, et al. Peptidergic (substance P)
nerves in the genito-urinary tract. Neuroscience 1978;
3:419425.
37. Maggi CA, Barbanti G, Santicioli P, et al. Cystometric
evidence that capsaicin-sensitive nerves modulate the
afferent branch of the micturition reflex in humans. J
Urol 1989; 142:150154.
38. Gu J, Restorick J, Blank MA, et al. Vasoactive intestinal
polypeptide in the normal and unstable bladder. Br J Urol
1983; 55:645647.
39. Alm P, Alumets J, Hakenson R, et al. Peptidergic (vasoactive intestinal peptide) nerves in the genito-urinary tract.
Neuroscience 1977; 2:751754.
40. Bennett BC, Kruse MN, Roppolo JR, et al. Neural control of
urethral outlet activity in vivo: role of nitric oxide. J Urol
1995; 153:20042009.
41. Andersson KE, Arner A. Urinary bladder contraction and
relaxation: physiology and pathophysiology. Physiol Rev
2004; 84:935986.
42. Fetscher C, Fleichman M, Schmidt M, et al. M(3) muscarinic receptors mediate contraction of human urinary bladder.
Br J Pharmacol 2002; 136:641643.
43. Somogyi GT, Zernova GV, Yoshiyama M, et al. Frequency
dependence of muscarinic facilitation of transmitter release
in urinary bladder strips from nerally intact or chronic
spinal cord transected rats. Br J Pharmacol 1998; 125:
241246.
44. Fergusson DR, Kennedy I, Burton TJ. ATP release from
rabbit urinary bladder epithelial cells by hydrostatic pressure change a possible sensory mechanism. J Physiol
1997; 505:503.
45. de Groat WC. Spinal cord projections and neuropeptides
in visceral afferent neurons. Prog Brain Res 1986; 67:
165188.
46. Hawthorn MH, Chapple CR, Cock M, et al. Urotheliumderived inhibitory factor(s) influences on detrusor muscle
contractility in vitro. Br J Pharmacol 2000; 129:416419.
47. de Groat WC. The urothelium in overactive bladder:
passive bystander or active participant? Urology 2004;
(suppl 1):711.
48. Lincoln J, Burnstock G. Autonomic innervation of the
urinary bladder and urethra. In: Maggi CA, ed. Nervous
Control of the Urogenital System. London: Harwood Academic Publishers, 1993:3368.
49. Habler HJ, Janig W, Koltzenburg M. Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat. J Physiol 1990;
425:545562.

50. Wen J, Morrison JFB. Sensitisation of pelvic afferent neurones from the rat bladder. J Auton Nerv Sys 1996; 58:187.
51. Nathan PW. Sensations associated with micturition. Br J
Urol 1956; 28:126131.
52. Nathan PW. The central nervous connections of bladder.
In: Chisholm GD, Williams DI, eds. Scientific Foundation
of Urology. London: Heinemann, 1976.
53. MacMahon SB, Morrison JFB. Spinal neurones with long
projections activated from the abdominal viscera of the cat.
J Physiol 1982; 332:120.
54. Sillen U. Central neurotransmitter mechanisms involved in
the control of urinary bladder function. Scand J Urol
Nephrol Suppl 1980; 58:145.
55. Barrington FJF. The effect of lesions on the hind and
midbrain on micturition in the cat. Q J Exp Physiol 1915;
15:181202.
56. Blok BFM, Holstege G. The central control of micturition
and continence: implications for urology. BJU Int 1999;
83(suppl 2):16.
57. Kavia R, Dasgupta R, Fowler CJ. Functional imaging and
central control of the bladder. J Comp Neurol 2005; 493:2732.
243
58. Hess WR. The Functional Organisation of the Diencephalon. London: Grune & Stratton, 1957.
59. Andrew J, Nathan PW. Lesions of the anterior frontal lobes
and disturbances of micturition and defecation. Brain 1964;
87:233261.
60. Tang PC, Ruch TC. Non-neurogenic basis of bladder tonus.
Am J Physiol 1955; 181:249257.
61. de Groat WC. Physiology of the urinary bladder and
urethra. Ann Int Med 1980; 92:312315.
62. Denny-Brown D, Robertson EG. On the physiology of
micturition. J Physiol 1933; 56:149190.
63. Lapides J. Structure and function of the internal vesical
sphincter. J Urol 1958; 80:341353.
64. Hutch J. A new theory of the anatomy of the internal
urinary sphincter and the physiology of micturition. Invest
Urol 1965; 3:3658.
65. Kuru M. Nervous control of micturition. Physiol Rev 1965;
45:425494.
66. Brindley GS, Craggs MD. The pressure exerted by the
external sphincter of the urethra when its motor nerve
fibres are stimulated electrically. Br J Urol 1974; 46:453462.
15
Physiological Properties of the Lower Urinary Tract
Christopher H. Fry
Postgraduate Medical School, University of Surrey, Guildford, U.K.
INTRODUCTION
Since the last edition of the Scientific Basis of Urology,
there has been considerable progress in our elemental
understanding of lower urinary tract (LUT) physiology.
The previous edition included two chapters LUT
physiologyone on detrusor physiology and the
other on detrusor pathophysiology. With advancing
knowledge it has become clear that it is increasingly
difficult to discuss one without the other. Moreover,
while detrusor smooth muscle behavior may be the
end organ, which generates measurable changes
such as overactivity or incontinence, the urothelium
and suburothelium appear to be at least as important as
the muscle itself in determining the contractile properties of the LUT.
THE UROTHELIUM, BARRIER FUNCTIONS,

AND BACTERIAL INFECTIONS
3.
The apical cells have several important characteristics.

1.
2.
Structure of the Urothelium and Suburothelium

The urothelium is a transitional epithelium and its
barrier function, to separate underlying tissues of the
bladder wall from urine, has long been appreciated.
The cells have a high turnover rate, therefore, if damaged, the barrier function is maintained. However, it is
more than a mere passive barrier and additionally has
transport as well as sensory/signaling (section Secretory and Signaling Properties of the Urothelium/
Suburothelium) functions. Figure 1 shows a crosssection of the bladder wall consisting of four layers:
urothelium, suburothelium, detrusor, and serosa. For
consideration of transport and barrier properties, the
urothelium can be considered alone; however, when
sensory/signaling properties are discussed the urothelium and suburothelium seem to form a single integrated structure. The inset in Figure 1 shows in more detail
the urothelium/suburothelium layer: in the suburothelium is embedded a functional syncitium of interstitial
cells observed as the cell network in the figure, as well
as a capillary network and unmyelinated and myelinated nerves.
The urothelium has three layers in
1.
2.
an apical (umbrella) cell layer with a very low

permeability to urine and pathogens,
an intermediate layer, and
a basal layer (lamina propria) that interacts with

the extracellular matrix of the suburothelial region
for structural support.
3.
70% to 90% of the apical surface area is covered by

protein plaques separated by lipid membrane
called the hinge area. Each plaque contains about
1000 subunits composed of an inner ring of six
large, and an outer ring of six smaller particles (1).
The subunits are formed from five proteins collectively called uroplakins, two of which have four
transmembrane domains (UPIa and UPIb), while
the others are type 1 transmembrane proteins
(UPII, UPIIIa, and UPIIIb). UPIa associates with
UPII, and UPIb associates with UPIIIa or UPIIIb.
Their cytoplasm has a high density of fusiform
vesicles, formed by two apposing plaques joined
together by hinge membrane. The vesicles and
surface membrane plaques are joined by a network of cytoplasmic filaments that attach to tight
junctions at the apical/lateral membrane interface,
and desmosomes in the basolateral membrane (2).
Their function is considered below.
Umbrella cells are joined by tight junctions. Solute
movement across the urothelium can be through
the cell (transcellular) or through the tight junction and the lateral intercellular space (paracellular) pathway. The tight junctions ensure that this
urothelial layer is the major impedance for the
movement of substances from urine to plasma.
Whether alterations in uroplakin expression

relate to urinary tract disease is unclear. Ablation of
the uroplakin-II gene in mice (3) or loss of uroplakin
expression in myelomeningocele patients (4) led to
hyperplastic urothelial growth and may interfere with
smooth muscle development. UPIII expression is also
a prognostic factor in patients with upper urinary tract
urothelial carcinoma (5).
Storage Properties of the Bladder:

Role of the Urothelium
Addition of cytoplasmic vesicles into the apical membrane during bladder filling and their removal during
Chapter 15: Physiological Properties of the Lower Urinary Tract
245
Figure 1 Cross-section of the bladder wall

showing the principal layers; right is an expanded
view of the suburothelial layer.
contraction (6,7) minimizes the surface to volume ratio

of the bladder and hence the flux of substances over
the urothelium. Recent studies have shown that stretch
to either apical or basolateral membrane exerts opposite effects (8). Stretch also increases protein secretion
into the urine, raises cell cAMP levels (7), and stimulates ATP release into the blood side (9) (the latter is
also considered in section Secretory and Signaling
Properties of the Urothelium/Suburothelium).
Release of ATP results in its binding to purinergic
(P2X) receptors on the basolateral membrane of the
apical cells and stimulates an increase in the intracellular [Ca2], [Ca2]i, which in turn stimulates vesicle
fusion (7,10). Raised [Ca2]i is also induced by capsaicin and blocked by the vanilloid receptor antagonists.
The molecular basis of vesicle fusion shares many
features with synaptic vesicle fusion. The apical membrane and the fusiform/discoidal vesicles contain
SNAP 23 and SNARESsynaptobrevin and syntaxin.
Another key component for synaptic vesicle fusion
is the Rab proteins, with Rab 27b found in umbrella
cells (11).
Urothelial Permeability and Transport

The urothelium is very impermeable to major ions in
urine (Na, K, and Cl!) (12), with an electrical
resistance some 15,000 to 20,000 time that of the
proximal tubule epithelium. Both the tight junctions
and transcellular routes contribute significantly to this
resistance. This low transport of ions means that urine
composition does not change greatly during storage.
Uroplakins may contribute to the low solute and
water permeability of the urothelium. With UPIII
knockout mice, water fluxes were twofold greater,
although transepithelial resistance, rate of ion transport, and urea fluxes were not different (13).
It has also been proposed that a glycosaminoglycan (GAG) layer on the apical membrane represents a
major permeability barrier to small electrolytes and
nonelectrolytes and that derangement of the barrier
contributes to bladder pathologies such as painful
bladder syndrome (14). The GAG layer is a multicharged anionic polysaccharide that is supposed to
trap water and hence hinder diffusion to the apical
cells. However, the evidence is not entirely convincing

for the following reasons:
l
There is no significant electrical resistance across

the GAG layer; the Na channel blocker amiloride
rapidly diffuses through the GAG layer and
blocks apical Na channels.
Alteration of luminal [Na] rapidly alters membrane transport.
Enzymatic cleavage of GAG does not alter ion
transepithelial permeability (13).
The mammalian urothelium has a transepithelial

potential (!20 to !120 mV, apical surface negative)
(12,15), with Na the predominant transported ion.
Na transport is blocked by the epithelial Na channel (ENaC) blocker amiloride, applied to the apical
face, and more slowly by ouabain on the basolateral
face to block the Na pump. The rate of transepithelial
Na transport mirrors the permeability of the apical
membrane to Na; amiloride decreases and aldosterone increases Na permeability. The presence of Na
channels in the apical membrane of mammalian urothelium as well as in cytoplasmic vesicles has been
confirmed by immunohistochemistry (16). The exit of
intracellular Na across the basolateral membrane via
the Na/K ATPase raises cell [K] to 70 to 90 mM
(17,18), which subsequently leaves through K channels in the same membrane (19).
In addition to the amiloride-sensitive Na channel, the apical membrane contains three other channels: a stretch-activated channel (20), permeable to
Na and K and blocked by high amiloride concentrations (>100 mM)two degradation products of the
amiloride-sensitive channel; a nonselective, amilorideinsensitive cation channel (21); and a separate nonselective ion channel. The basolateral membrane contains, in addition to the Na/K ATPase and K channel,
a large (64 pS) Cl! conductance (22) that is responsible
for the passive distribution of Cl! under physiological
conditions and is also permeable to HCO3. These cells
also regulate their volume during osmotic stress (23).
A raised basolateral extracellular osmolality results in
cell shrinkage and a decrease in basolateral membrane
K and Cl! permeability and activation of Na/H
and Cl!/HCO3! exchangers. The net effect, in concert
246
Fry
with Na/K ATPase activity, increases cell [K] and

[Cl!] and hence water flux to restore cell volume.
Water movement across the basolateral membrane is
also facilitated by aquaporins (24).
2.
Uropathogenic Bacteria
Uropathogenic Escherichia coli (UPEC) is the most
common causative agent for urinary tract infections
in women (5). UPEC have part of their pathogenic
cycle as an intracellular phase within urothelial cells
where replication and formation of bacterial communities occurs before exiting the host cell (25). Cellular
invasion is facilitated by adhesive fiberstype I pili
(26). Thus, UPEC can form intracellular bacterial
reservoirs that persist for several weeks and may be
resistant to antibiotics. Much of this work has been
carried out on animal models; however, intracellular
bacterial communities from exfoliated urothelial cells
have also been detected in the urine of patients (27).
and crucially depend on the volumes used to fill

the bladder. When normalized to wall tension
versus changes to bladder radius, the plots are
linearized, enabling more meaningful estimates of
tissue compliance or stiffness (28).
Estimation of active wall tension (muscle contractility)
from internal pressure changes: This has been
attempted from urodynamic measurements to
compare contractile properties of the bladder in
patients with different pathologies (29,30). The
approach is really only useful when the pressure
is generated isovolumically (31), that is, energy is
not also being used to move fluid. Figure 2A shows
calculations of the change of pressure during the
development of wall tension when a bladder is
emptying, or remaining at constant volume. In the
latter case more pressure develops as the bladder
empties, because the radius is reducing as a function of time, despite contractile effort being similar
in the two cases. Caution must be applied therefore
in ascribing changes of luminal pressure purely to
alterations of muscle contractility.
FUNCTIONS OF THE LOWER URINARY TRACT:

TENSION AND PRESSURE
The LUT has two functions: to store and periodically
void urine. To achieve these functions various regions
of the LUT have different physical states.
BIOMECHANICAL PROPERTIES
OF THE BLADDER WALL
Filling: The bladder wall is highly compliant (low wall

stress), coupled to a high outflow resistance.
Voiding: The bladder wall stress is high, coupled to a
low outflow resistance.
The bladder is composed of four major layers: urothelium; lamina propria; smooth muscle (detrusor) and an
outer serosal layer. The biomechanical properties of
the bladder wall are mainly determined by the connective tissues of the lamina propria and detrusor
layers, as well as the detrusor itself. The detrusor
layer, which is 60% to 70% of the thickness of the
normal bladder wall, is composed of smooth muscle
cells aligned in longitudinal and circumferential layers
that are highly variable in cross-section, length and
orientation and embedded in a collagen/elastin
matrix. The passive tissue characteristics result from
the viscoelastic properties of the collagen and elastin
fibers in the extracellular matrices of the lamina propria and detrusor as well as the detrusor muscle cells
themselves. The active properties result from contraction of the muscular structures within and surrounding the LUT and transmission of the resultant force via
the extracellular and cellular tissues. Whether the high
compliance of the filling bladder is merely an absence
of muscular contraction or is contributed by active
muscle relaxation remains a subject of debate.
These physical properties depend in large measure on changes to the physical (biomechanical) properties of LUT tissues.
Before considering the physiological background
to contraction and relaxation in the LUT, it is essential
to understand the relationship between wall tension
and intraluminal pressure. Muscles lining any hollow
organ exhibit a state of tension, T, because of either
physical stretch of the tissue (passive tension) or
contraction of the muscular elements (active tension).
The most important tensile component is that tangential to the wall of the organ. The manifestation of this
wall tension (stress) is a pressure, P, within the lumen.
However, the relationship between T and P is not
linear, but depends on the radius of the organ, r, and
the wall thickness, d (Laplaces law).
P 2Td=r
Thus, a large sphere generating the same unit

wall tension as a smaller sphere will produce a
smaller pressure change. This can lead to confusion
when changes of pressure, regardless of the vessel
size, are equated to changes of wall tension and
muscle performance. Two situations exemplify the
need to understand this interrelationship between
pressure and wall tension.
1.
Changes to internal pressure by passively filling the

bladder: When pressure-volume relationships are
generated during bladder filling plots are nonlinear
Passive and Active Properties
Passive Forces and Bladder Compliance

The stiffness of a body is the ratio of an applied force
to the resulting change of dimension; the inverse
parameter is compliance and is a measure of its
distensibility. In three dimensional terms pressures
and volumes are substituted for force and distension,
for example, bladder compliance (C) is the ratio of
change to bladder volume, V, per change to unit
intravesical pressure, P, that is, C = DV/DP. In principle, it is difficult to state a standard value for bladder
247
Figure 2 (A) Derived bladder pressure-time plots (1,2) as a result of an increase in wall tension, T, and calculated using Laplaces
relationship for a thin-walled sphere, equation (1). The two pressure-time plots correspond either to a bladder emptying along the
relationship shown in (i) curve 1, or at constant volume shown in (ii ) curve 2. The tension and volume relationships are scaled by the lefthand side vertical axes and the pressure relationships scaled by the right-hand side verical axis. (B) Upper plot: Ex vivo experimental
pressure-volume curves from three bladders, labeled stiff, control, and compliant. The stiff and compliant curves were obtained
from obstructed bladders and the control from an unobstructed bladder. Lower plot: Derived tension-length (circumference) curves from
the above data using Laplaces law (see text for details); circumference was calculated from bladder volume assuming a spherical shape.
Source: Adapted from Ref. 28.
compliance: bladder capacity increases with age

whereas intravesical pressures are less variable; intravesical pressure itself is a function of bladder volume
and thus compliance would vary as the bladder fills.
Furthermore, compliance is a steady-state property
and measurements must only be made when any
stress-relaxation has fully subsided. Several
approaches have been used to account for these confounding factors. Normalization factors have been
introduced in urodynamic studies to correct for values
obtained in different size bladders (32,33) and ex vivo,
when nonlinear pressure-volume relationships have
been transformed into linear stress-strain relationships (28), when more direct comparison can be
made with data from tissue strips (Fig. 2B) (28,34).
Outflow tract obstruction has variable effects on
bladder compliance: brief periods have little effect
(35), but after longer periods compliance may either
decrease (36,37) or increase (28,34,38). These different
effects may result from the severity and duration of
the obstruction. Decreased compliance will raise
intravesical pressures for a given amount of filling
and so initiate a micturition reflex at lower volumes. If
maintained a significantly raised bladder pressure
may cause upper tract damage (39). Conversely, if
compliance is increased detrusor muscle contractions
will be less effective in raising pressure and thus make
voiding more difficult.
Stress Relaxation
Stress-relaxation is a viscoelastic feature of the whole
bladder and isolated muscle strips and manifests as a
partial reduction of stress (pressure or tension) after a
rapid change of strain (volume or length). This is of
benefit to the filling bladder as steady-state changes of
pressure are minimized during filling. With urodynamic measurements it means that if a bladder is
rapidly filled then changes to intravesical pressure
may be greater than during slow-fill. Thus, rapid-fill
would underestimate compliance, as it is a steadystate property. Measurement of pressure or tension
should only be made when any stress-relaxation has
finished. Stress-relaxation may reside from a rearrangement of the cellular and extracellular elements.
In the over-compliant obstructed bladder the extent of
stress-relaxation diminishes in the same proportion as
steady-state stiffness (34). Because the proportion of
extracellular material increases in these bladders this
suggests that the phenomenon may reside in both the
cellular and extracellular components.
Collagen and the Extracellular Matrix

The most significant extracellular components in
determining the passive properties of LUT tissues
are collagen (especially types I and III) and elastin
248
Fry
(40). In obstructed bladders collagen content is raised,

particularly in the detrusor muscle layer, with an
increased ratio of type III to type I reported in some
(4143). Collagen-I forms large fibers and predominates in tissues with high tensile strength, while
collagen III, forming smaller fibers, imparts increased
tissue flexibility and an ability to rearrange during
bladder filling (44), which may be of importance to
bladders undergoing hypertrophy. Collagen synthesis
by cells is upregulated by physical stretch, which may
provide the basic stimulus in bladder outflow obstruction (45). During bladder development the collagen
III:I ratio shows an inverse relation to compliance (46);
however, other geometrical factors, as mentioned
above, will also determine compliance so that changing collagen ratio per se is only one factor, among
many.
The nonenzymatic adduction (combination) of
sugar to protein (glycation) is associated with diabetes
and ageing and is a significant factor in damage to
extracellular and cellular proteins by promoting crosslinking and aggregation. The formation of advanced
glycation end-products increases tissue stiffness of
(47). However, several studies have shown that bladder compliance actually increases in animal models of
diabetes (48,49), so the role of glycation product
formation remains unclear.
THE ACTIVE PROPERTIES OF MUSCLES

IN THE LOWER URINARY TRACT
Interaction between contractile proteins in the muscle
cell generates active tension that manifests itself as
an increase of tension within the cell or a shortening of
the muscle. If a muscle generates tension without
shortening, this is termed an isometric contraction.
In a hollow organ, such as the bladder, such an

increase of tension would be manifest as a rise of
pressure (above). If the muscle shortens against a load,
the contraction is termed isotonic.
Isometric Contractions and Length-Tension

Relationships
All muscles exhibit a bell-shaped dependence for
active force on initial resting length, that is, there is
an optimum resting length, L0, at which active force is
maximal (Fig. 3A). However, this length-tension curve
extends over a greater range of initial lengths for
smooth compared with striated muscle and is particularly true of detrusor where passive extension much
beyond L0 does not greatly reduce force (50). Thus,
large changes in bladder circumference will still permit adequate force development by individual myocytes and, when normalized to L0, L-T relationships
are similar in control and pathological organs (51).
Isotonic Contractions and Force-Velocity

Relationships
In this case the muscle shortens against an opposing
load, and observation shows that the heavier the load
(and hence the force, F, opposing shortening) the
slower is the initial velocity of shortening, v. The
relationship between force and velocity was described
by Hill (52) and is shown in Figure 3B:
F a & v b F0 a & b
where a and b are constants. Note that where the curve

crosses the abscissa (F axis), the opposing load is so
large that the muscle cannot shorten, and contraction
becomes isometric. A characteristic of this curve is the
Figure 3 (A) Length-tension curve for a detrusor smooth muscle strip, in vitro. Active (peak twitch) force; passive, resting force and the
sum of the two forces (total) are shown for contractions elicited at 16-Hz stimulation. Muscle forces are measured over a range of resting
muscle lengths, normalized to the length (L0) at which maximum active force was generated. (B) Force-velocity curve for a shortening
smooth muscle strip subject to different loads, F. The load is normalized to the maximum load, F0, above which shortening can no longer
occur (i.e., an isometric contraction). Shortening rate is expressed in muscle lengths per second. The line is a fit of equation (2) and
extrapolated to the v and F/F0 axes.
extrapolation to a maximum velocity of shortening at

zero load, V0, as this is related to the maximum
frequency of cross-bridge cycling (see below). This
curve bears a resemblance, but only superficial, to
the pressure-flow plots obtained in urodynamics (53)
but have been used by some to estimate the contractile
state of the bladder (54). However, such extrapolations
should be treated with caution as the relationship
between pressure-flow measurements and forcevelocity determinations is not linear for the same
dimensional reasons as discussed above when considering Laplaces law.
The Contractile State

There is lack of agreement whether absolute force
(normalized to cross-sectional area) varies in samples
from normal and pathological bladders. This is important, as it is necessary to know if conditions such as
detrusor underactivity or overactivity are mirrored by
changes to the contractile state of detrusor muscle.
Alternatively, these conditions may be because of
other causes such as alteration to the proportion of
muscle in a bladder sample, the extent of functional
innervation, or the physical properties of the extracellular matrix. Therefore, in this respect care must be
exercised in the interpretation of experimental data, as
contractile responses evoked by agents such as muscarinic receptor agonists may give different responses
compared with nerve-mediated responses. Some animal models of bladder obstruction suggest contractile
failure (34,55), but not in all cases (28) and it is
possible that failure is a feature of later growth
when a decompensated, more compliant bladder is
generated by obstruction. The most obvious clinical
correlate for this is the valve bladderin which all
the clinical manifestations expected from the above
changes can be seen, and in a few patients, a progression may be demonstrable. It remains far more difficult to predict those in whom progression, or not, will
occur or those in whom the diseased bladder function
plateaus. With human detrusor there is little evidence
that detrusor contractility is different in normal and
overactive bladders (56).
Energetics of Contraction and Bladder

Blood Flow
Compared with skeletal and cardiac (striated) muscle,
smooth muscle exhibits high economy, as contractions
can be maintained for a relatively long period with a
lower ATP consumption than in the initial stages of
force development. However, to perform external
work (work = force ' distance), smooth muscle has
a lower efficiency than striated muscle. Efficiency is
the work produced per unit change of free energy for
the driving chemical reaction, that is, ATP hydrolysis,
and is about 5 compared with 20 kJ/mol for smooth
and striated muscle, respectively (57). One reason for
this decreased efficiency may be that in smooth muscle ATP is needed not just for cross-bridge cycling but
also to phosphorylate the myosin light chain (see
below) (58).
249
A particular feature of smooth muscle metabolism is that oxidative metabolism and lactate production occur together and their respective rates are not
correlated. In some smooth muscles, force development and O2 consumption are closely correlated,
whereas lactate production is associated with other
ATP-consuming processes such as the Na pump (59).
This has led to the suggestion that metabolism is
functionally compartmentalized, with O2 consumption above basal levels directed toward meeting the
needs for ATP consumption associated with contraction. The relevance of this metabolic division to detrusor muscle has not been tested.
A decrease in bladder blood flow occurs during
filling, and is associated with tissue hypoxia and
acidosis (60,61). It is assumed that these changes
occur because the bladder wall is stretched and the
blood vessels occluded. In the hypertrophied bladder,
angiogenesis does not keep pace with increased muscle growth so that such ischemic conditions would be
exacerbated. Tissue hypoxia has complex effects on
detrusor contractility, which may contribute to overactive behavior. In the short term, there is an increase
of detrusor contractility before a longer-term reduction (62). The increased contractility is probably
because of an intracellular acidosis that increases
muscle contractility, in contrast to striated muscle
(63). In the long term, the reduction of aerobic metabolism will limit the ability of the cell to regulate
intracellular [Ca2] and so generate the conditions
for spontaneous activity.
DETRUSOR SMOOTH MUSCLE: CONTRACTILE

MECHANISMS
Contractile Proteins and Intracellular Ca2
Smooth muscle cells of the bladder are spindleshaped, single nucleated cells organized into bundles
separated by connective tissue. The thin filaments are
composed of a- and b-actin, which are attached to
dense bodies on the cell membrane, and provide
binding sites for the myosin thick filaments. There
are four smooth muscle myosin isoforms (SM1A, 1B,
2A, 2B) that exhibit different contractile properties.
Adult bladders are composed of nearly equal amounts
of SM1B and SM2B (64). SM1 produces more force
than SM2; SM-A types are more slowly contracting
than SM-B. In obstruction, there is a shift to more
SM1A, which results in slower and more forceful
contractions to overcome increased resistance (65).
An increase in the sarcoplasmic [Ca2], [Ca2]i,
from a basal level of 50 to 100 nM is required to
initiate detrusor contraction; half maximal activation
is achieved at about 1 mM (66). The source of Ca2 can
be extracellular, via L- and T-type Ca2 channels
(67,68), or from intracellular stores (69). Release from
intracellular stores may be separately mediated by
activation of IP3 receptors, as it can be blocked by
receptor inhibitors, or via ryanodine receptors (70).
The increase in [Ca2]i is transient and Ca2 are either
removed from the cell via Na/Ca2 exchange (71), or
reaccumulated in intracellular stores via a SERCA
250
Fry
of ROCK activity, such as Y-27632 and HA-1077,

attenuate agonist-induced contractions, but do not
affect depolarization-mediated [with high (KCl)] contractures (75), which suggests that the rho-kinase
pathway plays a role in the contractile state of the
bladder.
Detrusor Activation and Muscarinic Mechanisms
Figure 4 Schematic diagram of myosin activation (phosphorylation, P) and inactivation (dephosphorylation) by the calciumcalmodulin complex and associated kinase and phosphatase
reactions.
pump; the activity of the latter is modulated by

intracellular proteins, such as phospholamban (72).
As with other smooth muscles, the contractile proteins
are activated by phosphorylation of myosin by a
myosin light chain kinase (MLCK), which in turn is
activated by a Ca24-calmodulin complex. Relaxation
will occur if the myosin light chain is dephosphorylated, by a myosin light chain phosphatase (MLCP).
The sensitivity of the contractile system can therefore
be altered by modulating the activities of MLCK or
MLCP. A schematic diagram of contractile activation
is shown in Figure 4. Intracellular messengers that can
modulate the activity of these enzymes and hence
contractile activity, in response to receptor activation
by extracellular neurotransmitters and other signaling
compounds, are described below.
MLCH activity can be decreased itself, being
phosphorylated via a number of kinases including
CaM kinase II, mitogen-activated protein (MAP)
kinase, cAMP-dependent kinase (protein kinase A,
PKA), and p21-activated kinase (73). An example of
the relevance of such modulation is by consideration
of the action of caffeine, application of which causes
near-maximum release of Ca2 from intracellular
stores, but evokes little or no tension. The absence of
contractile activation is probably through its actions as
a phosphodiesterase (PDE) inhibitor that will enhance
levels of intracellular cAMP, and hence activation of
PKA.
MLCP activity can also be reduced by phosphorylation, which increases the Ca2 sensitivity of the
contractile system. Of significance is inhibition of
MLCP activity by rho-associated kinase (ROK or
ROCK) (74), which in turn is activated by small G
proteins of the rho-family. In detrusor two isoforms
of ROCK (I and II) have been identified (74). Inhibitors
Acetylcholine (ACh) is released not just from parasympathetic and somatic motor nerves to smooth and
skeletal muscle targets, respectively, but also from
nonneuronal sources such as the urothelium. In the
normal human bladder ACh is the sole neurotransmitter eliciting contraction, that is, there are no atropine-resistant contractions, while in many pathologies
associated with bladder overactivity ATP is an additional activator (56,76,77). With animal bladders,
except for old-world monkeys, a dual muscarinicpurinergic activation is present.
Muscarinic receptors are one of the main targets
for ACh and they are expressed throughout the LUT.
There are five subtypes of muscarinic receptors based
on molecular (m15) and pharmacological (M15) characteristics. In detrusor, immunoprecipitation analyses
show that m2 and m3 subtypes are expressed, with m2
receptors in three- to ninefold excess (78). In normal
human detrusor the minor M3 fraction is responsible
for contractile activation (79). More recently, the role of
M2 receptors has been reevaluated and it has been
proposed that M2 receptors exert a more significant
role in certain pathological conditions (e.g., denervated
or hypertrophied bladders), or when M3 receptors are
desensitized (80). One possibility is that M2-dependent
actions may derive from the urothelium. It has also
been proposed that M2 receptors in the normal bladder
facilitate the function of other receptors, such as M3
receptors, or counteract the relaxant effect of b-adrenoceptor agonists (81,82). However, there is little difference on overactive bladder function between the
effect of more selective M1/M3-selective blockers and
those with a less specific action, although the side
effect profiles are different (83).
M3 receptors are coupled to Gq/11 proteins,
which importantly activate the enzyme phospholipase
C (PLC) to convert membrane phosphoinositides to
the second messengers, inositol trisphosphate (IP3)
and diacylglycerol (DAG). IP3 in turn releases Ca2
from intracellular stores, after binding to an IP3 receptor, to activate the contractile proteins. There is a body
of experimental evidence to support the relevance of
this pathway in detrusor: muscarinic agonists generate a rise in [Ca2] independent of membrane potential, and its release is blocked by IP3 receptor blockers
(70); the potency of the muscarinic receptor agonist
carbachol is reduced by other IP3 receptor blockers
such as heparin and PLC inhibitors (84). Moreover
inositol phosphate production mirrors tension generation in detrusor strips exposed to muscarinic agonists
(85). However, other work suggests that this is not the
exclusive pathway, in part because of the relative
ineffectiveness of other PLC inhibitors to reduce
carbachol-induced tension. It has been suggested that

activation of the rho-kinase pathway and of protein
kinase C by G protein activation and DAG, respectively, reduces the activity of myosin light chain
phosphatase to increase the Ca2 sensitivity of the
contractile proteins; the rise of intracellular Ca2 is
explained by activation of nonspecific cation channels
coupled to L-type Ca2 channel activation (86). Several attempts to reconcile this controversy have been
attempted, and it may be that there are considerable
species differences in the relative importance of inositol phosphate and other pathways (87).
M2 receptors are coupled to Gi protein that
reduces cAMP production by its influence on adenylate cyclase activity. It has been proposed that M2
receptor activation inhibits the effect of other agonists
that increase cAMP production, such as b-receptor
stimulation.
Recent reviews summarize the role of muscarinicdependent pathways in the bladder and their relevance
to contractile activation (79,88): M3 pathways are summarized in Figure 5 and M2 pathways in Figure 6.
Presynaptic muscarinic receptors have also been
described where it is proposed that they modulate
transmitter release in either a negative (M2/4) or positive (M1) feedback mode. Knockout experiments indicate that M 4 , rather than M 2 , receptors inhibit
transmitter release (89).
Desensitization of muscarinic receptors results in
detrusor smooth muscle being less sensitive to nerve
251
Figure 6 Schematic diagram of the intracellular signaling pathways activated after muscarinic (M2) and adrenergic (b3) receptor activation. Abbreviations: Gi/o1 and Gs, G proteins; camp,
cyclic adenosine monophosphate; PKA, protein kinase A.
stimuli. This is mediated by phosphorylation of the

muscarinic receptor by guanosine phosphatebinding
G proteincoupled receptor kinase (GRK) (90); m2 and
m3 GRK2 mRNAs have been described. Protein expression of GRK2 is significantly reduced in detrusor from
patients with benign prostatic hyperplasia (91) and
may therefore contribute to detrusor overactivity.
Purinergic Transmission
Figure 5 Schematic diagram of the intracellular signaling

pathways activated after purinergic (P2X) and muscarinic (M3)
receptor activation, by adenosine triphosphate (ATP) and acetylcholine (ACh), respectively. Abbreviations: IP3, inositol trisphosphate; PLC, phospholipase C; PIP 2 , phosphatidylinositol
4,5-bisphosphate; Gq/11, a G protein; s.r., sarcoplasmic reticulum; DAG, diacylglycerol; PKC, protein kinase C; rho-A, Ras
homologue gene family, member Aa small GTPase protein;
rho-kinase, rho-associated, coiled-coil containing protein kinase
1 (ROCK); CICR, Ca2-induced Ca2 release; Cam, calmodulin;
MLC, myosin light chain (P-phosphorylated form).
The role of ATP as an extracellular signaling molecule

is now well accepted. In most mammalian species,
ATP is coreleased with ACh from parasympathetic
nerves, which activates purinergic receptors to initiate
detrusor contraction. This is in contrast to healthy
human bladders where contraction is predominately
mediated by ACh. However, purinergic nervemediated contraction is increased in a number of bladder
pathologies including hypertrophy, idiopathic overactivity, interstitial cystitis, and neurogenic damage. The
increase in atropine resistance in bladder disorders
may be because of increased sensitivity of detrusor
cells to ATP, increased release from motor nerves, or
reduced ATP hydrolysis within the neuromuscular
junction. The three possibilities have been investigated using human detrusor samples from stable and
overactive bladders. Only the last hypothesis was
considered a possibility as ectonucleotidase (ATPase)
activity in overactive human bladder samples is significantly reduced (92).
ATP binds to purinergic, P2, receptors that are in
turn divided into P2X and P2Y families on the basis of
pharmacological and molecular studies (93,94). P2X
receptors are ionotropic ligandgated nonspecific
252
Fry
cation channels, while P2Y receptors are metabotropic

G protein coupled. Currently, seven P2X receptors
subtypes have been cloned and characterized (P2X17).
P2X1 labeling is present on detrusor muscle (95) and
activation generates an inward, depolarizing current
sufficient to activate L-type Ca2 channels to generate
an action potential (AP) and Ca2 influx to initiate
contraction (96).
There are eight subtypes of P2Y receptors
(P2Y 1,2,4,6,1114 ) linked either to G q/11 (P2Y 1,2,6 ),
Gi (P2Y1214) or several proteins: Gq/11/Gi (P2Y4);
Gq/11/Gs (P2Y11) (97,98). P2Y receptors, although not
a specific subtype, have been implicated in relaxation
of smooth muscle, possibly via cAMP-dependent PKA
activity.
Metabolism of Neurotransmitters in the

Neuromuscular Junction
A general principle of neuromuscular transmission is
that neurotransmitter concentrations are locally high
and postjuntional receptors have a relatively low affinity. Thus, the transmitter can fully activate and rapidly
dissociate from its receptor. This, coupled to an efficient enzymatic removal of the transmitter, ensures
that the action is transient and thus in a muscle
contributes to a transient activation of contraction.
ACh is broken down by acetylcholinesterases
(AChE), and the chief product, choline, is taken up
by the nerve terminal to act as a substrate for further
transmitter production. Several agents, such as physostigmine, attenuate AChE activity and prolong and
enhance detrusor contractile activation. ATP is also
broken down by extracellular endonucleotidases, and
in human detrusor where ATP is functionally ineffective as an excitatory transmitter, it is assumed that it
is completely hydrolyzed before it can activate the
muscle cell. This is corroborated by the observation
that in human detrusor samples displaying atropineresistant contractions, endonucleotidase activity was
reduced (92).
Nitrergic Mechanisms
There are three nitric oxide synthase (NOS) isoforms,
encoded by separate genes, named for the tissue from
which they were first isolated from, or the order in
which the genes were cloned: neuronal NOS (nNOS,
NOS 1); inducible NOS from macrophage (iNOS,
NOS 2); and endothelial NOS (eNOS, NOS 3); there
is also a form of nNOS that is localized within
mitochondria (mtNOS). Each of these enzymes can
be found in every cell type of the LUT. The expression
of several factors determine whether there is a relaxatory effect to nitric oxide (NO): NOS; the NO receptor, soluble guanylate cyclase (sGC); and PDE, the
enzyme that degrades cGMP, the product of sGC
activity. There are 11 PDE isoforms so far identified:
PDE15 are described to be present in the bladder
(103). PDE5-selective inhibitors such as sildenafil
(Viagra) and vardenafil are structural analogs of
cGMP and competitively inhibit PDE. NO donors
have a small relaxatory effect on detrusor (104), but
PDE inhibitors, such as vardenafil, relaxed precontracted detrusor (105), suggesting a relatively high
endogenous PDE activity. These findings are supported by the beneficial effects of PDE5 inhibitors
with LUTS, when used to treat erectile dysfunction
(106). The cellular pathways in relaxation that are
mediated by NO are shown in Figure 7.
Nicotine and Nicotinic Receptors

Transmission at parasympathetic pelvic ganglia is
mediated largely by nicotinic receptors, and these
receptors may also be involved at other sites in the
LUT. Nicotine can evoke ACh release from motor
OTHER MODULATORS OF DETRUSOR

CONTRACTILE FUNCTION
Adrenergic Mechanisms
There are five distinct adrenergic receptor typesa1,
a2, b1, b2, and b3with each being further divided into
subtypes. All three b-subtypes are expressed in detrusor muscle with the b3-receptor the most highly
expressed (99). Activation of b2- and b3-receptors can
cause significant relaxation of detrusor smooth muscle
(100). Thus, there is much interest in specifically
targeting b3-receptors for treatment of detrusor overactivity (101) and for producing an agent suitable for
human use. The b-receptor-mediated relaxatory mechanism is thought to involve a rise in cAMP (Fig. 6) and
modulation of large conductance Ca2-activated K
channels. There is a comprehensive survey of the in
vitro and in vivo actions of b-receptor modulators
(102).
Figure 7 Schematic diagram of the intracellular signaling pathways activated after NO (nitric oxide) exposure. Pathways that
cause contraction are shown by solid lines, those that cause
relaxation by dotted lines. Abbreviations: GTP, guanosine triphosphate; cGMP, cyclic guanosne monophosphate; PDE-5,
phosphodiesterase, type 5.
nerves, which itself may be upregulated by tachykinins acting via NK2 receptors (107). This contractile
effect of nicotine is blocked by the nicotine receptor
antagonist hexamethonium. The nicotinic receptor is a
pentameter of subunits, with 17 different subunits
identified so far (a110, b14, g, d, e). Mutation studies
suggest that the a3 and b4 subunits are required for
bladder function (108).
Adenosine Receptors
While purinergic, P2, receptors have received much
attention, the pyrimidine P1 receptor family is less
well studied. Four subtypes have been clonedA1,
A2A, A2B, and A3and all are G proteincoupled
receptors. A1/3 receptors are negatively coupled to
adenylyl cyclase activity, while A2 receptors are positively coupled. Adenosine relaxes bladder preparations precontracted by carbachol through an A 2
(possibly A2B) receptor mechanism (109). Adenosine
also reduces nerve-mediated contractions, possibly by
reducing neurotransmitter release via A1 receptors (M
Hussain, Y Ikeda, CH Fry, unpublished data). The
relevance of P1-receptor activation is evidenced by the
fact that adenosine is a breakdown product of ATP by
the action endonucleotidases. Thus, P1-receptors exert
a negative-feedback effect on the excitatory action of
the neurotransmitter ATP.
Neuropeptides
Various neuropeptides, including calcitonin gene
related peptide (CGRP), substance P, neurokinin A,
vasoactive intestinal polypeptide (VIP) and pituitary
adenylate cyclase-activating peptide (PACAP), are
released in the bladder from efferent and afferent
(110) nerves and urothelial cells. These peptides may
also be released by noxious stimuli and promote
inflammation. CGRP inhibits spontaneous activity
and relaxes ACh-induced tension. Tachykinins (substance P and neurokinin A) are prototypic of endogenous agonists of specific G proteincoupled receptors:
tachykinin NK1 and NK2. NK1 receptors have been
found in blood vessels and the urothelium of all
species thus far examined, whereas their expression
in muscle cells seems restricted to only a few animal
species [rats, guinea pigs (111)]. The stimulation of
NK1 receptors activates PLC, leading to inositol phosphate accumulation, and is linked to smooth muscle
contraction. Substance P also stimulates detrusor
smooth muscle. NK2 receptors are localized on detrusor muscle of all mammalian species studied, including humans (112). The stimulation of NK2 receptors is
coupled to inositol phosphate accumulation and has a
contractile effect on the bladder. VIP and PACAP
receptors (VPAC1/2 and PAC1) are G proteincoupled
receptors on neurons and smooth muscle. They are
coupled to several signal transduction pathways,
including activation of adenylate cyclase and elevation of cyclic guanylate monophosphate levels in
tissues (65). VIP release evokes relaxation of detrusor
and urethra smooth muscle.
253
Endothelin
The three isoforms of endothelin (ET-1,2,3) mediate
their actions via ETA and ETB receptors (113): ETA
receptors have a dominant role in human detrusor,
but their function is unclear. Because they initiate only
a slow rise of tension, it has been proposed that they
may potentiate nerve-mediated responses (114). ETA
receptors might contribute to premicturition contractions, and receptor antagonists, such as YM598, may
have ameliorating effects in patients with bladder
overactivity associated with obstruction (115).
SPONTANEOUS ACTIVITY
Significance of Spontaneous Activity
Nonneuronal contractions, resistant to the neurotoxins
such as tetrodotoxin (TTX), occur in the detrusor and
have several synonyms: intrinsic, autonomous, phasic,
rhythmic, nonmicturition, spontaneous or transient
activity, and micromotion. This activity was first
reported by Sherrington in cats, as transient rises in
bladder pressure seen during filling. Spontaneous
smooth muscle contractions could also stimulate afferent fibers to generate centrally mediated reflex bladder contractions.
Investigation of the significance of phenomena,
such as spontaneous activity, in human bladder function is often difficult to gauge for practical reasons and
animal models can provide valuable insight. In neonatal rats, spontaneous, TTX-resistant, activity is
absent at birth, increases in amplitude by week two,
then changes from high-amplitude low-frequency to
adult-like low-amplitude high-frequency activity by
week six (116) (Fig. 8). At this time, micturition in the
neonate is mediated by a somato-bladder spinal reflex
pathway, activated by the mother licking the perineum. As development progresses, this primitive reflex
Figure 8 Pressure transients recorded ex vivo in bladders from

a normal neonatal rat (upper trace); a normal adult rat (middle
trace); an adult rat subjected to spinal cord transection to obtain
a functional bladder outlet obstruction (lower trace). Source:
From Ref. 118.
254
Fry
is replaced by supraspinal mechanisms that control

brain-to-bladder reflexes and voluntary voiding.
Thus, developmental changes in the central control
of voiding occur in concert with changes in peripheral
neurotransmission and the spontaneous properties of
the bladder smooth muscle (117). In bladders from
spinal cordtransected (SCT) rats (118) and outletobstructed (119) bladders, there is a reemergence of
high-amplitude low-frequency spontaneous activity
similar to that observed in neonatal bladders. The
question therefore arises if the pattern of spontaneous
activity is modulated by peripheral nervous control in
the human bladder.
Origin of Spontaneous Activity

Detrusor overactivity is likely to be a multifactorial
process with several possible origins.
1.
2.
3.
4.
5.
The neurogenic hypothesis: Reduced peripheral or

central inhibition increases activation of the micturition reflex and contractions associated with
detrusor overactivity (120).
The myogenic hypothesis: Changes in the excitability
and coupling of smooth muscle cells with other
myocytes or interstitial cells leads to the generation of uninhibited contractions (121).
The urotheliogenic hypothesis: Changes in the sensitivity and coupling of the suburothelial myofibroblast network enhances spontaneous detrusor
activity (122).
The autonomous hypothesis: Structures within the
bladder wall coordinate to drive spontaneous
contractions, which somehow become enhanced
in pathology (123).
A leak of transmitter from motor fibers causes
small local contractions or increases tone (124).
Neurogenic detrusor overactivity, explained by

hypothesis 1, may pertain to a distinct subset of
patients. Muscle samples taken from such bladders
are similar to those from stable bladders (56).
Increased spontaneous activity is recorded from
isolated muscle strips and cells obtained from other
overactive (e.g., idiopathic) bladders. Contractions are
resistant to neurotoxins but labile to Ca2 channel
blockers or K channel openers (65). While upregulated activity in isolated cells may be present, this
alone will not explain spontaneous activity in multicellular preparations that requires intercellular coordination of responses within and between muscle
bundles. Two possibilities have been proposed for
increased intercellular communication.
1.
Increased intercellular coupling through gap junctions. Gap junctions are composed of the connexin
(Cx) family of proteins. In human detrusor,
expression of the main intermuscular connexin,
Cx45, is actually less in samples from idiopathically overactive bladders, correlated with a higher
gap junction resistance in such samples (125).
Other groups, however, suggest that another connexin isoform, Cx43, is upregulated in overactive
bladders (125).
2.
Cx43 antibodies label interstitial cells in the detrusor layer, which exist between muscle bundles.
These cells are also characterized by their labeling
for the tyrosine kinase receptor protein c-kit and
the generation of spontaneous and carbacholevoked Ca2 and electrical activity (126). It is
postulated that rather than initiate spontaneous
activity in the detrusor syncitium, interstitial cells
modulate its activity (127), possibly by coordinating activity in different muscle bundles. However,
these cells could form a control point for regulation of spontaneous activity; they are innervated
by afferent nerves labeling for NOS (128), and
they also express cGMP activity. However, the
origin of spontaneous activity in muscle bundles
remains unclear.
The urotheliogenic hypothesis is premised on a

urothelialmyofibroblast network connected by gap
junctions supporting pacemaker-driven spontaneous
activity where bladder pathology, because of spinal
cord transection for example, leads to an upregulation
of gap junctions in the urothelium and myofibroblast
network. This, in turn, leads to an increasingly functional myofibroblast syncytium with focal pacemaker
activity that drives spontaneous contractions.
The autonomous hypothesis takes into account
the potential role of interstitial cells within the bladder
wall but does not clearly indicate a specific mechanism
to generate spontaneous activity. It probably represents
a qualitative description of a combined myogenic/
urotheliogenic hypothesis. As with the urotheliogenic
hypothesis spontaneous bladder contractions are
enhanced by low-dose muscarinic stimulation, via M3
receptors on suburothelial myofibroblasts and several
agents such as ATP, substance P, nicotinic receptor
agonists, noradrenaline, and NO (129).
SECRETORY AND SIGNALING PROPERTIES OF

THE UROTHELIUM/SUBUROTHELIUM
Urothelium/Nerve Interactions
The urothelium releases many chemical factors, such
as ATP (9), ACh (130), NO (131), and substance P
(132), in several cases in response to stimuli such as
stretch and changes in local osmolarity (9,133). The
urothelial/suburothelial region is well supplied with
sensory neurons that label for receptors to some of
these releasates and suggests that these neurones
interact with the urothelium to detect changes in
bladder fullness. Urothelial cells also express receptors typically found on afferent nerves, such as purinergic receptors (134,135), members of the vanilloid
receptor group TRPV1,2,4 (136), TRPA1 (137), TRPM8
(138), B1,2 bradykinin receptors (139), and adrenergic
receptors (131,140), suggesting a complex interaction
between urothelium and sensory nerves.
ATP Release
Most work has been applied to ATP release: it is
hypothesized that when the urothelium is stretched
ATP is released and targets purinergic (P2X3) receptors on sensory neurons. This hypothesis is inferred
from data using P2X3 knockout mice, which exhibited
a reduced afferent firing and micturition reflex
(141,142) on bladder filling. A layer of suburothelial
interstitial cells (ICs, myofibroblastssee below) is
also in intimate association with these nerve endings,
and ICs also generate excitatory responses to exogenous ATP through P2Y receptor activation. It has been
proposed that these cells modulate the urothelium/
neurone interaction (143,144). In pathologies associated with bladder overactivity and enhanced bladder
sensation ATP release, IC number and neuronal P2X3
labeling are increased, and the latter is decreased
when overactivity is reduced, as with bladders treated
with botulinum toxin (Botox-A) (145147). Therefore,
it may be further hypothesized that increased release
of urothelial ATP contributes to afferent sensitization
through enhanced activation of suburothelial nerves
and/or ICs.
Acetylcholine Release
ACh is also released by the urothelium in response to
stretch, increasing with age and estrogen status
(130,148,149). Release may be through a nonvesicular
mechanism (150), and thus is different from vesicular,
neural release from parasympathetic nerves in stimulating bladder contraction. Urothelial-derived ACh,
like ATP, may also have a role in promoting sensory
activation. Hence, it is hypothesized that anticholinergics exert their effect not on detrusor muscle but on
urothelial muscarinic receptors. All five subtypes of
muscarinic receptors are expressed throughout the
urothelial layers (151). There was specific localization
of the M2 subtype to the umbrella cells and M1 to the
basal layer, with M3 receptors more generally distributed. There is some evidence that receptor subtype
ratios may change (increased M2:M3) in detrusor
overactivity but the significance of this remains
unclear (152). The mechanism by which ACh modulates urothelial secretory activity is also unclear, but
blockade of urothelial muscarinic receptors with atropine inhibits stretch-induced ATP release (153).
Stretch-released ACh may therefore act in a feedback
mechanism to induce urothelial ATP release.
Suburothelial Interstitial Cells

A network of interstitial myofibroblast-like cells (ICs)
has been identified in the suburothelium; these cells
have demonstrable cell markers including vimentin
(an intracellular filament protein seen in cells of
mesodermal origin) and c-kit (a cell surface marker).
They are connected by gap junctions containing
connexin, Cx43, and are closely apposed to unmyelinated nerve endings (154,155). ICs may mediate urothelium-derived ATP responses by generating
propagating depolarizing Ca2 waves across the IC
network, through P2Y6 activation, thus amplifying
local responses to exogenous ATP (144,155). The ability of myofibroblasts to form functional networks can
255
involve mechanisms other than via gap junctions: if

two isolated cells are pushed together, each cell demonstrates enhanced responses to ATP without the
obvious formation of gap junctions. The cellular adhesion molecule, Cadherin-11, has been demonstrated
on myofibroblast membranes (156) and the activation
of the membranes by intercellular adhesion may offer
a mechanism. This enhancement of response is abolished by the c-kit receptor ligand glivec.
These purinergic responses are mimicked by
extracellular acidosis and attenuated by capsaicin
and NO donors. The latter effect is in keeping with
the demonstration of NOS/GC activity in these cells
(128). The effect of extracellular acidosis and capsaicin
is of significance as it proposes a mechanism for the
response of the bladder wall to local ischemiaa
feature of bladder fillingespecially in the presence
of outflow obstruction or reduced compliance
(60,157,158).
Muscarinic M2 and M3 receptor labeling has also
been localized to suburothelial ICs and is increased in
samples from idiopathic overactive bladders and
painful bladder syndrome (159). Isolated ICs do not
respond to exogenous muscarinic receptor agonists by
a rise of intracellular [Ca2], so the intracellular signaling mechanism(s) remains unknown (143). However, these observations may be significant as sensory
C-fiber and Ad-fiber afferent firing in response to
bladder filling was reduced by M3-selective antimuscarinics (160), or less-selective agents such as oxybutynin (161) or tolterodine (162).
Interactions Between Urothelium/Suburothelium

and Detrusor
Several lines of evidence indicate that the urothelium/
suburothelium directly affects detrusor function,
through both inhibitory and excitatory mechanisms.
With in vitro detrusor preparations the potency and
maximum contractile responses to ACh, but not KCl,
are reduced if the urothelium is intact. The active
substance is unknown, but diffusible, and may
involve activation of a b-adrenoreceptor (163,164). A
similar phenomenon is present in ureter, which in this
case may involve a cyclo-oxygenase product (165).
Optical imaging of transverse sections of the
bladder wall shows propagating Ca2 and membrane
potential waves in the suburothelial layer in response
to physical stretch or very low concentrations of the
muscarinic agonist carbachol. After a delay these
responses may spread to the detrusor layer initiating
activity there (166) (Fig. 9A). Optical imaging of
bladder sheets, with the urothelial surface uppermost,
showed similar propagating waves of Ca2 transients
but only on the surface where the mucosa was intact
(Fig. 9B). These waves may be initiated by stretch,
muscarinic agonists, or purines such as UTP. UTP was
chosen as the purine elicits excitatory responses from
suburothelial ICs, but not directly from detrusor muscle. Isolated strips of detrusor generate spontaneous
activity, especially if the urothelium/suburothelium is
intact, and such activity is also upregulated by exogenous UTP (167). These observations are of interest as
256
Fry
Figure 9 (A) Upper panel: Photograph of a section through the bladder wall, with a superimposed grid. Lower panel: Isochronal maps
for the spread of Ca2 transients after mechanical focal stimulation of the suburothelial layer at the red star. The darker the shading the
longer the isochrones after the initial evoked Ca2 transient. Conduction initially occurs along the suburothial space, and only after a
delay is there a limited wave in the detrusor layer (left, single-headed arrow). A separate activation source is also present in the detrusor
layer (bottom, double-headed arrow). (B) Upper panel: Tension transients recorded from bladder sheets dissected from a spinal cord
transected (top) and normal (bottom) rat bladders. Lower panel: Ca2 transients recorded from a spinal cordtransected rat bladder from
which half the mucosa was removed (solid, black box region). Separate transients are recorded simultaneously from each grid square.
An individual transient from each half of the preparation is also shown. Source: From Refs. 70 and 122.
they indicate that spontaneous activity could originate

in the suburothelium region of the bladder wall.
Furthermore, the specificity of site of action of some
activators offers particular targets that may modulate
such activity.
Thus, there is evidence that a suburothelial population of ICs is an intermediate stage in the sensory
response to bladder wall stimuli. It acts as a variable
amplifier, mediating signals between the urothelium
and sensory afferents or the detrusor smooth muscle
layer. Moreover, the increase in myofibroblast numbers in conditions associated with bladder overactivity
(117,168) suggests that this cell layer may offer a
therapeutic target to alleviate the condition. Agents
that modulate IC activity, such as glivec, also reduce
spontaneous activity in the bladder (169,170). These
agents offer an alternative therapeutic avenue to manage detrusor overactivity.
ELECTRICAL ACTIVITY AND ACTION

POTENTIALS
Cellular Activation and Propagation
Detrusor smooth muscle is an electrically excitable
tissue capable of generating evoked and spontaneous
APs (Fig. 10). The function of electrical activity
remains unclear in the human bladder, but could

have two functions.
1.
2.
To initiate cellular events such as contractile activation

To propagate to adjacent regions of the tissue and
so coordinate activity of a multicellular tissue
The role of APs in initiating and maintaining

contractile function in detrusor muscle remains equivocal, as many excitatory neurotransmitters have significant actions that are independent of changes to the
membrane potential. However, the presence of electrical activity means that APs should at least exert a
modulatory role over contractile function, and may
assume a greater significance during certain bladder
pathologies when ionotropic neurotransmitters, such
as ATP, assume a more important role.
It is unclear whether APs propagate extensively
throughout detrusor tissue. In true functional syncitia,
such as myocardium, APs propagate from cell to cell
through gap junctions, composed of different isoforms
of the protein connexin (Cx), and concentrated in
intercalated disks. Gap junctions, as observed under
the electron microscope, are sparser in detrusor. This
has led many to conclude that APs do not freely
propagate. However, electrophysiological experiments
show electrical coupling between cells, and the
257
Figure 10 (A) APs recorded from an isolated human detrusor cell. The major currents contributing to the different phases of the AP are
shown. (B) Voltage-clamp traces from an isolated human detrusor cell. Membrane current is recorded in response to a step
depolarization from !60 to 0 mV. Inward current is carried by an influx of cations and outward current by an efflux of cations. Two
traces are superimposed: in the absence and presence of the L-type Ca2 channel blocker verapamil (10 mM). Note both inward current
(mainly Ca2 entry through L-type Ca2 channels) and outward current (mainly K efflux through Ca2 activated BKCa channels) are
attenuated by verapamil.
connexin isoform Cx45 has been identified (171). The

relatively poor electrical coupling, compared with
detrusor, and the failure of Cx45 to be found in intercalated disk-like structures may account for the relative
difficulty in observing gap junctions in detrusor.
Ion Channels
The membrane potential is sufficiently negative (!40
to !50 mV) for regenerative APs to be initiated. The
AP upstroke phase is carried by Ca2 influx, predominantly through L-type Ca2 channels, and repolarization is mediated by K efflux through several K
channels (67,172). Such Ca2 influx is sufficient to
elicit further release from intracellular stores to sustain contractions. T-type Ca2 channels have also been
described in detrusor muscle and the proportion of
total inward Ca2 current is increased in cells from
overactive bladders (173). Because T-type channels are
activated at more negative membrane potentials, they
may contribute to increased spontaneous activity in
the overactive bladder. Several receptor modulators
that alter detrusor contractility also affect the L-type
Ca2 current. Antimuscarinic agents such as propiverine attenuate L-type Ca2 current (174), an effect
probably mediated via M3 receptors. b-agonists also
attenuate Ca2 current by a cAMP/ protein kinase
Adependent mechanism (175).
The most significant K channel in detrusor is
the Ca2-activated large conductance K channel
(BKCa). This channel has physiological roles in determining membrane potential, AP repolarization (176),
and regulating contractile events (70): channel opening is coupled to intracellular Ca2 sparks emanating
from intracellular Ca stores via ryanodine receptors
(176). Outward current is also modulated by Ca2
current influx through L-type and T-type Ca2 channels. In the former case this has been proposed as a
mechanism to regulate Ca2 influx into the myocyte
(70), and in the latter case as a basis for spontaneous

fluctuations of membrane potential (177). Reduction
in BK channel activity may contribute to myogenic
bladder overactivity, as deletion of the slo-gene that
encodes for the channel protein enhances muscle
sensitivity to cholinergic and purinergic agonists
(178); conversely injection of slo-cDNA reduced overactivity (179). BK channel activity is regulated by
phosphorylation of the pore-forming a-subunit, or
associated proteins (180), and affords a mechanism
whereby cAMP and cGMP, through PKC, can regulate
channel function. Conversely, the Ca2-dependent
phosphatase, calcineurin, decreased BKCa conductance (181) so that overall Ca2 exerts a complex
control of channel function.
Intracellular ATP-gated K channels (KATP) have
also been described in detrusor smooth muscle, and
channel openers hyperpolarize the cell and reduce
spontaneous activity. A problem with the use of
these channel modulators is that of tissue specificity,
as many are as potent, if not more, in generating
similar responses in vascular smooth muscle.
Stretch-activated channels could serve a dual
purpose in the detrusor myocyte: to permit cation
influx to depolarize the cells and thus cause contraction to counter an initial stretch; and to initiate intracellular signaling cascades that may initiate cellular
reconfiguration or growth (182,183). Physical stretch
of detrusor myocytes opens nonselective cation channels, depolarizes the cell, and augments Ca2 influx
through Ca2 channels (184). However, stretch also
increases K channel activity (185). The net effect
would be to increase spontaneous activity.
THE BLADDER BASE

The bladder base consists of the trigone, ureterovesical junction, and deep detrusor, and the anterior
bladder wall may be considered to form a functional
258
Fry
entity to provide an effective reflux mechanism as

well as control urine outflow during filling and voiding (186). It is defined as the triangular region between
the ureteral orifices and the bladder outlet and plays a
crucial role in ureterovesical function, continence and
micturition. The trigone is believed to develop, with
the ureter, from an outgrowth of the mesonephric
duct, with a common mesodermal origin of the vesical
neck musculature, the trigone, and the ureterovesical
junction. More recent studies, however, suggest that
the trigone is formed predominantly from bladder
muscle, and the contribution from ureteral longitudinal fibers at the lateral edges is more limited (187).
In general, the bladder base is thought to provide a rather stable structure against which the bladder dome can contract and relax during the
micturition cycle. This might be the reason for the
relatively high amount of connective tissue and greater spontaneous activity within individual myocytes,
compared with detrusor. The spontaneous activity is
mainly driven by ion fluxes through membrane
L-type Ca2-channels and Ca2-actvated Cl! channels. Extensive gap junction coupling ensures electrical propagation and coordination of function
throughout the tissue (188).
The bladder base and trigone represents an area
of dual sympathetic/parasympathetic innervation.
Both systems significantly control contractile function
(102,189), demonstrated from experiments using
adrenergic and muscarinic receptor agonists and
antagonists. The a1D-adrenoceptor seems to be the
most abundant subtype in humans; muscarinic receptor representation is similar to the in detrusor (102). It
is generally agreed, through animal studies, that sympathetic activation is active during the storage phase
and induces contraction of the bladder base, as well as
the internal urethral smooth muscle sphincter. The
role of the parasympathetic innervation is more controversial, even that there may be a relaxatory component through the generation of NO during micturition
(190). However, there is considerable synergy of
action between the adrenergic and muscarinic systems
in the trigone, with respect to contractile activation.
The two systems probably act through different cellular pathways; the adrenergic system primarily operates through Ca2 sensitization of the contractile
machinery, while muscarinic-dependent activation is
a [Ca2]i-dependent process (189,191). This synergistic
mechanism may contribute to the prevention of urinary reflux into the ureter and increase outflow closure pressure, as the trigone is sited to regulate both
functions.
However, during micturition the trigone may
relax and cause a funneling effect to facilitate voiding. As in the urethra, animal experiments have shown
that NO is the key relaxing factor in the bladder base
(190). NO effectively relaxes isolated smooth muscle
preparations from the outflow region, suggesting that
it may be involved in the decrease in intraurethral
pressure observed at the start of normal micturition.
This NO-based relaxation provides an effective mechanism to allow the bladder base and trigone to switch
from a closed to open state, especially in combination
with the adrenergic control of the Ca2 sensitization of

the contractile machinery.
MUSCLES OF THE URETHRA

Role of the Musculature
During voiding, relaxation of the bladder outlet precedes detrusor contraction and during filling the
outlet is contracted. Several processes contribute to
these functions, mediated through urethral smooth
and skeletal muscle as well as the mechanical properties of the lamina propria. Smooth muscle is arranged
in an outer circular and an inner longitudinal layer.
Contraction of the circular muscle should maintain
continence, and the longitudinal muscle may shorten
during micturition. It has been proposed that the
pelvic organs also provide a support for the intraabdominal portion of the urethra, especially in
women. However, up to half of total urethral pressure
in women is because of smooth muscle tone (192,193).
The urethra is innervated by both the sympathetic and
parasympathetic systems. Activity in pelvic nerve
parasympathetic fibers relaxes urethral smooth muscle, especially in the proximal portion, and therefore
the outflow region; sympathetic fibers (T10-L2) generate contraction.
Urethral Smooth Muscle

Sympathetic and parasympathetic branches of the
autonomic nervous system exert control over uretheral smooth muscle. Sympathetic control is mediated by a1 receptors, mainly the a1A/L subtype in both
human and animal tissue (194,195) and about half of
the urethral pressure is maintained by adrenergic
receptor activation (190). Partial a1A/L agonists, such
as Ro 115-1240, have been proposed as agents that
may be used to manage stress urinary incontinence
(SUI) in women without significant cardiovascular
side effects (196).
Urethral relaxation is believed to be because of
release of NO (190) and prejunctional muscarinic
receptors may also limit noradrenaline release from
sympathetic fibers (197). Urethral relaxation can be
mediated by b-receptor activation, predominantly the
b2 subtype. However, this mechanism is probably less
important than in detrusor (102). The b2 agonist
clenbuterol also increased urethral skeletal muscle
contraction, raising the possibility that it may have a
role in the treatment of urinary incontinence by inhibiting detrusor contraction and augmenting external
urethral sphincter activity (198). NO-mediated relaxation is because of production of cGMP and activation
of cGMP-dependent protein kinase, cGK. Electrical
field stimulation generates relaxations that are abolished by inhibitors of NO production and are absent
in mice lacking cGK (199). CO can also exert relaxation
through a rise of cGMP, which may be equivalent in
magnitude to the effect of NO (200). Derangements of
the NO system have been demonstrated in several
disorders associated with LUT function, such as
diabetes, bladder outlet obstruction, or bladder

inflammation (201,202).
Sex hormones play an important role in modulating urinary tract smooth muscle function, including
urethral muscle. Lack of estrogen following menopause may contribute to decreased urethral tone,
urothelial integrity, and incontinence, and the earlier
papers suggested that estrogen supplementation may
be beneficial. However, recent clinical studies indicate
that supplemental estrogen lowers collagen content in
the periurethral connective tissue and decreases urethral closure pressure (203), thereby worsening the
symptoms of incontinence (204).
Urethral smooth muscle exhibits spontaneous
electrical and mechanical activity that contributes to
the overall muscular tone. Electrical activity occurs
because of bursts of spikes superimposed on a slower
more rhythmic activity, and could be initiated by
autonomic transmitters (205). Two types of Ca2
currentsL type and T typehave been recorded in
isolated myocytes. Blockade of the former type
reduced the number of spikes in each burst; the
frequency within bursts was attenuated by blockade
of T-type current (206). However, both channels represent targets that may modulate spontaneous activity. The muscle cells are closely associated with
interstitial cells that may at least modify their activity
(207). The observation that interstitial cells are closely
associated with NOS synthasecontaining nerves suggests that they may be intermediaries between nerves
and urethral smooth muscle (208).
Urethral Skeletal Muscle

Urethral skeletal muscle (rhabdosphincter) forms an
incomplete ring around the urethral conduit (209). In
human tissue three fiber types have been described:
fast twitch fatigue sensitive, fast twitch fatigue resistant, and slow twitch, with the majority fatigue resistant (210). Muscle bulk decreases with age and with
parity in women, and SUI may be associated with a
loss of muscle mass (211). A decrease in motor nerve
function may also contribute to SUI: In a mouse model
vaginal distension reduced leak point pressures and
the number of urethral nerves, without affecting muscle mass (212). The decline of sphincter, and striated
muscle, function has motivated the development of
myoblast implants that may improve continence (213),
or the use of basic fibroblast growth factor to facilitate
muscle cell generation (214). When using implants,
muscle fiber cells showed improved results over the
use of fibroblasts (215).
Selective inhibitors of serotonin (5-HT) and noradrenaline (NA) uptake, such as duloxetine, are potential agents for the therapeutic management of SUI
because 5-HT and NA terminals are present in Onufs
nucleus that supplies the rhabdosphincter with motor
nerves (216). Clinical trials suggest that the effect of
duloxetine on alleviating USI is small, but significant
(217)clinical uptake of this drug has been slow.
The striated muscle of the urethral sphincter
may undergo abnormal activity, resulting in urinary
259
retention, Fowlers syndrome (218). The origin of the

condition is unknown, but one hypothesis is that it is
because of ephaptic (i.e., direct cross talk) electrical
transmission between cells, much as can occur
between nerve axons under certain conditions (219).
Neuromodulation may be effective in restoring voiding activity, but there remain significant complication
rates (220).
REFERENCES
1. Walz T, Haner M, Wu X R, et al. Towards the molecular
architecture of the asymmetric unit membrane of the
mammalian urinary bladder epithelium: a closed twisted ribbon structure. J Mol Biol 1995; 248:887900.
2. Minsky BD, Chlapowski FJ. Morphometric analysis of the
translocation of lumenal membrane between cytoplasm
and cell surface of transitional epithelial cells during
the expansioncontraction cycles of mammalian urinary
bladder. J Cell Biol 1978; 77:685697.
3. Schlager TA, Grady R, Mills SE, et al. Bladder epithelium
is abnormal in patients with neurogenic bladder due to
myelomeningocele. Spinal Cord 2004; 42:163168.
4. Ohtsuka Y, Kawakami S, Fujii Y, et al. Loss of uroplakin
III expression is associated with a poor prognosis in
patients with urothelial carcinoma of the upper urinary
tract. BJU Int 2006; 97:13221326.
5. Ronald A. The etiology of urinary tract infection: traditional and emerging pathogens. Dis Mon 2003; 49:7182.
6. Lewis SA, de Moura JL. Incorporation of cytoplasmic
vesicles into apical membrane of mammalian urinary
bladder epithelium. Nature 1982; 297:685688.
7. Truschel ST, Wang E, Ruiz WG, et al. Stretch-regulated
exocytosis/endocytosis in bladder umbrella cells. Mol
Biol Cell 2002; 13:830846.
8. Yu W, Khandelwal P, Apodaca G. Distinct apical and
basolateral membrane requirements for stretch-induced
membrane traffic at the apical surface of bladder umbrella
cells. Mol Biol Cell 2009; 20:282295.
9. Ferguson DR, Kennedy I, Burton TJ. ATP is released from
rabbit urinary bladder epithelial cells by hydrostatic
pressure changesa possible sensory mechanism?
J Physiol 1997; 505:503511.
10. Apodaca G. The urothelium: not just a passive barrier.
Traffic 2004; 5:112.
11. Khandelwal P, Ruiz WG, Balestreire-Hawryluk E, et al.
Rab11a-dependent exocytosis of discoidal/fusiform
vesicles in bladder umbrella cells. Proc Natl Acad Sci
U S A 2008; 105:1577315778.
12. Lewis SA, Diamond J. Active sodium transport by mammalian urinary bladder. Nature 1975; 253:747748.
13. Tzan CJ, Berg JR, Lewis SA. Mammalian urinary bladder
permeability is altered by cationic proteins: modulation
by divalent cations. Am J Physiol 1994; 267:C1013C1026.
14. Parsons CL. The role of the urinary epithelium in the
pathogenesis of interstitial cystitis/prostatitis/urethritis.
Urology 2007; 69(4 suppl):916.
15. Wickham JE. Active transport of sodium ion by the
mammalian bladder epithelium. Invest Urol 1964; 2:
145153.
16. Smith PR, Mackler SA, Weiser PC, et al. Expression and
localization of epithelial sodium channel in mammalian
urinary bladder. Am J Physiol 1998; 274:F91F96.
17. Lewis SA, Wills NK. Apical membrane permeability and
kinetic properties of the Na pump in rabbit urinary
bladder. J Physiol 1983; 341:169184.
260
Fry
18. Lewis SA, Wills NK, Eaton DC. Basolateral membrane

potential of a tight epithelium: ionic diffusion and electrogenic pumps. J Membr Biol 1978; 41:117148.
19. Lewis SA, Hanrahan JW. Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium.
Pflugers Arch 1985; 405:S83S88.
20. Wang EC, Lee J M, Johnson JP, et al. Hydrostatic pressure-regulated ion transport in bladder uroepithelium.
Am J Physiol 2003; 285:F651F663.
21. Lewis SA, Ifshin MS, Loo DDF, et al. Studies of sodium
channels in rabbit urinary bladder by noise analysis.
J Membr Biol 1984; 80:135151.
22. Hanrahan JW, Alles WP, Lewis SA. Single anion-selective
channels in basolateral membrane of a mammalian tight
epithelium. Proc Natl Acad Sci U S A 1985; 82:77917795.
23. Donaldson P, Lewis SA. Effect of hyperosmotic challenge
on basolateral membrane potential in rabbit urinary bladder. Am J Physiol 1990; 27:C248C257.
24. Spector DA, Wade JB, Dillow R, et al. Expression, localization, and regulation of aquaporin-1 to -3 in rat urothelia. Am
J Physiol 2002; 282:F1034F1042.
25. Wright KJ, Seed PC, Hultgren SJ. Development of intracellular bacterial communities of uropathogenic Escherichia
coli depends on type 1 pili. Cell Microbiol 2007; 9:22302241.
26. Anderson GG, Palermo JJ, Schilling JD, et al. Intracellular
bacterial biofilm-like pods in urinary tract infections.
Science 2003; 301:105107.
27. Rosen DA, Hooton TM, Stamm WE, et al. Detection of
intracellular bacterial communities in human urinary
tract infection. PLoS Med 2007; 4:e329.
28. Farrugia MK, Godley ML, Woolf AS, et al. Experimental
short-term fetal bladder outflow obstruction: II effects on
bladder stiffness and detrusor contractility. J Paediatr
Urol 2006; 2:254260.
29. Bross S, Braun PM, Michel MS, et al. Bladder wall tension
during physiological voiding and in patients with an
unstable detrusor or bladder outlet obstruction. BJU Int
2003; 92:584588.
30. Bross S, Braun PM, Michel MS, et al. Preoperatively
evaluated bladder wall tension as a prognostic parameter
for postoperative success after surgery for bladder outlet
obstruction. Urology 2003; 61:562566.
31. Thiruchelvam N, Godley ML, Fry CH. Comment on:
bladder wall tension during physiological voiding and
in patients with an unstable detrusor or bladder outlet
obstruction. BJU Int 2004; 93:11171118.
32. Wahl EF, Lerman SE, Lahdes-Vasama TT, et al. Measurement of bladder compliance can be standardized by a
dimensionless number: clinical perspective. BJU Int 2004;
94:898900.
33. Wahl EF, Lerman SE, Lahdes-Vasama TT, et al. Measurement of bladder compliance can be standardized by a
dimensionless number: theoretical perspective. BJU Int
2004; 94:895897.
34. Thiruchelvam N, Wu C, David A, et al. Neurotransmission and viscoelasticity in the ovine fetal bladder after in
utero bladder outflow obstruction. Am J Physiol 2003;
284:R1296R1305.
35. Farrugia MK, Woolf AS, Fry CH, et al. Radiotelemetered
urodynamics of obstructed ovine fetal bladders: correlations with ex vivo cystometry and renal histopathology.
BJU Int 2007; 99:15171522.
36. Liao LM, Schaefer W. Cross-sectional and longitudinal
studies on interaction between bladder compliance and
outflow obstruction in men with benign prostatic hyperplasia. Asian J Androl 2007; 9:5156.
37. Shaw MB, Herndon CD, Cain MP, et al. A porcine model of
bladder outlet obstruction incorporating radio-telemetered
cystometry. BJU Int 2007; 100:170174.
38. Kohan AD, Danziger M, Vaughan ED, Jr, et al. Effect of

aging on bladder function and the response to outlet
obstruction in female rats. Urol Res 2000; 28:3337.
39. Ghoniem GM, Bloom DA, McGuire EJ, et al. Bladder
compliance in meningomyelocele children. J Urol 1989;
141:14041406.
40. Macarak EJ, Ewalt D, Baskin L, et al. The collagens and
their urologic implications. Adv Exp Med Biol 1995;
385:173177.
41. Tekgul S, Yoshino K, Bagli D, et al. Collagen types I and
III localization by in situ hybridization and immunohistochemistry in the partially obstructed young rabbit bladder. J Urol 1996; 156:582586.
42. Kim JC, Yoon JY, Seo SI, et al. Effects of partial bladder
outlet obstruction and its relief on types I and III collagen
and detrusor contractility in the rat. Neurourol Urodyn
2000; 19:2942.
43. Thiruchelvam N, Nyirady P, Peebles D, et al. Urinary
outflow obstruction increases apoptosis and deregulates
Bcl-2 and Bax expression in the fetal ovine bladder. Am J
Pathol 2003; 162:12711282.
44. Chang SL, Howard PS, Koo HP, et al. Role of type III
collagen in bladder filling. Neurourol Urodyn 1998;
17:135145.
45. Coplen DE, Macarak EJ, Howard PS. Matrix synthesis by
bladder smooth muscle cells is modulated by stretch
frequency. In Vitro Cell Dev Biol Anim 2003; 39:157162.
46. Baskin LS, Contantinescu S, Duckett JW, et al. Type III
collagen decreases in normal fetal bovine bladder development. J Urol 1994; 152:688691.
47. Furber JD. Extracellular glycation crosslinks: prospects for
removal. Rejuvenation Res 2006; 9:274278.
48. Poladia DP, Bauer JA. Functional, structural, and neuronal alterations in urinary bladder during diabetes: investigations of a mouse model. Pharmacology 2005; 74:
8494.
49. Daneshgari F, Liu G, Imrey PB. Time dependent changes in
diabetic cystopathy in rats include compensated and
decompensated bladder function. J Urol 2006; 176:380386.
50. Malmqvist U, Arner A, Uvelius B. Mechanics and Ca2
sensitivity of human detrusor muscle bundles studied in
vitro. Acta Physiol Scand 1991; 143:373380.
51. Ekstrom J, Uvelius B. Lengthtension relations of smooth
muscle from normal and denervated rat urinary bladders.
Acta Physiol Scand 1981; 112:443447.
52. Hill AV. The forcevelocity relation in shortening muscle.
In: First and Last Experiments in Muscle Mechanics.
Cambridge: Cambridge University Press, 1970:2341.
53. Griffiths DJ. The mechanics of the urethra and of micturition. Br J Urol 1973; 45:497507.
54. Malone-Lee J, Wahedna I. Characterisation of detrusor
contractile function in relation to old age. Br J Urol 1993;
72:873880.
55. Williams JH, Turner WH, Sainsbury GM, et al. Experimental model of bladder outflow tract obstruction in the
guinea-pig. Br J Urol 1993; 71:543554.
56. Bayliss M, Wu C, Newgreen D, et al. A quantitative study
of atropine-resistant contractile responses in human
detrusor smooth muscle, from stable, unstable and
obstructed bladders. J Urol 1999; 162:18331839.
57. Paul RJ. Smooth muscle energetics. Ann Rev Physiol 1989;
51:331349.
58. Hai CM, Murphy RA. Ca2, crossbridge phosphorylation,
and contraction. Ann Rev Physiol 1989; 51:285298.
59. Paul RJ, Bauer M, Pease W. Vascular smooth muscle:
aerobic glycolysis linked to NaK transport processes.
Science 1979; 206:1414.
60. Azadzoi KM, Pontari M, Vlachiotis J, et al. Canine bladder
blood flow and oxygenation: changes induced by filling,
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
contraction and outlet obstruction. J Urol 1996; 155:

14591465.
Bellringer JF, Ward J, Fry CH. Intramural pH changes in
the anaesthetised rabbit bladder during filling. J Physiol
1994; 480:82P83P.
Thomas PJ, Fry CH. The effects of cellular hypoxia on
contraction and extracellular ion accumulation in isolated
human detrusor smooth muscle. J Urol 1996; 155:726731.
Liston TG, Palfrey EL, Raimbach SJ, et al. The effects of
pH changes on human and ferret detrusor muscle function. J Physiol 1991; 432:121.
Martin AF, Bhatti S, Pyne-Geithman GJ, et al. Expression
and function of COOH terminal myosin heavy chain
isoforms in mouse smooth muscle. Am J Physiol Cell
Physiol 2007; 293:C238C245.
Andersson KE, Arner A. Urinary bladder contraction and
relaxation: physiology and pathophysiology. Physiol Rev
2004; 84:935986.
Wu C, Kentish JC, Fry CH. Effect of pH on myofilament
Ca2-sensitivity in a-toxin permeabilized guinea-pig
detrusor muscle. J Urol 1995; 154:191194.
Montgomery BS, Fry CH. The action potential and net
membrane currents in isolated human detrusor smooth
muscle cells. J Urol 1992; 147:176184.
Sui GP, Wu C, Fry CH. A description of Ca2 channels in
human detrusor smooth muscle. BJU Int 2003; 92:476482.
Fry CH, Gallegos CRR, Montgomery BS. The actions of
extracellular H on the electro-physiological properties of
isolated human detrusor smooth muscle cells. J Physiol
1994; 480:7180.
Wu C, Sui, GP, Fry CH. The role of the L-type Ca2
channel in refilling functional intracellular Ca2 stores
in guinea-pig detrusor smooth muscle. J Physiol 2002;
538:357369.
Wu C, Fry CH. Evidence for Na/Ca2 exchange and its
role in intracellular Ca2 regulation in guinea-pig detrusor smooth muscle cells. Am J Physiol Cell Physiol 2001;
280:C1090C1096.
Nobe K, Sutliff RL, Kranias EG, et al. Phospholamban
regulation of bladder contractility: evidence from genealtered mouse models. J Physiol 2001; 535:867878.
Yamaguchi O. Response of bladder smooth muscle cells
to obstruction: signal transduction and the role of mechanosensors. Urology 2004; 63(3 suppl 1):1116.
Kimura K, Ito M, Amano M, et al. Regulation of myosin
phosphatase by Rho and Rho-associated kinase (Rhokinase). Science 1996; 273:245248.
Durlu-Kandilci NT, Brading AF. Involvement of Rho
kinase and protein kinase C in carbachol-induced calcium
sensitization in beta-escin skinned rat and guinea-pig
bladders. Br J Pharmacol 2006; 148:376384.
Sjogren C, Andersson KE, Husted S, et al. Atropine
resistance of transmurally stimulated isolated human
bladder muscle. J Urol 1982; 128:13681371.
Palea S, Artibani W, Ostardo E, et al. Evidence for
purinergic neurotransmission in human urinary bladder
affected by interstitial cystitis. J Urol 1993; 150:20072012.
Wang P, Luthin GR, Ruggieri MR. Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J Pharmacol
Exp Ther 1995; 273:959966.
Hegde SS. Muscarinic receptors in the bladder: from basic
research to therapeutics. Br J Pharmacol 2006; 147:S80S87.
Braverman AS, Doumanian LR, Ruggieri MR, Sr. M2 and
M3 muscarinic receptor activation of urinary bladder
contractile signal transduction. II. Denervated rat bladder.
J Pharmacol Exp Ther 2006; 316:875880.
Ehlert FJ, Griffin MT, Abe DM, et al. The M2 muscarinic
receptor mediates contraction through indirect mechanisms
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
261
in mouse urinary bladder. J Pharmacol Exp Ther 2005;

313:368378.
Ehlert FJ, Ahn S, Pak KJ, et al. Neuronally released
acetylcholine acts on the M2 muscarinic receptor to
oppose the relaxant effect of isoproterenol on cholinergic
contractions in mouse urinary bladder. J Pharmacol Exp
Ther 2007; 322:631637.
Chapple CR, Khullar V, Gabriel Z, et al. The effects of
antimuscarinic treatments in overactive bladder: an
update of a systematic review and meta-analysis. Eur
Urol 2008; 54:543562.
Braverman AS, Tibb AS, Ruggieri MR Sr. M2 and M3
muscarinic receptor activation of urinary bladder contractile signal transduction. I. Normal rat bladder. J Pharmacol Exp Ther 2006; 316:869874.
Harriss DR, Marsh KA, Birmingham AT, et al. Expression
of muscarinic M3-receptors coupled to inositol phospholipid hydrolysis in human detrusor cultured smooth
muscle cells. J Urol 1995; 154:12411245.
Frazier EP, Braverman AS, Peters SL, et al. Does phospholipase C mediate muscarinic receptor-induced rat
urinary bladder contraction? J Pharmacol Exp Ther
2007; 322:9981002.
Wuest M, Hiller N, Braeter M, et al. Contribution of Ca2
influx to carbachol-induced detrusor contraction is different in human urinary bladder compared to pig and
mouse. Eur J Pharmacol 2007; 565:180189.
Frazier EP, Peters SL, Braverman AS, et al. Signal transduction underlying the control of urinary bladder smooth
muscle tone by muscarinic receptors and beta-adrenoceptors. Naunyn Schmiedebergs Arch Pharmacol 2007;
377:449462.
Zhou H, Meyer A, Starke K, et al. Heterogeneity of
release-inhibiting muscarinic autoreceptors in heart atria
and urinary bladder: a study with M2- and M4-receptordeficient mice. Naunyn Schmiedebergs Arch Pharmacol
2002; 365:112122.
Pals-Rylaarsdam R, Xu Y, Witt-Enderby P, et al. Desensitization and internalization of the m2 muscarinic acetylc h o l ine r e c e p t o r a r e d ir ec t ed b y i n d e p e n d en t
mechanisms. J Biol Chem 195; 27048:2900429011.
Furuya Y, Araki I, Kamiyama M, et al. Decreased expression of G protein-coupled receptor kinases in the detrusor
smooth muscle of human urinary bladder with outlet
obstruction. Int J Urol 2006; 13:12261232.
Harvey RA, Skennerton DE, Newgreen D, et al. The
contractile potency of adenosine triphosphate and ectoadenosine triphosphatase activity in guinea pig detrusor
and detrusor from patients with a stable, unstable or
obstructed bladder. J Urol 2002; 168:12351239.
Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 1998; 50:413492.
Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 2007; 87:659797.
Elneil S, Skepper JN, Kidd EJ, et al. Distribution of P2X1
and P2X3 receptors in the rat and human urinary bladder.
Pharmacology 2001; 63:120128.
Wu C, Bayliss M, Newgreen D, et al. A comparison of the
mode of action of ATP and carbachol on isolated human
detrusor smooth muscle. J Urol 1999; 162:18401847.
IUPHAR receptor database. Available at: http://www.
iuphar-db.org/index_ic.jsp.
Abbracchio MP, Burnstock G, Boeynaems JM, et al. International Union of Pharmacology LVIII: update on the P2Y
G protein-coupled nucleotide receptors: from molecular
mechanisms and pathophysiology to therapy. Pharmacol
Rev 2006; 58:281341.
Yamaguchi O. Beta3-adrenoceptors in human detrusor
muscle. Urology 2002; 59(5 suppl 1):2529.
262
Fry
100. Badawi JK, Uecelehan H, Hatzinger M, et al. Relaxant

effects of beta-adrenergic agonists on porcine and human
detrusor muscle. Acta Physiol Scand 2005; 185:151159.
101. Leon LA, Hoffman BE, Gardner SD, et al. Effects of the
beta3-adrenergic receptor agonist CL-316243 on bladder
micturition reflex in spontaneously hypertensive rats.
J Pharmacol Exp Ther 2008; 326:178185.
102. Michel MC, Vrydag W. Adrenoceptors in the lower
urinary tract. Br J Pharmacol 2006; 147:S88S119.
103. Uckert S, Stief CG, Mayer M, et al. Distribution and
functional significance of phosphodiesterase isoenzymes
in the human lower urinary tract. World J Urol 2005;
23:368373.
104. Qiu Y, Kraft P, Craig EC, et al. Identification and functional study of phosphodiesterases in rat urinary bladder.
Urol Res 2001; 29:388392.
105. Filippi S, Morelli A, Sandner P, et al. Characterization and
functional role of androgen-dependent PDE5 activity in
the bladder. Endocrinology 2007; 148:10191029.
106. Mulhall JP, Guhring P, Parker M, et al. Assessment of the
impact of sildenafil citrate on lower urinary tract symptoms in men with erectile dysfunction. J Sex Med 2006;
3:662667.
107. Shinkai M, Takayanagi I. Effect of tachykinins on the
acetylcholine output stimulated by nicotine from guinea-pig bladder. Regul Pept 1993; 46:399401.
108. de Biasi M, Nigro F, Xu W. Nicotinic acetylcholine receptors in the autonomic control of bladder function. Eur J
Pharmacol 2000; 393:137140.
109. Nicholls J, Hourani SM, Kitchen I. Characterization of P1purinoceptors on rat duodenum and urinary bladder. Br J
Pharmacol 1992; 105:639642.
110. Fahrenkrug J, Hannibal J. Pituitary adenylate cyclase
activating polypeptide immunoreactivity in capsaicinsensitive nerve fibres supplying the rat urinary tract.
Neuroscience 1998; 83:12611272.
111. Candenas L, Lecci A, Pinto FM, et al. Tachykinins and
tachykinin receptors: effects in the genitourinary tract.
Life Sci 2005; 76:835862.
112. Templeman L, Sellers DJ, Chapple CR, et al. Investigation
of neurokinin-2 and -3 receptors in the human and pig
bladder. BJU Int 2003; 92:787792.
113. Davenport AP, Battistino B. Classification of endothelin
receptors and antagonists in clinical development. Clin Sc
(Lond) 2002; 103(suppl 48):1S3S.
114. Donoso MV, Salas C, Sepulveda G, et al. Involvement of
ETA receptors in the facilitation by endothelin-1 of nonadrenergic non-cholinergic transmission in the rat urinary
bladder. Br J Pharmacol 1994; 111:473482.
115. Ukai M, Yuyama H, Noguchi Y, et al. Participation of
endogenous endothelin and ETA receptor in premicturition
contractions in rats with bladder outlet obstruction.
Naunyn Schmiedebergs Arch Pharmacol 2006; 373:197203.
116. Ng YK, de Groat WC, Wu HY. Smooth muscle and neural
mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development. Am J Physiol Regul Integr Comp
Physiol 2007; 292:R2100R2112.
117. Szell EA, Somogyi GT, de Groat WC, et al. Developmental
changes in spontaneous smooth muscle activity in the
neonatal rat urinary bladder. Am J Physiol Regul Integr
Comp Physiol 2003; 285:R809R816.
118. Ikeda Y, Fry CH, Hayashi F, et al. The role of gap
junctions in spontaneous activity of the rat bladder. Am
J Physiol Renal Physiol 2007 293:F1018F1025.
119. Chua WC, Liu L, Mansfield KJ, et al. Age-related changes of
P2X1 receptor mRNA in the bladder detrusor from men
with and without bladder outlet obstruction. Exp Gerontol
2007; 42:686692.
120. de Groat WC. A neurologic basis for the overactive

bladder. Urology. 1997; 50:3652.
121. Brading AF. A myogenic basis for the overactive bladder.
Urology 1997; 50:5767.
122. Ikeda Y, Kanai A. Urotheliogenic modulation of intrinsic
activity in spinal cord transected rat bladdersthe role of
mucosal muscarinic receptors. Am J Physiol Renal Physiol
2008; 295:F454F461.
123. Drake MJ, Hedlund P, Harvey IJ, et al. Partial outlet
obstruction enhances modular autonomous activity in
the isolated rat bladder. J Urol 2003; 170:276279.
124. Andersson KE. Antimuscarinics for treatment of overactive bladder. Lancet Neurol 2004; 3:4653.
125. Christ GJ, Day NS, Day M, et al. Increased connexin43mediated intercellular communication in a rat model of
bladder overactivity in vivo. Am J Physiol Regul Integr
Comp Physiol 2003; 284:R1241R1248.
126. McCloskey KD, Gurney AM. Kit positive cells in the
guinea pig bladder. J Urol 2002; 168:832836.
127. Hashitani H, Yanai Y, Suzuki H. Role of interstitial cells and
gap junctions in the transmission of spontaneous Ca2
signals in detrusor smooth muscles of the guinea-pig urinary bladder. J Physiol 2004; 559:567581.
128. Smet PJ, Jonavicius J, Marshall VR, et al. Distribution of
nitric oxide synthase-immuno-reactive nerves and identification of the cellular targets of nitric oxide in guinea-pig
and human urinary bladder by cGMP immunohistochemistry. Neuroscience 1996; 71:337348.
129. Gillespie JI, Drake MJ. The actions of sodium nitroprusside and the phosphodiesterase inhibitor dipyridamole on
phasic activity in the isolated guinea pig bladder. BJU Int
2004; 93:851858.
130. Yoshida M, Inadome A, Maeda Y, et al. Non-neuronal
cholinergic system in human bladder urothelium. Urology 2006; 67:425430.
131. Birder LA, Apodaca G, De Groat WC, et al. Adrenergicand capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am J Physiol
1998; 275:F226F229.
132. Marchand JE, Sant GR, Kream RM. Increased expression
of substance P receptor encoding mRNA in bladder
biopsies from patients with interstitial cystitis. Br J Urol
1998; 81:224228.
133. Sun Y, Chai TC. Augmented extracellular ATP signaling
in bladder urothelial cells from patients with interstitial
cystitis. Am J Physiol Cell Physiol 2006; 290:C27C34.
134. Wang EC, Lee JM, Ruiz WG, et al. ATP and purinergic
receptor-dependent membrane traffic in bladder umbrella
cells. J Clin Invest 2005; 115:24122422.
135. Birder LA, Ruan HZ, Chopra B, et al. Alterations in P2X
and P2Y purinergic receptor expression in urinary bladder from normal cats and cats with interstitial cystitis. Am
J Physiol Renal Physiol 2004; 287:F1084F1091.
136. Birder LA, Kanai AJ, de Groat WC, et al. Vanilloid
receptor expression suggests a sensory role for urinary
bladder epithelial cells. Proc Natl Acad Sci U S A 2001;
98:1339613401.
137. Andrade EL, Ferreira J, Andre E, et al. Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder. Biochem Pharmacol 2006; 72:104114.
138. Stein RJ, Santos S, Nagatomi J, et al. Cool (TRPM8) and
hot (TRPV1) receptors in the bladder and male genital
tract. J Urol 2004; 172:11751178.
139. Chopra B, Barrick SR, Meyers S, et al. Expression and
function of bradykinin B1 and B2 receptors in normal and
inflamed rat urinary bladder urothelium. J Physiol 2005;
562:859871.
140. Birder LA, Nealen ML, Kiss S, et al. Beta-adrenoceptor
agonists stimulate endothelial nitric oxide synthase in rat
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
urinary bladder urothelial cells. J Neurosci 2002; 22:

80638070.
Cockayne DA, Hamilton SG, Zhu QM, et al. Urinary
bladder hyporeflexia and reduced pain-related behaviour
in P2X3-deficient mice. Nature 2000; 407:10111015.
Vlaskovska M, Kasakov L, Rong W, et al. P2X3 knock-out
mice reveal a major sensory role for urothelially released
ATP. J Neurosci 2001; 21:56705677.
Wu C, Sui GP, Fry CH. Purinergic regulation of guinea pig
suburothelial myofibroblasts. J Physiol 2004; 559:231243.
Sui GP, Wu C, Fry CH. Characterization of the purinergic
receptor subtype on guinea-pig suburothelial myofibroblasts. BJU Int 2006; 97:13271331.
Sun Y, Keay S, De Deyne PG, et al. Augmented stretch
activated adenosine triphosphate release from bladder
uroepithelial cells in patients with interstitial cystitis.
J Urol 2001; 166:19511956.
Salas NA, Somogyi GT, Gangitano DA, et al. Receptor
activated bladder and spinal ATP release in neurally
intact and chronic spinal cord injured rats. Neurochem
Int 2007; 50:345350.
Khera M, Somogyi GT, Kiss S, et al. Botulinum toxin A
inhibits ATP release from bladder urothelium after chronic spinal cord injury. Neurochem Int 2004; 45:987993.
Yoshida M, Miyamae K, Iwashita H, et al. Management of
detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging.
Urology 2004; 63(3 suppl 1):1723.
Yoshida M, Masunaga K, Satoji Y, et al. Basic and clinical
aspects of non-neuronal acetylcholine: expression of nonneuronal acetylcholine in urothelium and its clinical
significance. J Pharmacol Sci 2008; 106:193198.
Hanna-Mitchell AT, Beckel JM, Barbadora S, et al. Nonneuronal acetylcholine and urinary bladder urothelium.
Life Sci 2007; 80:22982302.
Zarghooni S, Wunsch J, Bodenbenner M, et al. Expression
of muscarinic and nicotinic acetylcholine receptors in the
mouse urothelium. Life Sci 2007; 80:23082313.
Mansfield KJ, Liu L, Moore KH, et al. Molecular characterization of M2 and M3 muscarinic receptor expression in
bladder from women with refractory idiopathic detrusor
overactivity. BJU Int 2007; 99:14331438.
Birder LA, Barrick SR, Roppolo JR, et al. Feline interstitial
cystitis results in mechanical hypersensitivity and altered
ATP release from bladder urothelium. Am J Physiol Renal
Physiol 2003; 285:F423F429.
Davidson RA, McCloskey KD. Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons.
J Urol 2005; 173:13851390.
Sui GP, Wu C, Fry CH. Electrical characteristics of suburothelial cells isolated from the human bladder. J Urol
2004; 171:938943.
Kuijpers KA, Heesakkers JP, Jansen CF, et al. Cadherin-11
is expressed in detrusor smooth muscle cells and myofibroblasts of normal human bladder. Eur Urol 2007;
52:12131221.
Greenland JE, Brading AF. The effect of bladder outflow
obstruction on detrusor blood flow changes during the
voiding cycle in conscious pigs. J Urol 2001; 165:245248.
Kershen RT, Azadzoi KM, Siroky MB. Blood flow, pressure and compliance in the male human bladder. J Urol
2002; 168:121125.
Mukerji G, Yiangou Y, Grogono J, et al. Localisation of M2
and M3 muscarinic receptors in human bladder disorders
and their clinical correlations. J Urol 2006; 176:367373.
Iijima K, de Wachter S, Wyndaele JJ. Effects of the M3
receptor selective muscarinic antagonist darifenacin on
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
263
bladder afferent activity of the rat pelvic nerve. Eur Urol

2007; 52:842849.
de Laet K, de Wachter S, Wyndaele JJ. Systemic oxybutynin decreases afferent activity of the pelvic nerve of
the rat: new insights into the working mechanism of
antimuscarinics. Neurourol Urodyn 2006; 25:156161.
Hedland P, Streng T, Lee T, et al. Effects of tolterodine on
afferent neuro-transmission in normal and resiniferotoxin
treated conscious rats. J Urol 2007; 178:326331.
Hawthorn MH, Chapple CR, Cock M, et al. Urotheliumderived inhibitory factor(s) influences on detrusor muscle
contractility in vitro. Br J Pharmacol 2000; 129:416419.
Murakami S, Chapple CR, Akino H, et al. The role of the
urothelium in mediating bladder responses to isoprenaline. BJU Int 2007; 99:669673.
Mastrangelo D, Iselin CE. Urothelium-dependent inhibition of rat ureter contractile activity. J Urol 2007; 178:
702709.
Kanai A, Roppolo J, Ikeda Y, et al. Origin of spontaneous
activity in neonatal and adult rat bladders and its
enhancement by stretch and muscarinic agonists. Am J
Physiol Renal Physiol 2007; 292:F1065F1072.
Sui GP, Wu C, Roosen A, et al. Modulation of bladder
myofibroblast activity: implications for bladder function.
Am J Physiol 2008; 295:F688F697.
Kubota Y, Hashitani H, Shirasawa N, et al. Altered
distribution of interstitial cells in the guinea-pig bladder
following bladder outlet obstruction. Neurourol Urodyn
2008; 27:330340.
Biers SM, Reynard JM, Doore T, et al. The functional
effects of a c-kit tyrosine inhibitor on guinea-pig and
human detrusor. BJU Int 2006; 97:612616.
Kubota Y, Biers SM, Kohri K, et al. Effects of imatinib
mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the
guinea-pig urinary bladder. Neurourol Urodyn 2006;
25:205210.
Sui GP, Coppen SR, Dupont E, et al. Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders. BJU Int 2003;
92:297305.
Imaizumi Y, Torii Y, Ohi Y, et al. Ca2 images and K
current during depolarization in smooth muscle cells of
the guinea-pig vas deferens and urinary bladder. J Physiol 1998; 510:705719.
Sui GP, Wu C, Severs N, et al. The association between Ttype Ca2-current and outward current in isolated human
detrusor cells from stable and overactive bladders. BJU
International 2007; 99:436441.
Zhu HL, Brain KL, Aishima M, et al. Actions of two main
metabolites of propiverine (M-1 and M-2) on voltagedependent L-type Ca2 currents and Ca2 transients in
murine urinary bladder myocytes. J Pharmacol Exp Ther
2008; 324:118127.
Kobayashi H, Miwa T, Nagao T, et al. Negative modulation of L-type Ca2 channels via beta-adrenoceptor stimulation in guinea-pig detrusor smooth muscle cells. Eur J
Pharmacol 2003; 470:915.
Herrera GM, Heppner TJ, Nelson MT. Voltage dependence of the coupling of Ca2 sparks to BK(Ca) channels
in urinary bladder smooth muscle. Am J Physiol Cell
Physiol 2001; 280:C481C490.
Yanai Y, Hashitani H, Kubota Y, et al. The role of Ni2sensitive T-type Ca2 channels in the regulation of spontaneous excitation in detrusor smooth muscles of the
guinea-pig bladder. BJU Int 2006; 97:182189.
Werner ME, Knorn AM, Meredith AL, et al. Frequency
encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation
264
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
Fry
by BK channels. Am J Physiol Regul Integr Comp Physiol
2007; 292:R616R624.
Christ GJ, Day NS, Day M, et al. Bladder injection of
naked hSlo/pcDNA3 ameliorates detrusor hyperactivity in obstructed rats in vivo. Am J Physiol Regul Integr
Comp Physiol 2001; 281:R1699R1709.
Tian L, McClafferty H, Chen L, et al. Reversible tyrosine
protein phosphorylation regulates large conductance
voltage- and calcium-activated potassium channels via
cortactin. J Biol Chem 2008; 283:30673076.
Loane DJ, Hicks GA, Perrino BA, et al. Inhibition of BK
channel activity by association with calcineurin in rat
brain. Eur J Neurosci 2006; 24:43344341.
Adam RM, Eaton SH, Estrada C, et al. Mechanical stretch
is a highly selective regulator of gene expression in
human bladder smooth muscle cells. Physiol Genomics
2004; 20:3644.
Aitken KJ, Block G, Lorenzo A, et al. Mechanotransduction
of extracellular signal-regulated kinases 1 and 2 mitogenactivated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix
metalloproteinases. Am J Pathol 2006; 169:459470.
Wellner MC, Isenberg G. Stretch effects on whole-cell
currents of guinea-pig urinary bladder myocytes. J Physiol 1994; 480:439448.
Baker SA, Hennig GW, Han J, et al. Methionine and its
derivatives increase bladder excitability by inhibiting
stretch-dependent K channels. Br J Pharmacol 2008;
1530:12591271.
Tanagho EA. The ureterovesical junction. In: Chisholm
GD, Williams DI, eds. Scientific Foundations of Urology.
London: Heinemann, 1982:395404.
Viana R, Batourina E, Huang H, et al. The development of
the bladder trigone, the center of the anti-reflux mechanism. Development 2007; 134:37633769.
Roosen A, Wu C, Sui GP, et al. Characteristics of spontaneous activity in the bladder trigone. Eur Urol 2009 56(2):
346153.
Roosen A, Wu C, Sui GP, et al. Synergistic effects in
neuromuscular activation and calcium-sensitisation in
the bladder trigone. BJU Int 2008; 101:610614.
Andersson KE, Wein AJ. Pharmacology of the lower
urinary tract: basis for current and future treatments of
urinary incontinence. Pharmacol Rev 2004; 56:581631.
Roosen A, Sui GP, Fry CH, et al. Adreno-muscarinic
synergism in the bladder trigone: calcium-dependent
and -independent mechanisms. Cell Calcium 2009 45(1):
1117.
Tanagho EA, Meyers FH, Smith DR. Urethral resistance:
its components and implications. I. Smooth muscle component. Invest Urol 1969; 7:136149.
Awad SA, Downie JW. Relative contributions of smooth
and striated muscles to the canine urethral pressure
profile. Br J Urol 1976; 48:347354.
Nishimatsu H, Moriyama N, Hamada K, et al. Contractile
responses to alpha1-adrenoceptor agonists in isolated
human male and female urethra. BJU Int 1999; 84:
515520.
Bagot K, Chess-Williams R. Alpha(1A/L)-adrenoceptors
mediate contraction of the circular smooth muscle of the
pig urethra. Auton Autacoid Pharmacol 2006; 26:
345353.
Musselman DM, Ford AP, Gennevois DJ, et al. A randomized crossover study to evaluate Ro 115-1240, a selective
alpha(1A/1L)-adrenoceptor partial agonist in women
with stress urinary incontinence. BJU Int 2004; 93:7883.
Mattiasson A, Andersson K E, Sjogren C. Adrenoceptors
and cholinoceptors controlling noradrenaline release from
198.
199.
200.
201.
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
adrenergic nerves in the urethra of rabbit and man. J Urol

1984; 131:11901195.
Morita T, Kihara K, Nagamatsu H, et al. Effects of
clenbuterol on rabbit vesicourethral muscle contractility.
J Smooth Muscle Res 1995; 31:119127.
Persson K, Pandita RK, Aszodi A, et al. Functional characteristics of urinary tract smooth muscles in mice lacking
cGMP protein kinase type I. Am J Physiol Regul Integr
Comp Physiol 2000; 279:R1112R1120.
Schroder A, Hedlund P, Andersson KE. Carbon monoxide relaxes the female pig urethra as effectively as nitric
oxide in the presence of YC-1. J Urol 2002; 167:18921896.
Ho MH, Bhatia NN, Khorram O. Physiologic role of nitric
oxide and nitric oxide synthase in female lower urinary
tract. Curr Opin Obstet Gynecol 2004; 16:423429.
Yang Z, Dolber PC, Fraser MO. Diabetic urethropathy
compounds the effects of diabetic cystopathy. J Urol 2007;
178:22132219.
Jackson S, James M, Abrams P. The effect of oestradiol on
vaginal collagen metabolism in postmenopausal women
with genuine stress incontinence. BJOG 2002; 109:339344.
DuBeau CE. Estrogen treatment for urinary incontinence:
never, now, or in the future? JAMA 2005; 293:9981001.
Callahan SM, Creed KE. Electrical and mechanical activity of the isolated lower urinary tract of the guinea-pig. Br
J Pharmacol 1981; 74:353358.
Bradley JE, Anderson UA, Woolsey SM, Thornbury KD,
McHale NG, Hollywood MA. Characterization of T-type
calcium current and its contribution to electrical activity
in rabbit urethra. Am J Physiol Cell Physiol 2004; 286:
C1078C1088.
McHale N, Hollywood M, Sergeant G, Thornbury K.
Origin of spontaneous rhythmicity in smooth muscle.
J Physiol 2006; 570:2328.
Lyons AD, Gardiner TA, McCloskey KD. Kit-positive
interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle. BJU Int 2007;
99:687694.
Strasser H, Ninkovic M, Hess M, et al. Anatomic and
functional studies of the male and female urethral sphincter. World J Urol 2000; 18:324329.
Creed KE, van der Werf BA. The innervation and properties of the urethral striated muscle. Scand J Urol Nephrol
Suppl 2001; 207:811.
Strasser H, Tiefenthaler M, Steinlechner M, et al. Age
dependent apoptosis and loss of rhabdosphincter cells.
J Urol 2000; 164:17811785.
Lin YH, Liu G, Daneshgari F. A mouse model of simulated birth trauma induced stress urinary incontinence.
Neurourol Urodyn 2008; 27:353358.
Furuta A, Jankowski RJ, Honda M, et al. State of the art of
where we are at using stem cells for stress urinary
incontinence. Neurourol Urodyn. 2007; 26:966971.
Takahashi S, Chen Q, Ogushi T, et al. Periurethral injection of sustained release basic fibroblast growth factor
improves sphincteric contractility of the rat urethra denervated by botulinum-a toxin. J Urol. 2006; 176:819823.
Kwon D, Kim Y, Pruchnic R, et al. Periurethral cellular
injection: comparison of muscle-derived progenitor cells
and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence.
Urology 2006; 68:449454.
Thor K. Serotonin and norepinephrine involvement in
efferent pathways to the urethral rhabdosphincter: implication for treating stress urinary incontinence. Urology
2003; 62(suppl 4A):39.
Mariappan P, Ballantyne Z, NDow JM, et al. Serotonin
and noradrenaline reuptake inhibitors (SNRI) for stress

urinary incontinence in adults. Cochrane Database Syst
Rev 2005; 20(3):CD004742.
218. Fowler CJ, Christmas TJ, Chapple CR, et al. Abnormal
electromyographic activity of the urethral sphincter, voiding dysfunction, and polycystic ovaries: a new syndrome?
BMJ 1988; 297:14361438.
265
219. Sanders DB. Ephaptic transmission in hemifacial SPASM:

a single-fiber EMG study. Nerve Muscle 1989; 12:690694.
220. Datta SN, Chaliha C, Singh A, et al. Sacral neurostimulation for urinary retention: 10-year experience from one
UK centre. BJU Int 2008; 101:192196.
16
Scientific Basis of Urodynamics
Michael Craggs and Sarah Knight
London Spinal Cord Injuries Centre, Royal National Orthopaedic Hospital, Stanmore, U.K.
INTRODUCTION
The lower urinary tract comprises the bladder, urethra
and striated sphincter, which act as a single functional
unit under nervous control. The bladder has two
principal functions; first, to be a secure low-pressure
storage reservoir for the entire urine output from the
kidneys and, second, to contract efficiently and empty
at socially convenient times. The normal person voids
on average about 300 mL completely in about 40 seconds
six times a day with no leakage and little sensation of
bladder filling in between. However, if these functions
are ever compromised, as, for example, in patients
presenting with symptoms such as incontinence,
urgency, frequency or obstructed voiding, it is important that we are able to assess the operation of the
lower urinary tract objectively during both the storage
and voiding phases.
Such investigations form the basis of urodynamics,
a study that attempts to measure and determine the
relationship between bladder volume, bladder pressure (cystometry) and urine flow (flowmetry) under
the best physiological conditions possible. The physiological basis and coordination of normal bladder
and sphincter function are described fully elsewhere
(chap. 15).
Much is said about the lack of correlation
between symptoms and urodynamic measures in
some patients; for example, it is not always possible
to observe unstable contractions of the detrusor in the
untreated patient complaining of urge incontinence.
This sort of finding has made some urologists skeptical of the value of invasive urodynamics (pressure
monitoring by catheter per urethra) to the extent that
they believe the method is unnatural, offers little to
the diagnosis of a patients problems and introduces
infection to the bladder. On the other hand, there are
many clinicians who do accept urodynamics as a
useful investigative tool but only as a practiced art
requiring years of experience and careful evaluation.
However, there is an important scientific basis of
urodynamics, which, if properly standardized and
interpreted, can lead to significant findings for making
a proper diagnosis of a patients problems. An example of this is in the derived measure of urethral
resistance, where an analysis relating detrusor pressure to flow can objectively define obstructed voiding,
a condition not always accurately diagnosed from flow

measurement alone (1). Therefore, to transform urodynamics from an art form to a science, we must have a
full understanding of the principles of the technique,
the underlying physics and mechanics, the derivation
of parameters and an appreciation of the extent and
limitations of urodynamics as a diagnostic tool.
PHYSICS AND MECHANICS OF THE LOWER

URINARY TRACT
From an engineering point of view, the bladder and
urethra act as a single functional unit, which can be
modeled as a system comprising reservoir, valves and
connecting tubes, with pressure, flow and volume as
measured variables. To understand the working of
this unit during the normal micturition cycle, it is
easiest to consider storage and voiding separately, but
before that we should be clear about the definition of
the measures, specifically, pressure.
Pressure is defined as the force acting per unit
area, and force is a vector quantity, which has a
direction; however, pressure is scalar and has no
direction. In urodynamics, pressure is usually quoted
in the units of cmH2O. The standard international (SI)
unit is the kilopascal (kPa), which is approximately
equal to 10 cmH2O.
Storage Phase
Bladder filling is an essentially passive process during
which the normal functional capacity of 300 to 500 mL
should be reached with only a small increase in pressure and before sensations of bladder fullness are
perceived. It is possible to account for the bladder as
a low-pressure, high-capacity reservoir in purely
physical terms.
The small pressure rise within the bladder as it
fills is mainly the result of elastic forces generated
within the bladder wall. If the bladder is assumed to
behave as a thin-walled sphere, its pressure-volume
characteristics can be described by Laplaces law (P
2T/R) where Ppressure, Rradius, and Ttension
per unit width of bladder wall (Fig. 1). As the bladder
fills and the volume increases (R:), the wall tension
increases (T:), but the ratio T/R remains constant,
Chapter 16: Scientific Basis of Urodynamics
267
Figure 1 The physics of the bladder as a low-pressure reservoir. The bladder modeled as a thin-walled sphere obeying the law of
Laplace. Relationships between volume, pressure, radius and tension in a thin-walled sphere.
resulting in little change in intravesical pressure until

the bladder is almost full.
The use of Laplaces law to determine wall tension and pressure is based on the assumption that the
bladder is a thin-walled sphere during the whole of
the filling phase. However, the bladder actually progresses from a relatively thick-walled irregular shape
when empty to a thin-walled multicurved vessel
when full, but the model is a sufficient approximation
for a relatively full bladder.
The property of the bladder wall that allows a
large increase in volume with only a small associated
pressure rise is known as compliance, which is
defined as the change in volume divided by the
corresponding change in pressure (DV/DP). The wall

also exhibits the property of elasticity, as it can return
to its normal size after voiding with no permanent
deformation (plastic) changes. The bladder wall is
composed of three layers: an outer covering of connective tissue (serosa), a smooth muscle layer (detrusor)
and a mucous membrane (urothelium) which lines the
interior of the bladder. These layers comprise collagen
and elastin fibers as well as muscle. The layers in
combination make the bladder very compliant until
the structural capacity of the bladder is reached, and
further increases in volume cause increased wall stiffness as the properties of the collagen fibers in the wall
dominate and reduce compliance (Fig. 2).
Figure 2 Structure, compliance and capacity of the bladder. Bladder musculature and graph showing relationship between bladder
volume and pressure with respect to structural properties.
268
Craggs and Knight
that of the bladder pressure (Pves) to ensure no leakage

of urine (Pura > Pves). The urethral closure pressure
may be divided into passive and active forces (3).
Passive forces are maintained by the mucosal seal,
which occurs when the urethral walls oppose each
other. Active forces are exerted by the contraction of
the urethral and periurethral muscles.
The urethral pressure increases as the bladder
fills and during periods when the intra-abdominal
pressure increasesfor example, as a result of postural change or coughing and sneezingthus maintaining continence.
Voiding Phase
Figure 3 Viscoelastic properties of the bladder wall. The effect

of bladder fill rate on measured detrusor pressure. A typical
spring and dashpot model of the viscoelastic behavior of the
bladder wall.
In addition to compliance and elasticity, the bladder exhibits the time-dependent property of viscoelasticity in which the bladder wall tension and extension
are dependent on rate of filling. When the bladder wall
is stretched rapidly, as during fast filling, it appears
stiffer than when filled more slowly; however, when
the filling is stopped, the wall exhibits relaxation in an
exponential manner. Viscoelasticity is often modeled as
a combination of spring (purely elastic) and dash-pot
(purely viscous) elements (Fig. 3) (2).
These properties of the bladder wall during
storage are likely to be purely passive physical mechanisms attributable to the properties of the tissues,
although there may be some active neuromodulatory
effects brought about by reflex contractions of the
detrusor muscle.
Throughout the storage phase, the urethra must
remain closed with a pressure (Pura), which exceeds
Voiding is initiated voluntarily following sensation of

bladder fullness and is preceded by relaxation of the
urethral sphincter. The detrusor muscle contracts in
response to uninhibited parasympathetic stimulation,
but urine does not flow until the bladder pressure
exceeds the urethral pressure (Pves > Pura). This initial
detrusor contraction is isometric since there is no
significant change in bladder volume and no external
work done in passing urine.
As soon as the bladder pressure exceeds the
urethral closure pressure, the detrusor contraction
becomes isotonic, and there is shortening of the
detrusor muscle, producing increased external work
as the flow reaches a maximum. The isotonic contraction is sustained by reflex activation mediated by
increased tension in the receptors in series with the
contracting muscle fibers until it falls below a threshold as the detrusor begins to relax at the end of
voiding (Fig. 4).
As with any fluid flow, the rate of urine output
(Qura) is dependent on both the driving force (Pves), and
the resistance to flow (Rura) (Qura ? Pves/Rura). Other
fluid properties, such as viscosity, are normally
constant and relatively unimportant, but the use of
radio-opaque substances, which can be quite viscous,
for X-ray imaging of the urinary tract during urodynamics may have small effects on flow. Physiologically,
Figure 4 Pressure relationships from storage to voiding, indicating the transition from isometric to isotonic detrusor contraction.
269
Figure 5 Force-length curve and force-velocity curve for detrusor muscle contraction.
the isometric part of the detrusor contraction should

follow the standard force-length (or volume) and forcevelocity curves that can be demonstrated for other
muscles (Fig. 5) (4).
If the bladder is overstretched or not sufficiently
full, a submaximal isometric contraction will be produced and reduced flow rate will be achieved. The
optimal contraction of the detrusor will be at bladder
volumes within the normal functional capacity. The
isotonic contraction of the detrusor, that is, when the
bladder is emptying, is related to the velocity of shortening of the detrusor muscle. As the force changes from
isometric to isotonic, the maximum speed of shortening increases with decreasing load until, in the theoretical limit, no force is required when the muscle
becomes unloaded. The resistance to flow is dependent
on both active and passive factors in the urethra,
including urethral lumen shape and length, active
contraction of the sphincter, and intrinsic muscle and
anatomical features. These factors may change
throughout the voiding phase; therefore, a simple
interpretation of urethral resistance may be difficult
to evaluate, and it may be more useful to describe these
changes in terms of impedance that includes a timedependent element. Although the viscosity of urine can
be approximated as a constant, the type of urine flow is

likely to alternate between laminar and turbulent,
depending on changes in the urethral impedance.
Understanding these basic principles of physics
and mechanics is important if we are to develop good
scientific methodology for urodynamic investigations,
the measurement of and relationship between pressure, flow and volume during the storage and voiding
phases of bladder function.
METHODOLOGY, PRACTICE, AND PROBLEMS

The basic aim of urodynamics is to provoke the lower
urinary tract in the relatively controlled environment
of a clinical laboratory so as to provide an objective
basis for a patients symptoms, lead to a clear diagnosis of the dysfunction and, hopefully, give a guide
to rational treatment. Perhaps the greatest failure
in this endeavor is poor practice and the misinterpretation of urodynamic tests. There are a number of
techniques available for the practice of urodynamics
depending on whether the individual patient has a
voiding or storage problem, or a combination of both,
these are summarized in Table 1. In standard
Table 1 Techniques Used in Urodynamics for Measurement of Lower Urinary Tract Function
Technique
Function
Indication
Cystometry
Measurement of bladder storage and voiding

function, including sensation
Measurement of voiding function
Measurement of urethral closure pressures
Behavior of bladder and urethra during activities of
daily living
Measurement of bladder storage and voiding in
conjunction with imaging of bladder morphology
Neurophysiological assessment of urethral
sphincter and pelvic floor function
Incontinence and dysfunction of detrusor
Flowmetry
Urethral pressure profilometry
Ambulatory urodynamics
Videourodynamics
Neurophysiological
Incontinence and voiding dysfunction

Incontinence due to sphincter incompetence
Incontinence
Multifactorial incontinence or suspected
abnormalities in lower urinary tract
Incontinence or dyssynergic voiding
270
Craggs and Knight
urodynamic testing, we usually measure only pressure, flow and volume and leave other physiological
measures, such as the electromyography of sphincters,
too more specialized investigations. To interpret accurately the results of urodynamic tests and carry them
out in a scientific and reproducible manner, free from
technical artefact, it is important to have a full understanding of the physical principles of these measurements, as described in the previous section.
Pressure can be measured in a number of ways;
simple manometry is based on the height of a fluid
column, with the pressure proportional to the height
of the column. Alternatively, the fluid column can be
connected (e.g., via a fluid-filled catheter) to an external strain gauge pressure transducer in which the
pressure is proportional to the electrical signal output
from the transducer. This is the most commonly used
pressure measurement technique in urodynamics.
Microtip catheter transducers utilize a miniature
pressure sensor mounted in the tip (or along the
side) of a catheter, which can be inserted into the
bladder.
Bladder pressure is usually expressed with reference to atmospheric pressure; therefore, the correct
zeroing of transducers to atmospheric pressures is
very important. In addition, the position of the catheter tip transducer within the bladder can affect the
pressure recording, as the pressure measured at the
top of the bladder is approximately 10 cmH2O lower
than that at the base because of the height of the water
within the bladder itself, and the fluid-filled catheters
are very dependent on relative height of external
transducer and tip of catheter (Fig. 6). The standard
zero reference is the level of the symphisis pubis
[International Continence Society (ICS)].
Figure 6 Effect of catheter position in bladder on recorded pressure. (A) Effect of intravesical catheter position of microtip pressure transducer. (B) Effect of relative position of external pressure
transducer.
Cystometry
The aim of cystometry is to investigate the pressurevolume relationship of the bladder during the filling
and storage phases. To measure pressure, a catheter is
introduced into the bladder either per urethra or, if
access is available, suprapubically. In addition to
measuring bladder pressure, it is important to measure intra-abdominal pressure, usually through a
catheter placed in the rectum. In this way, the intraabdominal pressure (Pabd) can be subtracted from the
intravesical pressure (Pves) to give a true detrusor
pressure (Pdet) free from artefacts caused by extravesical pressure increases, such as coughing or straining.
However, it must be noted that in addition to abdominal pressure changes, Pabd will also reflect intrinsic
bowel activity (Fig. 7).
During a filling cystometrogram, fluid is retrogradely instilled into the bladder in a physiological
manner. In the ideal situation, this means using isotonic saline at body temperature, at a rate close to that
of natural diuresis from the kidneys ("10 mL/min).
However, because of constraints of time and convenience, filling rates are normally much faster
(10100 mL/min), though these rates may be too provocative for some overactive bladders, and may lead
to overfilling in others that are hypoactive. As
described earlier, a normal bladder shows very little
change in pressure during filling (high compliance),
but because of possible decreases in the viscoelastic
nature of the bladder wall (e.g., in the neuropathic
bladder), it may be necessary to fill much more slowly
in some patients. A further matter is the position of
the patient during cystometrylying down, sitting or
standing may be important if we are to replicate the
conditions in which patients symptoms are revealed,
but this cannot always be practical. Most filling
Figure 7 Subtraction cystometry with detrusor pressure calculated from difference between vesical and rectal pressure measurement. (A) Cough appears in both Pves and Pabd and
therefore no Pdet. (B) Intrinsic bowel contraction appears in
Pabd only; therefore, Pdet shows inverse trace. (C) Bladder contraction activity appears in Pves alone and therefore in Pdet.
271
cystometry is performed with the patients lying either

supine or semirecumbent and then raised to sitting or
standing position for voiding cystometry (see later in
this section).
Urethral Pressure Profilometry

This technique is used to determine the intraluminal
pressure along the length of the urethra, during the
storage phase. This investigation has lost favor among
some clinicians, probably as a result of its relative
complexity and inaccuracy if not performed correctly.
However, knowledge of urethral resistance can be
useful in identifying urethral incompetence (as in
anatomical obstruction by stricture, benign prostatic
hyperplasia (BPH), or active dyssynergic sphincters).
There are two principal techniques for measuring
UPP: fluid perfusion profilometry (5) and solid-state
microtip pressure transducers.
The perfusion technique is based on the pressure
required to perfuse fluid through a catheter at a constant rate; this is best achieved with a syringe driver
rather than a peristaltic pump. A catheter with side
holes is introduced into the urethra and zeroed in the
standard way, and the catheter is attached via a threeway tap to a pressure transducer and an infusion
pump. The pressure recorded is defined as the resistance the urethral wall produces to the perfusing
fluid. The catheter is then withdrawn slowly along
the length of the urethra, thus giving a pressure profile. A profile can also be obtained by withdrawing a
solid-state pressure tip transducer at a constant rate
(Fig. 8).
There are a number of factors that must be taken
into account when performing UPP to ensure accuracy. As described earlier, pressure has no direction,
and therefore measuring in a particular orientation
along the urethral wall may introduce errors because
of position of side holes or transducer sensors. In
addition, bladder volume, patient position and artefacts caused by movement or abdominal straining can
also introduce errors.
Urethral pressure profiles can be measured in
steady-state or static conditions, or, alternatively,
under conditions of stress such as coughing or
straining.
Figure 8 Brown-Wickham perfusion technique for measurement of urethral pressure profile.
Flowmetry
Of all urodynamic tests, this is the least invasive and
easiest to perform with modern flow rate recorders
and should certainly be the first line of investigation
for suspected voiding problems. As described in the
previous section, the flow rate of urine from the
bladder is dependent on both the driving force of
the pressure generated in the bladder and the resistance of the urethra. A simple flow rate measurement
cannot accurately distinguish the relative importance
of these two factors; therefore, accurate diagnosis is
often not possible without measuring both bladder
pressure and flow rate simultaneously. Even then, the
effect of urethral resistance may be an important
additional factor.
There are a number of techniques available for
measuring urine flow, based on the different principles described in Table 2.
Modern electronic devices can output a number
of parameters based on the voiding characteristics.
However, a single flow rate on a single occasion is
unlikely to be of much diagnostic value, and a number
of factors should be taken into account when performing meaningful flowmetry. The bladder volume at
Table 2 Methods of Measuring Urine Flow

Type of flowmeter
Principle
Weight transducer
Weight of urine voided measured and differentiated with

respect to time to calculate flow rate.
A servomotor rotates the disc at a constant rate, but rate of
urine flow tends to slow the disc, requiring greater power
to keep at a constant speed. Power consumption of
servomotor is proportional to flow rate and can be
integrated for voided volume.
A capacitor comprised of a metal strip is placed vertically in
the collecting vessel. As volume of urine increases, the
level of urine on the strip changes and capacitance
decreases. The signal is proportional to voided volume
and can be differentiated for flow rate.
Rotating disc
Capacitance
Comments
Cruising (moving urine stream over

disc and sides of collecting container)
can cause artifact.
Urinating on to capacitance strip may

cause artifact.
272
Craggs and Knight
The ideal situation in which to carry out flowmetry is in conjunction with cystometry, so that voiding pressure and flow rate can be measured
simultaneously, although the delay in urine flow exiting the bladder and reaching the flow meter must be
taken into account. Pressure-flow studies can, for
example, help to identify whether a low flow rate is
due to a urethral obstruction or a hypocontractile
bladder; this information could not be inferred from
a flow rate alone.
INTERPRETATION: ART OR SCIENCE?

Figure 9 The relationship between maximum flow rate out of
the bladder and bladder volume, demonstrating the reduction in
flow rate at bladder volumes significantly larger or smaller than
the functional volume.
which the flow rate was measured is extremely important, especially with respect to the force-length curve
of the detrusor muscle. An over- or understretched
bladder will not be capable of generating the maximal
force to expel urine and thus may give a lower than
optimal flow rate (Fig. 9). The urethral resistance may
also change with bladder volumes and also give rise
to different flow rates.
It is important to estimate the total bladder volume not only from the voided volume but also taking
into account any residual volume in the bladder. This
can be calculated by ultrasound or by catheter. In
unfamiliar surroundings, a patient may feel uncomfortable, be inhibited or strain during voiding, and
this may lead to an unnatural flow pattern and incomplete voiding. Technical artefacts can occur if the
stream of flow is not aimed directly into the flow
meter, and cruising or moving the stream across the
flow meter can lead to abnormal traces. Flowmetry
can also now be carried out with portable devices at
home. This can lead to more accurate results, as the
patient is more comfortable and multiple measurements can be recorded.
Interpretation of urodynamic investigations is very

much dependent on both a proper understanding of
the physiological factors controlling and affecting the
lower urinary tract and the technical aspects of urodynamic technique. This is particularly important if
the interpreters did not undertake the investigations
themselves, Table 3 summarizes some important factors which may influence urodynamic findings.
The diagnostic potential of urodynamic studies
relies on a number of measures, which should
be quantified and standardized to minimize subjective
interpretation. The ICS Standardization Committee
has been pivotal in introducing procedures and
guidelines for the standardization of terminology of
lower urinary tract function, dysfunction and rehabilitation, including derived parameters and indices of
function (6). However, it should be appreciated that
rather little of this standardization has been based on
comparative control data from healthy volunteers.
Therefore, interpretation of urodynamic data on
patients, in the hope of providing definitive diagnosis,
can still remain unclear.
Filling Cystometry
From the filling cystometrogram, we can determine
information about bladder compliance, the incidence
of unstable detrusor activity, and the sensations
perceived by subjects as the bladder volume increases
to capacity.
As described in the previous section, bladder
compliance is defined as the ratio of volume change
Table 3 Potential Factors That May Lead to Inaccuracies in Urodynamic Findings

Problem
Artefact and possible effects
Transducer zeroing and position

Poor subtraction
Incorrect pressure with respect to reference and poor subtraction of Pves and Pabd.
Incorrect Pdet recording, best checked by continuous coughing, which should show
equal pressure changes on Pves and Pabd.
May not reproduce patients symptoms and give false pressure.
May cause irritation, increasing sensations and possibly provoking overactivity.
Filling too fast may provoke unstable contractions and underestimate compliance.
A large catheter may cause irritation and partial obstruction.
This will cause a damped pressure recording and incorrect pressure values.
Will not represent true Pves or Pdet; that is, catheter being expelled during voiding or
coughing.
May reduce ability to void, especially in patients with reduced sensation or outflow
obstruction.
May increase urgency symptoms.
May affect symptoms.
Patient position
Filling medium, temperature, viscosity, pH
Filling rate
Catheter size
Air bubbles in water-filled lines
Misplaced catheter
Overfilling of bladder
Presence of infection
Concomitant medication
to pressure changeDV/DP. For a normal healthy

bladder, the maximum capacity should be reached
with only a small change in bladder pressure, to give a
high value for compliance (about 40 mL/cmH2O). As
the bladder becomes stiffer, particularly toward maximum capacity, the pressure per unit volume can
increase, and the compliance becomes smaller in a
nonlinear fashion. It is important to recognize the
effect that the rate that bladder filling can have on
compliance, and in some cases it may be necessary to
stop the fill and wait until the bladder pressure has
returned to its stress-relaxed state before calculating a
value for compliance. This might be particularly
important in the pathologically small bladder where
fibrosis, a reduction in elasticity and other factors
might be expected to reduce compliance severely.
Indeed, in the neurogenic bladder of spinal cord
injury, the rate of filling cystometry is usually kept
low at 10 to 20 mL/min.
During filling, the normal bladder should
remain stable up to full bladder capacity, showing
little or no activity. Unlike the slow rises in detrusor
pressure associated with lowered bladder compliance,
detrusor overactivity is characterized by phasic events
which rise relatively and then fall again within a few
tens of seconds. Bladder activity can also be provoked
by rapid filling (e.g., 10 mL/sec) or ice water, and
these may be more useful provocative tests than the
standard filling cystometrogram in some patients,
such as those with a neurological cause for their
overactive detrusor, as in spinal cord injury. Although
it is not common practice in routine urodynamic
laboratories, quantifying detrusor overactivity is
important if a proper assessment of interventions is
to be made. Various approaches have been adopted,
including measuring the peak pressure, calculating
the area under the unstable contraction and recording
the frequency of events. For many urodynamic tests,
overactivity presents itself as a single end-fill event,
which occurs when the bladder reaches its maximum
cystometric volume (Fig. 10).
273
The sensations recorded by subjects during a

filling cystometrogram usually receive much less
attention than the occurrence of abnormal pressure
changes. The standard technique for monitoring bladder sensations is the use of markers for the first
sensation (FS), first desire to void (FDV), and strong
desire to void (SDV) (7), which is sometimes described
as strong urge and occasionally associated with severe
discomfort and pain, though it is not always associated with changes in detrusor pressure. However,
these markers may be very subjective, variable and
susceptible to prompting by the investigator. Bladder
sensations may also be confused because of interference from catheterization, nonphysiological fill rates
and embarrassment.
With the development of a new technique for
investigating bladder sensations during cystometry, it
may now become possible to determine the relevance
of urodynamics and symptomatology more objectively particularly when determining the effects of
interventions such as drugs or neuromodulation (8).
This will be especially useful in conditions such as
urgency, frequency and urge incontinence where
overactivity cannot always be reliably demonstrated
even at full capacity.
The maximum capacity of the bladder is
described as the volume at which patients feel they
can no longer delay micturition. This measure can be
very different in the same individual in everyday life
and probably depends on many factors. Of course,
functional bladder capacity, rather than maximum
capacity, may be more accurately reflected in
frequency-volume diaries. However, in the relatively
controlled environment of the urodynamics laboratory, repeated cystometries can give a reasonable indication of a persons maximum capacity. Of course this
is more difficult to determine in patients with poor or
absent sensation such as those with a neurological
cause for their bladder problems. In these patients, it
is possible to get a reasonable estimate of maximum
capacity by filling until detrusor overactivity occurs.
Interestingly, patients who present with intractably
small bladder capacities are sometimes found to have
relatively normal capacities when tested under general anesthesia.
Urethral Pressure Profiles
Figure 10 Typical filling cystometrogram traces illustrating

(A) normal bladder, (B) bladder with loss of compliance at end
fill and (C) instability and end-fill loss of compliance.
A number of parameters can be derived from the

static urethral pressure profile, including maximum
urethral pressure (MUP), maximum urethral closure
pressure (MUCP), and functional profile length (FPL)
(Fig. 11). Although the FPL may not be diagnostically
useful in itself, it can be used to calculate a further
index by multiplying it with MUP. This index has
been used to diagnose stress incontinence.
The stress UPP can be used to determine the
decrease in transfer of abdominal pressures to the
urethra during maneuvers such as the Valsalva and
cough to determine degree of stress incontinence.
However, it has been shown that the position of
the patient during these studies can dramatically alter
274
Craggs and Knight
Figure 11 Typical urethral pressure profiles in (A) female and

(B) male subjects.
the results. Diagnostic interpretations of urethral function can also be made from the characteristic shape of
the UPP. However, it should be noted that there are a
number of differences caused by both age and sex that
may lead to inaccuracies in these interpretations. For
example, the MUP often decreases with age, and, in
women, it often decreases, especially after the menopause. The MUP is often lower in patients with stress
incontinence, though the findings are not always consistent with symptoms reported by the patient, and
there is a significant overlap in the values found in
incontinent and continent subjects.
patients (9). The shape of the normal flow curve is

near Gaussian and deviations from this shape may
suggest pathology; for example, a low flat curve may
be indicative of a stricture, while an intermittent flow
with low flow rate may indicate obstruction. It is
also important during uroflowmetry to determine
the residual volume; this can be calculated most
accurately by catheterizing the patient, or by
ultrasonography.
Pressure-flow studies are carried out to determine whether the low flow rate recorded during
flowmetry is attributable to a high urethral resistance
or a low bladder pressure. The interrelationship
between these variables means that accurate diagnosis
of dysfunction requires simultaneous measurement of
flow rate and detrusor pressure. The most common
variables measured are the maximum flow rate (Qmax)
and the detrusor pressure at this flow rate (PdetQmax).
As for simple flowmetry, a number of nomograms
have been derived for these variables to identify
patients with obstructive disorders (Fig. 12) (10,11).
It is important to note that nearly all of these
nomograms have a large equivocal region, which
makes a definitive diagnosis impossible.
If a low flow rate is also associated with a low
detrusor pressure, it is may be necessary to determine
the contractility of the detrusor muscle. Detrusor contractility is an important parameter that is relatively
complicated to measure and understand. An isometric
detrusor contraction can be produced by asking a
patient to stop voiding as soon as the maximum
flow rate is reached. As the detrusor muscle continues
to contract against a closed outlet, the contraction is
assumed to be isometric, the resulting pressure is
defined as Pdet.iso. A single stop test is often used,
and a single value for Pdet.iso is derived from this.
However, the definition of contractility includes a
dependence on the muscle length. The serial stop
Flowmetry and Voiding Cystometry

The most useful voiding study involves the simultaneous measurement of voiding pressure and flow
rate. This allows the derivation of a number of factors
that may be useful in the diagnosis of lower urinary
tract dysfunction, particularly obstructive disorders.
However, if flowmetry alone is undertaken, there are
a number of factors that are measured, including
maximum flow rate, average flow rate, flow time,
voiding time, voided volume and time to maximum
flow. Several nomograms have been developed for
determining the degree of obstruction from flow rate
alone. These include the Liverpool nomogram, which
was constructed after analyzing flow rates and bladder volumes collected from many hundreds of
Figure 12 Nomogram for determining bladder outlet obstruction.
275
Figure 13 Graphs illustrating derived bladder parameters Watts factor (WF) and bladder output relationship (BOR).
test has therefore been suggested as a more accurate

method for determining a true contractility curve
(12,13).
In this way, the detrusor force at each given
volume can be determined for each stop test. It should
be noted that patients with severe stress incontinence
may not be able to stop urine flow sufficiently, and
there is the theoretical possibility that repeated cessation may cause an inhibition of detrusor contraction.
The Watts factor (WF) developed by Griffiths
(14) is an alternative method for deriving a continuous
calculation of detrusor contractility. The WF is defined
as the power per unit area of bladder and is numerically equal to isometric detrusor pressure throughout
voiding (Fig. 13).
ADVANCED TECHNIQUES
AND NEW DEVELOPMENTS
In some centers, more specialized techniques to assist
diagnosis have been introduced into the practice of
urodynamics. Of these, the videocystometrogram
(VCMG), in which X-ray imaging of the urinary tract
with a contrast medium is combined with urodynamic
measurements, is the most common (Fig. 14).
Although it can yield valuable information about
bladder morphology, urethral dysfunction (e.g., in
assessing dyssynergic sphincters or bladder neck
obstruction) and ureteral reflux (important to identify
and eliminate for the preservation of renal function),
which cannot be obtained with urodynamics alone, it
has the danger of overexposure to X-rays and is relatively expensive, thus precluding frequent use. However, some patient groups, for example, those with a
neurogenic bladder will particularly benefit from the
additional information video urodynamics can yield.
Another technique gaining much favor is ambulatory urodynamics, where bladder pressure, rectal
pressure and leakage can be monitored continuously
over a long period on a portable data-logger to assess
the effects of normal activities on lower urinary
Figure 14 Videourodynamics from a case of spinal cord injury.
tract function. Considerable technical back up is

required for this type of study, and much time and
care are needed in data analysis, especially for the
identification and elimination of artefacts. Although
expensive, it is said to be a valuable aid in diagnosing
conditions such as idiopathic instability where standard urodynamics has been unsuccessful.
Newer methods are now being investigated
which may overcome some of the problems associated
with conventional voiding cystometry for investigating obstructive disorders such as benign prostatic
hypertrophy. As we have seen, bladder pressure is
conventionally measured by catheterization with the
attendant risk of traumatizing the urethra and introducing infection into the bladder (estimated to occur
in about 15% of urodynamic investigations), so by
eliminating the need for catheters in the bladder, this
morbidity could be reduced. One such technique to do
this is noninvasive urodynamics first demonstrated in
an objective test of the effects of atropine on the
276
Craggs and Knight
Figure 15 The principles of noninvasive urodynamics using the Mediplus CT3000. Isometric bladder pressure is indirectly measured
by monitoring the pressure of an inflatable cuff, applying compression to the urethra, at the point where urine flow ceases.
bladder in man where an inflating cuff around the

penis was used to indirectly measure isometric bladder pressure at the point when urine flow stopped
(15), similar to reverse sphygmomanometry. This
early technique has more recently been developed
into a practical clinical test (Fig. 15) (16), which is
now commercially available (CT3000Mediplus Ltd.,
U.K.). Another technique along similar lines uses a
condom and external catheter, the urine flow is interrupted by an external valve and isovolumetric bladder
pressure measured (17). Such noninvasive bladder
pressure measuring techniques could provide a
more cost-beneficial diagnostic method for selecting
patients for surgery as well as lowering morbidity
through infection.
Ultrasonography has been used more widely in
a number of clinics. The most common use is as a
measure of residual urine although reliability can be
very variable. Measurements of bladder wall thickness and bladder weight have also been reported
as a diagnostic tool for determining bladder outlet
obstruction (18).
Future developments include the use of functional magnetic resonance imaging (fMRI) to produce
high-quality anatomical characterization of the function of the bladder, urethra and pelvic floor. However,
the use of MRI combined with urodynamics is still in
an experimental phase (19), but it has the potential for
giving three-dimensional views of the bladder and
urethra during filling and voiding. More recently,
positron emission tomography (PET) studies have
been used to determine which areas of the brain are
responsible for sensations of bladder filling (20). It is
likely that this technique will remain a research tool
for the foreseeable future but the results will help to
increase our knowledge and understanding of the
control of the micturition cycle at a cerebral level.
SYMPTOMS AND URODYNAMICS

Although we may now have a good scientific understanding of the basis of urodynamics, the question still
remains: What exactly is the relationship between
urinary symptomatology and urodynamics? There
have been many studies and papers written about this
topic but without a firm conclusion as to whether they

do correlate in any urological problem.
In a recent study of women questioned about
their complaints of urinary incontinence, it was found
that not only were the subjective answers by patients
not helpful in differentiating the etiology of incontinence but also that almost no questions were helpful
in predicting which patients would have normal video
urodynamics (21). Interestingly, questions about nocturia, frequency, urgency and urge incontinence,
among others, were not statistically significant for
any group. If there is so little correlation between
symptoms and urodynamics, what is the purpose of
urodynamics? If it is the symptoms that drive patients
to seek help in the first place, what additional information is urodynamics providing? Are the rather
nonphysiological techniques used in urodynamics
part of the problem?
Perhaps for some urological problems, such as
urge incontinence, the difficulty in urodynamics has
to do with measuring the sensations during bladder
filling and urge itself rather than looking just for
instability as a cause for their symptoms. Diaries
show that these patients void frequently, suggesting
that they may be having strong sensations of urge (or
perhaps more correctly urgency?) and the desire to
void at relatively low bladder volumes.
During urodynamics, patients are asked through
dialogue to report on any sensations; for example, the
standard measures are FS of filling, FDV, and,
finally, SDV. Prompting the patient for answers
probably helps to undermine the value of these measures of sensation, and this may be very important if
we are trying to get an accurate picture of the bladder
volume at the time. Perhaps we should pay more
attention to the sensations experienced by patients
during urodynamics and get accurate measures of
bladder volumes for each level of reported sensation
(Fig. 16) (8).
So, in urge (urgency?) incontinence at least, there
may be a more positive correlation between symptoms and the semi-objectively measured sensations
during urodynamics, which would be important
information when we are assessing the impact of
interventions such as neuromodulation (22).
277
Figure 16 The urge keypad is shown on the right. Operating independently of investigator prompting, subjects press buttons
indicating five levels on the scale, 0 (none), (i) (slight; FSF), (ii) (moderate; first desire to void), (iii) (strong; strong desire to void), and (iv)
(desperate), as their subjective sensations change during bladder filling. Only one button can be pressed at a time. An electrical output
from the keypad for each level of sensation can be recorded alongside the other cystometrogram traces shown on the left.
CONCLUSION: URODYNAMICSART OR
SCIENCE?
This chapter has outlined the scientific basis of urodynamics and how simple physical principles can be
applied to explain the functions of the bladder during
filling and voiding cystometry. The techniques for
measuring and recording pressure changes and flow
accurately are described together with how we can
relate these measurements to some functions of the
lower urinary tract. For the urologist, it is important to
know when and in what ways the lower urinary is
dysfunctional, so comparisons with normal function
are crucial. There are some tabulated examples of
normal versus patient data in the form of nomograms
or charts, such as flow versus voided volume (23) or
detrusor pressure (24). From these charts, clinicians
can relate their findings on urodynamics to the variation of the normal population and draw conclusions
about departure of their patients from normality. Even
more sophisticated graphs have evolved which
describe the contractility and urethral resistance (25)
calculated as derivations from pressure, volume and
flow measured during urodynamics. Applying these
graphs blindly and without full appreciation of
urodynamic techniques, their errors and their artefacts
is very likely to lead to false information about a
patients true lower urinary tract function.
Finally, if we are to relate some types of urinary
symptoms with urodynamics, we must include more
objective ways of recording the sensations of filling
and urge in our studies (7). Reading urodynamic
records in an intelligent way is both an art form and
a science requiring considerable experience to reach a
high standard of interpretation.
FURTHER READING
Abrams P. Urodynamics. 3rd ed. London: Springer, 2006.
Chapple CR, MacDiarmid SA, Patel A. Urodynamics
Made Easy. 3rd ed. London: Churchill Livingstone Elsevier,
2009.
Griffiths D. The Mechanics and Hydrodynamics of the Lower

Urinary Tract. Medical Physics Handbooks 4. Bristol: Adam
Hilger Limited, 1980.
Mundy AR, Stephenson TP, Wein AJ, eds. Urodynamics: Principles, Practice and Application. 2nd ed. New York: Churchill
Livingstone, 1994.
Sand PK, Ostergard DR. Urodynamics and the Evaluation of
Female Incontinence. London: Springer-Verlag, 1997.
REFERENCES
1. Schafer W. Urethral resistance. Urodynamic concepts of
physiological and pathological bladder outlet function
during voiding. Neurourol Urodyn 1985; 4:161201.
2. van Mastrigt R, Coolsaet BL, van Duyl WA. Passive
properties of the urinary bladder in the collection phase.
Med Biol Eng Comput 1978; 16:471482.
3. Zinner NR, Sterling AM, Ritter RC. Role of inner urethral
softness in urinary continence. Urology 1980; 16:115117.
4. Hill AV. The heat of shortening and the dynamic constants
of muscle. Proc R Soc Lond B Biol Sci 1938; 126:136195.
5. Brown M, Wickham JE. The urethral pressure profile. Br J
Urol 1969; 41:211217.
6. Abrams P, Cardozo L, Fall M, et al. The standardisation of
terminology in lower urinary tract function: report from
the standardisation sub-committee of the International
Continence Society. Urology 2003; 61:3749.
7. Wyndaele JJ. The normal pattern of perception of bladder
filling during cystometry studied in 38 young healthy
volunteers. J Urol 1998; 160:479481.
8. Oliver SE, Fowler CJ, Mundy AR, et al. Measuring the
sensations of urge and bladder filling during cystometry in
urge incontinence and the effects of neuromodulation.
Neurourol Urodyn 2003; 22:716.
9. Haylen BT, Parys BT, Anyaegbunam WI, et al. Urine flow
rates in male and female urodynamic patients compared
with the Liverpool nomograms. Br J Urol 1990; 65:483487.
10. Griffiths D, Hofner K, van Mastrigt R, et al. Standardisation of terminology of lower urinary tract function: pressure-flow studies of voiding, urethral resistance, and
urethral obstruction. International Continence Society
Sub-Committee on Standardisation of Terminology of
Pressure-Flow Studies. Neurourol Urodyn 1997; 16:1.
11. Schafer W. Analysis of bladder outlet function with the
linearized passive urethral resistance relation, linPURR
278
12.
13.
14.
15.
16.
17.
18.
Craggs and Knight

and a disease specific approach for grading obstruction:
from complex to simple. World J Urol 1995; 13:47.
Susset JG, Brissot RB, Regnier RB. The stop-flow technique:
a way to measure detrusor strength. J Urol 1982; 127:
489494.
Craggs MD, Knight SL, McFarlane JA. Detrusor force
measured by serial stop-tests in healthy volunteers. Neurourology Urodyn 1998; 17:12.
Griffiths DJ. Mechanics of micturition. In: Yalla SV,
McGuire EJ, Elbadawi E, et al., eds. Neurology and Urodynamics: Principles and Practice. New York: Macmillan,
1988.
Brindley GS, Craggs MD. The effect of atropine on the
urinary bladder of the baboon and of man. J Physiol 1975;
256:55.
Griffiths CJ, Rix D, MacDonald AM, et al. Noninvasive
measurement of bladder pressure by controlled inflation of
a penile cuff. J Urol 2002; 167:13441347.
Rikken B, Pell JJ, van Mastrigt R. Repeat noninvasive
bladder pressure measurements with an external catheter.
J Urol 1999;162(2):474479.
Oelke M, Hofner K, Jonas U, et al. Diagnostic accuracy of
noninvasive tests to evaluate bladder outlet obstruction in
men: detrusor wall thickness, uroflowmetry, postvoid
19.
20.
21.
22.
23.
24.
25.
residual urine, and prostate volume. Eur Urol 2007; 52(3):

827834.
Simmons A, Williams SC, Craggs M, et al. Dynamic multiplanar EPI of the urinary bladder during voiding with
simultaneous detrusor pressure measurement. Magn
Reson Imaging 1997; 15:295300.
Athwal BS, Berkley KJ, Hussain I, et al. Brain responses to
changes in bladder volume and urge to void in healthy
men. Brain 2001; 124(pt 2):369377.
Amundsen C, Lau M, English SF, et al. Do urinary symptoms correlate with urodynamic findings? J Urol 1999;
161:18711874.
Craggs MD. Objective measurement of bladder sensation:
use of a new patient-activated device and respons to
neuromodulation. BJU Int 2005; 96(suppl 1):2936.
Haylen BT, Ashby D, Sutherst JR, et al. Maximum and
average urine flow rates in normal male and female populations the Liverpool nonograms. Br J Urol 1989; 64(1):3038.
Abrams PH, Griffiths DJ. The assessment of prostatic
obstruction from urodynamic measurements and from
residual urine. Br J Urol 1979; 51:129134.
Schaefer W. Basic principles and clinical application of
advance analysis of bladder voiding function. Urol Clin
N Am 1990; 17:553566.
17
Scientific Basis of Male Hypogonadism
Thang S. Han and Pierre-Marc G. Bouloux
Department of Endocrinology, Royal Free and University College Hospital Medical School,
Royal Free Hospital, London, U.K.
BACKGROUND
Definition of Male Hypogonadism
Male hypogonadism is a multisystem clinical syndrome that results from subphysiological circulating
levels of testosterone because of disruption of one or
more levels of the hypothalamic-pituitary-testicular
(HPT) axis (1). Defective androgen receptor (AR) or
specific converting enzymes at target organs also
results in hypogonadism.
An Overview of Key Factors Controlling

Hypothalamic-Pituitary-Testicular Axis
The HPT axis is a closed loop system interlinking the
central hypothalamic-pituitary components to the testis by means of various hormones. It is considered to
be the core of the reproductive endocrine system,
regulating not only classic sexual function but also a
host of vital bodily functions. The axis is activated by
constant changes of ambience signaled by neuronal
and hormonal activities (Fig. 1). Centrally, hypothalamic production of gonadotrophin-releasing hormone
(GnRH) is regulated by cortical neurotransmitters,
including g-aminobutyric acid (GABA), catecholamines, and endorphins. Pulsatile GnRH secretion
stimulates pituitary synthesis and secretion of gonadotrophins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). In turn, gonadotrophins act on
the testis to initiate biosynthesis of testosterone and
spermatogenesis. Testicular hormones produced in the
Sertoli and Leydig cells, in turn, exert a negative
feedback control on the central components to complete the closed loop of HPT axis.
Testicular hormones exert their effects soon after
conception, stimulating the formation of both external
and internal male sex organs while inhibiting the
differentiation of the internal female Mullerian structures (Fig. 2). During puberty they induce secondary
sexual characteristics and, in adulthood, are responsible for sexual function as well as maintaining longterm health of vital organs and general well-being.
Defects of the HPT axis lead to variable clinical
features of hypogonadism, depending on the timing
of disease onset. Correct diagnosis of the defect
is important for appropriate treatment: optimal
development in the young and ensuring of longterm health in adults.
ANATOMY AND PHYSIOLOGY OF THE TESTIS

Anatomy of the Testis
The testes function as both an endocrine and exocrine
organ, and lie within the scrotum, encapsulated by
tunica albuginea. Each testis is divided into approximately 300 lobules by thin connective tissue septa.
Each lobule contains two to three very convoluted
seminiferous tubules, in which gametes are produced
by spermatogenesis. In the intertubular regions lie the
steroid-producing Leydig cells. Tubuli recti are the
terminal ends of the seminiferous lobules, which
extend into the mediastinum of the testis, anastomosing to form the rete testis (Fig. 2). From the rete testis,
a series of 6 to 12 fine efferent ducts join to form the
duct of the epididymis. The distal pole of the epididymis gives rise to the vas deferens, which ends as the
ampulla of the vas at the posterior aspect of the
bladder, where it joins the seminal vesicular duct,
forming the ejaculatory duct that passes through the
prostate to enter the prostatic urethra. Together the
seminal vesicles and prostate contribute approximately
90% to 95% of the volume of the ejaculate. In health,
each testis can produce between 10 and 200 million
gametes a day.
Spermatogenesis
Spermatogenesis involves a complex series of cell
divisions, transforming spermatogonia to mature
spermatozoa (Fig. 3). Until spermiation, the gametes
are nursed by Sertoli cells, which regulate the internal
environment of the seminiferous tubule under the
influence of FSH and testosterone. This environment
is created by the formation of inter-Sertoli cell junctions where processes of Sertoli cell cytoplasm from
adjacent cells merge. The junctions are predominantly
located at the basolateral regions of the cell to create
occluding-type junctions, the anatomical basis of the
blood-testis barrier. As a result, intercellular transport
between the Sertoli cell and spermatogonia is possible,
but this barrier is impermeable to macromolecules.
280
Han and Bouloux
Figure 1 Diagram of the feedback control of the hypothalamopituitary-testicular axis. Source: Adapted from Ref. 2 with
permission.
During the fetal stage of sexual differentiation, the

Sertoli cells produce the polypeptide anti-Mullerian
hormone (AMH) that inhibits the formation of internal
female genitalia (uterus and Fallopian tubes) from the
Mullerian duct. Sertoli cells also produce the hormones inhibin and activin, which exert feedback
action on FSH secretion.
ENDOCRINE REGULATION OF MALE

REPRODUCTIVE SYSTEM
Regulation and Action of GonadotrophinReleasing Hormone
Embryologically, GnRH neurons are born in the nasal
pit. Under the influence of adhesion molecules they
migrate alongside the vomeronasal nerve into their
final destination in the septopreoptic area of the
anterior hypothalamus. Their axons innervate the
median eminence and secrete pulsatile GnRH into
the pituitary portal system. GnRH is initially produced as a 92 amino acid (AA) pre-pro-GnRH (Fig. 4),
which is encoded by the GnRH gene mapped to
8p21-p11.2. Posttranslational cleavage by prohormone
convertase-1 of the leading 23 AAs (pre portion) and

the supporting gonadotrophin-associated peptide
(GAP) 56 AAs (pro portion) results in the decapeptide GnRH.
GnRH neurons discharge at an intrinsic frequency of 90 to 120 minutes, releasing GnRH in a pulsatile
manner. The amplitude and frequency of discharge
are modulated by cortical neurotransmitters (Fig. 4).
The a-adrenergic impulses have a stimulatory action
and b-adrenergic, dopamine, and endorphins
impulses have an inhibitory action on GnRH secretion. Testosterone and progesterone, probably via
b-endorphins, attenuate pulse frequency.
It is well recognized that stress can have an
adverse effect on reproductive function. Many factors
are involved in this process, including corticotrophinreleasing hormone (CRH), which inhibits GnRH
through direct neuronal contact between the paraventricular nucleus and preoptic region. Elevated prolactin levels, in response to stress, further suppress
GnRH pulsatility. Observations in a wide variety of
species indicate that intermediary cytokines such as
interleukin 1 (IL-1) or tumor necrosis factor-a (TNF-a)
may also have an inhibitory effect (3,4).
Leptin, a hormone produced by adipocytes
whose gene was cloned by Friedmans group, is
another hormone that influences GnRH secretion
probably indirectly via neuropeptide Y by intervening
with the feedback mechanism (5). It acts on the
hypothalamus via specific leptin receptors to signal
the nutritional status (adipose tissue reserve). Leptin
may be involved in the initiation of puberty. In
anorexia nervosa, low leptin levels are associated
with reduced GnRH levels, an effect that can be
reversed by leptin administration (6).
The G proteincoupled receptor 54 (GPR54, also
known as KISS1R) and its natural ligand kisspeptin,
are encoded by KISS1R and KiSS1 genes (7). The
kisspetin/GPR54 system is expressed in the arcuate
and anteroventral periventricular nuclei of the forebrain and implicated in the neuroendocrine regulation
of reproduction. GPR54 / mice and compound heterozygote missense mutations of the GPR54 in
humans have hypogonadotrophic hypogonadism
(8,9). A study of healthy male volunteers has shown
that kisspeptin administration increases levels of
gonadotrophins and testosterone (10). Hypothalamic
levels of KiSS1 and GPR54 mRNA increase dramatically at puberty, suggesting that kisspeptin signaling
could mediate the neuroendocrine events that trigger
its onset. A study of primary culture of human fetal
GnRH-secreting neuroblasts (FNC-B4) has shown that
kisspeptin-induced GnRH secretion and kisspeptin
per se are decreased by oestradiol and stimulated by
androgens. Furthermore, leptin was also shown to
increase KiSS1/GPR54 expression in FNC-B4 (11).
Together, these observations indicate that interactions
between metabolic and sexual hormones may trigger
the KiSS1/GPR54 signaling to GnRH neurons to regulate reproductive activity.
GnRH has specific receptors (G proteinbound
receptors with seven transmembrane loops) on pituitary gonadotrophs. Binding of GnRH to these specific
Figure 2 Schematic overview of the differentiation of the internal male and female (for comparison) reproductive tracts from the
Wolffian and Mullerian ducts. Source: Adapted from Ref. 2 with permission.
281
282
Han and Bouloux
Figure 3 Diagram of the structural organization of the human

seminiferous tubule and the stages of spermatogenesis. (A)
Spermatogonia push their way through the tight junctions
between adjacent Sertoli cells and become primary spermatocytes. (B) In the first meiotic division (1) each of the 46 chromosomes doubles up and then forms a pair with another doubled-up
chromosome during which time there is exchange of genetic
material (crossing over). The cell divides and each daughter cell
contains one of the pairs of chromosomes. In the second meiotic
division (2) each double chromosome simply separates producing genetically identical secondary spermatocytes. Source:
Adapted from Ref. 2 with permission.
receptors initiates gene expression of a- and b-chains

of FSH and LH and promotes their secretion by
inducing inositol-1,4,5,-triphosphate (IP3) and diacylglycerol (DAG), resulting in mobilizing of intracellular
calcium and protein kinase C (PKC), respectively.
Pulsatile exposure of gonadotrophs to GnRH is
essential for gonadotrophin secretion: Continuous
exposure to GnRH attenuates gonadotrophin secretion because of GnRH receptor downregulation. This
is exploited in the treatment of steroid-dependent
conditions such as endometriosis, prostate carcinoma,
Figure 4 Synthesis of GnRH and its actions on pituitary

gonadotrophs. GnRH is synthesized as a large prohormone
and released with gonadotrophin-associated peptide. It acts on
the gonadotroph via a G proteinlinked receptor that activates
phospholipase C, which stimulates the inositol signaling pathway. (A) GnRH is secreted in a pulsatile manner with one pulse
occurring approximately each hour. (B) Administration of longacting agonist analogues induces an initial stimulation of LH
(and follicle-stimulating hormone) but over a few days causes
complete desensitization of the pituitary gonadotroph. (C) Loss
of endogenous GnRH secretion induces loss of GnRH receptors
and the LH response to a bolus injection of GnRH is very low.
Abbreviations: PIP2, phosphatidylinositol 4,5-bisphosphate; IP3,
inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein
kinase C; GnRH, gonadotrophin-releasing hormone; LH,
luteinizing hormone. Source: Adapted from Ref. 2 with permission.
and precocious puberty where long-acting potent

GnRH analogues are used. In contrast, correct pulsatile administration of GnRH induces LH and FSH
release and thus normalizes reproductive function,
forming the basis of one form of treatment for secondary hypogonadal conditions such as Kallmanns syndrome (KS).
Regulation and Action of Luteinizing Hormone

Like thyroid-stimulating hormone (TSH) and human
chorionic gonadotrophin (hCG), LH and FSH are large
glycoproteins. They have a common noncovalent
Chapter 17: Scientific Basis of Male Hypogonadism
binding of two peptide chains (a and b) to form a

heterodimer. The a-chain is identical in all four glycoprotein hormones, the b-chain only mediating the
biological effect. The a-chain is encoded by a gene
on 6q12.21, FSH-b on 11p13 and LH-b and hCG-b on
19q13.32. The genes of LH and FSH receptors both
locate to chromosome 2.
LH binds to specific GPRs on the surface of
Leydig cells, activating adenyl cyclase and generating
the intracellular second messenger cyclic adenosine
monophosphate (cAMP), which, acting via PKA, leads
to steroid biosynthesis. LH influences the conversion
of cholesterol to pregnenolone, the most critical step of
testosterone biosynthesis, via two mechanisms:
cAMP-mediated stimulation of the synthesis and
activity of cytochrome P450scc (side chain cleavage)
enzyme, and a PKC-mediated increase in the production of cholesterol by activation of hydrolase.
Regulation and Action of Follicle-Stimulating

Hormone
FSH binds to specific receptors on Sertoli cells to
promote spermatogenesis. Sertoli cells stimulate the
activity of aromatase, an enzyme catalyzing the conversion of testosterone to oestradiol. The hormones
inhibin B and activin are formed in Sertoli cells in an
FSH-dependent manner. Inhibin B is a heterodimeric
glycoprotein hormone comprising a and b subunits;
the latter has two variants (bA and bB). Homodimers of
the b-chains are called activin A (bA-bA) and activin B
(bB-bB). On one hand, inhibin B exerts a specific negative feedback inhibition on pituitary FSH secretion.
Loss of inhibin B in conditions such as Sertoli-cellonly syndrome, or following radiotherapy or chemotherapy, augments FSH levels while LH remains
unchanged. On the other hand, both activins A and
B stimulate FSH secretion. A structurally unrelated
protein termed follistatin has the capacity to suppress
FSH, probably through the binding and neutralizing
the actions of activin. Testosterone and oestradiol inhibit
FSH either directly or through GnRH suppression.
Testosterone and Androgen Effect

Testosterone Biosynthesis
Central to the male reproductive system is testosterone, secreted by Leydig cells of the testis at about 5 to
7 mg a day. The regulation of testosterone biosynthesis and metabolism involves a complex system interlinking the HPT axis and target organs. Testosterone
synthesis per se requires multienzymatic action and
its bioavailability is modulated by circulating binding
proteins such as sex hormonebinding globulin
(SHBG) and albumin and the sensitivity of the target
organs.
Leydig cells have a large endoplasmic reticulum
and high density of mitochondria. The precursor of
testosterone synthesis is cholesterol, synthesized
mainly by the Leydig cells with a very small amount
taken up from the circulation and stored as esters in
283
Leydig cell fat vacuoles. The 29 carbon atom cholesterol is hydrolyzed to the 19 carbon atom testosterone,
through five enzymatic stages (Fig. 5). Defects in any
of these steps result in primary hypogonadism.
The most significant rate-limiting step is the
conversion from cholesterol to pregnenolone, which
takes place on the inner mitochondrial membrane,
where cytochrome P450scc catalyzes in three consecutive stages: first hydroxylation on atom C20, then C22
and thereafter, cleavage between C20 and C22 to
produce pregnenolone and isocaproic acid. Cytochrome P450scc (also known as 20,22-desmolase),
encoded by a gene on chromosome 15, is the crucial
enzyme in all steroid-producing tissues, such as the
adrenal gland and ovary. Pregnenolone is the parent
substance of all biologically active steroid hormones.
It diffuses across the mitochondria into the endoplasmic reticulum where further processing occurs. There
are two pathways (D4 or D5) depending on whether
the double bond is located in ring A or B. The D5
synthesis pathway is preferred in human. Accordingly,
hydroxylation frequently occurs first in position
C17 by the action of P450c17 (17a-hydroxylase), to
17a-hydroxypregnenolone. The weak androgens
dehydroepiandrosterone (DHEA) and androstenediol
are produced by the enzymes 17,20-desmolase and
17b-hydroxysteroid dehydrogenase consecutively. A
further important step is the conversion of the less
biologically active D5 steroids 17a-pregnenolone,
DHEA and androstenediol, to the corresponding
more effective D4 steroids 17a-progesterone, androstenedione and testosterone. This step is catalyzed by
the enzyme 3b-hydroxysteroid dehydrogenase, and
comprises oxidation of the 3b-hydroxyl group to a
ketone group, with subsequent transfer of the double
bond from C5-C6 on ring B to C4-C5 on ring A (D5D4
isomerization). The majority of testosterone produced
this way is immediately released into the circulation
via the spermatic vein, with small amounts via lymphatic system.
Testosterone and its metabolite oestradiol exert
negative feedback action on LH secretion, by both
inhibiting GnRH production and action through diminution of GnRH pulse frequency, but not pulse
amplitude. Downregulation of GnRH receptors also
occurs. Oestradiol has an inhibitory effect predominantly on the pituitary where it decreases LH pulse
amplitude without changing pulse frequency.
Transport of Testosterone in the Circulation
The bioavailability of testosterone is modulated by its

binding to circulating SHBG (10 9 M affinity binding)
and albumin (10 6 M affinity binding) and the sensitivity of the target organs. Testosterone, a lipophilic
molecule, diffuses freely across Leydig cell membranes into the circulation where 60% is bound with
high affinity to SHBG and 38% is loosely bound to
albumin, leaving only 2% free testosterone available
for biological activity.
Most of the SHBG is produced by liver, with
small amounts derived from prostate and mammary
glands. SHBG is a large glycoprotein (92.5 kD)
284
Han and Bouloux
Figure 5 The major human testicular steroidogenic pathway.
encoded by a gene located on 17p13.1. It circulates in

the serum as homodimer, and carries two binding
sites for sex steroids with high affinity for testosterone
and dihydrotestosterone (DHT), and relatively lower
affinity for oestradiol. Circulating SHBG concentrations determine free testosterone levels: As a corollary,
reproductive function is greatly influenced by various
conditions that affect SHBG levels (Table 1).
Table 1 Factors Altering Concentrations of Serum SHBG
Elevation of SHBG
Suppression of SHBG
Estrogen use
Androgen deficiency
Growth hormone deficiency
Hyperthyroidism
Hepatitis
Liver cirrhosis
Phenytoin
Androgen therapy
Obesity
Acromegaly
Hypothyroidism
Nephrotic syndrome
Corticosteroids
Hyperinsulinism
Gestagens
Abbreviation: SHBG, sex hormonebinding globulin.
Metabolism of Testosterone
Testosterone can also act as a prohormone for DHT and

oestradiol (Fig. 5), and diffuses into target cells where
conversion to 5a-DHT or 17b-oestradiol occurs, depending on the presence of metabolizing enzymes. There
are two high homology isoenzymes of 5a-reductase
responsible for testosterone conversion to DHT. Type I
5a-reductase is encoded by a gene on chromosome 5,
expressed mainly in skin and liver. A gene on chromosome 2 encodes type II 5a-reductase, which is
expressed predominantly in prostate, adrenal gland,
seminal vesicle, genital skin, hair follicles, and cerebral cortex. About 80% of circulating DHT is produced
peripherally and 20% directly secreted by the testes.
Testosterone and DHT bind to the identical AR,
but DHT is more potent, having a 10-fold greater
affinity for the receptor and slower dissociation.
About 30 mg of oestradiol a day is generated by
extratesticular aromatization of testosterone and
androstenedione in adipose tissue, osteocytes, and
285
Figure 6 The human androgen receptor gene. Reprinted with permission from WWW.ENDOTEXT.ORG, the free on-line Endocrinology
textbook, in Androgen Physiology: Receptor and Metabolic Disorders by AO Brinkmann, L J De Groot, Editor, Version November 2009,
published by MDTEXT.COM, South Dartmouth, MA, USA.
prostate. An additional 10 mg of oestradiol is secreted

directly by Leydig cells. In physiological concentrations, testosterone and its metabolites complement
one another during normal sexual development and
virilization at puberty.
Testosterone and DHT are catabolized in the
liver first by oxidation, reduction, or hydroxylation,
and second by conjugation with glucuronic acid or
sulfation at positions C3 or C17. Elimination takes
place via urine as the corresponding 17-ketosteroid
and sulfate (e.g., androsterone, etiocholanolone, epiandrosterone, and epitestosterone). Despite being
bound to transport proteins in the circulation, elimination of testosterone from the serum by liver is very
efficient as reflected by a short half-life (10 minutes) of
free testosterone in the blood.
the receptor contains a polymorphic polyglutamine

(CAG) tract with variable repeat sequences (normally
835 repeats) lying inside a split N-terminal transactivation domain (Fig. 7). The number of CAG
repeats affects the transcription intensity of the receptor. Longer CAG repeats are associated with weaker
activation of the AR-mediated transcription (12).
Coactivators and androgens tend to bind more
Structure and Function of the Androgen Receptor
The AR belongs to the steroid and thyroid hormone

receptor family and encoded by eight exons of a gene
located proximal to the centromere on the long arm of
the X chromosome (Xq1112) (Fig. 6). It encodes a 919
AA polypeptide with a conceptual molecular weight of
98.5 kD. AR is a DNA-binding protein and its activity
is ligand dependent and transcription regulating.
AR has three domains with different functions.
The amino-terminal (N-terminal) segment (exon A) of
Figure 7 Variation of the sequences of CAG repeats in the gene

encoding androgen receptor. Reprinted with permission from
WWW.ENDOTEXT.ORG, the free on-line Endocrinology textbook, in Androgen Physiology: Receptor and Metabolic Disorders
by AO Brinkmann, L J De Groot, Editor, Version November 2009,
published by MDTEXT.COM, South Dartmouth, MA, USA.
286
Han and Bouloux
Figure 8 DNA-binding domain of androgen receptor. Reprinted

with permission from WWW.ENDOTEXT.ORG, the free on-line
Endocrinology textbook, in Androgen Physiology: Receptor and
Metabolic Disorders by AO Brinkmann, L J De Groot, Editor,
Version November 2009, published by MDTEXT.COM, South
Dartmouth, MA, USA.
strongly to an AR with short CAG sequences, resulting

in stronger androgen effects. Short CAG repeat sequences (less than 22) are associated with increased risk of
prostate carcinoma (13), whereas sequences greater
than 38 have been shown to relate to androgen resistance and Kennedys syndrome, an X-linked degenerative spinal/bulbar muscular atrophy (14) associated
with gynecomastia, testicular atrophy, impotence, and
remarkably high incidence of diabetes (15).
The centrally located hydrophilic DNA-binding
domains (DBDs), exons B and C, carry two zinc
fingers, which bind to specific sections of DNA in or
next to androgen-sensitive genes and thus influence
transcription (Fig. 8). The DBDs of the steroid hormone receptors are largely similar (4090%), gene
specificity being conferred by the ligand-binding site.
The carboxy-terminal (C-terminal) end (exons (DH) carries the hydrophobic androgen-binding domain
responsible for testosterone and DHT binding. This
domain has 40% to 50% similarity with the AA
sequence of the corresponding domains of the progesterone, mineralocorticoid, and corticosteroid receptors.
However, there is only a slight similarity with the
ligand-binding domain of the estrogen receptor.
The unliganded AR is stabilized by its association
with a heat shock protein (HSP 90) acting as a molecular
chaperone. Binding of testosterone or DHT to AR induces a conformational change, causing AR to dissociate
from HSP 90, thereby becoming activated. The ligandbound AR dimerizes, which increases its DNA-binding
activity. The dimerized AR tandems target specific
DNA sections of androgen response elements (AREs)
in the promoter region of the androgen-sensitive genes,
thereby initiating gene transcription. The first zinc finger
is responsible for specificity of DNA binding. The
second zinc finger stabilizes AR binding. Binding of
AR to DNA influences transcription of androgen-sensitive genes, which lie downstream (30 ) of the AREs. The
Figure 9 Molecular mechanism of testosterone action on target

cell. Reprinted with permission from WWW.ENDOTEXT.ORG, the
free on-line Endocrinology textbook, in Androgen Physiology:
Receptor and Metabolic Disorders by AO Brinkmann, L J De
Groot, Editor, Version November 2009, published by MDTEXT.
COM, South Dartmouth, MA, USA.
transcriptional activity of the AR involves recruitment of

coactivators that link AR to components of the transcription machinery such as RNA-polymerase II (RNA-Pol
II), TATA box-binding protein (TBP), TBP-associating
factors, and general transcription factors. This communication triggers mRNA and protein synthesis, which
consequently results in an androgen response (Fig. 9).
Biological Effects of Testosterone
Testosterone is essential for development of the male

phenotype and optimal male function. In addition to
its role in reproductive function, testosterone activates
many other tissues, including bone, bone marrow,
kidney, liver, breast, skin, skeletal muscle, and brain.
The actions of testosterone depend on the stage of
development. During sexual differentiation, testosterone induces development and growth of the Wolffian
duct structures (epididymis, vas deferens, and seminal
vesicle) (Fig. 10). During puberty, testosterone induces
virilization, leading to development of secondary sexual characteristics. In the adult male, testosterone
maintains the male phenotype, sexual function and
exerts anabolic effects. It has an indirect effect on the
prostate and external genitalia, following conversion to
DHT (Fig. 10). If no testosterone is produced, these
organs regress, with failed seminal vesicle and prostate
development, leading to aspermia.
In the embryo, growth of the external genitalia is
dependent on the concentration of DHT generated in
situ from testosterone. A lack of DHT due to reduced
testosterone production or 5a-reductase enzymatic
conversion to DHT leads to an intersexual state,
with associated absent phallic development. During
puberty, increase in androgens causes penile growth.
After puberty, negligible androgen-induced penile
growth occurs because of downregulation of ARs
(16). Conversely, androgen deficiency in adult does
not result in reduction of penile size.
287
sexual development with insufficient masculinization

of male external genitalia, ranging from a completely
female phenotype to external male genitalia with
distal hypospadias. During fetal development, androgen deficiency also results in malpositioning of the
testes and microphallus.
Before Puberty
Figure 10 Upon entering the target cells, testosterone (T) binds

directly to the AR and the complex T-AR binds to specific DNA
sequences and regulates gene transcription, resulting in initiation
and regulation of spermatogenesis and in differentiation and
development of the Wolffian duct. Testosterone is also metabolized to DHT by 5a-reductase type II, which binds directly to the
AR and the complex DHT-AR interacts to specific DNA sequences and regulates gene transcription, resulting in differentiation
and development of the prostate and external genitalia. Abbreviation: AR, androgen receptor; DHT, dihydrotestosterone.
Source: Adapted from Ref. 2 with permission.
Testosterone and Cardiovascular System
Androgens may play an important role in the

increased risk of cardiovascular disease in men. Testosterone is an important determinant of cardiovascular health, having positive effects on several features of
the metabolic syndrome, inducing a reduction in waist
circumference, lipids, and inflammatory markers (17).
Although testosterone overreplacement in hypogonadal men reduces high-density lipoprotein (HDL) and
elevates low-density lipoprotein (LDL) cholesterol, this
effect may be counteracted by reduction in plasminogen activator inhibitor favoring increased fibrinolysis.
Testosterone and Central Nervous System
ARs are found in the hypothalamus, pituitary, cerebellar tonsil, and septum of the central nervous system (18). In the hypothalamus and pituitary,
androgens exert a negative feedback effect on GnRH
and gonadotrophin secretion. In addition, aromatase
and 5a-reductase are expressed in all regions of the
brain at varying density. Androgens exert a number of
effects on higher function, including drive, libido,
and possible effects on visuospatial recognition. Conversely, testosterone deficiency is associated with
depression and lack of motivation.
DEVELOMENT OF MALE HYPOGONADISM

Clinical Presentation of Male Hypogonadism
Fetal Stage
Androgen deficiency during sexual differentiation

(9th14th week of gestation) leads to a disorder of
Testosterone deficiency occurring during this period

leads to inadequate virilization, resulting in the syndrome of eunuchoidism with typical long limbs and
increased arm span due to late epiphyseal fusion of
the appendicular skeleton. The rate of long bone
growth is faster than that of the spine, resulting in a
high ratio of lower body to upper body length. Maldevelopment of other androgen-dependent organs
such the larynx (high-pitched voice) occurs and
there is reduced facial and pubic hair growth and
testes remain small.
Adulthood
Although voice, body proportions, and size of the

penis are unaffected by androgen deficiency at this
stage, body and facial hair growth dwindles, requiring
infrequent shaving. Symptoms include decreased sexual potency, loss of libido, and infertility. Testicular
size may be reduced. Long-term health complications
include osteoporosis because of a lack of stimulation
of osteoblasts by androgens and estrogen, and anemia
as a result of decreased stimulation of erythropoiesis
by testosterone. Quality of life is also impaired
because of loss of strength from muscle atrophy and
reduced vitality. Subfertility occurs because of loss of
testosterone action on spermatogenesis.
Etiology of Male Hypogonadism

Hypogonadism can be primary (testicular in origin:
hypergonadotrophic hypogonadism) or secondary,
(hypothalamic or pituitary in origin: hypogonadotrophic hypogonadism). This classification has therapeutic implications, because fertility may be restored with
appropriate hormonal stimulation in patients with
secondary hypogonadism, though not in primary.
Fertility options for men with primary testicular failure may include the use of donor sperm, adoption, or
intracytoplasmic sperm injection (ICSI). Either type of
hypogonadism may be caused by congenital or
acquired conditions (Table 2). Other causes of male
hypogonadism result from defects of AR or in the
enzymes that metabolize testosterone within the target
organ.
Congenital Causes of Hypogonadotrophic Hypogonadism
Idiopathic hypogonadotrophic hypogonadism and

Kallmanns syndrome. Both idiopathic hypogonadotrophic hypogonadism (IHH) and KS are caused by
insufficient secretion of hypothalamic GnRH, leading
to a loss of stimulation of the pituitary production of
gonadotrophins. As a consequence, androgen production and spermatogenesis by the testes are impaired.
288
Han and Bouloux
Table 2 Etiology of Male Hypogonadism

I. Hypogonadotrophic hypogonadism
Congenital
Idiopathic hypogonadotrophic hypogonadism
Kallmanns syndrome
Fertile eunuch syndrome
Genetic defects of gonadotrophin subunits
Prader-Willis syndrome
Laurence-Moon-Bardet-Biedls syndrome
Defective genes of pituitary differentiation (PIT-1 and PROP-1)
Mutations and deletions genes of gonadotrophin-releasing hormoneproducing neurons (DAX-1 gene)
Acquired
Tumor: Pituitary adenomas, residual cell tumors (craniopharyngiomas, epidermoid tumors, Rathkes pouch tumors), gamete tumors
(germinomas, teratomas, dysgerminomas), metastases
Infiltrative: Sarcoidosis, hemochromatosis, histiocytosis-X, tuberculosis
Infection: Tuberculosis, HIV/AIDS, fungal, syphilis
Trauma: Contusion, skull fracture, pituitary stalk transection
Vascular: Ischemia, Sheehans syndrome
Stress: Excessive exercise, mental stress, severe dieting (anorexia nervosa, anorexia bulimia), malnourishment
Illness: Diabetes, nephrotic syndrome, obesity, primary hypothyroidism, critical illness, sickle cell disease, thalassemia
Medical use and abuse: Radiation, anabolic steroids, glucocorticoids, narcotics, alcoholism, hypophysectomy
Aging
II. Hypergonadotrophic hypogonadism
Congenital
Klinefelters syndrome (47, XXY)
47,XYY syndrome
Testicular dysgenesis
Noonans syndrome
Defective androgen synthesis (enzyme deficiency):
20a-Hydroxylase
17,20-Demolase
3b-Hydoxysteroid dehydrogenase
17-Hydroxylase
17-Ketosteroid reductase (also known as17b-hydroxysteroid dehydrogenase)
Acquired
Infection: Mumps orchitis, HIV/AIDS, tuberculosis
Medical use and abuse: Opiates, chemotherapy, radiation, glucocorticoids, alcoholism, castration (gender reassignment)
Neoplastic: Testicular cancer
Trauma: Injury, orchidoplexy, crypto-orchidism, vanishing testes syndrome, seminiferous tubule failure
Illness: Cirrhosis, chronic renal failure, myotonic dystrophy, hyperthyroidism, autoimmune disease, Sertoli cell syndrome, diabetes,
metabolic syndrome, cystic fibrosis, sickle cell disease, thalassemia major
Others: Idiopathic
III. Defects in target organs
5a-Reductase (pseudovaginal perineoscrotal hypospadias)
Aromatase deficiency
Complete androgen insensitivity syndrome
Partial androgen insensitivity syndrome:
Rosewaters syndrome, Reifensteins syndrome, Gilbert-Dreyfus syndrome, Lubs syndrome
KS is a congenital disorder inherited as a sporadic

condition or as an X-linked recessive trait occurring in
about 1 in 10,000 male births. The gene KALIG-1
located in the regions of Xp22.3 for classic KS (19)
and associated anosmia has been cloned (20).
The KALIG-1 gene encodes anosmin 1, a cell
adhesion protein involved in the coordinated migration of GnRH-producing neurons and olfactory neurons. Born in the nasal pit, GnRH neurons migrate to
the hypothalamus during early fetal development
under the influence of anosmin 1. This process is
impaired in KS because of inactivating mutations of
the KALIG-1 gene (21). The concurrent underdeveloped
olfactory tract can be identified by magnetic resonance

imaging (MRI) scanning (22). Manifestations of X-linked
KS include bimanual synkinesis and unilateral renal
agenesis. Other defects such as cerebellar dysfunction,
cleft palate, and congenital deafness may also be present in autosomal dominant forms of KS because of
mutations of FGFR1. Recent studies have revealed
other candidate genes for KS including NELF, FGF8,
PKR2, and PK2 (23). Failure of gonadotrophin-induced
testosterone production results in testicular maldescent.
Partial pubertal development may be present in
patients with partial defects, making early diagnosis
difficult.
GnRH-producing neurons also appear to be

affected by mutations and deletions of the DAX-1
gene (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome
gene 1). DAX-1 is located on Xp21 and expressed in
the hypothalamus, pituitary, adrenal gland, gonads,
and gonadal structures during fetal development. It
encodes a 470 AA orphan receptor related to the
steroid receptor family. It regulates expression of
hormones involved in sexual differentiation. Mutations in the C-terminal end of DAX-1 lead to hypogonadotrophic hypogonadism as well as adrenal
hypoplasia congenita (24).
Fertile eunuch syndrome. This syndrome is a
variant of IHH, also known as Pasqualinis syndrome.
Patients have reduced GnRH secretion, though just
enough to stimulate pituitary FSH and a minute
amount of LH secretion. The small amount of LH is
sufficient to raise intratesticular testosterone levels but
not extratesticular testosterone; thus, these patients
are fertile, as spermatogenesis is intact, but they
acquire eunuchoid body segments.
Prader-Willis syndrome. Prader-Willis syndrome (PWS) results from a genetic imprinting defect
caused by a seven-exon gene deletion or inactivation
of the paternally inherited chromosome 15 (15q1113),
whereby the maternally inherited copy is silent. PWS
may also be caused by uniparental disomy from
inheritance of two copies of the inactive maternal
gene. The incidence is 1 in 15,000 to 20,000 live births.
The syndrome is characterized by hypotonia at birth,
hypogonadism, short stature, facial dysmorphism,
learning difficulties, hyperphagia, and obesity (25).
Hypogonadism in such patients is due to a disturbance in GnRH secretion, resulting in undescended
testes and microphallus at birth, as well as failure of
onset of puberty. Hyperphagia leads to childhood
obesity, and up to 10% go on to develop diabetes (26).
Laurence-Moon-Bardet-Biedls syndrome. LaurenceMoon-Bardet-Biedls syndrome (LMBBS) is rare autosomal recessive heterogeneous genetic disorder (prevalence <1:100,000). At least 12 of the genes (BBS)
responsible for LMBBS have been identified. LMBBS
is characterized by learning difficulties, retinitis pigmentosa, polydactyly, obesity, diabetes, renal abnormalities, and hypogonadism secondary to
hypothalamic dysfunction. There has been longstanding uncertainty as to whether the Laurence-Moons
syndrome and the Bardet-Biedls syndrome are two
separate conditions. Klein and Ammann suggested
that the patients of Laurence and Moon represented
a distinct disorder with paraplegia and without
polydactyly and obesity (27), but a 22-year prospective cohort study of 46 patients from 26 Newfoundland families conducted by Moore et al. found no
correlation between clinical/dysmorphic features
and genotype (28).
Defective genes of pituitary differentiation. The
pituitary transcription factor-1 (PIT-1) and prophet
of PIT-1 (PROP1) play an important role in the ontogenesis, differentiation, and function of somatotrophic, lactotrophic, thyrotrophic and gonadotrophic
cells. Mutations in these factors result in pituitary
289
hormone deficiencies (gonadotrophins, growth hormone, prolactin, and TSH) and delayed puberty and
hypogonadism (29). Other factors that regulate pituitary differentiation include LHX3, a homeodomain
transcription factor, and HESX-1 gene expressed in
embryonic brain and pituitary. Mutations of HESX-1,
a transcription factor and homeobox gene, cause
septo-optic dysplasia (30), a syndrome characterized
by panhypopituitarism, optic nerve atrophy, and midline defects of the cerebrum (corpus callosum agenesis). Another genetic cause of pituitary failure is due to
inactivating mutations of LH b-chain, which results in
LH deficiency and thus androgen deficiency and
infertility.
Acquired Causes of Hypogonadotrophic Hypogonadism
Pituitary disorders. Benign adenoma of the anterior lobe of the pituitary is the most common cause
of pituitary insufficiency. Prolactinoma is the most
frequent, followed by nonfunctioning adenomas.
Growth hormone and ACTH-secreting tumors are
less common hormone-secreting adenomas causing
acromegaly and Cushings disease, respectively.
Gonadotrophin and thyrotrophin-secreting tumors
are even rarer causes. Pituitary disease may also
occur with granulomatous and infiltrative disorders,
cranial trauma with or without pituitary stalk transection, irradiation, and hypophysitis.
Serious illness, acquired immune deficiency syndrome, and stress. Transient hypogonadotrophic
hypogonadism may occur in patients with serious
disorders or malnutrition. Testosterone levels in
patients with (acquired immune deficiency syndrome)
AIDS may be suppressed and associated with low
gonadotrophin levels (suggestive of hypothalamicpituitary involvement) or elevated gonadotrophin levels (suggestive of primary testicular disease).
Aging. The concept of a male andropause is
debatable (31). There is evidence to suggest that
some aging males with reduced production of testosterone acquire clinical features of hypogonadism,
increased risk of myocardial infarction, and osteoporosis. Levels of SHBG need to be taken into account as
they usually increase with advancing age, probably
related to higher serum oestradiol levels from increased
adiposity and from a fall in IGF-1. Often the FSH and
LH levels are mildly increased, suggesting that a
primary testicular disorder may also be present. The
circadian variation in serum testosterone levels may be
lost with aging. Management of such individuals may
be guided by recommendations prepared together by
several groups of endocrinologists and urologists (32).
Congenital Causes of Hypergonadotrophic Hypogonadism
Klinefelters syndrome. This condition is most

commonly caused by an extra X chromosome as the
result of nondysjunction during the first meiotic division of the maternal gamete, and occurs in 1 in 400 live
births (33), the incidence increasing with older maternal age. Up to 90% of men with Klinefelters syndrome
have the classic 47,XXY karyotype, but mosaicism is
occasionally present (34). Less than 5% are due to
290
Han and Bouloux
nondysjunction during zygotic mitosis. Others variants may be due to an additional Y chromosome
such as 48,XXXY or 48,XXYY.
Patients with Klinefelters syndfrome present
with small (<5 mL) firm testes, gynecomastia, eunuchoid habitus, and raised gonadotrophin levels. Despite
low testosterone production, high levels of SHBG may
result in normal range testosterone levels in about
40% of patients. Azoospermia is common, but those
with mosaicism may have some spermatogenesis and
be potentially fertile. Histologically, there is hyaline
degeneration of the germinal epithelium with few
intact gametes (35) and immature Leydig cells (36).
Virilization, libido, and sexual potency may develop
initially, but wane by the age of 25 to 35 years as Leydig
cells begin to fail. High gonadotrophin levels hyperstimulate testicular aromatase activity leading to elevated oestradiol levels, causing gynecomastia in 50% of
patients. Enlarged breasts are unsightly and patients
are predisposed to a 3% to 5% risk of breast cancer.
Patients are usually eunuchoid and have an increased
risk of osteoporosis, learning disabilities as manifested
by dyslexia, and attention deficit disorder. There is
increased incidence of autoimmune disorders, such as
systemic lupus erythematosus, rheumatoid arthritis,
and Sjogrens syndrome. The underlying cause may
be due to lower autoimmune-protecting testosterone
and higher autoimmune-promoting estrogen levels in
Klinefelters patients. These autoimmune disorders
may become attenuated following initiation of testosterone therapy (37).
47,XYY syndrome. An extra Y chromosome
from paternal meiosis results in 47,XYY syndrome,
occurring in about 0.1% of newborn males. There are
few clinical signs other than a relatively tall stature.
Testicular function is mildly impaired with normal
Leydig cell function, and thus the levels of testosterone and LH are within reference range. Although,
most patients are fertile, a proportion may be azoospermic because of germinal epithelium maturation
arrest. Serum FSH levels are usually increased, and
cognitive abilities may be reduced in some men. It was
originally thought that patients with 47,XYY syndrome have aggressive behavior (38), but there has
been no consistent evidence to support this notion.
Testicular dysgenesis. Testicular (gonadal) dysgenesis is a heterogeneous disorder with both Y-linked
and non-Y-linked forms. The underlying sex chromosome abnormality includes XO, mixed XO/XY karyotype, or pure XY karyotype with streak gonads (39).
Swyers syndrome is a condition of pure testicular

dysgenesis in association with an XY karyotype (40).
Eighty percent of patients with sporadic or familial 46,
XY gonadal dysgenesis do not have a mutation or
deletion of the SRY gene, indicating that other autosomal or X-linked genes have a role in sex determination.
In about 10% of these patients, defects are present in the
gene on the Y chromosome responsible for differentiation of the testes (SRY) (40,41). Gonadal dysgenesis has
also been shown to be caused by duplication (41) or
deletion mutation (42) of DAX-1 on Xp21.2. Steroidogenic factor 1 (SF1/AdBP4/FTZF1, NR5A1) is a nuclear
receptor transcription factor involved in the regulation
of adrenal and gonadal development, steroidogenesis,
and reproduction (43). A recent study identified heterozygous missense mutations in NR5A1 in 4 out of 30
individuals with XY gonadal dysgenesis. These mutations (V15M, M78I, G91S, L437Q) were shown to impair
transcriptional activation through abnormal DNA binding (V15M, M78I, G91S), altered subnuclear localization
(V15M, M78I), or disruption of the putative ligandbinding pocket (L437Q) (44). Such patients usually
have ambiguous genitalia, requiring surgical repair to
the assigned gender (45). Streak gonads are associated
with an increased risk of gonadoblastoma, and gonadectomy is usually performed at an early age with
immediate estrogen replacement therapy as these
patients are often raised as females (46).
Enzyme defects of testosterone biosynthesis. Biosynthesis of testosterone from cholesterol is catalyzed
in the Leydig cells by five different enzymes (Fig. 5).
Defects in any of these enzymes lead to failure of
testosterone biosynthesis (Table 3). Defect of an enzyme
characteristically leads to reduction of testosterone with
an excessive production of its substrate. The Wolffian
duct fails to differentiate into the male sexual organs
and feminization of the external genitalia occurs. The
phenotype of the individual depends on the residual
activity of the defective enzyme. If a complete enzyme
block occurs, female genitalia are formed with a blind
vaginal pouch, absent uterus, Fallopian tubes, and
ovaries because of the expression of anti-Mullerian
hormone. The condition is found occasionally when
patients undergo investigations for hernias but turn
out to be abdominal or inguinal testes. The enzyme
defect is normally discovered during investigations for
primary amenorrhea. As testicular and adrenal steroid
biosynthesis shares many enzymes in common, defects
may lead to symptoms of hypogonadism and adrenocortical insufficiency simultaneously.
Table 3 Enzyme Defects Affecting Androgen Biosynthesis

Enzymatic defect
Cortisol
Androstenedione
Testosterone
Pregnenolone
Progesterone
17a-Hydroxyprogesterone
;
;
$
;
$
;
;
;
;
:
;
;
;
;
;
;
;
$ or :
:
$
;
;
$ or :
:
$
;
;
$ or :
;
$
20a-Hydroxylase
17,20-Demolase
3b-Hydoxysteroid dehydrogenase
17-Hydroxylase
17b-Hydroxysteroid
dehydrogenasea
5a-Reductase
a
Also known as 17-ketosteroid reductase.
Acquired Causes of Hypergonadotrophic Hypogonadism
Myotonic dystrophy. Myotonic dystrophy is an

autosomal dominant disorder, characterized by hypogonadism, muscle weakness, and frontal balding.
Repeat sequences of CTG in an untranslated region
of the DMPK gene have been identified in many of
these patients, but the disorder seems to be genetically
and phenotypically heterogeneous. Testicular failure
usually occurs after age 40 years; thus, they often have
children who may inherit the disease. Testosterone
levels may be variably decreased in the presence of
azoospermia and high gonadotrophin levels.
Cryptorchidism. Cryptorchidism may occur unilaterally or bilaterally in about 3% to 4% at birth. Since
most testes descend with age, the one-year incidence is
reduced to less than 1%. Because normal testicular
descent requires normal pituitary function and DHT
levels, the incidence of cryptorchidism is increased in
patients with KS. It is vital to distinguish cryptorchidism
from retractile testes so that the treatment with hCG or
orchidoplexy is applied correctly. The aim is to bring
down the undescended testes into the scrotum before
one to two years of age to improve fertility potential. In
prepubertal boys, a four-week trial of hCG may be used
to determine whether testicular descent can occur
before considering surgical intervention.
Vanishing testes syndrome (congenital anorchism or
prepubertal functional castrate). The initial presentation of vanishing testes syndrome is sexual immaturity in a male patient with impalpable testes. The
condition may arise from fetal testicular torsion after
sufficient testosterone exposure to complete reproductive tract development. The levels of testosterone are
low with elevated gonadotrophins. If the LH levels are
only minimally increased, hCG stimulation testing of
the gonad may help diagnosis. An absent testosterone
response to hCG stimulation suggests the vanishing
testes syndrome. A response to hCG stimulation may
indicate the presence of intraabdominal testes and
requires further assessment by MRI scanning.
Other acquired causes of hypergonadotrophic hypogonadism. These include trauma, mumps orchitis,
radiation and chemotherapy, and Sertoli-cell-only syndrome. Recent evidence has suggested that in utero
exposure to maternal toxic substances such as cigarette
smoking and alcohol may have long-lasting adverse
effects on the reproductive system, including reduced
testicular size and sperm production in adult males (47)
and cryptorchidism in newborn boys (48). The underlying mechanism is not clear, but toxins and hypoxia
may be important factors interfering with androgen
activity and/or their modulators. In rats, androgen
action blockade by flutamide results in cryptorchidism
and hypospadias, but only within a critical programming window for reproductive tract masculinization,
equating to 8 to 14 weeks of gestation in humans (49).
Hypogonadism Secondary to Target Organ Defects
Target organ defects are disorders in which testicular

hormones testosterone and/or its metabolites (DHT
and oestradiol), and AMH do not function, despite
291
intact testicular function. The disorders may be due to

defects of the corresponding receptor or the enzymes
(5a-reductase or aromatase).
Androgen insensitivity syndrome (AIS) is caused
by sporadic or familial loss of function mutations of the
AR and affects 1 in 20,000 to 90,000 births. More than
600 mutations have been documented in the AR gene,
located on Xp11 to 12, 200 in exons B-H carrying
steroid and DBDs. Although there are only 23 mutations in exon A, a complete loss of receptor function
occurs compared with slight receptor dysfunction in
mutations of other exons. The N-terminal section (exon
A) of the AR is very variable, containing a polymorphic
polyglutamine (CAG) repeat sequences comprising 8 to
35 repeats (Fig. 7). Excess number of CAG repeats (>38)
results in androgen resistance because coactivators and
androgens bind less strongly to AR with long CAG
sequences. The severity of androgen resistance is
reflected by the variety of clinical phenotype of affected
individuals. Testosterone levels may be within or
slightly above the male testosterone reference range
with modestly elevated gonadotrophin levels.
Complete androgen insensitivity syndrome. Complete AIS is the most severe form of AR resistance.
These patients have a 46(XY) karyotype and are phenotypically female, with normal pubertal development
including unambiguous female external genitalia, but
lack Mullerian duct structures and androgen-dependent body hair. They typically present with inguinal
hernia during infancy or with primary amenorrhea in
adolescence. The testes may reside in the abdomen,
groin, or labia majora, and secrete androgen and
AMH. Testosterone has no effects on target organs;
thus, the Wolffian structures fail to differentiate, leading to the absence of epididymis, seminal vesicle, duct,
and prostate. Testicular AMH inhibits the differentiation of Mullerian structures resulting in the absence of
the uterus, Fallopian tubes and ovaries, and a short
blind pouch vagina. Osteopenia or osteoporosis occurs
in about a quarter of individuals with complete AIS
(46) and estrogen replacement has been shown to
improve bone mineral density (BMD).
Reifensteins syndrome. Partial AR defect in this
disorder results in a male phenotype at birth with
variable pseudohermaphroditism. The testes are often
not descended completely and small in size with
resultant atrophy of the germinal epithelium and
azoospermia. The lack of feedback inhibition of the
pituitary by testosterone results in elevated LH levels,
and as a consequence, hyperstimulation of Leydig
cells occurs. FSH may be normal or slightly increased.
The external genitalia are insufficiently masculinized,
ranging from incomplete labioscrotal fusion with formation of a pseudovagina, through perineoscrotal
hypospadia, to slight penile hypospadia. Sometimes,
the only signs are revealed at puberty, with gynecomastia and azoospermia. The Wolffian duct structures
may be normal or hypoplastic. Virilization may be
delayed with normal pubic and axillary hair distribution, but body hair is sparse and the voice may not
break. Gynecomastia in these patients, unlike those
with Klinefelters syndrome, is not predisposed to
increased risk of breast cancer.
292
Han and Bouloux
5a-Reductase deficiency. This is an autosomal

recessive condition caused by various types of mutations
including substitution and deletion of the 5a-reductase
type 2 (SRD5A2) gene, leading to 5a-reductase deficiency in the background of a XY genotype (50). The
degrees of masculinization defects correlate weakly
with genotype defects. External female genitalia are
present at birth and these individuals are often raised
as girls until puberty, when the underlying condition
is revealed by developing male secondary characteristics. The diagnosis is based on clinical manifestations and an increased testosterone to DHT ratio, both
after puberty and in response to hCG before puberty.
Sexual reassignment is a major issue, and patients
would benefit from clinical psychological assessment
before proceeding to corrective surgery.
TESTOSTERONE THERAPY
Goals of Testosterone Therapy
Ideally, long-term testosterone replacement therapy
(TRT) should be safe, potent with a high tissue-specific selectivity, convenient and inexpensive with longacting properties. Maximal efficacy within adequate
safety limits should be taken into consideration and
cost-effectiveness compared with other drugs. The
aim of treating testosterone deficiency is to restore
and maintain stable, physiological concentrations of
serum testosterone for prolonged periods using convenient formulations that facilitate compliance, and
avoid wide fluctuations of androgen levels. Ultimately, the outcome of treatment is to improve mortality,
but it appears that attaining morbidity benefits is
more realistic, aiming to improve quality of life,
symptoms of hypogonadism, prevention of longterm health sequelae, development of secondary sex
characteristics, and restoration of fertility in cases of
hypogonadotrophic hypogonadism. The adverse
effects of testosterone therapy should be considered
(Table 4). Because of the stimulatory effects on androgen-dependent tumor growth, treatment with testosterone, pulsatile GnRH, and gonadotrophin are
contraindicated in those with prostate cancer, male
breast cancer, or untreated prolactinoma. Relative
contraindications are applied to those with sleep
apnea and polycythemia because of the risk of hyperviscosity. Because testosterone treatment tends to
reduce sperm counts and testicular size, it is not
recommended for men who are seeking fertility.
Figure 11 Testosterone and its derivatives.
Testosterone Therapy in Adult Males

with Hypogonadism
For reasons of safety and ease of monitoring, testosterone rather than synthetic androgens is used. It has a
short circulating half-life or transit time and low oral
bioavailability due to rapid hepatic oxidation and
glucuronidation to biologically inactivate metabolites.
This has led to the development of parenteral depot
formulations (injectable, implantable, and transdermal), products bypassing the hepatic portal system
(sublingual, buccal, gut lymphatic absorption) and
orally active synthetic androgens (51). Testosterone
is the prototype androgen with a 19-carbon, fourring steroid structure and two oxygens (3-keto, 17bhydroxy) including a D4 nonaromatic A ring (Fig. 11).
Testosterone therapy aims to attain a physiological range of serum testosterone (942 nmol/L), DHT
and oestradiol levels, to gain optimal virilization and
normal sexual function. Testosterone therapy can be
used in the male patient with hypogonadism who is
not seeking fertility or is not able to achieve fertility
and late teenage males with delayed puberty. The
approved preparations of testosterone for clinical use
include long-acting and short-acting intramuscular
preparations, scrotal and transdermal patches, transdermal gel, and orally administered agents (Table 5).
Orally administered testosterone such as testosterone
undecanoate is available, but it undergoes hepatic
metabolism rapidly (52), thus requiring frequent dosing. As a result, the levels of testosterone are often
erratic. For patients with hypogonadotrophic hypogonadism requiring fertility, hCG with or without
Table 4 Common Adverse Effects of Testosterone Replacement Therapy and Recommended Monitoring Tests During the Course of
Therapy
System/organ
Adverse effect
Monitoring during therapy
Prostate
Hematological
Lipids
Spermatogenesis
Respiratory
Benign prostate hypertrophy

Polycythemia, hyperviscosity
Dyslipidemia
Reduced sperm counts
Sleep apnea
Digital rectal examination, prostate specific antigen

Hemoglobin, hematocrit
Fasting lipid profile
Sperm counts
Clinical symptoms, sleep study
293
Table 5 Currently Available Testosterone Preparations

Route
Generic name
Trade name
Dosage
Adverse effects
Transdermal
Testosterone patch
TTS scrotal
Androderm
Testoderm
2 $ 5 mg/day
1 membrane/day
Contact dermatitis
Supraphysiological
dihydrotestosterone levels
Testosterone gel
Testosterone gel
Testosterone enanthate
Testogel, Androtop gel

Testim
Testosterone Depot
Testosterone undecanoate
Nebido
2550 mg/day
50 mg/day
1 ampoule (250 mg)
every 23 wk
1 ampoule (1 g) every 12 wk
Implants
Testosterone
Testosterone implants
6 implants (1.2 g) every 6 mo
Oral
Buccal
Testosterone undecanoate
Testosterone
Andriol Testocaps
Striant
24 capsules (40 mg) daily

1 tablet (30 mg) twice daily
Parenteral
human menopausal gonadotrophin (hMG) or pulsatile GnRH therapy with or without assisted reproduction are considered (53).
Transdermal Testosterone
Pharmacogenetics and metabolism. About 10% to

15% of the transdermal testosterone is absorbed and
the remaining 85% to 90% is lost, some of which may
be because of metabolism by 5-a reduction, while
factors such as variation in the clearance rate of
testosterone, which could be a function of skin blood
flow, skin temperature, or perspiration may also contribute. Short-term application (714 days) of 5 to 10 g
of 1% transdermal testosterone gel (5 g gel contains
50 mg testosterone as active ingredient) results in peak
serum testosterone levels between 18 and 24 hours
(54), with a steady state achieved after application of
the testosterone gel after 7 to 14 days. Studies have
shown that DHT and oestradiol levels increased steadily after the first application (55). Long-term evaluation
over one, three, and six months of treatment showed
steady serum testosterone, DHT, and oestradiol levels
similar to that of short-term pharmacokinetics, with
only small and variable peaks of serum testosterone
after each daily application. Suspension of testosterone
application leads to a gradual fall in serum testosterone levels between 48 and 96 hours, indicating the
presence of a testosterone reservoir that is a unique
feature of this form of delivery (54). With continued
application of testosterone gel, accumulation rates
showed no further increases, providing further reassurance on the safety of this delivery system (55).
Serum DHT concentrations increase threefold
after application of 50 mg of testosterone in 5 g gel
and nearly fivefold with 100 mg of testosterone in 10 g
gel, a higher increase than that observed with patches
(56). This could be the consequence of both a higher
delivery of bioavailable testosterone and a higher
conversion of testosterone to DHT in the skin where
high 5-a reductase activity is expressed as well as the
larger area of skin surface exposed to the testosterone
gel compared with testosterone patch. DHT levels are
nonetheless much lower than that observed with
scrotal patches.
Potential partner transfer

Fluctuating symptoms
Deep and slow intragluteal
injection
Specialist minor surgical
implantation
Variable testosterone levels
Gum/buccal irritation
Clinical efficacy. The average recommended dose

of transdermal testosterone is 50 mg (in 5 g of gel)/day,
but about a fifth of patients may not achieve stable
serum testosterone concentration in the young adult
reference range (300100 ng/dL or 10.434.7 mmol/L).
It is uncertain whether lower responders are less compliant or are biologically different in that they might
have low absorption and/or high clearance of testosterone. In such patients, a trial of injectable testosterone
may be desirable (57).
A dose of 1% testosterone gel has been shown to
have positive benefits on sexual function, mood, muscle strength, body composition, and BMD in men with
various forms of hypogonadism over a considerable
time (54,58). Increases of about 1% to 2% in BMD after
six months of treatment appeared to relate to the
testosterone level achieved (58). In patients with treatment-resistant depression, 1% testosterone gel added
to existing antidepressant regimens resulted in
reduced symptoms of depression (59). After about
three years of treatment, levels of total LDL and
HDL cholesterol and triglycerides remained
unchanged (54). For this reason, testosterone gel is
considered an appropriate therapy for patients with
hypogonadism as well as age-related male hypogonadism (60).
Scrotal testosterone patches are available in 40
and 60 cm2 sizes, delivering 4 and 6 mg of testosterone
daily, respectively. Scrotal skin absorbs testosterone
better than does nonscrotal skin, but may be reduced
if the scrotum is small or has abnormal skin surface.
Because genital skin contains high concentrations of
5a-reductase, DHT levels are often persistently elevated (61). The HDL to total cholesterol ratio does not
change by this route of testosterone therapy. The longterm potential effects of increased levels of DHT are
currently unknown but may cause benign prostate
hypertrophy (BPH). The cost of using the scrotal
patch is expensive but is convenient to use.
Parenteral Testosterone Preparations
Enanthate and cypionate testosterone. The absorption of these esters is prolonged by suspension in oil.
After intramuscular injection, peak levels occur in
294
Han and Bouloux
about 72 hours and a slow decline occurs during the

following one to two weeks. A steady state of plasma
testosterone between injections can be achieved by
doses between 50 and 100 mg of testosterone administered intramuscularly at 7 to 10 days intervals.
Higher doses of 100 to 150 mg of testosterone every
two weeks can also be used, but the use of 300 mg
injections every three weeks is associated with wider
fluctuations of testosterone levels, wider swings
between peak and nadir, resulting in fluctuating
symptoms.
Lower doses of testosterone are used in adult
male patients with prepubertal onset of hypogonadism
who are going through puberty for the first time during
therapy. The starting testosterone dose of 50 mg every
three to four weeks with a gradual increment during
subsequent months until full replacement is achieved
within 12 months. It may take three to four years to
attain full virilization. Men with BPH who have hypogonadism and prostatic symptoms may be given 50 to
100 mg of testosterone every two weeks as an initial
regimen with careful monitoring of urinary symptoms
and prostate examinations.
Undecanoate preparations. Nebido1 is a testosterone undecanoate preparation suspended in castor
oil for intramuscular injection. This testosterone ester
had been used in oral capsules since the 1970s, but
had obviously already been applied as intramuscular
injections in China for several years, with a long halflife (62). The vehicle was changed from Chinese teaseed oil to castor oil in Europe (61,63,64). When
compared with testosterone enanthate, serum testosterone levels remain in the normal range for long
periods of time (64). Some patients have now been
followed for up to eight years, with good substitution
and no serious side effects (65). Treatment necessitates
1000 mg injections every 10 to 14 weeks, following a
loading dose at 6 weeks (32,66). Adjustments of injection intervals may be scheduled according to serum
testosterone levels determined from serum samples
taken just before the next injection. In addition to its
use in hypogonadism, testosterone undecanoate has
become a valuable asset in male contraception trials
and, as such, has outlived testosterone enanthate and
buciclate. Testosterone undecanoate has the potential
to be combined with norethisterone enanthate (NETE)
into one contraceptive regimen, and in combination
with subcutaneous etonorgestrel implants, it is currently being tested in Europe by Schering and
Organon in multicenter trials for male contraception
(67). Since this preparation in teaseed oil has a shorter
half-life than its European variant in castor oil, it
needs to be injected more frequently than Nebido.
However, a trial involving 308 couples in China, using
monthly injections of 500 mg testosterone undecanoate, resulted in 97% of men showing azoospermia
with complete contraceptive protection.
Gonadal Stimulation in Hypogonadotrophic

Hypogonadism
Because gonadotrophin or GnRH therapy is effective
only in hypogonadotrophic hypogonadism, this
diagnosis must be established before consideration

of therapy. Although these agents may also be used
to induce puberty in boys and to treat androgen
deficiency in hypogonadotrophic hypogonadism, the
major use of these preparations is in the initiation and
maintenance of spermatogenesis in hypogonadotrophic men who desire fertility.
Gonadotrophin Therapy in Testosterone Deficiency
Because of its ability to bind to LH receptors on

Leydig cell, hCG can stimulate the production of
testosterone. Peripubertal boys with hypogonadotrophic hypogonadism and delayed puberty can be
treated with hCG instead of testosterone to induce
pubertal development. The initial regimen of hCG is
usually 1000 to 2000 IU administered intramuscularly
once to twice a week. The clinical response is monitored, and testosterone levels are measured about
every two to three months for optimal dose adjustments. High doses of hCG may reduce testicular
stimulation by downregulating the end organ. The
advantages of hCG over testosterone include its long
half-life and the stimulation of testicular growth, and
maintenance of greater stability of testosterone levels,
resulting in fewer fluctuations in hypogonadal symptoms. In addition, hCG treatment is necessary for
stimulating enough intratesticular testosterone to
allow the initiation of spermatogenesis. The disadvantages of hCG include the need for more frequent
injections and greater cost.
Gonadotrophin Therapy for Induction of Spermatogenesis
Male patients with onset of hypogonadotrophic hypogonadism before completion of pubertal development
may have testes generally smaller than 5 mL. These
patients usually require therapy with both hCG and
hMG (or FSH) to induce spermatogenesis. Men with
partial gonadotrophin deficiency or who have previously (peripubertally) been stimulated with hCG may
initiate and maintain production of sperm with hCG
therapy alone. Men with postpubertal acquired hypogonadotrophic hypogonadism and who have previously had normal production of sperm can also
generally initiate and maintain spermatogenesis with
hCG treatment alone. Fertility may be possible at much
lower sperm counts (less than 1 million/mL) than
what would otherwise be considered fertile. Therapeutic dose of hCG starts at 1000 to 2000 IU intramuscularly twice a week with monthly monitoring of
testosterone levels for appropriate dose adjustments,
which may take two to three months to achieve. When
normal levels of testosterone are attained, monthly
assessment of testicular growth and sperm counts is
carried out during a 12-month period. Because of the
high cost of hMG (or FSH) preparations, hCG usually is
the initial therapy of choice for at least 6 to 12 months.
In general, the response to hCG therapy is greater in
those with large initial testicular volume. In completely
hypogonadotrophic men, combining purified FSH and
testosterone without LH or hCG does not stimulate
spermatogenesis. If spermatogenesis has not been initiated by the end of 6 to 12 months of therapy with hCG
or LH, administration of an FSH-containing preparation is initiated in a dosage of 225 IU intramuscularly

three times a week along with the hCG injections. If
pregnancy occurs, the patients regimen can be
switched to hCG only to allow continued spermatogenesis for subsequent potential pregnancies. If no further
pregnancies are desired, testosterone therapy can be
used, or long-term hCG therapy can be continued with
appropriate contraceptive measures. Rarely, antibodies
against hCG may arise, rendering therapy ineffective;
in such situations, human LH may be used as
alternative.
Gonadotrophin-Releasing Hormone Therapy
In patients with hypothalamic hypogonadism (intact

pituitary gland), synthetic GnRH can be given in a
pulsatile fashion subcutaneously through a pump
every two hours. Response to GnRH therapy reflected
by two weekly LH, FSH, and testosterone levels.
GnRH therapy can be used to initiate pubertal development, maintain virilization and sexual function, and
initiate and maintain spermatogenesis. In most
patients, these effects may take from 3 to 15 months
to achieve sperm production. Fertility can be achieved
with very low sperm counts (<1 million/mL). GnRH
may be more effective than gonadotrophin stimulation
in increasing testicular size and initiating spermatogenesis in many patients with hypogonadotrophic
hypogonadism.
Assisted Reproductive Technology
ICSI directly into the egg by a single sperm or immature form retrieved from the testis is sufficient to
fertilize an egg. In vitro fertilization with ICSI may
be a viable option in many men with hypogonadism
who cannot otherwise be induced to produce enough
sperm as well as in the presence of a female factor that
may further make pregnancy by the couple impossible. The procedure is expensive and the low-cost
intrauterine insemination may be more suitable
when the patient has mild to moderate oligospermia.
Pituitary Tumors
Patients with acquired hypogonadotrophic hypogonadism may require assessment for a possible pituitary
tumor with appropriate pituitary imaging studies, such
as MRI, and assessment of full pituitary function to
provide the correct diagnosis and therapy. If a prolactinoma is present, medical therapy with bromocriptine,
pergolide, or cabergoline would be the first approach to
reduce prolactin levels sufficiently to allow gonadal
function to resume or allow stimulation with gonadotrophins. Even when prolactin levels cannot be normalized, hCG therapy alone or combined with hMG (or
FSH) therapy may stimulate spermatogenesis. Surgical
therapy may be considered for significant pituitary
tumors that are not prolactin-secreting microadenomas,
and only those with prolactin-secreting microadenomas
who cannot tolerate medical treatment because of
adverse effects.
295
FUTURE RESEARCH AND DEVELOPMENT

Testosterone Therapy in Combination with
Phosphodieterase Type 5 Inhibitors
The use of testosterone to treat human erectile dysfunction (ED) has proved controversial because of
conflicting findings. For example, it has been found
that erectile function remains intact in some severely
hypogonadal men who have reduced libido; on the
other hand, testosterone therapy in hypogonadal men
with ED leads to improvement of erectile function
(68). Testosterone probably has a complex interaction
with the penile system through its influences on the
metabolism of mediators such as nitric oxide (NO)
and cyclic guanosine monophosphate (cGMP), as well
as its action on phosphodieterase type 5 (PDE5). The
normal erectile function involves the synthesis of the
neurotransmitter NO and the subsequent accumulation of cGMP (69). The NO formation, which leads to
erection, is regulated by the activity of NO synthase
(NOS) isoenzymes, whereas cGMP degradation,
which promotes smooth muscle tone and terminates
erection, is specifically controlled by PDE5. The regulation of the activity of these two counteracting
enzymes allows the penis to be contracted for the
majority of the time. Androgens play a pivotal role
in these mechanisms by regulating both NOS and
PDE5 activity (70). In castrated mice testosterone
replacement induces de novo DNA synthesis in the
smooth muscle cells and blood vessels, with a pancellular proliferative effect in the penis and endothelial NOS expression (71). Testosterone also induces
second messenger signal transduction cascades,
including increases in cytosolic calcium and activation
of PKA, PKC, and mitogen-activated protein kinase
(MAPK), leading to smooth muscle relaxation, neuromuscular and junctional signal transmission, and neuronal plasticity (72).
PDE5 inhibitors such as sildenafil enhance vasodilatation by blocking the inactivation of cGMP,
resulting in increased smooth muscle relaxation and
better erections in the presence of sexual stimulation.
It has been shown that the efficacy of sildenafil is
improved by the presence of testosterone (69,71). The
idea of combining therapy of testosterone with PDE5
inhibitors is based on several reasons. First, there is a
large proportion (up to 50%) of hypogonadal men with
ED who do not respond to monotherapy of PDE5
inhibitor (73), and second, the presence of a consensus
sequence for the AR in the 50 -flanking region of the
PDE5 promoter suggests that testosterone could regulate PDE5 expression (72). In a clinical setting, it has
been demonstrated that TRT improves ED in partial
androgen-deficient aging men (74), renal transplant or
dialyzed patients (75), and in patients with type 2
diabetes (76) who fail monotherapy of sildenafil.
Pharmacogenetics
Original research on X-linked spinal and bulbar muscular atrophy (Kennedys disease) suggested a mutation at the N-terminus of the AR gene exon 1, involving
296
Han and Bouloux
Table 6 Diseases Related to Abnormally Short or Long

Polymorphic Polyglutamine (CAG) Sequences of the N-Terminal Section (Exon A) of the Androgen Receptor
Consequences of short CAG
sequences
Prostate hypertrophy
Prostate cancer
Coronary artery disease
Breast cancer
Increased erythropoiesis
Reference
Zitzmann et al. (80)
Bratt et al. (81)
Alevizaki et al. (82)
Suter et al. (83)
Zitzmann (84)
Consequences of long CAG

sequences
Androgen insensitivity
Osteoporosis
Crabbe et al. (85)

Zitzmann et al. (86)
amplification of CAG (polyglutamine) repeats as the

underlying cause (77), probably because of the neurotoxicity of the polyglutamine-expanded gene products
(78). Subsequent studies have identified a number of
candidate genes for this CAG triplet polymorphism of
the AR (79). It appears that a normal length of CAG
repeats is between 8 and 35. Abnormally shorter
sequences or longer sequences than this range are
associated with increased risk of diseases that are
androgen dependent (Table 6) (8086). This relationship has been explained by the fact that the length of
the CAG repeats of the AR determines the transactivation activity of the receptor (87), such that shorter CAG
repeats are associated with stronger and longer CAG
repeats with weaker activation of the transcription
triggered by the AR. The underlying mechanism may
lie in the unfolded state of the sequence when it
exceeds a critical length (87,88).
The potential for pharmacogenetic tailoring of
TRT dose to an individuals genetic background of
androgen sensitivity and potential adverse effects may
be based on CAG triplets and metabolic characteristics
of each patient to enhance effectiveness and avoid
unwanted effects (12,84). Testosterone treatment of
hypogonadal men with shorter CAG repeat lengths
exhibits a more pronounced increase in prostate volume and hematocrit than men with longer CAG
repeat lengths. Furthermore, patients with long CAG
repeats have been shown to respond better to endocrine therapy (81). On the basis of this evidence, it
would be plausible to select a shorter-acting formulation to treat hypogondal men with a shorter CAG
repeat length, as they give more flexibility for monitoring and dose adjustments. The dose of testosterone
in a particular formulation may also have an impact.
For example, higher dose of testosterone may benefit
men with a longer CAG repeat length (more resistant
to testosterone) but contraindicated in those with a
shorter CAG repeat length (increased risk of prostate
cancer and polycythemia).
Synthetic Androgens and Elective Androgen

Receptor Modulators
In the past 40 years, more than a thousand testosterone derivatives have been synthesized to enhance
intrinsic androgenic potency, prolong duration of
action, and/or improve oral bioavailability of synthetic androgens. Major structural modifications of testosterone include 17b-esterification, 19-nor methyl, 17-a
alkyl, 1-methyl, 7-a methyl, and D-homo-androgens
(Fig. 11); most of these are unavailable for clinical use
because of hepatotoxicity (89), especially 17a-alkyl
and 7a-methyl derivatives. Only a few synthetic
androgens are currently being investigated in clinical
trials, including 7a-methyl-19-nortestosterone
(MENT), which is not hepatotoxic (90). MENT is a
nandrolone derivative with high tissue-specific selectivity as it is preferably metabolized by aromatization
rather than 5a-reduction. MENT is more potent than
testosterone; it is therefore being considered as transdermal formulation or for long-term use in subdermal
implants as well as for male contraception. Evidence
from preclinical trials has demonstrated that it has less
effects on the prostate than testosterone but has little
effects on BMD in hypogonadal males (91).
Current research on steroids with tissue specificity is being extended to the search for selective androgen receptor modulators. The compounds are
structurally modified so that the nonsteroidal molecules avoid intrinsic aromatization or 5a-reduction
(92) and may produce agonistic effects in desired
target tissues, with minimal unwanted adverse effects.
Such compounds have a potential for treating certain
groups of patients such as hypogonadal men with
osteoporosis, BPH, or hyperviscosity.
REFERENCES
1. American Association of Clinical Endocrinologists. American Association of Clinical Endocrinologists Medical
Guidelines for clinical practice for the evaluation and
treatment of hypogonadism in adult male patients2002
update. Endocr Pract 2002; 8:440456.
2. Nussey SS, Whitehead SA. Endocrinology: an integrated
approach. Oxford: BIOS: Taylor & Francis, 2001.
3. Kalra PS, Sahu A, Kalra SP. Interleukin-1 inhibits the
ovarian steroidinduced luteinizing hormone surge and
release of hypothalamic luteinizing hormone-releasing
hormone in rats. Endocrinology 1990; 126:21452152.
4. Yoo MJ, Nishihara M, Takahashi M. Tumor necrosis factora mediates endotoxin induced suppression of gonadotropin-releasing hormone pulse generator activity in the rat.
Endocr J 1997; 44:141148.
5. Zhang Y, Proenca R, Maffei M, et al. Positional cloning of
the mouse obese gene and its human homologue. Nature
1994; 372:425432.
6. Baranowska B, Baranowska-Bik A, Bik W, et al. The role of
leptin and orexins in the dysfunction of hypothalamopituitary-gonadal regulation and in the mechanism of
hyperactivity in patients with anorexia nervosa. Neuro
Endocrinol Lett 2008; 29:3740.
7. Kotani M, Detheux M, Vandenbogaerde A, et al. The
metastasis suppressor gene KiSS-1 encodes kisspeptins,
the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001; 276:3463134636.
8. de Roux N, Genin E, Carel JC, et al. Hypogonadotrophic
hypogonadism due to loss of function of the KiSS1-derived
peptide receptor GPR54. Proc Natl Acad Sci U S A 2003;
100:1097210976.
9. Seminara SB, Messager S, Chatzidaki EE, et al. The GPR54
gene as a regulator of puberty. N Engl J Med 2003;
349:16141627.

10. Dhillo WS, Chaudhri OB, Thompson EL, et al. Kisspeptin54 stimulates gonadotropin release most potently during
the preovulatory phase of the menstrual cycle in women.
J Clin Endocrinol Metab 2007; 92(10):39583966.
11. Morelli A, Marini M, Mancina R, et al. Sex steroids and
leptin regulate the first kiss (KiSS 1/G-protein-coupled
receptor 54 system) in human gonadotropin-releasinghormone-secreting neuroblasts. J Sex Med 2008; 5:
10971113.
12. Zitzmann M, Nieschlag E. Androgen receptor gene CAG
repeat length and body mass index modulate the safety of
long-term intramuscular testosterone undecanoate therapy
in hypogonadal men. J Clin Endocrinol Metab 2007;
92:38443853.
13. Ferro P, Catalano MG, DellEva R, et al. The androgen
receptor CAG repeat: a modifier of carcinogenesis? Mol
Cell Endocrinol 2002; 193:109120.
14. Dejager S, Bry-Gauillard H, Bruckert E, et al. A comprehensive endocrine description of Kennedys disease revealing androgen insensitivity linked to CAG repeat length.
J Clin Endocrinol Metab 2002; 87:38933901.
15. Berkhoff M, Sturzenegger M, Spiegel R, et al. X-chromosomal bulbospinal muscular atrophy (Kennedy syndrome).
Schweiz Med Wochenschr 1998; 128:817823.
16. Baskin LS, Sutherland RS, DiSandro MJ, et al. The effect of
testosterone on androgen receptors and human penile
growth. J Urol 1997; 158:11131118.
17. Saad F, Gooren L, Haider A, et al. A dose-response study
of testosterone on sexual dysfunction and features of the
metabolic syndrome using testosterone gel and parenteral
testosterone undecanoate. J Androl 2008; 29:102105.
18. Sarkey S, Azcoitia I, Garcia-Segura LM, et al. Classical
androgen receptors in non-classical sites in the brain.
Horm Behav 2008; 53:753764.
19. Hardelin JP, Levilliers J, Young J, et al. Xp22.3 deletions in
isolated familial KS. J Clin Endocrinol Metab 1993; 76:
827831.
20. Franco B, Guioli S, Pragliola A, et al. A gene deleted in KS
shares homology with neural cell adhesion and axonal
path-finding molecules. Nature 1991; 353:529536.
21. Layman LC. Genetics of human hypogonadotrophic hypogonadism. Am J Med Genet 1999; 89:240248.
22. Klingmuller D, Dewes W, Krahe T, et al. Magnetic resonance imaging of the brain in patients with anosmia and
hypothalamic hypogonadism (Kleinfelter syndrome).
23. Cadman SM, Kim SH, Hu Y, et al. Molecular pathogenesis
of Kallmanns syndrome. Horm Res 2007; 67:231242.
24. Calvari V, Alpigiani MG, Poggi E, et al. X-linked adrenal
hypoplasia congenita and hypogonadotrophic hypogonadism: report on new mutation of the DAX-1 gene in two
siblings. J Endocrinol Invest 2006; 29:4147.
25. Gunay-Aygun M, Cassidy SB, Nicholls RD. PraderWilli
and other syndromes associated with obesity and mental
retardation. Behav Genet 1997; 27:307324.
26. Nagai T, Mori M. PraderWilli syndrome, diabetes mellitus and hypogonadism. Biomed Pharmacother 1999;
53:452454.
27. Klein D, Ammann F. The syndrome of LaurenceMoon
BardetBiedl and allied diseases in Switzerland. Clinical,
genetic and epidemiological studies. J Neurol Sci 1969;
9:479513.
28. Moore SJ, Green JS, Fan Y, et al. Clinical and genetic
epidemiology of BardetBiedl syndrome in Newfoundland: a 22-year prospective, population-based, cohort
study. Am J Med Genet A 2005; 132:352360.
29. Pfaffle RW, Blankenstein O, Wuller S, et al. Combined
pituitary hormone deficiency: role of Pit-1 and Prop-1.
Acta Paediatr Suppl 1999; 88:3341.
297
30. Dattani MT, Martinez-Barbera JP, Thomas PQ, et al. Mutations in the homeobox gene HESX1/Hesx1 associated with
septo-optic dysplasia in human and mouse. Nat Genet
1998; 19:125133.
31. Morales A. Andropause (or symptomatic late-onset hypogonadism): facts, fiction and controversies. Aging Male
2004; 7:297303.
32. Nieschlag E. Testosterone treatment comes of age: new
options for hypogonadal men. Clin Endocrinol 2006;
65:275281.
33. Hamerton JL, Canning N, Ray M, et al. A cytogenetic
survey of 14,069 newborn infants. I. Incidence of chromosome abnormalities. Clin Genet 1975; 8:223243.
34. Huckins C, Bullock LP, Long JL. Morphological profiles of
cryptorchid XXY mouse testes. Anat Rec 1981; 199:
507518.
35. Paulsen CA, Gordon DL, Carpenter RW, et al. Klinefelters
syndrome and its variants: a hormonal and chromosomal
study. Recent Prog Horm Res 1968; 24:321363.
36. Nistal M, Paniagua R, Abaurrea MA, et al. 47,XXY Klinefelters syndrome with low FSH and LH levels and absence
of Leydig cells. Andrologia 1980; 12:426433.
37. Bizzarro A, Valentini G, Di Martino G, et al. Influence of
testosterone therapy on clinical and immunological features of autoimmune diseases associated with Klinefelters
syndrome. J Clin Endocrinol Metab 1987; 64:3236.
38. Santen RJ, DeKretser DM, Paulsen CA, et al. Gonadotrophins and testosterone in the XYY syndrome. Lancet 1970;
2(7668):371.
39. Sarafoglou K, Ostrer H. Clinical review 111. Familial sex
reversal: a review. J Clin Endocrinol Metab 2000; 85:483493.
40. Hawkins JR, Taylor A, Goodfellow PN, et al. Evidence for
increased prevalence of SRY mutations in XY females with
complete rather than partial gonadal dysgenesis. Am J
Hum Genet 1992; 51:979984.
41. Barbaro M, Oscarson M, Schoumans J, et al. Isolated 46,XY
gonadal dysgenesis in two sisters caused by a Xp21.2
interstitial duplication containing the DAX1 gene. J Clin
Endocrinol Metab 2007; 92:33053313.
42. Smyk M, Berg JS, Pursley A, et al. Male-to-female sex
reversal associated with an approximately 250 kb deletion
upstream of NR0B1 (DAX1). Hum Genet 2007; 122:6370.
43. Lin L, Gu WX, Ozisik G, et al. Analysis of DAX1 (NR0B1)
and steroidogenic factor-1 (NR5A1) in children and adults
with primary adrenal failure: ten years experience. J Clin
44. Lin L, Philibert P, Ferraz-de-Souza B, et al. Heterozygous
missense mutations in steroidogenic factor 1 (SF1/Ad4BP,
NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol
Metab 2007; 92:991999.
45. Minto CL, Liao KL, Conway GS, et al. Sexual function in
women with complete androgen insensitivity syndrome.
Fertil Steril 2003; 80:157164.
46. Han TS, Goswami D, Trikudanathan S, et al. Comparison
of bone mineral density and body proportions between
women with complete androgen insensitivity syndrome
and women with gonadal dysgenesis. Eur J Endocrinol
2008; 159:179185.
47. Jensen TK, Jrgensen N, Punab M, et al. Association of in
utero exposure to maternal smoking with reduced semen
quality and testis size in adulthood: a cross-sectional study
of 1,770 young men from the general population in five
European countries. Am J Epidemiol 2004; 159:4958.
48. Damgaard IN, Jensen TK, Petersen JH, et al. Cryptorchidism and maternal alcohol consumption during pregnancy. Environ Health Perspect 2007; 115:272277.
49. Welsh M, Saunders PT, Fisken M, et al. Identification in
rats of a programming window for reproductive tract
298
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
Han and Bouloux

masculinization, disruption of which leads to hypospadias
and cryptorchidism. J Clin Invest 2008; 118:14791490.
Hackel C, Oliveira LE, Ferraz LF, et al. New mutations,
hotspots, and founder effects in Brazilian patients with
steroid 5alpha-reductase deficiency type 2. J Mol Med
2005; 83:569576.
Korbonits M, Slawik M, Cullen D, et al. A comparison of a
novel testosterone bioadhesive buccal system, striant, with
a testosterone adhesive patch in hypogonadal males. J Clin
Frey H, Aakvaag A, Saanum D, et al. Bioavailability of
testosterone in males. Eur J Clin Pharmacol 1979; 16:345349.
Pitteloud N, Hayes FJ, Dwyer A, et al. Predictors of
outcome of long-term GnRH therapy in men with idiopathic hypogonadotrophic hypogonadism. J Clin Endocrinol Metab 2002; 87:41284136.
Swerdloff RS, Wang C, Cunningham G, et al. Long-term
pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab 2000; 85:45004510.
Mazer N, Bell D, Wu J, et al. Comparison of the steady-state
pharmacokinetics, metabolism, and variability of a transdermal testosterone patch versus a transdermal testosterone gel
in hypogonadal men. J Sex Med 2005; 2:213226.
Dean JD, Carnegie C, Rodzvilla J, et al. Long-term effects of
Testim1 1% testosterone gel in hypogonadal men. Rev
Urol 2004; 6(suppl 6):S22S29.
Jockenhovel F. Testosterone supplementation: what and
how to give. Aging Male 2003; 6:200206.
Wang C, Cunningham G, Dobs A, et al. Long-term testosterone gel (AndroGel) treatment maintains beneficial
effects on sexual function and mood, lean and fat mass,
and bone mineral density in hypogonadal men. J Clin
Pope HG, Jr, Cohane GH, Kanayama G, et al. Testosterone
gel supplementation for men with refractory depression: a
randomized, placebo-controlled trial. Am J Psychiatry
2003; 160:105111.
Han TS, Bouloux PMG. Transdermal testosterone gel treatment of hypogonadal men. Aging Health 2008; 4:517528.
Behre HM, Abshagen K, Oettel M, et al. Intramuscular
injection of testosterone undecanoate for the treatment of
male hypogonadism: phase I studies. Eur J Endocrinol
1999; 140:414419.
Wang L, Shi DC, Lu SY, et al. The therapeutic effect of
domestically produced testosterone undecanoate in Klinefelter syndrome. N Drugs Mark 1991; 8:2832.
Nieschlag E, Buchter D, von Eckardstein S, et al. Repeated
intramuscular injections of testosterone undecanoate for
substitution therapy in hypogonadal men. Clin Endocrinol
1999; 51:757763.
Schubert M, Minnemann T, Hubler D, et al. Intramuscular
testosterone undecanoate: pharmacokinetic aspects of a
novel testosterone formulation during long-term treatment
of men with hypogonadism. J Clin Endocrinol Metab 2004;
89:54295434.
Zitzmann M, Saad F, Nieschlag E. Longterm experience of
more than 8 years with a novel formulation of testosterone
undecanoate (Nebido) in substitution therapy of hypogonadal men. Endocr Abstr 2006; 11:178.
Morales A, Nieschlag E, Schubert M, et al. Clinical experience with the new long-acting injectable testosterone undecanoate. Report on the educational symposium on the
occasion of the 5th World Congress on the Aging Male,
912 February 2006, Salzburg, Austria. Aging Male 2006;
9:221227.
Aaltonen P, Amory JK, Anderson RA, et al. 10th Summit
Meeting consensus: recommendations for regulatory
approval for hormonal male contraception. J Androl
2007; 28:362363.
68. Aversa A, Isidori AM, Spera G, et al. Androgens improve

cavernous vasodilation and response to sildenafil in
patients with erectile dysfunction. Clin Endocrinol 2003;
58:632638.
69. Morelli A, Vignozzi L, Filippi S, et al. Erectile dysfunction:
molecular biology, pathophysiology and pharmacological
treatment. Minerva Urol Nefrol 2005; 57:8590.
70. Aversa A, Isidori AM, De Martino MU, et al. Androgens
and penile erection, evidence for a direct relationship
between free testosterone and cavernous vasodilation in
men with erectile dysfunction. Clin Endocrinol 2000;
5:517522.
71. Burnett AL. The role of nitric oxide in erectile dysfunction:
implications for medical therapy. J Clin Hypertens (Greenwich) 2006; 8(suppl 4):5362.
72. Heinlein CA, Chang C. The roles of androgen receptors
and androgen-binding proteins in nongenomic androgen
actions. Mol Endocrinol 2002; 16:21812187.
73. Hwang TI, Chen HE, Tsai TF, et al. Combined use of
androgen and sildenafil for hypogonadal patients unresponsive to sildenafil alone. Int J Impot Res 2006; 18:
400404.
74. Shamloul R, Ghanem H, Fahmy I, et al. Testosterone
therapy can enhance erectile function response to sildenafil
in patients with PADAM: a pilot study. J Sex Med 2005;
2:559564.
75. Chatterjee R, Wood S, McGarrigle HH, et al. A novel
therapy with testosterone and sildenafil for erectile dysfunction in patients on renal dialysis or after renal transplantation. J Fam Plann Reprod Health Care 2004; 30:8890.
76. Kalinchenko SY, Kozlov GI, Gontcharov NP, et al. Oral
testosterone undecanoate reverses erectile dysfunction
associated with diabetes mellitus in patients failing on
sildenafil citrate therapy alone. Aging Male 2003; 6:9499.
77. La Spada AR, Wilson EM, Lubahn DB, et al. Androgen
receptor gene mutations in X-linked spinal and bulbar
muscular atrophy. Nature 1991; 352:7779.
78. Thomas PS, Jr, Fraley GS, Damian V, et al. Loss of endogenous androgen receptor protein accelerates motor neuron
degeneration and accentuates androgen insensitivity in a
mouse model of X-linked spinal and bulbar muscular
atrophy. Hum Mol Genet 2006; 15:22252238.
79. Butland SL, Devon RS, Huang Y, et al. CAG-encoded
polyglutamine length polymorphism in the human
genome. BMC Genomics 2007; 8:126.
80. Zitzmann M, Depenbusch M, Gromoll J, et al. Prostate
volume and growth in testosterone-substituted hypogonadal men are dependent on the CAG repeat polymorphism
of the androgen receptor gene: a longitudinal pharmacogenetic study. J Clin Endocrinol Metab 2003; 88:20492054.
81. Bratt O, Borg A, Kristoffersson U, et al. CAG repeat length
in the androgen receptor gene is related to age at diagnosis
of prostate cancer and response to endocrine therapy, but
not to prostate cancer risk. Br J Cancer 1999; 81:672676.
82. Alevizaki M, Cimponeriu AT, Garofallaki M, et al. The
androgen receptor gene CAG polymorphism is associated
with the severity of coronary artery disease in men. Clin
Endocrinol 2003; 59:749755.
83. Suter NM, Malone KE, Daling JR, et al. Androgen receptor
(CAG)n and (GGC)n polymorphisms and breast cancer risk
in a population-based casecontrol study of young women.
Cancer Epidemiol Biomarkers Prev 2003; 12:127135.
84. Zitzmann M. Mechanisms of Disease: pharmacogenetics of
testosterone therapy in hypogonadal men. Nat Clin Pract
Urol 2007; 4:161166.
85. Crabbe P, Bogaert V, De Bacquer D, et al. Part of the
interindividual variation in serum testosterone levels in
healthy men reflects differences in androgen sensitivity
and feedback set point: contribution of the androgen

receptor polyglutamine tract polymorphism. J Clin Endocrinol Metab 2007; 92:36043610.
86. Zitzmann M, Brune M, Kornmann B, et al. The CAG repeat
polymorphism in the androgen receptor gene affects bone
density and bone metabolism in healthy males. Clin Endocrinol 2001; 55:649657.
87. Walcott JL, Merry DE. Trinucleotide repeat disease. The
androgen receptor in spinal and bulbar muscular atrophy.
Vitam Horm 2002; 65:127147.
88. Chen YW. Local protein unfolding and pathogenesis of
polyglutamine-expansion diseases. Proteins 2003; 51:
6873.
299
89. Kopera H. The history of anabolic steroids and a review of

clinical experience with anabolic steroids. Acta Endocrinol
Suppl (Copenh) 1985; 271:1118.
90. Edelstein D, Dobs A, Basaria S. Emerging drugs for hypogonadism. Expert Opin Emerg Drugs 2006; 11:685707.
91. Qoubaitary A, Swerdloff RS, Wang C. Advances in male
hormone substitution therapy. Expert Opin Pharmacother
2005; 6:14931506.
92. Bhasin S, Calof OM, Storer TW, et al. Drug insight:
testosterone and selective androgen receptor modulators
as anabolic therapies for chronic illness and aging. Nat
Clin Pract Endocrinol Metab 2006; 2:146159.
18
Male Sexual Function
Giulio Garaffa, Suks Minhas, and David J. Ralph
Department of Urology, University College London Hospitals, London, U.K.
INTRODUCTION
This chapter describes the development of the male
reproductive system and the role of the male hormone, Testosterone. The physiological mechanisms of
erection, causes and treatment of erectile dysfunction
(ED) and male fertility are also reviewed.
THE DEVELOPMENT OF THE MALE

REPRODUCTIVE SYSTEM
The establishment of the male reproductive system
involves the formation of the testis and the maintenance and differentiation of the Wolffian duct. Both
events are crucial for normal sexual function.
The presence of the Y chromosome, and in particular of the sex determining region of the Y chromosome
(SRY) gene, activates a cascade of molecular and cellular events leading to the differentiation of cells of the
primitive genital ridge into Leydig and Sertoli cells and
the organization of the testis structure (Figs. 1 and 2).
Furthermore, increasing evidence indicates that
SRY induces Sertoli cells differentiation via another
transcription factor, the SRY box containing gene 9
(SOX9). On the other hand, Leydig cells start to
differentiate after Sertoli cells appear. This suggests
that their differentiation is regulated indirectly by
paracrine factors secreted by Sertoli cells.
Sertoli cells ensure the production of Mullerian
inhibitory factor (MIF), which is responsible for the
regression of the Mullerian duct, the precursor of the
female reproductive tract.
The Leydig cells produce testosterone and insulin-like factor 3 (ILF3). Androgens are important for
differentiation of the Wolffian duct, also known as
mesonephric duct, to form the body and tail of the
epididymis, the vas deferens and the seminal vesicles.
Moreover, androgens and ILF3 are required for testicular descent (1,2).
Besides male sexual differentiation and the development of testicular tissue, the descent of the testes from
an intra-abdominal into a scrotal position is an essential
prerequisite for spermatogenesis to occur. Cryptorchdism (Greek: hidden testis) is regarded the most frequent
pediatric complication and affects 3% of the male newborns. While this decreases to 1% in boys aged one year,
the prevalence rate of this condition is 30% in premature
boys. Conditions like low birth weight, small size for
gestational age, maternal exposure to estrogens during
the first trimester of pregnancy is associated with a
higher frequency of undescended testicle.
Genetic factors also play an important role in this
condition, as demonstrated in inherited X chromosomelinked anomalies associated with cryptorchisdism (Table 1) (3). If left untreated, cryptorchisdism
may lead to disturbed spermatogenesis, impaired
fertility, and a higher incidence of testicular cancer
(up to 33-fold for bilateral cryptorchisdism), which is
likely a consequence of exposure of the testis to the
increased intra-abdominal temperatures (4).
Testicular descent can be subdivided in two separate phases, an intra-abdominal and inguino-scrotal
phase. The first phase is initiated at about 10 to 14
weeks of gestation and lasts to about week 20 to 23.
This phase is mediated by ILF3 factor, that is, structurally closely related to relaxin and that stimulates
gubernaculum mesenchymal cells. However, androgens don not play any role during the phase of intraabdominal descent (5). The second phase of descent is
usually completed by week 35 in the human and is
mediated by the action of androgens on the gubernaculum and the cranial suspensory ligament of the testis.
Above all, the androgen-mediated regression of the
cranial suspensory ligament is a condition sine qua
non for a complete testicular descent. The androgens
also affect the second phase of the testicular descent by
inducing the masculinization of the sensory nucleus of
the genitofemoral nerve. Furthermore, the sensory
branch of this nerve, when adequately stimulated by
androgens, acts via the calcitonin gene related transmitter inducing the migration of the gubernaculums
testis and transection of this nerve causes ipsilateral
cryptorchisdism (6).
The male external genitalia originate from the
genital tubercle that develops following the formation
of the urethral plate and urethral tube (see chapter).
The genital tubercle consists of the lateral plate mesoderm, surface ectoderm, and endoderm urethral
Chapter 18: Male Sexual Function
301
Figure 1 The Y chromosome is critical for normal male

sexual determination.
Figure 2 Male genital development in the fetus.
Table 1 Syndromes Associated with Cryptorchisdism

Chromosomal location
Name of the syndrome
Xq 11-q 12
Xq 12-q 21.31
Xq 25-q 27
Xp 21.1
Xq 28
Xp 22.31
Xp 11.3-q 11.2
Xq 28
Xp 22.32
Xq 26.1
Xp 21.1-p 11.22
Xq 27-q 28
Xp 11.4-p 21.2
1p 22-p 21
3p 22-p 26
Testicular feminization
FG syndrome
Dandy Walker syndrome
Aarskorg Scott syndrome
Torcicollis, renal dysplasia, cryptorchisdism
X-linked Kallmann syndrome
Arthrogryphosis multiplex congenital
Frontometaphyseal dysplasia
Ichthyosis
Lowe oculocerebrorenal syndrome
X-linked dysmorphism
Lenz dysplasia
11p 13
15q 11-q13
16p 13
WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, mental retardation)

Prader-Willy
Rubinstein-Taybi syndrome
Zellweger syndrome
Fanconi anemia
Denys-Drash syndrome
epithelium derived from the urogenital sinus. The first

phase of the genital tubercle growth is androgen
independent and relies on the activity of a growth
factor known as sonic hedgehog (Shh). Shh orchestrates, directly or indirectly, the activity of numerous
genes that play a role in the outgrowth and differentiation of the genital tubercle (7).
After Shh has promoted the formation of a

sexually dimorphic genital tubercle, starting from
the seventh to eighth gestational week, exposure to
androgens masculinizes the genitalia. The enzyme
5a-reductase converts testosterone in dihydrotestosterone (DHT), the most potent androgen to cause
virilization of the developing external genitalia in
302
Garaffa et al.
Table 2 Causes of Male Pseudohermaphroditism

Defect in the formation of the testis
Hypoplastic testicular Leydig cells
Insufficient secretion of LH
Insufficiency of the LH receptor
P450 deficiency
3b-Hydroxysteroid-dehydrogenase type II deficiency
17a-Hydroxylase/17,20-lyase deficiency
17b-Hydroxysteroid-dehydrogenase type III deficiency
5a-Reductase deficiency
Functional androgen receptor deficiency
the human fetus. DHT induces rapid elongation of the

genital tubercle to form a phallus, the closure of the
opposing urethral folds to form the penile urethra and
the formation of the scrotum by fusion of the genital
swelling.
Disruption of androgen signaling can result in
feminization of the genitalia, which may include
hypospadias, which affects one in every 125 male
births and accounts for the most common genital
malformation (8). More severe disturbances in androgen biosynthesis result in the clinical condition of
pseudohermaphroditism, which is characterized by a
genotypic sex masked by a phenotypic appearance
closely resembling the other sex. Causes of male
pseudohermaphroditism are reported in Table 2 (9).
THE HYPOTHALAMIC-PITUITARY-TESTICULAR
AXIS
The hypothalamus interacts with the pituitary gland
via a portal vascular system and by neural pathways
of particular relevance are the preoptic area and the
medial basal region of the hypothalamus that contain
neurons that secrete gonadotropin-releasing hormone
(GnRH) in a pulsatile fashion, the so-called GnRH
pulse generator. Several hormones, neurotrasmitters
and cytokines modulate GnRH secretion.
GnRH, whose precursor gene has been identified
and mapped on the chromosome 8p, is a decapeptide
released into the pituitary-portal system every 90 to
140 minutes.
The pulsatile secretion of GnRH in turn stimulates pulsatile secretion of luteinizing hormone (LH)
and follicle-stimulating hormone (FSH) by the gonadotrophs of the anterior pituitary. The pulsatile
release of GnRH is essential for the stimulation of
LH and FSH release; in fact a continuous of GnRH or
GnRH agonists inhibits gonadotropin release.
GnRH binds to cell surface receptors on the
pituitary and enhances the secretion of both LH and
FSH by a calcium dependent mechanism that is independent of cyclic AMP (cAMP).
LH and FSH are composed of two glycoproteins
chains and have a molecular weight of about 30,000 d.
The a subunit of the two hormones is identical and the
distinct immunological and functional characteristics
of the two hormones are conveyed by the unique b
subunits.
LH interacts with cell membrane receptors on
Leydig cells to stimulate adenyl cyclase and enhance
Figure 3 The metabolic pathway for androgen production.
the formation and release of cAMP. This second

messenger binds to the regulatory subunit of a protein
kinase and activates the production of testosterone
from cholesterol. The conversion of cholesterol to
pregnenolone by the activity of the cytochrome p450
is the rate-limiting step in the synthesis of testosterone
(Fig. 3). However, in the vast majority of cases, the rate
of conversion is determined by the rate of delivery of
cholesterol to the enzyme in the inner mitochondrial
membrane by the steroidogenic acute regulatory protein (StAR), a process that may be the main site of action
of LH (10). Additional factors that may influence Leydig
cell function include insulin-like growth factor I, transforming growth factor a and b, epidermal growth factor
and fibroblast growth factor.
Despite of a pulsatile release of FSH, LH and
testosterone, FSH and testosterone pulses are not
apparent in normal men because of the slower secretion of newly synthesized rather than stored hormone,
and the longer circulating half lives.
FSH interacts with receptors on the basal aspect
of the Sertoli cell membrane and activates the production of cAMP that as a second messenger activates
protein kinase activity. The end result is an increased
secretion of several Sertoli cell proteins, including
androgen-binding protein (ABP) and the aromatase
enzyme (CYP 19) that converts testosterone in estradiol.
The mechanism by which FSH regulates spermatogenesis is still poorly understood.
LH secretion is regulated primarily by negative
feedback independently by testosterone and estradiol.
estradiol instead appears to be the primary steroid
regulator of FSH secretion.
Testosterone exerts his action of negative feedback directly at the level of the hypothalamic pulse
generator and of the pituitary. These effects may be
mediated by the local conversion of testosterone in
estradiol. Estrogens instead exert their negative feedback by decreasing GnRH pulse frequency at the level
of the hypothalamus and by reducing the amplitude
of LH pulses via decreased pituitary responsiveness to

GnRH (11).
The feedback regulation of the secretion of FSH
instead involves both steroid hormones and the
gonadal peptide inhibin. The latter, is a peptide produced by the germ cells starting from puberty and
consists of two subunits (a and b). Since the b subunit
can exist in two different forms, there are two forms of
inhibin, inhibin A and inhibin B. Inhibin B is the active
form and exerts its activity by inhibiting the release of
FSH form the pituitary; the level of inhibin B correlates with the presence of germ cells in early spermatogenesis in the adult men and with the presence of
Sertoli cells before puberty (12).
The hypothalamic-pituitary-gonadal axis is also
influenced by physical stress, such as illness and fasting.
In case of stress, the increased levels of corticotropins and
cytokines exert an inhibitory effect on the axis, with
consequent reduction of the testosterone levels. In particular, on fasting, the inhibition of the axis is consequence
to the reduced plasmatic levels of leptins that are
required for a normal pulse generator activity (13).
THE EFFECTS OF TESTOSTERONE

Testosterone is the most important circulating androgen in men and is bound to sex hormonebinding
globulin (SHBG) and albumin. Usually around 2% of
testosterone is free, 60% is bound to SHBG and 38% to
albumin. Albumin binds with low affinity to all steroids whereas SHBG has a high affinity for testosterone. Because of the low affinity for steroids, all the
testosterone bound to albumin is available for tissue
uptake, and therefore, the bioavailable testosterone in
plasma approximates the sum of free and albumin
bound hormone (14).
Testosterone can be converted to 5a-DHT, which
promotes male sexual differentiation and virilization, or
can be aromatized to estrogens by aromatase enzymes.
Conversion of testosterone in DHT occurs mainly
in the target tissues and accounts for 6% to 8% of
testosterone metabolism; the process is carried out by
the group of 5a-reductase isoenzymes. Isoenzyme 1 is
present in the liver and in the skin with the exception of
the genital area. The isoenzyme 2 instead is expressed
in the urogenital tract and skin and liver and is defective
in subjects with 5a-reductase deficiency.
About 85% of the conversion of testosterone to
estradiol is carried out in the peripheral tissue and the
remaining in the testis. The process is catalyzed by
aromatase enzyme (CYP 19) that is primarily
expressed in adipose tissue. Furthermore, the conversion of testosterone to estradiol increases with increasing adipose tissue and advancing age.
In addition to regulating the hypothalamic-pituitary system via negative feedback, Androgens initiate
and maintain spermatogenesis, promote the formation
of the male phenotype during embryogenesis and are
responsible for sexual maturation at puberty and its
maintenance thereafter.
Moreover testosterone exerts an important function on protein synthesis and bone maturation.
303
Both testosterone and DHT bind to the same

high affinity androgen receptor (AR) that is located
in the target cells nucleus and that is coded by a gene
located in the long arm of the X chromosome. The
binding activates the AR that interacts and with a
variety of cofactors and stimulates the transcription of
genes under androgen control (15). Almost all effects
of androgens are exerted through the same high
affinity androgen receptor.
DHT creates a more amplified androgen signal,
when compared with testosterone; this is probably
due to a higher affinity of DHT for the AR (16).
However, even if the vast majority of the effects
of the androgens are mediated by direct interaction
with the AR, androgens may also indirectly influence
the expression of a variety of genes. These effects may
involve modulation the expression and activity of
secondary transcription factors, the production of
growth factors, or changes in the expression of other
hormones (17).
THE PHYSIOLOGICAL MECHANISMS

OF ERECTILE FUNCTION AND THE
PATOPHYSIOLOGY OF ERECTILE
DYSFUNCTION
Penile erection is a complex neurovascular process
involving relaxation of the smooth muscle inside the
corpora cavernosa, increased arterial inflow into the
penis, and restricted venous outflow from the organ.
The corporeal bodies of the penis are surrounded by
both a superficial and deep layer of fascia that is
continuous with the fascia of the perineum. The
deeper layer is known as Bucks fascia and is encircled
by a soft connective layer, the Dartos fascia and by the
skin (Figs. 46).
The arterial blood supply to the penis is derived
from the internal pudendal arteries, branch of the
hypogastric arteries. The pudendal arteries trifurcate
into the spongiosal artery, cavernosal artery, and dorsal
artery, which supply the spongiosum, corpora cavernosa, and glans, respectively. The cavernosal arteries
course through each corpus cavernosum longitudinally
and form helicine arteries, which are responsible for the
flow in the vascular spaces of the corpora cavernosa,
causing engorgement leading to erection. The dorsal
arteries of the penis travel under Bucks fascia giving
circumflex branches to the corpora cavernosa and
spongiosa. The venous drainage of the penis can be
divided in superficial and deep system. The superficial
system drains all the tissue superficial to the Bucks
fascia into the saphenous vein. The deep venous system
instead drains into the pudendal plexus. The deep
dorsal vein that runs on the dorsal aspect of the penis
drains the blood from the corpora cavernosa, the spongiosum, and the glans. In particular, the venous drainage from the corpora cavernosa occurs through the
subtunical veins that are compressed and occluded by
the expansion of the sinusoidal spaces during erection.
The compression of the subtunical veins is condition
sine qua non for an adequate erection and is called
veno-occlusive mechanism (Fig. 7).
304
Garaffa et al.
Figure 5 Cross-sectional anatomy of the human penis.
Figure 4 The structure of the penis.
Figure 6 Arterial supply and venous drainage of the human penis.
The Phases of Erection

During erection, the relaxation of the smooth muscle
of the arterioles supplying the sinusoidal spaces
results in an increase in blood flow into the corpora
cavernosa. Penile detumescence is also an active process that requires the contraction of the cavernosal
smooth muscle under the influx of sympathetic stimulation. All these changes in the smooth muscle tone
translate into modifications in the flow in the cavernosal arteries that can be recorded with an eco color
ultrasound (ECDUS). Ultrasonography is the ideal
investigation to study systolic and diastolic velocities
and spectral waveform changes before and during
305
Figure 7 Veno-occlusive mechanism. During sexual stimulation, the smooth muscle surrounding the sinusoidal spaces relaxes leading
to engorgement of the spaces with blood. This leads to compression of subtunical venules and emissary veins, causing penile erection.
erection because of its ability to visualize small vessels

with low flow.
The variation in systolic and diastolic velocities
and the consequent modification of spectral waveform
allow the subdivision of erection in the following
phases (Fig. 8).
Phase 1
This phase is characterized by a progressive relaxation

of the arterial smooth muscle with a resulting rapid
inflow of blood into the corpora cavernosa. The inflow
is continuous for the duration of the cardiac cycle and
leads initially to an isometric filling (phase 1A) and
then to a progressive increase of intracorporeal pressure from 11 mmHg, the normal value during the
flaccid state, to around 25 mmHg (phase 1B).
The arterial blood flow is characterized by a
peak systolic velocity that progressively increases up
to 35 cm/sec and by a positive end diastolic velocity.
During this phase there is only a slight elongation and fullness of the penis.
Phase 2
As the inflow of blood continues, it becomes associated with increasing cavernous pressure, which grows
from 25 mmHg to approximately 40 mmHg. The onset
of this phase is heralded by a progressive decrease in
the end diastolic velocity because of the increased
intracorporeal pressure. Ultimately, the end diastolic
velocity becomes 0 when the intracavernosal pressure
equals the diastolic pressure. There is also a decrease
in the peak systolic velocity.
Phase 3
Figure 8 The hemodynamic phases of penile erection.
This phase is identified when end diastolic flow

reaches 0. The continued inflow of blood and the
progressive distension of the sinusoidal spaces compress the subtunical venous plexuses against the noncompliant tunical albuginea. This phenomenon, which
306
Garaffa et al.
is also known as the veno-occlusive mechanism,

impedes the outflow of blood through the emissary
veins and leads to a further increase of intracavernosal
pressures. This leads to a progressive reduction in the
duration of systolic inflow and peak systolic velocity
during this phase that translates into narrower, sharperpeaked and lower waveforms at the ECDUS.
Phase 4
This phase is characterized by reversed diastolic flow

that initially develops at the end of diastole as the
intracavernosal pressure becomes higher than diastolic pressure. As intracorporeal pressure increases, the
reverse flow becomes evident throughout diastole.
During this phase the systolic waveform becomes
narrower and sharply peaked, and its duration continues to reduce.
Phase 5
This phase is characterized by the progressive loss of

both systolic and diastolic flow signals. The loss
becomes complete when the intracavernosal pressure
reaches the systolic pressure. The first half of this
phase is defined by the loss of both forward systolic
and reverse diastolic flow components. Loss of reversal flow is initially noted during end diastole, while
velocity and duration of the systolic component
decrease continuously.
The second half of the phase is the end stage of
the flow cycle and is characterized by the loss of both
systolic and diastolic flow and by a further increase of
the intracavernosal pressure de to a contraction of the
ischiocavernosus muscles. During this half of the
phase, the corpora cavernosa become functionally
dead spaces with hardly any inflow or outflow (1820).
The Phases of Detumescence

Detumescence is initiated by the relaxation of the
ischiocavernosus muscles and is consequence of the
active contraction of the cavernosal smooth muscle
under sympathetic nervous stimulation.
Detumescence can be subdivided into three
phases; the first is characterized by a transient
increase of intracavernosal pressure, because of the
contraction of the smooth muscle against a closed
venous system. The second phase instead is characterized by a progressive slow decrease of pressure
leading to the slow reopening of the venous channels
with resumption of the basal level of arterial inflow.
The last phase is characterized by a fast reduction of
intracavernosal pressure because of a fully restored
venous outflow capacity (21).
The Neurophysiology of Penile Erection

The penis receives a complex autonomic and somatic
innervation. The autonomic innervation, which can be
subdivided in sympathetic and parasympathetic,
derives from the neurons in the spinal cord and
peripheral ganglia (Fig. 9). Autonomic nerves form
Figure 9 The autonomic and somatic innervation of the penis.
the cavernous nerves, which enter the corpora cavernosa and the corpus spongiosum to initiate the neurovascular events that lead to erection and detumescence.
Somatic innervation, instead, consists of sensory fibers
that carry sensory stimuli from the genitalia and of
motor fibers that induce the contraction of bulbocavernosus and ischiocavernosus muscles.
The sympathetic pathway arises from the eleventh thoracic to the second lumbar spinal segment
(T11-L2) and its fibers are carried through the white
rami to reach the sympathetic chain ganglia. Some of
these fibers then travel via the lumbar splanchnic
nerves to the superior and inferior hypogastric plexuses. Some fibers originated from the superior hypogastric plexus form the hypogastric nerve and reach
the pelvic plexus.
In the vast majority of cases, the T10-T12 segments are the origin of the sympathetic fibers, and the
chain ganglia cells projecting to the penis are located
in the sacral and caudal ganglia.
The parasympathetic pathway originates from
neurons located in the intermediolateral cell columns
of the second, third, and forth sacral spinal cord segments. The preganglionic fibers form the pelvic nerves
that reach the pelvic plexus, where they join the sympathetic nerves derived from the superior hypogastric
plexus. From the pelvic plexus originate the cavernous
nerves that innervate the corpora cavernosa and spongiosa. These nerves are located in close proximity to the
prostatic capsule and are easily damaged in case of
radical excision of rectum, bladder and prostate.
A parasympathetic input along the cavernous
nerves induces relaxation of the cavernosal smooth
muscle and ultimately an erection. On the other hand,
the stimulation of the hypogastric nerve or of the
sympathetic trunk induces contraction of the cavernosal
smooth muscle and leads to penile detumescence.

Table 3 Central Neurotransmitters Involved in Penile Erection
Neurotransmitter
pathways
Effect on erection
Dopamine
Serotonin
Noradrenaline
GABA
Oxytocin
Nitric oxide
Excitatory
Mainly inhibitory
Excitatory
Inhibitory
Inhibitory
Excitatory
The cerebral impulses originate presumably

from the medial preoptic area (MPOA) that is under
the control of hypothalamic nuclei. In particular,
central dopaminergic neurons with projections to the
MPOA and paraventricular nucleus have been identified in hypothalamic nuclei. Dopaminergic hypothalamic neurons also project to the lumbar spine and
may be involved with the erectile process. The key
role played by dopamine in the central nervous system has been confirmed in the animal model by the
induction of the erections following intrathecal
administration of this neuromediator. Central noradrenergic neurons, largely represented in the MPOA,
seem to play also a key role in the regulation of
erections, but in contrast to peripheral neural pathways, appear to facilitate penile erection. Serotoninergic neurons are also well represented in the MPOA
and in the paraventricular areas of the hypothalamus
where they play a key role in the inhibition of sexual
drive. Central neurotrasmitters involved in penile
erection are reported in Table 3.
Cerebral impulses travel in sympathetic, parasympathetic and somatic pathways to regulate the
erectile process.
The somatosensory pathway originates from the
sensory receptors located in the penile skin, corpora,
glans and urethra. The fibers originated from the
receptors form the dorsal nerves of the penis that
then becomes part of the pudendal nerve. The stimulation of these receptors triggers the release of messages of pain, temperature, and touch that via the
pudendal nerve, the spinal cord and spinothalamic
tract reaching the thalamus and the sensory cortex.
Onufs nucleus that is located in the second to fourth
sacral spinal segment is the center of somatomotor
penile innervation. From this nucleus derive the nerve
fibers that travel in the sacral nerve and then in the
pudendal nerve to reach the ischiocavernosus and
bulbospongiosus muscles. The contraction of the
ischiocavernosus muscle is responsible of the onset
of the phase 5 of the erection while the rhythmic
contraction of the bulbocavernosus muscle is part of
the mechanisms of ejaculation.
The above described nervous structures are
responsible for the three types of erection: psychogenic, reflexogenic and nocturnal. Psychogenic erection is
the consequence of audiovisual stimuli and of the
fantasy and is consequence of the direct activation of
the cerebral nuclei that modulate the spinal cord
centers to activate the erectile process.
Reflexogenic erection is on the other hand as a
consequence of tactile stimuli to the genitals that reach
307
the spinal erection centers via the dorsal and pudendal nerve where they activate the autonomic nuclei.
The activation of the autonomic nuclei subsequently
induces the release of NO and acetylcholine from the
terminations of the cavernosal nerves and the consequent relaxation of the cavernosal smooth muscle.
Nocturnal erection instead occurs during the
rapid eye movement (REM) phase of the sleep and
its mechanisms are still obscure (22).
Neurotransmitters Involved in Penile Erection

and Detumescence
Detumescence and flaccidity are mainly mediated by
the release of norephinephrine by the adrenergic
nerve endings in the corporeal tissue. The vasoconstrictive effect of noradrenaline is mediated by its
interaction with adrenergic receptors that are well
represented in the cavernosal artery wall as well as
in the cavernous trabeculae (23,24). In particular,
among the adrenergic receptor subtypes, the a-adrenoreceptors seem to be the main responsible of the
smooth muscle contraction, since they are represented
ten fold more than their b-receptor counterpart in the
corpora cavernosa.
Despite noradrenaline playing the key role in the
control of smooth muscle contraction, many other
agents are known to induce detumescence.
Among those, endothelin, tromboxane A2, prostaglandin F2 and leukotrienes are potent vasoconstrictors produced by the endothelial lining of the vessels
that may play a role inducing smooth muscle contraction in the corpora cavernosa (25,26).
Vasoconstriction is partially mediated by the
activation of a GTPase, RhoA, and its downstream
effector, Rho kinase. The final result is a calcium
dependent activation of myosin light chain and
actin/myosin assembly.
Penile erection is a complex neurovascular process involving relaxation of the corpus cavernosal
smooth muscle (Fig. 10). The nerves, endothelium of
sinusoids and blood vessels, and smooth muscle cells
in the penis produce transmitters and modulators that
control the erect versus the flaccid state of the penis.
Acetylcholine and NO are of critical importance to the
physiological induction and maintenance of erections.
The key role played by acetylcholine is confirmed by
the fact that cholinergic nerve endings are well represented within the cavernosal smooth muscle and
surrounding penile arteries. Acetylcholine induces
relaxation of the smooth muscle directly via a stimulation of nicotinic receptors that, when activated
induce an intracellular cascade of second messengers.
However, acetylcholine acts in an indirect way by
stimulating the endothelium to release NO.
NO also plays a key role in the induction of the
relaxation of corporeal smooth muscle. In particular, this
molecule can be derived from different pathways. There
is a constitutive endothelial synthesis of NO via the
enzyme endothelial NO synthase (eNOS) and a neuronal
synthesis of the mediator via the neuronal NO synthase
(nNOS) located in the nerve endings of the nonadrenergic/noncholinergic (NANC) system. NO production can
308
Garaffa et al.
Figure 12 The cellular action of NO incorporal smooth muscle

(sGC-soluble guanylate cyclase).
Figure 10 Diagram depicting the functional anatomy of the

corpus cavernosum and the main vasoactive factors involved
in penile erection.
Figure 11 Biosynthesis of prostanoids.
be also induced by various stimuli that activate another

form of NOS, the inducible NOS (iNOS) (Fig. 11).
eNOS and nNOS are two isoforms of the same
enzyme and are tightly regulated and produce physiologically relevant levels of NO in the endothelial cells
and autonomic nerve endings of the penis. There is
increased evidence that both isoforms of the enzyme
play an important rule in the mechanisms of erection as

documented by the complex and delicate regulation of
eNOS and nNOS that involves multiple molecular
mechanisms that act in concert to both positively and
negatively affect the function of these enzymes (27).
eNOS is an important mediator of both physiological and pathological responses of the penis. In case
of normal endothelial function, NO has vasodilatatory
and counterbalances RhoA/Rho kinase mediated
vasoconstriction.
NO causes relaxation of the corporeal smooth
muscle via the production of cGMP, its active intracellular second messenger (Fig. 12). The activity of
cGMP is terminated by its conversion in GMP, which
is catalyzed by enzymes called phosphodiesterases
(PDE). A large variety of PDE isoenzymes have been
identified within human tissues, however the type 5
isoenzyme appears to be the most important in the
human penis (Table 4).
Other mediators that may play a role in smooth
muscle relaxation are the vasoactive intestinal peptide
(VIP), calcitonine gene related peptide (CGRP), histidine, methionine, and some prostaglandins. In particular, VIP immunoreactive fibers have been identified
within the corpus cavernosus trabeculae and surrounding penile arteries and the administration of VIP
Table 4 Pharmacology and Localization of Phosphodiesterases

Isoenzyme
Substrate
Tissue
PDE
PDE
PDE
PDE
PDE
PDE
PDE
PDE
PDE
PDE
PDE
cAMP/cGMP
cAMP/cGM
cAMP/cGMP
cAMP
cGMP
cGMP
cAMP
cAMP
cAMP
cAMP/cGMP
cAMP/cGMP
Brain, heart, vascular smooth muscle

Brain, heart, vascular smooth muscle, skeletal muscle, adrenal cortex
Smooth muscle, platelet, liver
Brain, heart, vascular smooth muscle, skeletal muscle, adrenal cortex, lung, testis
Vascular smooth muscle, platelet
Retina
Skeletal muscle, heart
Testis, ovary, intestinal tract
Spleen, intestinal tract
Brain, testis, thyroid
Vascular smooth muscle, brain, hearth
1
2
3
4
5
6
7
8
9
10
11
antagonists has been proven to prevent smooth muscle

relaxation (28).
Pathophysiology of Organic Erectile

Dysfunction
Erections are neurovascular phenomena modulated by
hormonal stimuli, local biochemical interactions and
biochemical mechanisms; as a consequence, all conditions that affect the hemodynamic of erectile response
may be responsible of the onset of ED that is defined as
the inability to achieve and maintain a penile erection
adequate for satisfactory sexual intercourse.
Since erection is a neurovascular event, ED can
be consequence of disease of the brain, spinal cord,
cavernous and pudendal nerves and receptors in the
terminal arterioles and cavernosal smooth muscles. In
particular, lesions affecting the brain, such as strokes,
Alzheimers or Parkinsons diseases, tumor, or trauma
cause ED through a derangement of the hypothalamic
centers or overinhibition of the spinal center. Dysfunction at the spinal cord level may affect either the
afferent or efferent nerve pathways. The peripheral
neuropathy seen in alcoholism, vitamin deficiency,
diabetes as well as nerve lesions typical of trauma
and pelvic surgery may disrupt the neural pathway.
With the exception of when the nerves are transacted
from radical surgery and injury, most neurological
dysfunctions are partial.
ED can be also consequence of disease of terminal aorta, hypogastric, pudendal and penile arteries.
Although trauma or a congenital anomaly may cause
arterial insufficiency, in the majority of cases arteriogenic impotence is a consequence of a generalized
atherosclerotic process. Associated risk factors include
hypercholesterolemia, obesity, smoking, diabetes,
radiation, hypertension, and perineal trauma. In case
of arteriogenic impotence, the initial symptoms are
characterized by a progressive delay in the onset of
erection and early detumescence. With progressive
reduction of luminal diameter, partial to complete
ED ensues.
ED can also be consequence of the lack of the
veno-occlusive mechanism due to the presence of
abnormal venous channels, tunical abnormalities or
functional impairment of the erectile tissue.
Endocrine factors play a role in approximately
3% of all organic ED cases. In particular, hypogonadism, hypothyroidism, hyperthyroidism, pituitary
tumors, and hyperprolactinemia are well known
causes of ED (29).
ED might also be consequence of impairment of
erectile tissue due to Peyronies disease, traumas,
fibrosis, diabetes, tumor infiltration and scleroderma.
Finally many medications may induce ED, in
particular some classes of antihypertensive drugs
and antiandrogens (Table 5). Hypnotics and major
tranquillizers may exert their effects by elevating
plasma prolactin as well as depressing the libido.
Centrally acting antihypertensives instead, are
thought to exert their effect on erectile function by
their central actions on a-receptors (Fig. 13).
309
Table 5 Drug That May Be Responsible for Erectile Dysfunction

Psychotropic drugs
Benzodiazepines, butyrophenones
Antidepressants
Tryciclics, monoamine oxidase inhibitors
Anthypertensives
b-blockers, spironolactone
Endocrine drugs
Cyprotherone acetate, LHRH analogues, estrogens
Anticholinergic drugs
Atropine
Others
Cimetadine, digoxin, alcohol, some recreational drugs
Figure 13 Pathophysiological mechanism of erectile dysfunction. Under physiological conditions, corporeal smooth muscle is
maintained by relaxatory factors acting in opposition to contractile
factors. In disease states, the balance is tipped to favor contractile
factors. This may be secondary to increased formation/uninhibited
activity of these factors or secondary to reduced production/activity
of relaxatory factors.
Pharmacological Treatment of Erectile

Dysfunction
The pharmacological treatment of ED has been revolutionized in recent years, particularly with the introduction of PDE inhibitors. The pharmacotherapy for
ED is based on the principles of stimulating central or
peripheral proerectile pathways or inhibiting antierectile mechanisms. In particular, drugs enhancing
peripheral nitrergic-mediated pathways have been
developed, and PDE inhibitors, such as Viagra,
deserve a special mention. The medications currently
available for treating ED are reported in Table 6.
310
Garaffa et al.
Table 6 Currently Available Pharmacological Treatments for Male Erectile Dysfunction

Oral treatments
Mechanism of action
Sildenafil citrate (ViagraTM)

Tadalafil (CialisTM)
Vardenafil hydrochloride
Apomorphine sublingual (UprimaTM)
Yohimbine
Phentolamine (VasomaxTM)
Prazosin and Doxazosin
L-arginine
Intracavernosal agents
PGE1 (Alprostadil)
Papaverine
Vasoactive intestinal polypeptide
Linsidomine chlorhydrate
Phentolamine
PDE 5 inhibitor
PDE 5 inhibitor
PDE 5 inhibitor
Dopamine agonist
a-Adrenoceptor antagonist
NO precursor
Mechanism of action
Increase in intracellular cAMP
Nonselective PDE inhibitor
NO donor
Topical agents used for the treatment of MED

Transdermal nitroglycerin
Intraurethral PGE1 (MUSE)
Topical PGE1
Mechanism of action
NO donor
Table 7 Pharmacokinetic Profiles of Available PDE 5 Inhibitors

Drug
Cmax (ng/mL)
Tmax (hr)
T1/2 (hr)
560
209
378
0.8
0.7
2.0
3.7
3.9
17.5
Sildenafil
Vardenafil
Tadalafil
Phosphodiesterase 5 Inhibitors
Drugs such as Sildenafil and the novel PDE 5 inhibitors Vardenafil and Tadalafil are referred to as competitive inhibitors of PDE 5, as such, they have a
similar chemical structure to cGMP. As mentioned
before, PDE inhibitors inhibit the breakdown of cGMP
to 5-GMP, leading to an increase in corporeal smooth
muscle relaxation.
Pharmacological Concepts of Potency and

Selectivity of a Drug
An ideal drug for the treatment of a medical condition
should be potent and have a high selectivity toward
its target tissue. In pharmacological terminology, drug
potency is defined as the concentration of a drug
required to inhibit an enzyme or receptor in vitro.
Potency under these conditions is expressed as the
IC50 of the drug; that is, the concentration of the drug
in vitro required to inhibit 50% of a measured
response, such as enzyme activity. Thus, in the case
of PDE 5 inhibitors, the lower the IC50 value, the more
potent the drug is in vitro. This is purely a pharmacological concept meaning that less of the drug is
required to produce a desired effect in vitro. In other
words, the lower IC50 value of a PDE inhibitor, the
more potent, theoretically, the drug is. However, a
low IC50 does not always indicate that a drug is more
potent and thus has greater efficacy. The efficacy of a
drug is a measure of its clinical or in vivo effects and
therefore does depend simply on the potency of the

drug in vitro, but also on its pharmacokinetic profile,
that is, its absorption, distribution, and metabolism
within the body (Table 7).
The selectivity of a drug is its relative activity
between different receptor types or isoenzymes. For
competitive antagonists such as the PDE inhibitors,
selectivity can be expressed by the ratio of IC50 values
for each PDE isoenzyme. Selectivity is the main factor
determining the side-effect profile of a drug (Table 8).
PDE inhibitors used for MED should have a high
selectivity for PDE 5, thereby minimizing their side
effects on other PDEs present in the body. The side
effects of PDE 5 inhibitors are related to their effects on
other PDEs around the body. This accounts for the
commonly observed adverse events ascribed to these
drugs, including headache, facial flushing and dyspepsia.
Table 8 Properties of the PDE 5 Inhibitors
Sildenafil
Tadalafil
Vardenafil
Time to onset
Duration of action
IC50 PDE 5 (nM)
60 min
48 hr
3.5
45 min
2436 hr
0.9
2540
Not known
0.7
Selectivity ratio
PDE 1
PDE 2
PDE 3
PDE 4
PDE 5
PDE 6
80
1000
1000
10 000
1
10
10 000
10 000
10 000
10 000
1
780
257
10 000
10 000
10 000
1
16

Table 9 Medical Treatment for Peyronies Disease
Medication
Mechanism of action
Vitamine E
Colchicine
Antioxidant and anti-inflammatory effect

Inhibition of deposition of collagene, antiinflammatory
Inhibition of deposition of fibrosis
Promotes cell regeneration
Induces the production of NOS, inhibition
of deposition of fibrosis
Antiestrogen inhibits the release of TGF1
Reduces the deposition of collagen
Increases collagenase activity
Anti-inflammatory, reduces the deposition
of collagen
Decrease proliferation of fibroblasts
POTABA
Carnitine
L-arginine
Tamoxifen
Pentoxyphilline
Verapamil
Corticosteroids
Interferons
Peyronies Disease
Peyronies disease (PD) is a condition that manifests
as a fibrous scar of the tunica albuginea that may
determine penile deformity, penile curvature, hinging, narrowing and painful erections. This condition is
also frequently associated with ED; it is still unclear
whether ED predisposes to PD or is a consequence of
the contracture of the tunica albuginea.
Although the exact mechanisms responsible of
the onset of this disease are still unclear, it is widely
accepted that PD is a disorder of wound healing that
derives from an imbalance between profibrotic and
antifibrotic agents. Among the profibrotic factors,
transforming growth factor 1 (TGF1), normally
released by neutrophils and macrophages during the
acute and proliferative phases of wound healing, is
overexpressed in PD plaques where it induces deposition of collagen. However, other profibrotic enzymes
may also play a role in the pathogenesis of PD
promoting the deposition of collagen or inhibiting
the breakdown of collagen mediated by the matrix
metalloproteinases (MMP).
Since the exact mechanisms of PD is not known,
there is still no effective medical treatment for this
condition and the only management is surgical The
type of surgical procedure varies according to the
degree of deformity, the quality of the erection and
the penile length. Surgery may involve plication of the
tunica albuginea, plaque incision/excision associated
with grafting of the consequent defect, a combination
of the previous techniques, or the insertion of a penile
prosthesis.
The medical treatments available for PD are
outlined in Table 9 (30).
FERTILITY
Development of the Testicle and
Spermatogenesis
Male fertility depends on the successful integration of
a series of physiological events, starting from sperm
production and ending with the successful fertilization of the oocyte. This multi step process is reported
in Table 10.
311
Table 10 Sequence of Events Leading to the Fertilization of the

Oocyte
Spermatogenesis
;
Acquisition of motility
;
Migration through female tract
;
Capacitation
;
Zona pellucida binding
;
Acrosome reaction
;
Zona pellucida penetration
;
Sperm head decondensation
;
Fusion with oocyte DNA
An adequate development of the testicles is a

condition sine qua non for the process of sperm
production.
During childhood, the testes are small and contain the seminiferous tubules, that at this stage appear
as solid, cord-like structures containing spermatocytes
and Sertoli cells. The seminiferous tubules progressively then acquire a lumen as the spermatocytes start
to differentiate under the influence of testosterone and
FSH. In particular, FSH acts through the Sertoli cells to
stimulate the germ cells to manufacture sperm. The
Sertoli cells, when spermatogenesis proceeds, are
responsible of the secretion of inhibin B that constitutes the negative feedback mechanism for the production of FSH.
Spermatogenesis is a complex process that starts
after puberty and involves two cell divisions, meiosis I
and II, that starting from a diploid cell, the spermatogonium, leads to the formation of a haploid cell,
known as the gamete.
In particular, each spermatogonium undergoes
cell division to produce type A spermatogonia, which,
in turn produce intermediate and then type B spermatogonia. Subsequently, each spermatogonia undergoes meiosis I to become primary spermatocytes. Each
spermatogonia gives rise to 16 primary spermatocytes.
Each primary spermatocyte enters meiosis II to give
rise to four spermatids and finally four spermatozoa.
Thus, each of the 3 million spermatogonia that begin
the process every day gives rise to 64 spermatozoa.
The spermatogenic cycle in the testis lasts approximately 16 days, however, the complete process, from
the commencement of spermatogenesis to emission,
lasts around 72 days.
Sertoli cells play a key role in the process of
spermatogenesis since they provide support and
nourishment for the germ cells. They are irregular
columnar cells that extend from the basal lamina to
the lumen to provide structural organization to the
tubule.
Important functions of Sertoli cells are the synthesis of ABP, which helps to maintain the high
312
Garaffa et al.
androgen levels inside the seminiferous tubules, and

the creation of the blood testis barrier, that subdivides
the seminiferous tubules into a basal compartment
containing spermatogonia, and a luminal compartment that contains spermatocytes and spermatids.
Hormonal Control of Spermatogenesis

FSH and testosterone are the main hormones that
control spermatogenesis; FSH receptors are well represented on Sertoli cells and spermatogonia while the
AR are located on Sertoli cells, Leydig cells and myoid
peritubular cells. FSH stimulates Sertoli cells to produce ABP and inhibin B. In particular, ABP maintains
testosterone levels approximately 100 times higher
than in the systemic circulation, conditions required
for meiosis II (31).
There is also in vitro evidence that local autocrine and paracrine factors such as neuropeptides,
vasoactive peptides, growth factors, and immune
derived cytokines play regulatory functions in the
seminiferous tubules (Fig. 14) (32,33).
Spermatozoa
Transformation of the human spermatid into a mature
sperm involves reorganization of the nucleus and
cytoplasm and the development of a flagellum.
Mature spermatozoa have an oval head, which contains a nucleus The anterior halve is surrounded
by the acrosomal cap, a midpiece, which is characterized by the presence of mitochondria, and a tail or
flagellum.
The acrosome contains lytic enzymes that are
essential for the penetration in the zona pellucida,
while the tail provides the progressive movement
that allows the sperm to reach the ampullar portion
of the tube.
The head is characterized by the presence of
condensed nuclear chromatin, which represents the
haploid chromosome complement. The nucleus relocates to occupy an eccentric position at the head of the
spermatid that is covered by an acrosomal cap. The
flagellum has a complex structure that consists of nine
outer microtubules, disposed in a circular fashion, and
two inner microtubules. This proteic core is surrounded by mitochondria, which provide the energy
necessary for movement, in the middle section, and
only by a cell membrane in the tail. Sperm motility is
consequence of the sliding action of the microtubules
in the axial structure of the tail thanks to the action of
the protein nexin that forms bridges between the
tubules via its dynein arms.
Male Infertility
Male infertility can be considered a syndrome since it
may result from a variety of congenital or acquired
conditions.
The ejaculate can be abnormal because of a
primary testicular dysfunction, to an impaired stimulation of otherwise normal testis or to an abnormal
development of the sperm or to a defect of migration.
Impairment of the seminal parameters might also be
consequence of inflammation or infection of the reproductive tract due to the detrimental effects of mediators of the inflammatory response and of the free
radicals on the semen. Moreover, men who produce
normal sperm may be infertile because o ejaculatory
or ED.
According to their etiopathogenesis, the disorders of spermatogenesis can be subdivided in the
following categories:
Idiopathic
The cause of infertility remains unexplained in up to

50% of infertile men. It is likely that the impairment of
the seminal parameters are consequence of an alteration in the process of maturation due to the presence
of high levels of reactive oxygen species (ROS) that
Figure 14 Hormonal regulation of testicular function.
leads to oxidative damage to the acrosome and

impairment in the motility (34).
Primary Testicular Failure
This condition, also known as hypergonadotropic

hypogonadism, is characterized by small size testicles,
impaired spermatogenesis and elevated levels of
FSH. The condition may be associated with androgen
deficiency.
Genetic Abnormalities
The incidence of genetic abnormalities, that is, less

than 0.1% in the normal population, is 2.2% in men
attending fertility clinics and raises to 15.4% in those
men that are azoospermic. The most common abnormality is Klinenfelthers syndrome that consist in a
disomy of the X chromosome (XXY) and that accounts
for almost 50% of all abnormalities. Also some
microdeletions of the Y chromosome are associated
with defective spermatogenesis. In particular, the lack
of the AZF gene is associated with total lack of
spermatogenesis.
313
Obstructive Azoospermia
This condition should be suspected in patients with

bilateral normal sized testis, normal FSH, LH and
testosterone. The obstruction can be anywhere in
between the testis and the ejaculatory ducts. In particular, obstruction of the epididymis might be congenital (Youngs syndrome) or consequence of recurrent
episodes of epididymitis. Obstruction of the vas can
be postinfective or iatrogenic. Accidental division of
the vas can has been reported during inguinal hernia
repair. The congenital absence of the vas can be
indicative of Wolffian duct abnormality; in this case
the condition is unilateral and can be associated with
malformation of the genitalia, in particular hypospadias, and with ipsilateral malformations of the upper
urinary tract such as absence of the renal aplasia.
Instead, bilateral absence of the vas deferens is a
common finding in patients carrier of the cystic fibrosis gene.
The congenital obstruction of the ejaculatory
ducts may be due to Mullerian ducts cysts or Wolffian
ducts abnormalities. Malignancy and lithiasis account
for the most common acquired causes.
Testicular Maldescent
This condition, also known as cryptorchisdism, might

be consequence of an intrinsic testicular defect or be
due to multiple environmental factors. Usually these
testicles have a normal number of Leydig cells and
therefore testosterone secretion is normal. The germinal epithelium instead tends to be partially or
completely atrophied.
Sertoli-Cell-Only Syndrome
This condition is characterized by the complete

absence of any spermatogenic epithelium. The only
cells present in the seminiferous tubules are the Sertoli
cells. The condition not necessarily involves the entire
testicle, since tubules with normal spermatogenesis
and with Sertoli only syndrome may coexist in the
same gonad.
Acquired Testicular Failure
Acquired testicular failure can be consequence of a

variety of condition. Testicular torsion, epididymoorchitis, radiation therapy and chemotherapy are
among the most common causes.
Secondary Testicular Failure
This condition, also known as hypogonadotrophic

hypogonadism, is consequence of a deficiency of
hypothalamic releasing or pituitary hormones. The
latter can be a complete deficiency of pituitary hormones, a combined loss of both FSH and LH, or an
isolated loss of LH or FSH. The condition may be
secondary to a direct damage of the pituitary gland by
a trauma, a vascular event or an expansive process, or
consequence of a hyper production of prolactin. Recognition of hypogonadotrophic hypogonadism is of
particular importance since it is the only cause of
azoospermia amenable to hormonal treatment.
Sperm Maturation
When they pass from rete testis to the caput of the
epididymis, spermatozoa are still functionally immature and immotile; their maturation occurs when they
pass through the epididymis. The epididymis secretes
a variety of substances and actively reabsorbs testicular fluid under the direct control of adrenergic and
androgenic stimuli. In particular, the secretion of
glycerophosphorylcholine, inositol, and carnitine create a favorable environment where spermatozoa can
complete their maturation, which involves acquisition
of motility and of fertilizing capacity. From a morphological point of view, the cytoplasmatic droplets
decrease in size and migrate distally along the midpiece, the acrosomal membrane swells and the epididymal glycoproteins are incorporated in the plasma
membrane. An increase in the S-S bonds in the flagellum and in the intracytoplasmatic concentration of
ATP is also noted (Fig. 15).
The normal site where mature sperms are stored
is the tail of the epididymis; spermatozoa are not
usually found in the seminal vesicles except in case
of ejaculatory duct obstruction.
Ejaculation
The process of ejaculation is probably triggered by the
hypothalamus and consists of two consecutive phases,
emission and ejaculation proper. Emission consists of
the propulsion of the semen in the bulbar urethra and
is a consequence of the sequential contraction of the
epididymis, vas, seminal vescicles, prostate, and bladder neck under the influence of thoracic sympathetic
outlets (T10-L2) through the hypogastric nerve.
Ejaculation proper instead consists in the expulsion of the semen from the bulbar urethra to the
outside. This process, which is an involuntary spinal
314
Garaffa et al.
Table 11 Advanced Semen Analysis Tests
A. Optional tests
Computer-aided semen analysis
Culture studies
Chemical studies
Chromatin studies
B. Sperm function tests
Sperm-mucus interaction
Sperm capacitation
Zona binding and acrosome reaction
Sperm-ovum interaction
evaluate the sperm concentration, motility, viability,

morphology and agglutination and search for the
presence of antibodies anti sperm and nonsperm cells.
Although the semen analysis represents the cornerstone of the evaluation of the infertile male, it is
generally inadequate to establish a definitive diagnosis or predict a prognosis. Furthermore, even if conducted according to World Health Organization
(WHO) criteria, the basic parameters measured in
this analysis, such as the sperm concentration, motility
and morphology are highly dependent on subjective
factors. Therefore a number of advanced sperm tests
have been developed in an attempt to overcome the
limitations and the drawback of the standard semen
analysis (Table 11). These advanced sperm tests
depend on the evaluation of sperm functions by
precise and objective techniques and their interactions
inside the female genital tract till the stage of fertilization. Unfortunately, there is still no single sperm
function test that can evaluate all sperm functions.
Currently, all the advanced sperm tests are still
considered optional or research test by the WHO.
Figure 15 A mature human spermatozoon.
REFERENCES
somatic reflex, is a consequence of the rhythmic
contraction of the pelvic floor and perineal muscles
ischiocavernosum and bulbocavernosum and is mediated by motor fibers of the pudendal nerve (S2S4).
The bulk of the ejaculate consists of secretions of
the seminal vesicles (65%) and prostate. The testicles
contribution accounts for less than 1% of the entire
ejaculate. The secretions of the seminal vesicles are
rich in fructose, coagulates and bicarbonates that are
responsible for the typical alkaline pH. On the other
hand, the prostatic secretions contain proteolytic
enzymes that liquefy the coagulated proteins of the
ejaculated semen, and are rich in zinc, citric acid and
acid phosphatase that confer the characteristic acid pH.
An obstruction of the seminal vesicles or bilateral vasal
aplasia can be easily recognized by the presence of acid
pH and by the absence of fructose in the ejaculate.
Semen Analysis
The standard semen analysis involves a chemical and
microscopic examination. The chemical examination
evaluates color, odor, volume, liquefaction, viscosity
and pH. While the microscopic examination aims to
1. Barsoum I, Hung-Chang YH. The road to maleness: from

testis to Wolffian duct. Trends Endocrinol Metab 2006; 17(6):
223228.
2. Foresta C, Bettella A, Vinanzi C, et al. Insulin like factor 3: a
novel circulating hormone of testicular origin in humans.
3. Saifi GM, Chandra HS. An apparent excess of sex- and
reproduction-related genes on the human X chromosome.
Proc Biol Sci 1999; 266(1415):203209.
4. Benson RC, Beard CM, Kelalis PP, et al. Malignant potential
of the cryptorchid testis. Proc Mayo Clin 1991; 66:372378.
5. Adham IM, Steding G, Thamm T, et al. The overexpression
of INSL3 in female mice causes descent of the ovaries. Mol
Endocrinol 2002; 16:244252.
6. Hrabovsky Z, Farmer PJ, Hutson JM. Undescended testis is
accompanied by calcitonin gene related peptide accumulation within the sensory nucleus of the genitofemoral nerve
in trans-scrotal rats. J Urol 2001; 165:10151018.
7. Haraguchi R, Mo R, Hui C, et al. Unique functions of Sonic
hedgehog signalling during external genitalia development. Development 2001; 128:42414250.
8. Boehmer ALM, Nijman RJM, Lammers BAS, et al. Etiological studies of severe o familiar hypospadias. J Urol
2001:12461254.
9. Klonisch T, Fowler PA, Hombach-Klonsch S. Molecular
and genetic regulation of testis descent and external genitalia development. Dev Biol 2004; 270:118.

10. Stocco DM, Clark BJ. The role of the steroidogenic acute
regulatory protein in steroidogenesis. Steroids 1997; 62(1):
2936.
11. Hayes FJ, Decruz S, Seminara SB, et al. Differential regulation of gonadotropin secretion by testosterone in the
human male: absence of a negative feed back effect of
testosterone on follicle-stimulating hormone secretion.
J Clin Endocrinol Metab 2001; 86(1):5358.
12. Andersson AM, Skakkebaek NE. Serum inhibin B levels
during male childhood and puberty. Mol Cell Endocrinol
2001; 180(12):103107.
13. Chan Jl, Heist K, DePaoli AM, et al. The role of falling
leptin in the neuroendocrine and metabolic adaptation to
short-term starvation in healthy men. J Clin Invest 2003;
111(9):14091421.
14. Pardridge WM, Landaw EM. Testosterone transport in
brain: primary role of plasma protein-bound hormone.
Am J Physiol 1985; 249(5):E534E542.
15. Lee DK, Chang C. Molecular communication between
androgen receptor and general transcription machinery.
J Steroid Biochem Mol Biol 2003; 84(1):4149.
16. Griffin JE, Mc Paul MJ, Russell DW, et al. The androgen
resistance syndromes: steroid 5-a-reductase 2 deficiency,
testicular feminization, and related disorders. In: Scriver
CR, Beaudet AL, Sly WS, et al., eds. The Metabolic and
Molecular Bases of Inherited Disease. 8th ed. New York:
McGraw-Hill, 2001:4117.
17. Heemers HV, Verhoeven G, Swinnen JV. Androgen activation of the sterol regulatory element binding protein pathway: current insights. Mol Endocrinol 2006; 20:2265.
18. Schwartz AN, Wang KY, Mack LA, et al. Evaluation of
normal erectile function with color flow Doppler sonography. AJR Am J Roentgenol 1989; 153:11551160.
19. Chiou RK, Pomeroy BD, Chen WS, et al. Hemodynamic
patterns of pharmacologically induced erection: evaluation
by color Doppler sonography. J Urol 1998; 59:109112.
20. Chung WS, Park YY, Kwon SW. The impact of aging on
penile hemodynamics in normal responders to pharmacological injection: a Doppler sonographic study. J Urol 1997;
157:21292131.
315
21. Bosh RJ, Bernard F, Aboseif SR, et al. Penile detumescence:

characterization of the three phases. J Urol 1991; 146:867871.
22. El Sakka AI, Lue TF. Physiology of penile erection. Sci
World J 2004; 4(S1):128134.
23. Hedlund H, Andersson K. Comparison of responders to
drug acting on adrenoreceptors and muscarinic receptors
in human isolated corpus cavernosum and cavernous
artery. J Auton Pharmacol 1985; 5:81.
24. Diederch W, Stief CG, Lue TF, et al. Norepinephrine
involvement in penile detumescence. J Urol 1990; 143(6):
12641266.
25. Hedlund H, Andersson K, Fovaeus M, et al. Characterization of contraction-mediating prostanoid receptors in
human penile erectile tissues. J Urol 1989; 141(1):182186.
26. Azadzoi KM, Kim N, Brown ML, et al. Endothelium
derived nitric oxide and cycloxygenase products modulate
corpus cavernosum smooth muscle tone. J Urol 1992; 147(1):
220225.
27. Musicki B, Burnett AL. eNOS function and dysfunction in
the penis. Exp Biol Med 2006; 231:154165.
28. Ottesen B, Wagner G, Virag R, et al. Penile erection:
possible role of for vasoactive intestinal polypeptide as a
neurotransmitter. Br Med J 1984; 288(6410):911.
29. Burnett Al. Erectile dysfunction. J Urol 2006; 175(S1):
S25S31.
30. Taylor FL, Levine L. Non-surgical therapy of Peyronies
Disease. Asian J Androl 2008; 10(1):7987.
31. De Gendt K, Swinnen JV, Saunders PT. A Sertoli cellsensitive knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci U S A 2004;
101:1327.
32. Gnessi L, Fabbri A, Spera G. Gonadal peptides as mediators of development and functional control of the testis: an
integrated system with hormones and local environment.
Endocr Rev 1997; 18(4):541609.
33. Schlatt S, einhardt A, Nieshlag E. Paracrine regulation of
cellular interactions in the testis: factors in search of a
function. Eur J Endocrinol 1997; 137(2):107117.
34. Iamarrone E, Balet R, Lower AM, et al. Male infertility. Best
Pract Res Clin Obstet Gynaecol 2003; 17(2):211229.
19
The Prostate and Benign Prostatic Hyperplasia
Mark Feneley and Anthony R. Mundy
Mark Emberton
Department of Urology, Division of Surgical and Interventional Sciences,
University College London, London, U.K.
THE PROSTATE
An average urologist spends about 30% of his or her
time dealing with problems related to the prostate.
Surprisingly, for a structure that attracts so much of
our attention, we know very little about why the
prostate is there and what it does. It is one of four
accessory sex glands or pairs of glands; the other three
are the seminal vesicles, Cowpers glands, and the
glands of Littre. If we know little about the prostate,
we know even less about the others. The seminal
vesicles, which are secretory glands and not storage
organs for semen as their name implies, contribute
substantially to the volume of seminal fluid and produce one or two substances that we know about,
notably fructose and glyceryl phosphocholine. However, the other two structures are something of a
mystery. The embryological development of these
organs reflects notable gender-specific differences
and contrasts substantial species-specific differences
in the structural and functional development of the
mammalian male genital tract.
We do know that the prostate is intimately
related anatomically to the bladder neck and plays
an integral part in ensuring antegrade ejaculation.
We know it contains a substantial amount of smooth
muscle as well as glandular tissue and that this
smooth muscle is under a-adrenergic control and is
thought somehow to be involved in the process of
seminal emission prior to ejaculation. We know that
the prostate contributes various substances to the
ejaculate, some of which are present in unusually
high concentrations, notably zinc, citrate, and polyamines. Prostatic-specific antigen is an enzyme
involved in the liquifaction of the seminal coagulum,
and has acquired great medical importance as a serum
marker for prostate cancer, as well as being a prognostic indicator in benign prostatic hyperplasia (BPH).
We know that the development and function of the
prostate are under hormonal control; in other words,
it is a secondary sex organ.
We do not know, however, what part if any the
prostate plays in continence in normal individuals.
The mechanism of emission and ejaculation is uncertain (in humans), and we do not know why the
prostatic secretion contains so much zinc, citrate and
polyamines, or what the roles are of these and the
various other substances that the prostate secretes. We
also do not understand why prostatic diseases of
benign hyperplasia and cancer occur naturally only
in the human and dog, or why these pathologies do
not develop in other male secondary sexual organs.
The Structure of the Prostate

The prostate has been described as having a lobar
structure, first, because of the endoscopic appearance
of lobes in patients with BPH and in the pathological specimen after retropubic prostatectomy, and second, on embryological grounds in which the prostate
is seen to develop from five distinct ductal systems (1).
More recently, the morphology of the prostate has
been described on the basis of the predisposition of
parts of its structure to various pathological processes,
and to McNeal we owe our current understanding of
the structure of the prostate (Fig. 1) (25).
McNeal noted that the nodules of BPH began in
the periurethral glandular area within the collar of the
preprostatic sphincter in the supramontanal part of
the prostate. Subsequently, he noted that these microscopic nodules were principally concentrated just
below the distal margin of the preprostatic sphincter,
and he named that area the transition zone. The transition zone only accounted for about 2% of the glandular tissue of the normal prostate but accounted for
much more as the hyperplastic nodules coalesced,
became macroscopic, and tended to displace the normal prostate away from the urethra.
McNeal noted that about 25% of prostatic cancer
originated within the transition zone, whereas 75% of
cancers and almost all instances of prostatitis arose in
the so-called peripheral zone. The peripheral zone
forms a posteriorly and inferiorly orientated cup
that encloses the central zone, through the center of
which are transmitted the ejaculatory ducts. McNeal
Chapter 19: The Prostate and Benign Prostatic Hyperplasia
317
Figure 1 Zones of the prostate.
noted the comparative absence of disease in the central zone by comparison with the other glandular
areas of the prostate and likened it to the seminal
vesicles, which are also comparatively free of disease.
He speculated further that both these structures might
arise embryologically from the Wolffian ducts and
noted some histological similarities between the central zone and the seminal vesicles to support this
contention (5).
Completing the anterior aspect of the substance
of the prostate where the peripheral zone peters out
on either side is the anterior fibromuscular stroma of
the prostate. Most of the glandular tissue of the prostate is in the peripheral zone, which accounts for 75%
of the total, the remaining 25% of the glandular tissue
of the prostate being in the central zone. It is not
known whether the three zones of glandular tissue
transition, central, and peripheralhave different
secretory functions.
Embryological Development of the Prostate

As mentioned above, the seminal vesicles (and possibly the central zone of the prostate) arise from the
Wolffian ducts along with the vasa and their ampullae
and the epididymes. Their embryological development is under the control of testosterone. Otherwise,
the rest of the prostate and other secondary sexual
organs develop by ductal outbudding from the urethra into surrounding urogenital sinus mesenchyme.
The embryological and pubertal development of the
prostate is in contrast principally under the control of
dihydrotestosterone. These distinctions in tissue
androgen sensitivity are evident from abnormalities
of prostatic and secondary sexual organ development
that occur with congenital 5-a reductase deficiency
and androgen insensitivity syndromes. Differences of
embryological origin and hormonal control may also

account for the observation that, whereas the prostate
is commonly involved in disease, the structures of
Wolffian duct origin rarely are.
Endocrinology of the Prostate

The normal prostate develops and functions in
response to dihydrotestosterone produced within
prostate cells from circulating testosterone (Fig. 2).
Circulating testosterone is derived principally from
testicular Leydig cell secretion; the testis produces
6 to 7 mg a day under the influence of luteinizing
hormone, which in turn is produced by the pituitary
in response to the pulsatile release of luteinizing
hormonereleasing hormone from the hypothalamus.
The adrenal glands secrete essential steroid hormones
including testosterone and also androgens of lesser
activity that alone or by peripheral interconversion
may be metabolically important in target tissues. Without sufficient testicular testosterone synthesis, however, the adult prostate undergoes partial involution.
Testosterone is insoluble in water and is carried
in the circulation bound principally to sex hormone
binding globulin, also somewhat loosely to albumen,
leaving a small but physiologically important fraction
of free circulating testosterone. Free testosterone is a
lipid-soluble molecule derived from cholesterol that
transfers across lipid cell membranes with ease.
Testosterone stimulates androgen signaling principally through binding the androgen receptor, though
it may also have cell surface interactions with intracellular signaling pathways. It also serves as a precursor for other steroid hormones through irreversible
metabolic conversion within target tissues, principally
to either dihydrotestosterone (by 5-a reductase) or
estradiol (by aromatase).
318
Feneley et al.
Figure 2 Endocrinological influences on the prostate. Pulsatile

release of LH-RH from the hypothalamus causes the release of
luteinizing hormone (LH) from the anterior pituitary, which circulates to the Leydig cells of the testis, which produce testosterone
(T). Testosterone circulates from the testis bound to sex hormone
binding globulin (SHBG) and is available to the prostate. Circulating testosterone has a negative feedback inhibition of the
hypothalamic release of LH-RH. Adrenal secretion and peripheral steroid metabolism are other sources of circulating androgens. Abbreviation: LH-RH, luteinizing hormonereleasing
hormone.
In the prostate, intracellular 5-a reductase irreversibly converts testosterone to dihydrotestosterone.

Dihydrotestosterone binds the androgen receptor with
higher affinity and stimulates greater receptor activation than its precursor testosterone. Prostatic dihydrotestosterone is predominantly synthesized in situ,
whereas circulating dihydrotestosterone is derived
principally from 5-a reductase type 1 activity in the
skin and liver. By an alternative pathway, testosterone
may be converted irreversibly to estradiol through
aromatization, a much less active pathway in the
prostate but may be relevant in the development of
BPH and prostatic inflammation.
There are two 5-a reductase isozymes, type 1 and
type 2. In the prostate, the type 2 isozyme is predominantly expressed and therefore the more important.
Finasteride and dutasteride are both 5-a reductase
inhibitors used for treatment of BPH. Finasteride specifically inhibits 5-a reductase type 2, whereas dutasteride inhibits both isozymes (6). It is a deficiency of
the type 2 isozyme that is responsible for the intersex
state first noted by Imperato-McInley in the Dominican Republic in which apparently normal girls change
to boys at puberty (7).
Figure 3 Testosterone (T) dissociates from its protein binding

and translocates across the cell membrane. Intracellularly, it is
converted to dihydrotestosterone (DHT) and binds to the androgen receptor (AR), from where it translocates to the nucleus.
In cells that express androgen receptor, intracellular androgen binds the androgen receptor, which
then translocates to the nucleus and forms a complex
that binds DNA to initiate transcription. Binding of
the androgen receptor with androgen involves various
coactivators, copressors, dimerization, and conformational changes of the receptor that determines its
molecular activity as a transcription factor. Through
binding of the receptor DNA-binding domain to specific target DNA, androgen-sensitive gene transcription is initiated (Fig. 3).
Within the prostate, there are intracellular differences in 5-a reductase and androgen receptor expression according to cell type and epithelial differentiation
reflecting dynamic cellular interactions that are
involved in tissue regulation and function. Both epithelial cells and stromal cells contain 5-a reductase
enzymes (8). The type 2 isozyme provides the majority
of this enzymatic activity, and is present predominantly
in stromal cells, which are therefore the principal source
of prostatic androgen, dihydrotestosterone. Small
amounts of the type 1 isozyme are expressed by glandular epithelial cells. Similarly, both epithelial and
stromal cells contain androgen receptors. Within glandular structures, androgen receptor is expressed at relatively low levels in the basal epithelial cell layer
(representing the stem cell and proliferating compartments), contrasting much greater expression in the
luminal secretory cells. This reflects the dependence of
glandular differentiation and epithelial secretion on
androgen signaling. Prostatic androgen receptor is
more consistently present in the stromal cells where
319
flatter, and without any secretory activity, and programmed cell death (apoptosis) is readily visible. In
relation to these different epithelial cell types, there
are differences in the underlying extracellular matrix
that probably reflect the driving force for epithelial
differentiation (12). In addition, there are the neuroendocrine cells described below.
Growth Factors in the Prostate
Figure 4 The effect of androgens in the stroma is to cause the

release of growth factors that have an autocrine effect on themselves and a paracrine effect on epithelial cells.
they stimulate transcription of various growth factors

that drive both the epithelial cells and the stromal cells
themselves to grow and develop (9,10). It is this interaction between the stroma and the epithelium that is
thought to account not only for development and for
normal function but also, when the interaction is
deranged, for the process leading to BPH.
Thus, several processes are active within the
prostate. The endocrine effect of testosterone leads to
the intracrine effect of dihydrotestosterone, which generates the autocrine and paracrine effects of growth
factors on both the stroma and the epithelium of the
prostate (Fig. 4). These growth factors, responsible for
the autocrine and paracrine effects, act principally during the G1 phase of the cell cycle and have various
effects, mainly stimulatory but in some instances inhibitory, leading generally to the entry of the cell into the
S phase and ultimately then to mitosis.
The normal adult prostate should not be thought
of as being an entirely static structure. There is a
turnover of cells throughout life and this turnover is
heterogeneous (11). Those glandular cells further out
in the periphery of the gland away from the urethra
show little in the way of secretion and most in the way
of mitotic activity within the prostate, resembling
basal or stem cells. At an intermediate level along
the prostatic duct, the epithelial cells show less mitosis
but almost all of the secretory activity of the prostate.
These two types of acinar cells are tall and columnar
and show no evidence of cell death. More centrally,
close to the urethra, the epithelial cells are lower,
It has been known for many years that prostatic cells

could only proliferate in vitro in the presence of
serum. The factors that were present in serum were
not present in plasma and so it seemed that whatever
was necessary to stimulate cell growth in vitro was
derived from the process of blood coagulation and
most probably liberated from platelets. Thus, one of
the first growth factors to be identified was called
platelet-derived growth factor. Various other factors
were subsequently identified as being growth factors
for various different cell types in vitro and have
derived their names from the circumstances under
which they were isolated. These substances may
have effects other than being growth factors, and
most have many different roles in stimulating cellular
growth both in vitro and in vivo (chapter 1).
Other factors that are known to be important in
the normal growth and development in the prostate
have been (a) derived from the mouse submaxillary
gland [epidermal growth factor (EGF)]; (b) shown to
have a very characteristic and strong tendency to bind
to heparin (fibroblast growth factor, one of the socalled heparin-binding growth factors); or (c) biochemically related to proinsulin [insulin-like growth
factor (IGF)]. Hence, the origin of names that are
somewhat confusing to the uninitiated.
Typically, growth factors are grouped into families of factors having similar effects and there is an
EGF family, a fibroblast growth factor family, a transforming growth factor b (TGF-b) family (TGF-a
somewhat confusinglyis a member of the EGF
family), and the IGF family. There are other growth
factor families as well, but these are the ones that are
thought to be important in the normal prostate.
Of these various growth factors, the most important stimulatory ones are EGF and TGF-a, both of
which are derived from the EGF family, and basic
fibroblast growth factor (bFGF) of the fibroblast
growth factor family. Of these, bFGF and EGF are
thought to account for 80% and TGF-a for 20% of
the stimulatory growth factor effect in health (13). The
other important growth factor is TGF-b, whose effects
are complex but generally inhibitory (14).
Other growth factors such as IGF are thought to
be principally permissive in the same sense that
androgens are permissive. IGF and testosterone have
to be present in vivo and in vitro, but adding more
and more of either does not produce a corresponding
parallel proliferation of the prostate in vitro, whereas
adding EGF, TGF, or bFGF does. The effects of some
of these factors are only or principally apparent at
different stages of development. EGF and bFGF are
predominant in the adult (developed) prostate. On the
320
Feneley et al.
other hand, TGF and another member of the fibroblast

growth factor family, acidic fibroblast growth factor,
are principally evident in fetal life (15).
Most of the growth factors alluded to above act to
increase gene expression, although the TGF-b family is
notable for generally producing inhibition. Most growth
factors act through single-pass transmembrane receptors with tyrosine kinase activity that, through a transduction pathway, induce mitogen-activated protein
kinases, which produce the increase in gene expression.
The Role of the Prostate in Continence

One could be forgiven for thinking that the prostate
has a major role in continence by virtue of its situation
around the bladder neck, but of course the female of
the species manages perfectly well without it.
In individuals of either sex there is a change in
the orientation of the smooth muscle bundles of the
detrusor as they approach the region of the bladder
neck, with a tendency to become more oblique in
disposition, smaller and more finely interspersed in
a more dense connective tissue framework (Fig. 5) (16).
In males, but not in females, there is a quite definitely
circular/oblique orientation of smooth muscle bundles around the area just below the bladder neck and
within the substance of the prostate, with a dense
adrenergic innervation that can be demonstrated by
immunofluorescence (Fig. 6) (17). Elsewhere in the
bladder in both sexes and in the female urethra, aadrenergic innervation is only seen in relation to
blood vessels, although a-adrenergic receptors may
be more widely distributed. This ring of smooth muscle around the supramontanal prostatic urethra,
between the bladder neck and the site of drainage of
the prostatic ducts and ejaculatory ducts into the
urethra, is called the preprostatic sphincter. It is present to ensure antegrade ejaculation (see below).
Elsewhere within the prostate, particularly
within the prostatic capsule but distributed throughout the stroma, there are smooth muscle cells that
Figure 5 The orientation of smooth muscle bundles at the

bladder neck to form the preprostatic sphincter.
Figure 6 Immunofluorescence photomicrograph to show the

presence of noradrenergic nerves interspersed between the
smooth muscle bundles at the bladder neck.
amount to about 50% of the total mass of the stroma of

the prostate (18). These cells also have (mainly) an aadrenergic innervation (19).
Classical pharmacological studies were able to
distinguish between a- and b-adrenergic activities
and, later, between a-1 receptors (postjunctional
a-adrenoceptors that mediate the effector response)
and a-2 receptors (prejunctional a-adrenoceptors that
regulate neurotransmitter release). Further, pharmacological a-1 subtypes are now distinguished by the
selective effects of antagonists. The International
Union of Pharmacology classification of a-receptors
clarified the nomenclature used to describe native as
distinct from cloned receptors (20). The majority of the
adrenergic receptors are of the a-adrenergic type, 98%
of which are located within the stroma, of which 90%
are of the a-1 subtype. Only 10% of a receptors and
neurons are of the a-2 subtype. Of the a-1, the a-1A
type accounts for 60%, and it is these that characterize
the cells that have been targeted pharmacologically by
drugs such as indoramin, terazosin, doxazosin, alfuzosin, and tamsulosin in an attempt to reverse some of
the features of bladder outflow obstruction due to
BPH (21).
This adrenergic innervation is not the only form
of innervation to the prostate or even the only innervation of prostatic smooth muscle cells. There is a
cholinergic innervation thought to control epithelial
secretion (22), and there are a number of different
nonadrenergic, noncholinergic nerve or receptor types
as well. The latter include serotonin (5-hydroxytryptamine), dopamine b hydroxylase, vasoactive intestinal
polypeptide, neuropeptide Y, leu-encephalin, metencephalin, calcitonin generelated peptide, and substance P (23). What these are all doing is by no means
clear.
Some of the nonadrenergic, noncholinergic substances listed above are present in so-called neuroendocrine cells (24). The principal neuroendocrine
cell types are cells that secrete serotonin and thyroidstimulating hormone. Other cells contain calcitonin or
the calcitonin generelated peptide and somatostatin.
It is not clear what these do, but they are thought to be
involved in the regulation of secretion or cell growth.
The function of the other neurons or receptors is
completely unknown; they may not actually have
any function. As has been pointed out elsewhere in
this volume, the presence of neurons does not necessarily imply the presence of receptors for those neurons and vice versa, and even if both are present, that
does not necessarily mean that there is a neural pathway, and if there is, it does not necessarily follow that
that pathway has a physiological significance even if it
has a pathological significance.
Thus, there is no evidence that the prostate plays
any active role in continence, although in disease it
may interfere with normal continence. There is no
evidence that the male bladder neck in the strict
sense is any different from the female bladder neck
except that it simply has a preprostatic sphincter
superimposed on the common bladder neck pattern.
All the evidence is that the prostatic smooth muscle
and its innervation and the other nerve supply to the
prostate are involved in glandular secretion and
the process of emission.
EMISSION AND EJACULATION

Ejaculation in human beings remains a mystery, in
part as it is so difficult to study because of the
problems of sexual sensibilities. In animals this is
less of a problem; indeed, because of the financial
implications of breeding in animal husbandry, ejaculation in some species has been studied in considerable detail.
We have known for some time from so-called
split ejaculate studies in humans that spermatozoa
and prostatic fluid are ejaculated first, followed by
seminal vesicle fluid later (25). We also know that ejaculation is antegrade in the presence of a competent
bladder neck despite the competence of the urethral
sphincter mechanism, which therefore must be overcome actively or reflexly to enable ejaculation to occur. It
is also common male experience that voiding is difficult
with an erection and for a while after ejaculation.
The latter has been shown to be because of
tightening of the bladder neckmore accurately,
of the preprostatic sphincteron erection, which
becomes more marked in the period leading up to
ejaculation. This has been shown both in urethral
pressure profile studies, which show enhancement
of the pressure zone produced by the bladder neck
(Fig. 7), and in transrectal ultrasound studies, which
demonstrate the preprostatic sphincter quite clearly
and which show it to become more marked in the preejaculatory period (26).
The next step, also shown on transrectal ultrasound (26) (and radiographically in some animal species), is the transportation of spermatozoa from the
ampullae of the vasa into the prostatic urethra. This
process of filling of the inframontanal prostatic urethra,
321
below the occluding preprostatic sphincter, is known as

emission, as distinct from the process by which the
seminal fluid is transported to the outside world, which
is ejaculation. The mechanism for this is not clear. Once
it occurs, ejaculation is imminent. Next is a contraction
of the prostatic smooth muscle including the preprostatic sphincter, which is presumably under a-1A adrenergic control.
Synchronously but not in any coordinated fashion, there is a sequence of five or six contractions of
the bulbospongiosus muscle. The first generally
occurs before any of the seminal fluid has entered
the bulbar urethra, and the last occurs after pulsatile
ejaculation ceases. Neither is the mechanism for this
clear, nor is the means by which the urethral sphincter
mechanism is overcome. It used to be thought that it
was overcome passively by the sheer pressure that
prostatic emission generates. But the recent transrectal
ultrasound studies referred to above suggest that this
is probably not the case. Urethral sphincter studies to
look for a relaxation mechanism have not been useful
in providing an alternative explanation. In any case, a
postulated urethral sphincter relaxation mechanism
would tend to flounder on the observation that after
transurethral resection, men are not incontinent during sexual activity when the bladder neck mechanism
has been ablated.
Once emission and ejaculation have begun, there
is further contraction of the prostate and of the seminal vesicles until the process is complete. There is
therefore an important role for the prostate in the
process of emission leading to ejaculation, irrespective
of the content of prostatic secretion added.
The Physiological Function

of Prostatic Secretion
In an average ejaculate, 2 mL is contributed by the
secretion of the seminal vesicles, 0.5 mL is contributed
by the secretion of the prostate, and Cowpers glands
and the glands of Littre contribute 0.1 mL (27). The
contribution of the sperm cells themselves is insignificant. Although the function of these various secretions is not clear, epididymal sperm can fertilize an
ovum but not as well as ejaculated sperm, so presumably their function is to maximize the potential for
fertilization. This may be by having a protective effect
during the onward journey of the sperm until its
contact with the ovum, or an effect to enhance motility
and sperm survival more directly, or a role to increase
the fertilizing effect of the sperm when it reaches the
ovum. There are various pieces of evidence to support
a role in each of these three areas, but details are
distinctly lacking.
These various secretions also have a protective
role in the lower urinary tract itself. The sheer presence of the fluid provides lubrication both of the
urethra itself and, through the pre-ejaculatory fluid,
for penetration, although penetration has often
occurred long before most human males produce
pre-ejaculatory fluid. Nonetheless, this may at least
be the role of the glands of Littre. It would be nice to
think they were there for some purpose!
322
Feneley et al.
Figure 7 (A) A urethral pressure profile of the bladder

neck (where x is the site of the bladder neck and y is the
site of the urethral sphincter mechanism). (B) A urethral
pressure profile during erection to show the enhanced
pressure zone at the bladder neck.
A protective effect on the lower urinary tract by

the biological effects of some of the components of
seminal fluid may be more important, indeed much
more important than just the mechanical washing of
the urethra. In fact, it has been argued that the principal function of the prostate and the other sexual
secondary sex organs is to protect the integrity of the
spermatozoa. Zinc has a strong antimicrobial action;
spermine less so. Immunoglobulins of various types

could have a similar biological role. All this is, however, unproven.
One substance known to enhance the fertilizing
capacity of ejaculated sperm cells after ejaculation is
fertilization-promoting peptide, which is structurally
similar to thyrotrophin-releasing hormone (28). Neither
is its mode of action nor whether there are other
compounds with a similar action is known. EGF has
a high concentration in seminal fluid, second only to
colostrum in fact, and it may be that this reflects a role
in fertilization.
Numerous compounds are produced by the
prostate, most of which have no obvious function.
Acid phosphatase splits glycerylphosphocholine produced by the seminal vesicles to produce glycerylphosphate ultimately, and it may be that glycerylphosphate is important in sperm protection. It may be
more than just coincidental that in a laboratory setting
glycerin is used for this sort of purpose. Polyamines
are the strongest known cationic substances in nature
and they may have an important role in transcription
and translation (29). Alternatively, these two observations may be no more than just coincidence. Prostatespecific antigen is a serine protease that has a role in
sperm liquefaction (30). Sperm coagulation and liquefaction have an important role in small mammals
such as rats and mice, but their role in humans is
unclear.
More interesting perhaps, because of their high
concentration within the prostate, are zinc and citrate.
Citrate is present in 240 to 1300 times the concentration found elsewhere (31), and zinc is present in about
30 times the concentration elsewhere; (32) it seems
likely that there is a reason for this and for the
uniquely high concentrations of polyamines. There
appears to be a close correlation between all three,
and it has long been suspected that citrate is there as a
ligand for zinc (33). It is thought that the zinc is there
to help maintain the quaternary structure of sperm
chromatin (34) in addition to the biological protective
effect mentioned above. It now seems likely that the
complex of zinc and citrate and polyamines forms a
structure that has electrochemical neutrality and
therefore buffers the citrate (35), although this does
seem an extremely energy-inefficient way of achieving
this effect. An alternative, or additional, explanation is
that the complex is there not just to protect the zinc
(so to speak) but to hold the citrate there (36). The
optimum pH for the activity of acid phosphatase is
much lower than the natural pH of seminal fluid, and
if acid phosphatase does indeed have an important
biological role, this may be facilitated by the availability of large amounts of citrate. This, however, is pure
speculation at present.
One of the problems is that most of these substances have only been investigated from the point of
view of their concentrations in disease to serve as a
marker for a disease state rather than to investigate
their physiological role. Until the thrust of research is
redirected, the function of these various components
of prostatic and seminal vesicular secretion will continue to remain obscure.
323
BENIGN PROSTATIC HYPERPLASIA

Having discussed some of the aspects of our current
understanding of the prostate in health, we now turn
to a consideration of the disorders of the prostate seen
in BPH.
The general impression generated in many
reviews is that BPH is a generalized and diffuse disease
of the prostate that occurs as a result of some sort of
hormonal derangement that leads to hyperplasia of the
prostate, producing enlargement of the gland as a
whole, which in turn leads to compression of the
prostatic urethra and a progressive occlusion of the
bladder outflow and the clinical syndrome of prostatism. None of this is true.
BPH is very unusual in that it only occurs in
human beings and dogs. The same is true of carcinoma of the prostate. Despite this and the fact that
both diseases are common, most authorities suggest
that there is no direct link between the two diseases
(37). Recently, however, there has been a suggestion
that the two are related: that a series of genetic hits
gives rise to BPH, and further hits to prostate cancer
(38). In this hypothesis, BPH would represent an early
stage in the development of prostate cancer; however,
benign hyperplasia itself is not a premalignant pathological abnormality.
BPH seems to be a disease (also like carcinoma of
the prostate) that any human adult male can expect to
get if he lives long enough with functioning testes and
if his prostate was normal to start off with. It is clearly
important, therefore, to distinguish between the clinical conditions associated with the histological disease
of BPH and other clinical conditions affecting the
lower urinary tract in aging males.
The recent interest in symptom scores and in the
effects of aging on the bladder in individuals of either
sex seems to make it clear that there are symptoms
arising from the lower urinary tract, and particularly
from the bladder, that are related to aging, which need
to be distinguished from those symptoms related to
histological BPH in males. Furthermore, many of the
features of the BPH clinical symptom complex are
difficult to explain in relation to histological BPH
alone. Acute retention can be explained when it is
secondary to urinary tract infection, severe constipation, or sympathetic stimulation in hypothermia and
psychological stress, but otherwise its nature is somewhat elusive, although it may be due to prostatic
infarction or some other prostatic vascular accident
(39). The urge symptom complex related to secondary detrusor instability is more easy to explain (40),
but bladder decompensation is more difficult given
our present knowledge of clinical BPH and the experimental effects of obstruction on bladder function (41),
unless this is related more to the coincidentally aging
bladder than to BPH per se. It is, in fact, difficult to
explain how BPH causes obstruction at all. Squeezing
on a hosepipe is a very inefficient way of stopping the
water emerging from the end, and a very marked
restriction of the caliber of the hosepipe has to be
produced before there is any overt change in flow.
Obstruction by urethral constriction is easily understood
324
Feneley et al.
in relation to urethral stricture disease, but it is much

more difficult to argue for a similar effect in the prostatic
urethra in BPH when a large resectoscope can be passed
through and into the bladder with ease. It may, therefore, be that it is distortion of the prostatic urethra that is
more important in producing outflow obstruction than
compression or constriction, as it is distortion of a
hosepipe by kinking that is more likely to stop flow.
Etiology
It has already been mentioned that the only proven
risk factors for developing BPH are aging and the
presence of functioning testes (42), assuming that the
prostate was normal to start off within other words,
if 5-a reductase is present to convert testosterone to
dihydrotestosterone, and functioning androgen receptors are available for binding the dihydrotestosterone.
Various factors have been investigated such as
dietary factors, alcohol, and cirrhosis of the liver (43),
all of which may indirectly support the hypothesis of
an endocrine imbalance in parallel with age-related
declining circulating androgen levels or a relative
decrease with respect to estrogen, but without substantive evidence at tissue level. Inherited factors may
also predispose to the development of the disease in
families and at a younger than usual age (44). More
recently, BPH has been shown to be associated
broadly with erectile and ejaculatory dysfunction,
with the possibility of common underlying mechanisms involving molecular signaling (particularly
upregulation of the RhoA/Rho-kinase pathway). Its
association with aging, endocrinological changes,

and vascular disease may also account for observed
associations with hypertension, metabolic syndrome,
and type II diabetes (45,46). The presence of nitric
oxide in prostate and bladder, and their deficiency in
outflow obstruction, as well as the responsiveness of
both voiding symptoms and erectile dysfunction to
a-receptor blockade (perhaps reflecting hyperadrenergic status), suggests certain commonalities in etiology and therapeutic opportunity at least in some
cases (47,48).
Pathogenesis
It has been pointed out that androgens (in other
words, functioning testes) are permissive for prostatic
growth and development in health, and the same is
true for the prostate in vitro. Androgens (and IGF and
other factors) have to be present for prostate cells to
grow, but once the critical permissive concentration
has been reached, there is no extra growth produced
by adding more.
Also discussed above was the concept of a
stromal-epithelial interaction in which testosterone
production by the epithelial cells leads to the elaboration of growth factors by the stromal cell that act in
both an autocrine and paracrine fashion to produce
further growth and differentiation in both the stroma
and the epithelium. This stromal-epithelial interaction
was most clearly demonstrated by Cunha (Fig. 8), who
implanted embryonic urogenital sinus mesenchyme
and adult bladder epithelial cells from a normal
Figure 8 Cunhas experiments showing the importance of stroma (in this case urogenital sinus mesenchyme) in epithelial differentiation.
Abbreviation: T fm, testicular feminization.
mouse under the renal capsule of a nude mouse and

showed that the urogenital sinus mesenchyme
induced prostatic epithelial differentiation of the bladder epithelium (10). Cunha also showed that normal
prostatic differentiation could be induced from the
bladder epithelium taken from a mouse with androgen receptor deficiency as long as the urogenital sinus
mesenchyme was taken from a normal mouse.
Prostatic epithelial differentiation did not occur
when the urogenital sinus mesenchyme was taken
from a mouse with androgen receptor deficiency,
whatever the androgen receptor status of the bladder
epithelium. In this way, Cunha demonstrated the
importance of the androgen receptor in the prostatic
stroma (derived from urogenital sinus mesenchyme)
as well as the importance of the stromal-epithelial
interaction in producing epithelial and glandular
development.
In BPH, the stromal:epithelial ratio increases (18).
Normally, it is something in the region of 2:1, but in
BPH it increases to 3:1 or 4:1. As mentioned above,
there is a substantial smooth muscle component to the
stroma in health and in BPH, but the majority of the
stroma is made up of connective tissue such that about
50% of BPH is connective tissue, 25% is smooth muscle, and 25% is epithelium (19).
The first discernible sign of BPH is the presence
of microscopic nodules of fibromuscular hyperplasia
with a variable epithelial cell component. These nodules are found initially in the transition zone just
below the smooth muscle collar of the preprostatic
sphincter (Fig. 9) (49). The transition zone is found on
either side of the urethra, and nodules in this area are
a mixture of glandular and epithelial hyperplasia. The
nodules of BPH also form, with lesser frequency, in
the periurethral glandular tissue within the smooth
muscle collar of the preprostatic sphincter. Here, these
nodules develop principally posteriorly and have no
epithelial or glandular component, consisting entirely
of connective tissue (50,51). Within these nodules,
most unusual changes can be seen, notably fibroblasts
transforming themselves into smooth muscle cells. It
was this observation that led McNeal to suggest that
the nodules of BPH, wherever they occurred, were the
Figure 9 The siting of early nodules in benign prostatic hyperplasia: just below and within the collar of the preprostatic sphincter (PS) in the general area of the transitional zone (TZ).
325
result of an embryonic reawakening as a consequence of localized changes in the normal stromalepithelial interaction (49,52).
The so-called lateral lobes of BPH are derived
from micronodule formation and coalescence and
further growth within the transition zone, whereas
the so-called middle lobe is derived from nodule
formation and development within the periurethral
glandular sleeve posteriorly. The remainder of the
prostateas has been well recognized for many
yearsis compressed outward to form a false capsule
posteriorly (53).
In other words, BPH is not a diffuse and generalized disease of the prostate but is a highly localized
disease of the smallest area of the prostate (the transition zone and the periurethral gland area) (54).
Whereas the description embryonic reawakening
may not be entirely accurate, the disease does seem
to be related to an abnormal stromal-epithelial interaction confined to this small area of the prostate,
in which both increased cell growth and reduced
cell deaththat is to say, programmed cell death
(apoptosis)are equally important components (55).
It was mentioned above that along the prostatic duct,
from the urethra out to the periphery, the most distal
glands were dividing while the most proximal ones
(nearest the urethra) were undergoing apoptosis. The
epithelial cells of the more distal or peripheral glands,
which have been noted to be more mitotically active,
seem to be related to underlying smooth muscle cells
that are vimentin positive, whereas those that are
proximal or central, which tend to show apoptosis,
are related to smooth muscles that are actin positive
(12,56). Perhaps vimentin-positive smooth muscle
cells tend to be more stimulatory, whereas those that
are actin positive produce inhibitory growth factors or
growth factors that tend to promote apoptosis. Alterations in growth factor expression have, indeed, been
identified in BPH, although it is not yet possible to say
with confidence that these growth factors are the
cause of this histological condition, and that their
effects are confined to the transition zone and not
present elsewhere within the prostate (57).
In the normal prostate, the main stimulatory
growth factors are EGF and TGF-a, the former being
principally active in adults and the latter in growth
and differentiation of the prostate. Neither of these
appears to play any part in BPH, although the EGF
expression appears to be reduced (58). Another
growth factor family with an important role in the
normal prostate is the fibroblast growth factor family.
Like TGF-a, acidic fibroblast growth factor is predominantly active in growth and differentiation (15). It is
bFGF that is the main stimulatory growth factor of this
group in adults. All members of the fibroblast growth
factor family have a variety of actions on a variety of
different cell types, including producing angiogenesis
and remodeling the extracellular matrix by producing
modulating proteases and inducing the synthesis of
fibronectin (14,59). Members of the fibroblast growth
factor family are abundant in the extracellular matrix,
where they bind heparin avidly. Basic fibroblast
growth factor is interesting because it is not secreted;
326
Feneley et al.
it is only released from cells by injury or cell death,

although it may, of course, have intracrine activity (60).
However, there is another member of this family,
called keratinocyte growth factor (KGF) (61). This is
a truly paracrine substance that is secreted only by
stromal cells but for which there are receptors only on
epithelial cells. It may be that some of the stimulatory
activity previously ascribed to bFGF is, in fact, more
related to KGF. Both bFGF and KGF appear to have a
role in BPH as both show increased expression (62).
The TGF-b family is the family of growth factors
that are generally inhibitory for epithelial cells. It is
more true to say that they may be inhibitory or
stimulatory depending on the cell type, the state of
differentiation, and the circumstances (14,63), but in
general they are inhibitory for epithelial cells and
stimulatory for stromal cells. They also increase angiogenesis and extracellular matrix formation (64,65).
This family is interesting, first because these growth
factors have receptors that are serine/threonine kinases rather than tyrosine kinases, which are usual with
growth factor receptors (65), and second, because they
can inhibit the transition from the G1 phase to the S
phase of the cell cycle and thereby override the action
of mitogens, including the growth factors alluded to
above (14,66).
There are five isoforms of TGF-a, but only three
have been found in mammals (a-1 a-3) and only a-1
and a-2 have been investigated in BPH. TGF-a-1 is
negatively regulated by androgen: a fall in androgen
leads to increased expression of the growth factor, and
vice versa (65). It also seems that bFGF and KGF
expression is regulated by TGF-b (14). As a result,
fibroblast growth factor expression is regulated by
androgen indirectly by the androgen effect on TGF-a
expression.
The fibroblast growth factorsbFGF and KGF
are stimulatory growth factors and TGFs a-1 and a-2
are inhibitory. Stromal cells produce both fibroblast
growth factors and TGF-a-1 (14,57). Epithelial cells
produce TGF-a-2 (67). bFGF acts on both epithelial
cells and stromal cells but principally on stromal cells
because that is where it is released. KGF, on the other
hand, can only act on epithelial cells because only
epithelial cells have receptors for it. The TGFs a-1 and
a-2 act on both epithelial and stromal cells (Fig. 10).
Thus, KGF stimulates and TGF-b inhibits epithelial cells, and bFGF stimulates and TGF-b inhibits
stromal cells; in the normal prostate, a steady state
exists in which each pair is in balance. It is believed (68)
that in BPH, the fibroblast growth factors tend to
override the TGF-a and there is therefore a proliferation of epithelial and stromal cells and, in addition,
because of the effect on extracellular matrix components, an increased activity there as well.
If one assumes that this hypothesis is true, there
is still a need to identify the cause of the changes
outlined above. It has been suggested that declining
androgen levels could account for the increasing
expression of TGF-a (69), but that would not explain
the localized nature of the disease process. An alternative explanation is that repeated microtrauma or
inflammation produced by repeated (lifelong) voiding
Figure 10 The interaction of growth factors in benign prostatic

hyperplasia. Abbreviations: bFGF, basic fibroblast growth factor;
IGF-II, insulin-like growth factor II; KGF, keratinocyte growth
factor; TGF-b, transforming growth factor b; TGF-a, transforming
growth factor a; EGF, epidermal growth factor.
and ejaculation, acting, as they do, on the point of

angulation of the prostatic urethra, which is where the
transition zone is situated, leads to cell damage at that
site, which in turn leads to the release of bFGF, which
starts the whole process off (70).
Whether or not the details given above and the
hypothesis outlined in the last few paragraphs prove
ultimately to be true, it is quite clear that BPH is not a
diffuse generalized disease of glandular epithelium
due to androgen imbalance within the glandular epithelium or due to systemic changes in the androgen:
estrogen ratio. It is a focal, stromal-induced disease
affecting the transition and periurethral zones, producing micronodule formation by a stromal-epithelial
interaction that appears to be mediated by growth
factors. Androgen appears to act through TGF-a
expression to regulate the expression of other growth
factors. The micronodules enlarge and coalesce, perhaps with an altered balance between cell growth and
programmed cell death, which is also mediated by
growth factor activity. This, in turn, produces an effect
on the prostatic urethra, possibly by distortion, to
produce bladder outflow obstruction. This, however,
is only one of the symptom complexes found in aging
patients who have histological BPH, and in some,
nonobstructive BPH may well produce symptoms by
means that are not entirely clear. Others, possibly as a
result of a vascular accident within the prostate or
otherwise because of sympathetic stimulation or superadded obstruction by external compression in constipation or by epithelial edema in urinary infection,
develop acute retention. Others develop detrusor
instability as a consequence of the secondary changes

induced by obstruction in the detrusor cells, although
many patients with BPH will have detrusor instability
coincidentally as a result of this. Still others, possibly as
a result of the coincidence of BPH in conjunction with
the impaired detrusor contractility that is commonly
seen with the aging bladder, develop detrusor decompensation and the symptom complex that leads ultimately to chronic retention and overflow incontinence.
In some of these patients, the obstructive element
causes high intravesical pressures, leading to structural
and functional abnormalities of the upper urinary tract
and thus to renal impairment and renal failure.
It should again be stated that some of the
changes outlined in the last paragraph are hypothetical and unproven, but are nonetheless more likely to
be accurate than the rather simplistic ideas hitherto
promulgated on the basis of prostatic enlargement,
urethral compression, and obstruction leading to
prostatism.
A more pragmatic approach considers the clinical impact of lower urinary tract symptoms (whether
directly or indirectly related to BPH, or due to other
disease or aging processes) in relation to the natural
history of BPH or its treatment. The clinical diagnosis
of BPH is made by demonstrating prostatic enlargement in the presence of typical symptoms, and exclusion of other significant urinary tract disease (71). Its
diagnosis represents opportunity for various management options with various efficacy, risks, and side
effects. Symptom scores that are valid for BPH tend
therefore to be used for initial assessment of symptoms but may in fact reflect changes due to various
underlying pathophysiologies. Flow rate and residual
volume assessment should be carried out according to
practice guidelines (72). Serum creatinine measurement is generally recommended in view of associated
chronic renal disease (73), and PSA is measured principally for prostate cancer risk assessment.
Therapeutic objectives including symptom reduction and improved quality of life are consistently
achieved in the majority of men with uncomplicated
BPH by pharmacological treatment, particularly a
blockade, 5-a reductase inhibition, or their combination
(74,75). The Medical Therapy of Prostatic Symptoms
(MTOPS) study was a landmark in demonstrating the
utility of combined a-blockade and 5-a reductase inhibition, particularly for men with higher symptom score
(mean IPSS 17), larger prostate volume (mean 32 cc),
and higher PSA level (>1.5 ng/mL) (74). The success of
these treatments lies in their achieving overall benefit,
and providing acceptable balance between therapeutic
convenience and its side effects (whether or not presenting symptoms were specifically obstructive or even
directly due to BPH). Recent interest has arisen in
alternative pharmacological interventions including
muscarinic blockade (76), botulinum toxin (77), and
other lower urinary tract receptor (78).
In recommending treatment, it is important to
understand the natural history of the untreated disease. The Olmsted County Study of Urinary Symptoms and Health Status Among Men was an
important longitudinal, community, cohort study
327
demonstrating progressive prostatic growth, decline

in flow rate, and increasing prostate-related symptoms with age (79). The development of prostatic
enlargement and its relationship with baseline PSA
level, with higher PSA levels predicting enlargement,
was shown in the Baltimore Longitudinal Study of
Aging (80). Prior historical studies of the natural history of untreated BPH had emphasized that clinical
deterioration in symptomatic men was by no means
inevitable, that the clinical course of BPH may be
episodic, and that spontaneous improvement may
occur in a significant proportion of those not proceeding to surgery (81). The trend for improvement may
also be seen with symptomatic improvement after
lifestyle modification, or with symptomatic improvement within the placebo arm of randomized trials, or
more simply with statistical regression toward the
mean when observing a highly selected population.
These considerations, taken together with an association between sexual dysfunction and BPH (82), indicate that some aspects of lower urinary tract function
relate to modifiable risk factors, for which lifestyle,
diet and physical exercise may be important. Their
modifications may also have a wider impact on mens
health, aging, cardiovascular disease, and autonomic
nervous system activity (83,84).
In recent years, increasing interest has focused
on the progressive course of benign hyperplasia and
its risk factors. In the Olmsted County Study and the
Health Professionals Followup Study, the risk of
retention was 6.8/1000 and 4.5/1000, respectively
(85,86). While recognizing the importance of observational community studies, present understanding has
been substantially influenced by placebo arms of
pharmacotherapy trials where outcomes of interest
include symptom deterioration, incontinence, acute
urinary retention, development of urinary tract infection or renal insufficiency, and need for surgical
treatment. Taking these as indicators of clinical progression, symptom deterioration is by far the most
frequent, representing around 80% of events (87).
Progression overall, however, is relatively uncommon, as defined by all indicators. In the MTOPS
study, the risk of clinical progression in the placebo
arm was 4.5 per 100 man-years, representing a total
risk of 17% at 4 years, including symptom deterioration in 14%, retention in 2%, and surgery in 5% (88).
Clinical progression in men with BPH is predicted by
baseline prostate volume, and serum PSA, impacting
risk of symptom deterioration, flow rate decline, urinary retention, and need for surgery (87,89). In contrast to treatment with the 5-a reductase inhibitor
finasteride that reduces all these measures of progression, alfuzosin reduces all except urinary retention (90).
Particular concern has focused on the risk of
developing acute urinary retention. Pharmaceutical
trials have emphasized that retention develops more
frequently when the clinical diagnosis of BPH is
associated with certain baseline characteristics that
include large prostate volume, elevated PSA not due
to prostate cancer, prostatic inflammation, lower maximum flow rate, higher postvoid residual volume,
328
Feneley et al.
previous acute retention, older age, and symptom

deterioration (86,88,9193). Review of reported pharmacotherapy trials indicates spontaneous acute urinary retention rates in placebo arms of between 0.9%
and 5.2%, though comparison or extrapolation of such
figures are of little validity owing to variability in trial
duration, individual enrollment, and follow-up (94).
Furthermore, the significance of acute retention may
vary as clinical outcomes and potential to restore
spontaneous voiding may relate to prostate size (95)
and whether retention is precipitated or spontaneous
spontaneous retention reportedly having a greater need
for surgery (96). Finally, the likelihood of successful
voiding trial after a first episode of retention may be
further modified by treatment, particularly with
a-blockers (97).
Treatment with a 5-a reductase inhibitor alone
may reduce the risk of retention or surgery by at least
55% (98), or more in high-risk individuals (89). The
clinical impact of 5-a reductase inhibition and combination therapy relates to prostate volume at baseline.
Medications with adrenergic or anticholinergic activity may increase the risk of acute urinary retention
(86). The association of inflammation with aging,
lower urinary tract symptoms, progression of benign
hyperplasia and its relationship to the development of
malignant prostatic disease is attracting interest
(91,99), and may indicate a role for estrogen, estrogen
receptors a and b, and selective estrogen receptor
modulators in the prostate (100,101).
A hypothesis proposing that clinical progression
is specifically attributable to progression of benign
hyperplasia would be supported where treatment
that specifically targets the prostate pathology also
reduces the risk of adverse clinical outcomes; in contrast, failure to prevent clinical progression might
indicate the impact of other pathophysiology. Indeed,
symptom deterioration on treatment with alfuzosin
has been shown to be associated with higher baseline
post-void residuals and their progressive worsening,
as well as a greater risk of BPH-related endpoints
(102). The failure of BPH therapy to prevent clinical
progression indicates the need for more precise
understanding of lower urinary tract pathophysiology
as well as optimal treatment for risk reduction and
prevention of retention (103,104).
Thus, there is an uncertain relation between
histological BPH, lower urinary tract symptoms,
response to treatments for BPH, and pathophysiological events underlying clinical progression. Advances
have principally been in symptom management rather
than overall or long-term strategic therapy for BPH.
Further research may identify markers for more efficacious strategies (105).
Urodynamic Aspects
It has already been suggested that BPH may be
asymptomatic or symptomatic, and that symptoms
may arise purely and simply from the condition itself
by virtue of its effects on the prostate alone or from its
secondary obstructive effect on the prostatic urethra.
Mention has been made of the secondary effects that
obstruction can have on the previously normal bladder, leading to detrusor instability, and on the aging
bladder, hastening the process begun by aging and
leading through progressive degrees of detrusor
decompensation to chronic retention with overflow;
and that in the latter category high intravesical pressures can lead to obstructive changes in the upper
tracts, leading ultimately to renal failure. Also mentioned have been those factors that can act in BPH to
cause acute retention.
Symptom severity, which is increasingly being
defined by symptom scores (106108), can be used as
selection criteria for surgery. When symptom scores
are high, 90% of men will experience substantial
improvement in symptoms after surgery, even in the
absence of proven urodynamic obstruction (109,110).
In addition, the symptoms of BPH in obstructed
patients seem to be relieved by thermotherapy and
other recent alternative treatments for this condition without any effect on flow (111).
Urinary symptoms may equally be the result of
detrusor instability that has developed as a consequence of obstruction, in which case the patient may
have both obstructive and irritative symptoms, and
they might equally be relieved by transurethral resection of the prostate if relief of the obstruction causes
the detrusor to return to normal function.
Unfortunately, those same urinary symptoms
may occur as a result of idiopathic detrusor instability that the patient has developed for some entirely
different reason, and the fact that he might coincidentally have histological BPH is irrelevant. Such a
patient will not benefit from transurethral resection
of the prostate because the instability will persist.
Similarly, a patient with a poorly contractile
bladder may have it in association with, or even,
perhaps, as a consequence of, outflow obstruction
due to BPH and might therefore benefit from transurethral resection of the prostate. But, equally, the
poorly contractile bladder may be an age-related phenomenon and coincidental BPH may not be causing
any symptoms, in which case transurethral resection
of the prostate would not be helpful (112,113).
Clearly, therefore, BPH is not always obstructive
but may nonetheless cause symptoms, generally of an
irritative nature, and, equally, those symptoms may
occur for other reasons than BPH, generally arising in
the bladder smooth muscle as an age-related or other
phenomenon, causing detrusor instability or impaired
detrusor contractility or a combination of the two.
Somehow, these different phenomena have to be
dissected out to determine the cause of a patients
symptoms and to decide how best to treat him, and
urodynamic studies of various different types are the
means by which this is done in clinical practice.
Historically, only bladder outflow obstruction
was considered of any significance. Indeed, 50 years
ago, treatment was only considered in the presence of
urinary retention or when there was evidence of
impaired function of the upper urinary tract and
kidneys. More recently, detrusor instability has been
recognized as an entity, but even so, detrusor instability and bladder decompensation leading to chronic
retention and overflow incontinence were both

thought of only as consequences of obstruction rather
than conditions that might arise per se. Irritative
symptoms in the absence of obstructive instability
were largely ignored. It is only the recent interest in
symptom scores, in alternative treatments for BPH
that reduce symptoms without any effect on urodynamic variables, and in the effect of aging on the
bladder that has led to a reconsideration of this historical attitude. Nonetheless, the attitude that only
obstruction mattered has established the primacy of
the pressure-flow relationship in the objective assessment of BPH in clinical practice.
Urinary obstruction can be regarded as being
present when intravesical pressure has to be raised to
maintain the urinary flow rate. When an elevated
intravesical pressure can no longer maintain the urinary flow rate, the flow rate begins to decline. Thus, in
the first stage of obstruction, voiding detrusor pressure is raised above the upper limit of normal (which
is about 50 cm H2O), but the peak urinary flow rate is
still greater than 15 mL/sec, which is the lower limit
of normal. In the second stage of obstruction, the peak
urinary flow rate drops below 15 mL/sec.
This relationship between pressure and flow was
likened by Griffiths (114) to the Hill equation, which
was a well-established and widely accepted means of
describing the relationship between the force of contraction and the speed of shortening of skeletal muscle. Griffiths, by relating detrusor pressure to flow rate
in what he called the bladder output relation,
showed that the two relationships were very similar.
He also introduced the concept of the urethral resistance relationship by relating flow rate to detrusor
pressure throughout the period of a voiding detrusor
contraction (115). In this way, he was able to distinguish graphically (Fig. 11) between an obstructed
system, in which pressure was high and flow was
low, and an unobstructed system, in which pressure
was low and flow was high. In practice, rather than
plot the continuous relationship graphically, Abrams
and Griffiths (116) devised a nomogram on which
could be plotted the single point of detrusor pressure
at maximum urinary flow rate (Fig. 12). Several other
physicists, notably Schafer, have devised more sophisticated techniques for the analysis of pressure-flow
data, but they are all based on the same principles
(117,118).
The two main reasons why physicists are continuing to analyze this pressure-flow relationship are,
first, to be able to express it in a single term rather
than as a relationship between two variables, particularly if this could be determined noninvasively, and,
second, to minimize the equivocal zone.
The desire to find a noninvasive way of extrapolating the pressure-flow relationship is obviously
commendable but has so far been unsuccessful. Nonetheless, in the interests of patient comfort and as it is a
quick, simple, cheap, and easy test to use, the problem
has been circumvented in routine clinical practice by
measuring flow rate alone and by inferring the pressureflow relationship from this measurement (116). In this
way, most unobstructed patients with normal flow
329
Figure 11 (A) A dynamic pressure-flow plot during voiding to

illustrate normal emptying with low pressure and high flow. (B) A
dynamic pressure-flow plot of so-called compressive bladder
outflow obstruction due to benign prostatic hyperplasiawith
high pressure and low flow.
rates can be saved from ineffective and inappropriate

obstruction-relieving surgery. Unfortunately, a low
flow rate is not exclusively due to high pressurelow
flow outflow obstruction; a low flow rate may also be
due to a poorly contracting detrusor.
Poor detrusor contractility also accounts for most
of the equivocal results on pressure-flow analysis.
Poor detrusor contractility characteristically shows
normal or low detrusor pressure and low flow, and
this is very common, with an incidence that increases
with age. Indeed, as a general rule, all men over the
age of 80 have a maximum flow rate of less than
10 mL/sec (119).
Thus, we appear to have three independent
variables determining the clinical picturesymptoms,
BPH, and bladder outflow obstructioneach of
330
Feneley et al.
Figure 12 An obstructive pressure-flow plot, similar to Figure 12

in Chapter 16, superimposed on the Abrams-Griffiths nomogram. Normally, only the detrusor pressure at maximum flow
(dot) would be marked on the plotthis figure illustrates the
principle.
which, as has been said, may be confounded by two

age-related abnormalities of detrusor contractility
impaired detrusor contractility and detrusor instability. Irritative symptoms in men with or without BPH
correlate strongly with the presence of detrusor instability on urodynamic evaluation (120), and this presumably accounts, at least in part, for the observation
that symptom scores are the same in an unselected
population of elderly women (in whom detrusor
instability is equally common) as they are in an
unselected population of elderly men (121). By contrast, objectively demonstrable obstruction correlates
poorly with symptoms (122), presumably because
obstructive symptoms are less bothersome,
although those with bothersome obstructive symptoms who have objectively demonstrable obstruction,
rather than low pressurelow flow bladders because
of impaired detrusor contractility, do best after transurethral resection of the prostate (123).
Detrusor instability is clearly an important factor
in the patient with BPH, whether it is a secondary
consequence of obstruction or coincidental. Nocturia
and daytime urgency and frequency are the commonest reasons why elderly men seek medical attention
(124), and although there are other causes of these
symptoms, detrusor instability is the commonest reason for all these symptoms being present together.
It is generally thought that most instances of
detrusor instability in patients with BPH are secondary to obstruction, and that 70% or so will improve
symptomatically after transurethral resection of the
prostate because the obstruction has been relieved,
allowing the detrusor to recover and the pathophysiological changes to reverse. Various experimental studies support this view (125), although they have failed
to show complete recovery after relief of obstruction,

only a tendency to improve. The alternative view is
that detrusor instability in these patients is an entirely
age-related phenomenon in both sexes that coincidentally develops at the same time as BPH in men, and
that the reason why so many men improve after
transurethral resection of the prostate is related to
denervation (or de-afferentation, to be more accurate)
produced by this surgical procedure (126,127).
Impaired detrusor contractility is more problematic. Like detrusor instability, this is common in the
elderly of both sexes and is a regular cause of dissatisfaction in male patients who have undergone transurethral resection of the prostate because they were
thought to be obstructed (128). Unlike detrusor instability, which regularly seems to resolve after this
operation, impaired detrusor contractility rarely, if
ever, improves. This is the first reason for suggesting
that impaired detrusor contractility is an independent
age-related condition rather than secondary to
obstruction, as has always been assumed. The second
reason is that experimental models of obstruction
cause a thick-walled, trabeculated, high-pressure,
unstable bladder, and not chronic retention (129).
Thus, the view that impaired contractilitycausing
residual urine initially, progressing eventually to
chronic retention with overflow incontinencearises
as a result of decompensation of the detrusor in the
face of continuing obstruction after an initial phase of
compensation seems flawed. It may be that if obstruction is superimposed on impaired detrusor contractility, the consequences are correspondingly more
severe, and it may be that these patients are prone
to high-pressure retention and renal impairment as a
result. Either way, impaired detrusor contractility is
common in elderly men and women, and may be
complicated by superadded detrusor instability in
some (130), suggesting that in many, if not all, males
with BPH, impaired contractility is an independent
variable unrelated to obstruction in its cause.
And so the diagnostic quandary persists.
Obstruction is still the only problem that can reliably
and predictably be treated, and urodynamic studies
that measure both detrusor pressure and urinary flow
during voiding are still the only way to diagnose
obstruction reliably. With a clinical problem that is
so common, this poses logistic problems and, as
obstruction is only one part of the picture, the problems are compounded. Nonetheless, a normal flow
rate will virtually exclude obstruction, and such
patients might be treated by any of the various nonsurgical treatments currently available and discussed
in detail elsewhere. Otherwise, these patients might
benefit from simple reassurance and discussion. Those
with a reduced flow rate and a little or no residual
urine can confidently be diagnosed as obstructed and
treated accordingly. Those with a substantial residual
urine volume (250 mL or more on repeated assessment) may have an obstructive component to their
symptoms but are more likely to have impaired
detrusor contractility, and the likelihood of this
increases as the residual urine volume and the patients
age increase, and the likelihood that transurethral
resection of the prostate will help these patients symptomatically or objectively decreases accordingly. There
are various caveats to these generalizations, but at least
they form a basis for considering the problems in clinical practice.
REFERENCES
1. Lowsley O. The development of the human prostate
gland with reference to the development of other structures at the neck of the urinary bladder. Am J Anat 1912;
13:299346.
2. McNeal JE. The zonal anatomy of the prostate. Prostate
1981; 2:3549.
3. McNeal JE. The prostate gland: morphology and pathology. Monogr Urol 1983; 4:36.
4. McNeal JE. The prostate gland: morphology and pathobiology. Monogr Urol 1988; 9:34.
5. McNeal JE. Normal histology of the prostate. Am J Surg
Pathol 1988; 12:619633.
6. Marberger M. Drug insight: 5-alpha-reductase inhibitors
for the treatment of benign prostatic hyperplasia. Nat Clin
Pract Urol 2006; 3(9):495503.
7. Wilson J, Griffin J, Russell D. Steroid five alpha reductase
(II) deficiency. Endocr Rev 1993; 14:577593.
8. Schweikert H, Totzauer P, Rohr H, et al. Correlated biochemical and stereological studies on testosterone metabolism in the stromal and epithelial compartment of human
benign prostatic hyperplasia. J Urol 1985; 134:403407.
9. Camps JL, Chang SM, Hsu TC, et al. Fibroblast-mediated
acceleration of human epithelial tumour growth in vivo.
Proc Natl Acad Sci U S A 1990; 87:7579.
10. Cunha GR, Battle E, Young P, et al. Role of epithelial
mesenchymal interactions in the differentiation and spatial organisation of visceral smooth muscle. Epithelial Cell
Biol 1992; 1:7683.
11. Bruchovski N, Lesser B, Van Doorn E, et al. Hormonal
effects on cell proliferation in rat prostate. Vitam Horm
1975; 33:61102.
12. Lee C, Sensibar JA, Dudek SM, et al. Prostatic ductal
system in rats: regional variation in morphological and
functional activities. Biol Reprod 1990; 43:10791086.
13. Begun FP, Story MT, Hopp KA, et al. Regional concentration of basic fibroblast growth factor in normal and
benign hyperplastic human prostates. J Urol 1995;
153:839843.
14. Story MT, Hopp KA, Meier DA, et al. Influence of transforming growth factor beta 1 and other growth factors on
basic fibroblast growth factor level and proliferation of
cultured human prostate-derived fibroblasts. Prostate
1993; 22:183197.
15. Taylor TB, Ramsdell JS. Transforming growth factor alpha
and its receptor are expressed on the epithelium of the rat
prostate gland. Endocrinology 1993; 113:13061308.
16. Gosling JA, Dixon DS. The structure and innervation of
smooth muscle in the wall of the bladder neck and proximal urethra. Br J Urol 1975; 47:549552.
17. Hedlund H, Anderson K, Larsson B. Alpha adrenoceptors
and muscarinic receptors in the isolated human prostate. J
Urol 1985; 134:12911293.
18. Bartsch G, Muller H, Oberholzer M, et al. Light microscopic and stereological analysis of the normal human
prostate and of benign prostatic hyperplasia. J Urol 1979;
122:487491.
19. Shapiro E, Hartanto V, Lepor H. The response to alpha
blockade in benign prostatic hyperplasia is related to the
percent area density of prostate smooth muscle. Prostate
1992; 21:297307.
331
20. Bylund DB, Eikenberg DC, Hieble JP, et al. International

Union of Pharmacology nomenclature of adrenoceptors.
Pharmacol Rev 1994; 46:121136.
21. Forray C, Bard JA, Wetzel JM, et al. The alpha-1 adrenergic receptor that mediates smooth muscle contraction in
the human prostate has the pharmacological properties of
the cloned human alpha-1c subtype. Mol Pharmacol 1994;
45:703709.
22. Lieberman C, Nogimori T, Wu CF, et al. TRH pharmacokinetics and nerve stimulation evoked prostatic fluid
secretion during TRH infusion in the dog. Acta Endocrinol (Copenh) 1989; 120:134142.
23. Gu J, Polak JM, Probert L, et al. Peptidergic innervation of
the human male genital tract. J Urol 1983; 130:386391.
24. Crowe R, Chapple C, Burnstock G. The human prostate
gland: a histochemical and immunohisto chemical study
of neuropeptides, serotonin, dopamine beta hydroxylase
and acetylcholinesterase in autonomic nerves and ganglia. Br J Urol 1991; 68:5361.
25. Brindley G. Pathophysiology of erection and ejaculation. In:
Hendry W, Whitfield H, eds. A Textbook of Genitourinary
Surgery. London: Churchill Livingstone, 1988:10831094.
26. Gil-Vernet J, Alvarez-Vijande R, Gil-Vernet J, Jr. Ejaculation in men: a dynamic endorectal ultrasonographical
study. Br J Urol 1994; 73:442448.
27. Tauber PF, Zaneveld LJD, Propping D, et al. Components
of human split ejaculate. J Reprod Fertil 1975; 43:249267.
28. Kennedy AM, Morrell JM, Siviter RJ, et al. Fertilisation
promoting peptide in reproductive tissues and semen of
the male marmoset. Mol Reprod Dev 1997; 47:113119.
29. Williams-Ashman HG, Cannellakis ZN. Polyamines in
mammalian biology and medicine. Perspect Biol Med
1979; 22:421453.
30. Lilja H. Structure and function of prostatic and seminal
vesicle-secreted proteins involved in the gelatin and liquefaction of human semen. Scand J Clin Lab Invest Suppl
1988; 48:1317.
31. Coffey DS. Physiology of male reproduction: the biochemical and physiology of the prostate and seminal
vesicles. In: Harrison JH, Gittes RF, Perlmutter AD,
et al., eds. Campbells Urology, Vol. 1. 4th ed. Philadelphia:
WB Saunders, 1978:6194.
32. Fair WR, Wehner N. The prostatic antibacterial factor:
identity and significance. In: Marberger H, ed. Prostatic
Disease, Vol. 6. New York: Alan R Liss, 1976:383.
33. Grayhack JT, Kropp KA. Changes with aging in prostatic
fluid: citric acid and phosphatase and lactic dehydrogenase concentration in man. J Urol 1965; 56:611.
34. Kvist U. Reversible inhibition of nuclear chromatin
decondensation (NCD) ability of human spermatozoa
induced by prostatic fluid. Acta Physiol Scand 1980;
109:7378.
35. Kvist U. Importance of spermatozoal zinc as temporary
inhibitor of sperm nuclear chromatin decondensation
ability in man. Acta Physiol Scand 1980; 109:7984.
36. Kvist U, Kjellberg J, Bjorndahl L, et al. Seminal fluid from
men with agenesis of the Wolffian ducts: zinc binding
properties and effects on sperm chromatin stability. Int J
Androl 1990; 13:245252.
37. Greenwald P, Kirmes V, Polan A, et al. Cancer of the prostate
among men with BPH. J Natl Cancer Inst 1974; 355:5356.
38. Carter H, Plantadosi S, Isaacs J. Clinical evidence for and
implications of the multistep development of prostate
cancer. J Urol 1990; 143:742746.
39. Spiro L, Labay G, Orkin L. Prostatic infarction: role in
acute urinary retention. Urology 1974; 3:345347.
40. Cuchi A. The development of detrusor instability in
prostatic obstruction in relation to sequential changes in
voiding dynamics. J Urol 1994; 51:13421344.
332
Feneley et al.
41. Levin RM, Longhurst PA, Monson FC, et al. Effect of

bladder outlet obstruction on the morphology, physiology
and pharmacology of the bladder. Prostate Suppl 1990;
3:926.
42. Glynn R, Campion E, Bouchard G, et al. The development
of benign prostatic hyperplasia among volunteers in the
normative aging study. Am J Epidemiol 1985; 121:7890.
43. Adlercreutz H. Western diet and Western diseases: some
hormonal and biochemical mechanisms and associations.
Scand J Clin Invest Suppl 1990; 50:323.
44. Sanda M, Beatty T, Stautzman R. Genetic susceptibility of
benign prostatic hyperplasia. J Urol 1994; 151:115119.
45. Rosen RC, Giuliano F, Carson CC. Sexual dysfunction and
lower urinary tract symptoms (LUTS) associated with
benign prostatic hyperplasia (BPH). Eur Urol 2005; 47(6):
824837.
46. McVary K. Lower urinary tract symptoms and sexual
dysfunction: epidemiology and pathophysiology. BJU
Int 2006; 97(suppl 2):2328.
47. Yassin A, Saad F, Hoesl CE, et al. Alpha-adrenoceptors
are a common denominator in the pathophysiology of
erectile function and BPH/LUTSimplications for clinical practice. Andrologia 2006; 38(1):112.
48. Giuliano F. Lower urinary tract symptoms and sexual
dysfunction: a common approach. BJU Int 2008; 101
(suppl 3):2226.
49. McNeal JE. Origin and evolution of benign prostatic
enlargement. Investig Urol 1978; 15:340345.
50. Eble JN, Tejada E. Prostatic stromal hyperplasia with
bizarre nuclei. Arch Pathol Lab Med 1991; 115:8789.
51. Leong SS, Vogt PF, Yu GM. Atypical stroma with muscle
hyperplasia of the prostate. Urology 1988; 31:163167.
52. McNeal JE. The pathobiology of nodular hyperplasia. In:
Bostwick DG, ed. Pathology of the Prostate. New York:
53. Franks LM. Benign nodular hyperplasia of the prostate: a
review. Ann R Coll Surg Engl 1954; 14:92106.
54. Partin AW, Oesterling JE, Epstein JI, et al. Influence of age
and endocrine factors on the volume of benign prostatic
hyperplasia. J Urol 1991; 145:405409.
55. Isaacs JT. Antagonistic effect of androgen on prostatic cell
death. Prostate 1984; 5:545547.
56. Sensibar JA, Griswold MD, Sylvester SR, et al. Prostatic
ductal system in rats: regional variation in localisation of
an androgen-repressed gene product, sulphated glycoprotein-2. Endocrinology 1991; 128:20912102.
57. Steiner MS. Review of peptide growth factors in benign
prostatic hyperplasia and urological malignancy. J Urol
1995; 153:10851096.
58. Gregory H, Willshire IR, Kavanagh JP, et al. Urogastrone
epidermal growth factor concentrations in prostatic fluid
of normal individuals and patients with benign prostatic
hypertrophy. Clin Sci 1986; 70:359363.
59. Folkman J, Klagsbrun M, Sasse J, et al. Heparin-binding
angiogenic proteinbasic fibroblast growth factoris
stored within the basement membrane. Am J Pathol
1988; 130:393400.
60. Ku PT, Damore P. Regulation of fibroblast growth factor
(bFGF) gene and protein expression following its release
from sublethally injured endothelial cells. J Cell Biochem
1995; 58:328343.
61. Yan G, Fukaborvi Y, Nikolaropoulos S, et al. Heparinbinding keratinocyte growth factor is a candidate for
stromal to epithelial andromedin. Mol Endocrinol 1992;
6:21232128.
62. Yan G, Fukabori Y, McBride G, et al. Exon switching and
activation of stromal and embryonic fibroblast growth
factor (FGF)FGF-receptor genes in prostate epithelial
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
cells accompany stromal independence and malignancy.

Mol Cell Biol 1993; 13:45134522.
Sporn MB, Roberts AB. TFT-beta: problems and prospects. Cell Regul 1990; 1:875882.
Brogli E, Wu T, Namiki A, et al. Indirect angiogenic
cytokines upregulate VEGF and bFGF gene expression
in vascular smooth muscle cells, whereas hypoxia
upregulates VEGF expression only. Circulation 1994; 90:
649652.
Roberts AB, Sporn MB. Physiological actions and clinical
applications of transforming growth factor-beta (TGF-b).
Growth Factors 1993; 8:19.
Timme TL, Truong LD, Merz VW, et al. Mesenchymal
epithelial interactions and transforming growth factor
beta expression during mouse prostate morphogenesis.
Endocrinology 1994; 134:10391045.
Millan FA, Denhez F, Kondaiah P, et al. Embryonic gene
expression patterns of TGF beta-1, beta-2 and beta-3 suggest different development functions in vivo. Development 1991; 111:131143.
Sporn MB, Roberts AB. Interactions of retinoids and
transforming growth factor beta in regulation of cell differentiation and proliferation. Mol Endocrinol 1991; 5:37.
Katz AE, Benson MC, Wise GJ, et al. Gene activity during
the early phase of androgen-stimulated rat prostatic
regrowth. Cancer Res 1989; 49:58895894.
Cho KS, Kim J, Choi YD, et al. The overlooked cause of
benign prostatic hyperplasia: prostatic urethral angulation. Med Hypotheses 2008; 70(3):532535.
Madersbacher S, Alivizatos G, Nordling J, et al. EAU 2004
guidelines on assessment, therapy and follow-up of men
with lower urinary tract symptoms suggestive of benign
prostatic obstruction (BPH guidelines). Eur Urol 2004; 46(5):
547554.
Gravas S, Tzortzis V, Melekos MD. Translation of benign
prostatic hyperplasia guidelines into clinical practice.
Curr Opin Urol 2008; 18(1):5660.
Rule AD, Lieber MM, Jacobsen SJ. Is benign prostatic
hyperplasia a risk factor for chronic renal failure? J Urol
2005; 173(3):691696.
McConnell JD, Roehrborn CG, Bautista OM, et al. The
long-term effect of doxazosin, finasteride, and combination therapy on the clinical progression of benign prostatic hyperplasia. N Engl J Med 2003; 349(25):23872398.
McVary KT. A review of combination therapy in patients
with benign prostatic hyperplasia. Clin Ther 2007; 29(3):
387398.
Kaplan SA, Roehrborn CG, Rovner ES, et al. Tolterodine
and tamsulosin for treatment of men with lower urinary
tract symptoms and overactive bladder: a randomized
controlled trial. JAMA 2006; 296(19):23192328.
Antunes AA, Srougi M, Coelho RF, et al. Botulinum toxin
for the treatment of lower urinary tract symptoms due to
benign prostatic hyperplasia. Nat Clin Pract Urol 2007; 4(3):
155160.
Andersson KE. LUTS treatment: future treatment options.
Neurourol Urodyn 2007; 26(6 suppl):934947.
Roberts RO, Jacobsen SJ, Jacobson DJ, et al. Longitudinal
changes in peak urinary flow rates in a community-based
cohort. J Urol 2000; 163(1):107113.
Wright EJ, Fang J, Metter EJ, et al. Prostate specific antigen predicts the long-term risk of prostate enlargement:
results from the Baltimore Longitudinal Study of Aging. J
Urol 2002; 167(6):24842487.
Ball AJ, Feneley RCL, Abrams PH. The natural history of
untreated prostatism. Br J Urol 1981; 53:613616.
Gur S, Kadowitz PJ, Hellstrom WJ. Guide to drug therapy
for lower urinary tract symptoms in patients with benign
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
prostatic obstruction: implications for sexual dysfunction.

Drugs 2008; 68(2):209229.
Parsons JK, Kashefi C. Physical activity, benign prostatic
hyperplasia, and lower urinary tract symptoms. Eur Urol
2008; 53(6):12281235.
McVary KT, Rademaker A, Lloyd GL, et al. Autonomic
nervous system overactivity in men with lower urinary
tract symptoms secondary to benign prostatic hyperplasia. J Urol 2005; 174(4 pt 1):13271433.
Jacobsen SJ, Jacobson DJ, Girman CJ, et al. Natural history
of prostatism: risk factors for acute urinary retention. J
Urol 1997; 158(2):481487.
Meigs JB, Barry MJ, Giovannucci E, et al. Incidence rates
and risk factors for acute urinary retention: the health
professionals followup study. J Urol 1999; 162(2):376382.
Roehrborn CG. Alfuzosin 10 mg once daily prevents
overall clinical progression of benign prostatic hyperplasia but not acute urinary retention: results of a 2-year
placebo-controlled study. BJU Int 2006; 97(4):734741.
Crawford ED, Wilson SS, McConnell JD, et al. Baseline
factors as predictors of clinical progression of benign
prostatic hyperplasia in men treated with placebo. J
Urol 2006; 175(4):14221426.
Roehrborn CG, McConnell JD, Lieber M, et al. Serum
prostate-specific antigen concentration is a powerful predictor of acute urinary retention and need for surgery in
men with clinical benign prostatic hyperplasia. PLESS
Study Group. Urology 1999; 53(3):473480.
Kaplan SA, McConnell JD, Roehrborn CG, et al. Combination therapy with doxazosin and finasteride for benign
prostatic hyperplasia in patients with lower urinary tract
symptoms and a baseline total prostate volume of 25 ml
or greater. J Urol 2006; 175(1):217220.
Nickel JC, Roehrborn CG, OLeary MP, et al. The relationship between prostate inflammation and lower urinary tract symptoms: examination of baseline data from
the REDUCE trial. Eur Urol 2008; 54(6):13791384.
Emberton M, Elhilali M, Matzkin H, et al. Symptom
deterioration during treatment and history of AUR are
the strongest predictors for AUR and BPH-related surgery in men with LUTS treated with alfuzosin 10 mg once
daily. Urology 2005; 66(2):316322.
Kefi A, Koseoglu H, Celebi I, et al. Relation between acute
urinary retention, chronic prostatic inflammation and
accompanying elevated prostate-specific antigen. Scand
J Urol Nephrol 2006; 40(2):155160.
Roehrborn CG. Reporting of acute urinary retention in
BPH treatment trials: importance of patient follow-up
after discontinuation and case definitions. Urology 2002;
59(6):811815.
McNeill AS, Rizvi S, Byrne DJ. Prostate size influences the
outcome after presenting with acute urinary retention.
BJU Int 2004; 94(4):559562.
Roehrborn CG, Bruskewitz R, Nickel GC, et al. Urinary
retention in patients with BPH treated with finasteride or
placebo over 4 years. Characterization of patients and
ultimate outcomes. The PLESS Study Group. Eur Urol
2000; 37(5):528536.
McNeill SA, Hargreave TB, Roehrborn CG. Alfuzosin
10 mg once daily in the management of acute urinary
retention: results of a double-blind placebo-controlled
study. Urology 2005; 65(1):8389.
McConnell JD, Bruskewitz R, Walsh P, et al. The effect of
finasteride on the risk of acute urinary retention and the
need for surgical treatment among men with benign
prostatic hyperplasia. Finasteride Long-Term Efficacy
and Safety Study Group. N Engl J Med 1998; 338(9):
557563.
333
99. Mishra VC, Allen DJ, Nicolaou C, et al. Does intraprostatic inflammation have a role in the pathogenesis and
progression of benign prostatic hyperplasia? BJU Int 2007;
100(2):327331.
100. Prins GS, Korach KS. The role of estrogens and estrogen
receptors in normal prostate growth and disease. Steroids
2008; 73(3):233244.
101. Sciarra A, Mariotti G, Salciccia S, Gomez AA, Monti S,
Toscano V, et al. Prostate growth and inflammation.
J Steroid Biochem Mol Biol 2008; 108(35):254260.
102. Roehrborn CG. BPH progression: concept and key learning from MTOPS, ALTESS, COMBAT, and ALF-ONE.
BJU Int 2008; 101(suppl 3):1721.
103. Khastgir J, Khan A, Speakman M. Acute urinary retention: medical management and the identification of risk
factors for prevention. Nat Clin Pract Urol 2007; 4(8):
422431.
104. Parsons JK. Modifiable risk factors for benign prostatic
hyperplasia and lower urinary tract symptoms: new
approaches to old problems. J Urol 2007; 178(2):395401.
105. Mullins C, Lucia MS, Hayward SW, et al. A comprehensive approach toward novel serum biomarkers for benign
prostatic hyperplasia: the MPSA Consortium. J Urol 2008;
179(4):12431256.
106. Boyarski S, Jones G, Paulson DF, et al. A new look at
bladder neck obstruction by the Food and Drug Administration regulators: guidelines for the investigation of
benign prostatic hypertrophy. Trans Am Assoc Genitourin Surg 1977; 68:2932.
107. Madsen PO, Iversen P. A point system for selecting
operative candidates. In: Hinman F Jr., ed. Benign Prostatic Hypertrophy. New York: Springer-Verlag, 1983:
763765.
108. Barry MJ, Fowler FJ, OLeary MP, et al. The American
Urological Association symptom index for benign prostatic hyperplasia. J Urol 1992; 148:15491557.
109. McConnell JD, Barry MJ, Bruskewitz RC, et al. Benign
prostatic hyperplasia: diagnosis and treatment. In: Clinical Practice Guidelines, No. 8. Rockville: U.S. Department
of Health and Human Services1994:99103.
110. Emberton M, Neal DE, Black N, et al. The effect of
prostatectomy on symptom severity and quality of life.
Br J Urol 1996; 77:233247.
111. Tubaro A, Ogden C, de la Rosette J, et al. The prediction
of clinical outcome from thermotherapy by pressure flow
study. Results of a European multicentre study. World J
Urol 1994; 12:352356.
112. Neal DE, Styles RA, Powell PH, et al. Relationships
between detrusor function and residual urine in men
undergoing prostatectomy. Br J Urol 1987; 60:560566.
113. George NJR, Feneley RCL, Roberts JBM. Identification of
the poor risk patient with prostatism and detrusor failure.
Br J Urol 1986; 58:290295.
114. Griffiths DJ. Urodynamics: the Mechanics and Hydrodynamics of the Lower Urinary Tract. Medical Physics
Handbook 4. Bristol: Adam Hilger, 1980.
115. Griffiths DJ. Urethral resistance to flow: the urethral
resistance relation. Urol Int 1975; 30:28.
116. Abrams PH, Griffiths DJ. The assessment of prostatic
obstruction from urodynamic measurements and from
residual urine. Br J Urol 1979; 51:129134.
117. Van Mastrigt R, Rollema HJ. Urethral resistance and
urinary bladder contractility before and after transurethral resection as determined by the computer program
CLIM. Neurourol Urodyn 1988; 7:226230.
118. Schafer W. Principles and clinical application of advanced
urodynamic analysis of voiding function. Urol Clin North
Am 1990; 17:553566.
334
Feneley et al.
119. Jorgensen JB, Jensen KME, Morgensen P. Age-related

variation in urinary flow variables and flow cure patterns
in elderly males. Br J Urol 1992; 69:265271.
120. Olssen CA, Goluboft ET, Chang DT, et al. Urodynamics
and the etiology of post-prostatectomy incontinence. J
Urol 1994; 151:20632065.
121. Lepor H, Machi G. Comparison of AUA symptom index
in unselected males and females between fifty-five and
seventy-nine years of age. Urology 1993; 42:3640.
122. Barry MJ, Cockett ATK, Holtgrewe HL, et al. Relationship
of symptoms of prostatism to commonly used physiological and anatomical measures of the severity of benign
prostatic hyperplasia. J Urol 1993; 150:351356.
123. Abrams P. In support of pressure flow studies for evaluating men with lower urinary tract symptoms. Urology
1994; 44:153155.
124. Roberts RO, Rhodes T, Panser LA. Natural history of
prostatism: worry and embarrassment of firm urinary
symptoms and health care-seeking behaviour. Urology
1994; 43:621628.
125. Malkowiez SB, Wein AJ, Elbadawi A, et al. Acute biochemical and functional alterations in the partially obstructed
rabbit urinary bladder. J Urol 1986; 136:13241329.
126. Susset JG. The effect of aging and prostatic obstruction on
detrusor morphology and function. In: Hinman C Jr., ed.
Benign Prostatic Hypertrophy. New York: Springer-Verlag,
1985:653665.
127. Luutzeyer W, Hannapel J, Schafer W. Sequential events in
prostatic obstruction. In: Hinman C Jr., ed. Benign Prostatic Hypertrophy. New York: Springer-Verlag, 1985:
693700.
128. Schafer W, Rubben H, Noppeney R, et al. Obstructed and
unobstructed prostatic obstruction. A plea for urodynamic
objectivation of bladder outflow obstruction in benign
prostatic hyperplasia. World J Urol 1989; 6:198203.
129. Dixon JS, Gilpin CJ, Gilpin SA, et al. Sequential morphologic changes in the pig detrusor in response to chronic
partial urethral obstruction. Br J Urol 1989; 64:385390.
130. Coolsaet BRLA, Blok C. Detrusor properties related to
prostatism. Neurourol Urodyn 1986; 5:435441.
20
Genetic and Biological Alterations in Cancer
Vincent J. Gnanapragasam
Uro-oncology Group, Department of Oncology, Hutchinson MRC Research Centre,
University of Cambridge, Cambridge, U.K.
INTRODUCTION
The hallmark of the cancer cell is its ability to escape
from the normal regulatory mechanisms that control
cellular biology. Thousands of research studies have
been published on the changes that occur in the cancer
cell that might facilitate this process. While some
changes are generic to many tumor types, others are
specific. Changes are known to occur at all levels of
the cell and can influence events from the DNA code
right up to its posttranslational modification into a
functional protein. The serial accumulation of genetic
and biological changes is thought to drive the transition from normal to dysplastic and eventually malignant cells. Collectively genes that increase the potential
for tumorigenesis at any level of cell function are
termed oncogenes while those whose loss facilitates
cancer are termed tumor suppressor genes. In this
chapter, only the principles of genetic and biological
changes will be discussed with salient examples in
urological cancers (Fig. 1). A full list of all the known
alterations would take many books to cover.
GENETIC CHANGES IN CANCER

The basis of all function in the cell is the data contained in the genome. It is clear that the coding
sequences, which are transcribed into mRNA represent only a part of the known human genome. The
non-coding DNA is now thought to contain putative
regulatory elements whose function in controlling
gene expression is not fully understood. Indeed, in
recent studies important predisposition loci for prostate cancer have been identified in so called gene poor
regions and their function in the cell is unclear (1).
Within the genome the genetic alterations that can
occur and predispose to cancer can include rearrangements and polymorphisms, which alter the normal
coding reading sequence.
Chromosomal Rearrangements
Chromosomal rearrangements include translocations,
deletions, duplications and inversions. Each of this
can be caused by breakage of DNA in the genome
followed by rejoining of the broken ends to produce a

new chromosomal structure. There are two types of
rearrangements. In balanced rearrangements the chromosomal gene order is changed but there is no duplication or removal of any of the DNA. Examples of this
include inversion and reciprocal translocations. In
imbalanced rearrangements there is a change in the
total number of genes in the affected chromosome
such as a loss of one copy or the addition of a copy.
Examples of these are deletions and duplications. In
bladder cancer the most well known is the loss of
chromosome 9 (either the p or q arm) (2). In clear cell
renal caner the loss of 3p by deletion or translocation
can be found in over two thirds of tumors (3). In
papillary renal cancers trisomies of chromosome
7,12,16,17 and 20 have all been shown to occur albeit
at low frequencies. In testicular germ cell tumors the
only consistent abnormality is iso-chromosome 12p
where an extra short arm is present. In prostate cancer
the most common genetic event to date are gene
fusions involving the 50 untranslated region of the
androgen dependent TMPRSS2 gene and the ETS
family of transcription factors. These changes are
though to occur in over 50% of clinical prostate
cancers (4).
DNA Polymorphisms
Polymorphisms are sequence variations in the DNA
code such as nucleotide substitutions, deletions, insertions and duplications. These alterations may or may
not result in changes in protein phenotype and function. Polymorphisms in genes that code for enzymes,
for example, have been associated with cancer risk
and progression. In bladder cancer polymorphisms in
the N-acetyltransferase 1 and 2 enzymes result in
amino acid changes that reduce their efficiency in
de-activating potential bladder carcinogens (5). Similar polymorphic changes in DNA repair genes can
change the cells ability to repair damaged DNA. The
accumulation of damaged DNA can then lead to genetic
instability and carcinogenesis. Examples of this include
polymorphisms in the XP (xeroderma pigmentosa) and
XRCC (X-ray repair cross-complementary) genes that
have been detected in bladder and prostate cancer (6).
336
Gnanapragasam
of high-grade cancers (8). Amplification of the androgen receptor (AR) gene is thought to occur in castration resistant prostate cancer and may be an important
mechanism by which prostate cancer cells respond to
very low levels of androgens (9).
EPIGENETIC CHANGES IN DNA

DNA Methylation
Figure 1 Summary chart of genetic and biological alterations

that can occur in cancer cells.
Loss of Heterozygosity
LOH describes the loss of the normal two heterogeneous allele configurations at a gene locus. This can
occur through chromosomal rearrangements or polymorphisms. LOH can be detected by using microsatellite analysis (with known simple sequence
repeats) or by looking for single nucleotide polymorphisms (SNP). This latter technique can now be done
using very large SNP arrays that can screen for thousands of polymorphisms simultaneously. A good
example of LOH in a urological cancer is the Wilms
tumor. The WT 1 gene is located on chromosome
11p13. Loss of 1 allele at 11p13 has been found in
over 40% of tumors suggesting that inactivation of this
gene is a critical step in Wilms tumor development
(7). In bladder cancer LOH at 3p, 8p, 13q and 17p have
been identified in carcinoma in situ as well as muscle
invasive tumors but not in superficial disease highlighting the differences in these tumor types (8).
Gene Copy Number Changes

Increases or loss of expression of a particular molecule
can be due to changes at any level. At the most basic is
an alteration in the number of gene copies available
for transcription. Gene copy number changes are well
described in many malignancies. The c-myc gene for
instance which encodes a nuclear transcription factor
is known to be amplified by an increase in copy
number in many tumors including pancreatic, breast
and brain cancer. In bladder cancer the ERBB2 receptor gene is known to be amplified at 17q in 10% to 14%
Methylation results in gene silencing by the process of

adding a methyl group on a cytosine in the context of
a Cytosine-Guanine rich DNA sequence (called CpG
islands). CpG islands are frequently found in the
promoter region of genes and are usually unmethylated to allow efficient gene transcription. Methylation
at CpG islands therefore results in transcriptional
silencing by preventing the binding of transcription
factors. Methylation also enhances chromatin folding
and condensation thus making the gene promoter
inaccessible to the transcription machinery. Both
abnormal hypermethylation and hypo-methylation
can occur in cancer cells. Many tumor suppressor
genes have been shown to be silenced by the mechanism of DNA methylation. Numerous studies have
been performed to investigate methylated targets in
urological cancers. In renal cell cancers a number of
genes have been shown to be inactivated by methylation including VHL and RASS1FA. These events
occur in between 20% and 30% of cancers (10). In
bladder cancer hypermethylation at 5 gene loci including RASS1FA and E Cadherin have been associated
with an increased risk of tumor progression (11).
Analysis of cell free serum DNA in men with bladder
cancer has also suggested that hypermethylation of
the APC gene was associated with a poorer clinical
outcome following radical cystectomy (12). Gene
methylations have also been identified in prostate
cancer. One of the most frequent is hypermethylation
of the GSTPI gene, which is involved in detoxification
by conjugation with glutathione. GSTP1 methylation
has been reported in biopsies of over 70% of prostate
cancers and also in the serum and urine of patients.
These results have prompted its potential use as a
tumor marker for prostate cancer detection (13).
Histone Acetylation
Efficient RNA transcription is dependent on the ability of the transcriptional machinery to gain access to
the tightly packed DNA. In the normal state DNA is
supercoiled around histone proteins which allow the
long DNA strand to be compacted. The combination
of DNA and histones together comprise the chromatin. Histones proteins are subject to posttranslational
modification, which can affect how efficiently the
DNA coiled around them is transcribed. Histones
consist of a globular domain and a charged NH2
terminal tail and it is this section that is the prime
site for protein modification. An important modification is the process of acetylation, which is mediated by
histone acetyl transferases (HAT), while deacetylation
Chapter 20: Genetic and Biological Alterations in Cancer
is mediated by histone deacetylases (HDAC). Acetylation of histone proteins at lysine residues removes
positive charges, and reduces the affinity between
histones and DNA. This then allows easier access of
the transcriptional machinery to gene promoter
regions. Histone acetylation therefore enhances transcription while deacetylation represses it. Upregulation of HDAC proteins has been demonstrated in
disease progression to androgen insensitive prostate
cancer and is associated with a shorter time to biochemical relapse following radical surgery (14,15).
Preclinical studies have shown good efficacy of
HDAC inhibitors in blocking cell proliferation and
inducing growth arrest as well as apoptosis in in
vitro and in vivo models of prostate cancer (16,17).
These promising results have prompted phase I and II
trials into the use of HDAC inhibitors in clinical
cancers.
TRANSCRIPTION FACTORS IN CANCER

The transcriptional machinery binds to the promoter
of genes and initiates the process of gene transcription. This process is facilitated by transcription factors,
which have cognate binding sites at the gene promoter. A complex of more than 20 individual transcription factors can make up the transcriptional
engine together with RNA polymerase II. Upregulation of transcription factors is a principal hallmark of
cancer biology and a major class of oncogenes. Activation of transcription factors is also the final end
point of many signal transduction and stress response
pathways. There are numerous families of transcription factors and considerable functional redundancy
between them. Specific factors however have been
strongly associated with individual urological cancers.
Hypoxia inducible factors (HIF) are transcription factors that function to promote cell survival in hypoxic
conditions by increasing expression of genes involved
in glycolysis as well as new vessel formation. HIF
factors are themselves regulated by the VHL protein,
which targets them for protein degradation. Mutations of VHL in renal cancer are common events
occurring in up to 70% of cases (18,19). This results
in increased stability of HIF transcription factors and
enhanced expression of its target genes.
Nuclear factor k B (NF-kB) is a family of at least
6 transcription factors which have an important role
in regulating cell proliferation and inhibiting apoptosis. They are activated by a range of mechanisms
including interleukin, growth factors and in stress
response. In the inactivate state they are maintained
at the cell membrane by inhibitors [inhibitors of k B
(IkBs)]. In bladder cancer the level of NF-kB expression is significantly associated with histological grade
and tumor stage (20). In renal cell cancers use of a NFkB inhibitor has shown promising results in blocking
tumor growth by inducing cell apoptosis (21). In
prostate cancer cell lines androgen-independent prostate tumor cells demonstrate constitutive activation
of NF-kB while IkB expression levels were low. In
contrast, androgen-sensitive prostate cell lines
337
expressed low levels of NF-kB (22). Early phase clinical studies with an NF-kB inhibitor (bortezomib) in
combination with docetaxel in men with prostate
cancer have shown early promise in reducing PSA
levels (23).
The ETS family is one of the biggest groups of
transcription factors and is involved in many key
cellular processes including differentiation, proliferation, migration and angiogenesis. Gene fusions of
members of the ETS family have been reported in
many tumor types with the best know being the
fusion of the FLI-1 transcription factor to the EWS
gene in Ewings sarcoma (24). In 2005 Tomlins et al.
published a seminal paper describing the fusion of the
androgen regulated TMPRSS2 gene with the ERG and
ETV-1 ETS members in a large number of prostate
cancers (4). Since then numerous variants of gene
fusions between TMPPRSS2 and ETS family members
have been reported (25,26). These fusions have been
found in between 40% to 60% of primary clinical
prostate cancers. This finding provides a strong link
between the androgen receptor and a transcription
factor in the initiation and progression of prostate
cancer. The TMPRS22: ETS fusion has also been proposed as a putative prognostic marker of a poorer
outcome following radical prostatectomy and a urinebased assay has shown promise as a potential cancer
detection tool (27,28).
ALTERNATIVE SPLICING IN CANCER CELLS

The first step in transcription is the production of
pre-mRNA which contains both intron and exon
sequences. Splicing occurs when introns are removed
and exons are joined to produce the final mRNA for
protein translation. Alternative (as opposed to constitutive) splicing allows pre-mRNA to be processed
into different final mRNA sequences and hence proteins which may have different functional effects.
The process of splicing is performed in the nucleus
by the spliceosome (a large multi-protein complex)
and is regulated by enhancers and silencers, which
identify the exon-intron boundary. It has become
recently evident that the process of splicing can be
significantly altered in the cancer cell. This may be
the result of a mutation in the enhancers and repressors of splicing or due to changes in the splicing
machinery itself (29). In bladder cancer, for example,
different FGFR3 transcript has been observed in
benign as opposed to cancer cells as a result of
alternate splicing at the pre-mRNA stage (30). Alternative splicing of fibroblast growth factors (FGF)
receptors has also been reported in prostate cancer
cells and clinical tissue (31). High throughput techniques have now been introduced to look for global
splicing changes in cancer ad other diseases. Li et al.
have recently determined mRNA isoform specific
signatures for prostate cancers using a large custom
made splicing array (32). The significance of splicing
changes and their exploitation for therapy is still at
an early stage but is an area of active interest among
cancer biologists.
338
Gnanapragasam
CHANGES IN PROTEIN TRANSLATION

IN CANCER
Translation is the process by which the information
encoded in the mRNA is used to produce the protein.
Alterations in the expression or function of the translational machinery can lead to marked changes in
protein synthesis, which ultimately is the functional
product of the gene. This process occurs at the ribosome where the translation initiation complex binds to
mRNA and reads the tri-nucleotide sequence. The
initiator complex consists of transfer RNA (tRNA),
40 and 60S ribosomal units and translation factors,
which assemble the complex at the 50 end of the
mRNA sequence. Key translation factors in this complex include the eIF family, which has five members
with each having a distinct role in translation. eIF4e in
particular has a crucial role in translational regulation.
Expression of eIF4e is regulated through many different signal transduction pathways including ERK and
p38. elF4e is also regulated by binding proteins such
as 4E-BP1, which inactivate its function. Phosphorylation of binding proteins reduces their interaction
with eIF4e and results in increased protein translation.
One important mechanism is through the mammalian
target of rapamycin (mTOR) protein kinase. Rapamycin which inhibits mTOR is used in transplant
immuno-suppression to block T cell activation. It has
been recently advocated as a promising new anticancer therapy (33). eIF4E itself has been shown to
be overexpressed in many cancers including prostate
cancer (34). Another mechanism of regulation is by the
phosphorylation of the ribosomal 40S protein by S6
kinase (S6K). Phosphorylation of S6K is regulated by
among others, the PI3K and mTOR pathways. Translation factors are therefore common targets of signal
transduction pathways, which are themselves dysregulated in cancer (see below). Finally protein translation in cancer can also be altered by inherent
mutations in the transcribed RNA. This can result in
a mutated protein or abnormal protein production. A
classic example of this is the BRCA1 protein. In malignant cells the transcript has a longer 50 untranslated
region compared with BRCA 1 in normal mammary
tissue. The modified transcript is less efficient during
translation and results in a reduction in BRCA1 protein expression.
MicroRNA
miRNA are small single stranded RNA molecules of
about 21 to 23 nucleotides. These do not code for
proteins and are members of the large family of noncoding RNA which have regulatory functions in the
cell. miRNA are a relatively new discovery in gene
regulation but are a fast growing family of molecules.
They function as regulators of translation by annealing to mRNA and inhibiting the translational
machinery. This is achieved through miRNA incorporation into a RNA induced silencing complex (35).
This complex then localizes to mRNA with a complimentary strand and cleaves it. This effectively blocks
protein translation either with or without associated
degradation of the mRNA. To date over 500 hundred

miRNAs have been discovered and this number is
likely to grow with increasing understanding of their
importance and function. miRNAs have been implicated in the regulation of many cellular processes
and their role in cancer is unsurprisingly is the subject of intense research. Urological cancers have all
been shown to harbour abnormal expression of miRNAs (36,37). Gottardo et al. have shown that 4 miRNAs (mir-28, miR-185, miR-27, and let-7f2) are
significantly increased in renal cell carcinoma. In
bladder cancer a panel of 10 miRNAs were shown
to be increased in comparison with normal controls.
In prostate cancer a microarray approach detected
51 miRNAs that were altered between benign and
malignant tissue with 37 downregulated (resulting in
increased target mRNA translation) and 14 upregulated (reduced target mRNA translation) (38). The
authors further proposed that patterns of miRNA
expression could distinguish between androgen
dependent and independent prostate cancers. The
field of cancer relevant miRNA (called OncomiRs)
is growing rapidly and is very likely to lead to new
therapeutic targets in the near future.
PEPTIDE GROWTH FACTORS IN CANCER

Peptide growth factors play an important role in
carcinogenesis and are currently the best exploited
in terms of new drug development. Ligands bind to
their cognate receptors at the cell membrane and
initiate a cascade of downstream signalling that is in
general promitogenic and antiapoptotic. The key
growth factors implicated in cancer include fibroblast
growth factors (FGF), epidermal growth factor (EGF),
insulin-like growth factor (IGF), vascular endothelial
growth factor (VEGF), platelet derived growth factor
(PDGF), and transforming growth factor (TGF). In
cancer cells, alterations at every level of growth factor
regulation have been found. In prostate cancer both
growth factor ligands and receptors have been shown
to be upregulated in carcinogenesis and tumor progression (3941). In the FGF axis for instance, ligands
including FGF1, 2, 8, and 17 have been shown to be
increased in high-grade and high-stage disease (42
44). FGF10 has been recently implicated as an important stromal-epithelial initiator of prostate tumor
development (45). FGF receptor (FGFR)1 is known to
be overexpressed as an early event in clinical prostate
cancer, and induced expression is causative in the
development and progression of tumor in a mouse
model (46,47). FGFR4 in contrast is preferentially
overexpressed in more advanced tumors.
In bladder cancer the EGF system has emerged
as an important target for therapy. EGF receptor (also
known as Her 1) is over-expressed in a large number
of tumors and expression is associated with tumor
grade and stage (48). Another member of the EGF
receptor family is the Her 2 receptor, which has no
known ligand (termed an orphan receptor) but functions by binding as a homodimer to other EGF receptors. Her 2 has been found to be overexpressed in
breast cancer and targeting this receptor (by the Her

2 antibody, Herceptin) has emerged as a promising
new therapy. Crucially it is primarily effective in
tumors that are known to be Her 2 positive. Her 2 is
also known to be over-expressed in bladder cancer but
to date the benefits of Herceptin in this tumor have
not been encouraging (49,50). Activating mutations of
FGFR3 have emerged as a common feature in many
bladder cancers. The principal mutation occurs in the
extracellular domain, which allows ligand independent dimerization and constitutive activation of the
receptor. Intriguingly these mutations are most commonly found in low-grade superficial tumors and are
much less apparent in muscle invasive disease. The
prognostic significance of FGFR mutations as well as
its therapeutic value is currently the subject of much
research (51).
In renal cancer VEGF has emerged as the most
important growth factor axis in the terms of novel
therapy. Up-regulation of VEGF is a key feature in
many renal cell cancers. Bevacizumab, an antibody
against the VEGF-A isoform has shown clinical efficacy
in advanced renal cancer and prolongs time to disease
progression in combination with interferon (52). The
multiple growth factors that are active in cancers have
made it difficult to gain therapeutic benefit from
blocking just one axis. This has led to the concept of
using inhibitors against multiple targets. An example
of this is the multi tyrosine kinase inhibitor sorafenib.
This drug is an inhibitor of Raf kinase, PDGF, VEGF
receptors 2 and 3 and the c Kit receptor. Initial studies
have shown that sorafenib can prolong progressionfree survival in patients with advanced clear-cell
renal-cell carcinoma in whom previous therapy has
failed (53). Sorafenib is currently being evaluated in a
multinational randomized trial coordinated by the
Medical Research Council in the U.K.
SIGNAL TRANSDUCTION CHANGES IN CANCER

The effect of extracellular stimuli in promoting cell
growth and differentiation is mediated through intracellular signalling pathways. These include among
others the mitogenic activated protein kinases
(MAPK), phosphoinositol 3 kinase (PI3K), and phospholipase C (PLC) pathways. The PI3K pathway is
involved in promoting cell survival and anti-apoptosis.
One of the key genes lost in prostate cancer, Phosphatase and tensin homologue deleted on chromosome 10
(PTEN) is an inhibitor of PI3K signalling. PTEN loss
has been shown to directly contribute to an aggressive
and invasive tumor phenotype through enhanced PI3
signalling (54). A principal target of PI3K is Akt,
which is turn is known to activate the serine threonine
kinase mTOR, which as previously discussed is an
important regulator of protein translation. The MAPK
family consist of 3 members; extracellular related
kinase (ERK), p38 and Jun N-terminal kinase (JNK)
also known as stress-activated protein kinase (SAPK).
The ERK pathway in particular has an important role
in tumor biology and is the principle mediator of the
mitogenic effect of many growth factors. In cancer
339
cells this pathway can be switched on inappropriately

by a constitutively active upstream component or by
continuous or enhanced exposure to the stimulus. A
good example of the former is the effect of the Ras
oncogenes. Ras is a small GTPase protein that is
membrane bound and an upstream component of
the ERK cascade. In the normal state it is activated
by guanine exchange factors, which are targets of
mitogenic stimuli (e.g., phosphorylated growth factor
receptors). Mutations in Ras can result in a constitutively active protein in the absence of a stimulus and
this induces sustained activation of ERK. Ras mutations have been detected in between 10% to 20% of
prostate cancers and 13% of bladder cancers (55,56).
Signalling pathways are however more commonly
hyper-activated through increased activity of exogenous stimuli, for example, increased growth factor
ligands and receptors as previously discussed. EGF
mediated ERK activation for instance is responsible
for reducing the cytotoxic effect of radiation and
increasing clonogenic survival and repopulation following irradiation (57). Inhibition of this pathway has
been shown to radiosensitize tumor cells and enhance
cell kill. Recently, a separate layer of endogenous
regulators of ERK pathway has been reported. These
act as negative feedback inhibitors of ERK activity and
serve a homeostatic function in normal cells to control
the intensity and duration of signalling. Members of
this group include Sprouty, similar expression to FGF
(Sef) and Spred. Recent work has shown that these
inhibitors are downregulated in urological cancers
and can result in unattenuated enhanced signalling
(58,59).
CELL CYCLE REGULATION CHANGES

IN CANCER
The normal cell cycle consists of mitosis (nuclear and
cytoplasmic division), G1 (First growth phase),
S (DNA synthesis and chromosome replication) and
G2 (second growth phase). When the cell is quiescent
it is said to enter the G0 phase (out of the cell cycle).
This process is controlled by cyclins in complex with
cyclin-dependent kinases (CDK) as well as other proteins, which regulates the progression through the cell
cycle. The retinoblastoma protein (Rb) functions as an
inhibitor of cell cycling by blocking progression into
the S phase. Its normal function is to prevent replication of damaged DNA. Loss of Rb therefore results in
increased progression through the cell cycle. In bladder cancer Rb deletions or mutations have been
detected in up to 30% of muscle invasive tumors (8).
In prostate cancer loss of Rb has been suggested as an
independent predictor of disease specific survival
(54). Other studies have suggested that levels of
Cyclin D1 (which phosphorylates and reduces Rb
activity) are also expressed at higher levels in prostate
cancer (60). p53 is a well-characterized tumor suppressor and has many known functions including
DNA repair, initiating apoptosis after DNA damage
and cell cycle progression. Its function in regulating
the cell cycle is achieved through transcriptional
340
Gnanapragasam
regulation of p21 (also known as CDK inhibitor 1A).

p21 inhibits cell cycling by forming a complex with
CDK 2 and preventing progression into the S phase of
the cycle. Mutations in the p53 gene results in
increased expression of a non-functional protein. As
a result of this there is reduced p21 expression and
increased progression through the cell cycle. In bladder cancer high levels of p53 have been positively
correlated with a poorer disease specific outcome for
muscle invasive disease (8). p21 loss has also been
reported as a frequent finding in aggressive bladder
cancers (61). Tumors with a coordinated dysregulation of different cell cycle regulators (such as combined loss of p53 and Rb) seem to exhibit even poorer
survival outcomes (62,63).
APOPTOSIS IN CANCER CELLS

There are two basic apoptotic pathways in the cell. In
the mitochondrial (or intrinsic) pathway cellular damage stimulates migration of the proapoptotic proteins
Bad and Bax to the mitochondrial surface where it
binds and inhibits antiapoptotic Bcl 2. This allows the
release of cytochrome C, which is the first step in the
assembly of the apoptosome complex. This complex
then activates caspase 9, which in turn triggers the
activation of other caspase and ultimately leads to
destruction of cellular proteins as well as DNA degradation. The second mechanism is through the activation of death receptors (extrinsic pathway). These
include the Fas, tumor necrosis factor (TNF) and
tumor necrosis factorrelated apoptosis inducing
ligand (TRAIL) receptors. Following binding of their
respective ligands these receptors mediate the assembly of a death-inducting signal complex (DISC), which
activates caspase 8 and in turn triggers the caspase
cascade resulting in cell death. Bcl 2 has been shown
to be reduced in androgen dependent prostate cancer
but is over-expressed in androgen-independent disease (64). These and similar findings in other cancers
has led the development of targeted therapy against
Bcl 2. Oblimersen is a Bcl 2 antisense oligonucleotide
and a potent inhibitor of its expression. Studies in
preclinical models have shown that cotreatment of
prostate cancer cells can enhance the cytotoxic effects
of chemotherapy (65). Phase II studies have also
shown promising results in combining oblimersen
with docetaxel for men with hormone refractory disease in prolonging survival (66). Bcl 2 has also been
tested as a target in renal cell cancers. Downregulation
in in vitro cultured renal cancer cell has been shown to
enhance sensitivity to Fas receptor-mediated apoptosis (67). A recent phase I/II trial of oblimersen as an
adjunct to interferon therapy in renal cell cancer however has failed to show any significant clinical benefit
(68). In the extrinsic pathway loss of FAS expression
has been demonstrated in bladder and renal tumors
(69,70). Decoy receptors are also known to exist for the
Fas and TRAIL axis. These are produced by tumor
tissue and block ligands from binding to the membrane bound receptors. High levels of Fas decoy
receptors have been reported in the serum of patients
with renal and prostate cancers (71,72). Osteoprotegerin can act as a decoy for TRAIL and has been
implicated in bladder and prostate cancer. In bladder
cancer low levels of Osteoprotegerin have been linked
to a favorable outcome in muscle invasive bladder
cancer while in prostate cancer high levels have been
associated with disease progression (73,74). There are
as yet no clinical trials exploiting the death receptors
in cancers. Of note, TRAIL is already an exploited
target as BCG immunotherapy is thought to act in part
by activating these receptors and inducing immune
mediated cell death (75).
CONCLUSION
Alteration can occur at every level in the cell processes
in cancer biology and it has only been possible here to
highlight important changes in urological cancers.
Common themes occur in different malignancies but
some alterations may be more relevant depending on
the tumor type. The goal for the next generation of
cancer drugs will be to define the key alterations that
are relevant for a specific tumor type and target these
using novel therapies. This is already occurring with
novel VEGF targeted therapy in renal cancers and
early clinical studies of HDAC inhibitors in prostate
cancers. It is highly likely however that a combination
of drugs directed at different targets will be most
likely to have the optimal therapeutic effect. Moreover
drug combinations may well be tailored for a specific
patient on the basis of the unique set of genetic and
biological alterations identified in their particular
tumor.
REFERENCES
1. Eeles RA, Kote-Jarai Z, Giles GG, et al. Multiple newly
identified loci associated with prostate cancer susceptibility. Nat Genet 2008; 40(3):316321.
2. Fadl-Elmula I. Chromosomal changes in uroepithelial carcinomas. Cell Chromosome 2005; 4:1.
3. Kardas I, Mrozek K, Babinska M, et al. Cytogenetic and
molecular findings in 75 clear cell renal cell carcinomas.
Oncol Rep 2005; 13(5):949956.
4. Tomlins SA, Mehra R, Rhodes DR, et al. Whole transcriptome amplification for gene expression profiling and
development of molecular archives. Neoplasia 2006;
8(2):153162.
5. Hung RJ, Boffetta P, Brennan P, et al. GST, NAT, SULT1A1,
CYP1B1 genetic polymorphisms, interactions with environmental exposures and bladder cancer risk in a high-risk
population. Int J Cancer 2004; 110:598604.
6. Franekova M, Halasova E, Bukovska E, et al. Gene polymorphisms in bladder cancer. Urol Oncol 2008; 26(1):18.
7. Huff V. Wilms tumor genetics. Am J Med Genet 1998;
79(4):260267.
8. Knowles MA. What we could do now: molecular pathology of bladder cancer. Mol Pathol 2001; 54:215221.
9. Gnanapragasam VJ, Robson CN, Leung HY, et al. Androgen receptor signalling in the prostate. BJU Int 2000;
86(9):10011013.
10. Morris MR, Hesson LB, Wagner KJ, et al. Multigene
methylation analysis of Wilms tumor and adult renal
cell carcinoma. Oncogene 2003; 22(43):67946801.

11. Yates DR, Rehman I, Abbod MF, et al. Promoter hypermethylation identifies progression risk in bladder cancer.
Clin Cancer Res 2007; 13(7):20462053.
12. Ellinger J, El Kassem N, Heukamp LC, et al. Hypermethylation of cell-free serum DNA indicates worse outcome in
patients with bladder cancer. J Urol 2008; 79(1):346352.
13. Harden SV, Guo Z, Epstein JI, et al. Quantitative GSTP1
methylation clearly distinguishes benign prostatic tissue
and limited prostate adenocarcinoma. J Urol 2003;
169:11381142.
14. Weichert W, Roske A, Gekeler V, et al. Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and
HDAC2 expression is associated with shorter PSA relapse
time after radical prostatectomy. Br J Cancer 2008;
98(3):604610.
15. Halkidou K, Gaughan L, Cook S, et al. Upregulation and
nuclear recruitment of HDAC1 in hormone refractory
prostate cancer. Prostate 2004; 59(2):177189.
16. Qian DZ, Wei YF, Wang X, et al. Antitumor activity of the
histone deacetylase inhibitor MS-275 in prostate cancer
models. Prostate 2007; 67(11):11821193.
17. Arts J, Angibaud P, Marien A, et al. R306465 is a novel
potent inhibitor of class I histone deacetylases with broadspectrum antitumoral activity against solid and haematological malignancies. Br J Cancer 2007; 97(10):13441353.
18. Wiesener MS, Munchenhagen PM, Berger I, et al. Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell
renal carcinomas. Cancer Res 2001; 61(13):52155222.
19. Kaelin WG Jr. The von Hippel-Lindau tumor suppressor
protein and clear cell renal carcinoma. Clin Cancer Res
2007; 13(2 pt 2):680s684s.
20. Levidou G, Saetta AA, Korkolopoulou P, et al. Clinical
significance of nuclear factor (NF)-kappaB levels in urothelial carcinoma of the urinary bladder. Virchows Arch
2008; 452(3):295304.
21. Sourbier C, Danilin S, Lindner V, et al. Targeting the
nuclear factor-kappaB rescue pathway has promising
future in human renal cell carcinoma therapy. Cancer
Res 2007; 67(24):1166811676.
22. Palayoor ST, Youmell MY, Calderwood SK, et al. Constitutive activation of IkappaB kinase alpha and NF-kappaB
in prostate cancer cells is inhibited by ibuprofen. Oncogene
1999; 18(51):73897394.
23. Price N, Dreicer R. Phase I/II trial of bortezomib plus
docetaxel in patients with advanced androgen-independent prostate cancer. Clin Prostate Cancer 2004; 3(3):
141143.
24. Sorensen PH, Lessnick SL, Lopez-Terrada D, et al. A second Ewings sarcoma translocation, t(21;22), fuses the EWS
gene to another ETS-family transcription factor, ERG. Nat
Genet 1994; 6(2):146151.
25. Tomlins SA, Laxman B, Dhanasekaran SM, et al. Distinct
classes of chromosomal rearrangements create oncogenic
ETS gene fusions in prostate cancer. Nature 2007;
448(7153):595599.
26. Helgeson BE, Tomlins SA, Shah N, et al. Characterization
of TMPRSS2:ETV5 and SLC45A3:ETV5 gene fusions in
prostate cancer. Cancer Res 2008; 68(1):7380.
27. Attard G, Clark J, Ambroisine L, et al. Transatlantic Prostate Group. Duplication of the fusion of TMPRSS2 to ERG
sequences identifies fatal human prostate cancer. Oncogene 2008; 27(3):253263.
28. Nam RK, Sugar L, Yang W, et al. Expression of the
TMPRSS2:ERG fusion gene predicts cancer recurrence
after surgery for localised prostate cancer. Br J Cancer
2007; 97(12):16901695.
29. Faustino NA, Cooper TA. Pre-mRNA splicing and human
disease. Genes Dev 2003; 17:419437.
341
30. Li HR, Wang-Rodriguez J, Nair TM, et al. Two-dimensional transcriptome profiling: identification of messenger
RNA isoform signatures in prostate cancer from archived
paraffin-embedded cancer specimens. Cancer Res 2006;
66(8):40794088.
31. Tomlinson DC, LHote CG, Kennedy W, et al. Alternative
splicing of fibroblast growth factor receptor 3 produces a
secreted isoform that inhibits fibroblast growth factorinduced proliferation and is repressed in urothelial carcinoma cell lines. Cancer Res 2005; 65(22):1044110449.
32. Kwabi-Addo B, Ropiquet F, Giri D, et al. Alternative
splicing of fibroblast growth factor receptors in human
33. Tolcher AW. Novel therapeutic molecular targets for prostate cancer: the mTOR signaling pathway and epidermal
growth factor receptor. J Urol 2004; 171(2 pt 2):S41S43.
34. De Benedetti A, Graff JR. eIF-4E expression and its role in
malignancies and metastases. Oncogene 2004; 23(18):
31893199.
35. Cho WC. OncomiRs: the discovery and progress of microRNAs
in cancers. Mol Cancer 2007; 6:60.
36. Gottardo F, Liu CG, Ferracin M, et al. Micro-RNA profiling
in kidney and bladder cancers. Urol Oncol 2007; 25(5):
387392.
37. Ozen M, Creighton CJ, Ozdemir M, et al. Widespread
deregulation of microRNA expression in human prostate
cancer. Oncogene 2008; 27(12):17881793.
38. Porkka KP, Pfeiffer MJ, Waltering KK, et al. MicroRNA expression profiling in prostate cancer. Cancer Res 2007; 67(13):
61306135.
39. Mimeault M, Pommery N, Henichart JP. New advances on
prostate carcinogenesis and therapies: involvement of
EGF-EGFR transduction system. Growth Factors 2003;
21(1):114.
40. Furstenberger G, Senn HJ. Insulin-like growth factors and
cancer. Lancet Oncol. 2002; 3(5):298302.
41. Kwabi-Addo B, Ozen M, Ittmann M. The role of fibroblast
growth factors and their receptors in prostate cancer.
Endocr Relat Cancer 2004; 11(4):709724.
42. Dorkin TJ, Robinson MC, Marsh C, et al. aFGF immunoreactivity in prostate cancer and its co-localization with
bFGF and FGF8. J Pathol 1999; 189(4):564569.
43. Gnanapragasam VJ, Robinson MC, Marsh C, et al. FGF8
isoform b expression in human prostate cancer. Br J Cancer
2003; 88(9):14321438.
44. Heer R, Douglas D, Mathers ME, et al. Fibroblast growth
factor 17 is over-expressed in human prostate cancer.
J Pathol 2004; 204(5):578586.
45. Memarzadeh S, Xin L, Mulholland DJ, et al. Enhanced
paracrine FGF10 expression promotes formation of multifocal prostate adenocarcinoma and an increase in epithelial
androgen receptor. Cancer Cell. 2007; 12(6):572585.
46. Sahadevan K, Darby S, Leung HY, et al. Selective overexpression of fibroblast growth factor receptors 1 and 4 in
clinical prostate cancer. J Pathol 2007; 213(1):8290.
47. Acevedo VD, Gangula RD, Freeman KW, et al. Inducible
FGFR-1 activation leads to irreversible prostate adenocarcinoma and an epithelial-to-mesenchymal transition. Cancer Cell 2007; 12(6):559571.
48. Sriplakich S, Jahnson S, Karlsson MG. Epidermal growth
factor receptor expression: predictive value for the outcome after cystectomy for bladder cancer? BJU Int 1999;
83(4):498503.
49. Latif Z, Watters AD, Dunn I, et al. HER2/neu gene amplification and protein overexpression in G3 pT2 transitional
cell carcinoma of the bladder: a role for anti-HER2 therapy? Eur J Cancer 2004; 40(1):5663.
50. Hussain MH, MacVicar GR, Petrylak DP, et al. Trastuzumab,
paclitaxel, carboplatin, and gemcitabine in advanced human
342
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
Gnanapragasam
epidermal growth factor receptor-2/neu-positive urothelial
carcinoma: results of a multicenter phase II National Cancer
Institute trial. J Clin Oncol 2007; 25(16):22182224.
Black PC, Agarwal PK, Dinney CP. Targeted therapies in
bladder canceran update. Urol Oncol 2007; 25(5):433438.
Escudier B, Pluzanska A, Koralewski P, et al. AVOREN
Trial investigators. Bevacizumab plus interferon alfa-2a for
treatment of metastatic renal cell carcinoma: a randomised,
double-blind phase III trial. Lancet 2007; 370(9605):
21032111.
Escudier B, Eisen T, Stadler WM, et al. TARGET Study
Group. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007; 356(2):125134.
van der Poel HG. Molecular markers in the diagnosis of
prostate cancer. Crit Rev Oncol Hematol 2007; 61(2):
104139.
Anwar K, Nakakuki K, Shiraishi T, et al. Presence of ras
oncogene mutations and human papillomavirus DNA in
human prostate carcinomas. Cancer Res 1992; 52:
59915996.
Jebar AH, Hurst CD, Tomlinson DC, et al. FGFR3 and Ras
gene mutations are mutually exclusive genetic events in
urothelial cell carcinoma. Oncogene 2005; 24(33):
52185225.
Dent P, Yacoub A, Fisher PB, et al. MAPK pathways in
radiation responses. Oncogene 2003; 22(37):58855896.
McKie AB, Douglas DA, Olijslagers S, et al. Epigenetic
inactivation of the human sprouty2 (hSPRY2) homologue
in prostate cancer. Oncogene 2005; 24(13):21662174.
Darby S, Sahadevan K, Khan MM, et al. Loss of Sef (similar
expression to FGF) expression is associated with high
grade and metastatic prostate cancer. Oncogene 2006;
25(29):41224127.
Han EK, Lim JT, Arber N, et al. Cyclin D1 expression in
human prostate carcinoma cell lines and primary tumors.
Prostate 1998; 35(2):95101.
Stein JP, Ginsberg DA, Grossfeld GD, et al. Effect of
p21WAF1/CIP1 expression on tumor progression in bladder cancer. J Natl Cancer Inst 1998; 90:10721079.
Grossman HB, Liebert M, Antelo M, et al. p53 and RB
expression predict progression in T1 bladder cancer. Clin
Cancer Res 1998; 4:829834.
Cordon-Cardo C, Zhang ZF, Dalbagni G, et al. Cooperative
effects of p53 and pRB alterations in primary superficial
bladder tumors. Cancer Res 1997; 57:12171221.
64. McDonnell TJ, Troncoso P, Brisbay SM, et al. Expression of

the protooncogene bcl-2 in the prostate and its association
with emergence of androgen-independent prostate cancer.
Cancer Res 1992; 52(24):69406944.
65. Leonetti C, Biroccio A, DAngelo C, et al. Therapeutic
integration of c-myc and bcl-2 antisense molecules with
docetaxel in a preclinical model of hormone-refractory
66. Tolcher AW, Chi K, Kuhn J, et al. A phase II, pharmacokinetic, and biological correlative study of oblimersen
sodium and docetaxel in patients with hormone-refractory
prostate cancer. Clin Cancer Res 2005; 11(10):38543861.
67. Kelly JD, Dai J, Eschwege P, et al. Downregulation of Bcl-2
sensitises interferon-resistant renal cancer cells to Fas. Br J
Cancer 2004; 91(1):164170.
68. Margolin K, Synold TW, Lara P, et al. Oblimersen and
alpha-interferon in metastatic renal cancer: a phase II study
of the California Cancer Consortium. J Cancer Res Clin
Oncol 2007; 133(10):705711.
69. Sejima T, Isoyama T, Miyagawa I. Alteration of apoptotic
regulatory molecules expression during carcinogenesis
and tumor progression of renal cell carcinoma. Int J Urol
2003; 10:476479.
70. Maas S, Warskulat U, Steinhoff C, et al. Decreased Fas
expression in advanced-stage bladder cancer is not related
to p53 status. Urology 2004; 63:392396.
71. Furuya Y, Nagakawa O, Fuse H. Prognostic significance of
serum soluble Fas level and its change during regression
and progression of advanced prostate cancer. Endocr J
2003; 50:629633.
72. Kimura M, Tomita Y, Imai T, et al. Significance of serumsoluble CD95 (Fas/APO-1) on prognosis in renal cell cancer patients. Br J Cancer 1999; 80:16481651.
73. Mizutani Y, Matsubara H, Yamamoto K, et al. Prognostic
significance of serum osteoprotegerin levels in patients
with bladder carcinoma. Cancer 2004; 101:17941802.
74. Eaton CL, Wells JM, Holen I, et al. Serum osteoprotegerin
(OPG) levels are associated with disease progression and
response to androgen ablation in patients with prostate
cancer. Prostate 2004; 59:304310.
75. Ludwig AT, Moore JM, Luo Y, et al. Tumor necrosis factorrelated apoptosis inducing ligand a novel mechanism
for Bacillus Calmette-Guerin-induced antitumor activity.
Cancer Res 2004; 64:33863394.
21
General Biology of Cancer and Metastasis
David E. Neal
CANCER
An understanding of cancer is important to the urologist, not only because it is common, but also because
its study provides insight into normal and abnormal
cellular function. One in five adults dies of cancer
(Tables 1 and 2), and about 30% to 50% of common,
solid epithelial tumors are advanced and incurable
when first detected clinically. So far as urological
tumors are concerned, prostate, bladder, and kidney
cancers are common, and while testis cancer is rare, it
is important because, even when advanced, it is
frequently curable and because it occurs in young
men with an otherwise full life expectancy.
It is thought that individual cancers arise from a
single cell following a set of genetically determined
events. The evidence for this monoclonal origin lies in
findings, first, that all cells in a tumor often contain
specific point mutations in genes, which would be
unlikely to arise by chance in several cells, and,
second, that in women, in whom one X chromosome
is inactivated in a mosaic and apparently random
fashion throughout the body, one particular X chromosome is inactivated throughout the tumor.
CHEMICAL CARCINOGENESIS
Many different chemicals have been shown to be
carcinogenic (Table 3). The best known historical
example is that of scrotal cancer, in which Percival
Pott demonstrated through epidemiological observations that boys who had been employed as chimney
sweeps were likely to develop this disease as adults.
He hypothesized that chemicals in soot caused the
problem. Tobacco use (mainly cigarette smoking)
was later shown to be clearly associated with cancers
of the mouth, larynx, trachea, lung, kidney, and
bladder.
Evidence of a chemical cause for bladder cancer
was found by Rehn in 1894, when he recorded a series
of such tumors occurring in workers in aniline dye
factories. Hueper was subsequently able to show that
2-naphthylamine was carcinogenic in dogs, and further investigation demonstrated that a variety of
chemicals may be carcinogenic.
Common factors in most human cancers
include damage to several genes (rather than one)
resulting from point mutations, insertions, or deletions in certain genes known as oncogenes or tumor
suppressor genes. Even recently, chemical causes of
urothelial cancers have been shown to be important.
Balkan nephropathy exists in certain defined parts of
the Balkans. A herb found in these places, and also
used widely in Chinese medicine, contains aristocholic acid and this chemical is nephrotoxic and
carcinogenic.
Occupations that have been reported to have a
significantly excess risk of bladder cancer are shown
in Table 4.
Historically, chemicals have been classified into
those that produced mutations in DNA on first application (initiators), but that themselves were usually
insufficient to cause cancer unless there was further
exposure to the initiator or unless initiator application
was followed by a promoter. Promoters are compounds that do not cause cancer, however, often
they are applied, but they will cause the development
of cancer when there has been previous application of
an initiator. Promoters include chemicals such as
phorbol esters, which stimulate protein kinase C
(PKC). PKC phosphorylates several proteins on serine
and threonine residues and activates MAP kinase,
which is also activated by the ras pathway (see Chapter 22 for Ras).
Most carcinogens are genotoxic and cause damage to DNA. It is thought that there is also a class of
nongenotoxic carcinogens that, in mice, cause peroxisome proliferation, and that may activate agents that
interfere with the cell cycle or apoptosis. It is uncertain
whether this mechanism is active in man.
GENETIC POLYMORPHISMS
Many genotoxic carcinogens are inactive and require
to be converted into active agents by acetylation or
hydroxylation (Fig. 1). Mammalian cells have developed a complex system for detoxifying external biologically active chemicals known as xenobiotics. These
enzymes, which also detoxify drugs and carcinogens,
include the following:
l
Hydroxylation by means of the cytochrome P450

system (CYP), which is found on the microsomal
fraction of cells (particularly in the liver)
344
Neal
Table 1 Common Causes of Human Cancer

l
l
l
l
l
l
Ionizing radiation (hematopoietic cells, bone)

Sunlight (malignant melanoma, squamous cell carcinoma of the skin)
Familial genetic causes (certain types of breast, colon, kidney, and prostate cancer)
Familial cancer predispositions due to altered activity in detoxifying or activating enzymes such as N-acetyltransferase 2 and
glutathione transferase M (bladder, lung, and colon cancer)
Chemicals from occupations and smoking (bladder, larynx, and lung cancer)
Chronic inflammation (squamous cell carcinoma arising in a chronic ulcer, schistosomal bladder cancer, tumors in ulcerative colitis,
and adenocarcinoma arising in Barrett esophagus)
Viral infection: DNA viruses: penile and cervical cancer (papovavirus), liver cancer (Hep B and C), Epstein-Barr virus (Burkitts
lymphoma, nasopharyngeal cancer); RNA viruses: human T-cell leukemia (HTLV-1), Kaposis sarcoma (HIV-1)
Table 2 Cancer Incidence and Death in the United States

Type of cancer
New cases per year
Percentage
Deaths per year
Percentage
1,170,000
29,800
152,000
24,000
27,700
170,000
183,000
32,000
185,000
22,000
13,500
31,000
52,300
21,200
93,000
18,250
5,100
8,000
100
3
13
2
2
15
16
3
14
2
1
3
4
528,300
7,700
57,000
13,600
25,000
149,000
46,300
6,800
35,000
13,300
4,400
5,700
9,900
8,300
50,000
12,350
210
4,150
100
1
11
3
5
28
9
1
7
3
1
1
2
Total
Mouth and pharynx
Colon and rectum
Stomach
Pancreas
Lung
Breast
Malignant melanoma
Prostate
Ovary
Cervix
Uterus
Bladder
Kidney
Hematopoietic
CNS
Testis
Sarcomas
8
2
1
9
2
1
Table 3 Compounds Associated with Bladder Cancer

2-Naphthylamine
4-Aminobiphenyl
Benzidine
Chlornaphazine
4-Chloro-O-toluidine
O-toluidine
4,49-Methylene bis (2-choloranliline)

Methylene dianiline
Benzidine-derived azo dyes
Table 4 Occupations Associated with Bladder Cancer

Textile workers
Dye workers
Tyre rubber and cable workers
Petrol workers
Leather workers
Shoe manufacturers and cleaners
Painters
Hairdressers
Glutathione transferases, which couple glutathione to water-insoluble chemicals and which

are classified into families (a, u, m, p)
N-acetyltransferase pathway (NAT-1 and NAT-2)
Some individuals are more at risk than others

after exposure to a carcinogen because of genetic polymorphisms. This arises because each individual carries
two alleles for each gene, but within a population there
may be many alleles. Some combinations will lead to
an individuals having enzymes that may be more
or less active than those found in other individuals.
Polymorphic genes of interest in bladder cancer include
N-acetyltransferase types 1 and 2 (NAT-1, NAT-2),
glutathione transferase M1 and p (GSTM1 and GSTp),
Lorry drivers
Drill press operators
Chemical workers
Rodent exterminators and sewage
workers
and several cytochrome P450s (CYP2D6: debrisoquine

hydroxylase; and CYP1A1). Individuals who have high
levels of NAT-2 or GSTM1 may be less likely to develop
cancer after exposure to smoking because they can
detoxify the mutagen, whereas those with high levels
of CYP2D6 may be more likely to develop cancer
because they convert inactive to active forms of the
mutagen. Similarly, some individuals may be generally
resistant to carcinoma formation because they have
polymorphisms that produce protective compounds. It
should be noted that some enzymes will have a dual
effect; for instance, NAT-2 in the liver will detoxify some
carcinogens, but may activate others. This explains the
paradox that fast acetylators are less likely to develop
bladder cancer, but may be more likely to develop colon
Chapter 21: General Biology of Cancer and Metastasis
345
Figure 1 Metabolic activation of a carcinogen. Many chemical carcinogens have to be activated by a metabolic transformation before
they will cause mutations by reacting with DNA. The compound illustrated here is aflatoxin B1, a toxin made from a mold (Aspergillus
flavus-oryzae) that grows on grain and peanuts stored under humid tropical conditions. It is thought to be a contributory cause of liver
cancer in the tropics and is associated with specific mutations of the p53 gene.
cancer. NAT-1 is found in high levels in the bladder

urothelium, and nontoxic metabolites produced by
NAT-2 in the liver and excreted in the urine can be
taken into the urothelial cells and converted by NAT-1
into active carcinogens that produce genotoxic damage
in the bladder cell.
Cancer Is a Multistep Process

Human cancer requires several mutations to occur
before a tumor develops clinically. One reason for
supporting this view is that cancer incidence increases
markedly with age, and this would be unlikely if
tumors simply arose from a single gene mutation.
Instead, tumorigenesis would be more likely to
occur randomly throughout life. Recent molecular
biological studies have supported the view that cancer
arises only when there has been an accumulation of
several genetic events.
Cancer Genes
Several tumors have a strong genetic component,
including retinoblastoma (Rb gene), Wilms tumor
(WT gene), familial forms of prostate cancer (unknown
gene, possibly on chromosome 1), breast cancer
(BRCA1 and BRCA2 genes and mutations in p53 in
Li-Fraumeni families), familial adenomatosis polyposis (APC gene), hereditary nonpolyposis colon cancer
(HNPCC) (MSH1 gene), and von HippelLindau disease (VHL gene). These tumors have proven to be
instructive because in most cases they appeared to be
inherited clinically in a dominant fashion. Nevertheless, biochemically, these genes act in a recessive fashion because, although the disease is caused by an
inherited mutation in one allele of a tumor suppressor
gene, cancer develops only when the other normal
allele becomes deleted or mutated by chance (Fig. 2).
Why tumors in such patients appear only in certain
Figure 2 The genetic mechanisms underlying retinoblastoma. In the hereditary form, all cells in the body lack one of the normal two
functional copies of a tumor suppressor gene, and tumors occur where the remaining copy is lost or inactivated by a somatic mutation. In
the nonhereditary form, all cells initially contain two functional copies of the gene, and the tumor arises because both copies are lost or
inactivated through the coincidence of two somatic mutations in one cell.
346
Neal
tissues is uncertain when tumor suppressor genes are

inactivated in most cells. Methylation in the prompter
of such genes can also, through epigenetic mechanisms, functionally inactivate such genes. The impact
of oncogenes and tumor suppressor genes is dealt with
in other chapters in this book.
Other tumors can arise in patients whose DNA
repair machinery is deficient (xeroderma pigmentosa,
ataxia telangiectasia, and HNPCC).
DNA Repair Genes

Mismatch Repair
Patients with HNPCC have tumors in which there are

a large number of microsatellites in the genome of the
tumor. Microsatellites are short (two to four nucleotides; e.g., CACA on one strand and GTGT on the
other), noncoding, tandem repeats of DNA, which are
found throughout the genome and which show pronounced polymorphism. Abnormalities in microsatellites have been found in Huntingtons chorea, the
fragile X syndrome, and spinobulbar atrophy, in
which, over several generations, there is a progressive
expansion of the nucleotide repeats in the genes of
interest. This leads to disease, but it is found that with
each succeeding generation there is an increased
severity of the disease because the microsatellites
become longer. This phenomenon is called genetic
anticipation. In HNPCC, microsatellite instability is
thought to be the result of failure to correct replication
errors that occur during cell division. This failure is
caused by hereditary defects in the enzymes responsible for the repair of these replication mismatch
errors. These enzymes include MSH2, MLH1, GTBP,
and PMS1.
Nucleotide Excision Repair
Abnormal nucleotide insertions can occur during

DNA replication and are excised by mechanisms
that are deficient in xeroderma pigmentosa.
CELLULAR CHARACTERISTICS OF CANCER

Not all features of cancer cells are caused directly by
abnormal genes; some abnormalities are due to failure
to regulate genetically normal genes (Table 5). These
epigenetic events include upregulation of enzymes
that can dissolve the basement membrane (such as
cathepsins and plasminogen activators) and may predispose the cell to metastasize; changes in expression
of cell surface molecules (such as HLA antigens) that
may allow the cancer cell to escape detection by the
bodys immunosurveillance; loss of molecules responsible for intercellular adherence, leading the cell to be
more likely to metastasize (such as E-cadherin); upregulation of growth factors or their receptors, allowing
the cell to become self-reliant (such as epidermal
growth factor and its receptor, EGFr); and upregulation of molecules that detoxify anticancer drugs (such
as the multitumor-suppressor geneMDR-1).
Table 5 Characteristics of the Transformed Cell

Plasma membrane abnormalities
Increased transmembrane transport (as for glucose and
calcium)
Excessive endocytosis and blebbing
Increased mobility
Adhesion molecules
Decreased adhesion
Failure of organization of actin into stress fibers
Reduced fibronectin expression
Increased expression of enzymes such as cathepsin,
plasminogen activators, and metalloproteinases
Growth
Growth to high density (lack of density-dependent inhibition)
Decreased requirement for added growth factors
Anchorage-independent growth
Immortal
Can cause animal tumors
ANGIOGENESIS
Angiogenesis is the process of new blood-vessel formation, which is found in a number of diseases,
including cancer, diabetic retinopathy, and inflammatory arthritides. The initiation of angiogenesis in
tumors is dependent on the coordinated expression
of several factors (Table 6). Angiogenesis requires
dilatation of vessels, breakdown of perivascular
stroma, migration and proliferation of endothelial
cells, and canalization of endothelial buds. Potent
inhibitors of angiogenesis include angiostatin (which,
paradoxically, is secreted by some primary tumors)
and thrombospondin (which is involved in the binding
of macrophages to apoptotic cells, and which is also
upregulated by normal p53). A potent stimulus of
angiogenesis is hypoxia, which upregulates the expression of vascular endothelial growth factor (VEGF).
This occurs through the upregulation of the intermediate hypoxia-inducible factors (HIF) (Fig. 3).
VEGF is a specific mitogen for endothelial cells.
It exists in four forms (121-, 165-, 189-, and 206-aa
peptides) and is secreted by a number of cell types.
VEGFr, which is a tyrosine kinase, is found on endothelial cells in two forms (Flt-1 and Flk-1).
RENAL CELL CANCER

A good example of how modern molecular biology
has helped to unravel the causes and different phenotypes of cancer is kidney cancer. Renal cancer is not
Table 6 Peptides Associated with Angiogenesis
Vascular endothelial growth factor (VEGF)
FGFs
PDGF
Tumor necrosis factor a
Angiogenin
EGF
TGF-a and TGF-b
Platelet-derived endothelial cell growth factor or thymidine
phosphorylase
347
Figure 3 Factors controlling angiogenesis. Hypoxia induces transcription of hypoxia-inducible factors (HIF) that act on HIF-responsive
elements in the promoter region of genes such as VEGF.
a single disease; it is comprised of several different

types of cancer, each with a different histology, a
different clinical course, and different genetic changes.
For these different tumor types, hereditary, familial,
and sporadic forms exist. Most of our current knowledge was originally derived from studies on familial
forms of the disease, but subsequent studies of sporadic cancers found similar changes. Recent developments in genetics and molecular biology have led to
an increasing knowledge of the origin and biology of
renal cell carcinoma (RCC).
Chromosome 3 Changes: the von HippelLindau

Tumor Suppressor
Clear cell RCCs are characterized by nonhomologous
chromatid exchange, leading to loss of genetic material from the short (p) arm of chromosome 3 and gains
of material from the long (q) arm of chromosome 5.
Loss of heterozygosity studies in sporadic RCCs identified three common regions of allelic loss: 3p1214,
3p2122, and 3p2526.
Loss of 3p and gain of 5q implicate genes within
these regions as being involved in clear cell RCC
tumorigenesis. Tumor suppressor genes are likely to
be found in areas of chromosomal loss (3p) and
oncogenes found in areas of chromosomal gain (5q).
The only gene definitively shown to be involved in the
development of such tumors is the VHL, located at
3p25, a documented tumor suppressor. Renal cell

cancers found in VHL follow Knudsons theory of
the two hit model of carcinogenesis. The VHL gene is
inactivated in patients with VHL and in almost 100%
of sporadic clear cell RCC.
The inactivation of VHL is accomplished by
mutation, deletion, or methylation. Mutations of the
VHL gene seen in sporadic RCC differ from those seen
in RCC associated with VHL disease. In sporadic
RCC, 45% of the mutations are clustered in the second
exon, although abnormalities are seen in all three
exons. Large deletions are not observed, and 48% of
the mutations are microdeletions or insertions, resulting in frame shifts of the protein-coding sequence.
VHL encodes two different protein isoforms.
Both isoforms are capable of suppressing RCC growth
in vivo and will be referred as pVHL. pVHL functions
as a component of an ubiquitin ligase complex (VBC
complex) and its best understood function relates to
its ability to target specific proteins for destruction.
pVHL forms stable complexes that contain other proteins called elongin B, elongin C, Cul2, and Rbx1.
These complexes are capable of directing the covalent
attachment of polyubiquitin tails to specific proteins,
which serves as a signal for such proteins to be
degraded by the proteasome. Several pVHL targets
have been identified, including the members of the
HIF-a family (HIF-1a, HIF-2a, and HIF-3a). HIF-1a
and HIF-2a, when bound to a HIF-b member (such as
348
Neal
Table 7 Main Protein Targets of the VHL Protein

Target protein
Function
Hypoxia-inducible factors (HIF-1 and HIF-2)

Vascular endothelial growth factor (VEGF)
Glucose transporter protein (Glut-1)
Platelet-derived growth factor (PDGF-b)
Transforming growth factor (TGF-a, TGF-b)
Intracellular fibronectin
Erythropoietin
Carbonic anhydrase 9 and 12
Chemokine receptor (CXCR4)
Protein transcription
Angiogenesis
Glucose transport
Angiogenesis, mitogenesis
Epithelial proliferation, mitogenesis
Epithelial proliferation
Erythropoiesis
Extracellular pH control
Metastasis
HIF-1b, also called ARNT), form a sequence-specific,

DNA-binding transcription factor called HIF. Ordinarily, the HIF-a members are highly unstable except
under low-oxygen conditions. In the presence of oxygen, these proteins become hydroxylated on conserved prolyl residues in a reaction catalyzed by
members of the EGLN family. pVHL recognizes the
hydroxylated HIF-a species and orchestrates their
destruction through ubiquitination (Table 7).
Since the 1990s, evidence has continued to accumulate demonstrating that hypoxia is a common consequence of the rapid growth of many solid tumors,
including tumors such as kidney cancer. Studies of
renal cell biology also provide evidence that hypoxia
is an important regulator of a network of gene expression with the ability to both stimulate and inhibit
individual genes and to effect their expression on
both posttranscriptional and posttranslational levels.
When there is normoxia, the VHL complex targets HIF
and degrades it. In cells that lack pVHL, or when
oxygen is limiting, the complex cannot degrade HIF;
HIF-1a and HIF-2a are upregulated through the phosphatidylinositol 4,5-bisphosphate-AKT-mammalian
target of rapamycin pathway (P13K-AKT-mTOR)
and Ras/Raf/MEK/mitogen-activated protein kinase
(MAPK) pathway and they then form the transcription factor HIF. HIF is free to accumulate intracellularly and activates the transcription of a cadre of genes
that encode proteins essential to cancer cell functions
under hypoxic conditions.
Included among these genes are genes that control glucose uptake and metabolism [such as the Glut1
(glucose transporter protein) and various glycolytic
enzymes], extracellular pH (such as carbonic anhydrase
IX), angiogenesis (such as VEGF), erythropoiesis (such
as erythropoietin), and mitogenesis [such as transforming growth factor-a (TGF-a) and platelet-derived
growth factor-b (PDGF-b)]. Signaling occurs through
various cell surface receptors, including EGFR, HER2,
VEGFR, and type I insulin-like growth factor receptor.
These considerations explain the frequent overproduction of HIF, as well as its downstream targets, in RCC.
pVHL binds to other cellular proteins, some of which
may also be polyubiquitinated or in some other way
modulated by pVHL. These proteins include the atypical protein kinase C family members, Sp1, heteronuclear ribonucleoprotein hnRNP A2, specific RNA Pol II
subunits (Rpb1 and hsRBP7), Jade-1, Vdu1/2, fibronectin, proteins associated with microtubules, nuclear factor kappa B, and metalloproteinases (MMPs).
Although our knowledge of the functions of

pVHL is still incomplete, studies conducted thus far
highlight its role as an inhibitor of HIF, and dysregulation of HIF is likely to contribute to the development
of RCC.
METASTASIS
This is the process by which cells from the primary
tumor enter the circulation and seed elsewhere in the
body to produce secondary tumors. It is the usual
cause of death in cancer. It is a series of linked
processes, but it is thought that only some subclones
of cells are capable of spreading. Most cells entering
the circulation do not survive. There are intriguing
features; some tumors have preferential sites for
metastasisthe prostate or bone, for example. Experimental data have shown that subclones of cells,
produced from the same tumor, may have very different capacities for metastasis (Fig. 4). The following
steps are thought to be important in metastasis:
l
l
l
l
l
l
l
l
Angiogenesis
Invasion and degradation
Altered adhesion
Migration and circulation
Tumor cell attachment
Invasion and degradation
Migration
Colonization of the secondary site
Degradation of Extracellular Matrix Barriers

Metastasis requires cancer cells to penetrate extracellular matrix barriers. This involves the proteolytic
degradation of extracellular matrix components.
MMPs are a family of zinc-dependent endopeptidases
(gelatinases) known to degrade extracellular matrix
components. Their activity is inhibited by tissue inhibitors of MMPs (TIMPs). High levels of TIMP-2 immunoreactivity, detected both in tumor cells and in
stroma, have been reported to be associated with
cancer-specific death. This is somewhat surprising,
given that TIMPs inhibit the degradation of the extracellular matrix. However, recent studies have shown
that, under certain circumstances, TIMP-2 can also
activate MMP-2. Other enzymes include the plasminogen activators. These enzymes may be expressed by
host stromal around a tumor merely as an indicator of
tissue remodeling (Fig. 5). Hence, they have not
shown consistent usefulness as prognostic markers.
349
Figure 4 Factors involved in metastasis.
Figure 5 Secretion of proteases (and their

inhibitors) by tumor and stroma.
Altered Cell Adhesion

Decreased intercellular adhesiveness favors detachment of tumor cells, and this may play a role in
regression to metastatic disease. At least four families
of cell adhesion molecules are thought to be involved
in cell-cell adhesion (cadherins, selectins, immunoglobulins, and integrins). The most widely studied have
been E-cadherin, a cell surface glycoprotein restricted
to epithelial tissue, which is involved in calciumdependent homotypic cell-cell adhesion, and the vascular cell adhesion molecules, E-selectin, vascular cell
adhesion molecule-1 (VCAM-1), and intercellular
adhesion molecule-1 (ICAM-1). The regulation of the
expression of E-cadherin on tumor cells is unclear.
Posttranslational modification of the protein product
may affect function. It is known that three molecules
350
Neal
(a, b, and c catenins) form bridges between the cytoplasmic tail of E-cadherin and the cytoskeleton, a
bridging that may be necessary for E-cadherin to
function normally.
Migration and Circulation

Tumor cells have increased motility that allows them
to insinuate between the components of the extracellular matrix. They then reach the basement membranes
of capillaries or lymph vessels, which they lyse to enter
the circulation. This process is likely to be controlled in
part by the secretion of cytokines, chemokines, and
growth factors by tumor or stromal cells.
Attachment and Colonization

Most tumor cells do not survive. From experimental
studies, we know that less than 1% survive and less
than 0.1% will grow and proliferate. We know that
growth in animal models is far more complex than
that produced by the same cells in tissue culture.
Attachment of tumor cells is controlled by various
factors. Shear forces at the bifurcation of vessels

increase the risk that tumor cells will become attached.
Certain tumors may induce the formation of distant
fibrin clots that makes it more likely that tumor cells
will stick. Tumor cells that possess the capacity to
invade and migrate are more likely to spread in secondary sites. Capillaries, lymph vessels, and veins are
more susceptible than arteries to invasion by tumor
cells, as they lack protease inhibitors.
The site of metastasis can be typical. For
instance, bone stroma is likely to be the site of secondary spread from prostate cancer. This occurs
because of the secretion of particular growth factors
that encourage the survival of prostate cells. Other
sites, such as the spleen, are very unlikely to be
invaded by tumor cells. The immunological factors
in tumor control are discussed elsewhere in the book.
ACKNOWLEDGMENT
Artwork reproduced from Alberts B, Johnson A,
Lewis J, et al., eds. Molecular Biology of the Cell. 4th
ed. New York: Garland Science, 2002.
22
Tumor Suppressor Genes and Oncogenes
David E. Neal
ONCOGENES
G Proteins and Ras
What have sometimes been referred to as recessive

oncogenes are now more commonly called tumor
suppressor genes. Many DNA viruses produce proteins that directly perturb the function of tumor suppressor genes (e.g., large T antigen of SV40 binds p53
and Rb; E6 and E7 of the papilloma virus bind p53 and
Rb; the E1A protein of adenovirus binds Rb).
The term oncogene was originally used to
describe those genes carried by viruses (most of
them being retroviruses), which were found to be
the cause of transmissible forms of cancer in animals.
These retroviral oncogenes (v-oncs) were closely
related to normal host cellular genes called protooncogenes. Cellular oncogenes found in cancer
(c-oncs) were shown to be normal proto-oncogenes
that had become activated by a variety of mechanisms, including point mutations, deletion, insertional
mutagenesis, translocation, and overexpressionoften
associated with gene amplification. The initial techniques involved in identifying these genes (transfection
of tumor DNA into NIH 3T3 fibroblasts) tended to
select dominantly acting transforming genes, which
are now generally known as oncogenes.
As the number of known oncogenes increased
and their functions became known, they were classified into families defined by their normal cellular
counterparts. These include growth factors and their
receptors, nuclear regulators of gene transcription and
DNA replication, and signal transduction proteins,
which couple cell surface receptors and the nucleus.
More than 60 oncogenes have now been identified.
The types of oncogenes and some of their functions
are shown in Figure 1. Their transfection into cells
causes transformation (Table 1).
A cellular proto-oncogene can be converted to an
oncogene in the following ways:
Proteins that bind and hydrolyze guanidine triphosphate (GTP) are found commonly in the cell and play a
crucial role in cell signaling. When GTP (which is
found in large excess in the cell compared with GDP)
is bound to such G proteins, the protein becomes
activated and initiates a cascade of events. However,
GTP-binding proteins also rapidly hydrolyze GTP to
GDP, thereby rendering themselves inactive. Other
proteins can control the activity of GTP binding to
such proteins, including one called GTPase-activating
protein (GAP), which binds to G proteins, inducing
them to convert GTP to GDP and become inactive.
Another protein called guanine nucleotidereleasing
protein (GNRP) binds to G proteins, inducing them
to release GDP and bind GTP (which is present in large
amounts in the normal cell), converting it into an active
form. G proteins are classified into the following:
l
l
Insertion of a section of DNA into the promoter region

of the gene (insertional mutagenesis, e.g., Wnt-1)
Deletion [conversion of epidermal growth factor
receptor (EGFr) to v-erbB-2]
Translocation or chromosomal rearrangement
(Philadelphia chromosome) (Fig. 2)
Point mutation (H-ras)
Gene amplification (EGFr in brain tumors)
l
l
Monomeric G proteins (such as ras, rac, and rho)

Heterotrimeric G proteins (often directly coupling
cell surface receptors to intracellular receptors, such
as adrenoreceptors or muscarinic acetylcholine
receptors to adenyl cyclase and phospholipase C,
respectively)
Ras Family
The human ras gene family consists of three closely

related genes: H-ras, K-ras, and N-ras, which encode
21-kDa signal transduction G proteins involved in the
transmission of signals from cell surface receptors. Ras
proteins belong to the monomeric family of G proteins
as distinct from the trimeric family that couple cell
surface receptors (such as adrenergic receptors) to
intracellular events. Other monomeric G proteins
include the rho and rac family, which, like ras proteins, are involved in the relay of signals from the cell
membrane. Activation of ras by mutation occurs as a
result of a single amino acid change as a consequence
of single nucleotide mutations. Activated H-ras has
been found in bladder carcinomas, Ki-ras in lung and
colon carcinomas, and N-ras in hematological malignancies. H-ras is a monomeric G protein. In its
mutated (H-ras) form, it contains a single-point mutation, leading to the conversion of a glycine to a valine
352
Neal
Figure 1 The activities and cellular locations of the products of the main classes of known proto-oncogenes. Some representative
proto-oncogenes in each class are indicated in brackets.
Table 1 Some Oncogenes

Oncogene
Proto-oncogene
Abl
Tyrosine kinase
erbB
Tyrosine kinase (EGFr, c-erbB2, etc.)
Fes
Tyrosine kinase
Fms
Tyrosine kinase (M-CSM factor)
Fos
AP-1 protein
Jun
AP-1 protein
raf (MAP kinase kinase kinase) Serine kinase activated by ras
Myc
Transcription factor
H-ras
GTP (G)-binding protein
K-ras
G protein
Rel
NFkB transcription factor
Sis
PDGF
Src
Tyrosine kinase
Source of virus
Virus-induced tumor
Mouse
Chicken
Cat
Cat
Mouse
Chicken
Chicken
Chicken
Rat
Rat
Turkey
Monkey
Chicken
Leukemia
Erythroleukemia
Sarcoma
Sarcoma
Osteosarcoma
Fibrosarcoma
Sarcoma
Sarcoma
Sarcoma
Sarcoma
Reticuloendotheliosis
Sarcoma
Sarcoma
Figure 2 The translocation between chromosomes 9

and 22 responsible for chronic myelogenous leukemia.
The smaller of the two resulting abnormal chromosomes is called the Philadelphia chromosome, after
the city where the abnormality was first recorded.
Chapter 22: Tumor Suppressor Genes and Oncogenes
353
Figure 3 The serine/threonine phosphorylation cascade

activated by ras and C kinase. In the pathway activated by
receptor tyrosine kinases via ras, the MAP kinase kinase
kinase is often a serine/threonine kinase called Raf, which is
thought to be activated by the binding of activated ras. In the
pathway activated by G proteinlinked receptors via C
kinase, the MAP kinase kinase kinase can either be Raf or
a different serine/threonine kinase. A similar serine/threonine
phosphorylation cascade involving structurally and functionally related proteins operates in yeasts and in all animals that
have been studied, where it integrates and amplifies signals
from different extracellular stimuli. Receptor tyrosine kinases
may also activate a more direct signaling pathway to the
nucleus by directly phosphorylating, and thereby activating,
gene regulatory proteins that contain SH2 domains.
residue at codon 12. Mutated H-ras is constitutively

active whether GTP is bound or not.
Ras helps to link activated tyrosine kinase receptors to downstream events by acting as a molecular
switch. It is in the off position when bound to GDP
and in the on position when bound to GTP. Some
tyrosine kinase receptors phosphorylate themselves
on tyrosine residues, which then bind to other proteins that have SH2 domains that dock to phosphorylated tyrosine residues. These proteins with SH2
domains then link to other proteins (such as Sos)
that activate ras. Activated ras initiates a serine/
threonine phosphorylation cascade, which eventually
activates a kinase called mitogen-activated protein
kinase (MAP kinase). Eventually, such activation stimulates the action of a number of transcription factors,
including jun and elk-1 (Fig. 3). Figure 3 shows
another important point: namely, that protein kinase
C can also activate MAP kinase.
Heterotrimeric G Proteins
A large number of membrane-bound receptors function by activating downstream trimeric G proteins,

which initiate a phosphorylation cascade stimulating
certain enzymes. The trimeric G proteins consist of
three parts (a, b, and g) that disassemble when activated. The a-subunit bound to GTP activates nearby
enzymes such as adenyl cyclase, which synthesizes
cyclic AMP (Fig. 4), or phospholipase C, which forms
inositol triphosphate (IP3), and diacylglycerol from
inositol biphosphate found in the cell membrane
(Fig. 5). IP3 causes calcium release from various intracellular stores and diacylglycerol activates protein
kinase C, which is a serine/threonine kinase (Fig. 6)
that, as pointed out above, can also stimulate MAP
kinase.
Examples of receptors linked by G proteins to
phospholipase C include the muscarinic receptor. Activation of the muscarinic receptor causes an increase in
transmembrane calcium flux and also release of IP3,
which further releases calcium from the endoplasmic
reticulum. Receptors linked by G proteins to adenyl
cyclase include the b-adrenergic receptor (stimulation
of cyclic AMP) and the a2-receptor (inhibition of
cyclic AMP). The a1-receptor stimulates phospholipase C as well as inhibits adenyl cyclase. Thus,
although a wide variety of receptors are coupled to
different G proteins, the exact consequences of receptor activation depend on the specific G protein, which
couples the receptor to downstream intracellular signaling mechanisms.
354
Neal
Figure 4 Two major pathways by which G protein

linked cell surface receptors generate small intracellular
mediators. In both cases, the binding of an extracellular
ligand alters the conformation of the cytoplasmic
domain of the receptor, causing it to bind to a G protein
that activates (or inactivates) a plasma membrane
enzyme. In the cAMP pathway, the enzyme directly
produces cAMP. In the Ca2 pathway, the enzyme
produces a soluble mediator (inositol trisphosphate)
that releases Ca2 from the endoplasmic reticulum.
Like other small intracellular mediators, both cAMP
and Ca2 relay the signal by acting as allosteric effectors: they bind to specific proteins in the cell, altering
their conformation and thereby their activity. Abbreviation: cAMP, cyclic AMP.
Tyrosine Kinase Growth Factor Receptors

Many cell surface receptors for growth factors contain
a tyrosine kinase domain as part of the protein, which
is similar to the src family of oncogene tyrosine kinase
(including Src, Yes, Fgr, Lck, Lyn, Hck, and Blk) and
contain SH2 domains facing the internal portion of the
cell. Other growth factor receptors are closely associated with separate tyrosine kinase proteins that are
not an intrinsic part of the receptor protein itself. Most
of the transforming growth factor receptors [EGFr,
insulin-like growth factor I receptor (IGF-Ir), nerve
growth factor receptor (NGFr), platelet-derived
growth factor receptor (PDGFr), fibroblast growth
factor receptor (FGFR), and vascular endothelial
growth factor receptor (VEGFr)] contain an intrinsic
tyrosine kinase domain, with an exception for transforming growth factor-b (TGF-b), which contains a
serine/threonine kinase. When activated by ligand
binding, tyrosine kinase receptors autophosphorylate
at several phosphorylation sites and can then act as
docking sites for a small set of intracellular proteins
that recognize tyrosine-associated phosphate sites via
their SH2 domains (such as SOS and GrB2). Activation
of the EGFr produces dimerization of the receptor,
autophosphorylation of tyrosine residues, and phosphorylation of target proteins. EGFr activation is
linked to the ras signal transduction pathway via
two proteins (Sos and Grb2), which, when EGFr is
activated, link via Raf (MAP kinase kinase kinase) to
the downstream signals of the ras pathway such as
MAP kinase.
In many growth factor receptors, ligand binding

induces dimerization, a conformational change takes
place, and the tyrosine kinase domain is activated.
Some mutated growth factor receptors are constitutively active, whereas other receptors can become
activated in the absence of ligand if the receptor protein is found at very high levels. Such receptors can
also form heterodimers [such as c-erbB-2 with c-erbB3 or with c-erbB-1 (EGFr)]. It should be noted that
herceptin has recently been introduced as cancer treatment in advanced breast cancer; it is a humanized
antibody against c-erbB2. It is proof of the principle
that novel treatments against some of these targets can
be clinically useful.
Epidermal Growth Factor and the EGF Receptor
EGF is a 53amino acid peptide with mitogenic activity whose action is mediated by binding to a membrane-bound receptor. EGF was originally isolated
from murine submaxillary gland extracts, and its distribution is widespread, with high levels of milk,
prostatic fluid, and urine. The detection of high levels
of EGF in urine prompted studies of EGFr levels in
bladder cancer.
EGFr is a 175-kDa transmembrane protein with
an extracellular EGF-binding domain, a small hydrophobic region that spans the plasma membrane, and
an intracellular domain, which has tyrosine kinase
activity as well as target tyrosine residues for autophosphorylation. EGFr (c-erbB-1 gene) has considerable sequence homology with the gp65erbB protein
Figure 5 The hydrolysis of PIP2. Two intracellular mediators are produced when PIP2 is hydrolyzed: inositol triphosphate (IP3), which
diffuses through the cytosol and releases Ca2 from the ER, and diacylglycerol, which remains in the membrane and helps activate the
enzyme protein kinase C. There are at least three classes of phospholipase Cb, g, and dand it is the b class that is activated by G
proteinlinked receptors. We shall see later that the g class is activated by a second class of receptors, called receptor tyrosine kinases,
that activate the inositol phospholipid signaling pathway without an intermediary G protein.
Figure 6 The two branches of the inositol phospholipid pathway. The activated receptor binds to a specific trimeric G protein (Gq),
causing the a subunit to dissociate and activate phospholipase C-b, which cleaves PIP2 to generate IP3 and diacylglycerol. The
diacylglycerol (together with bound Ca2 and phosphatidylserinenot shown) activates C kinase. Both phospholipase C-b and C kinase
are water-soluble enzymes that translocate from the cytosol to the inner face of the plasma membrane in the process of being activated.
The effects of IP3 can be mimicked experimentally in intact cells by treatment with Ca2 ionophores, while the effects of diacylglycerol
can be mimicked by treatment with phorbol esters, which bind to C kinase and activate it.
355
356
Neal
from the avian erythroblastosis virus. EGFr is also the

target for the peptide growth factor TGF-a, which is
synthesized by a wide range of common epithelial
tumors, including transitional cell carcinomas (TCC)
of the urinary bladder, renal, and prostate.
EGFr is distributed throughout the body and is
present on normal fibroblasts, corneal cells, kidney
cells, basal prostate epithelium, and basal urothelium.
Increased expression of EGFr protein is transforming
in some cell lines, and some human solid tumors,
including TCC, have increased levels of EGFr protein.
This appears to be achieved by a variety of mechanisms, including gene amplification, upregulation of
mRNA, and increased translation or posttranslational
modification of the protein.
C-erbB-2
The proto-oncogene c-erbB-2 (also known as neu or

HER-2) encodes a transmembrane glycoprotein,
which is related to the EGFr. Several groups have
reported a candidate ligand for the c-erbB-2 receptor,
and elucidation of its structure (neu differentiation
factor or heregulin-a) reveals it to be an additional
member of the EGF family. Two further members of the
erbB receptor family are HER3/p160erbB-3 and HER4/
p180erbB-4. Recent evidence suggests, however, that heregulin is the physiological ligand for c-erbB-3 and that for
c-erbB-2 is as yet unidentified.
Fibroblast Growth Factors

The FGFs are a family of peptides related by sequence
similarity that have been implicated in the regulation
of cell proliferation, angiogenesis, embryonal development, differentiation, and motility. The family
includes FGF (aFGF; FGF-1), basic FGF (bFGF; FGF-2),
int-2 (FGF-3), hst (FGF-4), FGF-5, hst2 (FGF-6), keratinocyte growth factor (KGF; FGF-7), androgen-inducible
growth factor (AIGF; FGF-8), and FGF-9. They bind
with varying degrees of specificity to a family of
tyrosine kinase receptors (FGFRs). bFGF has been
shown to transform mouse fibroblasts in vitro, int-2
(FGF-3) is the human homologue of one of the common
integration sites of mouse mammary tumor virus, and
hst (FGF-4) is the most frequently detected human
transforming gene after ras in the NIH 3T3 assay.
These two genes were reported to be amplified in 3/
43 (7%) of bladder tumors and 41/238 (17%) of breast
carcinomas.
Basic FGF has been identified in both benign and
malignant human prostate. Immunohistochemistry
has identified FGF-2 predominantly in not only prostatic stroma but also epithelial cells. FGF-1 is also
present in the prostate but at a lower level than FGF-2.
In addition, in a prostate stromal cell culture model,
there is evidence for the interaction of FGF-2 and TGFb1, resulting in positive and negative proliferative
effects, respectively. Besides potential autocrine loop
activation, FGFs may also contribute to the pathophysiology of the prostate via paracrine activity.
FGF-7, or KGF, is synthesized and secreted by stromal
cells, being thought to act on FGF receptorbearing
epithelial cells, resulting in cellular proliferation. It is

also interesting to note that, in a transgenic mouse
system, overexpression of FGF-3 or int-2, alterations to
the prostate of the male progeny, were dramatic and
involved only hyperplasia, without evidence of malignant transformation.
The FGFrs share structural similarities in both
the extracellular domains that bind FGFs and the
intracellular domains that activate the signal
transduction pathway. The four FGFRs, FGFR-1 to
FGFR-4, display different binding affinities for the
different FGFs. FGF-1 binds to all four receptors,
while FGF-2 is more restricted and binds only to
the first three FGFRs. Receptor-receptor interactions
are known to occur and may have a role in the
development of both benign hyperplasia and carcinoma of the prostate. To date, the expression of FGFs
and their receptors is not fully understood, but there
does appear to be a major difference between benign
and malignant prostate in the activity of the FGF
system. Splice variants occur in the FGFRs. It is likely
that subtle changes occur in the FGF system during
malignant progression. This has been demonstrated
elegantly in a mouse prostate cancer model. During
progression from the benign to the malignant phenotype, the prostate epithelial cells display a switch
of expression of FGFR splice variants, conferring a
switch of high-affinity binding from FGF-7 to FGF-2.
FGF-2 was also concomitantly upregulated along
with FGF-3 and FGF-5. Further evidence supporting
a role for FGFs in the development of malignancy is
that transformation of BHK-21 (baby hamster kidney)
cells with plasmids carrying the bFGF coding
sequence results in cells that exhibit the transformed
phenotype. The prostatic cancer cell lines DU145 and
PC-3 produce active FGF-2 and express large
amounts of FGFR-2. However, only the DU145 cell
line, and not the PC-3 cell line, has been shown to
respond to exogenous FGF-2.
The interaction between the androgen receptor
and growth factor systems is important. It is known
that certain growth factors, including FGF-7/KGF,
IGFs, and EGF, may activate androgen-dependent
gene expression in the absence of androgen, and this
may represent a mechanism for the development of
hormone insensitivity in prostate tumors.
Insulin-Like Growth Factors

IGF-I is a 70-amino acid polypeptide with functional
homology to insulin. The mitogenic effect of IGF-I is
due to its ability to facilitate the transfer of cells from
the G1 phase to the S phase in the cell cycle. IGF-I and
the closely related IGF-II are present in biological
fluids and tissue extracts, and are usually bound to
an IGF-binding protein (IGF-BP). There are two types
of receptors for the IGFs. The type 1 receptor is a
tyrosine kinase and binds both IGF-I and IGF-II. The
type 2 receptor is structurally distinct and binds primarily IGF-II. The majority of the mitogenic effects of
the IGFs appear to be mediated via the type 1 IGF
receptor.
The growth of normal prostatic epithelial cells in

culture is dependent on the presence of IGF-I and II.
The response is most marked with IGF-I. The type 1
IGF receptor has been identified in normal, benign,
and malignant prostatic tissues. It is preferentially
expressed in the basal cells. Production of IGF-II has
been demonstrated by prostatic fibroblasts, but IGF-I
has not been identified as being produced by either
prostatic fibroblasts or epithelial cells. However, IGF-I
mRNA has been identified in stromal cells from
benign prostatic hyperplasia (BPH) specimens. The
mechanism of IGF-II-mediated stimulation of prostatic epithelial cells in BPH appears to be similar to
that in normal prostate, although the expression of
IGF-II mRNA is 10-fold higher in fibroblasts from
BPH tissues. This led to the hypothesis that such
cells may be reverting to a fetal-like state, where
IGF-II expression is normally high, causing stimulation of proliferation and the development of BPH.
357
often associated with predisposition to a wide range of

sporadic tumors of other tissues led to the further
suggestion that somatic mutation or loss of these
same genes was probably involved in the genesis of a
wide range of cancers, including bladder cancer. This
also provided a possible explanation for the observation of the nonrandom chromosome deletions seen in
sporadic tumors. This concept in turn led to the identification of the p53 gene as a tumor suppressor gene.
Cytogenetic studies have shown that frequent
nonrandom chromosome alterations occur in cancer.
For instance, in bladder cancer, these involve chromosomes 1p, 3p, 9p, 10q, 11, 13q, 17p, and 18q. Loss of
one copy of chromosome 9p was found as an early
event in low-stage, low-grade tumors, unlike other
chromosome losses, which appeared to be more prevalent in advanced tumors. The high frequency of
chromosome 9p losses in bladder cancer is not seen
in other tumor types, suggesting the possible location
of a tumor suppressor gene specific to bladder cancer.
Transforming Growth Factor b

TGF-b1 is the predominant species in the TGF-b superfamily, which includes several modulators of growth,
differentiation, and morphogenesis (including bone
morphogenetic proteins). TGF-b 1 is classically
regarded as a stimulator for mesenchyme cells and
an inhibitor for epithelial cells. Five isoforms of TGF-b
have so far been identified, of which only the first
three occur in mammals. TGF-b1 is a homodimer of
two 112-amino acid subunits linked by disulfide
bonds. Cellular receptors for TGF-b1 have been identified in the rat ventral prostate where they are negatively regulated by androgens and involved in the
mechanism of castration-induced prostatic cell death.
Receptors for TGF-b1 have been identified in the
human prostate cancer cell lines DU145 and PC-3.
TGF-b1 has been shown to have an inhibitory effect
on human prostatic fibroblasts and epithelial cells in
culture. It has been suggested that TGF-b1 causes
inhibition of proliferation by preventing phosphorylation of the protein product of the retinoblastoma
gene (pRB) by upregulation of p15, which is a cyclindependent kinase inhibitor, and by stabilization of the
p27 protein. The TGF-b receptor is a serine protease
and links to downstream signals, including SMAD2.
TUMOR SUPPRESSOR GENES

Evidence for the existence of tumor suppressor genes
has come from several sources, although the significance of the observations was not appreciated at the
time. Experiments dating back to the 1960s, involving
the fusion of normal and transformed cells, had
shown that normal genes can suppress transformation
and malignancy in cell lines.
The second line of evidence came from an analysis of the inheritance of rare hereditary childhood
cancers. On the basis of retinoblastoma, Knudson proposed a two-hit inactivation process in which both
copies of a critical gene had to be inactivated for the
disease to be manifest. That these hereditary cancers are
The p53 Tumor Suppressor Gene

The human p53 gene, located on the short arm of
chromosome 17, encodes a nuclear phosphoprotein,
which binds to specific DNA sequences in the human
genome and appears to play a key role in the control
of DNA replication and hence cellular proliferation.
Nonrandom chromosome 17 losses involving the p53
locus are commonly found in human tumors, including bladder cancer, and the loss of one copy of the p53
gene has been found to be frequently accompanied by
mutation of the remaining allele. Transfection studies
have shown that the wild-type protein is able to suppress cell proliferation and transformation. It thus acts
in such circumstances as a tumor suppressor gene,
which can contribute to tumor formation by deletions
or loss of function mutations. Germline mutations of
the p53 gene have been found in certain inherited
predispositions to cancer (Li-Fraumeni syndrome, in
which there is clustering of soft tissue and bone sarcomas at any age; and cancers under the age of 45 in
breast, brain, adrenal cortex, and lung) and somatic
cell mutations have frequently been detected in a wide
range of common sporadic tumors, including breast,
colon, brain, and lung tumors, as well as bladder
cancer. These mutations are predominantly of the
point missense type and occur at many different
locations within four highly conserved regions of the
gene (codons 117142, 171181, 234258, and 270286)
located in exons 4 to 9 of the p53 gene.
The p53 protein has three parts: a central DNAbinding domain in which mutations are usually of the
missense type, a transcription domain (N-terminus)
and a regulatory domain (C-terminus) in which mutations result in nonsense or stop codons. The protein
murine double minute-2 (mdm-2) binds near the
N-terminus of the protein. One of the genes activated
by p53 is the p21 protein (WAF1), which inhibits the
activity of cyclin-dependent kinases and which also
binds to PCNA. Other genes include gadd45 and the
protein bax (which promotes apoptosis).
358
Neal
The p53 protein has a short half-life, measured

in the order of minutes, and is normally present at
low levels not detectable by immunohistochemistry.
One of the remarkable features of the p53 gene is
that a broad spectrum of mutations can lead to an
altered conformation and inactivation of the protein
product with an increased cellular half-life, resulting in accumulation in the cell to levels that are
readily detectable by immunohistochemical staining. Although this initially led to suggestions that
positive detection of p53 by immunohistochemistry
was synonymous with the detection of mutations,
this is now realized not to be the case. Moreover,
some mutations do not result in accumulation of the
altered protein product, and these have to be
screened for by alternative complementary methods
such as single-strand conformation polymorphism
assays to obtain a more complete picture. However,
even combined, these techniques are, at best, a way
of trawling for mutations rather than a way of systematically screening for all possible mutations.
The definitive method of establishing the presence
of a mutation in a sample is by DNA-sequencing
procedures.
Inactivation of Normal p53 in Tumors
Some proteins have been shown to bind to p53 and

inactivate it. For instance, in sarcomas, it has been
shown that p53 mutations are rare, but there is
increased expression of a protein called mdm-2,
which, like the large T antigen of the SV40 virus,
binds to p53, inactivates it, and stabilizes it. This
confirms the frequent involvement of the p53 pathway
in many human tumors.
p53 and G1 Arrest
Irradiation of cells has been shown to result in marked

upregulation of the wild-type p53 protein. It may be
that the primary event is DNA repair following radiotherapy, which then activates p53. However, upregulation of p53 switches on downstream genes such as
p21, which is a potent inhibitor of cell division, and
places them into G1 arrest, allowing completion of
DNA repair if this is possible.
p53 and Apoptosis
Some authors believe that increased levels of p53 can,

if DNA damage is too severe to repair during G1
arrest, force the cell into apoptosis. There is no doubt
that with severe DNA damage there is an initial
attempt at DNA repair followed by upregulation of
p53, stimulation of p21, and cell cycle arrest. With
more severe DNA damage or with less severe damage
in certain types of cells (such as thymocytes), this
process may be followed by apoptosis, but, currently,
it is controversial as to whether p53 is directly responsible for apoptosis in such circumstances. Certainly,
p53 can upregulate bax (a promoter of apoptosis), but
it is also certain that in some cells apoptosis can occur
without recourse to the p53-bax pathway.
mdm-2 Oncogene
The mdm-2 gene is located on chromosome 12q1314
and encodes a 90-kDa nuclear protein. The mdm-2
protein may act as an oncoprotein when overexpressed, by binding to the guardian of the genome,
wild-type p53 protein, and abrogating its functions.
Retinoblastoma Gene
The Rb1 gene encodes a 105- to 115-kDa nuclear phosphoprotein, which binds to DNA and appears to be
involved in growth regulation in a wide variety of cell
types. There are related p107 and p130 proteins, whose
function in man has not been elucidated but which may
have overlapping actions. The Rb1 gene product binds
to the transforming proteins of several DNA tumor
viruses, including adenovirus E1A, SV40 large T antigen, and human papilloma virus E7 proteins. Abnormalities in the Rb gene were first reported in patients
with inherited retinoblastoma who had one abnormal
copy of the gene in all retinal cells; it is thought that
subsequent spontaneous alterations in the remaining
copy of the Rb gene causes tumor formation.
The Rb protein is central to the function of the
cell cycle; it is inactive when phosphorylated or when
bound to inactivating proteins. It is the target of
cyclin-dependent kinases and cyclin D. When it is
activated, it stimulates the E2F family of transcription
factors (myc and jun are among them): it is a negative
regulator of the cell cycle. Disruption of this pathway
is central to many tumors. The cyclin-dependent kinase inhibitor p16 is particularly linked to Rb, so when
p16 is upregulated, the activity of cdk2 is inhibited.
The Rb protein is not phosphorylated, so it binds to
DNA and inhibits transcription, and the E2F family of
transcription factors is switched off.
A wider role for the Rb1 gene was suggested by
the observation that those who inherit the mutant allele
have a higher incidence of not only a wide range of
nonocular second tumors, particularly osteosarcomas,
but also lung melanoma and bladder cancer.
Initial studies with the Rb1 cDNA probe, based
mainly on tumor cell lines, reported frequent structural
abnormalities of the gene and absence of Rb1 mRNA.
Subsequent extensions of these studies with polyclonal
antisera indicated that absence or abnormal forms of
the Rb protein are almost universal in small-cell lung
cancer cell lines and present in one-third of bladder
carcinoma cell lines, while being infrequent in colonic,
breast, and melanoma cell lines. Studies on primary
bladder tumors have shown that loss of heterozygosity
for Rb1-linked markers is associated with the development of invasive lesions. The overall frequency of Rb1
allelic loss in bladder cancer was 3/63 (5%) for superficial (pTa/pT1) and 30/58 (53%) for invasive (T2T4)
tumors. Major rearrangements of the remaining Rb1
gene were detected in only a small proportion of these
cases, suggesting that generally more subtle small
deletions or point mutations are involved that have
yet to be characterized. Altered Rb protein expression
is associated with a poor outcome in several tumor
types, including bladder.
359
von HippelLindau Disease
Genes Associated with Chemoresistance
This is inherited as a dominant abnormality and

consists of retinal cysts, cerebellar hemangioblastomas, pancreatic cysts, pheochromocytomas, renal
cysts, and renal cancer. The VHL gene is situated on
chromosome 3p and also affects many sporadic renal
cancers. It encodes a small protein (213 aa) with three
exons, and in the human gene there are eight pentapeptide repeats in exon 1. Most mutations in VHL are
found at the carboxy terminus of exon 1 and at the
beginning of exon 3. Similar mutations are found in
sporadic tumors, but, in addition, mutations are found
in exon 2, a rare event in VHL. The VHL protein binds
to the elongation factors elongin B and C, which help
in the synthesis of mRNA; it is thought that the VHL/
elongin B and C complex moves from the nucleus into
the cytoplasm as a response to cell/cell contacts. VHL
also upregulates the peptide VEGF (see next section),
a feature that may be responsible for angiogenesis in
renal cancer
Chemotherapy with epirubicin or mitomycin C is

increasingly used for recurrent superficial papillary
tumors. In muscle-invasive disease, adjuvant and neoadjuvant chemotherapy regimens are currently being
tested in clinical trials. Molecular mechanisms that mediate cellular drug resistance may play a role in the
differential responsiveness of individual bladder tumous
to chemotherapy. Transcript levels of the multidrug
resistance (mdr1) gene have been found to vary 34-fold
in high-grade muscle-invasive bladder tumors. In addition, renal cell carcinomas have very high levels of this
gene, perhaps explaining why it is chemoresistant.
ACKNOWLEDGMENT
Artwork reproduced from Alberts B, Johnson A,
Lewis J, et al., eds. Molecular Biology of the Cell. 4th
ed. New York: Garland Science, 2002.
23
The Molecular Genetics and Pathology
of Renal Cell Carcinoma
Maxine G. B. Tran
Department of Urology, Addenbrookes Hospital, Cambridge, U.K.
Tim OBrien
Department of Urology, Guys and St Thomas Hospital, London, U.K.
Patrick H. Maxwell
Division of Medicine, University College Hospital, London, U.K.
INTRODUCTION
Renal cell carcinoma (RCC) is the commonest malignancy of the kidney and represents 2% to 3% of all
adult cancers. Each year in the United Kingdom, more
than 6600 patients are diagnosed with kidney cancer,
and over half will die of the disease. The highest
prevalence is in the 6th decade of life, with a male:
female ratio of approximately 2:1.
Although RCC is a relatively rare malignancy,
the incidence is steadily increasing at a rate of approximately 2% per year worldwide, in part but not
entirely due to increased use of imaging modalities
leading to recognition of tumors that were previously
undiagnosed (1,2). Incidentally discovered renal
masses now account for 48% to 66% of RCC diagnoses
(3). In the United States the incidence rates in black
Americans have increased rapidly surpassing the incidence rate of white Americans. Large epidemiological
studies have established cigarette smoking, western
style diet, obesity and hypertension as main risk
factors in both sexes for the development of RCC;
while recreational exercise is associated with
decreased risk (1,4). Diuretic use is associated with
an increased risk in females, and alcohol consumption
is inversely associated with risk in men (5,6). A positive family history of RCC is associated with a two- to
threefold increase in risk, while the inherited forms of
RCC account for 2% of all cases.
THE PATHOLOGICAL AND GENETIC

CLASSIFICATION OF ADULT RENAL
EPITHELIAL NEOPLASMS
A workshop held in Heidelberg in 1996 culminated
in the widely accepted Heidelberg classification of
adult renal epithelial neoplasms (7) (Table 1). The
Union Internationale Contre le Cancer (UICC) and

the American Joint Committee on Cancer (AJCC)
held a workshop in Minnesota the following year
and proposed a very similar classification system (8).
More recently, the World Health Organisation
(WHO) in 2004 introduced a new classification of
adult renal epithelial neoplasms that is more comprehensive and includes new diagnostic categories (9)
(Table 2).
These new classification systems reflect our
increased understanding of the molecular pathology
of renal neoplasms, the close correlation between
specific genetic abnormalities and histological subgroups, and differ from the older systems, which
were essentially based on architectural and cytological
subtypes (10). Importantly, they allow the majority of
cases to be classified by routine H&E light microscopy, with more specialized molecular genetic studies
reserved for a minority of atypical cases; in addition
the classification systems have prognostic utility
(11,12).
Having once been considered a single disease
entity, it is now clear that there are a number of
different types of kidney cancer, each with a different
histological pattern, clinical behavior, prognosis, and
each caused by abnormalities of different genes
(11,13).
The major clinical and histopathological features
of the main adult renal epithelial neoplasms will be
reviewed in this chapter. We will then consider our
current understanding of the molecular and genetic
basis of renal neoplasm development, how these
relate to the current classification of renal epithelial
neoplasms, and finally how this improved insight has
led to the development of new management strategies
in renal cancer.
Chapter 23: The Molecular Genetics and Pathology of Renal Cell Carcinoma
Table 1 The Heidelberg Classification of Adult Renal Epithelial
Neoplasms
Benign tumors
Malignant tumors
Papillary renal adenoma

Metanephric adenoma
Oncocytoma
Conventional renal cell carcinoma

Papillary renal cell carcinoma
Chromophobe renal cell carcinoma
Collecting duct carcinoma (including
renal medullary carcinoma)
Renal cell carcinoma, unclassified
Table 2 WHO Classification of Kidney Tumors

Familial renal cancer
Renal Cell tumors
Malignant
Clear cell renal cell carcinoma
Multilocular clear cell renal cell carcinoma
Papillary renal cell carcinoma
Chromophobe renal cell carcinoma
Carcinoma of the collecting ducts of Bellini
Renal medullary carcinoma
Xp11 translocation carcinomas
Carcinoma associated with neuroblastoma
Mucinous tubular and spindle cell carcinoma
Renal cell carcinoma unclassified
Benign
Papillary adenoma
Oncocytoma
Metanephric tumors
Metanephric adenoma
Metanephric adenofibroma
Metanephric stromal tumors
Mixed mesenchymal and epithelial tumors
Cystic nephroma
Mixed epithelial and stromal tumor
Synovial sarcoma
Nephroblastic tumors
Nephrogenic rests
Nephroblastoma
Cystic partially differentiated nephroblastoma
Neuroendocrine tumors
Carcinoid
Neuroendocrine carcinoma
Primitive neuroectodermal tumor
Neuroblastoma
Phaeochromocytoma
Other tumors
Mesenchymal tumors
Hematopoietic and lymphoid tumors
Germ cell tumors
Metastatic tumors
THE CLINICAL AND HISTOPATHOLOGICAL

FEATURES OF ADULT RENAL EPITHELIAL
NEOPLASMS
Benign Renal Tumors
Papillary Renal Adenoma: the Macroscopic and Microscopic
Appearances of Papillary Renal Adenoma and Its
Association with Papillary Adenocarcinoma
Papillary adenomas are very common lesions, affecting 40% of the general population and are usually an
361
incidental finding at autopsy or in nephrectomies.

They are almost always multiple in number, averaging eight lesions per kidney in specimens removed for
papillary renal cell carcinoma (PRCC) (14). Papillary
adenomas are histologically, immunohistochemically
and cytogenetically very similar to low-grade PRCCs;
being differentiated principally by size so that an
adenoma must be less than 5 mm in maximum diameter. Lesions greater than 5 mm are considered as
having malignant potential. Histologically, papillary
adenomas are characterized by a tubulopapillary
architecture formed by cuboidal cells with scant basophilic cytoplasm. There should not be any evidence of
nuclear pleomorphism, significant mitotic activity or
vasculolymphatic invasion (15). Because of the similarities between papillary adenomas and PRCCs, it
has been proposed that they may represent a continuum of a malignant process (16), although it is unclear
what percentage of renal adenomas will progress to
carcinomas.
Metanephric Adenoma: Epidemiology, Macroscopic
and Microscopic Appearances
Metanephric adenomas are rare tumors of the kidney

that have an invariably benign clinical course. They
are twice as common in females compared with males,
with a mean age at presentation of 41 years (15). They
are usually an incidental finding, although they can
present with the classical triad of hematuria, loin pain
and palpable mass. An unusual paraneoplastic feature
of this tumor is polycythaemia, due to erythropoietin
and other cytokines produced by the metanephric
tumor cells (17). The erythrocytosis resolves following
tumor resection.
Histologically, metanephric adenomas are composed of predominantly small tubules lined by cuboidal epithelial cells, and glomeruloid bodies
reminiscent of embryonic metanephric tissue are also
seen. In the past they have been confused with and
misclassified as Wilms tumor, nephroblastomatosis,
or variants of tubular carcinoma or PRCC.
Oncocytoma: Epidemiology, Macroscopic
Renal oncocytomas was first reported by Zippel in

1942 (18), and account for approximately 2% to 7% of
renal neoplasms. There is a male to female ratio of
2.5:1, with a mean age of 65 years at presentation.
Renal oncocytomas are usually asymptomatic and are
diagnosed incidentally during investigations for other
medical problems. Historically they are said to have a
characteristic radiological appearance of a cartwheel
peripheral vascular arrangement with a central star on
angiography, although a recent small series has
reported that this could also be a feature of chromophobe RCC and is thus unreliable (19).
Oncocytomas, by and large, are discrete unilateral
benign masses, although bilateral and multifocal tumors
have been reported (20). Metachronous tumor development has been described to occur in 5% of patients, and
they coexist with RCC in 10% (20). On gross pathological
362
Tran et al.
examination, oncocytomas appear solid, spherical, are

well circumscribed, un-encapsulated, homogenous,
tan-brown in color and range in size averaging
about 7 cm. A central, stellate, fibrous scar can be
found in approximately a third of cases (21), and
hemorrhage can occasionally be seen, though cystic
change and necrosis is rare (Fig. 1A).
Oncocytomas are thought to arise from the intercalated cells of the collecting duct (22). Histologically,
the classic oncocyte consists of polygonal or round
cells with abundant eosinophilic cytoplasm, arranged
in nests or trabeculae separated by hypocellular,
hyalinized stroma (Fig. 1B). By definition, areas of
papillary or clear cell differentiation are not allowed.
The nuclei appear smooth and round with evenly
dispersed chromatin. There is a minimal degree of
nuclear atypia and mitotic activity. Ultrastructurally
the tumor cells are packed with mitochondria
accounting for the eosinophilic appearance of the
cells seen on light microscopy.
Occasionally oncocytomas may show atypical
features including nuclear polymorphism, prominent
nucleoli, and adjacent renal parenchymal and perinephric fat involvement. Oncocytomas are benign
tumors and prognosis following resection is excellent
(21,23). Malignant variants of oncocytomas have been
reported in the literature, however many of these case
reports predate the recognition and distinction of
chromophobe carcinoma, and it is likely that the
majority of the previously so-called malignant oncocytomas would be reclassified as eosinophilic variants
of chromophobe or conventional renal cell carcinoma
(CRCC).
Malignant Tumors (Renal Cell Carcinoma)

Conventional RCC (CRCC): Epidemiology,
Macroscopic and Microscopic Appearances
CRCC is the most common form of renal cancer,

accounting for approximately 75% of cases (7).
CRCC is also known as clear cell renal cell carcinoma (CCRCC) and hypernephroma. There is a
male to female ratio of 2:1. The great majority of
CRCC are diagnosed incidentally by imaging for
nonurological symptoms. The classic presentation
with the triad of hematuria, flank pain and loin
mass is now rare. Notwithstanding this, one third
of patients have metastases on presentation and are
considered incurable by surgery alone. Most CRCC
are solitary cortical neoplasms although multicentricity (4%) and bilaterality (0.53%) may occur (9). The
peak incidence of CRCC is ten years earlier than
prostate or bladder cancer, occurring most commonly in the sixth decade.
Architecturally, CRCCs commonly have both
solid and cystic regions and are typically uniformly
golden yellow in color, but larger tumors may have a
variegated appearance with a mixture of viable,
necrotic and hemorrhagic areas (Fig. 1C).
Historically, CRCC is thought to arise from the
proximal tubule, although this is now disputed with
recent evidence pointing to a distal tubular origin
(24,25). The great majority of CRCC are comprised

predominantly of neoplastic cells with abundant clear
cytoplasm, a low nuclear/cytoplasm ratio, and nuclei
with prominent nucleoli (Fig. 1D). The clear cytoplasm is due to the presence of intracytoplasmic
lipid and glycogen. The cells are usually organized
in sheets and trabeculae separated by a rich vascular
fibrous stroma, or are arranged in a pseudoglandular
or cystic pattern. Multilocular cystic renal cell carcinoma (MCRCC) is a subtype of CRCC, where the
tumor is composed almost entirely of cysts.
The cysts are of variable size and are lined by a single
layer of clear cells (9), they are separated by fibrous
septa which may also contain nests of clear cells.
MCRCC has an excellent prognosis (26) and is recognized as a separate entity in the current WHO classification (27) (Table 2).
Sarcomatoid change occurs in approximately 5%
of CRCC cases (28). On immunohistochemistry, CRCC
cells stain positive for vimentin, CD10, Glutathione Stransferase a (GST-a) and Cytokeratin 8 and 18, which
can be helpful in the differential diagnosis between
different subgroups of RCC (29,30).
Papillary Adenocarcinoma: Epidemiology, Macroscopic and
Microscopic Appearances
PRCC is the second commonest form of renal cancer,

comprising 10% to 15% of cases in most surgical
series, and has been known as chromophil carcinoma and tubulopapillary carcinoma in the past.
PRCC is commoner in males with a male to female
ratio of 1.5:1. Presentation can occur over a wide age
range, though most cases present in the sixth decade.
The most important clinical feature of PRCC is that
they are often multifocal and bilateral, and frequently
are accompanied by multiple renal adenomata in the
ipsilateral or contralateral kidney (31). Patients with
end-stage kidney disease (ESKD) have an increased
risk of developing RCC, and the incidence of PRCC is
significantly higher (1.4% compared with 0.04% and
57% compared with 10%, respectively) compared with
the general population (32). Radiologically, PRCCs are
often hypovascular and so contrast enhancement on
CT imaging may be minimal (<10 Hounsfield units)
(33,34). Tumor calcification also occurs more frequently in PRCC than in other subtypes.
Macroscopically, PRCCs are usually small
(<4cm) and are typically well circumscribed and
organ confined (Fig. 1E). Larger tumors tend to have
areas of necrosis and hemorrhage rather than being a
solid tumor mass.
Histologically, these tumors are characterized by
the presence of lipid-laden macrophages (foamy
macrophages) and small calcific concretions (psammomma bodies) in the papillary cores. By definition,
over 70% of the tumor must exhibit papillary or
tubulopapillary architecture (35) (Fig. 1F).
Two distinct subtypes with prognostic implications have been described; type 1 PRCC consists of
basophilic cells with scant cytoplasm covering thin
papillae and type 2 PRCC consists of eosinophilic cells
with abundant granular cytoplasm, larger nuclei and
363
Figure 1 Macroscopic and histological appearances of adult renal epithelial neoplasms. (A) Macroscopic appearance of renal
oncocytoma, showing tan-brown cut surface and prominent central fibrous scar. (B) Typical histological appearance of oncocytoma,
with nests of eosinophilic cells in hyalinized stroma. (C) Macroscopic appearance of CRCC with prominent cystic degeneration and
hemorrhage. (D) Histology of conventional RCC with clear cell morphology. (E) Macroscopic image of nephrectomy specimen bearing
multiple PRCCs. (F) Histological appearance of type 1 PRCC with tubulopapillary architecture. Collections of foamy macrophages were a
prominent feature in this case. (G) Macroscopic appearance of chromophobe RCC with a beige cut surface. (H) Chromophobe RCC
characterized by large polygonal cells with prominent cell borders.
364
Tran et al.
pseudostratification. Type 1 tumors are twice as common and are associated with a better prognosis than
type 2 PRCC (36). CK7 immunoreactivity has been
reported in the vast majority of PRCC and can be a
useful diagnostic tool. Overall, considering stage for
stage and grade for grade, PRCC has a better five-year
survival than CRCC (37).
Chromophobe RCC: Epidemiology, Macroscopic
Chromophobe RCC is the third most common carcinoma of the kidney and accounts for approximately
5% in most surgical series. They are slow growing
tumors, are usually organ confined at presentation
and have a better prognosis than CRCC. There is a
slight male predominance, with a mean age of presentation in the sixth decade although there is a wide
age range of 31 to 75 years (38). Chromophobe RCCs
average 8 cm in size, with a range from 1.3 to 20 cm.
Grossly, the cut surface is solid, and less variegated
than CRCC, and is usually beige/light brown or gray
in color (Fig. 1G). Histologically, the cells have variable amounts of pale or eosinophilic cytoplasm staining blue with Hales colloidal iron stain. Two variants
of chromophobe RCC are recognized: classical and
eosinophilic. The classical type is composed of large
polygonal tumor cells with an abundant fine granular
cytoplasm that tends to condense near the cell membrane, arranged in a compact solid architecture. This
classical form of chromophobe RCC can mimic CRCC
phenotypically. The eosinophilic variant is composed
of small tumor cells with abundant eosinophilic granular cytoplasm and shares a similar phenotype with
renal oncocytoma. Diagnostic distinction between the
two can be made with Hales colloidal iron stain or
cytokeratin (7) immunohistochemistry. The relationship between chromophobe RCC and oncocytoma is
still being investigated with oncocytomas postulated
to be the benign counterpart. Both are thought to arise
from the intercalated cells of the collecting duct and
both have mitochondrial abnormalities.
Ultrastructurally, the cytoplasm of the cells contain numerous microvesicles 150 to 300 nm in diameter, that are oval or round in shape and often have
the complex appearance of a vesicle within a vesicle.
Genetically, chromophobe RCCs display characteristic monosomy of multiple chromosomes (1,2,6,10,
13,17,21).
Collecting Duct Carcinoma: Epidemiology,
Macroscopic and Microscopic Appearances
Collecting duct carcinoma (CDC) is a rare tumor and

accounts for less than 1% of renal malignancies in
surgical series. There is a slight male predominance
and average age at presentation is 55 years. These
tumors arise from the collecting ducts, especially the
papillary ducts (Bellini ducts) hence they are also
called Bellini duct carcinomas. They are centrally
located in the kidney, averaging 5 cm though range
in size from 2.5 to 12 cm, and the cut surface appears
solid with a gray-white color. Patients may present
with hematuria, flank pain and mass. Urine cytology
may be positive (39) and imaging studies may suggest

urothelial malignancy.
Histologically, the tumor is composed of infiltrating glands lined by pleomorphic cells which lie in
a desmoplastic stroma (40). These tumor cells show
positive immunoreactivity to epithelial membrane
antigen, vimentin, Leu-1, highmolecular weight cytokeratins (CK8 and CK19) and mucin (4042).
Renal medullary carcinoma is an aggressive
variant of CDC that is predominantly seen in young
males with sickle cell trait. These are very poorly
differentiated tumors and have a poor prognosis (27).
Sarcomatoid Change in Renal Cell Carcinoma
Sarcomatous change can occur in all histological subtypes of renal carcinoma, and is now recognized as
dedifferentiation of carcinoma cells and not a distinct
histological entity. The incidence of finding a sarcomatoid component is approximately 5% to 8% in CRCC,
3% in PRCC and 9% in chromophobe RCC. The significance of finding sarcomatoid change in RCC is that
after adjusting for stage, tumor size and presence of
necrosis, sarcomatous differentiation portends a worse
prognosis (43,44). It is not clear whether the primary
histological subtype affects the poor clinical outcome.
THE MOLECULAR GENETICS OF RENAL

EPITHELIAL NEOPLASIA DEVELOPMENT
Inherited Cancer Predisposition Syndromes
Identify Genes Involved in RCC Development
Recent years have seen a significant increase in our
understanding of the molecular genetics of renal epithelial neoplasia development. While 98% of RCCs are
sporadic in nature, 2% of cases arise in individuals
with an inherited predisposition and it is studies on
these individuals and rare families at high risk of
developing kidney cancer that has provided significant insights into the molecular mechanisms of RCC
tumorigenesis which are also relevant to sporadic
kidney cancer. Strikingly, mutations in completely
different genes are now implicated in different histological types of kidney cancer (13). These provide
good illustrations of different genetic mechanisms
contributing to cancer development, including loss
of function of tumor suppressor genes (e.g., VHL in
CRCC), and also activation of proto-oncogenes (e.g.,
c-Met in type 1 papillary renal cell carcinoma).
Von HippelLindau Disease and Familial CRCC
Von Hippel-Lindau (VHL) disease is an autosomal

dominant inherited multicancer syndrome that affects
approximately 1 in 36,000 of live births. Affected
individuals have a very high lifetime risk (c.70%) of
developing renal cancer, which is always CRCC, and
is commonly multifocal, bilateral and of early onset
compared with sporadic cases. Other aspects of the
syndrome include hypervascular tumors of the CNS
and retina, pheochromocytomas, endolymphatic sac
tumors, pancreatic cysts, pancreatic islet cell tumors

and epididymal cystadenomas.
The first description of this syndrome was probably by Treacher Collins in 1894, who detailed his
observations of bilateral retinal angiomas in two siblings (45). Ten years later, von Hippel, a German
ophthalmologist noted retinal angiomas in two of his
patients (46). Later still, in 1927, the Swedish pathologist Arvid Lindau described the association of retinal
lesions with increased risk of developing haemangioblastomas of the brain and spinal cord (47) and the
eponym von HippelLindau disease was finally
coined by Charles Davison in 1936 (48).
The protean manifestations of VHL mean that
patients can present in a variety of ways. Common
symptoms include: loss of vision, neurological deficits, paroxysmal raised blood pressure and local pain.
Individuals with a positive family history of VHL can
be diagnosed as affected on the basis of a single
typical VHL tumor such as a retinal or cerebellar
hemangioma, CRCC or pheochromocytoma (49). The
presence of endolymphatic sac tumors and multiple
pancreatic cysts which are uncommon in the general
population are also highly suggestive of VHL disease
in the context of a positive family history. In contrast,
renal and epididymal cysts occur very frequently in
the general population are therefore not reliable indicators of VHL status.
In the absence of a positive family history, clinical diagnosis of VHL disease can be made with the
detection of two or more haemangioblastomas, or a
single hemangioblastoma in association with a visceral manifestation (49). Diagnosis of VHL can be
confirmed by molecular genetic analysis of the VHL
gene which is informative in virtually all VHL families
(50). Family members of confirmed VHL patients are
offered genetic counseling and testing, and are
enrolled into clinical monitoring programs if found
to be carriers of a germline mutation in VHL.
Clinically, VHL families can be broadly grouped
on the basis of the absence (type 1) or presence (type 2)
of a risk of developing pheochromocytoma. Type 2
VHL families are further subdivided into type 2A (low
risk of developing CRCC), type 2B (high risk of developing CRCC), and type 2C (risk of developing pheochromocytoma alone). Recently a third subtype of
VHL disease has been recognized, type 3 VHL disease,
also known as Chuvash polycythaemia, as it was
originally described as being a condition endemic in
the Chuvash region of Central Russia. Type 3 disease
differs from the other variants of VHL disease by the
autosomal recessive rather than dominant mode of
inheritance, and is characteristically not associated
with CRCC, or other manifestations of classical VHL
disease (Table 3) (51).
Identification of the VHL Tumor Suppressor Gene
Linkage analysis of VHL kindreds mapped the VHL

locus to the short arm of chromosome 3 in 1988, and
the VHL gene was cloned in 1993 (3p2526) after a
concerted international effort (52). Improved molecular technology means that a germline VHL mutation or
365
deletion can be demonstrated in virtually all individuals with a clinical diagnosis of VHL disease (50).
Approximately 20% to 37% of patients have large or
partial germline deletions, 30% to 38% have missense
mutations, and 23% to 27% have nonsense or frameshift mutations (49,50). Intriguingly, clear genotypephenotype correlations have emerged indicating that
different VHL mutations predispose to particular
types of tumor development. Type 1 VHL disease
families tend to have deletions and truncations, and
these patients have a low risk of developing pheochromocytoma (53,54). Families affected by type 2
VHL disease, with a high risk of pheochromocytoma,
have missense mutations in the VHL gene (54), while
type 3 VHL families (autosomal recessive erythrocytosis) usually have a specific 598C>T mutation (51). It
is rare for specific groups of mutations to be so well
correlated with phenotypic manifestations and distinguishes VHL from many other tumor suppressor gene
syndromes, where a mutation causes loss of tumor
suppressor function with a single phenotypic result,
for example, retinoblastoma.
The great majority of affected individuals have
inherited an inactivated VHL allele, with de novo
mutations accounting for only about 20% of cases
(55). Somatic inactivation of the remaining wild-type
VHL allele is necessary to initiate the tumorigenic
process in VHL disease. Thus at a cellular level,
VHL disease is recessive requiring a second hit mutation, which more often than not is a deletion (56), for
phenotypic manifestations to occur.
The VHL Tumor Suppressor Gene
in Sporadic CRCC Tumorigenesis
Soon after the VHL gene was isolated from studying

VHL families (52), it was shown that the VHL gene is
mutated in 50% to 60% of sporadically occurring
CRCC (57) and silenced by hypermethylation in a
further 19% of cases (58). Human renal cancer cell
lines isolated from sporadic CRCC usually have
defects in VHL function, and reexpression of a wildtype VHL gene is effective at suppressing their growth
as tumors in nude mice (59). Thus, VHL is a classical
two-hit gatekeeper tumor suppressor gene (TSG),
conforming to Knudsons two-hit criteria, and is conclusively implicated in the majority of sporadic and
hereditary CRCCs (Table 4).
The Function of the VHL Tumor Suppressor Gene Product
The VHL gene contains three exons, the mRNA is

ubiquitously expressed and encodes two proteins
with relative molecular masses of approximately
30kDa (pVHL30) and 19kDa (pVHL19). The smaller
polypeptide arises as a result of an alternate translation initiation site at Met 54 of pVHL30 (60,61).
The VHL proteins can be found in both the
nucleus and the cytoplasm, where they have been
variously reported to be associated with the endoplasmic reticulum, microtubules and mitochondria (6264).
A larger proportion of pVHL19 is found in the nucleus
compared with pVHL30, although the relevance of
366
Tran et al.
Table 3 Familial RCC: Syndrome, Gene Implicated, Renal Tumor Subtype, and Clinical Features
Syndrome
Gene
Gene location
Renal tumor subtype
Other clinical features
Von HippelLindau
VHL
3p25
CRCC
Retinal hemangiomas, CNS

haemangioblastomas,
pheochromocytoma
Chromosomal 3
translocations
FHIT
3p14
CRCC
RASSF1A
unknown
c-MET
3p21
unknown
7q31.1-4
CRCC
CRCC
PRCC (type 1)
FH
1q42-43
PRCC (type 2)
BDH
17p11.2
unknown
1q21-31
chromophobe RCC
oncocytoma
PRCC
TSC1
TSC2
9q34
16p13
CRCC
CRCC
Familial CRCC
Hereditary papillary RCC
(HPRC1)
Hereditary Leimyomatosis
RCC (HPRC2)
Birt-Hogg-Dube
Hyperparathyroidism-jaw
tumor
Tuberous sclerosis
Uterine leimyosarcoma,
cutaneous leiomyomata
Multiple lipoma
Pulmonary cysts
Parathyroid adenoma/carcinoma
Ossifying jaw tumors
Angiomyolipoma
seizures, mental retardation
Table 4 Common Chromosomal Abnormalities Detected in Sporadic RCC

RCC subtype
DNA losses
DNA gains
Chromosomal rearrangements
Conventional (rearrangements)
Papillary
Chromophobe
Oncocytoma
3, 14
Y
1,2,6,10,13,17,21,Y
1
3q, 7,12,16,17,20
-
3p (translocations), 5q
t(X;1) translocation
11q13 region (translocation)
subcellular localization and/or trafficking of VHL is

not yet clear. Both forms of pVHL are biologically
active and behave similarly in most biochemical
assays.
The complex genotype-phenotype associations
observed in VHL disease (see above) indicate that
the VHL gene product has multiple and tissue-specific
functions (65). It is clear that the tumor suppressor
role of VHL is that of a gatekeeper. It satisfies genetic
criteria since inactivation of VHL is a rate-limiting
step in CRCC development, and it also satisfies functional criteria as reintroduction of a wild-type VHL
allele suppresses the ability of VHL-defective cells to
form tumor xenografts in nude mice (59).
The best-established function of VHL is its
central role in cellular oxygen sensing (66). The
VHL gene product has been shown to be the recognition component of a multiprotein complex composed of elongins B/C, cul-2 and Rbx (6770). In
normoxic conditions, this functions as an E3 ubiquitin ligase that binds and targets the prolylhydroxylated hypoxia-inducible factor (HIF) a subunits for polyubiquitination. The polyubiquitintagged HIF a subunits are subsequently captured
by the 26S proteosome for degradation.
The transcription factor HIF is a master regulator
of the cells adaptive response to reduced oxygen
availability and has a key role in the regulation of
genes involved in glycolytic pathways and angiogenesis (71). A HIF complex consists of a heterodimer of
an oxygen regulated a subunit and a constitutively
expressed stable b subunit (72). Inactivation of
pVHL results in elevated intracellular levels of HIFa,
allowing dimerization with the b subunit and formation of an active HIF complex that can then drive the
transcription of target genes including VEGFA and
EPO (7375). This results in broadly adaptive changes
in gene expression that normally occur in response to
hypoxia, including promoting glucose uptake and
angiogenesis (66). There is an abundance of evidence
indicating that the HIF transcriptional pathway is
involved in mediating at least some significant aspects
of renal cancer tumorigenesis (24,7680). Consequences of HIF activation include constitutively high levels
of glycolytic enzyme expression and angiogenic
growth factor signaling thus providing a plausible
explanation for the highly vascular nature of VHL
associated tumors.
There are two main isoforms of HIF, HIF1a and
HIF2a. Interestingly, the role of HIF2a seems to be
unusually prominent relative to HIF1a in VHLdefective renal cancer (78,80,81). Potential insight
into HIFs role in CRCC development also comes
from genotype-phenotype studies. To date, all CRCC
associated VHL mutations disrupt the ability of pVHL
to regulate HIF (53). However, some mutations which
deregulate HIF carry a low risk of CRCC (type 2A or
type 3 VHL disease), suggesting that HIF dysregulation may be necessary but not sufficient to drive
CRCC development.
Other putative VHL pathways have been shown
to be involved in the tumor suppressor action of VHL
in the kidney. For example, pVHL has been shown to
have a direct role in fibronectin matrix assembly (82),
and interacts with the oncogenic b-catenin signaling
pathway (83). Biochemical studies have suggested that
367
Figure 2 Schematic diagram showing the role of VHL in HIF regulation and other putative interactions such as with Atypical protein
kinase C, VDU 1 and 2, RNA polymerase II subunits, fibronectin assembly, and the b-catenin signaling pathway.
pVHL has other protein substrates such as atypical

protein kinase C, de-ubiquinating enzymes (VDU-1
and VDU-2) and RNA polymerase II subunits (8486).
The function of the VHL protein is summarized in
Figure 2.
Chromosome 3 translocations, VHL-competent
CRCC and non-VHL-familial CRCC. An inherited predisposition to CRCC has been associated with balanced
translocations involving the short arm of chromosome
3, although this is rare. Several reports have identified a
variety of chromosomal breakpoints such as t(3;8)(p14,
q24) (87), t(3;8)(p13,q24.1) (88), t(3;6)(p13;q25) (89),
t(2;3)(q35;q21) (90) and t(3;12)(q35;q21) (89). Analysis
of tumors from patients with chromosome 3 translocations has demonstrated loss of the derivative chromosome by random nondisjunction, and somatic
mutation of the VHL gene in the remaining chromosome, rendering these tumors VHL null (90,91).
A common feature of CRCC, regardless of VHL
status, is chromosome 3p allele loss (92). Loss of
heterozygosity analysis in sporadic CRCC samples
has implicated several candidate TSG sites: 3p21.3
(lung cancer tumor suppressor gene region 1
LCTSGR1), 3p12 (LCTSGR2) and 3p14 (fragile histidine
triad gene-FHIT) (92,93). These studies are consistent
with experiments showing suppression of renal tumor
cell growth mediated by transfer of fragments of chromosome 3p regions not containing the VHL gene (94).
RAS association domain family 1A (RASSF1A)
gene which resides at 3p21.3 has been shown to be
frequently hypermethylated or epigenetically silenced

in both VHL-defective and VHL-competent CRCC
(9597). Interestingly, hypermethylation of RASSF1A
has also been reported in 40% of PRCC, in the absence
of deletions of Chromosome 3p (96). Taken together,
these findings indicate that additional non-VHL TSGs
residing on chromosome 3p have crucial roles in renal
cancer development in both VHL-defective and VHLcompetent CRCC.
Families with high risk of CRCC but without any
other manifestation of VHL disease and that do not
harbor VHL germline mutations, are very rare and they
are termed non-VHL familial CRCC (FCRC). They have
a dominantly inherited susceptibility to CRCC but the
molecular mechanisms underlying this are still unclear
and they do not have evidence of mutations in VHL,
MET, CUL2 or chromosome 3 translocations (98).
The MET proto-oncogene and familial type 1 papillary RCC (HPRC1). In a similar way to CRCC, studies
of families with hereditary forms of PRCC have provided insights into the underlying genetic pathways
(13,99). Hereditary papillary renal carcinoma (HPRC)
is a rare dominantly hereditary form of type 1 PRCC
of reduced penetrance, affecting approximately 1 per
10 million of the population. It is characterized by the
development of multiple type 1 PRCC, affecting both
kidneys. Genetic linkage studies on HPRC kindreds
led to the identification of the proto-oncogene c-MET
on chromosome 7 as the gene responsible for HPRC
(100). HPRC associated tumors are also characterized
368
Tran et al.
by trisomy 7 caused by nonrandom duplication of the

chromosome bearing the mutated MET gene (101).
Activating missense mutations have been found in the
tyrosine kinase domain of c-MET in both the germline
of affected individuals with HPRC, and in 13% of
patients with sporadic type 1 PRCC (102).
c-MET acts as a cell-surface receptor for the hepatocyte growth factor/scatter factor (HGF/SF), and normally is involved in diverse biological events such as
cell growth, migration and morphogenesis. Upon stimulation by HGF/SF, the c-MET receptor kinase undergoes autophosphorlyation on multiple tyrosine residues
and recruits a number of signal transducers and adapter
proteins to its cytoplasmic domain and multiple docking sites (such as growth factor receptor-bound protein 2
(Grb2), Gab1, phosphatidylinositol 3-kinase (PI3K),
phospholipase C-g, Shc and Src) (103). This results in
the activation of several downstream signaling pathways resulting in pleotropic effects. For example, the
direct binding of Grb2 to c-MET activates the Ras/
mitogen-activated pathway regulating cell-cycle progression (103) and Gab1 is involved in initiating morphogenesis, cell motility and apoptosis (104).
The Fumurate Hydratase Gene and Hereditary Leiomyomatosis
Renal Cell Carcinoma/Familial Type 2 Papillary RCC (HPRC2)
Hereditary leiomyomatosis renal cell carcinoma

(HLRCC) is an autosomal dominant hereditary form
of type 2 PRCC accompanied by uterine fibroids and
cutaneous leiomyomatosis lesions. HLRCC patients in
contrast to VHL and HPRC1 patients may have solitary tumors, although bilaterality and multifocality
has recently been reported (105). The renal tumors in
HLRCC are significantly more aggressive than other
hereditary forms of kidney cancer, with even small
tumors being associated with nodal and metastatic
disease.
HLRCC is caused by mutations in the gene that
codes for the Krebs cycle enzyme, fumarate hydratase
(FH) (106). The FH gene has been mapped to chromosome 1q42.3-q43 and mutations are thought to be
inactivating and include both deletions and missense
mutations (107). FH satisfies some important criteria
of a tumor suppressor gene, as germline mutations are
found in HLRCC kindreds and loss of the second
allele has been detected in kidney tumor tissue from
patients with HLRCC (108).
The molecular mechanism underlying HLRCC
tumorigenesis and the role of FH mutations in sporadic kidney cancer is not yet defined, although FH
has recently been shown to increase the stability of the
transcription factor HIF suggesting a VHL and hypoxia independent mechanism for HIF accumulation in
HLRCC renal tumors (109).
Birt-Hogg-Dube gene and familial chromophobe
RCC/renal oncocytoma. Birt-Hogg-Dube (BHD) syndrome is an autosomal dominant hereditary cancer
syndrome, in which affected individuals have susceptibility to develop hamartomatous tumors of the hair
follicle (fibrofolliculloma), pulmonary cysts, and bilateral, multifocal renal tumors (110112). The renal
tumors that can occur in BHD are varied, they may
be chromophobe RCC (33%), mixed chromophobe/

oncocytic (50%), oncocytoma (7%) or conventional
RCC (5%). Genetic linkage studies in BHD kindreds
led to the localization and identification of the BHD
gene on chromosome 17p11.2 in 2002 (113). BHD
mutations can be variable and include frameshift or
nonsense mutations, although insertion or deletion of
a cytosine in exon 11 has been detected in over half of
BHD families (114). Most mutations are predicted to
truncate the BHD protein, folliculin, which is highly
conserved across species. Somatic BHD mutations or
loss of heterozygosity at the BHD locus has been
detected in 70% of RCCs from BHD patients supporting its probable tumor suppressor function (115).
Further studies on the genotype-phenotype correlations and characterization of BHD mutations are
required to better understand the molecular mechanisms of BHD tumorigenesis.
Familial PRCC and hyperparathyroidism-jaw tumor
syndrome/familial papillary thyroid cancer. Familial
hyperparathyroidism-jaw tumor syndrome is an autosomal dominant inherited disease characterized by
primary hyperparathyroidism due to either parathyroid adenoma or carcinoma, and ossifying fibroma of
the jaw, and the gene has been mapped to chromosome
1q21-q31 (116). Affected individuals commonly have
renal cysts, but renal cortical adenoma and type 1
PRCC has also been described in a large kindred (117).
Malchoff et al. (118) described PRCC arising in
two family members with familial papillary thyroid
cancer, and linkage analysis in this family mapped the
gene to chromosome 1q21. Therefore, evidence from
these two syndromes suggests the presence of an as
yet unidentified PRCC susceptibility locus in the 1q.21
region.
Tuberous sclerosis. Tuberous sclerosis (TSC) is an
autosomal dominant inherited syndrome characterized by seizures, mental retardation, and multiple
hamartomas. Renal manifestations of TSC occur in
over half of affected individuals and include angiomyolipoma (AML) in 85%, renal cysts in 45%, and
renal cell carcinoma in 4% (119). AMLs are yellow and
gray in color, and composed microscopically of adipocytes, sheets of smooth muscle and blood vessels.
Two genes implicated in TSC have been identified,
TSC1 and TSC2, and these have been mapped to
chromosomes 9q34 (120) and 16p13.3, respectively
(121). TSC1 encodes a protein called hamartin, which
is involved in cell adhesion through the ezrin-radixinmoesin (ERNM) family of actin binding proteins (122),
and TSC2 encodes tuberin, a GTPase activating protein for Rap1, a member of the Ras-related superfamily (123). Both AML and renal cysts are more
common and numerous in patients with TSC2 mutations compared with TSC1 (119).
Genetic Insights and Molecular

Targeting Enable the Development
of New Therapeutic Strategies in RCC
RCC has an excellent prognosis if detected in the early
organ-confined stages, with surgical excision usually
being curative therapy in stages I-III. However,
approximately a third of patients present with metastatic disease, which has a dismal prognosis with a
median survival of 6 to 10 months and 10% to 20%
survival at two years (124). RCC is notorious for being
largely chemotherapy and radiotherapy resistant.
Many systemic agents have been trialled, including
capecitabine, thalidomide, fluorouracil and medroxyprogesterone acetate, and have yielded disappointing
results (125,126). This is thought to be due to high
levels of expression of the multidrug resistant gene
(MDR) product P-glycoprotein by renal tumor cells.
Radiotherapy is ineffective as suggested by in vitro
studies which have shown renal cancer cells to be the
least radiosensitive of 76 human cell types (127), so its
use is mainly limited to palliative treatment.
Until very recently, IFN-a and/or IL-2 have been
the only widely established effective treatment in
advanced renal cancer, with high-dose IL-2 being
shown to have an overall response rate of 15% to 20%
(128,129).
Improved understanding of the genetic basis and
the activating mechanisms underlying renal cancer
has contributed to the development of much needed
new approaches in the management of advanced
RCC. There has been an abundance of research since
the elucidation of the role of the VHL-HIF pathway in
renal tumorigenesis. Activation of the transcription
factor HIF mediates the transcription of target genes
normally involved in the cellular response to hypoxic
stress, including VEGFA, EGFR, TGF-a and EPO. HIF
has also been shown to be upregulated by other
growth factor and cell signaling pathways, including
the PI3K/AKT/mTOR and Ras/Raf/MAPK pathways. Various treatment strategies targeting both
upstream and downstream of these signaling pathways have yielded exciting results. Bevacizumab is a
neutralizing antibody to VEGFA, and was the first of
the new line of molecular therapies that has been
shown to be effective in advanced RCC, significantly
prolonging the time to progression (4.8 months compared with 2.5 months placebo) (130). Sunitinib is an
inhibitor of multiple tyrosine kinases including
VEGFR and PDGF. It has the added advantage of
being orally administered, and has shown encouraging results in a recent large phase III trial, with a mean
progression-free survival of 11 months compared with
5 months in the IFN treated patients (131). Sorafenib is
another multitarget kinase inhibitor, having VEGFR,
PDGF and Raf signaling inhibitory effects; and has
also shown positive results in a large randomized
phase III trial, with significant progression-free survival benefit of 24 weeks in treated patients compared
with 12 weeks in the placebo group (132). Other
promising new treatments include Temsirolimus and
the orally active Everolimus, which are inhibitors of
the mTOR pathway which is upstream of HIF (133).
SUMMARY
Advances in molecular techniques have allowed the
elucidation of specific genetic events involved in the
development of RCC. Our increased understanding of
369
the genetic basis of kidney cancer has enabled the

diagnostic classification of renal tumors into specific
subtypes on the basis of clinical, histopathological,
and genetic criteria. It is now clear that each RCC
subtype represents a distinct entity characterized
by specific genetic alterations leading to dysregulation
of critical molecular pathways underlying their
tumorigenesis.
Further progress in dissecting the genetic basis
of renal tumorigenesis will no doubt identify other
putative RCC genes, and clarifying their functional
roles will increase our understanding further. So far,
our studies of familial renal tumors have provided
significant insight into the molecular pathways
involved in both inherited and sporadic RCC. This
has already resulted in the development of exciting
therapeutic strategies in the form of small molecular
inhibitors in the management of advanced RCC, and
will undoubtedly continue to direct future novel
therapies which will ultimately improve our diagnosis
and management of familial and nonfamilial RCC.
REFERENCES
1. Murai M, Oya M. Renal cell carcinoma: etiology, incidence
and epidemiology. Curr Opin Urol 2004; 14(4):229233.
2. Chow WH, Devesa SS, Warren JL, et al. Rising incidence
of renal cell cancer in the United States. JAMA 1999;
281(17):16281631.
3. Volpe A, Jewett MA. The natural history of small renal
masses. Nat Clin Pract Urol 2005; 2(8):384390.
4. Bergstrom A, Pisani P, Tenet V, et al. Overweight as an
avoidable cause of cancer in Europe. Int J Cancer 2001; 91(3):
421430.
5. McLaughlin JK, Lipworth L, Tarone RE. Epidemiologic
aspects of renal cell carcinoma. Semin Oncol 2006; 33(5):
527533.
6. Setiawan VW, Stram DO, Nomura AM, et al. Risk factors
for renal cell cancer: the multiethnic cohort. Am J Epidemiol 2007; 166(8):932940.
7. Kovacs G, Akhtar M, Beckwith BJ, et al. The Heidelberg
classification of renal cell tumours. J Pathol 1997; 183(2):
131133.
8. Storkel S, Eble JN, Adlakha K, et al. Classification of renal
cell carcinoma: Workgroup No. 1. Union Internationale
Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997; 80(5):987989.
9. Eble JN, Sauter G, Epstein JI, et al. Pathology and Genetics. Tumors of the Urinary System and Male Genital
Organs. Lyon: IARC Press 2004.
10. Thoenes W, Storkel S, Rumpelt HJ, et al. Cytomorphological typing of renal cell carcinomaa new approach.
Eur Urol 1990; 18 (suppl 2):69.
11. Moch H, Gasser T, Amin MB, et al. Prognostic utility of
the recently recommended histologic classification and
revised TNM staging system of renal cell carcinoma: a
Swiss experience with 588 tumors. Cancer 2000; 89(3):
604614.
12. Amin MB, Amin MB, Tamboli P, et al. Prognostic impact
of histologic subtyping of adult renal epithelial neoplasms: an experience of 405 cases. Am J Surg Pathol
2002; 26(3):281291.
13. Linehan WM, Walther MM, Zbar B. The genetic basis of
cancer of the kidney. J Urol 2003; 170(6 pt 1):21632172.
14. Xipell JM. The incidence of benign renal nodules (a
clinicopathologic study). J Urol 1971; 106(4):503506.
370
Tran et al.
15. Grignon DJ, Eble JN. Papillary and metanephric adenomas of the kidney. Semin Diagn Pathol 1998; 15(1):4153.
16. Wang KL, Weinrach DM, Luan C, et al. Renal papillary
adenomaa putative precursor of papillary renal cell carcinoma. Hum Pathol 2007; 38(2):239246.
17. Yoshioka K, Miyakawa A, Ohno Y, et al. Production of
erythropoietin and multiple cytokines by metanephric
adenoma results in erythrocytosis. Pathol Int 2007; 57(8):
529536.
18. Zippel L. Zur Kenntnis der Oncocytem. Virch Arch 1942;
308:360.
19. Kondo T, Nakazawa H, Sakai F, et al. Spoke-wheel-like
enhancement as an important imaging finding of chromophobe cell renal carcinoma: a retrospective analysis on
computed tomography and magnetic resonance imaging
studies. Int J Urol 2004; 11(10):817824.
20. Dechet CB, Bostwick DG, Blute ML, et al. Renal oncocytoma: multifocality, bilateralism, metachronous tumor
development and coexistent renal cell carcinoma. J Urol
1999; 162(1):4042.
21. Perez-Ordonez B, Hamed G, Campbell S, et al. Renal
oncocytoma: a clinicopathologic study of 70 cases. Am
Jo Surg Pathol 1997; 21(8):871883.
22. Storkel S, Pannen B, Thoenes W, et al. Intercalated cells as
a probable source for the development of renal oncocytoma. Virchows Arch B Cell Pathol Incl Mol Pathol 1988;
56(3):185189.
23. Amin MB, Crotty TB, Tickoo SK, et al. Renal oncocytoma: a reappraisal of morphologic features with clinicopathologic findings in 80 cases. Am J Surg Pathol
1997; 21(1):112.
24. Mandriota SJ, Turner KJ, Davies DR, et al. HIF activation
identifies early lesions in VHL kidneys: evidence for sitespecific tumor suppressor function in the nephron. Cancer Cell 2002; 1(5):459468.
25. Esteban MA, Tran MG, Harten SK, et al. Regulation of
E-cadherin expression by VHL and hypoxia-inducible
factor. Cancer Res 2006; 66(7):35673575.
26. Webster WS, Thompson RH, Cheville JC, et al. Surgical
resection provides excellent outcomes for patients with
cystic clear cell renal cell carcinoma. Urology 2007; 70(5):
900904; discussion 4.
27. Lopez-Beltran A, Scarpelli M, Montironi R, et al. 2004
WHO classification of the renal tumors of the adults. Eur
Urol 2006; 49(5):798805.
28. Renshaw AA. Subclassification of renal cell neoplasms: an
update for the practising pathologist. Histopathology
2002; 41(4):283300.
29. Avery AK, Beckstead J, Renshaw AA, et al. Use of antibodies to RCC and CD10 in the differential diagnosis of
renal neoplasms. Am J Surg Pathol 2000; 24(2):203210.
30. Liu L, Qian J, Singh H, et al. Immunohistochemical
analysis of chromophobe renal cell carcinoma, renal
oncocytoma, and clear cell carcinoma: an optimal and
practical panel for differential diagnosis. Arch Pathol Lab
Med 2007; 131(8):12901297.
31. Renshaw AA, Corless CL. Papillary renal cell carcinoma.
Histology and immunohistochemistry. Am J Surg Pathol
1995; 19(7):842849.
32. Farivar-Mohseni H, Perlmutter AE, Wilson S, et al. Renal
cell carcinoma and end stage renal disease. The Journal of
urology 2006; 175(6):20182020; discussion 21.
33. Blei CL, Hartman DS, Friedman AC, et al. Papillary renal
cell carcinoma: ultrasonic/pathologic correlation. J Clin
Ultrasound 1982; 10(9):429434.
34. Press GA, McClennan BL, Melson GL, et al. Papillary
renal cell carcinoma: CT and sonographic evaluation. AJR
Am J Roentgenol 1984; 143(5):10051009.
35. Bostwick DG, Murphy GP. Diagnosis and prognosis of

renal cell carcinoma: highlights from an international
consensus workshop. Semin Urol Oncol 1998; 16(1):4652.
36. Pignot G, Elie C, Conquy S, et al. Survival analysis of 130
patients with papillary renal cell carcinoma: prognostic
utility of type 1 and type 2 subclassification. Urology
2007; 69(2):230235.
37. Amin MB, Corless CL, Renshaw AA, et al. Papillary
(chromophil) renal cell carcinoma: histomorphologic
characteristics and evaluation of conventional pathologic
prognostic parameters in 62 cases. Am J Surg Pathol 1997;
21(6):621635.
38. Crotty TB, Farrow GM, Lieber MM. Chromophobe cell
renal carcinoma: clinicopathological features of 50 cases.
J Urol 1995; 154(3):964967.
39. Nguyen GK, Schumann GB. Cytopathology of renal collecting duct carcinoma in urine sediment. Diagn Cytopathol 1997; 16(5):446449.
40. Kennedy SM, Merino MJ, Linehan WM, et al. Collecting
duct carcinoma of the kidney. Hum Pathol 1990;
21(4):449456.
41. Srigley JR, Eble JN. Collecting duct carcinoma of kidney.
Semin Diagn Pathol 1998; 15(1):5467.
42. Kim MK, Kim S. Immunohistochemical profile of common epithelial neoplasms arising in the kidney. Appl
Immunohistochem Mol Morphol 2002; 10(4):332338.
43. Cheville JC, Lohse CM, Zincke H, et al. Sarcomatoid renal
cell carcinoma: an examination of underlying histologic
subtype and an analysis of associations with patient outcome. Am J Surg Pathol 2004; 28(4):435441.
44. de Peralta-Venturina M, Moch H, Amin M, et al. Sarcomatoid differentiation in renal cell carcinoma: a study of
101 cases. Am J Surg Pathol 2001; 25(3):275284.
45. Collins E. Intra-ocular growths (two cases, brother and
sister, with peculiar vascular new growth, probably retinal, affecting both eyes). Trans Ophthalmol Soc UK 1894;
14:141149.
46. Hippel Ev. Ueber eine sehr seltene Erkrankung der
Nethaut. Graefe Arch Ophthalmol 1904; 59:83106.
47. lindau A. Zur frage der angiomatosis retinai und ihrer
hirncomplikation. Acta Ophthalmol 1927; 4:193226.
48. Davison C BS, Cyke CG. Retinal and central nervous
haemangioblastomatosis with visceral changes (von
Hippel-Lindaus disease). Bull Neurol Inst New York
1936; 5:7293.
49. Maher ER, Kaelin WG Jr. von Hippel-Lindau disease.
Medicine (Baltimore) 1997; 76(6):381391.
50. Stolle C, Glenn G, Zbar B, et al. Improved detection of
germline mutations in the von Hippel-Lindau disease
tumor suppressor gene. Hum Mutat 1998; 12(6):417423.
51. Ang SO, Chen H, Gordeuk VR, et al. Endemic polycythemia in Russia: mutation in the VHL gene. Blood Cells Mol
Dis 2002; 28(1):5762.
52. Latif F, Tory K, Gnarra J, et al. Identification of the von
Hippel-Lindau disease tumor suppressor gene. Science
1993; 260(5112):13171320.
53. Clifford SC, Cockman ME, Smallwood AC, et al. Contrasting effects on HIF-1alpha regulation by diseasecausing pVHL mutations correlate with patterns of
tumourigenesis in von Hippel-Lindau disease. Hum
Mol Genet 2001; 10(10):10291038.
54. Chen F, Kishida T, Duh FM, et al. Suppression of growth
of renal carcinoma cells by the von Hippel-Lindau tumor
suppressor gene. Cancer Res 1995; 55(21):48044807.
55. Sgambati MT, Stolle C, Choyke PL, et al. Mosaicism in
von Hippel-Lindau disease: lessons from kindreds with
germline mutations identified in offspring with mosaic
parents. Am J Hum Genet 2000; 66(1):8491.
56. Glasker S, Sohn TS, Okamoto H, et al. Second hit deletion
size in von Hippel-Lindau disease. Ann Neurol 2006; 59(1):
105110.
57. Gnarra JR, Tory K, Weng Y, et al. Mutations of the VHL
tumour suppressor gene in renal carcinoma. Nat Genet
1994; 7(1):8590.
58. Herman JG, Latif F, Weng Y, et al. Silencing of the VHL
tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 1994; 91(21):97009704.
59. Iliopoulos O, Kibel A, Gray S, et al. Tumour suppression
by the human von Hippel-Lindau gene product. Nat Med
1995; 1(8):822826.
60. Iliopoulos O, Ohh M, Kaelin WG Jr. pVHL19 is a biologically active product of the von Hippel-Lindau gene
arising from internal translation initiation. Proc Natl Acad
Sci U S A 1998; 95(20):1166111666.
61. Schoenfeld A, Davidowitz EJ, Burk RD. A second major
native von Hippel-Lindau gene product, initiated from
an internal translation start site, functions as a tumor
suppressor. Proc Natl Acad Sci U S A 1998; 95(15):
88178822.
62. Shiao YH, Resau JH, Nagashima K, et al. The von HippelLindau tumor suppressor targets to mitochondria. Cancer
Res 2000; 60(11):28162819.
63. Schoenfeld AR, Davidowitz EJ, Burk RD. Endoplasmic
reticulum/cytosolic localization of von Hippel-Lindau
gene products is mediated by a 64-amino acid region.
Int J Cancer 2001; 91(4):457467.
64. Hergovich A, Lisztwan J, Barry R, et al. Regulation of
microtubule stability by the von Hippel-Lindau tumour
suppressor protein pVHL. Nat Cell Biol 2003; 5(1):6470.
65. Clifford SC, Maher ER. Von Hippel-Lindau disease: clinical and molecular perspectives. Adv Cancer Res 2001;
82:85105.
66. Maxwell PH, Wiesener MS, Chang GW, et al. The tumour
suppressor protein VHL targets hypoxia-inducible factors
for oxygen-dependent proteolysis. Nature 1999; 399(6733):
271275.
67. Iwai K, Yamanaka K, Kamura T, et al. Identification of the
von Hippel-lindau tumor-suppressor protein as part of an
active E3 ubiquitin ligase complex. Proc Natl Acad Sci U S
A 1999; 96(22):1243612441.
68. Kamura T, Koepp DM, Conrad MN, et al. Rbx1, a component of the VHL tumor suppressor complex and SCF
ubiquitin ligase. Science 1999; 284(5414):657661.
69. Lisztwan J, Imbert G, Wirbelauer C, et al. The von HippelLindau tumor suppressor protein is a component of an E3
ubiquitin-protein ligase activity. Genes Dev 1999; 13(14):
18221833.
70. Cockman ME, Masson N, Mole DR, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von
Hippel-Lindau tumor suppressor protein. J Biol Chem
2000; 275(33):2573325741.
71. Maxwell PH. Hypoxia-inducible factor as a physiological
regulator. Exp Physiol 2005; 90(6):791797.
72. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible
factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A
1995; 92(12):55105514.
73. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular
endothelial growth factor gene transcription by hypoxiainducible factor 1. Mol Cell Biol 1996; 16(9): 46044613.
74. Beck I, Ramirez S, Weinmann R, et al. Enhancer element
at the 30 -flanking region controls transcriptional response
to hypoxia in the human erythropoietin gene. J Biol Chem
1991; 266(24):1556315566.
75. Semenza GL, Nejfelt MK, Chi SM, et al. Hypoxiainducible nuclear factors bind to an enhancer element
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
371
located 30 to the human erythropoietin gene. Proc Natl

Acad Sci U S A 1991; 88(13):56805684.
Krieg M, Haas R, Brauch H, et al. Up-regulation of
hypoxia-inducible factors HIF-1alpha and HIF-2alpha
under normoxic conditions in renal carcinoma cells by
von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 2000; 19(48):54355443.
Kondo K, Klco J, Nakamura E, et al. Inhibition of HIF is
necessary for tumor suppression by the von HippelLindau protein. Cancer Cell 2002; 1(3):237246.
Turner KJ, Moore JW, Jones A, et al. Expression of
hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene
mutation. Cancer Res 2002; 62(10):29572961.
Zimmer M, Doucette D, Siddiqui N, et al. Inhibition of
hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors. Genetic and clinical aspects of
familial renal neoplasms. The molecular basis of von
Hippel-Lindau disease. Mol Cancer Res 2004; 2(2):8995.
Raval RR, Lau KW, Tran MG, et al. Contrasting properties
of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von
Hippel-Lindau-associated renal cell carcinoma. Mol Cell
Biol 2005; 25(13):56755686.
Kondo K, Kim WY, Lechpammer M, et al. Inhibition of
HIF2alpha is sufficient to suppress pVHL-defective tumor
growth. PLoS Biol 2003; 1(3):E83.
Ohh M, Yauch RL, Lonergan KM, et al. The von HippelLindau tumor suppressor protein is required for proper
assembly of an extracellular fibronectin matrix. Mol Cell
1998; 1(7):959968.
Peruzzi B, Athauda G, Bottaro DP. The von HippelLindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells. Proc
Natl Acad Sci U S A 2006; 103(39):1453114536.
Okuda H, Saitoh K, Hirai S, et al. The von Hippel-Lindau
tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. J Biol Chem 2001; 276(47):
4361143617.
Kuznetsova AV, Meller J, Schnell PO, et al. von HippelLindau protein binds hyperphosphorylated large subunit
of RNA polymerase II through a proline hydroxylation
motif and targets it for ubiquitination. Proc Natl Acad Sci
U S A 2003; 100(5):27062711.
Li Z, Wang D, Na X, et al. Identification of a deubiquitinating enzyme subfamily as substrates of the von HippelLindau tumor suppressor. Biochem Biophys Res Commun 2002; 294(3):700709.
Cohen AJ, Li FP, Berg S, et al. Hereditary renal-cell
carcinoma associated with a chromosomal translocation.
N Engl J Med 1979; 301(11):592595.
Melendez B, Rodriguez-Perales S, Martinez-Delgado B, et
al. Molecular study of a new family with hereditary renal
cell carcinoma and a translocation t(3;8)(p13;q24.1). Hum
Genet 2003; 112(2):178185.
Kovacs G, Hoene E. Loss of der(3) in renal carcinoma cells
of a patient with constitutional t(3;12). Hum Genet 1988;
78(2):148150.
Bodmer D, Eleveld MJ, Ligtenberg MJ, et al. An alternative route for multistep tumorigenesis in a novel case of
hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation. Am J Hum Genet 1998; 62(6):
14751483.
Schmidt L, Li F, Brown RS, et al. Mechanism of tumorigenesis of renal carcinomas associated with the constitutional chromosome 3;8 translocation. Cancer J Sci Am
1995; 1(3):191195.
Clifford SC, Prowse AH, Affara NA, et al. Inactivation of
the von Hippel-Lindau (VHL) tumour suppressor gene
372
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
Tran et al.
and allelic losses at chromosome arm 3p in primary renal
cell carcinoma: evidence for a VHL-independent pathway
in clear cell renal tumourigenesis. Genes Chromosomes
Cancer 1998; 22(3):200209.
Martinez A, Fullwood P, Kondo K, et al. Role of chromosome 3p12-p21 tumour suppressor genes in clear cell
renal cell carcinoma: analysis of VHL dependent and
VHL independent pathways of tumorigenesis. Mol Pathol
2000; 53(3):137144.
Sanchez Y, el-Naggar A, Pathak S, et al. A tumor suppressor locus within 3p14-p12 mediates rapid cell death
of renal cell carcinoma in vivo. Proc Natl Acad Sci U S A
1994; 91(8):33833387.
Dreijerink K, Braga E, Kuzmin I, et al. The candidate
tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis. Proc
Natl Acad Sci U S A 2001; 98(13):75047509.
Morrissey C, Martinez A, Zatyka M, et al. Epigenetic
inactivation of the RASSF1A 3p21.3 tumor suppressor
gene in both clear cell and papillary renal cell carcinoma.
Cancer Res 2001; 61(19):72777281.
Yoon JH, Dammann R, Pfeifer GP. Hypermethylation of
the CpG island of the RASSF1A gene in ovarian and renal
cell carcinomas. Int J Cancer 2001; 94(2):212217.
Woodward ER, Clifford SC, Astuti D, et al. Familial clear
cell renal cell carcinoma (FCRC): clinical features and
mutation analysis of the VHL, MET, and CUL2 candidate
genes. J Med Genet 2000; 37(5):348353.
Zbar B, Tory K, Merino M, et al. Hereditary papillary
renal cell carcinoma. J Urol 1994; 151(3):561566.
Schmidt L, Duh FM, Chen F, et al. Germline and somatic
mutations in the tyrosine kinase domain of the MET
proto-oncogene in papillary renal carcinomas. Nat Genet
1997; 16(1):6873.
Zhuang Z, Park WS, Pack S, et al. Trisomy 7-harbouring
non-random duplication of the mutant MET allele in
hereditary papillary renal carcinomas. Nat Genet 1998;
20(1):6669.
Schmidt L, Junker K, Nakaigawa N, et al. Novel mutations of the MET proto-oncogene in papillary renal carcinomas. Oncogene 1999; 18(14):23432350.
Zhang YW, Vande Woude GF. HGF/SF-met signaling in
the control of branching morphogenesis and invasion.
J Cell Biochem 2003; 88(2):408417.
Rosario M, Birchmeier W. How to make tubes: signaling
by the Met receptor tyrosine kinase. Trends Cell Biol 2003;
13(6):328335.
Grubb RL 3rd, Franks ME, Toro J, et al. Hereditary
leiomyomatosis and renal cell cancer: a syndrome associated with an aggressive form of inherited renal cancer.
J Urol 2007; 177(6):20742079; discussion 980.
Delahunt B, Eble JN. Papillary renal cell carcinoma: a
clinicopathologic and immunohistochemical study of 105
tumors. Mod Pathol 1997; 10(6):537544.
Tomlinson IP, Alam NA, Rowan AJ, et al. Germline
mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell
cancer. Nat Genet 2002; 30(4):406410.
Toro JR, Nickerson ML, Wei MH, et al. Mutations in the
fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. Am
J Hum Genet 2003; 73(1):95106.
Isaacs JS, Jung YJ, Mole DR, et al. HIF overexpression
correlates with biallelic loss of fumarate hydratase in
renal cancer: novel role of fumarate in regulation of HIF
stability. Cancer Cell 2005; 8(2):143153.
Birt AR, Hogg GR, Dube WJ. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch
Dermatol 1977; 113(12):16741677.
111. Toro JR, Glenn G, Duray P, et al. Birt-Hogg-Dube syndrome: a novel marker of kidney neoplasia. Arch Dermatol 1999; 135(10):11951202.
112. Zbar B, Alvord WG, Glenn G, et al. Risk of renal and
colonic neoplasms and spontaneous pneumothorax in the
Birt-Hogg-Dube syndrome. Cancer Epidemiol Biomarkers
Prev 2002; 11(4):393400.
113. Nickerson ML, Warren MB, Toro JR, et al. Mutations in a
novel gene lead to kidney tumors, lung wall defects, and
benign tumors of the hair follicle in patients with
the Birt-Hogg-Dube syndrome. Cancer Cell 2002;
2(2):157164.
114. Schmidt LS, Nickerson ML, Warren MB, et al. Germline
BHD-mutation spectrum and phenotype analysis of a
large cohort of families with Birt-Hogg-Dube syndrome.
Am J Hum Genet 2005; 76(6):10231033.
115. Vocke CD, Yang Y, Pavlovich CP, et al. High frequency of
somatic frameshift BHD gene mutations in Birt-HoggDube-associated renal tumors. J Natl Cancer Inst 2005;
97(12):931935.
116. Szabo J, Heath B, Hill VM, et al. Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine tumor
gene HRPT2 maps to chromosome 1q21-q31. Am J Hum
Genet 1995; 56(4):944950.
117. Haven CJ, Wong FK, van Dam EW, et al. A genotypic and
histopathological study of a large Dutch kindred with
hyperparathyroidism-jaw tumor syndrome. J Clin Endocrinol Metab 2000; 85(4):14491454.
118. Malchoff CD, Sarfarazi M, Tendler B, et al. Papillary
thyroid carcinoma associated with papillary renal neoplasia: genetic linkage analysis of a distinct heritable
tumor syndrome. J Clin Endocrinol Metabol 2000;
85(5):17581764.
119. Rakowski SK, Winterkorn EB, Paul E, et al. Renal manifestations of tuberous sclerosis complex: incidence, prognosis, and predictive factors. Kidney Int 2006;
70(10):17771782.
120. Fryer AE, Chalmers A, Connor JM, et al. Evidence that the
gene for tuberous sclerosis is on chromosome 9. Lancet
1987; 1(8534):659661.
121. Kandt RS, Haines JL, Smith M, et al. Linkage of an
important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. Nat Genet
1992; 2(1):3741.
122. Lamb RF, Roy C, Diefenbach TJ, et al. The TSC1 tumour
suppressor hamartin regulates cell adhesion through
ERM proteins and the GTPase Rho. Nat Cell Biol 2000;
2(5):281287.
123. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 1993; 75(7):13051315.
124. Campbell SC, Flanigan RC, Clark JI. Nephrectomy in
metastatic renal cell carcinoma. Curr Treat Options
Oncol 2003; 4(5):363372.
125. Yagoda A, Petrylak D, Thompson S. Cytotoxic chemotherapy for advanced renal cell carcinoma. Urol Clin
North Am 1993; 20(2):303321.
126. Motzer RJ, Russo P. Systemic therapy for renal cell carcinoma. J Urol 2000; 163(2):408417.
127. Deschavanne PJ, Fertil B. A review of human cell radiosensitivity in vitro. Int J Radiat Oncol, Biol, Phys 1996;
34(1):251266.
128. Fyfe G, Fisher RI, Rosenberg SA, et al. Results of treatment of 255 patients with metastatic renal cell carcinoma
who received high-dose recombinant interleukin-2 therapy. J Clin Oncol 1995; 13(3):688696.
129. Yang JC, Sherry RM, Steinberg SM, et al. Randomized
study of high-dose and low-dose interleukin-2 in patients
with metastatic renal cancer. J Clin Oncol 2003; 21(16):
31273132.
130. Yang JC, Haworth L, Sherry RM, et al. A randomized trial
of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med
2003; 349(5):427434.
131. Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus
interferon alfa in metastatic renal-cell carcinoma. N Engl J
Med 2007; 356(2):1151124.
373
132. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in

advanced clear-cell renal-cell carcinoma. N Engl J Med
2007; 356(2):125134.
133. Atkins MB, Hidalgo M, Stadler WM, et al. Randomized
phase II study of multiple dose levels of CCI-779, a novel
mammalian target of rapamycin kinase inhibitor, in
patients with advanced refractory renal cell carcinoma.
J Clin Oncol 2004; 22(5):909918.
24
Transitional Cell Carcinoma of the Bladder
T. R. Leyshon Griffiths
Urology Group, Department of Cancer Studies and Molecular Medicine,
University of Leicester, Leicester, U.K.
Nick Mayer
University Hospitals of Leicester, Leicester, U.K.
INTRODUCTION
Each year in the United Kingdom, approximately
10,100 people are diagnosed with bladder cancer
and 4700 people die from the disease. The male to
female ratio is 5:2. Although it is ranked the fourth
most common cancer in men and the eleventh most
common cancer in women (1), bladder cancer is an
enormous burden on the health economy because of
its prevalence and duration. In the United Kingdom it
has been estimated that the cost of treatment for
bladder cancer to the National Health Service is
approximately 200 million per annum, making it
the most expensive cancer to treat (2).
The age-specific bladder cancer incidence rates
in Great Britain show a consistent decrease since the
early 1990s for all age groups under age 85. While
some of this can be attributed to coding changes,
reduction in smoking and reduced exposure to occupational carcinogens may also have played a role,
especially as mortality rates have also decreased (3).
In the developed world, transitional cell carcinoma
(TCC), rather than squamous or adenocarcinoma, is
responsible for 90% of bladder cancers. About 25% of
newly diagnosed cancers are muscle invasive (T2T4);
the remainder are nonmuscle invasive (70%) and classified as noninvasive (pTa), lamina propriainvasive
(pT1), or in situ (Tis [CIS, carcinoma in situ]5%).
ETIOLOGY
Smoking
Smoking cigarettes is the principal preventable risk
factor for bladder cancer in both men and women. In
Europe, it is estimated that up to half the bladder
cancer cases in men and a third in women are caused
by cigarette smoking (4,5). A causal relationship has
been established between an exposure to tobacco and
cancer studies in which chance, bias, and confounding
factors can be controlled with reasonable confidence
(6). Current smokers have around three times the risk
of never-smokers of developing bladder cancer, while
ex-smokers have double the risk of never-smokers (7).

Risk positively associates with both increasing dose
and duration of smoking (4,5). Passive smoking may
also increase risk. In a recent European study, exposure
to environmental tobacco during childhood (but not
adulthood) increased the risk of bladder cancer (8).
Smoking cessation reduces risk by 40% within 1
to 4 years and reaches 60% after 25 years (4,5). The
alleged carcinogenic constituents of tobacco smoke
include arylamines (particularly 4-aminobiphenyl),
polycyclic aromatic hydrocarbons (PAHs), N-nitroso
compounds, heterocyclic amines, and various epoxides.
Studies show that risk varies by type of tobacco, with a
higher risk for black air-cured than blond fluecured tobacco. Smoking also leads to higher mortality
from bladder cancer during long-term follow-up (9).
Occupation
Occupational exposure to chemicals in Europe
accounts for up to 10% of male bladder cancers. Most
carcinogens have a latent period of 15 to 20 years
between exposure and the development of tumors.
The proportion may be higher in countries with lessregulated industrial processes. Bladder cancer has an
important place in the history of occupational disease.
In 1895, Rehn reported cases of bladder cancer in a
German aniline dye factory. In 1938, Hueper produced the first experimental evidence showing that
the aromatic amine, b-naphthylamine, could induce
bladder cancer in dogs (10). Following this and other
reports, a full epidemiological survey conducted by
Case showed that exposure to a-naphthylamine,
b-naphthylamine, or benzidine, rather than to aniline
itself, was the main factor associated with the development of bladder cancer (11). Aromatic amines were
widely used in the manufacture of dyes and pigments
for textiles, paints, plastics, paper and hair dyes, in
drugs, pesticides and as anticipated in the rubber
industry. In 1952, production of b-naphthylamine
ceased in the United Kingdom, and in 1953 bladder
cancer became a prescribed industrial disease (12).
Chapter 24: Transitional Cell Carcinoma of the Bladder
It is calculated that approximately 4% of bladder

cancer cases in European men are because of exposure
to PAHs, by-products of the catabolic process (13).
Hair Dyes
Occupational studies of hairdressers have produced
conflicting results. However, a recent study in Sweden
suggested that modern hair dyes are not carcinogenic
(14). Within the European Union, the Scientific Committee on Cosmetic Products and Non-food products
(ISCCNFP) aims to set up a high-risk permanent
and semipermanent register of hair dye formulations.
Drugs
In the 1950s and 1960s, analgesic abuse was rife in
Australia and New Zealand. Both upper tract and
bladder TCC were linked to the aniline derivative,
phenacetin. Cyclophosphamide has also been shown
to induce bladder cancer; the increased risk of TCC
has been calculated as ninefold (15). In comparison to
other carcinogenic agents, the latency period is relatively short. Acrolein, a metabolite of cyclophosphamide, is responsible for this increase, which is
independent of the occurrence of hemorrhagic cystitis.
Pelvic Irradiation
Patients who are treated with pelvic radiotherapy for
cervical carcinoma have a two- to fourfold increased
risk of developing bladder cancer (16). In patients
treated for prostate cancer, the incidence of bladder
cancer was significantly higher in patients treated
with external beam radiotherapy than those treated
with radical prostatectomy (17).
Food and Drink

Several dietary factors have been related to bladder
cancer, but the results of different studies have been
controversial. A meta-analysis of 38 articles supported
the hypothesis that vegetable and fruit intake reduced
the risk of bladder cancer (18).
Genetic Polymorphisms
Drug- and carcinogen-metabolizing enzymes are
important in the processing of lipophilic chemicals
to products that are more water soluble and can be
excreted. These enzymes systems are partly controlled
by genetic polymorphism. In the liver, chemicals are
oxidized by the cytochrome P450 superfamily and
detoxified by N-acetylation, predominantly by Nacetyltransferases (NAT). Aromatic amines are usually detoxified by NAT2. Certain allelic combinations
result in the slow acetylation phenotype. Studies have
suggested that people who carry the slow variant
are at increased risk of bladder cancer (relative risk
1.4), and this may be especially true in smokers (19).
Approximately 50% of Caucasians and 25% of Asians
are slow acetylators. Glutathione S-transferase (GST)
375
is the product of the GSTM1 gene and is involved in

the detoxification of PAHs. Approximately 50% of
Caucasians and Asians have a homozygous deletion
of GSTM1 gene, which is associated with a relative
risk of 1.4 (20).
PATHOLOGY
The urinary collecting system, which extends from the
renal calyces to the urethra, is lined by a specialized
epithelium with unique morphological and ultrastructural features known as transitional epithelium or
urothelium.
Tumors of the bladder are roughly 50 times more
common than those of the upper tracts, the vast majority (>90%) are TCCs that are usually pure but can
show a range of divergent differentiation including
squamous (20%), glandular (6%), and rarer forms
such as trophoblastic. Pure squamous carcinoma and
adenocarcinoma (urachal and nonurachal) also occur
but are rare. Primary bladder adenocarcinoma has to
be distinguished from direct or metastatic spread from
a variety of other sites, including the colorectum, prostate, ovary, breast, and stomach. This distinction usually requires clinical and radiological correlation.
Tumor Grade
For more than three decades the preferred grading
system in the United Kingdom for invasive and noninvasive TCC has been the World Health Organization
(WHO) 1973 classification (21), which has been repeatedly validated and shown to be of clinical relevance
for treatment and prognosis. WHO 1973 divides TCC
into three grades on the basis of cytological and
architectural disorder: grade 1 being well differentiated, grade 2 moderately differentiated, and grade 3
poorly differentiated. However, the classification has
been criticized for imprecise definitions, resulting in
poor reproducibility between pathologists and for
having the majority of tumors in the middle grade
(grade 2), which as a group therefore shows considerable variability in clinical behavior. In 1998, the
WHO/International Society of Urological Pathology
(ISUP) consensus classification was published by a
group of expert uropathologists (22), notably without
input from urologists, which was intended to replace
the original WHO 1973 classification. The 1998 classification has subsequently been adopted in the most
recent WHO 2004 classification (23). The main differences are two grades of carcinoma (high grade and
low grade) in the WHO 2004 classification and the
introduction of the term papillary urothelial neoplasm
of low malignant potential, to replace the best differentiated grade 1 tumors, avoiding the term carcinoma
for tumors with low risk of either recurrence or progression (Fig. 1). However, there has been considerable resistance in the United Kingdom to adopting the
WHO 2004 classification, which was not prospectively
validated prior to its introduction and has subsequently not demonstrated either improved reproducibility or clinical relevance over WHO 1973 (25). A
376
Griffiths and Mayer

Table 1 TNM Classification of Urinary Bladder Cancer (2002)
Figure 1 World Health Organization (WHO) 1973 versus 2004

classifications. Source: Adapted from Ref. 24.
particular uncertainty is whether all patients in the

high-grade group will benefit from intravesical BCG
therapy or be exposed to unnecessary side effects.
Current reporting guidelines therefore recommend
providing the urologist with both classifications
(26,27).
Tumor Stage
Bladder tumors are often separated into nonmuscle
invasive (CIS, pTa, pT1) or muscle invasive (T2, T3,
T4) to aid clinical management. Clinical stage is based
on a combination of histopathological assessment,
bimanual clinical examination, and imaging studies.
The International Union Against Cancer (UICC) 2002
is the most recent pathological TNM staging system
(Table 1) (28). For transurethral (TUR) resection specimens, showing lamina propria invasion (pT1), the
pathologists should provide some indication of the
extent and/or depth of invasion, as this correlates
with outcome and may influence whether to perform
a reresection. It is important for the pathologists to
comment on whether detrusor muscle is present in
pT1 tumors. Those lacking detrusor and/or those who
are high-grade normally require reresection because
of the significant risk of understaging. It is useful for
the urologists to send the deep or base biopsies in a
separate specimen pot, to aid the histological identification of detrusor muscle in TUR specimens. In
patients being considered for cystectomy with orthotopic urinary reconstruction, TUR biopsies from the
prostatic urethra are also usually submitted to exclude
CIS. It is of interest that of those apparently organconfined (clinical stage T2) tumors, 40% to 50% are
more advanced on pathological examination of the
radical cystectomy specimen.
Histological Variants
Several histological variants of TCC are now well
described that are relevant to prognosis and treatment. The micropapillary variant of TCC was first
described in 1994 (29) and histologically resembles
T (Primary tumor)
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
Ta Noninvasive papillary carcinoma
Tis Carcinoma in situ (flat tumor)
T1 Tumor invades subepithelial connective tissue
T2 Tumor invades muscle
T2a Tumor invades superficial muscle (inner half)
T2b Tumor invades deep muscle (outer half)
T3 Tumor invades perivesical tissue:
T3a Microscopically
T3b Macroscopically (extravesical mass)
T4 Tumor invades any of the following: prostate, uterus, vagina,
pelvic wall, abdominal wall
T4a Tumor invades prostate, uterus, or vagina
T4b Tumor invades pelvic wall or abdominal wall
N (Lymph nodes)
NX Regional lymph nodes cannot be assessed
N0 No regional lymph node metastasis
N1 Metastasis in a single lymph node 2 cm or less in greatest
dimension
N2 Metastasis in a single lymph node more than 2 cm but not
more than 5 cm in greatest dimension, or multiple lymph
nodes, none more than 5 cm in greatest dimension
N3 Metastasis in a lymph node more than 5 cm in greatest
dimension
M (Distant metastasis)
MX Distant metastasis cannot be assessed
M0 No distant metastasis
M1 Distant metastasis
ovarian serous carcinoma. It is rare, accounting for

about 1% of TCC with a strong male predominance. It
can involve either the surface or invasive components
of the tumor (Figs. 2 and 3). The micropapillary
phenotype is often associated with advanced clinical
stage at presentation, including a propensity for
lymph node metastasis (30). There is evidence that
for nonmuscle invasive micropapillary disease, BCG
therapy is often ineffective (31) and therefore radical
treatment should be offered early.
The nested variant of TCC also shows a marked
male predominance. It is unusual in having very
bland cytomorphology, particularly in the superficial
tumor (Fig. 4). However, it is usually clinically aggressive and often muscle invasive at presentation (32).
The sarcomatoid variant of TCC is a biphasic
tumor displaying malignant epithelial and mesenchymal morphologies. The latter can include divergent
differentiation into tissues not normally found in the
bladder such as bone, cartilage, and skeletal muscle,
so-called heterologous differentiation. There is sometimes a history of previous external beam radiotherapy or cyclophosphamide treatment. These tumors are
often advanced at presentation and are associated
with poor survival (33).
Small cell carcinoma (SCC) of the bladder is
morphologically identical to its pulmonary counterpart and usually neuroendocrine differentiation is
demonstrable with immunohistochemistry (Fig. 5).
In roughly half of cases there is coexistent TCC
Figure 2 Noninvasive micropapillary TCC showing the characteristic filiform arrangement of micropapillae, without fibrovascular cores. Note the high-grade cytomorphology that is typical of
this variant; there is also focal necrosis. Abbreviation: TCC,
transitional cell carcinoma.
Figure 3 Invasive micropapillary TCC comprising small compact aggregates of cells infiltrating the lamina propria and
demonstrating prominent lacunae (retraction spaces around
the invasive nests), which should not be confused with lymphovascular space invasion. Abbreviation: TCC, transitional cell
carcinoma.
and/or CIS and this has been cited as evidence for a

transitional cell origin (34), although others have
proposed alternative origins from stem cells or
bladder neuroendocrine cells. Metastatic disease is
usually present (*70% cases) at diagnosis, and the
disease is generally managed as a systemic disease
377
Figure 4 Nested variant of TCC. In small biopsies, because of

the bland cytological features, the invasive nests of malignant
cells in the lamina propria can be confused histologically with
hyperplastic Von Brunns nests or other benign transitional cell
proliferations. The overlying flat urothelium here is normal; note
that the cells in the invasive nests are very similar in size. There
is usually more obvious cellular atypia in the more deeply
invasive tumor, but this is not always represented in superficial
resections. Abbreviation: TCC, transitional cell carcinoma.
Figure 5 Small cell carcinoma. The cells have negligible cytoplasm, and as a consequence the nuclei are closely packed and
show prominent nuclear moulding. There is a high mitotic and
apoptotic rate, and although it is not demonstrated here, necrosis
is usually prominent. The tumors are typically positive with the
neuroendocrine immunohistochemical markers synaptophysin
and chromogranin-A and also with CD56 and TTF-1.
with similar cisplatin-based chemotherapy regime as

for pulmonary SCC. There is no apparent survival
difference for control of local disease between radiotherapy and radical cystectomy (35,36).
378
Griffiths and Mayer
Premalignant Conditions
Keratinizing squamous metaplasia (KSM) is rare and
sometimes referred to clinically as leukoplakia. It is
usually associated with some form of chronic inflammatory process, for example, infections (usually
Escherichia coli, Streptococcus faecalis, or Proteus),
indwelling catheters, stones, or parasitic eggs. In
contrast to its nonkeratinizing counterpart, KSM is
a risk factor for the development of invasive malignancy, which is usually squamous cell carcinoma.
The more extensive the changes found at cystoscopy,
the greater the risk of malignant transformation (37).
Small areas of KSM can be managed by TUR resection and cystoscopic follow-up; extensive disease,
particularly in younger patients, merits radical
cystectomy.
Urothelial dysplasia (low-grade intraurothelial
neoplasia) is defined morphologically as cytological and architectural changes felt to be preneoplastic
but which fall short of carcinoma in situ (23).
Urothelial dysplasia is most commonly diagnosed
in mucosa adjacent to invasive and high-grade TCC.
Although it has been argued that dysplasia represents a risk factor for recurrence and progression
(38), it is not clear whether dysplasia develops before
or concomitantly with clinically manifest TCC. Furthermore, in many clinical studies that have shown
risk of progression, dysplasia has not been separated
from CIS.
CLINICAL FEATURES
Natural History
The natural history of bladder cancer can be classified
as follows:
l
l
No further recurrence
Local recurrence, which can occur on a single
occasion or on multiple occasions; it can involve
single or multiple tumor recurrences, but recurrent tumors are usually of the same stage and
grade as the primary tumor
Local progressionan increase in local stage with
time, the appearance of distant metastases, and
subsequent death
Predicting Recurrence/Progression in
NonMuscle Invasive (pTa, pT1) Tumors
Of newly diagnosed nonmuscle invasive tumors,
approximately 30% are multifocal at presentation.
The classic way to categorize patients with pTa/pT1
tumors is to divide them into risk groups on the basis
of prognostic factors derived from univariate analyses
and in some studies multivariate analyses (3944). In
each of these studies, multifocality is consistently a
predictor of recurrence. Prior recurrence rate (43,44)
and positive three-month check cystoscopy (39,40) are
also key predictors. Histological grade is consistently
a predictor of progression to muscle invasive disease
(4144). The poor prognosis of pT1G3 TCC is well
described, with 50% progression rate if associated
with concomitant CIS (41,45).
A British study proposed a cystoscopic followup plan based on the risk of recurrence determined
from two objective parameters: solitary or multifocal
at presentation and tumor-free/recurrence at first
three-month check cystoscopy (46) (Table 2). Sixty
percent of nonmuscle invasive tumors are low-risk,
30% intermediate-risk, and 10% high-risk for recurrence. In a meta-analysis of seven randomized trials
with one immediate postoperative instillation of chemotherapy, ideally within 6 hours but if not within
24 hours of TUR bladder tumor, the risk of recurrence
was decreased by 39%. The benefit was confirmed in
both solitary and multifocal tumors (47). However, of
those with multifocal tumors, 65% still had a recurrence at a median follow-up of 3.4 years despite immediate-dose chemotherapy. For these patients, European
Association of Urology (EAU) Guidelines recommend
additional adjuvant intravesical instillations. The duration and frequency of additional instillations remain
controversial. In a multivariate study, patients were
divided into three groups at low-, intermediate-, and
high-risk of recurrence and progression (42). However,
this did not differentiate the risk of recurrence and
progression. Some patients may have a high risk of
recurrence but not progression, whereas others may
have a high risk of recurrence and progression.
To separately predict the short-term and longterm risks of recurrence and progression in individual
patients, the European Organisation for Research and
Treatment of Cancer (EORTC) developed a scoring
system (Table 3) and risk tables (Table 4) (48). The
Table 2 Prognostic Groups, Their Relationship to Risk of Recurrence and Recommended Management Plans for pTa and pT1 (G1
and G2) (WHO, 1973) Tumors
Prognostic groups
Cystoscopic findings
Management plan
Group 1
Solitary tumor at presentation, no tumor recurrence

at 3 mo (20% risk of recurrence at 1 yr)
Solitary tumor at presentation, tumor recurrence at
3 mo; multiple tumors at presentation, no tumor
recurrence at 3 mo (40% risk of recurrence at 1 yr)
Multiple tumors at presentation, tumor recurrences
at 3 mo (90% risk of recurrence at 12 yrs)
Followed up safely by annual flexible

cystoscopy
Followed up 3-monthly by flexible
cystoscopy for the first year; then
annually if no recurrence
3-monthly rigid cystoscopic assessment
under general anesthesia for 2 yr;
then annually if no recurrence
Group 2
Group 3
Source: From Refs. 39, 46.

Table 3 Weighting Used to Calculate Recurrence and Progression Scores
Factor
Recurrence
Progression
Number of tumors
Single
27
8
0
3
6
0
3
3
Tumor diameter
<3 cm
3 cm
0
3
0
3
Prior recurrence rate

Primary
!1 recurrence/year
>1 recurrence /year
0
2
4
0
2
2
Category
Ta
T1
0
1
0
4
Concomitant CIS
No
Yes
0
1
0
6
Grade (1973 WHO)

G1
G2
G3
0
1
2
0
0
5
017
023
Total score
379
Muscle Invasive Disease

Over 50% of patients with muscle invasive disease have
occult metastatic disease at presentation; its presence is
strongly related to initial tumor stage, being most frequent in T4 disease. Among patients with muscle
invasive bladder cancer thought to have negative
nodes on preoperative computerized tomography,
around one-fifth are found to have metastatic nodal
disease when managed by radical cystectomy and bilateral lymphadenectomy alone. The presence of concomitant CIS and/or upper tract dilatation is associated
with less good outcomes following radiotherapy, and
consequently these patients are usually offered radical
cystectomy. Neoadjuvant chemotherapy has become
standard in many centers prior to radical cystectomy
or radiotherapy following the publication of two metaanalyses, one showing a 5% overall survival benefit (50),
the other showing a 6.5% survival benefit (51).
MOLECULAR PATHOGENESIS
OF BLADDER TCC
Molecular biology is paramount to our understanding
of bladder cancer pathogenesis. Although great
insights have been made into our ability to predict
recurrence and progression using established prognostic markers, they are not sufficiently sensitive or
specific to predict individual prognosis. Understanding the molecular profile of individual patients could
usher us into an era of improving prediction of the
natural history of the disease and providing a more
personalized and tailored treatment. It should also
facilitate the development of novel targeted treatments. Multiple genetic and epigenetic alterations
have been described in bladder cancer, including
those that affect signal transduction, the cell cycle,
invasion, angiogenesis, and apoptosis.
Abbreviation: CIS, carcinoma in situ.

basis for these tables was the EORTC database, which

provided individual data for 2591 patients diagnosed
with pTa/pT1 tumors who were randomized in
seven EORTC trials. Patients with primary CIS were
excluded.
Primary CIS
Patients presenting with bladder pain, dysuria, and
positive urine cytology are likely to harbor primary
CIS. If primary CIS is diffuse, 50% of these patients die
of metastatic TCC within a year or two if aggressive
intravesical therapies are not instituted (49). Currently, these patients are usually offered maintenance
intravesical BCG.
Model for Bladder TCC Progression

Current evidence suggests that genetic alterations on
chromosome 9 are an early event in bladder tumor
formation. Chromosome 9 loss of heterozygosity
Table 4 Probability of Recurrence and Progression According to Total Score

Probability of recurrence at 1 yr
Probability of recurrence at 5 yr
Recurrence score
(95% CI)
(95% CI)
Recurrence risk group
0
14
59
1017
15
24
38
61
(1019)
(2126)
(3541)
(5567)
31
46
62
78
(2437)
(4249)
(5865)
(7384)
Low
Intermediate
Intermediate
High
Probability of progression at 1 yr
Probability of progression at 5 yr
Progression score
(95% CI)
(95% CI)
Progression risk group
0
26
713
1423
0.2
1
5
17
(00.7)
(0.41.6)
(47)
(1024)
0.8
6
17
45
(01.7)
(58)
(1420)
(3555)
Low
Intermediate
High
High
380
Griffiths and Mayer
Figure 6 A simplified genetic model for bladder

tumor development. Minus signs indicate copy
number losses or loss of heterozygosity.
(LOH) is found in more than 50% of all bladder tumors,

regardless of stage and grade. It has also been identified in hyperplastic urothelium. Low-grade pTa tumors
usually have alterations in the RAS-MAP kinase signal
transduction pathway, the most frequent being fibroblast growth factor receptor 3 (FGFR3) mutations on
chromosome 4p (52). Most genetic events to date have
been identified in high-grade and muscle invasive
tumors. Many of these events are also found in CIS,
confirming the likely progression to muscle invasive
TCC via CIS. Muscle invasive tumors and CIS frequently have alterations in the p53 and retinoblastoma
(pRb) pathways that control the cell cycle. Although
almost certainly too simplistic, this model does provide
a useful basis for further genetic studies (Fig. 6). There
are clearly some gaps in our knowledge. For example,
no significant molecular differences have yet been
found among CIS, muscle invasive TCC, and the
metastases that develop from them. Moreover, we are
unsure whether pT1 tumors represent a distinct group
or are merely caught in their journey to muscle invasion.
Molecular Basis of Metachronous

and Synchronous Bladder TCC
Two theories have arisen from the observation that
patients with bladder cancer often present with metachronous tumors that develop at different times and
sites within the bladder: the field change (oligoclonal)
and monoclonal theories (53). The field change theory
attributes multifocality to individual transformation
of urothelial cells at a number of sites where there is
dysplasia or CIS. In contrast, at least in some cases,
molecular biological studies support a common clonal

origin for concomitant urothelial tumors. This was
first demonstrated in methylation studies of the
X chromosome (54). For each of four female patients
with multiple bladder tumors, it was found that all
the tumors from a given individual had inactivation of
the same X chromosome. Lateral intraepithelial
spread and dispersal of transformed cells are possible
mechanisms underlying the monoclonal theory.
Current evidence suggests that oligoclonal tumors
might be more common in precursor lesions and
early tumor stages. The frequent monoclonality
found in patients with advanced tumors could be
because of outgrowth of one tumor clone with specific
genetic alterations (53).
SPECIFIC GENETIC ALTERATIONS

IDENTIFIED IN TCC
The key genetic alterations identified in bladder TCC
are in the following subsections.
Oncogenes
Several known oncogenes are altered in TCC. They
contribute to the malignant phenotype in a dominant
manner. This is achieved either by overexpression of
the gene product, or, less commonly, by expression of
the mutant protein product with altered function.
FGFR3, RAS, and PI3 Kinase
The FGFR3 gene found on chromosome 4 (4p16)

encodes a glycoprotein that belongs to a family of

Table 5 Frequency of FGFR3 Mutations in Bladder Tumors
Tumor
Papilloma
Ta
T1
CIS
Muscle invasive
FGFR3 mutation
frequency (%)
*75
>60
2025
0
*16
Abbreviation: CIS, carcinoma in situ.

structurally related tyrosine kinase receptors. Activating point mutations of FGFR3 have been identified
in approximately 40% of bladder tumors overall
(Table 5). The only other malignancy in which significant involvement of FGFR3 has been reported is
multiple myeloma. A large study of more than
700 tumors recently found that FGFR3 mutation was
associated with a high recurrence rate in TaG1 tumors
(55). Mutant FGFR3 is predicted to activate the RASMAP kinase pathway. FGFR3 and RAS gene mutations are mutually exclusive in bladder cancer, as
expected if each has the same signaling consequences.
RAS mutations are found in 13% of bladder TCC and
85% of low-grade pTa tumors were found to have a
RAS or a FGFR3 mutation (56).
RAS can also activate the PI3 kinase pathway.
Mutations of the a-catalytic subunit of PI3 kinase have
recently been identified. Interestingly, 26% of FGFR3
mutant tumors were also PI3 kinase mutant compared
with 7% of FGFR3 wild-type tumors. The functional
relationship between these events is not yet known (57).
EGFR, ErbB2 (HER2)
Overexpression of genes that encode receptor tyrosine

kinases such as EGFR (epidermal growth factor receptor) and the ErbB2 receptor have been reported in
TCC of the bladder. In a prospective study, the overexpression of EGFR correlated not only with higher
tumor stage and grade but also with patient survival
(58). Mechanisms underlying EGFR overexpression in
bladder cancer are not clear, although gene amplification is not the principal mechanism.
The ErbB2 gene encodes a 185-kDa cell surface
glycoprotein with extensive homology to EGFR (59).
Overexpression of the gene in the absence of gene
amplification has been described (59). Although associations between gene amplification, protein overexpression, and death from bladder cancer have been
reported, the prognostic significance of ErbB2 overexpression has not been fully explored.
Tumor Suppressor Genes

and Cell Cycle Regulators
In the mammalian cell cycle, functional inactivation of
these genes either by mutation, deletion, or DNA
hypermethylation contributes to the development of
cancer. The key tumor suppressor genes altered in
bladder cancer are TP53 and RB1. The p53 pathway
381
markers are p53, p21, MDM2, and p14. The pRb

pathway markers are pRb, E2F1, cyclins, cyclin kinases, p27, and p16.
TP53
The TP53 gene has been mapped to chromosome

17q13. TP53 gene mutations are associated with
high-grade and high-stage TCC. Around 50% of muscle invasive TCCs harbor TP53 gene mutations (60).
Overall, p53 protein accumulation is associated with
TP53 gene mutation. Mutant p53 has an increased
half-life and can be detected with ease, whereas normal physiological concentrations of the wild-type protein are undetectable. Most studies have therefore
used immunohistochemical detection of p53 protein
as a surrogate for TP53 gene mutation. However,
microdissection studies of p53-immunopositive and
immunonegative regions have demonstrated that
p53 accumulation is not a reliable method of screening
for TP53 gene mutations in TCC (61). Clearly p53
accumulation can arise by alternate methods of p53
inactivation or by upregulation of functional wildtype p53. One such mediator of p53 inactivation in
TCC is the MDM2 protein.
Despite studies correlating p53 immunohistochemistry with progression, meta-analysis of 117
studies concluded that there is insufficient evidence
to infer that p53 by itself is a good prognostic marker
for bladder cancer, previously because of small study
cohorts and heterogeneity in laboratory and statistical
methods (62). Finally, a recent prospective trial found
no clinical utility for p53 expression in predicting
survival in high-risk patients with pT1 tumors
although this may have been affected by the low
event rate related to treatment efficacy (63).
RB1
The RB1 gene located at chromosome 13q14 was the

first tumor-suppressor gene isolated. Structural alterations to the RB1 gene by mutation or deletion are
associated with muscle invasive TCC and have been
reported in up to half of these tumors. Studies have
shown that alterations in p53 and pRb pathways act in
cooperative or synergistic ways to promote bladder
cancer progression (64).
Tumor Invasion and Metastasis

As an initial step, cancer cells must detach from their
original site before they can invade the surrounding
tissue and metastasize to distant organs. The main
families of adhesion molecules are the cadherins, integrins, members of the immunoglobulin superfamily,
and selectins. This diverse system of transmembrane
glycoproteins mediates cellextracellular matrix adhesion and intercellular matrix adhesion.
Cadherins are the main mediators of cell-cell
adhesion in epithelial tissues, being major components of both the adherens junction and desmosomes.
Adhesion is achieved by homodimeric interactions
between the extracellular domains of classical
382
Griffiths and Mayer
cadherins (E-cadherin, P-cadherin, and N-cadherin) on

neighboring cells. Catenins (a-, b-, and g-catenin and
p120) anchor the cadherins to the cell cytoskeleton. In
bladder TCC, reduced E-cadherin and b-catenin
expression is associated with high grade and stage.
Although E-cadherin has been shown to be an independent prognostic factor on multivariate analysis (65),
many other studies have disputed its independent
prognostic value for progression. Mutations, hypermethylation, and transcriptional repression of the Ecadherin gene may play a role in reduced expression.
The ability of tumor cells to degrade tissue
extracellular matrix and basement membrane components is a prerequisite for invasion and metastasis.
Such functions can be performed by a family of zincdependent proteolytic enzymes called matrix metalloproteinases (MMPs). A balance is maintained by
tissue inhibitors of metalloproteinases. In bladder
cancer, MMP-2 and MMP-9 expression is associated
with higher tumor stage and grade. MMP-2 expression is associated with decreased survival (66).
are integral to the cell structure and repair processes.

Activated caspase-3 can inhibit the cyclin-dependent
kinase activity of p27 by cleaving it into two fragments.
A recent study evaluated p53, Bcl-2, caspase-3,
and survivin in patients treated with radical cystectomy. Multivariate analysis showed that changed
expression of all four markers has an independent
prognostic value for recurrence and survival (71).
Angiogenesis
New Urinary Biomarkers
Angiogenesis, the growth of new blood vessels from

existing vessels, is essential to meet the metabolic
requirements for tumor growth. Angiogenesis is controlled by the balance of pro- and antiangiogenic
signals. Promoters of angiogenesis in bladder cancer
include vascular endothelial growth factor receptor
(VEGFR), thymidine phosphorylase, acidic and basic
fibroblast growth factors (FGF1 and 2), and cyclooxygenase (COX)-2. An inhibitor of angiogenesis is
thrombospondin. Both promoters and inhibitors have
been shown to have an impact on clinical outcomes
such as recurrence, progression, and survival (67).
Increased microvessel density, a histological surrogate
for angiogenesis, is a significant independent prognostic indicator of recurrence and poor survival (68).
COX is a key enzyme in the synthesis of prostaglandins from arachidonic acid. The inducible form of
COX, COX-2, produces prostaglandins E1 and E2,
which are considered to be angiogenic stimulators.
COX-2 is rarely detected in normal urothelium.
Although elevated COX-2 expression correlates with
high-grade and muscle invasive TCC (69), COX-2
appears to have limited prognostic potential (70).
Novel urinary biomarkers have been developed to aid

diagnosis of bladder cancer and to reduce the check
cystoscopic burden (72). However, none to date has
had sufficient sensitivity to replace cystoscopy at diagnosis. Moreover, no single marker has been shown to
aid the decision making with regard to the frequency
of surveillance cystoscopies. Current FDA-approved
tests are nuclear matrix protein-22 (NMP-22), UroVysionTM/fluorescence in situ hybridization (FISH),
ImmunoCytTM, BTA stat, BTA TRAK, and fibrin-fibrinogen degradation products. Other key biomarkers
under investigation include microsatellite analysis,
telomerase, hyaluronic acid and hyaluronidase,
BLCA-4, cytokeratins, and survivin.
At present the three most commonly used novel
biomarkers are NMP-22 (sensitivity 4965%; specificity
4087%), UroVysion (sensitivity 6987%; specificity
8996%), and ImmunoCyt (sensitivity 38100%; specificity 7384%). NMP-22 is an important regulator of
mitosis. In tumor cells, the nuclear mitotic apparatus is
elevated and NMP-22 is released from cells in detectable levels; it is a point-of-care test. The UroVysion test
utilizes FISH technology with probes for chromosome
3, 7, 17 and a locus for 9p21. In the ImmunoCyt assay,
three fluorescently marked antibodies label two
mucin-like proteins and a high molecular weight
form of carcinoembryonic antigen.
The development of urinary markers for the
detection of bladder cancer is a dynamic field, but
the perfect marker with high sensitivity, specificity,
and point-of-care results is still to be found. An important issue will be the financial cost-benefit ratio.
Apoptosis
Apoptosis is a unique form of cell death in which the
cell activates a self-destruct mechanism, causing its
death. Suppression of apoptosis can facilitate accumulation of gene mutations. Genes responsible for the
regulation of apoptosis interact in a cascade that can be
divided into initiation, regulation, and degradation
phases. Initiators include p53 and cytokines such as
tumor necrosis factor-a and fas ligand. The key regulators are the Bcl-2 family of proteins and survivin. The
degradation stage of apoptosis is primarily mediated
by a family of 10 or more proteolytic enzymes called
caspases. They are expressed as precursors that are
activated when cleaved. Caspases target proteins that
URINARY BIOMARKERS OF TCC

Urine Cytology
Urine cytology was first described by Papanicolaou
and Marshall in 1945. The median specificity is >95%
when obvious cancer cells are identified. However,
the median sensitivity of cytology is only 35% (72).
The main limitation is that low-grade tumors often
shed cytologically normalappearing cells. Urine
cytology is more sensitive in patients with highgrade tumors and CIS.
TRIALS ASSESSING INHIBITORS OF

MOLECULAR TARGETS AND COX-2
Trials are ongoing or planned for tyrosine kinase
inhibitors, targeting EGFR and ErbB2 (such as lapatinib), VEGFR (such as sorafenib and sunitinib), and
farnesyl transferase (such as SCH66336). Monoclonal

antibody studies include those using cetuximab that
targets EGFR, trastuzumab that targets the ErbB2
receptor, and bevacizumab that targets VEGFR (73).
Most targeted treatments are predominantly cytostatic, resulting in stable disease in some cases for
variable periods if given alone. There is therefore great
interest in combining targeted treatments with combination chemotherapy regimens or radiotherapy.
In vitro studies suggest that precise scheduling of
targeted agents and chemotherapy is important and
is likely to influence outcome (74).
The BOXIT trial sponsored by the National Cancer Research Institute (NCRI) has commenced in the
United Kingdom. The primary objective of this
phase 3 trial is to determine if the addition of the
oral COX-2 inhibitor celecoxib to standard therapy is
more effective in terms of disease recurrence at three
years compared with standard therapy alone for the
treatment of nonmuscle invasive TCC of the bladder
in intermediate or high-risk groups.
NEW TECHNOLOGIES IN THE DIAGNOSIS

OF BLADDER CANCER
Photodynamic Diagnosis
The use of white light cystoscopy alone at the time of
TUR bladder tumor may miss lesions that are present
but not visible. Fluorescence cystoscopy is performed
using blue-violet light after intravesical instillation
of photosensitizer, usually 5-aminolevulinic acid (5ALA) or hexaminolevulinate (HAL). 5-ALA fluorescence cystoscopy has been shown to detect more
CIS and papillary tumors (75). In a randomized trial,
5-ALA fluorescence cystoscopy at the time of diagnostic TUR bladder tumor was associated with reduced
residual disease (76). Of those who underwent fluorescence cystoscopy at initial TUR bladder tumor, 4.5%
had residual disease compared with 25% who underwent white light cystoscopy. In the same trial, fluorescence cystoscopy at diagnostic TUR bladder tumor
reduced the recurrence rate. Eight years after initial
TUR bladder tumor, 29% of those who underwent 5ALA fluorescence cystoscopy developed a recurrence
compared with 55% in the white light cystoscopy arm.
HAL fluorescence cystoscopy has also been shown to
improve the detection of CIS and papillary tumors.
Although very promising, its value remains to be
proven with regard to reducing progression rates and
improving survival. Moreover, although 5-ALA fluorescence cystoscopy reduced recurrence rates by 64%,
patients did not routinely receive a single dose of intravesical chemotherapy within 24 hours of initial TUR
bladder tumor in this trial (76). The question therefore
remains whether photodynamic diagnosis gives added
value if immediate-dose intravesical chemotherapy is
administered. The additional costs of the equipment
need to be considered in any health economic model.
Principles
Fluorescence occurs when a molecule absorbs one

color of light, and emits another color; this is termed
383
Stokes shift. The molecule absorbs the photon; an

electron is elevated to an excited state and then
returns to its resting state. The photon is then emitted
with less energy and so has a longer wavelength.
Porphyrins absorb violet-blue light and emit red light.
5-ALA is an initial substrate of heme biosynthesis. Exogenous application of 5-ALA induces an accumulation of fluorescent porphyrins, predominantly
protoporphyrin IX (PPIX) in epithelial tissue. Using
a blue-violet light with a wavelength of 400 nm, PPIX
appears as fluorescent red while normal urothelium
appears blue. This is because PPIX accumulates up to
10 times more in neoplastic cells than in normal tissue.
The mechanism of accumulation of fluorescent PPIX
in urothelial cancer is unclear. Several theories,
including a difference in the metabolic rate of neoplastic tissue, hyperproliferation, and inflammationinduced increased permeability to ALA, have been
proposed. These are supported by the observations
that increased PPIX can be detected in urothelial
hyperplasia, inflammation, and granulation tissue.
5-ALA absorption is limited because of its positive
electric charge. The esterification of 5-ALA as HAL
makes ALA more lipophilic, which enables it to cross
the cell membrane more easily. A consequence of this
is more rapid cellular uptake and higher fluorescence
than ALA. HAL therefore needs to be administered
only one hour before cystoscopy compared with two
hours before for 5-ALA. The procedure requires special telescopes with a filter to reduce autofluorescence,
and a specific light source.
THE FUTURE
Technologies such as high-throughput transcript
profiling, microarrays, and proteomics are facilitating
the comprehensive identification of molecular pathways and targets that are active in bladder cancer.
This will also enable the development of novel targeted treatments. Future management of bladder cancer will employ molecular profiling that will be able to
provide accurate predictions of prognosis and chemotherapeutic response in individual patients.
REFERENCES
1. CancerStats. Available at: http://info.cancerresearchuk.
org/cancerstats/.
2. Whelan P. Bladder cancercontemporary dilemmas in its
management. Eur Urol 2008; 53(1):2426.
3. Pelucchi C, Bosetti C, Negri E, et al. Mechanisms of disease: the epidemiology of bladder cancer. Nat Clin Pract
Urol 2006; 3:327340.
4. Brennan P, Bogillot O, Cordier S, et al. Cigarette smoking
and bladder cancer in men: a pooled analysis of 11 casecontrolled studies. Int J Cancer 2000; 86:289294.
5. Brennan P, Bogillot O, Greiser E, et al. The contribution of
cigarette smoking to bladder cancer in women (pooled
European data). Cancer Causes Control 2001; 12:411417.
6. IARC Working Group on the Evaluation of Carcinogenic
Risks to Humans. Tobacco smoke and involuntary smoking. IACR Monogr Eval Carcinog Risks Hum 2004; 83:1438.
384
Griffiths and Mayer
7. NICE Guidance on Cancer Services. Improving Outcome

in Urological Cancers. National Institute for Clinical Excellence, 2002.
8. Bjerregaard BK, Raaschou-Nielsen O, Srensen M, et al.
Tobacco smoke and bladder cancerin the European Prospective Investigation into Cancer and Nutrition. Int J
Cancer 2006; 119:24122416.
9. Aveyard P, Adab P, Cheng KK, et al. Does smoking status
influence the prognosis of bladder cancer? A systematic
review. BJU Int 2002; 90:228239.
10. Hueper WC, Wiley FH, Wolfe HD. Experimental production of bladder tumours in dogs by administration of betanaphthylamine. J Ind Hyg Toxicol 1938; 20:4684.
11. Case RAM, Hosker ME. Tumour of the urinary tract as an
occupational disease in the rubber industry in England and
Wales. Br J Prev Soc Med 1954; 8:3950.
12. BAUS Subcommittee on Industrial Bladder Cancer. Occupational bladder cancer: a guide for physicians. Br J Urol
1988; 61:183191.
13. Kogevinas M, t Mannetje A, Cordier S, et al. Occupation
and bladder cancer among men in Western Europe. Cancer
Causes Control 2003; 14:907914.
14. Czene K, Tiikkaja S, Hemminki K, et al. Cancer risks in
hairdressers: assessment of carcinogenicity of hair dyes
and gels. Int J Cancer 2003; 105:108112.
15. Fairchild WV, Spence CR, Solomon HD, et al. The incidence of bladder cancer after cyclophosphamide therapy. J
Urol 1979; 122:163164.
16. Duncan RE, Bennett DW, Evans AT, et al. Radiationinduced bladder tumours. J Urol 1977; 118:4345.
17. Boorjian S, Cowan JE, Konety BR, et al. Cancer of the
prostate Strategic Urologic Endeavor Investigators. Bladder cancer incidence and risk factors in men with prostate
cancer: results from Cancer of the Prostate Strategic Urologic Research Endeavor. J Urol 2007; 177:883887.
18. Steinmaus CM, Nunez S, Smith AH. Diet and bladder
cancer: a meta-analysis of six dietary variables. Am J
Epidemiol 2000; 151:693702.
19. Garcia-Closas M, Malats N, Silverman D, et al. NAT2 slow
acetylation, GSTM1 null genotype, and risk of bladder
cancer: results from the Spanish Bladder Cancer Group
and meta-analyses. Lancet 2005; 366:649659.
20. Engel LS, Taioli E, Pfeiffer R, et al. Pooled analysis and
met-analysis of glutathione S-transferase M1 and bladder
cancer: a HuGe review. Am J Epidemiol 2002; 156:95109.
21. Mostofi F. International histologic classification of
tumours. A report by the Executive Committee of the
International Council of Societies of Pathology. Cancer
1973; 33:14801484.
22. Epstein JI, Amin MB, Reuter VR, et al. The World Health
Organisation/International Society of Urological Pathology consensus classification of urothelial (transitional
cell) neoplasms of the urinary bladder. Bladder Consensus
Conference Committee. Am J Surg Pathol 1998; 22:
14351448.
23. Eble JN, Sauter G, Epstein JI, et al. World Health Organisation Classification of Tumours. Pathology and Genetics
of Tumours of the Urinary System and Male Genital
Organs. Lyon, France: IACR Press, 2004:89158.
24. Epstein JI, Amin MB, Reuter VE. Bladder Biopsy Interpretation. Philadelphia: Lippincott, Williams and Wilkins,
2004:40.
25. Harnden P. A critical appraisal of the classification of
urothelial tumours: time for a review of the evidence and
a radical change? BJU Int 2007; 99:723725.
26. Lopez-Beltran A, Montironi R. Non-invasive urothelial
neoplasms: according to the most recent WHO classification. Eur Urol 2004; 46:170176.
27. Standards and datasets for reporting cancers. Dataset for

tumours of the urinary collecting system (renal pelvis, ureter,
bladder, and urethra). Royal College of Pathologists, January
2007. Available at: http://www.rcpath.org/resources/pdf/
G044TumoursurinaryCollectingSystemFINAL.pdf.
28. Sobin DH, Witteking CH, eds. TNM Classification of
Malignant Tumours. 6th ed. New York: Wiley-Liss, 2002.
29. Amin AB, Ro JY, el-Sharkawy T, et al. Micropapillary
variant of transitional cell carcinoma of the urinary bladder. Histologic pattern resembling ovarian papillary serous
carcinoma. Am J Surg Pathol 1994; 18:12241232.
30. Kamat AM, Dinney CP, Gee JR, et al. Micropapillary
bladder cancer: a review of the University of Texas M.D.
Anderson Cancer Center experience with 100 consecutive
patients. Cancer 2007; 110:6267.
31. Kamat AM, Gee JR, Dinney CP, et al. The case for early
cystectomy in the treatment of nonmuscle invasive micropapillary bladder carcinoma. J Urol 2006; 175:881885.
32. Drew PA, Furman J, Civantos F, et al. The nested variant of
transitional cell carcinoma: an aggressive neoplasm with
innocuous histology. Mod Pathol 1996; 9:989994.
33. Wright JL, Black PC, Brown GA, et al. Differences in
survival among patients with sarcomatoid carcinoma, carcinosarcoma and urothelial carcinoma of the bladder. J
Urol 2007; 178:23022306.
34. Wang X, MacLennan GT, Lope-Beltran A, et al. Small cell
carcinoma of the urinary bladderhistogenesis, genetics
diagnosis, biomarkers, treatment and prognosis. Appl
Immunohistochem Mol Morphol 2007; 15:818.
35. Sved P, Gomez P, Manoharan, et al. Small cell carcinoma of
the bladder. BJU Int 2004; 94:1217.
36. Abol-Enein H, Kava BR, Carmack AJ. Nonurothelial cancer
of the bladder. Urology 2007; 69:93104.
37. Khan MS, Thornhill JA, Gaffney E, et al. Keratinising
squamous metaplasia of the bladder: natural history and
rationalisation of management based on review of 54 years
experience. Eur Urol 2002; 42:469474.
38. Cheng L, Cheville JC, Neumann RM, et al. Natural history
of urothelial dysplasia of the bladder. Am J Surg Pathol
1999; 23:443447.
39. Parmar MK, Freedman LS, Hargreave TB, et al. Prognostic
factors for recurrence and follow-up policies in the treatment of superficial bladder cancer: report from the British
Medical Research Council Subgroup on Superficial Bladder Cancer (Urological Cancer Working Party). J Urol 1989;
142:284288.
40. Fitzpatrick JM, West AB, Butler MR, et al. Superficial
bladder tumours (stage pTa, grades 1 and 2): the importance of recurrence pattern following initial resection. J
Urol 1986; 135:920922.
41. Heney NM, Ahmed S, Flanagan MJ, et al. Superficial
bladder cancer: progression and recurrence. J Urol 1983;
130:10831086.
42. Millan-Rodriguez F, Chechile-Toniolo G, Salvador-Bayarri
J, et al. Primary superficial bladder cancer risk groups
according to progression, mortality and recurrence. J
Urol 2000; 164:680684.
43. Kurth KH, Denis L, Bouffioux C, et al. Factors affecting
recurrence and progression in superficial bladder tumours.
Eur J Cancer 1995; 31A:18401846.
44. Lutzeyer W, Rubben H, Dahm H. Prognostic parameters in
superficial bladder cancer: an analysis of 315 cases. J Urol
1982; 127:250252.
45. Jakse G, Loidl W, Seeber G, et al. Stage T1 grade G3
transitional cell carcinoma of the bladder: an unfavourable
tumour? J Urol 1987; 137:3943.
46. Hall RR, Parmar MK, Richards AB, et al. Proposal for
changes in cystoscopic follow-up of patients with bladder
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
cancer and adjuvant intravesical chemotherapy. BMJ 1994;

308:257260.
Sylvester RJ, Oosterlinck W, van der Meijden AP. A single
immediate postoperative instillation of chemotherapy
decreases the risk of recurrence in patients with stage Ta
T1 bladder cancer: a meta-analysis of published results of
randomised clinical trials. J Urol 2004; 171:21862190.
Sylvester RJ, Oosterlinck W, van der Meijden AP, et al.
Predicting recurrence and progression in individual
patients with stage TaT1 bladder cancer using EORTC
risk tables: a combined analysis of 2596 patients from
seven EORTC trials. Eur Urol 2006; 49:466477.
Utz DC, Farrow GM. The management of carcinoma in situ
of the urinary bladder: the case for surgical management.
Urol Clin North Am 1980; 7:533541.
Advanced Bladder Cancer (ABC) Meta-analysis Collaboration. Neoadjuvant chemotherapy in invasive bladder
cancer: update of a systematic review and meta-analysis
of individual patient data advanced bladder cancer
(ABC) meta-analysis collaboration. Eur Urol 2005;
48:202205.
Winquist E, Kirchner TS, Segal R, et al. Genitourinary
Cancer Disease Site Group, Cancer Care Ontario program
in Evidence-based Care practice Guidelines Initiative. Neoadjuvant chemotherapy for transitional cell carcinoma of
the bladder: a systemic review and meta-analysis. J Urol
2004; 171:561569.
Knowles MA. Novel therapeutic targets in bladder cancer:
mutation and expression of FGF receptors. Future Oncol
2008; 4:7183.
Hafner C, Knuechel R, Stoehr R, et al. Clonality of multifocal urothelial carcinomas: 10 years of molecular genetic
studies. Int J Cancer 2002; 101:16.
Sidransky D, Von Eschenbach A, Oyasu R, et al. Clonal
origin bladder cancer. N Engl J Med 1992; 326:737740.
Hernandez S, Lopez-Knowles E, Lloreta J, et al. Prospective study of FGFR3 mutations as a prognostic factor in
non-muscle invasive urothelial bladder carcinomas. J Clin
Oncol 2006; 24:36643671.
Jebar AH, Hurst CD, Tomlinson DC, et al. FGFR3 and Ras
gene mutations are mutually exclusive genetic events in
urothelial cell carcinoma. Oncogene 2005; 24:52185225.
Lopez-Knowles E, Hernandez S, Malats N, et al. PIK3CA
mutations are an early genetic alteration associated with
FGFR3 mutations in superficial papillary bladder tumours.
Cancer Res 2006; 66:74017404.
Mellon K, Wright C, Kelly P, et al. Long-term outcome
related to epidermal growth factor receptor status in bladder cancer. J Urol 1995; 153:919925.
Sauter G, Moch D, Moore P, et al. Heterogeneity of erbB-2
gene amplification in bladder cancer. Cancer Res 1993;
53:21992203.
Spruck CHR, Ohneseit PF, Gonzalez-Zulueta M, et al. Two
molecular pathways to transitional cell carcinoma of the
bladder. Cancer Res 1994; 54:784788.
Abdel-Fattah R, Challen C, Griffiths TRL, et al. Alterations
of TP53 in microdissected transitional cell carcinoma of the
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
385
human bladder: high frequency of TP53 accumulation in

the absence of detected mutations is associated with poor
prognosis. Br J Cancer 1998; 77:22302238.
Malats N, Bustos A, Nascimento CM, et al. P53 as a
prognostic marker for bladder cancer: a meta-analysis
and review. Lancet Oncol 2005; 6:678686.
Dalbagni G, Parekh DJ, Ben-Porat L, et al. Prospective
evaluation of p53 as a prognostic marker in T1 transitional
cell carcinoma of the bladder. BJU Int 2007; 99:281285.
Chatterjee SJ, Datar R, Youssefzadeh D, et al. Combined
effects of p53, p21, and pRb expression in the progression
of bladder transitional cell carcinoma. J Clin Oncol 2004;
22:10071013.
Byrne RR, Shariat SF, Brown R, et al. E-cadherin immunostaining of bladder transitional cell carcinoma, carcinoma in situ and lymph node metastases with long-term
follow-up. J Urol 2001; 165:14731479.
Papathoma AS, Petraki C, Grigorakis A, et al. Prognostic
significance of matrix metalloproteinases 2 and 9 in bladder cancer. Anticancer Res 2000; 20:20092013.
Charlesworth PJ, Harris AL. Mechanisms of disease: angiogenesis in urologic malignancies. Nat Clin Pract Urol 2006;
3:157169.
Bochner BH, Cote RJ, Weidner N, et al. Angiogenesis in
bladder cancer: relationship between microvessel density
and tumour prognosis. J Natl Cancer Inst 1995; 87:16031612.
Yoshimura R, Sano H, Mitsuhashi M, et al. Expression of
cyclooxygenase-2 in patients with bladder carcinoma. J
Urol 2001; 165:14681472.
Shariat SF, Matsumoto K, Kim J, et al. Correlation of
cyclooxygenase-2 expression with molecular markers,
pathological features and clinical outcome of transitional
cell carcinoma of the bladder. J Urol 2003; 170:985989.
Karam JA, Lotan Y, Karakiewicz PI, et al. Use of combined
apoptosis biomarkers for prediction of bladder cancer
recurrence and mortality after radical cystectomy. Lancet
Oncol 2007; 8:128136.
Vrooman OP, Witjes JA. Urinary markers in bladder cancer. Eur Urol 2008; 53:909916.
Sonpavde G, Ross R, Powles T, et al. Novel agents for
muscle-invasive and advanced urothelial cancer. BJU Int
2008; 101:937943.
McHugh LA, Kriajevska M, Mellon JK, et al. Combined
treatment of bladder cancer cell lines with lapatinib and
varying chemotherapy regimensevidence of scheduledependent synergy. Urology 2007; 69:390394.
Hungerhuber E, Stepp H, Kriegmair M, et al. Seven years
experience with 5-aminolevulinic acid in detection of transitional cell carcinoma of the bladder. Urology 2007;
69:260264.
Denzinger S, Burger M, Walter B, et al. Clinically relevant
reduction in risk of recurrence of superficial bladder cancer
using 5-aminolevulinic acid-induced fluorescence diagnosis: 8-year results of prospective randomized study. Urology
2007; 69:675679.
25
Prostate Cancer
Freddie C. Hamdy
Nuffield Department of Surgery, University of Oxford, Oxford, U.K.
Craig N. Robson
Northern Institute for Cancer Research, The Medical School, University of Newcastle,
Newcastle, U.K.
As the eyes grow dim, the trunk bends, the cartilages ossify,
and the arteries change in their coats, so the prostate is
supposed to grow large and hard. . .
James Miller, 1864
INTRODUCTION
ETIOLOGY
Prostate cancer is the most common malignancy in

Western countries. In the United States, 186,320 cases
were diagnosed in 2008 and there were 28,660 deaths
from the disease (1). In England and Wales, there were
13,481 cases registered in 1990, rising to 34,302 new
cases in 2005 with 10,000 prostate cancer deaths (2).
Public awareness about the disease is clearly on the
increase, partly because of media interest and growing
general interest in mens health issues. While incidence
has been increasing gradually, this has not corresponded to a paralleled increase in death rates, but
there is a striking reduction, both in the United States
and England and Wales, which appears to be of slightly
higher magnitude in the United States compared to the
United Kingdom (3). This observation is difficult to
interpret, and although screening using prostate-specific antigen (PSA) testing in asymptomatic men has
been shown recently to reduce prostate cancerspecific
mortality (4), it alone cannot explain this reduction
because of the very low opportunistic PSA-testing
patterns in the United Kingdom (5). The reasons are
undoubtedly multifactorial, such as screening, the earlier detection and aggressive treatment of nonmetastatic locally advanced disease, and improved diet as
well as general environmental issues.
Evidence of carcinoma has been found in postmortem studies in approximately 30% of men aged 50
years, and 70% of men over the age of 80 years (6), but
only a small proportion of these tumors progress to
become clinically significant. With well-conducted
screening programs, it is now possible to detect
small volume tumors amenable to cure. However,
clinicians are still unable to predict which tumor is
likely to progress and which will remain quiescent.
Prostate cancer results from a complex and yet unclear

interaction between aging, genetic factors, hormones,
growth factors, and the environment, including an
increasing body of evidence incriminating dietary fat
(7).
Prostate cancer can be sporadic: familial with
clustering of disease within families and exposure to
common risk factors; or hereditary, with typical characteristics of early age onset and an autosomal dominant inheritance pattern. The latter is likely to be
triggered by a single gene passed along families, yet
to be discovered and possibly located in chromosome
1 (8). It has been estimated that men who have three
first-degree relatives have a 10.9-fold increase in risk
of developing the disease (9). An increased risk of
prostate cancer has also been associated with familial
breast cancer (7). It is generally accepted that prostate
cancer is not divided into latent and clinically significant tumor, but that its very long natural history,
coupled with cumulative genetic and biological
changes, eventually leads to progressive disease. It is
the length of this natural history that allows more men
to die with the disease than from it.
PATHOLOGY
Adenocarcinoma arising from the prostatic epithelium
accounts for about 95% of prostatic malignancies and
is usually composed of small glandular acini that
infiltrate in an irregular haphazard manner. The critical feature in prostatic adenocarcinoma is absence of
the basal cell layer (Fig. 1), which may be detected
immunohistochemically by using monoclonal antibodies against highmolecular weight cytokeratin.
Chapter 25: Prostate Cancer
387
Figure 1 (A) A photomicrograph of the prostate demonstrating high-grade prostatic intraepithelial neoplasia with dysplastic changes,
nuclear enlargement, hyper chromatism, prominent nuclei, cellular crowding, overlapping nuclei, and epithelial hyperplasia *H&E
staining; magnification approximately 400"). (B) A photomicrograph of the prostate demonstrating adenocarcinoma with small glandular
acini infiltrating in an irregular haphazard manner. The acini are composed of a single layer of cells showing nuclear enlargement with
prominent nucleoli. The critical diagnostic feature in prostatic adenocarcinoma is absence of the basal layer (H&E staining; magnification
approximately 400").
Perineural and microvascular invasion may be seen,

the latter correlating with histological grade.
Prostatic adenocarcinoma originates in the
peripheral zone in approximately 75% of cases, with
the rest originating in the transition zone (10). The
tumors arising from these separate zones have different pathological features and clinical behavior. Transition zone tumors arise in or near foci of benign
prostatic hyperplasia (BPH) and are usually smaller
and better differentiated (Gleason pattern 1 and 2).
Peripheral zone cancers are often less well differentiated (Gleason pattern 2, 3, or 4), larger in volume then
transition zone tumors, and are frequently associated
with greater stromal fibrosis, extracapsular extension,
seminal vesicle invasion, and lymph node metastases.
Histological Grading
There are numerous grading systems, but the
accepted standard is that developed by Gleason (11).
The system is based on the degree of architectural
differentiation, and individual cell cytology does not
play a role. The system identifies five patterns that are
often seen in prostatic adenocarcinoma, and to accommodate this, a primary and secondary pattern are
assigned and the Gleason score is given as their

sum, ranging from 2 to 10, with the dominant pattern
recorded first, for example, 3 4 7 (Fig. 2). Gleason
score correlates strongly with crude survival, tumorfree survival, and cause-specific survival, and is a
significant predictor of time to recurrence following
radical prostatectomy (12).
Grading errors are common in needle biopsy
specimens of the prostate, with underestimation of
the grade in 40% of cases, and overestimation in 25%
when compared with the whole specimen following
prostatectomy. This occurs more readily in biopsies
containing small foci and low-grade cancers, reflecting
sampling error and tumor heterogeneity. Nevertheless, useful predictive information can be provided by
Gleason grading needle biopsies (13). More recently,
tertiary Gleason grades were introduced, and defined
as Gleason grade pattern 4 or greater for Gleason score
6 and Gleason grade pattern 5 for Gleason score 7 or 8.
They are not at present consistently reported by
pathologists, and although advocated as an important
set of information in the evaluation of a patients
disease, to date consensus has not been reached
regarding its interpretation and impact on management (14).
388
Hamdy and Robson
Figure 2 The Gleason system of grading adenocarcinoma of the prostate.
Table 1 TNM Classification of Prostate Cancer

Tumor
Tx
T0
T1
T1a
T1b
T1c
T2
T2a
T2b
T3
T3a
T3b
T4
Nodes
Metastasis
Nx
N0
N1
Mx
M0
M1
M1a
M1b
M1c
Primary tumor cannot be assessed

No evidence of primary tumor
Tumor clinically unapparent, not palpable nor visible by imaging
Incidental finding following TURP in up to 5% of tissue
Incidental finding following TURP in more than 5% of tissue
Tumor identified by needle biopsy (e.g., because of elevated PSA)
Tumor confined to the prostate, palpable or visible by imaging
Tumor involves one lobe
Tumor involves both lobes
Locally advanced tumor
Extracapsular extension
Invasion of the seminal vesicle
Invasion into the prostatic apex or into (but not beyond) the prostatic capsule is not classified as T3,
but as T2
Tumor is fixed or invades adjacent structures other than seminal vesicles; i.e., bladder neck,
external sphincter, rectum, levator muscles, and/or pelvic wall
Regional lymph nodes cannot be assessed
No regional lymph node metastases
Regional lymph node metastases
Presence of distant metastases cannot be assessed
No distant metastases
Distant metastases present
Nonregional lymph nodes
Skeletal metastases
Other sites
Tumor Staging
The TNM system is the most common classification
used worldwide (Table 1). Clinical staging is limited by
a number of factors including understaging with digital
rectal examination (DRE) and TURP specimens, limited
accuracy of imaging, and the wide pathological variation of tumors identified on needle biopsy (stage T1c).
The inaccuracy of clinical staging is especially important when comparing nonsurgical treatment methods
(observation or radiotherapy) with pathologically
staged disease following radical prostatectomy.
Prostate cancer commonly spreads to the pelvic
lymph nodes, bones, especially the axial skeleton, and
lung. Unlike most other malignancies, skeletal metastases from prostate cancer are osteoblastic in over 80%
of cases, and despite the increase in bone formation
they lead to disturbance in the normal skeletal architecture and subsequent pathological fractures if left
untreated.
Putative Premalignant Lesions of the Prostate

Prostatic Intraepithelial Neoplasia and
Atypical Small Acinar Proliferation
Prostatic intraepithelial neoplasia (PIN) is believed to

be the preinvasive end of a morphological continuum
of cellular proliferation affecting prostatic ducts, ductules, and acini. It tends to be multifocal and occurs in
the peripheral zone, as does prostate cancer. PIN is
divided into two groups: low and high grade. The
continuum from normal prostatic epithelium through
low- and high-grade PIN to invasive cancer is characterized by increased epithelial dysplasia within the
luminal secretory cell layer. The dysplastic changes
with increasing grade of PIN include nuclear enlargement, hyperchromatism, prominent nucleoli, cellular
crowding with overlapping nuclei, and epithelial
hyperplasia (Fig. 1). The basal cell layer remains intact
though there may be some disruption in high-grade
PIN. There is strong clinical, histological, and molecular evidence linking high-grade PIN with prostate
cancer. High-grade PIN is seen in up to 16% of needle
biopsies in men over 50 years of age. In malignant
prostates, PIN is more frequent and of higher grade
than in glands without cancer. The incidence of PIN
increases with age, with low-grade PIN occurring in
men in their third and fourth decade and high-grade
PIN occurring in their fifth decade. Its association
with cancer when repeat biopsies are performed is
in the range of 0% to 24% (15). Atypical small acinar
proliferation, on the other hand, does not appear to be
a specific entity but a diagnostic category, including a
number of atypical lesions that are suspicious for but
not diagnostic of prostate cancer. The incidence of
ASAP on prostate biopsy has been reported to be
between 2.5% and 9.0% with a firm diagnosis of cancer in 30% to 60% of those cases (16).
389
(Hybritech Inc., California, U.S.) demonstrated a sensitivity of 90% in diagnosing prostate cancer in the
total PSA range of 2.6 to 4.0 ng/mL, while sparing
approximately 18% of patients from having prostatic
biopsies (23). The use of f/t PSA ratios in routine
clinical practice remains to be determined. If measured before the age of 50 years, PSA levels appear to
be a strong predictor of the incidence of prostate
cancer later in life (24). PSA kinetics is determined
as changes of PSA over time, represented either as
PSA velocity, or PSA doubling time. PSA kinetics
appears to be associated with diagnosis and disease
recurrence/progression after treatment, although its
interpretation remains unclear in monitoring patients
who receive active monitoring and attempts are made
to adjust changes observed to rising concentration for
noncancerous changes such as age and BPH (25). For
many years, the normal PSA threshold below which
men were not recommended prostate biopsy varied
between 3 and 4 ng/mL in an age group starting at
approximately 50 years. However, results from the
Prostate Cancer Prevention Trial (PCPT) in men
receiving the 5-a reductase inhibitor finasteride versus
placebo for prostate cancer chemoprevention showed
through end-of-study biopsies that 15% of men had
PSA levels less than 4 ng/mL (26). There is therefore
no PSA threshold below which a man can be told that
he does not have prostate cancer histologically. Furthermore, it has been demonstrated that using lower
PSA threshold such as 1.5 ng/mL and a lower cutoff
age of 45 years, the positive predictive value of PSA
was 21.3%, with half the detected patients exhibiting
clinically significant disease (27).
Prostate-Specific Antigen
PSA is a serine protease and organ-specific glycoprotein (molecular weight 34,000) that originates in the
cytoplasm of ductal cells of the prostate. It belongs to
the family of glandular kallikreins of which 15 are
known to date and are clustered in a locus on chromosome 19q133-4 (17). PSA is responsible for liquefaction of seminal coagulation. The measurement of
serum PSA concentrations is now well established as a
useful investigation in the diagnosis and follow-up of
patients with prostate cancer (18). The greatest limitation of PSA is that it is tissue and not tumor specific in
the prostate. However, PSA concentrations are the
best overall predictor of bone scan findings and can
be used as a screening test for prostate cancer (19,20).
PSA circulates in blood mainly bound to protease
inhibitors, including a-1-antichymotrypsin (ACT)
and a-2-macroglobulin (AMG); only a small fraction
of the total PSA exists in a free state. While AMG
encapsulates all epitopes of the PSA protein, ACT
leaves some exposed; therefore, immunoassay techniques have been developed to assess free PSA and PSA
bound to ACT but not to AMG (21). It has been
demonstrated that the free/total (f/t) PSA ratio in
patients with BPH is significantly higher than in prostate cancer, but its role is not established in diagnosing
the disease. Several studies report various optimal
cutoff levels, largely because of the different nature
of assays used (22). A study using the Hybritech assay
Circulating Tumor Cells

Clinically localized prostate cancers are frequently
understaged in over 50% of cases, with resulting positive surgical margins, extracapsular extension, and
potential treatment failure. This has stimulated
researchers to detect circulating micrometastases
prior to the establishment of overt secondaries, to
avoid unnecessary radical treatment. Metastasis does
not rely on the random survival of cells released from
the primary tumor, but from the selective growth of
specialized subpopulations of highly metastatic cells
endowed with properties that will allow them to
successfully complete each step of the metastatic cascade (28,29). Over the years, technologies such as flow
cytometry and the reverse transcription polymerase
chain reaction (RT-PCR) have given researchers the
opportunity of detecting circulating tumor cells
(CTCs) with high levels of sensitivity. In that sense,
prostate cancer has a significant advantage over other
malignancies, because of the specificity of PSA as a
reliable marker, although additional identifying
markers are being used, including cytokeratins. However, it has to be emphasized again that PSA is not
tumor specifica major drawback of all techniques
attempting to detect CTCs. A PSA-positive cell in the
peripheral blood, therefore, does not necessarily mean
a prostate tumor cell, but a cell expressing PSA, which
is likely to be of prostatic origin, in particular if the cell
390
Hamdy and Robson
is found to express the gene constitutively, that is,

mRNA for PSA. On the basis of these principles,
analytical flow cytometry and RT-PCR have been
used in an attempt to detect and isolate CTCs from
patients with prostate cancer. Studies have shown that
although quantification of circulating PSA-positive
cells by FC was a better predictor of skeletal metastases than isotope bone scanning, the majority of these
cells were not of prostatic origin, raising important
questions regarding the role of nonprostatic circulating PSA-positive cells in patients with prostate cancer
(30,31). RT-PCR methods, on the other hand, are considerably more sensitive in detecting circulating PSApositive cells, relying on the identification of mRNA for
PSA, an unequivocal proof that the cells are of prostatic
origin. A number of studies demonstrated the ability of
RT-PCR to detect circulating prostate cells in patients
with apparently localized disease, undergoing radical
prostatectomy, and some found a strong correlation
between a positive PCR reaction, capsular tumor penetration, and positive surgical margins, suggesting the
potential of this technique to be used for molecular
staging of prostate cancer (32). Other workers used
nested RT-PCR to compare the sensitivity of PSA with
a different marker, prostate-specific membrane antigen
(PSMA), in the detection of circulating prostatic cells
(33). The authors of all these studies assume that
circulating PSA-positive cells are endowed with metastatic propensity, despite the fact that the results only
demonstrate the presence of cells of prostatic origin.
Furthermore, the specificity of PSA mRNA in identifying cells of prostatic origin has been questioned (34).
More recently, novel sensitive methods of capturing
these cells were developed, using immunomagnetic
bead technology, such as CellSearch and CellTracks
systems (Veridex, Raritan, New Jersey, U.S.) (35), and
enumerating CTCs has been advocated as a useful
prognostic marker in castration-resistant patients (36).
As technology advances further, it will be possible to
determine the phenotype, genotype, and biological
significance of CTCs in the future.
SCREENING FOR EARLY PROSTATE CANCER

Primum non nocere
Hippocrates
There has been an apparent consensus, on the basis of
the evidence, that there is no justification to introduce
population screening for prostate cancer. However,
this consensus has been put to question by recently
published results from the European Randomised
Study of Screening for Prostate Cancer (4). In this
large study with a median follow-up of 9 years,
72,952 men aged 55 to 69 years were randomized to
screening and there were 89,435 age-matched controls.
At follow-up to date, there was a relative risk reduction of 0.80 for dying of prostate cancer, and in absolute terms, 1410 men needed to be screened, and, of
these, 48 men needed to receive treatment to prevent
one man from dying of prostate cancer. So despite the
fact that screening has now been shown to reduce
prostate cancer mortality, it appears to be at a significant cost of overdetection and overtreatment.

Until such time that methods of active monitoring/surveillance are refined to prevent low-risk
patients from being overtreated, and reliable biomarkers are identified to stratify patients into appropriate risk categories, it will be difficult for any health
provider to advocate PSA-testing as a mass screening
public health policy (37). The classic triad of detection
tests consists of serum PSA measurement, DRE and
transrectal ultrasound (TRUS) of the prostate and
biopsy, which detect up to 6% of a screened population as having prostate cancer. However, methods of
screening are changing rapidly, and when the results
of these studies are available, it may well be that
urologists will be detecting a different category of
disease altogether compared with current screening
modalities.
We found the patient complaining of excruciating pains in various parts of the body, which
could be compared to nothing except the pains
under which persons afflicted with carcinoma
occasionally labour. He could void no urine
without the assistance of a catheter. The prostate
gland, examined by the rectum was found to be
much enlarged and of a stony hardness. I continued to visit him in consultation for nearly a
year, at the end of which time he suddenly lost
the use of the muscles of his lower limbs and
died a fortnight afterwards.
Sir Benjamin Brodie, 1842
Early prostate cancer is asymptomatic. The above
description by Benjamin Brodie, over 150 years ago,
illustrates all the relevant symptoms in advanced
prostate cancer. Patients with symptomatic disease
can present in a variety of ways, including bladder
outflow obstruction, irritative bladder symptoms secondary to trigonal involvement, and hematuria. Metastatic disease may present with skeletal pain, spinal
cord compression secondary to collapsed vertebrae,
and pathological fractures; or with general systemic
manifestations, including weight loss, weakness, and
anorexia. With locally advanced disease, prostate cancer may manifest itself as renal failure secondary to
bilateral ureteric orifices involvement.
TREATMENT
The more resources we have, and the more
complex they are, the greater are the demands
on our clinical skill. These resources are calls
upon our judgment, and not substitutes for it.
Sir Francis Walsh
For the sake of simplicity, prostate cancer can be
classified into early organ-confined and advanced
disease when treatment is discussed.
Organ-Confined Prostate Cancer
Advanced Prostate Cancer
Over the past decade, it has become evident that many

cancers diagnosed through PSA testing in asymptomatic men are of low risk, with little potential to
progress and become lethal disease. Patients with
newly diagnosed clinically localized prostate cancer
can thus be categorized into low-, intermediate-, and
high-risk disease, following several analyses of data
by a number of researchers (38). However, most data
concern disease that has been already treated rather
than patients receiving observation only. Historical
cohort studies have been undertaken in the United
States and Europe, confirming the observation that
competing morbidity is an important factor in many
men with early prostate cancer, linked to their age,
serum PSA at diagnosis, and Gleason grading (39).
Conventional curative treatments of localized
disease includes surgery in the form of radical prostatectomy through the open retropubic and perineal
routes, laparoscopic and more recently robot-assisted
laparoscopic intraperitoneal or extraperitoneal
approaches; radiotherapy with its variations, including
external beam conformal approaches, intensity modulated techniques, mostly in combination with neoadjuvant androgen suppression for a period of three to six
months; and radioactive seed implantation otherwise
known as brachytherapy. These treatments carry relatively low morbidity in experienced hands and a high
cure rate. Active monitoring also known as active surveillance remains a reasonable option in many men with
low-risk disease. Radical interventions are not recommended for men who are likely to have a life expectancy
of less than 10 years because of competing morbidities
and the likelihood that treatment will not benefit the
patient. Studies indicate that selected groups of men may
benefit from radical intervention, particularly those who
are youngest and fittest, and have high-grade tumors.
High-level evidence concerning the effectiveness
of treatments is limited at present to one randomized
controlled trial of watchful waiting versus radical prostatectomy performed in Scandinavia, which showed
successively a reduction in mortality from prostate cancer in favor of radical prostatectomy at 6-, 8-, and 10-year
median follow-up, with a decrease in disease progression and development of metastases (4042). However,
the study was performed before the PSA era, and most
tumors treated were of higher volume and stage than
contemporary series. Addressing the same issues,
PIVOT (The Prostate Cancer Intervention Versus
Observation Trial) in the United States has closed to
recruitment, comparing watchful waiting to surgery,
as well as the ProtecT study in the United Kingdom,
comparing active monitoring, surgery, and external
beam radiotherapy (43,44). Results are awaited. In
observational studies, survival following treatment
for localized prostate cancer is good for all modes of
treatment: 85% to 90% for radical prostatectomy, 65%
to 90% for radiotherapy, and 70% to 90% for conservative management. This major issue of treatment efficacy in early prostate cancer is likely to be resolved
once the results from both the PIVOT and ProtecT
studies are reported in due course.
Locally Advanced Disease
391
For many years, external beam irradiation has been

widely accepted in treating locally advanced prostate
cancer, and its combination with androgen suppression for three years has been shown to improve survival (45). A study by the Medical Research Council has
closed to recruitment recently, comparing androgen
suppression alone and in combination with radiotherapy. The results are awaited. To determine the benefits
of immediate versus deferred treatment in asymptomatic men with nonmetastatic advanced prostate cancer, a further study by the Medical Research Council
suggested that there is a small but significant advantage in treating these patients early, albeit to delay or
prevent the advent of serious morbidity and complications from progressive disease. (46). Downstaging of
extracapsular disease has also been attempted in a
number of studies, whereby androgen ablation is performed for three to six months prior to radical prostatectomy. Although the incidence of positive margins
decreases in clinically T2 tumors, there is no apparent
change in T3 disease, and disease-free survival is not
affected (47,48). In general, radical prostatectomy is not
recommended for nonorgan confined disease, but
there appears to be an increasing tendency to offer
surgery to patient with high-risk disease and/or clinically operable extracapsular tumors, although the evidence of benefits to such patients from surgical
intervention remains to be demonstrated through
well-conducted clinical trials.
Metastatic Disease
In 1940, Charles Huggins discovered the beneficial

effects of androgen deprivation in patients with metastatic prostate cancer (49). Since then, hormonal
manipulation has remained the mainstay of treatment
in advanced stages of the disease. It is still expected
that about 80% of all patients with advanced prostate
cancer will respond to androgen blockade. Patients
will show both subjective and objective responses,
manifested by considerable and rapid symptomatic
improvement, particularly in metastatic skeletal pain,
together with local and distant regression of the disease. This is complemented by normalization of serum
tumor markers. Relapse, however, is common at a
mean interval of two years following initiation of
treatment. The disease is then hormone resistant and
prognosis becomes extremely poor. Methods of hormonal manipulation include bilateral orchidectomy
and estrogen preparations, which are rarely prescribed nowadays, in view of the serious cardiovascular side effects encountered with the recognized
3 mg daily dose, to achieve castrate levels. The use
of the smaller dose of 1 mg daily remains controversial, as castrate levels are not reached in 30% of
patients. Alternative therapy includes the use of analogues of the hypothalamic luteinizing hormonereleasing hormone (LHRH). These occupy the receptors of LHRH in the pituitary, initially stimulating the
release of luteinizing hormone and then blocking the
392
Hamdy and Robson
subsequent stimulation of the receptors by the endogenous pulsatile secretion of luteinizing hormone. More
recently, a new class of drugs, the GnRH antagonists,
has been shown to immediately block the GnRH receptor and thus produce rapid AD without the ensuing
testosterone surge, removing the need for administering antiandrogens to prevent testosterone flare. To
date, results from randomized controlled trials comparing analogues with antagonists of LHRH report equivalent results in achieving castrate testosterone levels
(50). In the late 1980s, total androgen blockade has been
advocated to prevent the effect of the nontesticular
circulating testosterone formation by the adrenals.
After initial enthusiasm, further studies failed to
show any survival advantage in patients treated with
maximum androgen blockade (51,52), although a
recent study from Japan reports that overall survival
in such patients is indeed improved with maximum
blockade compared with monotherapy using LHRH
analogues (53). But this was achieved in a relatively
small series of patients, and the debate continues.
In men with castration-resistant prostate cancer,
taxanes have been shown to improve survival and
quality of life in two randomized controlled trials
undertaken simultaneously in North America
(54,55). Docetaxel has now been adopted in men
with these advanced stages of prostate cancer and
low comorbidity as a palliative treatment option.
More recently, a novel agent, abiraterone acetate,
which is a potent, selective, and orally available inhibitor of CYP17 and the key enzyme in androgen and
estrogen biosynthesis, has been shown to be of significant benefit in men with castration-resistant prostate
cancer (56), and is being investigated further.
Awareness about the effects of androgen ablation
on the skeleton has increased considerably over recent
years, in particular loss of bone mineral density, which
appears to occur mostly in the first 12 months of
treatment, and could lead to osteopenia and osteoporosis, which may compound the effect of metastatic
disease (57). Bone protection agents are therefore being
tested to prevent these complications, such as
bisphosphonates and RANK-ligand inhibitors that
both act on reducing osteoclast activity. A recent
study of a RANK-L inhibitor showed that it reduces
the incidence of skeletal events in such patients (58). In
men with skeletal metastases and castration-resistant
disease, potent bisphosphonates such as zoledronic
acid were shown to reduce and delay the incidence
of skeletal-related events if given irrespective of the
patients symptoms with a sustained effect over time
(59,60).
Novel Therapies in Prostate Cancer

The development of new therapeutic approaches in
prostate cancer will rely on continuing progress made
in three specific areas: (i) imaging of the prostate and
identification of cancerous lesions; (ii) the delivery of
different forms of energy to achieve safe and targeted
tissue ablation; and (iii) understanding the biology of
prostate cancer from its early genetic alterations to the
molecular changes responsible for tumor progression.
On the basis of modern TRUS technology, two distinct

modes of energy delivery systems for tissue ablation
have been revived in recent years: cryotherapy and
high-intensity focused ultrasound (HIFU).
Cryotherapy
The in situ destruction of tumors by the application of

low temperatures was first developed in the 1970s to
treat localized prostate cancer with reasonable success.
Cryoablation had a number of advantages over other
forms of treatment, but suffered from many limitations.
Equipment was cumbersome, probes were placed
mostly transurethrally under digital rectal guidance,
temperature control was poor, and damage to adjacent
tissue was common. The ability of real-time TRUS to
guide cryoprobe placement and accurately monitor the
freezing process, in addition to the development of
urethral warming devices, encouraged clinicians to
attempt again the destruction of prostate cancer by
freezing. A number of studies on humans followed the
animal work, and results are slowly emerging (61,62).
High-Intensity Focused Ultrasound
The technique consists of delivering ultrasonic energy

with resultant heat and tissue destruction to a discrete
point without damaging intervening tissue. Much
higher temperatures are generated at the focal point
using HIFU (approximately 988C) than diffuse ultrasound hyperthermia (approximately 428C), leading to
complete tumor necrosis. After evaluating the technique initially in animal models and in vitro using cell
lines, its use was reported in the treatment of BPH
without significant side effects as a minimally invasive therapeutic option in symptomatic patients. The
relative safety of HIFU coupled with its documented
ability to cause targeted tissue necrosis prompted
researchers to extrapolate its application to the treatment of prostate cancer, as an alternative to surgery
and radiotherapy (63).
Photodynamic Therapy
Gene Therapy
Since the discovery of DNA and its structure by James

Watson and Francis Crick in 1953, our knowledge of
the molecular basis and human genetics in health and
disease has made giant steps forward, bringing closer
the double helix to the bedside of patients where
every other conventional treatment may be failing.
Advances in molecular biology, particularly in
recombinant DNA technology, have paved the way
to unlimited possibilities in predicting, controlling,
and preventing disease at its molecular origin.
The prostate is a prime target in the development of successful gene therapy for the following
reasons: (i) prostate cancer is slow growing; (ii)
tumor burden can be significantly reduced by surgery;
and (iii) the prostate expresses unique antigens,
including PSA and PSMA.
The most significant limitation of gene therapy is
the difficulty in gene delivery to the relevant cells.
Gene transfer can be achieved in vitro or in vivo.

In vitro methods can be chemical through calciumphosphate transfection, physical through electroporation or microinjection, by fusion with liposomes,
through receptor-mediated endocytosis, or using
recombinant viruses. In vivo methods include direct
injection of DNA either naked, contained in liposomes,
conjugated to a carrier (e.g., antibodies to a specific cell
surface protein), or by particle bombardment (64).
Gene-directed enzyme prodrug therapy
(GDEPT) is based on the potential use of prodrugs
that are essentially inert, but can be converted in vivo
to highly toxic, active species with the aim of specifically destroying tumor cells. Activation can be the
result of metabolism by an enzyme that is either
unique to the target organ, or present at much higher
concentrations compared to other tissues. Tumor
destruction involves two essential steps: (i) specific
targeting of malignant cells with the gene encoding
enzyme, which can also be under the control of a
specific promoter (e.g., PSA or PSMA in the prostate),
and (ii) administration of the prodrug, which will be
activated into its toxic derivative by the appropriate
enzyme within the target tissue concerned, with little
or no systemic consequences (65,66).
Tumor vaccines represent a different potential
treatment modality. There are three basic approaches
to construct cancer vaccines: (i) whole tumor cells or
nonpurified cellular extracts preparations, in an
attempt to include relevant tumor antigens that can
stimulate protective immune responses; (ii) partially
purified preparations, enriched in the cellular fraction
most likely to contain relevant tumor antigens; and
(iii) preparations from highly purified tumor antigens.
The development of these vaccines depends on
expression of a family of genes reported to encode
antigens recognized by autologous cytotoxic T lymphocytes. Such genes have now been identified in
melanoma cells (MAGE-1, MAGE-3, BAGE, GAGE) as
well as in head and neck tumors, nonsmall cell lung
cancers, and bladder carcinomas. Work from Chen
et al. (67) demonstrated the presence of two genes
(PAGE and GAGE-7) expressed in the LNCaP cell line
that may be specific to prostate cancer and serve as a
potential target for tumor immunization.
Every technique mentioned above has specific
advantages and disadvantages, the details of which
are beyond the scope of this chapter. Experimental
393
studies of gene therapy in prostate cancer are emerging in the literature at an increasing frequency.
ANDROGEN REGULATION
Androgens are important male sex hormones that, in
addition to being essential for the growth and differentiation of all male sex accessory organs, are strongly
associated with the development and progression of
prostate cancer. Androgen action in prostate cancer is
mediated through the androgen receptor (AR), a
ligand-dependent transcription factor that is a member of the steroid/thyroid hormone receptor gene
superfamily. The mitogenic effects of androgens on
prostatic growth appear to be mediated through the
action of soluble peptide growth factors, acting in
either an autocrine or paracrine manner. Various
model systems for prostate cancer have shown fibroblast growth factor 7 (FGF-7) and transforming
growth factor-b1 (TGF-b1) to be paracrine mediators
of androgen action and FGF-2 to be an androgenmediated autocrine growth factor.
Androgen depletion prolongs the disease-free
interval for prostate cancer patients, indicating that
the cancer cells are androgen sensitive for growth.
However, this treatment is only palliative because
androgen-insensitive clones of cancer cells expand
and progress. This observation has been made in
almost every case of prostate cancer. These androgen-insensitive (or castrate-resistant) cells acquire the
ability to proliferate in the absence of androgen
through genetic mutations. Mutations may result in
changes in the function/expression of AR protein or
growth factors and their receptors.
Androgen Receptor
The AR can be structurally divided into three domains:
a transcriptional activation domain, a DNA-binding
domain, and a ligand-binding domain (68) (Fig. 3).
Cellular signaling occurs following androgen binding to
the AR and translocation of the receptor to the nucleus.
This activated complex associates with androgen
responsive elements contained in the DNA sequence
of a number of target genes to affect their transcriptional
activity. The possible presence in vivo of alternate
AR isoforms, the extent of AR phosphorylation, the
Figure 3 Functional organization of the androgen

receptor.
394
Hamdy and Robson
association with other proteins, and the presence of

polymorphic glutamine and glycine regions may provide additional levels of control for AR action.
Radioligand-binding studies and immunohistochemistry have been used to detect AR protein expression. Both primary and metastatic prostate cancer
show elevated AR by ligand-binding assays when
compared to nonmalignant prostate tissue (69). Immunohistochemistry supports elevated AR in prostate
cancer with strong nuclear staining, mostly of a heterogeneous nature in hormone-relapsed and in primary and metastatic hormone-refractory prostate
cancer (70).
A number of studies have suggested that a high
frequency of amino acid substitutions occur in the AR
protein (2550%) for advanced prostate cancer and in
hormone-relapsed tumors from primary and metastatic sites. Functional analysis of these mutations
has revealed alterations in ligand binding and transcriptional activation. Additionally, AR gene amplification has been identified in 30% of recurrent prostate
tumors (71). Examination of the corresponding primary tumor prior to initiation of hormone therapy
showed no evidence of amplification, suggesting that
amplification occurred during androgen deprivation,
conferring a growth advantage on the prostate cancer
cells. Variation in the length of a polyglutamine
stretch in the N-terminal domain of the AR protein,
causing an alteration in AR function, has been suggested as a contributory factor toward an increased
lifetime risk for the development of prostate cancer.
heterogeneous nature of prostate tumors have further

complicated data analysis. However, mRNA molecular profiling of the full human genome using microdissected tumor material has identified small groups
of genes, so-called gene signatures, that correlate with
the Gleason grading system, providing several potential targets for therapeutic intervention. Increased
expression of the AR is consistently associated with
progression to hormone refractory prostate cancer,
highlighting the important, central role of the AR in
the hormone refractory phenotype (74,75).
Micro-RNAs are a group of small, noncoding,
single-stranded RNAs of approximately 22 nucleotides that repress protein production by targeting specific mRNAs. Recent research has highlighted the
widespread altered expression of these micro-RNAs
in cancer. Several hundreds of different micro-RNAs
have been identified; each micro-RNA can repress the
expression of multiple proteins. In common with
many cancers, many micro-RNAs have been demonstrated to be overexpressed in prostate cancer (76,77).
Two micro-RNAs, miR-15a and miR-16-1, are shown
to act as tumor suppressor genes in prostate cancer by
controlling proliferation, invasion, and cell survival
(78). Interestingly, a number of micro-RNAs have
been shown to be androgen regulated, again highlighting the important role of the AR signaling pathway in prostate cancer (79).
RECURRENT GENE REARRANGEMENTS IN

PROSTATE CANCER
Apoptosis
In late 2006, Arul Chinnaiyans research group identified the first recurrent gene fusions in the majority of
prostate cancers (72). The major fusion identified
comprised a fusion of the 50 promoter region of the
TMPRSS2 gene (a prostate-specific, androgen-responsive gene) to an Ets family (oncogenic transcription
factor) gene. The resulting fusions result in the overexpression of an oncogenic Ets family transcription
factor. These findings have been confirmed by several
research groups, and additional, less common gene
rearrangements have further been identified (73).
These recurrent fusions appear to correlate with prostate cancer development and progression. Circulating
prostate cancer cells from serum have been identified
using FISH to detect these fusion genes, opening up
many opportunities to exploit these gene rearrangements as powerful biomarkers to study prostate cancer response to therapy.
MOLECULAR PROFILING
IN PROSTATE CANCER
Extensive investigations have been conducted to profile the genes that are aberrantly expressed in prostate
cancer to help elucidate those oncogenes and tumor
suppressor genes that contribute toward development
and progression of the disease. The complexity and
APOPTOSIS-REGULATING GENES
IN PROSTATE CANCER
Hormone ablation in prostate cancer achieves its effect

by activation of apoptosis (programmed cell death)
(80,81), which is a distinct mode of cell death that
occurs in both normal physiological conditions and
the diseased state, and is frequently dysregulated in
disease, including cancer (82). Apoptosis is an active
process mediated by a family of cysteine proteases
(caspases) and is characterized by distinct morphological changes in individual cells, with compaction
and margination of nuclear chromatin, cytoplasmic
condensation, and convolution of nuclear and cell
outlines (83). Later changes involve nuclear fragmentation and budding of the cell with the development
of membrane-bound apoptotic bodies, which are
removed by phagocytosis. In contrast to necrosis,
which is a passive process, there is no associated
inflammation.
A number of genes are involved in the control of
apoptosis including the proto-oncogene Bcl-2 on chromosome 18q21 (84,85) and the tumor suppressor gene
p53 on chromosome 17p13 (86).
Bcl-2
The Bcl-2 gene was initially identified in B cell lymphomas (87), and the gene product was shown to act
by inhibiting cell death without directly affecting cell
proliferation. The Bcl-2 protein family has at least

20 members, comprising two functionally antagonistic
groups controlling the balance between cell death and
survival. Bax and Bak are proapoptotic Bcl-2 family
members that induce mitochondrial outer membrane
permeabilization, promoting release of caspaseactivating proteins and other cell death mediators.
The antiapoptotic proteins, including Bcl-2, oppose
this action and preserve the integrity of the outer
membrane (88,89).
In the prostate Bcl-2 is normally expressed in
basal epithelial cells, seminal vesicles, and ejaculatory
ducts. In primary prostate cancer Bcl-2 is expressed in
around 25% of cases (90). Bcl-2 overexpression is
associated with increasing tumor stage and the
development of hormone refractory disease (91,92).
In high-grade prostatic intraepithelial neoplasia
(HGPIN) the reported expression of Bcl-2 has shown
a wide variation from 0% to 100%. The largest study
reported Bcl-2 expression in 4/24 (17%) cases of
HGPIN (90,93,94).
p53
Inactivation of the tumor suppressor gene p53 is the
most common mutation identified in human cancers
(95). Functional (wild-type) p53 protein has DNAbinding properties and forms a key part of the mechanism by which mammalian cells undergo growth
arrest or apoptosis in response to DNA damage. Mutation of p53 may result in loss of its normal function.
Mutations in the p53 gene are most frequently
detected in the highly conserved exons 5, 6, 7, and 8.
In most cases one allele is completely deleted with a
missense mutation in the remaining allele. Mutant p53
protein has a prolonged half-life compared to the wildtype p53 protein, and its nuclear accumulation is
detectable by immunohistochemistry.
In benign prostatic epithelium p53 positivity is
absent. In primary prostate cancer p53 nuclear positivity is present in around 20% of cases (96,97). p53
protein accumulation appears to be a late event, being
associated with advanced stage, high Gleason tumor
grade, hormonal resistance, poor survival, DNA aneuploidy, and high cell proliferation rate (98101). A
good correlation is observed between p53 immunoreactivity in prostate cancer and direct evidence of
gene mutation using the polymerase chain reaction,
single-strand conformational polymorphism and
direct sequencing (102). p53 positivity is infrequent
in HGPIN, the largest study showing strong nuclear
staining to be 14% (103).
Wild-type p53 may participate with Bcl-2 and
Bax in a common pathway, regulating cell death by
decreasing the expression of Bcl-2 while simultaneously increasing the expression of Bax, resulting in
apoptosis (104). The combination of Bcl-2 overexpression and nuclear accumulation of p53 protein in
human prostate cancer has been shown to correlate
with the development of hormone refractory disease
(96), and are independent prognostic markers for
postradical prostatectomy recurrence (92,105).
395
ANGIOGENESIS
Microvessel Density
The ability of a tumor to grow and metastasize depends
on the induction of a tumor vasculature, referred to as
the angiogenic switch. The quantification of new
microvessels within a tumor is commonly performed
using antibodies against factor VIII to identify endothelial cells. Increasing microvessel density correlates
with increasing Gleason score, presence of metastases,
and is an independent predictor of progression after
radical prostatectomy for Gleason score 5 to 7 tumor
(106,107).
Vascular Endothelial Growth Factor

Angiogenesis is controlled by a group of substances
termed angiogenic factors. Vascular endothelial
growth factor (VEGF) is a potent inducer of endothelial cell growth and is expressed in a variety of
tumors. VEGF expression is increased in prostate
cancer compared to benign prostatic epithelium
(108). Targeting the tumor vasculature with angiogenic inhibitors presents an attractive therapy to
limit growth of the primary cancer and prevent cancer
recurrence or metastases (109).
GROWTH FACTORS
Growth factors may act as positive or negative effectors of various cellular processes, including proliferation, differentiation, and cell death. Interaction occurs
with specific membrane receptors, which results in the
transmission of signals through an intracellular protein cascade and the activation or the repression of a
number of target genes. Several growth factors have
been associated with prostatic growth, including TGFa and TGF-b, FGFs, insulin-like growth factors (IGFs),
epidermal growth factor (EGF), nerve growth factor,
and various cytokines.
Growth factors act primarily over short distances
in either an autocrine or paracrine manner. In addition, growth factors may act through an endocrine
pathway affecting target cells at distant sites. Many
growth factors possess a mitogenic activity that is
mediated through a membrane-bound receptor. Interaction occurs with an extracellular ligand-binding
domain leading to a change in receptor conformation,
resulting in the activation of an intracellular tyrosine
kinase domain. Tyrosine phosphorylation of specific
intracellular proteins is responsible for the mitogenic
signal. In many cases the protein components of the
intracellular cascade remain to be elucidated (110).
Aberrant signaling may result from mutation in
growth factors or their downstream effector proteins,
leading to either loss of growth factor function, that is,
switching off the signaling pathway, or uncontrolled
expression or activation, that is, permanently
switched on signaling pathway. Such changes are
commonly associated with the malignant state and
the aggressive phenotypes of cancer cells. Given the
important role of growth factor receptor signaling in
396
Hamdy and Robson
cell proliferation and their frequent alterations in cancer, the receptors provide attractive targets for therapeutic intervention using small molecule inhibitors
and antibodies (111).
Transforming Growth Factor-b1

TGF-b1 belongs to a superfamily of structurally related
regulatory polypeptides that includes activins/inhibins
and bone morphogenetic proteins (BMPs). TGF-b1 is a
pleiotropic growth factor that acts through type I and II
receptor kinases to regulate multiple cellular mechanisms including proliferation, angiogenesis, immune
response, and cell differentiation. Generally, TGF-b1
functions as a mitogen for various mesenchymal cells
and a potent growth inhibitor of lymphoid, endothelial,
and epithelial cells. TGFb-1 and -2 have been implicated in the development of prostatic disease (112).
TGF-b1 has been detected using immunohistochemistry in both human prostatic stromal and epithelial cells,
and TGF-b2 mRNA has been identified in normal and
malignant human prostate. Addition of TGF-b1 to cultured prostatic epithelial and stromal cells inhibits
proliferation (113).
Bone Morphogenetic Proteins

The term BMP refers to an activity derived from bone
that induces ectopic bone formation in vivo (114). The
BMPs belong to the TGF-b superfamily that have the
capability of inducing ectopic and de novo bone formation in vivo, as well as having roles in chondrogenesis, differentiation, and development. To date,
few efforts were made to link BMP activity with the
development and progression of cancer. This is not
surprising because the majority of bony secondaries
result in osteolytic lesions, with increased bone
resorption and osteoclastic activity, unlike prostatic
secondaries that are mostly osteoblastic. A number of
studies have shown an association between BMP
expression and skeletal metastases in prostate cancer.
BMP-6, in particular, is expressed in the majority of
primary prostate cancers with established skeletal
secondaries, and rarely in localized disease. Overexpression of BMP-6 is associated with a more invasive
phenotype (115). Primary and secondary prostate cancer expresses BMP-6, which is found infrequently in
skeletal metastases from other human malignancies.
BMP-6 may have a role in the initiation of skeletal
secondaries, and the osteoblastic reaction commonly
seen in these deposits (116).
Fibroblast Growth Factors

The FGF family of polypeptide growth factors has
diverse physiologic and pathologic functions including development, wound healing, angiogenesis, and
tumorigenesis (117). The human FGFs comprise at
least 23 highly conserved genes and the receptor
family comprises four members, FGFR-1 to -4. Multiple ligands and receptors allow interaction between a
single receptor and several ligands, and between different receptor monomers through heterodimerization
following activation by FGF (118) to modulate a range
of biologic effects including mitogenesis, motility,
invasion, and differentiation. Multiple FGFs are
expressed at elevated levels in prostate cancer, including FGF-1, -2, -6, and -8 (119) acting as both paracrine
and autocrine growth factors.
Basic FGF (FGF-2) is secreted by prostatic fibroblasts in response to androgen and acts in an autocrine
fashion to stimulate fibroblast cell growth (120).
Stromal-derived keratinocyte growth factor (KGF/
FGF-7) is upregulated in hormone-resistant prostate
cancer and has a role as a potential paracrine growth
factor on epithelial cells (121). KGF has a potent mitogenic action on epithelial cells and is proposed to act as
an androgen-regulated mediator of epithelial cell
growth (122). A similar paracrine action applies to
FGF-8 (androgen-induced growth factor), which is
secreted in response to androgens and can stimulate
growth of epithelial and fibroblast cells. FGFR-1 to -3
can undergo alternative splicing to generate two alternative exons (IIIb and IIIc) that generate receptor isoforms with variant binding specificity. Differential
expression of the four FGFRs and a switch in the
expression of alternate spliced variants of FGFR2 are
documented (123). Given the importance of FGF signaling in mediating epithelial-stromal interactions during prostate carcinogenesis (124) and the aberrant
expression of many components of the FGF-FGFR system in prostate cancer, this highlights the great potential for therapeutic intervention in this pathway (125).
Insulin-Like Growth Factors

IGF-1 and -2 are potent mitogens that mediate normal
and neoplastic cell growth. The IGFs bind to specific
receptors, designated type I and II IGF receptors
(IGFR). Type I IGFR is a transmembrane heterotetramer tyrosine kinase that primarily mediates the
mitogenic actions of IGFs. IGFs are two of the most
abundant growth factors in bone (126), the preferential site for metastatic prostate cancer. Type I IGFR is
expressed by prostate cancer cells that could facilitate
the development of bone metastases, and several population studies suggest that elevated serum IGF-1
levels increase the risk of prostate cancer (127).
IGFs also have high affinity for a family of at
least six IGF-binding proteins (IGFBPs) that act to
regulate their bioavailability (128). The levels of circulating IGFBPs are regulated by endocrine factors and
by specific proteases that cleave IGFBPs to small
inactive peptides. IGFBPs are believed to modulate
proliferative and mitogenic effects of IGFs as well as
modulating cell growth independently of IGF.
Although all IGFBPs have high affinity for IGFs,
IGFBP-3 is the major transporter of IGFs in serum.
A number of studies have suggested that IGFBPs may
be involved in growth modulation of prostate malignancy. One study showed that IGFBP-2 was an androgen-dependent predictor for prostate cancer survival
(129).
Epidermal Growth Factor

Binding of EGF to the extracellular domain of its
receptor, EGFR, results in activation of the receptors
cytoplasmic tyrosine kinase, phosphorylation of substrate proteins, and stimulation of cell proliferation.
Members of the EGF family play a role in modulation
of prostatic growth. Withdrawal of androgen from the
rodent prostate leads to reduced expression of EGF,
which is a potent mitogen for epithelial cells (130).
Thus, the continued presence of androgens within the
prostate helps maintain epithelial cell proliferation
mediated through the expression of EGF. Increased
EGF receptor signaling has been demonstrated in
prostate cancer and progression (131), and more
recently missense mutations of the EGF receptor tyrosine kinase domain have been identified that have
oncogenic properties (132).
CELL ADHESION
Cell adhesion is of fundamental importance in establishing and maintaining tissue form and function.
Several adhesion molecules, including cadherins,
integrins, selectins, and members of the immunoglobulin superfamily, are involved in the mechanisms by
which a cell maintains contact with other cells and
interacts with the extracellular matrix (133,134). Fibronectin, collagen, laminin, and vitronectin are major
components of this complex extracellular matrix that
interact with their cognate receptors, most of which
are integrins. Integrins function as heterodimeric
membrane glycoproteins, the combination of a and b
subunits determining the ligand specificity. Differential expression of members of the large integrin family
allows the cell to modulate its interaction with other
cells and the extracellular matrix. Integrins are important components of cellular signal transduction, mediating cell-matrix interactions, while cadherins
principally mediate intercellular interactions.
Cadherins are a large family of calcium-dependent morphoregulatory proteins. The best studied
proteins within this family are E- and N-cadherin.
Membrane-associated cadherins require members of
the catenin family of proteins to mediate their interaction with the cytoskeleton. Catenins probably act by
oligomerizing proteins to which they bind and/or to
attach them to the actin cytoskeleton.
E-cadherin
The E-cadherin gene plays a critical role in embryogenesis and organogenesis, and membrane-bound Ecadherin protein mediates epithelial cell-cell recognition and adhesion (135). E-cadherin complexes are
important in maintaining the normal differentiated
phenotype of epithelial cells. Downregulation of Ecadherin is observed in many cancers, and for prostate
cancer dysregulation of E-cadherin is associated with
an invasive phenotype (136). The intracellular domain
of E-cadherin is anchored to the actin cytoskeleton via
three cytoplasmic proteins, a-, b- and g-catenin, and the
integrity of the cadherin-catenin complex is regulated
397
by phosphorylation. Protein kinase D1 (PKD1) phosphorylates E-cadherin and stabilizes cadherin-catenin

complexes (137). Downregulation of PKD1 is observed
in prostate cancer (138), and its effects on E-cadherin
likely contribute toward metastatic progression.
Aberrant E-cadherin staining is a powerful predictor of poor outcome, both in terms of disease progression and patient survival (136).
CD44
The CD44 gene is an integral transmembrane glycoprotein involved in specific cell-cell and cellextracellular matrix interactions (139). The gene is
encoded by 20 exons, at least 10 of which are differentially expressed because of alternative splicing of
mRNA (140). CD44 is expressed on the plasma membrane of prostatic glandular cells. It is involved in cell
adhesion because it acts as a receptor for the extracellular matrix components hyaluronic acid and osteopontin. CD44 is believed to play a major role in tumor
metastases; alternative splice variants of the receptor
differ in their capacity to enhance or decrease metastatic potential. In human prostate cancer CD44 downregulation is correlated with high tumor grade,
aneuploidy, and distant metastases (141).
PROLIFERATION
The hallmark of malignancy is uncontrolled growth. It
is therefore logical to assume that measuring proliferative capacity within a tumor may indicate its invasive
potential and ability to progress. Several methods are
available to assess proliferation, including determination of S-phase fraction by flow cytometry, labeling of
replicating DNA with bromodeoxyuridine (BrdU),
and application of immunohistochemistry using antibodies against proliferation-related antigens (Ki67 and
MIB-1) (142,143). Their use in clinical practice, however, remains to be determined.
TUMOR PLOIDY AND NUCLEAR

MORPHOMETRY
Nuclear DNA content, otherwise known as ploidy,
can be studied by flow cytometry and image or static
cytometry. Tumors can be broadly classified as diploid, tetraploid, or aneuploid, with accompanying
variations in view of the well-documented heterogeneity of prostatic adenocarcinoma. Several reports
have correlated DNA ploidy with prognosis in prostate cancer. The results are conflicting, and only half
the studies published confirm ploidy to be an independent prognostic marker (144). A more modern and
sensitive method of assessing ploidy has been developed, using fluorescent (FISH) and nonfluorescent
DNA in situ hybridization of interphase cells. These
techniques visualize individual chromosomes by specific binding of a labeled probe to a particular DNA
sequence, mostly localized at the centromere region.
There is good correlation with flow cytometry, but
FISH appears to be more sensitive (145). FISH analysis
398
Hamdy and Robson
is beginning to impact on clinical practice, for example, in assessing the HER2 gene copy number in breast
cancer stratification of patients.
GENETIC FACTORS
Genetic alterations are important contributory events
in neoplasia. A variety of genes have been identified
that are associated with predisposition or progression
for most of the common epithelial neoplasms. Most
known oncogenes and suppressor genes have been
screened for their importance in primary prostate
cancer, but no common mutations have been identified. p53 mutations are rare in early prostate cancer
but have been observed in almost 50% of advanced,
metastatic disease (100). Mutations in the retinoblastoma (Rb) gene and deletion or methylation of
p16INK4a (CDKN2), two genes intimately linked to
cell cycle progression, have been described in a few
prostate tumors and cell lines.
Allelic loss, defined by the absence of one of the
two copies of an autosomal locus present in somatic
cells, commonly occurs in prostate cancer. Loss of
heterozygosity and comparative genomic hybridization analyses have revealed frequent loss of genetic
material from chromosome regions 7q, 8p, 10pq, 13q,
16q, 17p, and 18q in primary and metastatic prostate
cancer. Specific gene loci have also been identified as
metastatic suppressors in prostate cancer. Introduction of the genes for KAI1 (chromosome 11p11.2), Ecadherin (chromosome 16q22), or CD44 (chromosome
11p13) into prostate cancer cells have been shown to
suppress metastatic ability.
Hereditary prostate cancer has been reported to
account for some 9% of all prostate cancer and more
than 40% of early onset disease (146). A number of
studies revealed a familial clustering for prostate cancer, supporting a genetic predisposition to the disease.
A risk factor of between 2 and 3 has been indicated in
first-degree relatives of affected men. This is further
increased when they are diagnosed at an earlier age
(147).
More recent work using genome-wide association
analysis of thousands of men investigating the frequency of small nucleotide polymorphisms have identified multiple genetic loci (located on chromosomes 2,
3, 6, 7, 10, 11, 17, 19, and X) that are associated with
susceptibility to developing prostate cancer (148150).
MATRIX METALLOPROTEINASES AND

THEIR INHIBITORS
Tumor invasion and metastasis represent key events in
the natural history of cancer. To complete the different
steps involved in the metastatic cascade of events, a
malignant cell has to overcome a number of natural
barriers, including extracellular matrix and basement
membranes. This can be partly achieved through
excess production of proteolytic enzymes either by
the tumor cells themselves, or through stimulation of
surrounding stromal cells to secrete such enzymes
(151). The matrix metalloproteinases (MMPs) are a
large family (23 in humans) of extracellular zinc

enzymes that mediate a number of tissue-remodeling
processes, and have been strongly associated with
cancer invasion and metastasis because of their capacity to degrade the extracellular matrix. A number of
malignancies including prostate cancer have been
shown to differentially express MMPs that correlated
strongly with aggressive disease (152154). MMPs are
tightly regulated by their inhibitors called tissue inhibitors of metalloproteinases that bind in a 1:1 stoichiometry to inactivate the MMPs. Past clinical evaluation of
small molecule inhibitors of the MMPs has been unrewarding, partly a consequence of treating patients with
advanced, metastatic disease. However, several novel
inhibitors of MMPs have been synthesized and they
may still provide a useful therapeutic approach for
prostate tumors that have not metastasized (155).
IN VITRO AND IN VIVO MODELS

OF PROSTATE CANCER
To investigate the biology of prostate cancer, a number of models have been developed over the years.
The three most widely used cell lines are LNCaP, an
AR-positive epithelial prostate cancer cell line originating from a metastatic lymph node; PC-3, an ARnegative epithelial prostate cancer line originating
from metastatic bone secondaries; and DU-145, an
AR-negative epithelial prostate cancer line originating
from metastatic brain secondaries (156158). A wide
range of murine in vivo experimental models are used
to study tumor initiation, growth, and metastatic progression in prostate cancer. These models frequently
employ gene knockout, knock-in or conditional
regulation of individual gene expression (oncogenes,
growth factors, cell cycle regulators), or combinations
of genes (159,160). These models are extremely useful
in studying a variety of factors thought to affect the
development and progression of prostate cancer (161).
The recent ability to alter the expression of a specific
gene in a single tissue cell type in a temporal fashion
will greatly facilitate our studies to accurately model
sporadic tumor formation for this disease in a whole
animal (162).
REFERENCES
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics 2008. CA
Cancer J Clin 2008; 58:7196.
2. Cancer Research UK statistics. Available at: http://info.
cancerresearchuk.org/cancerstats/types/prostate/.
3. Collin SM, Martin RM, Metcalfe C, et al. Prostate-cancer
mortality in the US and UK in 19752004: an ecological
study. Lancet Oncol 2008; 9:445452.
4. Schroder FH, Hugosson J, Roobol MJ, et al. ERSPC
Investigators. Screening and prostate-cancer mortality in
a randomized European study. N Engl J Med 2009; 360
(13):13201328.
5. Melia J, Moss S, Johns L. Rates of prostate-specific antigen
testing in general practice in England and Wales in
asymptomatic and symptomatic patients: a cross-sectional study. BJU Int 2004; 94:5156.

6. Muir CS, Nectoux J, Statzsewski J. The epidemiology of
prostatic cancer. Acta Oncol 1991; 30:133140.
7. Pienta KJ, Esper PS. Risk factors for prostate cancer. Ann
Intern Med 1993; 118:793803.
8. Smith JR, Freije D, Carpten JD, et al. Major susceptibility
locus for prostate cancer on chromosome 1 suggested by a
genome-wide search. Science 1996; 274:13711374.
9. Bova GS, Beaty TH, Steinberg GD, et al. Hereditary prostate cancer: epidemiologic and clinical features. J Urol
1993; 150:797802.
10. McNeal JE, Redwine EA, Frieha FS, et al. Zonal distribution of prostatic adenocarcinoma: correlation with histologic pattern and direction of spread. Am J Surg Pathol
1988; 12:897906.
11. Gleason DF. Histologic grading of prostate cancer: a perspective. Hum Pathol 1992; 23: 273279.
12. Humphrey PA, Frazier HA, Vollmer RT, et al. Stratification of pathologic features in radical prostatectomy specimens that are predictive of elevated initial postoperative
serum prostate-specific antigen levels. Cancer 1993;
71:18211827.
13. Bostwick DG. Gleason grading of prostatic needle biopsies: correlation with grade in 316 matched prostatectomies. Am J Surg Pathol 1994; 18:796803.
14. Trock BJ, Guo CC, Gonzalgo ML, et al. Tertiary Gleason
patterns and biochemical recurrence after prostatectomy:
proposal for a modified Gleason scoring system. J Urol
2009; 182(4):13641370.
15. Epstein JI. Precursor lesions to prostatic adenocarcinoma.
Virchows Arch 2009; 454(1):116.
16. Girasole CR, Cookson MS, Putzi MJ, et al. Significance
of atypical and suspicious small acinar proliferations,
and high grade prostatic intraepithelial neoplasia on
prostate biopsy: implications for cancer detection and
biopsy strategy. J Urol 2006; 175(3 pt 1):929933; discussion 933.
17. Lundwall A, Clauss A, Olsson AY. Evolution of kallikrein-related peptidases in mammals and identification of
a genetic locus encoding potential regulatory inhibitors.
Biol Chem 2006; 387:243249.
18. Lilja H, Ulmert D, Vickers AJ. Prostate-specific antigen
and prostate cancer: prediction, detection and monitoring.
Nat Rev Cancer 2008; 8(4):268278.
19. Chybowski FM, Larson Keller JJ, Beerstralh EH, et al.
Predicting radionuclide bone scan findings in patients
with newly diagnosed, untreated prostate cancer: prostate
specific antigen is superior to all other clinical parameters.
J Urol 1991; 145:313318.
20. Catalona WJ, Ritchie JP, Ahmann FB, et al. Comparison of
digital rectal examination and serum prostate specific
antigen in the early detection of prostate cancer: results
of a multicentre clinical trial of 6,630 men. J Urol 1994;
151:12831290.
21. Christensson A, Bjork T, Nilsson O, et al. Serum prostate
specific antigen complexed to alpha-1-antichymotrypsin as
an indicator of prostate cancer. J Urol 1993; 150:100105.
22. Leung H, Lai L, Day J, et al. Serum free PSA in the
diagnosis of prostate cancer. Br J Urol 1997; 80:256259.
23. Catalona WJ, Smith DS, Ornstein DK. Prostate cancer
detection in men with serum PSA concentrations of 2.6
to 4.0 ng/mL and benign prostate examination: enhancement of specificity with free PSA measurements. JAMA
1997; 227:14521455.
24. Lilja H, Ulmert D, Bjork T, et al. Long-term prediction of
prostate cancer up to 25 years before diagnosis of prostate
cancer using prostate kallikreins measured at age 44 to 50
years. J Clin Oncol 2007; 25:431436.
25. Tilling K, Garmo H, Metcalfe C, et al. Development of a
new method for monitoring prostate-specific antigen
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
399
changes in men with localised prostate cancer: a comparison of observational cohorts. Eur Urol 2009; 57:446452.
Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence
of prostate cancer among men with a prostate-specific
antigen level < or 4.0 ng per milliliter. N Engl J Med
2004; 350(22):22392246.
Lane JA, Howson J, Donovan J, et al. Prostate cancer
detection in an unselected young population: experience
of the ProtecT study. BMJ 2007; 335:11391143.
Fidler IJ. Metastasis: quantitative analysis of distribution
and fate of tumour emboli labelled with 125I-5-iodo-20 deoxyuridine. J Natl Cancer Inst 1970; 45:773782.
Fidler IJ, Hart IR. Biological diversity in metastatic
neoplasms: origins and implications. Science 1982;
217:9981003.
Hamdy FC, Lawry J, Anderson JB, et al. Circulating
prostate-specific antigen-positive cells correlate with
metastatic prostate cancer. Br J Urol 1992; 69:392396.
Fadlon EJ, Rees RC, Lawry J, et al. Detection of circulating
PSA-positive cells in patients with prostate cancer by flow
cytometry and reverse transcription polymerase chain
reaction. Br J Cancer 1996; 74:400405.
Katz AE, Olsson CA, Raffo AJ, et al. Molecular staging of
prostate cancer with the use of an enhanced reverse
transcriptase-PCR assay. Urology 1994; 43:765774.
Israeli RS, Miller WH, Su SL, et al. Sensitive nested
reverse transcription polymerase chain reaction detection
of circulating prostatic tumor cells: comparison of prostate-specific membrane antigen and prostate-specific antigen-based assays. Cancer Res 1994; 54:63066310.
Smith MR, Biggar S, Hussain M. Prostate specific antigen
messenger RNA is expressed in non-prostate cells: implications for the detection of micrometastases. Cancer Res
1995; 55:26402644.
Tibbe AG, de Grooth BG, Greve J, et al. Optical tracking
and detection of immunomagnetically selected and
aligned cells. Nat Biotechnol 1999; 17:12101213.
Scher HI, Jia X, de Bono JS, et al. Circulating tumour cells
as prognostic markers in progressive, castration-resistant
prostate cancer: a reanalysis of IMMC38 trial data. Lancet
Oncol 2009; 10(3):233239.
Neal DE, Donovan JL, Martin RM, et al. Screening for
prostate cancer remains controversial. Lancet 2009; 374
(9700):14821483.
DAmico AV, Whittington R, Malkowicz SB, et al. Biochemical outcome after radical prostatectomy, external
beam radiation therapy, or interstitial radiation therapy
for clinically localized prostate cancer. JAMA 1998; 280:9.
Albertsen PC, Hanley JA, Fine J. 20-year outcomes following conservative management of clinically localized
prostate cancer. JAMA 2005; 293(17):20952101.
Holmberg L, Bill-Axelson A, Helgesen F, et al. Scandinavian Prostatic Cancer Group Study Number 4. A randomized trial comparing radical prostatectomy with watchful
waiting in early prostate cancer. N Engl J Med 2002; 347
(11):781789.
Bill-Axelson A, Holmberg L, Filen F, et al. Scandinavian
Prostate Cancer Group Study Number 4. Radical prostatectomy versus watchful waiting in localized prostate cancer:
the Scandinavian Prostate Cancer Group-4 randomized
trial. J Natl Cancer Inst 2008; 100(16):11441154.
Bill-Axelson A, Holmberg L, Ruutu M, et al. Scandinavian
Prostate Cancer Group Study No. 4. Radical prostatectomy versus watchful waiting in early prostate cancer.
N Engl J Med 2005; 352(19):19771984.
Wilt TJ, Brawer MK, Barry MJ, et al. The Prostate cancer
Intervention Versus Observation Trial: VA/NCI/AHRQ
Cooperative Studies Program #407 (PIVOT): design and
baseline results of a randomized controlled trial
400
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
Hamdy and Robson

comparing radical prostatectomy to watchful waiting for
men with clinically localized prostate cancer. Contemp
Clin Trials 2009; 30(1):8187.
Donovan J, Mills N, Smith M, et al. Improving the design
and conduct of randomised trials by embedding them in
qualitative research: the ProtecT study. Br Med J 2002; 325
(7367):766770.
Bolla M, de Reijke TM, Van Tienhoven G, et al. EORTC
Radiation Oncology Group and Genito-Urinary Tract
Cancer Group. Duration of androgen suppression in the
treatment of prostate cancer. N Engl J Med 2009; 360(24):
25722574.
Adib RS, Anderson JB, Ashken MH, et al. Immediate
versus deferred treatment for advanced prostatic cancer:
initial results of the Medical Research Council trial. Br J
Urol 1997; 79:235246.
Abbas F, Scardino PT. Why neoadjuvant androgen deprivation prior to radical prostatectomy is unnecessary.
Urol Clin North Am 1996; 23(4):587604.
Witjes WP, Schulman CC, Debruyne FM. Preliminary
results of a prospective randomized study comparing
radical prostatectomy versus radical prostatectomy associated with neoadjuvant hormonal combination therapy
in T2-T3 N0 M0 prostatic carcinoma. The European study
group on neoadjuvant treatment of prostate cancer. Urology 1997; 49:(3A suppl):6569.
Huggins C, Hodges CV. Studies on prostate cancer. The
effect of castration, of oestrogen and of androgen injection
on serum phosphatase in metastatic carcinoma of the
prostate. Cancer Res 1941; 1:293297.
Klotz L, Boccon-Gibod L, Shore ND, et al. The efficacy and
safety of degarelix: a 12-month, comparative, randomized,
open-label, parallel-group phase III study in patients with
prostate cancer. BJU Int 2008; 102:15311538.
Maximum androgen blockade in advanced prostate cancer: an overview of 22 randomised trials with 3283 deaths
in 5710 patients. Prostate Cancer Trialists Collaborative
Group. Lancet 1995; 346:265269.
Caubet JF, Tosteson TD, Dong EW, et al. Maximum
androgen blockade in advanced prostate cancer: a metaanalysis of published randomized controlled trials using
nonsteroidal antiandrogens. Urology 1997; 49:7178.
Akaza H, Hinotsu S, Usami M, et al. Study Group for the
Combined Androgen Blockade Therapy of Prostate Cancer. Combined androgen blockade with bicalutamide for
advanced prostate cancer: long-term follow-up of a phase
3, double-blind, randomized study for survival. Cancer
2009; 115(15):34373445.
Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel
and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J
Med 2004; 351(15):15131520.
Tannock IF, de Wit R, Berry WR, et al. TAX 327 Investigators. Docetaxel plus prednisone or mitoxantrone plus
prednisone for advanced prostate cancer. N Engl J Med
2004; 351(15):15021512.
Attard G, Reid AH, AHern R, et al. Selective inhibition of
CYP17 with abiraterone acetate is highly active in the
treatment of castration-resistant prostate cancer. J Clin
Oncol 2009; 27(23):37423748.
Shahinian VB, Kuo YF, Freeman JL, et al. Risk of fracture
after androgen deprivation for prostate cancer. N Engl J
Med 2005; 352(2):154164.
Smith MR, Egerdie B, Hernandez Toriz N, et al. Denosumab HALT Prostate Cancer Study Group. Denosumab in
men receiving androgen-deprivation therapy for prostate
cancer. N Engl J Med 2009; 361(8):745755.
Saad F, Gleason DM, Murray R, et al. Zoledronic Acid
Prostate Cancer Study Group. A randomized, placebo-
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
controlled trial of zoledronic acid in patients with hormone-refractory metastatic prostate carcinoma. J Natl
Cancer Inst 2002; 94(19):14581468.
Saad F, Gleason DM, Murray R, et al. Zoledronic Acid
Prostate Cancer Study Group. Long-term efficacy of zoledronic acid for the prevention of skeletal complications in
patients with metastatic hormone-refractory prostate cancer. J Natl Cancer Inst 2004; 96(11):879882.
Pisters LL, Leibovici D, Blute M, et al. Locally recurrent
prostate cancer after initial radiation therapy: a comparison of salvage radical prostatectomy versus cryotherapy.
J Urol 2009; 182(2):517525.
Finley DS, Osann K, Skarecky D, et al. Hypothermic nervesparing radical prostatectomy: rationale, feasibility, and
effect on early continence. Urology 2009; 73(4):691696.
Ahmed HU, Zacharakis E, Dudderidge T, et al. Highintensity-focused ultrasound in the treatment of primary
prostate cancer: the first UK series. Br J Cancer 2009; 101
(1):1926.
Culver KW. Gene Therapy. A Handbook for Physicians.
New York: Liebert MA, 1994.
Connors TA. The choice of prodrugs for gene directed
enzyme prodrug therapy of cancer. Gene Ther 1995;
2:702709.
Both GW. Recent progress in gene-directed enzyme prodrug therapy: an emerging cancer treatment. Curr Opin
Mol Ther 2009; 11(4):421432.
Chen ME, Sikes RA, Troncoso P, et al. PAGE and GAGE-7
are novel genes expressed in the LNCaP prostatic carcinogenesis model that share homology with melanoma associated antigens. J Urol 1996; 155:642A.
OMalley B. The steroid receptor superfamily: more
excitement predicted for the future. Mol Endocrinol
1990; 4:363369.
Brolin J, Skoog L, Elman P. Immunohistochemistry and
biochemistry in detection of androgen, progesterone, and
estrogen receptors in benign and malignant human prostatic tissue. Prostate 1992; 20:281295.
Masai M, Sumiya H, Akimoto S, et al. Immunohistochemical study of androgen receptor in benign hyperplastic and
cancerous human prostates. Prostate 1990; 17:293300.
Visakapori T, Hyytinen E, Koivisto P, et al. In vivo
amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 1995; 9:401406.
Tomlins SA, Mehra R, Rhodes DR, et al. TMPRSS2:ETV4
gene fusions define a third molecular subtype of prostate
cancer. Cancer Res 2006; 66(7):33963400.
Kumar-Sinha C, Tomlins SA, Chinnaiyan AM. Recurrent
gene fusions in prostate cancer. Nat Rev Cancer 2008; 8(7):
497511.
True L, Coleman I, Hawley S, et al. A molecular correlate to
the Gleason grading system for prostate adenocarcinoma.
Proc Natl Acad Sci U S A 2006; 103(29):1099110996.
Tamura K, Furihata M, Tsunoda T, et al. Molecular
features of hormone-refractory prostate cancer cells by
genome-wide gene expression profiles. Cancer Res 2007;
67(11):51175125.
Ozen M, Creighton CJ, Ozdemir M, et al. Widespread
deregulation of microRNA expression in human prostate
cancer. Oncogene 2008; 27(12):17881793.
Porkka KP, Pfeiffer MJ, Waltering KK, et al. MicroRNA
expression profiling in prostate cancer. Cancer Res 2007;
67(13):61306135.
Bonci D, Coppola V, Musumeci M, et al. The miR-15amiR-16-1 cluster controls prostate cancer by targeting
multiple oncogenic activities. Nat Med 2008; 14(11):
12711277.
Ambs S, Prueitt RL, Yi M, et al. Genomic profiling of
microRNA and messenger RNA reveals deregulated
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
microRNA expression in prostate cancer. Cancer Res

2008; 68(15):61626170.
Kyprianou N, English HF, Isaacs JT. Programmed cell
death during regression of PC-82 human prostate cancer
following androgen ablation. Cancer Res 1990; 50:
37483753.
Colombel M, Symmans F, Gil S, et al. Detection of the
apoptosis-suppressing oncoprotein bc1-2 in hormonerefractory human prostate cancers. Am J Pathol 1993;
143:390400.
Kerr JFR, Winterford CM, Harmon BV. Apoptosis. Its
significance in cancer and cancer therapy. Cancer 1994;
27:20132026.
Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 2004; 5
(11):897907.
Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an
inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990; 348:334336.
Lu QL, Abel P, Foster CS, et al. bcl-2: role in epithelial
differentiation and oncogenesis. Hum Pathol 1996;
27:102110.
Lane DP. p53, guardian of the genome. Nature 1992;
358:1516.
Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to
immortalize pre-B cells. Nature 1988; 335(6189):440442.
Kroemer G. The proto-oncogene Bcl-2 and its role in
regulating apoptosis. Nat Med 1997; 3:614620.
Yip KW, Reed JC. Bcl-2 family proteins and cancer.
Oncogene 2008; 27(50):63986406.
Bauer JJ, Sesterhenn IA, Mostofi FK, et al. Elevated levels
of apoptosis regulator proteins p53 and bcl-2 are independent prognostic biomarkers in surgically treated clinically localised prostate cancer. J Urol 1996; 156:15111516.
McDonnell TJ, Troncoso P, Brisbay SM, et al. Expression
of the proto-oncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate
cancer. Cancer Res 1992; 52:69406944.
Apakama I, Robinson MC, Walter NM, et al. bcl-2 overexpression combined with p53 protein accumulation correlates with hormone-refractory prostate cancer. Br J
Cancer 1996; 74:12581262.
Krajewski M, Krajewski S, Epstein JI, et al. Immunohistochemical analysis of bcl-2, bax, bcl-X and mcl-1 expression in prostate cancers. Am J Pathol 1996; 148:15671576.
Stattin P, Damber JE, Karlberg L, et al. Bcl-2 immunoreactivity in prostate tumourigenesis in relation to prostatic
intraepithelial neoplasia, grade, hormonal status, metastatic growth and survival. Urol Res 1996; 24:257264.
Vogelstein B, Kinzler KW. p53 function and dysfunction.
Cell 1992; 70:523526.
Mellon K, Thompson S, Charlton RG, et al. p53, c-erbB-2
and the epidermal growth factor receptor in the benign
and malignant prostate. J Urol 1992; 147:496499.
Thomas DJ, Robinson M, King P, et al. p53 expression
and clinical outcome in prostate cancer. Br J Urol 1993;
72:778781.
Visakorpi T, Kallioniemi OP, Heikkinen A, et al. Small
subgroup of aggressive, highly proliferative prostatic
carcinomas defined by p53 accumulation. J Natl Cancer
Inst 1992; 84:883887.
Kallakury BV, Figge J, Ross JS, et al. Association of p53
immunoreactivity with high Gleason tumor grade in
prostatic adenocarcinoma. Hum Pathol 1994; 25:9297.
Heidenberg HB, Sesterhenn JP, Gaddipati JP, et al. Alteration of the tumour suppressor gene p53 in a high fraction
of hormone refractory prostate cancer. J Urol 1995;
154:414421.
401
101. Porkka KP, Visakorpi T. Molecular mechanisms of prostate cancer. Eur Urol 2004; 45(6):683691.
102. Navone NM, Troncoso P, Pisters LL, et al. p53 protein
accumulation and gene mutation in the progression of
human prostate carcinoma. J Natl Cancer Inst 1993;
85:16571669.
103. Humphrey PA, Swanson PE. Immunoreactive p53 protein
in high-grade prostatic intraepithelial neoplasia. Pathol
Res Pract 1995; 191:881887.
104. Miyashita T, Reed JC. Tumor suppressor p53 is a direct
transcriptional activator of the human bax gene. Cell 1995;
80:293299.
105. Moul JW, Bettencourt MC, Sesterhenn IA, et al. Protein
expression of p53, bcl-2 and KI-67 (MIB-1) as prognostic
biomarkers in patients with surgically treated, clinically
localized prostate cancer. Surgery 1996; 120:159166.
106. Weidner N, Carroll PR, Flax J, et al. Tumour angiogenesis
correlates with metastasis in invasive prostate carcinoma.
Am J Pathol 1993; 143:401409.
107. Silberman MA, Partin AW, Veltri RW, et al. Tumour angiogenesis correlates with progression after radical prostatectomy but not with pathologic stage in Gleason sum 5 to 7
adenocarcinoma of the prostate. Cancer 1997; 79:772779.
108. Ferrer FA, Miller LJ, Andrawis RI, et al. Vascular endothelial growth factor (VEGF) expression in human prostate cancer: in situ and in vitro expression of VEGF by
human prostate cancer cells. J Urol 1997; 157:23292333.
109. Faivre S, Demetri G, Sargent W, et al. Molecular basis for
sunitinib efficacy and future clinical development. Nat
Rev Drug Discov 2007; 6(9):734745.
110. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001; 2(2):127137.
111. Sachdev D, Yee D. Disrupting insulin-like growth factor
signaling as a potential cancer therapy. Mol Cancer Ther
2007; 6(1):112.
112. Shariat SF, Shalev M, Menesses-Diaz A, et al. Preoperative
plasma levels of transforming growth factor beta(1)
(TGFbeta(1)) strongly predict progression in patients
undergoing radical prostatectomy. J Clin Oncol 2001;
19:28562864.
113. Byrne RL, Leung H, Neal DE. Peptide growth factors in
the prostate as mediators of stromal epithelial interaction.
Br J Urol 1996; 77(5):627633.
114. Urist MR. Bone formation by autoinduction. Science 1965;
150:893899.
115. Darby S, Cross SS, Brown NJ, et al. BMP-6 over-expression in prostate cancer is associated with increased Id-1
protein and a more invasive phenotype. J Pathol 2008;
214(3):394404.
116. Hamdy FC, Autzen P, Wilson Horne CH, et al. Immunolocalization and mRNA expression of bone morphogenetic protein-6 in human benign and malignant prostate
tissue. Cancer Res 1997; 57:44274431.
117. Basilico C, Moscatelli D. The FGF family of growth factors
and oncogenes. Adv Cancer Res 1992; 59:115165.
118. Leung HY, Hughes CM, Kloppel G, et al. Expression and
functional activity of fibroblast growth factors and their
receptors inhuman pancreatic cancer. Int J Oncol 1994;
4:12191223.
119. Kwabi-Addo B, Ozen M, Ittmann M. The role of fibroblast
growth factors and their receptors in prostate cancer.
Endocr Relat Cancer 2004; 11(4):709724.
120. Story MT. Regulation of prostate growth by fibroblast
growth factors. World J Urol 1995; 13:297305.
121. Leung HY, Mehta P, Gray LB, et al. Keratinocyte growth
factor expression in hormone insensitive prostate cancer.
Oncogene 1997; 15:11151120.
122. Tanaka A, Miyamoto K, Matsuo H, et al. Human androgen-induced growth factor in prostate and breast cancer
402
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
Hamdy and Robson

cells: its molecular cloning and growth properties. FEBS
Letters 1995; 363:226230.
Sahadevan K, Darby S, Leung HY, et al. Selective overexpression of fibroblast growth factor receptors 1 and 4 in
clinical prostate cancer. J Pathol 2007; 213(1):8290.
Abate-Shen C, Shen MM. FGF signaling in prostate
tumorigenesisnew insights into epithelialstromal
interactions. Cancer Cell 2007; 12(6):495497.
Gowardhan B, Douglas DA, Mathers ME, et al. Evaluation
of the fibroblast growth factor system as a potential target
for therapy in human prostate cancer. Br J Cancer 2005; 92
(2):320327.
Yoneda T, Sasaki A, Mundy GR Osteolytic bone metastasis in breast cancer. Breast Cancer Res Treat 1994; 32:
7384.
Oliver SE, Gunnell D, Donovan J, et al. Screen-detected
prostate cancer and the insulin-like growth factor axis:
results of a population-based case-control study. Int J
Cancer 2004; 108(6):887892.
Gennigens C, Menetrier-Caux C, Droz JP. Insulin-like
growth factor (IGF) family and prostate cancer. Crit Rev
Oncol Hematol 2006; 58(2):124145.
Inman BA, Harel F, Audet JF, et al. Insulin-like growth
factor binding protein 2: an androgen-dependent predictor of prostate cancer survival. Eur Urol 2005; 47(5):
695702.
Denmeade SR, Lin XS, Isaacs JT. Role of programmed
(apoptotic) cell death during the progression and therapy
for prostate cancer. Prostate 1996; 28:251265.
Ratan HL, Gescher A, Steward WP, et al. ErbB receptors:
possible therapeutic targets in prostate cancer? BJU Int
2003; 92(9):890895.
Cai CQ, Peng Y, Buckley MT, et al. Epidermal growth
factor receptor activation in prostate cancer by three novel
missense mutations. Oncogene 2008; 27(22):32013210.
Cavallaro U, Christofori G. Cell adhesion and signalling
by cadherins and Ig-CAMs in cancer. Nat Rev Cancer
2004; 4(2):118132.
Guo W, Giancotti FG. Integrin signalling during tumour
progression. Nat Rev Mol Cell Biol 2004; 5(10):816826.
Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991; 251:14511455.
Umbas R, Isaacs WB, Bringuier PB, et al. Decreased Ecadherin expression is associated with poor prognosis in
patients with prostate cancer. Cancer Res 1994; 54:39293933.
Jaggi M, Rao PS, Smith DJ, et al. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is
associated with altered cellular aggregation and motility
in prostate cancer. Cancer Res 2005; 65(2):483492.
Jaggi M, Rao PS, Smith DJ, et al. Protein kinase C mu is
down-regulated in androgen-independent prostate cancer. Biochem Biophys Res Commun 2003; 307(2):254260.
Gunthert U, Stauder R, Mayer B, et al. Are CD44 variant
isoforms involved in human tumour progression? Cancer
Surv 1995; 24:1942.
Ponta H, Sherman L, Herrlich PA. CD44: from adhesion
molecules to signalling regulators. Nat Rev Mol Cell Biol
2003; 4(1):3345.
De Marzo AM, Bradshaw C, Sauvageot J, et al. CD44 and
CD44v6 downregulation in clinical prostatic carcinoma:
relation to Gleason grade and cytoarchitecture. Prostate
1998; 34(3):162168.
Cattoretti G, Becker MHG, Key G, et al. Monoclonal
antibodies against recombinant parts of Ki-67 antigen
detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992; 168:357363.
143. Noordzij MA, van der Kwast TH, van Steenbrugge GJ, et al.
Determination of Li-67 in formalin-fixed, paraffinembedded prostatic cancer tissues. Prostate 1995; 27:154159.
144. Adolfsson J. Prognostic value of deoxyribonucleic acid
content in prostate cancer: a review of current results. Int J
Cancer 1994; 58:211216.
145. Persons DL, Takai K, Gibney DJ, et al. Comparison of
fluorescence in situ hybridisation with flow cytometry
and static image analysis in ploidy analysis of paraffinembedded prostate adenocarcinoma. Hum Pathol 1994;
25:678683.
146. Carter BS, Beaty TH, Steinberg GD, et al. Mendelian
inheritance of familial prostate cancer. Proc Natl Acad
Sci U S A 1992; 89:33673371.
147. Edwards SM, Eeles RA. Unravelling the genetics of prostate cancer. Am J Med Genet C Semin Med Genet 2004;
129C(1):6573.
148. Eeles RA, Kote-Jarai Z, Giles GG, et al. Multiple newly
identified loci associated with prostate cancer susceptibility. Nat Genet 2008; 40(3):316321.
149. Gudmundsson J, Sulem P, Rafnar T, et al. Common
sequence variants on 2p15 and Xp11.22 confer susceptibility to prostate cancer. Nat Genet 2008; 40(3):281283.
150. Thomas G, Jacobs KB, Yeager M, et al. Multiple loci
identified in a genome-wide association study of prostate
cancer. Nat Genet 2008; 40(3):310315.
151. Liotta LA. Tumor invasion and metastasesrole of the
extracellular matrix: Rhoads Memorial Award lecture.
Cancer Res 1986; 46(1):17.
152. Hamdy FC, Fadlon EJ, Cottam DW, et al. Matrix metalloproteinase-9 expression in human prostatic adenocarcinoma and benign prostatic hyperplasia. Br J Cancer 1994;
69:177182.
153. Wood M, Fudge K, Mohler JL, et al. In situ hybridization
studies of metalloproteinases 2 and 9, and TIMP-1 and
TIMP-2 expression in human prostate cancer. Clin Exp
Metastasis 1997; 15:246258.
154. Still K, Robson C, Neal DE, et al. The ratio of tissue matrix
metalloproteinase-2 (MMP-2) to tissue inhibitor of matrix
metalloproteinase (TIMP-2) is increased in high-grade
prostate cancer. J Urol 1997; 157:25A.
155. Overall CM, Kleifeld O. Towards third generation matrix
metalloproteinase inhibitors for cancer therapy. Br J Cancer 2006; 94(7):941946.
156. Kaighn ME. Establishment and characterization of a
human prostatic carcinoma cell line (PC-3). Invest Urol
1979; 17:1623.
157. Stone KR, Mickey DD, Wunderli H, et al. Isolation of a
human prostate carcinoma cell line (DU-145). Int J Cancer
1978; 21:274281.
158. Horoszewicz JS, Leong SS, Kawinski E, et al. LNCaP
model of tumor prostatic carcinoma. Cancer Res 1983;
43:18091818.
159. Ahmad I, Sansom OJ, Leung HY. Advances in mouse
models of prostate cancer. Expert Rev Mol Med 2008;
10:e16.
160. Majumder PK, Grisanzio C, OConnell F, et al. A prostatic
intr neoplasia-dependent p27 Kip1 checkpoint induces
senescence and inhibits cell proliferation and cancer progression. Cancer Cell 2008; 14(2):146155.
161. Abate-Shen C, Shen MM. Mouse models of prostate
carcinogenesis. Trends Genet 2002; 18(5):S1S5.
162. Jonkers J, Berns A. Conditional mouse models of sporadic
cancer. Nat Rev Cancer 2002; 2(4):251265.
26
Testis Cancer
Danish Mazhar
Department of Medical Oncology, Addenbrookes Hospital, Cambridge, U.K.
Michael Williams
Department of Clinical Oncology, Addenbrookes Hospital, Cambridge, U.K.
INTRODUCTION
Testis cancer is a malignancy whose management has
been transformed over the last 40 years. This chapter
provides the scientific background to these clinical
developments. Germ cell malignancies of the testis
are categorized into seminoma and nonseminoma
germ cell tumor (NSGCT). In addition there are rarer
stromal cell tumors and lymphoma, which are not
discussed further.
Testis cancers occur predominantly between the
ages of 20 and 40 and are the commonest malignancy
in men of that age group. However, exquisite sensitivity to cytotoxic chemotherapy means that this
malignancy has a limited impact in terms of mortality.
In the United Kingdom, there were 2005 cases diagnosed in the year 2000 but only 74 deaths recorded in
2002 (1). The five yearage standardized relative survival was 95% in 1996 to 1999 and has shown a
progressive improvement since 1986 (1).
NSGCT has long been recognized as an anomalous malignancy because it manifests spontaneous
remission and because disseminated metastatic disease has been found to be curable both with wide-field
radiotherapy and with single-agent chemotherapy.
There have been a number of reports of spontaneous
remission (2). These were based on the diagnostic
standards of the day (3) and histological proof of
metastatic disease has rarely been obtained. It has
been argued that the level of proof required should
be the same as that required to initiate treatment (4).
Before the development of effective chemotherapy, radiotherapy was used to treat para-aortic nodal
metastases (5,6); local control and cure rates depended
on the bulk of disease treated (7). More surprisingly,
selected patients with three or fewer lung metastases
were successfully treated with whole lung radiotherapy and a boost to the tumor nodules. For these highly
selected patients, this policy resulted in a 40% cure
rate (5,8).
Nevertheless, for the majority of patients metastatic disease was a death sentence, even though it had
become clear in the 1960s that the disease was chemosensitive. The first major breakthrough occurred when
Samuels described combination therapy with vinblastine and bleomycin. This treatment was extremely
toxic by the standards of the time with a substantial
mortality but a complete remission rate of 35% (9). The
next major advance was the addition of cisplatin to
develop PVB chemotherapy (platinum, vinblastine,
bleomycin), first described by Einhorn (10). The BEP
regimen (bleomycin, etoposide, platinum) was
described by Peckham (11) and was subsequently
shown in a clinical trial to be less toxic and at least
as efficacious as PVB, and possibly more so in adverse
subgroups (12).
Patients with stage I disease and no demonstrable metastases have a high risk of relapse. In Europe
they were managed with adjuvant radiotherapy
(5,6,13) but in North America retroperitoneal lymph
node dissection (RPLND) was the favored approach
(14). The development of effective chemotherapy led
to the investigation of surveillance with chemotherapy
held in reserve (15). This study transformed the management of stage I patients in Europe and they are
now usually offered surveillance. There have been a
number of studies to define the risk of relapse in such
patients (16,17) and adjuvant chemotherapy has been
offered to such patients resulting in very low relapse
rates (18), but exposure to significant short-term
toxicity and to the long-term hazards of chemotherapy
(19,20). In North America RPLND continues to be the
standard therapy for clinical stage I nonseminoma,
although it is accepted that the options to be discussed
with the patient should include surveillance or nerve
sparing RPLND (14). The short-term morbidity of
RPLND has improved with modern techniques (21),
and, of those found histologically to have lymph node
involvement (stage II), 50% to 75% are cured by surgery alone (14).
Seminoma of the testis occurs in older men with
a peak age of incidence of about 30 years. It has a
lower metastatic potential than NSGCT. The predominant site of metastasis is the para-aortic region and
adjuvant radiotherapy is an effective treatment, which
has been refined in a series of trials (22,23). In many
centers, this treatment has now been replaced by a
404
Mazhar and Williams
Figure 1 Trends in reproductive health in the United States. Source: From Ref. 34.
single dose of carboplatin (24). This does not have the

well-established carcinogenic risks of wide-field
radiotherapy which carries a 1.4 fold relative risk of
second cancer approximating to an increase in incidence of 2% per decade of follow-up (25,26). However, although carboplatin has been in use in the
management of seminoma for 15 years, there are
limited data on long-term late effects (27). This has
led some authors to continue to recommend radiotherapy despite its known long-term carcinogenic
hazards (28). In addition, continued follow-up is
required as late relapse can occur and has been
reported up to four years later (29).
other Eastern European countries. It has been speculated that this may imply etiological factors affecting
the early years of life or even growth in utero (33).
EPIDEMIOLOGY/RISK FACTORS
The effect of social class is complex: some studies have

shown an association with higher social class (35), but
the relationship has diminished in the West. Studies in
Finland have shown a reduction from a five-fold risk
for social class one in the early 1970s (compared with
social classes four and five) down to a two-fold risk
more recently (36). It has been hypothesized that there
may be a dietary basis for this (37) or else that it may
relate to the earlier age of puberty (38,39). There are
also links to congenital abnormalities, infertility and
low amounts of exercise (39).
Epidemiology
The incidence of testis cancer in the United Kingdom
is 6/100,000 population (1). In the United Kingdom, it
has shown a gradual increase over the last 20 years
and more dramatic changes have been seen in some
other countries particularly Denmark and Sweden
(30,31). The incidence varies between different countries and races. Black populations show a low prevalence (1/100,000) both in Africa and the United States
indicating the predominance of genetic rather than
environmental factors (32). Complex changes occurring
in the years before the Second World War (19351945)
have been reported in some Nordic countries but not in
Environmental Factors
Figure 1 shows changes in the incidence of breast and

testicular cancer in the United States over the past
30 years. This has led to speculation that these changes
are related to increasing levels of exogenous oestrogens or exposure to other synthetic man-made chemicals in environment (34).
Social Class
Family History
A large number of studies have suggested a link to

family history. A British study found that having a
Chapter 26: Testis Cancer
brother with the condition is associated with a relative

risk of eight and having a father with the condition
with a relative risk of four (40). This issue is discussed
in detail later in this article.
Testicular Maldescent
There is a well-established association between testicular maldescent and testis cancer. The testis cancer risk
applies to both testes and not only the one that was
affected by maldescent. Although only 1% to 2% of
cryptorchid boys go on to develop testis cancer, the
relative risk is increased about fivefold. In a U.K. study
the relative risk was 7.5 and did not decrease with
younger age at orchidopexy. In addition, testicular
biopsy was identified as a strong risk factor for the
subsequent development of a tumor (41). A more
recent Swedish study has shown that those who underwent orchidopexy before 13 years of age had a relative
risk of 2.2 but for those operated on later the relative
risk was 5.4 (42). It is now recommended that the
procedure is performed on patients before the age of
two years, or even as young as six months old (4346).
Testicular Atrophy
Testicular atrophy is associated with maldescent,

infertility and testis cancer. In patients who have
already had one testicular cancer, the overall risk of
second malignancy in the contralateral testis is 2% to
3% (47,48). However, in patients who have a remaining testis of 12 mL or less, onset of the first malignancy
below the age of 31 and a history of maldescent, the
risk has been shown in one study to be 34% and
contralateral testicular biopsy has therefore been
advocated to detect carcinoma in situ (49). Testicular
microlithiasis has been shown to be associated with
carcinoma in situ in subfertile men and this may
provide an additional tool for the early diagnosis of
this disease (50).
TUMOR MARKERS
A high proportion of patients with testicular tumors
show abnormal proteins in the plasma. This does not
necessarily indicate that they have metastatic disease
because production may be solely from the primary
tumor in the testis. These proteins are useful in the
clinical management of the disease and 80% of
patients with NSGCT will show abnormal levels of
either AFP or hCG (13).
a-Fetoprotein
Raised levels of AFP are found in 60% of NSGCT
patients (13). Physiologically, large amounts of AFP
are produced by yolk sac and by the liver during
gestation, but production ceases after birth. Adult
levels are usually designated as 1 to 10 kU/L.
It is important to be aware that there is a subgroup of patients who have congenital elevation of
AFP up to a level of about 25 (5153). This has no
significance but may cause diagnostic confusion.
405
In addition, liver injury can cause elevation of

AFP. This can be caused by the chemotherapy used to
treat the patient or by other toxins, for example, alcohol (53). Such effects can lead to inappropriate recommendations for further treatment whereas a period of
observation will allow the level to fall to normal (53).
Other causes of elevated AFP levels are hepatocellular carcinoma and some gastro-intestinal tumors.
In addition, AFP may be raised in patients with liver
metastases from any primary tumor. With these caveats in mind, a raised AFP level can be diagnostic in a
patient with metastatic malignancy from an unknown
primary.
Human Chorionic Gonadotropin

hCG comprises an a and a b subunits. The a subunit is
identical to that of lutenizing normal, follicular stimulation hormone and thyroid stimulating hormone.
This can have importance in interpreting elevated
levels in some patients depending on the assay used.
The b subunit is specific to hCG and can be secreted
either bound to the a subunit or as free b. This small
molecule has a high clearance rate. This is important
in a complete understanding of a patients tumor
markers as discordant responses between different
hCG assays have been reported (54). hCG is elevated
in 55% of NSCGT patients and in 20% of seminoma.
Levels of greater than 500 IU/L in patients with
seminoma warrant careful clinical consideration as
this may indicate the presence of nonseminomatous
elements (55).
Congenital elevation of hCG has not been
reported but it can be produced by some tumors of
nongerm cell origin, particularly bladder and pancreatic cancers and colon carcinoma. It may rarely be
produced in inflammatory bowel disease.
Lactate Dehydrogenase
Elevated LDH is a nonspecific marker of cell turnover
and is of prognostic significance in the staging of
lymphoma (56). It is not specific for testis cancer but
it has a strong association with prognosis and is a
key element of the staging system devised by the
International Germ Cell Cancer Collaborative Group
(IGCCCG) (57) (Table 1). It is of limited value in
follow-up because of limited sensitivity, specificity
and a low positive predictive value for detecting
relapse of testicular germ cell tumors (GCTs); falsepositive increases are common (58). It does not add to
the early detection of relapse in surveillance of stage 1
patients (59).
Placental Alkaline Phosphatase

PLAP staining can be useful histologically in confirming the germ cell origin of a tumor. Elevated levels of
serum PLAP are seen in about half of patients with
metastatic seminoma but it is not a reliable test and
elevations are also seen in smokers (60). This serum
marker is therefore little used clinically.
406
Mazhar and Williams
Table 1 IGCCCG Prognostic Scoring System for NSGCT

Including Marker Levels
Good prognosis
Nonseminoma
l
l
l
Testis/retroperitoneal primary
No nonpulmonary visceral metastases
Good markersall of:
a-Fetoprotein less than 1000 ng/mL
Human chorionic gonadotropin less than 5000 IU/mL
(1000 ng/mL)
Lactate dehydrogenase less than 1.5 the upper limit of
normal
Seminoma
l
l
l
Any primary site

Normal AFP, any hCG, any LDH
Intermediate prognosis
Nonseminoma
l
l
l
Testis/retroperitoneal primary
Intermediate markersany of:
AFP 1,000 to 10,000 ng/mL
hCG 5,000 IU/L to 50,000 IU/L
LDH 1.5-10 upper normal level
Seminoma
l
l
l
Any primary site

Nonpulmonary visceral metastases
Normal AFP, any hCG, any LDH
Poor prognosis
Nonseminoma:
l
l
l
Mediastinal primary
Nonpulmonary visceral metastases
For markersany of:
AFP more than 10,000 ng/mL
hCG more than 50,000 IU/mL (10,000 ng/mL)
LDH more than 10 the upper limit of normal
Seminoma
l
No patients are classified as poor prognosis
Prognostic Significance
Staging by anatomical site of involvement is still used
clinically but its prognostic significance has become
much less with the development of effective chemotherapy. IGCCCG performed a multivariate analysis
on 5862 patients and used the level of AFP, hCG and
LDH as key elements in categorizing patients into
good, intermediate and poor risk cases (57). This has
been extremely important in providing a reliable
framework for clinical trials (Table 1), separating
patients with five-year progression free survival of
91% (good), 79% (intermediate) and 48% (poor) (57).
Rate of Tumor Growth

It has been shown in a small study of 58 patients, in
whom sequential pretreatment measurements were
available, that marker production doubling time is a
surrogate for tumor growth rate and carries a poor
prognosis in patients with nonseminoma treated with

BEP chemotherapy (61). The practical difficulties of this
technique mean that it has not been taken forward.
Response to Therapy
In patients who are ultimately shown to have stage I
disease with no distant metastases then after orchidectomy AFP should fall with a half-life of five to seven
days (13) and hCG with a half-life of 24 to 36 hours
(13). A prolonged half-life suggests the possibility of
metastatic disease but it is now widely accepted that
relapse should only be diagnosed when there is sustained and progressive elevation of tumor marker
levels (4).
Occasional patients present with surges of
marker levels following pulses of chemotherapy (62).
It is an attractive hypothesis to assume that the
rate of fall of tumor markers may have prognostic
significance and this has been shown in one study
(63). However, other studies have found that marker
half-life does not predict patients at high risk of progression after front-line therapy and that it is a poor
guide to long-term prognosis (64).
Persistently elevated tumor markers after the
completion of induction chemotherapy may indicate
residual active disease, but the alternative causes
indicated above need to be considered. In addition,
it is now widely accepted that a sustained and progressive rise in tumor markers should be documented
before second-line chemotherapy is offered. Patients
with residual cystic masses may have elevated tumor
markers, which return to normal after RPLND (65).
By contrast, patients with normal markers can
have enlarging masses of growing teratoma. These
should be managed surgically (66).
MOLECULAR BIOLOGY OF TESTICULAR GERM

CELL TUMORS
GCTs are a heterogeneous group of neoplasms that
arise mainly in the gonads and more rarely from
extragonadal sites along the midline including the
retroperitoneum, sacrum, mediastinum and pineal
gland. This pattern of distribution is thought to reflect
the route of migratory primordial germ cells to the
genital ridge.
It is possible to define three diverse groups of
testicular germ cell tumors (TGCTs). The first group
includes teratomas and yolk sac tumors of neonates
and infants. The second group includes seminomas
and nonseminomatous GCTs in postpubertal patients,
by far the commonest form of TGCT, and occurring
usually between the ages 15 and 40 years. The final
group is spermatocytic seminoma, which usually
occurs in an older group.
Carcinoma in situ or intratubular germ cell neoplasia (ITGCN) is commonly found in testes containing a GCT, and is regarded as a premalignant lesion.
In patients with ITGCN, the cumulative probability
for development of a testicular GCT is 70% after seven
years (67). Five percent of patients with testicular
tumors harbor ITGCN within the contralateral testis,

detectable by open biopsy in 99% of cases. For patients
with testicular volume <12 mL and an age at diagnosis <30 years, the risk of ITGCT in the contralateral
testis is >34% (49,68,69). Contralateral testicular
biopsy should be discussed with patients at high
risk of ITGCT. If it is to be performed, it should be
carried out preferably at the time of orchidectomy.
Biopsies to identify ITGCT must be preserved in
Strieves or Bouins solution (70).
There is debate as to the initiating genetic events
that lead to ITGCN and subsequent invasive GCT.
One theory postulates the most likely target cell for
transformation is the zygotene-pachytene spermatocyte where there appears to be a recombination
checkpoint. At this stage, there is replicated DNA
and upregulated p53, the latter to allow prolongation
of the phase and recombination. If unresolved, DNA
strand breaks occur and the elevated wild-type p53
can trigger apoptosis. Aberrant chromatid exchange
events associated with crossing-over may lead to
increased chromosome 12p copy number. Possibly
through the oncogenic affect of cyclin D2, this aberrant genomically unstable cell is now able to escape
the apoptotic effects of p53 and may reenter the cell
cycle, now as a neoplastic cell (71).
The second and more widely accepted hypothesis, based on immunohistochemical data linking gonocytes to ITGCN, developmental abnormalities
associated with GCT and epidemiological data, suggests that primordial germ cells may undergo abnormal cell division (polyploidization) because of mostly
environmental factors in utero and give rise to ITGCN
(40,72,73).
The three distinct categories of GCT described
above, have separate genetic characteristics. Most is
known about the adult seminomas and nonseminomas. The DNA content of ITGCN and seminoma is
hypertriploid and that of nonseminoma is hypotriploid. The characteristic genetic abnormality of these
tumors is excess genetic material of the short arm of
chromosome 12, usually in the form of an isochromosome i12p (74,75). Approximately 80% of cases
have i12p and the remainder have excess 12p genetic
material in derivative chromosomes (76). The consistent gain of 12p genetic material is supported by gene
array and comparative genomic hybridization data
(77,78). The presence of i12p in ITGCN is a matter of
controversy with most investigators suggesting it is
not present (79,80), supporting the hypothesis that
polyploidization is an initiating event followed by
i12p formation in the invasive phenotype.
The consistent gain of genetic material from
chromosome 12 seen in these tumors suggests that it
has a crucial role in their development. Some investigators have identified an amplified region at
12p11.2-12.1. Several genes of interest are located in
this region including SOX5, JAW1 and K-RAS (79).
However, data suggests that this minimally deleted
region applies preferentially to seminomas, suggesting that the search for putative relevant genes should
not be restricted to this region. For example, Houldsworth et al. have implicated cyclin D2, which is
407
mapped to region 12p13, as the most likely gene to

be involved (81).
A few studies have suggested the presence of
relevant tumor suppressor genes but the data have not
been consistent. Extensive molecular analyses have
identified genomic and/or functional (expression)
loss of several known tumor suppressor genes such
as RB1, WT1 and p16, although no firm data are
available to date linking these genes with tumor
development and/or progression (79,80). Mutation
of p53 is a rare event in GCT, though some have
suggested this event in some chemotherapy-resistant
tumors (82).
GCTs in infants and adults are genetically distinct (80,8387). While virtually all-adult cases are
aneuploid, pediatric cases are mostly diploid, particularly, teratomas, although yolk sac tumors may be
nondiploid. Amplification of i12p and 12p are rare in
childhood GCTs. On the other hand, such tumors are
characterized by deletions of 1p, particularly region
p36, loss of 6q, and structural abnormalities of chromosome 2 and 3p. Published reports suggest a significant heterogeneity in the structural chromosomal
constitution of infantile teratomas. Spermatocytic
seminoma may be either diploid or aneuploid, and
have losses of chromosome 9 rather than i12p (80).
Given the observation that amongst the two
strongest associations with testis cancer are having a
brother (relative risk 8) and having a father (relative
risk 4) (40,8890) with the condition, investigators
have sought to find a heritable susceptibility gene. A
genetic component of TGCT is supported by demonstration of a higher risk to monozygotic twins of TGCT
patients than to dizygotic twins (91) and by the observation that the frequency of bilateral disease is higher
among cases with a family history than those without
(88,89).
A segregation analysis using Scandinavian
TGCT patients has suggested that the best model for
a TGCT susceptibility locus is a major gene with a
recessive mode of inheritance, an estimated gene frequency of 3.8% and a lifetime risk of developing
TGCT of 43% among homozygous men (92). A recessive model was also suggested by Nicholson and
Harland (93) who performed an analysis based on
age at onset of TGCT and the frequency of bilateral
disease. However, in both studies genetic heterogeneity and other models of inheritance could not be
excluded.
In 1994, the International Testicular Cancer Linkage Consortium was established to collect a sufficiently large set of multiple case families for linkage
analysis. This group includes essentially all groups in
the world working in the field of TGCT susceptibility.
A genomewide linkage study for TGCT susceptibility
loci on 237 pedigrees with two or more cases of the
disease was recently completed, which found no statistically significant regions of linkage (94). Moreover,
a previous report of linkage to a region of Xq27 was
not replicated (95). The results demonstrate that TGCT
susceptibility cannot be caused by a single major gene.
A U.K. collaborative genetics of testicular cancer study
is now underway which aims at collecting samples
408
Mazhar and Williams
from 3000 patients with TGCT and their parents, and

performing genomewide single nucleotide polymorphism analysis.
INTRATUBULAR GERM CELL NEOPLASIA

ITGCN as a preinvasive malignancy was first suggested by Skakkebaek in 1972 (96). It is present in up
to 4% of cryptorchid patients, in up to 5% of contralateral gonads in patients with unilateral GCT and in up
to 1% of patients biopsied for oligospermic infertility.
Its association with TGCT arising in prepubertal
patients is still a source of controversy (97,98).
Microscopically, ITGCN is composed of atypical
germ cells situated at the periphery of seminiferous
tubules. It is rarely demonstrated immunocytochemically in tubules showing spermatogenesis, presumably reflecting pagetoid spread (i.e., along the
basement membrane). The abnormal germ cells comprising ITGCN have ballooned clear cytoplasm (containing fat and glycogen) and a large hyperchromatic
nucleus, which may show mitotic figures. These
appearances are identical in ITGCN adjacent to seminoma and nonseminomatous GCT.
The age at which the morphological features of
ITGCN can be recognized is another area of contention. Observation that ITGCN in adults displays
markers and some ultrastructural features seen in
fetal germ cells has been interpreted as evidence that
ITGCN originates in the fetus. However, markers seen
in the fetus may be displayed in adult tumors in the
absence of intervening lesions. Also, in contrast to the
high incidence of ITGCN seen in adult GCTs, it is a
rare observation in childhood tumors (98,99), and was
not identified in the orchidectomy specimens from
pubertal GCT patients (97,100). In a review of biopsies
from mal-descended testes, ITGCN was identified
only in the testis of a 16-year-old and not from
biopsies taken from children aged 9, 10, and 14 years
in whom GCT subsequently developed (101).
Placental alkaline phosphatase (PLAP) and
CD117 (c-kit) are used in diagnostic histopathology.
Positive staining is seen in a paranuclear and cytoplasmic membrane distribution. Other antibodies,
which immunoreact with ITGCN but are rarely used
in practice, include M2A and 43-F (102,103). Oct 3/4 is
also expressed in ITGCN (104).
Microlithiasis (microcalcification) was identified
in one study in 39% of specimens showing ITGCN
compared with an incidence 2% of 429 nonmalignant
testicular disorders (105). In a sonographic series of
528 symptomatic patients, 48 (9%) had microlithiasis
of which 13 (27%) had testicular cancer (106). Of the
remaining 480 patients without microlithiasis, 38 (8%)
had cancer. Thus, of those with cancer, about 25%
have microlithiasis, and of those with microlithiasis, a
similar proportion will have cancer.
However, the situation in healthy men is different. A prospective study of testicular sonography on
1504 healthy men aged 18 to 35 years revealed microlithiasis in 84 (5.6%). For black Americans the prevalence was 14%, and this higher proportion in a low
risk population would seem to deny a close association between microlithiasis and TGCT (107).
About one-third of patients with extragonadal
germ cell cancer harbor ITGCN within one or both
testicles which otherwise appear normal. The cumulative risk of developing a metachronous testicular cancer 10 years after diagnosis and treatment of
extragonadal GCTs is only 10%, and higher among
patients with nonseminomatous histology or retroperitoneal location than among patients with pure seminomatous histology (1.4%) or primary mediastinal
location (6.2%) (48). However, since all patients with
extragonadal germ cell cancer will receive platinumbased chemotherapy which will eliminate a substantial
percentage of ITGCN, a routinely performed bilateral
testicular biopsy is not recommended. Nevertheless, if
a biopsy is planned in patients with a higher risk for
ITGCT following an extragonadal GCT, this should be
preferably performed prior to chemotherapy. If performed thereafter, testicular biopsy should not be considered earlier than six months after the completion of
chemotherapy (108113).
HISTOLOGICAL EXAMINATION OF
GERM CELL CANCER
TGCTs are a heterogeneous group of neoplasms with
diverse histopathology and variable clinical course
and prognosis. This diversity is reflected in various
systems offered to classify these tumors. Currently the
most comprehensive and widely accepted system of
classification is the one proposed by the World Health
Organization (114), which is summarized in Table 2.
TGCTs are broadly divided into seminoma and nonseminoma for treatment planning because seminomatous types of testicular cancer are more sensitive to
radiation therapy.
Table 2 World Health Organization Histological Classification
of Germ Cell Testis Tumors (2004)
Intratubular germ cell neoplasia, unclassified
Tumors of 1 histological type (pure forms)
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
Seminoma
Seminoma with syncytiotrophoblastic cells
Spermatocytic seminoma
Spermatocytic seminoma with sarcoma
Embryonal carcinoma
Yolk sac tumor
Trophoblastic tumors
Choriocarcinoma
Trophoblastic neoplasms other than choriocarcinoma
Monophasic choriocarcinoma
Placental site trophoblastic tumor
Teratoma
Dermoid cyst
Monodermal teratoma
Teratoma with somatic type malignancies
Tumors of more than 1 histological type (mixed forms)

l
l
l
l
Mixed embryonal carcinoma and teratoma

Mixed teratoma and seminoma
Choriocarcinoma and teratoma/embryonal carcinoma
Others
It is recommended to completely laminate the

testicular specimen in transverse sections. Additional
sections have to be taken from the spermatic cord. For
a full histological examination of the tumor, it is
necessary to obtain one block per centimeter of
tumor, not less than a total of three blocks, as well
as blocks from the peritumoral region and of remote
testicular tissue. Further samples have to be taken
from the resection margin and from the spermatic
cord with 1 cm distance to the testicle. Immunohistology can include cytokeratin for the detection of nonseminomatous elements, PLAP for the identification
of ITGCN, CD31/factor VIII for the identification of
vessel endothelium as well as AFP and b-HCG.
ITGCN may be identified using H&E preparations,
PLAP staining or by semithin-section technique.
Histopathological reporting should address the
following issues: localization and size of the tumor,
multiplicity, tumor extension (rete testis, tunica albuginea, tunica vaginalis, epididymis, spermatic cord,
scrotum), pT category according to the UICC classification (115), histological type (WHO-ICD-O), the presence or absence of ITGCN, as well as the presence or
absence of invasion of blood or lymphatic vessels.
After chemotherapy for metastatic nonseminomatous GCT, if tumor markers are within normal limits
but residual masses remain, resection is performed as
a diagnostic and therapeutic procedure. Postoperative
disease course depends on a number of factors including number and site of metastases (116), completeness
of resection as assessed intraoperatively (117119).
The content of the mass, and the presence of viable
GCT (undifferentiated teratoma/embryonal carcinoma, yolk sac tumor and trophoblastic tumor) is
prognostic (118120).
In nine series each reporting residual masses from
over 50 patients, malignant GCT components were
found in 6% to 28% (mean 17%), fibrosis and necrosis
only in 22% to 65% (mean 45%), and differentiated
teratoma (plus or minus necrosis/fibrosis) in 22% to
57% (mean 36%) (119). The two-year progression free
survival rate for patients whose masses included
malignant GCT elements was 12.5% compared with
88% in patients where these components were absent
(119). Nonoperative predictors have been sought with a
view to leaving appropriate masses in situ. It has been
demonstrated that shrinkage of the mass during chemotherapy to a size of " 1.5 cm, high serum LDH, and
normal serum AFP and HCG levels before chemotherapy, and absence of differentiated teratoma from the
primary are indicative of necrotic tissue only in the
residual mass (121,122). The development of non-GCT
malignancies (such as sarcoma, adenocarcinoma and
squamous carcinoma) within the resected specimen is
associated with a poor prognosis (123).
STAGING PROCEDURES AND PROGNOSTIC

INFORMATION
To define the clinical stage of a patient with a gonadal
GCT the TNM classification of the UICC should be
used (115). In addition, most patients with metastatic
409
disease are classified according to the classification of

IGCCCG (57), which is also incorporated into the TNM
classification. The individual treatment strategy is
based on both the TNM classification and the IGCCCG
prognostic factorbased classification which includes
histology, location of primary tumor, location of metastases and level of AFP, b-HCG and LDH as prognostic
markers to categorize patients into good, intermediate, and poor prognosis groups (Table 1). For
patients presenting with primary extragonadal GCTs,
a different prognostic outcome model has been developed (124) but is not in routine use. For clinical stage 1
seminoma, although no prognostic classification system has been prospectively validated so far, there is
evidence that the size of the primary tumor (<4 cm vs.
#4 cm) and the infiltration of the rete testis are independent prognostic indicators for occult metastases
(125). Patient age (<34 vs. #34 yr) and the presence
of vascular invasion (VI) are of equivocal prognostic
relevance (125128). For clinical stage 1 nonseminoma,
infiltration of the primary tumor by venous blood
vessels or lymphatic infiltration (vascular invasion,
VI) are the most important prognostic indicators for
occult metastases (129134). Without adjuvant treatment 48% of patients with VI will develop metastases
while only 14% to 22% of those without will relapse
(135). The proliferation rate, as well as the percentage
of embryonal carcinoma in relation to the total tumor
volume, are further prognostic indicators (133,135,136).
However, these markers do not contribute independent
prognostic information in addition to the factor of
vascular invasion.
Computerized tomography (CT), with oral and
intravenous contrast, of the chest, abdomen and pelvis
is required for initial staging. For the evaluation of the
lung and mediastinum, chest CT is more sensitive
than plain X-ray films (137139). Ultrasonography of
the retroperitoneum is less sensitive than CT (140).
Magnetic resonance imaging (MRI) of the abdomen
and pelvis, does not provide additional information
and should be restricted to patients to whom intravenous contrast media cannot be (141,142). The role of
PET is controversial (143), and such scans are not
recommended outside clinical trials as part of routine
initial staging. Bone scans should be obtained in
patients with elevated levels of alkaline phosphatase
or if bone metastases are clinically suspected. Imaging
of the brain by CT or MRI is required in patients with
clinical signs potentially indicating brain metastases,
or in patients with extensive lung and/or retroperitoneal disease.
BASIS FOR CHEMOSENSITIVITY AND

CHEMORESISTANCE OF GERM CELL TUMORS
Germ cell cancer has become the model for a curable
solid malignancy. Around 95% of newly diagnosed
GCT patients are cured, and in patients with
advanced disease requiring initial cisplatin-based chemotherapy, close to 80% are cured. The molecular
basis for this exquisite sensitivity to cytotoxic treatment probably resides in inherent biological features
410
Mazhar and Williams
involved in cell growth, cell death, differentiation

pathways, and cellular response to DNA damage.
Normal tissues react to DNA damage by increasing the amount of p53 in cells. This induces the transcription of other proteins, notably Waf-1 and Mdm-2.
Waf-1 is a potent inhibitor of cyclin-dependent kinases
(144,145) and can cause cell cycle arrest in G1. G1
arrested cells have the opportunity to recover from
genotoxic damage. Alternatively, particularly when
myc, myb, or E2F are overexpressed, an elevated
p53 expression may lead to apoptosis (146,147).
Normal germ cells contain high levels of wildtype p53. GCT are similar and do not have mutations
in the p53 gene (148150). When GCT lines are
exposed to etoposide, p53 protein is increased, but
Waf-1 only modestly so, and apoptosis ensues (151).
This is different to many other cancers, where either
the p53 protein remains at low level, possibly because
of the presence of a p53 mutation, or p53 induction
takes place and the associated Waf-1 induction
causes G1 arrest. The sensitivity of GCT to cytotoxic
agents is therefore associated with the apoptotic
response to genotoxic damage. This, in turn, is in
part attributed to the high levels of wild-type p53, the
absence of p53 mutations and only a modest level of
Waf-1 induction.
GCT cell lines have relatively high levels of
apoptosis-promoting proteins Bax and low levels of
the suppressors of apoptosis, Bcl-2 (151). The Bax:Bcl
ratio may be of importance in determining whether
the response to cell damage is apoptosis or repair.
The role of p53 in affecting this ratio in GCT is
uncertain.
The basis for cisplatin sensitivity observed in
germ cell cancers has been the subject of investigation.
Exposure of cells to cisplatin causes crucial intrastrand
cross-links, and this is followed by nuclear excision
repair (NER). Testis cancer cells have been shown to be
deficient in this process. NER is complex and includes
the recognition of DNA damage by high mobility
group (HMG) proteins. There is a testis-specific HMG
protein. When this has been transfected into HeLa cells,
the amount of apoptosis following cisplatin exposure
was increased in some cases (152). Of the other proteins
involved in the NER, the xeroderma pigmentosum
group A (XPA) protein and the ERCC1-XPF endonuclease complex are present in low levels in testis cancer
cells. When these proteins were added to testis tumor
cell extracts, full NER capacity was restored (153),
suggesting a pivotal role for these proteins.
Sensitive testis cancer cells do not have higher
levels of p53 than resistant ones, though, since it is
likely that resistant cell lines had been derived from
tumors heavily exposed to chemotherapeutic agents
in vivo, other resistance mechanisms may be operative. Studies have implicated failure to activate
caspase-9 (154) or failure to induce expression of
FAS and recruitment of FADD and caspase-8 to FAS
(155) as a possible means by which GCTs subvert the
inherent capacity of these cells to rapidly activate cell
death pathways. High-level gene amplification, a
genetic feature often associated in other tumor types
with a poorer prognosis, has been implicated (156).
Amplification of genetic material at multiple sites

other than 12p was observed in 5 of 17 GCTs not
cured by cisplatin-based therapy, but not in 17 cured
GCTs. Expression studies aimed at identifying
markers of resistance not restricted to regions identified by molecular genetic studies provide an exciting
opportunity to utilize a group of expression identifiers
together with clinical parameters for risk stratification
of patients.
REFERENCES
1. Toms JR, ed. CancerStats Monograph 2004. London: Cancer Research UK, 2004.
2. Huddart R, Moore NR, Williams MV, et al. Difficulties in
the diagnosis of metastatic testicular teratoma. Br J Radiol
1990; 63:569572.
3. Franklin CIV. Spontaneous remission of metastases from
testicular tumours. A report of six cases from one centre.
Clin Radiol 1977; 28:499502.
4. Benstead K, Williams MV, Dixon A. Spontaneous regression of metastatic teratoma teratoma (letter). Clin Oncol
1991; 3:357.
5. Van der Werf-Messing B. Radiotherapeutic treatment of
testicular tumours. Int J Radiat Oncol Biol Phys 1976;
1:235248.
6. Peckham MJ. An appraisal of the role of radiation therapy
in the management of nonseminomatous germ cell
tumors of the testis in the era of effective chemotherapy.
Cancer Treat Rep 1979; 63:16531658.
7. Tyrell CJ, Peckham MJ. The response of lymph node
metastases of testicular teratoma to radiation therapy.
Brit J Urol 1976; 48:363370.
8. Van der Werf-Messing B. The treatment of pulmonary
metastases of malignant teratoma of the testis. Clin Radiol
1973; 24:121123.
9. Samuels ML, Lanzotti VJ, Holoye PY, et al. Combination
chemotherapy in germinal cell tumours. Cancer Treat Rev
1976; 3:185204.
10. Einhorn LH, Donohue J. Diamminedichloroplatinum,
Vinblastine and Bleomycin combination chemotherapy
in disseminated testicular cancer. Ann Intern Med 1977;
87:293298.
11. Peckham MJ, Barrett A, Liew KH, et al. The treatment of
metastatic germ-cell testicular tumours with Bleomycin,
Etoposide and Cis-platin (BEP). Br J Cancer 1983; 47:
613619.
12. Williams SD, Birch R, Einhorn LH, et al. Treatment of
disseminated germ-cell tumors with Cisplatin, Bleomycin
and either Vinblastine or Etoposide. N Engl J Med 1987;
316:14351440.
13. Peckham MJ, ed. The Management of Testicular Tumours.
London: Edward Arnold, 1981.
14. Foster RS, Donohue JP. Retroperitoneal lymph node
dissection for the management of clinical stage I nonseminoma. J Urol 2000; 163:17881792.
15. Peckham MJ, Barrett A, Husband JE, et al. Orchidectomy
alone in testicular stage I non-seminatous germ cell
tumours. Lancet 1982; 2:678680.
16. Freedman LS, Parkinson MC, Jones WG, et al. Histopathology in the prediction of relapse of patients with stage I
testicular teratoma treated by orchidectomy alone. Lancet
1987; 2(8554):294298.
17. Alexandre J, Fizazi K, Mahe C, et al. Stage I nonseminomatous germ-cell tumours of the testis: identification of a subgroup of patients with a very low risk of
relapse. Eur J Cancer. 2001; 37:576582.

18. Cullen MH, Stenning SP, Parkinson MC, et al. Short-course
adjuvant chemotherapy in high-risk stage I nonseminomatous germ cell tumors of the testis: a Medical Research
Council report. J Clin Oncol 1996; 14:110613.
19. Huddart RA, Norman A, Shahidi M, et al. Cardiovascular
disease as a long-term complication of treatment for
testicular cancer. J Clin Oncol 2003; 21(8):15131523.
20. van den Belt-Dusebout AW, de Wit R, Gietema JA, et al.
Treatment-specific risks of second malignancies and cardiovascular disease in 5-year survivors of testicular cancer. J Clin Oncol 2007; 25:43704378.
21. Beck SDW, Peterson MD, Bihrle R, et al. Short-term morbidity of primary retroperitoneal lymph node dissection in a
contemporary group of patients. J Urol 2007; 178:504506.
22. Fossa SD, Horwich A, Russell JM, et al. Optimal planning
target volume for stage I testicular seminoma: a Medical
Research Council randomised trial. J Clin Oncol 1999;
17:11461154.
23. Jones WG, Fossa SD, Mead GM, et al. Randomised trial of
30 versus 20 Gy in the adjuvant treatment of stage I
testicular seminoma: a report on Medical Research
Council Trial TE18, European Organisation for the
Research and Treatment of Cancer Trial 30942 (ISRCTN
18525328). J Clin Oncol 2005; 23:12001208.
24. Oliver RTD, Mason MD, Mead GM, et al. Radiotherapy
versus single-dose Carboplatin in adjuvant treatment of
stage I seminoma: a randomised trial. Lancet 2005;
366:293330.
25. Travis LB, Curtis RE, Storm H, et al. Risk of second
malignant neoplasm among long-term survivors of testicular cancer. J Natl Cancer Inst 1997; 89:14291439.
26. Zagars GK, Ballo MT, Lee AK, et al. Mortality after cure of
testicular seminoma. J Clin Oncol 2004; 22:640647.
27. Powles T, Robinson D, Shamash J, et al. The long-term
risks of adjuvant carboplatin treatment for stage I seminoma of the testis. Ann Oncol 2008; 19:443447.
28. Loehrer PJ, Bosl GJ. Carboplatin for stage I seminoma and
the sword of Damocles. C Clin Oncol 2005; 23:85668569.
29. Raj S, Williams MV, Dixon AK, et al. Late relapse of stage 1
seminoma following single-agent carboplatin. Oncology
2008; 73(56):419421.
30. Moller H. Clues to the aetiology of testicular germ cell
tumours from descriptive epidemiology. Eur Urol 1993;
23:815.
31. Bergstrom R, Adami HO, Mohner M, et al. Increase in
testicular cancer incidence in 9 European countries: a birth
cohort phenomenon. J Natl Cancer Inst 1996; 88:727733.
32. Ekbom A, Akre O. Increasing incidence of testis cancer: a
birth cohort effect. In: Rajpert De Meyts E, Grigor KM,
Skakkebaek NE, eds. Neoplastic transformation of testicular germ cells. APMIS 1998; 106:225229.
33. Freeman A, Harland SJ. Testis cancer. In: Mundy AR,
Fitzpatrick JM, Neal DE, et al. eds. Scientific Basis of Urology. 2nd ed., London: Taylor and Francis, 2004.
34. Sharpe RM, Irvine DS. How strong is the evidence of a link
between environmental chemicals and adverse effects on
human reproductive health? BMJ 2004; 328:447451.
35. Ross RK, McCurtis JW, Henderson BE, et al. Descriptive
epidemiology of testicular and prostatic cancer in Los
Angeles. Br J Cancer 1979; 39:284292.
36. Pukkala E, Wiederpass E. Socio-economic differences in
incidence rates of cancers of the male genital organs in
Finland, 19711995. Int J Cancer 2002; 102:643648.
37. Prattala R, Berg MA, Puska P. Diminishing or increasing
contrasts? Social class variation in Finnish food consumption patterns, 19791990. Eur J Clin Nutr 1992; 46:279287.
38. Tanner JM. Trend towards earlier menarche in London,
Oslo, Copenhagen, The Netherlands and Hungary.
Nature 1973; 243:9697.
411
39. Forman D, Pike MC, Davey G, et al. Aetiology of testicular

cancer: association with congenital abnormalities, age at
puberty, infertility and exercise. BMJ 1994; 308:13931399.
40. Forman D, Gallagher R, Moller H, et al. Aetiology and
epidemiology of testicular cancer: report of consensus
group. Prog Clin Biol Res 1990; 357:245253.
41. Swerdlow AJ, Higgins CD, Pike MC. Risk of testicular
cancer in cohort of boys with cryptorchidism. BMJ 1997;
314:15071511.
42. Pettersson MD, Lorenzo R, Nordenskjold A, et al. Age at
surgery for undescended testis and risk of testicular cancer. N Engl J Med 2007; 356:18351841.
43. Timing of elective surgery on the genitalia of male children with particular reference to the risks, benefits and
psychological effects of surgery and anaesthesia. Paediatrics 1996; 97:590594.
44. Hutson JM, Hasthorpe S. Abnormalities of testicular
descent. Cell Tissue Res 2005; 322:155158.
45. Hutson JM, Hasthorpe S. Testicular descent and cryptorchidism: the state of the art in 2004. J Paediatr Surg
2005; 40:297302.
46. McCabe JE, Kenny SE. Orchidopexy for undescended
testis in England: is it evidence based? J Paediatr Surg
2008; 43:353357.
47. Sokal M, Peckham MJ, Hendry WF. Bilateral germ cell
tumours of the testis. Br J Urol 1980; 52:158162.
48. Hartmann JT, Fossa SD, Nichols CR, et al. Incidence of
metachronous testicular cancer in patients with extragonadal germ cell tumors. J Natl Cancer Inst 2001; 93:
17331738.
49. Harland SJ, Cook PA, Fossa SD, et al. Intratubular germ
cell neoplasia of the contralateral testis in testicular cancer: defining a high risk group. J Urol 1998; 160:13531357.
50. de Gouveia Brazao CA, Pierik FH, Oosterhuis JW, et al.
Bilateral testicular microlithiasis predicts the presence of
the precurs testicular germ cell tumours in subfertile men.
J Urol 2004; 171:158160.
51. Schefer H, Mattman S, Joss RA. Hereditary persistence of
alpha-fetoprotein. Case report and review of the literature. Ann Oncol 1998; 9:667672.
52. Greenburgh F, Alpert E, Rose E. Hereditary persistence of
alpha fetoprotein. Gastroenterology 1990; 1998:10831085.
53. Pandha HS, Wasan HS, Harrington K, et al. The failure of
normalisation of alpha fetoprotein concentration after
successful treatment of teratoma. BMJ 1995; 311:434435.
54. Summers J, Wraggatt P, Pratt J, et al. Experience of discordant beta-hCG results by different assays in the management of non-seminomatous germ cell tumours of the
testis. Clin Oncol 1999; 11:388392.
55. Bjurlin MA, August CZ, Weldon-Linne M, et al. Histologically pure stage I seminoma with an elevated betahCG of 4497 IU/I. Urology 2007; 70:1007.e131007.e15.
56. A predictive model for aggressive Non-Hodgkins Lymphoma. The International Non-Hodgkins Lymphoma
Prognostic Factors Project. N Engl J Med 1993; 329:
987994.
57. International Germ Cell Cancer Collaborative Group.
International germ cell consensus classification: a prognostic factor-based staging system for metastatic germ cell
cancers. J Clin Oncol 1997; 15:594603.
58. Venkitaraman R, Johnson B, Huddart RA, et al. The utility
of lactate dehydrogenase in the follow-up of testicular
germ cell tumours. BJU Int 2007; 100:3032.
59. Ackers C, Rustin GJ. Lactate dehydrogenase is not a
useful marker for relapse in patients on surveillance for
stage I germ cell tumours. Br J Cancer 2006; 94:12311232.
60. Stinghen ST, Moura JF, Zancanella P, et al. Specific
immunoassays for placental alkaline phosphatase as a
tumor marker. J Biomed Biotechnol 2006; 2006(5):56087.
412
Mazhar and Williams
61. Price P, Hogan SJ, Bliss JM, et al. The growth rate of
metastatic non-seminatomous germ cell testicular
tumours measured by marker production doubling
time II. Prognostic significance in patients treated by
chemotherapy. Eur J Cancer 1990; 26:453457.
62. Al-Karim HA, Bryce C, Mulhall P, et al. Repeated AFP
surge: an unusual and potentially misleading tumour
marker phenomenon. Clin Oncol 2002; 14:294295.
63. Toner GC, Geller NL, Tan C, et al. Serum tumor marker
half-life during chemotherapy allows early prediction of
complete response and survival in non-seminomatous
germ cell tumours. Cancer Res 1990; 50:59045910.
64. Stephens MJ, Norman AR, Dearnaley DP, et al. Prognostic
significance of early serum tumour marker half-life and
life in metastatic testicular teratoma. J Clin Oncol 1995;
13:8792.
65. Beck SDW, Patel MI, Sheinfeld J. Tumour markers levels in
post-chemotherapy cystic masses: clinical implications for
patients with germ cell tumours. J Urol 2004; 171:168171.
66. Logothetis CJ, Samuels ML, Trindade A, et al. The growing teratoma syndrome. Cancer 1982; 50:16291635.
67. Skakkebaek NE, Berthelsen JG, Muller J. Carcinoma in
situ of the undescended testis. Urol Clin North Am 1982;
9:377385.
68. von der Maase H, Rorth M, Walbom-Jorgensen S, et al.
Carcinoma in situ of contralateral testis in patients with
testicular germ cell cancer: study of 27 cases in 500
patients. Br Med J (Clin Res Ed) 1986; 293:13981401.
69. Dieckmann KP, Loy V. Prevalence of contralateral testicular intraepithelial neoplasia in patients with testicular
germ cell neoplasms. J Clin Oncol 1996; 14:31263132.
70. Giwercman A, Andrews PW, Jorgensen P, et al. Immunohistochemical expression of embryonal marker
TRA-1-60 in carcinoma in situ and germ cell tumors of
the testis. Cancer 1993; 72:13081314.
71. Chaganti RS, Houldsworth J. Genetics and biology of
adult human male germ cell tumours. Can Res 2000;
60:14751482.
72. Grigor KM, Skakkebaek NE. Pathogenesis and cell biology of germ cell neoplasia: general discussion. Eur Urol
1993; 23:4653.
73. Looijenga LH, de Munnik H, Oosterhuis JW. A molecular
model for the development of germ cell cancer. Int J
Cancer 1999; 83:809814.
74. Bosl GJ, Ilson DH, Rodriguez E, et al. Clinical relevance of
the i(12p) marker chromosome in germ cell tumors. J Natl
Cancer Inst 1994; 86:349355.
75. Rodriguez E, Mathew S, Mukherjee AB, et al. Analysis of
chromosome 12 aneuploidy in interphase cells from
human male germ cell tumors by fluorescence in situ
hybridization. Genes Chromosomes Cancer 1992; 5:2129.
76. Rodriguez E, Houldsworth J, Reuter VE, et al. Molecular
cytogenetic analysis of i(12p)-negative human male germ
cell tumors. Genes Chromosomes Cancer 1993; 8:230236.
77. Rodriguez S, Jafer O, Goker H, et al. Expression profile of
genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification
at 12p11.2p12.1. Oncogene 2003; 22:18801891.
78. Kraggerud SM, Skotheim RI, Szymanska J, et al. Genome
profiles of familial/bilateral and sporadic testicular germ
cell tumors. Genes Chromosomes Cancer 2002; 34:
168174.
79. Skotheim RI, Lothe RA. The testicular germ cell tumour
genome. APMIS 2003; 111:136150; discussion 5051.
80. Oosterhuis JW, Looijenga LH. Current views on the
pathogenesis of testicular germ cell tumours and perspectives for future research: highlights of the 5th Copenhagen Workshop on Carcinoma in situ and Cancer of the
Testis. APMIS 2003; 111:280289.
81. Houldsworth J, Reuter V, Bosl GJ, et al. Aberrant expression of cyclin D2 is an early event in human male germ
cell tumorigenesis. Cell Growth Differ 1997; 8:293299.
82. Houldsworth J, Xiao H, Murty VV, et al. Human male
germ cell tumor resistance to cisplatin is linked to TP53
gene mutation. Oncogene 1998; 16:23452349.
83. Schneider DT, Schuster AE, Fritsch MK, et al. Multipoint
imprinting analysis indicates a common precursor cell for
gonadal and nongonadal pediatric germ cell tumors.
Cancer Res 2001; 61:72687276.
84. Bussey KJ, Lawce HJ, Himoe E, et al. Chromosomes 1 and
12 abnormalities in pediatric germ cell tumors by interphase fluorescence in situ hybridization. Cancer Genet
Cytogenet 2001; 125:112118.
85. Silver SA, Wiley JM, Perlman EJ. DNA ploidy analysis of
pediatric germ cell tumors. Mod Pathol 1994; 7:951956.
86. Jenderny J, Koster E, Borchers O, et al. Interphase cytogenetics on paraffin sections of paediatric extragonadal
yolk sac tumours. Virchows Arch 1996; 428:5357.
87. Stock C, Strehl S, Fink FM, et al. Isochromosome 12p and
maternal loss of 1p36 in a pediatric testicular germ cell
tumor. Cancer Genet Cytogenet 1996; 91:95100.
88. Forman D, Oliver RT, Brett AR, et al. Familial testicular
cancer: a report of the UK family register, estimation of
risk, and an HLA class 1 sib-pair analysis. Br J Cancer
1992; 65:255262.
89. Heimdal K, Olsson H, Tretli S, et al. Familial testicular
cancer in Norway and southern Sweden. Br J Cancer 1996;
73:964969.
90. Hemminki K, Li X. Familial risk in testicular cancer as a
clue to a heritable and environmental aetiology. Br J
Cancer 2004; 90:17651770.
91. Swerdlow AJ, De Stavola BL, Swanwick MA, et al. Risks
of breast and testicular cancers in young adult twins in
England and Wales: evidence on prenatal and genetic
aetiology. Lancet 1997; 350:17231728.
92. Heimdal K, Olsson H, Tretli S, et al. A segregation
analysis of testicular cancer based on Norwegian and
Swedish families. Br J Cancer 1997; 75:10841087.
93. Nicholson PW, Harland SJ. Inheritance and testicular
cancer. Br J Cancer 1995; 71:421426.
94. Crockford GP, Linger R, Hockley S, et al. Genome-wide
linkage screen for testicular germ cell tumour susceptibility loci. Hum Mol Genet 2006; 15:443451.
95. Rapley EA, Crockford GP, Teare D, et al. Localization to
Xq27 of a susceptibility gene for testicular germ cell
tumours. Nat Genet 2000; 24:197200.
96. Skakkebaek NE. Possible carcinoma in situ of the testis.
Lancet 1972; 2:516.
97. Manivel JC, Simonton S, Wold LE, et al. Absence ofintratubular germ cell neoplasia in testicular yolk sac
tumors in children. A histochemical and immunohistochemical study. Arch Pathol Lab Med 1988; 112:641645.
98. Hu LM, Phillipson J, Barsky SH. Intratubular germ cell
neoplasia in infantile yolk sac tumor. Verification by
tandem repeat sequence in situ hybridization. Diagn
Mol Pathol 1992; 1:118128.
99. Stamp IM, Barlebo H, Rix M, et al. Intratubular germ cell
neoplasia in an infantile testis with immature teratoma.
Histopathology 1993; 22:6972.
100. Soosay GN, Bobrow L, Happerfield L, et al. Morphology
and immunohistochemistry of carcinoma in situ adjacent to
testicular germ cell tumours in adults and children: implications for histogenesis. Histopathology 1991; 19:537544.
101. Ramani P, Yeung CK, Habeebu SSM. Testicular intratubular germ cell neoplasia in children and adolescents
with intersex. Am J Surg Pathol 1993; 17:11241133.
102. Giwercman A, Lindenberg S, Kimber SJ, et al. Monoclonal
antibody 43-9F as a sensitive immunohistochemical
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
marker of carcinoma in situ of human testis. Cancer 1990;

65:11351142.
Marks A, Sutherland DR, Bailey D, et al. Characterization
and distribution of an oncofetal antigen (M2A antigen)
expressed on testicular germ cell tumours. Br J Cancer
1999; 80:569578.
Looijenga LH, Stoop H, de Leeuw HP, et al. POU5F1
(OCT3/4) identifies cells with pluripotent potential in
human germ cell tumors. Cancer Res 2003; 63:22442250.
Kang JL, Rajpert-De Meyts W, Giwercmann A, et al. The
association of testicular carcinoma in situ with intratubular microcalcifications. J Urol Pathol 1994; 2:235242.
Bach AM, Hann LE, Hadar O, et al. Testicular microlithiasis: what is its association with testicular cancer?
Radiology 2001; 220:7075.
Peterson AC, Baumann JM, Light DE, et al. The prevalence
of testicular microlithiasis in an asymptomatic population
of men 18 to 35 years old. J Urol 2001; 166:20612064.
Bassetto MA, Pasini F, Franceschi T, et al. Extragonadal
germ cell tumour: a clinical study. Anticancer Res 1995;
15:27512754.
Casella R, Rochlitz C, Sauter G, et al. Burned out
testicular tumour: a rare form of germ cell neoplasias.
Schweiz Med Wochenschr 1999; 129:235240.
de Takats PG, Jones SR, Penn R, et al. a-Foetoprotein
heterogeneity: what is its value in managing patients with
germ cell tumours? Clin Oncol (R Coll Radiol) 1996;
8:323326.
Dueland S, Stenwig AE, Heilo A, et al. Treatment and
outcome of patients with extragonadal germ cell tumours:
the Norwegian Radium Hospitals experience 19791994.
Br J Cancer 1998; 77:329335.
Hartmann M, Pottek T, Bussar-Maatz R, et al. Elevated
human chorionic gonadotropin concentrations in the testicular vein and in peripheral venous blood in seminoma
patients. An analysis of various parameters. Eur Urol
1997; 31:408413.
van de Gaer P, Verstraete H, De Wever I, et al. Primary
retroperitoneal extragonadal germ cell tumour. J Belge
Radiol 1998; 81:221222.
Eble JN, Sauter G, Epstein JI, et al. World Health Organization Classification of Tumours: Pathology and Genetics
of Tumours of the Urinary System and Male Genital
Organs. Lyon, France: IARC Press, 2004:218249.
Sobin LH, Wittekind CH, eds. UICC: TNM Classification
of Malignant Tumours. 6th ed. New York: Wiley-Liss,
2002.
Steyerberg EW, Keiser HJ, Zwartendijk J, et al. Prognosis
after resection of residual masses following chemotherapy
for metastatic non-seminomatous testicular cancer: a multivariate analysis. Br J Cancer 1993; 68:195200.
Hendry WF, AHern RP, Hetherington JW, et al. Paraaortic lympadenectomy after chemotherapy for metastatic
non-seminomatous germ cell tumours: prognostic value
and therapeutic benefits. Br J Urol 1993; 71:208213.
Gerl A, Clemm C, Schmeller N, et al. Outcome analysis
after post-chemotherapy surgery in patients with nonseminomatous germ cell tumours. Ann Oncol 1995; 6:
483488.
Stenning SP, Parkinson MC, Fisher C, et al. Postchemotherapy residual masses in germ cell tumour patients:
content, clinical features, and prognosis. Medical
Research Council Testicular Tumour Working Party, Cancer 1998; 83:14091419.
Hendry WF. Decision-making in abdominal surgery following chemotherapy for testicular cancer. Eur J Cancer
1995; 31A:649650.
Toner GC, Panicek DM, Heelan RJ, et al. Adjunctive surgery after chemotherapy for non-seminomatous testicular
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
413
cancer: recommendations for patient selection. J Clin Oncol

1990; 8:16831694.
Steyerberg EW, Keizer HJ, Stoter G, et al. Predictors of
residual mass histology following chemotherapy for
metastatic non-seminomatous testicular cancer: a quantitative overview of 996 resections. Eur J Cancer 1994;
30A:12311239.
Little JS, Foster RS, Ulbright TM, et al. Unusual neoplasms
detected in testis cancer patients undergoing post-chemotherapy retroperitoneal lymphadenectomy. J Urol 1994;
152:11441149.
Hartmann JT, Nichols CR, Droz JP, et al. Prognostic
variables for response and outcome in patients with
extragonadal germ-cell tumors. Ann Oncol 2002; 13:
10171028.
Warde P, Specht L, Horwich A, et al. Prognostic factors
for relapse in stage I seminoma managed by surveillance.
J Clin Oncol 2002; 20:44484452.
Warde P, Gospodarowicz MK, Banerjee D, et al. Prognostic factors for relapse in stage I testicular seminoma
treated with surveillance. J Urol 1997; 157:17051710.
Weissbach L, Bussar-Maatz R, Lohrs U, et al. Prognostic
factors in seminomas with special respect to HCG: results of
a prospective multicenter study. Eur Urol 1999; 36:601608.
Horwich A, Alsanjari N, AHearn R, et al. Surveillance
following orchiectomy for stage I testicular seminoma. Br
J Cancer 1992; 65:775778.
Albers P, Siener R, Kliesch S, et al. Risk factors for relapse
in clinical stage I non-seminomatous testicular germ cell
tumors: results of the German Testicular Cancer Study
Group trial. J Clin Oncol 2003; 21:15051512.
Bohlen D, Borner M, Sonntag RW, et al. Long term results
following adjuvant chemotherapy in patients with clinical
stage I testicular non-seminomatous malignant germ cell
tumors with high risk factors. J Urol 1999; 161:11481152.
Klepp O, Dahl O, Flodgren P, et al. Risk-adapted treatment of clinical stage I non-seminoma testis cancer. Eur J
Cancer 1997; 33:10381044.
Kratzik Ch, Holtl W, Albrecht W, et al. Risk adapted
management for NSGCT stage 1: long-term results of a
multicenter study. J Urol 1996; 157:547A.
Ondrus D, Matoska J, Belan V, et al. Prognostic factors in
clinical stage I non-seminomatous germ cell testicular
tumors: rationale for different risk-adapted treatment.
Eur Urol 1998; 33:562566.
Pont J, Holtl W, Kosak D, et al. Risk-adapted treatment
choice in stage I non-seminomatous testicular germ cell
cancer by regarding vascular invasion in the primary
tumor: a prospective trial. J Clin Oncol 1990; 8:1619.
Read G, Stenning SP, Cullen MH, et al. Medical Research
Council prospective study of surveillance for stage I
testicular teratoma. Medical Research Council Testicular
Tumors Working Party. J Clin Oncol 1992; 10:17621768.
Albers P, Ulbright TM, Albers J, et al. Tumor proliferative
activity is predictive of pathological stage in clinical stage
A non-seminomatous testicular germ cell tumors. J Urol
1996; 155:579586.
White PM, Howard GC, Best JJ, et al. The role of computed
tomographic examination of the pelvis in the management
of testicular germ cell tumours. Clin Radiol 1997; 52:124129.
White PM, Adamson DJA, Howard GCW, et al. Imaging
of the thorax in the management of germ cell testicular
tumours. Clin Radiol 1999; 54:207211.
Meyer CA, Conces DJ. Imaging of intrathoracic metastases of non-seminomatous germ cell tumors. Chest Surg
Clin N Am 2002; 12:717738.
Krug B, Heidenreich A, Dietlein M, et al. Lymphknotenstaging maligner testikularer Keimzelltumoren. Fortschr
Rontgenstr 1999; 171:8794.
414
Mazhar and Williams
141. Bellin M, Roy C, Kinkel K, et al. Lymph node metastases:

safety and effectiveness of MR imaging with ultrasmall
superparamagnetic iron oxide particles: initial clinical
experience. Radiology 1998; 207:799808.
142. Hogeboom WR, Hoekstra HJ, Mooyart EL, et al. Magnetic
resonance imaging of retroperitoneal lymph node metastases of non-seminomatous germ cell tumors of the testis.
Eur J Surg Oncol 1993; 19:429437.
143. Lassen U, Daugaard G, Eigtved A, et al. Wholebody
positron emission tomography (PET) with FDG in
patients with stage I non-seminomatous germ cell
tumours (NSGCT). Eur J Nucl Med Imaging 2003; 30:
396402.
144. El-Deiry WS, Harper JW, OConnor PM, et al. WAF1/CIPI
is induced in p53-mediated G1 arrest and apoptosis.
Cancer Res 1994; 54:11691174.
145. Canman CE, Gilmer TM, Coutts SB, et al. Growth factor
modulation of p53-mediated growth arrest versus apoptosis. Genes Dev 1995; 9:600611.
146. Wagner AJ, Kokontis JM, Hay N. myc-mediated apoptosis requires wild-type p53 in a manner independent of cell
cycle arrest and the ability of p53 to induce p21 waf1/
cip1. Genes Dev 1994; 8:28172830.
147. Lin D, Fiscella M, OConnor PM, et al. Constititive expression of B-myb can bypass p53-induced Waf1/Cip1mediated G1 arrest. Proc Natl Acad Sci USA 1994; 91:
1007910083.
148. Peng HQ, Hogg D, Malkin D, et al. Mutations of the p53
gene do not occur in testis cancer. Cancer Res 1993;
53:35743578.
149. Heindal K, Lothe RA, Lystad S, et al. No germline TP53

mutations detected in familial and bilateral testicular
cancers. Genes Chromosomes Cancer 1993; 6:9297.
150. Fleischhacker M, Strohmeyer T, Imao Y, et al. Mutations
of the p53 gene are not detectable in human testicular
tumours. Mod Pathol 1994; 7:435439.
151. Chresta CM, Masters JR, Hickman JA. Hypersensitivity of
human testicular tumours to etoposide-induced apoptosis
is associated with functional p53 and a high Bax: Bcl-2
ratio. Cancer Res 1996; 56:18341841.
152. Zamble DB, Mikata Y, Eng CH, et al. Testis-specific HMGdomain proteinalters the responses of cells to cisplatin.
J Inorg Biochem 2002; 91:451462.
153. Koberle B, Masters JRW, Hartley JA, et al. Reduced repair
of cisplatin-induced DNA damage in testicular germ cell
tumours due to a specific protein defect. Curr Biol 1999;
9:273276.
154. Mueller T, Voigt W, Simon H, et al. Failure of activation of
caspase-9 induces a higher threshold for apoptosis and
cisplatin resistance in testicular cancer. Cancer Res 2003;
63:513521.
155. Spierings DC, de Vries EGE, Vellenga E, et al. Loss of
drug-induced activation of the CD95 apoptotic pathway
in a cisplatin-resistant testicular germ cell tumor cell line.
Cell Death Differ 2003; 10:808822.
156. Rao PH, Houldsworth J, Palanisamy N, et al. Chromosomal amplification is associated with cisplatin resistance
of human male germ cell tumours. Cancer Res 1998; 58:
42604263.
27
Radiotherapy: Scientific Principles and Practical
Application in Urogical Malignancies
Angela Swampillai, Rachel Lewis, Mary McCormack, and Heather Payne
Department of Oncology, University College Hospitals, London, U.K.
INTRODUCTION
Radiotherapy is the therapeutic use of ionizing radiation. X rays were first used in the treatment of cancer
over 100 years ago by Freund, a German surgeon.
Since that time, our understanding of the effects of
ionizing radiation on malignant and normal tissues
has progressed through the field of radiobiology. In
parallel with this, our knowledge of radiation physics
has advanced together with significant technological
developments in treatment planning and delivery.
This chapter outlines the underlying physical
and radiobiological principles of radiotherapy and
discusses the scientific practice of radiotherapy as
applied to urological tumors.
RADIATION PHYSICS
Radiation used therapeutically is called ionizing radiation, as it causes its effects through the ionization of
intracellular molecules. Ionizing radiation can be classified as electromagnetic or particulate.
Electromagnetic Radiation
The two types of electromagnetic radiation used in
radiotherapy are X rays and g rays. They are physically
identical, but known by different names to distinguish
their means of production. g Rays are produced from
the nuclear decay of radioactive isotopes. X rays are
produced by interactions that occur outside the nucleus.
Both types of radiation have short wavelengths, high
frequencies and carry high energies that enable them to
break chemical bonds and produce biological effects
(Fig. 1). The term photon is another name that can be
used to describe both X rays and g rays.
X rays are usually produced artificially by electrical means, accelerating electrons to a high energy
and then abruptly stopping them in a heavy metal
target. Part or all of the kinetic energy of the electrons
is converted into X rays. The energies necessary to
generate therapeutic X-ray beams capable of penetrating human tissues are in the megavoltage (MV) range
and are usually produced by a machine called a linear
accelerator. This type of treatment is an example of
teletherapy, where the source of radiation is distant

from the body and is often referred to as external
beam radiotherapy.
As the energy of the treatment beam increases so
does the penetration depth, as the X rays are less able to
interact with superficial tissues (Fig. 2). Modern radiotherapy techniques use MV photons with beam energies typically greater than 8 MV to treat urological
tumors such as carcinoma of the prostate or bladder.
Prior to the introduction of MV treatment (cobalt
machines and linear accelerators), the only available
treatment energies were up to 300 kV, which gave
inadequate penetration for the treatment of deep target
tissues. The term deep X-ray therapy (DXT) is often
mistakenly applied to all radiotherapy but historically
refers specifically to energies in the 250 to 300 kV range.
These beams deposit their maximum dose in the skin
and have a therapeutic penetration of only 3 to 4 cm.
Lower energy beams (90300 kV) are now used mainly
to treat lesions in the skin and superficial tissues.
The other common types of therapeutic radiation, g rays, are produced by the nuclear decay of
radioactive elements and represent the excess energy
that is emitted as an unstable nucleus decays into a
more stable form. The higher energy g rays emitted
from isotopes such as cobalt (60Co) or cesium (137Cs)
can be used therapeutically if harnessed into beams
for external radiotherapy treatment. Cobalt machines
are still used in some centers, but since they produce a
relatively low energy beam, their uses are limited for
urological treatments.
Other isotopes such as radium (226Ra), iridium
192
( Ir) and iodine (125I) can be used as temporary or
permanent implants, a process known as brachytherapy. Sealed isotope sources can be placed into body
cavities (intracavity brachytherapy) or directly into
tissues (interstitial brachytherapy).
The main physical advantage of brachytherapy
is that it allows a high tumor dose and considerable
sparing of surrounding normal tissues. This occurs
because of the inverse square law that governs the
intensity of all electromagnetic radiation. As the distance from a radiation source doubles, the intensity
falls to one-quarter, producing a rapid decline in effect
that can be exploited therapeutically in brachytherapy.
416
Swampillai et al.
of radioactive strontium for the palliation of painful

bone metastases in metastatic prostate cancer.
Strontium is taken up into bone, and the radioactive
decay produces local tumor cell kill, resulting in pain
relief.
Particulate Radiation
Figure 1 Illustration of the electromagnetic spectrum.
The isotopes used in brachytherapy are known as

sealed sources because they are contained within protective coverings to stop physical contact between the
body and the radioactive material while still allowing
the radiation to escape. Radium is now no longer used
because of the difficulty of ensuring adequate radiation protection. It decays into radon gas, which is
difficult to contain and emits a particles, a highly
damaging form of particulate radiation (see below).
A third therapeutic use for g rays is as unsealed
sources in which an isotope is exploited because of
its metabolic effect on biological tissues. The isotope is
usually incorporated into tissues because of its physical properties, radioactivity being released as the
isotope decays. An example of this is administration
Particulate radiation consists of subatomic particles,

including electrons, protons and a particles. In current
radiotherapy, electrons are the only commonly used
type of particulate radiation. Electrons are also generated by a linear accelerator, but instead of the beam
hitting a target to generate X rays, the electrons are
allowed to penetrate directly into the tissues to produce the therapeutic effect.
Electron beams differ from those of photons in
that there is a much sharper decline in radiation dose
(Fig. 2), sparing any underlying tissues. These properties make electron beams ideal for treating superficial
tumors where a high dose if needed at or just below
the skin surface, but it is necessary to spare the
underlying tissues.
Linear accelerators are usually able to produce
a range of electron beam energies of 4 to 20 MeV.
The effective treatment depth in centimeters is about
one-third of the beam energy in MeV (e.g., 4 cm for a
12-MeV electron beam). The electron energy used for
treatment is chosen so that the tumor will typically
receive 90% of the maximum dose (known as the 90%
isodose). This allows for the best homogeneity of
treatment dose across the tumor.
Interactions of X Rays with Matter

The process by which photons are absorbed depends
principally on their energy and the chemical composition of the absorbing material. Three distinct
Figure 2 Depth dose curves for different energies of electrons and mV photons.
Chapter 27: Radiotherapy: Scientific Principles and Practical Application in Urogical Malignancies
clinically relevant absorption mechanisms are recognized: the photoelectric effect, the Compton effect and
pair production. As energy is being absorbed in these
interactions, the SI unit of dose in radiotherapy measures the amount of energy in joules absorbed per unit
mass in kilograms. The unit is the Gray (Gy) and 1 Gy
is equal to 1 J of energy absorbed per kilogram of
mass. To illustrate the amount of radiation energy
absorbed in biological tissues, Hall has calculated that
the same amount of energy is transferred in drinking
one sip of hot coffee as is absorbed in a lethal 4-Gy
dose of whole body radiation.
The absorption process for low energy X rays is
called the photoelectric effect. The incident X ray
interacts with an inner orbital electron shell of an
atom within the absorbing material. The energy of
the X ray is transferred to the electron as kinetic
energy that causes the electron to be ejected from its
orbit shell and the X ray to be dissipated. The ejected
electron is now known as a fast electron. These fast
electrons can ionize other atoms in the absorbing
material, breaking chemical bonds and initiating a
sequence of changes that is ultimately expressed as
biological damage. Mathematically, in this interaction,
the energy absorbed is proportional to the cube of the
atomic number (Z) of the material through which the
beam passes (Fig. 3A).
The photoelectric effect is the major X-ray
absorption process for diagnostic radiology. Absorption is preferential in bone because of its relatively
high atomic number (Z), and this gives the characteristic white X-ray appearance of bone on standard
diagnostic films (as X rays fail to reach and blacken
the photoemulsion). This characteristic of the photoelectric effect is a disadvantage when used for radiotherapy, as bone and cartilage have a relatively high Z,
and preferential absorption in these structures can
cause radionecrosis. Because of the combination of
poor penetration in tissue, the maximum deposition of
energy in the skin and the preferential absorption in
bone and cartilage, these lower-energy X rays are no
longer routinely used except for the treatment of some
skin and superficial cancers.
The absorption interaction for photons in the
commonest therapy range of 1 to 10 MV is the
Compton effect. In this interaction, the incident photon interacts with a more loosely bound outer electron
in the atoms of the absorbing material. The photon
gives up part of its energy to the electron as kinetic
energy as before, and, deflected from its original path,
proceeds through the material with corresponding
decreased energy. The result of this interaction is
also an ejected fast electron, which can ionize other
atoms in the absorber. The deflected photon continues
on its new path, interacting with further outer orbital
electrons until all its energy is dissipated. Unlike the
photoelectric effect the Compton process is independent of the atomic number of the absorbing material,
and photons are not preferentially absorbed in bone
and cartilage (Fig. 3B).
At treatment energies in excess of 10 MV, pair
production predominates. The incident photon interacts with the nucleus of an atom, giving up all its
417
Figure 3 Interactions of photons with matter; (A) photoelectric

effect, (B) Compton scatter, (C) pair production. Source: Adapted
from Ref. 1.
energy in the process. This results in the production of

a positron and a fast electron. The fast electron can go
on to produce tissue ionizations, and the positron is
quickly annihilated. Positron annihilation occurs as a
further interaction with an adjacent electron of the
absorbing material. This creates two new photons of
0.51 MeV energy each, both capable of producing
additional tissue ionizations. Pair production, like
the photoelectric effect, is also dependent on atomic
number (Fig. 3C).
The resultant biological damage from fast electrons produced by all three different processes occurs
in two ways. Direct ionization of atoms within target
molecules may be produced, leading to chemical bond
breakage and initiating the chain of events that leads
to biological change. This is the dominant process
when neutrons or a particles are used to irradiate
418
Swampillai et al.
tissues. X rays and g rays interact mainly with target

biological molecules in an indirect way. The interaction is initially with other atoms and molecules within
the cells to produce free radicals. These are highly
reactive molecules with an unpaired electron in the
outer orbital shell and are able to diffuse far enough to
reach and damage the critical targets.
Since approximately 80% of a cell is composed of
water, it is necessary to consider what happens when
radiation interacts with a water molecule. As a result
of the interaction with a photon, the water molecule
may become ionized.
H2O
H2O e
H2O is an ion radicalit is an ion because it is

charged and a free radical because it has an unpaired
electron in its outer shell. It is a highly reactive species
with a very short lifespan in the order of nanoseconds.
The ion radical can then interact with another water
molecule to form the highly reactive hydroxyl radical
(OH).
H2O H2O
H3O OH
It is estimated that about two thirds of the damage to biological targets from X rays is produced by
hydroxyl radicals. Free radicals can be inactivated
by combining with sulphydryl molecules or they
may interact with oxygen and form a highly reactive
product, and by so doing, the damage may be fixed
(i.e., made permanent). The interaction process is
summarized in Figure 4.
Regardless of whether radiation effects occur
because of the direct or indirect interaction, the main
biological target in tissues is believed to be DNA. Such

interactions result in changes in DNA chemistry, leading to breaks within one or both helical strands. A
single-strand break within DNA may be recognized
and repaired as the complementary DNA strand is
still present to act as a template. When several ionizations occur in close proximity, a double-strand DNA
break may occur. It is thought that double-strand
breaks are the lethal lesions that result in cell kill
because of the inability of the cell to repair both
damaged strands of DNA simultaneously. Apart
from the physical characteristics of radiation beams,
there are a number of other important features relating to cells and tissues themselves that influence their
response to radiotherapy. These features are discussed
below.
RADIOBIOLOGY
Radiobiology is the study of the actions of ionizing
radiation on living things. The effects of radiation
on both normal and malignant tissues evolve through
a series of steps. This begins with the physical absorption of energy and ends with the biological effect,
which results principally from damage to DNA, Classical radiobiology promotes the four Rs of the radiation response: repair, redistribution, reoxygenation,
and repopulation (2). The development of tissue culture techniques has contributed enormously to our
understanding of radiobiology in ways that are not
possible to achieve in patients. With such techniques, it
is possible to observe the effects of radiation on cells in
vitro. The cell survival curve is a cornerstone of in vitro
radiobiology and describes the relationship between
radiation dose and the proportion of cells surviving
(Fig. 5). Survival refers to the ability of a single cell to
proliferate indefinitely to produce a large clone or
colony. Such a cell is said to be clonogenic. Tumor
cells are clonogenic, and if one cell survives after a
course of radiotherapy, this may be sufficient for the
tumor to regrow.
Repair
Figure 4 Summary of interaction of X rays or photons. Source:

Adapted from Ref. 1.
Radiation damage to mammalian cells can be divided

into the following three categories.
Lethal damagethis is irreversible and irreparable leading irrevocably to cell death.
Sublethal damage (SLD)this is damage that
under normal circumstances can be repaired over a
number of hours. SLD repair is inferred by the increase
in survival observed when a dose of radiation is split
into two fractions separated by a time interval.
Potentially lethal damage (PLD)this refers to
the component of radiation damage that can be modified by postirradiation environmental conditions.
The ability of cells to repair SLD was recognized
by Elkind and colleagues, who demonstrated that the
survival of Chinese hamster cells treated with a single
dose of radiation was lower than that of cells given the
same total dose of radiation, but split into two fractions separated by 30 minutes (Fig. 6) (3). Mechanisms
419
Figure 6 Survival of Chinese hamster cells exposed to two

fractions of X rays with various time intervals between the two
and incubated at room temperature. As the time interval is
increased the surviving fraction increases until at about two
hours, a plateau is reached corresponding to a surviving fraction
of 0.02. Source: Courtesy of Ref. 3.
Figure 5 Clonogenic cell survival curve following radiation. A

typical surviving fraction at 2 Gy is 0.6 with higher doses
producing a lower surviving fraction. Source: Courtesy of
Dr Gillian Duchesne.
of DNA repair include nonhomologous end-joining

and homologous recombination.
Redistribution
The radiosensitivity of cells also varies with the
phases of the cell cycle (Fig. 7). Radiation damage to
DNA is frequently not expressed until cell division.
Cells that are in the G0 (resting) phase or only in a
very slow cell cycle will not express damage immediately. The length of the cell cycle for most rapidly
dividing tumors is approximately 24 hours. In such
tumors, the interval between fractions of radiation
may be critical to the cell kill achieved, as radiation
sensitivity varies by at least two fold through the
cycle. Cells located in the S phase of the cell cycle
are said to be relatively radioresistant, while those in
the G2 or M phase are said to be radiosensitive. In the
experiments by Elkind et al. (3) described above, most
of the surviving cells from the first dose of radiation
were located in the S phase. When a second dose of
radiation was given after an interval of six hours, this
cohort of cells had progressed around the cell cycle
and was now in the G2 or M phase (more sensitive)
at the time of the second dosea process called
Figure 7 Survival of Chinese hamster cells exposed to two

fractions of X rays with various time intervals between the two
doses. This also illustrates three of the four Rs of radiobiology.
Source: Courtesy of Refs. 1 and 3.
420
Swampillai et al.
redistribution (or reassignment). If the increase in

radiosensitivity in moving from late S to the G2-M
period exceeds the effect of SLD repair, the surviving
fraction will fall. In human tissues, the time intervals
for cell cycles and SLD repair are generally less well
defined, but maximal recovery appears to be complete
after four to six hours.
While this phenomenon is relatively easy to
demonstrate and manipulate in vitro, it has been
difficult to exploit in routine clinical practice.
Reoxygenation
The importance of a good blood supply for radiosensitivity was first recognized by Mottram in 1924
(4). Further work by Thoday and Reed (5) demonstrated that the proportion of chromosome aberrations
resulting from irradiation of root tips in oxygen was
three times greater than those seen when irradiation
was carried out in nitrogen. They concluded that the
availability of oxygen is a very important factor in the
production of radiation-induced chromosome aberrations. Experiments by Thomlinson and Gray (6) and
Tannock (7) led to the conclusion that cells were
present in only two states, which have radiobiological
significanceoxygenated and hypoxic. Tumor cells
closest to blood vessels are generally well oxygenated,
and those at a distance greater than the oxygen-diffusion capacity are hypoxic.
Acute as well as chronic hypoxia occurs within
tumors; intermittent blood flow within abnormal
tumor vasculature was shown to be a cause of acute
hypoxia and variability in radiation response by
Chaplin et al. in 1986 (8). The proportion of hypoxic
cells within solid tumors may vary considerably
depending on the tumor type. Although a dynamic
process, the hypoxic fraction is thought to remain relatively constant within a given tumor. Immediately after
a radiation exposure, remaining viable cells are more
likely to be hypoxic, as the well-oxygenated cells die.
Over the following hours, oxygen diffuses into these
previously hypoxic areas and reoxygenation occurs.
The cells are now more sensitive to the next radiation
exposure and the whole process is repeated again. Over
a fractionated radiotherapy schedule of several weeks, it
is likely that most cells will receive some radiation
exposure while in the oxygenated state. Efforts to overcome tumor hypoxia have dominated radiobiological
research for the past 40 years. The approaches investigated have included hyperbaric oxygen and hypoxiccell radiosensitizing drugs, such as misonidazole, but
they have met with only limited success.
Repopulation
During a course of fractionated radiotherapy, viable
tumor cells continue to divide and repopulate the
tumor. Repopulation is an important process for
early-responding normal tissues, such as the gut and
bone marrow. A protracted, fractionated regimen helps
to spare these normal tissues but may compromise
tumor cure. In the above experiment, when the interval
between the split doses is 10 to 12 hours, there is an

increase in the surviving fraction because of cell division (repopulation) because this time interval exceeds
the length of the cell cycle of these rapidly growing
cells.
Treatment with radiation or cytotoxic drugs can
trigger surviving clonogens in a tumor to divide faster
than tumor cells in an unperturbed state. This is
known as accelerated repopulation. There is evidence
that this phenomenon may occur in human tumors.
Withers et al. (9) used the published literature on
radiotherapy for head and neck cancer to estimate
the dose of radiation required to achieve local control
in 50% of cases [tumor control dose 50 (TCD 50)].
The analysis suggests that clonogen repopulation in
this type of cancer accelerates at about day 28 after
treatment begins. A dose increment of about
0.6 Gy/day is thought to be required to compensate
for this repopulation. In clinical practice the dose of
radiation per fraction is not usually increased after
day 28 provided there are no unscheduled gaps over
the course of the treatment. Cancers of the bladder,
head and neck and cervix are tumors where gaps or
delays in treatment are thought to have considerable
significance for overall tumor control probability. It
has been recommended that, if gaps in treatment
occur in these types of tumors some form of compensation is required to ensure tumor control is not
compromised (10). This may involve increasing the
dose per fraction for part or all of the remaining
treatment or hyperfractionation, where two fractions
are given on the same day with an interfraction interval of at least six hours.
The Linear Quadratic Model

The duration of the cell cycle and the ability of cells to
repair SLD, varies not only between different normal
tissues but also between normal tissues and tumors.
Normal tissues may be divided into those that are
considered early responding, such as skin, mucosal
epithelium and bone marrow and those that are late
responding, such as the spinal cord and central nervous system tissue. Most tumors behave as earlyresponding tissues. Early-responding tissues are triggered to proliferate within a few weeks of starting a
fractionated course of radiotherapy and tissue damage, such as skin desquamation or tumor shrinkage,
will become apparent during the course of treatment.
However, a conventional course of radiotherapy of six
to seven weeks is never long enough to trigger proliferation in late-responding tissues; consequently,
radiation effects on these tissues may not become
apparent for months to years after the treatment.
Various mathematical models have been used to
describe the shape of the cell survival curve and are
useful for the theoretical interpretation of observed
experimental changes. The linear quadratic (a/b)
model is one model where a measures the probability
of causing lethal damage by a single event (such as a
double-stranded DNA break), and b estimates the
probability of cell death from the interaction between
two separate sublethal events. (a is a measure of the
421
more likely to be overcome. The systemic agents

may also radiosensitize tumor tissue and may favorably alter the tumor biology, for example, by reoxygenation, cell cycle redistribution, inhibiting DNA repair
and impairing accelerated repopulation. This concept
is being evaluated for bladder cancer in a randomized
controlled trial comparing radical radiotherapy with
radiotherapy plus concomitant 5 FU/mitomycin C.
Theoretically, 5 FU would kill radioresistant cells in
the S phase of the cell cycle. In addition, radiation
induces the expression of thymidine phosphorylase
in cancer tissues, which is the first enzyme involved in
the activation of 5 FU (12).
PRINCIPLES OF RADIOTHERAPY TREATMENT

Figure 8 Cell survival curve for low and high a/b ratio.
width of the shoulder of the curve, while b indicates the

curviness of the curve.) It is the ratio of a/b [i.e., the dose
at which cell killing by linear (a) and quadratic (b)
components is equal], rather than the individual values
of each, which determines how tissues behave to
changes in fractionation. Early-responding tissues and
most tumors have a high a/b ratio (1030 Gy), indicating
that they are relatively insensitive to changes in radiotherapy fractionation. Late-responding tissues have a
low a/b (14 Gy) indicating a higher degree of sensitivity to fractionation changes. By altering fractionation, it
is possible to produce a differential in cell kill between
tumors and late-responding normal tissues, modifying
the therapeutic index (i.e., the tumor response for a fixed
level of normal tissue damage) (Fig. 8).
Most tumors arise from epithelial cells and have
a fairly high a/b ratio (1025 Gy). Tissues that give
rise to late reactions (months or years) in radiotherapy
have relatively low a/b ratios and changes in fractionation can be made that improve the therapeutic ratio.
However, adenocarcinoma of the prostate has
been estimated to have a low a/b ratio (approximately
1.5 Gy). This is even lower than the estimated a/b ratio
of late bowel effects (3 Gy). If this data is correct, a
hypofractionated schedule of radiotherapy (>2 Gy per
fraction) could potentially achieve improved tumor
control without increasing toxicity. The CHHIP [conventional or hypofractionated high-dose intensitymodulated radiotherapy (IMRT) for prostate cancer]
trial (11) is a phase III randomized controlled trial
comparing different fractionation regimes (60 Gy/20
fractions of radiotherapy and 57 Gy/19 fractions)
against the standard (74 Gy/37 fractions) to look at
local control rates and long-term side effects, and will
also yield further radiobiological data.
Radiation is an important modality for the treatment of

cancer and can be used with radical, adjuvant or palliative intent. To justify treatment with radiotherapy it
is important that patients have histological confirmation of cancer and relevant imaging. They should have
all their treatment options discussed, be aware of treatment intentions, side effects and consent to treatment.
Radical radiotherapy aims to deliver a tumoricidal treatment dose to a well-defined target volume
with curative intent, sparing the surrounding normal
tissues as much as possible. Currently, most departments use 3-D conformal radiotherapy, which is
achieved using modern linear accelerators with multileaf collimators. More recently a further development
has been IMRT. This technique uses computer-assisted
technology to modify and shape the intensity of radiotherapy beams during treatment to deliver very precise
coverage of the target area, with even greater conformity. Radical radiotherapy is given as a fractionated
course, usually over six to eight weeks for localized
tumors of the bladder, prostate and penis.
Adjuvant radiotherapy is used to reduce the risk
of tumor recurrence after primary surgery. The aim of
treatment is to eradicate occult micrometastatic disease that cannot be demonstrated on imaging as
occurs in a significant percentage of patients. An
example of this is prophylactic, para-aortic radiotherapy after orchidectomy for stage 1 seminoma. Adjuvant treatment in this setting reduced relapse rates
from approximately 20% to 3% to 4% (13).
Palliative radiotherapy can be used to alleviate
symptoms of local disease (such as hematuria) or
distant metastases (such as bone pain). Treatment is
usually given with a small number of fractions, and
effective results can also be achieved with a single
fraction of radiotherapy.
The planning and delivery of radiotherapy is
discussed below:
Combined Modality Treatment
RADIOTHERAPY PLANNING
The combination of chemotherapy or biological agents

with radiotherapy is increasingly used to try and
improve local control rates and eradicate micrometastases. The effects of multimodality treatment can be
additive, synergistic or independent. By using two
treatment modalities, resistance mechanisms are
The initial phase of any radiotherapy is treatment

planning, during which the following parameters are
defined:
l
l
Patient position and immobilization

Definition of treatment volumes
422
l
l
Swampillai et al.
Choice of technique
Calculation of dose distribution
Patient Position and Immobilization

The treatment position of the patient must be accurately reproducible throughout radiotherapy. Variation in daily positioning is a result of daily treatment
setup and internal organ movement and as a result
can alter external and internal anatomy of the patient.
Ultimately, this can lead to underdosage of the tumor
or an overdosage of the surrounding normal tissues.
To limit patient setup error they are immobilized
in a comfortable position thereby minimizing movement. Treatment position varies with different tumor
sites but for urological tumors, the patient is supine.
Accurate patient setup on the planning machine is
achieved by aligning fixed, wall mounted lasers to
certain anatomical reference points such as bony landmarks or permanent skin tattoos applied during planning. An identical alignment system is then used on
the treatment machine, ensuring that patient setup is
fully reproducible.
There is also a need to limit variation in internal
organ movement. For example, the position of the
prostate is dependent on the relative positions of
the bladder and rectum. Therefore tumors of the
prostate are usually treated with a full bladder to
reduce the volume of bladder tissue irradiated and
also to move the small intestine out of the field.
Patients are given dietary advice/ laxatives to keep
the rectal volume as small as possible. To limit normal
tissue toxicity it is also necessary to determine if any
normal tissues need to be shielded. For example, in
penile cancer, the penis is placed in a block and this
rests on a lead shield, which protects the underlying

testis and skin.
Definition of Treatment Volumes

In delivery of radiotherapy treatment, parameters
such as volume and dose have to be specified for
the purposes of prescription, recording and reporting.
This has been standardized by the International Committee of Radiation Units and Measurements (ICRU),
allowing national and international comparison of
treatments between different centers (14). This report
defines the concepts of gross tumor volume (GTV),
clinical target volume (CTV), and planning target
volume (PTV).
The GTV is the demonstrable macroscopic extent
of the tumor. To encompass subclinical or microscopic
disease, one adds a margin to the GTV and this
volume is defined as the CTV. During a course of
radiotherapy there may be technical and physiological
variations, which can cause movement of the CTV. To
ensure that the prescribed dose is delivered to the
CTV for every fraction of treatment, a further margin
is added to form the PTV. This is a combination of a
setup margin (SM) to allow for variation in patient
positioning and an internal margin (IM) to account for
normal organ movement.
Localization of the Target Volume

The target volume for internal tumors is usually
localized by plain X rays on a machine called a simulator (conventional planning) or a computerized
tomography (CT) scanner with an integrated computerized planning system (CT planning) (Fig. 9).
Figure 9 A simulator. This is a diagnostic X-ray unit which has an image intensifier linked to a closed-circuit television for the purpose of
screening. It can reproduce treatment conditions.
For radical radiotherapy the target volume is

localized using a CT planning scan and the data from
this is sent to the radiotherapy planning computer for
outlining. This has the advantage of providing anatomical detail about the tumor and surrounding normal tissues, allowing for 3-D conformal therapy or
IMRT.
A simulator is a diagnostic X-ray machine used
to reproduce the exact treatment arrangement for any
MV therapy machine. It has the facility for screening
by an image intensifier linked to a close-circuit television. In the simulator, the target volume is localized
by using both clinical information (staging results,
surface anatomy and palpable masses) and radiological information (image intensifier views and staging
diagnostic X rays). The conventional method of planning a bladder cancer, for example, involves the introduction of contrast into the bladder through a urinary
catheter. Anteroposterior and lateral X-ray films are
then taken on the simulator and these are then used to
outline the target volume.
Choice of Technique
In addition to the target volumes defined above, the
other important considerations in the choice of technique are the types of normal tissues within the
proposed treatment volume. During a course of radiotherapy it is inevitable that both the tumor and normal
tissues will be irradiated. There is considerable variation in the tolerance of different normal tissues to
radiotherapy, and the Therapeutic Index, which is the
tumor response for a fixed level of normal tissue
damage, is a major determination of both the total
dose that can be delivered and the treatment technique. Therefore, it is important to outline and identify the important normal tissues within the target
volume as well as the tumor.
The simplest technique for treatment is a single
radiation field (Fig. 10). This is often used for palliative therapy, such as treatment of bone metastases.
Another simple arrangement is the use of two opposing parallel fields (anterior and posterior or two lateral
fields). This produces a more even dose distribution
throughout the irradiated area than a single field and
the combination of fields allows greater penetration at
Figure 10 Electron isodose chart.
423
depth. This technique is used for the prophylactic

treatment of the para-aortic lymph nodes in stage 1
seminoma (Fig. 11).
For radical treatment of the prostate or bladder it
is necessary to use higher treatment doses and often
three or four fields are used to allow the greatest
sparing of normal tissues while maximizing the
tumor dose (Fig. 12). This can be achieved with 3-D
conformal radiotherapy, which allows closer, more
accurate irradiation of irregularly shaped tumor volumes. Treatment beams are produced within the
machine, which conform much more closely to the
shape of the target volume. This produces greater
sparing of normal tissues allowing higher and potentially more effective doses of radiation to be delivered
to the tumor without increasing side effects (15).
An emerging technique is the use of highprecision radiotherapy in the form of IMRT. This
allows closer matching of treatment doses to tumor
volumes. Rather than having a single or a few radiation beams, the radiation with IMRT is effectively
broken into thousands of tiny pencil-thin beams.
These enter the body from many different angles
and all converge on the tumor. This produces a high
tumor dose and a lower dose to surrounding normal
tissues. An example of this is the use of IMRT in the
treatment of pelvic lymph nodes in prostate cancer.
Calculation of the Dose Distribution

Once the PTV and important normal organs are localized, a dose distribution across the target volume is
calculated and a plan is produced. This is done using a
computer software that manipulates dose data measured directly from treatment machines. On reviewing
the plan, the oncologist will ensure that the target is
covered by the 95% isodose line and according to
ICRU that there is a maximum variation of dose distribution of from 5% to 7% in dose across the PTV
(Fig. 13). The dose to the normal tissues is also checked
to ensure that their tolerance is not exceeded.
Treatment Delivery
Once an acceptable treatment plan has been agreed
on, planning information is transferred to the treatment machine and therapy is started. An essential part
of all treatment is verification of the planning setup,
which is achieved by comparison of X rays or scans
taken during simulation and treatment. Each center
will define an accepted degree of variation between
the images taken at simulation and those taken during
treatment. For radical plans of the pelvis this is usually between 3 and 5 mm.
Therapy doses may also be checked directly
during treatment by measurements from the patient.
The entire process from planning to verification of
treatment delivery is governed by the principles of
quality assurance. These define procedures that test
all the technical aspects of radiotherapy systems to
eliminate any inaccuracy or deficiency leading to
suboptimal treatments and must be a routine part of
treatment.
424
Swampillai et al.
Figure 11 An AP film from the planning CT scanner, showing field margins for para-aortic lymph node irradiation.
Side Effects of Treatment
Figure 12 A three-field plan of the prostate showing an anterior

and two posterior oblique wedged fields.
The side effects of radiotherapy may be divided into

those that occur during or within a few weeks of
treatment (acute reactions) and those that occur
months to years later (late reactions). The severity of
side effects depends principally on the normal tissues
present in the treatment field, total dose and dose per
fraction of treatment given. It is important to realize
that the radiobiological principles defined in the previous section of the chapter are the basis for both the
desirable therapeutic effects and the undesirable side
effects that occur during a course of treatment.
During radical radiotherapy for prostate or bladder cancer patients principally experience a range of
side effects to the normal bladder, bowel and skin
within the treatment volume. These may include deterioration of urinary outflow symptoms, hematuria,
proctitis and diarrhea. They are generally the effects
of radiation on the normal acute-reacting tissues in the
treatment volume. Acute side effects such as these are
usually self-limiting and generally resolve within six
to eight weeks after completing treatment. They can
usually be successfully managed with supportive care
during radiotherapy.
425
Brachytherapy
Brachytherapy has been used in the treatment of urological cancer for over 90 years, but it experienced a
renaissance for the treatment of prostate cancer in the
1980s (16). This was due to improved prostatic imaging with transrectal ultrasound and technological
advances in computerized planning systems, allowing
better dosimetry and more accurate treatment.
The planning principles for brachytherapy are
the same as for external beam treatment volumes
(i.e., patient immobilization, definition of treatment
volumes, choice of technique and calculation of the
dose distribution). Selecting patients for brachytherapy is a combination of tumor characteristics and the
ability of the patient to comply with radioprotection
issues.
Brachytherapy can be used as a single modality
to deliver a radical treatment alone (such a slow dose
rate 125I seed brachytherapy) or in combination with
external beam radiotherapy to deliver a boost treatment (such as high doserate 192Ir brachytherapy).
Permanent Iodine Seed Brachytherapy

Treatment with 125I seeds is indicated for patients with
low grade, organ confined tumors with a small volume (less than 50 cm3) and no previous transurethral
surgery (Fig. 14). Treatment has two stages, planning
and therapy, both are carried out with the patient in
the lithotomy position under anesthetic. Planning
involves a transrectal ultrasound guided prostatic
volume study that is done with reference to a fixable
perineal positioning template. The prostate is positioned within the template grid and the urethra is
centered in the middle row. Serial ultrasounds are
taken from the apex to the base and the prostate is
outlined. This information is sent to the planning
Figure 13 Isodose curves for a 6-MV linear accelerator (A) open

field (B) wedged field.
The late effects of radiotherapy are usually more

serious and may be irreversible. For pelvic radiotherapy, these local late effects include bladder fibrosis,
chronic proctitis and impotence. Radiation carcinogenesis is another late effect that occurs within the
site of the treatment volume and is believed to be due
to radiation-induced genetic damage to normal tissues. It tends to occur after other late effects. In all
radiotherapy treatments, the balance to be achieved
lies between the desired beneficial effects (cure or
palliation) and the level of acute and late effects that
can be tolerated by the treated individual.
Figure 14 AP simulator film showing the distribution of 125I seeds

within the prostate.
426
Swampillai et al.
computer and the relationship of the prostate to the

surface template allows the exact number and position
of each iodine seed to be planned and calculated with
precision. In the second stage of the treatment applicator needles are inserted transperineally through the
template into the corresponding planned position
within the prostate. The iodine seeds are then inserted
and the applicator needles withdrawn. A typical
implant involves 60 to 120 seeds depending on prostatic volume and individual seed activity.
The aim of treatment is to deliver a dose of
145 Gy to the prostate plus margin and to limit
the maximum rectal dose to < 200 Gy and urethral
dose <220 Gy. The half life of 125I is 59.4 days,
with dose accumulation over the first few weeks and
then a dose fall off to very low levels (17).
HighDose Rate
Figure 16 A patient in the lithotomy position attached to the

microselectron unit while undergoing 192Ir high dose rate after
loading brachytherapy.
192
Ir Brachytherapy
192
A temporary Ir implant can be used in combination

with external beam radiotherapy to escalate the dose of
radiation to the prostate without significantly increasing
normal tissue morbidity (Figs. 15 and 16) (18). The
aim is improved local tumor control, and this treatment is mainly used for higher grade or locally
advanced tumors. The implant procedure is similar
to iodine seed therapy with a perineal template and
applicator needles. In this case, instead of iodine
seeds, which deliver the dose of radiation over
many days, a very high activity (high dose rate)
radioactive iridium source is used to deliver treatment
over a period of minutes. Following insertion of the
applicator needles a CT scan is done. The target volume and organs at risk are outlined on the planning
system.
For treatment, flexible tubes link the template and

applicator needles to a remote after-loading machine
containing the iridium source. The iridium is driven
under computer control into each successive applicator
needle and moved within the needle in small steps for
predetermined time periods (dwell times). This allows
the total dose of treatment to the prostate to be built up
over three to four fractionated treatments usually given
over two days. There is also an advantage in allowing
the dose to be optimized for the final resting position of
each applicator needle in relation to all the others. At
completion of treatment, the needles and template are
removed and the patient proceeds to external beam
radiotherapy after a 2-week break (Fig. 16).
CONCLUSION
Radiotherapy has an important role in both the radical
and palliative treatment of urological tumors. The
basic principles of physics and radiobiology are crucial to the optimization of treatment schedules and
delivery. The science of radiation treatment continues
to evolve with the aim of allowing higher and more
effective doses of radiation to be delivered with minimal toxicity. The full potential of radiotherapy either
alone or in combination with other therapies perhaps
has yet to be fully realized.
REFERENCES
Figure 15 AP film of needles implanted in the prostate for

high dose rate after loading brachytherapy.
192
Ir
1. Hall EJ. The physics and chemistry of radiation absorption.

In: Hall EJ, ed. Radiobiology for the Radiologist. Philadelphia: Lippincott Williams and Wilkins, 1994:114.
2. Withers HR. The four Rs of radiotherapy. Adv Radiat
Biol 1975; 5:241247.
3. Elkind MM, Sutton-Gilbert H, Moses WB, et al. Radiation
response of mammalian cells in culture. V. Temperature
dependence of the repair of X-ray damage in surviving
cells (aerobic and hypoxic). Radiat Res 1965; 25:359376.
4. Mottram JC. On the skin reactions to radium exposure and
their avoidance in radiotherapy: an experimental investigation. Br J Radiol 1924; 29:18.
5. Thoday JM, Reed J. Effect of oxygen on the frequency of
chromosome aberrations produced by X-rays. Nature 1947;
160:119.
6. Tomlinson RH, Gray LH. The histological structure of
some human lung cancers and the possible implications
for radiotherapy. Br J Cancer 1955; 9:59549.
7. Tannock IF. The relation between cell proliferation and the
vascular system in a transplanted mouse mammary
tumour. Br J Cancer 1968; 22:258273.
8. Chaplin DJ, Durand RE, Olive PL. Acute hypoxia in
tumours: implications for modifiers of radiation effects.
Int J Radiat Oncol Biol Phys 1986; 12:12791282.
9. Withers HR, Taylor JMG, Maciejewski B. Treatment volume and tissue tolerance Int J Radiat Oncol Biol Phys 1988;
14:751759.
10. Royal College of Radiologists. Guidelines for the Management of the Unscheduled Interruption or Prolongation of a
Radical Course of Radiotherapy. London, 1996.
11. CHHiP trial Conventional or Hypofractionated High dose
Intensity Modulated Radiotherapy for Prostate cancer.
Chief Investigator Professor David Dearnaley, Royal
Marsden Hospital. Sponsored by Institute of Cancer
Research.
12. Huddart RA, Hall EJ, James ND, et al. BC2001: a multicentre phase III randomized trial of standard versus
reduced volume radiotherapy for muscle invasive bladder
cancer. J Clin Oncol 2009; 27(suppl; abstr 5022):15s.
13. Fossa SD, Horwich A, Russell JM, et al. Medical Research
Council Testicular Tumour Working Party. Optimal planning target volume for stage I testicular seminoma; an
MRC randomised trial J Clin Oncol 1999; 17:11461154.
427
14. International Commission on Radiation Units and Measurements. Prescribing, recording and reporting photon
beam therapy. ICRU 50. Washington DC: International
Commission on Radiation Units and Measurements, 1993.
15. Dearnaley DP, Khoo VS, Norman AR, et al. Comparison of
radiation side-effects of conformal and conventional radiotherapy in prostate cancer: a randomised trial. Lancet 1999;
353:267272.
16. Holm HH, Juul N, Pedersen JF. Transperineal 125-iodine
seed implantation in protatic cancer guided by transrectal
ultrasonography. J Urol 1983; 10:283286.
17. Hoskin P, Coyle C. Radiotherapy in Practice: Brachytherapy: Oxford: Oxford University Press, 2005:107122
18. Mate TP, Gottesman JE, Hatton J, et al. High dose rate after
loading iridium 192 prostate brachytherapy: feasibility
report. Int J Radiat Oncol Biol Phys 1998; 41:525533.
FURTHER READING
Radiation physics:
Williams JR, Thwaite DI, eds. Radiotherapy Physics in Practice.
Oxford: Oxford University Press, 2000.
Khan FM. The Physics of Radiation Therapy. Linppincott
Williams and Wilkins, 2003.
Radiobiology:
Steel GG. Basic Clinical Radiobiology. London: Arnold, 1997.
Radiotherapy planning:
Dobbs J, Barrett A, Ash D. Practical Radiotherapy Planning.
London: Arnold, 1999.
28
Principles of Systemic Cancer Therapy
Christina Thirlwell and John Bridgewater
University College London Cancer Institute, University College London
Medical School, London, U.K.
Judith Cave
Southampton General Hospital, Southampton, U.K.
INTRODUCTION
Two-thirds of patients with cancer will develop metastatic disease during their illness. The most appropriate management for metastatic disease is systemic
treatment, and this is most commonly chemotherapy
or, more recently targeted, biological therapies. The
potential benefit of systemic therapy for cancer has
been proposed for sometimein 1875 Cutler and Bradford induced remission in a patient with chronic myeloid leukemia by treatment with arsenic (1). During the
First World War, soldiers who died of mustard gas
poisoning were noted to have aplastic bone marrow
suggesting a cytotoxic effect of mustard compounds (2).
In 1946, Goodman and colleagues (3) successfully demonstrated that the cytotoxic effects of nitrogen mustard
could treat a mouse transplanted with lymphosarcoma.
The first human cancer to be cured by chemotherapy
was choriocarcinoma, which has been cured with the
drug methotrexate since the 1950s.
In this chapter the pharmacological, molecular,
and biological basis of systemic anticancer treatment
will be discussed, as well as some of the practical
issues surrounding the administration of chemotherapy. Recent research has concentrated on improving
response rates by the development of new molecularly targeted drugs and better supportive care.
CYTOKINETICS
The study of cytokinetics is central to oncological
practice because of the observation that cells that are
dividing more rapidly are usually more sensitive to
chemotherapy (4). Choriocarcinomas and teratomas
double in cell number in less than one month and are
cured in more than 80% of cases, but colonic carcinomas double in over three months and are often resistant to chemotherapy (57). The relationship between
cell doubling time and prognosis is not simple but for
many types of tumor markers of high cell proliferation
correlate with poor prognosis.
Tumor Growth
The relationship between cell cycle time and tumor
doubling time is complex (Fig. 1). A proportion of cells
will be in G0 or resting phase, either because they have

differentiated into nondividing cells or because they
are necrotic or apoptotic or because they are resting.
Doubling time is therefore influenced by the growth
fraction, the cells lost, and the cell cycle time.
Tumor growth has been modeled using tissue
cultures and animals. The simplest model of tumor
growth is exponential cell growth, which assumes that
tumor doubling time is constant and therefore tumor
size will increase exponentially with time (Fig. 2). An
alternative is Gompertzian growth. In this model the
growth fraction decreases as the tumor size increases,
taking into account the effect of increased tumor size
on blood supply, nutrients, and physical constraints.
A Gompertzian growth curve is sigmoid in shape (8)
and is a better reflection of the clinical scenario.
Cell Killing
There are numerous models of cell killing by treatment,
and these are relevant to therapeutics. The first model
is fractional cell kill, also known as the Skipper
SchabelWilcox model (9). Assuming that all cells in a
tumor are equally sensitive to chemotherapy and that
the tumor is growing exponentially, a given dose of a
given drug will kill a constant fraction of the cells. If the
treatment is repeated, or if another drug is given alongside the first drug, then the two fractional cell kills will
be multiplied (Fig. 3). Adjuvant treatment (discussed
later) of micrometastases after surgical removal of primary tumors is based on this hypothesisthe less the
tumor load at the start of chemotherapy the higher
the possibility of eradicating all the tumor and therefore curing a proportion of patients.
Unfortunately, it is not always true that adequate
doses of chemotherapy will eradicate all tumor cells.
The failure to completely eliminate even very small
tumors with chemotherapy is largely due to drug
resistance. If all cancers are clonal in origin (10), one
would expect uniform sensitivity to drugs. However,
as a tumor divides, spontaneous mutations occur and
some of the cells will develop drug resistance. Consequently, most chemotherapy regimes include combinations of drugs in an attempt to avoid selecting out
resistant cells in a heterogeneous tumor (Fig. 4).
Chapter 28: Principles of Systemic Cancer Therapy
Figure 1 The cell cycle. Cells cycle between synthesis of DNA

(S-phase) and mitosis or M-phase with variable gaps between
protein synthesis and cell division (Gap 1 or G1 and Gap 2 or
G2). Withdrawal into resting phase (G0) can be reversible or
permanent. Progression around the cell cycle is dependent on
cyclins and cyclin-dependent kinases.
429
Figure 3 Cell kill.
is primarily hand-foot syndrome. When combination

regimens are being designed, drugs with different
mechanisms of action, resistance, and dose-limiting
toxicities should be combined. All drugs in a regime
should be active as single agents, and interactions
should be considered.
Cytotoxic drugs have various mechanisms of
action, and some of these are listed in Table 1. Some
drugs act only on cells that are cycling and may even
be specific to cells in a particular phase of the cell
cycle. For example, microtubule poisons such as taxanes act during mitosis and cause metaphase arrest,
but alkylating agents are cell cycle independent. Drug
action is not always confined to one class; for example,
mitomycin C is primarily an antitumor antibiotic, but
it can also alkylate DNA.
Figure 2 Exponential tumor growth.
DRUG RESISTANCE
PHARMACOLOGY
Scheduling and combining drugs correctly is crucial to
the success of chemotherapy regimes. For example,
some drugs work better when intermittently dosed
than when given as continuous infusions (1215).
Intermittent dosing allows time for normal tissue
recovery between doses, it is therefore both feasible
and logistically sensible. In general, drugs with a short
half-life (T1/2) are more effective when given as an
infusion to maintain steady state concentration, such
as 5-fluorouracil (5-FU), which has a short half-life of
seven minutes. In contrast, drugs with a longer T1/2
such as cisplatin (T1/2 7 hours) are given as a shorter
infusion. Toxicity also changes with scheduling. When
5-FU is given as a bolus, its toxicity is predominantly
mucositis and neutropenia, whereas as an infusion it
Tumor cells may be resistant to drugs for reasons

relating to the tumor, the drug, or the host. Some
parts of a tumor may have such a poor blood supply
that adequate doses of drug cannot reach the cells;
nonreplicating cells may escape the cytotoxic effects of
some drugs. In addition, there are sanctuary sites in
the body where cytotoxic drugs cannot penetrate,
classically the central nervous system and the testes.
Individual patient characteristics can also cause tumor
resistance (16); for example, if drugs are metabolized
very efficiently or not absorbed adequately, then the
dose reaching the tumor will not be effective. Inability
of the patients to tolerate sufficient doses of drugs
because of toxicity may also lead to ineffective dosage
and lack of efficacy.
True resistance to adequate concentrations of
drug can be either intrinsic or acquired. This often
manifests clinically with disease progression while
undergoing treatment.
430
Thirlwell et al.
Figure 4 (A) Selection of a resistant clone by single agent cyclophosphamide chemotherapy. (B) Use of combination chemotherapy to
overcome drug resistance. Source: Part A from Ref. 11.
Table 1 Mechanisms of Action of Chemotherapy

Class of drug
Examples
Target
Mechanism of action
Alkylating agents
Cyclophosphamide, thiotepa,
carmustine, cisplatin
Methotrexate, cytarabine,
flourouracil, fludarabine
DNA
Vinca alkaloids
Taxanes
Irinotecan/topotecan
Microtubules
Etoposide
Topoisomerase II
Doxorubicin (and other

anthracyclins)
Bleomycin
Mitomycin
DNA
Create DNA cross-links that cannot be

repaired
Resemble normal substrates for RNA and DNA
synthesis. Protein synthesis is aborted
leading to cell death
Disrupt/stabilize microtubules leading to
metaphase arrest
Binds to topoisomerase IDNA cleavable
complex and prevents re-ligation after
cleavage
Produces double strand breaks in DNA an
prevents entry of cells into mitosis
Intercalation into DNA causes untwisting of DNA
helix and strand breaks
Single- and double-strand DNA breaks
Cross-links DNA preventing function
Antimetabolites
Mitotic inhibitors
Topoisomerase
drugs
Antibiotics
Antifolates, purine, and

pyrimidine analogues
Topoisomerase I
DNA
DNA
Multidrug Resistance
Once a tumor is resistant to one drug, it is often also
resistant to many other drugs. This discovery has led
to the concept of multidrug resistance, the best
studied mechanism of which is P-glycoprotein overexpression. P-glycoprotein is a cell membrane glycoprotein pump coded for by the multidrug resistance
(MDR-1) gene, which removes drugs from the cell
(17). It is a naturally occurring glycoprotein that forms
part of secretory epithelial structures similar to biliary
canaliculi. Increased expression of MDR-1 (18) occurs
when cells are cultured in the presence of cytotoxic
drugs. MDR1a knockout mice, who cannot produce
P-glycoprotein, have tumors that do not develop drug
resistance. Similarly P-glycoprotein overexpression in
vivo correlates with reduced responsiveness to drugs
and reduced survival in some series (19).
It is possible to block P-glycoprotein and prevent

the transmembrane protein from pumping drugs out
of cells. Drugs available for this purpose include
verapamil, quinidine, and cylcosporin A. Clinical trials using these drugs have demonstrated some success in altering pharmacokinetics (20), but in general
clinical efficacy has not improved survival. It is likely
that overexpression of P-glycoprotein is only one of
many mechanisms involved in multidrug resistance
(2123).
Reduced apoptosis of DNA damaged cells may
be an important mechanism of drug resistance. Apoptosis in tumor cells is under the control of a complex
pathway, which includes p53 and the Bcl-2 family of
proteins (24). Cells with mutant p53 are more likely to
be drug resistant than those with wild-type p53
(2527), and there is interest in restoring wild-type
p53 to cells in order to stimulate apoptosis after

chemotherapy. Overexpression of the antiapoptotic
protein Bcl-2 also contributes to chemoresistance,
and modulation of Bcl-2 function may increase chemosensitivity (28). Bcl-2 antisense oligonucleotide
therapy has also been tested in patients with nonHodgkins Lymphoma (29).
Resistance to Specific Drugs

Resistance mechanisms can be specific to a particular
drug, a selection of these are outlined in Table 2. For
example, anthracyclines and epipodophyllotoxins stabilize the cleavable complex formed by topoisomerase
II and DNA. Topoisomerase II expression can be
upregulated or topoisomerase II can mutate and
change its intracellular location to avoid this effect
(30,31). These mechanisms collectively are known as
Topo IIrelated drug resistance.
TOXICITY
Toxicities are generally divided into early and late,
and while the majority are early toxicities, such as hair
loss, which are entirely reversible, some late toxicities
are not. The toxicity of a treatment often defines the
doses at which a drug can be given, and therefore can
be a key factor in limiting efficacy. Management of
toxicity is clearly vital to the optimal delivery of
chemotherapy.
Early Toxicity
Because stem cells in the bone marrow are dividing
rapidly, all chemotherapy drugs have the potential to
cause temporary bone marrow suppression. Marrow
431
toxicity is very often the dose-limiting factor for

chemotherapeutic agents, and both high-dose chemotherapy and prolonged chemotherapy can cause longterm marrow dysfunction.
Most chemotherapy regimes are given once
every 3 weeks, and in general, bone marrow stem
cells will recover within 10 days to 3 weeks of the dose
being given. Because the life span of a circulating
white blood cell is 4 to 5 days, the effects of bone
marrow suppression on the peripheral blood are seen
4 to 12 days after the dose is given (the nadir or low
point). Platelet nadirs are also drug specific, but as the
lifespan of platelets is longer (910 days), they tend to
occur later in the chemotherapy cycle. Red blood cells
survive in the peripheral circulation for about three
months, and so anemia is usually only seen with
prolonged courses of chemotherapy, primarily with
platinum-based compounds.
During periods of neutropenia, patients are
prone to bacterial infections (32). Many studies have
shown that broad-spectrum antibiotics covering aerobic gram-negative bacilli, especially Pseudomonas aeruginosa, should be introduced at the first sign of fever.
Treatment should not be delayed until physical signs
of infection are present or an organism is identified
(33). The use of prophylactic oral quinolones has been
shown to reduce the incidence of infections (34). Injection of recombinant granulocyte colony-stimulating
factors (G-CSF) will reduce the duration and level of
the neutropenia and the risk of developing a fever
while neutropenic. To date, studies have failed to
prove a survival advantage or reduction in significant
infection rates for the use of G-CSF (35).
Other early side effects of chemotherapy are
shown in Table 3.
Cytotoxic drugs are all potentially teratogenic,
especially if given in the first trimester. Folate
Table 2 Mechanisms of Resistance to Chemotherapy

Drug class
Examples
Method of resistance
Topoisomerase I inhibitors
Spindle poisons
Decreased expression of topoisomerase I

Reduced binding to microtubules
Platinum
Alkylating agents
Irinotecan
Vinca alkaloids
Taxanes
Cisplatin
Cyclophosphamide
Antimetabolites
Methotrexate
Increased DNA repair

Drug inactivation by aldehyde dehydrogenase
Increased DNA repair
Decreased reduced folate carrier expression
Increased production of dihydrofolate reductase
Increased thymidylate synthase levels
5-Fluorouracil
Table 3 Early Toxicities of Chemotherapy

Toxicity
Drugs
Comments
Alopecia
Anthracyclines and taxanes
Mouth ulcers
Diarrhea
Constipation
Nausea
5-Flourouracil
Capecitabine
Vinca alkaloids
Most
Starts 23 wk after chemotherapy initiated. Reversible, usually starts

growing back within 12 mo of end of treatment
Can be exacerbated by fungal or viral infections
Often associated with ulceration elsewhere in the GI tract
Vinca alkaloids can cause gut neuropathy
Chemotherapy drugs can be directly emetogenic, but constipation and
GI ulceration can exacerbate this. Usually well controlled with
serotonin subtype 3 receptor blockers
432
Thirlwell et al.
antagonists such as methotrexate have the most consistently poor record (36). Observational studies of
patients given chemotherapy during pregnancy reveal
that chemotherapy can be administered relatively
safely in the second and third trimester, but it is
possible that miscarriage, intrauterine growth retardation, and premature labor may be increased (37).
The incidence of long-term side effects among adults
who have received chemotherapy while in utero are
not known.
Late Toxicity
Male and female fertility are commonly reduced after
chemotherapy (38), and there is a substantial literature
on the effects of individual drugs (39). In the male,
sperm production takes place constantly, and chemotherapy reduces or stops production by damaging the
germinal epithelium. Azoospermia is therefore not
always associated with diminished or absent hormone
production. During intensive treatment, 96% of men
will become azoospermic (40). Depending on the
regime, a proportion of them will regain fertility
after chemotherapy. In women, as ovarian germ cells
are present at birth, the mechanism of infertility is
different. Estrogen levels are depleted by treatment,
and an early menopause can be induced. The extent of
the toxicity is related to the underlying diagnosis,
dose and type of drugs used, and age of patient at
time of treatment (39).
Because chemotherapeutic drugs work by damaging DNA, they carry a risk of latent carcinogenesis.
Alkylating agents are associated with a significant risk
of myelodysplasia and acute myeloid leukemia (41),
and after combination chemotherapy, the risk of many
solid malignancies is increased: After treatment for
Hodgkins disease, the relative risk of any cancer is 6.4
times population risk, and the relative risk of AML is
144 (42,43). The risk of second malignancy is highest
when chemotherapy and radiotherapy are used in
combination.
Drug-Specific Toxicities
All chemotherapy drugs have specific side effects in
addition to the generic effects described (Table 4).
Specific side effects can either be related to cumulative
dose (e.g., anthracyclines and cardiac toxicity) or be
idiosyncratic. The mechanisms of toxicity vary but are
not necessarily directly related to the cytotoxic action
of the drugs. As management of toxicity improves, for
example, with the use of peripheral blood stem cell
rescue, bone marrow suppression is less likely to be
the dose-limiting toxicity and some of the toxicities
mentioned in Table 4 may become dose limiting.
This list is not intended to be comprehensive, but to
indicate the wide range of possible side effects of
chemotherapy.
The toxicity of certain drugs can be reduced by
techniques to protect normal tissue. For example,
cyclophosphamide and ifosfamide produce acrolein,
which is an irritant metabolite excreted through the
Table 4 Drug-Specific Toxicities

Drug
Examples of toxicities
Anthracyclines
Cytosine arabinoside
Methotrexate
Cardiotoxicity
Cerebral/cerebellar dysfunction
Hepatitis
Nephrotoxicity
Neurotoxicity
Constipation
Palmar-plantar erythrodysesthesia
Neurotoxicity
Hypersensitivity or anaphylaxis
Pulmonary fibrosis
Skin pigmentation
Hemorrhagic cystitis due to excretion
of metabolites
Bladder tumors
Nephrotoxicity
Deafness
Vinca alkaloids
5-Flourouracil
Taxanes
Bleomycin
Cyclophosphamide
Cisplatin
bladder. Mesna is a thiol compound that acts as a

reducing agent, and when given with cyclophosphamide reacts with acrolein in the urinary tract and
helps prevent chemical cystitis (44).
MEASURING EFFICACY
Before prescribing a course of chemotherapy, a
method of assessing the success or failure of the
treatment should be identified. The method will obviously depend on the tumor type and site. For tumors
that produce markers (e.g., prostate-specific antigen
for prostate cancer and alpha-fetoprotein for germ cell
tumors), serum levels can be readily measured. Symptomatic improvement can also often indicate response,
for example, by a reduction in analgesic requirement
by the patient. However, even in the palliative setting,
symptomatic improvement alone is rarely considered
adequate to prove response, and for solid tumors,
demonstration of radiological response is the gold
standard.
The common practice in assessing radiological
response is to obtain an image of a measurable tumor
site before and after treatment. The World Health
Organization, the National Cancer Institute, and the
European Organisation for Research and Treatment of
Cancer have issued guidelines for tumor response
criteria (45).
Response to treatment is a reasonable surrogate
for the only true measure of treatment efficacy, overall
survival.
Minimal Residual Disease

At a complete radiological response (or after radical
surgery), there will be no evidence of disease as measured by any standard marker. Unfortunately, there
will often be residual viable tumor cells present in
these patients (Fig. 5). Currently, imaging does not
allow us to detect microscopic disease, but using realtime quantitative polymerase chain reactions (PCR),
433
Figure 5 Minimal residual disease. Source:

Modified from Ref. 9.
residual disease at the molecular level can be detected.

The absence of minimal residual disease (MRD) could
theoretically be used as an end point for radical therapy. In acute promyelocytic leukemia (APML), evidence of MRD in the bone marrow correlates well
with outcome (46). It is likely that the absence of
evidence of t15:17 in the bone marrow, as measured
by real-time PCR, will also correlate with long-term
survival after chemotherapy.
INTENSIFYING TREATMENT
In vitro evidence suggests that for some drugs such as
cisplatin, there is a clear relationship between dose of
chemotherapy and proportion of cells killed (16,47). In
potentially curable tumors, there has been a great deal
of interest in escalating treatment doses to improve
survival, but, as mentioned above, dose is commonly
limited by bone marrow suppression. Supportive care
has improved significantly in recent years, and since
the 1970s, it has been possible to perform peripheral
blood stem cell rescue (48,49). This is a procedure
whereby the patients stem cells are collected from
the peripheral blood and stored so that they can be
returned after a high dose of chemotherapy, thus
shortening the duration of neutropenia. The introduction of 5-HT3 antagonists has enabled nausea to be
controlled more effectively (enabling the majority of
chemotherapy regimens to be given as outpatient),
and for some drugs the use of colony-stimulating
factors alone is enough to significantly increase the
tolerated dose (50).
NEW ANTICANCER DRUGS

Understanding of the molecular biology of malignant
cells has increased dramatically in recent years. Therefore, there is scope for developing drugs that are
targeted at malignant cells (51,52). The principle is
that drugs are developed that target abnormal biology
specific to tumors, thus increasing efficacy and
reducing normal tissue damage. This section aims to

mention some of the new drugs that are either in use
in the clinic or are undergoing trials, to illustrate the
range of possible new therapeutic strategies becoming
available.
Immune Modulators
Immune modulation is an attractive anticancer therapeutic option. It has been noted that prolonged periods
of immunosuppression predispose to malignancies (53)
and also that spontaneous regression of metastatic
tumors (classically renal cell cancers) can occur (54).
What is more, regression of tumors can occasionally be
induced by deliberate inoculation with pus or bacterial
toxins (55). The immune system can be stimulated
nonspecifically or by generation of specific antitumor
immunity.
Cytokines
The immune system can be nonspecifically stimulated
with supraphysiological doses of naturally occurring
cytokines. Examples include tumor necrosis factor
(TNF), interferons, and interleukins. This method
has been shown to have some efficacy in the treatment
of renal cell carcinoma, melanoma, and chronic
leukemias (5658).
Antibody Therapy
In the 19th century, investigators attempted to treat
patients with the sera of animals that had been inoculated with human cancers (59), and since the discovery of the monoclonal antibody in 1975 (60), it has
been possible to produce antibodies to specific tumor
antigens.
The exact means by which monoclonal antibodies mediate cell killing is complex and not completely
understood. Nevertheless, significant advances
have been made with the recent introduction of
434
Thirlwell et al.
anti-c-erbB2 (trastuzumab) for breast cancer and antiCD20 (rituximab) for B-cell lymphoma (61). The
human epidermal growth factor receptor 2 (HER2neu)
antigen is overexpressed on the cell surface of 25% to
30% of breast cancers (62) and is associated with a
poor prognosis (63). Patients can be tested for the
presence of c-erbB2 on the tumor cell surface, and if
the antigen is found, anti c-erbB2 antibodies induce
responses in patients who have failed treatment with
conventional chemotherapy, or in whom conventional
chemotherapy is contraindicated (64,65). There is a
higher response rate to chemotherapy plus antic-erbB2 antibodies than to chemotherapy alone (66).
The side effect profile of monoclonal antibodies is very
favorable when compared with conventional chemotherapy, and antibodies to other cell surface markers are
under investigation. Unfortunately, resistance to monoclonal antibodies invariably develops, and discovering
the mechanism of this resistance is going to be vital in
terms of improving immune-based therapies.
Targeting Mediators of Malignancy

Receptors on the cell surface require intracellular signaling pathways to translate receptor ligand binding
into cell proliferation or other end points. These intracellular signaling pathways are potential targets for
anticancer drugs. For example, the epidermal growth
factor receptor (EGFR also known as HER-1) is overexpressed in many cancers and seems to be responsible for malignant behavior of tumor cells (6769). The
EGFR has an intracellular tyrosine kinase (TK) domain
that is activated by phosphorylation. TK activation
can be blocked by specially designed small molecules.
Over the past five years, TK inhibitors (TKIs) and
monoclonal antibodies have been introduced for treatment of several solid cancer types including renal,
colorectal cancer, squamous cell carcinoma of head
and neck, and breast and ovarian cancer. Currently,
there are monoclonal antibodies directed against the
extracellular domain of the EGFR receptor (cetuximab

and panitumumab) and also the intracellular TK
domain (erlotinib, lapatinib, and gefitinib).
A potential problem with blocking intracellular
signaling pathways is that the effect may be shortlived if the cell can bypass the point in the intracellular pathway cascade at which the drug is acting.
This can either occur by upregulation of alternative
pathways or by mutation of target proteins. Individuals whose tumors harbor k-RAS mutations do not
respond to EGFR inhibition as the downstream pathway is continuously switched on by the mutated
k-ras oncogene (70).
Anti-Angiogenesis Agents
The ability to metastasize is a defining feature of
malignant tissue, and metastasis cannot occur without
angiogenesis. Angiogenesis is under the control of
multiple cytokines, enzymes, and proteins (71). The
concept of angiogenesis as a therapeutic target was
suggested by Folkman in 1972 (72). Thalidomide is an
anti-angiogenesis drug currently in use, originally
marketed as a sedative but was withdrawn because
of teratogenic effects, primarily amelia and phocomelia. It was proposed that these effects were due to
degeneration of major limb arteries (73), and it was
subsequently found that thalidomide inhibits the
angiogenic cytokines, basic fibroblast growth factor
(bFGF) and vascular endothelial growth factor (VEGF)
(74,75). Clinical efficacy has been variable (76,77).
VEGF has been identified as a particularly
important angiogenic cytokine (78).
A significant impact on both the overall survival
and time to progression in renal and colorectal cancer
has been achieved by using VEGF inhibitors alongside
mTOR (hypoxia-driven rapamycin pathway) and RAF
inhibitors (79). Several TKIs now inhibit more than
one pathway (see Table 5 for an overview of molecularly targeted drugs). It is now accepted that VEGF
Table 5 Overview of Molecularly Targeted Therapies Used in Solid Tumors

Cancer type
Agent
Molecular targets
Trial phase
Breast
Trastuzumab (Herceptin)
Lapatinib
HER-2 mAb
III
Colorectal
Cetuximab
Panitumumab
Bevacizumab (Avastin)
Erlotinib
Bevacizumab (Avastin)
Sorafenib
EGFR TKI
EGFR mAb (IgG1)
EGFR mAb (IgG2 humanized)
VEGF
EGRF TKI (intracellular)
VEGF mAb
VEGFR2,3, PDGFR, FLT-3,
BRAF, CRAF
VEGF2, PDGFR, FLT-3, c-kit
mTOR
mTOR
VEGFR1, 2, PDGFR, c-kit
VEGFR13, PDGFR
EGFR mAb
II/III
II/III
II/III
II/III
II,III
II,III
II,III
II,III
II
II
II
II
II/III
Nonsmall cell lung cancer

Renal
Squamous cell carcinoma of

head and neck
Sunitinib
Temsirolimus
Everolimus
Axitinib
Pazopanib
Cetuximab
Abbreviations: mAb, monoclonal antibody; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor; BRAF, B-raf proto-oncogene
serine/threonine protein kinase; CRAF, C-raf proto-oncogene serine/threonine protein kinase; EGFR, epidermal growth factor receptor.
Source: Adapted from Ref. 80.
inhibition should be the first-line therapy in advanced

and metastatic renal carcinoma; outcome data are
awaited for the use of these therapies in the adjuvant
setting.
Toxicities and Scheduling of Targeted Therapy

Novel targeted therapies have resulted in novel toxicities. Common side effects of EGFR inhibition include
fatigue, facial acneiform rash, and diarrhea. VEGF inhibition can lead to hypertension, renal impairment, and
thromboembolic events. These toxicities are generally
managed symptomatically. In the majority of cases
these side effects are managed through treatment
breaks and dose reduction where necessary.
Scheduling is critical in the use of targeted therapies. The recommended scheduling of sunitinib in renal
cell carcinoma is four-weeks-on therapy followed by
two weeks off to give the body time to recover between
cycles. However, it is also possible to take daily sunitinib
at a lower dose without significant toxicity.
Mechanisms of Resistance
to EGFR and VEGF Inhibitors
Resistance to targeted agents is a significant clinical
problem. Preclinical studies have determined several
mechanisms of developing resistance to angiogenesis
inhibition (VEGF/mTOR pathways) namely, upregulation of bFGF, overexpression of matrix metalloproteinase 9, increased levels of stromal cellderived
factor-1a and hypoxia-induced factor-1a, and recruitment of bone marrow CD45 cells.
Studies in development of resistance to EGFR
inhibition have found that several processes are
involved, including activation of alternative TKIs
that bypass the EGFR pathway (c-MET and IGF-1R),
increased angiogenesis, activation of downstream
mediators (PTEN and k-RAS), and the presence of
EGFR mutations (81).
CONCLUSIONS
Conventional chemotherapy along with radiotherapy
has formed the mainstay of curative and palliative
treatment of solid malignancies in the past 40 years.
All conventional cytotoxic drugs damage normal tissue as well as having a therapeutic effect by killing
tumor cells. Combining different types and doses of
cytotoxic drugs has lead to significant improvements
in survival from some important cancers in the last
50 years.
Over the past five years, novel molecularly targeted agents have emerged through improved understanding of the molecular basis of many cancers.
Molecularly targeted agents are now in routine use
as single therapies or more commonly alongside chemotherapy and radiotherapy. The introduction of
molecularly targeted inhibition of the VEGF, RAS,
BRAF, and mTOR pathways has had a significant
impact on the time to progression and overall survival
in renal cancer.
435
It is expected that in the future, systemic treatment of cancer will include targeted therapies alongside chemotherapy and radiotherapy with associated
improval in survival rates and reduction in associated
toxicities. It will also be possible to personalize cancer therapy through the identification of individuals
who are likely to respond to certain therapies.
REFERENCES
1. Burchenal JH. The historical development of cancer
chemotherapy. Semin Oncol 1977; 4(2):135146.
2. Krumbhaar EB, Krumbhaar HD. The blood and bone
marrow in yellow gas (mustard gas) poisoning: changes
produced in bone marrow of fatal cases. J Med Res 1919;
40:497.
3. Goodman LS, Wintrobe MM, Dameshek W, et al. Nitrogen
mustard therapy: use of methyl-bis(beta-chloroethyl)amine
hydrochloride and tris(bets-chloroethyls)amine hydrochloride for Hodgkins disease, lymphosarcoma, leukaemia and certain allied and miscellaneous disorders. JAMA
1946; 132:126132.
4. Twentyman PR, Bleehen NM. Changes in sensitivity to
cytotoxic agents occurring during the life history of monolayer cultures of a mouse tumour cell line. Br J Cancer
1975; 31(4):417423.
5. Steel GG, ed. Growth Kinetics of Tumours. Oxford: Oxford
University Press, 1977.
6. Shackney SE, McCormack GW, Cuchural GJ Jr. Growth
rate patterns of solid tumors and their relation to responsiveness to therapy: an analytical review. Ann Intern Med
1978; 89(1):107121.
7. Modulation of fluorouracil by leucovorin in patients with
advanced colorectal cancer: evidence in terms of response
rate. Advanced Colorectal Cancer Meta-Analysis Project.
J Clin Oncol 1992; 10(6):896903.
8. Schabel FM Jr. The use of tumor growth kinetics in planning curative chemotherapy of advanced solid tumors.
Cancer Res 1969; 29(12):23842389.
9. Skipper HE. Biochemical, biological, pharmacologic, toxicologic, kinetic and clinical (subhuman and human) relationships. Cancer 1968; 21(4):600610.
10. Friedman JM, Fialkow PJ. Cell marker studies of human
tumorigenesis. Transplant Rev 1976; 28:1733.
11. Goldie JH, Coldman AJ. The genomic origin of drug resistance in neoplasms: implications for systemic therapy.
Cancer Res 1984; 44(9):36433653.
12. Selawry OS, Henanian J, Wolman IJ. New treatment schedule with improved survival in childhood leukaemia. Intermittent parenteral versus daily oral administration of
methotrexate for maintenance of induced remission.
Acute leukaemia group B. JAMA 1965; 194(1):7581.
13. Reiter A, Schrappe M, Ludwig WD, et al. Chemotherapy in
998 unselected childhood acute lymphoblastic leukemia
patients. Results and conclusions of the multicenter trial
ALL-BFM 86. Blood 1994; 84(9):31223133.
14. Devita VT Jr., Serpick AA, Carbone PP. Combination chemotherapy in the treatment of advanced Hodgkins disease. Ann Intern Med 1970; 73(6):881895.
15. Wolfrom C, Hartmann R, Fengler R, et al. Randomized
comparison of 36-hour intermediate-dose versus 4-hour
high-dose methotrexate infusions for remission induction
in relapsed childhood acute lymphoblastic leukemia. J Clin
Oncol 1993; 11(5):827833.
16. Teicher BA, Herman TS, Holden SA, et al. Tumor resistance to alkylating agents conferred by mechanisms operative only in vivo. Science 1990; 247(4949 pt 1):14571461.
436
Thirlwell et al.
17. Riordan JR, Deuchars K, Kartner N, et al. Amplification of

P-glycoprotein genes in multidrug-resistant mammalian
cell lines. Nature 1985; 316(6031):817819.
18. Gottesman MM, Hrycyna CA, Schoenlein PV, et al. Genetic
analysis of the multidrug transporter. Annu Rev Genet
1995; 29:607649.
19. Pogliani EM, Belotti D, Rivolta GF, et al. Anthracycline
drugs and MDR expression in human leukemia. Cytotechnology 1996; 19(3):229235.
20. Belpomme D, Gauthier S, Pujade-Lauraine E, et al. Verapamil increases the survival of patients with anthracyclineresistant metastatic breast carcinoma. Ann Oncol 2000; 11
(11):14711476.
21. Solary E, Witz B, Caillot D, et al. Combination of quinine as
a potential reversing agent with mitoxantrone and cytarabine for the treatment of acute leukemias: a randomized
multicenter study. Blood 1996; 88(4):11981205.
22. Wilson WH, Bates SE, Fojo A, et al. Controlled trial of
dexverapamil, a modulator of multidrug resistance, in
lymphomas refractory to EPOCH chemotherapy. J Clin
Oncol 1995; 13(8):19952004.
23. Wishart GC, Bissett D, Paul J, et al. Quinidine as a resistance modulator of epirubicin in advanced breast cancer:
mature results of a placebo-controlled randomized trial. J
Clin Oncol 1994; 12(9):17711777.
24. Reed JC. Bcl-2 family proteins. Oncogene 1998; 17
(25):32253236.
25. Cho Y, Gorina S, Jeffrey PD, et al. Crystal structure of a p53
tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 1994; 265(5170):346355.
26. Chin KV, Ueda K, Pastan I, et al. Modulation of activity of
the promoter of the human MDR1 gene by Ras and p53.
Science 1992; 255(5043):459462.
27. Lowe SW, Ruley HE, Jacks T, et al. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell
1993; 74(6):957967.
28. Konopleva M, Zhao S, Hu W, et al. The anti-apoptotic
genes Bcl-X(L) and Bcl-2 are over-expressed and contribute
to chemoresistance of non-proliferating leukaemic CD34
cells. Br J Haematol 2002; 118(2):521534.
29. Waters JS, Webb A, Cunningham D, et al. Phase I clinical
and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkins lymphoma.
J Clin Oncol 2000; 18(9):18121823.
30. Pommier Y, Leteurtre F, Fesen MR, et al. Cellular determinants of sensitivity and resistance to DNA topoisomerase
inhibitors. Cancer Invest 1994; 12(5):530542.
31. Beck WT, Danks MK, Wolverton JS, et al. Resistance of
mammalian tumor cells to inhibitors of DNA topoisomerase II. Adv Pharmacol 1994; 29B:145169.
32. Bodey GP, Buckley M, Sathe YS, et al. Quantitative relationships between circulating leukocytes and infection in patients
with acute leukemia. Ann Intern Med 1966; 64(2):328340.
33. Schimpff S, Satterlee W, Young VM, et al. Empiric therapy with
carbenicillin and gentamicin for febrile patients with cancer
and granulocytopenia. N Engl J Med 1971; 284(19):10611065.
34. Engels EA, Lau J, Barza M. Efficacy of quinolone prophylaxis in neutropenic cancer patients: a meta-analysis. J Clin
Oncol 1998; 16(3):11791187.
35. Messori A, Trippoli S, Tendi E. G-CSF for the prophylaxis
of neutropenic fever in patients with small cell lung cancer
receiving myelosuppressive antineoplastic chemotherapy:
meta-analysis and pharmacoeconomic evaluation. J Clin
Pharm Ther 1996; 21(2):5763.
36. Doll DC, Ringenberg QS, Yarbro JW. Antineoplastic agents
and pregnancy. Semin Oncol 1989; 16(5):337346.
37. Berry DL, Theriault RL, Holmes FA, et al. Management of
breast cancer during pregnancy using a standardized protocol. J Clin Oncol 1999; 17(3):855861.
38. Chapman RM, Sutcliffe S. The effects of chemotherapy

and radiotherapy on fertility and their prevention. In:
Whitehouse JMA, Willams CJW, ed. Recent Advances in
Clinical Oncology. Edinburgh: Churchill Livingston,
1986:239251.
39. Sieber SM, Adamson RH. Toxicity of antineoplastic agents
in man, chromosomal aberrations antifertility effects, congenital malformations, and carcinogenic potential. Adv
Cancer Res 1975; 22:57155.
40. Drasga RE, Einhorn LH, Williams SD, et al. Fertility after
chemotherapy for testicular cancer. J Clin Oncol 1983; 1
(3):179183.
41. Reimer RR, Hoover R, Fraumeni JF Jr., et al. Acute leukemia after alkylating-agent therapy of ovarian cancer. N
Engl J Med 1977; 297(4):177181.
42. Tucker MA. Solid second cancers following Hodgkins
disease. Hematol Oncol Clin North Am 1993; 7(2):389400.
43. Henry-Amar M, Dietrich PY. Acute leukemia after the
treatment of Hodgkins disease. Hematol Oncol Clin
North Am 1993; 7(2):369387.
44. Brock N, Pohl J, Stekar J. Detoxification of urotoxic oxazaphosphorines by sulfhydryl compounds. J Cancer Res Clin
Oncol 1981; 100(3):311320.
45. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors.
European Organization for Research and Treatment of
Cancer, National Cancer Institute of the United States,
National Cancer Institute of Canada. J Natl Cancer Inst
2000; 92(3):205216.
46. Venditti A, Buccisano F, Del Poeta G, et al. Level of minimal residual disease after consolidation therapy predicts
outcome in acute myeloid leukemia. Blood 2000; 96
(12):39483952.
47. Skipper HE, Schabel FM. Quantitative and cytokinetic
studies in experimental tumour systems. In: Holland JF,
Frei FE, ed. Cancer Medicine. Philadelphia: Lea and
Febiger, 1988:663684.
48. Gorin NC, Najman A, Salmon C, et al. High dose combination chemotherapy (TACC) with and without autologous bone marrow transplantation for the treatment of
acute leukaemia and other malignant diseases: kinetics of
recovery of haemopoiesis. A preliminary study of 12 cases.
Eur J Cancer 1979; 15(9):11131119.
49. Appelbaum FR, Deisseroth AB, Graw RG Jr., et al. Prolonged complete remission following high dose chemotherapy of Burkitts lymphoma in relapse. Cancer 1978;
41(3):10591063.
50. Gianni AM, Bregni M, Siena S, et al. Granulocyte-macrophage colony-stimulating factor or granulocyte colonystimulating factor infusion makes high-dose etoposide a
safe outpatient regimen that is effective in lymphoma and
myeloma patients. J Clin Oncol 1992; 10(12):19551962.
51. Garrett MD, Workman P. Discovering novel chemotherapeutic drugs for the third millennium. Eur J Cancer 1999;
35(14):20102030.
52. Gibbs JB. Anticancer drug targets: growth factors and
growth factor signaling. J Clin Invest 2000; 105(1):913.
53. Penn I. Cancers complicating organ transplantation.
N Engl J Med 1990; 323(25):17671769.
54. Oliver RT, Nethersell AB, Bottomley JM. Unexplained
spontaneous regression and alpha-interferon as treatment
for metastatic renal carcinoma. Br J Urol 1989; 63(2):
128131.
55. Coley-Nauts HC, Fowler GA, Bogatko RN. A review of the
influence of bacterial infection and of bacterial products
(Coleys toxins) on malignant tumours in man. Acta Med
Scand Supp 1953; 274:2997.
56. Rosenberg SA, Yang JC, Topalian SL, et al. Treatment of
283 consecutive patients with metastatic melanoma or
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
renal cell cancer using high-dose bolus interleukin 2.

JAMA 1994; 271(12):907913.
Bukowski RM, Goodman P, Crawford ED, et al. Phase II
trial of high-dose intermittent interleukin-2 in metastatic
renal cell carcinoma: a Southwest Oncology Group study.
J Natl Cancer Inst 1990; 82(2):143146.
Quesada JR, Reuben J, Manning JT, et al. Alpha interferon
for induction of remission in hairy-cell leukemia. N Engl J
Med 1984; 310(1):1518.
Hericourt J, Richet C. De la serotherapie dons la traitment
du cancer. Comptes Rendues du lAcademie des Sciences
1895; 121:567569.
Kohler G, Milstein C. Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature 1975;
256(5517):495497.
Coiffier B, et al. CHOP chemotherapy plus rituximab
compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346(4):
235242.
Hynes NE, Stern DF. The biology of erbB-2/neu/HER-2
and its role in cancer. Biochim Biophys Acta 1994; 1198
(23):165184.
Slamon DJ, Godolphin W, Jones LA, et al. Studies of the
HER-2/neu proto-oncogene in human breast and ovarian
cancer. Science 1989; 244(4905):707712.
Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational
study of the efficacy and safety of humanized anti-HER2
monoclonal antibody in women who have HER2overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin
Oncol 1999; 17(9):26392648.
Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and
safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer.
J Clin Oncol 2002; 20(3):719726.
Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for
metastatic breast cancer that overexpresses HER2. N Engl
J Med 2001; 344(11):783792.
Aaronson SA. Growth factors and cancer. Science 1991; 254
(5035):11461153.
Salomon DS, Brandt R, Ciardiello F, et al. Epidermal
growth factor-related peptides and their receptors in
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
437
human malignancies. Crit Rev Oncol Hematol 1995; 19

(3):183232.
Tysnes BB, Haugland HK, Bjerkvig R. Epidermal growth
factor and laminin receptors contribute to migratory and
invasive properties of gliomas. Invasion Metastasis 1997;
17(5):270280.
Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras
mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008; 359(17):17571765.
Risau W. Mechanisms of angiogenesis. Nature 1997; 386
(6626):671674.
Folkman J. Anti-angiogenesis: new concept for therapy of
solid tumors. Ann Surg 1972; 175(3):409416.
Parman T, Wiley MJ, Wells PG. Free radical-mediated
oxidative DNA damage in the mechanism of thalidomide
teratogenicity. Nat Med 1999; 5(5):582585.
DAmato RJ, Loughnan MS, Flynn E, et al. Thalidomide is
an inhibitor of angiogenesis. Proc Natl Acad Sci U S A 1994;
91(9):40824085.
Kruse FE, Joussen AM, Rohrschneider K, et al. Thalidomide inhibits corneal angiogenesis induced by vascular
endothelial growth factor. Graefes Arch Clin Exp Ophthalmol 1998; 236(6):461466.
Baidas SM, Winer EP, Fleming GF, et al. Phase II evaluation of thalidomide in patients with metastatic breast cancer. J Clin Oncol 2000; 18(14):27102717.
Figg WD, Dahut W, Duray P, et al. A randomized phase II
trial of thalidomide, an angiogenesis inhibitor, in patients
with androgen-independent prostate cancer. Clin Cancer
Res 2001; 7(7):18881893.
Ferrara N. Vascular endothelial growth factor: molecular
and biological aspects. Curr Top Microbiol Immunol 1999;
237:130.
Motzer RJ, Basch E. Targeted drugs for metastatic renal cell
carcinoma. Lancet 2007; 370(9605):20712073.
Chowdhury S, Larkin JM, Gore ME. Recent advances in the
treatment of renal cell carcinoma and the role of targeted
therapies. Eur J Cancer 2008; 44(15):21522161.
Dempke WC, Heinemann V. Resistance to EGF-R (erbB-1)
and VEGF-R modulating agents. Eur J Cancer 2009; 45(7):
11171128.
29
Embryology
David F. M. Thomas
Department of Paediatric Urology, St Jamess University Hospital, Leeds, U.K.
INTRODUCTION
In recent years Embryology has undergone a remarkable transformation from being a largely descriptive
science to its current status at the forefront of innovative therapeutic research. Many of the advances in this
field, including the cloning of mammalian embryos,
can be traced back to early work on assisted conception and in vitro fertilization (IVF), but perhaps the
greatest impetus for embryo research came from the
isolation and culture of pluripotent embryonic stem
cells from early embryonic tissue (1). The ambitious
and costly embryo and stem cell research programs
that are now being undertaken across the world are
underpinned by the expectation that this work will
ultimately pave the way to the development of a wide
range of clinical applications, including cell transplantation, tissue regeneration, and tissue engineering.
More controversially, research is also underway to
explore the feasibility of creating artificial gametes
using genetic material derived from somatic cells. The
creation of humananimal hybrid embryos is being
explored as a potential source of (human) pluripotent
embryonic stem cells to circumvent the problems created by the scarcity of donated human oocytes. In some
controversial areas the pace of scientific advance threatens to outstrip the ability of legislators to adapt regulatory frameworks in a way that is acceptable to the
scientific community and society as a whole. In the
United Kingdom, responsibility for regulating embryo
and stem cell research lies with the Human Fertilisation
and Embryology Authority (HFEA).
A more detailed consideration of the current
status of the embryo and stem cell research would
be outside the scope of this chapter and, in any event,
would almost certainly be rapidly overtaken by further advances. However, this chapter will encompass
some of the relevant advances in less controversial
areas of developmental biology that are shedding new
light on the genetic and environmental basis of congenital malformations.
The advent of research methods such as polymerase chain reaction and fluorescent in situ hybridization have greatly facilitated the study of the genetic
mechanisms regulating the complex and tightly
ordered anatomical sequence that characterizes normal embryological development. As well as mapping
and sequencing genes, research methodology is identifying many of the gene products responsible for
implementing the genetic program encoded on
DNA at a cellular and molecular level. Moreover,
the function of specific genes is being extensively
studied in experimentally induced null or knockout
gene deletions in experimental rodents. Relevant
examples will be cited throughout the chapter.
With the benefit of information being derived
from new methodology, it is becoming increasingly
apparent that the traditional account of the embryological development of the urinary tract and other systems
was oversimplified. For example, on the basis of this
interpretation of sequential serial sections at different
stages in development, concepts such as the division of
the foregut and partition of the cloaca by the descent
of the urorectal septum and lateral ingrowth are now
thought to be a conceptual artifact. There is growing
evidence that many of the structural changes observed
in the developing embryo reflect a process of dynamic
remodeling in situ, which is a consequence of simultaneous growth, differentiation, and apoptosis, rather
than three-dimensional interactions between different
structures. Nevertheless, for the clinician, as opposed to
the embryologist, the conventional three-dimensional
descriptive accounts of embryology simplify understanding of the development of the genitourinary
tract. In turn, these descriptive models can be invoked
to explain many of the congenital abnormalities
encountered in clinical practice. For this reason, this
chapter provides a largely conventional, practical, and
clinically orientated account of developmental anatomy
of the genitourinary tract, accompanied, where appropriate, by consideration of genetics and relevant
aspects of developmental biology.
FERTILIZATION AND EARLY DEVELOPMENT

OF THE HUMAN EMBRYO
Human gestation spans the period from fertilization
to birth, which averages 38 weeks. Obstetricians conventionally subdivide pregnancy into three 3-month
trimesters. Embryogenesis, the formation of organs
and systems, occurs principally between the 3rd and
10th weeks. Subsequent development throughout the
Chapter 29: Embryology
439
fertilized zygote has undergone a series of cell divisions termed cleavage. By the fourth day, sequential
cleavage has created a 32-cell embryo, classically likened to a mulberry (hence the Latin-derived term
morula). Further cell division is accompanied by
structural differentiation and the formation of a
sphere-like blastocyst. It is at this stage in development
that the embryo is implanted into the uterine endometrium, six days after fertilization.
Figure 1 Key stages in the five to six days from fertilization to

implantation.
rest of fetal life is characterized by the processes of

differentiation, branching, maturation, and growth.
Fertilization is defined by fusion of the nuclear
material of the fertilizing spermatozoon and the oocyte.
The process of gametogenesis, whereby spermatozoa
and oocytes develop from their germ cell precursors, is
considered hereafter. In males, spermatogenesis is a
continuing process initiated at puberty whereas the initial phase of female gametogenesis occurs in fetal life,
and primary oocytes remain dormant in the prophase of
the first meiotic division until the onset of puberty.
During each ovulatory cycle, under the influence
of follicle-stimulating hormone, a small number of primary oocytes resume meiotic activity, but, of these, only
one usually progresses to maturity as a Graafian follicle.
At the mid-point of the menstrual cycle, the primary
oocyte destined for ovulation transforms into a secondary oocyte by proceeding into the remaining phases of
the long-arrested first meiotic division. Protected by the
zona pellucida, the secondary oocyte is extruded from
the surface of the ovary, from where it is drawn into the
reproductive tract by the fimbriae of the fallopian tube.
From an ejaculate totaling perhaps 40 to 100 million
spermatozoa, only a few hundred are destined to complete the journey up the female reproductive canal to
come into potential contact with the ovulated oocyte.
Penetration by the fertilizing spermatozoon of
the zona pellucida surrounding the secondary oocyte
triggers the second meiotic division, which results in
the formation of the definitive oocyte and an aggregate of nonfunctional DNA termed the polar body.
The normal human karyotype comprises a total of
23 pairs of chromosomes, that is, 22 pairs of autosomes
and one pair of sex chromosomes, either XX (female) or
XY (male). As a result of the two meiotic divisions, each
gamete (the spermatozoon or oocyte) carries only half
the normal complement of chromosomes, that is, 22
unpaired autosomes and either an X or Y sex chromosome. Fusion of the haploid nuclei of the two gametes
at the time of fertilization imparts diploid status (i.e., 22
pairs of autosomes plus 2 sex chromosomes) to the
nucleus of the fertilized zygote. From the time of fertilization, the journey down the fallopian tube to the
site of implantation in the primed uterine endometrium
takes five to six days (Fig. 1). During this time, the
CHROMOSOMAL ABNORMALITIES: CLINICAL

CONSIDERATIONS
A detailed account of the pathogenesis and clinical
manifestations of chromosomal abnormalities arising
during gametogenesis and cleavage is outside the
scope of this chapter. Major chromosomal abnormalities are common, but mostly result in the death of the
embryo and spontaneous abortion at an early stage in
pregnancy. The most serious chromosomal abnormalities consistent with survival to term are the trisomies,
notably trisomy 21 (Downs syndrome.) A trisomy
state occurs when an additional chromosome or portion of a chromosome becomes incorporated into the
nucleus of a gamete by nondisjunction or translocation. Nondisjunction refers to the faulty separation of
a pair of chromosomes during meiotic or mitotic cell
division. In translocation, paired chromosomes separate completely, but one of the pair, or a fragment
thereof, becomes inadvertently attached to another
unrelated chromosome and is thus retained within
the haploid nucleus. Trisomic states are also associated with the formation of a gamete that is lacking a
copy of the affected chromosome, and any zygote
arising from fertilization will therefore have only
one copy of this chromosometermed monosomy.
Although complete monosomic states involving autosomes are uniformly lethal, some partial monosomies
created by deletion of part of a chromosome are compatible with survival. Some have been identified in
association with specific syndromes; for example,
deletion of a particular portion of chromosome11 has
been implicated in the etiology of some cases of Wilms
tumor. Apart from translocation and nondisjunction,
other structural defects involving identifiable segments of chromosomes include deletion, inversion,
duplication, and substitution.
Nondisjunction of the X and Y sex chromosomes
during gametogenesis accounts for a number of genetically determined syndromes, notably Klinefelters syndrome (47 XXY) and Turners syndrome (45X).
Complete monosomic forms of Turners syndrome
(45X) account for approximately 50% of cases, while
mosaicism (45X/46XX) is present in approximately
30% of cases and a structural deletion on one of the X
chromosomes accounts for the remaining 20% of cases.
Unsurprisingly, the gross genetic imbalance created by the presence of additional replicated DNA
within the zygote nucleus results in profound disturbances of embryogenesis, affecting a number of
systems including the genitourinary tract. The incidence of coexistent renal anomalies ranges from
440
Thomas
Table 1 Chromosome Defects Associated with Urinary Tract Anomalies

Chromosome defect or syndrome
Frequency (%)
Genitourinary anomalies
Turners syndrome 45X
6080
Horseshoe kidney
Duplication
Trisomy 18 (Edwards syndrome)
70
Horseshoe kidney
Renal ectopia
Duplication
Hydronephrosis
Trisomy 13 (Patu syndrome)
6080
Cystic kidney
Hydronephrosis
Horseshoe kidney
Ureteric duplication
4p (WolfHirschorn syndrome)
33
Hypospadias
Cystic kidney
Hydronephrosis
Trisomy 21 (Downs syndrome)
37
Renal agenesis
Horseshoe kidney
approximately 5% in Downs syndrome (trisomy 21)

to 75% in Turners syndrome (45 X) (Table 1).
Nondisjunction and translocation anomalies are
not confined to gametogenesis, but can also occur
during the early mitotic cell divisions in the process
of cleavage. In the resulting state, termed mosaicism,
the embryonic tissues contain a varying ratio of cell
lines with differing karyotypes depending on the
phase of cleavage at which nondisjunction occurred,
such as two-cell, four-cell, and eight-cell embryo.
Abnormalities of the sex chromosomes are often
found in mosaic form. Ovotesticular disorders of sex
development (previously termed true hermaphroditism) (2) can be explained on this basis. Affected
individuals possess both ovarian (XX) and testicular
(XY) tissues that coexist in streak-like gonads termed
ovotestes. Gonadal mesenchyme carrying a Y chromosome differentiates as testicular tissue, whereas
tissue derived from the original population of nonY
embryonic cells differentiates passively down the
female (ovarian) pathway. Karyotypes show a varied
pattern, including 45 X/46 XY and 46 XX/47 XXY.
Turners syndrome and Klinefelters syndrome are

often associated with mosaic karyotypes.
IMPLANTATION AND EARLY EMBRYONIC

DEVELOPMENT
On the fifth or sixth day after fertilization, the blastocyst implants into the endometrium of the uterine
cavity in which the endometrium has been primed
by progesterone secreted by the corpus luteum. Over
the ensuing 10 days, two cavities develop within the
spherical mass of rapidly proliferating embryonic
cells. The embryonic disc, from which the early
embryo is derived, forms in the three-layered interface
between the amniotic cavity and definitive yolk sac.
The amniotic surface of the trilaminar embryonic disc
gives rise to the ectodermal tissues of the embryo,
whereas the endodermal derivatives originate from
the yolk sacderived surface. The intraembryonic
mesoderm is formed by the inpouring of cells on the
amniotic surface of the disc via the primitive streak
(Fig. 2). The layer of intraembryonic mesoderm
Figure 2 The embryonic disc at 16 days. Inpouring of cells at the primitive streak creates the intra-embryonic mesoderm from which the
genitourinary tract is formed.
441
Figure 3 Timescale of key events in the development of the upper and lower urinary tracts.
created by the inpouring process soon subdivides into

three components, comprising the paraxial mesoderm,
intermediate mesoderm and lateral plate mesoderm. It
is from the blocks of intermediate mesoderm on either
side of the midline that much of the genitourinary
tract is ultimately derived. The third and fourth weeks
of gestation are dominated by the processes of segmentation and somite formation that characterize all
vertebrate embryogenesis, which are closely regulated
by expression of the Hox group of genes. At this stage,
folding of the expanding embryonic disc imparts recognizable shape to the growing embryo.
UPPER URINARY TRACT (FIG. 3)

The kidneys develop from the caudal zones of the
paired columns of intermediate mesoderm on either
side of the midline. The formation of the metanephros,
the precursor of the definitive kidney, is preceded by
the formation of the pronephros and mesonephros in
the cephalad and mid-zones, respectively. During the
fourth week of gestation, the primitive pronephros
appears in the cervical (cephalad) portion of the intermediate mesoderm but then rapidly regresses and
disappears. Later in the fourth week, primitive nephron-like tubular structures appear in the mesonephros
(Fig. 4A).
Linear rod-like condensations of mesenchyme
develop concurrently in areas lateral to the developing
mesonephros, and these then canalize to give rise to
the mesonephric ductswhich advance caudally to
fuse with the cloaca (the terminal portion of hindgut
destined to give rise to the bladder by a process
termed compartmentalization). Canalization of the

mesonephric ducts, which are in communication
with the primitive mesonephric tubules, creates a
patent excretory unit that is believed to function
transiently between 6 and 10 weeks. The beginning
of the fifth week sees the appearance of the ureteric
buds (Fig. 4B), arising from the most distal portion of
the paired mesonephric ducts.
Normal embryological development of the
upper urinary tract is crucially dependent on the
role of the ureteric bud. Faulty or failed interaction
between the ureteric bud and metanephric mesenchyme results in renal agenesis or differing patterns
of renal dysplasia, whereas abnormal budding results
in ureteric duplication (which in the case of complete
duplication may also be associated with dysplasia in
the renal relevant moiety). Abnormalities of early
ureteric development are also implicated in the etiology of vesicoureteric reflux (VUR) and vesicoureteric
junction obstruction.
At around 32 days, the advancing ureteric bud
makes contact with the metanephros, thus initiating
the interactive process of nephron formation (nephrogenesis). During the sixth to ninth weeks, the lobulated embryonic kidneys ascend up the posterior
abdominal wall from their caudal sites of origin to
their definitive lumbar position (Fig. 5).
In human, the process of nephron formation
(nephrogenesis) within the embryonic and fetal kidney spans a period of approximately 30 weeks, from
weeks 6 to 36 of gestation, whereas in some other
species nephrogenesis continues postnatally. The ureteric bud and the metanephric mesenchyme each
make specific contributions to the definitive structure
442
Thomas
Figure 4 (A) Following the regression of the pronephros, the mesonephros assumes prominence before the emergence of the
metanephros as the definitive embryonic kidney. (B) At around 28 days, the ureteric buds appear and advance toward the metanephric
mesenchyme.
Figure 5 At Five to seven weeks: penetration of the metanephric

mesenchyme promotes nephrogenesis by a process of mutual
induction. Mesonephric tissues support the development of the
embryonic gonads in the genital ridges. The paramesonephric
ducts develop in the mesenchyme adjacent to the mesonephric
ducts.
of the kidney. Sequential budding and branching of

the ureteric bud gives rise to the renal pelvis, the
major calices, the minor calices, and the collecting
ducts. Within the metanephric mesenchyme, differentiating cells aggregate to form the glomeruli, convoluted tubules, and loop of Henle. At around the
10th week, the distal convoluted tubules (derived
from metanephric mesenchyme) establish continuity

with the collecting ducts (of ureteric bud origin) to
form functional excretory units. The definitive architecture of the kidney, with cortex and medulla, is
established by 15 weeks gestation although new generations of nephrons continue to appear within the
cortex up to 36 weeks. The rate of nephrogenesis is
greatest toward the end of this period. At around
36 weeks nephrogenesis ceases and the number of
nephrons then remains fixed throughout life, ranging
between 0.7 and 1.4 million per kidney. The process of
tubular differentiation within the metanephric mesenchyme is initiated by contact with the ureteric bud.
Similarly, proliferative budding and branching of the
ureteric bud tissues are dependent on induction by the
metanephric mesenchyme. Nephrogenesis represents
one of many examples of reciprocal induction occurring in embryological development. When cultured
independently neither ureteric bud tissue nor metanephric has the capacity to differentiate into nephronlike structures. However, when cultured together,
nephrogenesis is observed.
MOLECULAR BIOLOGY OF NORMAL AND

ABNORMAL UPPER TRACT DEVELOPMENT
In view of the pivotal role played by the ureteric bud
and embryonic ureter in regulating normal and abnormal upper tract development, it is not surprising that
the genes expressed in these structures and the identity of the gene products responsible for mediating the
cell-to-cell signaling at a molecular level have been
intensively studied. The methodology employed
includes immunohistochemistry, scanning electron
microscopy, tissue culture and organ culture, and
the use of targeted mutations in knock out mice.
Embryonic urothelium has been shown to induce

differentiation of adjacent mesenchyme by secretion
of sonic hedgehog (Shh), a signaling growth factor
that orchestrates the activity of a number of other
proteins and transcription factors (3). Secretion of
Shh and similar gene products by embryonic urothelium stimulates differentiation of the adjacent mesenchyme into detrusor muscle and ureteric smooth
muscle. Urinary tract abnormalities attributed to
faulty development of ureteric and bladder smooth
muscle are observed in knock out mice lacking Shh.
Similar phenotypes arise from deletion of the gene for
the transcription factor teashirt 3 (4).
Uroplakin 3 (UP3) is one of the structural proteins expressed in the asymmetric unit membrane on
the urothelial surface, and targeted ablation of the
UP3 gene in embryonic mice results in high-grade
VUR associated with dilated kidneys (5). The UP3
knock out mouse provides the most convincing
experimental model for VUR yet describedraising
the possibility that this gene might be implicated in
the etiology of familial VUR in humans. However,
genetic screening studies to identify possible UP3
mutations in patients with nonsyndromic VUR
have been uniformly negative (6) and likewise no
evidence of abnormal UP3 expression is present in
the urothelium of patients undergoing surgery for
high-grade VUR (7).
Nevertheless, UP3 mutations have been identified in a small number of patients with a combination
of VUR and severe renal dysplasia (8). Similarly
mutations of the PAX 2 gene have been implicated
in rare familial cases of VUR (9). The renin-angiotensin system (10) is also known to play an important role
in regulating early differentiation and development of
the urinary tract. Two types of receptor are responsible for mediating the actions of angiotensin II in target
tissues. Stimulation of the angiotensin 1 (AGTR 1)
receptor in the embryo promotes cellular proliferation,
matrix deposition, and the release of growth factors in
the mesenchymal precursors of the kidney and ureter.
By contrast, the angiotensin 2 (AGTR2) receptor,
which is expressed principally in the embryo and
fetus, is responsible for inducing apoptosis and
decreased cell growth. Knockout deletions of the
AGTR1 and AGTR2 genes in mice result in different
patterns of urinary tract malformations. While deletion of the AGTR1 gene is characterized by progressive dilatation of the collecting system in postnatal
life, deletion of AGTR2 gene gives rise to varying
degrees of congenital renal hypoplasia, dysplasia,
and ureteric dilatation. Some of the many genes now
known to be involved in early ureteric development
include Gdnf, c-ret, Rara, Agtr2, L1-CAM, Bmp4,
Foxc1, Foxc2, Slit 2 Slit 3, Robo, and Pax2 (11).
The process of nephron formation within the
kidney has also been extensively studied and a large
number of gene products have already been demonstrated to play a role in regulating the different
phases. The actions of positive growth factors, such
as fibroblast growth factor 2 and glial cell linederived
neurotrophic factor (GDNF), are balanced by other
molecules, such as tumor necrosis factor, that act as
443
negative growth factors by promoting apoptosis (programmed cell death).

Transcription factors constitute another important group of signaling molecules. These DNAbinding proteins are involved in regulating gene
expression. Examples of transcription factors closely
involved in nephrogenesis include the gene products
of the Pax2 gene and Wilms tumor suppressor gene
(WT1 gene), both of which play a central role in
branching of the ureteric bud and in the induction of
metanephric mesenchyme. Other signaling molecules
include those responsible for cell-to-cell adhesion and
cell-to-cell matrix adhesion, such as laminins and
integrins.
CONGENITAL RENAL MALFORMATIONS:

CLINICAL CONSIDERATIONS
For convenience, congenital anomalies of the kidney
and urinary tract (CAKUT) can be arbitrarily grouped
in the following broad morphological categories:
1.
2.
3.
4.
Renal agenesistotal absence of the kidney

Renal dysplasiaa kidney is present, although it
is often reduced in size. The internal architecture
is disordered and the histological appearances are
characterized by immature, primitive, undifferentiated tubules, and the inappropriate presence
of tissues such as cartilage and fibromuscular
tissue within the renal parenchyma
Cyst formation
Gross developmental anomalies, such as duplication, horseshoe kidney, and pelvic kidney
Ureteric duplication can be explained by bifurcation of the ureteric bud, and according to the level of
the original bifurcation, the duplication may be complete or incomplete. In cases of complete ureteric
duplication, the upper pole ureter paradoxically
enters the urinary tract at a more distal (caudal)
level than the lower pole ureter. In the accepted conceptual model this anatomical pattern (described by
the Meyer Weigert law) arises when the mesonephric
duct separates from the embryonic ureter and its
terminal portion descends toward the primitive posterior urethra, drawing the upper pole ureter with it.
Gross renal anomalies such as horseshoe kidney,
pelvic kidney, and crossed ectopia date from the 6th to
10th week of gestation when the developing kidneys
may fuse (horseshoe kidney), cross the midline
(crossed fused ectopia), or fail to ascend (pelvic
kidney).
EMBRYOLOGY OF THE LOWER URINARY

TRACT (BLADDER AND URETHRA) (FIG. 3)
The bladder and urethra originate from the anterior
portion of the cloaca, the common terminal section of
hindgut into which the mesonephric ducts and
embryonic ureters drain. The cloaca also contributes
the urogenital sinuswhich in turn contributes to the
vagina. Conventionally the urorectal septum has been
likened to a shutter that descends to subdivide the
444
Thomas
Figure 6 Descent of the urorectal septum between

four and six weeks divides the cloaca into an anterior
urogenital compartment, the precursor of the bladder,
urethra, prostate and distal vagina, and a posterior
anorectal canal.
cloaca into the urogenital canal anteriorly and the

anorectal canal posteriorly between the fourth and
sixth weeks of gestation (12). This process is aided
by the lateral ingrowth of the folds of Rathke (Fig. 6).
Our understanding of this mechanism is based on
historical interpretation of sequential sections of
embryos at different stages in early gestation. More
recently, however, this mechanism (and indeed the
very existence of the urorectal septum and Rathkes
folds) has been called into question by the results of
studies employing modern methodology such as scanning electron microscopy and immunostaining for
apoptosis. It is now thought that partitioning of the
cloaca results from in situ deposition of mesenchyme
in the cloaca and urogenital sinus coupled with simultaneous apoptosis and changes in the curvature of the
adjacent abdominal wall and spine (13).
As the bladder becomes more clearly defined in
the upper portion of the urogenital canal, the points of
entry of the ureter and mesonephric ducts begin to
separate (Fig. 7). When the distance between them
increases, the mesonephric ducts descend caudally to
open into the developing posterior urethra. By contrast, the ureters retain their original position with
respect to the developing bladder. Although the triangular plate of mesoderm enclosing the ureteric
orifices and openings of the mesonephric ducts is
covered by the endodermal lining of the urogenital
canal, its outline is retained as the trigone. It should be
noted, however, that as with the compartmentalization of the cloaca the conventional account of the
translocation of the mesonephric ducts and formation

of the trigone has been challenged by studies employing modern methodology (14). It now seems more
probable that apparent changes in relative anatomy
are a consequence of remodeling because of laying
down of new tissue and apoptosis in situ rather than
actual three-dimensional movement of anatomical
structures in the early embryo. The most distal (perineal) portion of the urogenital canal gives rise to the
entire length of the female urethra and to the posterior
urethra in the male. The anterior portion of the male
urethra is created by closure of the urogenital groove,
described below. Meanwhile the allantois, an elongated diverticulum protruding from the dome of the
fetal bladder into the umbilicus, is gradually obliterated but persists as the median umbilical ligament.
CLINICAL CONSIDERATIONS
Despite recent changes in our understanding of the
process of compartmentalization of the cloaca, the concept of failed or incomplete descent of the urorectal
septum, nevertheless, provides a simple and readily
understood model to account for the spectrum of
anomalies that includes urogenital sinus and complete,
persistent cloaca. The etiology of bladder exstrophy and
epispadias is less well understood, but these anomalies
are believed to reflect underlying defects of the cloacal
membrane. Persistence of all or part of the allantois
gives rise to the various urachal abnormalities.
445
Figure 7 (A, B): Although the ureters maintain

their position in relation to the developing bladder,
the mesonephric ducts are drawn distally to enter
the proximal urethra. (C) Testicular descent
causes the mesonephric ducts (vasa deferentia)
to loop over the ureters.
FUNCTIONAL DEVELOPMENT OF THE FETAL

URINARY TRACT
Around the 9th to 10th week of gestation, fetal urine is
excreted into the amniotic cavity. At this stage the
urothelium adopts the morphology associated with its
barrier function, notably the expression of uroplakins
on the luminal surface. The embryonic urothelium
also induces smooth muscle differentiation in surrounding pelvic and ureteric mesenchyme by secretion of Shh and other signaling molecules. Initially,
urine composition closely resembles that of plasma
filtrate. In the fetus, the excretory and homeostatic
functions of the kidney are fulfilled by the placenta,
and the principal role of the fetal kidneys is to contribute urine to amniotic fluid. The volume of amniotic fluid reaches its maximum of around 1000 mL at
38 weeks gestationat which point 90% of the amniotic fluid is derived from fetal urine. In addition to
providing a protective fluid environment for the fetus,
amniotic fluid plays a vital role in fetal lung development. Reduction in fetal urine output as a consequence of either renal agenesis or infravesical
obstruction results in oligohydramnios, which in
turn is associated with pulmonary hypoplasia. Molding or compression deformities such as talipes and
characteristic facial molding (Potters facies) occur as a
consequence of the compressive effect of the surrounding uterus.
Figure 8 Migration of the primordial germ cells from the yolk

sac across the coelomic cavity to colonize the mesoderm of the
genital ridges during the fifth week.
446
Thomas
Figure 9 (A) Timescale of key events in the development of the female genital tract. (B) Timescale of key events in the development of
the male genital tract.
GENITAL TRACTS
The internal and external genitalia of both sexes share
identical embryonic precursors, and it is only from the
6th week onward that differences become apparent. In
both sexes, the formation of the gonads and genital
tracts is initiated by the migration of primordial germ
cells from the base of the yolk sac, across the coelomic
cavity to condensations of mesenchyme flanking the
midline of the lumbar region of the embryo (the genital ridge) (Fig. 8). By a process of reciprocal induction
(analogous to nephrogenesis), the interaction of the
germ cells and surrounding mesenchyme results in
the formation of the primitive sex cords within the
embryonic gonad. It is around this time that a second
pair of genital ducts (the paramesonephric ducts)
makes its appearance. Derived from condensations
of coelomic epithelium and lying lateral to the mesophric ducts, these ducts fuse distally at their point of
attachment to the urogenital canal. From the sixth
week onward, the paths of male and female differentiation diverge and will be considered separately in
the following subsections;
The timescale of key events in the development
of both female and male genital tracts is summarized
Figure 9A and B.
Female (Fig. 9A)

Internal Genitalia
It is widely accepted that without the genetic information carried by the testis-determining gene (SRY),
the gonads and genital tract of the embryo are
447
Figure 10 Gametogenesis. Mitotic division of the primordial germ cells in the male embryo is suppressed by the Sertoli cells of the
embryonic germinal epithelium. Gametogenesis recommences at puberty. The gametes (spermatozoa) are the product of two meiotic
divisions, that is, primary spermatocyte to secondary spermatocyte to spermatozoa. Immediately after the second meiotic division, the
immature haploid gametes are termed spermatids. The morphological maturation to spermatozoa occurs during passage through the
seminiferous tubule. In the female, the initial phases of gametogenesis occur in the first five months of fetal life. Primary oocytes remain
in arrested meiotic division until meiosis recommences in the maturing Graafian follicle. The two meiotic divisions culminate in the
production of a single definitive oocyte and three polar bodies (P).
programmed to differentiate down a female pathway.

However, it is probably an oversimplification to view
differentiation of the female gonads as an entirely
passive or default process. For example, females
with Turner syndrome (45X) have dysplastic or streak
gonadsindicating that normal ovarian development
requires the presence of two X chromosomes and is
not simply determined by the absence of genetic
material carried on the Y chromosome.
Within the genital ridges the primitive sex cords
degenerate, but secondary sex cords derived from
mesoderm enclose the primordial germ cells to form
the primitive follicles. In the female, gametogenesis
commences in intrauterine life with the transition
from primordial germ cells to primary oocytes being
completed in the fetal ovary. These primary oocytes
then proceed into the first phase of meiosis before
entering a long phase of arrested division, which only
resumes again after puberty (Fig. 10). Persistence of
the mesonephric ducts in male embryos is dependent
on local stimulation by testosterone; therefore, in the
absence of androgen stimulation in the female, the
mesonephric ducts regress (with the exception of vestigial remnants of the epoophoron and paroophoron
and Gartners cysts) (Fig. 11). The paramesonephric

ducts persist and develop to become the fallopian
tubes, and their fused distal portions give rise to the
uterus and upper two-thirds of the vagina. At their
point of attachment to the urogenital sinus, the fused
tips of the paramesonephric ducts induce a condensation of tissuethe sinuvaginal bulb. Downward
growth of the sinuvaginal bulb in the direction of the
fetal perineum between weeks 10 and 20 has the effect
of separating the vagina from the urethra. The vagina is
initially represented by a solid plate of paramesonephric tissue, but this canalizes to create the vaginal lumen
before the 20th week. The distal third of the vagina is
derived from the urogenital sinus (endoderm), whereas
the introitus and external genitalia are ectodermal in
origin (Fig. 12).
External Genitalia
The external genitalia of the embryo and fetus are

programmed to differentiate passively down a
female pathway unless exposed to androgenic stimulation. Thus, the genital tubercle forms the clitoris, the
introitus and vestibule of the vagina are derived from
the urogenital sinus, the urogenital folds evolve into
Figure 11 (A) The genital tract of both male and female embryos remains in the same undifferentiated state until the sixth week of
gestation. (B) Paramesonephric duct a b derivatives in the female embryo.
Figure 12 Development of the lower female genital tract between 10 and 20 weeks. Migration of the sinuvaginal bulb toward the fetal
perineum results in formation and elongation of the vaginal plate and separation of the vagina from the urethra.
448
449
Figure 13 (A) Undifferentiated precursors of male and female external genitalia. (BD) Androgen (testosterone-derived dihydrotestosterone)-induced differentiation of the male external genitalia between 12 and 16 weeks. (E) In the absence of androgenic stimulation,
the external genitalia are programmed to differentiate along a female pathway.
the labia minora, and the labioscrotal folds persist as

the labia majora (Fig. 13).
Male (Fig. 9B)

Internal Genitalia
Although many aspects of the male differentiation

pathway are mediated by downstream genes
located on the Y chromosome and on autosomes, the
process is initiated by the testis-determining gene
(SRY) located on the Y chromosome. In experiments
in transgenic mice, the insertion of DNA carrying the
SRY gene into the genotype of an XX (female) mouse
results in differentiation of a male genital phenotype
(15). The SRY gene product (a transcription factor)
stimulates the medullary sex cords of the embryonic
gonad to differentiate into secretory Sertoli cells. From
the seventh week onward, the Sertoli cells secrete a
glycoproteinanti-Mullerian hormone or Mullerianinhibiting substance (MIS)that switches differentiation down the male pathway. At least three key
functions have been ascribed to MIS:
1.
2.
3.
It causes regression of the paramesonephric ducts,

which disappear completely in the male with the
exception of vestigial remnants (the appendix,
testis, and the utriculus).
It stimulates the Leydig cells of the embryonic
testis to produce testosterone from the ninth week
of gestation.
It induces the first stage of testicular descent by its
action on the gubernaculum, which anchors the
testis in the vicinity of the developing inguinal

canal.
Further division of the primordial germ cells is
inhibited in the male embryonic gonad. Until recently,
it was believed that the testis is quiescent throughout
childhood and that the subsequent sequence of gametogenesis is resumed again only at puberty. This concept is now being challenged by evidence pointing to
the occurrence of two maturational steps in the prepubertal testis. The first, occurring around two to
three months of age, consists of the switch from
gonocytes (fetal stem cells) to adult-type spermatogonia (the adult stem cell pool). The second step, at
approximately 5 years of age, is marked by a transient
phase of meiosis, which can be detected histologically
by the appearance of primary spermatocytes. Information obtained from biopsy studies of undescended
testes has revealed that cryptorchidism has a major
impact on prepubertal germ cell maturationwith
obvious implications for the timing of orchidopexy.
Regression of the paramesonephric ducts in
response to MIS is accompanied by the development
of mesonephric duct derivatives under the influence
of testosterone secreted by the fetal testis (Fig. 14A).
During the period between the 8th and 12th week of
gestation, the mesonephric ducts give rise to the
epididymis and rete testis, vas deferens, ejaculatory
ducts, and seminal vesicles (Fig. 14B). Within the
testis, the seminiferous tubules take their origin from
the primitive sex cords of the genital ridge mesoderm
with the rete testis and epididymis being derived from
the mesonephric tubules.
450
Thomas
Figure 14 (A) Differentiation of male internal genitalia. Secretion of Mullerian-inhibiting substance (MIS) by pre-Sertoli cells induces
regression of the paramesonephric ducts between 8 and 10 weeks. (B) MIS also stimulates the embryonic Leydig cells to secrete
testosterone, which in turn stimulates the mesonephric ducts to differentiate into the definitive male internal genitalia.
The development of the prostate gland provides

another example of reciprocal induction. At around
week 12, in response to androgenic stimulation, a
condensation of mesenchyme in the fetal pelvis induces the outgrowth of endodermal buds on the adjacent
region of the developing urethra (16). In the experimental setting it has been shown that transitional
epithelium from the adult rat can be induced to differentiate into prostatic glandular epithelium when
cultured in contact with the appropriate embryonic
mesenchyme. In normal fetal development, however,
the endodermal proliferation and branching that give
rise to the ducts and glandular acini of the prostate
gland cease after the 15th week. The glandular tissue
of the prostate is derived from the urethral endoderm
(urogenital sinus); the capsule and smooth muscle are
mesenchymal in origin, whereas the mesonephric
ducts form the intraprostatic vas deferens and ejaculatory ducts.
External Genitalia (Fig. 13)
Prior to the compartmentalization of the cloaca to

form the urogenital and anorectal canals between the
sixth and seventh weeks, the primitive perineum
consists of little more than the cloacal membrane
and genital tubercle. Separation of the cloaca into the
urogenital canal and anorectal canal is accompanied
by subdivision of the cloacal membrane into the
urogenital membrane anteriorly and the anal membrane posteriorly. Urogenital folds surround the urogenital membrane, flanked by the labioscrotal folds.
From the seventh week onward, the urogenital sinus
advances onto the perineum anteriorly and onto the
penis as the urethral groove. Ingrowth of the urethral
groove creates a solid urethral plate, which subsequently canalizes to form the definitive urethra. Differentiation of the male external genitalia is
dependent, first, on the enzymatic conversion of testosterone to dihydrotestosterone and also requires the
presence of the appropriate receptors in the target
tissues. During the 12th to 14th week, the external

genitalia begin to adopt a distinctively male configuration in response to androgenic stimulation (17).
Closure of the urethra is complete by around
15 weeks, the terminal portion being formed by
ingrowth of ectoderm from the tip of the glans.
Testicular Descent
As already described, the fetal testis originates from

the interaction of primordial germ cells, with the
mesenchyme of the genital ridge and tubular mesonephric duct derivatives. Testicular descent occurs in
two phases. The first stage occurs in response to
exposure to MIS, the second being stimulated by
testosterone. Between weeks 8 and 15, the cord-like
gubernaculum extending down from the testis
enlarges at its distal end in the region of the labioscrotal swellings (Fig. 15A). Because the length of the
gubernaculum remains relatively fixed during a
period of active fetal growth, this has the effect of
anchoring the testis in the region of the future inguinal
canal (Fig. 15B). The second, more active, phase of
testicular descent occurs around 25 to 30 weeks of
gestation when testosterone causes the gubernaculum
to shrink and contract, thus guiding the testis down the
inguinal canal into its final scrotal position (Fig. 15C)
(18). On its route of descent, the testis is preceded by a
sac-like protrusion of peritoneum, the processus vaginalis, which normally closes spontaneously prior to
delivery or in the early months of life.
GENITAL ANOMALIES: CLINICAL

CONSIDERATIONS
A number of mechanisms can give rise to abnormalities of genital development, including chromosomal
defects, abnormalities of gonadal development, and
impaired synthesis of sex hormones or defects of their
corresponding receptors.
451
Figure 15 (A) Formation of the gubernaculum. (B) 10 to 15 weeks: enlargement of the caudal end of the gubernaculums (in response to
MIS) anchors the fetal testis at the developing inguinal canal. (C) 25 to 30 weeks: testosterone-induced second stage of a testicular
descent.
Defective development of the paramesonephric

ducts results in a variety of clinical manifestations. At
its most fundamental level the unilateral absence of
paramesonephric duct derivatives, which is also associated with ipsilateral unilateral renal agenesis, points
to a fundamental defect dating from the original
intermediate mesoderm. Agenesis of the upper twothirds of the vagina (Rokitansky syndrome) results
from failure of the paramesonephric ducts to fuse
distally and merge with the urogenital sinus. Incomplete fusion of the distal ends of the paramesonephric
ducts accounts for anomalies such as bicornuate
uterus, while defective canalization of the vaginal
plate results in transverse vaginal septa and short
atretic segments.
Approximately 80% of infants with ambiguous
genitalia are 46 XX females with congenital adrenal
hyperplasia whose external genitalia have virilized in
response to high levels of circulating androgens of
adrenal origin. In this condition, however, the internal
reproductive tract develops normally down the

female pathway. Individuals with a 46 XY karyotype
and complete androgen-insensitivity syndrome (previously termed testicular feminization) have a
female external genital phenotype because of a genetically determined peripheral receptor insensitivity to
dihydrotestosterone, which causes the external genitalia to fail to virilize despite exposure to androgens
secreted by the fetal testes.
Incomplete closure of the urethral groove
accounts for moderate and severe forms of hypospadias, while distal (glanular) hypospadias probably
represents failure of ectodermal ingrowth from the
glanular tip. Severe forms of proximal hypospadias
are often associated with cryptorchidism and a persistent utriculus (Mullerian remnant). While these
features point to the presence of an underlying virilization defect, attempts to identify specific endocrinopathies in boys with severe hypospadias have
generally proved unrewarding.
452
Thomas
There is some epidemiological evidence to suggest that the incidence of hypospadias and crypt
orchidism is increasing and exposure of male fetuses
to estrogenic or antiandrogenic environmental pollutants has been postulated as a possible cause.
Cryptorchidism affects 1% to 2% of the male
infant population, but there is no single unifying
etiology. Endocrinopathy or imbalance of the pituitary-gonadal axis can be implicated in cases of bilateral cryptorchidism, particularly when associated
with hypospadias. By contrast local mechanical factors (possibly related to the gubernaculum or the
processus vaginalis) are likely to play a more important role in unilateral cryptorchidism. In the rare,
genetically determined syndrome of MIS deficiency,
intra-abdominal testes are found in conjunction with
persistent paramesonephric duct structures, including
fallopian tubes and uterus. Although deficiency of
MIS accounts for a proportion of cases, this condition
is misnamed since it is more commonly because of a
receptor defect for MIS.
Simple mechanical factors provide the most
obvious explanation for unilateral cryptorchidism,
but the reported finding of histological abnormalities
in the contralateral descended testis suggests that
endocrinopathy may also play a role in unilateral
cryptorchidism. The presence of a blind-ending vas
and spermatic vessels in most cases of testicular
agenesis is widely interpreted as evidence of intrauterine testicular torsion rather than a defect of
embryogenesis.
THE GENETIC BASIS OF GENITOURINARY

MALFORMATIONS (TABLE 2)
Malformations of the urinary and genital tracts are
relatively common and are estimated to account for
one-third of all congenital anomalies. Although the
importance of genetic factors is being increasingly
recognized, it remains the case that most CAKUT
occur on a sporadic basis resulting from defective
embryogenesis or faulty development in fetal life.
The adult and infant forms of polycystic renal
disease provide the most convincing examples of
genetically determined structural renal disease.
Genetic factors have also been implicated in the
etiology of renal agenesis, VUR, and upper tract
duplication. Genitourinary malformations with a

recognized familial tendency are most commonly
inherited as autosomal traits of variable penetrance
and expression. Although some conditions, such as
polycystic kidney disease, have their origins in a
single gene mutation, the etiology of most structural
anomalies of the urinary tract is more complexas
illustrated by the fact that upper tract anomalies are
often expressed unilaterally or asymmetrically even
when genetic factors are clearly implicated. When
genitourinary anomalies occur within the context of
a recognized syndrome, it is probable that the
affected gene (or genes) encodes for gene products
that play a fundamental molecular role in the regulation and development of a number of systems. For
example, the KAL gene implicated in X-linked Kallmanns syndrome (microphallus, renal agenesis,
and anosmia) is known to code for an adhesion
molecule that is expressed during the migration of
olfactory neurons in the brain and also in the developing genitor urinary tract. Other examples include
renalcoloboma syndrome (VUR and the ophthalmic anomaly, coloboma), which is because of a
mutation of the PAX-2 gene encoding for a transcription factor.
IN VITRO FERTILIZATION AND OTHER

APPLICATIONS OF DEVELOPMENTAL
BIOLOGY IN UROLOGY
The availability of techniques for IVF has given many
urological patients the potential for parenthood
whereas previously their outlook for fertility would
have been bleak. Examples of patients who might
benefit from IVF techniques include men with oligospermia or azoospermia because of cryptorchidism
and men with impaired ejaculation associated with a
history of posterior urethral valves or bladder exstrophy. In the technique of intracytoplasmic sperm
injection, spermatozoa are harvested directly from the
testis or epididymis and a single spermatozoan is
selected and injected into an oocyte. The fertilized
zygote is then implanted into the uterus. Other variants of IVF such as gamete intrafallopian transfer
(GIFT) or zygote intrafallopian transfer (ZIFT) may
also be applicable for urological patients with a range
of congenital or acquired causes of infertility.
Table 2 The Genetic Basis of Common Urinary Tract Abnormalities

Adult polycystic kidney disease
Autosomal dominant
Infantile polycystic kidney disease

Vesicoureteric reflux
Autosomal recessive
Autosomal dominant, variable
penetrance and expression
Various, i.e., autosomal dominant,
recessive and X-linked patterns
identified
Autosomal dominantvariable
penetrance and expression
When familial behaves as autosomal
dominant
Renal agenesis
Ureteric duplication
Pelviureteric junction obstruction
Chromosome 16p
Chromosome 4
(Pax-2 gene in renalcoloboma syndrome)
Documented familial evidence as isolated anomaly or

pelviureteric junction obstruction in association with
specific syndromes (e.g., SchinzelGiedion and
JohansonBlizzard syndromes)
453
Embryo Research
REFERENCES
The concept of therapeutic cloning is based on aspects

of the methodology originally developed for IVF. The
key steps are as follows:
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic

stem cell lines derived from human blastocysts. Science
1998; 282:11451147.
2. Houk CP, Hughes IA, Ahmed SF, et al. Summary of
consensus statement on intersex disorders. International
Intersex Consensus Conference. Pediatrics 2006;
118(2):753757.
3. Haraguchi R, Motoyama J, Sasaki H, et al. Molecular
analysis of coordinated bladder and urogenital organ formation by Hedgehog signalling. Development 2007;
134:525533.
4. Caubit X, Lye CM, Martin E, et al. Teashirt 3 is necessary
for ureteral smooth muscle differentiation downstream of
SHH and BMP4. Development 2008; 135(19):33013310.
5. Hu P, Deng FM, Liang FX, et al. Ablation of uroplakin III
gene results in small urothelial plaques, urothelial leakage,
and vesicoureteral reflux. J Cell Biol 2000; 151(5):961972.
6. Jiang S, Gitlin J, Deng FM, et al. Lack of major involvement
of human uroplakin genes in vesicoureteral reflux: implications for disease heterogeneity. Kidney Int 2004; 66:
1019.
7. Garthwaite MA, Thomas DFM, Subramaniam R, et al.
Urothelial differentiation in vesicoureteric reflux and
other urological disorders of childhood: a comparative
study. Eur Urol 2006; 49:154160.
8. Jenkins D, Bitner-Glindzicz M, Malcolm S, et al. Mutation
Analysis of Uroplakin II in children with renal tract
malformations. Nephrol Dial Transplant 2006; 21:
34153421.
9. Feather S, Woolf AS, Gordon I, et al. Vesicoureteric
refluxis it all in the genes? Lancet 1996; 348:725728.
10. Yerke EB. The new renal angiotensin system and its impact
on upper urinary tract development. Dialogues Pediatr
Urol 2000; 23:45.
11. Stahl DA, Koul HK, Chacko JK, et al. Congenital anomalies
of the kidney and urinary tract (CAKUT): a current review
of cell signaling processes in ureteral development.
J Pediatr Urol 2006; 2:29.
12. Thomas DFM. Cloacal malformations: embryology, anatomy and principles of management. In: Spitz L, Wurnig P,
Angerpointner TA, eds. Progress in Paediatric Surgery.
Vol 23. Berlin: Springer-Verlag, 1989:135143.
13. Pennington EC, Hutson JM. The absence of lateral fusion in
cloacal partition. J Pediatr Surg 2003; 38(9):12871295.
14. Oswald J, Schwentner C, Lunacek A, et al. Reevaluation of
the fetal muscle development of the vesigal trigone. J Urol
2006; 176:11661170.
15. Larsen WJ. Development of the urogenital system. In:
Larsen WJ, ed. Human Embryology. 3rd ed. Philadelphia:
16. McNeal JE. Anatomy and embryology. In: Fitzpatrick JM,
Kane RJ, eds. The Prostate. Edinburgh: Churchill Livingstone, 1989:39.
17. Kurzrock AE, Jeyatheesan P, Cunha GR, et al. Urethral
development in the fetal rabbit and induction of hypospadias: a model for human development. J Urol 2000;
164(5):17861792.
18. Hutson JM, Terada M, Baiyun Z, et al. Normal Testicular
Descent and the Aetiology of Cryptorchidism. Advances in
Anatomy, Embryology and Cell Biology. Vol. 132. Berlin:
Springer-Verlag, 1996:
1.
2.
3.
The nucleus of a harvested or donated oocyte is

removed by micropuncture. A diploid nucleus
extracted from a somatic cell is then injected into
the enucleated oocyte. For the purposes of therapeutic cloning, it is envisaged that the somatic cell
would be derived from the patient.
The cell thus created is induced to divide, as for
example, by an electrical charge.
A sequence of doubling divisions then ensues,
which mirrors that of the fertilized zygote. Proteins within the cytoplasm of maternal oocyte are
thought to be responsible for inducing the cloned
cell to behave as a fertilized zygote rather than the
mature somatic cell from which its nucleus was
derived.
Human embryo research has focused on the

extraction of pluripotent embryonic stem cells from
early embryonic tissue. Tissue derived by inducing
differentiation of pluripotent stem cells would share
the same genetic identity as the somatic nucleus used
to create the cloned embryo and would therefore not
provoke rejection by the immune system when reintroduced into the individual (patient) from whom the
nuclear material had been obtained.
Clinical indications envisaged for such forms of
treatment include tissue reconstruction, the treatment
of degenerative diseaseparticularly neurodegenerative disorders such as Parkinsons diseaseand islet
cell transplants to treat type 1 diabetes.
At present the potential application of early
human embryonic tissue for therapeutic purposes is
constrained by practical considerations such as the
scarcity of donated oocytes and the invasive nature of
oocyte donation. One of the more controversial proposals to overcome this hurdle envisages injecting a
human diploid nucleus into an enucleated oocyte
derived from another species to create a humananimal
hybrid embryo from which (human) stem cells would
be extracted at a very early stage to create stem cell
lines in tissue culture. Another approach being
explored would obviate the use of oocytes by manipulating differentiated cells in vitro to cause them to
revert to an undifferentiated state to create induced
pluripotent stem cells.
In the light of current knowledge, it seems most
likely that within the field of Urology the applications
of embryo research will lie mainly in reproductive
medicinenotably in the development of innovative
treatments for male infertility. By contrast, the treatment of renal impairment by reactivating nephrogenesis to stimulate new nephron formation in poorly
functioning kidneys or alternatively the creation of
tissue engineered kidneys for use in transplantation
must be seen as highly futuristic prospects.
30
Urological and Biochemical Aspects of
Transplantation Biology
David Talbot
Department of Hepatobiliary and Transplant Surgery, Freeman Hospital, Newcastle upon Tyne, U.K.
Naeem Soomro
Department of Urology, Freeman Hospital, Newcastle upon Tyne, U.K.
INTRODUCTION
The first successful human renal allograft was performed
in Boston by Merrill and Murray on December 23, 1954.
Preceding this had been many unsuccessful animal and
even human transplants. Only two things had been
learned from this early work; firstly, how vessels could
be successfully anastomosed, and, secondly, that transplantation between nonrelated individuals was impossible. The Boston transplant was successful because it was
between identical twin brothers, and it was not until
immunosuppressive drugs became available that transplantation could really develop.
ORGAN DONATION
The early transplants were all performed from live
donors; then, with the advent of immunosuppression,
nonrelated cadaver donors could be used. These initially
were simply cadavers and therefore were nonheart
beating donors, but later, with legislation and acceptance of brain death, heart-beating but brain-dead donors
were used. More recently, with the limited supply of
brain-dead donors, there has been significant expansion
of both live donation and nonheart beating donation.
BRAINSTEM DEATH
Though death is an unavoidable aspect of life, all
countries have their own procedures and legislation
connected with it. With the advent of transplantation
and possible use of organs, most countries were
forced to consider and thereby distinguish between
cardiac and brain death. In some countries, such as
Austria, donation was extremely easy because legislation had been introduced many years previously
giving the state more roles after death so postmortems were compulsory to minimize health risks
to the living. In many countries, criteria were established for the diagnosis of brain death. In the United
Kingdom, the tests had to be performed on two
occasions by two experienced medical practitioners.
Certain prerequisite conditions had to be fulfilled;
namely, normothermia and absence of sedative

drugs. Furthermore, the subject had to be shown to
be ventilator dependent with elevated carbon dioxide
levels and have absent cranial nerve reflexes, notably
the gag, corneal and vestibulocochlear reflexes, by two
experienced medical practitioners on two occasions
(1). When the patient was diagnosed as being brain
dead, the issues of donation and transplant could be
discussed with the relatives. The posts of transplant
coordinators evolved in the mid-1980s, in part to act as
bereavement counselors and to coordinate the multiple teams involved in a multiorgan donor. Their role
has been essential in maintaining donor numbers in
the face of reduced death rates from road traffic
accidents and improved outcome of neurosurgery.
NONHEART BEATING DONATION

Because of the increasing shortage of kidneys for
donation, there is a need to increase the pool of
organs. Therefore, the option of nonheart beating
donoration, which had been utilized before brain
death legislation, was explored again. These donors
have been subjected to a varying amount of primary
warm ischemia, during the time between cardiac
arrest and perfusion with cold preservation solution.
Different categories can be defined (Maastricht),
which have implications for the degree of warm
ischemia, simply these are controlled (awaiting cardiac arrest, Maastricht III and IV) and uncontrolled
(failed resuscitation in the emergency setting,
Maastricht I and II) (2). Because there is damage to
the organs after cardiac arrest (warm ischemia), care
must be taken to establish which kidneys are safe to
transplant (3). If such steps are taken and the kidneys
are used, delayed graft function is common (4).
However, after a time, the function recovers, and
the long-term outlook is the same as for kidneys
transplanted from brain-dead donors (4,5) Livers
and pancreata have all been transplanted from such
donors, but greater care must be taken, as delayed
graft function, if it occurs, in a liver transplant is
usually fatal (6).
Chapter 30: Urological and Biochemical Aspects of Transplantation Biology
The proportion of kidneys from such donors is

increasing year on year such that in 2007 20.4% of
kidney donors were from nonheart beating donors.
The increase has in the main been because this is a
different source of cadaver donors but to a lesser
extent there have been brain-dead donors that have
come to donation via the nonheart beating route for
different reasons.
LIVE RELATED AND UNRELATED DONATION

As cold ischemia is considerably reduced in live
donation, the long-term graft survival is better than
with cadaveric donor kidney grafts. Formerly, centers
with large cadaver-kidney programs have generally
had small live donor programs, presumably indicating that supply is sufficient for demand. However,
with the generally declining cadaver programs, live
donor numbers have increased. Commercialization of
transplantation has created a huge ethical issue, and
many countries have solved this by banning the
practice.
There are countries that embrace commercialization such as Iran where it is usual to keep a panel of
potential donors who are called up when a potential
recipient is identified the donors being subsequently
compensated financially. Most Western countries
though they have made donation for money illegal
often use some financial support such as for transport
costs but compensation for lost income is difficult. In
the United States, tax concessions for donors and a
reduction their health insurance have been proposed.
Regulation of live donor transplantation has evolved
but is country specific, so, for example, the United
Kingdom formerly required proof of first degree
relatives by Tissue typing/ DNA analysis or approval
by Unrelated Live Transplant Regulatory Authority
(ULTRA) for all others. This has recently changed
with the establishment of the Human Tissue Authority (HTA). This means that any potential live donor
after they have been assessed and are judged to be
suitable have to have an assessment by an independent assessor. These assessors have all been trained
and are approved by the HTA and establish that the
potential donor has not been coerced. All documents
are then submitted and approval granted by the HTA.
This mechanism means that all related and unrelated
are managed the same. It has also enabled paired
donation (swopping donor/recipient combinations
when the transplant cannot occur between pairs, e.
g., with ABO incompatible combinations or donor
specific antibodies) and completely altruistic donation. Full details on the legal requirements within
the United Kingdom for live donation can be found
at www.hta.gov.uk.
Kidney donation from live donors carries a low
mortality rate and is preferably conducted in parallel
with the recipient surgery, which minimizes ischemia.
It is also useful for the occasional difficult recipient
such that the kidney is not removed until the recipient
vessels are confirmed to be suitable. From the live
donors perspective undoubtedly the preferable
455
method of donation is with a laparoscopic approach.

From the recipients point of view the warm ischemic
time to the kidney is slightly longer (usually about five
minutes) during the kidney extraction but this has
been shown to have no significant affect on outcome.
However, laparoscopic right-kidney donation carries
some risk of caval or duodenal injury and should be
done routinely only after considerable experience has
been obtained from left-kidney donation.
ORGAN PRESERVATION
In the early days of renal transplantation, there was
considerable uncertainty as to how to store the organ.
The two methods were simple static cold storage and
dynamic machine perfusion, which was also performed at cold temperature. Most groups stopped
machine preservation when it was discovered that,
generally, the longer the kidney remained in storage,
the worse was the outcome (7). Certain groups, principally Belzer et al. from the United States, persisted
with machine perfusion, and when the solutions were
improved, it was found that the kidney preservation
was better with this method than with static storage
(8). This was principally the case with marginal donor
kidneys, and this method had the added benefit of
allowing the surgeons to improve their viability
assessment by assessing the resistance and flow
through the kidney (3,9).
The holy grail of preservation, however, is
being able to do it at normal temperature, as this allows
more accurate viability assessment and recovery of the
organ. However, it is extremely difficult because of the
metabolic demands of the organ, and only limited
success has been possible so far (10).
PRESERVATION SOLUTIONS
The first aim of preservation fluid is to replace blood,
which, if allowed to stand within the organ, will clot
and thereby render it useless. The second aim is to
reduce the temperature of the organ to near freezing
point to minimize the metabolic demands of the
organ (hence, the solutions are kept cold). At cold
temperatures, the normal cell-membrane electrolyte
pumps fail, and cellular swelling occurs. The early
solutions were essentially hyperosmolal mannitol,
and though they prevented swelling, the intracellular
electrolyte concentrations changed; therefore, acute
tubular necrosis was common, particularly when
cold storage was prolonged. The solutions were
therefore modified to preserve the intracellular
milieu; hence, Marshalls, Collins, and Euro-Collins
were developed. Though acute tubular necrosis still
occurred with these solutions, it was not as common.
However, other organs, notably the liver, that had to
work immediately on transplantation had to have a
very restricted cold ischemic time with these solutions. This improved with the development of
University of Wisconsin, or Belzer I, solution that contained the inert sugar lactobionate and starch, which
was very effective in preventing cell swelling (11). Both
456
Talbot and Soomro
kidneys and livers preserved with this solution could

be cold stored for longer with an increased chance of
primary function on reperfusion (12). After cold storage, reperfusion with blood generates oxygen free
radicals, and these further damage the graft. Though
the University of Wisconsin solution contains glutathione, which is a free radical scavenger, its slightly
acid pH means that the glutathione is not stable, and
so damage on reperfusion is a problem. Other solutions have been developed, notably histidine, tryptophane-ketoglutarate, and celsior, all of which contain
inert buffers and free radical scavengers that could
confer some advantages on reperfusion (13).
ASSESSMENT OF THE LIVE DONOR

Live donation in the United Kingdom is now governed by the 2004 Human Tissue Act, which became
law from September 2006. There is a legal requirement
in setting the transplant up (see above) and they can
only be performed in registered establishments.
Initial Assessment
This is usually performed by the nephrologist and
transplant coordinator, the former often referring the
potential donor to another nephrologists to avoid
conflict of interest. At the first consultation the following are established:
Medical, surgical and anesthetic fitness for surgery
Ensure the potential donor has two kidneys
Identify risk factors for the future development of
renal disease
Exclude the presence of diseases transmissible to the
recipient (e.g., infection or malignancy)
Provide information preferably both verbal and written on the procedure
Determine blood group compatibility (transplanting
across ABO barrier is possible but difficult)
Human leukocyte antigen (HLA) typing and crossmatching of the donor and recipient (determination
of donor specific antibodies present in the donor)
Subsequent Assessment
The perioperative mortality rate for living donor
nephrectomy is reported as between 1 in 1600 and
1 in 3300. The major perioperative complication rate
for donor nephrectomy is 2%.
Informed consent is undertaken with discussion
about the potential risks of donor nephrectomy,
including death and the general complications of surgery with specific mention of venous thromboembolism, intra-abdominal bleeding and infection,
chest complications, urinary tract infections, and the
possible need for blood transfusion.
The primary aim of the donor evaluation process
is to ensure the suitability, safety and well-being of the
patient. All potential donors have their ABO and HLA
compatibility with the donor checked. In addition, the
Figure 1 Magnetic resonance angiography for donor vessel

assessment.
recipients serum is tested against the donor cells by

cross-match.
In the past, renal anatomy was defined in the
donor by abdominal ultrasound, intravenous urography and renal angiography. This meant several hospitals visit with potential morbidity from anaphylaxis
and vascular damage from angiography. Now, in
most centers, either spiral computerized tomography
(CT) or magnetic resonance (MR) angiography with
3-D reconstruction has replaced the need for all these
tests. The procedure requires one visit to the hospital,
and the visualization of the renal venous anatomy is
excellent (Fig. 1).
Assessment of glomerular function rate (GFR) is
done in all the potential donors. Serum creatinine is
often used as a surrogate marker, and the CockcroftGault equation is widely used.
Male GFR (mL/min) 5 (140, age) 3 ideal body
weight (kg)/0.8 3 serum creatinine (lmol/L)
(Female GFR 3 0.85)
A creatinine clearance of 80 mL/min/73 m2 is a
reasonable lower limit for kidney donation. The radioisotope glomerular filtration rate obtained with 51Cr
EDTA is a more reliable measure, and this test can
also provide information about split renal function. A
minimum level of acceptable GFR is stipulated to
prevent the donor from being put at risk) (Table 1).
This level depends on the age, and it has to be above
two standard deviations below the mean for the age
(BTS guidelines) (Fig. 2).
The absolute and relative contraindications for
live donations are tabulated in Table 2 (1416).
ASSESSMENT OF CADAVERIC DONOR
Table 1 Acceptable GFR Prior to Donation

Donor age (yr)
GFR (mL/min/1.73m2)
Up to 40
50
60
70
80
86
77
68
59
50
On the basis of British Transplant Society guidelines (www.BTS.org.uk/

standards).
Figure 2 Diagram showing the variation with age of mean GFR

(solid black line). The outer dashed lines show the 2 population
standard deviation limits. GFR is constant up to the age of 40
years and then declines at the rate of 9mL/min/1.73 m2 per
decade. The reference plot is based on an analysis of data for
428 live renal transplant donors who had 51Cr-ETDA GFR
measurements performed according to the method described
in the British Nuclear Medicine Society GFR guidelines. The solid
gray line shows the safety limit of 86 mL/min/1.73 m2 for young
adults declining to 50 mL/min/1.73 m2 at age 80. For transplant
donors with preoperative GFR values above the solid gray line
the GFR of the remaining kidney will still be greater than
37.5 mL/min/1.73 m2 at age 80. Source: From Ref. 2.
Table 2 Contraindications to Donation for Renal Transplantation

Absolute contraindications
Evidence of coercion
Inability to give informed consent
Most malignancies
HIV
Major respiratory or cardiovascular disease
Hypertensive end-organ damage
Body-mass index 35 kg/m2
Pregnancy
Intravenous drug use
Thrombophilia
Renal disease
Relative contraindications
Age below 18 yr
Age over 70 yr
Body-mass index of 3035 kg/m2
Cigarette smoking
Psychiatric disorder
Risk factors for type 2 diabetes
Hepatitis B infection
Hypertension
457
In the United Kingdom, cadaveric donor rates are

24 per million population. This is the largest source
of kidneys for transplantation. The number of potential donors is much higher, but lack of awareness
among the staff and donors relatives about donation
means that these numbers have not increased.
The two main goals of donor evaluation are to
exclude donors with severe disease, which can be
transmitted to the recipient and to exclude kidneys
with severe anatomical or functional changes, which
could compromise allograft function.
All donors with malignancy are excluded except
those who have cutaneous basal cell carcinoma or
primary brain tumors with no metastases. Because of
the risk of transmitting choriocarcinoma, chorionic
gonadotrophin is estimated in all females of childbearing age who have died from cerebral hemorrhage.
The presence of HIV should preclude the use of
organs. Donors with active hepatitis B and C infection
are generally excluded, although organs can be transplanted safely into recipients who have already been
exposed to these viruses. Measuring viral gene loads
can be useful to assess the donors infectivity. Any
possibility of new variant Creutzfeldt-Jacob disease
always precludes donation. Donors who have had
adequate treatment of bacterial or fungal infection
can be used, but a cautious approach is mandatory.
Kidneys from old (<70 yr) or young (<2 yr)
donors or those with a body weight of less than
15 kg perform poorly. Prolonged hypotension, profound use of inotropic drugs, disseminated intravascular coagulation and diabetes mellitus in donors are
relative contraindications. Those with high serum creatinine levels can be used safely as kidney donors provided they have a reversible cause of their kidney
failure, such as hepatorenal failure. When the renal
impairment is established it is unwise to transplant the
kidney into a high demand recipient, that is, young
recipient, particularly male, with a large muscle mass.
Male donor to elderly female is more appropriate and
dual transplant better still. In general, the GFR of the
recipient will not be better than the donor and is likely
to be worse.
ASSESSMENT OF THE RECIPIENTS

Ideally, patients with chronic renal failure should
receive a transplant at precisely the time that they
require a transplant. Unfortunately, this is rarely possible. The timing of the transplant work-up is based on
the assumption that most patients would wait for
cadaveric transplant for up to two years, and it takes
approximately three months to prepare for a living
donor transplant.
The assessment aims to look at recipient suitability to undergo a surgical procedure, and to identify any underlying disease or condition that may
delay or would need to be rectified prior to transplantation. It is also an opportunity for the recipient to
know what transplantation involves and the pitfalls
and possible side effects of immunosuppression.
458
Talbot and Soomro
Cancer is generally a contraindication to immunosuppression, and patients who have active or

recent evidence of malignancy, except for some skin
cancers, may need to postpone or cancel transplant
evaluation. It would be prudent to wait for some time
to exclude patients who would develop a recurrence.
Delay of longer than five years would exclude 87% of
patients who would develop a recurrence, whereas a
two-year wait would eliminate about 53% of recurrences. Generally, in most malignancies, patients
should be free from recurrence for two years before
transplantation, but a longer waiting time is required
for malignant melanomas, breast cancer and colorectal cancer. No waiting time may be necessary in
most skin cancers.
Cardiovascular disease is one of the leading
causes of death after renal transplantation. Specific
indicators of underlying cardiac disease are a definite
history of acute myocardial infarction within the past
six months, angina, ventricular arrhythmia, or left
bundle branch block. Unfortunately, the ability of
these indicators to predict a postsurgical cardiac
event is not reliable.
The accepted risk factors for cardiovascular disease are as follows:
1.
2.
3.
4.
5.
6.
7.
Family history of premature coronary artery disease

Hypertension
Diabetes
Men over the age of 45
Women over the age of 55
Decreased high-density lipoprotein
Increased low-density lipoprotein
For asymptomatic patients who are at high risk or

have a history of IHD, a noninvasive test is quite
useful to screen patients who might require coronary
artery angiography. Thallium scintigraphy and dobutamine echocardiography have been used to screen
such patients. All patients who have critical lesions
on coronary angiography should undergo revascularization before transplantation.
Patients with a history of transient ischemic
attacks or other evidence of cerebral vascular disease
within the previous six months should have further
evaluation. Patients with autosomal dominant polycystic kidney disease (ADPKD) have an increased risk
of ruptured intracranial aneurysms. They should have
MR angiography, and patients who have an aneurysm
larger than 7 mm would probably benefit from prophylactic surgery.
Hepatitis A and E are not known to cause
chronic liver disease in renal transplant recipients.
However, recipients who are hepatitis B surface antigen positive, or are e antigen positive (HbeAG), or
who have serological evidence of active viral replication are at a higher risk of disease progression in the
posttransplant period. Patients who are hepatitis C
positive have an increased incidence of chronic active
hepatitis.
Extensive work-up is recommended for children
and also adults who have a history of urological
abnormalities or urinary tract infections. Renal ultrasonography and video-urodynamic studies may be
indicated to look into the possibility of reflux and

bladder physiology.
The risk of recurrent disease to the transplant
kidney is real, but only around 5% graft loss is
attributed to recurrent disease. There is a 10% to
25% risk of recurrence of hemolytic uremic syndrome
and 10% to 30% risk of recurrence of focal segmental
glomerulosclerosis; 40% to 50% of patients with recurrence will experience graft loss, and there is a 10% to
25% risk of recurrence in recipients whose original
disease was membranous glomerulonephritis. In some
cases, the type of immunosuppression used can affect
the probability of disease recurrence; for example,
calcineurin antagonists (ciclosporin and tacrolimus)
promote recurrence of hemolytic uremic syndrome.
NB it is important to have conducted genetic testing
for HUS as disease recurrence can be inevitable no
matter what the immunosuppression in which case
there are a growing number of patients offered combined liver and kidney transplant for this condition to
minimize any chance of this happening.
When recipients have met the criteria, they are
put on the waiting list to receive a cadaveric organ
through the national sharing scheme. At the time of
the availability of a suitably matched kidney, a final
cross-match is done before transplantation.
MATCHING DONOR AND RECIPIENTS

From the first successful renal transplant, it has been
known that for the best results the recipient should
have tissue similarities with the donor. This tissue
similarity is essentially the ABO blood group and
the major histocompatibility complex also known as
the HLA (carried by chromosome 6). For major blood
group matching, the same rules apply as blood transfusion, as the endothelium of the kidney is thrombogenic in an ABO mismatched combination. The major
histocompatibility complex is subdivided into classes
I and II, which, in man, comprise the HLAs A, B, C (I),
and D (II), respectively. Though class I is expressed by
all cells, the role of the professional antigen-presenting
cells is more important, and these express class II.
Therefore, matching for class II between donor and
recipient is more important. Matching has a significant impact on long-term graft survival; therefore,
most organ-sharing schemes have this as the principal
philosophy. However, paradoxically, in live unrelated
donation, usually spouse to spouse, where cold ischemic time is minimized, the normal milieu of the kidney has minimal disruption, and therefore the
recipients immune system does not become aware
that any change has occurred. As a consequence,
maximal mismatched kidney transplants can be tolerated without inevitable rejection. This state of affairs
is also improved by the use of more successful modern immunosuppressive drugs.
Prior to transplantation, the recipients are tested
for their immune sensitivity to the donor. This is done
by incubating the donor lymphocytes with the recipients sera to determine whether or not antibodies are
present. This is usually detected by the addition of
459
complement and viability stains for cell death. Flow

cytometry can also be used to detect lower concentrations of antibody and to determine the type of
antibody. When present, IgG is suggestive of memory
and therefore has alloreactive potential, whereas IgM
is more nonspecific. To transplant in the face of a
positive cytotoxic cross-match carries the risk of
hyperacute rejection when intravascular thrombosis
can occur within minutes of reperfusion.
RENAL TRANSPLANTATION
The surgical technique of transplantation is quite
standard and uses the extraperitoneal iliac fossa
approach. The vessels are accessible at this point,
and the ureter can be kept short, thereby minimizing
the risk of ischemia. With careful surgical technique,
no ureteric stent is required, but in large transplant
units with many surgeons of different grades, most
units utilize a ureteric stent, as it guarantees at least a
minimal level of anastomotic technique. It also means
the incidence of leak is extremely low. The external
iliac artery is the commonest artery used, but the
internal and common iliac arteries are also used
(Figs. 3 and 4). In children with small vessels, it is
important to utilize the common iliac artery or aorta to
ensure a good pressure inflow, as arterial thrombosis
is a common reason for graft failure. In those recipients of numerous previous renal allografts, an intraperitoneal approach and even an orthotopic approach
with native nephrectomy at the time of transplant
have been used successfully.
IMMUNOSUPPRESSION
Transplantation could not develop until the immune
system was tamed by drugs. These initially were very
nonspecificradiotherapy, steroids and 6-mercaptopurinebut when used in conjunction with good
donor-recipient matching, they produced 10-year
graft survival not dissimilar to those achieved today.
Figure 3 Anastomosing the transplant renal vein to the recipient external iliac vein.
Figure 4 Anastomosing the transplant renal artery to the recipient external iliac artery.
Azathioprine, derived from 6-mercaptopurine, was

developed in the 1960s. It is an antimetabolite and
therefore produces marrow suppression. Antithymocyte globulin is a polyclonal antibody initially derived
from the horse and later from the rabbit. This antibody
was introduced in 1968; it lyses human leukocytes and
thereby produces a cytokine storm, giving the recipient a self-limiting temperature and rigors. This was
found to be of particular benefit in steroid-resistant
rejection. OKT3, which is a monoclonal antibody
directed against T lymphocytes, was developed to
supersede ATG. Lymphoma, recognized as a particular problem of strong immunosuppression, was
slightly more common with OKT3. The calcineurin
antagonists ciclosporin and later tacrolimus were
derived from soil organisms and were found to be
very effective in minimizing the immunological
response; that is, IL2 production was inhibited. These
drugs produced a spectacular improvement in graft
survival up to five years. Beyond this, chronic graft
dysfunction produced steady graft attrition, so that at
10 years, graft survival was similar to that seen with the
more primitive agents. Mycophenolate mofetil and
mycophenolic acid inhibit the purine pathway, most
cells except lymphocytes have an escape pathway, so
lymphocyte proliferation is inhibited. Animal work has
suggested that these agents have benefit in chronic
graft dysfunction, though human data have not yet
confirmed this. Rapamycin, which is also derived from
soil organisms, downregulates IL2 production, but in a
slightly different way from ciclosporin and tacrolimus,
and it therefore is not complicated by the calcineurin
nephropathy that may in part contribute to chronic
graft dysfunction. Rapamycin could possibly therefore
also have benefits for long-term graft function.
In most transplant units combination therapies
of the above agents are used; increasingly popular is
antibody induction using IL2 receptor blockers, which
allows the oral medication to be used more sparingly.
Serious bacterial infection is not a common problem now as it was in early transplant experience,
460
Talbot and Soomro
where excessive immunosuppression was often used.

Viraemia is now more of an issue as a consequence of
immunosuppression, particularly with Epstein-Barr
virus or herpes 6, the former giving rise to neoplasia
and the latter to Karposis sarcoma. BKV and JCV,
which are related to SV40, and belong to the polyoma
virus group, can be troublesome after transplant. BKV
causes interstitial nephritis and ureteric strictures,
while JCV can cause leucoencephalopathy in the
immunosuppressed patient.
POSTTRANSPLANT MANAGEMENT
In the early postoperative period, fluid management
and blood pressure are the most important aspects, as
vascular thrombosis and pulmonary edema are serious consequences of both extremes of fluid state.
Immunosuppression should be modified to obtain
suitable levels of calcineurin antagonists. The patients
have generally been on antihypertensive agents, and
these may not be suitable after a transplant. ACE
inhibitors can often be used safely later, but renal
impairment at this time complicates the recipients
management. Antiviral prophylaxis is common for
cytomegalovirus (CMV), especially if the donor has
been exposed to this infection before and the recipient
has not. Gangciclovir, valacyclovir, or valganciclovir
can be used in this situation, though many units
choose to monitor for CMV by PCR and treat only
when viremia develops.
The recipients are supported until such time as
the kidney starts to work, and graft dysfunction is
monitored by graft biopsy to identify acute rejection
early. Acute rejection is usually treated by augmented
steroids, with or without altering the baseline regimen. Steroid-resistant rejection is still often managed
by poly- or monoclonal antibody therapy. Urinary
tract infections are managed with appropriate antibiotics, and the ureteric stent is often removed early in
this situation.
LONG-TERM MANAGEMENT
This is essentially a balancing act, retaining renal
function while trying to minimize potentially harmful
medication and avoiding rejection. Early after transplantation, immunosuppression is maximal. The
primary aim at this time is to reduce steroid augmentation without incurring rejection to baseline levels.
Fluid management is usually only relevant in the first
few weeks before it stabilizes, unless other problems
arise. Blood pressure needs to be optimized initially
by non-ACE inhibitor drugs, though they can be used
later, especially if proteinuria is detected.
Hypercholesterolemia can be a problem, particularly in recipients receiving ciclosporin or rapamycin, and they may require treatment with statins.
Diabetes mellitus, if present, usually worsens with
steroids. In addition, diabetes can develop where it
was not present before; this is especially the case
with tacrolimus, and it can be reversed by switching
immunosuppression to rapamycin. There is a great
propensity in the early phase for recipients to put on

weight by excessive eating. This occurs because they
are suddenly recovering from renal failure and feel
fitter, and the dietary restrictions of fluid and potassium are lifted. In addition, steroids increase their
appetites. Good dietary care is needed in this early
phase because reversing excessive weight gain later is
difficult.
The renal function must be monitored together
with immunosuppressive drug levels. Graft dysfunction must be investigated after elimination of obvious
courses by ultrasound scans and biopsy. Ureteric
stenosis should be managed appropriately, usually
by, initially, ureteric stent and later ureterotomy or
reimplantation, though there is a role for revolving
ureteric stents.
Acute rejection should be managed by steroid
augmentation and drug manipulation if necessary.
Consideration should always be given to patient compliance with immunosuppression, as this is a significant cause of graft failure, particularly in adolescent
recipients that are graduating to adult clinics. Osteoporosis and atherosclerosis from protracted steroid
use give rise to significant morbidity later; therefore,
steroids should be limited as much as is safe to do.
Similarly the use of steroids in child hood can have
irreversible affects on long bone development. The
small stature of children with renal failure though still
a problem is not as acute as was the case formerly due
to the common use of growth hormone.
Vigilance for skin tumors, cervical cancer,
Karposis sarcoma, and hypernephroma, particularly
in polycystic kidneys and posttransplant lymphoproliferative disease, should always be practiced.
RESULTS
In the early days of renal transplantation in the 1950s,
the only successful transplants were those between
identical twins, obviously a very select group. With
the advent of azathioprine and steroid, one-year outcome was of the order of 35% to 40%. This figure
improved dramatically with the advent of ciclosporin.
Since that time, a one-year recipient death rate of 5%
and graft survival of greater than 80% have become
usual. These improved figures extend to 10 years after
the transplant, but because of a steady attrition rate
from chronic rejection, the outcome after 10 years is
similar with modern immunosuppressive agents to
that seen in the preciclosporin era (17) at 40%. The best
results are generally seen in the recipients of live
donor kidneys with minimal cold ischemia, among
whom there is a graft half-life of 15 years. Those grafts
which experience initial delayed function have a significantly worse long-term outcome. In addition,
donor morbidity, such as hypertension, and death
from cerebrovascular disease have a negative impact
on graft survival. Poor tissue match between donor
and recipient, and recipient hypertension and smoking habits all have a negative impact on outcome. The
hidden issues of noncompliance with immunosuppressive medication could potentially have an
enormous impact on outcome (18), though one can

take the contrary view that certain drugs promote the
cytokine TGFb, which promotes fibrosis and therefore
potentially chronic rejection (19).
REFERENCES
1. Security DoHaS. Cadaveric organs for transplantation. A
code of practice including the diagnosis of brain death.
London: Health Departments of Great Britain and Northern Ireland, 1983.
2. Transplantation. EBPGfR. Nephrol Dial Transplant 2000;
15(suppl 7):4647.
3. Balupuri S, Buckley P, Snowden C, et al. The trouble with
kidneys derived from the non heart beating donor: a single
centre 10 year experience. Transplantation 2000; 69:842846.
4. Gok M, Buckley P, Shenton B, et al. Long-term renal
function in kidneys from non-heart-beating donors: a single-center experience. Transplantation 2002; 74:664669.
5. Nicholson M, Metcalfe MS, White SA, et al. A comparison
of the results of renal transplantation from non-heartbeating, conventional cadaveric and living donors. Kidney
Int 2000; 58:25852591.
6. DAlessandro A, Hoffmann R, Knechtle S, et al. Successful
extrarenal transplantation from non-heart-beating donors.
Transplantation 1995; 59:977982.
7. Newman C, Baxby K, Hall R, et al. Machine-perfused
cadaver kidneys. Lancet 1975; 2:614.
8. Burdick J, Rossendale J, McBride M, et al. National impact
of pulsatile perfusion on cadaveric kidney transplantation.
9. Light J. Viability testing in the non-heart-beating donor.
Transplant Proc 2000; 32:179181.
461
10. Brasile L, Stubenitsky B, Booster M, et al. Overcoming

severe renal ischemia: the role of ex vivo warm perfusion.
11. Hoffman R, Stratta R, DAlessandro A, et al. Combined
cold storage-perfusion preservation with a new synthetic
perfusate. Transplantation 1989; 47:3237.
12. Groenewoud A, Thorogood J. Current status of the Eurotransplant randomized multicenter study comparing kidney graft preservation with histidine-tryptophaneketoglutarate, University of Wisconsin and EuroCollins.
Transplant Proc 1993; 25:15821585.
13. Muhlbacher F, Langer F, Mittermayer C. Preservation
solutions for transplantation. Transplant Proc 1999;
31:20692070.
14. Association BTSaR. United Kingdom Guidelines for Living
Donor Kidney Transplantation, 2000.
15. Kasiske B, Ravenscraft M, Ramos E, et al. The evaluation of
living renal transplant donors: clinical practice guidelines.
J Am Soc Nephrol 1996; 7:22882313.
16. Kasiske B, Ramos E, Gaston R, et al. The evaluation of renal
transplant candidates. Clinical practice guidelines. Patient
Care and Education Committee of the American Society of
Transplant Physicians. J Am Assoc Nephrol 1995; 6:134.
17. Beveridge T, Calne R. Cyclosporin (Sandimmun) in cadaveric renal transplantation. Ten-year follow up of a European
multicenter trial group. Transplantation 1995; 59:15681570.
18. de Geest S, Borgermans L, Gemoets H, et al. Incidence,
determinants, and consequences of subclinical noncompliance with immunosuppressive therapy in renal transplant
recipients. Transplantation 1995; 59:340347.
19. Mohamed M, Robertson H, Booth T, et al. TGF-b expression in renal transplant biopsies. A comparative study
between cyclosporin-A and tacrolimus. Transplantation
2000; 69(5):10021005.
31
Tissue Transfer in Urology
Daniela E. Andrich and Anthony R. Mundy
INTRODUCTION
Two types of tissue transfer are common in urology,
firstly, the use of skin and buccal mucosa for urethral
reconstruction and, secondly, the use of bowel for
bladder reconstruction. The three factors that require
some consideration in relation to the subject of tissue
transfer in these contexts are the general subject of
wound healing, the use of grafts and flaps, and the
consequences of incorporating bowel into the urinary
tract.
The final section of this chapter will give a brief
overview of tissue engineering. In recent years there
has been a great deal of progress in tissue engineering
in urology with the aim of developing alternative
tissue sources for urethral and bladder reconstruction,
amongst other things. The most recent and exciting
developments in this field overall have been in the use
of pluripotent stem cells although their application in
urology is still in its infancy.
WOUND HEALING
This is the replacement of destroyed tissue by living
tissue. In certain reptiles, it includes the ability to
replace a tail or limb. This occurs because of the ability
of cells to dedifferentiate and then redifferentiate (1),
which does not occur in human adults.
Healing in adults is limited to the fibrous obliteration of dead space and limited regeneration. In the
embryo, scarless healing has been noted and this is
currently the focus of intense research. Fetal wounding shows less inflammatory infiltrate with less content of the inflammatory cytokine transforming
growth factor b (TGF-b) compared with the adult (2)
and fetal fibroblasts have been shown to be of particular importance in scarless wound healing (3).
The Phases of Wound Healing

Wound healing is traditionally divided into three
phasesan inflammatory phase, a proliferative
phase and a maturation phase. These phases overlap
to some degree, but as a general rule the inflammatory
phase lasts for about the first five days, the proliferative phase lasts from about day 3 to about day 14,
and the maturation phase starts on about day 8 and
lasts about three months. Each of these phases is

controlled and regulated by growth factors.
The first phase of wound healing is the hemostatic response to the injury itself and the inflammation
that follow immediately afterward. At the site where
blood vessels and the surrounding tissues are injured,
circulating blood comes into contact with extracellular
collagen and platelets aggregate and are activated and
the intrinsic component of the coagulation cascade is
activated as well. The platelets release the contents of
their granules, including cytokines, growth factors,
fibronectin, thromboxane and serotonin. The thromboxane and serotonin promote vasoconstriction at the
site of the injury, whereas vasodilatation occurs around
that site to allow other important healing factors to get
to the wound. The vasodilatation is mediated by histamine, which is also released from platelets but other
cells as well such as mast cells and basophils.
At the same time, both the intrinsic and the
extrinsic coagulation systems are activated, leading
to the formation of a fibrin clot. This serves as a
scaffolding to allow inflammatory cells such as neutrophils, macrophages and lymphocytes to move into
the injured area. The neutrophils arrive first. Selectin
receptors on the endothelial cell surface cause neutrophil adhesion to the endothelium, and integrin receptors on the neutrophil cell surface bind to the
extracellular matrix, thus bringing the neutrophils
into the damaged area. Neutrophils, however, are
not essential to wound healing except to deal with
bacterial contamination when that exists. The next
cells to arrive, the macrophages, are essential. They
arrive on about the third day after injury and act as the
principal cell controlling the wound-healing process.
The main functions are as follows:
1.
2.
3.
4.
5.
An antimicrobial effect caused by the release of

oxygen radicals and nitric oxide, and by phagocytosis
Wound debridement by phagocytosis and by the
release of various enzymes
Matrix synthesis regulation by the release of growth
factors, cytokines, enzymes and prostaglandins
Cell recruitment and activation by the release of
growth factors, cytokines and fibronectin
Angiogenesis by the release of growth factors and
cytokines
Chapter 31: Tissue Transfer in Urology
The principal cells recruited and activated are

fibroblasts and lymphocytes, which migrate in toward
the end of the inflammatory phase. The principal
enzyme released is collagenase. The principal growth
factors released are TGF-b, EGF, PDGF and the angiogenesis growth factors bFGF and VEGF (4). The principal cytokines are TNF-a, IL-1, IFN-c and ILC.
Lymphocytes are attracted by the cytokines
released by activated macrophages, and these in
turn release interferons and interleukins. They also
produce nitric oxide. The arrival of the fibroblasts
heralds the second, or proliferative phase. Their
number increases, so the number of neutrophils
decreases.
Fibroblasts, together with macrophages, then
start the process of matrix formation and collagen
synthesis. At the same time, endothelial cells proliferate from the margin of the wound to form new
capillaries by the process of angiogenesis (5). At this
stage, the inflammatory phase is over, and any
remaining inflammatory mediators are inactivated
and removed from the wound by diffusion or by the
macrophages. The fibroblasts are activated by PDGF
and FGF, and also by complement and fibronectin,
which binds fibroblasts and helps them to move into
the wound in the same way that integrins in the
extracellular matrix bound neutrophils in the early
inflammatory phase.
As activated fibroblasts begin the process of
matrix deposition, largely by replacing the fibrin and
fibronectin originating from hemostasis and macrophages with collagen, epithelial cells start to proliferate from the wound edges. Again, macrophages are
important, producing appropriate cytokines and
growth factors. As the proliferative phase comes to
an end and the maturation phase becomes established,
macrophages (and neutrophils) start to leave the area.
The cardinal feature of the maturation phase is
the deposition of collagen. It is the rate, quality and
total amount of collagen deposition that determines
the strength of the scar, and most of the problems of
healing that arise are secondary to poor collagen
deposition.
Initially, in the inflammatory phase, the matrix
deposited in the wound was composed principally of
fibrin and fibronectin that originated from hemostasis
and from macrophages, respectively. Other extracellular micoproteins, such as glycosaminoglycans and
proteoglycans, come next. Collagen follows after this.
In the intact dermis, collagen I predominates,
forming 80% to 90% of the total collagen. The remaining 10% to 20% is collagen III. In the granulation tissue
of the maturation phase, there is rather more type III
collagenas much as 30%although this reduces in
time to about 10%. Collagen III production actually
peaks during the inflammatory phase, while collagen I
production, which starts about the first day after
injury, continues for about four to five weeks. Initially,
the collagen is laid down in thin collagen fibers, parallel to the skin and organized along the stress line of
the wound. In time, the collagen fibers become
thicker, but they never develop the basket-weavelike pattern characteristic of normal dermis. It is
463
perhaps for this reason that the breaking strength of

a scar never quite equals the breaking strength of
normal skin. At one week, the breaking strength of a
wound is about 3% to 5% of its final strength; by three
weeks, it has reached 20% of its final strength; by three
months, it reaches 80% of the strength of unwounded
skin, and this is about as strong as it ever gets.
Wound contraction is the name given to the final
approximation of the wound edges and the shortening
of the scar that is typically seen. In the process of
healing by first intentionwhich is what we have
been describing so farcontraction is less marked
than in healing by second intention. In both circumstances, the process is the same and poorly understood. One theory proposes that the myofibroblasta
special type of fibroblastis responsible for this (6).
The other main theory is that the movement of cells
with reorganization of the cellular cytoskeleton is
responsible for contraction. In summary, the exact
mechanism is not clear.
Healing by secondary intention (where the
wound edges are widely separated) differs in two
respects.
1.
2.
Epithelialization occurs from the basal cells of the

epidermis at the margins of the wound. These
cells lose their attachment to the basement membrane and send out cytoplasmic projections,
becoming flatter as they do so. As they change,
they become more like phagocytes in appearance.
Within a day or two of injury, the basal cells show
mitosis at an increasing rate and continue to
replicate until the defect is covered. When coverage is complete and the epidermis (or other epithelium) has reconstituted itself, the basal cells
regain their normal appearance and synthesize
their own basement membrane constituents.
Contraction plays a far more important role in
healing by secondary intention. Animal models
have demonstrated contraction of up to 80% (7).
However, the pathological process involved in
contraction is the same.
Complications of Wound Healing

Complications include infection, wound dehiscence,
excessive granulation, keloid, pigmentation, pain,
weak scars, circatrization, implantation cyst and neoplasia. Factors influencing healing are listed in Table 1.
Table 1 Factors Influencing Wound Healing
Local factors
General
Poor blood supply

Adhesion to underlying structures
Direction of wound
Infection/foreign body
Movement
Drying
Neoplasia
Age
Nutrition (protein, vitamin C, zinc)
Hormones
Temperature
464
Andrich and Mundy
GRAFTS AND FLAPS

Most surgical trainees gain their first experience of
grafting in the use of thin split-skin grafts to cover a
granulating open wound. The open wound, which is
secreting collagen and actively generating small new
vessels, is covered by a nutrient-rich layer of serum,
from which the thin split-skin graft gains its nutrition
by a process known as imbibition. This is sufficient
to nourish a split-skin graft for a day or two, but to
nourish it beyond that time, as well as to fix it and
incorporate it into the healing wound, there must be a
link-up between the capillary bed on the undersurface
of the graft and the developing capillaries in the open
wound, together with interlinking of collagen, and
this process is known as inosculation. During the
48 hours of imbibition and the 48 hours of inosculation, the graft must be immobilized and if the host bed
is well vascularized, then the graft will take. At the
end of 96 hours, blood flow into the graft should be
established. Eventually, lymphatic vessels will grow
in as well. The tie-over dressing commonly used for
immobilizing a skin graft clearly produces some pressure. However, too much pressure, sufficient to inhibit
completely the formation of small hematomas and
seromas, would probably be sufficient to impair
blood flow in the host bed (8,9). In other words, the
dressing is to immobilize the graft; it is not supposed
to be hemostatic.
Thus far, we have been considering the simple
split-skin graft, and at this stage we should draw
attention to the difference between grafts and flaps.
A graft is tissue transferred from a donor site to a
recipient site without a blood supply, whereas a flap
is tissue that is transferred from a donor site with its
own blood supply intact, although one type of flap
is the free flap in which tissue is transferred with its
blood supply disconnected and then reconnected by
means of microvascular surgery at the recipient site.
In urology we are principally concerned with
split-thickness skin grafts, full-thickness skin grafts,
buccal mucosal grafts, bladder epithelial grafts and
flaps of local genital skin for urethral reconstruction.
In addition, the urologist may wish to use a dermal
graft for the correction of Peyronies disease, and
some have used a graft of tunica vaginalis for this
purpose (911).
Other than the use of genital skin flaps for urethral reconstruction, simpler advancement, rotation
and transposition flaps are commonly used for
wound closure, although we do not often think of
them as such, except in the specific case of the Z-plasty.
Less commonly, flaps may be used for perineal
and genital reconstruction, and in this way a simple
scrotal flap may be used to cover a perineal defect, or
the infinitely more complex radial or ulnar forearm
flap may be transferred by microvascular techniques
for phalloplasty (12).
Grafts
Skin has an epidermal layer and a dermal layer, under
which lies a subcutaneous, fatty layer. At the interface
between the dermis and the subcutaneous layer is the

subdermal plexus, which is fed from deeper segmental vessels and which in turn feeds the dermal and
epidermal layers above through a second, more superficial, plexus within the dermis itself, called the intradermal plexus (Fig. 1). The outermost layer of the
epidermis is the cornified layer, and the inner layer
the epidermis propercontains the skin appendages,
some of which extend into the dermal layer. The
principal skin appendages are the sweat glands, sebaceous glands and hair follicles, and in urological
practice it is worth noting that the glans and prepuce
in the male and the skin of the labia minora and
introitus in the female are free of skin appendages.
The same areas also have skin in which the dermis is
thin. The dermis consists of the more superficial
papillary or adventitial dermis and the deeper
reticular dermis. The papillary dermis has a fairly
constant thickness, and fibroblasts and capillaries predominate. The deeper reticular dermis is thicker but
more variable and here collagen and elastin predominate. The subdermal plexus lies on the deep aspect of
the reticular dermis, and the intradermal plexus lies
between the reticular and the papillary dermis.
A full-thickness skin graft includes both the epidermis and the dermis with the vessels of the subdermal plexus exposed on its undersurface, and it is to
these vessels that the new vessels developing within
the host bed must connect by inosculation.
Full-thickness skin grafts, because of their high
content of dermal collagen, contract very little during
healing and tend to give the most satisfactory longterm results as a consequence, but there are two
problems associated with their use. Firstly, the
amount of suitable skin is restricted and, secondly,
the subdermal plexus is less extensive than the intradermal plexus and, because of this and also because
the graft is relatively thick, it is only slowly revascularized and takes only under the most favorable conditions. A postauricular full-thickness skin graft (or in
fact any facial skin graft harvested from above the jaw
line) has a denser subdermal plexus and therefore
takes more readily than other extragenital skin.
The split-thickness skin graft contains the epidermis and a portion of the adventitial dermis with
the vessels of the intradermal plexus exposed on its
undersurface. The graft is thinner and the vessels of
the intradermal plexus are more plentiful and so a
split-thickness graft is more likely to take and will do
so under less favorable circumstances than a fullthickness skin graft. On the other hand, because
there is less dermal collagen, a split-thickness graft
tends to contract and, lacking the collagen and elastin
of the reticular dermis, the graft tends to be more
brittle and fragile than a full-thickness graft.
Bladder epithelial grafts and buccal mucosal
grafts are harvested and behave as full-thickness
grafts. Bladder epithelium has the disadvantage that
it is very prone to desiccation, which leads to hypertrophic changes when, for example, it is used at the
urethral meatus (13). Buccal mucosa is far more resistant to desiccation, and there is the additional advantage that it is easier to open the mouth than the
465
Figure 1 Cross-sectional diagram of the

skin. Abbreviations: STSG, split-thickness
skin graft; FTSG, full-thickness skin graft.
bladder. The particular advantages of buccal mucosa

are its thickness and toughness (which contrast markedly with the thinness and fragility of bladder epithelium), and the density of the intradermal and
subdermal plexuses (14) (Fig. 2).
Grafts were the first sort of tissue to be used for
urethral reconstruction but fell into disfavor because
of their unpredictability until just recently, with the
advent of buccal mucosal grafting for patch urethroplasty of strictures that are too long for excision
and end-to-end anastomosis (15). Until then, grafts
had become virtually restricted to the use of meshed
split-skin grafts for the salvage repair of extensive
urethral strictures (16,17). This is a technique that is
only rarely indicated nowadays. A free preputial skin
graft might be used by specialist reconstructive surgeons for anterior urethroplasty, particularly for
hypospadias repair, but grafting had become almost
entirely superseded by the use of flaps for hypospadias repair and for urethroplasty in the penile urethra,
largely under the influence of Duckett (18). Preputial
and postauricular full-thickness skin grafts are now
being used more frequently again, particularly for the
two-stage reconstruction of the penile urethra (19).
Postauricular full-thickness skin grafts have a particularly rich subdermal plexus and, although the
amount of tissue is relatively restricted, the take is
far more predictable than with other extragenital skin.
Thus, preputial or postauricular full-thickness skin

grafts or buccal mucosal grafts, which behave like
full-thickness grafts, may be used to reconstruct the
anterior urethra. Buccal mucosal grafts are particularly useful for the repair of urethral strictures because
of lichen sclerosus (also known as balanitis xerotica
obliterans or BXO) as buccal mucosa is not commonly
affected by this skin disease. Buccal mucosal grafts
and bladder epithelial grafts have also been used as
tube grafts in the anterior urethra (20) although tube
grafts of any sort are unreliable because of difficulties
with circumferential revascularization.
Flaps
The use of skin flaps in reconstructive surgery dates
back to northern India at about 600 BC, when Susruta
developed forehead rhinoplasty after a traumatic
nasal amputationa form of punishment at that
time (21). Then, in the 16th century, Gaspare Tagliacozzi from Bologna successfully transposed skin
pedicles from the arm to the face to reconstruct the
nose of patients who had lost theirs because of syphilis. Tagliacozzis work was disregarded by his peers
and it was not until World War One and Two when
surviving war veterans with shrapnel wounds, traumatic amputations and severe burns provided both
the opportunity and the need for advancements in
466
Andrich and Mundy
Figure 2 Subdermal plexus of buccal mucosal graft compared

with skin graft. (A) BMG. Dense subdermal capillary plexus. (B)
Skin. Less extensive subdermal capillary plexus, separate intradermal plexus.
Figure 3 Diagrammatic representation of: (A) a random peninsular flap; (B) an axial peninsular flap; (C) an axial island flap;
and (D) a free microvascular flap.
reconstructive surgery that led to the redevelopment

of such skin flap techniques (22) During the First
World War, Harold Gillies laid the foundations of
reconstructive plastic surgery. His cousin Archibald
McIndoe continued to perfect reconstructive techniques during World War 2, culminating (urologically)
in the first phallic reconstruction in the 1960s using a
staged tubed pedicled skin flap technique (23).
In urology we are principally concerned with
skin flaps for urethral and genital reconstruction,
omental and labial fat pad flaps to help support
vesicovaginal fistula closure, gracilis muscle flaps to
support urorectal fistula closure and intestinal flaps
for bladder reconstruction. Flaps may be classified in
two ways (8,9). Firstly, according to their method of
elevation, into: peninsular flaps, in which the base of
the flap remains in continuity; island flaps, in which
the flap is only in continuity through the arteriovenous pedicle; and free flaps, in which all continuity of
the flap is interrupted and the vascularity is reestablished at the recipient site. The second way of classifying flaps is by vascular classification, which
distinguishes between: random flaps, in which there
is no identifiable arteriovenous pedicle and survival is

dependent entirely on the dermal and intradermal
plexuses; axial flaps, which have an identifiable artery
and vein entering through the base; and two special
forms of axial flap known as myocutaneous and
fascio-cutaneous flaps. Random flaps are restricted
in use by a length to width ratio whereas axial flaps
are not, being restricted in length only by the nature of
the feeder vessels. A peninsular flap may be either a
random or an axial flap, but island flaps are all axial
flaps (Fig. 3). Peninsular flaps are used for advancement, rotation and transposition but are relatively
restricted in scope. Axial island flaps have a much
greater range of application. Island flaps are somewhat fragile and have to be handled with care, as the
vascular pedicle provides both structural and vascular
continuitywith such a fragile structure that vascular
continuity is threatened unless great care is taken.
Sometimes, however, the continuity of an island flap
is maintained through a muscle or fascial flap, giving
rise to the two particular forms of axial flap referred to
above. In the myocutaneous flap, the arteriovenous
pedicle is within the muscle itself and the skin island
(or paddle) retains its attachment to the muscle.

Examples include gracilis and rectus abdominis muscle flaps, both of which have applications in urology
(24,25). Similarly, the fasciocutaneous flap transmits
the arteriovenous pedicle in close relation to a defined
layer of fascia to which the skin paddle is attached. In
both the myocutaneous and the fasciocutaneous flaps,
the muscle and fascia, respectively, support and protect the arteriovenous pedicle, making the flap more
robust.
Free flaps, to be transferred by microvascular
anastomosis to the recipient site, can be elevated as
axial cutaneous, axial myocutaneous, or axial fasciocutaneous flaps and are the most versatile flaps of all
because they can be transferred anywhere. The limitations in their use are firstly technicalthey are difficult to learn how to performand they tend to leave
large donor sites that may leave ugly scars (26)
although this is usually more than offset by a satisfactory end result from the flap itself. Most flaps are
mobilized with a defined sensory nerve to provide
sensibility to the flap. The best example of the use of a
free flap in urology is the ulnar forearm flap used for
phalloplasty (12). A radial artery flap is easier to raise
but has considerable donor-site morbidity because the
many tendons in the forearm are exposed and, even if
these can be covered, the donor site leaves an
unsightly scar. The ulnar forearm flap exposes
fewer, if any, tendons and the donor site is more
easily concealed and has the additional advantage
that the skin on the ulnar side of the forearm is
much less hirsute, but the flap is more difficult to
raise. Both flaps are innervated by the lateral and
medial antebrachial cutaneous nerves, which are easily identified and mobilized with the flap to provide
sensibility.
Gracilis and rectus myocutaneous flaps have
also been used for phallic reconstruction, but are
more commonly used for covering large skin defects
in and around the groins and perineum. The gracilis
muscle can also be used purely as a muscle flap for
filling dead space and holding suture lines apart, in
the same way that the labial fat pad and omentum are
used.
Intestinal Flaps in Bladder Reconstruction

These days there are probably more urologists using
bowel for bladder reconstruction or replacement by
continent or conduit urinary diversion than there are
urologists using skin for urethral reconstruction.
Bowel is always carried on its blood supply and,
as long as there is no undue tension on the vascular
pedicle, the bowel heals well in its new circumstances
and ischemia is rare, except when the bowel and its
blood supply are compromised by previous radiotherapy. The purpose of this section is not to discuss the
more technical aspects related to mobilizing gut segments for use in the urinary tract, but to discuss the
metabolic, infective, histological and other consequences of incorporating the gut into the urinary tract.
Such matters have been extensively studied by certain
467
groups in experimental models (27), and the clinical

consequences have been extensively scrutinized by
other groups in humans, but it is still not yet clear
how we should apply the theoretical knowledge
gleaned from such studies in clinical practice, as that
knowledge is currently very incomplete.
Bowel has been used in urology for many years
and this has engendered an attitude that it can be
freely deployed within the urinary tract without significant consequence. On the other hand, intestine was
never meant to serve either as a conduit for urine or as
a storage vessel for it, and a more likely explanation
for the fact that it appears to be capable of being used
freely without complication is that in most circumstances only a small section of bowel is incorporated
and, in most patients in whom such procedures are
performed, there are compensatory mechanisms to
deal with any adverse consequences that might
otherwise result. Furthermore, many of these patients
have only a short life expectancy, as when an ileal
conduit or substitution cystoplasty is used after cystectomy for bladder cancer, and the short duration of
use in a patient who is expected to die before long in
any case might lead one to overlook consequences that
might be apparent in younger patients with longer life
expectancy.
Even with this proviso, it was recognized many
years ago that patients with ureterosigmoidostomy
were prone to the specific metabolic problem of
hyperchloraemic acidosis from the absorption of urinary constituents by the colon (28), and prone also to
the development of tumors at the site of implantation
of the ureters into the colon (29). For these reasons,
ureterosigmoidostomy fell into relative disuse some
years ago (30). Other than the consequences of incorporating a segment of gut into the urinary tract, there
may be consequences from removing it from its natural situation. In practice, the only problem that commonly arises is a degree of bowel dysfunction (31), for
reasons that are not entirely clear. There is, in any
case, a strong association between detrusor instability
and the irritable bowel syndrome (32) and neuropathic bowel and neuropathic bladder dysfunction,
(33) and it is these two groups that are most prone to
notice disturbance of bowel function after augmentation cystoplasty. Bowel dysfunction is far less commonly noted after substitution cystoplasty for
conditions such as bladder cancer.
Removal of the terminal ileum or of the ileocaecal valve can interfere with the absorption of bile salts
(34) and lead to bacterial colonization of the terminal
ileum, both of which may give rise to diarrhea. For
this reason, the terminal ileum is avoided whenever
possible during such surgery. There has been concern
that the removal of the terminal ileum can give rise to
vitamin B12 deficiency and other forms of anemia, but
these rarely seem to occur in practice. It is not clear
how much ileum has to be removed from the intestinal tract before bile acid malabsorption is sufficiently
severe to cause malabsorption of fat and fat-soluble
vitamins, but malnutrition and steatorrhea are rarely,
if ever, seen in clinical practice. Diarrhea may occur
more frequently in patients after ileal conduit
468
Andrich and Mundy
diversion or continent diversion after previous bowel

excision or when the bowel is diseased or deficient in
any other way. Removing the right side of the colon
may cause diarrhea, although this does not seem to be
a long-term problem. Removal of the sigmoid colon
may be a problem in neuropathic patients in whom
the sigmoid colon is an important storage organ, but it
does not usually cause bowel problems otherwise. In
short, other than in those with previously abnormal
bowel, problems rarely arise from taking the bowel
out of continuity, and all we need really consider are
the problems that arise from incorporating it into the
urinary tract.
THE CONSEQUENCES OF INCORPORATING

THE BOWEL INTO THE URINARY TRACT
The first point to make is that bowel continues to
behave like bowel even when it is in the urinary tract
and has been there for many years. It continues to
produce mucusthe only overt abnormality that most
patients are aware ofand it continues to secrete and
absorb just as it does in its natural situation. There is a
tendency for both the ileal and colonic epithelium to
atrophy with time (35), and it is not clear whether this
represents the consequence of loss of the normal
stimulation that it receives during fecal transport in
its normal situation or whether, alternatively, it represents some toxic effect of urine. However, the atrophy is less when the intestine is in contact with urine
than when it is not in contact with anything at all, so it
appears that urine tends to maintain ileal integrity, if
anything, albeit less so than does the fecal stream. The
atrophic appearance is therefore likely to represent a
true, albeit partial, atrophy rather than a toxic effect.
Metabolic Changes
The principal metabolic abnormality is a tendency to a
respiratory-compensated metabolic acidosis, usually
of mild degree (36). This may influence bone metabolism to a sufficient degree to cause demineralization
of the skeleton in growing children (37). Changes
otherwise tend to be subtle, particularly, it is thought,
if renal function is normal. Thus, only young
patients are prone to any sort of problems in most
circumstances.
Why people seem to suffer less from metabolic
acidosis these days with a substituted intestinal bladder than they used to suffer with a ureterosigmoidostomy is almost certainly related, at least in part, to the
recurrent sepsis that they also suffered with refluxing
ureterosigmoidostomies (38). The nature and severity
of the electrolyte anomalies that patients can suffer
from depend on the segment of bowel used in the
urinary tract. If stomach is used, hypochloremic metabolic alkalosis can occur. This is rarely significant in
the presence of normal renal function, but may be
when renal function is severely limited.
When jejunum is used, hyponatremic, hyperchloraemic, hyperkalemic metabolic acidosis occurs,
and this is both common and potentially serious,
Table 2 Metabolic Changes Using Different Gut Segments for

Bladder Reconstruction or Replacement
Stomach
Jejunum
Ileum, colon
Hypochloraemic metabolic alkalosis

Hyponatremic, hyperchloremic,
hyperkalemic metabolic acidosis
Hyperchloraemic acidosis
resulting in lethargy, nausea, vomiting, dehydration,

muscular weakness and an elevated body temperature and, if uncorrected, death. This is the jejunal
conduit syndrome (39). If ileum or colon is used,
hyperchloraemic acidosis may occur (Table 2).
The reported incidence varies and to a certain
extent depends on how it is looked for. If the plasma
chloride level is the parameter used to make the
diagnosis, then about 15% of patients suffer; if arterial
blood gas analysis is performed, then a respiratorycompensated metabolic acidosis is found far more
commonlyindeed, in the majority (36). The mechanism of the metabolic acidosis when ileum or colon is
interposed in the urinary tract is not entirely clear.
What is known is largely derived from the work of
McDougall and coworkers (27). Water transport
across the intestinal epithelium normally follows its
osmotic gradient and is dependent on the permeability of the intercellular junctions of the luminal cells. If
these junctions are tight, there is very little leakage;
when they are not so tight, water follows its osmotic
gradients. Generally, the tightness of the intercellular
junctions increases the further down the intestinal
tract the segment is taken from. Stomach is very
leaky, but there are bidirectional fluxes that cancel
each other out. Jejunum is leaky and jejunal conduits
lose large amounts of water. Ileum is better but is still
somewhat leaky. Colon is the most efficient segment
of gut because its intercellular junctions are tighter,
and colon segments therefore have a much lower
tendency to lose water.
Most electrolyte shifts in the gut are transcellular, although paracellular movement of ions
does occur. Furthermore, most electrolyte movements
in one direction are coupled with movement of other
electrolytes in the opposite direction. Thus, when
sodium is absorbed, hydrogen is excreted and when
chloride is absorbed, bicarbonate is excreted. Under
certain circumstances, these transport processes can
be reversed.
Sodium absorption is much the same in the
ileum and in the colon and, in general, ileum absorbs
less chloride but more potassium than colon. In
neither ileal nor colonic segments is either sodium or
potassium loss a problem, although in extreme metabolic acidosis, hypokalemia can occur, as can hypocalcaemia and hypomagnesaemia.
The mechanism for the development of hyperchloraemic acidosis appears to be primarily because of
ammonia absorption. Ammonium ions may dissociate
into ammonia and hydrogen, in which case the ammonia diffuses into the cell and the hydrogen ion is
actively absorbed in exchange for sodium. Alternatively, ammonium may be absorbed as a substitute for
potassium through potassium channels. Either way,

ammonium enters the ileal or colonic luminal cell and
this is balanced by chloride absorption in exchange for
bicarbonate secretion. Thus, ammonium and chloride
are absorbed, causing hyperchloraemic acidosis, and
bicarbonate is lost.
When hypokalemia does occur it is usually in
ureterosigmoidostomy (40) or as a result of osmotic
diuresis. Hypocalcaemia is usually due to bone
demineralization. Hypomagnesaemia may be associated with hypocalcaemia but is more commonly due
to nutritional depletion or renal wasting.
In most patients this metabolic acidosis is of little
consequence but growing children with intestine
incorporated into the urinary tract have been shown
to be more prone to orthopedic problems and to
pathological fractures, and there was a tendency for
them to drop off the growth curve that they were
previously following (41). Exactly why acidosis
should lead to skeletal demineralization in growing
children and not in adults is not clear, but postmenopausal women who might also be prone to skeletal
demineralization do not show this abnormality after
enterocystoplasty or urinary intestinal diversion.
However, this is controversial, as more recently a
large cohort study of 123 children followed up into
their adolescence after childhood enterocystolasty.
The distribution of percentile positions before and
after enterocystoplasty showed normal configuration
and 85% of children either remained on the same or
reached a higher centile (42).
Again, the mechanism by which acidosis causes
demineralization of the skeleton is not clear, but
chronic acidosis is buffered predominantly by muscle
protein and by bone. In bone, in chronic metabolic
acidosis, hydrogen ions are buffered in exchange for
calcium. The main buffer is thought to be bicarbonate
or carbonate derived from skeletal carbon dioxide
stores, and the utilization of this buffer system is
accompanied by an efflux not only of calcium but of
divalent phosphate as well, because of dissolution of
the mineral phase. Calcium efflux is dependent on the
bicarbonate concentration as well as on the pH, and
compensated chronic metabolic acidosis, in which
there is a systemically reduced bicarbonate concentration, will have an adverse effect on skeletal mineralization even though the pH is within the normal range
because of the compensation mechanism.
It should again be emphasized that it is growing
children who are vulnerable to skeletal demineralization in this way. Once the skeleton has reached maturity, it appears to be resistant to this mechanism. In
practice, it means that growing children should have
any identifiable metabolic acidosis corrected, but in
adults it need not be corrected if it is asymptomatic
(37). It is frequently reported that patients with renal
function below a certain level should not undergo any
form of enterocystoplasty or urinary intestinal diversion because metabolic problems are far more likely.
Although frequently reported, there is little evidence
to support this except in ureterosigmoidostomy
patients who have continuing fecal reflux to contend
with as well as hyperchloraemic acidosis. In practice,
469
most patients with impaired renal function undergoing enterocystoplasty or urinary diversion have an
obstructive nephropathy, either due to frank outflow
obstruction at sphincter level or to high intravesical
pressure. If these two problems are eliminated by
enterocystoplasty, whatever the level of renal function, there will be at least a temporary stabilization, if
not an overt improvement in renal function, and this
usually amounts to a window of 18 months to two
years before intrinsic renal disease causes further
deterioration in renal function on the downward
slope to renal replacement therapy.
Urinary Infection and Malignant Transformation

Bacterial colonization of the urinary tract is very common after bowel interposition. Although one or two
authors report sterile urine in their patients, the majority of surgeons find that the urine is usually, if not
always, colonized.
Very often this is with a mixed growth of bacteria and less than the usual 100 000 organisms/mL
associated with a classic urinary infection. Not only
is this bacteriuria common, there is also an increased
incidence of both local and systemic infection in such
patients. Average reported figures are that 80% of
patients suffer bacteriuria (43) with a diverse bacterial
flora and that 15% of patients will suffer acute pyelonephritis at some stage (44). The highest incidence
appears to be during the first year. This incidence is
higher than in those who are on clean, intermittent
self-catheterization with a normal bladder (41). Unlike
urothelium, bowel does not attempt to sterilize its
luminal content. When bowel is distended, it becomes
relatively ischemic and the mucosal barrier preventing
bacterial translocation from mucosa to blood is disrupted. Urine from a substitute bladder is less bacteriostatic because the concentration of urea is lower and
the pH higher than in normal urine. These three
points probably account for the increased incidence
of infection in such patients.
This infection is difficult, if not impossible, to
eliminate and most patients suffer asymptomatic bacteriuria even if they are completely unaware of it.
Although asymptomatic, it may be nonetheless important in the long run as there is a theoretical link
between infection and cancer.
Patients with ureterosigmoidostomy were noted
to have an incidence of cancer at the ureterointestinal
anastomosis of between 6% and 29%, with a mean of
11% (29). Generally speaking, there is a lag time of 10
to 20 years between the surgery and the development
of the tumor. These tumors are extremely aggressive
and one-third of patients die from them. They appear
to be adenocarcinomas in the main, although other
histological types have been reported. Overall, this
represents a 500-fold increase in the incidence of
tumors in the sigmoid colon in this patient group,
and it has been reported that, in patients diverted by
this means before the age of 25, the increased risk is
7000-fold (45). The etiology of these tumors is not
clear. Presumably, as most are adenocarcinomas,
they arise in intestinal epithelium adjacent to the
470
Andrich and Mundy
anastomosis, although adenocarcinoma has been

shown to arise from transitional epithelium exposed
to feces in experimental animals (46). Furthermore,
histological assessment of the ureters in patients with
ureterosigmoidostomies shows a high incidence of
dysplasia as judged by biopsies or surgical specimens
from close to the anastomosis (47). Stewart suggested
that these tumors arose as a result of the conversion of
nitrates and secondary amines in the urine into nitrosamines due to the action of fecal bacteria (48). Nitrosamines are known carcinogens and certainly patients
with ureterosigmoidostomies show high concentrations of nitrosamines in the fecal/urinary slurry that
they pass.
Patients with enterocystoplasties or urinary
diversions show dysplastic changes, and malignant
change has been reported, albeit infrequently (49).
Most patients show chronic inflammatory changes in
their bladder on biopsy, and nitrosamines are present
in the urine, in this case produced by the bacteriuria
that they have rather than by exposure to feces. The
greater the degree of the bacteriuria, the greater
the degree of the pyuria associated with it, the higher
the nitrosamine levels in the urine and the higher the
incidence of histological changes, which in some
instances would be classified as premalignant if they
were found in the bowel in its natural situation. Such
changes include keratinizing squamous metaplasia
and transitional epithelial mucin distribution
changes similar to the alterations in colonic mucin
secretion demonstrated in patients with ureterosigmoidostomy. Other factors have been suggested for
the development of these tumors, including superoxide radicals and epidermal growth factor receptor
proliferation, induced by the urothelial-colonic juxtaposition. Whatever the cause, there seems to be little
doubt that some patients, at least, will develop tumors
in the long run. Patients should therefore not be lost to
follow-up so that such tumors can be detected at the
earliest possible moment. Patients usually are enrolled
to surveillance investigations around 10 years after
their enterocystoplasty. Traditionally cystsocopy is
used for this purpose, but recently MRI cystogram
has shown the advantage that this noninvasive imaging technique has adequate resolution to pick up small
bladder lesions and at the same time screens the
upper tact for incidental pathology (50).
Robertson has shown that metabolic and dietary

factors are present in stone forming enterocystoplasty
patients, which may be as important in the etiology
of stone formation as the already recognized factors of
infection and poor reservoir drainage. The majority of
enterocystoplasty patients have risk factors for at least
three different types of stone although 86% are of
infective origin. Stone formers have a higher pH,
and sodium and protein excretion and a lower calcium and citrate excretion (51). Nonstone formers
have a higher fluid intake, higher citrate excretion, a
lower urinary pH and higher dietary magnesium and
phosphate intake (52).
However, it should be noted that patients with
ileal conduits and with orthotopic substitution cystoplasties that empty their bladders spontaneously and
with little or no residual urine have a low incidence of
stone formation. An orthotropic substitution that has to
be emptied by clean intermittent self-catheterization
has a higher incidence. The highest incidence of all
occurs in patients with continent diversions being
emptied by clean intermittent self-catheterization
from the top of the bladder via a Mitrofanoff channel,
rather than from the bottom through the urethra
suggesting that stagnation is the most important factor
in stone formation, with mucus presumably acting as a
nidus and bacterial colonization as the catalyst.
Other Problems
It has already been noted, when discussing metabolic
abnormalities, that the primary abnormality leading to
acidosis is the increased absorption of ammonia. This
ammonia is normally converted into urea by the liver.
If the liver is in any way abnormal, then hyperammonemic encephalopathy may result (53), and is particularly common in patients with chronic liver disease
such as cirrhosis. In healthy patients however, the
hepatic reserve to clear ammonia is high, so this is
not a common problem unless liver disease is fairly
advanced.
Certain drugs may be absorbed from the urine
when bowel is interposed, and phenytoin (54) and
certain antimetabolites such as methotrexate (55) have
been reported to reach toxic levels in such patients.
Perhaps more importantly, glucose can be reabsorbed
from the urine in diabetic patients and control of their
diabetes may be made more difficult as a result (56).
Stones
Patients with ileal conduits were found to have a 20%
incidence of renal calculus formation if followed for
more than 20 years (41). Up to one-third of patients
with Kock pouches were found to have stones in their
pouch, in this instance related to nonreabsorbable
staples. More recently, stones have been noted in the
absence of foreign material in patients with diversions
and orthotopic substitutions and these have been
attributed to acid renal tubular fluid, an increased
excretion of calcium, and a high incidence of infection
with urease-producing bacteria, as well as to the
presence of mucus and almost universal bacteriuria.
TISSUE ENGINEERING IN UROLOGY

Tissue engineering is defined as an interdisciplinary
field which applies the principles of engineering and
life sciences toward the development of biological
substitutes that aim to maintain, restore or improve
tissue function (57). There are three current strategies
for tissue engineering: integrating biomaterials such
as cell-seeded or nonseeded matrices into diseased
tissues to promote the development of functional new
tissue; nuclear transfer for reproductive or therapeutic
cloning; and stem cell induced tissue regeneration.
Cell-Seeded or Cell-Nonseeded Matrices

There are different classes of biomaterials: natural
derived materials, such as collagen and alginate; tissue matrices, such has bladder submucosa and small
intestinal submucosa (cell-seeded or nonseeded); and
synthetic polymers such as polyglycolic acid, polylactic
acid and polylactic-co-glycolic acid. These biomaterials
slowly disintegrate and are replaced by extracellular
matrix relying on ingrowths of healthy cells and
remodeling into regenerated tissue. Several nonseeded
biomaterials have been used in animal models for
urethral reconstruction (5861).
In clinical practice, nonseeded materials have
been used with some success in substitution urethroplasty for relatively short strictures (62) potentially
replacing buccal mucosal grafts in similar circumstances, but have failed in longer strictures requiring
circumferential repair (63), presumably because the
maximum distance cells can travel from the healthy
urethral edges is only only 1 cm. For longer strictures,
matrices seeded with urethral epithelial or buccal
mucosal cells are required, but the larger the mass of
cells, the more it becomes important to have an adequate vascular network to deliver nutrients and oxygen to each cell. To overcome this problem, it has been
proposed to prevascularize the matrix before seeding
the cells onto the scaffold (64).
The application of tissue-engineered tissues in
bladder replacement is also still experimental and
results of studies using acellular matrices have been
disappointing resulting in graft contraction and loss of
capacity (6567). Bladder augmentation with cellseeded matrices appear to be more promising achieving a composite graft of normal bladder architecture
(68) but the goal of a bio-engineered bladder becoming
a clinical reality is still some way into the future (69).
Nuclear Transfer for Reproductive or

Therapeutic Cloning
Cloning involves transfer of a donor nucleus with its
genetic material into an enucleated oocyte, which will
produce an embryo with identical genetic material as
its cell source (reproductive cloning). The embryo can
be implanted into a female to develop into a clone of
the donor, as demonstrated with Dolly the sheep in
1997. Therapeutic cloning (somatic cell nuclear transfer) is used to only produce autologous embryonic
stem cells for tissue regeneration after a patients
nuclear DNA from a somatic cell was transferred
into an enucleated (donated) oocyte. Therapeutic cloning is in its infancy but a degree of renal tissue
regeneration has been achieved in a large animal
model (70).
Stem CellsInduced Tissue Regeneration

Ethical issues concerning the use of cloned human
embryos for the derivation of stem cells have stimulated the search for methods of harvesting stem cells
or reprogramming differentiated cells into pluripotent
stem cells.
471
There are several sources of stem cells provision,

principally autologous adult stem cells harvested
mainly from bone marrow biopsy (but also from adipose and other tissues) and human embryonic stem
cells, which are ethically more controversial. Both
have the potential to differentiate into derivatives of
all three embryonic germ layers (71). Pluripotent
embryonic stem cells are separated during the blastocyst stage at five days after fertilization and if used
clinically would have the disadvantage of requiring
immunosuppression apart from the potential risk to
transdifferentiate into a malignant phenotype. Adult
autologous stem cells avoid these problems but the
quantity of harvested cells is limited. Another source
are fetal stem cells derived from amniotic fluid or
placental tissue which could be stored for later potential future self-use, avoiding immunosuppression and
malignant transformation (72).
New strategies to generate a source of patientspecific pluripotent stem cells is focusing on reprogramming technologies which requires resetting of the
gene expression program of a somatic cell to a state
consistent with embryonic development (73). Amazing progress had been made recent years in cell and
developmental biology. Undoubtedly further developments in finding the ideal source of stem cells for
tissue regeneration will evolve in the not too distant
future (74).
REFERENCES
1. Brockes JP, Kumar A. Appendage regeneration in adult
vertebrates and implications for regenerative medicine.
Science 2005; 310(5756):19191923.
2. Adzick NS, Lorenz HP. Cells, matrix, growth factors, and
the surgeon. The biology of scarless fetal wound repair.
Ann Surg 1994; 220(1):1018.
3. Lorenz Hp, Lin RY, Longaker MT, et al. The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast
Reconstr Surg 1995; 96(6):12511259.
4. Kingsnorth AN, Slavin J. Peptide growth factors and
wound healing. Br J Surg 1991; 78:12861290.
5. Furcht LT. Critical factors controlling angiogenesis: cell
products, cell matrix and growth factors. Lab Invest 1986;
55:505509.
6. Skalli O, Gabbiani G. The biology of the myofibroblast
relationship to wound contraction and fibrocontractive
disease. In: Clark RAF, Henson PM, eds. The Molecular
and Cellular Biology of Wound Healing. New York:
Plenum Press, 1988:373.
7. Blair GH, Slome D, Walter JB. Review of experimental
investigations on wound healing. In: Ross JP, ed. British
Surgical Practice: Surgical Progress. London: Butterworth,
1961.
8. Converse JM, McCarthy JG, Brauer RO, et al. Transplantation of skin. Grafts and flaps. In: Reconstructive Plastic
Surgery. Vol. 1. 2nd ed. Philadelphia: WB Saunders,
1977:152182.
9. Grabb WC, Smith JW. Plastic Surgery. 2nd ed. Boston:
Little Brown, 1973:1122.
10. Devine CJ Jr, Horton CE. Surgical treatment of Peyronies
disease with dermal graft. J Urol 1989; 142:12231226.
11. Perlmutter AD, Montgomery BT, Steinhardt G. Tunica
vaginalis free graft for the correction of chordee. J Urol
1985; 134:311313.
472
Andrich and Mundy
12. Gilbert DA, Horton CE, Terzis J, et al. New concepts in

phallic reconstruction. Ann Plast Surg 1987; 18:128136.
13. Ehrlich RM, Reda EF, Koyle MA, et al. Complications of
bladder mucosal graft. J Urol 1989; 142:626627.
14. Baskin LS, Duckett JW. Mucosal grafts in hypospadias and
stricture management. AUA Update Series 1994.
15. Burger RA, Muller SC, El-Damanhoury H, et al. The buccal
mucosal graft for urethral reconstruction: a preliminary
report. J Urol 1992; 147:662664.
16. Horton CE, Devine CJ Jr. A one stage repair of hypospadias cripples. Plast Reconstr Surg 1970; 45:425430.
17. Schreiter F, Noll F. Meshgraft urethroplasty using split
thickness skin graft of foreskin. J Urol 1989; 142:12231226.
18. Duckett JW. The island flap technique for hypospadias
repair. Urol Clin North Am 1981; 8:503511.
19. Morehouse DD. Current indications and technique of two
stage repair for membraneous urethral strictures. Urol Clin
North Am 1989; 16:325328.
20. Mundy AR. Results and complications of urethroplasty
and its future. Br J Urol 1993; 71:322325.
21. Brain DJ. The early history of rhinoplasty. Facial Plast Surg
1993; 9:8189.
22. Backstein R, Hinek A. War and medicine: the origins of
plastic surgery. Univ Toronto Med J 2005; 3:217219.
23. Williams P, Harrison T. McIndoes Army. 2nd ed. London:
Sphere, 1981.
24. McCraw J, Massey F, Shaiklin K, et al. Vaginal reconstruction with gracilis myocutaneous flaps. Plast Reconstr Surg
1976; 58:176183.
25. Horton CE, Sadore RC, Jordan GH, et al. Use of the rectus
abdominis muscle and fascia flap inreconstruction of epispadias/exstrophy Clin Plast Surg 1988; 15:393397.
26. Taylor GI, Daniel RK. The anatomy of several free flap
donor sites. Plast Reconstr Surg 1975; 56:243253.
27. Koch MO, McDougal WS, Thompson CO. Mechanisms of
solute transport following urinary diversion through intestinal segments: an experimental study with rats. J Urol
1991; 146:13901394.
28. Fern DO, Odel HM. Electrolyte pattern of the blood after
bilateral ureterosigmoidostomy. JAMA 1949; 142:634641.
29. Zabbo A, Kay R. Ureterosigmoidostomy and bladder
extrophy: a long-term follow-up. J Urol 1986; 136:396398.
30. Fisch M, Wammack R, Muller SC, et al. The Mainz 11
(sigma rect um pouch). J Urol 1993; 149:258263.
31. Singh G, Thomas DG. Bowel problems after enterocystoplasty. Br J Urol 1997; 79:328332.
32. Whorwell PJ, Lupton EW, Erdiran D, et al. Bladder smooth
muscle dysfunction in patients with irritable bowel syndrome. Gut 1986; 27:10141017.
33. Spirnak SP, Caldamone AA. Ureterosigmoidostomy. Urol
Clin North Am 1986; 13:285.
34. Durrans D, Wujanto R, Carrol RN, et al. Bile acid malabsorption: a complication of conduit surgery. Br J Urol 1989;
64:485488.
35. Dean AM, Woodhouse CRJ, Parkinson MC. Histological
changes in ileal conduits. J Urol 1984; 132:11081111.
36. Nurse DE, Mundy AR. Metabolic complications of cystoplasty. Br J Urol 1989; 63:165170.
37. Mundy AR, Nurse DE. Calcium balance, growth and skeletal mineralisation in patients with cystoplasties. Br J Urol
1992; 69:257259.
38. Wear JB Jr, Barquin OP. Ureterosigmoidostomy. Urology
1973; 1:192200.
39. Klein EA, Montie JE, Montague DK, et al. Jejunal conduit
urinary diversion. J Urol 1989; 64:412.
40. Geist RW, Ansell JS. Total body potassium after ureteroileostomy. Surg Gynaec Obstet 1961; 113:585589.
41. McDougall WS, Koch MO. Impaired growth and development and urinary intestinal interposition. Trans Am Assoc
Genitourin Surg 1991; 105:3.
42. Gerharz EW, Preece M, Duffy PG, et al. Enterocystoplasty
in childhood: a second look at the effect on growth. BJU Int
2003; 91:7983.
43. Guinan PD, Moore RH, Neter E, et al. The bacteriology of
ileal conduit urine in man. Surg Gynaec Obstet 1972;
134:7882.
44. Schwarz GR, Jeffs RD. Ileal conduit urinary diversion in
children: computer analysis of follow-up from 2 to 16 years.
J Urol 1975; 114:285288.
45. Husmann DA, Spence HM. Current status of tumour of the
bowel following ureterosigmoidostomy: a review. J Urol
1990; 144:607610.
46. Aaronson IA, Constantinides CG, Sallie LP, et al. Pathogenesis of adenocarcinoma complicating ureterosigmoidostomy. Experimental observations. Urology 1987;
29:538543.
47. Aaronson IA, Sinclair-Smith CC. Dysplasia of ureteric
epithelium: a source of adenocarcinoma in ureterosigmoidostomy? Z Kinderchir 1984; 39:364367.
48. Stewart M, Hill MJ, Pugh RC, et al. The role of N-nitrosamine in carcinogenesis at the uretero-colic anastomosis.
Br J Urol 1981; 53:115118.
49. Filmer RB, Spencer JR. Malignancies in bladder, augmentations and intestinal conduits. J Urol 1990; 143:671678.
50. Andrich DE, Hirst JP, Kirkham APS, et al. Feasibility of
Magnetic Resonance Cystography in the surveillance of augmentation and substitution cystoplasty. BJU Int 2008; 101:45.
51. Robertson WG, Woodhouse CR. Metabolic factors in the
causation of urinary tract stones in patients with enterocystoplasties. Urol Res 2006; 34:231238.
52. Hamid R, Robertson WG, Woodhouse CR. Comparison of
biochemistry and diet in patients with enterocystoplasty
who do and do not form stones. BJU Int 2008; 101:14271432.
53. McDermott WV Jr. Diversion of urine to the intestines as a
factor in ammoniagenic coma. N Engl J Med 1957; 256:
460462.
54. Savarirayan F, Dixey GM. Synope following ureterosigmoidostomy. J Urol 1969; 101:844845.
55. Bowyer GW, Davies TW. Methotrexate toxicity associated
with an ileal conduit. Br J Urol 1987; 60:592.
56. Sridhar KN, Samuell CT, Woodhouse CRJ. Absorption of
glucose from urinary conduits in diabetics and nondiabetics. Br Med J 1983; 287:13271329.
57. Atala A. Tissue engineering and cell therapy: perspectives
for urology. In: Wein AJ, Kavoussi LR, Novick AC, et al.,
eds. Campbell-Walsh Urology. Vol. 1. 9th ed. Philadelphia:
Saunders Elsevier, 2007:553573.
58. Bazeed MA, Thurhoff JW, Schmidt RA, et al. New treatment for urethral strictures. Urology 1983; 21:5357.
59. Kropp BP, Ludlow JK, Spicer D, et al. Rabbit urethral
regeneration using small intestinal submucosa onlay
grafts. Urology 1998; 52:138142.
60. Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a
possible off the shelf biomaterial for urethral repair.
Urology 1999; 54:407410.
61. Sievert KD, Bakircioglu ME, Nunes L, et al. Homologous
acellular matrix graft for urethral reconstruction in the
rabbit: histological and functional evaluation. J Urol 200;
163:19581965.
62. Atala A, Guzman L, Retik A. A novel inert collagen matrix
for hypospadias repair. J Urol 1999; 162:11481151.
63. DeFilippo RE, Yoo JJ, Atala A. Urethral replacement using
cell-seeded tubularized collagen matrices. J Urol 2002;
168:17891793.

64. Fontaine M, Schloo B, Jenkins R, et al. Human hepatocyte
isolation and transplantation into an athymic rat, using
prevascularized cell polymer constructs. J Pediatr Surg
1995; 30:5660.
65. Kropp BP, Sawyer BD, Shannon HE, et al. Characterization
of small intestinal submucosa regenerated canine detrusor:
assessment of reinnervation, in vitro compliance and contractility. J Urol 1996; 156:599607.
66. Sutherland RS, Baskin LS, Hayward SW, et al. Regeneration of bladder urothelium, smooth muscles, blood vessels,
and nerves into an acellular tissue matrix. J Urol 1996;
156:571577.
67. Yoo JJ, Meng J, Atala A. Bladder augmentation using
allogenic bladder submucosa seeded with cells. Urology
1998; 51:221225.
68. Oberpenning F, Meng J, Yoo J, et al. De novo reconstitution
of a functional urinary bladder by tissue engineering. Nat
biotechnol 1999; 17:149155.
473
69. Bolland F, Southgate J. Bio-engineering urothelial cells for

bladder tissue transplant. Expert Opin Biol Ther 2008;
8:10391049.
70. Lanza RP, Chung HY, Yoo JJ, et al. Generation of histocompatible tissues using nuclear transplantation. Nat Biotechnol. 2002; 20:689696.
71. Tomson JA, Kalishman J, Golos TG, et al. Isolation of a
primate embryonic stem cell line. Proc Natl Acad Sci U S A
1995; 92:78447848.
72. Siddiqui MM, Atala A. Amniotic fluid-derived pluripotential cells. In: Lanza RA, ed. Handbook of Stem Cells. Vol. 2.
Philadelphia: Academic Press, 2004:175179.
73. Jaenisch R, Young R. Stem cells, the molecular circuitry of
pluripotency and nuclear reprogramming. Cell 2008;
132(4):567582.
74. Hipp J, Atala A. Sources of stem cells for regenerative
medicine. Stem Cell Rev 2008; 4(1):311.
32
Energy Sources in Urology
Andy Symes and Ken M. Anson
Department of Urology, St Georges Hospital, London, U.K.
INTRODUCTION
In every operating theater around the world, an energy
source of some description will be available to help the
surgeon perform procedures with as limited morbidity as possible. The move toward less invasive therapeutic interventions has also resulted in energy
sources moving out of the operating theater into the
outpatient department. Advances in energy delivery
have arisen alongside and often in tandem with
advances in surgery. The introduction of extracorporeal shock wave lithotripsy (ESWL) remains the most
potent example of the impact of technology upon
surgical practice. The liaison between industry and
health care professionals remains critically important
both to the understanding of present day devices, and
for the development of the surgical tools of the future.
It is our aim in this chapter to present the physical principles that underpin the various energy sources that are currently commercially available to
urologists. It is hoped that this will provide a working knowledge that will help the clinician not only to
choose the tool best suited to the job at hand, but also
to be able to use it appropriately and safely. We have
divided the energy sources into four broad families:
electrosurgical, electromagnetic, acoustic, and
mechanical (Table 1). We shall examine the physical
characteristics of each, explain their tissue interactions, outline the delivery systems available, and provide a brief description of their clinical applications.
ELECTROSURGICAL ENERGY
Cauterizing wounds to stop bleeding was recorded as
early as HippocratesThose diseases which medicines do not cure, iron cures; those which iron cannot
cure, fire cures; and those which fire cannot cure, are
to be reckoned wholly incurable (1). It was not until
the 19th century that a physicist called Becquerel first
demonstrated electrocautery. He showed that passing
electrical current through a needle generated heat,
which could be used as a controllable source of heat
to stop bleeding via coagulation (the process by which
blood forms solid clots). Jacques-Arsene dArsonoval
(1891) subsequently discovered that high-frequency
alternating electric current could be passed through
the body without an electric shock, creating heat but

not neuromuscular excitation (2). The term diathermy,
heating through, was coined in 1907 by German
physician Carl Franz Nagelschmidt, and one year
later, Vincenz Czerny used this process to cut tissue
(3). The first electrosurgical device, the surgical Bovie
knife, was not developed commercially until 1928 (4).
The general principles of this device apply today and
surgical diathermy remains an essential tool for all
surgeons.
Basic Principles
To understand how diathermy works, it is important
to be aware of the basic principles governing any
electrical circuit (Table 2). Electric current flows
when electrons pass from one atom to another, with
flow expressed in amperes (A). The greater the current, the larger are the number of electrons that are
moving, and to allow flow an electric circuit must
be formed between a positive and negative electrode.
Voltage, measured in volts, is the force that enables
electrons to move around a circuit. If electrons meet
resistance (impedance) to their flow, then heat is
generated at that point and resistance is measured in
ohms. The overall power of an electric circuit is measured in watts and is an interaction between current,
voltage, resistance, and time. Standard mains electric
current is alternating (flows in both directions) and
oscillates at around 60 cycles per second. The speed of
oscillation is measured in hertz (Hz) (Fig. 1).
Applied Physics
A generator is the source of flow and voltage to an

electrosurgical circuit. In clinical practice the completed
circuit comprises a generator, the active electrode
(handpiece), the patient and the return electrode, with
the connecting cables in between. The patients tissue
impedes flow of the current, leading to the production
of heat. At 60 Hz, electricity interferes with the normal
conduction of nerves and leads to electrocution: ventricular fibrillation, nerve stimulation, and muscular
seizures (5). However, nerve and muscle stimulation
cease at around 100,000 Hz, and it is then possible to
pass high power through the body without causing
Chapter 32: Energy Sources in Urology

Table 1 Energy Sources in Urology
Electrosurgical energy
Electromagnetic energy
Acoustic energy
Mechanical energy
Diathermy
Radio waves
Microwaves
Lasers
Extracorporeal shock wave lithotripsy
Ultrasound
Kinetic energy
Table 2 The Properties of Electricity

Current
Circuit
Voltage
Resistance
Flow of electrons during a period of time

(measured in amperes)
Pathway for the uninterrupted flow of electrons
Force pushing current through the resistance
(measured in volts)
Obstacle to the flow of current, measured in
ohms (impedance resistance)
electrocution. Modern electrosurgical generators produce current at greater than 300,000 Hz.
There are currently two ways in which an electrosurgical circuit can be formedbipolar or monopolar circuits.
Bipolar electrosurgery. In bipolar electrosurgery
both the active and return electrodes are enclosed in
one instrument, usually a pair of forceps. The two
tines of the forceps perform both the active and return
electrodes. The current enters the patients body only
in the tissue between the two tines. No patient return
electrode is therefore required and because only a
475
small amount of tissue is exposed to the current a

relatively low power can be used.
Monopolar electrosurgery. Monopolar electrosurgical circuits are the most commonly used. The active
and return electrodes are separated by a part of the
patients body, with the active electrode usually an
electrosurgical probe or pair of forceps, and the return
electrode in the form of a much larger, flat plate in
contact with the patients skin. The current flows
through the circuit and is concentrated (current density) at the narrow active electrode, resulting in heat
deposition at this point.
Tissue Interactions
The only variable that determines the effect electrosurgical current has on tissues is the rate at which it
produces heat. High levels of heat generated rapidly
will cut tissues; low levels of heat generated slowly will
coagulate tissues and result in hemostasis. The ability
of the electrosurgical current to produce heat in the
tissues can be changed by manipulating several factors:
current density, power, time, and waveform.
1.
Electrosurgical generators are able to produce a

variety of electrical waveforms. As the waveform
changes, the effects on surgical tissue alter and a
different surgical effect is produced. The three
waveforms are traditionally termed CUT,
COAG, and BLEND, and cause the surgical effects
of cutting, fulguration (destruction of tissue by
high-frequency electric current), and desiccation
(Fig. 2).
CUT waveform: This is a continuous high-current,
low-voltage waveform. The high current helps it
Figure 1 Electrosurgery differs from electrocautery in its use of alternating current.
476
Symes and Anson
Figure 2 Alteration of power and waveform affects tissue interaction.
to heat tissues quickly and the low voltage prevents

the formation of sparks, which would dissipate the
heat. The tissues heat quickly, swell, and explode
and the effect can be very clean, with the electrode
acting like a scalpel.
COAG waveform: COAG is an intermittent lowcurrent, high-voltage waveform. The low current
causes a slow heating effect and the high voltage
causes the production of sparks, dissipating the
heating effect over a large area. At low power this
leads to desiccation or drying out of the tissues,
with formation of a coagulum. At higher powers the
sparks cause a burning effect, leading to necrosis
and fulguration of tissues. This is sometimes
referred to as spray. At very high power the
COAG waveform will produce enough current density for cutting.
BLEND waveform: This combines the effect of COAG
and CUT by altering the parameters of the waveform, with an increasing coagulation effect and a
relative reduction in the cutting effect.
2.
3.
Current density is the ratio of the power of the

current to the area of the tissue it affects. Thus,
current density will be high at the active electrode
and low at the return electrode for a given power
of current.
Power of the current can affect the surgical effects
it produces. For example, a high-power COAG
waveform can be used to cut tissue; similarly, a
low-power CUT waveform can be used very effectively to desiccate tissue.
The size and position of electrode will also

impact on current density. A small electrode will
produce a higher current density than a large electrode. Eschar is relatively high in resistance to current;
therefore, keeping electrodes clean and free of eschar

will enhance performance by maintaining lower resistance within the surgical circuit.
Delivery Systems and Clinical Applications
Argon-enhanced electrosurgery. Electrosurgical

current easily ionizes argon gas, making it more conductive than air. As argon gas is inert and noncombustible, it is a safe medium through which to pass
current. Applying a stream of argon gas to the electrosurgical current creates a bridge between the electrode
and tissue, which allows use in a noncontact fashion.
This reduces surrounding tissue damage, causes
less smoke and odor, and is said to produce a flexible
eschar.
Vessel-sealing technologyTM. Vessel-sealing technology combines electrosurgicalTMenergy and pressure
to create a seal. The LigaSure device (ValleyLab,
Colorado, U.S.) utilizes a specialized generator/
instrument system that can be used in open or minimally invasive surgery. Vessels (<7 mm) or tissue
bundles are placed between the jaws of the instrument, which are closed under pressure, and a bipolar
current is applied (6). Upon release of the blades, the
coagulated/sealed tissue is divided by sharp dissection. Thermal spread is reduced and seals have been
proven to withstand 3" normal systolic blood pressure. The potential benefits of this technology are
reduced blood loss, time saving compared to suturing,
reduced needle stick injuries, avoidance of thermal
damage to surrounding structures, and avoidance of
foreign bodies (sutures).
Bipolar resection. The GyrusTM bipolar resectoscope applies bipolar electrosurgical current across a
specialized TUR loop, with the active and return
electrode on the same axis, separated by a ceramic
insulator (7). The Olympus system also utilizes a

bipolar system,
allowing transurethral resection in
TM
saline (TURis ). Benefits include better hemostasis,
and avoidance of dilutional hyponatremia, as isotonic
saline is the irrigation solution used. Measurable outcomes appear similar to conventional monopolar
TURP (7,8), although long-term data are awaited.
Safety
When first introduced in the 1920s, surgical diathermy

machines converted grounded mains electricity to
grounded electrosurgical current. The current would
return to ground, which was the common earth, via
the return electrode. If the return electrode were to fail,
then the current would find an alternative way to earth
via a contact between the patient and any other
earthed object. This was called current division and
could lead to an accidental patient burn. Isolated
electrosurgical systems were introduced in 1968 with
the isolated generator, creating a separate circuit not
referenced to the common earth but with its source in
the generator itself. In other words the circuit is
completed not by the ground but by the generator
itself. Current division is therefore minimized and the
potential for alternative site burns reduced. If the
circuit is brokenthe return electrode falls offthen
current will cease to flow. Return electrode burns,
however, may still occur and burns still occur through
inappropriate use. All operators should therefore be
familiar with the equipment in use in theater. Electrosurgery should not be used in the presence of flammable agents, and oxygen-enriched environments
should be avoided, as there is a very real risk of
ignition. When not in use, active electrodes should
be kept in a clean, dry, and well-insulated nonconductive holster. Cords should not be wrapped around
metal instruments, nor bundled together. This can
lead to reactive coupling, generating electromagnetic
energy [radio frequency (RF)], which is not always
confined by insulation.
Minimally invasive surgery. Electrosurgery used
with minimally invasive surgery has its own hazards:
insulation failure, direct coupling of current, and
capacitively coupled current (9). Breaks in insulation
can create an alternate route for current to flow, and
this is often not apparent with minimally invasive
surgery where the views may be limited. Direct coupling occurs when the user activates the generator
near another metal instrument. The secondary instrument becomes energized, with current seeking a pathway back to the return electrode, with potential for
patient injury.
A capacitor occurs when a nonconductor separates two conductors, and may create an electrostatic
field between them, thus passing current between
them. During minimally invasive surgery, an inadvertent capacitor may be created by the surgical
instruments. The conductive active electrode is surrounded by nonconductive insulation, which in turn
is surrounded by a conductive metal cannula. Burns
may occur from the outer metal cannula, and plastic
sheaths are now favored.
477
Smoke. Surgical smoke is created when tissue is

heated and cellular fluid is vaporized by the thermal
action of the energy source. Viral DNA, bacteria,
carcinogens, and irritants are known to be present in
electrosurgical smoke, and where possible a smoke
evacuation system should be used (10).
Tissue density feedback. Modern electrosurgical
generators measure tissue impedance at the electrode contact and adjust settings according to the
tissue involved. Voltages are kept lower and theater
staff are not required to frequently change power
settings.
Return electrode monitoring (REMTM, ValleyLab).
Pad site burns are caused by inadequate contact of the
return electrode on the patient. REM-equipped generators actively monitor the amount of impedance at
the patient/pad interface. If the generator detects a
high level of impedance at the interface, then the
system will deactivate.
ELECTROMAGNETIC ENERGY
Many urological instruments use sources that provide
energy in the form of electromagnetic radiation
(EMR). At first sight, they appear to be very disparate
devices with varied delivery systems, but, on closer
inspection, they share many common principles that
arise from the use of the electromagnetic wave source.
Their tissue effects may also seem unrelated but in fact
are similar (i.e., the deposition of heat within tissues),
and often they can be separated only by the speed and
depth of energy absorption. However, some of these
sources (particularly lasers) have very specific tissue
effects, which are wavelength dependent and vary
greatly from one wavelength to another.
EMR is a self-propagating wave in space with
electric and magnetic components. These components
oscillate at right angles to each other and to the
direction of propagation, and are in phase with each
other. EMR is classified into types according to the
frequency of these waves: in order of increasing frequency, radio waves, microwaves, terahertz radiation,
infrared radiation, visible light, ultraviolet radiation,
X rays, and gamma rays (Fig. 3). EMR carries energy
and momentum, which may be imparted when it
interacts with matter.
Basic Science
Visible light, X rays, microwaves, radio waves, and
lasers are all part of the electromagnetic spectrum and
are members of the same family. All travel through
space at the same velocity (v) of 3 " 108 m/sec (the
speed of light (c)about 186,000 miles per second).
When they travel through a material medium, their
speed is reduced but with no evidence of movement
of the medium to indicate their passage. There is a
constant relationship between velocity (v c, the
speed of light through an empty space), wavelength
(k), and frequency (f), such that v fk. It follows that
the relationship between wavelength and frequency is
inversely proportional.
478
Symes and Anson
Figure 3 The electromagnetic spectrum.
Quantum PhysicsWave-Particle Duality

Theories about light stem back to the fifth to sixth
century BC, when ancient Indians, Greeks, and
Romans all put forward theories bearing striking
resemblance to the modern theories of lightThe
light and heat of the sun; these are composed of minute
atoms which, when they are shoved off, lose no time in
shooting right across the interspace of air in the direction imparted by the shove (11). In the 15th and 16th
centuries, Gassendi, and then Robert Hooke and
Christian Huygens, proposed opposing theories of
light, as either particulate or wave. In 1845, Michael
Faraday discovered that the angle of polarization of a
beam of light could be altered by a magnetic field as it
passed through a polarizing material, an effect now
known as Faraday rotation (12). This was the first
evidence that light was related to electromagnetism.
Faraday proposed in 1847 that light was a high-frequency electromagnetic vibration, which could propagate even in the absence of a medium such as the
ether (12). This wave theory was successful in
explaining nearly all optical and electromagnetic phenomena, and was a triumph of 19th century physics. By
the late 19th century, a handful of experimental anomalies remained that could not be explained by or were in
direct conflict with the wave theory. Experimental
evidence led to three main observations: first, when
light was directed at a metal surface, electrons were
instantaneously ejected; second, when light intensity
was increased, more electrons were ejected, but their
velocity remained the same; and third, changing the
color of the light toward the blue end of the visible
spectrum (increasing the frequency) increased the
maximum velocity of the ejected electrons (the photoelectric effect) (13). The wave theory of light could not
explain these phenomena, as metals would have to
reach high temperatures before electrons were ejected,

and some time delay would be required to allow sufficient energy transfer. Similarly, increasing the intensity would increase the speed of the ejected electrons,
and the frequency of light would not be expected to
change these findings, since intensity, and not color,
determines the energy of waves. In 1905, Einstein
solved this puzzle by resurrecting the particle theory
of light to explain the observed effects (14). Einsteins
ideas were met initially by great skepticism, but his
explanation of the photoelectric effect would triumph,
ultimately forming the basis for wave-particle duality
and much of quantum mechanics.
These findings led to the quantum theory of
light, which states that light (or any other EMR) exists
in the form of quanta known as photons, which are
tiny packets of energy with no mass traveling
through space at the speed of light (3 " 108 m/sec).
The energy of each photon is given as E hf, where
E is the energy in joules, f is the frequency, and h
is a universal constant (Plancks constant, 6.63 "
10#34 J/sec). The fundamental concept that energy is
dependent on the frequency of the radiation applies to
all parts of the electromagnetic spectrum. Therefore, it
can be seen that as the frequency increases, so does the
energy of the EMR. Thus, microwaves and radio
waves are of low energy, while X-ray photons have
a high energy enabling them to ionize matter that they
come into contact with.
Applied Physics
Although electromagnetic waves share many physical
characteristics, varying only in frequency and wavelength, their effect upon living tissue is very different.
For example, the human body is transparent to radio
waves, opaque to visible light, and mostly transparent

to X rays. The quantum theory states that to raise an
atom from ground to excited state, a photon of a given
frequency and hence given energy (DE hf, where DE
is the energy difference) is required. Thus, for a photon to be absorbed (to transfer its energy), it must
interact with an atom that will be excited only by that
corresponding packet of energy. Radio waves can pass
through the body with little effect, while microwaves
cause molecular rotation and torsion, and induce tissue heating. The infrared band causes molecular
vibration, and X rays cause tissue ionization.
At the microscopic level, the electrical response
of soft tissues to EMR is dependent on the frequency
of the energy. This phenomenon is known as the
dielectric property of tissues. At low frequencies
[i.e., below several hundred kilohertz (kHz)], conductivity is determined by electrolytes in the extracellular
space, and tissues have a high permeability to EMR at
these frequencies. In the low-kilohertz range, tissues
exhibit a dispersion (the alpha dispersion) because of
several processes. These include polarization of
counter ions near charged surfaces and membranebound structures within tissues. In the RF band, the
tissues exhibit a dispersion (the beta dispersion) centered on the 0.1 to 10-MHz range, because of charging
of cell membranes through the intracellular and
extracellular media. At microwave frequencies
(above 1 GHz and centered around 20 GHz), gamma
dispersion occurs because of rotational relaxation of
tissue water.
Tissue Interactions
In urological practice, nonlaser EMR is commonly
used to induce thermal damage in tissues. Radio
wave and microwave generators have been designed
to heat the prostate, bladder, and kidney to different
temperatures to achieve varying degrees of tissue
damage. Some laser techniques have also been developed to obtain similar thermal effects. In each case, the
energy inherent within the EMR is converted to heat
within tissues. Each different energy source will vary
between the amount of energy that can be delivered
and the depth to which that energy can reach, but the
basic tissue effect desired is the same. The response of
tissues to heating varies with the temperature
achieved at the target area. At temperatures around
458C, tissue retracts because of macromolecular conformational changes, bond destruction, and membrane alterations. Beyond 608C, protein denaturation
occurs, and this is commonly called coagulation
and results in coagulative necrosis. Carbonization of
tissues occurs at approximately 808C along with membrane permeabilization and collagen denaturation.
Finally, at temperatures in excess of 1008C, vaporization of water occurs, and in combination with carbonization, yields decomposition of tissue constituents.
Below temperatures of 458C, there is virtually no
histological change in tissue following heating. The
World Health Organization (WHO) has defined heat
treatments as hyperthermia for temperatures of 378C
479
to 458C, and as thermotherapy when temperatures

exceed 458C.
Many other factors influence the degree of heat
deposition at a particular area including the locoregional blood supply, the thermal conductivity of the
target and adjacent tissue, and the histopathological
nature of the target tissue.
Radio waves and Microwaves

Microwave and RF radiation is EMR that is lower in
frequency and therefore longer in wavelength than
infrared radiation. Radio frequency is the name
given to that section of the electromagnetic spectrum
from frequencies of 300 kHz to 300 GHz. In general,
the section of the electromagnetic spectrum from
frequencies of approximately 300 MHz to 300 GHz
and wavelengths of approximately 1 m to 1 mm are
called microwaves. As a result of its low energy, RF,
unlike other forms of nonionizing EMR, requires
direct contact with tissues to exert an effect. As the
RF energy attempts to pass into tissue, transference of
energy occurs in a concentrated area. RF energy is
efficient with minimal wastage; consequently, lowwattage energy (up to 15 W) can achieve temperatures
approaching 1008C within a short time (typically
5 minute) without the need for interruption and cooling. RF devices are therefore often small and compact.
Because of the low energy of RF, adjacent structures
are relatively spared from heat dissipation.
Microwaves occupy the electromagnetic band at
frequencies of 300 MHz to 300 GHz. These higher
frequencies result in higher energy, and therefore
greater tissue penetration than RF. The effect of microwave energy on tissue is predominantly because of
molecular torsion and rotation (oscillation) and polarization of small molecules, especially water, resulting
in tissue heating. In clinical practice, frequencies of
915 or 1296 MHz are used.
Clinical Applications
Transurethral Needle Ablation of the Prostate
Transurethral needle ablation (TUNA) of the prostate

is a procedure used to treat benign prostatic hyperplasia (BPH). It is performed by placing interstitial RF
needles through the urethra and into the lateral lobes
of the prostate, causing heat-induced coagulation
necrosis. The tissue is heated to 1108C at an RF
power of 456 kHz for approximately 3 minutes per
lesion. A coagulation defect is created. The TUNA
device consists of an RF generator and monitor where
energy, ablation time, lesion temperature, urethral
temperature, and tissue impedance are displayed. In
this device, the 465 to 490-kHz RF signal is transmitted
directly into the prostate via two needles delivered via
the TUNA catheter (Fig. 4). The delivery system has a
disposable cartridge, a resterilizable handpiece, and a
zero-degree fiberoptic lens. The TUNA cartridge contains two retractable needles, each surrounded by a
protective retractable Teflon shield, except at the needle tips. When extended, the needles leave the body of
480
Symes and Anson
Figure 4 TUNA catheter with retractable needles and sheaths.
the catheter and diverge at an angle of 408 between

each other and perpendicular to the catheter. Thermocouples are located at the tips of the protective shields
and near the tip of the catheter for temperature
monitoring of the lesion and prostatic urethra, respectively. The catheter tip is clear in order to allow
visualization of the needles during penetration into
the prostate. The procedure can be performed under
local anesthesia and sedation (15). Lesions produced
by the TUNA device are well-defined, ellipsoid
necrotic lesions as a result of coagulative necrosis.
The exact mechanism whereby relief of symptoms
occurs is debated. The concept of anatomical debulking is flawed, as prostate volume changes following
TUNA treatment are minimal (16). There is increasing
evidence that the therapeutic effect of TUNA is
explained by intraprostatic neuromodulation, which
alters the physiologic function of voiding (dynamic
component of BPH). Thermal neural ablation, including surgical alpha-receptor blockade, has been demonstrated by various researchers (17,18). There has
been concern about the durability of this procedure,
with up to 20% of patients requiring a TURP within
two years (19). One study has provided good longterm clinical improvement at five-year follow-up with
more than 75% of the patients not requiring additional
treatment (20).
Radio Frequency Ablation of Renal Tumors

This technology was initially developed for treating
primary and metastatic liver lesions (21). Zlotta et al.
(22) first described the use of RF ablation (RFA) as the
primary treatment for small renal tumors. It is used in
patients with small renal tumors who have poor renal
reserve, multiple bilateral renal cell carcinomas (RCC)
in von HippelLindau or hereditary RCC, or in
those who are poor surgical candidates (23). A highfrequency electrical current is transmitted through
an electrode placed directly into the renal tumor
either percutaneously, under USS/CT/MRI guidance, or under direct vision laparoscopically (24,25).
Alternating current delivered through the probe causes

ions in the surrounding tissues to vibrate, creating
frictional heat that results in heat-induced tissue damage. Target temperatures of 1058C are achieved by
generators of greater than 200 W (Cool-Tip ValleyLab,
Radionics). The heat generated by RFA causes tissue
destruction in three phases. Immediately postablation,
molecular friction produces destruction of cellular
structure, protein denaturation, membrane lipid melting, and cellular vaporization (26). Coagulative necrosis follows days later with surrounding areas of cellular
edema and inflammation leading to tumor destruction
(26). The final evolution of the ablated tissue is reabsorption of the necrotic foci with the resulting fibrotic
scar nonenhancing on contrast imaging (27). For the
cellular changes to occur as described earlier, temperatures above 508C must be achieved. The success of
tumor ablation with RFA depends on factors including
probe temperature, generator power, temperature distribution, and targeting of the tumor (28).
Microwave Prostatic Therapies
Microwave energy may be applied transrectally and

transurethrally to the prostate, and may induce hyperthermia or thermotherapy, depending on the target
tissue temperature achieved. Transrectal hyperthermia, originally described in the treatment of prostate
cancer (29) and subsequently BPH (30), has failed to
show relief of the obstructive component of BPH and
has largely been abandoned. Transurethral microwave thermotherapy (TUMT) aims to achieve high
temperatures (45808C) in the prostate and involves
the insertion of a specially designed Foley-type catheter into the bladder, allowing a microwave antenna
to be positioned within the prostatic fossa (Fig. 5
ProstaLund Operations AB, Lund, Sweden; marketed
by ACMI). Microwaves are then passed into prostate
tissue to induce thermal coagulative necrosis.
Energy sources may be monopolar or bipolar.
The ProstatronTM device uses a monopolar antenna.
The initial software program, Prostasoft 2.0, was a
481
risk) multiple and recurrent superficial bladder

tumors, when other treatment strategies have failed,
and/or when cystectomy is contraindicated or the
patient refuses to undergo radical surgery (34,35).
The Synergo1 system employs a 915-MHz microwave
applicator inserted into the bladder via a special catheter. Thermocouples are also inserted to control the
temperature of the bladder wall layers, which are
heated to a temperature of 428C ($28). Intravesical
Mitomycin is pumped out and reinstilled, to avoid
overheating. Each treatment lasts one hour, and no
anesthesia is required. Side effects are minimal and
largely related to the sensation of heat, which is temporary, and the effects of mitomycin.
Lasers
Figure 5 The ProstaLund CoreTherm 1 catheter (PLFT

ProstaLund Feedback Treatment).
low-energy protocol with maximum energy of 60 W.

Treatment took 60 minutes, and noticeable symptomatic but not objective improvement occurred. Later,
the higher-energy Prostasoft 2.5 was introduced,
which allowed a stepwise increase in energy without
interruptions to achieve intraprostatic temperatures of
758C (1678F), and used urethral cooling with 208C
(688F) water. The treatment also took 60 minutes,
and results were better than with the initial software
(31). The most current and powerful software is
Prostasoft 3.5 (32). Only 30 minutes of treatment is
required. Rectal temperature is monitored, and an
alarm system is set that causes a cutout if excessive
rectal temperatures occur. The therapy is contraindicated in patients with prostate or bladder cancer,
urethral stricture, or claudication, or in patients with
a pacemaker, defibrillator, or other metallic implants.
Studies have compared TUMT with TURP, revealing
greater symptomatic improvement with TURP than
TUMT, as well as better objective response as measured by flow rate (33). Complications were lower
with TUMT and it may be an alternative to surgery in
patients who are deemed a high surgical or anesthetic
risk and not controlled with pharmacotherapy.
Microwave Bladder Therapies
Human and animal studies have proven that microwave-induced hyperthermia combined with intravesical mitomycin C is a feasible, effective, and safe
conservative approach for those patients with (high
The invention of the laser, an acronym for light

amplification by stimulated emission of radiation,
can be dated to 1958 with the publication of Infrared
and Optical Masers, by Schawlow (36). It was Albert
Einstein, however, in his 1917 paper Zur Quantentheorie der Strahlung, who laid the theory for the
invention of the laser and its predecessor the maser.
His theoretical model postulated that when a photon
interacted with an excited atom, another photon
would be released with identical wavelength. In
1960, Theodore Maiman invented the ruby laser considered to be the first successful optical or light laser
(37). He used a ruby rod, excited by a helical flash
lamp, to produce a beam of red laser light. A number of
lasers were subsequently produced in the 1960s including the carbon dioxide (CO2) and the neodymium:
yttrium-aluminium-garnet (Nd:YAG) laser, which
remain the most popular lasers used in medicine
today. Initially called an invention looking for a job,
the laser now represents an entire scientific field and a
multibillion dollar industry.
Basic Principles
Absorption and spontaneous emission. Atoms consist of a positively charged core (nucleus), which is
surrounded by negatively charged electrons. The electrons can be thought of as orbiting the nucleus, with
those with the largest energy orbiting furthest from
the nuclear core. There are many energy levels that an
electron within an atom can occupy; however, electrons usually rest at lower energy levels. Normally,
when a photon of light is absorbed by an atom in
which one of the outer electrons is initially in a lowenergy state, the energy of the atom is raised to an
upper energy level, and remains in this excited state
for a very short period. It then spontaneously returns
to the lower state, with the emission of a photon of
light. These common processes of absorption and
spontaneous emission cannot give rise to the amplification of light. The best that can be achieved is that for
every photon absorbed, another is emitted.
Stimulated emission. This is a very uncommon
process in nature but it is central to the operation of
lasers. If a photon of light interacts with an excited
atom, at a higher energy state, it can stimulate a return
482
Symes and Anson
to the lower state. One photon interacting with an

excited atom results in two photons being emitted.
Furthermore, the two emitted photons are said to be in
phase. Stimulated emission is the process that can give
rise to the amplification of light and results in the laser
beam produced having the property of coherence.
Under most conditions, stimulated emission does
not occur to a significant extent, as under conditions
of thermal equilibrium, there will be far more atoms in
the lower energy level than in the higher level.
Absorption, therefore, will be much more common
than stimulated emission. For stimulated emission to
predominate there must be more atoms in the higher
energy state than in the lower one. This unusual
condition is referred to as a population inversion
and is necessary for laser action to occur.
Population inversion. Population inversion is the
state of a medium where a higher-lying electron level
has a higher population than a lower-lying level.
Finding substances (gain medium) in which a population inversion can be set up is central to the development of new kinds of laser. The first material used
was synthetic ruby, and lasers today use argon, CO2,
semiconductors, Nd:YAG, among others. To stimulate
most of the electrons to the high-energy states, an
initial energy is delivered to the atoms in the laser
medium in the form of light, heat, or electricity.
Laser Design
Once stimulated emission occurs, photons are generated exponentially provided population inversion is
maintained. The light from a typical laser emerges in an
extremely thin beam with very little divergence.
Another way of saying this is that the beam is highly
collimated. The high degree of collimation arises
from the fact that the cavity of the laser has very nearly
parallel front and back mirrors, which constrain the
final laser beam to a path perpendicular to those
mirrors. The back mirror is made almost perfectly
reflecting while the front mirror is about 99% reflecting, letting out about 1% of the beam (Fig. 6). This 1% is
the output beam, which you see. The light has passed
back and forth between the mirrors many times in
order to gain intensity by the stimulated emission of
more photons at the same wavelength. The light from a
laser typically comes from one atomic transition with a
single precise wavelength. So the laser light has a single
Table 3 Properties of Laser Light

Coherent (in phase)
Collimated (nondivergent)
Monochromatic (of a single wavelength)
spectral color and is almost the purest monochromatic

light available. This wavelength is entirely dependent
on the laser medium present.
Applied Physics
These three unique properties of laser light (beam

coherence, collimation, and monochromaticity)
(Table 3) separate it from the widely divergent, confused, and multiple wavelength light emitted from
conventional light sources. Because of the high spatial
coherence of laser light, the beam is remarkably
collimated and therefore of low divergence. This
property allows the full power of the generated light
energy to be coupled into, and transmitted down, fine
fiberoptic cables to the operative site. As a result, very
high-power densities (power per unit cross-sectional
area) can be achieved at the tissue surface. Furthermore, the beam can be focused to produce even
greater power densities with more pronounced tissue
effects. The interaction of the light with tissue is
dependent both on the laser wavelength and the
color of the tissue irradiated; thus, the single wavelength nature of the beam confers a high selectivity of
action. One single laser will not be suitable for all
clinical applications. A number of different laser
wavelengths are available for clinical use. They are
named after the medium used to generate the laser
energy (Table 4).
Laser energy can be continuous or pulsed.
Pulsing of lasers delivers a higher peak power (the
maximum rate of energy delivery during the time
of the pulse) and is commonly used to fragment
calculi [pulsed dye, holmium:YAG, and frequencydoubled potassium-titanyl-phosphate (KTP)/Nd:
YAG]. Pulsing is achieved either by applying intermittent energy to the active medium or by a process
known as Q switching, in which a fast shutter is
placed between the active/using medium and the
exit mirror (partial reflector). When the shutter is
closed, the photons are trapped within the active
medium, resulting in a rapid buildup of energy.
When the shutter opens, there is an instantaneous
release of energy with a very large peak power.
Tissue Interactions
Figure 6 Basic laser design.
Boulnois has proposed a classification for the possible

types of photomedical processes that can occur
when laser light interacts with biological tissue (38).
The four photobiological laser processes are thermal,
photochemical, electromechanical, and photoablative.
As with the use of microwaves and RF, the predominant tissue effect with lasers in urology is thermal.
Thermal interactions. When laser light interacts
with tissue, it is reflected, transmitted, scattered, and
absorbed, depending on the tissue type. When
483
Table 4 Characteristics of Medical Lasers
Mode
Wavelength
(nm)
Visibility
Tissue
penetration Tissue
(mm)
absorption
Carbon
dioxide
CW
10600
Invisible
0.1
Argon
CW
Blue
Nd:YAG
CW P
488
514(dual
wavelength)
1064
Invisible
KTP-532
CW P
532
Holmium:YAG
Pulsed dye
laser
(coumarin
green)
Laser
type
Effects
Clinical Uses
Water, proteins,
nucleic
acids, fat
Hemoglobin,
melanin
Intense carbonization, Superficial skin lesions

vaporization.
(penile warts).
Minimal penetration
Bowens disease
Tissue coagulation,
Nephron-sparing
vaporization
surgery, laparoscopy
38
Protein
Green
0.31
Hemoglobin
2100
Invisible
0.5
Water
504
Green
Minimal
Hemoglobin, but
pulses too
short for HB
to absorb
Semiconductor
diode lasers
CW
800900
Invisible
37
Protein
Thulium
CW
2013
Invisible
0.002
Water
Protein denaturation, BPH & TCC therapy,

coagulative necrosis
interstitial therapy to
solid tumors
Tissue coagulation,
Photoselective
vaporization
vaporization prostate
(PVP)
Mechanical pressure Urinary tract calculi.
Enucleation of
wave fragmentation
prostate
Thermal
Pressure effects.
Urinary tract calculi
Plasma formation.
Minimal damage
to urothelium; little
thermal effect
Protein denaturation, BPH therapy, interstitial
coagulative necrosis, therapy to solid
vaporization
tumors
Tissue coagulation,
BPH therapy,
vaporization
vaporization prostate
CW, continuous wave; P, pulsed.
Figure 7 Laser wavelength absorption by hemoglobin and water.
absorbed, the electromechanical energy of the incident beam is converted into thermal energy within the
tissues. Scattering of the beam results in widespread
distribution of the energy at all angles to the incident
beam; the energy is subsequently absorbed and converted into thermal energy. Both the wavelength of
the interacting light and the color of the tissue irradiated determine the relative amount of absorption and
scattering within tissue components (Fig. 7). The CO2
laser has a wavelength of 10,600 nm and is therefore
highly absorbed by water, proteins, nucleic acids, and

fat; thus, its depth of penetration is very small at
0.1 mm. The high absorption by water makes this
laser light unsuitable for endoscopic procedures (39),
and it is used for the treatment of superficial lesions
(skin/external genitalia). There is intense and instantaneous tissue vaporization and carbonization with a
very shallow thermal damage front, and healing
is achieved with minimal scarring. In contrast, the
Nd:YAG wavelength (1064 nm) has little effect on
484
Symes and Anson
water, but proteins absorb it. It is, therefore, widely

scattered to a depth of up to 0.8 cm, as the predominantly water-containing tissue is transparent. The
scattering results in a wider distribution of the energy
at all angles away from the incident beam. The energy
is therefore absorbed within a larger volume of tissue.
The power of a laser is related to the power density,
that is, the power per surface area, and is given as
PD W/pr2 [W (watts) is the power emitted from the
laser]. The total energy delivered multiplies the power
density by duration of exposure; thus, total energy
Ws/pr2 (measured in joules). It can be seen that
doubling of the spot size reduces the power fourfold.
The rate of ablation varies with the total energy
delivered; the higher the total energy, the faster is
the ablation. At low power, heat is conducted into
deeper tissues. Consequently, at a higher power density, there is less thermal damage to underlying
tissues. A highly focused beam results in a shallow
zone of thermal damage below the target tissue, the
thermal damage front, and results in minimal scarring. A defocused beam results in a reduction of
power density at the lesion, but a thick zone of thermal damage front beneath the targets surface.
Photoablative and electromechanical interactions.
Pulsing of lasers (such as holmium:YAG) has the
additional benefit of a very high peak pressure, causing fragmentation by erosion, shattering, and plasma
bubble formation with cavitation. When the pulse is
very short, thermal damage is minimized (39),
although the holmium:YAG laser will damage ureteric
tissue if in contact (40). This property endows the laser
with a dual purpose, as it can be used to incise tissue
such as the prostate and bladder neck (HoLEP) (41).
Photochemical effectsphotodynamic therapy. Lowpower laser light can photochemically activate a
photosensitizing dye such as a hematoporphyrin
derivative. This dye is chemically inert until exposed
to specific laser wavelengths; this exposure results in
energy release in the form of free radicals that are
cytotoxic (42). Tissues that take up these dyes can be
destroyed when laser energy is directed at them.
Endothelial cells are most sensitive to the effects of
free radicals and have increased density within
tumors. The injured endothelium also releases interleukin and tumor necrosis factor, causing additional
damage to malignant epithelium. This technique is
being used in the treatment of carcinoma in situ of the
bladder and is being investigated in the treatment of
both benign and malignant prostatic disease (42).
Tissue-welding effects. Laser energy can induce
interdigitation of collagen, resulting in a tissuewelding effect. Albumin has been used as a tissue
solder, particularly in vasovasostomy (43).
Laser Delivery Systems
Laser light can be focused to diameters near 50% of its

wavelength. This allows laser light to be transmitted
along single quartz fiberoptic fibers. Laser energy is
conducted along the fiber by a process known as
internal reflection where the light is reflected from
one side to the other, as the beam travels along the
fiber. The beam must be reflected at a specific angle,

known as the critical angle of reflection. If the fiber is
displaced, or bent too far, the beam will no longer
strike the sidewall at the critical angle and will escape
from the fiber, causing injury to the user, the endoscope, or both. Once the beam exits the fiber tip, it is
no longer coherent, as it is driven out of phase during
internal reflection, and divergence occurs (about 17%,
assuming a flat fiber tip). Therefore, the highest power
density is at the tip of the fiber.
The Nd:YAG laser has been used to cause widespread

coagulative necrosis within the benign prostate (endoscopic laser ablation of the prostate) (44), and it can
also be used to coagulate superficial bladder tumors.
Q-switching of Nd:YAG lasers may be used to fragment calculi, although it is less effective than the
holmium laser, since it relies primarily on the photochemical mechanism for lithotripsy. Addition of a
KTP crystal doubles the frequency (therefore halving
the wavelength532 nm) and causes the beam to
become visible in the green band of visible light. Its
use has recently become popular in photoselective
vaporization of the prostate (45). This wavelength is
highly absorbed by oxyhemoglobin and penetrates the
prostatic tissue only 1 to 2 mm deep. These important
characteristics allow the laser energy to be confined in
a small volume of tissue, eliminating the risks caused
by excessive coagulation. The high-power KTP laser
instantly removes tissue by vaporization of cellular
water, leaving only a thin 1- to 2-mm rim of coagulated tissue remaining. The KTP laser is thought to
ablate prostate tissue more efficiently than the
Nd:YAG laser. The Nd:YAG laser can also be used
for ablation of skin lesions (including penile carcinoma), and in removal of hair post urethral reconstruction. However, the significant depth of penetration
means that there is a risk of injury to the underlying
tissues and subsequent scar/stricture formation.
The holmium:YAG laser is widely used in endoscopic surgery both for stone fragmentation (46) as well
as resection of the prostate (HoLEP) (47). The beam falls
in the near infrared portion of the electromagnetic spectrum (2100 nm). The Ho:YAG laser has 350-msec pulse
duration. The laser energy is absorbed in <0.5 mm of
fluid, making it an ideal surgical laser for endourological applications such as laser lithotripsy. The laser
energy is delivered to the surface of stones using flexible
quartz fibers with multiple different diameters (200, 365,
550 mm). During the pulse of laser energy, there is a
microscopic air bubble on the tip of the fiber that allows
the laser energy to travel further than through the fluid
medium directly transmitting the laser energy into the
stone. In addition, the Ho:YAG laser has been used to
incise urethral strictures, ureterocoeles, and obstructing
pelviureteric junctions.
The Indigo laser is a diode laser with a wavelength
between 800 and 850 nm. It penetrates deep into tissues,
up to 3 mm, and thus produces thermal lesions several
centimeters in diameter. It has been used primarily for
prostate tissue ablation via interstitial laser coagulation.
The CO2 laser is an infrared laser with a very

long wavelength (10,640 nm), which results in a relatively short depth of penetration (40 to 240 mm). Its
zone of thermal tissue injury is small and it is used to
treat superficial skin lesions, such as condyloma.
Laser Safety
Strict protocols regarding the use of lasers in the theater are mandatory. All personnel in the theater must
wear protective goggles. Different goggles are
required for different wavelengths, and the wearer
should check that the goggles protect against the
wavelength in use. The area where the laser is being
used must be clearly marked with warning notices and
the theater doors locked when the laser is in use. An
illuminated light must be on during laser firing. Black
curtains should be applied above eye level to all
windows. The retina is susceptible to laser light in
the range 400 to 1400 nm. Accidental exposure can
lead to injury, ranging from small scotomata to optic
nerve burns. Wavelengths beyond 1400 nm may cause
corneal injuries. There is a greater chance of an accidental burn to the skin than the eye. Laser safety rules
should be available in all areas where lasers are in use,
and the local laser safety officer will be able to advise
on these.
ACOUSTIC ENERGY
The word acoustic is derived from the ancient Greek
word akousto c , meaning pertaining to hearing.
Audible sound waves travel at frequencies of 50 to
20,000 Hz. Frequencies above this range (30,000 Hz to
1 MHz) are termed ultrasonic, with frequencies below
termed infrasonic (100 kHz to 0.1 Hz). The word
acoustic" refers to the entire frequency range without
limit. The introduction of ESWL and high-intensity
focused ultrasound (HIFU) into clinical practice has
ushered in a new generation of treatment modalities
for a variety of benign (stones) and malignant (prostate)
conditions. Shock wave and ultrasonic therapies will be
discussed here with HIFU mentioned elsewhere.
Basic Principles
Waves carry vibrations through a medium (solid, liquid, gas) and are created whenever an object moves
within a fluid (either gas or liquid). Waves have a
measurable speed, wavelength, and frequency and are
created when an object moves, compressing molecules
adjacent to it. This compression is in turn transmitted
to further adjacent molecules and so on. The compression is therefore relieved in the original region but is
propagated onward to a new region with the formation of a wave. The speed of this wave is dependant on
the characteristics and temperature of the medium
within which it travels (solids > liquids > gases). It
is independent of pressure, frequency, and amplitude.
Acoustic waves differ from electromagnetic waves in
that individual molecules do not travel with acoustic
waves. Electromagnetic energy displays wave-particle
duality with particles (photons) physically traveling
485
through space. Acoustic energy therefore requires a

medium where as electromagnetic energy does not:
light can travel through a vacuum but sound cannot.
When an object moves within a medium, it
moves to (compresses) and moves away from the
medium (rarefaction) creating a compressive and tensile wave. In most cases these waves propagate similarly and move at the same sound speed. Wave forms
may be sinusoidal in nature; however, many are not,
for example, shock waves.
Extracorporeal Shock Wave Lithotripsy

Shock waves are high-energy waves with high amplitude, characterized by extremely short buildup times.
Using shock waves, the first kidney stone was
successfully disintegrated in Munich in 1980, and
now ESWL is accepted as a first-line treatment in
the management of renal and ureteric stones (48).
Lithotripsy has undergone numerous technological
advances; however, the fundamentals of shock wave
generation and delivery have changed little.
Applied Physics
A typical shock wave is shown in Figure 8. It is

characterized by a short wave of about 5 msec with a
near-instantaneous leap to a peak-positive pressure of
about 40 MPa (compressive phase) (MPa, MegaPascal
the metric unit of pressure). This is known as a shock.
The pressure falls rapidly to zero and then below with a
peak-negative pressure of 10 MPa (tensile phase),
which is always much less than the positive pressure.
The majority of lithotripters produce a similar shock
wave; however, the peak pressures may vary (peak
positive: 30 to 110 MPa; peak negative: #5 to #15 MPa).
Thus, the main difference is the amplitude of wave.
When a shock wave encounters a medium, part
of the wave will continue (transmitted wave) and part
will be reflected (reflected wave). The density and
sound speed of a medium determine its specific acoustic impedance. The transmission from water to tissue
is very efficient (99%), as is water to stone (7595%).
The transmission from water to air is very poor and
99.9% of energy will be reflected. Shock wave generators in lithotripsy are therefore water filled, with both
patient and generator in water (most efficient), or with
water-filled pads directly applied to the patient,
with care taken to eliminate air from the contact.
Thus, the flank provides the best acoustic window
with a pure tissue path to the kidney.
The shock wave is focused onto the stone to
concentrate acoustic energy and minimize damage to
the surrounding tissues. This is achieved by differing
means depending on machine. The acoustic pressure
is greatest at one point and is surrounded by an area
of high amplitude. This is known as the focal zone,
which is ellipsoid in shape and ranges from a few to
tens of millimeters. The focal length is the distance
from the mouth of the therapy head to the focus
(stone). The power of a lithotripter is related to the
volume and peak pressure at the focal point. The
energy delivered is measured in joules and is greatest
486
Symes and Anson
Figure 8 Acoustic shock wave.
for spark-gap lithotripters, because of their large focal

points and high peak pressures (49).
Tissue Interactions and Stone Fragmentation
There are thought to be numerous ways in which

shock waves fragment stones. When applied to a
stone, the positive pressure phase part of the shock
wave is reflected at the stone surface, causing erosion
at the entry and exit points because of a high-pressure
gradient. A compressive wave continues through the
stone, causing shattering (Fig. 9). Shear stress on the
stone will be generated by both shear and compressive waves as they enter the stone. Superfocusing is
the amplification of stresses inside the stone either by
refraction or diffraction of the shock wave dependent
on the geometry of the stone. Squeezing occurs
because of differing sound speeds of waves between

the stone and surrounding fluid, resulting in tensile
stress and stone breakage. During the negative-pressure phase cavitation occurs (50), causing rapid
expansion of gas bubbles in the liquid medium at
the stone surface (Fig. 10). This phenomenon occurs
when a negative pressure greater than the ambient
pressure exists in a liquid, which then fails under
stress (50). These unstable bubbles collapse, forming
microjets that strike the stone surface. Microjet velocities range from 130 to 170 m/sec.
Lithotripter Design
Lithotripters require an energy source, a focusing

mechanism for the shock wave, a coupling medium,
and a stone localization system.
Figure 9 (A) Positive-pressure phase effect of a shock wave upon a calculus. Surface erosion occurs at entry and exit points.
(B) Negative-pressure phase effect of a shock wave upon a calculus. The effect of cavitation.
487
Figure 10 Model stone located at the focal point. Cavitation

bubbles exist 540 msec after the shock wave is released.
Figure 12 Electromagnetic lithotripter. Application of an electric

current to the coil results in a strong magnetic field that repels a
metallic disk upward to strike a fixed metal plate. Shock waves
are focused by an acoustic lens or parabolic reflector.
Figure 11 Spark-gap lithotripter. A spark, generated at F1,

causes a hydrodynamic pressure wave that is focused at a set
distance to the target area (F2) by an ellipsoid reflector.
Electrohydraulic lithotripters. Spark-gap lithotripters use an underwater spark that generates a hydrodynamic pressure wave (Fig. 11). The point of spark
generation (F1) is placed in front of an ellipsoid mirror, which focuses the pressure waves at a set distance
to the target area (F2). Position of the spark relative to
the ellipse is critical, with displacement by only 1 mm
resulting in loss of focusing and lengthening and
broadening of the focal zone (51). Although frequent
changing and or replacement of the electrodes can
overcome this, there can still be significant variability
from shock to shock.
Electromagnetic lithotripters. Electromagnetic lithotripters require the application of an electric current
to a coil. This generates a strong magnetic field that
repels and thrusts a metallic disk upward to strike a
fixed metallic plate, thus generating an acoustic shock
wave (Fig. 12). The shock waves are focused either by an
acoustic lens (such as the Siemens LITHOSTAR1) or by
a parabolic reflector (such as the Storz MODULITH1).

Electromagnetic shock wave sources can deliver several
hundred thousand shock waves before servicing, in
contrast to spark-gap machines.
Piezoelectric lithotriptor. Piezoelectric lithotriptors
have a number of piezoceramic crystals aligned on a
fixed metallic concave surface (Fig. 13). The application of simultaneous high-voltage electrical current to
the piezoceramic crystals causes a sudden conformational change that generates an acoustic shock
wave. As a result of the focusing mechanism, piezoceramic lithotriptors produce shock waves with not
only a very high-peak pressure at F2 but also small
focal points. This type of lithotriptor is said to be safe
for use in patients with pacemakers.
As previously mentioned the shock wave is
coupled to the body using water, which results in
efficient transfer of energy. The first-generation lithotripters (e.g., Dornier HM3) used an open water
bath in which the patient was immersed and had
excellent results. Most current machines have the
shock wave head mounted in a therapy head, which
is filled with water. A thin rubber membrane is
pressed against the patient with a coupling jelly or
oil to reduce air. This design is more convenient for
the clinic; however, it is less effective at allowing
energy transfer because of reflection from the rubber
and the inevitable small air bubbles.
488
Symes and Anson
subject of much study (58,59). ESWL has been shown

to cause cellular damage regardless of the cells doubling time. The mitochondria are most sensitive to its
effect (59), although damage also occurs at the cell
membrane, within the nucleus, along the endoplasmic
reticulum, and in other cell organelles (such as lysosomes). Cellular damage may be mechanical or
because of the generation of free radicals (60,61)
caused by homolytic cleavage of the molecule within
the collapsing bubble. Extracorporeal shock wave
techniques have been described in the treatment of
Peyronies plaques (62) although outcomes are mixed.
Ultrasonic Probe for Stone Fragmentation
Figure 13 Piezoceramic lithotripter. Application of an electric

current to piezoceramic crystals causes a sudden conformational
change, resulting in the generation of an acoustic shock wave.
Localization is essential for effective lithotripsy

and currently either ultrasound or X rays are used.
Ultrasound is either in-line or off-line with relation to
the therapy head. In-line is more accurate than off-line,
where the targeted zone may not correspond with the
treated zone. Ultrasound is preferable to X ray localization, as it allows real-time monitoring and avoids
radiation exposure. It is poor at localizing upper and
mid-ureteric stones where fluoroscopy is preferred.
The ultrasound probe used to fragment calculi consists of an ultrasound energy source coupled to a long
metal probe (Fig. 14). The application of about 100 W
to the piezoceramic elements results in oscillation at
a frequency of 23 to 27 MHz (63). The probe oscillates
in both transverse and longitudinal directions with a
displacement of 20 mm. At displacements of >50 mm,
soft-tissue disintegration occurs, and such machines
may be useful in renal sparing surgery. Heat is
generated at the point of stone disintegration, and
therefore irrigation is required for cooling. This heat
can reach temperatures of 508C; therefore, ultrasound
contact lithotripsy is unsuitable for use in the ureter,
although its direct effects on tissue are minimal. The
metal probe is usually hollow to allow suction of
irrigant and small stone fragments. Some hard stones
are resistant to ultrasound lithotripsy, but lithotripsy
appears particularly useful for soft stones that
are difficult to treat with mechanical intracorporeal
lithotripters.
Harmonic Scalpel
Tissue Damage
Severe adverse effects are well documented with ESWL

and therefore it is not true that shock waves pass
harmlessly through the body (52). Focal zones from
lithotripters may reach up to 5 cm and thus the entire
kidney may be subject to mechanical forces. Tissue is,
however, far less susceptible to damage than kidney
stones as shock waves pass through tissue (like water)
without significant reflection resulting in far less tensile
forces. Cavitation driven by negative pressure has been
shown to cause vascular trauma to the kidney and is
thought to be responsible for tissue damage (53). There
is evidence that patients with impaired renal function
treated with ESWL may experience late-onset hypertension and worsening of renal function (54), and this is the
subject of an on going Food and Drug Administration
(FDA) sponsored multicenter study.
ESWL is well described in the fragmentation of urinary

calculi, but its effect on both benign and malignant
tissue has also been studied. The effects of ESWL on in
vitro and in vivo tumor cell lines (5557), and in particular, whether enhancement of chemotherapeutic
effect occurs on ESWL-treated tissue, have been the
The Harmonic ScalpelTM is a device consisting of an

ultrasonic source that causes vibration of forceps or a
blade. Typically, the displacement is 50 to 80 mm, and
the vibrational frequency approaches 55,500 Hz (64).
Heat is generated, with tissue temperatures ranging
from 508C to 1008C and a depth of penetration of 1 to
2 mm. Coagulation occurs by means of protein denaturation when the blade couples with protein, forming
a coagulum that seals small coapted vessels. When the
effect is prolonged, secondary heat is produced that
seals larger vessels. Blood and tissue are desiccated
and oxidized (charred), forming eschar that covers
and seals the bleeding area. Rebleeding can occur
when blades removed during electrosurgery stick to
tissue and disrupt the eschar. The surgeon controls the
precision of cutting and coagulation by adjusting
the power level, blade edge, tissue traction, and
blade pressure. This device has proved particularly
useful in laparoscopic surgery.
MECHANICAL ENERGY
The adjective kinetic to the noun energy has its roots
in the Greek word for motion (kinesis). Lord Kelvin is
credited with coining the term kinetic energy circa
489
Figure 14 Ultrasound probe. Piezoceramic elements oscillate, causing longitudinal and transverse vibration of the probe.
1849. In Urological surgery there are a few devices that

utilize this source of energy; one of the most widely
available is the Swiss LithoclastTM.
Basic Principles
Work is a force acting upon an object to cause displacement. When work is done upon the object, that
object gains energy. Mechanical energy is the energy
possessed by an object because of its motion (kinetic
energy) or position (potential energy). An object that
possesses mechanical energy is able to do work, by
being able to apply a force to another object to cause it
to be displaced.
Mechanical Lithotripsy
The Swiss Lithoclast was developed by the departments of medical electronics and urology at the University Teaching Hospital in Lausanne, Switzerland
(65). The device utilizes compressed air, derived from
the operating theater supply (66,67). This pressure, in
the range 3 to 5 bar, is delivered in a pneumatic blast
of short duration to the inside of a hollow metal
cylinder. This causes a metal projectile to be rapidly
fired to the opposite end of the cylinder, where it
collides and transfers kinetic energy to a metal probe
(Fig. 15) (68). The distal end of the probe is in direct
Figure 15 Mechanical lithotripsy. A compressed-air jet propels the metal projectile to strike the base of the probe (pneumatic
lithotripsy). An electromagnetic field may be substituted for compressed air (electrokinetic lithotripsy).
490
Symes and Anson
contact with the stone. Transference of kinetic energy

from probe to stone causes highly effective fragmentation. Analogy has been made with the jackhammer
used to fragment parts of the pavement during road
construction. The device is simple, cheap, and very
effective. One major problem is the propulsion effect
upon stones, which is much more marked with this
form of energy than with laser lithotripsy. More
recently the StoneBreakerTM (LMA Urology, Gland,
Switzerland) device has been released, offering a
more compact and ergonomic design, as well as
greater contact pressures. Early reports are encouraging (69).
CONCLUSION
It is hard to think of an area of urological practice that
does not involve an energy source. With the increasing number of technologies available (each with their
own unique advantages and disadvantages), it is
important that clinicians understand the basic principles of the equipment that they wish to use. This
chapter aims to provide the reader with the relevant
information to enable them to make the best use of the
technology available today to offer safe and efficacious surgery for our patients.
REFERENCES
1. Cruse JM. History of medicine: the metamorphosis of
scientific medicine in the ever-present past. Am J Med
Sci 1999; 318:171180.
2. Culotta CA. Dictionary of Scientific Biography. New York:
Charles Scribners Sons, 1970:302.
3. Sachs M, Sudermann H. [History of surgical instruments:
7. The first electrosurgical instruments: galvanic cauterization and electric cutting snare]. Zentralbl Chir 1998;
123:950954.
4. OConnor JL, Bloom DA, William T. Bovie and electrosurgery. Surgery 1996; 119:390396.
5. Fraser-Darling A. Electrocution, drowning, and burns.
Br Med J (Clin Res Ed) 1981; 282:530531.
6. Heniford BT, Matthews BD, Sing RF, et al. Initial results
with an electrothermal bipolar vessel sealer. Surg Endosc
2001; 15:799801.
7. Botto H, Lebret T, Barre P, et al. Electrovaporization of
the prostate with the Gyrus device. J Endourol 2001; 15:
313316.
8. Dunsmuir WD, McFarlane JP, Tan A, et al. Gyrus bipolar
electrovaporization vs transurethral resection of the prostate: a randomized prospective single-blind trial with 1 y
follow-up. Prostate Cancer Prostatic Dis 2003; 6:182186.
9. Tucker RD, Voyles CR. Laparoscopic electrosurgical complications and their prevention. AORN J 1995; 62:5153, 55,
5859 passim; quiz 7477.
10. Giordano BP. Dont be a victim of surgical smoke. AORN J
1996; 63:520, 522.
11. Brown PM. Lucretius: De Rerum Natura. Bristol: Bristol
Classic Press, 1984.
12. Hamilton J. Faraday: The Life. London: Harper Collins,
2002.
13. Serway RA. Physics for Engineers and Scientists. Philadelphia:
Saunders College Publishing, 1990.
14. Greiner W. Quantum Mechanics: An Introduction. Berlin:
Springer-Verlag, 2001.
15. Issa MM. Transurethral needle ablation of the prostate:

report of initial United States clinical trial. J Urol 1996; 156:
413419.
16. Isa MM, Wojno KJ, Oesterling JE, et al. Histopathologic
and biochemical study of the prostate following transurethral needle ablation (TUNA): insights to the mechanisms
of improvement in BPH symptoms. J Endourol 1996;
10: 109.
17. Zlotta AR, Raviv G, Peny MO, et al. Possible mechanisms
of action of transurethral needle ablation of the prostate on
benign prostatic hyperplasia symptoms: a neurohistochemical study. J Urol 1997; 157:894899.
18. Perachino M, Bozzo W, Puppo P, et al. Does transurethral
thermotherapy induce a long-term alpha blockade? An
immunohistochemical study. Eur Urol 1993; 23:299301.
19. Schatzl G, Madersbacher S, Djavan B, et al. Two-year
results of transurethral resection of the prostate versus
four less invasive treatment options. Eur Urol 2000; 37:
695701.
20. Hill B, Belville W, Bruskewitz R, et al. Transurethral needle
ablation versus transurethral resection of the prostate for
the treatment of symptomatic benign prostatic hyperplasia:
5-year results of a prospective, randomized, multicenter
clinical trial. J Urol 2004; 171:23362340.
21. Lau WY, Leung TW, Yu SC, et al. Percutaneous local
ablative therapy for hepatocellular carcinoma: a review
and look into the future. Ann Surg 2003; 237:171179.
22. Zlotta AR, Wildschutz T, Raviv G, et al. Radiofrequency
interstitial tumor ablation (RITA) is a possible new modality for treatment of renal cancer: ex vivo and in vivo
experience. J Endourol 1997; 11:251258.
23. Gervais DA, McGovern FJ, Wood BJ, et al. Radio-frequency
ablation of renal cell carcinoma: early clinical experience.
Radiology 2000; 217:665672.
24. Pavlovich CP, Walther MM, Choyke PL, et al. Percutaneous radio frequency ablation of small renal tumors: initial
results. J Urol 2002; 167:1015.
25. Jacomides L, Ogan K, Watumull L, et al. Laparoscopic
application of radio frequency energy enables in situ renal
tumor ablation and partial nephrectomy. J Urol 2003;
169:4953; discussion 53.
26. Crowley JD, Shelton J, Iverson AJ, et al. Laparoscopic and
computed tomography-guided percutaneous radiofrequency ablation of renal tissue: acute and chronic effects
in an animal model. Urology 2001; 57:976980.
27. Matsumoto ED, Watumull L, Johnson DB, et al. The radiographic evolution of radio frequency ablated renal tumors.
J Urol 2004; 172:4548.
28. Rehman J, Landman J, Lee D, et al. Needle-based ablation
of renal parenchyma using microwave, cryoablation,
impedance- and temperature-based monopolar and bipolar radiofrequency, and liquid and gel chemoablation:
laboratory studies and review of the literature. J Endourol
2004; 18:83104.
29. Mendecki J, Friedenthal E, Botstein C, et al. Microwave
applicators for localized hyperthermia treatment of cancer
of the prostate. Int J Radiat Oncol Biol Phys 1980; 6:
15831588.
30. Yerushalmi A, Fishelovitz Y, Singer D, et al. Localized
deep microwave hyperthermia in the treatment of poor
operative risk patients with benign prostatic hyperplasia.
J Urol 1985; 133:873876.
31. Ahmed M, Bell T, Lawrence WT, et al. Transurethral
microwave thermotherapy (Prostatron version 2.5) compared with transurethral resection of the prostate for the
treatment of benign prostatic hyperplasia: a randomized,
controlled, parallel study. Br J Urol 1997; 79:181185.
32. Gravas S, Laguna P, Kiemeney LA, et al. Durability of
30-minute high-energy transurethral microwave therapy
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
for treatment of benign prostatic hyperplasia: a study of

213 patients with and without urinary retention. Urology
2007; 69:854858.
Hoffman RM, Monga M, Elliot SP, et al. Microwave
thermotherapy for benign prostatic hyperplasia. Cochrane
Database Syst Rev 2007:CD004135.
Gofrit ON, Shapiro A, Pode D, et al. Combined local
bladder hyperthermia and intravesical chemotherapy for
the treatment of high-grade superficial bladder cancer.
Urology 2004; 63:466471.
Colombo R, Da Pozzo LF, Salonia A, et al. Multicentric
study comparing intravesical chemotherapy alone and with
local microwave hyperthermia for prophylaxis of recurrence of superficial transitional cell carcinoma. J Clin
Oncol 2003; 21:42704276.
Schawlow AL. Lasers. Science 1965; 149:1322.
Maiman T. Stimulated optical radiation in ruby. Nature
1960; 187:493494.
Boulnois JL. Photophysical processes in revent medical
laser developments: a review. Lasers Med Sci 1985; 1:4766.
Dretler SP. Laser lithotripsy: a review of 20 years of
research and clinical applications. Lasers Surg Med 1988;
8:341356.
Zorcher T, Hochberger J, Schrott KM, et al. In vitro study
concerning the efficiency of the frequency-doubled double-pulse Neodymium:YAG laser (FREDDY) for lithotripsy of calculi in the urinary tract. Lasers Surg Med
1999; 25:3842.
Dushinski JW, Lingeman JE. Urologic applications of the
holmium laser. Tech Urol 1997; 3:6064.
Fried NM. Therapeutic applications of lasers in urology: an
update. Expert Rev Med Devices 2006; 3:8194.
Mingin GC, Ditrolio JV. Vasovasostomy using albumisol
solder with an argon laser. Br J Urol 1998; 81:628629.
Anson K, Nawrocki J, Buckley J, et al. A multicenter,
randomized, prospective study of endoscopic laser ablation versus transurethral resection of the prostate. Urology
1995; 46:305310.
Te AE, Malloy TR, Stein BS, et al. Photoselective vaporization of the prostate for the treatment of benign prostatic
hyperplasia: 12-month results from the first United States
multicenter prospective trial. J Urol 2004; 172:14041408.
Matsuoka K, Iida S, Nakanami M, et al. Holmium: yttriumaluminum-garnet laser for endoscopic lithotripsy. Urology
1995; 45:947952.
Le Duc A, Gilling PJ. Holmium laser resection of the
prostate. Eur Urol 1999; 35:155160.
Lingeman JE, Kim SC, Kuo RL, et al. Shockwave lithotripsy: anecdotes and insights. J Endourol 2003; 17:687693.
Pfister RC, Papanicolaou N, Yoder IC. Urinary extracorporeal shock wave lithotripsy: equipment, techniques, and
overview. Urol Radiol 1988; 10:3945.
Crum LA. Cavitation microjets as a contributory mechanism for renal calculi disintegration in ESWL. J Urol 1988;
140:15871590.
491
51. Cathignol D, Mestas JL, Gomez F, et al. Influence of water

conductivity on the efficiency and the reproducibility of
electrohydraulic shock wave generation. Ultrasound Med
Biol 1991; 17:819828.
52. Evan AP, Willis LR, Lingeman JE, et al. Renal trauma and
the risk of long-term complications in shock wave lithotripsy. Nephron 1998; 78:18.
53. Ueda S, Matsuoka K, Yamashita T, et al. Perirenal hematomas caused by SWL with EDAP LT-01 lithotripter.
J Endourol 1993; 7:1115.
54. Bataille P, Cardon G, Bouzernidj M, et al. Renal and
hypertensive complications of extracorporeal shock wave
lithotripsy: who is at risk? Urol Int 1999; 62:195200.
55. Russo P, Stephenson RA, Mies C, et al. High energy shock
waves suppress tumor growth in vitro and in vivo. J Urol
1986; 135:626628.
56. Randazzo RF, Chaussy CG, Fuchs GJ, et al. The in vitro and
in vivo effects of extracorporeal shock waves on malignant
cells. Urol Res 1988; 16:419426.
57. Kohri K, Uemura T, Iguchi M, et al. Effect of high energy
shock waves on tumor cells. Urol Res 1990; 18:101105.
58. Berens ME, Welander CE, Griffin AS, et al. Effect of acoustic
shock waves on clonogenic growth and drug sensitivity of
human tumor cells in vitro. J Urol 1989; 142:10901094.
59. Clayman RV, Long S, Marcus M. High-energy shock
waves: in vitro effects. Am J Kidney Dis 1991; 17:436444.
60. Morgan TR, Laudone VP, Heston WD, et al. Free radical
production by high energy shock wavescomparison with
ionizing irradiation. J Urol 1988; 139:186189.
61. Suhr D, Brummer F, Hulser DF. Cavitation-generated free
radicals during shock wave exposure: investigations with
cell-free solutions and suspended cells. Ultrasound Med
Biol 1991; 17:761768.
62. Lebret T, Loison G, Herve JM, et al. Extracorporeal shock
wave therapy in the treatment of Peyronies disease: experience with standard lithotriptor (Siemens-multiline). Urology 2002; 59:657661.
63. Marberger M. Disintegration of renal and ureteral calculi
with ultrasound. Urol Clin North Am 1983; 10:729742.
64. Lee SJ, Park KH. Ultrasonic energy in endoscopic surgery.
Yonsei Med J 1999; 40:545549.
65. Lanquetin JM, Jichlinski P, Favre R, et al. The Swiss
lithoclast. J Urol 1990; 143:179A.
66. Denstedt JD, Eberwein PM, Singh RR. The Swiss Lithoclast: a new device for intracorporeal lithotripsy. J Urol
1992; 148:10881090.
67. Schulze H, Haupt G, Piergiovanni M, et al. The Swiss
lithoclast: a new device for endoscopic stone disintegration. J Urol 1993; 149:1518.
68. Vorreuther R, Klotz T, Heidenreich A, et al. Pneumatic
v electrokinetic lithotripsy in treatment of ureteral stones.
J Endourol 1998; 12:233236.
69. Rane A, Kommu SS, Kandaswamy SV, et al. Initial clinical
evaluation of a new pneumatic intracorporeal lithotripter.
BJU Int 2007; 100:629632.
33
Instrumentation in Urology
Rashmi Singh
Department of Urology, Kingston and St. Georges Hospital, London, U.K.
Ken M. Anson
Department of Urology, St. Georges Hospital, London, U.K.
INTRODUCTION
The remarkable developments in endoscopy over the
years have allowed access to virtually all areas of
the genitourinary system. Advances in technology
and miniaturization have enabled diagnostic and
therapeutic options which up until recently were
unimaginable.
The urologist was the first surgical specialist to
explore and evaluate the field of minimal access surgery. Long before other surgeons awoke to the possibilities and impact of minimally invasive surgery,
urologists had demonstrated and established the
unquestionable benefits of transurethral bladder and
prostate surgery compared with open surgery. The
progress in technology during the late 20th century
made laparoscopic surgery and minimally invasive
surgery a reality. These technologies included development of the charge coupling device (CCD) chip that
allowed the transmission of high-resolution video
images, high-intensity light sources that enhanced
visualization of the surgical field and improved
instrumentation design for endoscopic and laparoscopic approaches. The advent of laser and fiberoptic technology together with advances in endoscope
miniaturization have revolutionized upper tract
endoscopy, resulting in increased efficiency and safety
of these procedures.
The phenomenal advances in minimally invasive
surgery have resulted in a shift toward day case and
laparoscopic surgery. Recent years have seen the
advent of robotic and computer-assisted surgery,
which in the future promises to facilitate complex
endoscopic procedures through voice control over
the networked operating room, enhancement of dexterity and development of virtual simulator trainers.
ENDOSCOPY
History of Endoscopy
The history of endoscopy (1) dates back to the 19th
century when surgeons such as Phillip Bozzini developed a long thin funnel attached to a candle proximally
to provide a source of illumination. The instrument

known as the Lichtleiter or light conductor (Fig. 1)
could be used to inspect a number of anatomical
cavities including the vagina, bladder and rectum,
using funnels of varying sizes. Segalas and Desormeaux
tried to refine the illumination of this instrument further
by using either mirrors to focus the light or alcohol and
turpentine burning kerosene lamps. Despite their
attempts, the use of these instruments remained limited
because of the problems associated with externally
placed illumination.
The invention of the light bulb in 1880 by Thomas
Edison enabled German urologist Nitze to overcome
this problem. He developed the electric light cystoscope which had a small lamp mounted at the end
which could then be used to visualize the inside of
the bladder (Fig. 1). During the 20th century, further
modifications to the optical system of the cystoscope
continued. Otis produced the spherical prism in collaboration with Wappler and Bausch and Lomb, which
enabled the wide-angle lens cystoscope. In 1907 the
Amici prism was launched which allowed a vertical,
brighter and sharper image.
Despite these various modifications, limitations
in image quality and light intensity still compromised
safety. It was the work of Harold Hopkins that revolutionized endoscopy. His development of the rod
lens system in the late 1940s enabled the transmission
of light to the end of the endoscope, thus illuminating
the lumen of interest and simultaneously allowing
transmission of the image from the end of the endoscope to the eyepiece of the scope for viewing.
Despite significant improvements in light transmission with the rod lens system, illumination still
depended on a filament lamp at the tip of the cystoscope. Hopkins therefore joined forces with Karl Storz
of Germany who had developed a system of transmitting light by glass fibers called the cold light. In 1967
the Storz-Hopkins instrument was launched which
combined the Hopkins optical system and the Storz
cold light.
Paralleling these advances in light technology
were developments in instrument design (Fig. 1).
The original cystoscope was modified by adding a
Chapter 33: Instrumentation in Urology
493
Figure 1 Historical development of endoscopes. (A) Bozzini Lichtleiter, (B) late 1880s cystoscope, (C) rod lens modern day
cystoscope, (D) flexible ureterorenoscope, and (E) distal mounted CCD videocystoscope.
working sheath separate from the lens system, the

incorporation of irrigation systems, and the addition
of a deflecting (Albarran) mechanism. Brown developed the first 24-F double-catheterizing cystoscope
and in 1906 Bransford Lewis designed the universal
cystoscope through which operating instruments
could be passed. Buerger, an American urologist
combined many of the features developed by his
predecessors including the 24-F Brown sheath, the
Otis wide-angle lens and the Albarran deflecting
lever to produce the Brown-Buerger cystoscope
which became the most popular instrument for performing cystoscopy in the United States. This cystoscope was further refined with time to enable
fulguration via a probe. The use of cautery for treatment of bladder tumors was illustrated by surgeons
such as Nitze and Bottini, while Beer pioneered the
use of monopolar fulguration under water. Wappler
further advanced the use of electrocautery with the
development of a dual generator capable of producing
both cutting and coagulation current.
Early methods described for removal of prostatic
tissue include the Young cold punch technique.
However, it was Maximilian Stern who developed
the first resectoscope, which enabled electroresection.
Joseph McCarthy designed the lever mechanism for
controlling the resection loop, which formed the prototype for the resectoscope as we know it today. The
Sachse cold knife optical urethrotome was designed
with a similar handle mechanism to the resectoscope
but had a reverse thrust.
Attention then turned to the development of

camera systems to allow a record of the surgery to
be taken. Although camera systems had been in use as
early as 1875, it was Nitze who advanced the technique of endoscopic photography and first published
his images. The next step was color photography
40 years later followed by the launch of television
cameras.
The late 20th century heralded the digital revolution. Significant advances in digital imaging technology in combination with modern endoscopes have
made it possible to now attain images with unparalleled detail and clarity.
Rod Lens, Fiber-Optic and Digital Technology

The original cystoscopes used between 1886 and 1951
were made up of a series of thin glass lenses positioned within a long narrow tube separated by annular spacers which held the lenses apart at measured
distances. The limitations in precisely grinding these
lenses meant that stray images occurred from their
surface resulting in degradation of image quality and
poor contrast. The light was provided by tiny lamps,
which had a short life and often burnt out midprocedure particularly if there was a surge in current.
To replace the lamp, the scope would have to be
removed from the bladder and then reinserted resulting in longer operating times and also ran the risk of
an electric shock to the patient. Hopkins overcame
some of these problems by using long solid rod-shaped
494
Singh and Anson
Figure 2 Rod lens cystoscope.
glass cylinders within the telescope, which could be

ground and aligned with greater precision within
the scope. The air gaps in between were the lenses.
Adding an antireflective coating to both ends of the rod
significantly increased the light available at the eyepiece (Fig. 2).
The introduction of fiber-optics in the 1960s led
to the next revolution in endoscope design. Hopkins
developed a method of transmitting images and light
down a bundle of transparent fibers assembled as a
cable, which are then coated with another transparent
material with a different refractive index. The light
then undergoes multiple total internal reflection. The
fiber bundles can be either coherent whereby the
position of a fiber at its proximal end mirrors its
position at the distal end or incoherent where many
fibers are assembled in a random arrangement and
transmit high-density light throughout the cable.
In the early fiber-optic endoscopes, the illumination channel was coupled to an external xenon light
source, while the imaging channel transmitted an
image created by a lens at the distal end of the endoscope to a connected camera unit or display device
(Fig. 3).
Figure 3 Cross-section of flexible cystoscope.
The Digital Revolution

One of the most significant advances in imaging technology has been the process of digitalization. The
improved image quality with digital technology is
secondary to the unique ability to adjust the characteristics of an image on a pixel-by-pixel basis. This is in
contrast to older analogue systems where changes in
image variables affected other image characteristics
simultaneously causing analogue signals to be prone
to interference. Digital images are processed by a CCD
consisting of a silicone chip that absorbs light energy
(Fig. 4). The CCD converts the various light intensities
within an image into corresponding continuous voltage signals. A digital converter then captures each
voltage signal and converts the voltage values into
discreet numbers such as 0 or 1. The encoded numbers
for each image element or pixel include information
on color, contrast or light intensity. These stable variables can then be modified using image-processing
software and can recombine on the video screen to
create an exact replica. They can also be stored on
computer hard drives or digital tape drives.
With the advent of digital technology and miniature CCDs, endoscope designers have moved to
placing the imaging chip at the distal tip of the endoscope just behind the objective lens system. Because of
the higher pixel count produced compared with fiberoptic bundles these chip on the tip endoscopes are
able to produce higher resolution images. Unlike fiberoptic scopes, these endoscopes do not have a lens
system running through them. The image is captured
by the chip and transmitted via cables running through
the scope to the camera. Digital video systems (2) are
being applied in several areas in urology in particular
flexible cystoscopy, laparoscopy and ureterorenoscopy. Miniature complementary metal oxide semiconductors (CMOS) imaging chips as small as 1 mm
are now emerging as competitors to CCD chips (Fig. 4)
because they are simple, their cost is decreasing, they
are inherently all-digital and they provide comparable
or possibly superior image quality. The advantages of
digital flexible scopes with distal CMOS video sensors
and built in LEDs for illumination are that a separate
snap-on camera head with white balancing and
495
Figure 5 Cross-sectional view of distal end of continuous flow

resectoscope.
Resectoscopes
Figure 4 (A) CMOS chip, (B) distal sensor digital flexible

ureterorenoscope, and (C) CCD chip.
focusing issues is no longer necessary and there is no

bulky light source requiring connection and adjustment. This enables systems like the Gyrus ACMI
DUR-D to be an all-in one plug and play device,
which is user friendly (Fig. 4).
Unlike fiber-optic scopes, images from distal
sensor digital scopes appear not to be affected by
pixelation, glare or Moire effect and there is minimal
loss of information during image transmission (3).
Rigid and Flexible Instruments

The entire upper and lower urinary tract can now be
accessed endoscopically using either rigid endoscopes
(which are based on the rod lens system) or flexible
scopes which employ fiber-optic technology. Chip on
the tip technology is rapidly being incorporated into
these endoscopes.
The continuous flow resectoscope was first described

by Iglesias in 1975 and was developed in response to
the concerns that TURP syndrome is related to high
intraoperative intravesical pressures. Unlike the standard intermittent flow resectoscopes, it has a fixed
inlet channel for continuous irrigation and an outlet
channel to which low-pressure continuous suction can
be applied (Fig. 5). This allows continuous, simultaneous irrigation and suction, reducing the need for
intermittent bladder emptying that is required with
standard resectoscopes and enables sterile and safe
evacuation of fluids (3). Studies of the continuous flow
resectoscope have shown it to be superior to the
conventional resectoscope in terms of operating rate
and time, blood loss and irrigant absorption (4). Other
advantages include less interruption during the procedure, better endoscopic vision due to a continuous
clear inflow of more than 600 mL/min and low intravesical pressures of less than 10 mmHg. Since the
entire amount of irrigating fluid is collected blood loss
can be calculated and the amount of absorption can be
determined. Despite these reported advantages, preference of the surgeon still tends to dictate the choice of
system used for TURP with many urologists still using
intermittent flow resectoscopes.
Ureteroscopes
Hugh Hampton Young performed the first endoscopy of a dilated ureter in a child in 1912 using a
cystoscope. The first rigid ureteroscopy was performed in the 1970s by Goodman. The tips of the
earliest ureteroscopes were as large as 13 to 16 F. The
second-generation ureteroscopes became smaller at
8.5 to 11 F but often still required dilation of the
ureteric orifice to enable atraumatic intubation. The
developments in fiber-optic technology in the 1960s
together with the demands of proximal upper tract
anatomy led to the development of the modern
496
Singh and Anson
small-caliber ureteroscopes used today, which are

usually between 6 and 8.5 F. These instruments
have moved away from being fully rigid to semirigid because of the subtle flexibility of the metallic
shaft, which allow gentle deflection without deterioration in vision. Other design enhancements include
an offset eyepiece to provide a straight working
channel for rigid lithotripsy and beveled tips to
minimize trauma to the ureteric orifice and mucosa.
As the shaft progresses proximally, its caliber gradually increases giving the ureteroscope more strength
and stability.
The need to access the proximal ureter and
pelvi-calyceal system both for diagnosis and therapy
led to the creation of flexible ureterorenoscopes.
Design of these instruments has rapidly evolved
over the years, particularly with regards to miniaturization to produce a generation of flexible ureteroscopes which are steerable and fully flexible through
to 2708 with nearly one to one torque and limited loss
of image clarity at a magnification of 30 to 50 times.
The present scopes are 6.8 F in diameter with a 3.6 F
working channel for irrigation and the introduction
of working elements such as guidewires, baskets,
graspers, laser fibers and biopsy forceps. Their
bi-directional active deflection near the distal tip
significantly aids maneuverability particularly for
accessing lower pole calyces. Deflection can either
be primary active controlled by a lever or secondary
passive. Dual active deflection ureteroscopes have an
active secondary deflection mechanism controlled by
a second lever instead of a passive mechanism
enabling up to 3608 downward deflection. Miniaturization of ancillary devices such as baskets and laser
fibers has improved the efficacy of flexible ureterorenoscopy bringing the inner recesses of the pelvicalyceal system into focus for both diagnostic and
therapeutic interventions.
The latest flexible ureteroscopes such as the
DUR-D by Gyrus/ACMI have the added advantage
of chip on the tip technology with integrated camera and light within the scope. It has a CMOS imaging
sensor at its distal tip replacing the previous generations highly susceptible and lower resolution fiberoptics. It also employs a cool light LED, which is
associated with fewer burns and lasts longer than
xenon lamps.
However, an important limiting factor remains
the fragility of these instruments. Their small diameter sheaths with even smaller working channels
and deflection mechanisms are at risk of degradation and damage. Limited durability of the scopes
(approximately 4050 uses prior to significant damage) has significant economical implications. Common causes for damage include degradation through
repeated use, excessive torquing/twisting, inadvertent firing of the laser fiber within the working
channel, improper advancement of ancillary devices
through a deflected ureteroscope and inadvertent
damage during cleaning/processing. The use of
nitinol instruments, smaller laser fibers and ureteral
access sheaths can help to improve the life of the
scopes.
Nephroscopes
As with other rigid endoscopes in urology, the design
of the nephroscope is based on a stainless steel sheath,
a port for light transmission, eyepiece for direct vision,
irrigation ports and a working channel. The main
difference is the large caliber of the sheath, which is
usually 24 to 26 F and the large 13.5-F working channel. The nephroscope can either be used with an outer
working sheath to enable continuous irrigation or
more commonly without the outer sheath but using
an Amplatz sheath for irrigation. Most nephroscopes
have an angled offset eyepiece to accommodate rigid
instruments through the working channel.
Flexible nephroscopes, similar in design to the
flexible ureterorenoscopes but shorter and larger are
becoming available to further enhance access during
PCNL.
Laparoscopes
The most commonly used laparoscopes have either 08
or 308 lenses and similarly to cystoscopes are composed of an objective lens, a rod lens system and a
fiber-optic cable. An offset working laparoscope is
also available which comprises a working channel
for the passage of basic laparoscopic instruments.
The advantage of this scope is that it allows the
surgeon to work in direct line with the image and
potentially reduces the number of ports required.
However, the space taken up by the working channel
compromises the optical system resulting in inferior
image quality.
The size of the earlier laparoscopes ranged from
2.7 to 12 mm with a usual size of 10 mm. The larger
laparoscopes provided a wider view, better optical
resolution and brighter image. Now miniaturization
has resulted in the development of new generation
laparoscopes of 2 to 5 mm, which provide comparable
visualization to the original 10-mm laparoscopes.
Other advances in laparoscope technology include
flexible mobile tips, chip on the tip technology
and all-in-one EndoEye (Olympus) laparoscopes.
IMAGING
Light Sources
The quality of the image obtained in endoscopy is
very dependent on the quantity and quality of light
available. A typical light source consists of a lamp, a
heat filter, a condensing lens, an intensity control unit
and a light guide cable.
Lamps
The lamp or bulb is the most important component of
the light source and the quality of light will depend on
the type of lamp used. The main types of lamp in use
are quartz halogen, xenon, incandescent bulbs, and
metal halide vapor arc lamps. The former two are the
most frequently used in the commercially available
light sources.
Halogen bulbs produce a highly efficient, almost

crisp white light source with excellent color rendering
(ability of a light source to reproduce the colors of an
object compared with an ideal or natural light source).
The electrodes in halogen lamps are made of tungsten.
These bulbs are low voltage and use halogen gas,
which enables them to burn more intensely with a
long life (average 2000 hours).
Xenon lamps consist of a spherical or ellipsoidal
envelope made of quartz glass, which can withstand
high thermal loads and internal pressure. For superior
image quality, highest-grade clear fused silica quartz
is used. It is typically doped, although not visible to
the human eye, to absorb harmful UV radiation during use. The light emitted by a xenon lamp is more
natural compared with halogen, but the colors
obtained with xenon tend to have a slightly bluish
tint. Most modern cameras, however, analyze and
compensate for these variations by automatic equalization of whites enabling the same image to be
obtained with both light sources. A proper white
balancing at the start of the procedure is therefore
critical for obtaining a natural color. White light is
composed of equal proportions of red, blue, and green
colors. At the time of white balancing using a white
target, the camera sets its digital coding for these
primary colors to equal proportion. If during the
white balance, the telescope is not seeing a perfectly
white object then the setup of the camera will be
impaired resulting in poor color perception.
Heat Filter
For 100% of energy consumed, a normal light source
uses approximately 2% in light and 98% in heat. A
heat filter is therefore needed to prevent the light cable
from becoming extremely hot. The newer xenon light
sources are defined as cool light, in other words the
heat to light ratio is lowered by creating more light.
They are still not completely heat free and still have
ignition hazards. Precautions must be taken to reduce
such risks including avoiding contact between active
light cables and drapes or the patients skin and using
standby mode wherever possible.
Condensing Lens
The purpose of the condensing lens is to converge the
light emitted by the lamp to the area of the light cable
input. In most light sources it is used for increasing
the light intensity per square centimeter area.
Manual or Automatic Intensity Control Circuit

Manual adjustment allows the light source to be
adjusted to a power level defined by the surgeon.
This may be useful sometimes when close-up viewing
is hampered by too much light or distant views are too
dark.
Modern light systems use automatic intensity
adjustment technology. Electronic signals produced
497
by the camera are coded to be transported. Coding

dissociates the luminance and chrominance of the
image. It is the luminance (quantity of light signal)
that dictates the quality of the final image. When there
is too much light for the image, for example, when the
endoscope is very close to tissue, the luminance signal
increases. Conversely, when the light is low (distant
view or blood in the field), the luminance is low and
the electronic signal is much weaker. A good quality
luminance signal is calibrated to 1 mV. Overexposed
images will cause the electronic signal to increase to
greater than one, while underexposed images drop
the signal to less than one. Light sources analyze these
signals and appropriately lower or increase their
intensity to bring the signal back to the reference
point.
Light Guide Cables

Light guide cables transmit the light output of the
light source to the viewing endoscope. They consist of
flexible glass fiber bundles encased in a cable jacket
with appropriate connectors on either end. The efficiency of light transmission is reduced as the length of
the cable increases. The cables need to be handled
with care as if mishandled or broken, damage to the
fiber bundles or outer coating can result in poor
quality transmission and loss of light. When assessing
the quality of a light guide cable, one must never look
directly at the light with the naked eye, but instead
shining the light on the ceiling or floor can indicate the
number of damaged fibers. Damaged/old cables are
relatively cheap to replace and should be done as
required otherwise the quality of endoscopy will be
significantly compromised.
Camera Systems
The early tube cameras used for endoscopy before
the 1990s were based on cathode ray tube technology
and were heavy and cumbersome to use. They have
now been replaced with chip cameras, which are
based on CCD and analogue technology. A CCD is
an electrical silicon device that is used to create
images of objects and to store information. It converts
light or electrical input into an electronic signal (the
output). The output is then processed and reconstructed on the television monitor into a video
image. The high resolution achieved is related to the
large number of pixels in the CCD array-the more
pixels the finer the detail. Typically, modern CCDs
contain anything from 1000 to 500,000 pixels.
A three-chip camera contains three separate
CCDs, each one taking a separate measurement of
red, green, and blue lights. Light coming into the lens
is split by a trichroic prism assembly, which directs
the appropriate wavelength ranges of light to their
respective CCDs. By taking a separate reading of red,
green, and blue values for each pixel, three-CCD
cameras achieve much better precision than singleCCD cameras. Although three CCD cameras are
498
Singh and Anson
generally regarded to provide superior image quality

to one CCD cameras in terms of color, crispness of
image and resolution, they are bulky and cannot be
coupled directly to the endoscope because of size.
They are also generally more expensive. Such cameras
tend to be used predominantly in laparoscopic surgery. For most other procedures, urologists tend to
employ single-chip cameras, which are small enough
to couple directly with the endoscope without making
it bulky to use.
High-Definition Video Technology

High-definition (HD) imaging systems are an exciting
new development and take digital technology a step
further. The main feature that makes them superior to
standard-definition (SD) systems is the ability to capture and display more imaging data enabling much
more clarity and detail (5).
Image resolution describes the detail an image
holds. It quantifies how close structures can be to
each other and still be visibly apart. An established
measuring unit regarding image resolution is the
number of pixels that make up the image. Pixel
counts can be expressed as a single number, for
example, 3-megapixel digital camera, which nominally has three million pixels or a pair of numbers, as
in a 640 by 480 computer display, which has
640 pixels from side to side and 480 from top to
bottom. The more pixels used to represent an image,
the closer the result can resemble the original.
Increasing the number of pixel lines increases the
quality of the picture and subsequently provides the
surgeon with a more detailed and realistic impression. Pixel density or pixels per inch (PPI) is a measurement of the resolution of a computer/television
display, related to the size of the display in inches
and the total number of pixels in the horizontal and
vertical directions. For example, a display that is 11
inches wide by 8.5 inches high, capable of a maximum 1024 by 768 pixel resolution, can display about
93 PPI in both the horizontal and vertical directions.
This figure is determined by dividing width (or
height) of the display area in pixels, by width (or
height) of the display area in inches. The apparent
PPI of a monitor therefore depends on the screen
resolution (number of pixels) and the size of the
screen in use; a monitor in 800 by 600 mode has a
lower PPI than the same monitor at 1024 by 768
mode.
A key component of HD systems is the highcapacity CCD chips, which capture much more data,
thus producing more pixels, which with the benefit of
digital output result in a more reliable picture with
significantly greater detail and clarity.
The other key component of HD technology is
high-resolution video screens. HD pixels are smaller
and squarer than SD pixels so are sharper and more of
them can be fitted onto the screen. HD screens have
four times the resolution of standard SD screens
enabling them to show all of the fine detail that the
high-capacity chips can deliver (Fig. 6).
Figure 6 Image seen with (A) HDTV and (B) SDTV.
HD imaging has also been reported to offer

better-depth perception, which is particularly important in laparoscopic intracorporeal suturing.
Fluorescent Cystoscopy
Fluorescent cystoscopy forms the basis of the photodynamic diagnosis and therapy of bladder tumors.
When light is targeted at a tissue, some of the photons
will be absorbed by the molecules in the tissue. If the
energy produced by the photon results in the movement of an electron to a higher energy shell, when the
electron returns to it original position, energy is emitted in the form of light (fluorescence). This fluorescent
light can then be detected either with the naked eye as
a change in color or by a sensor. Exogenous fluorescence involves the use of an external chemical to
induce fluorescence (6). One such agent is the photosensitizing agent 5-aminolevulinic acid (5-ALA). 5ALA is the starting point of the heme biosynthesis
pathway. The metabolite immediately before heme in
this pathway is protoporphyrin IX (PPIX), which is
fluorescent, appearing red under blue-violet light.
Photodynamic diagnosis works on the basis that
malignant cells accumulate up to ten times more
PPIX than normal cells. During fluorescent cystoscopy,
exogenous 5-ALA is instilled into the bladder via a
urethral catheter two hours pre cystoscopy but can also
be given intravenously. The procedure uses the D-light
imaging system (Karl Storz) which includes a short-arc
xenon light source and filter turret capable of generating light in three operational modes: conventional
white light, fluorescence and autofluorescence mode.
It is possible to manually change from blue to white
light during the procedure. Specially modified cystoscopes with lenses that maximally enhance the contrast
between benign tissue autofluorescence and tumor
fluorescence are used. Papillary tumors are seen to
appear intensely red under blue light as do areas of
mucosa involved with CIS (Fig. 7).
Figure 7 Cystoscopic views seen under (A) white and (B) blue
light.
Narrow Band Imaging

Narrow band imaging (NBI) is a high-resolution
endoscopic technique using optical filter technology.
It enhances the fine structure of mucosal surfaces, for
example, vascularity by optimizing the absorbance
and scattering characteristics of light. It is based on
the phenomenon that the depth of light penetration
depends on wavelength-the longer the wavelength,
the deeper the penetration. Blue light penetrates only
superficially while red light is able to reach the deeper
layers. The first prototype NBI system (Olympus) is
based on a light source with sequential red-green-blue
(RGB) illumination. White light from a xenon source is
passed through a rotatory RGB filter which separates
the white light into the three separate colors, which
are then used to sequentially illuminate the mucosa.
The red-, green-, and blue-reflected light is detected
separately by a monochromatic CCD at the tip of the
endoscope and the three images are integrated into a
single color image by the video processor. In addition
to the conventional RGB filters for white light endoscopy (WLE), the narrow band imaging system has
special RGB filters of which the band-pass ranges
have been narrowed and the relative contribution of
blue light has been increased. This narrow bandwidth
is strongly absorbed by hemoglobin thus increasing
the clarity of vascular structures, for example, capillaries. The technology has been widely used in gastroscopy, where results have been reported as superior to
WLE especially for the diagnosis of Barretts esophagus. A small study looking at the application of NBI in
cystoscopy showed increased detection rate of urothelial carcinoma compared with WLE (7).
Although the technique is still in its infancy and
the details of further larger studies are needed, the
future appears promising.
499
normal vision, information on depth perception is

interpreted by the visual cortex in response to binocular horizontal disparity or stereoscopy. This occurs
when the left and right eyes view an object from
slightly different angles. The difference between the
resulting images transmitted to the two retinas is
interpreted in the cortex as depth information. During
laparoscopic surgery, both eyes see exactly the same
image and stereoscopic depth information is lost. With
time and experience, surgeons are able to overcome
this by extracting pictorial depth cues from conventional images though this does not substitute for full
3-D perception of the operating field. Stereoscopy,
stereoscopic imaging or 3-D video imaging is any
technique capable of recording three-dimensional
visual information or creating the illusion of depth
in an image thus restoring depth perception by simulating binocular horizontal disparity. The illusion of
depth in a two-dimensional image is created by presenting a slightly different image to each eye. Images
are acquired via a single or dual lens/camera system
and then presented to the surgeon on a head-mounted
display (HMD) or on a video monitor. With a 3-D
video monitor, the two images are separated by displaying a sequence of alternating left and right images
which is synchronized with the surgeons vision in the
left and right eyes, enabling depth perception.
Synchronization can be via liquid crystal shutter
glasses worn by the surgeon, which will let light
through in synchronization with the images on the
display. Alternatively synchronization can be
achieved if the surgeon wears polarized 3-D glasses
whereby a polarizing filter rotates the alternating left
and right images by 908 to achieve image separation.
With an HMD system a small display optic in front of
one (monocular HMD) or each eye (binocular HMD)
displays images (Fig. 8). Head-mounted displays may
also be coupled with head-tracking devices, allowing
the user to look around the operative field by
moving their head which can help overcome the loss
of hand-eye axis which can occur during complex
maneuvers.
Second generation 3-D imaging systems include
autostereoscopy, which obviates the need for HMDs
or glasses altogether. Eye tracking systems are used to
automatically adjust the displayed images and 3-D
field by tracking the surgeons pupils.
Virtual retinal displays create images by scanning low-power laser light directly onto the retina.
ANCILLARY INSTRUMENTATION
Instrument Channels
Three-Dimentional Imaging
The advances in HD and digital technology discussed
earlier have been successfully applied to laparoscopic
surgery resulting in improved image technology and
clarity. The introduction of three-dimentional (3-D)
video imaging systems or stereoscopic imaging in
the last decade has the potential for safer, faster
laparoscopy with a reduced learning curve (8). During
Instrument channels enable the influx and efflux of

irrigant solutions and the delivery of ancillary devices
into the operating field for therapeutic and diagnostic
interventions. The first generation ureteroscopes had
no working channel unlike the modern endoscopes,
which have been designed to have as large an instrument channel as is practically possible or two separate
working channels. Fiber-optic technology and
500
Singh and Anson
to enhance the view particularly when performing

laser procedures.
As flexible ureteroscopes have only one working
channel for irrigation and instrumentation, poor flow
can impede visibility. The use of small (<3 F) instruments can help overcome this limitation.
Ancillary Devices
Guidewires
Figure 8 Head-mounted display for 3-D imaging.
increased miniaturization has enabled the incorporation of instrument channels as large as 75% of the total
diameter of the scope. As a result it is possible to pass
a grasping device and lithotripsy probe simultaneously through the single channel of some scopes.
Although the ideal instrument channel should be as
large as possible, the larger the instrument passed
through the channel, the greater the reduction in flow
of irrigant resulting in impaired visibility. To overcome this limiting factor, as for resectoscopes, some
semi-rigid ureteroscopes now have dual channel functionality allowing a continuous flow irrigation system
to be set up. By keeping the outlet open, it is possible
to balance the hydrostatic pressure within the ureter
Guidewires are pivotal to any endourological procedure. Wires vary in material composition, size, tip
design, rigidity, surface coating, and length (9).
Wires are composed of an inner stainless steel
core, known as the mandrel, which is covered by a
tightly coiled thin spring wire (spring guide). The
mandrel may be round or flat in cross-section.
Round mandrels have a standard stiffness, while flat
mandrels are used for stiffer wires providing greater
rigidity for the same diameter. The size and stiffness
of the mandrel determines the rigidity and strength of
the wire, while the spring guide acts as a track for the
smooth passage of scopes and catheters. Recently
wires have been developed with a mandrel made of
nickel titanium alloy (Nitinol). Unlike stainless steel
these wires are kink resistant and can be designed
with a stiffer core. They also have the added advantage of memory enabling coil and recoil as will be
discussed in more detail below.
The typical range for guidewires is 0.018 (1.4 F)
to 0.038 inch (2.9 F) in diameter and 80 to 260 cm in
length. Tips vary in their length, shape and flexibility.
The distal tip is designed to be soft and flexible
(floppy tip) to minimize trauma to the ureter.
The frictional resistance of the wire is determined by its stiffness and coefficient of friction. The
coating of the wire determines the friction along its
surface. The standard coating on most wires is PTFE
(Teflon), which reduces the coefficient of friction by
50%. The friction coefficient of hydrophilic wires is
only 16% enabling almost frictionless passage. These
latter wires are therefore particularly useful for negotiating an impacted calculus or tortuous ureter.
Some of the newer wires combine the properties
of both standard and hydrophilic wires, for example,
Sensor1 (Boston Scientific) making them universal for
almost all endourological procedures (Fig. 9). The
Sensor wire has a distal hydrophilic tip, which enables
negotiation in difficult situations, combined with a
Nitinol PTFE coated body, which makes the wire kink
resistant.
Retrieval Devices
A variety of baskets and graspers are available for stone
retrieval during endoscopic stone surgery (Fig. 10).
They vary in their size, design and opening mechanisms. They range in size from 1.9 to 7 F with an
average size of 3 F to fit down the working channel
of most ureteroscopes. The device consists of a handle
and a plastic sheath. Within the sheath is a thin shaft of
metal, which forms the basket and comes in various
501
Figure 9 Guide wires. (A) Stiff PTFE guidewire, (B) hydrophilic

guidewire, and (C) PTFE guidewire with hydrophilic tip.
shapes. The handle controls the opening mechanism of

the basket and can be detachable.
Baskets are made from either stainless steel or
nitinol usually with an outer PTFE sheath. In certain
baskets, the sheath can be detached to reduce the
overall size of the instrument of the basket in the
working channel. Most baskets have a variable length
tip extending beyond the basket. Hollow core baskets
have a central channel to accommodate a laser fiber or
guide wire. All baskets have a minimum of three
wires, and the configuration of the wires affects its
retrieval properties.
Similar to baskets, graspers are also made from
stainless steel or nitinol wires, contained in an outer
sheath and controlled by a release handle. They are
typically three-pronged with distal hooks and vary in
diameter from 1.9 to 5 F. Forceps/grasping devices have
teeth to enable grip. They can be reusable or single use
and semi-rigid or flexible depending on where they are
being deployed within the collecting system.
Shape Memory Alloys

Nitinol is known as a shape memory alloy (SMA), in
other words an alloy that remembers its shape. SMAs
Figure 10 Baskets and graspers for stone retrieval.
are able to undergo a molecular rearrangement in the

solid phase (Fig. 11). This means that during a solidstate phase change a molecular rearrangement occurs,
but the molecules remain closely packed so that the
substance remains a solid (10). These phase changes,
known as martensite and austenite, involve the rearrangement of the position of particles within the crystal structure of the solid. Below the transition
temperature, Nitinol is in the martensite phase and
can be bent into various shapes. To fix the parent
shape, the metal must be held in position and heated
to about 5008C. The high temperature causes the
atoms to arrange themselves into the most compact
and regular pattern possible resulting in a rigid cubic
502
Singh and Anson
Figure 11 Shape memory alloy molecular rearrangements:

transformation from the austenite to the martensite phase and
shape memory effect.
tissue, thus decreasing trauma to the tissues as they

enter the abdominal cavity. Studies examining the
benefits of radially expanding, noncutting trocar systems have documented decreased pain, less port-site
bleeding, shorter wound scars and higher patient satisfaction. However, noncutting trocar systems typically require greater force to insert into the abdominal
cavity, potentially increasing the risk of vascular
injury.
In addition to the issue of cutting versus noncutting, the shape of the trocar tip has also been
shown to be important. Conical tips pose less risk to
intra-abdominal structures during insertion compared
with pyramidal tips. Thus the optimal trocar system
seems to be a noncutting trocar with a conically
shaped tip.
In recent years disposable radially dilating noncutting complex trocar systems have been marketed
but cost is a significant issue (Fig. 12). A further
advancement has been the use of optical dilating
bladeless trocars, which have an additional visual
component thus combining radial dilatation with
visual control. This allows safe and controlled placement of the first port during laparoscopy.
MECHANICAL ASSISTANCE DEVICES

arrangement known as the austenite phase. Above the
transition temperature, Nitinol reverts from the martensite to the austenite phase that changes it back into
its parent shape. This cycle of shape deformation and
recovery can be repeated multiple times. The use of a
SMA in urology is well illustrated by the MemokathTM range of stents.
In addition to its excellent shape memory, Nitinol
is thinner and more elastic than conventional stainless
steel. This has led to the commercial introduction of
nitinol as an exciting technology in endourology
enabling greater miniaturization of devices. This in
turn means greater irrigant flow and less limitation to
scope deflection during flexible ureterorenoscopy.
Port Technology
One of the critical areas of importance in laparoscopy
has been the development of an optimal access port
system. Characteristics of an ideal trocar include
decreased tissue trauma and bleeding, decreased
pain at the trocar site, ease of entry, a tight fascial
seal to prevent frequent dislodgement during the
operation, low risk of herniation and elimination of
time-consuming wound closures. In the infancy of
laparoscopy, most trocar systems employed metal,
cutting tips and were reusable. Ease of entry through
tissue was one of the main advantages. However,
reports started to emerge describing complications
associated with trocar systems. These included vascular and visceral injury, abdominal wall hematomas,
trocar site pain and hernias.
As a result of these complications, new trocar
systems were developed to try and minimize some of
these complications. Recent systems have employed
noncutting obturator tips, which dilate or separate
Robotic Technology
Urology has been one of the leading specialities to
pioneer robotic surgery, particularly with the DaVinci
robot for radical prostatectomy. The technique is rapidly being taken up and developed internationally.
The first described use of robotic technology in
the United Kingdom was in the 1980s by the urologist
John Wickham at Guys hospital (11). He designed the
PROBOT. This was essentially a TURP robotic frame,
which could carry out automated transurethral prostate resection. The frame supported a six-axis Unimate
Puma robot together with a Wickham Endoscope
liquidizer, which rotated at 40,000 rpm and an aspirator. Although the technique was proven to be safe,
effective and successful it was not embraced or developed any further by the urological world. The next
generation of robot to follow was the Automated
Endoscopic System for Optimal Positioning (AESOP)
in the 1990s. In this system a voice or pedal-controlled
robotic arm holds and navigates a laparoscopic camera
in response to commands. The EndoAssist is similar
but is controlled by infrared signals from the surgeons
headset. Though cheaper than the AESOP it requires
more space to set up in the operating theater.
The ZEUS and the DaVinci (Fig. 13) robotic
devices were the next master-slave systems to
become commercially available. It is the latter system
that has been widely adopted in urology, with an
increasing number of prostatectomies being performed robotically in the United States and more
recently in the United Kingdom. The system consists
of a remote operating console from which the surgeon
controls the robots arms and energy sources, for
example, diathermy using manual or pedal controls
(12). The robot only translates the surgeons
503
Figure 13 Robotic surgery.
movements as opposed to following preprogrammed

set movements. At the console the surgeon can adopt
a more suitably ergonomic position than is often possible when operating at the patients side. Hand and
foot controls allow more intuitive rather than fulcrum
movements over the instruments as occurs in laparoscopy. This has led to the theory that the learning
curve for robotic surgery is shorter and less demanding than for laparoscopy making the transition from
open surgery to robotics easier. Up to 10 magnification, 3-D stereoscopic vision and motion scaling
greatly enhance the image projected at the console.
Motion scaling and the EndoWrist technology minimize tremor enabling smooth precise surgical movements. The other advantage of the EndoWrist
instruments is that they enable seven degrees of freedom of movement compared with only four with
standard laparoscopy, thus reproducing natural
human wrist movements more accurately. The other
components of the system include a patient side-cart
which supports the robotic arms and the 3-D endoscope and a stack system housing the camera unit,
recording equipment, television screen and a laparoscopic insufflator.
The main disadvantages of the DaVinci system
at present include purchase and maintenance costs,
long setup time and lack of haptic (tactile) feedback.
However, it is anticipated that as the technique is
increasingly embraced and refined over the next few
years, these issues may be less of a problem particularly as surgeons increasingly incorporate virtual reality (VR) simulators into their training.
FUTURE TRENDS
Virtual Endoscopy
Figure 12 (A) Laparoscopy trochar, (B) optical trocar system,

and (C) noncutting radially dilating trochar system.
Virtual endoscopy (VE) is a technique in which computer-simulated 3-D viewing of hollow structures
based on high-resolution anatomical imaging is conducted. VE is based on the careful integration of three
504
Singh and Anson
Figure 14 (A) Virtual colonoscopy image and (B) conventional

colonoscopy image showing 8-mm polyp.
componentsimage acquisition, processing, and

analysis (13). Image acquisition obtains a volumetric
data set using various radiological modalities such as
ultrasound, MRI, or helical CT. Uninterrupted image
volumes are required which are free from motion
artefact and have a high degree of spatial resolution.
Volumetric data are then transferred to a workstation
via a fiber-optic connection for processing images
using perspective projection and real-time 3-D rendering computer techniques. The 3-D images created are
then carefully analyzed. using an endoscopic flythrough simulation where a computer mouse is
used to control the speed and direction of image
presentation. The lumen wall can be made transparent
to view adjacent structures. In this way VE images
enable endoluminal navigation through hollow
organs, simulating conventional endoscopy (Fig. 14).
A specific point in the lumen wall can be targeted and
axial, coronal and sagittal images displayed simultaneously to define the anatomy in more detail. Software programs can also be used to calculate wall
thickness in combination with convexity and curvature information. Thus it is possible to identify hotspot regions, which may harbor pathology.
VE has been used to inspect a number of anatomical regions in particular in the gastrointestinal
tract where virtual colonoscopy has been extensively
studied and has recently received NICE approval. The
virtual colonoscopy experience has shown the technique to be well tolerated, requiring no sedation and is
noninvasive or minimally invasive (14). The applications of VE in urology, which have been described to
date include virtual CT ureteroscopy in the diagnosis
of ureteric disease and virtual CT cystoscopy for
mapping bladder wall thickness. Several clinical studies have validated the use of virtual cystoscopy in the
diagnosis of bladder tumors with high sensitivity and
specificity (15). Although encouraging, the published
experience in this area of urology remains sparse at
present.
continue at an explosive pace with increased miniaturization of equipment and digitalization of images.
Robotic surgery brings the potential for telesurgery and telementoring from a remote, even international site via portable laptop control stations and
wireless connectivity (16). The ability to communicate
with colleagues and experts during procedures
increases training possibilities and patient safety.
VR simulators will become central to the future
training of endoscopic surgeons. Although currently
in its infancy, with refinements in software enabling
tactile as well as visual reality, VR simulation models
will become more lifelike. The operating theater of the
future is likely to undergo radical change to accommodate the shift toward new-age surgery (17). Current operating rooms are cluttered by stack systems
and trolleys bearing insufflators, light sources and
camera control units. Further electrical and biological
hazards are posed by tubing, video cables, light leads,
diathermy sources and foot pedals scattered through
the theater.
Dedicated minimally invasive operating suites
are now available with design elements to facilitate
endoscopic surgery and overcome some of these problems (Fig. 15). Procedures performed in dedicated
minimally invasive surgical suites have been shown
to be shorter and more efficient compared with a
standard operating theater (18). Poor ergonomics has
always been one of the major limiting factors in endoscopic surgery. Use of retractable arms, pendant
workstations and voice command navigation systems
to control equipment will improve these ergonomic
issues. Further development of intraoperative imaging
including real-time 3-D reconstructions of patients
Other Developments
Urological surgery is continuing to evolve rapidly
particularly in the realms of minimally invasive
surgery. It is likely that technological advances will
Figure 15 State of the art theater.
505
REFERENCES
Figure 16 Wireless capsule endoscopy.
and computer-assisted surgery offer surgeons the

opportunity for more accurate surgical planning.
Over the next decade, nanotechnology may see
the development of nanorobots with tiny integrated
circuit processors measuring just 50 mm, which can
operate on individual cells and vessels rather than
organs. Advancements in microchip and wireless technology may also allow the development of implantable
cameras (Fig. 16), sensors and datasets as well as
magnetically controlled implants that can be navigated
remotely. The technology has arrived and carries exciting potential. Future research is likely to focus on the
delivery of diagnostic and therapeutic modalities
through natural orifices so that truly noninvasive
surgery can become a reality.
ACKNOWLEDGMENT
We would like to thank the many manufacturers, in
particular Olympus/ACMI who contributed material
toward this chapter.
1. Shah J. Endoscopy through the ages BJU Int 2002;

89(7):645652.
2. Borin J, Abdelshehid C, Deane L, et al. The distal sensor
digital flexible ureteroscope: an optical evaluation.
Urotrends 2006; 11(2):4.
3. Notley RG. Transurethral resection of the prostate with
and without continuous irrigation. J R Soc Med 1982;
75(11):871874.
4. Iglesias JJ, Sporer A, Gellman AC, et al. New Iglesias
resectoscope with continuous irrigation, simultaneous suction and low intravesical pressure. J Urol 1975; 114(6):
929933.
5. Olympus. Home page. Available at: www.keymed.co.uk/
optimised.
6. Zaak D, Karl A, Knuchel R, et al. Diagnosis of urothelial
carcinoma of the bladder using fluorescence endoscopy.
BJU Int 2005; 96(2):217222.
7. Bryan RT, Billingham LJ, Wallace DM. Narrow-band imaging flexible cystoscopy in the detection of recurrent urothelial cancer of the bladder. BJU Int 2008; 101(6):702705.
8. Pietrzak P, Arya M, Joseph JV, et al. Three-dimensional
visualization in laparoscopic surgery. BJU Int 2006;
98(2):253256.
9. Patel U, Ghani K, Anson K. Equipment used in endourology. Chapter 3 in Endourology A Practical Handbook.
Taylor and Francis Group.
10. NDC. Home page. Available at: http://www.nitinol.com.
11. Challacombe BJ, Khan MS, Murphy D, et al. The history of
robotics in urology. World J Urol 2006; 24(2):120127.
12. Murphy D, Challacombe B, Khan MS, et al. Robotic technology in urology. Postgrad Med J 2006; 82:743747.
13. Assimos DG, Vining DJ. Virtual endoscopy. J Endourol
2001; 15(1):4751.
14. David Burling, James E East, Stuart A Taylor. Investigating
rectal bleeding. BMJ 2007; 335:12601262.
15. Tsampoulas C, Tsili AC, Giannakis D, et al. 16-MDCT
cystoscopy in the evaluation of neoplasms of the urinary
bladder. AJR Am J Roentgenol 2008; 190(3):729735.
16. Agarwal R, Levinson AW, Allaf M, et al. The RoboConsultant: telementoring and remote presence in the operating room during minimally invasive urologic surgeries
using a novel mobile robotic interface. Urology 2007;
70(5):970974.
17. Reijnen MM, Zeebregts CJ, Meijerink WJ. Future of operating rooms. Surg Technol Int 2005; 14:2127.
18. Hsiao KC, Machaidze Z, Pattaras JG. Time management in
the operating room: an analysis of the dedicated minimally
invasive surgery suite. JSLS 2004; 8(4):300303.
34
Minimally Invasive Technologies in the Treatment
of Renal and Prostate Cancer
Hashim U. Ahmed, Caroline Moore, Manit Arya, and Mark Emberton
Department of Urology, Division of Surgical and Interventional Sciences, University
College London, London, U.K.
INTRODUCTION
The use of minimally invasive ablative therapies in
localized prostate and renal cancer offers the potential to reduce side effects and the healthcare burden
associated with traditional surgical modalities. This
article reviews outlines the therapeutic dilemmas
facing patients with localized low volume prostate
cancer and renal tumors and provides a comprehensive review of the basic science of cryotherapy, highintensity focused ultrasound (HIFU), radiofrequency
ablation and emerging technologies such as photodynamic therapy. These ablative technologies can
deliver a minimally invasive, day case treatment
with low morbidity. This chapter reviews the results
of each modality in each of prostate cancer and renal
tumors both in terms of side effects and cancer
control.
Renal Tumors
Renal parenchymal tumors represents approximately
3.5% of all malignancies, but are the third most common cancer of the urinary tract. In the United States,
there was an estimated incidence of 51,190 cases in
2007 with 12,890 renal cell carcinoma related deaths
(1). In the United Kingdom, the incidence was 4622
with 3777 deaths in 2005. Worldwide, there are about
208,500 new cases per year (2). The rising incidence is
exemplified in the United Kingdom with the number
of new cases rising from about 5 per 100,000 population in 1975 to about 10 per 100,000 population in
2005. Similar patterns have been shown in the United
States (3,4). This has predominantly been due to the
increased use of diagnostic imaging for those who
have abdominal symptoms leading to incidentally
discovered small renal masses (5). Incidentally diagnosed renal tumors now account for between half and
two thirds of renal cancer diagnoses (6) and have lead
to a stage migration to lower risk tumors. However,
although there has been an associated increase in
surgical intervention there has been no concomitant

increase in cancer-specific survival (CSS) (7).
Advances in the speed and precision of crosssectional and ultrasound imaging have led to greater
use in investigating abdominal symptoms. Ultrasound, computed tomography (CT), and magnetic
resonance imaging (MRI) are currently being used
for evaluation of abdominal complaints, virtual colonography, lung cancer screening, and follow-up of
various benign and malignant conditions. This has
brought with it a rise in the detection of small (<4 cm)
solid renal masses, such that the majority of newly
diagnosed renal masses are incidentally found (8). As
a result, according to the U.S. SEER (Surveillance,
Epidemiology, and End Results) database over the
last 18 years, renal tumor size at presentation has
steadily and consistently decreased (9). The majority
(6580%) of these tumors are renal cell carcinomas
(RCCs) when histology is evaluated (10). Although
radical nephrectomy was routinely offered for small
renal masses, a nephron-sparing approach is increasingly offered. Despite this, extirpative surgery is not
without shortcomings. Open partial nephrectomy for
instance requires a large flank incision and has a long
postoperative recovery period with complications in
up to one-third (11). Recent publications on laparoscopic partial nephrectomy have reported complication rates that are not too dissimilar to the open
approach (12,13). Nonetheless, the standard of care
for clinically localized RCC remains surgical resection
because of the favorable prognosis associated with
surgery and the relative ineffectiveness of systemic
therapy to give adequate clearance. Patients undergoing radical or partial nephrectomy for tumors, which
are localized and 4 cm or less in size have demonstrated five-year CSS rates in excess of 95% (14).
Laparoscopic approaches to nerve-sparing surgery
have shown equally good early results (15).
However, because of the nature of incidental
tumors, which are small and likely to progress
slowly, observation or active surveillance of selected
small renal masses in elderly populations may be a
Chapter 34: Minimally Invasive Technologies in the Treatment of Renal and Prostate Cancer
recommended strategy. A meta-analysis of clinically

localized renal tumors showed an overall median
growth rate of 0.28 cm/yr for observed lesions across
multiple series (16). While growth rates vary considerably among reports, only 1% of observed lesions in
this meta-analysis progressed to metastatic disease
after a median follow-up of almost three years.
Prostate Cancer
The management of localized prostate cancer has
centered on surveillance or radical therapy such as
prostatectomy or radiotherapy. Furthermore, because
of the reduction in disease severity as a result of early
detection it is likely that the small absolute risk
reductionof approximately 5% over 10 years that
has been demonstrated in a randomized controlled
trial comparing surgery with watchful waitingin
men with low to moderate risk disease is likely to be
reduced even further (17). The overtreatment problem is further exemplified by two recent randomized
controlled trials assessing the role of PSA screening.
The U.S. PLCO trial showed no difference in mortality between the screened and nonscreened arms with
a mean follow-up of 7 years, while the European
ESPRC study showed that 1400 men would need to
be screened and 48 men treated to save one life
within 10 years. The advent of active surveillance
with selective delayed intervention is also likely to
make this difference in mortality between surveillance and radical therapy less significant. As radical
treatments carry significant morbidity with operative
complications (wound infection, hemorrhage, hospital stay) and can cause significant long-term toxicity
(incontinence, impotence, rectal problems) there has
been a demand to develop ablative therapies that
attempt to reduce treatment burden while retaining
cancer control and avoiding the psychological morbidity associated with surveillance. Although a number of minimally invasive therapies have been
describedcryosurgery, HIFU, photodynamic therapy (PDT)each one is at a different stage in its
evaluation and diffusion into clinical practice.
MINIMALLY INVASIVE TECHNOLOGIES:

BASIC SCIENCE
Cryotherapy
Cryotherapy is the ablation of tissue by extremely cold
temperatures. The first written report of its use was in
19th-century London, where Arnott applied ice-salt
mixtures to breast and cervical cancers (18). Cryotherapy exerts its effects via a number of pathways,
namely,
1.
2.
3.
4.
direct cytolysis through extracellular and intracellular ice crystal formation,

intracellular dehydration and pH changes,
ischemic necrosis via vascular injury,
cryoactivation of antitumor immune responses,
5.
6.
7.
507
induction of apoptosis,
endothelial damage leads to platelet aggregation
and microthrombosis, and
injury also occurs during warming because of
osmotic cellular swelling and vascular hyperpermeability.
A number of factors affect the efficiency of tissue

destruction, namely,
1.
2.
3.
4.
5.
6.
velocity of cooling,
nadir temperature,
freezing duration,
velocity of thawing,
number of freeze-thaw cycles, and
presence or otherwise of large blood vessels,
which can act as heat sinks.
Overall, a minimum freezing temperature of

408C for duration of three minutes is sufficient for
tumor eradication (19). It has also been demonstrated
that complete cell death is unlikely at temperatures
greater than 208C, although cells not destroyed by
initial freezing to 208C were destroyed with a second
freeze cycle (20). Histopathological changes after cryotherapy in the prostate is divided into an early degenerative phasebecause of coagulative necrosisand a
later phase of repairfibrosis, calcification and hyalinization (21,22).
Renal tissue needs exposure to or below
19.48C. Clinical protocols err on the side of caution
and freeze to 408C with ultrasound monitoring of
the evolving cryolesion. For renal tumors, in the
absence of direct puncture into the collecting system
with the cryoprobe, the collecting system heals without leading to urinary fistulas. This is in contrast to
radiofrequency ablation, which has been associated
with a greater risk of urinary leakage.

Ultrasound refers to mechanical vibrations above the
threshold of human hearing (16 kHz) and has the
ability to interact with tissue to produce biological
changes. Ultrasound is generated by applying an
alternating voltage across a piezoelectric material
such as lead zirconate titanate. These materials oscillate at the same frequency as the alternating current
causing ultrasound wave that can propagate through
tissues. This in turn causes alternating cycles of
increased and reduced pressure (compression and
rarefaction, respectively). Diagnostic ultrasound usually uses frequencies in the range of 1 to 20 MHz, but
therapeutic HIFU uses frequencies of 0.8 to 3.5 MHz
with delivery of energy within the ultrasound beams
that are several times greater than the energy levels
within diagnostic ultrasound. Therapeutic ultrasound can be conveniently divided into two broad
categories: low intensity (0.1253 W/cm 2) and
high intensity (>5 W/cm2). The former can stimulate normal physiological responses to injury, and
accelerate other processes such as the transport of
drugs across the skin. High-intensity ultrasound can
508
Ahmed et al.
selectively destroy tissue if delivered in a focused

manner (23).
HIFU relies on the physical properties of ultrasound, which allow it to be brought into a tight focus
either using an acoustic lens, bowl shaped transducer
or electronic phased array. As ultrasound propagates
through a tissue, zones of high and low pressure are
created. When the energy density at the focus is
sufficiently high (during the high pressure phase),
tissue damage occurs. The volume of ablation (or
lesion) following a single HIFU pulse or exposure is
small and varies according to transducer characteristics. It is typically shaped like a grain of rice or
cigar with dimensions in the order of 1 to 3 mm
(transverse) " 8 to 15 mm (along beam axis). To ablate
larger volumes of tissue for the treatment of solid
cancers, these lesions are placed adjacent to each
other. The two predominant mechanisms of tissue
damage are by the conversion of mechanical energy
into heat and inertial cavitation. If tissue temperatures are raised above 568C, then immediate thermal
toxicity can occur provided the temperature is maintained for at least one second. This will lead to irreversible cell death from coagulative necrosis. In fact,
during HIFU the temperatures achieved are much
greater than this, typically above 808C, so even short
exposures, can lead to effective cell death. Inertial
cavitation occurs at the same time but is neither as
controllable nor predictable. It occurs because of the
alternating cycles of compression and rarefaction. At
the time of rarefaction, gas can be drawn out of
solution to form bubbles, which then collapse rapidly.
The mechanical stress and a degree of thermal injury
induce cell necrosis (24). Histologically, the tissue
changes that occur are homogeneous coagulative
necrosis, with an inflammatory response that follows
leading to formation of granulation tissueindicated
by the presence of immature fibroblasts and new
capillary formationat the periphery of the necrotic
area at about a week after treatment. Polymorphonuclear leukocytes migrate deep into the treated tissue
and then at two weeks, the boundary of the treated
region is replaced by proliferative repair tissue. The
repair process has not been investigated in detail at
the cellular level beyond this time, but imaging techniques using contrast enhanced ultrasound or magnetic resonance imaging show an eventual shrinkage
of treated volumes indicating that the necrotic area
has been replaced by fibrous scar tissue.
The placement of the small HIFU lesions
requires precise planning for an entire tumor to be
ablated reliably. Furthermore, patient movement can
lead to areas of viable malignant tissue remaining
after treatment and even in ideal situations other
factors can prevent a successful treatment. The most
important of these include the heat sink effect and
calcification. The heat sink effect relates to one area
that overheats in the HIFU pulses pathway and thus
prevents adequate ultrasound propagation to the targeted areasuch a phenomenon occurs if the time
between HIFU pulses is inadequate for tissue cooling
or if an area is high in water content, such as a cyst. In
addition, highly vascularized tissues might be more
resistant to thermal ablation owing to the heat sink

effect of their blood supply. Calcification simply leads
to reverberation and shielding of the targeted area
from parts of the HIFU pulse leading to inadequate
heating of the tissue.
Radiofrequency Ablation
RFA acts by converting RF waves to heat, resulting in
thermal damage. High-frequency current flows from
the needle electrode to target tissue with the resultant
ionic agitation and heat-producing molecular friction,
denaturation of proteins and cell membrane disintegration. The cellular and tissue effects of RFA vary
with the duration of ablation and the local temperature achieved. This temperature-time dependence was
demonstrated by in vitro studies in which irreversible
cell injury of benign and malignant human cell lines
were heated to 458C for 60 minutes, 558C for 5 minutes,
and 708C for 1 minute. These changes take four to six
minutes at temperatures >508C and occur almost
immediately above 608C. Temperatures >1058C result
in vaporization of tissue, resulting in gas formation and
inefficient creation of RF lesion. The goal of RFA is to
induce temperatures of 508C to 1008C throughout the
tumor. Histological analysis after RFA demonstrates
typical coagulative necrosis characterized by cell membrane disruption, protein denaturation and vascular
thrombosis. Exophytic tumors that are surrounded by
perirenal fat are better treated than central tumors in
which vascular structures can act as a heat sink.
In practice, a grounding pad is placed on the
patient, and the radiofrequency probe is inserted in
the ablation zone. A computer-controlled generator
provides an alternating current in the radiowave frequency of the electromagnetic spectrum. Bipolar RFA
decreases the risk of accidental burns associated with
monopolar RFA. The impedance of the tissue to this
monopolar current leads to local tissue hyperthermia,
which is the basis for cell kill effect. The temperatures
reached during RFA depend on the generators power,
tissue impedance, heat conductivity and heat dissipation via the local circulation. Commercially available
RFA units are classified into temperature-based or
impedance-based systems. This means that the computer-controlled generator provides energy to the
probe on the basis of either the average temperature
achieved or the measured impedance of the tissue
monitored during ablation. Impedance rises toward
infinity when tissues are desiccated during ablation or
when there is charring. RFA technology can also be
classified by dry and wet RFA. The latter allow constant
infusion of saline during ablation to reduce the degree
of charring and thus premature rise in impedance.
Photodynamic Therapy
PDT uses a photosensitizing drug activated, after a
given drug light interval, by light of a specific wavelength. It requires tissue oxygen for the treatment
effect, with the activated drug forming reactive
509
Figure 1 Diagrammatic representation of the action of photodynamic therapy.
oxygen species, which are directly responsible for

damage to the treated volume (Fig. 1). The photosensitizing drugs are activated while either in the
tissue or in the vasculature. Tissue activated drugs
have long drug light intervals (typically hours to days)
which means that the drug and light are given in
separate treatment sessions. They usually take a long
time to be cleared from the body, and can accumulate
in the skin, requiring patients to be covered from
sunlight (which could activate the drug and cause a
sunburn like reaction) for a few weeks. Some of the
tissue-activated photosensitizers accumulate preferentially in tumor tissue. This includes amino levulinic
acid (ALA), which is used in the diagnosis of bladder
tumors, and has also been assessed for use in the
treatment of prostate cancer. Vascular activated drugs
have the advantage of a short drug light interval (e.g.,
minutes), which allows the whole treatment to be
done in a single session. They are usually cleared
rapidly from the circulation, without accumulation
in the skin, such that light restrictions are not necessary after a few hours.
Light delivery for prostate cancer, along with
other interstitial tumors, uses low power laser light
directed to the treatment site by optical fibers. These
fibers can deliver light only at the end of the fiber (like
a torch) or along a cylindrical diffuser (like a striplight). For prostate cancer, a transperineal approach is
currently used, with hollow plastic needles placed in
the prostate using transrectal ultrasound imaging.
Cylindrical diffusers of the desired length are then
placed within the hollow plastic needles, and low
power laser, at a wavelength determined by the individual photosensitizer, is delivered to the prostate.
Other approaches that have been used are transurethral light delivery, and open insertion of fibers at
laparotomy.
PROSTATE CANCER: CLINICAL OUTCOME

Cryotherapy
Cryotherapy Devices
In 1966, probes cooled by liquid nitrogen in a closed

circulation was the advent of modern cryotherapy and
one of the very first applications of probe delivered
cryotherapy was transurethral treatment for benign
prostatic hyperplasia, soon followed by prostate cancer treatment using an open perineal approach (25).
The transperineal percutaneous route came about a
few years later (Fig. 2). The first-generation cryosurgery techniques for prostate cancer ablation were
carried out without transrectal ultrasound guidance
and urethral warmers. As a result of accurate monitoring of needle placement and ice ball, this inevitably led
to a high incidence of complications (urinary incontinence, urethral sloughing, and rectourethral fistulae)
(26). Transrectal ultrasound guidance and urethral
warmers were introduced in second-generation cryosurgery leading to more precise probe placement as
well as real-time monitoring of the ice ball. The urethral warmers decreased the risk of urethral sloughing.
Third-generation devices has seen the introduction of
probes in which pressurized gas is used to both freeze
(argon gas) and then actively thaw (helium gas),
allowing for smaller probes (17-gauge) which could
enable more precise treatment a reduction in secondary structure damage, such as the external sphincter.
Clinical Cryotherapy Series
Thirteen series reporting extractable outcome data

on the treatment of primary prostate cancer using
cryosurgery were identified (Table 1) (2739).
These series have a wide range of patients treated
510
Ahmed et al.
overall outcome data with mean number of sessions

ranging from 1.14 to 1.2.
Morbidity data demonstrate a similar variation
in reporting with five series not reporting potency
rates and four series not reporting incontinence rates.
Where these were mentioned, most series did not
clearly outline definitions of potency used, while
incontinence grading was similarly poor. Impotence
rates ranged from 49% to 95% and incontinence rates
(all types) ranged from 0.4% to 16.7%. Fistulae rates
varied from 0% to 0.5%. Other side effects and complications of treatment were not reported consistently
with only three series demonstrating clear morbidity
data and others either not reporting such outcomes or
doing so selectively.

High-Intensity Focused Ultrasound Devices
Figure 2 Diagram demonstrating percutaneous prostate

cryoablation.
(N range 22975) with a wide range of follow-up

(mean range nine months to 12 years). It was not clear
whether there was double reporting of series from
previous reports. Seven series reported on Damico
risk categories, while four reported on high and low
risk categories using a different definition with no
comment on why such criteria were used or whether
they were validated. Two reported no risk stratification. Generation of device also varied but this was
always reported. Hormonal use was reported on nine
series and varied from 0% to 91.5% and was usually in
the form of neoadjuvant treatment to reduce the size
of the gland although the reason for hormonal ablation was not always given. PSA outcome data showed
the greatest variation. A number of PSA nadirs were
used including <1 ng/mL, <0.5 ng/mL, <0.4 ng/mL,
and <0.2 ng/mL. Those series in which follow-up
allowed also reported biochemical control according
to the American Society for Therapeutic Radiology
and Oncology (ASTRO) criteria. Three series also had
salvage cryosurgery cases embedded within the data
and it was not always possible to differentiate
between the two salvage and primary outcomes.
One series modified the ASTRO criteria to determine
failure (PSA < 0.5 ng/mL with increase of at least
0.2 ng/mL on two consecutive occasions). Again,
there were no reasons given as to why these criteria
were used. Biopsy data was extractable from only nine
series, and in those where biopsies were carried out
the majority of series justified this on the basis of a
clinically significant PSA increase or an inability to
obtain a satisfactory PSA nadir rather than doing
biopsies for all men after treatment. Three series
reported on cryosurgery retreatments as part of the
Currently, there are two commercially available transrectal devices that can treat the prostate gland.
Ablatherm high-intensity focused ultrasound device.
The Ablatherm1 device (Edap-Technomed, Lyon,
France) and the Sonablate 1 500 (Focus Surgery,
Indiana, U.S.). The Ablatherm1 device until very
recently had separate imaging (7 MHz) and therapy
transducers (3 MHz) which had a fixed focal length of
4 cm. Prostate imaging during treatment was not
possible but performed between treatment zones
by inserting the imaging transducer through the therapeutic transducer. The latest modification to the
Ablatherm combines treatment and planning probes
so that visual feedback is possible during treatment.
However, because the Ablatherm uses algorithmdriven treatment protocols with preset energy levels,
individual pulse energy levels cannot be modified by
the operator. Other features include the incorporation
of the probe into a table, which holds the pump and
cooling mechanism and on which the patient is placed
in the right lateral position. Treatment is to each lobe
in turn and performed anterior to posterior within a
complete block that incorporates the full anteriorposterior height of the prostate. A number of safety
features that monitor the rectal wall energy deposition
are in place to prevent damage to this area. Many
centers that use the Ablatherm combine transurethral
resection of the prostate or bladder neck incision to
reduce gland size and stricture formation.
Sonablate 500 high-intensity focused ultrasound
device. The Sonablate1 system consists of a rectal
probe (containing the transducer) with an operating
frequency of 4 MHz that attempts to optimize the
combined imaging and therapy roles of the transducer
(Fig. 3). This has the advantage of allowing visualization of treatment effect following each pulse of the
treatment cycle. Degassed water is pumped through
the system and is chilled to temperatures of 178C to
208C to prevent rectal wall injury by heat build-up.
Rectal wall monitoring features are also in place with
this probe. Treatment planning, execution and monitoring are controlled using an user interface which
allows the surgeon to precisely target the area of
2nd
N 204
3rd
N 50
3rd
N 416
3rd
N 31
3rd
N 65
3rd
N 122
3rd
N 22
2nd
N 54
2nd/3rd
N 590
Cohen et al. (34)
Cresswell et al.
(37)
Prepelica et al.
(30)
Han et al. (32)
2nd
N 975
2nd
N 141
2nd
N 383
Long et al. (28)
12 mo
Low 48.4%; high 51.6%b
Not reported
Low 25%; inter 34%;

high 41%
Low 12.5%; high 87.5%b
Low 17%; inter 30%;

high 52%c
21 mo
3 yr
24 mo
5.1 yr
5.43 yr
58 mo
13 mo
35 mo
High 100%b
Primary group not

reported separately
Low 24%; inter 24%;
high 52%
Low 15.9%; inter 30.3%;
high 53.7%
9 mo
Not reported
18%
14%
13%
Not reported
separately
28%
6977% (PSA < 1 ng/mL); 18%

4060% (PSA < 0.4 ng/
mL)
89.5% (old ASTRO); 76%

(PSA < 1 ng/mL); 62%
(PSA < 0.5 ng/mL)
4877% (PSA < 0.3 ng/
mL); 7589% (PSA
< 1 ng/mL); 1.3%
metastases; 1.3%
mortality
63% (PSA < 1 ng/mL);
52% (PSA < 0.5 ng/mL)
49%d
Primary group not

reported separately
35% (PSA < 1 ng/mL)
1 positive biopsy
(only one
biopsied)
81.7% (old ASTRO); 50% 8 patients
(PSA < 4 ng/mL); 35%
biopsied,
(PSA < 1 ng/mL)
1/8 positive
75% (PSA < 0.4 ng/mL)
Not reported
79% (PSA < 0.5 ng/mL);

3% metastases
79.7% < 0.4 ng/mL; 79.6% 10.1%

4-yr biochemical
diseasefree survivala
18 mo
20.4 mo
Positive biopsy
56% (old ASTRO); 62.4% 25.6%

(nadir plus 2 ng/mL)
90% (PSA < 0.5 ng/mL)
4%
Biochemical control
12.55 yr
Low 55%; high 45%b
Low 18%; inter 39%;

high 43%
Low 72%; inter 18%;
high 10%
Low 39.5%; inter 39.5%;
high 21%
Median
follow-up
80% overall
87%
Not reported
0.4%
Not reported
Not reported
7.5% (not graded) 93%

Not reported
Not reported
Fistulae
Perineal fistula 0.2%

Urethrorectal
fistula 0.4%
Not reported
0.5%
0.004%
2%
0
100% impotent
after procedure.
49% predicted
probability of
impotence
86%
0
Not reported
Not reported
Erectile
dysfunction
16.7% mild
72%
2% severe
95%
15.9% (stress
incontinence)
4.3% (pad usage)
1.3%
53%
3% (pad use)
5% (no pads)
Not reported
3.1% (any daily

pad use)
Not reported
3.7% (12 pads/

day)
3.4% (stress
incontinence)
0.6% (total
incontinence)
Not reported
Incontinence
Old ASTRO modified: three consecutive rises with final PSA > 1 ng/mL
Low risk: PSA < 10 ng/mL and Gleason grade $7; high risk: PSA > 10 ng/mL or Gleason grade >7
Low risk: Gleason $6, PSA < 10 ng/mL, stage $ T2a; intermediate risk: any one of Gleason %7 or PSA%10 ng/mL or stage % T2b; high risk: two to three of moderate risk factors
d
PSA nadir %0.5 ng/mL or PSA nadir <0.5 ng/mL with increase of at least 0.2 ng/mL on two consecutive occasions
Cohen et al. (27)
Koppie et al. (39)
3rd
N 76
Donnelly
et al. (38)
Bahn et al. (31)
Aus et al. (29)
Cytron et al. (33)
Polascik et al.
(35)
Ellis et al. (36)
Type, N
Study
Damico risk groups

(unless otherwise
specified)
Table 1 Biochemical and Biopsy Outcome in Series Reporting Treatment of Localized Prostate Cancer Using Cryosurgery
511
512
Ahmed et al.
treatment, adjust the focal length of the transducer

(currently 3 cm, 4 cm, or 4.5 cm) and alter the power
intensity delivered to each focal zone individually.
Rather than a protocol driven treatment, the power
intensity of each pulse is guided by greyscale changes
within the targeted area that represent steam, so that
greater certainty about cell kill is obtained (Fig. 4) (40).
In other words, the power is raised to obtain what we
have deemed Uchida changes (or greyscale popcorning), named after the Japanese urologist who
pioneered work using the Sonablate 500. The Sonablate 500 delivers treatment to the prostate in three
separate blocks. The anterior portion of the prostate is
treated initially, followed by midzone and then posterior gland (Fig. 5). The probe requires adjustment
between each of these blocks. The posterior block is
always treated using the 3-cm focal length and with
lower energy levels. Within each zone, multiple overlapping lesions are created to enhance ablation.
Other high-intensity focused ultrasound devices
include extracorporeal, transperineal, and transurethral.
Clinical High-Intensity Focused Ultrasound Series
Figure 3 The Sonablate 500 transrectal HIFU probe and transducer. Source: Courtesy of UKHIFU Ltd., U.K.
The use of HIFU for the treatment of prostate cancer

has until very recently been nested within a small
number of enthusiasts over the last ten years. These
have refined the indications, developed the technology
Figure 4 Screen capture demonstrating what the operator sees during a Sonablate 500 treatment. Source: Courtesy of UKHIFU Ltd.,
U.K.
Figure 5 Diagrammatic representation of HIFU pulses delivered. Rows of pulses adjacent to each other are used to ablate
larger areas of prostate. Source: Courtesy of UKHIFU Ltd., U.K.
and disseminated their early experience. Fourteen

series have so far reported outcome data, with four
using the Sonablate 500 and the remainder using the
Ablatherm device (Table 2) (4154). There was double
reporting of a number of series, but this was not always
made explicit in the papers. The number of patients
treated ranged from 30 to 402, and mean follow-up
ranged from 11 to 76.8 months. Morbidity data was
inconsistently reported (Table 1). Incontinence rates
were 0.5% to 15.4% and impotence rates ranged from
13% to 53%. These did not always grade the incontinence and where grading was used, these differed and
were therefore not comparable across series. Definitions of impotence and potency had similar problems.
Fistulae rates ranged from 0% to 2%, although most of
the fistulae were nested in the early experience of most
users with prototype machines. Other symptoms were
inconsistently and selectively reported in most series.
Neoadjuvant hormonal therapy was primarily used for
cytoreduction of gland size but represented a possible
confounding factor for cancer control rates. Damico
risk stratification was reported in six series, while one
series categorized all patients as high risk using another
definition (stage % T3a, Gleason score 810, PSA > 20
ng/mL). Biochemical outcome was reported using
either mean PSA values at longest follow-up, PSA
nadir values (<1 ng/mL, <0.5 ng/mL, <0.2 ng/mL,
<0.3 ng/mL, and 0.1 ng/mL), old ASTRO criteria as
well as ASTRO Phoenix criteria (validated for radiotherapy only). Biopsies were not carried out in all men
in all series, but only one series did not report on
biopsy data. Most series included HIFU retreatments
within the overall outcome data with the mean HIFU
sessions ranging from 1.17 to 1.4 (Table 1). Second
HIFU therapies were regarded as part of the treatment
protocol rather than treatment failures per se, since one
of the advantages of HIFU has been purported to be its
repeatability sometimes up to three times for men who
have residual prostate cancer.
The exact follow-up protocol to be used and
criteria for success or failure are yet to be ascertained
in HIFU therapy of prostate cancer, as is the case with
513
cryosurgery. A number of groups have demonstrated

that the use of gadolinium contrast enhanced MRI
may be of use in the early determination of cancer
control. Areas of necrosis are obvious through lack of
enhancement and areas of residual undertreated tissue can be shown as early as two weeks after treatment with good correlation to biochemical outcome.
The exact role for contrast enhanced MRI in follow-up
of patients after HIFU is yet to be determined (55,56).
The natural history of prostate cancer prevents
the use of mortality as an outcome measure in most
short to medium term reports of prostate cancer therapy, so surrogates in the form of biochemical failure
have emerged. However, the optimal definition of
biochemical failure is far from clear. Indeed, because
of this lack of certainty the reporting of minimally
invasive modalities has shown little consistency. The
variability in biochemical outcome is demonstrated by
the differing PSA nadirs used to define successful outcome with groups using any one of PSA < 1 ng/mL,
< 0.5 ng/mL, 0.4 ng/mL, < 0.3 ng/mL, < 0.2 ng/mL,
and < 0.1 ng/mL. PSA of <0.2 ng/mL has evidence
within radical prostatectomy series demonstrating its
effectiveness to predict long-term outcome but such
evidence is insufficient for HIFU (57). A number of
series use three successive PSA rises to define treatment failure according to the old ASTRO criteria to
define biochemical failure after radiotherapy (58). This
has its own drawbacks since the old ASTRO criteria are
not appropriate for evaluating PSA elevations sooner
than three to five years after treatment and were only
developed for use in radiotherapy. Indeed, with the
emergence of the ASTRO Phoenix criteria (nadir 2
ng/dL) (59), the old definition is in itself questionable,
although some HIFU series have attempted to use this
new definition. Indeed, the Phoenix definition of failure is being used by the Federal Drug Administration
as the key determinant of success against which HIFU
will be assessed in FDA Phase II/III trials in the United
States, so this may need to be adopted as a reasonable
standard for the foreseeable future.
Equally, morbidity data are reported poorly.
Most series do not define incontinence or impotence,
while the majority show inconsistent reporting of
other perceived minor complications. As the premise
of minimally invasive ablative therapies is to reduce
the morbidity of therapy for men with localized prostate cancer while retaining cancer control and thus
overcome the therapeutic dilemma that such men
presently face, such lack of open reporting regarding
morbidity data is disappointing. Although such data
are difficult within the constraints of retrospective
case series, it is important that a minimum dataset is
agreed on for reporting complications.
Assessing health technology outcomes is difficult because of the hardware and software developments that occur as well as improved treatment
delivery from overcoming the initial learning curve.
For this reason, comparison with other series utilizing
transrectal HIFU and other modalities is problematic,
since not all publications make such changes explicit.
The impact of these changes as well as the introduction of rectal cooling is evident by a reduction in the
Sonablate 500
N 163
Sonablate 200/500
N 63
Sonablate 200/500
N 181
Ablatherm
N 163
Ablatherm
N 119
Ablatherm
N 140
Ablatherm
N 227
Ablatherm
N 30
Ablatherm
N 30
Ablatherm
N 146
Ablatherm
N 402
Ablatherm
N 271
Ablatherm
N 102
Mearini et al. (42)
Uchida et al. (43)
Blana et al. (45)
Misra et al. (46)
Poissonnier et al.
(53)
Ficarra et al. (54)
Thuroff et al. (49)
Chaussy and
Thuroff (51)
Gelet et al. (50)
18
22
23.8
12
Mean
follow-up
(mo)
19
19
Not available
Not specified
11
22
Not specified
Low 28%; inter 48%;

high 23.6%
20
12
High 100%b
Not specified
27
Not reported
Low 55%; inter 42%;

46.8
high 3%
Low 51.4%; inter 48.6% 76.8
Low 51.5%; inter 48.5% 57.6
Low 35%; inter 41%;

high 24%
Low 29%; inter 45%;
high 26%
Low 49%; inter 29%;

high 9%
Low 28%; inter 38%;

high 34%
Damico risk groups

(unless otherwise
specified)
Any positive biopsy or a PSA > 1 ng/mL with three consecutive rises
Clinical stage of %T3a or Gleason score 810, or total PSA > 20 ng/mL
Vallancien et al.
(47)
Blana et al. (48)
Blana et al. (52)
Uchida et al. (44)
Sonablate 500
N 172
Ahmed et al. (89)
Study
Device, number/
sessions
66% (3 consecutive
increases in PSA
velocity >0.75/yr or
positive biopsy);
8084% (ASTRO)
8084% (old ASTRO)
92% (PSA < 1);

83% (PSA < 0.5);
56% (PSA < 0.1)
Median PSA 0.6; mean
PSA 1.8
68.4% (PSA < 0.5);

66% and 59% at 5
and 7 yr (Phoenix)
84% (PSA < 0.5); 66%
disease-free survival
at 5 yra
90% (PSA < 0.3);
100% (PSA < 1)
Mean PSA 0.9
84%, 80%, and 78%

[at 1, 3, and 5 yr
(ASTRO)]
75% (Phoenix)
86% (PSA < 1.0)
64% (PSA < 0.2)
56.3% (Phoenix)
92.4% no evidence of
residual disease
61% (PSA < 0.2)
83% (PSA < 0.5)
78.2% (Phoenix)
median PSA
nadir 0.15
70.3% (PSA < 0.4)
75% (ASTRO)
Biochemical control
(PSA, ng/mL)
6.1% (grade 1); 1.8%

(grade 2); 0
(grade 3)
Not reported
7.3% (vital
prostate
cancer)
65%
25%
2934%
43%
32%
Not reported
39%
53%
55.3% (erections
sufficient for
intercourse)
Not reported
17% (sexually
active)
13% (erectile
dysfunction)
Not evaluable
10.6% grade 1;
2.5% grade 2;
1.5% grade 3
15.4% pre-HIFU TURP; Not reported
6.9% HIFU only
Not available
Not available
6% (5% grade I,
0.7% grade II)
7%
13%
3%
7%
14%
23%
14%
5.8% grade 1.
No grade 2/3
incontinence
12% grades 1 and 2;
1% grade 3
0.5% (grade 1)
Not reported
13.6%
2% grade 1
13%
IIEF decreased
from 16 to 12
Not available
Not reported
1%
0.6%
Not reported
1%
2%
0.6%
16% mild, mixed;

0.6% grade 3
Fistulae
33.9%
Erectile
dysfunction
7.0% (grade 1, no pads) 70% (sufficient for 0

0.6% (grade 3)
penetrative sex)
IIEF-15 33.828.1
Incontinence
7.6%
Positive
biopsy
Table 2 Biochemical and Biopsy Outcome in Series Reporting Treatment of Localized Prostate Cancer Using HIFU
514
Ahmed et al.
recto-prostatic fistula rate. It was not always clear,

especially from the HIFU series whether software
modifications occurred and to what proportion of
patients it was applied. Using the Sonablate 500, our
own group has demonstrated that by changing power
levels in a real-time fashion so as to visualize greyscale changes (so called visually directed HIFU)
within each focal treatment zone leads to significantly
lower PSA levels postoperatively in the primary setting (60). This was the first attempt at standardizing
HIFU treatment. Future reports should report outcome data from just one clearly defined technique,
device and software and to state otherwise if they
have to report with numerous variables in the technique. Ideally, outcome data should be reported for
the various modifications if possible.
Another difficulty in comparing HIFU with
other treatments for localized prostate cancer is best
illustrated by examining the demographic features of
published trials. Most HIFU reports include patients
in their late seventies and mid-eighties as the technology was initially used on men who were not suitable
for other radical therapies. Heterogeneity of risk categories also exists in the cryosurgical groups and
many series report risk categories using criteria that
have yet to be validated. A number of studies do not
include such data, but do include Gleason scores and
PSA level. Such heterogeneity makes interpretation of
disease-free survival and overall survival between
studies difficult. The majority of published series on
HIFU used in a primary setting include patients who
were medically unfit for surgery or for a variety of
reasons did not wish to have standard radical therapies. This group may be the group that benefit least
from treatment and suffer a greater amount of morbidity from ablative techniques.
Photodynamic Therapy Series

The first report of PDT for prostate cancer was in a letter
to the Lancet in 1990 (61). Two patients, whose prostate
cancer was discovered after transurethral resection of
the prostate, had a second resection, to ensure as complete a resection as possible. The residual prostate was
then treated, using a transurethral approach, with a
tissue based photosensitizer (either hematoporphyrin
derivative or the polyporforin Photofrin). The drug light
interval was either 48 or 72 hours. One patient died six
months later of a previously undiagnosed lung tumor,
and prostate histology at post mortem showed no evidence of residual disease. The second patient had a
reduction in PSA and follow-up biopsy at three months
showed no evidence of residual disease. The next
reported use of PDT is as a salvage treatment following
the failure of external beam radiotherapy. Nathan and
colleagues report the treatment of 14 patients using the
tissue based photosensitizer meso-tetra-hydroxyphenyl-chlorin (mTHPC, Foscan) (62). This was a
phase I/II study at given drug dose (0.15 mg/kg),
with a range of drug light intervals (35 days), and a
variety of light doses, using both bare fibers and cylindrical diffusers. Post procedure CT and MRI scanning
showed devascularized areas, which correlated with the
515
volume of intended treatment. One patient had a rectal

biopsy to assess an area of abnormal rectal mucosa,
following the procedure, and developed a rectourethral
fistula, which required surgical intervention. Other side
effects included urinary retention in three patients, and
temporary stress incontinence in a further two patients.
PSA was reduced in ten out of 14 treatments, by up to
96%. The same group reports primary treatment in a
group of six men, who received a total of ten treatments
(63). Each treatment was designed to treat either the
right or left lobe, depending on biopsy and imaging
results. The treatments were well tolerated, with side
effects including irritative voiding symptoms for two
weeks, temporary recatheterization (for less than two
weeks) after two treatments, and one episode of gramnegative sepsis, despite prophylactic antibiotics. Posttreatment imaging showed devascularized areas in up
to 51% of the gland. PSA levels fell after eight out of ten
treatments.
A group in Germany have looked at the use of
ALA in prostate cancer (64). Their initial work
assessed the distribution of the tissue based photosensitizer ALA following oral administration, four
hours prior to radical prostatectomy. Fluorescence
distribution studies showed that the ALA was present
in areas of prostate cancer, but not in benign tissue.
Following this, they went on to perform photodynamic therapy in six patients, with previously
untreated prostate cancer, using either a transurethral,
transperineal or open surgical approach. No side
effects are reported. Posttreatment monitoring was
done using PSA, which fell by an average of 55%;
unfortunately, there is no report of posttreatment
imaging or biopsies. Hahn and colleagues have
assessed another photosensitizer, motexafin lutetium
(MLu, LuTex) in patients requiring salvage treatment
following either external beam radiotherapy or brachytherapy (6567). The study of 17 men used a variety of
drug doses, drug light intervals and light doses (Table 3).
Those men receiving high-dose PDT (2 mg/kg MLu,
drug light interval of three hours and 150 J/cm light
dose), showed a marked increase, followed by a
decrease, in PSA. The effects were much less marked,
and PSA rises post baseline were earlier, in the men
receiving low-dose PDT (e.g., 0.5 mg/kg MLu, 24-hour
drug light interval and 25 J/cm light dose). Side effects
included mainly grade I urinary symptoms, with catheter related grade II urinary symptoms in one patient
only. Again, no posttreatment imaging is reported.
A vascular acting photosensitizer, (Tookad or
WST-11), one of the palladium bactereopheophorbide
family, has been reported in both primary and salvage
groups. A Canadian group initially performed a drug
dose escalation study, followed by a light-dose escalation study, in men who had previously had external
beam radiotherapy (6871). They showed that around
60% of men receiving whole-gland treatment at the
maximal drug and light dose, had a complete response
to treatment, as shown by almost complete nonenhancement of the prostate on one week posttreatment
dynamic gadolinium enhanced MRI, and prostate biopsies negative for prostate cancer at six months. This
group used computer-aided light delivery planning
48 hr,
72 hr
Transurethral,
15 J/cm2,
638 nm
WST-09 (Tookad) Perineal template,

0.12 mg/kg IV
computer-aided
light-dose
planning
Weersink et al.
(68) Trachtenberg et al.
(69, 70) Haider
et al. (71)
25 days Less than

whole
gland
Less than
whole
gland
Post TURP
remnant
Target
volume
10 min
Whole gland
in multi
fiber
patients
1 cm CDperineal 4 hr
Variable
(N 2);
transurethral
(N 3), 1 at RP,
633 nm
Pinthus et al. (65); Motexafin lutetium Perineal template, 3/6/24 hr Whole gland
25150 J/cm2,
(MLu, LuTex)
Verigos et al.
0.5/1/2 mg/kg IV
(66) Patel
732 nm
et al. (67)
Computer-aided
light-dose planning
ALA 20 mg/kg
orally
Zaak et al. (64)
Freehand
transperineal
insertion; 50 or
100 J or J/cm,
652 nm
mTHPC 0.15 mg/

kg IV
Transperineal,
3 days
freehand
insertion, 20 or
50 J/cm2, 652 nm
Drug
light
interval
Light delivery:
route, dose,
wavelength
Moore et al. (63)
Windahl et al. (61) Hematoporphyrin

derivative (1.5
mg/kg);
Photofrin 2.5
mg/kg, IV
Nathan et al. (62) mTHPC 0.15 mg/
kg IV
Study
Photosensitizer
(drug dose)
Table 3 Studies Using Photodynamic Therapy in the Treatment of Prostate Cancer
Adverse effects
Organ-confined 24 (2 light
fibers); 28
recurrence
multiple
after definitive
fibers
radiotherapy
6
Primary:
Gleason sum
58, PSA
4.910.6 ng/
mL
17
Post
radiotherapy
(8 external
beams,
9 brachytherapies)
Transient rise;
posttreatment PSA
fall with high-dose
PDT only
Yes
Posttreatment
imaging not
reported
Complete
response (MRI
and biopsy) in
60% at high
drug/light dose
1/14 patients
grade II urinary
urgency
(catheter
related); grade I
GU symptoms
in many
2 rectourethral
fistuale;
intraoperative
hypotension;
decreased
urinary function
until 6/12
6 (10)
Gleason 3 3
in all; PSA
1.915;
6 primary,
4 repeat
treatments
10/14 PSA reduction

(up to 96%)
Rectourethral
fistula after
rectal biopsy;
2 stress
incontinence;
3 acute
retention
$51 cm3 necrosis; PSA reduction in 8/10 1 gram-negative
treatments
sepsis; irritative
necrosis/fibrosis
voiding
on biopsy;
symptoms for
residual cancer
2 wk; 2 recathin all
eterization
(1 mild stress/
urge
incontinence)
None reported
Yes (average decrease None
55%)
Up to 91%
necrosis on
cross-sectional
imaging; 3/14
negative
biopsies
14
Reduction (102.5 ug/L; None reported

6.00.2 ug/L)
PSA response
Post
radiotherapy
for T2/3
cancer; PSA
pre-RT up to
37
Imaging results
Not assessed
No. patients
2
Post TURP
(primary)
Patient
characteristics
(Gleason, PSA,
primary/
salvage)
516
Ahmed et al.
software to help to determine the optimal positions of

the fibers on the basis of pretreatment imaging and
average optical properties of the prostate. Side effects
included two rectourethral fistulae, one of which
required surgical intervention. Urinary side effects
tended to last for up to six months. The same photosensitizer has been used by a group in London, in men
with no previous treatment for prostate cancer (72). The
first part of the study involved one optical fiber in each
of the right and left lobes, to assess the effect of different light doses. The second part assessed the effect of
multiple optical fibers. Posttreatment MRI showed
nonenhancing lesions in the treatment volume, for
those patients receiving optimal light doses. Publication of the full results is awaited.
A number of ways of improving PDT for prostate cancer have been suggested, including the modification of photosensitizers to allow more accurate
targeting of cancer within the gland and more complex ways of monitoring and adjusting the treatment
in real time (by assessment of intraprostatic drug, light
and oxygen levels). Further studies will show whether
these developments will allow the development of
photodynamic therapy from its current status as an
experimental procedure, to one that is an established
technique for both primary and salvage treatment of
organ-confined prostate cancer.
FUTURE PERSPECTIVES: FOCAL THERAPY

Focal therapy involves treatment directed only at the
cancer focus and a margin of tissue surrounding the
cancer (73,74). Most treatment related side effects are due
517
to injury to the immediate surroundings of the prostate

and not due to treatment of the prostate per se. Damage
to prostate capsule, pelvic nerves/ganglia, bladder neck,
bladder, seminal vesicles, rhabdosphincter, Denonvilliers
fascia, and rectum cause erectile dysfunction, ejaculatory
dysfunction, stress-related urinary incontinence, urgerelated urinary incontinence, reduced bladder functional
capacity, urethral or bladder neck strictures and bowel
dysfunction (75,76).
Since unifocal or unilateral disease is present in
up to one-third of men who are diagnosed in the PSAscreened era, it is feasible to attempt focal therapy.
The modalities of cryosurgery, HIFU and photodynamic therapy are ideally placed to take on this new
therapeutic strategy since they are capable of creating
localized focal necrosis within the prostate of a predetermined size in a relatively controlled manner.
To date, only case series carried out in single
institutions have reported as full publications although
prospective trial data is starting to emerge at the time of
writing (Tables 4 and 5). Onik et al. (86) first reported
their results on hemiablation using cryotherapy. These
results have recently been updated with the results of
55 men completing at least one-year follow-up (77).
95% (52/55) had a stable PSA (as defined by ASTRO
criteria) and of the 51 potent prior to the procedure,
44 (86%) remained potent after hemiablation. Four (7%)
required retreatment because of cancer remaining in
the untreated area. Transperineal template biopsies
were used to verify unilateral cancer and data using
transperineal 5 mm spaced template biopsies shows
that just under half of patients deemed to have unilateral disease by TRUS biopsy actually have cancer in
Table 4 Series Evaluating Focal Therapy of Prostate Cancer Using Cryotherapy
Number
Therapy
Biopsy
Mean PSA (ng/mL)
Gleason score
Potency
Incontinence
F/U (mean, mo)
Disease control
Onik et al. (77)

(Endocare)
Ellis et al. (78)

(Endocare)
Lambert et al.
(79) (Oncura)
Bahn et al.
(80) (Endocare)
Crawford/Barqawi
(81) (Endocare)
COLD registry
(82) (Endocare)
112
Hemi
Template
8.3
$6
85%
0%
43.2
93% NED
60
Hemi
TRUS
7.2 / 4.7
</ 8
70.6%
3.6%
15.2
76.7% (biopsy)
25
Hemi
TRUS
6 (range 113)
</ 7
70.8%
0%
28
88% (>50% nadir
reduction)
31
Hemi
TRUS Doppler
4.95
</ 7
89%
0%
70
96% (biopsy)
92% (ASTRO)
100
Focal
Template
5.2 / 4.1
</ 7
83%
97% (biopsy at
12/12)
795
Focal/partial
TRUS
</ 8
65%
2.8%
12
4.5% (36/295)
25% (36/199)
83% (ASTRO)
Table 5 Series Evaluating Focal Therapy of Prostate Cancer Using HIFU
Number
Therapy
Biopsy
Mean PSA (ng/mL)
Gleason score
Potency
Incontinence
Disease control
Muto et al. (83)

(Sonablate 500)
Barret (84)
(Ablatherm)
29
Hemiablation
TRUS biopsy
5 (range 225)
</ 8
Not reported
Not reported
76.5% (biopsy)
12
Hemiablation
TRUS biopsy
<10
</ 7
Not reported
0%
58% (10 yr)
Ahmed/Emberton (85) (Sonablate 500)

20
Hemiablation
Template
<15
</ 7
95%
5%
10% (any cancer)
(0% significant cancer) (biopsy)
518
Ahmed et al.
both lobes (87,88). Bahn et al. (80) have recently

reported another series in which hemiablation was
carried out using cryosurgery. At a mean follow-up
of 70 months, biochemical disease-free status, according to the ASTRO definition, was maintained by 92.8%
of patients (26/28) and a 96.0% negative-biopsy rate
(24/25) was observed. Potency was retained by a total
of 88.9% of those potent prior to treatment. The investigators used color doppler-guided TRUS biopsies to
verify unilateral disease as well as posttreatment success. Since this technique does not carry the necessary
accuracy for cancer detection significant under-treatment was a major problem. Another group demonstrated in 60 men who underwent focal cryosurgery
followed by penile rehabilitation with a vacuum
device, that 73% of those potent prior to treatment
maintained potency alongside a low incontinence rate
of 3.6% (78). After therapy, 35 patients underwent
biopsy, with 14 showing positive findings (40.0%) at a
mean of 12.0 months post treatment. Thirteen out of
fourteen were actually from the untreated side. This
was not surprising since this group do not report on
using any further evaluation beyond TRUS biopsy to
establish location of cancer. Lambert et al. (2007)
reported on a small series of 25 who underwent hemiablation cryosurgery for presumed unilateral disease,
although again this was based on TRUS biopsy at
diagnosis only (79). Of the 24 patients potent prior to
hemiablation cryosurgery, 17 (71%) were potent postoperatively. There was no rectal toxicity or incontinence. Recurrent cancer was detected in 3, with two
recurrences on the contralateral side and one recurrence on the ipsilateral side of the cryosurgery. All
were retreated successfully. Finally, Muto et al. (2008)
report on hemiablation using the Sonablate 500 HIFU
device (83). 29 patients who were found to have unilateral disease on the basis of TRUS biopsy were treated
to ablation of both peripheral zones and one half of the
transition zone. 10% (3/28) had positive biopsies at six
months whereas 23.5% (4/17) had further positive
biopsies at 12 months. There was no significant change
in the IPSS score for urinary symptoms, although there
was one urethral stricture and one urinary tract infection. Although demonstrating feasibility of focal therapy using a transrectal HIFU device, the reporting in
this series was very poor and the group tried to make
comparison with a nonrandomized group that were
treated with whole-gland HIFU. Indeed, they made no
comment on the erectile function rate.
This very limited data from uncontrolled case
series suggests that treatment related toxicity could
be reduced by treating malignant areas while preserving a significant amount of prostate tissue. Equally, the
data suggests it might be possible to obtain clinically
important periods of remissions from disease progression. Nonetheless, what is now required is a standardized assessment of a focal therapy intervention in a
well-characterized group of men with a follow-up regimen under ethical prospective trial conditions. Two
prospective National Cancer Research Network (U.K.)
HIFU trials using the Sonablate 500 device evaluating
the role of hemiablation of unilateral disease and focal
ablation of bilateral, low volume disease verified by
template transperineal prostate biopsies, are being

undertaken at our center (87). At the time writing,
interim results have demonstrated preservation of genitourinary function in 95% of men with residual
low volume cancer in the treated areas of approximately 10% (89). Prospective ethically approved trials
evaluating hemiablation cryosurgery are underway
in Colorado, U.S.A. (90), Memorial Sloan Kettering
Cancer Center and MD Anderson Cancer Center.
Focal therapy can be delivered in a patientspecific manner as described below:
Hemiablation of Unilateral Cancer

The entire lobe affected, regardless of volume or
position of cancer foci.
Focal Ablation of Unilateral or Bilateral Disease

Only cancer areas will be ablated with a margin (23 mm)
of normal tissue and at least one neurovascular bundle
will be preserved. Focal ablation, rather than hemiablation, of unilateral disease will be used if the cancer is
confined to just one quadrant.
Index Lesion Ablation

Ablation of the largest focus of cancer alone with a
2- to 3-mm margin, provided the remaining lesions are
small (total volume <0.2 cc) and low grade (Gleason
score $6) (Figs. 6 and 7A, B).
What is the justification for the inclusion of the
third intervention, index lesion ablation? First, there is
good evidence that disease progression is determined
by the volume of a tumor and that this volume equates
to about 0.5 cc. Second, about 80% of men have a
Figure 6 Whole-mount section of a prostate showing an index

lesion in the right peripheral zone with a smaller low volume
(<0.5 cc) lesion with Gleason score 6. This would be a typical
example of a man who may have been suitable for index lesion
ablation.
519
to propose that ablation of the dominant lesion will

give rise to disease control provided the remaining
lesions can be well characterized in the pretreatment
evaluation. We would propose a lower volume threshold (<0.2 cc) to ensure that this minimizes the risk from
such a therapeutic strategy.
RENAL TUMORS
Cryotherapy
There are currently four cryoablation manufacturers:
Endocare (California, U.S.), Galil Medical (Yokneam,
Israel), Oncura (Illinois, U.S.), and Cryomedical
Sciences (Maryland, U.S.). The first three-use argon
gas to cause rapid freezing at the probe tip was based
on the Joule-Thomson effect. The Cryomedical
Sciences unit is a nitrogen-based system and is currently only used for prostate and liver applications.
The recent development of third-generation cryotechnology using argon/helium gas circulation and ultrathin 17-gauge needles has improved the procedure
with reduced traumatic penetration of the renal capsule and precise insertion of the cryoprobes into the
tumor under intraoperative ultrasound guidance.
Multiple small probes can also be placed into the
tumor so minimizing blood loss when the probes are
removed as would happen with larger probes. Currently, renal cryoablation is performed laparoscopically or percutaneously. Surgeon preference and
experience are crucial for choosing the optimal
approach because each has advantages and limitations. Laparoscopic renal cryoablation offers the
advantage of precise cryoprobe positioning and monitoring of the ice ball under real-time ultrasound as
well as vision. Ultrasound shows the ice ball as a
hyperechoic advancing area with a posterior acoustic
shadow. Anterior or medial tumors, which would be
difficult to target percutaneously can be approached
laparoscopically. Percutaneous renal cryoablation is
performed with the use of open gantry magnetic resonance imaging or CT guidance, although there has
been a report of percutaneous cryoablation under
general anesthesia with ultrasound guidance alone.
Figure 7 (A) Pretreatment dynamic gadolinium contrast

enhanced axial MRI demonstrating a malignant lesion in right
peripheral zone of prostate at the midgland. Template transperineal biopsies with sampling every 5 mm demonstrated two
biopsies positive in that area (Gleason 3 4) with 1 mm Gleason
3 3 in one biopsy from the left side of the gland. (B) Post-HIFU
ablation of the right peripheral zone index lesion dynamic contrast enhanced axial MRI at the same level demonstrating necrosis (dark area).
dominant tumor such as this, which also gives rise to

most of the extracapsular extension. Third, 80% seem
to have a dominant lesion with the remaining lesions of
low grade ($Gleason score 6) and a total volume of
secondary lesions <0.5 cc. It seems reasonable therefore
RFA
Open, laparoscopic, and percutaneous techniques
have been used for renal RFA. Laparoscopic RFA
has the advantage of mobilization of the tumor and
avoidance of adjacent organ damage, and placement
of the probe under direct vision, whereas percutaneous RFA, which is most popularly used, is better
tolerated, can be performed under sedation on an
outpatient basis and fits the minimally invasive criteria more closely. Probes are inserted under ultrasound, CT, and MRI guidance although crosssectional imaging is by far more recommended since
formation of microbubbles and the hyperechoic rim
make ultrasound for real-time imaging of RFA problematic. The success of RFA is usually assessed typically by CT scan one month after treatment by
520
Ahmed et al.
demonstrating the absence of enhancement in the

ablated tissue as early scans usually show a false
positive enhancing rim.
RFA is suitable for tumors that are <4 cm which
are >1 cm from the pelviureteric junction, and >1 cm
from segmental renal vessels without abutting the
pyelocalyceal system. Tumor location is the most
important determinant for the choice of surgical
approach since posterior or laterally based tumors
are best served with the percutaneous approach and
anterior tumors using a laparoscopic method. The
feasibility of RFA for the treatment of renal tumors
was reported in 1997 with only three patients who
then underwent surgical resection after ablation. Several case series using percutaneous RFA have been
published, but with large variation in patient selection, technique, follow-up and outcomes.
A meta-analysis conducted in 2008 evaluated
forty-seven studies (1375 kidney lesions in total)
treated by cryoablation or RFA. Cryoablation usually
was performed laparoscopically (65%), whereas 94%
of lesions treated with RFA were approached percutaneously. The study found no differences between
ablation modalities with regard to mean patient age,
tumor size or duration of follow-up. However, pretreatment biopsy was performed significantly more
often for cryoablated lesions (82.3%) compared with
RFA (62.2%; P < 0.0001), while unknown pathology
occurred at a significantly higher rate for small renal
masses that underwent RFA (40.4%) as opposed to
cryoablation (24.5%; P < 0.0001). Repeat ablation treatment was required more often after RFA (8.5% vs.
1.3%; P < 0.0001) and the rates of local tumor progression were significantly higher for RFA (12.9% vs.
5.2%; P < 0.0001) compared with cryoablation. Metastasis was reported less frequently for cryoablation
(1.0%) versus RFA (2.5%) although this did not reach
statistical significance P 0.06). The meta-analysis
concluded that short-term data on tumor ablation,
retreatment and local tumor progression supported
cryoablation over RFA although development of
metastases and long-term survival were yet to be
shown to be superior, especially with lack of randomized comparative trials.
SUMMARY
The therapeutic dilemma between surveillance and
radical therapy for those with early low risk prostate
cancer and renal tumors combined with the significant
morbidity associated with radical therapies has led to
development of minimally invasive procedures that
attempt to achieve effective cancer control while
reducing the treatment burden. HIFU, RFA and cryosurgery have emerged as forerunners in this field and
mature datasets now exist showing effective cancer
control in the medium term with good side-effect
profiles. PDT may follow suit. However, there is still
much to determine. There is yet to be any agreement
on what constitutes a successful treatment until longterm data are available.
CONFLICT OF INTERESTS
Hashim Uddin Ahmed and Mark Emberton receive
funding from the Medical Research Council, Pelican
Cancer Foundation, The Prostate Research Campaign,
U.K. (charity) and Prostate Cancer Research Centre,
U.K. (all charities) for work in focal therapy of prostate
cancer. In addition, Mark Emberton and Caroline
Moore receive funding and are Consultants for
Negma Lerads, France (manufacturers of TOOKAD,
a photodynamic agent used in prostate cancer therapy). Mark Emberton is a Consultant for Misonix/
Focus Surgery/UKHIFU (manufacturers and distributors of the Sonablate 500 HIFU device). Mark Emberton
and Hashim Ahmed receive funding from the Pelican
Cancer Foundation charity (U.K.) and Misonix/Focus
Surgery/UKHIFU (manufacturers and distributors of
the Sonablate 500 HIFU device). Mark Emberton and
Hashim Ahmed are approved proctors for training
other surgeons in the use of the Sonablate 500 machine
and have received fees for this activity.
Manit Arya has no conflict of interest.
REFERENCES
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2007. CA
Cancer J Clin 2007; 57:4366.
2. Lindblad P, Adami HO. Kidney Cancer in Textbook of
Cancer Epidemiology. New York: Oxford University Press,
2002:467485.
3. Office for National Statistics, Registrations of cancer diagnosed in 2005, England 2008.
4. Chow WH, Devesa SS, Warren JL, et al. Rising incidence of
renal cell cancer in the United States. JAMA 1999;
281(17):16281631.
5. Jayson M, Sanders H. Increased incidence of serendipitously discovered renal cell carcinoma. Urology 1998;
51:203205.
6. Volpe A, Panzarella T, Rendon RA, et al. The natural
history of incidentally detected small renal masses. Cancer
2004; 100:738745.
7. Hollingsworth JM, Miller DC, Daignault S, et al. Rising
incidence of small renal masses: a need to reassess treatment effect. J Natl Cancer Inst 2006; 98:13311334.
8. Lightfoot N, Conlon M, Kreiger N, et al. Impact of noninvasive imaging on increased incidental detection of renal
cell carcinoma. Eur Urol 2000; 37:521527.
9. Nguyen MM, Gill IS, Ellison LM. The evolving presentation of renal carcinoma in the United States: trends from
the Surveillance, Epidemiology and End Results program.
J Urol 2006; 176(6 pt 1):23972400.
10. Frank I, Blute ML, Cheville JC, et al. Solid renal tumors: an
analysis of pathological features related to tumor size.
J Urol 2003; 170(6 pt 1):22172220.
11. Pasticier G, Timsit MO, Badet L, et al. Nephron-sparing
surgery for renal cell carcinoma: detailed analysis of complications over a 15-year period. Eur Urol 2006; 49:485490.
12. Matin SF, Gill IS, Worley S, et al. Outcome of laparoscopic
radical and open partial nephrectomy for the sporadic
4 cm or less renal tumor with a normal contralateral
kidney. J Urol 2002; 168:13561360.
13. Ramani AP, Desai MM, Steinberg AP, et al. Complications
of laparoscopic partial nephrectomy in 200 cases. J Urol
2005; 173:4247.
14. Frank I, Blute ML, Leibovich BC, et al. Independent validation of the 2002 American Joint Committee on cancer primary tumor classification for renal cell carcinoma using a
large, single institution cohort. J Urol 2005; 173:18891892.
15. Moinzadeh A, Gill IS, Finelli A, et al. Laparoscopic partial
nephrectomy: 3-year follow up. J Urol 2006; 175:459462.
16. Kunkle DA, Egleston BL, Uzzo RG. Excise, ablate or
observe: the small renal mass dilemmaa meta-analysis
and review. J Urol 2008; 179(4):12271233.
17. Bill-Axelsen A, Holmberg L, Mirrja Ruuth, et al. Watchful
waiting and prostate cancer. NEJM 2005; 352:19771984.
18. Arnott J. On the Treatment of Cancer by the Regulated
Application of an Anesthetic Temperature. London:
Churchill, 1851.
19. Hoffmann NE, Bischof JC. The cryobiology of cryosurgical
injury. Urology 2002; 60(2 suppl 1):4049.
20. Tatsutani K, Rubinsky B, Onik G, et al. Effect of thermal
variables on frozen human primary prostatic adenocarcinoma cells. Urology 1996; 48:441447.
21. Borkowski P, Robinson MJ, Poppiti RJ Jr, et al. Histologic
findings in postcryosurgical prostatic biopsies. Mod Pathol
1996; 9:807811.
22. Grampsas SA, Miller GJ, Crawford ED. Salvage radical
prostatectomy after failed transperineal cryotherapy: histologic findings from prostate whole-mount specimens
correlated with intraoperative transrectal ultrasound
images. Urology 1995; 45:936941.
23. Hill CR, ter Haar GR. Review article: high intensity
focused ultrasoundpotential for cancer treatment. Br J
Radiol 1995; 68(816):12961303.
24. Kennedy JE. High-intensity focused ultrasound in the
treatment of solid tumours. Nat Rev Cancer 2005;
5(4):321327.
25. Soanes WA, Gonder MJ. Use of cryosurgery in prostatic
cancer. J Urol 1968; 99(6):793797.
26. Merrick GS, Wallner KE, Butler WM. Prostate cryotherapy:
more questions than answers. Urology 2005; 66(1):915.
27. Cohen JK, Miller RJ, Rooker GM, et al. Cryosurgical ablation of the prostate: two-year prostate-specific antigen and
biopsy results. Urology 1996; 47:395401.
28. Long JP, Bahn D, Lee F, et al. Five-year retrospective,
multi-institutional pooled analysis of cancer-related outcomes after cryosurgical ablation of the prostate. Urology
2001; 57:518523.
29. Aus G, Pileblad E, Hugosson J. Cryosurgical ablation of the
prostate: 5-year follow-up of a prospective study. Eur Urol
2002; 42:133138.
30. Prepelica KL, Okeke Z, Murphy A, et al. Cryosurgical
ablation of the prostate: high risk patient outcomes. Cancer
2005; 103:16251630.
31. Bahn DK, Lee F, Badalament R, et al. Targeted cryoablation
of the prostate: 7-year outcomes in the primary treatment
of prostate cancer. Urology 2002; 60:311.
32. Han KR, Cohen JK, Miller RJ, et al. Treatment of organ
confined prostate cancer with third generation cryosurgery: preliminary multicenter experience. J Urol 2003;
170:11261130.
33. Cytron S, Paz A, Kravchik S, et al. Active rectal wall
protection using direct transperineal cryo-needles for histologically proven prostate adenocarcinomas. Eur Urol
2003; 44:315321.
34. Cohen JK, Miller RJ Jr, Ahmed S, et al. Ten-year biochemical disease control for patients with prostate cancer
treated with cryosurgery as primary therapy. Urology.
2008; 71(3):515518.
35. Polascik TJ, Nosnik I, Mayes JM, et al. Short-term cancer
control after primary cryosurgical ablation for clinically
localized prostate cancer using third-generation cryotechnology. Urology 2007; 70(1):117121.
521
36. Ellis DS, Manny TB Jr, Rewcastle JC. Cryoablation as

primary treatment for localized prostate cancer followed
by penile rehabilitation. Urology 2007; 69(2):306310.
37. Cresswell J, Asterling S, Chaudhary M, et al. Third-generation cryotherapy for prostate cancer in the UK: a prospective study of the early outcomes in primary and recurrent
disease. BJU Int 2006; 97(5):969974.
38. Donnelly BJ, Saliken JC, Ernst DS, et al. Prospective trial of
cryosurgical ablation of the prostate: five-year results.
Urology 2002; 60:645649.
39. Koppie TM, Shinohara K, Grossfeld GD, et al. The efficacy
of cryosurgical ablation of prostate cancer: the University
of California, San Francisco experience. J Urol 1999;
162:427432.
40. Haar GT, Coussios C. High intensity focused ultrasound:
physical principles and devices. Int J Hyperthermia 2007;
23(2):89104.
41. Zacharakis E, Ahmed HU, Dudderidge T, et al. Transrectal
High Intensity Focused Ultrasound in the treatment of
localised prostate cancer the first UK series. J Urol 2008;
179(4 suppl 1):493.
42. Mearini L, DUrso L, Collura D, et al. Visually directed
transrectal high intensity focused ultrasound for the treatment of prostate cancer: a preliminary report on the italian
experience. J Urol 2009; 181(1):105111.
43. Uchida T, Ohkusa H, Nagata Y, et al. Treatment of localized prostate cancer using high-intensity focused ultrasound. BJU Int 2006; 97:5661.
44. Uchida T, Ohkusa H, Yamashita H, et al. Five years experience of transrectal high-intensity focused ultrasound
using the Sonablate device in the treatment of localized
prostate cancer. Int J Urol 2006; 13(3):228233.
45. Blana A, Rogenhofer S, Ganzer R, et al. Eight years experience with high-intensity focused ultrasonography for
treatment of localized prostate cancer. Urology 2008;
72(6):13291333.
46. Misra V, Roupret M, Chartier-Kastler E, et al. Oncologic
control provided by HIFU therapy as single treatment in
men with clinically localized prostate cancer. World J Urol
2008; 26(5):481485.
47. Vallancien G, Prapotnich D, Cathelineau X, et al. Transrectal
focused ultrasound combined with transurethral resection
of the prostate for the treatment of localized prostate cancer:
feasibility study. J Urol 2004; 171:22652267.
48. Blana A, Walter B, Rogenhofer S, et al. High-intensity
focused ultrasound for the treatment of localized prostate
cancer: 5-year experience. Urology 2004; 63:297300.
49. Thuroff S, Chaussy C, Vallancien G, et al. High intensity
focused ultrasound and localized prostate cancer: efficacy
results from the European multicentric study. J Endourol
2003; 17:673677.
50. Gelet A, Chapelon JY, Bouvier R, et al. Transrectal high
intensity focused ultrasound for the treatment of localised
prostate cancer: factors influencing the outcome. Eur Urol
2001; 40:124129.
51. Chaussy C, Thuroff S. The status of high-intensity focused
ultrasound in the treatment of localized prostate cancer
and the impact of a combined resection. Curr Urol Rep
2003; 4:248252.
52. Blana A, Murat FJ, Walter B, et al. First analysis of the longterm results with transrectal HIFU in patients with localised prostate cancer. Eur Urol 2008; 53(6):11941201.
53. Poissonnier L, Chapelon JY, Rouvie`re O, et al. Control of
prostate cancer by transrectal HIFU in 227 patients. Eur
Urol 2007; 51(2):381387.
54. Ficarra V, Antoniolli SZ, Novara G, et al. Short-term outcome after high-intensity focused ultrasound in the treatment of patients with high-risk prostate cancer. BJU Int
2006; 98(6):11931198.
522
Ahmed et al.
55. Kirkham AP, Emberton M, Hoh IM, et al. MR imaging of

prostate after treatment with high-intensity focused ultrasound. Radiology 2008; 246(3):833844.
56. Rouvie`re O, Lyonnet D, Raudrant A, et al. MRI appearance
of prostate following transrectal HIFU ablation of localized
cancer. Eur Urol 2001; 40(3):265274.
57. Ganzer R, Rogenhofer S, Walter B, et al. PSA nadir is a
significant predictor of treatment failure after high-intensity focussed ultrasound (HIFU) treatment of localised
prostate cancer. Eur Urol 2008; 53(3):547553.
58. American Society for Therapeutic Radiology and Oncology
Consensus Panel: consensus statement: guidelines for PSA
following radiation therapy. In J Radiat 1997; 37:10351041.
59. Roach M 3rd, Hanks G, Thames H Jr, et al. Defining
biochemical failure following radiotherapy with or without
hormonal therapy in men with clinically localized prostate
cancer: recommendations of the RTOG-ASTRO Phoenix
Consensus Conference. Int J Radiat Oncol Biol Phys 2006;
65(4):965974.
60. Illing RO, Leslie TA, Kennedy JE, et al. Visually directed
HIFU for organ confined prostate cancer a proposed
standard for the conduct of therapy. BJU Int 2006;
98(6):11871192.
61. Windahl T, Andersson So, Lofgren L. Photodynamic therapy of localised prostate cancer. Lancet 1990; 336:1139.
62. Nathan TR, Whitelaw DE, Chang SC, et al. Photodynamic
therapy for prostate cancer recurrence after radiotherapy: a
phase I study. J Urol 2002; 168:14271432.
63. Moore CM, Nathan TR, Lees WR, et al. Photodynamic
therapy using meso tetra hydroxyl phenyl chlorin
(mTHPC) in early prostate cancer. Lasers Surg Med 2006;
38:356363.
64. Zaak D, Sroka R, Hoppner M, et al. Photodynamic therapy
by means of 5 ALA induced protoporphyrin IX in human
prostate cancerpreliminary results. Med Laser Appl
2003; 18:9195.
65. Pinthus JH, Bogaards A, Weersink R, et al. Photodynamic
therapy for urological malignancies: past to current
approaches. J Urol 2006; 175:12011207.
66. Verigos K, Stripp DC, Mick R, et al. Updated results of a
phase I trial of motexafin lutetium mediated interstitial
photodynamic therapy in patients with locally recurrent
prostate cancer. J Environ Pathol Toxicol Oncol 2006;
25:373388.
67. Patel H, Mick R, Finlay J, et al. Motexafin lutetium-photodynamic therapy of prostate cancer: short- and long-term
effects on prostate-specific antigen. Clin Cancer Res 2008;
14(15):48694876.
68. Weersink RA, Forbes J, Bisland S, et al. Assessment of
cutaneous photosensitivity of TOOKAD (WST09) in preclinical animal models and in patients. Photochem Photobiol 2005; 81:106113.
69. Trachtenberg J, Weersink RA, Davidson SR, et al. Vasculartargeted photodynamic therapy (padoporfin, WST09) for
recurrent prostate cancer after failure of external beam
radiotherapy: a study of escalating light doses. BJU Int
2008; 102(5):556562.
70. Trachtenberg J, Bogaards A, Weersink RA, et al. Vascular
targeted photodynamic therapy with palladium-bacteriopheophorbide photosensitizer for recurrent prostate cancer
following definitive radiation therapy: assessment of safety
and treatment response. J Urol 2007; 178(5):19741979.
71. Haider MA, Davidson SR, Kale AV, et al. Prostate gland:
MR imaging appearance after vascular targeted photodynamic therapy with palladium-bacteriopheophorbide.
Radiology 2007; 244(1):196204.
72. Moore CM, Hoh I, Mosse C, et al. Vascular-targeted photodynamic therapy in organ confined prostate cancerreport
of a novel photosensitiser. European Association of Urology
Annual Meeting abstract, 2006, AM06-1094.
73. Ahmed HU, Pendse D, Illing R, et al. Will focal therapy
become a standard of care for men with localized prostate
cancer? Nat Clin Pract Oncol 2007; 4(11):632642.
74. Eggener SE, Scardino PT, Carroll PR, et al. International
Task Force on Prostate Cancer and the Focal Lesion Paradigm. Focal therapy for localized prostate cancer: a critical
appraisal of rationale and modalities. J Urol 2007; 178(6):
22602267.
75. Nilsson S, Norlen BJ, Widmark A. A systematic overview
of radiation therapy effects in prostate cancer. Acta Oncol
2004; 43(4):316381.
76. Meraney AM, Haese A, Palisaar J, et al. Surgical management of prostate cancer: advances based on a rational
approach to the data. Eur J Cancer 2005; 41(6):888907.
77. Onik G, Vaughan D, Lotenfoe R, et al. "Male lumpectomy":
focal therapy for prostate cancer using cryoablation. Urology 2007; 70(6 suppl):1621.
78. Ellis DS, Manny TB Jr, Rewcastle JC. Focal cryosurgery
followed by penile rehabilitation as primary treatment for
localized prostate cancer: initial results. Urology 2007; 70(6
suppl):915.
79. Lambert EH, Bolte K, Masson P, et al. Focal cryosurgery:
encouraging health outcomes for unifocal prostate cancer.
Urology 2007; 69(6):11171120.
80. Bahn DK, Silverman P, Lee F Sr, et al. Focal prostate
cryoablation: initial results show cancer control and
potency preservation. J Endourol 2006; 20(9):688692.
81. Barqawi AB, Crawford ED. The current use and future
trends of focal surgical therapy in the management of
localized prostate cancer. Cancer J 2007; 13(5):313317.
82. Jones JS, Rewcastle JC, Donnelly BJ, et al. Whole gland
primary prostate cryoablation: initial results from the cryo
on-line data registry. J Urol 2008; 180(2):554558.
83. Muto S, Yoshii T, Saito K, et al. Focal therapy with highintensity-focused ultrasound in the treatment of localized
prostate cancer. Jpn J Clin Oncol 2008; 38(3):192199.
84. Barret E, Prapotnich D, Cathelineau X, et al. Focal therapy
with HIFU for prostate cancer in elderly: feasibility study
with 10 years follow up. P-68. Focal therapy and imaging
in prostate cancer workshop, Amsterdam, 2009.
85. Ahmed HU, Emberton M. Is focal therapy the future for
prostate cancer? Future Oncol 2010; 6(2):261268.
86. Onik G, Narayan P, Vaughan D, et al. Focal nervesparing cryosurgery for treatment of primary prostate
cancer: a new approach to preserving potency. Urology
2002; 60:109114.
87. Ahmed HU, Stevens D, Barbouti O, et al. Prostate cancer
risk stratification and cancer mappingtemplate transperineal prostate mapping biopsies. European Association of
Urology Annual Meeting, 2008.
88. Barqawi A, Lugg J, Wilson S, et al. The role of 3dimensional systematic mapping biopsy of the prostate in men
presenting with apparent low risk disease based on
extended transrectal biopsy. 439, AUA 2008.
89. Ahmed HU, Sahu M, Govindaraju SK, et al. High intensity
focused ultrasound (HIFU) hemiablation trial in localised
unilateral prostate cancer: interim results. Abstract 863.
European Association of Urology Annual Meeting, 2009.
90. Barqawi AB, Lugg JE, Crawford ED. Target Focal therapy
in early prostate cancer: initial single institution results.
ASCO annual meeting, 2007.
35
Stratified Risk Assessment for Urological Surgery
Thiru Gunendran
Department of Urology, University Hospital of South Manchester, Manchester, U.K.
Nicholas A. Wisely
Department of Anaesthesia, University Hospital of South Manchester, Manchester, U.K.
Manchester, U.K.
INTRODUCTION
Urological surgery covers a wide range of interventions and operations that may be offered to a broad
spectrum of patients with a variable ability to
respond to the stresses induced by such treatment.
Hence, one of the most challenging aspects of
surgerysetting aside technical ability and competence to undertake the procedureis correctly to
select and match the patients pathophysiological
reserves to the stresses inherent in any planned
operative intervention. Thus, while a total cystectomy with neobladder formation would be a reasonable proposal in a 58-year-old, the same
procedure would demand a very different risk analysis in a patient 20 years older. Even minor interventions are subject to the same constraints: an
overweight diabetic patient requiring minor groin
surgery might well be considered unsuited to otherwise routine day-case surgery.
A stratified risk assessment implies therefore
an accurate appraisal of both the patients initial
reserves and the ability to respond to any particular
stress as well as a detailed understanding of the
basic physiological requirements that will be necessary for any organ or system to regain normal function following operative interventions. Clearly, a
successful outcome to the intervention can be anticipated only by careful matching of these parameters
(Fig. 1). In this chapter, the generic response to stress
will initially be reprised followed by a consideration
of risk according to the severity of the surgical procedure (low, intermediate, or high); finally risk
assessment will be examined in terms of systembased criteria (i.e., cardiovascular, pulmonary),
which will determine safe intervention either as a
day case or as an inpatient for minor, intermediate,
or major surgery.
Although there are many (often counter-productive)
pressures on the modern NHS it will not be forgotten
that the primary purpose of risk assessment is

patient safety; hospital efficiency, and other logistical considerations (e.g., same day admission for
major surgery) must never override the absolute
requirement to manage the patient so as to minimize
risk (e.g., previous day admission for critical preoperative measurements). For all types of patient, minimal risk is paramount.
STRESS RESPONSE TO SURGERY (1,2)

The stress response is the name given to the hormonal
and metabolic changes that follow any injury or
trauma. This response can also be initiated by acute
blood loss, shock, hypoxia, tissue ischemia, acidosis,
hypothermia, pain, and altered immune function. It
can be both adaptive and destructive. The physiologic
response that ensues following injury encompasses a
wide range of neuroendocrinologic, hematologic, and
immunologic effects. There is also simultaneous activation of the sympathetic nervous system. The magnitude and duration of the response are proportional
to the surgical insult and the development of complications such as sepsis. This response to injury has
classically been grouped into the ebb phase and the
flow phase (Fig. 2).
The initial ebb phase, typically characterized by
clinical features of shock, develops quickly and is
primarily due to autonomic and neuroendocrine
mechanisms. The features are related to preservation
of blood volume and redirection of blood flow to
reduce tissue ischemia. During the flow phase that
follows, there is an increase in catabolic activity and
consumption of body fuels. This helps promotes
wound healing and resolution in inflammation by
transferring amino acids and fuel from muscle and
adipose tissue to the site of injury. In the recovery phase
attempts are made to restore the bodys depleted
stores. This process is slow and prolonged.
524
Gunendran et al.
Figure 1 Various factors affecting outcome of

surgery.
Figure 2 Physiologic response to injury.
Neuroendocrine Response to Surgery

This is characterized by increased secretion of pituitary
hormones (hypothalamic-pituitary-adrenal axis) and
activation of the sympathetic autonomic nervous system (Fig. 3). It is regulated by the central nervous
system that functions as part of a complex reflex arc.
In the initial phase (ebb phase) catecholamines, cortisol,
aldosterone, growth hormone, and glucagon secretion
increases, while insulin levels may decline. The suppression of insulin secretion is presumed to be secondary to the effects of catecholamines and autonomic
stimulation of the pancreas. After some time (flow
phase), blood levels of catecholamines also decline
despite relatively maintained levels of the other hormones. A degree of insulin resistance may also follow
despite elevated glucose levels resulting in increased
oxygen requirement. The overall metabolic effect is one
of increased catabolism with substrate mobilization,
muscle protein loss coupled with retention of sodium
and water. The resultant breakdown of polysaccharides,
lipids, proteins, and nucleic acids into smaller units
provides energy sources necessary for maintenance of
cell growth and repair. Generally, the magnitude of this

response is proportional to the severity of the surgical
trauma. The increase in metabolic rate varies from 10%
in elective surgery to 60% with multiple trauma and
150% in major burns.
Immunological and Hematological Response to

Surgery
The local release of cytokines has a major role in the
inflammatory response to surgery and tissue trauma.
Cytokines are produced from activated leucocytes, fibroblasts, and endothelial cells as an early response to tissue
injury. The activated cells release interleukin-1 and
tumor necrosis factor-a. This then stimulates the production of more cytokines, in particular interleukin-6,
which then mediates the acute phase response. Cytokines can broadly be grouped into pro-inflammatory
and anti-inflammatory cytokines (Fig. 4). They can
influence cell proliferation, differentiation, and survival, and actively promote wound healing. Proinflammatory cytokines promote the inflammatory
Chapter 35: Stratified Risk Assessment for Urological Surgery
525
Figure 3 Neuroendocrine response in the initial phase of surgery.
Pro-inflammatory cytokines
Anti-inflammatory cytokines
Tumor necrosis factor-a

Interleukin-1
Interleukin-2
Interleukin-6
Interleukin-8
Interleukin-12
Interleukin-18
Interferon-g
Granulocyte-macrophage
colony-stimulating factor
Tumor necrosis factor-b

Interleukin-4
Interleukin-10
Interleukin-13
Interleukin-16
Interferon- a
Positive acute phase

proteins
Negative acute phase

proteins
Fibrinogen
Serum amyloid A
C-reactive protein
Complement factors
Alpha 1-antitrypsin
Alpha 1-antichymotrypsin
Alpha 2-macroglobulin
Ferritin
Albumin
Transferrin
Insulin growth factor 1
Retinol-binding protein
Figure 5 Positive and negative acute phase proteins.

Figure 4 Pro-inflammatory cytokines and anti-inflammatory
cytokines.
response to trauma and infection, while anti-inflammatory

cytokines may be associated with immunosuppression and enhanced susceptibility to infections. The
net effect of an inflammatory response is determined by the balance between pro-inflammatory
cytokines and anti-inflammatory cytokines.
the plasma concentrations of various acute phase

proteins (Fig. 5). These proteins act as inflammatory
mediators to prevent ongoing tissue damage, isolate
and destroy the infective organism, and activate the
repair process.
RISK STRATIFICATION
The Acute Phase Response
The acute phase response (APR) is initiated in
response to inflammation, infection, or tissue trauma,
resulting in the production of acute phase proteins by
the liver. One of the key mediators of this response is
related to the stimulation of hepatocytes in the liver
by cytokines, in particular interleukin-6. The APR is
characterized by fever, granulocytosis, and changes in
Surgery results in tissue trauma, which initiates many

of the responses mentioned in the preceding text.
Although the stress response can be beneficial as it
leads to wound healing, restoration, and repair, it can
be quite morbid for older patients and those with a
compromised physiological reserve. This can be particularly significant when major abdominal, thoracic,
or pelvic surgery is being performed. In most cases
526
Gunendran et al.
however, where operations are minor and the procedure is short with minimal tissue injury, limited blood
loss, or fluid shifts, the accompanying physiologic
response is mild and transient. Hence by looking at
the surgical stress of the procedure and identifying the
physiological reserve of the patient, a decision can be
made on the ideal setting (e.g., day case, short stay, or
inpatient) of surgery and the necessary preoperative
intensification that needs to ensue.
SURGICAL STRESS OF PROCEDURE

The key pathogenic factor is the surgical stress of the
procedure and likelihood for increased demands on
organ function and tissue oxygen delivery. The choice
of the surgical technique according to the patients
risk profile is also important.
Low-Risk Procedures
There is minimal tissue damage with no significant
fluid shifts, negligible blood loss, low risk of postoperative complications, minor postoperative pain, and
little requirement for postoperative oxygen demand.
Patients who fall into the low-risk group probably require minimal preoperative investigations.
Generally, for the majority of endoscopic and inguinoscrotal cases, routine tests would suffice on the
basis of set hospital criteria. Most patients would
tolerate a short procedure under general or regional
anesthesia.
Intermediate to High-Risk Procedures

These include procedures that involve moderate to
extensive tissue damage, significant intra- and
extracellular fluid shifts, increased demand for postoperative oxygen consumption, and procedures
where moderate pain is expected or recovery is
unpredictable.
When a more demanding procedure is planned,
baseline fitness and coexisting illness will determine
what further tests are necessary to establish if the
patient will be able to tolerate the physiological
stresses associated with surgery. Despite reducing
tissue trauma, laparoscopic procedures carry a number of challenges because of the effects of pneumoperitoneum and patient positioning.
Laparoscopic- and Robotic-Assisted

Laparoscopic Surgery
Laparoscopic surgery has gained increasing popularity
within the urological community. It aims to reduce the
surgical stress response by avoiding large incisions and
minimizing tissue trauma, which correlates to reduction in pain, blood loss, postoperative pulmonary complications, and hospital stay. Laparoscopic surgery
causes less tissue injury than conventional procedures
with a lesser rise in the levels of interleukin-6, tumor
necrosis factor-a, interferon-g, and acute phase proteins
(3,4). However, this is at the expense of increased

intra-abdominal pressure, systemic absorption of carbon dioxide, and reduced venous return, which if
prolonged carries its own risk of cardiopulmonary
and thrombotic complications. Additionally, one has
to predict how the patient might react to positive
pressure ventilation, fluid challenges, and other
mechanisms normally employed to counter rises in
intra-abdominal pressure (5). The steep head down
position in robotic-assisted laparoscopic surgery carries the added risk of cerebral edema, intraocular
edema, and pulmonary interstitial edema. Some
experts view a history of significant cardiovascular
comorbidity, pulmonary disease, or cerebrovascular
disease as contraindications for laparoscopic pelvic
procedures such as prostatectomy and cystectomy
due to the head down position (6). Selection for such
procedures will thus depend on clinical judgment
and assessment of the patients ability to withstand
the physiological changes associated with patient
positioning.
PHYSIOLOGICAL RESERVE OF THE PATIENT

Clearly, it will be necessary initially to estimate and if
indicated later to measure objectively the patients
physiological functional capacity and reserves. For
example, the cardiopulmonary reserve is determined
by the patients cardiopulmonary functional capacity
and the ability to support postoperative demand for
increased oxygen consumption. Cardiac output and
myocardial ischemia are important determining factors in this equation. For these purposes assessment
protocols are widely utilized, and frequently these
may be found to vary significantly between departments and hospital units. Such variation however is
not of concern as long as the proforma addresses
specific risk associated with each type of surgery, for
example, day case or intermediate inpatient surgery.
The requirements for construction of a valid proforma
to cover differing orders of clinical risk for various
forms of surgery are detailed by physiological system
as noted later.
RISK ASSESSMENT FOR DAY-CASE SURGERY

Patients with reasonable cardiopulmonary functional
capacity and physiological reserve undergoing lowrisk procedures should ideally be undertaken as a day
case. Such patients should be selected according to
their physiological status and not limited by arbitrary
restrictions such as age and weight (7). In fact, day
surgery should be the norm unless there is a specific
contraindication. Assessment should be performed
well in advance of their surgery to correct any abnormalities and allow the patient to be adequately
informed and prepared for surgery.
In the United Kingdom, the Department of
Health has stated its aim for 75% of elective procedures to be performed on a day-case basis within the
next decade (8). In 2001 the Audit Commission produced a list of urological procedures thought to be
suitable for day surgery (orchidopexy, circumcision,

meatotomy, and transurethral resection of bladder
tumor) (9,10), while the British Association of Day
Surgery (BADS) (11) proposed that at least 50% of
urethrotomies, bladder neck incisions, and laser prostatectomies could be done as day case. Ideally any
quick, simple procedures with minimal risk of prolonged bleeding, little analgesic requirements, and
prompt recovery would fall into this category. There
is some recent evidence suggesting that slightly more
complex, minimally invasive surgical procedures,
such as laparoscopic nephrectomy, can safely and
successfully be undertaken on a day-case basis (12).
However, this is significantly dependant on the institution, surgeons expertise, appropriate staffing support, and facilities for overnight stay. A number of
factors may deem day-case surgery to be inappropriate, unsafe or impossible although ultimately such
decisions are at the discretion of the anesthetist, urologist, and local hospital policy.
Exclusion for Day Surgery (7,9,13,14)

Operation Factors
l
l
Moderate to high risk of postoperative hemorrhage

Significant risk of airway compromise following
surgery
Analgesic requirements likely to be moderate or
uncontrolled with oral analgesia
Continued requirement for intravenous fluids
postoperatively
Long recovery period is likely
Patient Factors
l
Respiratorypoorly controlled asthma or chronic

obstructive pulmonary disease (COPD), for example, needing oral steroids often or within last three
months; dyspnea at rest or mild exertion; obstructive sleep apnea; requirement for home oxygen or
non invasive ventilation
Cardiovascularmyocardial infarction (MI)
within last six months; uncontrolled or poorly
controlled angina, cardiac failure, or hypertension
(blood pressure greater than 100 to 110 mmHg
diastolic, or systolic greater than 170 to 180 mmHg
regardless of diastolic pressure); orthopnea, significant valvular or congenital heart disease
Cerebrovascularrecent cerebrovascular accident
or transient ischemic event
Hematologicalhemophilia; coagulopathy;
patients on warfarin (relative contraindication);
sickle cell disease (not trait)
Endocrinepoorly controlled diabetics (HbA1c
>10% or 86 mmol/mol); insulin-dependent diabetics (although many day surgery units now
consider stable diabetics maintained on oral hypoglycemic drugs or insulin suitable for most daysurgery procedures); untreated thyrotoxicosis;
adrenal insufficiency
l
l
527
Renalmoderate to severe renal failure; patients

on dialysis
Liveradvanced liver failure
Neurologicaluncontrolled epileptics or history
of recent fits; myasthenia gravis; muscular dystrophies
Anesthetic factorssuxamethonium apnea;
malignant hyperpyrexia; difficult intubation; previous anesthetic reactions
Otherspregnant patients
Social Factors
l
Lives alone or no responsible person at home for

24 hours following surgery (excludes local anesthetic procedures)
Transport problemsno suitable transport home
(must be non-public transport for general anesthetic procedure/regional anesthesia)
No ready access to a telephone
RISK ASSESSMENT FOR INPATIENT SURGERY

Patients deemed unsuitable for day case will have to
be admitted for their procedure as inpatients. Other
factors may also need to be taken into account
depending on their social circumstances, coexisting
illness, and cooperation. Increasingly, short-stay
wards with a 23-hour model of care have been developed as an intermediate between true day-case surgery and inpatient surgery. Suitable patients have
been carefully selected as appropriate cases and are
treated using anesthetic techniques that allow rapid
recovery with minimal pain. The episode of care can
generally be delivered within an envelope of 23 hours
during which time patients may require pain relief,
some intravenous fluids, and monitoring in a supervised setting until fit for discharge.
PREOPERATIVE AND SYSTEM SPECIFIC

ASSESSMENT
Cardiovascular Assessment and Optimization
(15)
Assessment of the cardiovascular reserve is predominantly aimed at looking for the presence of congestive
heart failure, valvular heart disease, myocardial ischemia, uncontrolled hypertension and arrhythmias.
Patients with vascular impairment often have coronary artery disease risk factors and the usual symptomatic presentation of angina in these patients may
be obscured by exercise limitations imposed by intermittent claudication. Ideally, surgery should be
delayed until the cardiac condition has been optimized, either medically or surgically. In patients
with angiographically proven coronary artery disease,
revascularization should be undertaken first if moderate- to high-risk elective urological surgery is contemplated (16,17). By comparison, patients undergoing lowrisk operations only have a very small risk of postoperative death (<1%) or MI that is unaffected by prior
coronary treatment (17).
528
Gunendran et al.
METs
Equivalent type of activity
< 4 METs (poor)
Activities of daily living (e.g.

eating, dressing), house work
Climbing two flights of stairs,
gardening, cycling
Running, squash, singles tennis
47 METs (moderate)
> 7 METs (excellent)
Figure 6 Assessment of functional capacity using metabolic

equivalents (METs).
The early assessment of surgical risk was

directed at indentifying individuals at high risk of
perioperative ischemic events with transurethral prostate resections being considered intermediate risk (17).
Preoperative cardiac risk assessment was initially
popularized by Goldman et al. (18) and later modified
by Detsky et al. (19). It was proposed that MI within
six months, unstable angina, recent pulmonary
edema, critical aortic stenosis, and emergency surgery
carried a significant risk of perioperative cardiac complications. Although highly specific, they had a low
sensitivity. The Revised Goldman Cardiac Risk Index
was much simpler but still misleading and could not
always reliably identify patients with severe cardiopulmonary abnormalities.
Prior to surgery, the subjective assessment of
patients exercise tolerance is thought to be a reasonable indicator of fitness for anesthesia. A patients
functional capacity can be measured in metabolic
equivalents (METs), although assessment is limited
by mobility (Fig. 6). A patient with an exercise tolerance of four METs or more is thought to be at low risk
of perioperative morbidity.
However, METs do not provide an accurate or
objective measure of cardiopulmonary functional
capacity. Stress testing (exercise electrocardiogram
monitoring) has also probably outlived its time as
the most useful discriminator for predicting adverse
cardiac events. This is because such assessments are
unable to predict the ability of the cardiopulmonary
system to deliver oxygen to the tissues under conditions of surgical stress. Cardiopulmonary exercise
(CPX) testing is being used increasingly to assess
patients fitness prior to major urological surgery.
CPX can risk stratify patients into low, higher, and
unacceptably high-risk groups prior to surgery. Lowrisk groups can be safely managed on the ward after
surgery leaving more critical care capacity to manage
the higher risk patients. The objective data provided
by CPX allow those patients with unacceptably high
risk to be counseled into nonsurgical management,
and the test may also identify cardiac or respiratory
problems that can be optimized prior to surgery.
Further details about CPX testing is described on
page 529.
Disease Modifying Interventions
Recent myocardial infarction. MI may be the first

presenting feature of ischemic heart disease in up to
50% of patients. Patients with a previous MI have an
overall risk of perioperative reinfarction of 30% to 40%

within three months and 15% to 25% at three to six
months (20,21). The mortality rate with reinfarction
can be 30% to 50% (20). As such, elective urological
procedures should be postponed for at least six
months after an MI where possible (22). Uncontrolled
angina is also associated with a high risk of intraoperative MI if the symptom is not controlled preoperatively. Following MI, a b-blocker can be beneficial
as it protects the ischemic myocardium, promotes
recovery, and lowers the risk of death by decreasing
the hearts demand for oxygen (23).
Congestive cardiac failure. Patients with diastolic
heart failure or a left ventricular ejection fraction of
less than 30% on echocardiography are at an increased
risk of cardiovascular events during surgery. Angiotensin-converting enzymes are increasingly being
used to manage congestive cardiac failure as they
increase cardiac output, improve exercise tolerance,
and prolong survival (24). These drugs may, however,
precipitate renal failure in patients with marked renal
artery stenosis by reducing the efferent arteriole vascular resistance with a resultant fall in glomerular
filtration pressure.
Valvular heart disease.
1. Aortic stenosisThere is risk of myocardial ischemia due to increased demand from the hypertrophied ventricle. Intra-operative hypotensive
periods can be very poorly tolerated in severe
stenosis because of fixed cardiac output, and
such patients should be referred for valve surgery
(replacement) first prior to major surgery.
2. Mitral stenosisThe risk of pulmonary edema is
increased in patients undergoing elective surgery
depending on the severity of stenosis.
3. Aortic regurgitation and mitral regurgitation
Generally surgery is reasonably better tolerated
than with valve stenosis.
4. Endocarditis prophylaxisAntibiotic prophylaxis
against infective endocarditis is no longer recommended for people undergoing genitourinary
tract procedures (25).
Cardiomyopathy. A cardiological opinion should
be sought if hypertrophic obstructive cardiomyopathy
is present.
Pacemakers. Pacemaker checks should be verified. Single or dual chamber pacemakers can be
reprogrammed in fixed rate mode to avoid risks
associated with diathermy use. Bipolar diathermy is
generally safe, but if monopolar diathermy is used it
should be employed in short bursts and the patient
plate sited so as to avoid transmission through the
thorax.
Implantable converter defibrillator. Any pacemaker
that has the ability to cardiovert or overpace should be
deactivated prior to surgery to avoid erroneous discharge in case diathermy noise is interpreted as a
tachyarrhythmia; reactivation is required immediately
following the procedure.
Hypertension. Hypertension is the leading cause
of congestive heart failure, and in untreated patients,
the five-year mortality rates approach 50% (26).
529
Figure 7 Recommendations for control of hypertension prior to surgery.
Treatment has been shown to be associated with

decreased death rates from stroke and coronary
heart disease in a nonsurgical setting. Hypertension
should be treated to the currently recommended target of 140/90 mmHg or lower (27). Patients with
relevant comorbidities, for example, diabetes or
renal disease, should be treated to a lower blood
pressure target. Blood pressures greater than recommended values on assessment should be referred to
the general practitioner or physician for control prior
to surgery (Fig. 7). Approximately 3% to 5% of hypertensive patients have a surgically correctable cause for
their elevated blood pressure such as renal artery
stenosis, Cushings disease, pheochromocytoma or
aldosteronoma. Phaeochromocytoma patients often
have severe hypertension and carry an operative
death rate in excess of 50% if untreated.
Laparoscopy in cardiac disease. Patients with
severe congestive cardiac failure and valvular insufficiency are more prone to develop cardiac complications than patients with ischemic heart disease (28).
CARDIOPULMONARY TESTING
Although cardiovascular and respiratory assessment
is often undertaken as a separate entity comprising of
individual system assessment, it is probably of much
greater relevance if they are assessed as a combined
unit. CPX testing is a cost-effective, noninvasive and
objective method of evaluating the pathophysiology of
both the cardiovascular and respiratory systems. It
classifies cardiac failure on the basis of oxygen
consumption at the anaerobic threshold (AT) and
the maximal aerobic capacity. As postoperative mortality following major surgery is closely related to
preexisting cardiac failure, CPX is arguably now the
gold standard for evaluation of cardiopulmonary
function. CPX-derived data of cardiovascular and
respiratory limitation have repeatedly emerged as
precise predictors of survival rate and CPX concentrates on measurement of ventricular function, respiratory function, and cellular function via measurement
of gas exchange, as well as detection of myocardial
ischemia.
During the test a patient performs an increasing
amount of work on an electronically braked cycle
ergometer while their oxygen consumption (VO2),
carbon dioxide production (VCO2), and tidal volumes
(Vt) are measured through a tight fitting mask or
mouthpiece. A 12-lead electrocardiogram (ECG) is
also analyzed throughout the test to detect ECG evidence of exercise-induced myocardial ischemia and
arrhythmias. After a two-minute period of rest, the
subject starts to cycle at around 60 revolutions per
minute (rpm) with no added load for three minutes.
The work load is increased at the rate of between 5
and 20 W/min depending on the fitness of the subject.
The test should last between 8 and 15 minutes and
stops when the subject is unable to maintain 60 rpm.
Critical Data Derived from the Test

Anerobic Threshold
AT is the oxygen consumption of the subject at the

point when the work that he or she is performing just
exceeds his or her bodys capacity to achieve this
without the accumulation of lactic acid in the blood.
It is also referred to as the lactate threshold. AT is
reproducible and is effort independent (29). Work
above AT results in increasing anerobic metabolism,
lactic acidosis, and is nonsustainable. Carbon dioxide
production increases relative to oxygen uptake at
work levels in excess of AT as more carbon dioxide
is generated from bicarbonate buffering of lactic acid.
The AT can be identified by plotting VCO2 against
VO2 in the V slope method (Fig. 8). The AT is the VO2
at the break point in the relationship between VCO2
and VO2.
Peak VO2
Peak VO2 is the highest value of VO2 during the test. It

is clearly effort dependent but values of over 20 mL/
kg/min have been associated with a reduced mortality after major surgery.
VE/VCO2
VE/VCO2 is the ventilatory efficiency for carbon dioxide. It reveals how efficient the lungs are at removing
carbon dioxide and is increased in disorders that
increase the amount of lung dead space such as
COPD, cardiac failure, and pulmonary vascular diseases.
DVO2/Dwork rate
DVO2/Dwork rate is the relationship between oxygen

uptake and work. The normal subject will increase
their oxygen consumption by 10 mL/min/watt. Cardiorespiratory pathology will reduce this.
530
Gunendran et al.
Figure 8 V-slope method for determining the VO2 anaerobic threshold (AT). VCO2 (mL/min) is plotted against VO2 (mL/min) as
represented by the solid blue points. Initially the slope is unity up to the AT as marked by the dashed green line. The gradient of the slope
then increases from this deflection point as more CO2 is produced from bicarbonate buffering of lactic acid. The VE/VO2 as shown by the
purple circles also rises after AT is passed.
O2 pulse
O2 pulse is the oxygen consumption per heart beat

and is the product of stroke volume and the arterialvenous oxygen difference. It is reduced in conditions
such as coronary artery disease, heart failure, and
pulmonary vascular disorders.
AT and mortality in elderly patients
Olders group in Melbourne found that if patients

above 60 years had an AT over 11 mL/kg/min then
they had a much lower postoperative mortality compared to those with an AT below 11 mL/kg/min (30).
Postoperative mortality was particularly high at 42% if
myocardial ischemia was identified from the ECG
prior to the patient reaching their AT. They went on
to use CPX testing in elderly patients for major surgery to determine where they would be best managed
postoperatively. Those patients deemed fit with an AT
above 11 mL/kg/min and sent to the general ward
had a 0% cardiovascular mortality (31). Hence, CPX
testing is increasingly being used to assess fitness and
predict mortality following major urological surgery
and is currently the most accurate indicator of physiological reserve.
Respiratory Assessment and Optimization

Postoperative pulmonary complications are an
important cause of perioperative morbidity, mortality, and increased length of stay. The incidence of
complications depends on coexisting risk factors,
type, and site of surgery as well as mode of anesthesia (Fig. 9) (21,32).
A chest radiograph is indicated if active or
advanced lung disease is suspected. Peak flow measurement can be useful in COPD or asthma. A
baseline blood gas can also help predict the likelihood for postoperative ventilation following major
surgery. It should be checked if the patient is breathless on minimal exertion or at rest, has an oxygen
saturation of less than 95% on room air or is cyanosed. A resting PaCO2 >6.0 kPa suggests respiratory failure and is predictive of pulmonary
complications.
Chronic lung disease

Anatomical chest wall deformity e.g., kyphoscoliosis, ankylosing
spondylitis
Physiological restrictive lung disease e.g., neuromuscular
disease
Congestive heart failure
Heavy smoking
Malnutrition
Severe obesitythis tends to reduce ventilatory compliance and
functional residual capacity. Gas exchange is also impaired by
altering the ventilation/perfusion relationship in the lungs.
Obstructive sleep apneathis can result in difficulty with airway
management
Duration of surgeryprolonged operations carry a higher risk
although the definition of prolonged surgery ranges from two
to four hours
Surgical incision sitethoraco-abdominal incisions carry a much
higher risk followed by upper and lower abdominal incisions
Figure 9 Factors influencing respiratory complications following
major surgery.
Pulmonary function tests (PFTs) have long been

used in an attempt to stratify pulmonary risk in
patients with significant respiratory disease. While
no single test can effectively predict morbidity and
mortality from pulmonary complications, the forced
expiratory volume in the first second of forceful exhalation (FEV1) can be a useful tool. When the FEV1 is
greater than 2 L or 50% of predicted, major complications are rare. However, routine pulmonary function
testing prior to abdominal surgery has been shown to
be of no additional benefit when compared with a
thorough history and physical examination (33).
Moreover, one of the most useful surrogate markers
for respiratory outcome is adequate postoperative
analgesia with movement and maintaining the ability
to cough. Protecting and facilitating this ability is
more valid than PFT or any other clinical markers in
predicting outcome. The analgesia provided by a thoracic epidural analgesia is preferable especially if an
upper abdominal or thoracoabdominal incision is
utilized.
Disease Modifying Interventions
Smoking cessation. Smoking has been linked to

peri- and postoperative complications with increased
risk of respiratory, cardiac, wound, and infectious
complications (3436). Cessation of smoking 10 to
20 hours preoperatively lowers carboxyhemoglobin
concentrations and may reduce heart rate, blood pressure, and catecholamine concentrations. The incidence
of chest infections can be reduced by 50% if the patient
stops smoking eight weeks before surgery with the
risk falling to that of nonsmokers if cessation was for
six months prior to surgery. Sputum production also
declines over a six-week period if smoking is stopped.
An effective smoking intervention program applied
six to eight weeks before surgery has been shown to
halve the frequency of postoperative complications
especially those related to wound healing and the
531
cardiovascular insufficiency (37). Prescription of nicotine replacement therapy (transdermal patch, gum,
nasal spray, and sublingual tablets) increases the rate
of quitting by 50% to 70%, regardless of setting.
Medical therapy. In patients with chronic lung
disease and poorly controlled asthma, the use of preoperative inhalers, nebulized bronchodilators, and/or corticosteroids may be beneficial. It may also help prevent
respiratory exacerbations in the postoperative period.
Chest physiotherapy. Preoperative physiotherapy
helps clear sputum and secretions. It can also educate
patients to undertake regular postoperative breathing
exercises.
Oxygen delivery. There is reasonable evidence
supporting the benefits of preoperative oxygenation in
high-risk surgical patients. When oxygen delivery
falls, tissue oxygenation becomes physiologically
inadequate. Optimizing such high-risk patients with
fluids and inotropes can also enhance tissue perfusion
and promote oxygen delivery prior to surgery (38).
Currently, only about 5% of all planned elective surgical admissions to intensive care are admitted preoperatively (39).
Laparoscopy and respiratory disease (5,40). Laparoscopy results in multiple postoperative benefits allowing for quicker recovery and shorter hospital stay.
However, severe lung disease is generally believed to
be a contraindication to laparoscopic surgery, partly
due to concerns over impaired gas exchange, hypercarbia, and pneumoperitoneum. The partial pressure of
carbon dioxide in the blood (PaCO2) increases because
of carbon dioxide absorption from the peritoneal cavity. In compromised patients, cardiopulmonary disturbances aggravate this increase in PaCO2. Adequate
pain control with a thoracic epidural, careful attention
to ventilation parameters, and the combination of an
experienced anesthesiologist and advanced laparoscopic surgeon can enable the safe management of
patients with end-stage lung disease (41).
Renal Disorders and Assessment

Renin is synthesized and stored in the juxtaglomerular
apparatus of the kidney. Fall in blood pressure,
ACTH, ADH, glucagon, potassium, and prostaglandins can all increase renin secretion. Through the
generation of angiotensin II, it helps preserve extracellular fluid volume. Patients with renal impairment
however may experience problems with fluid and
electrolyte imbalance and metabolic waste product
elimination. The lower the glomerular filtration rate
(GFR), the more problems to be anticipated. Impaired
immune function, delayed wound healing, as well as
coagulation defects may also be encountered. Every
effort should be made to optimize the renal function
leading up to surgery.
Individuals on renal replacement therapy should
undergo preoperative dialysis to improve their electrolyte and fluid balance. Close liaison with the patients
renal physician is vital. These patients often have a
creatinine clearance of less than 15 mL/min resulting
in fixed renal excretion of salt and water, which does
532
Gunendran et al.
not cope with acute changes. A sudden increase in

fluid volume or salt concentration can lead to hypertension and congestive cardiac failure. With such a low
number of functional nephrons, thiazide monotherapy
is ineffective and loop diuretics are often indicated.
Drugs that may potentially affect or worsen the
renal function should be utilized with caution, for
example, angiotensin-converting enzyme inhibitors,
nonsteroidal anti-inflammatory drugs, radiocontrast
agents, metformin, and aminoglycosides. Drug doses
will need to be carefully titrated on the basis of the
GFR, and, as noted in the preceding text, careful
attention to fluid balance and asepsis will be required
to avoid deterioration in remaining renal function.
normalize within a few days. Thyroid hormones stimulate the oxygen consumption of tissue resulting in
increased metabolic rate and heat production. Surgery
in the presence of uncontrolled thyroid disease can
precipitate a thyrotoxic crisis or thyroid storm, which
can be fatal. Postoperative complications (e.g., sepsis,
persistent tachycardia, hypertension, hemorrhage,
pyrexia) can mimic the thyrotoxic state and a high
index of suspicion coupled with early recognition is
paramount. In an emergency setting, the use of
b-adrenergic blockers for tachycardia and a-adrenergic
blockers for hypertension may be necessary.
Hypothyroidism may sometimes be difficult to
identify clinically but if present should be treated by
replacement therapy with thyroxine before surgery.
Metabolic Assessment and Optimization

Diabetes
Insulin is a key anabolic hormone. After induction of

anesthesia and during surgery (ebb phase), insulin
secretion is suppressed despite rising blood glucose
presumably due to the effects of catecholamines and
a-adrenergic inhibition of b-cell secretion within the
endocrine pancreas. During the postoperative period
however, in the flow phase, expression of insulin
increases and insulin levels rise. Tolerance for exogenous glucose is reduced and in general, glucose levels
are higher than normal resulting in the so called
insulin resistance. The resistance to insulin during
the flow phase is thought to be secondary to the effect
of glucocorticoids.
In diabetics undergoing surgery, optimal control
of blood glucose is crucial. Hyperglycemia is associated
with electrolyte disturbances, altered collagen formation, abnormalities in leukocyte function, reduced granulocyte adherence, impaired phagocytosis, delayed
chemotaxis, depressed bactericidal capacity, and suppressed immune function. Ketoacidosis may develop,
leading to increased carbon dioxide production and
ventilatory requirements. Recent evidence suggests
that perioperative hyperglycemia is the main risk factor
for the development of postoperative infection in critically ill surgical patient and that intensive glucose
control lowers this risk (42). Tight glycemic control in
critically ill patients has also been shown to reduce
mortality and hospital length of stay (43).
Adrenal Failure
The daily basal dose of corticosteroid is equivalent to

7.5 mg of prednisolone or 30 mg of hydrocortisone
(44). In patients with a normal endocrine response,
cortisol secretion usually rises immediately after the
start of surgery and can be modified by anesthetic
intervention. In primary or secondary adrenal failure,
the bodys physiological response to stress is
impaired, and increases in cortisol levels will not
occur. Patients who have been on long-term high
dose steroids will also lack such a response because
of suppression of the hypothalamic corticotropinreleasing hormone and pituitary adrenocorticotropic
hormone secretion. Such individuals may have mild
symptoms of adrenal insufficiency such as fatigue,
malaise, arthralgia, depression, nausea, and low-grade
fever. These patients usually respond poorly to stress.
Failure of cortisol secretion may result in the circulatory collapse and hypotension characteristic of an
Addisonian crisis.
Pre- and perioperative steroid cover will be
required in the following group of patients:
1.
2.
Hyper- and Hypothyroidism
Patients who are found to be thyrotoxic must be

deferred until adequate control of the disease is
achieved by medical or surgical therapy. Under normal
circumstances, concentrations of total and free triiodothyronine (T3) usually fall after surgery and
3.
Patients who are currently on daily dose of 10 mg

prednisolone (or equivalent). Equivalent doses
include methylprednisolone 4 mg, dexamethasone
6 mg, and hydrocortisone 20 mg.
Patients who have received longer than one-week
course of 10 mg prednisolone daily in the last
three months. This is because adrenal suppression
can occur after one week and may take up to three
months to recover (45).
Patients on high-dose inhaled corticosteroids, for
example, beclomethasone 1.5 mg daily)
Equivalent daily doses are also recommended in

the postoperative period until the patient is able to
resume their usual oral medication (Fig. 10).
Figure 10 Suggested regime for steroid replacement

in adrenal insufficiency.
Liver Disorders and Assessment

One of the most useful indicators of surgical risk in
liver failure patients is the Child-Pugh score (46,47)
(Figs. 11, 12), although the newer MELD (Model for
End-Stage Liver Disease) score takes into account
serum creatinine as well and may be a better predictor of survival (48). The cornerstones of perioperative management are medical treatment of the
complications of liver disease, including coagulopathy, ascites, encephalopathy, and malnutrition.
These individuals are at an increased risk of bleeding, sepsis, acute renal failure, respiratory distress
syndrome, and postoperative hepatic decompensation including hepatic coma or death. Therefore, the
decision to perform surgery in these patients must be
carefully weighed.
Measure
Points (per field)
Bilirubin (mmol/l)
Albumin (g/L)
INR
Ascites
Neurological state
< 34
> 35
< 1.7
None
None
3450
3035
1.702.20
Slight
Minimal
> 50
< 30
> 2.20
Moderate
Coma
Figure 11 Relative risk of a surgical procedure for patients with

liver cirrhosis (Child-Pugh score). Abbreviation: INR: International
normalized ratio.
Total score
56
79
1015
Operative mortality
05%
1015%
> 25%
Figure 12 Prediction of operative mortality based on ChildPugh scores.
Neurological Disorders and Assessment

Specific consideration should be given to assessment
of any associated respiratory impairment including
weakness of the respiratory muscles and the inability
to clear secretions. Patients who suffer from autonomic dysfunction may have a labile blood pressure
and in such patients, urinary retention, arrhythmias,
delayed gastric emptying, and sudden cardiac arrest
may be triggered by sympathetic stimulation, blood
loss, and postural change. Useful bedside tests of
autonomic function include orthostatic hypotension
(a drop of >30 mmHg systolic pressure indicates
autonomic dysfunction) and reduced heart rate in
response to the Valsalva maneuver.
Patients with dementia, learning difficulties, and
Alzheimers disease can become extremely confused,
agitated, and disorientated in a surgical ward. They
may also not be able to give a coherent history and
533
attempts to speak to relatives, next of kin or carers are

mandatory.
Musculoskeletal Disorders and Assessment

Patients with rheumatoid arthritis can have unstable
necks, limited neck movement, or reduced mouth
opening, and flexion/extension radiographs of the
neck are required before general anesthesia. Similarly,
patients with severe ankylosing spondylitis can experience problems with intubation, and the anesthetist
should be warned in advance in case a fiberoptic
intubation is necessary.
Muscular dystrophy can pose a problem postoperatively because of excessive secretions and respiratory problems. Preoperative chest physiotherapy
and nebulizers may be beneficial if major surgery is
planned.
Hematological Disorders and Assessment

A careful history may reveal features of excessive
bleeding after previous surgery, nose bleeds, spontaneous bruising, or a family history of bleeding disorders. A coagulation screen is necessary for all such
patients. If this is normal and the index of suspicion is
high for an inherited or acquired defect of coagulation, more sophisticated clotting studies may be
required, including a factor VII assay.
In a study of patients with idiopathic thrombocytopenic purpura, bleeding after trauma was
uncommon unless the platelet count was less than
60,000/mL. Surgical bleeding due solely to a reduced
platelet count does not generally occur until the
platelet count is less than 50,000/mL, while clinical
or spontaneous bleeding does not occur until the
platelet count is less than 10,000 to 20,000/mL (49).
Patients with hematological malignancy, hemoglobinopathies (e.g., sickle cell disease, thalassemia),
or other blood dyscrasias may require no additional
intervention prior to surgery but if in doubt a hematological opinion should be sought. In the case
of known multiple red cell antibodies, appropriately
cross-matched blood products will be required
well in advance of any procedure where significant
blood loss is likely. If a patient is known to have
severe clotting factor deficiencies, 4 to 6 units of fresh
frozen plasma should be administered prior to
surgery.
Most anesthetists would regard a preoperative
hemoglobin of greater than 10 g/dL as adequate,
although lower levels may be accepted in chronic
disease or procedures where minimal blood loss is
expected. One alternative would be to consider
autologous predeposit (blood banking) if the
patient is fit enough and there is a greater than
50% chance of significant blood loss requiring blood
products. Arrangements for intraoperative red
blood cell salvage during major surgery offer an
alternative to allogeneic or predonated autologous
blood transfusion.
534
Gunendran et al.
In patients who object to blood transfusion on

religious or other grounds, the use erythropoietin may
be considered. It should be administered at least four
weeks before elective surgery and requires careful
monitoring.
Immunological Disorders and Assessment

Immunological conditions like immune thrombocytopenic purpura, GuillainBarre syndrome, and
LambertEaton (myasthenic) syndrome require discussion with the anesthetist and responsible physician
before surgery. Pre-anesthetic treatment with intravenous immunoglobulins may be advised in some cases.
Immunosuppressed patients, either as a result of disease or medical therapy, are potentially at higher risk
of sepsis. They may require additional antibiotic prophylaxis in the pre-, peri-, and postoperative period
depending on the surgical procedure undertaken.
Microbiological Assessment and Antibiotic

Prophylaxis
Urine
Routine preoperative testing and treatment of urine is

advisable in patients undergoing endoscopic, percutaneous, or open stone surgery. However, bladder urine
culture has been found to correlate poorly with infection
in the upper urinary tract (50), while positive stone
culture and renal pelvic urine culture are better predictors of potential urosepsis (51). The antibiotic of choice
depends on surgeon preference and local protocol.
During the last decade, community-acquired
extended-spectrum b-lactamase (ESBL)-producing
Escherichia coli have been increasingly recognized in
the community. These organisms are most often isolated from the urinary tract but have also been isolated from the blood. Advancing age, female gender,
foreign travel, requirement for hemodialysis, urinary
incontinence, bladder cancer, prostate cancer, urolithiasis, urethral catheter, diabetes mellitus, prior ESBL
colonization, and recent hospitalization are important
risk factors for developing ESBL-producing bacterial
infections (52,53). In addition, previous urological
operations and fluoroquinolone or cephalosporin use
during the last three months were found to be independent risk factors (53). This is of great importance in
planning empirical antibiotic therapy.
Methicillin-Resistant Staphylococcus aureus
In United Kingdom, the Department of Health has

indicated that methicillin-resistant staphylococcus
aureus (MRSA) screening for all elective admissions
should be introduced by March 2009 (54). This
excludes elective day-case endoscopic procedures.
The specific sites have not been stated although all
screening must include the nose with consideration to
groin, perineum, axilla, urine from catheterized
patients and skin lesions or wounds. Ideally, this
should be undertaken at the preoperative clinic at
least six to nine weeks before admission.
Antibiotic Prophylaxis (55)
Endoscopic surgery. No prophylaxis is required

for uncomplicated procedures unless there is a history
of genitourinary infection, recent instrumentation,
institutionalization, bacterial colonization, or other
risk factors like diabetes mellitus, malnutrition, and
immunosuppression. All patients undergoing transurethral resection (TUR) of the prostate, ureteroscopy
for proximal or impacted stone and percutaneous
nephrolithotomy should be given antibiotic prophylaxis peroperatively. Antibiotics may also be used in
patients who have an indwelling urinary catheter,
ureteral stent, or nephrostomy tube. Routine, uncomplicated procedures like cystoscopy, bladder tumor
resection, and ureteroscopy for distal stone do not
typically require antibiotic cover.
Open and laparoscopic surgery. No prophylaxis is
required for clean procedures. Surgery involving
opening of the urinary tract should receive a single
or one-day dosage of antibiotic prophylaxis, while a
slightly longer course is recommended in procedures
using bowel segments or implantation of prosthetic
devices.
Typically, cephalosporins, fluoroquinolones, and
aminoglycosides are used, although the choice of
antimicrobials should be based on procedure type,
knowledge of the local pathogen profile, antibiotic
sensitivity, and patient allergies.
Nutritional Status Assessment

All patients should have their height and weight
measured to calculate their body mass index (BMI).
Obesity may be defined as a BMI >30 kg/m2, while
morbid obesity refers to those with a BMI >40 kg/m2.
These patients may have problems with reduced
respiratory functional residual capacity and desaturation, obstructive sleep apnea, impaired glucose tolerance (or undiagnosed diabetes), difficult intubation,
and uncontrolled hypertension (56). They are also at
much higher risk of thromboembolism and should
receive both mechanical and pharmacological thromboprophylaxis. A supervised exercise program and
dietician referral may be beneficial, leading up to
surgery.
On the other hand, poor nutritional status is
often a neglected condition that is only addressed in
the postoperative period. A weight loss of more than
10% is a risk factor for increased postoperative morbidity and mortality rates. Chronic hypoalbuminemia
with serum albumin levels less than 30 g/L are
associated with visceral protein mass depletion. Malnutrition can predispose to delayed ambulation, respiratory complications, wound infection, and poor
wound healing. It is important to identify at-risk
patients early to facilitate recovery following major
abdominal and pelvic surgery. Perioperative nutritional support in these patients decreases postoperative complications such as wound infections and
sepsis (57), and preoperative nutritional support
should also be considered in patients with loss of
>20% of their normal body weight. In general, enteral
feeding is preferred to total parenteral nutrition

(TPN). Dietician referral in conjunction with highcalorie build-up drinks prior to surgery may be beneficial in mild to moderately malnourished patients.
OTHER SPECIFIC CONSIDERATIONS

Thromboprophylaxis (58,59)
Patients undergoing urological surgery can be stratified according to low, medium, or high risk of developing venous thromboembolism (60) (Fig. 13).
Contributory risk factors include increasing age,
high BMI, poor mobility, multiple medical problems,
and surgical factors. Open pelvic, perineal, and
abdominal surgery of longer than 30 minutes duration
carry significantly higher risk than a short inguinal,
535
scrotal, or endoscopic case. Patients who have been on

long haul travel of more than four hours duration
within the last week as well as those on the combined
oral contraceptive are also at higher risk. Patients
should be advised to stop such hormone medication
four weeks before elective surgery if major surgery is
contemplated.
Depending on the patients risk category several
modalities can be utilized to reduce the chance of a
thromboembolic event. Most hospitals will have a
local policy that can be consulted. Below is a suggested guideline (Fig. 14).
In a recently published guideline, the American
Urological Association recommends early ambulation
for the vast majority of patients undergoing transurethral procedures, with the use of compression stockings,
mechanical prophylaxis, and low-dose unfractionated
Figure 13 Risk factors and incidence of postoperative thromboembolism in patients undergoing

elective surgery. Abbreviations: DVT, deep vein
thrombosis; PE, pulmonary embolism. Source:
Modified from Ref. 60.
Figure 14 Risk stratification and surgical thromboprophylaxis.
536
Gunendran et al.
Hypercoagulable state (Factor V Leiden mutation, deficiencies

in protein S, protein C and antithrombin III, prothrombin gene
mutation, anti-phospholipid antibody syndrome)
Recent thromboembolic episode within the last 1 to 3 months
Mechanical heart valve
Atrial fibrillation with a history of cardioembolism
Recurrent pulmonary emboli or deep vein thrombosis
Figure 15 High-risk patients where bridging from warfarin to
subcutaneous low molecular weight heparin or intravenous
unfractionated heparin is required.
heparin (LDUH) or low molecular weight heparin

(LMWH) reserved for high-risk patients (61). For open
urologic surgery, complex anti-incontinence, and pelvic
reconstructive surgery, a combination of mechanical and
pharmacologic prophylaxis (LDUH or LMWH) is recommended. However, due to some concerns about the
risk of retroperitoneal bleeding at time of urologic
laparoscopic and/or robotically assisted surgery, the
use of mechanical prophylaxis is recommended intraoperatively other than in the high risk group where
additional pharmacologic prophylaxis may be useful.
Anticoagulation Therapy Dicontinuation

The peak effects of warfarin occur two to three days
after a dose with the overall effect lasting up to five
days. In atrial fibrillation, the risk of stroke doubles
when international normalized ratio (INR) falls to
subtherapeutic levels. A systematic review from 2003
showed that discontinuation of warfarin without
administration of intravenous unfractionated heparin
carries a 0.6% risk of perioperative thromboembolic
event (62). High-risk patients should be administered
subcutaneous LMWH once the INR falls below therapeutic levels (Fig. 15). Bridging from warfarin to
intravenous heparin is recommended in patients
with mechanical heart valves and those with a recent
venous thromboembolic episode (within the previous
4 weeks) (63). Where possible, surgery should be
postponed for three to six months if there has been a
recent thromboembolic event. In low risk patients,
warfarin can be stopped three to five days before
surgery and resumed the same day once surgery is
complete unless there is excessive bleeding.
Antiplatelet Therapy Discontinuation

The use of antiplatelet agents in primary and secondary prevention of MI is increasingly common. The
effect of antiplatelets are irreversible for the life of
the platelet (14 days), and its function returns to
normal only once new platelets are released into the
circulation.
Current guidelines published by the U.K.
National Institute for Health and Clinical Excellence
(NICE) recommend that antiplatelet therapy (aspirin
and clopidogrel) should be continued for one year
after a non-ST-segment elevation acute coronary syndrome and for four weeks after an ST elevation MI
(64). This combination should also be continued for
one year following any percutaneous coronary intervention (e.g., stenting, angioplasty), as it reduces risk
of death, MIs, in-stent thrombosis, and stroke (65).
Clopidogrel is regarded as mandatory until the coronary stents are fully endothelialized, which takes
3 months for bare metal stents, but up to 1 year for
drug-eluting stents. Patients who stop clopidogrel
within 30 days of stent placement are 10 times more
likely to die especially as stent thrombosis carries a
20% risk of mortality (66). Late stent thrombosis rates
are relatively low with short-term discontinuation of
clopidogrel in patients with drug-eluting stents providing aspirin therapy is maintained (67).
There is currently insufficient evidence to suggest that low-dose aspirin (75 mg) significantly
increases morbidity or mortality after TURP or other
urological procedures. One study has, however,
shown that aspirin at a dose of 150 mg daily carries
a higher risk of bleeding after TURP and recommends
that it should be discontinued 10 days before surgery
(although transfusion rates were not significantly
higher) (68).
The risk of bleeding with dipyridamole (antiplatelet and vasodilator) is thought to be less and probably
not clinically significant. There was no statistically significant difference between the incidences of major
(combination 3% vs. aspirin alone 4%) or minor (both
12%) bleeding (69).
In all cases, antiplatelet therapy should be
restarted as soon as it is safe so to do. Early aspirin
initiation after lower urinary tract surgery has not been
shown to carry an increased risk of postoperative bleeding. In summary, the decision to discontinue these
agents needs to be weighed against the risk of serious
or life-threatening cardiac events (70). Such judgment
depends on the type of surgery and merits discussion
between the surgeon, anesthetist, and cardiologist.
Substance Abuse
Patients with a history of substance abuse (drugs or
alcohol) undergoing surgery are likely to experience
withdrawal symptoms while in hospital. All patients
should be asked about the number of units of alcohol
consumed in a typical week. The current medically
recommended units of alcohol per week are 14 for
women and 21 for men. A more detailed history
should be taken if their intake is above that recommended. A simple, quick method is to use the Fast
Alcohol Screening Test (FAST) that consists of four
simple questions (71). Signs of alcohol withdrawal
such as tremor, sweating, intoxication, confusion, or
stigmata of liver disease should heighten awareness
and under these circumstances appropriate, timely
inpatient prescription of benzodiazepines can be
extremely useful in managing the condition. A reducing chlordiazepoxide regime over a three- to five-day
period may be necessary if prolonged recovery and
inpatient stay is likely.
Allergy and Drug Intolerance

The estimated risk of anaphylaxis in the general population is 1% to 2% for insect stings and foods, with a
lower reported prevalence for drugs and latex (72),
but in spite of being so uncommon intraoperative
anaphylactic reactions are a constant concern to the
anesthesiologist. The incidence of such reactions in
anesthetized patients ranges between 1 per 5000 and
1 per 25,000, mortality being 3% to 4% (73). A thorough assessment includes adequate knowledge of
known allergies, drug sensitivities, and past reactions
to anesthetics. There may be a documented allergy to
anesthetic drugs, analgesics, local anesthetics, muscle
relaxants, or latex. A family history of malignant
hyperpyrexia, pseudocholinesterase deficiency, and
porphyria may warn the anesthetist of future potential
problems. Allergy history may be obtained from a
number of sources, for example, the patient or their
carers, general practitioners, care homes, and hospital
notes. This should be clearly recorded according to
trust documentation policy.
The incidence of latex allergy is slightly higher in
health care workers, rubber industry workers, and
patients with neural tube defects (including spina
bifida) due to recurrent bladder catheterizations.
Patients with latex allergy require special preparation
of the operation theater and such cases should be
undertaken first on the morning list to allow latex
dust to settle. Depending on the number of air changes
within the room, sufficient time should be allowed to
reduce any aerosolized natural rubber latex proteins in
the atmosphere. Adequate coordination of the entire
surgical staff together with replacement of any anesthetic and surgical material containing latex are all
mandatory to prevent severe hypersensitivity reactions.
Pregnancy
In a large study of nonobstetric surgery undertaken
during pregnancy, an operative rate of 0.75% was
reported (74). Surgery and anesthesia in pregnancy
increase the risk of miscarriage in the first trimester
from 5% to 8%, and the risk of premature labor from
5% to 7.5% (75). Where possible, it is sensible to delay
nonurgent surgery until the second trimester.
RISK ASSESSMENT FOR EMERGENCY

SURGERY
Assessment of patients requiring emergency surgery
depends on their current presenting condition and
associated background comorbidities. As time is
often the greatest limiting factor for successful optimization of any risk factors present, two questions
need to be addressed from the outset.
l
Can surgery be delayed until their medical condition has been optimized?
Do the risks of surgery outweigh the benefits?
Clearly if the risk of significant morbidity or

death from the patients condition outweighs the
537
risk of surgical related mortality, then it may be

deemed appropriate to proceed with surgery providing the surgeon, anesthetist, and patient are all in
agreement. Often, a degree of medical optimization
can occur prior to surgery, and the conditions that can
be improved by medical management should be identified and targeted. Particular attention to adequate
oxygenation, optimal fluid resuscitation, and correction of electrolyte and blood gas abnormalities (e.g.,
acidosis, hypercarbia) will be required and it is helpful to inform and involve the critical care team (and
the anesthetist) from the outset if resuscitation with
invasive monitoring is required. Such patients are best
managed in the high dependency or intensive care
unit. Diabetic patients require tight glycemic control
with a sliding scale to avoid ketoacidosis, reduce
sepsis, and decrease mortality. In patients with renal
failure, there is no evidence to suggest that measures
used to protect the kidneys during the perioperative
period are beneficial. There is no difference in morbidity (renal failure) or mortality following the use of
various perioperative interventions including the use
of dopamine and its analogues, diuretics, calcium
channel blockers, angiotensin-converting inhibitors,
or hydration fluid (76). Immediate assessment and
prompt recognition of comorbidities coupled with
timely intervention will enable the patient to tolerate
the hemodynamic insults imposed by surgery as
much as possible, and this can make the difference
between a successful outcome and mortality.
SPECIFIC SURGICAL PROBLEMS

Transurethral Resection Syndrome
TUR syndrome is most commonly associated with
transurethral resections of the prostate and has an
incidence of 0.5% to 2%. It can also occur following
large bladder tumor resections and percutaneous
nephrolithotomy. It occurs because of the adverse
effects of excessive irrigation fluid absorption when
glycine 1.5% is used. Prolonged resection time (>6090
minutes), large vascular gland resection, presence of
capsular perforation, and continuous flow resection
are thought to predispose to this complication.
The osmolality of 1.5% glycine is 230 mosm/L
compared to serum osmolality of 290 mosm/L and
hence dilutional hyponatremia and fluid overload will
occur with excessive glycine absorption (Fig. 16).
Glycine itself is toxic to the heart, central nervous
system, and retina and may lead to hyperammonemia.
The inclusion of 1% ethanol in the glycine bags allows
detection of excessive absorption by measuring the
ethanol concentration in expired breath (77). Although
a rising breath ethanol level suggests absorption, it is
unpredictable and does not prevent TUR syndrome.
Early signs of glycine absorption include prickling
sensations and facial warmth. A spectrum of symptoms and signs can follow ranging from nausea,
tachycardia, restlessness, dyspnea, arrhythmias,
hyper- or hypotension, transient blindness, confusion,
seizures, coma and death (78).
538
Gunendran et al.
Figure 16 Pathophysiology of TUR syndrome.
Investigations
Low serum sodium can confirm the diagnosis; levels

below 120 mmol/L commonly cause symptoms and a
rapid fall is most likely to lead to problems. At sodium
levels below 115 mmol/L, electrocardiography may
show QRS widening, ST segment elevation and
T wave inversion. A low-serum osmolality and high
anion gap may be present.
Management
If possible, stop further resection and control the

bleeding. Patients may need to be sedated and ventilated if awake. Slow correction of hyponatremia with
intravenous furosemide to promote a diuresis is recommended although some authorities maintain that
intravenous hypertonic saline is the treatment of
choice (79). The serum sodium should be increased
by about 1 mmol/L/hr as rapid correction has been
implicated with central pontine myelinolysis. In the
presence of intractable seizures or encephalopathy,
the sodium level may be corrected more rapidly at a
rate of up to 8 to 10 mmol/L/hr, and this level should
be checked every few hours.
Undoubtedly, the best management is prevention: limiting resection time, careful surgical technique, and appropriate patient selection are important
factors to consider. Newer techniques, such as bipolar
resection, laser ablation, and photoselective vaporization, which use saline as the irrigating fluid, can prevent
the risk of TUR syndrome.
Sepsis
Sepsis following urological procedures may present
with unexplained hypotension, rigors, shock, or systemic inflammatory response syndrome (leucocytosis
or leucopenia, fever or hypothermia, tachycardia,
tachypnea). Early recognition is paramount as these
signs may be the first event in a cascade to multiorgan
failure. Once established, aggressive fluid resuscitation in conjunction with early transfer to a high
dependency or intensive care unit for invasive monitoring, inotropic support, and organ support can be
the difference between life and death. Mortality is
considerably increased when severe sepsis or septic

shock are present (up to a 50%).
Gas Embolism
Laparoscopic urological surgery has increasingly
become accepted as an alternative to open surgical
techniques. Carbon dioxide (CO2) is the most commonly
used gas to create a pneumoperitoneum and inadvertent
rapid insufflation directly into the blood stream or solid
organ can result in gas embolism. Most cases occur
immediately after the pneumoperitoneum is established.
A large embolus can cause a gas lock resulting in
ineffective right ventricular contraction, arrhythmias,
profound hypotension, and cardiac arrest. Although
rare it is potentially fatal with a reported incidence of
15 per 100,000 cases per year (80). Signs include cyanosis, hypoxia, a mill-wheel cardiac murmur and sudden hypotension in the setting of reduced end-tidal CO2,
and decreased chest compliance. Transesophageal echocardiography may help confirm the diagnosis. The
mainstay of treatment is to remove the pneumoperitoneum, hyperventilate with 100% oxygen, and place the
patient in a head down left lateral decubitus position. A
central venous catheter should be placed into the right
atrium if not already present and blood withdrawn
(typically foamy blood) to aspirate the embolus.
Problems Due to Inappropriate Patient

Positioning
The lithotomy position may cause nerve compression,
dislocation of hip prostheses, lower leg compartment
syndrome, and respiratory compromise in patients
with preexisting lung disease. Postoperative injuries
to the common peroneal nerve (especially from pressure effects exerted by the stirrups), ulnar nerve, sciatic
nerve, brachial plexus, obturator nerve, and lateral
cutaneous nerve of the thigh have also been described.
The steep head down Trendelenburg position
utilized during robotic prostatectomy can cause shoulder and nerve palsies. If shoulder braces are used to
prevent the patient sliding, the clavicle can be pushed
up into the brachial plexus. Also when the hip is
abducted more than 308, obturator nerve strain and
leg pain is more likely to occur although the addition

of 458 hip flexion or greater can alleviate the strain
(81). Rhabdomyolysis has been reported after prolonged positioning in the extended lateral decubitus
position for nephrectomies. The use of a subaxillary
roll is important to prevent excessive traction of the
brachial plexus. Care must be taken to avoid excessive
arm abduction and rotation and to ensure that all
pressure points are adequately padded (81).
Intraoperative Priapism
Priapism during a cystoscopic procedure can occur
because of surgical stimulation when depth of anesthesia is inadequate. It is usually sufficient to stop the
stimulus for a few minutes and deepen the general
anesthesia. A small dose of intravenous ketamine or
b-blocker (e.g., propranolol 1 mg) can also be useful. If
such measures fail, it may be necessary to aspirate
corporeal blood or administer a small intracavernosal
dose of an alpha-adrenergic agonist (250 mg of phenylephrine). Resolution usually occurs within 5 to
20 minutes.
Obturator Spasm
Obturator spasm occurs when the obturator nerve is
directly stimulated by diathermy current. The nerve
runs adjacent to the lateral walls of the bladder, and
resection of lateral wall bladder tumors can cause
direct stimulation of the nerve resulting in sudden,
violent adduction of the hip (adductor jerk). This
increases the risk of bladder perforation, incomplete
tumor resection, and obturator hematomas. Reducing
the diathermy current, transpositioning of inactive
electrode from buttock to thigh, using combined general anesthesia and muscle relaxants, resection of
tumor in smaller chips, shifting to saline irrigation
and preventing over distension of the bladder can all
help reduce the magnitude of adductor spasm. Obturator nerve blocks (3 cm lateral and 3 cm inferior to
the pubic tubercle) can also be beneficial.
REFERENCES
1. Palmer QB. Physiologic responses to surgery and injury.
In: John DC, Robin CNW, eds. Surgery. London: Mosby,
2001:section 1:3.13.18.
2. Desborough JP. The stress response to trauma and surgery.
Br J Anaesth 2005; 85:109117.
3. Kehlet H. Multimodal approach to control postoperative
pathophysiology and rehabilitation. Br J Anaesth 1997;
78:606617.
4. Burgos FJ, Linares A, Pascual J, et al. Modifications of renal
blood flow and serum interleukin levels induced by laparoscopic and open living donor nephrectomies for kidney
transplant: an experimental study in pigs. Transplant Proc
2005; 37:36763678.
5. Conacher ID, Soomro NA, Rix D. Anaesthesia for laparoscopic urological surgery. Br J Anaesth 2004; 93:859864.
6. Vallencian G, Guillonneau B, Fournier G, et al. Laparoscopic Radical Prostatectomy: Technical Manual. Paris: Les
Editions, 2002.
539
7. Department of Health. Day surgery: Operational Guide.

Waiting, Booking and Choice. London: DoH, 2002.
8. The NHS PlanA plan for investment, A plan for reform.
HMSO, The Copyright Unit, St Clements House, 216
Colegate, Norwich NR3 1BQ, 2000.
9. Audit Commission. Day surgery: Review of national findings. Audit Commission, London 2001.
10. Cahill CJ. Basket cases and trolleysday surgery proposals for the millennium. J One Day Surg 1999; 9:1112.
11. British Association of Day Surgery. BADS directory of
procedures. London: British Association of Day Surgery,
2007.
12. Golash A, Luscombe C, Rajjayabun P, et al. The expansion
of laparoscopic day case major urological procedures; an
initial experience. J One Day Surg 2007; 17(suppl):B1.
13. Department of Health. NHS Modernisation Agency. Day
surgery: A Good Practice Guide. London: DoH, 2004.
14. Department of Health. NHS Modernisation Agency.
National Good Practice Guidelines on Pre-Operative
Assessment for Day Surgery. London: DoH, 2002.
15. Fleisher LA, Beckman JA, Brown KA, et al. ACC/AHA
2007 Guidelines on Perioperative Cardiovascular Evaluation and Care for Noncardiac Surgery: Executive Summary: A Report of the American College of Cardiology/
American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines on
Perioperative Cardiovascular Evaluation for Noncardiac
Surgery) Developed in Collaboration With the American
Society of Echocardiography, American Society of Nuclear
Cardiology, Heart Rhythm Society, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular
Angiography and Interventions, Society for Vascular Medicine and Biology, and Society for Vascular Surgery. J Am
Coll Cardiol 2007; 50:17071732.
16. Foster ED, Davis KB, Carpenter JA, et al. Risk of noncardiac operation in patients with defined coronary disease: The Coronary Artery Surgery Study (CASS) registry
experience. Ann Thorac Surg 1986; 41:4250.
17. Eagle KA, Rihal CS, Mickel MC, et al. Cardiac risk of
noncardiac surgery: influence of coronary disease and
type of surgery in 3368 operations. CASS Investigators
and University of Michigan Heart Care Program. Coronary
Artery Surgery Study. Circulation 1997; 96:18821887.
18. Goldman L, Caldera DL, Nussbaum SR, et al. Multifactorial index of cardiac risk in noncardiac surgical procedures.
N Engl J Med 1977; 297:845850.
19. Detsky AS, Abrams HB, Forbath N, et al. Cardiac assessment for patients undergoing noncardiac surgery. A multifactorial clinical risk index. Arch Intern Med 1986;
146:21312134.
20. Tarhan S, Moffitt EA, Taylor WF, et al. Myocardial infarction after general anesthesia. Anesth Analg 1977;
56:455461.
21. Rao TL, Jacobs KH, El-Etr AA. Reinfarction following
anesthesia in patients with myocardial infarction. Anesthesiology 1983; 59:499505.
22. Falter F, Martinelli G. Perioperative care of the patient with
cardiovascular disease undergoing non-cardiac surgery.
Surgery (Oxford) 2005; 23:246250.
23. Radford MJ, Krumholz HM. Beta-blockers after myocardial infarctionfor few patients, or many? N Engl J Med
1998; 339:551553.
24. Foex P. Pharmacology of cardiovascular drugs. Clin
Anaesthesiol 1989; 3:131161.
25. Richey R, Wray D, Stokes T. Prophylaxis against infective
endocarditis: summary of NICE guidance. BMJ 2008;
336:770771.
26. Kannel WB, Castelli WP, McNamara PM, et al. Role of
blood pressure in the development of congestive heart
540
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Gunendran et al.
failure. The Framingham study. N Engl J Med 1972;
287:781787.
National Collaborating Centre for Chronic Conditions.
Hypertension: Management in Adults in Primary Care:
Pharmacological Update. London: Royal College of Physicians, 2006.
Midgley S, Tolley D. Anaesthesia for Laparoscopic Surgery
in Urology. EAU-EBU Update Series. 2006; 4:241245.
Kothmann E, Danjoux G, Owen SJ, et al. Reliability of the
anaerobic threshold in cardiopulmonary exercise testing
of patients with abdominal aortic aneurysms. Anaesthesia
2009; 64:913.
Older P, Smith R, Courtney P, et al. Preoperative evaluation of cardiac failure and ischemia in elderly patients by
cardiopulmonary exercise testing. Chest 1993; 104:701704.
Older P, Hall A, Hader R. Cardiopulmonary exercise testing as a screening test for perioperative management of
major surgery in the elderly. Chest 1999; 116:355362.
Smetana GW, Lawrence VA, Cornell JE. Preoperative pulmonary risk stratification for noncardiothoracic surgery:
systematic review for the American College of Physicians.
Ann Intern Med 2006; 144:581595.
Lawrence VA, Page CP, Harris GD. Preoperative spirometry before abdominal operations. A critical appraisal of its
predictive value. Arch Intern Med 1989; 149:280285.
Morton HJV. Tobacco smoking and pulmonary complications after operation. Lancet 1944; 1:368370.
Jorgensen LN, Kallehave F, Christensen E, et al. Less
collagen production in smokers. Surgery 1998; 123:450455.
Bluman LG, Mosca L, Newman N, et al. Preoperative
smoking habits and postoperative pulmonary complications. Chest 1998; 113:883889.
Moller AM, Villebro N, Pedersen T, et al. Effect of preoperative smoking intervention on postoperative complications: a randomised clinical trial. Lancet 2002; 359:114117.
Wilson J, Woods I, Fawcett J, et al. Reducing the risk of
major elective surgery: randomised controlled trial of preoperative optimisation of oxygen delivery. BMJ 1999;
318:10991103.
Intensive Care National Audit and Research Centre.
Annual report from the national case mix programme
database. London: Intensive Care National Audit and
Research Centre, 1998.
Kaba A, Joris J. Anaesthesia for laparoscopic surgery.
Current Anaesthesia and Critical Care 2001; 12:159165.
Linden PA, Gilbert RJ, Yeap BY, et al. Laparoscopic fundoplication in patients with end-stage lung disease awaiting transplantation. J Thorac Cardiovasc Surg 2006;
131:438446.
Ramos M, Khalpey Z, Lipsitz S, et al. Relationship of
perioperative hyperglycemia and postoperative infections
in patients who undergo general and vascular surgery.
Ann Surg 2008; 248:585591.
van den Berghe G, Wouters P, Weekers F, et al. Intensive
insulin therapy in the critically ill patients. N Engl J Med
2001; 345:13591367.
Byyny RL. Preventing adrenal insufficiency during surgery. Postgrad Med 1980; 67:219225, 228.
LaRochelle GE Jr, LaRochelle AG, Ratner RE, et al. Recovery of the hypothalamic-pituitary-adrenal (HPA) axis in
patients with rheumatic diseases receiving low-dose prednisone. Am J Med 1993; 95:258264.
Child CG, Turcotte JG. Surgery and portal hypertension.
In: The liver and portal hypertension. Philadelphia: Saunders, 1964:5064.
Pugh RN, Murray-Lyon IM, Dawson JL, et al. Transection
of the oesophagus for bleeding oesophageal varices. Br J
Surg 1973; 60:646649.
48. Malinchoc M, Kamath PS, Gordon FD, et al. A model to

predict poor survival in patients undergoing transjugular
intrahepatic portosystemic shunts. Hepatology 2000;
31:864871.
49. Lacey JV, Penner JA. Management of idiopathic thrombocytopenic purpura in the adult. Semin Thromb Hemost
1977; 3:160174.
50. Mariappan P, Tolley DA. Endoscopic stone surgery: minimizing the risk of post-operative sepsis. Curr Opin Urol
2005; 15:101105.
51. Mariappan P, Smith G, Bariol SV, et al. Stone and pelvic
urine culture and sensitivity are better than bladder urine
as predictors of urosepsis following percutaneous nephrolithotomy: a prospective clinical study. J Urol 2005;
173:16101614.
52. Laupland KB, Church DL, Vidakovich J, et al. Communityonset extended-spectrum beta-lactamase (ESBL) producing
Escherichia coli: importance of international travel. J Infect
2008; 57:441448.
53. Yilmaz E, Akalin H, Ozbey S, et al. Risk factors in community-acquired/onset urinary tract infections due to
extended-spectrum beta-lactamase-producing Escherichia
coli and Klebsiella pneumoniae. J Chemother 2008; 20:
581585.
54. MRSAOperational Guidance (Gateway reference 10324),
Department of Health, Crown copyright. 2008. Available
at: http://www.dh.gov.uk/en/Publicationsandstatistics/
Lettersandcirculars/Dearcolleagueletters/DH_086687.
55. Grabe M, Bishop MC, Bjerklund-Johansen TE, et al. Guidelines on the management of urinary and male genital tract
infections: Peri-operative antibacterial prophylaxis in urology. Arnhem, The Netherlands: European Association of
Urology (EAU), 2008:9099.
56. The Association of Anaesthetists of Great Britain and Ireland. Peri-operative management of the morbidly obese
patient, 2007.
57. Torosian MH. Perioperative nutrition support for patients
undergoing gastrointestinal surgery: critical analysis and
recommendations. World J Surg 1999; 23:565569.
58. Hill J, Treasure T. Reducing the risk of venous thromboembolism (deep vein thrombosis and pulmonary embolism) in inpatients having surgery: summary of NICE
guidance. BMJ 2007; 334:10531054.
59. Fitzmaurice DA, Murray E. Thromboprophylaxis for
adults in hospital. BMJ 2007; 334:10171018.
60. Salzman EW, Hirsh J. Prevention of venous thromboembolism. In: Colman RW, Hirsh J, Marder V, et al., eds.
Hemostasis and thrombosis: basic principles and clinical
practice. New York: Lippincott, 1982:986.
61. Forrest JB, Clemens JQ, Finamore P, et al. AUA Best
Practice Statement for the prevention of deep vein thrombosis in patients undergoing urologic surgery. J Urol 2009;
181:11701177.
62. Dunn AS, Turpie AG. Perioperative management of
patients receiving oral anticoagulants: a systematic review.
Arch Intern Med 2003; 163:901908.
63. Kearon C, Hirsh J. Management of anticoagulation before
and after elective surgery. N Engl J Med 1997; 336:
15061511.
64. National Institute for Health and Clinical Excellence. Clinical guideline 48: MI: secondary prevention in primary and
secondary care for patients following a myocardial infarction. London: NICE, 2007.
65. Steinhubl SR, Berger PB, Mann JT, et al. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial.
JAMA 2002; 288:24112420.

66. Jones JS. Urologists: be aware of significant risks to stopping anticoagulants in patients with drug-eluting coronary
stents. BJU Int 2007; 99:13301331.
67. Eisenberg MJ, Richard PR, Libersan D, et al. Safety of
short-term discontinuation of antiplatelet therapy in
patients with drug-eluting stents. Circulation 2009;
119:16341642.
68. Nielsen JD, Holm-Nielsen A, Jespersen J, et al. The effect of
low-dose acetylsalicylic acid on bleeding after transurethral prostatectomy-a prospective, randomized, doubleblind, placebo-controlled study. Scand J Urol Nephrol
2000; 34:194198.
69. Halkes PH, van Gijn J, Kappelle LJ, et al. Aspirin plus
dipyridamole versus aspirin alone after cerebral ischaemia
of arterial origin (ESPRIT): randomised controlled trial.
Lancet 2006; 367:16651673.
70. Spahn DR, Howell SJ, Delabays A, et al. Coronary stents
and perioperative anti-platelet regimen: dilemma of bleeding and stent thrombosis. Br J Anaesth 2006; 96:675677.
71. Hodgson R, Alwyn T, John B, et al. The FAST Alcohol
Screening Test. Alcohol Alcohol 2002; 37:6166.
72. American Academy of Allergy and Immunology Board of
Directors Position Statement. J Allergy Clin Immunol 1994;
94:666668.
541
73. Withington DE. Allergy, anaphylaxis and anaesthesia. Can

J Anaesth 1994; 41:11331139.
74. Mazze RI, Kallen B. Reproductive outcome after anesthesia
and operation during pregnancy: a registry study of 5405
cases. Am J Obstet Gynecol 1989; 161:11781185.
75. Wheeler D. Perioperative management of challenging
comorbid conditions. Surgery (Oxford) 2006; 23:262266.
76. Zacharias M, Conlon NP, Herbison GP, et al. Interventions
for protecting renal function in the perioperative period.
Cochrane Database Syst Rev 2008; (4):CD003590.
77. Hahn RG. Ethanol monitoring of irrigating fluid absorption in transurethral prostatic surgery. Anesthesiology
1988; 68:867873.
78. Olsson J, Nilsson A, Hahn RG. Symptoms of the transurethral resection syndrome using glycine as the irrigant.
J Urol 1995; 154:123128.
79. Hahn RG. Fluid absorption in endoscopic surgery. Br J
Anaesth 2006; 96:820.
80. Orebaugh SL. Venous air embolism: clinical and experimental considerations. Crit Care Med 1992; 20:11691177.
81. Van Appledorn S, Costello AJ. Complications of robotic
surgery and how to prevent them. In Patel VR ed. Robotic
urologic surgery. New York: Springer, 2007:169178.
36
Screening in Urology
Manchester, U.K.
INTRODUCTION
Screening has been defined as the identification of
unrecognized disease or defect by the application of
tests, examinations, or other procedures that can be
applied rapidly. The process may be broadly divided
into one-shot screening exercises and procedures
applied to chronic diseases or conditions that may be
repeated at intervals. The second category may be
further subdivided into mass screening, selective
screening, and case finding. In the context of urological surgery, the debate concerning screening programs chiefly concerns the search for chronic
disease, typically urological neoplasia, usually by
means of case finding or, at best, selective screening
programs.
ONE-SHOT SCREENING
One-off screening procedures are most frequently
employed to detect defects typified by congenital
malformation or inherited metabolic disorders, such
as phenylketonuria and galactosemia, for which treatments are available. Screening for disorders that are
untreatable has generally been avoided (see sect.
Fundamentals of Screening), although this basic
concept has been challenged over the last 15 years
(1,2). Programs involving sophisticated tests for rare
diseases (3,4) may not, at a superficial glance, seem
cost-effective; however, the long-term costs of supporting the patient to adulthood with undetected disease may be the crucial factor in determining publichealth policy (5).
Single-shot screening of a blood specimen
originally replaced the nappy test for phenylketonuria 40 years ago and introduced the possibility of
detecting galactosemia, cystic fibrosis, and others
although testing for congenital hypothyroidism
remained the only other official recommendation in
the early days. Recent advances in tandem mass
spectrometry with DNA extraction from dried blood
spots in the newborn have greatly expanded the analytical possibilities although major questions relating
to laboratory technique, quality control, health policy,
and ethics have not been fully addressed (6,7). As
ever, the development of effective treatments for
hitherto untreatable disorders remains at the heart of

this debate.
SCREENING FOR CHRONIC DISEASE

Mass (Population) Screening
Mass screening is the identification of preclinical disease within the population by procedures that have
been carefully assessed and validated as part of public
health policy.
Mass-screening programs demand a precise
cost-benefit analysis closely linked to the ethical and
cultural ethos of the country and government concerned. In general, programs initiated by the departments of health in the West address only health issues
that are both common and, at least in part, preventable, such as heart disease or certain types of neoplasia. Within this politico-medical process, certain
groups, such as young women with disease, inevitably attract more political support than other groups
such as older men with disease.
Selective Screening
In an attempt to boost the effectiveness of screening
programs in relation to cost, the specified test may be
directed at selected groups within the overall population. In fact, nearly all programs are selective in terms
of age and sex, as in the case of breast-cancer screening, where high-risk groups of women are targeted in
terms of age and menopausal status.
Genetic and racial variables are further examples
of factors that may be selectively targeted in screening
programs. Within urology, the risks of prostate cancer
are known to be increased in certain families with a
history of the disease, and epidemiological studies
clearly demonstrate the differing racial incidence of
the disease both worldwide and, most dramatically,
within the United States itself.
Selective screening may also be undertaken by
targeting groups exposed to various industrial or
social risks. The lifelong follow-up of workers from
aniline dye factories and the association of various
diseases with heavy smoking are examples of this
type of selectivity.
Chapter 36: Screening in Urology
Case Finding
Case finding is widely interpreted as screening by
both the general public and the medical profession. In
essence, case finding is no more than sporadic attempts
by interested doctors and patients to detect disease or
establish that disease is not present. As will be described
below, chronic urological disease does not, in general,
attain criteria sufficient to support full public-health
screening programs. In the absence of such programs
often interpreted by critics as a lack of interest by the
Department of Healthcase finding becomes the predominant mode of preclinical disease identification,
usually widely, and often inaccurately, reported by
both the lay and medical press. Case finding is, however, a genuine and valid attempt by concerned doctors
and patients to solve a medical dilemma, but the process
should not be confused with scientifically designed and
validated mass-screening programs.
In this chapter, the issues and principles of
screening will be described in general terms. The
process will then be analyzed with reference to the
generally accepted criteria for screening, and finally
the application of these principles to chronic urological disease will be discussed.
LIFETIME EVENTS AND SCREENING

TERMINOLOGY
When mass-screening programs for chronic diseases
are developed, it is common practice to describe
episodes that occur during the patients lifetime by
an accepted terminology that describes and defines
the evolving events up to the point of death. These
lifetime periods may be illustrated as in Figure 1.
543
Assuming initial health, the first event is the

initiation of a biological disease process. This continues until, in the absence of a screening program,
symptoms appear, leading to presentation and eventual clinical diagnosis.
The time from disease initiation to clinical diagnosis is defined as the total preclinical phase (TPCP) (8).
Clearly, the TPCP is a theoretical concept, as the time at
which the disease is initiated can never be known with
certainty; indeed, with reference to neoplasia, it is likely
that multiple events are required to establish the cancerous process, making it almost impossible to define
precisely the start time of the disease.
If a mass-screening test has been developed for a
disease, it becomes possible to detect its presence
during the TPCP. The period between the earliest
time at which the disease is detectable by the test
and the time at which clinical symptoms become
apparent is defined as the detectable preclinical
phase (DPCP), also known as the sojourn time (9).
Clearly, the length of the DPCP depends on the sensitivity of the screening test, and this is likely to be
different for each disease under consideration. It is
also apparent that different forms of screening tests
for any one disease will be associated with different
DPCP times; thus, breast self-examination will have a
shorter DPCP than mammography, and digital rectal
examination will have a shorter DPCP than a prostatespecific antigen (PSA) screening test. Naturally, the
effectiveness of any proposed mass-screening program will be determined by the ratio of DPCP to
TPCPthe longer the detectable phase, the more
effective the program. Occasionally, DPCP is mistakenly understood to stand for precancerous phase. This
is misleading, as a variable portion of the correctly
Figure 1 Lifetime events and terminology of screening programs. DPCP: detectable preclinical phase; TPCP: total preclinical phase.
Depending on the sensitivity of the test, screening may be undertaken at any time t during the DPCP. Lead-time bias is defined as the
time between the initiation of the test and the time of symptomatic clinical presentation. The death of the patient may occur earlier or later
depending on whether the screening process is harmful or beneficial to the individual.
544
George
Figure 2 Length bias. Solid lines indicate duration of detectable preclinical phase. Biologically active tumors develop and present more
quickly than indolent tumors that progress over long periods. Some tumors may never present clinically. Screening events (vertical
dotted lines) automatically select a preponderance of the more indolent tumors. Source: Refs. 12 and 13.
defined DPCP may be related to invasive (i.e., not

precancerous) yet asymptomatic cancerous growth.
Hence, screening for diseases that have a significant
invasive asymptomatic period during the DPCP (perhaps breast) is likely to be less effective than screening
for diseases with extensive noninvasive periods during the DPCP (perhaps prostate).
The detection of disease during the DPCP by the
screening test itself results in difficulties when
attempts are made to judge the effectiveness of any
single program. The time between the positive result
of the screening test and the time at which clinical
symptoms would have appeared is defined as the
lead-time bias. It will be evident that if treatment for
the disease is ineffective, the patient will die at the
same point as if it had originally been detected
because of clinical symptoms. Survival, however,
will apparently have been increased by the amount
of time between the positive screening test and the
clinical presentation; hence, it is necessary to account
for lead-time bias when assessing the efficacy of
screening for any particular disease (10). For severe
chronic disease such as cancer, the elimination of leadtime bias is best achieved by randomized controlled
trials of the screening program, as described below.
The death of the patient is an event that is determined by the severity of the disease and the efficacy of
treatment. As noted above, it is necessary to demonstrate that a screening program results in prolonged
survival after lead-time bias and other distortions have
been taken into account. It should not be forgotten that
screening programs may also lead to a shortening of
the patients lifeif, for instance, death occurs because
of septicemia following transrectal biopsy for presumed prostate cancer suspected on the basis of a
screening test, the program (depending on the frequency of the complication) will be judged as being
of questionable value with regard to public health.
A further distortion that may be observed when
mass screening for chronic disease is described as
length bias (11). In the case of neoplasia, different
tumors grow at different rates, and, thus, fast-growing
cancers are characterized by a short DPCP. A screening
test undertaken during the DPCP will, of necessity,
detect more slow-growing cancers than fast-growing
cancers (Fig. 2); thus, the disease so detected will have
a more favorable outcome than the disease detected by
standard clinical tests. This length-bias effect may be
reduced by repeat screening, but once again it can be
seen that improved prognosis in the screening group
may not necessarily be related to a real improvement in
survival for the disease in question.
It is often difficult to persuade the general public
and occasionally the medical professionthat earlier
detection of disease is not automatically associated
with a better prognosis. Figures 1 and 2 well illustrate
the complexity of the issues raised when mass screening is undertaken for chronic disease.
FUNDAMENTALS OF SCREENING
Before describing other issues concerned with massscreening programs, one should emphasize that there
are certain situations in which it is not possible or
necessary to offer a screening test.
l
If there is no preclinical phase (TPCP), it is clearly

not possible to offer a test which can detect that
phase of disease.

l
If there is no possible treatment for the disease,

conventional wisdom suggests that screening
should not be offered, as, by definition, no
improvement in morbidity and mortality will be
achieved. Interestingly, this principle has recently
been questioned in relation to single-shot screening of the newborn for rare inherited disorders.
Duchennes muscular dystrophy is an X-linked
lethal disorder of delayed onset with no effective
treatment. It has been argued (14) that screening
for the disease should be undertaken on the
grounds that it would lessen parental distress by
alerting them to risks before the disease became
apparent in their older children. Clearly, such a
proposition raises highly complex ethical arguments that need to be carefully considered before
any such program could be adopted (14).
If it is possible to cure a disease when it presents
clinically, there is no need to offer a screening test
for the disease. Examples of neoplastic diseases
that conform to this definition are clearly few and
far between, but testicular tumor might be cited as
an example of a cancer that is curable if efficiently
detected in stage one by the patient himself.
ETHICAL CONSIDERATIONS AND ATTITUDES

TO SCREENING
It will be realized that in a mass-screening program for
disease, different forces are at work that depend on
individual expectation of the enterprise. It is often
assumed that satisfactory outcomes would, by definition, be acceptable to everybody concerned with the
program. To assess this matter, it is necessary to look at
any screening program from the point of view of both
the Department of Health, the screener, and the
individual member of the general public, the screenee, who is required to undertake the specified test.
The attitude and perspective of those offering a
mass-screening programusually the Department of
Healthare principally influenced by their primary
directive to improve the health of the nation as a
whole. The screener does not promise or imply that
every individual screened will be better off as a result
of taking the test, although this point is rarely emphasized in the accompanying publicity that suggests that
screening must be good for you. In this context, the
death of a patient following prostatic biopsy (as noted
above) would be an acceptable if regrettable incident
were mass screening for prostate cancer to be confirmed as a beneficial program for the nation as a
whole. The individual suffering disadvantage, as
inevitably occurs in the best-validated screening programs, will have a different perspective on the matter.
The dichotomy between these two points of view is in
part related to the generalized belief noted above that
early diagnosis must be goodcatch the disease
early. It is perhaps understandable how this
entrenched view of health and disease may lead to
disappointment both for the screener (15) and the
screenee who suffers an adverse event following the
screening test.
545
Cost issues are also a major factor for those

considering ways of improving the public health. In
developing countries, it is by no means certain that
screening for certain diseases would be of greater
advantage to the population than money put into
other aspects of public health such as water supplies
and sewage systems. In the West, cost-benefit analysis
raises highly complex issues requiring scientific resolution, often in an atmosphere made obscure by the
prominent opinions of vested-interest groups. An
example, discussed below, is the ongoing controversy
surrounding prostate cancer screening. Critical analysis
of diagnostic and mortality data shows that this disease
is not the commonest cause of cancer death in males
(12.6%, 1:8 cancer deaths) and particularly for younger
males a case might be made for expending valuable
resource on other noncancer causes of mortality (Figs. 6
and 7 )the debate continues in both medical and lay
press.
The expectations and aspirations of those
screened are very different. Persons selected by a
program or those who elect to go to their general
practitioner naturally hope that disease will not be
detected. Asymptomatic persons who visit their practitioner do so in the expectation that they will be
found to be healthywhat normal person would
attend a physician in the expectation that cancer
would be found? The screener is looking for disease,
but the screenee expects a healthy outcome. These
attitudes explain in part the problems that occur when
a screening test goes wrongwhen either the test
induces morbidity or the test reveals disease. Success
or failure of the process is a value judgment based on
point of view. In practice, of course, the vast majority
of people who undergo screening tests are found to be
negative (true negativesee below). When diseases
are of low prevalence, a negative predictive value is
both very accurate and very reassuring for the person
concerned. A negative screening test for a rare but
serious disease is, in fact, one of the greatest benefits
that can accrue from a screening program.
Ethical considerations in screening programs
are of major importance to both health departments
and the population as a whole. Clinicians readily
acknowledge ethical problems in routine medical
practice, but in this case the patients have almost
invariably sought the help of the physician to deal
with their disease. In the case of mass screening,
selected people who consider themselves to be
healthy have been approached by physicians on
behalf of the Department of Health and asked to
undertake tests which, as has been seen, will usually,
but not necessarily, be of benefit to them. Initiation of
the screening process deems that the ethical burden
on the screener is as great or greater than that resting
on colleagues in clinical medicine.
VALIDITY OF SCREENING
Conventionally, the validity of screening is measured
in terms of both the test, or tests, utilized in the
procedure and the validity of the program itself in
546
George
Figure 3 Sensitivity and specificity of a screening test and the effect of variation of the cut off value. (A) high sensitivity, low specificity;
(B) low sensitivity, high specificity.
its entirety. Each of these aspects may be measured by

means of two indicators: Sensitivity relates to the
positive identification of preclinical disease, and specificity is associated with establishment of healthy
persons within the general population. Naturally,
the validity of screening in terms of sensitivity and
specificity can be known only if the true disease rate in
the population is knowna follow-up diagnostic test
is required to confirm or refute the result of the
screening test.
The Validity of Screening Tests

In terms of the test, sensitivity is defined as the percentage of persons with a positive test among those
who are later found to have the disease. Specificity is
defined as the percentage of persons screening negative among people who are genuinely free of disease.
The relationship between the indicators and the presence or absence of disease is usually depicted as in
Table 1. As noted above, the calculations demand that
an independent diagnostic test is able to determine
which of the screened population has or does not have
the disease itself. It has already been seen that application of this diagnostic test may induce significant
morbidity in persons who are other wise genuinely
well. Sensitivity is the ratio between true positives and
true positives with false negatives. Specificity is the
ratio between true negatives and false positives with
true negatives.
Table 1 Possible Outcomes of Screening Examinations
Diagnosis
Screening Test Result
Disease present
Disease absent
Positive
A
(True positive)
b
(False positive)
Negative
C
(False negative)
d
(True negative)
Sensitivity a/a c; specificity d/d b; positive predictive value a/a b;

negative predictive value d/d + c.
It is unusual for the screening test itself to

demarcate exactly between those with and without
disease. Almost invariably, the population groups
(healthy and diseased) overlap considerably; hence,
variably specified test results will give rise to different
levels of calculated sensitivity and specificity.
Conventionally, these difficulties are depicted by
means of a bimodal graph, as illustrated in Figure 3.
The screening test scale is shown on the horizontal
axis, and the distribution of the healthy and diseased
populations overlaps to a greater or lesser extent.
Selection of different discriminant values of the test
results (known as the cutoff point) demonstrates the
inverse association between the sensitivity and specificity of the screening test. Selection of cutoff point A
determines that the test will have high sensitivity and
low specificityall those with the disease will be
identified by the test, but a high proportion without
disease will also be identified (false positive). Altering
the cutoff point to B results in a test with low sensitivity but high specificitya small number of persons
with the disease are missed (false negative), although
the great majority of people without disease are
correctly identified as screen negative.
Selection of an appropriate cutoff point and
hence particular values of sensitivity and specificity
is a complex process involving considerable degrees
of subjective judgment related to the disease in question. Some disorders, such as cervical cancer, can
afford a high sensitivity for the screening test, as
diagnostic clinical confirmation is reliable and relatively inexpensive, while in others, high specificity
would be of greater desirability. Value judgments
concerning the disease in question, the cost of diagnostic tests, and its place in society are required, and
the results of such judgments will determine the levels
of false-positive and false-negative screening tests that
will be tolerated.
Urologists well appreciate the effect a variable
cutoff may have on the sensitivity and specificity of
disease. Difficulties in calculating effective cutoffs
for prostate cancer screening have been widely

reported, and the advent of newer, more sophisticated
PSA tests (free and bound) have not lessened the
debate on the ideal discriminant value between
benign and malignant disease.
A further attempt to refine and optimize values
of sensitivity and specificity involves the construction
of a receiver operating characteristic (ROC) curve.
Such curves can be drawn if the screening test result
547
is both quantitative and variable. Sensitivity on the

vertical axis and 1/- specificity on the horizontal axis
are plotted for a range of values (Fig. 4A), and the
optimal point is that which lies furthest from the
diagonal. Although, strictly speaking, ROC curves
are difficult to construct for subjective tests such as
digital rectal examination, this form of analysis reinforces the impression that PSA is the best predictor of
the presence of prostatic carcinoma (16).
Figure 4 (A) Receiveroperator characteristic

curve for serum PSA, transrectal ultrasonography
(TRUS) and digital rectal examination (DRE).
Source: Data from Ref. 16. (B) Receiveroperator
characteristic curve analysis from 553 men suggesting that addition of a logistic regression algorithm could further enhance prediction of biopsy
outcome over and above serum PSA and urine
PCA3 alone. Source: Data from Ref. 17.
548
George
Checking the Sensitivity of a Test

The screening test has already been defined as a
measurement that is able to identify disease in the
preclinical period (DPCP). Unfortunately, because
the true prevalence of disease during this stage
must, by definition, be unknown, the sensitivity of
the test cannot with certainty be calculated. To overcome this problem, screening tests are occasionally
tested on populations with known clinical disease.
Experience has shown, however, that this may be
highly misleading, in that the test may perform
exceptionally well when offered such a relatively
gross challenge but will perform indifferently in the
true DPCP screening situation. Hence, sensitivity and
specificity may vary widely according to the stage of
the disease.
A further theoretical approach to the problem of
defining sensitivity might be to perform a diagnostic
test on every person offered the screening test.
Clearly, however, the morbidity of the diagnostic
test applied to large numbers of asymptomatic people
would preclude this approach on ethical grounds
alone. The estimation of test sensitivity therefore
rests imperfectly with the test itself and the diagnostic
measures that are subsequently undertaken to confirm
or deny the presence of disease. The emergence of
new clinical cases (called in cancer screening programs interval cancers) from a previously screened
population, as well as information gathered from
rescreening exercises, eventually leads to a reasonable
estimate of the sensitivity of a particular screening
test. By contrast, the estimation of specificity is less
problematical. As the ratio of people with disease to
the general population is usually very low, an accurate estimation may be obtained by calculating the
proportion of those testing negative to the sum of
those with negative or false-positive tests.
The predictive value is an important measure of a
screening test of interest to both the screener and the
screened population (Table 1). A positive predictive
value is the proportion of those with preclinical disease
relative to those with a positive screening test. A
negative predictive value is the proportion of persons
who are healthy among the population with a negative
test. Predictive values vary and relate not only to the
validity of the test itself (see above) but also to the
prevalence of the disease in the general population.
These effects are illustrated in Table 2. A high positive
predictive value clearly indicates satisfactory test performance, but as disease prevalence declines, so does
the positive predictive value until, with a disease of
Table 2 The Effect of Preclinical Prevalence of Disease on

Positive and Negative Predictive Values
Predictive value (%)
Prevalence (%)
10 (high)
1
0.1 (low)
Sensitivity: specificity: 95%.
Positive
Negative
68
16
2
99.4
99.9
99.99
Table 3 The Effect of Repeat Screening on the Prevalence of

Disease
Prevalence (per 103)
Cancer
Survey
Cervix
Females
Age 20+
Rescreen
<3 years
Females
Age 2064
Rescreen 1 year
Males
Age 4065
Rescreen 6 months
Breast
Lung
1st Pass
2nd Pass
3.9
1.3
2.7
1.5
1.0
0.4
Source: Abstracted from Ref. 1820.
low prevalence, only a very small percentage of those

with a positive test actually have preclinical disease
2% in the example given in Table 2. By contrast, under
the same circumstances of low prevalence, a negative test
clearly indicates that it is very unlikely indeed that the
person has the disease. As has been stated above, this
is an ideal result from the point of view of screenees,
who have attended in the hope that their impression
of good health will be supported by the test.
Effect of Repeat Screening

It might be expected that disease pickup rates
would be maximal during the first screening exercise
and reduced thereafter. Table 3 illustrates this effect
for early studies involving programs for cervix, breast
and lung cancers. More recently, similar effects have
been noted during screening for prostate cancer in
the United States where, additionally, the incidence of
metastatic disease in bone has fallen steadily, perhaps
indicating detection of disease earlier in the DPCP.
PROGRAM VALIDITY
The performance of the screening test itself is naturally but one part of the overall efficacy of any
particular screening program. Other factors that will
be crucial to the outcome of the enterprise include the
screening request response rate, the interval between
screens, and the ability to accurately process those
people found to be screen positive. Most recently, in
the United Kingdom, such problems have been vividly illustrated in the case of screening for cervical
carcinoma. Allegedly inexperienced and underfunded
laboratories have led to significant doubts concerning
the interpretation of large numbers of smear tests.
Clearly, the efficacy of a program is no greater than
the efficacy of its weakest link.
Overall evaluation of a screening program
depends therefore not only on sensitivity and specificity indicators but also on the entire screening infrastructure and most particularly on outcome measures
as judged by objectives laid down at the commencement of the program. Apart from these problems with
infrastructure, resources, diagnostic quality control,
etc., it has been noted that screening programs contain
549
Figure 5 Ideal design of a randomized controlled trial to evaluate the benefits or otherwise of a screening program. Refusers: people
who refuse to undertake screening tests despite randomization to the screening arm. Interval cancers: cancers arising by conventional
clinical symptoms between screening tests.
a degree of bias that may significantly affect outcome

measures, such that those persons identified with
screen-positive disease will almost invariably survive
longer than those detected by standard clinical criteria.
Some of these biases have already been mentioned but
are restated below to emphasize their importance in
the overall evaluation of screening programs:
Lead-time bias: Lead-time bias is that proportion of the
DPCP by which survival will be increased even if the
screening program has no effect on disease mortality.
Length bias: Length bias determines that the screening
process will naturally select persons with more
indolent or biologically inactive disease. Length
bias is most pronounced at the initial screen and is
in part mitigated by subsequent screening.
Selection bias. Despite attempts to screen broad sections of the community, the process itself is inevitably voluntary; thus, those who attend screening
programs are generally more interested in their own
personal health than those who refuse. As stated
above, these health-conscious people expect to be
told that they do not have serious disease and to this
end have usually spent their lives avoiding risk
factors such as unhealthy diets and smoking. Additionally, even if these persons are found to have
disease, their attitude to personal health means that
it is likely that they will have a better outcome from
the disease than those who neglect themselves.
Overdiagnosis bias. A number of chronic conditions,
particularly cancers, may have a semi-indolent
phase that may not lead to clinical disease in the
natural lifetime of the patient. Overdiagnosis of this
form of diseaseperhaps best illustrated by the case

of prostate cancerwill again lead to false estimates
of survival. This bias can be seen to be an extreme
form of length bias, as illustrated in Figure 2.
These forms of bias, acting individually and
together, almost invariably mean that patients with
screen-detected disease survive longer than those
detected in the normal way. To eliminate effectively,
the combined influence of these factors requires a
randomized controlled trial of the screening process
itself (15). The basic design for such a trial is illustrated in Figure 5. It can be seen that not only is the
population randomly allocated to a study or control
group, but also all cancers are followed whether
screen detected or refusal detected. Although it is
generally agreed that randomized trials offer the
most objective test of a screening process, ethical
issues may prevent establishment of a formalized
trial structure. While such trials have been established
for prostate cancer (see page 556), these issues may
cause significant problems for diseases such as breast
cancer, where randomization to the control arm may
meet with consumer resistance. To circumvent these
problems, a number of complex methodologies has
been proposed, the details of which are beyond the
scope of this account.
SCREENING AND CONTROL OF DISEASE

Screening is but one method of controlling disease in
the population. Others include prevention or the
application of curative methods once the disease has
550
George
become manifest. Of these options, screening is a

relative newcomer, and its role in the control of
mass disease remains somewhat controversial. It is
convenient to consider disease control in terms of both
mass screening and selective screening.
Encouraged by successes in eliminating chronic
inflammatory disease, health departments within the
past 25 years have turned their resources toward an
attempt to eradicate other chronic diseases such as
cancer. However, inspection of mortality-reduction targets shows that screening is expected to contribute only
a small proportion of the target totalit has gradually
become clear that the complexities and problems
involved in mass screening preclude utilization of this
methodology as a significant means of disease control.
Several factors underlie this failure to achieve
specified goals. The Wilson and Junger criteria
(described below) dictate that disease must be common and an important health problem. Yet, careful
analysis of mortality data (Figs. 6 and 7) demonstrates
clearly that this is often not the case despite public
perception to the contrary. Presently, programs are in
place for cervical and breast cancer and certain aspects
of cardiovascular disease (hypertension), although
organization of programs varies widely between
countries and, within time, in any one country.
There remain significant problems related to population compliance. No better example of the discrepancy
between expectation and achievement is provided by
the prostate cancer screening policy in the United
States. Despite enormous publicity and guidelines
provided by bodies such as the American Cancer
Society, significant screening test take-up rates are to
be found only within the Anglo-Saxon population.

Take-up within the black community, although
improving, is acknowledged to be low and difficult
to achieve. Black men have a 60% higher incidence of
the disease than whites and the disease presents at an
earlier age (23), often at a more advanced stage (24). In
a recent study by Catalona investigating high-risk
populations in Washington, Missouri, only 1200
black men were available to compare with nearly
16,000 whites (25). It is very clear that, in this group
of people, screening is presently of limited value as a
method of disease control.
However, selective screening has been more successful at reducing disease rates in the last few decades. Targeting populations exposed to risk factors
such as asbestos and tobacco has been in part successful in reducing disease because of these agents.
Nevertheless, prevention strategies are at least as
important as a means of disease control. Urine screening of workers who were in the past exposed to
aniline dyes continues, but, undoubtedly, the main
factor responsible for the reduction in industrial
diseaserelated transitional cell carcinoma is the
exclusion of carcinogenic substances from the manufacturing process in the mid-1950s.
In summary, mass screening for chronic disease
is effective as a means of disease control in certain
carefully defined groups of patients. For the majority,
however, other programs of disease control are
required. By contrast, single-shot screening of the
newborn is an effective method of disease containment, which, even in the case of rare disease, may
prove to be cost-effective in the long term.
Figure 6 System-specific cause of death for males England and Wales 2008. Source: Data from Ref. 21.
551
Figure 7 The 10 commonest causes of cancer death in men in the United Kingdom (2007). Prostate deaths (10,239) at any age
constitute 12.6% of the whole. Source: Data from Ref. 22.
GENERAL PRINCIPLES RELATING

SCREENING PROGRAMS
During the past 30 years, the general principles that
underpin a successful screening program have been
analyzed and refined. A broad consensus has
emerged from individuals and international workshops (26,27) regarding the criteria that should be
met to establish a successful screening program. Of
these criteria, the most widely quoted at the present
time are those published in the public health papers of
the World Health Organization (28) by Wilson and
Junger (Table 4). It is instructive to discuss each individual principle in turn.
The condition should be an important health
problem. Clearly, this statement refers to the point of
view of the health department of the country concerned. Accurate mortality statistics (Figs. 6 and 7)
will be required for that country, although these may
well reveal that problems considered by the general
public to be of major importance may well not
be significant in public health terms. Clearly, in the
northwest of England, cardiovascular disease is the
preeminent public-health problem, whereas in males
each organ-specific cancer site, with the possible
exception of lung, contributes little to the overall picture. For women, breast cancer assumes a significant
position in the public-health priority list, not only
because of the numbers involved but also because
the disease affects younger women whose economic
and social lives are important to the nation. By the
same token, disorders that affect older menfor
Table 4 Accepted Criteria for Successful Population Screening Programs

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
The condition sought should be an important health problem

The natural history of the condition should be adequately understood
There should be a detectable latent or preclinical stage
A test of suitable sensitivity and specificity should be available
There should be an accepted mode of treatment for patients discovered to have the disease
Adequate facilities for both diagnosis and treatment should be available
The tests must be acceptable to the population
The benefits should outweigh any adverse effects of screening
The cost of screening must be acceptable in relation to overall medical expenditure
Screening for chronic disease is an ongoing process requiring critical analysis and audit of results
552
George
Figure 8 Sensitivity and specificity as applied to PSA testing. Conventionally a cut off of 4 ng is accepted as the best compromise
although significant numbers of cancers may be identified with PSA values below that level. (A) Theoretical ideal sensitivity, poor
specificity; (B) good specificity, unacceptable sensitivity.
example, colorectal and prostate tumorsscore less

heavily in terms of being an important health
problem in the eyes of the Department of Health.
The controversy that surrounds prostate cancer screening at the present time is further discussed below.
The natural history of the condition should be
adequately understood. It is naturally very important
that the biological progress of the disease should be
known with reasonable certainty. This matter is not
always as simple as it might seem. In the case of
single-shot antenatal screening for hydronephrosis
and urinary tract obstruction, significant numbers of
babies with hydronephrosis were detected as the
sophistication of interuterine ultrasound increased.
Subsequently, it became clear that physiological
changes at and after birth restored many of these
upper urinary tracts to normal, and it was clear that
the screening test was overestimating the incidence
of significant pathology in these children. Prostate
cancer provides the best example of a chronic condition whose natural history is incompletely understood. Evidence from well-known postmortem
studies from the 1950s indicates that the disease is
extremely common in old agemen die with rather
than of prostate cancer. The inability to distinguish
indolent from aggressive cancers in younger men
continues to generate heated debate.
There should be a detectable latent or preclinical
stage. It has already been mentioned that it is impossible, by definition, to perform a screening test on a
disease that does not have a detectable preclinical
stage. Hence, while cervical dysplasia permits consideration of effective programs for cervical cancer
sputum, cytology and chest X ray have been found
wanting in terms of their ability to reduce mortality
from lung cancer (15).
A test of suitable sensitivity and specificity
should be available. The measures that describe the
validity of any particular screening test have been
described above, and the difficulties of agreeing an

acceptable cutoff point have been emphasized. This
problem is particularly well illustrated by reference
to PSA and the ability of this marker to distinguish
between benign and malignant prostate disease
(Fig. 8). Although 4 ng is generally accepted as an
adequate cutoff, significant numbers of men with
PSA results under this value are found to have
tumors, and some authorities argue that a lower
cutoff, as illustrated, should be employed.
There should be an accepted mode of treatment
for patients discovered to have the disease. Of course,
early diagnosis of an untreatable disease is not a costeffective public-health policy, although the debate
surrounding such conditions as Duchennes muscular
dystrophy has been noted. The proposed treatment
should be effective in terms of reducing mortality, and
the ideal methodology for evaluation of screening
programs has been described.
Adequate facilities for diagnosis and treatment
should be available. It is self-evident that there is no
point in establishing an expensive screening process if
facilities to confirm diagnosis and confer treatment are
not readily available.
The test must be acceptable to the population.
The poor take-up rate in some screening programs
testifies to the fact that there is a limit to the discomfort that the public is willing to suffer to detect
asymptomatic disease (from which, in any case, they
believe they are not suffering). Mammography is not
usually a comfortable procedure, and there is no
doubt that in North America the thought of a rectal
examination and perhaps transrectal ultrasound is the
primary reason for poor attendance at screening sessions by certain segments of society. The paradoxical
situation of the Afro-American in the United States
has already been mentioned.
The benefits should outweigh any adverse effect
of screening. Benefits in this context are taken to
refer to those as observed from the public-health point

of view. Clearly, from the individual standpoint, there
may be significant morbidity (and perhaps, rarely,
mortality) from a screening test; hence, personally,
the individual would be unlikely to recognize any
benefit. Judged overall, the benefits to the general
population should outweigh the adverse effects within
the same population, and outcome measures should
indicate a real advantage to the screened populations,
ideally in terms of increased survival times.
The cost of screening must be acceptable in
relation to overall medical expenditure. It has been
emphasized that screening is but one part of health
policy aimed at containing disease. Furthermore, disease containment is but one part of overall medical
strategy for the population, and, as such, screening
costs have to be considered side by side with all other
claims on the health-service purse.
Screening for chronic disease is an ongoing process requiring critical analysis and audit of results. Selfevidently, disease is an ever-present threat emerging
constantly during the life years of the population.
Hence, effective screening programs must be ongoing,
as must continuing audit and evaluation of the process.
In general, the Wilson and Junger criteria (28)
provide a very good framework for discussion of
disease as a matter of public-health policy. The principles are naturally more applicable to some diseases
than others, and the opportunity is now taken to
discuss screening programs in terms of organ-based
uropathology.
SCREENING FOR UROLOGICAL DISEASE

Single-Shot Screening
Congenital abnormalities of the urogenital system
are relatively common, as such abnormalities are not
invariably fatal for the fetus. Within the past 15 years,
553
increasingly sophisticated ultrasound technology has

been able to pick up urological abnormalities in utero
with increasing confidence. Most normal fetal kidneys
are visible before 20 weeks, and hydronephrosis may
be detectable as early as 16 weeks. With such technology, the incidence of congenital renal abnormalities
has been reported as approximately 1 in 800 live births
(29), which compares favorably with the 1 in 650
reported from autopsy series (30).
Good-quality pictures may be obtained of both
hydronephrosis and hydroureter when present
(Fig. 9A and B). Although not all cases of simple
hydronephrosis may need intervention after birth,
the screening process undoubtedly identifies at-risk
babies, allowing the pediatric service to concentrate
its efforts in a cost-effective and efficient manner.
SCREENING FOR UROLOGICAL ASPECTS

OF INFECTIOUS DISEASE
Screening During Pregnancy
Within the United Kingdom, testing is made available
to pregnant women (i.e., selective screening) for
rubella antibody, syphilis, HIV, and hepatitis B infections (31). Informed consent is required but the assays
are run from a single blood test that is also used for
other routine monitoring under pregnancy.
Screening for Infectious Disease

Overall, because of successful immunization and public health programs, the infectious diseases of the past
no longer constitute a major mortality threat to the
population. By contrast, the incidence of certain sexually transmitted diseases has increased dramatically
in recent years leading, if not to death, to chronic
morbidity, pelvic pathology, and infertility.
Figure 9 (A) Antenatal ultrasound of a fetus with hydronephrosis and hydroureter but a normal bladder. (B) Diagramatic representation
to illustrate scan.
554
George
Table 5 The Benefits and Disadvantages of Screening for
Cancer
Benefits
Disadvantages
Improved prognosis for some

cases detected by screening
Longer morbidity for cases

whose prognosis is unaltered
Less radical treatment needed

to cure some cases
Overtreatment of borderline
abnormalities
Reassurance for those with

negative test results
False reassurance for those

with false-negative results.
Unnecessary morbidity for
those with false-positive
results Hazard of screening
test
Resource costs
Resource saving
Figure 10 New diagnoses of Chlamydia trachomatis England

and Wales 19982007, totaling 113,585 cases in 2006. Rates in
women consistently greater than men but stabilizing in 2005
perhaps as a result of the NCSP. Effective screening in infectious
diseases might be expected to impact more rapidly when compared to screening for chronic (neoplastic) disease as discussed
in Ref. 47. Source: Data from Health Protection Agency. Available at: www.hpa.org.uk/infections/chlamydia.
The vaccination programs to counter the oncological effects of sexually transmitted HPV 16 and 18
are mentioned in chapter 9. The incidence of Chlamydia trachomatis in younger adults (Fig. 10) reached
113,585 cases per year in 2006, with girls (1624) and
men (1829) predominantly effected. It is acknowledged that the observed increase might be due not
only to increased prevalence but also to greater health
awareness, sporadic screening, more sensitive tests,
and more complete national statistics (32). To counter
this epidemic, an opportunistic screening program
was rolled out across England (NCSP, National Chlamydia Screening Programme) initially commencing at
certain locations in 2004 (33). Previous studies, however, had revealed patchy uptake with limited engagement in deprived areas, so there is concern that the
scheme might lead to further inequalities in health
(32). Nevertheless, compared to the earlier cumbersome culture techniques, the evolution of rapid point
of care tests for both men (VB1 urine) (34) and women
(vaginal swab) (35) has allowed immediate specific
treatment and, most valuably, rapid contact tracing.
SCREENING FOR CHRONIC UROLOGICAL

DISEASE
Screening programs are directed toward the eradication of serious chronic disease such as cancer
(Table 5). Therefore, if such programs are to be
applied to the urogenital system, neoplastic disease
is most likely to be the target of the enterprise. Serious
chronic inflammatory disease, such as tuberculosis, is

no longer a significant health problem from a screening point of view. However, as will be seen in terms of
numbers, urological cancers, with the possible exception of prostate, do not recommend themselves as
ideal subjects for screening protocols.
Renal Cancer
Kidney cancer is relatively uncommon. Ranked as the
11th most common neoplasm diagnosed in the United
Kingdom by crude rate, 7000 cases were observed in
2004, 2.5% of all detected neoplasms (excluding nonmelanoma skin cancer) (36). Although clearly not, in
these terms, a cancer of the highest political priority,
cancer-specific mortality nevertheless was 3500 cases
in 2005 (37) and the great majority of these deaths
were from advanced stage metastatic disease. Advances in uroradiological diagnosis suggest that a case
might be made for earlier stage detection of the disease by ultrasound, and while such a program would
not meet with strict political approval in terms of
Wilson and Junger criterion 1 (Table 4), other criteria
could be accepted by both screeners and screenees
who would hopefully benefit from diagnostic downstaging enabling active surveillance (38) followed by
excision as indicated utilizing modern laparoscopic
techniques. Such an approach might eventually eliminate or at least reduce significantly the need for the
hyperexpensive ethically challenging new oncological
therapies now associated with advanced stage disease. It would seem that a politically astute overall
cost-benefit analysis would be the key to population
screening for these diseases of apparently lesser
numerical importance yet escalating therapeutic complexity, particularly when the test is as innocuous and
as acceptable for the screenee as renal ultrasound.
A form of selective screening might apply to
patients with von HippelLindau disease, who have
multiple abnormalities, including a 30% incidence of
renal cell carcinoma. However, the symptoms and
signs of the disorder are so characteristic and the
association with kidney cancer so strong that the
term selective screening is unnecessary for what is
in effect a practical clinical surveillance program.
Bladder (Urothelial) Cancer

Although more common than kidney cancer, bladder
cancer is again not perceived as a critical public health
problem with 3% (4700) cancer deaths reported by
Cancer Research UK (CRUK) in 2005 (37). Hence, the
question as to whether symptom-free members of the
public should be population screened for bladder
cancer has not as yet been addressed other than in
the context of selective studies for particular at-risk
groups, that is, for workers previously exposed to
Aniline dyes.
It is accepted that bleeding usually arises from
established urothelial tumor rather than a preclinical
phase. The debate continues as to the ideal diagnostic
protocol that should be employed following such a
symptom bearing in mind that only approximately
three quarters of patients with bladder cancer have
hematuria (39) and the many other disorders that may
manifest as blood in urine. Urine cytology has been
widely utilized, but it is acknowledged that, while the
test has good specificity, it has a relatively low sensitivity for detecting low- and intermediate-grade transitional cell carcinoma (40). Cystourethroscopy is the
accepted gold standard for identifying urothelial
tumor, although even direct vision does not result in
perfect sensitivity and specificity for the test.
The imperfections of these modalities have led to
a search for additional novel markers and techniques to
improve detection rates in bladder cancer (4143). In
this context it is very important to distinguish between
test performance when used in the broader diagnostic mode (i.e., at the hematuria clinic) and performance when applied to the more selective screening
mode that involves patients already known to have the
disease (surveillance protocols). Not surprisingly, complicating conditions such as inflammation, being more
555
prevalent in the former group, have led to relatively

disappointing results as a diagnostic test (relating both
to sensitivity and specificity), and thus recent enthusiasm for the new markers has tended to concentrate on
their performance as part of surveillance protocols for
tumor recurrence (43,44). Few studies report test performance exclusively in the diagnostic screening
group, most series including both diagnostic and recurrent tumor patients (45). Hence, current urothelial
screening is a good example of the confounding
problems referred to earlier, whereby a potential
screening test is evaluated in patients who are already
known to suffer from the clinical disease (see page 548).
As has been emphasized, the real test of such screening
techniques occurs when the procedure is appropriately
applied to a truly unselected population.
Prostate Cancer
Prostate cancer is acknowledged at the present time to
be the second commonest cause of cancer death in
males, with 10,239 patients dying of the disease in
2007 (22) (Fig. 7). However, from the public-health
point of view, these figures are relatively unimpressive. Taken together, Figures 6 and 7 show that,
despite the marked increase (39%) in age standardized
incidence rate recorded between 1995 and 2004 (36),
such deaths only account for approximately 4% (12.6%
of 31%) of male mortality, and hence it is not surprising that health care policy targets excess mortality and
deprivation relating to lung cancer (8%) and cardiovascular/respiratory disease that together presently
account for nearly half (47%) of all male deaths in
England and Wales (21).
CRUK figures (22) show (Fig. 11) that the majority of these deaths occur in men over 65, 85% of cases
being over 70 years old; indeed, recorded death
Figure 11 Quinquennial deaths from prostate cancer in the UK (2007). Of 10,239 deaths, 85% occurred in men over 70 years old.
Source: Data from Ref. 22.
556
George
because of prostate cancer in the over 85 years group

has actually increased in recent years, probably
because of more accurate information on death certificates. Similarly, the median age of death because of
the disease in white Americans is 80 years (46). Ageadjusted mortality increased gradually in both the
United Kingdom and the United States up to the
early 1990s, but then fell in both countries, more so
in the United States by a factor of 4 (47)despite the
dramatic rise in incidence noted above. Hence, in
terms of public and thus political perception and
notwithstanding spirited efforts to raise the disease
profile (48), this image of an elderly disease with a
declining mortality is always likely to suffer compared
to a high impact disease (mortality), particularly in
younger members of the opposite sex.
Leaving politics and vested interest groups to one
side, it can be seen that a critical examination of prostate
cancer in terms of Wilson and Junger criteria raises two
fundamental questions that touch on most aspects of
any screening process. First, is it possible to design and
carry out a randomized controlled trial of screening (see
page 549) with the death rate from prostate cancer as
the primary outcome, and second, for those cases
identified, is there an effective treatment available?
The European Randomised Study of Screening
for Prostate Cancer (ERSCP) was instituted in the
early 1990s to answer the first question by means of

PSA-based screening (49) (Fig. 12A). 182,000 men
were identified in seven different European countries
including a core group of 162,387 men between 55 and
69 years of age. Variations in schedule, screening
interval, PSA cutoff value (50), and other factors
(Fig. 13) occurred between centers (51,52), but interim
analysis of the common core protocol identified 5990
cancers in the screened group and 4307 in the control
group, corresponding to a cumulative incidence of
8.2% and 4.8%, respectively (Fig. 12B).
In an intention to screen analysis after a median
follow-up of 9 years and an average screening interval
of 4 years, the study identified a relative reduction of
20% in the death rate from prostate cancer in men
between 55 and 69 years. It was calculated that to
prevent death, 1410 men would need to be screened
and 48 additional men would have to be treated.
While noting the positive outcome for the trial,
the authors commented on the major issues raised in
terms of overdiagnosis, overtreatment, quality of life,
overall cost, and cost-effectiveness, recalling memories of Schroders own earlier editorial comments on
overtreatment (53).
The Prostate, Lung, Colorectal and Ovarian
(PLCO) screening trial in the United States reported
interim results published in the same issue of the New
Figure 12 (A) Details of the Rotterdam randomized controlled trial, screening acceptance arm. Basic RCT design as illustrated in Figure
5. Source: Data from Ref. 50. (B) Enrolment and outcomes of ERSPC according to age group at randomization. Predefined core group of
162,387 males identified. Source: Data from Ref. 49.
557
Figure 13 Sensitivity, specificity, and cutoff values illustrated by early data obtained from the Rotterdam screening study. The difficulty
of establishing a cutoff between health and disease can be appreciated. Source: Data from Ref. 50.
England Journal (54). In a smaller study of the prostate

arm, 38,343 men were followed for 6 years with
annual PSA screening and a cutoff of 4 ng/mL. At
interim analysis the death rate was very low but did
not differ significantly between the study groups. The
authors considered that this lack of separation might
be due to a number of reasons: the screening protocol
with a 4-ng cutoff might not be effective; the high level
of peer pressure screening in the control group
might dilute any observable effect; pretrial PSA
tests might have already eliminated significant tumors
from the entire group (high background PSA testing
in the United States); and finally, the possibility that
therapeutic improvements during the period of the
study might have altered outcomes in both trial arms.
The simultaneous publication of these two conflicting, large trials (49,54) mandated a period of
reflection for the urological community (55): on one
hand an apparently negative result, on the other an
encouragingly positive outcome in terms of lives
saved yet tinged with the promise of significant overtreatment and questionable cost-effectiveness. More
than a decade of intensive investigation seemed to
have obscured rather than clarified the question as to
whether screening for prostate cancer does more good
than harm.
Originally dissatisfied with prospects for screening
and particularly with a poor evidence base for treatment
modalities, Neal, Donovan and coworkers (56,57)
addressed the 5th postulate relating to acceptable/
ideal treatment for screen-detected disease that, of
course, remained unanswered by the recently
reported studies. As a result of their evidence the
UK Health Technology Assessment Programme
funded another approach that sought to answer the
second question relating to efficacy of treatment. The
ProtecT (prostate testing for cancer and treatment)
feasibility study (58) is a multicenter trial in which
patients with suitable early disease are randomized to

radical surgery, radical radiotherapy, or watchful
waiting (active surveillance). The randomization and
counseling process is reported to take 20 minutes, but
in practice can last for much longer, a significant
problem that has led to reevaluation of the processes
involved in the trial (59). This and other studies of
patient preference (60) should eventually determine
whether screening for this disease will be valuable,
but it is apparent that even if ProtecT identifies an
ideal therapy the high overtreatment rate implied by
the ERSPC will confound the purpose of the public
health exercise.
As has been known for some time, to merely
identify histologic prostate cancer is not in itself an
adequate goal; to be of value the screening process
must be able to select out the minority subset of
disease that carries an excess mortality risk to the
patient within his own lifespan.
Testis Cancer
The rarity of this condition precludes a national
screening effort, but all urologists will be aware of
the importance of self-examination and self-referral in
preventing presentation of patients with advanced
disease. Publicity about self-examination may be likened to that surrounding breast disease, but, clearly,
diagnostic confirmation and treatment are both easier
and cheaper for cases of testis cancer, with the added
benefit of excellent long-term outcome measures.
CONCLUSION
Screening, as noted above, remains a relatively novel
and somewhat unproven mechanism for disease control. With time, the common-sense view that it must
558
George
be good for you appears to become more rather than

less controversial. Recent studies questioning the
value of screening for Downs syndrome (61) and
cervical cancer (62) have generated heated debate
while the latest trend of whole body screening with
computerized tomography (63) (the ideal gift for a
50th birthday) has led to accusations of a screening
industry feeding off simple-minded enthusiasma
charge clearly directed at the medical profession as a
whole. Nevertheless, the issues are and will remain
within the public domain, so the urologist must advise
according to the evidence taking care to ensure that
the patient is not deceived by the apparent simplicity
of the available information.
REFERENCES
1. Bowman JE. Screening new-born infants for Duchenne
muscular dystrophy. BMJ 1993; 306:349.
2. Parsons EP, Clarke AJ, Hood K, et al. Newborn screening
for Duchenne muscular dystrophy: a psycho-social study.
Arch Dis Child Fetal Neonatal 2002; 86:F91F95.
3. Bogart MH, Pandian MR, Jones OW. Abnormal maternal
serum chorionic gonadotrophin levels in pregnancies with
fetal chromosome abnormalities. Prenat Diagn 1987; 7:
623630.
4. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum
screening for Downs syndrome in early pregnancy. BMJ
1988; 297:883887.
5. Rosenberg T, Jacobs HK, Thompson R, et al. Cost effectiveness of neonatal screening for Duchenne muscular
dystrophy: how does this compare to existing neonatal
screening for metabolic disorders? Soc Sci Med 1993;
37:541547.
6. Downing M, Pollitt R. Newborn blood spot screening in
the UKpast, present and future. Ann Clin Biochem 2008;
45:1117.
7. Parsons EP, Clarke AJ, Hood K, et al. Newborn screening
for Duchenne muscular dystrophy: a psycho-social study.
Arch Dis Child Fetal Neonatal 2002; 86:F91F95.
8. Cole P, Morrison AS. Basic issues in cancer screening. In:
Miller AB, ed. UICC Technical Report Series, 40. Geneva:
UICC, 1978:7.
9. Day NE, Walter SD. Simplified models for screening: estimation procedures from mass screening programmes.
Biometrics 1984; 40:17.
10. Hutchison GB, Shapiro S. Lead time gain by diagnostic
screening for breast cancer. J Natl Cancer Inst 1968; 41:
665669.
11. Feinleib M, Zelen M. Some pitfalls in the evaluation of
screening programmes. Arch Environ Health 1969; 19:
412417.
12. Prorok PC, Connor RJ, Baker SG. Statistical considerations
in cancer screening programmes. Urol Clin North Am
1990; 17:699708.
13. Prorok PC, Connor RJ. Screening for the early detection of
cancer. Cancer Invest 1986; 4:225238.
14. Bradley DM, Parsons PE, Clarke AJ. Experience with
screening new-borns for Duchenne muscular dystrophy
in Wales. BMJ 1993; 306:357360.
15. Prorok PC, Chamberlin J, Day NE, et al. UICC workshop
on the evaluation of screening programmes for cancer. Int J
Cancer 1984; 34:14.
16. Ellis WJ, Chetner MP, Preston SD, et al. Diagnosis of
prostatic carcinoma: the yield of serum prostate specific
antigen, digital rectal examination and transrectal
ultrasonography. J Urol 1994; 152:15201525.
17. Deras Il, Aubin SMJ, Blase A, et al. PCA3: a molecular

urine assay for predicting prostate biopsy outcome. J Urol
2008; 179; 15871592.
18. Christopherson WM. The control of cervix cancer. Acta
Cytol 1966; 10:610.
19. Shapiro S. Evidence on screening for breast cancer from a
randomised trial. Cancer 1977; 39:27722782.
20. Brett GZ. The value of lung cancer detection by 6 monthly
chest radiographs. Thorax 1968; 23:414420.
21. Office of National Statistics: data from England and Wales,
2008.
22. Cancerresearch.org/cancerstats. 2007.
23. Smith DS, Carvalhal GF, Mager DE, et al. Use of lower
prostate specific antigen cut-offs for prostate cancer screening in black and white men. J Urol 1998; 160:17341738.
24. Powell IJ, Meyskens FL. AfricanAmerican men and
hereditary/familial prostate cancer: intermediate-risk populations for chemo-prevention trials. Urology 2001; 57:
178181.
25. Catalona WJ, Antenor JA, Roehl KA. Screening for prostate
cancer in high-risk populations. J Urol 2002; 168:19801984.
26. Whitby LG. Screening for disease: definitions and criteria.
Lancet 1974; 2:819821.
27. Miller AB. Screening in cancer: a report of the UICC
workshop in Toronto. UICC Technical Report Series 40.
Geneva: UICC, 1978.
28. Wilson J, Junger G. Principles and practice of screening for
disease. World Health Organization Public Health Paper
No. 34. Geneva: WHO, 1968.
29. Thomas DFM, Whitaker RH. Prenatal diagnosis. In:
Whitaker RH, ed. Current Perspectives in Paediatric Urology.
New York: Springer-Verlag, 1989:4553.
30. Ashleigh DJB, Mostofi FK. Renal agenesis and dysgenesis.
J Urol 1960; 83:211230.
31. Department of Health. Screening for infectious diseases in
pregnancy: standards to support the UK antenatal screening programme. Available at: http://www.dh.gov.uk/
dr_consum_dh/groups/dh_digitalassets/@dh/@en/
documents/digitalasset/dh_4092049.pdf
32. Macleod J, Salisbury C, Low N, et al. Coverage and uptake
of systematic postal screening for genital Chlamydia trachomatis and prevalence of infection in the United Kingdom
general population: cross-sectional study. BMJ 2005;
330:940943.
33. Low N, McCarthy A, Macleod J, et al. Epidemiological,
social, diagnostic and economic evaluation of population
screening for genital chlamydial infection. Health Technol
Assess 2007; 11(8):1165.
34. Nadala EC, Goh BT, Magbanua JP, et al. Performance
evaluation of a new rapid urine test for Chlamydia in
men: prospective cohort study. BMJ 2009; 339: b2655.
35. Mahilum-Tapayl, Laitila V, Wawrzyniak JJ, et al. New
point of care Chlamydia rapid testbridging the gap
between diagnosis and treatment: performance evaluation
study. BMJ 2007; 335:11901194.
36. Cancer Research UK. Cancer Stats London: Incidence,
2008.
37. Cancer Research UK. CancerStats London: Mortality, 2007.
38. Chawla SN, Crispen PL, Hanlon AL, et al. The natural
history of observed enhancing renal masses: meta-analysis
and review of the world literature. J Urol 2006; 175:
425431.
39. Boman H, Hedelin H, Jacobsson S, et al. Newly diagnosed
bladder cancer: the relationship of initial symptoms,
degree of micro haematuria and tumour marker status.
J Urol 2002; 168:19551959.
40. Pfister C, Shautard D, Devonec M, et al. Immunocyt test
improves the diagnostic accuracy of urine cytology: results
of a French multicentre study. J Urol 2003; 169:921924.

41. Parekattil SJ, Fisher HAG, Kogan BA. Neural network
using combined urine nuclear matrix protein-22, monocyte
chemoattractant protein-1 and urinary intercellular adhesion molecule-1 to detect bladder cancer. J Urol 2003;
169:917920.
42. Kipp BR, Halling KC, Campion MB, et al. Assessing the
value of reflex fluorescence in situ hybridisation testing in
the diagnosis of bladder cancer when routine urine cytological examination is equivocal. J Urol 2008; 179:
12961301.
43. van Rhijn BWG, van der Poel HG, van der Kwast TH.
Urine markers for bladder cancer surveillance: a systematic review. Eur Urol 2005; 47:736748.
44. Grossman HB, Soloway M, Messing E, et al. Surveillance
for recurrent bladder cancer using a point of care proteomic assay. JAMA 2006; 295:299305.
45. Konety BR. Molecular markers in bladder cancer: a critical
appraisal. Urol Oncol: seminars, original investigations
2006; 24:326337.
46. Ries LAG, Melbert D, Krapcho M, et al. SEER Cancer
Statistics Review 19752005. Bethesda, MD: National Cancer Institute, 2008.
47. Collin SM, Martin RM, Metcalfe C, et al. Prostate cancer
mortality in the USA and UK in 19752004: an ecological
study. Lancet Oncol 2008; 9:445452.
48. Kirby RS. Prostate cancer diagnosis: strengths and weaknesses of prostate specific antigen and evaluation of prostate cancer gene 3 (PCA3). 2009. Available at: http://
www.prostateuk.org/prostatenews/20090301_article_
pca3_rsk.htm.
49. Schroder FH, Hugossson JH, Roobol MJ, et al. Screening
and prostate cancer mortality in a randomised European
study. N Engl J Med 2009; 360:13201328.
50. Bangma CH, Rietbergen JBW, Schroder FH. Prostate specific antigen as a screening testThe Netherlands experience. Urol Clin North Am 1997; 24:307314.
51. de Koning HJ, Liem MK, Baan CA, et al. Prostate cancer
mortality reduction by screening: power and time frame
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
559
with complete enrolment in the ERSPC trial. Int J Cancer

2002; 98:268273.
Schroder FH, Roobol-Bouts M, Vis AN, et al. Prostate
specific antigen based early detection of prostate cancer
validation of screening without rectal examination. Urology
2001; 57:8390.
Schroder FH. Prostate cancerto screen or not to screen.
BMJ 1993; 306:407408.
Andriole GL, Grubb RL, Buys SS, et al. Mortality results
from a randomised prostate cancer screening trial. N Engl J
Med 2009; 360:13101319.
Barry MJ. Screening for prostate cancerthe controversy
that refuses to die. N Engl J Med 2009; 360:13511354.
Neal DE, Donovan JL. Prostate cancer: to screen or not to
screen? Lancet Oncol 2000; 1:1724.
Neal DE, Leung HY, Powell PH, et al. Unanswered questions in screening for prostate cancer. Eur J Cancer 2000;
36:13161321.
Donovan J, Hamdy F, Neal D, et al. Prostate testing for
cancer and treatment (ProtecT) feasibility study. Health
Technol Assess 2003; 7(14):188.
Donovan JL, Mills N, Smith M, et al. Quality improvement
report: improving design and conduct of randomised trials
by embedding them in qualitative research: ProtectT. BMJ
2002; 325:766770.
Sculpher M, Bryan S, Fry P, et al. Patient preferences for
management of non-metastatic prostate cancer: discrete
choice experiment. BMJ 2004; 328:382386.
Wellesley D, Boyle T, Barber J, et al. Retrospective audit of
different antenatal screening policies for Downs syndrome
in 8 district general hospitals in one health region. BMJ
2002; 325:1519.
Raffle AE, Alden B, Quinn M, et al. Outcomes of screening
to prevent cancer: analysis of cumulative incidence of
cervical abnormality and modeling of cases and death
prevented. BMJ 2003; 326:901904.
Editorial. Screening for cancer with computed tomography. BMJ 2003; 326:894895.
37
Evidence-Based Medicine
Kieran J. OFlynn
Department of Urology, Salford Royal Foundation Trust, Salford, Manchester, U.K.
INTRODUCTION
Evidence-based medicine is broadly defined as using
the best available evidence to guide decision making
in the care of patients (1). It has become a hugely
popular movement in medicine over the past decade
and the term evidence base is frequently used to
inform decision making in other disciplines. The rise
of the evidence-based movement reflects a number of
key changes in society over the past two decades.
First, there is a vast and daily growing medical literature that makes it virtually impossible for a dedicated clinician to keep up-to-date in his or her own
chosen field. Second, with increased investment in
health care in most economies, governments, health
care providers, and the public wish to be assured that
new monies are spent appropriately and not wasted
on treatments, whose efficacy is in doubt. Third, the
global increase in Internet access has meant that information, once limited to academic journals can now be
sourced through a suitable search engine by anyone
with access to a modem.
Sackett, regarded as one of the founding fathers of
the EBM movement, defined evidence-based medicine
as the conscientious, explicit, and judicious use of
current best evidence in making decisions about
the care of individual patients" (2). This definition is
appropriate for urologists as it emphasizes the importance of both diagnostic, clinical expertise and judgment,
which is so important in a craft specialty. Evidencebased medicine is meant to complement, not replace,
clinical judgment tailored to the individual patients,
recognizing that patients will potentially make different
choices, based on their own belief and prejudices, when
presented with unbiased accurate information (3).
EVIDENCE-BASED UROLOGY IN PRACTICE

There are many types of questions that a urologist needs
to ask (or be asked in turn by a patient or relative). These
may include questions concerning etiology, diagnosis,
prognosis, harm, effectiveness, and qualitative outcomes. Different questions require different study
designs. To find out what living with a condition (e.g.,
advanced prostate cancer) is like, a qualitative study
that explores the patient experiences is required. In
contrast, a qualitative study relying only on the subjective
experiences of individuals could be unhelpful when

trying to establish whether an intervention or treatment works. The best design for effectiveness is the
randomized controlled trial (RCT), which is discussed
later in the chapter. A hierarchy of evidence exists
(Fig. 1), published by the Oxford Centre for EvidenceBased Medicine, by which different methods of collecting evidence are graded as to their relative levels of
validity. The design of a study (such as a case report for
an individual patient or a double-blind randomized
control trial) and the end points measured (such as
survival or quality of life) affect the strength of the
evidence. A cross-sectional survey is a useful design to
determine the frequency of a particular condition. However, when determining an accurate prognosis for someone diagnosed with, say, lower urinary tract symptoms,
a cross-sectional survey (that observes people who have
the disease and describes their condition) can give a
biased result. A design more suited for a prognosis in
question is an inception cohorta study that follows up
a recently diagnosed patient and records what happens
to them over an extended period of time. The most up-todate hierarchy of evidence for the five themed areas
(diagnosis, therapy, prognosis, economic, and decision
analysis) can be accessed at www.cebm.net.
It is important to recognize that different questions
require different study designs for critical appraisal;
first, because you need to choose a paper with the
right type of study design for the question that you are
seeking to answer and, second, to realize that different
study designs are prone to different biases. Thus, when
critically appraising a piece of research it is important to
first ask: did the researchers use the right sort of study
design for their questions? It is then necessary to check
that the researchers tried to minimize the biases (i.e.,
threats to internal validity) associated with any particular study design. These differ between studies.
An evidence-based approach can be practiced in
any situation where there is doubt about an aspect of
clinical diagnosis, prognosis, or management. The
process comprises five distinct steps (2):
1.
2.
The information required is converted into an

answerable question.
The literature is searched with maximum efficiency for the best evidence with which to answer
the posed question. In an ideal world, the best
Chapter 37: Evidence-Based Medicine
Levels of
evidence
Therapy/Prevention/Etiology/Harm
1a
Systematic review with homogeneity of RCTs
1b
Individual RCT with narrow confidence interval
1c
All or none*
2a
Systematic review (with homogeneity) of cohort

studies
2b
Individual cohort study (including low quality

RCT; (e.g., follow-up <80%)
3a
Systematic review (with homogeneity) of case

controlled study
3b
Individual case control study
Case series (and poor quality cohort and case

control studies)
Expert opinion without explicit critical appraisal

or based on physiology, bench research or first
principles
*Met when all the patients died before the treatment became
available, but some now survive on it, or when some patients
died before the treatment became available, but now none die on it.
Figure 1 Levels of evidence for therapeutic studies. Source:
Adapted from http://www.cebm.net.
3.
4.
5.
quality evidence would come from properly conducted randomized trials and/or well constructed
meta-analyses.
The evidence obtained from the literature search
is critically appraised for its validity (closeness to
the truth) and usefulness (clinical applicability).
The results of the appraisal are implemented into
clinical practice.
The clinician evaluates his or her own performance.
FINDING THE EVIDENCE

Online Searching
Until recently clinicians depended on the printed word
for their information. Books, including this one, are
likely to be out of date by the time they are printed.
Scanning specialty journals offer only a limited sample
of new developments and is a particular problem for
the inquisitive urologist as the best clinical research is
most likely to be published in journals with a large
impact factor. It is impossible for a clinician to keep up
with all important new developments simply by reading a few journals. A urologist (even if he had the time),
reading most of the high-yield journals (e.g., New
England Journal of Medicine, Lancet, Journal of Urology, European Urology, Urology, British Journal of
Urology International) would still miss some of the
scientific and clinically relevant articles in the specialty.
561
Colleagues may not be available when advice is

required, and the information obtained tends to be
overtly reliant on personal experience, possibly biased,
with anecdote being no substitute for hard data!
Ideally, a busy clinician requires rapid access
(within minutes) so that the information can guide
clinical questions as they arise, and learning is promoted by the answers coming soon after the questions
(just in time learning). Ideally information should
be targeted to a specific clinical question, with potential solutions derived from the best and most current
research information.
The advent of the personal computers and personal digital assistants (PDAs) means that information
is easy to come by and the potential hits are almost
limitless. Clinicians who want to keep up with the
medical literature and source the most relevant information will need to invest time in developing their
electronic searching capabilities and learn to rapidly
appraise the information for its validity. Knowledge
management, the effective and efficient way of finding and organizing the best available information, is
an undisputable core skill for the 21st century clinician. Internet sites and resources that can be used
include the following:
Medline
Medline (Medical Literature Analysis and Retrieval

System Online) is a bibliographic database of life
sciences and biomedical information. It includes bibliographic information on articles from academic journals
covering the entire field of health care. Compiled by the
United States National Library of Medicine, MEDLINE
is freely available on the Internet. The quality of the
information retrieved depends on the search criteria
used, and the occasional user may be frustrated as most
searches will turn up information that is not clinically
relevant and important articles may be missed. Searching may be made more sensitive by using specific
MESH headings, although the process can be tedious.
Online tutorials are available and Medline can be
accessed at www.ncbi.nlm.nih.gov/pubmed/.
Google Scholar
Google Scholar is a freely accessible Web search

engine at www.scholar.google.co.uk that indexes the
full text of scholarly literature across an array of
publishing formats and disciplines. Released in
November 2004, the Google Scholar index includes
most peer-reviewed online journals of the worlds
largest scholarly publishers. A significant problem
with Google Scholar is the secrecy about its coverage.
Some publishers do not allow it to access their journals. Google Scholar does not publish a list of scientific journals crawled, and the frequency of its updates
is unknown. It is therefore impossible to know how
current or exhaustive searches are in Google Scholar.
Clinical Practice Guidelines
When done well, guidelines are comprehensive synthesis of the best available evidence, upon which the
562
OFlynn
guidance is based. In urology, important guidelines

have been produced by the European Urology Association (www.uroweb.org) and the American Urological
Association (www.auanet.org). Standards for the production of guidelines are discussed later and there
remains a great variation in how guidelines meet
these standards. With ever emerging new data, guidelines may quickly become out of date, and it is worthwhile checking when they were last revised.
The Cochrane Collaboration
The Cochrane Collaboration is a group of over 15,000

volunteers in more than 90 countries who review the
effects of health care interventions tested in biomedical
RCTs. It was founded in 1993 by Iain Chambers and
named after Archie Cochrane (19091988), a prominent
epidemiologist with the Medical Research Council in
Cardiff who argued for up to date systematic reviews
of all RCTs of clinical care. This collaboration comprises
an ever expanding international group of clinicians,
epidemiologists, statisticians, and consumers who have
come together to produce systematic reviews, which
are updated each time an important new trial is published. Abstracts of these reviews are made available
on the web at www.cochrane.org and complete reports
are available by subscription.
RSS
RSS (Really Simple Syndication) is a family of web

feed formats used to publish frequently updated works
in a standardized format allowing users to aggregate
information from multiple websites as it is published.
Examples of websites that might be useful to clinicians
are those for news, MEDLINE, AUA, EAU, other
Societies, Journals, and government supporting
agencies. By subscribing to any one of a large number
of software options, clinicians can get timely updates,
tailored to their needs, on a personal desktop computer.
UpToDate#
UpToDate# is an evidence-based, peer-reviewed information resource available via the Web, desktop computer, and PDA. Currently it covers more than 7700
topics in 15 medical specialties (including urology) and
includes more than 80,000 pages of text, graphics, links
to Medline abstracts, more than 260,000 references, and
a drug database. The content is peer reviewed and an
updated version is released every four months.
The National Institute for Clinical Excellence
The National Institute for Clinical Excellence

(accessed at www.nice.org.uk) was established in the
United Kingdom in April 1999. As one of the key
elements of the NHS in England and Wales, NICE
aims to provide patients and the health care industry
with authoritative, robust, and reliable guidance on
best-practice procedures in health care. Appraisal of
new and existing pharmaceutical interventions and
medical technologies is a key function of NICE, with
emphasis on the demonstration of the value that these
products and devices can bring to the NHS on the
basis of proven clinical and cost-effectiveness. NICE is

also involved in the development and implementation
of evidence-based guidelines for appropriate clinical
practice, and in the production of tools that can be
used for clinical audit within the NHS.
Journals of Secondary Publication
These journals make a useful contribution by identifying high-quality research from a number of different
sources. Articles in these publications are screened for
relevance to clinical practice and have passed critical
appraisal filters and are methodologically sound. ACP
Journal Club (www.acpjc.org) is published by the
American College of Physicians/American Society of
Internal Medicine in Annals of Internal Medicine. It
reviews the worlds English-language medical journals
in internal medicine, selects scientifically strong articles
by explicit criteria, and summarizes those meeting
these criteria as structured abstracts with commentary
by an expert, one article per page. Typically, a structured abstract of the paper occupies one page of the
journal along with a commentary from an established
clinician in the field, giving the bottom line. EvidenceBased Medicine, published by the BMJ, performs a similar function and has a greater emphasis on recently
published surgical trials.
APPRAISING THE EVIDENCE

Principles of Critical Appraisal
Urologists should have the ability to do an in-depth
analysis of research articles that are especially important to their practice. The basic elements of critical
reading are centered on three main areas: validity,
results, and clinical relevance.
Internal validity
Internal validity assesses if the results of clinical

research are correct for the patients in the study.
Internal validity is threatened by two processes, bias
and chance. Bias is any systematic error (e.g., in
assembling and allocating participants to comparison
groups, following them up, and measuring outcomes)
that might distort the observed result relative to the
true situation. Chance is a random error, inherent in all
observations. The probability of chance effects can be
minimized by studying a large number of patients and
is described by p values (the probability of a falsepositive result), power (the probability of a false-negative result), and by confidence intervals for the range
that is likely to include the true effect size.
The urologist will need to ask if the results of the
study apply to his patients? This concept of external
validity (generalizability) emphasizes the potential differences between study patients and those seen on a
daily basis in clinical practice. Study patients are typically highly selected. Trials are most commonly published from academic centers, with patients, who fulfill
clearly defined criteria, are rarely elderly, do not have
other coexisting diseases, and are willing to cooperate.
As part of the trial participants are encouraged to be
compliant with the treatment and will be reviewed

frequently by interested enthusiastic clinicians. As a
result treatment effectiveness reported from clinical
studies may be higher than that achieved in later routine clinical. In interpreting research findings, the
reader must make a well-informed judgment about
whether the study results can be applied in his own
practice.
Studies of the care of patients in many settings
have consistently shown a gap between the evidence
base, as derived from carefully performed trials or
observational studies, the recommendations of experts
in clinical practice guidelines, and actual practice (4).
Reasons include a lack of knowledge of research
results, clinician concern about applying the results
of large studies to individual patients, financial constraints, and failure to organize care in a way that
fosters use of evidence.
Assessing a Systematic
Review and Meta-Analysis
Any urologist attempting to keep abreast of current
developments must find ways of dealing with the
huge amount of published literature. One commonly
used solution to this problem is to track down and
appraise a review article. Reviews are frequently written by experts in their field and by virtue of this are
prone to be selective in their appraisal of the current
literature. Reviews may generate incorrect conclusions
and clinical recommendations, potentially delaying
the introduction of efficacious treatment. A systematic
review is an overview of primary studies in which the
authors have systematically searched for, appraised,
and summarized all of the medical literature for a
specific topic. Its goal is to minimize bias, by seeking
out published and unpublished reports in every language. Systematic reviews may include some statistical methods for combining the results of individual
studies. A meta-analysis is a systematic review that
uses quantitative methods to summarize the results.
The rationale for systematic reviews and meta-analysis
is based on a number of premises. Firstly, in some
therapeutic areas (e.g., drug treatment in the management of detrusor overactivity), there exist numerous
trials attempting to answer questions about clinical
efficacy. These trials are frequently carried out in
diverse settings and may show different net effects
or uncertainty (because of small trial size) with some
studies showing little effect (or harm) while others
showing benefit.
Sound methodology is at the heart of systematic
review and meta-analysis. In a systematic review, the
questions to be answered and the methods used must
be clearly stated. Thorough data collection is of vital
importance in the preparation of a systematic review,
whether or not a meta-analysis is a part of the review.
Complete identification of all relevant studies (published or unpublished) is particularly important and
where possible the original patient data should be
reassessed (5). Some studies may not have been published for reasons related to the findings. Authors are
less likely to submit RCTs with negative results for
563
publication and when submitted, trials with negative

results are less likely to be published. A comprehensive review of RCTs relying solely on Medline
searches will omit about half of the available studies.
The reasons for this are multifactorial. Part of this
problem results from inadequate indexing. For example, it was only in 1990 that the term randomized
control trial was introduced as a descriptor term and
in 1991 as a publication type. Further problems arise
in that some authors may not have described their
methodology clearly enough to allow accurate indexing. Retrieval of high-quality studies will depend on
the searching skills of the authors. A rigorous systematic review will include hand-searching journals, conference proceedings, theses, and the databases of
pharmaceutical companies.
Ideally the methods section of a systematic
review should include a section describing the inclusion criteria for papers in the systematic review and
the criteria used for assessing the validity of individual studies. A systematic review in which multiple
independent reviews of individual studies were carried out, with good agreement between the reviewers,
lends credence to the outcome. Where possible the use
of individual patient data (rather than summary data
or published reports) is important as it enables the
reviewer to look in greater detail at combined subgroups form different trials. This was used with great
effect in the meta-analysis of total androgen blockade
(TAB) in advanced prostate cancer where the individual details of 5710 patients were individually assessed
(6). This meta-analysis showed five-year survival rates
22.8% and 26.2% for castration and maximum androgen blockade, a nonsignificant improvement of 3.5%,
leading to a shift in patient management. A good
meta-analysis should allow readers to determine for
themselves whether the decisions taken by the
reviewers were reasonable and the likely impact on
the final estimate of effect size. There are many
checklists for the assessment of the quality of systematic reviews; the QUOROM statement (quality of
reporting of meta-analyses) provides a benchmark
against which a meta-analysis can be assessed (7).
Because of the many ways in which decisions taken
about selection, inclusion and aggregation of data may
affect the main findings, it is usual for authors to carry
out some sensitivity analysis on the meta-analysis. A
proper sensitivity analysis should explore, among
other things, the effect of excluding various categories
of studies; these might include unpublished studies
perhaps because of negative findings, or those of poor
quality. If a sensitivity analysis is not performed, the
reader is left guessing whether the excluded studies
might have led to a different conclusion.
Calculating Effect Sizes

Clinical trials commonly present their results as the
frequency of some outcome (e.g., stone passage in the
intervention groups and the control group). For metaanalysis, these are usually summarized as a ratio of
the frequency of the events in the intervention to that
in the control group. Either the odds ratio or the risk
564
OFlynn
Odds ratio (OR) The ratio of the odds of having the target disorder in the experimental group relative to the odds in favor of having the
target disorder in the control group (in cohort studies or systematic reviews) or the odds in favor of being exposed in subjects with the
target disorder divided by the odds in favor of being exposed in control subjects (without the target disorder). This is expressed as OR
AD/BC
Adverse event
Study Group
Totals
Did occur
Did not occur
New intervention
AB
Standard intervention
CD
Totals
Confidence Intervals (CI) quantifies the uncertainty in measurement. It is usually reported as 95% CI, which is the range of values
within which we can be 95% sure that the true value for the whole population lies. For example, for an NNT of 10 with a 95% CI of
5 and 15, we would have 95% confidence that the true NNT value was between 5 and 15. The width of the confidence interval
is determined by the sample size (larger samples will give more precise results with a narrower CI) and the variability of the
characteristic being measured (between subjects, within subjects, measurement error, etc.)
Homogeneity and heterogeneity Clinical homogeneity means that, in trials included in a review, the participants, interventions and
outcome measures are similar or comparable. Studies are considered statistically homogeneous if their results vary no more than might
be expected by chance. Clinical heterogeneity may arise as trials differ in their patient selection, disease severity, operative management
and duration of follow-up.
Figure 2 Commonly used terminology in meta-analysis. Source: Adapted from http://www.consort-statement.org.
ratio can be used (Fig. 2). Although they are technically different, the odds ratios and relative risks are
usually interpreted in the same way. A ratio of 2
implies that the defined outcome happens about
twice as often in the intervention group as in the
control group; an odds ratio of 0.5 implies around a
50% reduction in the defined event in the treated
group compared with the controls. The findings
from individual studies can be combined using an
appropriate statistical method. Separate methods are
used for combining odds ratios, relative risks, and
other outcome measures such as risk difference or
hazard ratio. The methods use a similar approach in

which the estimate from each study is weighted by the
precision of the estimate.
The results of meta-analysis tend to be presented
graphically in a Forest plot or blobbogram (Fig. 3).
Each trial is represented by a horizontal line, the
length of which represents the uncertainty of the
estimate of the treatment effect in that individual
study (the 95% confidence interval) of the estimate.
The blob in the middle represents the point estimate of the difference between the groups. The vertical line down the middle is the line of no effect,
Figure 3 Forest plot. In a forest plot, each trial is

represented by a horizontal line, the length of which is
the confidence interval for the study. The graph may be
plotted on a natural logarithmic scale when using odds
ratios, so that the confidence intervals are symmetrical
about the means from each study and to ensure undue
emphasis is not given to odds ratios greater than 1
when compared to those less than 1. The area of each
square is proportional to the studys weight in the metaanalysis. A vertical line representing no effect is also
plotted. If the confidence intervals for individual studies
overlap with this line, it demonstrates that at the given
level of confidence their effect sizes do not differ from
no effect for the individual study. The combined measure of effect is commonly plotted as a diamond, the
lateral points of which indicate confidence intervals for
this estimate.
Are the results valid?

Is this a systematic review of randomized trials?
How likely is it that relevant articles were missed?
Were the individual studies assessed for validity?
Were individual patient data (or aggregated data used in the
analysis?
Was the assessment of the results reproducible?
Were the results similar from study to study?
Are the valid results of the systematic review of therapy
important?
Are the results consistent across studies?
What is the magnitude of treatment effect?
How precise is the treatment effect?
Figure 4 Assessing an systematic reviews.
representing a relative risk of 1 (i.e., no difference

between the two groups). If the confidence interval of
the result crosses the vertical line, this means that
either the sample size was too small to detect a
difference between the two groups or there was no
significant difference between the treatments. The
aggregate effect size for certain sub-groupings and
the overall effect size are usually displayed in the
same figure. The tiny diamond below the horizontal
lines represents the pooled data from all the trials, the
lateral points of which indicate confidence intervals
for this estimate.
Systematic reviews have the advantage that they
are generally more reliable and accurate because of
the explicit methods used. Thus, large amounts of
information can be quickly assessed by the discerning
reader. Results of different studies can be formally
compared to see if the findings are consistent, and if
this is not the case, new hypotheses may be generated
about particular subgroups (Fig. 4).
On a cautionary note, the finding of some metaanalysis have later been contradicted by other later
systematic reviews or large RCTs. Eysenck (8) and
others argue that the results of meta-analysis are only
applicable if the data summarized is homogeneous,
that is, treatment, patients, and end points must be
similar or at least comparable. The key difficulty lies
in deciding which trials are combinable. Good metaanalyses will use explicit and objective criteria for
inclusion or rejection of studies on the grounds of
quality. Misleading meta-analysis may arise because
of the existence of publication bias and other biases
that are introduced in the process of finding, selecting,
and combining studies.
A good meta-analysis of badly designed studies
may result in a misleading outcome. Because of the
risk of publication bias, many meta-analyses now
include a failsafe N statistic that calculates the
number of studies with null results that would need
to be added to the meta-analysis in order for an effect
to no longer be reliable (9). Use of a funnel plot may
enable the identification of possible bias (10). Funnel
plots display the included trials in a meta-analysis in a
565
plot of size effect against sample size. As smaller

studies have greater heterogeneity, the expected picture is that of an inverted funnel. If the plot is asymmetrical, this may support the view that some trials
may have been missed in the original analysis.
Assessing a Randomized Controlled Trial

In an oft-quoted editorial in 1995, Richard Horton,
then editor of the Lancet castigated surgery for its
over-reliance on case series in the surgical literature,
with insufficient evidence derived from RCTs (11).
The situation has improved over the past decade, yet
many new surgical advances are not adequately scrutinized under trial conditions.
The purpose of randomization is to avoid selection bias and to generate groups that are comparable to
each other. For this reason, evidence from a randomized control trial is the soundest we can obtain about
causation (whether it concerns etiology, therapeutics,
or the outcome of a new surgical procedure). A number
of criteria must be met for the proper conduct of an
RCT; clinical equipoise (a genuine uncertainty of the
researcher as to whether there is a real difference
between the treatments), no foreknowledge, no bias
in patient management, no bias in the outcome assessment of the patient, and an intention to treat analysis of
the data, with no post randomization exclusions.
CONSORT (Consolidated Standards of Reporting Trials), first published in 1996, describes various
initiatives developed by the group to improve the
quality and standards of RCTs (12). The CONSORT
statement is an evidence-based, minimum set of recommendations for reporting randomized trials. It
offers a standard way for authors to prepare reports
of trial findings, facilitating their complete and transparent reporting, reducing the influence of bias on
their results, and aiding their critical appraisal and
interpretation. The CONSORT Statement comprises a
22-item checklist and a flow diagram, along with some
brief descriptive text (Fig. 5). The checklist items focus
on reporting how the trial was designed, analyzed,
and interpreted; the flow diagram displays the progress of all participants through the trial (Fig. 6).
Considering an evolving document, the CONSORT
Statement is subject to periodic changes as new
evidence emerges. The current definitive version of the
guidance may be accessed at www.consort-statement.
org/. The CONSORT statement is endorsed by most
of the worlds most cited medical journals.
In a properly controlled trial, the decision to enter
a patient is made, in ignorance of which of the trial
treatments the patient will be allocated (13). Known
prognostic factors should be recorded for all patients
before the treatment is allocated. There should be no
bias in patient management, although this can obviously pose significant problems in surgical trials. In an
intention to treat analysis, the randomization not only
decides the allocated treatment, but also how the
patients data will be analyzed (14). If a medical therapy is being compared with surgery, all the surgical
program is included (e.g., delay prior to surgery, death
566
OFlynn
CONSORT Checklist
Items to include when reporting a randomized trial
Item
PAPER SECTION
Description
TITLE & ABSTRACT
How participants were allocated to interventions (e.g., random allocation, randomized, or

randomly assigned).
INTRODUCTION
Scientific background and explanation of rationale.
METHODS Participants
Eligibility criteria for participants and the settings and locations where the data were collected.
Interventions
Precise details of the interventions intended for each group and how and when they were
actually administered.
Objectives
Specific objectives and hypotheses.
Outcomes
Clearly defined primary and secondary outcome measures and, when applicable, any methods
used to enhance the quality of measurements (e.g., multiple observations, training of assessors).
Sample size
How sample size was determined and, when applicable, explanation of any interim analyses
and stopping rules.
Randomization
Sequence generation
Method used to generate the random allocation sequence, including details of any restrictions
(e.g., blocking, stratification)
Randomization Allocation
concealment
Method used to implement the random allocation sequence (e.g., numbered containers or central
telephone), clarifying whether the sequence was concealed until interventions were assigned.
10
Randomization
Implementation
Who generated the allocation sequence, who enrolled participants, and who assigned
participants to their groups.
11
Blinding (masking)
Whether or not participants, those administering the interventions, and those assessing the
outcomes were blinded to group assignment. If done, how the success of blinding was
evaluated.
12
Statistical methods
Statistical methods used to compare groups for primary outcome(s); Methods for additional
analyses, such as subgroup analyses and adjusted analyses.
13
RESULTS
Participant flow
Flow of participants through each stage (a diagram strongly recommended). For each group
report the numbers of participants randomly assigned, receiving intended treatment, completing
the study protocol, and analyzed for the primary outcome. Describe protocol deviations from
study as planned, together with reasons.
14
Recruitment
Dates defining the periods of recruitment and follow-up.
15
Baseline data
Baseline demographic and clinical characteristics of each group.
16
Numbers analyzed
Number of participants (denominator) in each group included in each analysis and whether
the analysis was by intention-to-treat". State the results in absolute numbers when feasible
(e.g., 10/20, not 50%).
17
Outcomes and estimation
For each primary and secondary outcome, a summary of results for each group, and the
estimated effect size and its precision (e.g., 95% confidence interval).
18
Ancillary analyses
Address multiplicity by reporting any other analyses performed, including subgroup analyses
and adjusted analyses, indicating those pre-specified and those exploratory.
19
Adverse events
All important adverse events or side effects in each intervention group.
20
DISCUSSION
Interpretation
Interpretation of the results, taking into account study hypotheses, sources of potential bias or
imprecision and the dangers associated with multiplicity of analyses and outcomes.
21
Generalizability
External validity of the trial findings.
22
Overall evidence
General interpretation of the results in the context of current evidence.
Figure 5 CONSORT checklist. Adapted from www.consort-statement.org.
567
Figure 6 Consort flow diagram.
before surgery), and the data is analyzed irrespective of

whether or not the patient received the prescribed
treatment. In essence by the end of a trial there are
four groups of patients and the trial will compare the
total number assigned to receive one treatment compared with the other.
Although improving, there remains a relative
paucity of high quality RCTs in the surgical literature.
RCTs in surgery pose particular problems. By its very
nature, the practice of surgery is highly technical, and
surgeons invest a great deal of time and effort in
acquiring operative skills and may be reluctant to
trial a new technique that truly assesses its efficacy. In
urological surgery, the uptake of laparoscopic and robotic
surgery (despite the expense) had been enormous,
untroubled by an evidence base attesting to its
efficacy. In most medical trials, all patients receive
standardized treatments (e.g., a fixed dosage of a

drug). The treatment is independent of any particular
skill of the physician. The operating skill of the surgeon and his experience clearly affect the outcome,
and care needs to be taken that surgeons participating
in RCTs are adequately trained during a prerandomization period that should ideally last until the surgeon is fully conversant with the new technique.
The surgeon should have an acceptable rate of postoperative complications from the procedure. It follows
that whenever a new surgical technique, which
requires training, undergoes evaluation, it is unlikely
that any surgeon would be willing to randomize his
first few patients! These precautions, designed to
reduce bias in the surgical trial, may however lead
to a restriction in the number of participating surgeons. Yet, the more surgeons participate in a trial, the
568
OFlynn
more convincing the results, and ideally the trial

should aim at obtaining results that can be generalized. Proper randomization is pivotal in the conduct
and assessment of an RCT as nonrandomized trials
tend to overestimate the effects of treatment.
Clearly, it is ethical to randomize patients in
surgical trials when we are uncertain as to whether a
specific procedure might do more harm than good.
Conversely, patients cannot have their treatment
chosen at random, if either they or their surgeons
are already reasonably certain about what treatment
they prefer. Such an attitude has lead to the widespread acceptance of radical prostatectomy by
patients and doctors as the treatment of choice for
localized prostate cancer in the United States despite
good randomized controlled trial evidence. As a
consequence, the PIVOT trial designed to assess the
efficacy of radical prostatectomy versus watchful
waiting has struggled to enroll participants, since
its inception in 1994 (15).
When surgery is being compared to a nonsurgical treatment it is impossible to blind the carers or
the patients at the time. Unblinded studies overestimate the effects of treatment, and ideally the outcome
assessment should be performed by a clinician who is
unaware of the procedure, and the patient should not
divulge these details to the assessor. An attempt to
recruit a patient into an RCT may dramatically alter
the surgeon-patient relationship. Surgeons with their
extrovert and optimistic approach may have difficulty
explaining to their patients the basis for their uncertainty and the need for a randomized trial. Likewise
patients, used to the benign paternalism of the surgical fraternity, may not readily accept their surgeons
(well founded) clinical uncertainty.
A properly conducted RCT should have an
explicit methodology section, with evidence of credible patient enrolment, clear objectives, and clear outcome measures. It is important that the outcomes
being measured are important to doctors and/or
patients. Large studies with a prestudy calculation of
the required sample size are essential, as small studies
may not have sufficient size to show a true treatment
effect. Studies with large number of dropouts should
probably be treated with circumspection, as their
absence from the analysis my lead to an overestimation of the effect of the treatment. Most of the worlds
leading journals will no longer publish RCTs with less
than 80% follow-up.
A good RCT will have a clear statement adequately describing the statistical procedures used and
conform to the CONSORT guidance. It should be
remembered that a statistical significance of p < 0.05
is only 1:20. Any study where the authors choose a
single positive statistic out of many should probably
be discarded. Treatment effects are frequently
recorded as relative risk reductions (RRRs) (Fig. 7).
However, RRR fail to discriminate huge absolute
treatment effects from small ones. This is because
the RRR discards the underlying susceptibility or
baseline risk of patients entering randomized trials.
With increasing commercial pressures, there is a
pressing need to assess the efficacy of new (and
almost always more expensive) surgical interventions.

Despite its enhanced cost, approximately 35,000
robotic prostatectomies have been performed in the
United States, yet to date there is no RCT attesting to
its efficacy over conventional surgery. The message is
clear. New technology should be carefully assessed
and validated prior to its introduction into health care.
Assessing a Clinical Practice Guideline

Clinical Practice Guidelines
Clinical practice guidelines are documents produced

with the aim of guiding decisions and criteria regarding diagnosis, management, and treatment in specific
areas of health care. In contrast to previous approaches,
which were often based on tradition or authority, modern medical guidelines are based on an examination of
current evidence within the paradigm of evidencebased medicine. They usually include summarized consensus statements, but unlike the latter, they also
address practical issues.
The United States and other countries maintain
medical guideline clearinghouses. In the United
States, the National Guideline Clearinghouse maintains a catalog of high-quality guidelines published by
various organizations. In the United Kingdom, clinical
practice guidelines are published primarily by the
National Institute for Health and Clinical Excellence
(NICE). Guidelines are also produced by the American
Urological Association (AUA) and the European Association of Urology (EAU).
Modern clinical guidelines identify, summarize,
and evaluate the best evidence and most current data
about prevention, diagnosis, prognosis, therapy, risk
benefit, and cost-effectiveness. They should define the
most important questions related to clinical practice
and identify all possible decision options and their
outcomes (Fig. 8). Some guidelines contain decision or
computation algorithms to be followed. Thus, they
integrate the identified decision points and respective
courses of action to the clinical judgment and experience of practitioners. Many guidelines place the treatment alternatives into classes to help providers in
deciding which treatment to use. Additional objectives of clinical guidelines are to standardize medical
care, to raise quality of care, to reduce several kinds of
risk (to the patient, to the health care provider, to
medical insurers, and health plans), and to achieve the
best balance between cost and effectiveness. It has
been demonstrated repeatedly that the use of guidelines by health care providers such as hospitals is an
effective way of improving patient care.
Clinicians concerns about the legal status of
guidelines and potential litigation resulting from noncompliance may be a barrier to their implementation.
In the United Kingdom, mere deviation from a guideline is unlikely to be accepted as evidence of negligence by a court, unless the deviation itself were of a
type that no doctor acting under ordinary skill and
care would make. Doctors cannot be found negligent
simply because they follow a practice that is rejected
by another school of medical thought.
569
Calculating relative risk reduction

Relative risk reduction (RRR) is the proportional reduction in rates of bad outcomes between the experimental and control participants
in a trial, calculated below and accompanied by a 95% confidence interval. The RRR doesnt reflect the risk of the event without therapy
(the CER or baseline risk) and therefore cannot large treatment effects from small ones.
Absolute risk reduction is the absolute difference in rates of outcomes between the experimental and control group in a clinical trial.
ARR retains the underlying susceptibility of patients and provides more complete information than RRR
Number need to treat (NNT) is derived from 1/ARR and is the number of patients that we need to treat with the experimental therapy in
order to prevent one additional bad outcome
Disease
No disease
Total No of patients
Exposed
Number
Not exposed
Number
Experimental event rate (EER) A/A B

Control event rate (CER) C/C D
Relative risk EER/CER
Relative risk reduction EER#CER/CER expressed at a percentage
Absolute risk reduction (ARR) CER # EER
Number needed to treat 1/ARR ($100)
Control %
50
5
0.5
Outcome
Treatment %
40
4
0.4
Relative risk reduction %

20
20
20
Risk reduction
Absolute risk reduction %
10
1
0.1
No needed to treat
10
100
1000
Figure 7 Treatment effects in a randomized control trial.
Are the recommendations of the guideline valid?

Did the developers carry our a reproducible literature review?
Is each of the recommendations tagged by the level of evidence
upon which it is based and linked to a specific citation?
Will the recommendations help me care for my patient?
Is the burden of illness too low to warrant implemenatation?
Are the beliefs of the patient about the value of the intervention
incompatible with the guideline?
Are resources available to implement the guideline?
Figure 8 Assessing a guideline.
In assessing a guideline, it is important to know

the back round of the group who have developed it.
If the guideline has not been developed by a fully
multidisciplinary group that is representative of
those who will be using it, the result may be a lack
of ownership, with limited utilization. Recommendations that do not take due account of the evidence can
potentially result in suboptimal, ineffective, or harmful practice. There is often a paucity of high-quality
scientific information to guide clinical practice, and

the authors of a good guideline will document the
strength of their recommendation using the criteria
espoused by the Centre for Evidence-Based Medicine
(16) (Fig. 1).
Guideline development groups often lack the
time, resources, and skills to gather and assess the
evidence in detail. Value judgments made by a guideline group may be the wrong choice for individual
patients. Recommendations are naturally influenced
by the opinions, clinical experience, and composition
of the guideline group, and if it is not truly multidisciplinary or representative, incorrect conclusions
may be promoted. Patients needs may not be the
only priority in making recommendations; those of
doctors, risk managers or politicians may also be
involved. Conflicting guidelines from different professional bodies can confuse and frustrate patients and
clinicians alike. As of April 2009, the American
Urological Association advises that the prostatespecific antigen (PSA) test should be offered to wellinformed men aged 40 years or older who have a life
expectancy of at least 10 years, a view not currently
supported by the American Cancer Society.
570
OFlynn
In conclusion, the development of good clinical

guidelines should consider all relevant disciplines and
stakeholders as well as local circumstances. Guidelines should be firmly based on reliable evidence
relating to clinical effectiveness and cost-effectiveness,
and any recommendations should be linked to the
evidence, with references and a grading of the supporting evidence. Active educational intervention
should be adopted for the effective dissemination of
the results.
Assessing a Case Series

Many interventions in medicine cannot be evaluated
by a randomized control trial. The development of
surgery as a craft specialty has largely depended on
the reporting of carefully performed observational
studies. Case series abound in the urological literature
and although much derided occupy an important part
of the surgical literature. A case series is a medical
research study that tracks patients with a known
exposure, given similar treatment, or examines their
medical records for exposure and outcome. A case
series can be retrospective or prospective and usually
involves a smaller number of patients than more
powerful case-control studies or RCTs. Case series
may be consecutive or nonconsecutive, depending
on whether all cases presenting to the reporting
authors over a period of time were included, or only
a selection. Accordingly, case series may be confounded by selection bias, which limits statements
on the causality of correlations observed.
There is an important role for a carefully performed observational studies, which may be used to
show clinical uncertainty, to generate a new hypothesis.
Careful observation and reporting of the evaluation of
a new surgical procedure is valid evidence, as it may
reduce the learning curve of other surgeons eager to
improve their skills. Case series have the advantage
that they are cheap, quick and easy to perform. A case
series will frequently provide important preliminary
information, paving the way for a methodologically
sound randomized control trial, where feasible. Some
therapeutic interventions have an impact so large that
observational data alone are sufficient to show it (e.g.,
urinary catheterization for relief of pain associated
with urinary retention). Randomized control trials
are frequently time limited and observational data
provides a realistic means of assessing the long-term
outcome of medical or surgical intervention.
While often thought provoking, particularly
when describing an authors experience with a new
surgical technique, case series are prone to overinterpretation by the authors and provide the weakest
evidence for assessing the efficacy of a treatment as
they are subject to uncontrolled biases, particularly in
patient selection.
The reader of a case series will have to decide
whether the evidence is the strongest that could have
been obtained under the circumstances or if the intervention could have been assessed by means of a
controlled trial.
Assessing a Case-Control Study

In a case-control study, the subjects are selected on the
basis of whether they do or do not have the particular
disease under study. The groups are then compared
with respect to the proportion who have had a history
of exposure or characteristic of interest. If those cases
(patients) who have had an adverse outcome were
more likely to have undergone the treatment, this may
constitute some evidence that the treatment might
cause or precipitate the adverse outcome.
Some of the advantages of case control studies
are that they are easy to carry out and will become
even easier in the future as more and more patient
records are computerized. Case control studies also
provide a feasible method for the evaluation of rare
diseases and for assessing rare and late adverse effects
of drugs. This study design is efficient in both time
and costs relative to other analytical approaches.
For a case control study to provide sound evidence of whether there is a valid statistical link
between an exposure and disease, comparability of
cases and controls is essential. The major issues to be
considered in the design and assessment of a case
control study are the composition of the study group
and the information about exposure and disease. Strict
diagnostic criteria for the disease are mandatory. The
individuals with the condition must be identified.
Commonly, there are two sources, first, patients
treated at a particular hospital (hospital-based case
control study) and second, selecting persons in a
defined population during a given period of time
(population-based case control study). The advantage
of a population-based study is that it avoids bias from
whatever factor led the patient to be treated in a
particular hospital by a particular physician (referral
bias, medical expertise, etc.), and it allows a description of a disease in the population and direct assessment of rates of disease in exposed and nonexposed
individuals.
For the control group, the crucial requirement is
that they are comparable to the target population of
the cases and that any exclusions or restrictions
made in the identification of cases applies equally to
the controls and visa versa (2). Case control studies
cannot provide information about the absolute risk of
an event but only about the relative risk. However,
case control studies can provide useful information
and are useful when the disease outcome is rare or the
duration of follow-up is long.
Assessing an Article on Diagnosis

Making a clinical diagnosis is dependent on history
taking, physical examination, and diagnostic tests.
Urological practice is heavily reliant on diagnostic
tests. We use laboratories with ever increasing frequency to order tests, sometimes inappropriately, so
we should be concerned about their use and utility.
Diagnostic data is used for several different but interrelated reasons. Diagnostic information is necessary to
assess the severity of an illness and to predict the
subsequent clinical course and prognosis of the
Are the results valid?

Was there an independent, blind comparison with the reference
(gold) standard of diagnosis
Was the diagnostic test evaluated in an appropriate spectrum
of patients (like those it would be used in practice)?
Was the reference standard applied irrespective of the diagnostic
test result?
Is this valid evidence important?
Will the result help me in patient care?
Is the diagnostic test available, accurate and precise in my
practice?
Are the results applicable to my patients?
Will the test change my management?
Will my patients be better off because of this test?
Figure 9 Assessing an article on diagnosis.
condition (e.g., Gleeson grade in prostate cancer).

Such information may also help estimate the potential
for response to therapy (hormone manipulation) and
to determine the actual response to therapy.
Appraising an article on diagnosis should aim to
assess the validity and applicability of the test (Fig. 9)
(14). The diagnostic test should in the first instance
have been compared with the gold standard, that is, a
definitive diagnosis attained by biopsy, surgery,
autopsy, or other accepted standard to decide its
validity. The test should be applied to patients
known to have the target disorder and also applied
to a second group of patients known not to have the
target disorder, that is, all patients should have definitive verification of their disease status. The true
situation is given independently by the reference test,
so that the false-negatives and false-positive rates of
the screening test can be obtained (Fig. 10). This can
pose practical difficulties particularly in cancer screening.
Changing the cutoff value for a test (e.g., PSA) will
alter the sensitivity and specificity of the test. It may
Disease present
Disease absent
Test positive
True positive
(TP) a
False positive
(FP) b
Test negative
False negative
(FN) c
True negative
(TN) d
Sensitivity TP/TP FN
Positive predictive value TP/TP FP
Specificity TN/TN FP
Negative predictive value TN/TN FN
Pre-test probability TP FN/ TP FN FP TN
Likelihood ratios for a positive test result (LR )
Sensitivity
a=ac
1#specificity 1#d=bd
Likelihood ratios for a negative test result (LR#)
c=ac
1#sensitivity
Specificity d=bd
Figure 10 Calculating sensitivity, specificity, positive predictive

value, and likelihood ratios.
571
be impossible to say if a cancer is present or not in

those patients who are negative on screening, and
accordingly the false-negative rate for the test cannot
be directly measured. Where this occurs, positive samples may be over-represented in the verified sample
leading to inflated estimates of sensitivity (verification
bias). Some authors may get around this problem by
continuing to follow up the patients for a period of time
to identify cancers that may have been present despite
a negative result on screening. In this kind of assessment using follow-up, estimates of sensitivity will
decrease over time as more cases of cancer are discovered in patients who had negative test results; these are
then counted as false-negative results. Some authors
may make the erroneous assumption that all unverified patients are disease free and in situations where
the proponderance of unverified patients have negative test results, this may results in an inflated estimation of the tests specificity.
The primary determinants of a diagnostic test
benefit are its sensitivity and specificity. Sensitivity
may be defined as the proportion of people with the
target disorder who have a positive test and specificity
as the proportion of people without the target disorder
who have a negative test. Sensitivity and specificity
are characteristics of the test alone and will depend on
the artificial cutoff point that defines a positive and
negative test result (Fig. 11). For example, in screening
tests for prostate cancer, age specific serum PSA levels
are frequently used. Reducing the cutoff level would
increase the tests sensitivity but reduces the tests
specificity. In clinical practice, this will increase the
false test positives, creating anxiety for the patient
(and surgeon) as a work-up is performed to decide if
prostate cancer is present. Tests with high values of
sensitivity may be very useful as a negative result
effectively rule out the diagnosis. Similarly when a
test has a very high specificity, a positive result effectively rules in the diagnosis.
The likelihood ratio (LR) is sometimes cited in
articles on diagnostic tests (Fig. 10). It expresses the
odds that a given finding would occur in a patient
with, as opposed to without, the target disorder or
condition. With the LR above 1, the probability of the
condition or disease being present goes up; when it is
below 1 the probability of the disease being present
goes down and when the LR is 1, the probability is
unchanged. LR can also be calculated for the negative
as well as positive. Likelihood ratios can be used to
support or exclude a diagnosis and can be used to
generate posttest probability (i.e., the proportion of
patients with that particular test result who have the
target disorder). In assessing a diagnostic test, the
clinician must decide if the test is applicable to the
patients clinical situation and if the test result will
alter patient management.
Assessing an Article on Prognosis

Prognosis refers to the possible outcome of the disease
and the frequency with which it is expected to occur
(e.g., death, survival, etc.). Frequently, there are characteristics of the patient or the underlying condition
572
OFlynn
Figure 11 Importance of cutoff values on test performance. As the cutoff value is moved to the left, the true positive rate (sensitivity)
increases, but specificity decreases.
that we use to predict the patients eventual outcome.

Prognostic factors need not cause the outcome but
may be associated with it strongly enough to predict
their development. Ideally, the best study design to
identify the presence of an increased risk associated
with a prognostic factor is a cohort study. In an ideal
cohort study, investigators follow a group of individuals who have not yet had the adverse event and
monitor the number of outcome events over a long
period of time.
In assessing an article about prognosis, it is
important to determine if a well-defined sample of
patients at a similar point in the course of their illness
were included (Fig. 12). There are likely to be systematic differences in the results of population-based and
speciality clinicbased studies on the clinical course
and prognosis of a disease as referral may be influenced by disease severity. A hospitals reputation may
result from its particular expertise in a specialized
area of clinical care. Referral bias results from the
referral of patients with a particular condition to a
Is the evidence about prognosis valid?

Was a defined representative sample of patients assembled at
a common point in the course of their disease?
Was follow-up of study patients sufficiently long and complete?
Were objective outcome criteria applied in a blind fashion?
If subgroups were identified
. Was there adjustment for important prognostic factors?
Is the valid evidence about prognosis important?
How likely are the events over time?
How precise are the prognostic estimates?
Will the results help me care for my patient?
Were the study patients similar to my own?
Will this evidence make a clinically impact on decision about
what to offer or tell our patient?
Figure 12 Assessing an article on prognosis.
>
clinician or tertiary center with acknowledged expertise in the area concerned. This may increase the
likelihood of adverse or nonfavorable outcomes.
Inception cohorts at tertiary care centers yield useful
information to other clinicians who work in such
settings, but it may not be possible to generalize the
results to the wider population. To allow a true
assessment of outcome patients should be at a similar
well-defined point in the course of their disease. The
follow-up period of the study should be sufficiently
long to detect the outcome of interest (e.g., late recurrence of tumor). Under ideal circumstances, the
authors will report on all patients entered into the
study, but in practice this rarely occurs. Patients may
fail to return for follow-up for a wide variety of
reasons (death, ill health, relocation, etc.). The larger
the number of patients whose fate is unknown, the
greater the treat to the studys validity. In general,
fewer than 5% loss probably leads to little bias and
greater than 20% probably threatens the validity of the
study. The lower the risk of a prognostic outcome, the
greater the potential effect of patients who are lost to
follow-up (17).
The examination for important prognostic outcomes should be carried out by clinicians who were
blind to the other features of these patients. This
avoids bias on two counts. Firstly, a clinician who
knows the patient has a prognostic factor may carry
out a more detailed search for the relevant condition
(diagnostic-suspicion bias). Secondly, clinicians (pathologists, radiologists, and surgeons) may have their
judgment influenced by prior knowledge of the case
(expectation bias). Ideally in a published report on
prognosis, the diagnostic suspicion bias will have
been avoided by subjecting all patients to the same
diagnostic studies. In surgical studies on prognosis,
the occurrence of death is frequently a cited outcome.
Judging the cause of death is very prone to error
(especially when based on death certification) and
assigning a cause to death may be subject to both
diagnostic-suspicion and expectation biases.
Articles on prognosis in surgery frequently claim

an altered prognosis for different subgroups. These
groups prognostic factors may be age, gender, disease
grade, TNM stage, or comorbidity. It should be
remembered that these prognostic factors need not
cause the outcome, they may only be associated with
its development strongly enough to predict it. When a
new prognostic factor is identified, there is no
guarantee that it hasnt resulted from a quirk in
its distribution between patients with different prognoses. This initial group is called the training set. In
assessing articles on prognosis, there should be a
statement (in the methods section of a paper) of a
prestudy intention to examine this specific possible
prognostic factor (the test set). If this second independent study confirms the prognostic finding in the test
set, one can feel more confident about the validity of
the evidence. One final point in studying prognosis:
There are very few disorders for which medical interventions do not interfere with studying the natural
history of the condition, and it should be clear from the
publication what interventions the patients underwent.
CONCLUSION
Although evidence-based medicine has become as the
gold standard for clinical practice, there are a number of limitations and criticisms of its use. There are 3
to 5 million new biomedical publications each year,
and it is virtually impossible for a clinician to keep up
to date in his field without a clear strategy to guide his
reading. Large randomized double-blind placebocontrolled trials are expensive, so that funding sources
play a role in what gets investigated. Government
funding will inevitably flow toward high impact medical
interventions, while pharmaceutical companies will
fund studies intended to demonstrate the efficacy
and safety of particular drugs, often avoiding headto-head comparisons, when the outcomes may be in
doubt. Surgical trials are expensive to set up and run
and attract less funding. As a consequence many
newer surgical treatments have established a place
in the urologists practice, with grade 4 evidence at
best. Such is the position of emerging surgical techniques in urology that it might prove impossible to
evaluate them with RCTs.
Evidence-based guidelines do not remove the
problem of extrapolation of results to different populations or longer time frames. Even if several topquality studies are available, questions always remain
about how far, and to which populations, their results
can be applied. Furthermore, skepticism about results
may always be extended to areas not explicitly covered: for example, a drug may influence a secondary
endpoint such as test result (PSA, symptom score,
etc.) without having the power to show that it
decreases overall morbidity or mortality or in a population. The quality of studies performed varies;
certain groups have been historically under-researched
(racial minorities, the elderly and people with many
comorbid diseases). This makes comparison difficult
573
and the clinician may be unsure if the results will

apply to his patient.
It is recognized that not all evidence is made
accessible and that efforts to reduce various forms of
publication and retrieval bias and retrieval bias are
required. Failure to publish negative trials is the most
obvious gap, and moves to register all trials at the
outset, and then to pursue their results, are under
way. In response to the growing body of opinion in
favor of prospective registration of controlled trials,
Current Controlled Trials Ltd.) launched a website
(http://www.controlled-trials.com) in late 1998, aiming to increase the availability, and promote the
exchange, of information about ongoing RCTs worldwide. This project is committed to providing free and
open access to information about ongoing trials. The
rapid development of e-publishing should reduce the
difficulty of accessing a trial that concludes it did not
prove anything new, including its starting hypothesis.
For todays urologist, the laudable aim of making decisions based on evidence may be impaired by
the quality and scope of the published literature.
Tonelli (18) argued that the knowledge gained from
clinical research does not directly answer the primary
clinical question of what is best for the patient at
hand and suggests that evidence-based medicine
should not discount the value of clinical experience.
Although EBM asserts the primacy of meta-analysis
and randomized control trials over other evidence
sources, the astute clinician will balance these factors,
his personal experience and the patients wishes, in
deciding how best to treat the person in front of them.
REFERENCES
1. Evidence-Based Medicine Working Group. Evidence Based
Medicine. A new approach to teaching the practice of
medicine. JAMA 1992; 268:24202425.
2. Straus SE, Richardson WS, Glaziou P, et al. Evidence-based
medicine. How to practice and teach EBM. 3rd ed. Edinburgh: Churchill Livingstone, 2005.
3. Sackett DL, Rosenberg WM, Gray JA, et al. Evidence-based
medicine. What it is and what it isnt. BMJ 1996; 312:71.
4. Smith R. What information do doctors need? BMJ 1996;
313:10621068.
5. Dickersin K. The Existence of Publication Bias and Risk
Factors for Its Occurrence. JAMA 1990; 263(10):13851389.
6. Maximum androgen blockade in advanced prostate cancer:
an overview of the randomised trials. Prostate Cancer Trialists Collaborative Group. Lancet 2000; 355(9214):14911498.
7. Moher D, CookDJ, Eastwood S, et al., for the QUOROM
Group. Improving the quality of reports of meta-analyses
of randomised controlled trials: the QUOROM statement.
Lancet 1999; 354:18961900.
8. Eysenck HJ. Problems with meta-analysis. In: Chalmers I,
Altman DG. Sytematic Reviews. London: BMJ Publishing
Group, 1995:6474.
9. Egger M, Smith GD. Misleading meta-analysis. BMJ 1995;
311(7007):753754.
10. Egger M, Davey Smith G, Scheider M, et al. Bias in metaanalysis detected by a simple graphical test. BMJ 199;
315:629634.
11. Horton R. Surgical Research or comic opera: questions but
few answers. Lancet 1996; 347:984985.
574
OFlynn
12. Moher D, Schulz KF, Altman DG. The CONSORT statement: revised recommendations for improving the quality
of reports of parallel-group randomised trials. Lancet 2001;
357(9263):11911194.
13. Jadad AR, Moore RA, Carroll D, et al. Assessing the quality
of reports of randomized clinical trials: is blinding necessary? Control Clin Trials 1996; 17:112.
14. Sackett DL, Haynes RB, Guyatt GH, et al. Clinical Epidemiology. A Basic Science for Clinical Medicine. Little,
Brown and Company. 2nd ed. 1991;180181.
15. Wilt TJ, Brawer MK, Barry MJ, et al. The Prostate cancer
Intervention Versus Observation Trial:VA/NCI/AHRQ
Cooperative Studies Program #407 (PIVOT): design and
baseline results of a randomized controlled trial comparing

radical prostatectomy to watchful waiting for men with
clinically localized prostate cancer. Contemp Clin Trials
2009; 30(1):8187.
16. Guyatt G, Rennie D, eds. Users Guides to the Medical
Literature. A Manual for Evidence Based Clinical Practice.
Chicago: AMA Press, 2002.
17. Sackett DL, Haynes RB, Guyatt GH, et al. In Clinical Epidemiology, A Basic Science for Clinical Medicine. 2nd ed.,
1991:180181.
18. Tonelli MR. The limits of evidence-based medicine. Respir
Care 2001; 46(12):14351440.
Index
a-1-antichymotrypsin (ACT), 389

Ablatherm1 device (Edap-Technomed), 510
ABP. See Androgen-binding protein (ABP)
Absorption, of electromagnetic radiation, 481
ABU. See Asymptomatic bacteriuria (ABU)
ACE. See Angiotensin-converting enzyme (ACE)
Acetylation, 67
in histone proteins, 336337
Acetylcholine (ACh)
muscarinic mechanisms in, 250
neurotransmitter, 230, 232, 233
in penile erection, 307
in purinergic transmission, 251
release, 255
Acetylcholinesterase (AChE), 252
for cholinergic innervation, 229
AChE. See Acetylcholinesterase (AChE)
Acidosis, consequences of CKD, 217
Acid urine, 170
Acoustic energy
extracorporeal shock wave lithotripsy. See
Extracorporeal shock wave lithotripsy
(ESWL)
principles of, 485
ultrasonic probe for stone fragmentation, 488
ACP Journal Club, 562
Acquired immune deficiency syndrome (AIDS)
hypogonadotrophic hypogonadism and, 289
Acquired resistance, 134
ACR. See American College of Radiologists (ACR)
ACT. a-1-antichymotrypsin (ACT)
Action potential (AP)
in detrusor smooth muscle, 256257
Actrapid, potassium levels and, 205
Acute allograft rejection, 55
Acute inflammation. See Inflammation
Acute kidney injury (AKI)
ATN and, 206208
cause of, 203, 204
complications of, 205206
bleeding tendency, 205206
fluid overload, 205
hyperkalemia, 205
infection, 206
diagnosis of, 203205
blood tests for, 203204
imaging for, 204205
renal biopsy for, 205
urine testing for, 204
dialysis, 206
management of, 205
RIFLE criteria for, 203, 204
survival rate of, 203
symptoms, 203
Acute myeloid leukemia, 432
Acute-on-chronic renal failure, 212
Acute phase response (APR)
to urological surgery, 525
Acute promyelocytic leukemia (APML), 433
Acute renal failure (ARF), 87. See also Acute
kidney injury (AKI)
Acute tubular necrosis (ATN)
clinical course of, 206, 207
histological sequence of, 206
pathogenesis of, 206208
processes in, 206
recovery from, 208
renal hemodynamics, alterations in, 207208
tubular function alterations in, 208
urine testing and, 204
Acute urethral syndrome, 147
Acute urinary retention
BPH and, 327328
Adams, James, 84
Adaptive immunity, 4852
B-cell activation, 5152
CD4 T cells, 5051
CD8 T cells, 51
complement activation, classical
pathway of, 52
cytotoxicity of, 52
humoral immunity, 5152
immunoglobulins, 52
MHC, 49
opsonization, 52
T-cell, 4849
thymic tolerance, 48
Adenine, 2
Adenocarcinoma. See Prostate cancer
Adenosine, in contrast nephropathy, 207
Adenosine receptors, 253
Adenosine triphosphate (ATP), 3
excitatory neurotransmission and, 232
purinergic receptor and, 233
in purinergic transmission, 251252
release, 254255
ADH. See Antidiuretic hormone (ADH)
Adherens junctions, 10
Adhesins
with pathogenic strains of Escherichia coli, 137
types of, 136137
afimbrial adhesions, 136
fimbriae, 136137
Helicobacter pylori, 136
Adhesion, altered cell
in metastasis, 349350
Adhesion molecules, 5255
Adhesion theory, clinical aspect of, 143144
Adjuvant radiotherapy, 421. See also
Radiotherapy
ADPKD. See Autosomal dominant polycystic
kidney disease (ADPKD)
Adrenergic mechanism, 252
Adulthood
androgen deficiency in, 287
Adult renal epithelial neoplasms
classification of, 360
Heidelberg, 360, 361
WHO, 360, 361
clinical and histopathological features of
benign renal tumors, 361362
malignant tumors, 362, 364
histological appearances of, 363
macroscopic appearances of, 363
molecular genetics of, 364369
Adult(s)
GCT in, 407
obstruction, 104
Adventitial layer
of urinary bladder and urethra
inferior hypogastric wing and, 221
presacral fascia and, 221
puboprostatic ligaments, 222
pubourethral ligaments, 222, 225
vaginal wall and, anterior, 222
Adynamic bone disease, 218
AESOP. See Automated Endoscopic System for
Optimal Positioning (AESOP)
a-fetoprotein (AFP)
testis cancer and, 405
Afferent impulse reflexes, 241
Afimbrial adhesions, 136
AFP. See a-Fetoprotein (AFP)
Afterload, 186190
factors affecting in, 189, 190
management of, 194195
a-GAL-1-4-b-GAL, 140
Aging
BPH and, 324
hypogonadism and, 289
Agonal respiration, 193
AIGF. See Androgen-inducible growth factor
(AIGF)
AII. See Angiotensin II (AII)
AIS. See Androgen insensitivity syndrome (AIS)
AJCC. See American Joint Committee on Cancer
(AJCC)
AKI. See Acute kidney injury (AKI)
ALA. See Amino levulinic acid (ALA)
5-ALA. See 5-aminolevulinic acid (5-ALA)
Albumin
testosterone and, 303
Aldosterone
in sodium excretion, 69
Alfuzosin, for BPH, 328
Allantois, 444
Allergy
urological surgery and, 537
Allopurinol, 170
a-2-macroglobulin (AMG), 389
Ambulatory urodynamics, 275
American College of Radiologists (ACR), 86
American Joint Committee on Cancer
(AJCC), 360
American Society for Therapeutic Radiology and
Oncology (ASTRO) criteria, 510, 513
American Urological Association (AUA),
535, 568
AMG. See a-2-macroglobulin (AMG)
AMH. See Anti-Mullerian hormone (AMH)
Amici prism, 492
Amino acids reabsorption
in proximal tubule, 65
Amino levulinic acid (ALA), 509
use of, 515
5-aminolevulinic acid (5-ALA), 498
AML. See Angiomyolipoma (AML)
AMPdependent protein kinase (AMPK), 197
AMPK. See AMPdependent protein kinase
(AMPK)
Ancillary instrumentation, for ureteroscopy,
499502
guidewires, 500, 501
instrument channels, 499500
retrieval devices, 500501
shape memory alloy, 501502
trocar system, 502, 503
Androgen. See also Testosterone
deficiency, 287
synthetic, elective AR modulators and, 296
Androgen-binding protein (ABP)
on Sertoli cells, 311, 312
testosterone levels and, 312
Androgen-inducible growth factor (AIGF), 356
Androgen insensitivity syndrome (AIS), 291
Androgen receptor (AR)
functional organization of, 393
and prostate cancer, 393394
structure and function of, 285286
synthetic androgens and, 296
Anemia, consequences of CKD, 217218
Anerobic threshold (AT), 529
and mortality in elderly patients, 530
Angiogenesis, 346
in bladder TCC, 382
prostate cancer, 395
Angiomyolipoma (AML), 368
Angiotensin-converting enzyme (ACE), 70
inhibitors, 214215
576
Index
Angiotensin II (AII), 62
in ATN, 207
Angiotensin II receptor blockers (ARB)
candesartan, 215
in hypertension, 214
losartan, 215
in proteinuria, 215
Anterior fibromuscular stroma, of prostate, 317
Anthracyclines, 431
Anti-angiogenesis agents
for systemic anticancer treatment, 434435
Anticodon, 30
Antidiuretic hormone (ADH), 60
urine, concentration and dilution of, 7980
Antidiuretic hormone receptors
Antigen-presenting cells (APC), 42
Antimicrobial resistance, 134135
alteration of binding site, 135
altered permeability, 135
enzyme inactivation, 135
types of, 134
Anti-Mullerian hormone (AMH), 280
AP. See Action potential (AP)
APC. See Antigen-presenting cells (APC)
Apical cell layer, of urothelium, 244
Apical membrane, 63
APML. See Acute promyelocytic leukemia
(APML)
Apoptosis, 24, 198
in bladder TCC, 382
in BPH, 325
in cancer cell, 340
NF-kB in, 337
p53 gene and, 358
Apoptosis-regulating genes, in prostate cancer
apoptosis, 394
Bcl-2, 394395
p53, 395
Applied physics
electromagnetic energy, 478479
electrosurgical energy, 474475
extracorporeal shock wave lithotripsy, 485486
lasers, 482
APR. See Acute phase response (APR)
Aquaporins, 80
AR. See Androgen receptor (AR)
ARB. See Angiotensin II receptor blockers (ARB)
ARF. See Acute renal failure (ARF)
Arginine-glycine-aspartic acid motifs, 107
Arginine vasopressin (AVP), 109
Argon-enhanced electrosurgery, 476
ASAP. See Atypical small acinar proliferation
(ASAP)
Assisted reproductive technology, 295
Asymptomatic bacteriuria (ABU), 141142
AT. See Anerobic threshold (AT)
ATN. See Acute tubular necrosis (ATN)
ATP. See Adenosine triphosphate (ATP)
ATP-gated K channels (KATP), in detrusor
smooth muscle, 257
ATP-mediated innervation, 232
Atropine, frequency-response curve and, 231232
Attachment and colonization, 350
Attitudes, to screening, 545
Atypical small acinar proliferation (ASAP), 389
AUA. See American Urological Association (AUA)
AUG start codon, 31
Automated Endoscopic System for Optimal
Positioning (AESOP), 502
Autonomous hypothesis, 254
Autophagy, 198
Autosomal dominant polycystic kidney disease
(ADPKD), 458
AVP. See Arginine vasopressin (AVP)
Azoospermia, 432
obstructive, 313
Bacteraemia, 156
Bacterial infections, in urothelium, 244246
Bacterial killing, 3738
Bacterial translocation, 192
Bacteriocins, 131
Bacteriuria, 149150
BADS. See British Association of Day Surgery

(BADS)
Balanced rearrangements, of chromosomes, 335
Barrier functions, of urothelium, 244246
Bartters syndrome, 71
Base plate theory, 240
Basic fibroblast growth factor (bFGF), 319,
325326, 434
Baskets, 500501
Basolateral membrane, 63
B-cell activation
in adaptive immunity, 5152
BCG treatment
of bladder cancer, 5556
Bcl 2, antiapoptotic protein, 340
Bcl-2 gene
prostate cancer and, 394395
Benign prostatic hyperplasia (BPH), 387
clinical diagnosis of, 327
detrusor instability in, 327
epithelial and glandular development in,
324325
etiology, 324
growth factors in, 325326
outflow obstruction and, 323324
pathogenesis, 324328
patients, PSA ratio in, 389
PS and, 325
risk factors, 327
stromal epithelial ratio in, 325
symptoms, 323
treatment, 327, 328
urodynamic aspects, 328331
urogenital sinus mesenchyme, 325
Benign renal tumors, 361362
bFGF. See Basic fibroblast growth factor (bFGF)
BHD syndrome. See Birt-Hogg-Dube (BHD)
syndrome
Biofilm. See Glycocalix
Bipolar electrosurgery, 475
Bipolar resection, 476477
Birt-Hogg-Dube (BHD) syndrome, 368
Bladder, urinary
afferent innervation of, 233235
base, 257258
biomechanical properties of
collagen and extracellular matrix, 247248
passive and active, 246
passive forces and bladder compliance,
246247
stress relaxation, 247
blood flow
contractions and, 249
detrusor layers of, 246
embryonic development, 443444
neck, open, 239240
obstructive disorders, nomogram for, 274
physics of
storage phase, 266268
voiding phase, 268269
pressure, during filling, 237238
reconstruction, bowel and, 467468
sensation from, 233234
spinal cord, pathways of, 234
spontaneous activity of, 253254
storage properties of, 244245
wall tension
and intraluminal pressure, 246
Bladder cancer, 555. See also Transitional cell
carcinomas (TCC), of bladder
BCG treatment of, 5556
chemical cause for, 343, 344
etiology, 374375
occupations associated with, 343, 344
overview, 374
radical radiotherapy for, 424
smoking and, 374
Bladder compliance (C), 237
passive forces and, 246247
Bladder epithelial grafts, 464
Bladder filling-voiding cycle, 237
Bladder neck obstruction, 114
Bladder outflow obstruction, 328329
Bladder output relation, 329
Bladder surface mucin, 132133

Bladder TCC. See Transitional cell carcinomas
(TCC), of bladder
Bladder tumors
fluorescent cystoscopy and, 498
Bladder wall
effect of hypertrophy, 116
in high-pressure retention, 115116
Bleeding tendency, AKI and, 205206
BLEND waveform, 476
Bleomycin, 403
Blood tests, for AKI, 203204
BMP. See Bone morphogenetic protein (BMP)
BMP-7. See Bone-morphogenic protein-7 (BMP-7)
Bone morphogenetic protein (BMP)
Bone-morphogenic protein-7 (BMP-7), 110
Bovie knife, surgical, 474
Bowel
for bladder reconstruction, 467468
incorporation of, in urinary tract
effects of, 468470
Bowmans space, 60
BOXIT trial, 383
Bozzini, Phillip, 492
BPH. See Benign prostatic hyperplasia (BPH)
Brachytherapy, 391, 425
advantage of, 415
highdose rate 192Ir, 426
isotopes used in, 416
permanent iodine seed, 425426
planning principles for, 425
Bradykinin, 63
Brain control
lower urinary tract function and. See Cerebral
control, of voiding
Brain death
transplantation and, 454
BrdU. See Bromodeoxyuridine (BrdU)
British Association of Day Surgery (BADS), 527
Bromodeoxyuridine (BrdU), 397
Brown-Buerger cystoscope, 493
Brown-Wickham perfusion technique, 271
Buccal mucosa grafts, 464465
subdermal plexus of, 466
Bulb. See Lamps
Cable properties on smooth muscle fibers, 230
Ca2 channels, in detrusor smooth muscle, 257
Cadaveric donor, 457
Cadherin molecules, 10
Cadherins, 381382, 397
CAG. See Polymorphic polyglutamine (CAG)
Cajal-like cells, 105
CAKUT. See Congenital anomalies of the kidney
and urinary tract (CAKUT)
Calcitonin generelated peptide (CGRP),
contractile function and, 253
Calcium, renal handling of, 7677
Calcium oxalate, 164
Calcium resonium, potassium levels and, 205
Calcium stone disease, 173174
effect of bad Western diet, 174
epidemiological factors in, 176
medical conditions and, 174
risk factor model of, 174, 177
Cambrian explosion, 197
Cameras, for endoscopy, 497498
cAMP. See Cyclic adenosine-monophosphate
(cAMP)
Cancer, 156157
causes of, 344
cell cycle regulation changes in, 339340
cells
apoptosis in, 340
splicing in, alternative, 337
cellular characteristics of, 346
chemical carcinogenesis, 343
genes, 345
genetic changes in
chromosomal rearrangements, 335
DNA polymorphisms, 335
gene copy number changes, 336
LOH and, 336
Index
[Cancer]
genetic polymorphisms, 343346
immunotherapy, 55
incidence and death, 344
multistep process, 345
peptide growth factors in, 338399
prostate. See Prostate cancer
protein translation, changes in, 338
radiotherapy and. See Radiotherapy
renal cell, 346348
signal transduction changes in, 339
systemic therapy for, 428435
testis. See Testis cancer
transcription factors in, 337
Cancer Research UK (CRUK), 555
Cancer-specific survival (CSS), 506
Candesartan, for proteinuria, 215
Candida albicans, 146
Carbohydrates, 3
Carbon dioxide (CO2) laser, 482, 485
Carbonization, of tissues, 479
Carboplatin, 404
Cardiac output
factors affecting in, 191
Cardial contractility
factors affecting in, 189
Cardiogenic shocks, 183
Cardiopulmonary assessment
and urological surgery
data derived from tests, 529530
hematological disorders, 533534
immunological disorders, 534
liver disorders, 533
metabolic disorders, 532
microbiological assessment and antibiotic
prophylaxis, 534
musculoskeletal disorders, 533
nutritional status, 534535
renal disorders, 531532
respiratory disorders, 530531
Cardiovascular assessment
and urological surgery
cardiomyopathy, 528
congestive cardiac failure, 528
hypertension, 528529
implantable converter defibrillator, 528
laparoscopy in cardiac disease, 529
myocardial infarction, 528
pacemakers, 528
valvular heart disease, 528
Cardiovascular complications
of CKD, 219
Cardiovascular disease
renal transplantation and, 458
risk factors for, 458
Cardiovascular system
Case finding, 543
Castration-resistant prostate cancer
treatment of, 392
Catecholamines
CCD. See Charge coupling device (CCD);
Cortical collecting duct (CCD)
CCRCC. See Clear cell renal cell carcinoma
(CCRCC)
CDC. See Collecting duct carcinoma (CDC)
CD44 gene
cell adhesion and, 397
CD4 T cells, 5051
differentiation of, 51
presentation of antigen, 50
CD8 T cells
presentation of, 51
Cell
adhesion, 397
biology of
cellular behaviors, 710
molecules of life, 13
overview, 1
respond to environment, 37
growth, 8
membrane, 11
[Cell]
proliferation, 78
structure of, 1116
cell membrane, 11
endoplasmic reticulum, 1112
Golgi apparatus, 12
mitochondria, 1214
mitochondrial DNA, 1416
survival curve, 418, 419, 421
Cell cycle, 2124, 429
control of cell division, 2123
growth factors and, 2324
regulation changes in cancer, 339340
Cell death, 9
programmed, 24
Cell division, 1920
control of, 2123
growth factors and, 2324
Cell-matrix contact, 11
Cellular behaviors
cell death, 9
cell growth, 8
differentiation of, 910
migration, 10
proliferation, 78
Cellular immunity
Central nervous system
Central venous pressure (CVP), 185
Central zone, of prostate, 317
C-erbB-2, 356
Cerebral control, of voiding, 235236
Cerebral palsy
paracentral lobule in, 236
CFU. See Colony-forming units (CFU)
cGMP. See Cyclic guanosine monophosphate
(cGMP)
CGRP. See Calcitonin generelated peptide
(CGRP)
Charge coupling device (CCD), 492, 494
cameras, in endoscopy, 497498
high-definition imaging systems and, 498
Chemical carcinogenesis, 343
Chemokines, 39, 40
in leukocytes trafficking, 47
in RLR, 4548
stimulated leukocyte transendothelial
migration, 47
Chemotherapy
early toxicities of, 431
GCT and, 409410
mechanisms of action of, 429, 430
mechanisms of resistance to, 431
during pregnancy, 432
single agent cyclophosphamide, 430
Chest radiography, for AKI, 204205
Child-Pugh score, 533
Chinese hamster cells
survival of, 419
Chlamydia trachomatis, 554
diagnosis of, 154
Cholinergic innervation
of urethral smooth
muscle, 229230
Choriocarcinomas, 428
Chromatin packing, 19
Chromophobe RCC, 364
Chromosomal rearrangements
genetic changes in cancer and, 335
Chromosomes
translocation between, 351, 352
Chronic disease, screening for
case finding, 543
mass screening, 542
selective screening, 542
Chronic kidney disease (CKD)
cardiovascular complications of, 219
clinical approach to
acute vs. chronic, 212
complications, 212213
reversible factors, 212
underlying diagnosis, 212
clinical presentation, 211212
577
[Chronic kidney disease (CKD)]

consequences of
acidosis, 217
anemia, 217218
hyperkalemia, 216217
renal bone disease, 218
salt and water, 216
SHPT, 218
sodium, 216
uremia, 217
vitamin D deficiency, 218219
water, 216
diseases associated with, 211
epidemiology, 210211
etiology, 211
pathophysiology
dyslipidemia, 216
glomerulosclerosis, 213
hyperglycemia, 215
hypertension, 214
obesity, 215
proteinuria, 215
smoking, 216
tubulointerstitial scarring, 213
progression of
mechanisms, 212
risk factors, 214
slowing, 213
RRT in, 219
staging of, 210, 211
Chronic renal failure. See Chronic kidney disease
(CKD)
Cinacalcet, 219
Circulating tumor cells (CTC), 389390
Citric acid cycle
in mitochondria, 12, 15
CK. See Creatinine kinase (CK) levels
CKD. See Chronic kidney disease (CKD)
Class I major histocompatibility (MHC) antigens, 45
crystal structure of, 49
Clear cell renal cell carcinoma (CCRCC), 362
Clinical target volume (CTV), 422
Clostridium difficile, 146
CLR. See C-type lectin receptors (CLR)
CM. See Contrast media (CM)
CMOS. See Complementary metal oxide
semiconductors (CMOS)
CMV. See Cytomegalovirus (CMV)
CNS. See Congenital nephrotic syndrome (CNS)
CO2. See Carbon dioxide (CO2)
COAG waveform, 476
Cobalt machines, 415
Cochrane Collaboration, 562
Colicin V, 145
Colicky flank pain, 103
Collagen
and extracellular matrix, 247
Collecting duct
function of, 109
potassium transport in, 7274
sodium reabsorption in, 72
Collecting duct carcinoma (CDC), 364
Collins, Treacher, 365
Colonizing microorganisms, in health, 130131
Colony-forming units (CFU), 147
Color Doppler, 94
Commensal organisms
in lower urinary tract, 132
properties of, 131132
Commercialization
of transplantation, 455
Complement activation, classical pathway of
Complementary metal oxide semiconductors
(CMOS), 494, 495
Complement system, 39
Complete AIS, 291
Compton effect, 417
Computed tomography (CT), 8891
germ cell tumor and, 409
Hounsfield value, 90
maximum-intensity projections, 91
minimum-intensity projections, 91
578
Index
[Computed tomography (CT)]

multidetector, 91
multiphase imaging, 92
multiplanar reformats, 91
principles of, 89
spiral, 91
volume rendering, 9192
Condensing lens
as light sources in imaging, 497
Congenital anomalies of the kidney and urinary
tract (CAKUT), 443
Congenital anorchism, 291
Congenital nephrotic syndrome (CNS), 61
Connexin (Cx) expression, in human
detrusor, 254
Consolidated Standards of Reporting Trials
(CONSORT), 565
checklist, 566
flow diagram, 567
CONSORT. See Consolidated Standards of
Reporting Trials (CONSORT)
Continence
prostate role in, 320321
Continuous flow resectoscope, 495
Continuous venovenous haemofiltration
(CVVH), 206
Contractile proteins, 250
Contractile state, 249
Contractions
and bladder blood flow, 249
isometric
force-velocity relationships and, 248249
length-tension relationships and, 248
Contrast media (CM), 83, 86
adverse effects of, 8687
current recommendations, 8687
in gadolinium chelates, 99
MRI specific, 9899
nephropathy, 87
Contrast resolution, 83
Conventional RCC (CRCC), 362
familial, 364365
feature of, 367
Cortical collecting duct (CCD), 59
Corticotrophin-releasing hormone (CRH), 280
Costimulation, 5455
Cotransporters, 64
CpG islands. See Cytosine-Guanine rich DNA
sequence (CpG islands)
CRCC. See Conventional RCC (CRCC)
Creatinine kinase (CK) levels, 203
Creutzfeldt-Jacob disease, 457
CRH. See Corticotrophin-releasing hormone (CRH)
Cross-sectional imaging, 8895
color Doppler, 94
computed tomography, 8891
Doppler effect, 9394
duplex Doppler, 94
spectral Doppler, 94
therapeutic ultrasound, 95
ultrasound, 9293
contrast media, 9495
Cruising, 272
CRUK. See Cancer Research UK (CRUK)
Cryomedical Sciences (Maryland, U.S.), 519
Cryotherapy, 507
for prostate cancer, 392
background, 509
clinical series, 509510, 511
devices for, 509
percutaneous prostate cryoablation, 509
for renal tumors, 519
Cryptorchidism, 291, 300, 452
syndromes associated with, 301
Crystallization, 167
Crystalluria, 167
CSS. See Cancer-specific survival (CSS)
CT. See Computed tomography (CT)
CTV. See Clinical target volume (CTV)
C-type lectin receptors (CLR), 43
Current density, 476
Cutoff point
selection of, 546
CUT waveform, 475476
CVP. See Central venous pressure (CVP)

CVVH. See Continuous venovenous haemofiltration (CVVH)
Cx. See Connexin (Cx) expression
Cyanobacteria, 197
Cyclic adenosine-monophosphate
(cAMP), 109
Cyclic guanosine monophosphate (cGMP), 70
testosterone therapy and, 295
Cyclophosphamide
chemotherapy, 430
CYP. See Cytochrome P450 system (CYP)
CYP2D6
in cancer, 344
Cystine stone formation, 167169
Cystinuria, 65, 167
Cystitis
in females, 150151
Pap III/Forsmann antigen mechanisms, 144
Cystometry, 270271
filling, interpretation of urodynamics
and, 272273
flowmetry and, voiding, 274275
Cystoscope
cross-section of flexible, 494
rod lens, 493494
universal, 493
Cytochrome P450 system (CYP)
in cancer, 343344
Cytokines, 39
role in inflammatory response to urological
surgery, 524
Cytomegalovirus (CMV), 460
Cytopathic dysoxia, 192
Cytosine, 2
Cytosine-guanine rich DNA sequence
(CpG islands), 336
Cytotoxicity
Cytotoxic necrotizing factor 1, 135
Czerny, Vincenz, 474
dArsonoval, Jacques-Arsene, 474
DaVinci system
disadvantages of, 503
Davison, Charles, 365
DAX-1 gene, in hypogonadotrophic hypogonadism, 289
Day-case surgery, risk assessment for, 526527
DBD. See DNA-binding domains (DBD)
DCCT. See Diabetes Control and Complications
Trial (DCCT)
DCE MRI. See Dynamic contrastenhanced MRI
(DCE MRI)
Death-inducting signal complex (DISC), 340
Deep X-ray therapy (DXT), 415
Degranulation
in phagocytosis, 37
Dehydration, 169
Dendritic cells, 45
Dentin matrix protein-1 (DMP1), 78
Deoxyribonucleic acid (DNA), 23
arrangement of, 1618
double helix structure, 17, 25
epigenetic changes in, 336337
interact with histone acetylases, 20
modification of alter transcription, 29
polymerase, 18
polymorphisms, 335
replication, 18
structure of, 2426
triplet code for, 25
Dermis
deeper reticular, 464
papillary, 464
Desmosomes, 10
Detectable preclinical phase (DPCP), 543544
Detrusor, 222, 226
contractile function
modulators of, 252253
instability, in BPH, 327, 329330
layers, passive and active properties of, 246
[Detrusor]
smooth muscle
cellular activation and propagation, 256257
contractile proteins and intracellular Ca2
in, 249250
detrusor activation and muscarinic mechanisms in, 250251
electrical activity and action potentials,
256257
ion channels, 257
neurotransmitters in neuromuscular junction, metabolism of, 252
purinergic transmission in, 251252
Detumescence
neurotransmitters in, 307309
phases of, 306
DGC. See Dorsal gray commissure (DGC)
DHT. See Dihydrotestosterone (DHT)
Diabetes, 211, 215
Diabetes Control and Complications Trial
(DCCT), 215
Diabetes mellitus, 460
Diagnostic ultrasound, 93
Dialysis, in AKI, 206
Diarrhea
bladder reconstruction and, 467468
Diastolic velocity
erection, phases of, 305306
Diathermy, 474
Diclofenac
preventing colic, 105
DICOM. See Digital Imaging and Communications in Medicine (DICOM)
Dielectric property of tissues, 479
Diffusion-weighted imaging, 101
Digital Imaging and Communications in
Medicine (DICOM), 86
Digitalization, of endoscopy, 494495
Digital radiography (DR), 86
Digital rectal examination (DRE), 388
Dihydrotestosterone (DHT)
in testosterone
lack of, 286
metabolism, 284284
1, 25-Dihydroxycholecalciferol deficiency.
See Vitamin D deficiency
3-D imaging systems. See Three-dimensional
(3-D) imaging systems
Dipstick testing
in AKI, 204
in CKD, 212
DISC. See Death-inducting signal complex (DISC)
Disease control
screening and, 549551
Distal convoluted tubule
potassium transport in, 7274
Distal sphincter mechanism. See Urethral
sphincter mechanism
Distal tubular acidification, 109
Distal tubular hydrogen ion secretion, 7475
Distal tubule
calcium intake, 77
function of, 60
magnesium intake, 77
Distributive shocks, 183
Ditiazem
for proteinuria, 215
Diuretics
antihypertensive and, 214
DMP1. See Dentin matrix protein-1 (DMP1)
DNA. See Deoxyribonucleic acid (DNA)
DNA-binding domains (DBD), 5, 286
DNA repair genes
mismatch repair, 346
nucleotide excision repair, 346
Docetaxel, 392
Doderleins bacilli, 132
Dopamine, 195
in AKI, 205
Dopaminergic hypothalamic neurons, in
erection, 307
Doppler, Christian, 93
Doppler effect, 9394
Index
Dorsal gray commissure (DGC), 236
Downs syndrome, 440
DPCP. See Detectable preclinical phase (DPCP)
D-penicillamine, 169
DR. See Digital radiography (DR)
Dr adhesin family, 143
Drainage
experimental protocol for, 124
gravitational theory of, 124126
DRE. See Digital rectal examination (DRE)
Drug intolerance
Drug resistance, in tumor, 429, 430431
Drugs and risk factor of bladder TCC, 374
Duchennes muscular dystrophy, 545, 552
Duplex Doppler, 94
DXT. See Deep X-ray therapy (DXT)
Dynamic contrastenhanced MRI (DCE MRI),
99100
Dyslipidemia, CKD and, 216
EAU. See European Association of Urology (EAU)
E-cadherin
in cell-cell adhesion, 349350
E-cadherin gene
cell adhesion and, 397
ECF. See Extracellular fluid volume (ECF);
Intracellular fluid (ECF)
Echocardiography, 194
ECM. See Extracellular matrix (ECM)
ED. See Erectile dysfunction (ED)
Edison, Thomas, 492
Efferent limb
EFP. See End fill pressure (EFP)
EGF. See Epidermal growth factor (EGF)
EGFR. See Epidermal growth factor receptor
(EGFR)
eGFR. See Estimated GFR (eGFR)
EGFR, ErbB2 (HER2), 381
eIF2a. See Eukaryotic translation initiation factor
2a (eIF2a)
eIF4e, in translational regulation, 338
Einstein, Albert, 481
Ejaculation, 313314, 321
prostatic secretion, 321323
Elastography, 95
Elderly patients
AT and mortality in, 530
Electricity, 474
properties of, 475
Electrohydraulic lithotripters, 487
Electromagnetic lithotripters, 487
Electromagnetic radiation (EMR), 415416, 477
applied physics, 478479
lasers. See Lasers
radio frequency ablation of renal tumors,
480481
microwave bladder therapies, 481
microwave prostatic therapies, 480481
radio waves and microwaves, 479
tissue interactions, 479
transurethral needle ablation of the prostate,
479480
wave-particle duality, 478
Electromagnetic spectrum, 478
illustration of, 416
Electromechanical coupling, 230
Electrosurgical energy sources, in urology,
474477
principles of, 474477
delivery systems and clinical applications,
476477
safety, 477
Electrosurgical generators, 474475
Embryogenesis, 438439
E-cadherin gene and, 397
Embryology
chromosomal abnormalities, 439440
clinical considerations, 444
congenital anomalies of the kidney and
urinary tract (CAKUT), 443
[Embryology]
early development of, 438439, 440441
fertilization, 438439
genital tracts. See Genital tracts, embryonic
development
implantation, 440441
of lower urinary tract, 443444
overview, 438
of upper urinary tract, 441443
Embryonic disc, 440
Embryonic reawakening, 325
Emission, 321323
EMR. See Electromagnetic radiation (EMR)
ENaC. See Epithelial Na channel (ENaC)
blocker; Epithelial sodium channel
(ENaC)
Enanthate and cypionate testosterone, 293294
End fill pressure (EFP), 117
Endocare (California, U.S.), 519
Endocrine shock, 182
Endocrinology
of prostate, 317318
EndoEye (Olympus) laparoscopes, 496
Endogenous mediators, of inflammation,
3940
Endoplasmic reticulum (ER), 1112, 196
Endoscopes
fiber-optic, 494
flexible, 495
rigid, 495
Endoscopy
digitalization of, 494495
fiber-optic endoscopes, 494
flexible instruments, 495
history of, 492493
imaging systems in, 496499
camera systems, 497498
fluorescent cystoscopy, 498499
high-definition video technology, 498
light sources, 496497
narrow band imaging, 499
three-dimensional, 499
laparoscopes, 496
nephroscopes, 496
resectoscope, 495
rigid instruments, 495
rod lens cystoscope, 493494
ureteroscopes, 495496
virtual. See Virtual endoscopy (VE)
Endothelial NO synthase (eNOS)
Endothelin, contractile function and, 253
Endothelin-1, ischemic injury and, 207
End-stage kidney disease (ESKD), 362
End-stage renal failure (ESRF)
causes of, 211
diabetes and, 215
incidence of, 210, 211
prevalence of, 211
progression of, 214
proteinuria and, 215
smoking and, 216
End void pressure, measurement of, 116117
Energy depletion, 197198
eNOS. See Endothelial NO synthase (eNOS)
EORTC. See European Organisation for Research
and Treatment of Cancer (EORTC)
Epidermal growth factor (EGF), 7, 23, 110, 354
in cancer, 338339
family, 319, 325
Epidermal growth factor receptor (EGFR), 5, 354,
356, 434
mechanisms of resistance to, 435
Epinephrine, 195
Epipodophyllotoxins, 431
Epithelial layer
of urinary bladder and urethra, 226228
Epithelial Na channel (ENaC) blocker, of apical
membrane, 245
Epithelial sodium channel (ENaC), 72
Epithelial stress response, 107
EPO. See Erythropoietin (EPO)
ER. See Endoplasmic reticulum (ER)
579
Erectile dysfunction (ED)

drugs responsible for, 309
pathophysiology of, 309
PDE 5 inhibitors, 310
Peyronies disease and, 311
pharmacological treatment of, 309310
ERK. See Extracellular related kinase (ERK) pathways
ERSCP. See European Randomised Study of
Screening for Prostate Cancer (ERSCP)
Erythrocyte sedimentation rate (ESR)
in vasculitis, 204
Erythropoietin (EPO), peptide hormone
in anemia, 217
Escherichia coli strain, 130
expressing K-88 surface antigen, 141
E-selectin, 36
ESKD. See End-stage kidney disease (ESKD)
ESR. See Erythrocyte sedimentation rate (ESR)
ESRF. See End-stage renal failure (ESRF)
Estimated GFR (eGFR)
CKD staging and, 210, 211
Estrogen
urethral closure pressure and, 259
ESUR. See European Society of Urogenital
Radiology (ESUR)
ESWL. See Extracorporeal shock wave lithotripsy
(ESWL)
Ethical considerations
to screening, 545
ETS family, transcription factors, 337
Eukaryotic cells, 3
Eukaryotic translation initiation factor 2a
(eIF2a), 197
European Association of Urology (EAU), 568
European ESPRC study, 507
European Organisation for Research and Treatment of Cancer (EORTC), 378, 432
European Randomised Study of Screening for
Prostate Cancer (ERSCP), 390, 556
European Society of Urogenital Radiology
(ESUR), 87
Evidence
appraising
article on diagnosis, 570571
article on prognosis, 571573
case-control study, 570
case series, 570
clinical practice guidelines, 568570
effect sizes calculation, 563565
internal validity assessment, 562563
randomized controlled trials, 565568
systematic review and meta-analysis,
assessment of, 563
online searching
clinical practice guidelines, 561562
Cochrane Collaboration, 562
Google Scholar, 561
journals of secondary publication, 562
National Institute for Clinical
Excellence, 562
RSS (Really Simple Syndication), 562
UpToDate#, 562
Evidence-based medicine, 562
defined, 560
publications, 562
in urology, questions related to, 560
Excretory function
measured by gamma camera renography,
123124
Exons
mRNA processing, 2829
Exponential cell growth, 428, 429
External balance, 57
External magnet, effect of, 97
Extracellular fluid volume (ECF), 6770
Extracellular matrix (ECM), 107
barriers, degradation of, 348
collagen and, 247247
Extracellular related kinase (ERK) pathways, 339
Extracorporeal shock wave lithotripsy (ESWL),
104, 162, 474, 485487
580
Index
[Extracorporeal shock wave lithotripsy (ESWL)]

design of, 486488
effects of, 488
stone fragmentation, 486
Exudation, 3435
Familial breast cancer
Familial CRCC, 364365, 366
Familial hyperparathyroidism-jaw tumor
syndrome, 368
Faraday, Michael, 478
FAST. See Fast Alcohol Screening Test (FAST)
Fast Alcohol Screening Test (FAST), 536
Fast neurotransmitters, 230
FDA. See Food and Drug Administration (FDA)
Female genital tract
embryonic development
external genitalia, 447, 449
internal genitalia, 446447
timescale of, 446
FeNa. See Fractional excretion of
sodium (FeNa)
Fertile eunuch syndrome, 289
Fertility
ejaculation, 313314
male infertility, 312313
semen analysis, 314
spermatogenesis, 311312
spermatozoa, 312
sperm maturation, 313
testicle, development of, 311312
Fertilization, 438439
Fetal stage
androgen deficiency in, 287
FGF. See Fibroblast growth factor (FGF)
FGFR3 gene, 380381
FH. See Fumarate hydratase (FH)
Fiber-optic endoscopes, 493
Fibroblast growth factor (FGF), 23, 356
Fibroblast growth factors (FGF) family, 319
in BPH, 325326
in cancer, 338
Fibroblasts
wound-healing process and, 463
Fill-void observations, 117118
Fimbriae, 136137
type I, 137
type P, 138139
Fimbrinated status, historical relevance of, 140142
First desire to void (FDV), sensation
markers, 273
First sensation (FS), sensation markers, 273
Flaps, skin, 465467
axial island, 466
fasciocutaneous, 467
intestinal, in bladder reconstruction, 467468
myocutaneous, 466467
peninsular, 466
random, 466
Flexible ureterorenoscopy (FURS), 162
Flow cytometry, 459
Flowmetry, 271272
urine flow, measuring, 271
and voiding cystometry, 274275
Fluid overload, AKI and, 205
Fluorescence cystoscopy, 383, 498499
FMet-Leu-Phe. See Formylated methionineleucyl-phenylalanine (FMet-Leu-Phe)
Focal ablation
of unilateral/bilateral disease, 518
Focal therapy
Follicle-stimulating hormone (FSH), 279
HPT axis and, 302
regulation and action of, 283
Food and Drug Administration (FDA), 488
Force-velocity relationships
and isometric contractions, 248249
Forest plot, 564
Formation product, 165
Formylated methionine-leucyl-phenylalanine
(FMet-Leu-Phe), 39
Formylated peptides, 39
Fowlers syndrome, 259
FPL. See Functional profile length (FPL)
Fractional cell kill, 428
Fractional excretion of sodium (FeNa), 204
Frequency-response curve
atropine and, 231232
tetrodotoxin and, 231
Frontal lobe, voiding and, 236
FSH. See Follicle-stimulating hormone (FSH)
Full-thickness skin graft, 464
Fumarate hydratase (FH), 368
Functional profile length (FPL), 273
Furosemide, in AKI, 205
FURS. See Flexible ureterorenoscopy (FURS)
Gadolinium chelates
in contrast media, 99
GAG. See Glycosaminoglycan (GAG)
Galactosemia, 542
Galil Medical (Yokneam, Israel), 519
Gamete intrafallopian transfer (GIFT), 452
Gametogenesis, 447
Gamma camera renography
in excretory function, 124125
Gap junctions, 10
Gardinerella vaginalis, 146
G1 Arrest
p53 gene and, 358
Gas embolism
Gas lock, 538
Gastrointestinal ischemia, 191
Gating mechanism, in urinary bladder, 238
G-CSF. See Granulocyte colony-stimulating
factors (G-CSF)
GCT. See Germ cell tumor (GCT)
GDEPT. See Gene-directed enzyme prodrug
therapy (GDEPT)
GDNF. See Glial cell line-derived neurotrophic
factor (GDNF)
Gene copy number changes, in cancer, 336
Gene-directed enzyme prodrug therapy
(GDEPT), 393
Gene methylation, RASS1FA, 336
Genes
apoptosis-regulating, in prostate cancer
apoptosis, 394
Bcl-2, 394395
p53, 395
associated with chemoresistance, 359
in genetic mechanisms, 26
Gene therapy
Genetic abnormalities, and male infertility, 313
Genetic anticipation, 346
Genetic polymorphisms, in cancer, 343346
Genetics mechanism, 2630
control of transcription, 29
exons, 2829
genes, 26
introns, 2829
modification of DNA, 29
mRNA synthesis, 2628
posttranscriptional modifications in mRNA
stability, 2930
ribosomal RNA, 30
shRNA, 30
Genital tracts, embryonic development
abnormalities, 450452
female
external genitalia, 447, 449
male
external genitalia, 450
testicular descent, 450
Germ cell tumor (GCT)
in adults, 407
categories of, 407
chemosensitivity and chemoresistance of,
basis for, 409410
[Germ cell tumor (GCT)]

CT and, 409
histological classification of, 408409
in infants, 407
intratubular germ cell neoplasia, 406407, 408
prognostic information, 409
staging of, 409
testicular. See Testicular germ cell tumor
(TGCT)
GFR. See Glomerular filtration rate (GFR);
Glomerular function rate (GFR)
Gibbs-Donnan effect, 60
GIFT. See Gamete intrafallopian transfer (GIFT)
Gitelmans syndrome, 71
Glasgow scale, 193
Gleason grading system, of prostate cancer,
387388
Glial cell line-derived neurotrophic factor
(GDNF), 443
Glomerular capillary wall, 6061
filtration barrier, location of, 61
structure of, 6061
Glomerular filtration rate (GFR), 57, 6063
AKI and, 203, 205
calcium intake, 76
dynamics of, 6163
glomerular capillary wall, 6061
serum creatinine and, 210
Glomerular function rate (GFR), 457
Glomerulonephritis, CKD and, 211
Glomerulosclerosis, CKD and, 213
Glomerulotubular balance (GTB), 6667
Glomerulus, function of, 5960
Glucose reabsorption
Glutathione transferases (GSTM1 and GSTp)
in cancer, 344
Glycation, 248
Glycocalix, 136
Glycolysis, 12
Glycosaminoglycan (GAG), 132
layer, permeability barrier and, 245
Glycosuria
in hyperglycemia, 64
Glycosylation
of proteins, 12
GnRH. See Gonadotropin-releasing hormone
(GnRH)
Golgi apparatus, 12
Gompertzian growth, 428
Gonadotropin-releasing hormone (GnRH), 279
HPT axis and, 302
KS and, 287289
regulation and action of, 280, 282
CRH, 280
GPR54, 280
KiSS1/GPR54 expression, 280
leptin, 280
therapy in hypogonadotrophic
hypogonadism, 295
Goodpastures disease, 204, 205
Google Scholar, 561
GPR54. See G proteincoupled receptor 54 (GPR54)
G proteincoupled receptor 54 (GPR54), 280
G proteins
heterotrimeric, 351, 353
monomeric, 351
Grafts, 464465
bladder epithelial, 464
buccal mucosa, 464465
full-thickness skin, 464
postauricular full-thickness skin, 465
split-thickness skin, 464
Granulocyte colony-stimulating factors
(G-CSF), 431
Granulocytes, 35
Graspers, 500501
Gravitational theory
of drainage, 124126
hypothesis for open hydroureter, 126
Gray (Gy), 417
Grayscale ultrasound, 93. See also Ultrasound
Gross tumor volume (GTV), 422
Index
Growth factors
in BPH, 325326
peptide in cancer, 338339
in prostate, 319320, 396397
GSTM1 gene, 374
GSTPI gene methylation, 336
GTB. See Glomerulotubular balance (GTB)
GTV. See Gross tumor volume (GTV)
Guanine, 2
Guidewires, 500, 501
Gy. See Gray (Gy)
Halogen bulbs, 497
Harmonic Scalpel, 488
HAT. See Histone acetyl transferases (HAT)
hCG. See Human chorionic gonadotropin (hCG)
HDAC. See Histone deacetylases (HDAC)
HD imaging systems. See High-definition (HD)
imaging systems
Head-mounted display (HMD), 499
for 3-D imaging, 399, 400
Heart rate, 190191
Heat filter
Heat shock elements (HSE), 196
Heat shock factors (HSF), 196
Heat shock proteins (HSP), 195
in tubular regeneration, 208
HeLa. See HSE in human cells (HeLa)
Helicobacter pylori, 136
Helix-loop-helix proteins, 29
Helix-turn-helix proteins, 29
Hematological disorders, 533534
Hemiablation, 518
Hemidesmosomes, 10
Hemoglobin, 483
Hemolytic uremic syndrome (HUS), blood test
and, 203
HenleJGA, 67
Hepatocyte growth factor (HGF), 110
Hereditary leiomyomatosis renal cell carcinoma
(HLRCC), 368
Hereditary nonpolyposis colon cancer
(HNPCC), 346
Hereditary papillary renal carcinoma
(HPRC), 367
HER2neu antigen. See Human epidermal growth
factor receptor 2 (HER2neu) antigen
Her 2 receptor, in cancer, 338339
Heterochromatin, 29
Heterotrimeric G proteins, 351, 353
HFEA. See Human Fertilisation and Embryology
Authority (HFEA)
HGF. See Hepatocyte growth factor (HGF)
HGPIN. See High-grade prostatic intraepithelial
neoplasia (HGPIN)
HIF. See Hypoxia-inducible factors (HIF)
HIF 1. See Hypoxia-induced factor-1 (HIF 1)
HIFU. See High-intensity focused ultrasound
(HIFU)
High-definition (HD) imaging systems, 498
CCD chips and, 498
vs. standard-definition systems, 498
High-grade prostatic intraepithelial neoplasia
(HGPIN), 395
High-intensity focused ultrasound (HIFU), 95,
485, 507508
in prostate cancer, 392
clinical series, 512515
devices for, 510, 512
High mobility group (HMG) protein, 410
High-pressure bladders, radiologic assessment
of, 118120
High-pressure chronic retention, 120123
Hinge area, of urothelium, 244
Histone acetylation
DNA, epigenetic changes in, 336337
Histone acetyl transferases (HAT), 336
Histone deacetylases (HDAC), 337
Histone proteins, 18
HLA. See Human leukocyte antigen (HLA)
HLRCC. See Hereditary leiomyomatosis renal
cell carcinoma (HLRCC)
HMD. See Head-mounted display (HMD)
HMG protein. See High mobility group (HMG)

protein
HMRS. See Hydrogen magnetic resonance
spectroscopy (HMRS)
HNPCC. See Hereditary nonpolyposis colon
cancer (HNPCC)
Holmium (Ho):YAG laser, 484
Homeostasis, 5759
concept of external balance, 57
concept of renal clearance, 5859
Hooke, Robert, 478
Hopkins, Harold, 492
Hormonal manipulation, of prostate cancer, 391
Hormones
AMH, 280
CRH, 280
EPO, peptide, 217
FSH. See Follicle-stimulating hormone (FSH)
GnRH. See Gonadotropin-releasing hormone
(GnRH)
leptin, 280
LH. See Luteinizing hormone (LH)
Hounsfield, Sir Godfrey, 88
Hounsfield value, 90
Ho:YAG laser. See Holmium (Ho):YAG laser
HPRC. See Hereditary papillary renal carcinoma
(HPRC)
HPT. See Hypothalamic-pituitary-testicular
(HPT) axis
HPV. See Human papilloma viruses (HPV)
HSE. See Heat shock elements (HSE)
HSE in human cells (HeLa), 196
HSF. See Heat shock factors (HSF)
HSP. See Heat shock proteins (HSP)
HTA. See Human Tissue Authority (HTA)
Huggins, Charles, 391
Human chorionic gonadotropin (hCG)
for induction of spermatogenesis, 294295
in testosterone deficiency, 294
in vanishing testes syndrome, 291
Human embryo
early development of, 438439
fertilization of, 438439
research, 453
Human epidermal growth factor receptor 2
(HER2neu) antigen, 434
Human Fertilisation and Embryology Authority
(HFEA), 438
Human leukocyte antigen (HLA), 456
Human papilloma viruses (HPV), 156157
Human Tissue Authority (HTA), 455
Humoral immunity, 5152
HUS. See Hemolytic uremic syndrome (HUS)
Huygens, Christian, 478
Hydration, states of, 121
Hydrogen ion excretion, 7476
distal tubular hydrogen ion secretion, 7475
intracellular fluid buffers, 74
nonvolatile acids, 74
reabsorption of filtered bicarbonate, 74
urinary buffers, 7576
volatile acids, 74
Hydrogen magnetic resonance spectroscopy
(HMRS), 101
Hydronephrosis, 120123
Hydroureter, 120123
Hypercalciuria, 103
Hyperchloraemic acidosis, 468469
Hypercholesterolemia, 460
Hyperfiltration, 213
Hyperglycemia, 215
Hypergonadotrophic hypogonadism
acquired causes of, 291
congenital causes of, 289290
Hyperkalemia
in AKI, 205
in CKD, consequences of, 216217
Hyperlipidemia, 216
Hypernephroma, 362
Hypertension, 213, 214
Hyperuricosuria, 169
Hypoalbuminaemia, 66
Hypocalcaemia, 469
581
Hypogonadotrophic hypogonadism
acquired causes of, 289
congenital causes of, 287289
gonadotrophin therapy, 294295
Hypomagnesaemia, 469
Hypotension, 191192
Hypothalamic-pituitary-gonadal axis, 303
Hypothalamic-pituitary-testicular (HPT) axis,
302303
FSH and, 302
GnRH and, 302
key factors controlling, 279
LH and, 302
Hypothalamus, in brain, 236
Hypovolemic shocks, 183
Hypoxia, in renal cell cancer, 348
Hypoxia-induced factor-1 (HIF 1), 197
Hypoxia-inducible factors (HIF), 366
in angiogenesis, 346, 347
protein targets of VHL protein, 347348
role of VHL in, 367
transcription factors, 337
Hypoxic stress, 197198
IC. See Interstitial cells (IC)
ICAM. See Intercellular adhesion molecule (ICAM)
ICNARC. See Intensive Care National Audit and
Research Centre (ICNARC)
ICRU. See International Committee of Radiation
Units and Measurements (ICRU)
ICSI. See Intracytoplasmic sperm injection (ICSI)
ICU. See Intensive care unit (ICU)
Idiopathic hypogonadotrophic hypogonadism
(IHH), 287289
Idiopathic male infertility, 312313
IGCCCG. See International Germ Cell Cancer
Collaborative Group (IGCCCG)
IGF. See Insulin-like growth factor (IGF) family;
Insulin-like growth factors (IGF)
IGF-binding protein (IGFBP), 356, 396
IGFBP. See IGF-binding protein (IGFBP)
IGFR. See IGF receptors (IGFR)
IGF receptors (IGFR), 396
IHH. See Idiopathic hypogonadotrophic
hypogonadism (IHH)
IL-1. See Interleukin-1 (IL-1)
IM. See Internal margin (IM)
Image resolution, 498
Imaging systems, in endoscopy, 496499
fluorescent cystoscopy, 498499
high-definition, 498
light sources, 496497
narrow band imaging, 499
standard-definition, 498
three-dimensional, 499
Imbalanced rearrangements, of chromosomes, 335
Imbibition, 464
Immune modulation, 433
Immunoglobulins, 52
Immunoglobulin superfamily members, 54
Immunological disorders, 533534
Immunology
adaptive immunity, 4852
adhesion molecules, 5255
clinical diagnosis of, 5556
acute allograft rejection, 55
BCG treatment of bladder cancer, 5556
cancer immunotherapy, 55
innate immunity, 4348
organization of, 4243
overview, 42
structure of, 4243
Immunosuppression, 459460
Impaired detrusor contractility, 330
Importin-a, 16
IMRT. See Intensity-modulated radiotherapy
(IMRT)
Index lesion ablation, 518519
Indigo laser, 484
Inertial cavitation, 508
Infants
GCT in, 407
urinary tract infection in, 149
582
Index
Infections, in AKI, 206

Infection stones, 164
formation of, 170173
risk factor model for, 171
treatment of, 171172
Infectious disease, 553554
Inflammation
bacterial killing, 3738
causes of, 34
chemical mediators of, 3941
defects in neutrophil function, 38
endogenous mediators of, 3940
formation of exudates, 3437
leukocyte actions in, 3537
leukocyte activation in, 34
macrophages in, 40
microcirculation in, 34
monocytes in, 40
neutrophil activation, 3537
overview, 34
phagocytosis, 37
Inhibitory interneurons, urinary bladder
pressure and, 238
Innate immunity, 4348
C-type lectin receptors, 43
nod-like receptors, 4445
retinoid acidinducible gene-1 (Rig-1)-like
receptors, 4548
toll-like receptors, 44
Inosculation, 464
Inositol triphosphate (IP3), 353
Inpatient surgery, 527
Insulin-like growth factor (IGF) family, 319
Insulin-like growth factors (IGF), 22, 356357
Integrins, 5354
Intensity-modulated radiotherapy (IMRT),
421, 423
Intensive Care National Audit and Research
Centre (ICNARC), 182
Intensive care unit (ICU), 182
Interactive obstructive uropathy
biochemical studies of, 127129
bladder wall
effect of hypertrophy, 116
in high-pressure retention, 115116
clinical addendum, 129
end void pressure, measurement of, 116117
excretory function measured by gamma
camera renography, 123124
gravitational theory of drainage, 124126
high-pressure bladders, radiologic assessment
of, 118120
high-pressure chronic retention, 120123
historical perspective of, 113115
hydronephrosis, 120123
hydroureter, 120123
micturition cycle, 115
overview, 113
reconstructive surgery, 126127
urodynamic data, summary of, 117118
Intercellular adhesion molecule (ICAM), 36
Interleukin-1 (IL-1), 36, 192
Intermittent hemodialysis, 206
Internal margin (IM), 422
International Committee of Radiation Units and
Measurements (ICRU), 422
International Germ Cell Cancer Collaborative
Group (IGCCCG), 405
scoring system for NSGCT, 406
International Testicular Cancer Linkage
Consortium, 407
Interstitial cells (IC), suburothelium, 255
Interval cancers, 548
Interventional radiology (IR), 88
Intra-abdominal pressure (Pabd), 270
Intracellular fluid buffers, 74
Intracellular fluid (ECF), 57
Intracellular organelles, 11
Intracytoplasmic sperm injection
(ICSI), 295
Intradermal plexus, 464
Intraembryonic mesoderm, 440441
Intraluminal pressure, 246
Intraobstructive tubular dysfunction, 108109

collecting duct function, 109
distal tubular acidification, 109
potassium handling, 108109
sodium handling, 108109
Intratubular germ cell neoplasia (ITGCN),
406407, 408
Intravesical pressure (Pves), 270
Intrinsic resistance, 134
Intrinsic rhabdosphincter, 225226, 227228
somatic innervation of, 229
Introns, 17
mRNA processing, 2829
In vitro fertilization (IVF), 438, 452
Ion channels, in detrusor smooth muscle, 257
Ionizing radiation
defined, 415
electromagnetic, 415416
hazards of, 8788
particulate, 416
radiobiology, 418421
defined, 418
linear quadratic model, 420421
redistribution, 419420
reoxygenation, 420
repair of radiation damaged cells, 418419
repopulation, 420
IP3. See Inositol triphosphate (IP3)
IR. See Interventional radiology (IR)
Irritative symptoms, BPH and, 329, 330
Isodose chart, electron, 423
for 6-MV linear accelerator, 425
Isotopes, 415
used in brachytherapy, 416
ITGCN. See Intratubular germ cell neoplasia
(ITGCN)
IVF. See In vitro fertilization (IVF)
Jejunal conduit syndrome, 468
JGA. See Juxtaglomerular apparatus (JGA)
JNK. See Jun N-terminal kinase (JNK) pathways
Junk DNA, 18
Jun N-terminal kinase (JNK) pathways, 339
Juxtaglomerular apparatus (JGA), 59
KALIG-1 gene, in KS, 288
Kallikrein-Kinin system, 70
Kallmanns syndrome (KS), 282
CAG and, 286
IHH and, 287289
Karyotypes, 440
K capsular antigen, 145
Keratinizing squamous metaplasia (KSM), 378
Keratinocyte growth factor (KGF), 326, 356
Key signaling cascades, 7
KGF. See Keratinocyte growth factor (KGF)
Kidneys
blood-borne infection of, 146
congenital anomalies of, 443
functions of, 216
Kidney transplantation
cardiovascular disease and, 458
contraindications to donation for, 457
live donors and, 455
assessment of, 456457
Kinetic energy, 488490
mechanical lithotripsy, 489490
principles of, 489
KISS1R. See G proteincoupled receptor 54 (GPR54)
Klebsiella pneumoniae, 138
Klinefelters syndrome, 289290
Klotho, 78
Kochs postulates, 143
Krebs cycle, 198
KS. See Kallmanns syndrome (KS)
KSM. See Keratinizing squamous metaplasia (KSM)
b-Lactamase production, 135
Lactate dehydrogenase (LDH)
Lactobacillus acidophilus, 132
Lamps
types of, 496
Lancet, The, 141

LAP. See Left atrial pressure (LAP)
Laparoscopes, 496
Laparoscopic nephrectomy, 527
Laparoscopic partial nephrectomy, 506
Laparoscopy
and respiratory disease, 531
Laplaces law, 246
L-arginine, 110
Lasers
absorption, 481
characteristics of medical, 483
CO2, 485
delivery systems of, 484
design of, 482
holmium:YAG, 484
indigo, 484
invention of, 481
Nd:YAG, 481, 484
photodynamic therapy, 484
population inversion, 481482
properties of, 482
protocols regarding use of, 485
pulsing, 482, 484
spontaneous emission, 481
stimulated emission, 481482
Laurence-Moon-Bardet-Biedls syndrome
(LMBBS), 289
LDH. See Lactate dehydrogenase (LDH)
Lead-time bias, 549
Left atrial pressure (LAP), 193
Left ventricular end-diastolic pressure
(LVEDP), 193
Length bias, 549
Length-tension relationships
and isometric contractions, 248
Leptin, 280
Lesions
putative premalignant, of prostate
atypical small acinar proliferation, 389
prostatic intraepithelial neoplasia,
388389
Leukocytes
actions, 3537
activation, 34
trafficking, 47
Lewis, Bransford, 493
LH. See Luteinizing hormone (LH)
LHRH. See Luteinizing hormone-releasing
hormone (LHRH)
Liddles syndrome, 72
LigaSureTM device, 476
Light delivery, 509
Light guide cables, 497
Light sources, for imaging
condensing lens, 497
heat filter, 497
lamps, 496497
Linear accelerator, 415, 416
isodose curves for 6-MV, 425
Linear quadratic model, 420421
Lipids, 3
Lisinopril, for proteinuria, 215
Lithotomy position
Liver disorders
and assessment, for urological surgery, 533
LMBBS. See Laurence-Moon-Bardet-Biedls
Syndrome (LMBBS)
LMWH. See Low molecular weight heparin
(LMWH)
Local spinal reflex, 241
LOH. See Loss of heterozygosity (LOH)
Loop of Henle, 59, 60
calcium intake, 7677
Losartan, for proteinuria, 215
Loss of heterozygosity (LOH)
genetic changes in cancer and, 336
Low-dose unfractionated heparin (LDUH),
535536
Index
Lower urinary tract (LUT). See also Striated
sphincter; Urethra; Urinary bladder;
Urothelium
active properties of muscles in
contractile state, 249
contraction and bladder blood flow, 249
isometric contractions and length-tension
relationships, 248
isotonic contractions and force-velocity
relationships, 248249
functions of, 246
physics and mechanics of, 266269
pre-ejaculatory fluid in, 321
seminal fluid, 322
structure and function of. See Urinary bladder
and urethra, structure of
techniques in urodynamics for, 269
Lower urinary tract (LUT), host defence
mechanism, 132134
bladder surface mucin, 132133
commensal organisms, 132
genital skin, 132
immune response, 134
mucosal shedding, 134
Tamm-Horsfall protein, 133
urine flow, 132
Low molecular weight heparin (LMWH), 536
L-region. See Pontine storage center (PSC)
LUT. See Lower urinary tract (LUT)
Luteinizing hormone (LH), 279
HPT axis and, 302
regulation and action of, 282283
Luteinizing hormone-releasing hormone
(LHRH), 391392
LVEDP. See Left ventricular end-diastolic
pressure (LVEDP)
Lymphocytes, 463
Lysosomal fusion, 37
Lysozyme, 131
Macrophases, in inflammation, 40
chemical signal in, 4041
Magnesium, renal handling of, 77
Magnetic resonance imaging (MRI), 86, 95101
diffusion-weighted imaging, 101
dynamic contrast-enhanced, 99100
external magnet, effect of, 97
HMRS, 101
improving signal to noise ratio, 98
MR nuclei, 96
principles of, 97
radiofrequency radiation, application of, 98
resonance, 9798
Maiman, Theodore, 481
Major histocompatibility antigens (MHC), 45
crystal structure of, 49
resolution of, 49
Male genital tract
embryonic development
external genitalia, 450
testicular descent, 450
Male genital tract, infections of, 151154
Chlamydia trachomatis, diagnosis of, 154
localization of infection, 152
prostatic fluid, examination of, 152
prostatitis, 151152
antibiotic penetration in, 154
nonbacterial, 152154
Male hypogonadism. See also Hypergonadotrophic
hypogonadism; Hypogonadotrophic
hypogonadism
clinical presentation of, 287
definition of, 279
development of, 287292
etiology of, 287292
testosterone therapy in, 292294
Male infertility
azoospermia, obstructive, 313
genetic abnormalities and, 313
idiopathic, 312313
Sertoli-cell-only syndrome, 313
[Male infertility]
testicular failure
acquired, 313
primary, 313
secondary, 313
testicular maldescent, 313
Mammalian target of rapamycin (mTOR), 338
Mandrel, 500
Mannan-binding lectin (MBL), 43
MAP. See Mean arterial pressure (MAP)
MAPK. See Mitogenic activated protein kinases
(MAPK) pathways
MAP kinase. See Mitogen-activated protein
kinase (MAP kinase)
Mass screening, 542
Matrix metalloproteinases (MMP)
Maximum urethral closure pressure (MUCP), 273
Maximum urethral pressure (MUP), 273, 274
MBL. See Mannan-binding lectin (MBL)
McCarthy, Joseph, 493
MCRCC. See Multilocular cystic renal cell carcinoma (MCRCC)
mdm-2 gene, 358
MDR. See Multidrug resistant gene (MDR)
MDRD. See Modification of diet in renal disease
(MDRD)
MDR-1 gene. See Multidrug resistance (MDR-1)
gene
Mean arterial pressure (MAP), 186
Mechanosensation, in renal tubular cells, 106107
sensing of stretch, 107
Medial preoptic area (MPOA)
in erection, 307
Mediators, medullary vasoconstriction and, 207
Medical Research Council, 391
Medical Therapy of Prostatic Symptoms
(MTOPS), 327
Medline, 561
Megavoltage (MV) range, 415
MELD. See Model for End-Stage Liver Disease
(MELD)
MENT. See 7a-methyl-19-nortestosterone (MENT)
Mesangial cells, 63
Meso-tetra-hydroxyphenyl-chlorin (mTHPC,
Foscan), 515
Metabolic acidosis, 468, 469
Metabolic disorders
assessment and optimization
Metalloproteinases (MMP)
in metastasis, 348
Metanephric adenomas, 361
Metastasis, 348350
adhesion, altered cell, 349350
attachment and colonization, 350
extracellular matrix barriers, degradation of, 348
factors involved in, 349
migration and circulation, 350
proteases secretion, 348, 349
Metchnikoff, E., 35
Methicillin-resistant Staphylococcus aureus
(MRSA), 134, 534
Methotrexate, 470
Methylation, DNA, 336
7a-methyl-19-nortestosterone (MENT), 296
MHC. See Class I major histocompatibility
(MHC) antigens
Microbiological assessment
and urological surgery, 534
Microcirculation
changes in, 34
Microsatellites, 346
Microwaves, 479, 480481
Micturition cycle, 115
Migration, 10
and circulation in metastasis, 350
Minimally invasive surgery, 477
Minimally invasive therapies
cryotherapy. See Cryotherapy
HIFU. See High-intensity focused ultrasound
(HIFU)
PDT. See Photodynamic therapy (PDT)
RFA. See Radiofrequency ablation (RFA)
583
Minimal residual disease (MRD), 432433

MIS. See Mullerian-inhibiting substance (MIS)
Mismatch repair, 346
Mitochondria, 1214
Mitochondrial DNA, 1416
Mitogenic activated protein kinases (MAPK)
pathways, 339, 353
Mitosis, M phase, 1920
Mixed renal osteodystrophy, 218
MLCK. See Myosin light chain kinase (MLCK)
MLCP. See Myosin light chain phosphatase
(MLCP)
MMP. See Metalloproteinases (MMP)
Model for End-Stage Liver Disease (MELD), 533
Modification of diet in renal disease
(MDRD), 210
hypertension and, 214
Molecularly targeted therapies
scheduling of, 435
toxicities of, 435
Molecular profiling
in prostate cancer, 394
Monoclonal antibody therapy, 433434
Monocytes, in inflammation, 40
Monomeric G proteins, 351
Monopolar electrosurgery, 475
Monosomy, 439
Motexafin lutetium (MLu, LuTex), 515
MPOA. See Medial preoptic area (MPOA)
MRD. See Minimal residual disease (MRD)
M2 receptors, muscarinic mechanisms and,
250251
M3 receptors, muscarinic mechanisms and, 250
M-region, 235236
MRI. See Magnetic resonance imaging (MRI)
mRNA
protein translation in cancer and, 338
stability, posttranscriptional modifications
in, 2930
synthesis, 2628
MR nuclei, 96
MRSA. See Methicillin-resistant Staphylococcus
aureus (MRSA)
mTHPC. See Meso-tetra-hydroxyphenyl-chlorin
(mTHPC, Foscan)
MTOPS. See Medical Therapy of Prostatic
Symptoms (MTOPS)
mTOR. See Mammalian target of rapamycin
(mTOR); Rapamycin (mTOR)
Mucoproteinaceous matrix, 164
Mucosal shedding, 134
MUCP. See Maximum urethral closure pressure
(MUCP)
Mullerian-inhibiting substance (MIS), 449
Multidetector CT, 91
Multidrug resistance (MDR-1) gene, 430431
Multidrug resistant gene (MDR), 369
Multilocular cystic renal cell carcinoma
(MCRCC), 362
Multiphase CT imaging, 92
Multiplanar reformats, 91
Multiple organ dysfunction, 198201
molecular pathways mediating, 199
MUP. See Maximum urethral pressure (MUP)
Muscarinic mechanisms
detrusor activation and, 250251
Muscarinic receptors, 233, 250251
Muscular layer, of urinary bladder and urethra
detrusor, 222
levator ani, 224
preprostatic sphincter, 223
smooth muscle bundles, 222, 225
sphincter mechanism, 224
trigonal layer, 222
Musculoskeletal disorders
MV range. See Megavoltage (MV) range
Mycobacterium tuberculosis, 155
Myelodysplasia, 432
Myocardial contractility, 186
Myocardial ischemia, 191
584
Index
Myogenic hypothesis, 254

Myosin light chain kinase (MLCK), in contractile
state, 250
Myosin light chain phosphatase (MLCP), in
contractile state, 250
Myotonic dystrophy, 291
N-acetyltransferase pathway
in cancer, 344345
NADH. See Nicotinamide adenine dinucleotide
(NADH)
NADPH. See Nicotinamide adenine dinucleotide
phosphate (NADPH)
Nagelschmidt, Carl Franz, 474
Na/K ATPase and K channel, of apical
membrane, 245
2-naphthylamine, cancer and, 343
Narrow band imaging (NBI), 499
National Cancer Institute, 432
National Chlamydia Screening Programme
(NCSP), 154, 554
National Electrical Manufacturers Association
(NEMA), 86
National Health and Nutrition Examination
Survey (NHANES III), 210
National Institute for Clinical Excellence, 562
National Institute for Health and Clinical
Excellence (NICE), 568
NAT-1 pathway
in cancer, 344345
NAT-2 pathway
in cancer, 344345
Natriuresis, 68
Natriuretic peptides
Natural killer cells (NK), 45
NBI. See Narrow band imaging (NBI)
NCSP. See National Chlamydia Screening
Programme (NCSP)
Nd:YAG laser. See Neodymium: yttriumaluminium-garnet (Nd:YAG) laser
NEB. See Net external balance (NEB)
Necrosis, 198
Neisseria gonorrheae, 134
NEMA. See National Electrical Manufacturers
Association (NEMA)
Neodymium: yttrium-aluminium-garnet
(Nd:YAG) laser, 481, 484
Neonatal obstruction
effect of nephrogenic stage in, 110
Nephrectomy, 104
Nephrogenesis, 441442
Nephrolithiasis, 104
Nephron, functional segmentation of, 5960
distal tubule, 60
glomerulus, 5960
proximal tubule, 60
Nephroscopes, 496
Net external balance (NEB), 57
Net magnetic vector (NMV), 97
Neuroendocrine cells, 320321
Neurogenic hypothesis, 254
Neurological disorders
Neuronal NO synthase (nNOS)
Neuropeptides, contractile function and, 253
Neurotransmission, excitatory, 232
Neurotransmitters
acetylcholine, 230
in neuromuscular junction, metabolism of, 252
putative, 232
in vesicles, 230232
Neutrophil activation
in inflammation, 3537
Neutrophil function, defects in, 38
Neutrophils, 462
New England Journal, 556557
NFkb. See Nuclear transcription factors (NFkb)
NHANES III. See National Health and Nutrition
Examination Survey (NHANES III)
NICE. See National Institute for Health and
Clinical Excellence (NICE)
Nicotinamide adenine dinucleotide (NADH), 37

Nicotinamide adenine dinucleotide phosphate
(NADPH), 37
Nicotine and nicotinic receptors, 252253
Nicotinic acetylcholine receptor, 230, 233
Nitrergic mechanism, 252
Nitric oxide (NO)
in ATN, 207208
bleeding tendency and, 206
in urethral sphincter mechanism, 225, 239
Nitric oxide synthase (NOS), 252
testosterone therapy and, 295
NK. See Natural killer cells (NK)
NK1 receptors, contractile function and, 253
NK2 receptors, contractile function and, 253
NLR. See Nod-like receptors (NLR)
NLS. See Nuclear localization signal (NLS)
NMR. See Nuclear magnetic resonance (NMR)
NMV. See Net magnetic vector (NMV)
nNOS. See Neuronal NO synthase (nNOS)
Nocturnal erection, 307
Nod-like receptors (NLR), 4445
Nonbacterial prostatitis, 152154
Nondisjunction, as chromosomal abnormality,
439, 440
Nonheart beating donation, 454455
Noninvasive urodynamics, 275276
Nonseminoma germ cell tumor (NSGCT), 403
a-fetoprotein and, 405
human chorionic gonadotropin and, 405
IGCCCG prognostic scoring system for, 406
Nonvolatile acids (NVA), 74
Norepinephrine, 62, 195
Normal pyelourteric peristalsis, 104105
NOS. See Nitric oxide synthase (NOS)
Novel urinary biomarkers, 382
NPT. See Sodium phosphate cotransporters (NPT)
NSGCT. See Nonseminoma germ cell tumor
(NSGCT)
N-terminal residue, 39
Nuclear factor kB (NF-kB), transcription
factors, 337
Nuclear localization signal (NLS), 16
Nuclear magnetic resonance (NMR), 95
Nuclear transcription factors (NFkb), 44
Nucleoside, defined, 24
Nucleotides, 2
excision repair, 346
Nucleus, 1619
DNA arrangement of, 1618
DNA replication, 18
histone proteins, 18
telomerase, 1819
telomeres, 1819
Nucleus locus coeruleus, in brain, 235236
NVA. See Nonvolatile acids (NVA)
O-antigen, 145
Obesity, CKD and, 215
Obstructive nephropathy, 105106
emerging therapies in, 110
Obstructive shocks, 183
Obstructive symptoms, BPH and, 330
Obturator spasm, 539
Olmsted County Study of Urinary Symptoms
and Health Status Among Men, 327
Oncogenes
altered in TCC
EGFR, ErbB2 (HER2), 381
FGFR3, RAS, and PI3 kinase, 380381
cellular, 351
defined, 351
fibroblast growth factors, 356
G proteins and Ras, 351, 353354
insulin-like growth factors, 356357
retroviral, 351
transforming growth factor b, 357
tyrosine kinase growth factor receptors, 354356
c-erbB-2, 356
EGF, 354
EGFR, 354, 356
Oncura (Illinois, U.S.), 519
One-shot screening, 542, 553

Onufs nucleus, 226, 228
Opsonization, 37
O2 pulse, 530
Organ bath experiment, 231
Organ donation, 454
Organ ischemia, 191192
Organogenesis, 397
Osmolality, 7879
Osteitis fibrosa cystica, 218
Osteomalacia, 218
Osteoprotegerin, 340
Overdiagnosis bias, 549
Oxford Centre for Evidence-Based Medicine, 560
PAC. See Pulmonary artery catheter (PAC)
PACS. See Picture archiving and communication
systems (PACS)
Pair production, 417
Palliative radiotherapy, 421
PAOP. See Pulmonary artery occlusion pressure
(PAOP)
PAP. See Pulmonary artery pressure (PAP)
Papillary adenocarcinoma, 362, 364
Papillary dermis, 464
Papillary renal adenoma, 361
Papillary renal cell carcinoma (PRCC), 361,
362, 364
Paracalcitol, 219
Paracentral lobule, in brain, 236
Paramesonephric ducts, 449
defective development of, 451
Parasympathetic cholinergic activity, 229231
Parasympathetic innervation, 258
Parasympathetic neurons, 228229
varicosities in, 230
Parathyroid hormone (PTH) , 218
Parenteral testosterone
enanthate and cypionate testosterone, 293294
undecanoate preparations, 294
Parkinsons disease
paracentral lobule and, 236
Pars convoluta, 60
Pars recta, 60
Particulate radiation, 416
Pasqualinis syndrome. See Fertile eunuch
syndrome
Pathogen associated molecular patterns
(PAMP), 42
Pattern recognition receptors, 42
PCNL. See Percutaneous nephrolithotomy (PCNL)
PCPT. See Prostate Cancer Prevention Trial
(PCPT)
PCR. See Polymerase chain reactions (PCR)
PD. See Peyronies disease (PD)
PDA. See Personal digital assistants (PDA)
PDE. See Phosphodiesterase (PDE) inhibitors
PDE5. See Phosphodieterase type 5 inhibitors
(PDE5)
PDT. See Photodynamic therapy (PDT)
Peak VO2, 529
Pelvic fascia. See Urinary bladder and urethra
Penile erection, 307
neurophysiology of, 306307
neurotransmitters in, 307309
phases of, 304306
systolic and diastolic velocities in, 305306
physiological mechanisms of, 303309
Penis
anatomy of, 303304
arterial blood supply and venous drainage of,
303304
autonomic and somatic innervation of, 306
erection of, 304309
structure of, 303304
Pentose sugars, 24
Peptide growth factors, in cancer, 338339
Peptides, 346
Percutaneous nephrolithotomy (PCNL), 162
Percutaneous nephrolithotomy (PNL), 104
Periaqueductal gray matter (PAG), 236
Peripheral zone, of prostate, 316317
Index
Peritoneal dialysis, 206
Peritubular physical forces, 66
PERK. See PKR-like ER kinase (PERK)
Personal digital assistants (PDA), 561
Peyronies disease (PD), 311
PFT. See Pulmonary function tests (PFT)
p53 gene, 357358
and apoptosis, 358
cell cycle regulators, 339340
and G1 Arrest, 358
inactivation of normal, in tumors, 358
PHA1. See Pseudohypoaldosteronism type 1
(PHA1)
Phagocytic leukocytes, 45
Phagocytosis, 37
Pharmacogenetics, of TRT, 295296
Pharmacomechanical coupling, 230
Phenylketonuria, 542
Phenytoin, 470
PHEX. See Phosphateregulating gene with
homologies to genes for endopeptidases
on the X chromosome (PHEX)
Phosphatases, 6
Phosphate, 24
renal handling of, 7778
Phosphate reabsorption, 65
Phosphateregulating gene with homologies to
genes for endopeptidases on the X chromosome (PHEX), 78
Phosphodiesterase 5 (PDE 5) inhibitors, 310
testosterone therapy in combination with, 295
Phosphodiesterase (PDE) inhibitors, in nitrergic
mechanism, 252
Phosphoglycerides, 3
Phosphoinositol diphosphate (PIP2), 36
Phosphoinositol 3 kinase (PI3K) pathways, 339
Phospholipase C (PLC)
inhibitors, in muscarinic mechanisms, 250251
pathways, 339
Phospholipids, 3
Phosphorylation, 56
Phosphotyrosine-binding (PTB) domains, 4
Photodynamic diagnosis
of bladder cancer, 383
Photodynamic therapy (PDT), 484, 508509
for prostate cancer, 392393, 515517
Photoelectric effect, 417
Photons
interaction of, with matter, 416418
Photosensitizers, 515
Picture archiving and communication systems
(PACS), 86
Piezoelectric disk, 93
Piezoelectric lithotriptor, 487, 488
PI3K. See Phosphoinositol 3 kinase (PI3K)
pathways
Pili. See Fimbriae
PIN. See Prostatic intraepithelial neoplasia (PIN)
PIP2. See Phosphoinositol diphosphate (PIP2)
Pituitary
differentiation, defective genes of, 289
disorders, 289
tumors, gonadotrophin therapy for, 295
PIVOT trial, 568
Pixels, 498
Pixels per inch (PPI), 498
PKC. See Protein kinase C (PKC)
PKD1. See Protein kinase D1 (PKD1)
PKR-like ER kinase (PERK), 197
Placental alkaline phosphatase (PLAP)
testis cancer and, 405, 408
Plain radiography, 8384
Planning target volume (PTV), 422
PLAP. See Placental alkaline phosphatase (PLAP)
Platelet-endothelial cellular adhesion molecule
(PECAM)-1, 36
PLC. See Phospholipase C (PLC)
PLCO screening trial. See Prostate, Lung, Colorectal and Ovarian (PLCO) screening trial
PLD. See Potentially lethal damage (PLD)
Ploidy and prostate cancer, 397398
PNL. See Percutaneous nephrolithotomy (PNL)
Polymerase chain reactions (PCR), 156, 432
Polymorphic polyglutamine (CAG)

AR and, 285286, 296
shorter or longer sequences, diseases related
to, 296
Polymorphisms, DNA, 335
Pontine micturition center, 235236
Pontine storage center (PSC), 236
Population inversion, 482
Population screening, 542
Postauricular full-thickness skin grafts, 465
Postobstructive renal function, 109110
long-term renal injury in UUO, 110
nephrogenic stage in neonatal obstruction, 110
predicting functional recovery, 109110
tubular function, 110
Potasium reabsorption
DMP1 and, 78
Klotho and, 78
PHEX and, 78
phosphatonin and, 78
PTH and, 78
Potassium excretion, 7274
Potassium levels, AKI and, 203, 205
Potentially lethal damage (PLD), 418
PPI. See Pixels per inch (PPI)
PPIX. See Protoporphyrin IX (PPIX)
Prader-Willis syndrome (PWS), 289
PRCC. See Papillary renal cell carcinoma (PRCC)
Pregnancy
chemotherapy during, 432
screening during, 553
urological surgery in, 537
Preload, 183186
assessment and optimization of, 193194
conceptual analogy for, 184
Preprostatic sphincter (PS), 320
BPH and, 325
in emission and ejaculation, 321
Prepubertal functional castrate, 291
Preschool children
urinary tract infection in, 149
Pressure-flow relationship, 329
Priapism, intraoperative
Primary active transport, 63
program validity, of screening, 548549
Promoters, in cancer, 343
Prostaglandins
in ATN, 207
ProstaLund CoreTherm1 catheter, 481
Prostanoids, biosynthesis of, 307308
Prostate
cancer, 323
embryological development of, 317
endocrinology of, 31731
growth factors in, 319320
innervation of, 320
role, in continence, 320321
secretion, physiological function of, 321323
structure of, 316317
zones of, 316317
Prostate, Lung, Colorectal and Ovarian (PLCO)
screening trial, 556
Prostate cancer
advanced, 391392
locally, treatment of, 391
metastatic, treatment of, 391392
androgen receptor and, 393394
angiogenesis, 395
apoptosis-regulating genes in
apoptosis, 394
Bcl-2, 394395
p53, 395
castration-resistant, 392
cell adhesion, 397
circulating tumor cells, 389390
clinical presentation, 390
cryotherapy for
background, 509
clinical series, 509510, 511
devices for, 509
585
[Prostate cancer]
etiology, 386
focal therapy for, 517519
genetic factors, 398
Gleason grading system of, 387388
growth factors, 395397
hereditary, 398
HIFU in
clinical series, 512515
devices for, 510, 512
light delivery for, 509
linear quadratic model and, 421
management of, 507
matrix metalloproteinases, 398
micro-RNAs and, 394
molecular profiling in, 394
novel therapies in, 392
organ-confined, 391
overview of, 386
pathology, 386387
PDT for, 515517
photomicrograph of, 387
ploidy and, 397398
proliferation, 397
prostate-specific antigen, 389
putative premalignant lesions of
atypical small acinar proliferation, 389
prostatic intraepithelial neoplasia, 388389
radical radiotherapy for, 424
recurrent gene rearrangements in, 394
screening for, 390, 555557
TNM classification of, 388
novel therapies for, 392
photodynamic therapy for, 392393
in vitro and in vivo models of, 398
Prostate Cancer Prevention Trial (PCPT), 389
Prostate carcinoma, CAG and, 286
Prostate gland
development of, 450
transurethral needle ablation of, 479480
Prostate-specific antigen (PSA), 389
levels, 337, 389
for BPH, 327
testing, 386
Prostate-specific membrane antigen (PSMA), 390
Prostatic fluid, examination of, 152
Prostatic intraepithelial neoplasia (PIN), 388389
Prostatic secretion, physiological function of,
321323
Prostatisme vesicale, 113
Prostatitis, 151152
antibiotic penetration in, 154
nonbacterial, 152154
Prostatodynia, 151
Protein kinase C (PKC), 343
Protein kinase D1 (PKD1), 397
Protein leakage, 35
Protein-protein interactions, 45
Protein reabsorption, 6566
Proteins. See also Genes; Growth factors, prostate
cancer
with leucine zipper motif, 29
structure of, 12
Protein synthesis, 3033
final phase of, 32
initiation phase in eukaryotes, 32
tRNA, cloverleaf structure of, 30
Protein translation, changes in
in cancer, 338
Proteinuria, in CKD, 213, 215
Protoporphyrin IX (PPIX), 498
Proximal tubule, function of, 60, 6366
amino acid reabsorption, 65
calcium intake, 76
glucose reabsorption, 6465
phosphate reabsorption, 65
potassium reabsorption in, 72
protein reabsorption, 6566
reabsorption, effect of physical forces on, 64
secretion of organic anions, 66
sodium reabsorption in, 6364, 7072
water reabsorption in, 64
586
Index
PS. See Preprostatic sphincter (PS)

PSA testing. See Prostate-specific antigen (PSA),
testing
PSC. See Pontine storage center (PSC)
P-selectin, 36
Pseudohermaphroditism, male, 302
Pseudohypoaldosteronism type 1 (PHA1), 72
Pseudomonas aeruginosa, 431
PSMA. See Prostate-specific membrane antigen
(PSMA)
Psychogenic erection, 307
PTB. See Phosphotyrosine-binding (PTB)
domains
PTH. See Parathyroid hormone (PTH)
PTV. See Planning target volume (PTV)
Puberty, testosterone deficiency before, 287
Pudendal nerve
innervation of lower urinary tract, 229
Pulmonary artery catheter (PAC), 194
Pulmonary artery occlusion pressure (PAOP), 193
Pulmonary artery pressure (PAP), 193
Pulmonary function tests (PFT), 531
Pulsing, of lasers, 482
Pure uric acid stone, 164
Purinergic innervation, 232
Purinergic receptors, 233
Purinergic transmission, 251252
Purines, 24
Putative neurotransmitters, 232
PWCAM-1. See Platelet-endothelial cellular
adhesion molecule (PECAM)-1
PWS. See Prader-Willis syndrome (PWS)
P2X receptors
ATP and, 233, 251252
Pyelolithotomy, 104
Pyelonephritis, 151
P2Y receptors
ATP and, 233, 251252
Q-switching
of Nd:YAG lasers, 484
Quantum theory of light, 475, 476
Quinckes sign, 193
QUOROM statement, 563
RAAS. See Renin-angiotensin-aldosterone
system (RAAS)
Radiation therapy. See also Radiotherapy
and locally advanced prostate cancer, 391
Radical nephrectomy, 506
Radical radiotherapy, 421
Radiobiology, 418421
defined, 418
linear quadratic model, 420421
redistribution, 419420
reoxygenation, 420
repair of radiation damaged cells, 418419
repopulation, 420
Radiofrequency ablation (RFA), 508
laparoscopic, 519
of renal cell carcinomas, 480481
for renal tumors, 519520
Radiofrequency radiation, application of, 98
Radio frequency (RF) waves, 479
Radiological imaging, of urinary tract
contrast media, 86
cross-sectional imaging, 8895
digital radiography, 86
image, creation of, 83
ionizing radiation, hazards of, 8788
magnetic resonance imaging, 95101
overview, 83
PACS, 86
plain radiography, 8384
x-ray production of, 8486
Radiotherapy
adjuvant, 421
defined, 415
delivery of, 423
electromagnetic radiation used in, 415416
interactions of X rays with matter, 416418
palliative, 421
particulate radiation, 416
planning, 421423
[Radiotherapy]
principles of, 421
radical, 421
radiobiology, 418421
side effects of, 424425
Randalls plaque, 167
Randomized controlled trial (RCT), 560
assessing, 565568
RAP. See Right atrial pressure (RAP)
Rapamycin (mTOR), 8
RAS. See Renin angiotensin system (RAS)
Ras genes, 351, 353
Rats, bleeding tendency in, 206
Rb. See Retinoblastoma protein (Rb)
Rb gene. See Retinoblastoma (Rb) gene
RB1 gene, 381
RBP. See Retinolbinding protein (RBP)
RCC. See Renal cell carcinoma (RCC)
RCT. See Randomized controlled trial (RCT)
Reactive oxygen species (ROS), 9, 197
Receiver operating characteristic (ROC) curve, 547
Receptors
muscarinic, 233
neurotransmitters and, 230, 232233
nicotinic acetylcholine, 230, 233
purinergic, 233
P2X, 233
P2Y, 233
Recipients, transplantation
matching donor and, 458459
Reconstructive surgery, application, 126127
Red-green-blue (RGB) filters
in NBI, 499
5a-reductase
deficiency, 292, 317
inhibitor, for BPH, 327, 328
testosterone conversion, in prostate, 318
in testosterone metabolism, 284
Reflexogenic erection, 307
Reflux cystograms, 118
Reifensteins syndrome, 291
Relative supersaturation (RS) , 173174
REMTM. See Return electrode monitoring
(REMTM)
Renal biopsy
for AKI, 205
in CKD, 212
Renal bone disease, 218
Renal cell carcinoma (RCC), 346348
adult renal epithelial neoplasms. See Adult
renal epithelial neoplasms
chromophobe, 364
collecting duct carcinoma, 364
conventional (CRCC), 362
evaluation of, 506
overview, 360
radio frequency ablation of, 480481
sarcomatous change in, 364
sporadic, 365
chromosomal abnormalities in, 366
Renal clearance, 5859
Renal fibrosis, development of, 108
Renal function
calcium, renal handling of, 7677
extracellular fluid volume, 6770
glomerular filtration, 6063
glomerulotubular balance, 6667
growth and, 5758
homeostasis, 5759
hydrogen ion excretion, 7476
magnesium, renal handling of, 77
nephron, functional segmentation of, 5960
phosphate, renal handling of, 7778
potassium excretion, 7274
proximal tubular function, 6366
sodium excretion, 6770
sodium reabsorption beyond proximal
tubule, 7072
vitamin D, renal metabolism of, 78
Renal hemodynamics, alterations in, 207208
Renal oncocytomas, 361362
Renal pelvic pressure, changes in, 106
Renal plasma flow (RPF), 59

Renal replacement therapy (RRT)
in AKI, 205, 206
in CKD, 219
Renal tubular acidosis (RTA), 74
Renal tumors, 506507
cryotherapy for, 519
evaluation of, 506
incidence of, 506
RFA for, 519520
Renal ultrasound, for AKI, 205
Renin, 531
Renin-angiotensin-aldosterone system
(RAAS), 68
hypertension and, 214
Renin angiotensin system (RAS), 108
Reproductive system, male. See also Male
hypogonadism
development of, 300302
endocrine regulation of, 280287
Resectoscope, 495
Resonant frequency, 97
of hydrogen nuclei, 98
Respiratory burst, 37
Retinoblastoma protein (Rb), cell cycle
regulators
in cancer, 339
Retinoblastoma (Rb) gene, 358, 398
Retinoid acidinducible gene-1 (Rig-1)-like
receptors (RLR), 4548
chemokines, 4548
complement activation, 45
dendritic cells, 45
natural killer cells, 45
phagocytic leukocytes, 45
Retinolbinding protein (RBP), 65
Retroperitoneal lymph node dissection
(RPLND), 403
Return electrode monitoring (REMTM), 477
Reverse transcription polymerase chain reaction
(RT-PCR), 389390
RFA. See Radio frequency ablation (RFA)
RF waves. See Radio frequency (RF) waves
RGB filters. See Red-green-blue (RGB) filters
Rhabdomyolysis, 539
CK level in, 203
FeNa and, 204
Rhabdosphincter, 259
Rho-associated kinase (ROCK), in contractile
state, 250
Ribosomal RNA, 30
Ribosomes, 11
RIFLE criteria, for AKI, 203, 204
Right atrial pressure (RAP), 193
Right ventricular end-diastolic pressure
(RVEDP), 193
Right ventricular end-diastolic volume
(RVEDV), 193
RLR. See Retinoid acidinducible gene-1
(Rig-1)-like receptors (RLR)
RNA
splicing mechanism, 28
structure of, 2426
RNA polymerase, 26
Robotic technology, in urology, 502503
ROC curve. See Receiver operating characteristic
(ROC) curve
ROCK. See Rho-associated kinase (ROCK)
Rod lens
cystoscope, 493494
system, 492
Rontgen, Wilhelm, 84
ROS. See Reactive oxygen species (ROS)
Routes of infection, in urinary tract, 146
RPF. See Renal plasma flow (RPF)
RPLND. See Retroperitoneal lymph node
dissection (RPLND)
RRT. See Renal replacement therapy (RRT)
RS. See Relative supersaturation (RS)
RSS (Really Simple Syndication), 562
RTA. See Renal tubular acidosis (RTA)
RT-PCR. See Reverse transcription polymerase
chain reaction (RT-PCR)
Index
RVEDP. See Right ventricular end-diastolic
pressure (RVEDP)
RVEDV. See Right ventricular end-diastolic
volume (RVEDV)
Salbutamol, potassium levels and, 205
Salt and water, consequences of CKD, 216
SAPK. See Stress-activated protein kinase
(SAPK) pathways
SCC. See Small cell carcinoma (SCC)
Scheduling, 435
Schistosoma haematobium, 135
Screening
benefits, 553
for bladder (urothelial) cancer, 555
for chronic disease
case finding, 543
mass screening, 542
selective screening, 542
and control of disease, 549551
cost of, 553
defined, 542
ethical considerations and attitudes to, 545
fundamentals of, 544545
for infectious disease, 553554
lifetime events and terminology, 543544
detectable preclinical phase (DPCP), 543
total preclinical phase (TPCP), 543
one-shot, 542
during pregnancy, 553
programs
general principles, 551553
population, criteria for, 551
program validity, 548549
for renal cancer, 554
repeat, effect of, 548
for testis cancer, 557
tests, validity of, 546547
for urological disease, 553
validity of, 545548
SD systems. See Standard-definition (SD) systems
Secondary active transport, 63
Secondary hyperparathyroidism (SHPT)
in CKD, 218
vitamin D and, 218, 219
Selectins, 53
Selection bias, 549
Selective screening, 542
Semen analysis, 314
Senescence, 19
Sensation markers, 273
Sensitivity
defined, 546
of test, checking, 548
Sepsis
Septal and preoptic nuclei, in brain, 236
Septic shocks, 156
definition of, 183
Sertoli-cell-only syndrome, 313
Sertoli cells, 300, 449
FSH, regulation of, 283, 312
in spermatogenesis, 279280, 311312
Serum and AKI
potassium levels, 203, 205
urea, 203
Setup margin (SM), 422
Sex determining region of Y chromosome
(SRY), 300
Sex hormonebinding globulin (SHBG)
testosterone transport in circulation, 283284
Sex hormones
urinary tract smooth muscle function, 259
SH2. See Src-homology (SH)2 domains
Shape memory alloy (SMA), 501502
SHBG. See Sex hormonebinding globulin (SHBG)
Shh. See Sonic hedgehog (Shh)
Shh, secretion of. See Sonic hedgehog (Shh),
secretion of
Shocks
annual incidence of, 184
associated pathophysiology, 191193
[Shocks]
cellular responses to, 195201
central supply of substrate, 183191
clinical terms of, 182
cytosolic responses to, 195201
defined, 182183
hemodynamic pathophysiology, 183191
positive feedback cycle, 192
Shock wave, 485486
Short hairpin RNA (shRNA), 30
SHPT. See Secondary hyperparathyroidism
(SHPT)
shRNA. See Short hairpin RNA (shRNA)
SIADH. See Syndrome of inappropriate secretion
of antidiuretic hormone (SIADH)
Sialy Lewis X, 36
Signal to noise ratio (SNR), 83
Signal transduction
in cell membrane, 11
changes, in cancer, 339
Signal transduction, mechanism of, 47
acetylation, 67
key signaling cascades, 7
phosphorylation, 56
posttranslational modifications, 5
protein-protein interactions, 45
signal termination, 7
SUMOylation, 67
ubiquitylation, 6
Sildenafil
PDE inhibitors, 252
PDE5 inhibitors, 295
Silent afferents, 233
Simulator, 422, 423
Single nucleotide polymorphisms (SNP), 336
Single-shot screening, 542, 553
Skin
cross-sectional diagram of, 465
grafting of, 464465
layers of, 464
SkipperSchabelWilcox model, 428
SLD. See Sublethal damage (SLD)
SLE. See Systemic lupus erythematosus (SLE)
Slow neurotransmitters, 230
SM. See Setup margin (SM)
SMA. See Shape memory alloy (SMA)
Small cell carcinoma (SCC), 376
Small nuclear ribonucleoproteins (snRNP), 28
Small ubiquitin-related modifier (SUMO), 6
Smoking
cessation, 531
CKD and, 216
as risk factor of bladder TCC, 374
SNP. See Single nucleotide polymorphisms (SNP)
SNR. See Signal to noise ratio (SNR)
snRNP. See Small nuclear ribonucleoproteins
(snRNP)
Sodium excretion, 6770
Sodium phosphate cotransporters (NPT), 77
Sodium reabsorption
in proximal tubule, 6364, 7072
in collecting duct, 72
in distal convoluted tubule, 7172
loop of Henle, 71
Sojourn time, 543
Solute in exudation, 35
Sonablate1 500 (Focus Surgery), 510, 512
SONAR. See Sound navigation and ranging
(SONAR)
Sonic hedgehog (Shh), 301
secretion of, 443
Sonoelasticity, in prostate gland, 95
Sorafenib, for renal cancer, 339
Sound navigation and ranging (SONAR), 92
Spark-gap lithotripters, 487
Spatial resolution, 83
Specificity, defined, 546
Spectral Doppler, 94
Spermatogenesis, 279280, 311312
hormonal control of, 312
induction, gonadotrophin therapy for, 294295
Spermatozoa, 312
Sperm maturation, 313
587
Spinal cord
of pathways, lower urinary tract function, 234
Spiral CT, 91
Splicing, alternative
in cancer cells, 337
Split-thickness skin grafts, 464
Spontaneous activity
origin of, 254
significance of, 253254
Spontaneous emission, 481
Sporadic RCC
chromosomal abnormalities in, 366
Src-homology (SH)2 domains, 4
SRY. See Sex determining region of Y
chromosome (SRY)
Standard-definition (SD) systems, 498
high-definition imaging systems vs., 498
Staphylococcus aureus, 134
Starling forces
in GFR, 61
Stem cells, 471
Stereoscopy, 499
Stern, Maximilian, 493
Stimulated emission, 481482
Stone
composition of, 164
infections of, 164
recurrence, prevention of, 177178
STOP codon (UAG), 31
Storz, Karl, 492
Streptococcus mutans, 136
Stress
clinical manifestations of, 200
cytosolic response to, 195196
endoplasmic reticulum response to, 196197
hypoxic, 197198
mitochondrial responses to, 197198
nuclear responses to, 197
Stress-activated protein kinase (SAPK)
pathways, 339
Stress relaxation, of bladder, 247
Stress response
to urological surgery, 523524
acute phase response (APR), 525
ebb phase, 523
flow phase, 523
immunological and hematological, 524525
neuroendocrine, 524, 525
recovery phase, 523
Stress urinary incontinence (SUI), 258, 259
Stretch-activated channel
of apical membrane, 245
in detrusor smooth muscle, 257
Striated sphincter, 225226
Stroke volume (SV), 183191
afterload, 186190
heart rate, 190191
myocardial contractility, 186
preload, 183186
Stromal-epithelial interaction
in epithelial and glandular development,
324325
Strong desire to void (SDV), sensation
markers, 273
Sublethal damage (SLD), 418
Substance abuse
Substances solubility, 165
Suburothelium
detrusor and, 255256
secretory and signaling properties of, 254256
structure of, 244
SUI. See Stress urinary incontinence (SUI)
Sulfasalazine, 110
SUMO. See Small ubiquitin-related modifier
(SUMO)
SUMOylation, 67
Supersaturation, 165166
Surveillance, Epidemiology, and End Results
(U.S. SEER) database, 506
SV. See Stroke volume (SV)
Swiss Lithoclast, 489490
Swyers syndrome, 290
Sympathetic nerve, 229
588
Index
Symptom scores, BPH and, 328

Syndrome of inappropriate secretion of
antidiuretic hormone (SIADH), 70
Systemic anticancer treatment
anti-angiogenesis agents, 434435
cytokines for, 433
cytokinetics
cell killing, 428, 429
tumor growth, 428
drug resistance, 429, 430431
immune modulation for, 433
intensifying treatment, 433
measuring efficacy, 432433
molecularly targeted therapies for, 434
monoclonal antibody therapy for, 433434
pharmacology, 429
toxicity, 431432
Systemic inflammation, 192193
Systemic lupus erythematosus (SLE), blood test
in, 203, 204
Systolic velocity erection, 305306
Tachykinins, contractile function and, 253
Tagliacozzi, Gaspare, 465
TAL. See Thick ascending limb (TAL)
Tamm-Horsfall protein, 133
Tanslocation, as chromosomal abnormality,
439, 440
Target organ defects, 291292
TATA box, 26
TCC, of bladder. See Transitional cell carcinomas
(TCC), of bladder
T-cell antigen receptor (TCR), 48
T-cell antigens, 4950
T-cells, 48
recirculation, 4849
TCR. See T-cell antigen receptor (TCR)
Teletherapy, 415
Telomerase, 1819
Telomeres, 1819
Teratomas, 428
Terminal ileum
removal of, 467
Testicle, development of, 311312
Testicular atrophy, 405
Testicular dysgenesis, 290
Testicular failure, 313
Testicular germ cell tumor (TGCT)
histological examination of, 408409
molecular biology of, 406408
Testicular hormones, 279
Testicular maldescent, 313
Testis
anatomy of, 279
spermatogenesis, 279280
Testis cancer
epidemiology of, 404405
effect of social class, 404
environmental factors, 404
family history, 404405
testicular atrophy, 405
testicular maldescent, 405
germ cell tumor. See Germ cell tumor (GCT)
intratubular germ cell neoplasia, 408
overview, 403404
screening for, 557
testicular germ cell tumors
molecular biology of, 406408
tumor markers
a-fetoprotein, 405
human chorionic gonadotropin and, 405
lactate dehydrogenase and, 405
placental alkaline phosphatase and, 405
prognostic significance, 406
response to therapy, 406
tumor growth rate, 406
Testosterone
biological effects of, 286
biosynthesis, 283
enzyme defects of, 290
and cardiovascular system, 287
and central nervous system, 287
[Testosterone]
circulation, 283284, 317
conversion of, 303
deficiency
gonadotrophin therapy in, 294
before puberty, 287
effects of, 303
metabolism of, 284285
preparations, 293
synthesis of, 302
therapy, 292295
transport of, 283284
Testosterone replacement therapy (TRT)
in adult males with hypogonadism
parenteral testosterone, 293294
transdermal testosterone, 293
future research and development
PDE5 inhibitors and, 295
pharmacogenetics, 295296
synthetic androgens and elective androgen
receptor modulators, 296
goals of, 292
gonadal stimulation in hypogonadotrophic
hypogonadism, 294295
Testosterone undecanoate, 292
Tetrodotoxin, frequency-response curve and, 231
TFT. See Thin-film transistor (TFT) screen
TGCT. See Testicular germ cell tumor (TGCT)
TGF. See Transforming growth factor (TGF)
family
TGF-a. See Transforming growth factor
a (TGF-a)
TGF-b. See Transforming growth factor
b (TGF-b)
TGF-b1. See Transforming growth factor-b1
(TGF-b1)
The Lancet, 140
Therapeutic cloning, 453, 471
Therapeutic ultrasound, 95
Thermocouples, 481
Thermodynamic solubility product, 165
Thick ascending limb (TAL), 59
Thin-film transistor (TFT) screen, 86
Thoracolumbar reflex, 241
Three-dimensional (3-D) imaging systems, 499
head-mounted display for, 500
Three-stage theory of prostatism, 113114
Thromboprophylaxis, 535536
Thrombotic thrombocytopaenic purpura (TTP),
blood test and, 203
Thymic tolerance, 48
Thymine, 2
TIF. See Tubulointerstitial fibrosis (TIF)
TIMP. See Tissue inhibitors of MMPs (TIMP)
Tissue engineering, in urology, 470471
Tissue inhibitors of MMPs (TIMP), 348
Tissue transfer, in urology, 462463
TK. See Tyrosine kinase (TK)
TLR. See Toll-like receptors (TLR)
T-lymphocyte receptor, 36
T lymphocytes, 40
TMPRS22: ETS fusion, prostate cancers and, 337
TNF. See Tumor necrosis factor (TNF)
TNFa. See Tumor necrosis factor a (TNFa)
TNFR. See Tumor necrosis factor receptor
(TNFR)
TNF receptor-associated factor 2 (TRAF2), 196
Tobacco use, cancers and, 343
Toll-like receptors (TLR), 44
Tonicity of body fluid, 7879
Topoisomerase II, 431
Total preclinical phase (TPCP), 543
Toxicity, 431432, 435
Toxin, 135136
TPCP. See Total preclinical phase (TPCP)
TP53 gene, 381
TRAF2. See TNF receptor-associated factor 2
(TRAF2)
TRAIL. See Tumor necrosis factorrelated apoptosis inducing ligand (TRAIL) receptors
Trandolapril, for proteinuria, 215
Transcription, 29
Transdermal testosterone, 293
Transforming growth factor a (TGF-a), 23, 319,

325326
Transforming growth factor b (TGF-b), 319,
325326, 462
Transforming growth factor-b1 (TGF-b1),
107, 357
Transforming growth factor (TGF) family, 319
in BPH, 325326
Transient Receptor Potential Cationic Channel-1
(TRPC-1), 107
Transitional cell carcinomas (TCC), of
bladder, 356
case study, 374
clinical features, 378379
etiology, 374375
genetic alterations identified in, 380382
metachronous and synchronous, molecular
basis of, 380
molecular pathogenesis of, 379380
overview, 374
pathology
histological variants, 376377
premalignant conditions, 378
tumor grade, 375376
tumor stage, 376
photodynamic diagnosis, 383
progression, model for, 379380
smoking and, 374
trials, 383384
urinary biomarkers of, 382
Transition zone, of prostate, 316317
Translation, protein, 338
Transplantation, 454461
brain death and, 454
cadaveric donor and, assessment of, 457
commercialization of, 455
immunosuppression and, 459460
kidney
from live donors, 455
surgical technique of, 459
live donor, 455
long-term management of, 460
nonheart beating donation and, 454455
organ donation and, 454
organ preservation and, 455
solutions used for, 455456
postoperative management, 460
recipient, assessment of, 457458
Transrectal ultrasound (TRUS), 390
Transthoracic echocardiography, 194
Transurethral microwave thermotherapy
(TUMT), 480481
Transurethral needle ablation (TUNA), 479480
Transurethral resection in saline (TURisTM), 477
Transurethral resection (TUR) syndrome
pathophysiology of, 538
Trigone, 257258
Trisomy, 439
tRNA, cloverleaf structure of, 31
Trocar, 502, 503
TRPC-1. See Transient Receptor Potential
Cationic Channel-1 (TRPC-1)
TRT. See Testosterone replacement therapy
(TRT)
True detrusor pressure (Pdet), 270
TRUS. See Transrectal ultrasound (TRUS)
TSC. See Tuberous sclerosis (TSC)
TTP. See Thrombotic thrombocytopaenic
purpura (TTP)
Tuberculosis, 154156
clinical features of, 155
Tuberous sclerosis (TSC), 368
Tubular cells, mechanical insult on, 107108
Tubular function, alterations in, 208
Tubular proteinuria, 65
Tubuloglomerular feedback, 6667
Tubulointerstitial fibrosis (TIF), 108
Tubulointerstitial scarring, 213
Tumor-induced osteomalacia, 78
Tumor lysis syndrome, 205
Index
Tumor markers, testis cancer, 405406
Tumor necrosis factor a (TNFa), 39, 192
Tumor necrosis factor receptor (TNFR), 9
Tumor necrosis factorrelated apoptosis
inducing ligand (TRAIL) receptors, 340
Tumor necrosis factor (TNF), 433
Tumors
cell killing in, 428, 429
drug resistance in, 429, 430431
growth, 428
staging of, 388
Tumor suppressor genes, 357359
mdm-2 gene, 358
p53 gene, 357358
and apoptosis, 358
and G1 arrest, 358
inactivation of normal, in tumors, 358
RCC and, 347348
retinoblastoma gene, 358
von HippelLindau gene, 359
Tumor suppressor genes, in TCC, 381
Tumour necrosis factor (TNF), 340
TUMT. See Transurethral microwave
thermotherapy (TUMT)
TUNA. See Transurethral needle ablation
(TUNA)
TURisTM. See Transurethral resection in saline
(TURisTM)
Turners syndrome, 439, 440
TUR syndrome. See Transurethral resection
(TUR) syndrome
Type I fimbriae, 137
cellular invasion by organisms, 138
expression of, 138
Type I pili, 138
Type P fimbriae, 138139
structure and ultrastructure of, 139140
Type S pili, 143
Tyrosine kinase (TK), 434
growth factor receptors, 354356
Ubiquitylation, 6
UICC. See Union Internationale Contre le Cancer
(UICC)
UK Health Technology Assessment
Programme, 557
UKPDS. See United Kingdom Prospective
Diabetes Study (UKPDS)
ULTRA. See Unrelated Live Transplant
Regulatory Authority (ULTRA)
Ultrasound, 9293, 276
diagnostic range, 93
grayscale, 93
high-intensity focused (HIFU), 507508
image of renal stone, 95
overview, 507
for stone fragmentation, 488
Umbrella cell layer, of urothelium. See Apical
cell layer
Undecanoate preparations, 294
Unfolded protein response (UPR), 196
Unilateral UO (UUO), 103
blood flow, changes in, 106
long-term renal injury in, 110
macroscopic changes in, 105106
microscopic changes in, 105106
obstructive nephropathy in animal
models, 105
renal inflammation in, 107108
Union Internationale Contre le Cancer (UICC), 360
Unitary stretch receptor, 233
United Kingdom Prospective Diabetes Study
(UKPDS), 215
Universal cystoscope, 493
Unrelated Live Transplant Regulatory Authority
(ULTRA), 455
UO. See Ureteric obstruction (UO)
UP3. See Uroplakin 3 (UP3)
UP1a. See Uroplakin (UP1a)
UPEC. See Uropathogenic Escherichia coli
(UPEC)
UPJ. See Uretero-pelvic junction (UPJ)
UPP. See Urethral pressure profiles (UPP)
Upper urinary tract, embryonic development,

441443
Upper urinary tract obstruction
changes in renal pelvic pressure, 106
clinical presentation of, 103
effect of ureteric obstruction, 104105
etiology of, 103
incidence of, 103
intraobstructive tubular dysfunction, 108109
mechanical insult on tubular cells, 107108
mechanosensation in renal tubular cells, 106107
obstructive nephropathy, 105106
emerging therapies in, 110
postobstructive renal function, 109110
UPR. See Unfolded protein response (UPR)
UpToDate#, 562
Ureaplasma urealyticum, 146
Uremia
AKI and, 206
effects of toxins, CKD and, 217
Ureteric obstruction (UO), 103
effect of, 104105
Uretero-pelvic junction (UPJ), 103
Ureteroscopes, 495496
Ureteroscopy, 104
Uretero-vesicular junction (UVJ), 103
Urethra
muscles of, 258259
pressure, 238239
pressure profile of bladder neck, 321
Urethral pressure profiles (UPP), 271, 273274
Urethral pressure profilometry, 271
Urethral resistance relationship, 329
Urethral sphincter, 321
Urethral sphincter mechanism, 224226, 227
intrinsic rhabdosphincter, 225226
Onufs nucleus, 226, 228
smooth muscle, 224225
Urethral syndrome, 151
Uric acid calculi, 164
Uric acid stone
effect of bad Western diet, 176
formation of, 169170
treatment of, 170
Urinary biomarkers, of TCC, 382
Urinary bladder and urethra. See also Bladder,
urinary; Urethra
innervation of, 228229
afferent, 233235
functional aspects, 229232
pressure, synchronous, 237
structure of
adventitial layer, 221222
epithelial layer, 226228
muscular layer, 222224
Urinary buffers, 7576
Urinary catheter, for fluid balance, 205
Urinary obstruction, BPH and, 329
Urinary stone formation
calcium stone disease, 173174
chemical principles of, 165167
cystine stone formation, 167169
infected stone formation, 170173
overview, 162164
prevention of stone recurrence, 177178
stone, composition of, 164
theories of, 165
uric acid stone formation, 169170
Urinary symptoms, BPH and, 328
Urinary tract
abnormalities, 443
genetic basis of, 452
anomalies, chromosome defects associated
with, 440
bowel incorporation in, effects of, 468470
metabolic abnormality, 468469
stones, 470
urinary infection and malignant
transformation, 469470
589
[Urinary tract]
functional development of, 445
malformations of, 452
radiological imaging of. See Radiological
imaging, of urinary tract
upper. See Upper urinary tract, embryonic
development
Urinary tract infection (UTI)
clinical aspects of, 147158
introital question, 148
significance of, 147
colonizing microorganisms in, 130131
commensal organisms, properties of, 131132
defence machanisms, 131
in infants, 149
localization studies in, 157
lower urinary tract, host defence mechanism,
132134
of male genital tract, 151154
modifying factors, 131
in older patient, 150
organisms responsible for, 145146
overview, 130
in preschool children, 149
routes of infection, 146
by stones, 151
virulence factors in, 145
virulence mechanisms, 134135
against host, 135146
in young adult men, 150
Urine
bacterial counts in, 147
concentration and dilution of, 7880
cytology, 382
flow of, 132
pH levels in, 132
relative supersaturation of, 173174
Urine flow, measuring, 271
Urine testing, for AKI, 204
Urodynamic data, summary of, 117118
Urodynamics
advanced techniques, 275276
in BPH, 328331
influencing factors, 272
interpretation of, 272275
methodology, practice, and problems, 269272
cystometry, 270271
flowmetry, 271272
urethral pressure profilometry, 271
new developments, 275276
physics and mechanics
storage phase, 266268
voiding phase, 268269
symptoms and, 276
Uroepithelial cells, 134
Urogenital sinus mesenchyme, 324325
Urolithiasis, 104
Urological cancer, 425
Urological disease
screening for, 553557
Urological surgery, risk assessment for
allergy and drug intolerance, 537
anticoagulation therapy discontinuation and,
536
antiplatelet therapy discontinuation and, 536
cardiopulmonary assessment
data derived from tests, 529530
hematological disorders, 533534
immunological disorders, 534
liver disorders, 533
metabolic disorders, 532
microbiological assessment and antibiotic
prophylaxis, 534
musculoskeletal disorders, 533
nutritional status, 534535
renal disorders, 531532
respiratory disorders, 530531
cardiovascular assessment and optimization,
527529
challenging aspects of, 523
day-case, 526527
emergency, 537
gas embolism and, 538
inpatients, 527

1841846791

Uploaded by

Copyright:

Available Formats

1841846791

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

1841846791

Uploaded by

Copyright:

Available Formats

THE

The Scientific Basis of Urology

Anthony R. Mundy, MS, FRCP, FRCS

John M. Fitzpatrick, MCh, FRCSI, FRC(Urol), FRCSGlas, FRCS

David E. Neal, FMed Sci, MS, FRCS

Nicholas J. R. George, MD, FRCS

First published in 1999 by Isis Medical Media, Ltd, Oxford, UK

11. Pathophysiology and Management of Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

15. Physiological Properties of the Lower Urinary Tract

16. Scientific Basis of Urodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

18. Male Sexual Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Hashim U. Ahmed Department of Urology, Division of Surgical and

Department of Urology, St. Georges Hospital, London, U.K.

Manit Arya Department of Urology, Division of Surgical and Interventional

Beatson Institute for Cancer Research, Glasgow, U.K.

Pierre-Marc G. Bouloux Department of Endocrinology, Royal Free and

Southampton General Hospital, Southampton, U.K.

Michael Craggs London Spinal Cord Injuries Centre, Royal National

Faculty of Applied Sciences, University of Sunderland,

Neil G. Docherty The Conway Institute, University College Dublin, Ireland

Mater Misericordiae Hospital and University College

Postgraduate Medical School, University of Surrey,

Department of Urology, University College London Hospitals,

Nicholas J. R. George Department of Urology, Withington Hospital, University

Thiru Gunendran Department of Urology, University Hospital of South

Nuffield Department of Surgery, University of Oxford,

Academic Department of Paediatrics, Guys Hospital,

John A. Kirby Institute of Cellular Medicine, Newcastle University,

Beatson Institute for Cancer Research, Glasgow, U.K.

Rachel Lewis Department of Oncology, University College Hospitals, London,

Division of Medicine, University College Hospital,

University Hospitals of Leicester, Leicester, U.K.

Danish Mazhar Department of Medical Oncology, Addenbrookes Hospital,

Department of Oncology, University College Hospitals,

Department of Urology, University College London Hospitals,

Caroline Moore Department of Urology, Division of Surgical and

St Georges Hospital and Medical School, London, U.K.

Department of Oncology, University College Hospitals,

Department of Urology, University College London Hospitals,

William G. Robertson Physiology Department, Royal Free and University

Department of Urology, Kingston and St. Georges Hospital,

Rona Smith Cambridge University Hospitals NHS Foundation Trust,

Department of Oncology, University College Hospitals,

Department of Urology, St Georges Hospital, London, U.K.

David Talbot Department of Hepatobiliary and Transplant Surgery,

Department of Urology, Addenbrookes Hospital,

University College Hospital, London, U.K.

Department of Clinical Oncology, Addenbrookes Hospital,

Nicholas A. Wisely Department of Anaesthesia, University Hospital of South

Medical School Administration, University College London,

THE MOLECULES OF LIFE

the mechanisms that underlie cellular behavior, we

Bennett and Leung

into families on the basis of structural homology. As a

ribonucleic acid, also comprises these bases except

Figure 1 Chemical structures of some key classes of

Chapter 1: Introduction to Cell Biology

when protein-coding regions of DNA are copied into

of ATP, which contains three phosphate groups

HOW CELLS RESPOND TO THEIR