The Renin-Angiotensin-Aldosterone System - Methods and Protocols (PDFDrive)

Methods in
Molecular Biology 1614
Sean E. Thatcher Editor
The Renin-
Angiotensin-
Aldosterone
System
Methods and Protocols
Methods in Molecular Biology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
The Renin-Angiotensin-
Aldosterone System
Methods and Protocols
Edited by
Sean E. Thatcher
Department of Pharmacology and Nutritional Sciences, University of Kentucky,
Lexington, KY, USA
Editor
Sean E. Thatcher
Department of Pharmacology and Nutritional Sciences
University of Kentucky
Lexington, KY, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7028-5 ISBN 978-1-4939-7030-8 (eBook)
DOI 10.1007/978-1-4939-7030-8
Library of Congress Control Number: 2017937363
© Springer Science+Business Media LLC 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed
to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature

The registered company is Springer Science+Business Media LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface
The purpose of this book is to provide scientists with detailed protocols that will help in
measuring different components of the renin-angiotensin-aldosterone system (RAAS). This
book also helps in the use of new methods to measure angiotensin peptides and to apply
cutting-edge techniques to discern the influence of RAAS components on different aspects
of mammalian disease. Each chapter provides an in-depth focus on each experimental tech-
nique and gives the reader the best approach to examine how the RAAS might influence
his/her own experimental outcomes.
Lexington, KY, USA Sean E. Thatcher
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 A Brief Introduction into the Renin-Angiotensin-Aldosterone System:

New and Old Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sean E. Thatcher
2 A Color Segmentation-Based Method to Quantify Atherosclerotic
Lesion Compositions with Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Congqing Wu, Alan Daugherty, and Hong Lu
3 Assessment of Protein Carbonylation and Protein Tyrosine
Phosphatase (PTP) Oxidation in Vascular Smooth Muscle
Cells (VSMCs) Using Immunoblotting Approaches . . . . . . . . . . . . . . . . . . . . . 31
Sofia Tsiropoulou and Rhian M. Touyz
4 Methods for Studying the Role of RAAS in the Modulation of Vascular
Repair-Relevant Functions of Stem/Progenitor Cells . . . . . . . . . . . . . . . . . . . . 47
Yagna P.R. Jarajapu
5 Use of a Fluorescent Substrate to Measure ACE2 Activity in the Mouse
Abdominal Aorta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Yu Wang, Lisa A. Cassis, and Sean E. Thatcher
6 Measuring Blood Pressure Using a Noninvasive Tail Cuff Method in Mice . . . . 69
Yu Wang, Sean E. Thatcher, and Lisa A. Cassis
7 Blood Pressure Monitoring Using Radio Telemetry Method in Mice . . . . . . . . 75
8 Characterization and Functional Phenotyping of Renal Immune
Cells via Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Nathan P. Rudemiller and Steven D. Crowley
9 Assessment of the Renin–Angiotensin System in Cellular Organelle:
New Arenas for Study in the Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Bryan A. Wilson and Mark C. Chappell
10 Comprehensive Assessments of Energy Balance in Mice . . . . . . . . . . . . . . . . . . 123
Justin L. Grobe
11 In Vitro Assays to Determine Smooth Muscle Cell Hypertrophy,
Protein Content, and Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Katherine J. Elliott and Satoru Eguchi
12 A New Mouse Model for Introduction of Aortic Aneurysm
by Implantation of Deoxycorticosterone Acetate Pellets or Aldosterone
Infusion in the Presence of High Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Shu Liu, Ming C. Gong, and Zhenheng Guo
vii
viii Contents
13 Fluorescence-Based Binding Assay for Screening Ligands

of Angiotensin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Maiia E. Bragina, Nikolaos Stergiopulos, and Rodrigo A. Fraga-Silva
14 A Primer to Angiotensin Peptide Isolation, Stability, and Analysis
by Nano-Liquid Chromatography with Mass Detection . . . . . . . . . . . . . . . . . . 175
Mariola Olkowicz, Stefan Chlopicki, and Ryszard T. Smolenski
15 Analysis of Angiotensin Metabolism in the Kidney Using Mass Spectrometry . . . 189
Nadja Grobe and Khalid M. Elased
Erratum to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E1
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Contributors
Maiia E. Bragina • Laboratory of Hemodynamics and Cardiovascular Technology,

Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne,
Switzerland
Lisa A. Cassis • Department of Pharmacology and Nutritional Sciences,
University of Kentucky, Lexington, KY, USA
Mark C. Chappell • Hypertension and Vascular Research, Department of Surgery,
Cardiovascular Scineces Center, Winston-Salem, NC, USA
Stefan Chlopicki • Jagiellonian Centre for Experimental Therapeutics (JCET),
Jagiellonian University, Krakow, Poland; Department of Experimental Pharmacology,
Jagiellonian University Medical College, Krakow, Poland
Steven D. Crowley • Division of Nephrology, Department of Medicine, Durham VA
and Duke University Medical Centers, Durham, NC, USA
Alan Daugherty • Saha Cardiovascular Research Center, University of Kentucky,
Lexington, KY, USA; Department of Physiology, University of Kentucky, Lexington,
KY, USA
Satoru Eguchi • Department of Physiology and Cardiovascular Research Center,
Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA
Khalid M. Elased • Department of Pharmacology and Toxicology, Boonshoft School of
Medicine, Wright State University, Dayton, OH, USA
Katherine J. Elliott • Department of Physiology and Cardiovascular Research Center,
Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA
Ming C. Gong • Department of Physiology, University of Kentucky, Lexington, KY, USA;
Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA
Justin L. Grobe • Department of Pharmacology, Center for Hypertension Research, The
Obesity Research and Education Initiative, François M. Abboud Cardiovascular Research
Center, The Fraternal Order of Eagles’ Diabetes Research Center, University of Iowa,
Iowa City, IA, USA
Nadja Grobe • Department of Pharmacology and Toxicology, Boonshoft School of Medicine,
Wright State University, Dayton, OH, USA
Zhenheng Guo • Department of Physiology, University of Kentucky, Lexington, KY, USA;
Research and Development, Lexington Veterans Affairs Medical Center, University of
Kentucky, Lexington, KY, USA; Saha Cardiovascular Research Center, University of
Kentucky, Lexington, KY, USA; Department of Pharmacology and Nutritional Science,
University of Kentucky, Lexington, KY, USA
Yagna P.R. Jarajapu • Department of Pharmaceutical Sciences, College of Health
Professions, North Dakota State University, Fargo, ND, USA
Shu Liu • Department of Physiology, University of Kentucky, Lexington, KY, USA;
Research and Development, Lexington Veterans Affairs Medical Center, University
of Kentucky, Lexington, KY, USA; Saha Cardiovascular Research Center, University of
Kentucky, Lexington, KY, USA
Hong Lu • Department of Physiology, University of Kentucky, Lexington, KY, USA; Saha
Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA
ix
x Contributors
Mariola Olkowicz • Department of Biochemistry, Medical University of Gdansk, Gdansk,

Poland; Department of Biotechnology and Food Microbiology, Poznan University of Life
Sciences, Poznan, Poland
Nathan P. Rudemiller • Division of Nephrology, Department of Medicine,
Durham VA and Duke University Medical Centers, Durham, NC, USA
Rodrigo A. Fraga-Silva • Laboratory of Hemodynamics and Cardiovascular Technology,
Switzerland
Ryszard T. Smolenski • Department of Biochemistry, Medical University of Gdansk,
Gdansk, Poland
Nikolaos Stergiopulos • Laboratory of Hemodynamics and Cardiovascular Technology,
Switzerland
Sean E. Thatcher • Department of Pharmacology and Nutritional Sciences, University
of Kentucky, Lexington, KY, USA
Rhian M. Touyz • Institute of Cardiovascular and Medical Sciences, BHF Glasgow
Cardiovascular Research Centre, University of Glasgow, Glasgow, UK
Sofia Tsiropoulou • Institute of Cardiovascular and Medical Sciences, BHF Glasgow
Cardiovascular Research Centre, University of Glasgow, Glasgow, UK
Yu Wang • Department of Pharmacology and Nutritional Sciences, University of Kentucky,
Lexington, KY, USA
Bryan A. Wilson • McAllister Heart Institute, University of North Caroliina Chapel Hill,
Winston-Salem, NC, USA
Congqing Wu • Saha Cardiovascular Research Center, University of Kentucky, Lexington,
KY, USA
Chapter 1
A Brief Introduction into the Renin-Angiotensin-

Aldosterone System: New and Old Techniques
Sean E. Thatcher
Abstract
The renin-angiotensin-aldosterone system (RAAS) is a complex system of enzymes, receptors, and pep-
tides that help to control blood pressure and fluid homeostasis. Techniques in studying the RAAS can be
difficult due to such factors as peptide/enzyme stability and receptor localization. This paper gives a brief
account of the different components of the RAAS and current methods in measuring each component.
There is also a discussion of different methods in measuring stem and immune cells by flow cytometry,
hypertension, atherosclerosis, oxidative stress, energy balance, and other RAAS-activated phenotypes.
While studies on the RAAS have been performed for over 100 years, new techniques have allowed scien-
tists to come up with new insights into this system. These techniques are detailed in this Methods in
Molecular Biology Series and give students new to studying the RAAS the proper controls and technical
details needed to perform each procedure.
Key words Angiotensin, Historical, RAAS, Techniques, Methods
1 Introduction
The renin-angiotensin-aldosterone system (RAAS) is an important

endocrine system responsible for the control of blood pressure,
sodium levels, and extracellular fluid homeostasis. Since the discov-
ery of renin, by Tiegerstedt and Bergman in 1898, a tremendous
amount of work has been published in understanding the RAAS
and the involvement of this system in pathophysiology. There are
numerous reviews on the RAAS, and this review will focus on com-
ponents of the system and assays that are now typically used in the
study of the RAAS.
1.1 Renin Renin was the first component to be measured by Tiegerstedt and
Bergman when they injected kidney extracts into rabbits and noticed
that this induced hypertension in their model [1]. Since this time,
work on purification of renin (40 kilo-dalton (kDa)) and understand-
ing the role of renin activity in plasma of different animal models has
Sean E. Thatcher (ed.), The Renin-Angiotensin-Aldosterone System: Methods and Protocols, Methods in Molecular Biology, vol. 1614,
DOI 10.1007/978-1-4939-7030-8_1, © Springer Science+Business Media LLC 2017
1
2 Sean E. Thatcher
helped to discern the overall activation of the RAAS. Renin is an

aspartate protease that cleaves angiotensinogen to the decapeptide,
angiotensin I (AngI). It has been shown that low salt or use of an
angiotensin 1 receptor (AT1R) blocker/ACE inhibitor (ACEi) can
increase renin release [2, 3]. Renin release can also be induced by
anesthetics, such as pentobarbital, so it is important to be aware of
the specific drugs used as euthanasia for experimental models [4]. It
has also been shown that giving exogenous angiotensin II (AngII)
can inhibit renin release through a negative feedback mechanism [5].
While some measures of renin could include mRNA or protein via
real-time PCR or immunoblotting respectively, the best measure-
ment of renin is through renin activity. Typically, blood is drawn from
the animal with an inhibitor cocktail that contains ethylene diamino-
tetracetic acid (EDTA), and other inhibitors of RAAS components
for aminopeptidases, endopeptidases, and serine/cysteine proteases.
The blood is then spun by centrifugation and the plasma is measured
for renin activity. For mice, this is typically done through measure-
ment of AngI, before and after giving exogenous angiotensinogen,
typically from another source, such as rat. After a period of time, the
reaction is terminated by incubating at a boiling temperature and
then the samples are taken through a radioimmunoassay (RIA) for
AngI. Some will report plasma renin concentrations (PRC) as the
difference of AngI levels before and after giving exogenous angioten-
sinogen. However, this is not the best method [4]. PRCs would be
the AngI levels before giving any exogenous substrate. These con-
centrations would be different under the above conditions, such as a
salt diet. For plasma renin activity (PRA), this would be the value of
AngI formed under exogenous angiotensinogen at 37 °C for 30 min
to 1 h of incubation. The PRA value will then allow the determina-
tion if renin activity is altered under different experimental condi-
tions. Typically, results for PRA are given as nanograms/milliliter/
hour (ng/mL/h). It is important to note that performing this assay
requires the use of both positive and negative controls to ensure the
accuracy of the measurement. An example of a positive control would
be the use of an AT1R blocker in an animal to get a higher renin
measurement. An example of a negative control could be the mea-
surement of the sample with a renin inhibitor present, such as aliski-
ren. It is also important to note that while PRA can be used to
measure systemic renin activity, this assay can be utilized to measure
renin activity in kidney or other tissues. It is also possible that under
low renin concentrations, the incubation time may need to be
extended in order to get measurable AngI levels [6]. Since radiolabels
require the investigator to have specialized protocols for handling
and disposing of radioactive waste, more biomedical companies have
pursued other alternatives, such as colorimetric kits. Again, it is up to
the user to develop the proper positive and negative controls to
determine the specificity and accuracy of these kits.
Brief Introduction to the RAAS 3
1.2 ACE Angiotensin-converting enzyme 1 (ACE1, 150–180 kDa) is a

metalloprotease that cleaves two amino acids from AngI to form
angiotensin 1-8 (Ang-(1-8) or AngII) [7]. This dipeptidyl car-
boxypeptidase has a membrane-bound as well as a cleaved form
that can be found in the circulation [8–10]. It is a glycosylated
protein that can also metabolize other substrates such as bradyki-
nin, substance P, and angiotensin-(1-7) (Ang-(1-7)) [11–14].
ACE can be found in the lung and the endothelium of blood ves-
sels, and a shorter isoform can be found in the testis [15, 16].
ACE binds to two zinc atoms located within HEMGH domains
where the histidines help in coordination of the zinc atoms.
Typically, ACE activity is measured by using a peptide substrate,
such as hippuryl-His-Leu in a solution that contains zinc chloride
in a sodium borate buffer at an optimal pH [17]. Since EDTA is
a chelating agent, then ACE activity is measured in serum. In
using this assay, a set of duplicates are made with incubation of
the ACE inhibitor, captopril. The differences between the sam-
ples will allow the user to determine the ACE-dependent activity
in a sample. It should be noted that mRNA and protein can be
measured as well for ACE; however, the activity of ACE is the
best indicator for RAAS activation. It should be noted that ACE
is also thought to act as a mechano-sensor within the aorta [18].
ACE has been shown to have RGD domains that can bind to
integrins and can be mechano-sensors in shear stress or extracel-
lular matrix disruption [19]. Typically, increases in ACE mRNA
and protein can be found in aortas that are smooth muscle defi-
cient in fibuilin-4 [18]. These functions could be independent of
the catalytic function of ACE and should also be considered when
looking at cardiovascular disease.
1.3 ACE2 Angiotensin-converting enzyme 2 (ACE2, 90–120 kDa) is also a

metalloprotease, however unlike ACE, ACE2 cleaves only one
amino acid from AngII to make Ang-(1-7) [20–22]. ACE2 is also
insensitive to ACEi [8]. This monocarboxypeptidase can be cleaved
from the membrane to make a soluble form [23]. ACE2 can be
found in the kidneys, heart, adipose tissue, blood vessels, brain, and
sex organs [21]. ACE2 can be measured using either a radiolabeled
AngII or fluorogenic substrate. The ACE2 measurement by using
125
I-AngII requires the use of an HPLC so that the radiolabeled
Ang-(1-7) fraction can be separated from the other angiotensin
peptides. Use of a fluorogenic substrate requires less equipment and
money to test for ACE2 activity; however, the difference in sub-
strate affinities must be accounted for as well. In Chapter 6 of this
book, our lab uses the fluorogenic approach to measure ACE2
activity in saline or AngII-treated abdominal aortas of mice that are
wild type or ACE2-deficient. It must be noted that again in order
to determine ACE2 specific activity that either animal knockouts or
samples treated with an ACE2 inhibitor (e.g., MLN-4760) must be
4 Sean E. Thatcher
employed. ACE2 also has the ability to cleave other substrates such
as apelin, dynorphin A, and des-Arg9-bradykinin. Again, ACE2
mRNA and protein can be measured; however, the enzymatic activ-
ity is the best indicator for activation. ACE2 also has RGD domains
that can bind to integrins in cardiomyocytes [19], and overexpres-
sion of ACE2 in the heart can protect against ischemia-induced
pathophysiology [24].
1.4 Neprilysin Neprilysin (90–100 kDa) can process AngI directly to Ang-(1-7)

[14, 25–28]. Typically, this peptidase goes up with chronic ACE
inhibition and can play a significant role in heart failure [29].
Neprilysin requires zinc to function and also has other substrates
such as enkephalins, atrial natriuretic peptide, endothelin, sub-
stance P, and bradykinin. Neprilysin has been localized to the kid-
ney, heart, and blood vessels, and recently this endopeptidase has
been located in the mitochondria of the kidney [30]. In Chapter 9,
Dr. Chappell and colleagues discuss important pathways localized
to both the mitochondria and the nucleus for the RAS. Neprilysin
can be measured using 125I-AngI as a substrate and then measuring
125
I-Ang-(1-7) by HPLC. There are also fluorescent substrates for
measurement of neprilysin, but neprilysin inhibitors such as thior-
phan and phosphoramidon should be used in these measurements
to determine neprilysin-specific activity.
1.5 Angiotensinogen Angiotensinogen is roughly a 450 amino acid protein (55–60 kDa)

that when cleaved by renin forms the decapeptide, AngI. It is a
glycoprotein that requires species-specific renin to cleave angioten-
sinogen since mouse renin does not cleave human angiotensino-
gen. Angiotensinogen can be found primarily in the liver, but has
also been found in the kidney, adipose, and other tissues [31–34].
Angiotensinogen release can be stimulated by the acute inflamma-
tory response, insulin, estrogen, glucocorticoids, and thyroid hor-
mone [35–38]. Angiotensinogen can also be elevated in women
with pre-eclampsia, and it has been suggested that the oxidized
form of angiotensinogen can be readily cleaved by renin in com-
parison to the reduced form [39]. Since angiotensinogen is a pro-
tein and not an enzyme, then Western blot or enzyme-linked
immunosorbent assay (ELISA) are preferred methods.
1.6 AT1R, AT2R, The G-protein-coupled receptors (GPCRs), such as AT1R, AT2R,
and MasR (GPCRs) and MasR, have numerous functions in the cell spanning from
activating the contractile apparatus in smooth muscle cells, induc-
ing oxidative stress or influencing nitric oxide (NO) production,
and influencing cellular proliferation and apoptosis [40–42]. Two
independent labs were responsible for the initial cloning of the
AT1R [43, 44], and in rodents there are two subtypes of the
AT1R, AT1aR and AT1bR [45]. These receptors have similar
effects in mice with both receptors playing a role in growth and
blood pressure regulation [46–48]. Unfortunately, antibodies tar-

geting the AT1aR have been unsuccessful in development and
therefore most labs utilize real-time PCR for the measurement of
these receptors [49]. Another area of research is exploring ligands
for the AT1R and other angiotensin receptors to modulate down-
stream signaling. Beta-arrestin has been shown to couple to
AT1aR and can modulate angiotensin signaling [50–52]. In
Chapter 13 of this book, Bragina and colleagues have developed a
fluorescence-based binding assay for screening ligands to the
AT1R or other angiotensin receptors. This method allows for a
high throughput of compounds that can be screened initially as
well as mutational studies that can test for binding affinity of test
compounds.
AT2R was first described in fetal and neonatal brain tissue
[53]. While expression levels typically decrease after birth, it has
been shown that adult mice deficient in AT2R have increased
systolic blood pressure and low levels of bradykinin [54]. Recent
studies have also shown that agonists for the AT2R, such as
Compound-21, can induce natriuresis and lower blood pressure
[55]. The AT2R is thought to oppose the function of the AT1R
and has been found to reduce atherosclerosis [56], autoimmune
disease [57], obesity-hypertension [58], and stroke [59].
The MasR was first originally described as an angiotensin
receptor [60]. It was not until 2003 that Ang-(1-7) was identified
as the endogenous ligand for the MasR [61]. Since that time, there
have been numerous publications showing that the MasR is
involved in vasodilation [62], reproduction [63, 64], heart con-
tractility [65], and lipid profiles [66, 67]. While antibodies have
been used to target AT2R and MasR via Western blotting, more
evidence is needed to prove that these antibodies are specific to
these GPCRs.
1.7 Angiotensin AngII is considered the central peptide of the RAAS. Since its
Peptides initial discovery in 1940 [68, 69], numerous other peptides have
now been characterized to be a part of this system. In Chapter 14,
Olkowicz and colleagues have designed a protocol that helps in
the characterization of these peptides using nano-liquid
chromatography coupled with mass spectrometry. Peptides, such
as Ang-(1-7), Ang-(1-9), angiotensin A, and alamandine, have
been shown to be the counter-regulatory arm of the AngII/AT1R
pathway and have been manipulated to block the effects of AngII
[70–77]. Recent evidence has indicated that angiotensin peptides
have different compositions depending on the tissue localization
[33, 78–82]. In Chapter 15, Grobe and Elased developed a
method for the analysis of angiotensin peptides in the kidney using
mass spectrometry. Interestingly, you can see that different pep-
tides are generated in different areas of the kidney indicating spe-
cific localization of enzymes necessary for this complex pattern
6 Sean E. Thatcher
(Figure 4 of Chapter 15). These advanced techniques can help to

provide better information on the angiotensin peptide environ-
ment under certain pathophysiological conditions and bring about
other peptidase inhibitors that may provide better efficacy against
certain types of diseases.
1.8 Aldosterone The RAAS, as well as increases in extracellular potassium ions, can
increase aldosterone secretion. Aldosterone secretion from the
adrenals can then act upon the kidneys to conserve sodium and
excrete potassium. It also causes increases in water retention and
thereby can increase blood pressure. This increase in blood pres-
sure is slower in onset than AngII and the effects it can have on
vasoconstriction of blood vessels. If aldosterone secretion is high,
such as in primary aldosteronism, renin can then be suppressed in
the kidney. While this effect is RAAS-independent, hypertensive
patients typically exhibit an over-active RAAS. In Chapter 12, Liu
et al., have shown that aldosterone in the presence of salt can
increase blood pressure and cause abdominal aortic aneurysms in
aged C57BL/6J mice [83]. This new model gives investigators
new opportunities to study cardiovascular diseases independent of
AngII. Aldosterone is a steroid hormone that is derived from cho-
lesterol. Most assays use either mass-spectrometry or ELISA-based
methods for detection [84, 85]. Aldosterone binds to the miner-
alocorticoid receptor (MR) and this receptor has been localized to
tissues such as kidney, liver, brain, ileum, brown and white adipose
tissue, heart, and blood vessels [83, 86–93].
1.9 RAAS and Energy Obesity and metabolic disorders can be a result of an imbalance of
Balance energy intake versus energy expenditure. Recent work has shown
that leptin and the AT1aR interact in the regions of the brain, such
as the subfornical organ (SFO) [94, 95]. This reaction can induce
an increase in sympathetic nerve activity (SNA) which can result in
increased thermogenesis in brown adipose tissue and browning of
white adipose tissue (beige phenotype) [96, 97]. The creation of a
double transgenic mouse, referred to as the sRA mouse, expresses
human renin controlled by the neuron-specific synapsin promoter
and the human angiotensinogen gene controlled by its own pro-
moter. Since there is species-specific cleavage of angiotensinogen
by renin, this allows for increases in brain RAAS activity that is not
influenced by the peripheral RAAS [96, 98]. Recent data has
shown that the sRA mouse has elevated energy expenditure and
thermogenesis [96]. This effect has been shown to be mediated by
elevated sympathetic activity; however, there was no reported
increase in uncoupling protein-1 (UCP-1) [96]. These changes in
metabolic regulation and increased sympathetic drive could help to
explain some facets of obesity-hypertension and in Chapter 10,
Grobe describes in great detail the proper techniques needed to
examine energy balance in mice.
1.10 RAAS Early studies were first able to show that AngII could induce
and Oxidative Stress superoxide production in hypertensive rats and that extracellular
superoxide dismutase (SOD) mRNA production could be stabi-
lized by AngII [99, 100]. Studies have also shown that AngII can
activate hydrogen peroxide (H2O2) pathways, such as p38
mitogen-activated protein kinase (p38MAPK) that can increase
vascular hypertrophy [101]. These studies have been translated
to human vascular smooth muscle cells as well and indicate that
oxidative stress can be mediated through AngII and that inhibi-
tors of phospholipase D and NADPH oxidase can block these
effects [102]. Oxidative stress has also been linked to AngII-
induced inflammation [103]. Specifically, it has been shown that
AngII can induce JAK-STAT phosphorylation which results in
increases in p47 phox, a subunit of NADPH oxidase and interleu-
kin-6 (IL-6) transcription [103]. These effects could be blocked
with the use of an NADPH oxidase inhibitor (e.g., diphenylenei-
odonium, DPI) [103]. Whether through JAK-STAT signaling or
other types of tyrosine kinases, AngII can stimulate these path-
ways through redox-sensitive mechanisms [104–106]. Likewise,
ACE2 has been shown to inhibit oxidative stress through both
JAK-STAT and MAPK pathways [107]. In Chapter 3, Tsiropoulou
and Touyz have developed a method to measure protein carbon-
ylation and protein tyrosine phosphatase oxidation in vascular
smooth muscle cells. This technique should help to discern dif-
ferences between signaling of these redox-sensitive pathways in
AngII-related diseases.
1.11 RAAS The use of ACE inhibitors has been shown to reduce atheroscle-
and Atherosclerosis rosis in a number of animal models [108–110]. Likewise, it has
been shown that AngII signaling goes up with hypercholesterol-
emia in rabbit and rodent models [111–113]. Newly developed
drugs, such as aliskiren, have also shown decreases in atheroscle-
rosis [114, 115]. While these drugs have shown benefit in
humans, the use of combination therapy, through the use of an
ARB and ACEi, has not shown any additional benefit in cardio-
vascular outcomes [116]. Recently, the ACE2/Ang-(1-7)/MasR
pathway has also shown a beneficial role in decreasing atheroscle-
rosis in animal models [73, 117–119]. While Ang-(1-7) co-infu-
sion with AngII or through diet alone has shown decreases in
atherosclerosis [73, 120], the MasR has been shown to either be
detrimental [121] or no change [67] in the development of ath-
erosclerosis. Also, downstream components in this arm, such as
MrgD and alamandine, are also found in the aorta and involved
in vessel reactivity, respectively [122], however it still has not
been determined the role, if any, these components might play in
atherosclerosis progression or regression. Recent evidence does
suggest that the RAAS can also be detected in human atheroscle-
rosis [123–126]. Methodology to measure atherosclerosis can be
8 Sean E. Thatcher
performed in the aortic arch by en face analysis or in the aortic

root [127]. Quantification can be performed in the aortic root by
lesion area; however, this does not give any information on lesion
composition. In Chapter 2 of this book, Wu and colleagues use a
color segmentation method to quantify smooth muscle and mac-
rophage accumulation within specific lesions. This method should
also add benefit to determining plaque stability as well in these
mouse models of atherosclerosis.
1.12 RAAS and Stem It has been shown that a hematopoietic peptide called N-acetyl-
Cells seryl-aspartyl-lysyl-proline (Ac-SDKP) could be hydrolyzed
in vitro by ACE1 and that this peptide can block stem cell prolif-
eration [128, 129]. It was determined that the N-terminal active
site of ACE1 could hydrolyze Ac-SDKP 50 times faster than the
C-terminal active site [129]. This led to the idea of a local RAAS
in the bone marrow [130]. In 1997, Mrug et al., showed that
AngII could stimulate proliferation of CD34+ hematopoietic pro-
genitor cells and that this effect could be blocked by losartan
[131]. Since this time, other components such as renin [132],
ACE2 [133–135], angiotensinogen [136], MasR/Ang-(1-7)
[137], and AT1aR [138] have also been found in bone marrow.
Stem cells have gained tremendous attention as possible therapies
for a number of different diseases. In Chapter 4 of this book, new
methodology for studying the RAAS in stem cells and how to
properly gate the cells using flow cytometry are described by Dr.
Yagna Jarajapu.
1.13 RAAS The importance of the kidney and regulation of blood pressure
and Hypertension was first described by Harry Goldblatt [139, 140]. These experi-
ments were typically conducted in dogs, monkeys, and other
larger mammals, however with the development of gene knock-
out models in rodents, particularly mice; it became apparent that
technology was needed for blood pressure measurements in
smaller mammals. One of the first methods described for measur-
ing blood pressure in mice was by Hicks and colleagues in
Melbourne, Australia [141]. Since this time, procedures such as
tail-cuff and radiotelemetry have been used to measure blood
pressure in mice. In Chapters 6 and 7 of this book, Yu et al., have
outlined the basic procedures for both using tail-cuff platforms
and radiotelemetry for mice. These methods allow users to best
discern which methodology would work best for his/her given
experiments. Radiotelemetry is considered the “gold standard”
when measuring blood pressure in mice and allows the user to get
more information on activity and circadian rhythms of systolic
and diastolic blood pressure compared to tail-cuff measurements
[142]. A number of genetic knockout mouse models have shown
that the RAAS is involved in the maintenance of blood pressure
[31, 47, 143–146]. Stimuli, such as high salt, saturated fat diets,
and infusion of AngII, can be used to increase blood pressure and

activate the RAAS. When utilizing these methods, it is important
to acclimate the mice before recording the blood pressure, since
stress and glucocorticoid release can also influence the blood
pressure response [147, 148]. Other considerations that can
influence the blood pressure are age, mouse strain, and sex. In
fact, numerous studies have shown sex- dependent effects of
blood pressure with RAAS activation [145, 149–155]. While
these effects can be hormone driven, new evidence now indicates
that sex chromosomes can also influence AngII-induced hyper-
tension [156, 157]. It is important for the investigator to under-
stand all of these factors that might influence blood pressure so
that he/she can best set up the experiment.
1.14 RAAS AngII has also been found to be a pro-inflammatory peptide. Early
and Inflammation studies have shown that AngII can bind to immune cells in both
human and animal models and induce oxidative stress, NADPH
oxidase activity, release of cytokines, and increased expression of
adhesion markers [158–163]. AngII has also been shown to pro-
mote lymphocyte responses that can promote kidney injury [164].
Lymphocytes can be divided into either natural killer (NK) cells,
T-cells, or B-cells. T-cells, such as CD4+ or CD8+ lymphocytes,
are a part of the adaptive immune system, and can play a role in
hypertension, obesity, and diabetes [165–167]. Flow cytometry is
a powerful technique that can allow the separation of these immune
cells that can be further used in cell culture or for other molecular
techniques, such as real-time PCR or Western blotting. In Chapter
8 of this book, Rudemiller and Crowley detail a protocol for the
isolation of T-cells in the kidney and the appropriate markers for
classes of T-cells along with cytokine products, such as tumor
necrosis factor-alpha (TNF-α) and interferon gamma (INF-γ).
Recently, it has been shown that Cluster of Differentiation 70
(CD70), a protein expressed in antigen-presenting cells, and CD27
on T-cells play a critical role in the formation of T-memory cells
[168, 169]. These T-memory cells can then play an important role
in kidney injury and recurrent hypertension [168]. T-cells have
also been shown to play a role in RAAS-activated hypertension
[170, 171], obesity-associated diabetes [172], and autoimmune
disease [173]. The RAAS-activated immune system provides many
opportunities to further understand the role of these cells within
different types of human diseases.
This is but a brief introduction to the RAAS and different
techniques utilized to measure this complex system. It is impor-
tant to realize the limitations to any research technique and the
proper controls needed to insure accurate and reliable results. I
hope that the reader finds these chapters very useful in their
future studies.
10 Sean E. Thatcher
References
1. Tigerstedt R, Bergman PQ (1898) Niere und neurotensin by converting enzyme and neutral
Kreislauf1. Skand Arch Physiol 8(1):223–271. endopeptidase. Peptides 5(4):769–776
doi:10.1111/j.1748-1716.1898.tb00272.x 12. Kohara K, Brosnihan KB, Ferrario CM (1993)
2. Stokes GS, Oates HF, Weber MA (1975) Angiotensin(1-7) in the spontaneously hyper-
Angiotensin blockade in studies of the feed- tensive rat. Peptides 14(5):883–891
back control of renin release in rats and rab- 13. Campbell WG Jr, Donohue JA, Duket LH
bits. Clin Sci Mol Med Suppl 2:33s–36s (1980) Alterations in responses to bradykinin,
3. Zanchetti A, Stella A, Leonetti G, Morganti angiotensin I, and angiotensin II during the
A, Terzoli L (1976) Control of renin release: induction phase of one-kidney, one-wrapped
a review of experimental evidence and clinical hypertension and associated arterial disease in
implications. Am J Cardiol 37(4):675–691 rabbits. Am J Pathol 98(2):457–484
4. Chappell MC (2016) Biochemical evaluation 14. Rice GI, Thomas DA, Grant PJ, Turner
of the renin-angiotensin system: the good, AJ, Hooper NM (2004) Evaluation of
bad, and absolute? Am J Physiol Heart Circ angiotensin- converting enzyme (ACE), its
Physiol 310(2):H137–H152. doi:10.1152/ homologue ACE2 and neprilysin in angio-
ajpheart.00618.2015 tensin peptide metabolism. Biochem J 383(Pt
5. McDonald KM, Taher S, Aisenbrey G, De 1):45–51. doi:10.1042/BJ20040634
Torrente A, Schrier RW (1975) Effect of 15. Bernstein KE, Martin BM, Bernstein EA,
angiotensin II and an angiotensin II inhibi- Linton J, Striker L, Striker G (1988) The
tor on renin secretion in the dog. Am J Phys isolation of angiotensin-converting enzyme
228(5):1562–1567 cDNA. J Biol Chem 263(23):11021–11024
6. Hartman D, Sagnella GA, Chesters CA, 16. Howard TE, Shai SY, Langford KG, Martin
Macgregor GA (2004) Direct renin assay and BM, Bernstein KE (1990) Transcription of
plasma renin activity assay compared. Clin testicular angiotensin-converting enzyme
Chem 50(11):2159–2161. doi:10.1373/ (ACE) is initiated within the 12th intron
clinchem.2004.033654 of the somatic ACE gene. Mol Cell Biol
7. Skeggs LT Jr, Kahn JR, Shumway NP 10(8):4294–4302
(1956) The preparation and function of the 17. Brecher P, Tercyak A, Chobanian AV (1981)
hypertensin-converting enzyme. J Exp Med Properties of angiotensin-converting enzyme
103(3):295–299 in intact cerebral microvessels. Hypertension
8. Allinson TM, Parkin ET, Condon TP, Schwager 3(2):198–204
SL, Sturrock ED, Turner AJ, Hooper NM 18. Yamashiro Y, Papke CL, Kim J, Ringuette LJ,
(2004) The role of ADAM10 and ADAM17 in Zhang QJ, Liu ZP, Mirzaei H, Wagenseil JE,
the ectodomain shedding of angiotensin con- Davis EC, Yanagisawa H (2015) Abnormal
verting enzyme and the amyloid precursor mechanosensing and cofilin activation pro-
protein. Eur J Biochem 271(12):2539–2547. mote the progression of ascending aortic
doi:10.1111/j.1432-1033.2004.04184.x aneurysms in mice. Sci Signal 8(399):ra105.
9. Woodman ZL, Oppong SY, Cook S, Hooper doi:10.1126/scisignal.aab3141
NM, Schwager SL, Brandt WF, Ehlers MR, 19. Clarke NE, Fisher MJ, Porter KE, Lambert
Sturrock ED (2000) Shedding of somatic DW, Turner AJ (2012) Angiotensin convert-
angiotensin-converting enzyme (ACE) is ing enzyme (ACE) and ACE2 bind integrins
inefficient compared with testis ACE despite and ACE2 regulates integrin signalling. PLoS
cleavage at identical stalk sites. Biochem One 7(4):e34747. doi:10.1371/journal.
J 347(Pt 3):711–718 pone.0034747. PONE-D-11-26032 [pii]
10. Danilov SM, Gordon K, Nesterovitch AB, 20. Tipnis SR, Hooper NM, Hyde R, Karran
Lunsdorf H, Chen Z, Castellon M, Popova E, Christie G, Turner AJ (2000) A human
IA, Kalinin S, Mendonca E, Petukhov PA, homolog of angiotensin-converting enzyme.
Schwartz DE, Minshall RD, Sturrock ED Cloning and functional expression as a
(2011) An angiotensin I-converting enzyme captopril-insensitive carboxypeptidase. J Biol
mutation (Y465D) causes a dramatic increase Chem 275(43):33238–33243. doi:10.1074/
in blood ACE via accelerated ACE shedding. jbc.M002615200. M002615200 [pii]
PLoS One 6(10):e25952. doi:10.1371/jour- 21. Donoghue M, Hsieh F, Baronas E, Godbout K,
nal.pone.0025952 Gosselin M, Stagliano N, Donovan M, Woolf
11. Skidgel RA, Engelbrecht S, Johnson AR, Erdos B, Robison K, Jeyaseelan R, Breitbart RE,
EG (1984) Hydrolysis of substance p and Acton S (2000) A novel angiotensin-converting
enzyme-related carboxypeptidase (ACE2) con- Gong M, Cassis LA (2015) Deficiency of angio-

verts angiotensin I to angiotensin 1-9. Circ Res tensinogen in hepatocytes markedly decreases
87(5):E1–E9 blood pressure in lean and obese male mice.
22. Vickers C, Hales P, Kaushik V, Dick L, Gavin Hypertension 66(4):836–842. doi:10.1161/
J, Tang J, Godbout K, Parsons T, Baronas HYPERTENSIONAHA.115.06040
E, Hsieh F, Acton S, Patane M, Nichols A, 32. Yiannikouris F, Gupte M, Putnam K, Thatcher
Tummino P (2002) Hydrolysis of biological S, Charnigo R, Rateri DL, Daugherty A,
peptides by human angiotensin-converting Cassis LA (2012) Adipocyte deficiency of
enzyme-related carboxypeptidase. J Biol angiotensinogen prevents obesity-induced
Chem 277(17):14838–14843. doi:10.1074/ hypertension in male mice. Hyperten
jbc.M200581200. M200581200 [pii] sion 60(6):1524–1530. doi:10.1161/HYPER
23. Lambert DW, Yarski M, Warner FJ, Thornhill TENSIONAHA.112.192690
P, Parkin ET, Smith AI, Hooper NM, Turner 33. Dzau VJ, Brody T, Ellison KE, Pratt RE,
AJ (2005) Tumor necrosis factor-alpha con- Ingelfinger JR (1987) Tissue-specific regu-
vertase (ADAM17) mediates regulated lation of renin expression in the mouse.
ectodomain shedding of the severe-acute respi- Hypertension 9(6 Pt 2):III36–III41
ratory syndrome-coronavirus (SARS- CoV) 34. Gomez RA, Cassis L, Lynch KR, Chevalier
receptor, angiotensin-converting enzyme-2 RL, Wilfong N, Carey RM, Peach MJ (1988)
(ACE2). J Biol Chem 280(34):30113–30119. Fetal expression of the angiotensinogen
doi:10.1074/jbc.M505111200 gene. Endocrinology 123(5):2298–2302.
24. Der Sarkissian S, Grobe JL, Yuan L, Narielwala doi:10.1210/endo-123-5-2298
DR, Walter GA, Katovich MJ, Raizada MK 35. Itoh N, Matsuda T, Ohtani R, Okamoto H
(2008) Cardiac overexpression of angioten- (1989) Angiotensinogen production by rat hep-
sin converting enzyme 2 protects the heart atoma cells is stimulated by B cell stimulatory
from ischemia-induced pathophysiology. factor 2/interleukin-6. FEBS Lett 244(1):6–10
Hypertension 51(3):712–718. doi:10.1161/ 36. Murakami E, Hiwada K, Kokubu T (1980)
HYPERTENSIONAHA.107.100693 Effects of insulin and glucagon on produc-
25. Welches WR, Brosnihan KB, Ferrario CM tion of renin substrate by the isolated rat liver.
(1993) A comparison of the properties and J Endocrinol 85(1):151–153
enzymatic activities of three angiotensin process- 37. Krakoff LR, Eisenfeld AJ (1977) Hormonal
ing enzymes: angiotensin converting enzyme, control of plasma renin substrate; (angioten-
prolyl endopeptidase and neutral endopeptidase sinogen). Circ Res 41(4 Suppl 2):43–46
24.11. Life Sci 52(18):1461–1480
38. Dzau VJ, Herrmann HC (1982) Hormonal
26. Iyer SN, Ferrario CM, Chappell MC (1998) control of angiotensinogen production. Life
Angiotensin-(1-7) contributes to the antihy- Sci 30(7–8):577–584
pertensive effects of blockade of the renin-
angiotensin system. Hypertension 31(1 Pt 39. Zhou A, Carrell RW, Murphy MP, Wei Z, Yan
2):356–361 Y, Stanley PL, Stein PE, Broughton Pipkin
F, Read RJ (2010) A redox switch in angio-
27. Ferrario CM, Chappell MC, Dean RH, Iyer tensinogen modulates angiotensin release.
SN (1998) Novel angiotensin peptides regulate Nature 468(7320):108–111. doi:10.1038/
blood pressure, endothelial function, and natri- nature09505
uresis. J Am Soc Nephrol 9(9):1716–1722
40. Ryan MJ, Didion SP, Mathur S, Faraci
28. Ishida M, Ogawa M, Kosaki G, Mega T, FM, Sigmund CD (2004) Angiotensin
Ikenaka T (1983) Purification and charac- II-induced vascular dysfunction is mediated
terization of the neutral endopeptidase from by the AT1A receptor in mice. Hypertension
human kidney. J Biochem 94(1):17–24 43(5):1074–1079. doi:10.1161/01.HYP.
29. Lim GB (2014) Heart failure: LCZ696—a 0000123074.89717.3d
PARADIGM shift in treatment for heart 41. Mehta PK, Griendling KK (2007) Angiotensin
failure. Nat Rev Cardiol 11(11):618. II cell signaling: physiological and pathologi-
doi:10.1038/nrcardio.2014.139 cal effects in the cardiovascular system. Am
30. Wilson BA, Nautiyal M, Gwathmey TM, Rose J Physiol Cell Physiol 292(1):C82–C97.
JC, Chappell MC (2015) Evidence for a mito- doi:10.1152/ajpcell.00287.2006
chondrial angiotensin-(1-7) system in the kid- 42. Simoes e Silva AC, Silveira KD, Ferreira AJ,
ney. Am J Physiol Renal Physiol 00479:02015. Teixeira MM (2013) ACE2, angiotensin-(1-7)
doi:10.1152/ajprenal.00479.2015 and mas receptor axis in inflammation and
31. Yiannikouris F, Wang Y, Shoemaker R, Larian fibrosis. Br J Pharmacol 169(3):477–492.
N, Thompson J, English VL, Charnigo R, Su W, doi:10.1111/bph.12159
12 Sean E. Thatcher
43. Murphy TJ, Alexander RW, Griendling KK, by mechanical stress. Sci Signal 3(125):ra46.
Runge MS, Bernstein KE (1991) Isolation of doi:10.1126/scisignal.2000769
a cDNA encoding the vascular type-1 angio- 53. Nuyt AM, Lenkei Z, Palkovits M, Corvol P,
tensin II receptor. Nature 351(6323):233– Llorens-Cortes C (1999) Ontogeny of angio-
236. doi:10.1038/351233a0 tensin II type 2 receptor mRNA expression in
44. Sasaki K, Yamano Y, Bardhan S, Iwai N, fetal and neonatal rat brain. J Comp Neurol
Murray JJ, Hasegawa M, Matsuda Y, 407(2):193–206
Inagami T (1991) Cloning and expres- 54. Siragy HM, Inagami T, Ichiki T, Carey RM
sion of a complementary DNA encod- (1999) Sustained hypersensitivity to angioten-
ing a bovine adrenal angiotensin II type-1 sin II and its mechanism in mice lacking the
receptor. Nature 351(6323):230–233. subtype-2 (AT2) angiotensin receptor. Proc
doi:10.1038/351230a0 Natl Acad Sci U S A 96(11):6506–6510
45. Burson JM, Aguilera G, Gross KW, Sigmund 55. Kemp BA, Howell NL, Gildea JJ, Keller SR,
CD (1994) Differential expression of angio- Padia SH, Carey RM (2014) AT(2) recep-
tensin receptor 1A and 1B in mouse. Am tor activation induces natriuresis and lowers
J Phys 267(2 Pt 1):E260–E267 blood pressure. Circ Res 115(3):388–399.
46. Ito M, Oliverio MI, Mannon PJ, Best CF, doi:10.1161/CIRCRESAHA.115.304110
Maeda N, Smithies O, Coffman TM (1995) 56. Sales VL, Sukhova GK, Lopez-Ilasaca MA,
Regulation of blood pressure by the type 1A Libby P, Dzau VJ, Pratt RE (2005) Angiotensin
angiotensin II receptor gene. Proc Natl Acad type 2 receptor is expressed in murine athero-
Sci U S A 92(8):3521–3525 sclerotic lesions and modulates lesion evolution.
47. Oliverio MI, Best CF, Kim HS, Arendshorst Circulation 112(21):3328–3336. doi:10.1161/
WJ, Smithies O, Coffman TM (1997) CIRCULATIONAHA.105.541714
Angiotensin II responses in AT1A receptor- 57. Valero-Esquitino V, Lucht K, Namsolleck P,
deficient mice: a role for AT1B receptors in Monnet-Tschudi F, Stubbe T, Lucht F, Liu
blood pressure regulation. Am J Phys 272(4 M, Ebner F, Brandt C, Danyel LA, Villela
Pt 2):F515–F520 DC, Paulis L, Thoene-Reineke C, Dahlof B,
48. Poduri A, Owens AP 3rd, Howatt DA, Hallberg A, Unger T, Sumners C, Steckelings
Moorleghen JJ, Balakrishnan A, Cassis LA, UM (2015) Direct angiotensin type 2 recep-
Daugherty A (2012) Regional variation in tor (AT2R) stimulation attenuates T-cell
aortic AT1b receptor mRNA abundance is and microglia activation and prevents demy-
associated with contractility but unrelated to elination in experimental autoimmune
atherosclerosis and aortic aneurysms. PLoS encephalomyelitis in mice. Clin Sci (Lond)
One 7(10):e48462. doi:10.1371/journal. 128(2):95–109. doi:10.1042/CS20130601
pone.0048462 58. Ali Q, Wu Y, Hussain T (2013) Chronic AT2
49. Herrera M, Sparks MA, Alfonso-Pecchio receptor activation increases renal ACE2 activ-
AR, Harrison-Bernard LM, Coffman TM ity, attenuates AT1 receptor function and
(2013) Lack of specificity of commer- blood pressure in obese Zucker rats. Kidney Int
cial antibodies leads to misidentification 84(5):931–939. doi:10.1038/ki.2013.193
of angiotensin type 1 receptor protein. 59. Joseph JP, Mecca AP, Regenhardt RW,
Hypertension 61(1):253–258. doi:10.1161/ Bennion DM, Rodriguez V, Desland F, Patel
HYPERTENSIONAHA.112.203679 NA, Pioquinto DJ, Unger T, Katovich MJ,
50. Zhang J, Barak LS, Anborgh PH, Laporte SA, Steckelings UM, Sumners C (2014) The angio-
Caron MG, Ferguson SS (1999) Cellular traf- tensin type 2 receptor agonist compound 21 elic-
ficking of G protein-coupled receptor/beta- its cerebroprotection in endothelin- 1 induced
arrestin endocytic complexes. J Biol Chem ischemic stroke. Neuropharmacology 81:134–
274(16):10999–11006 141. doi:10.1016/j.neuropharm.2014.01.044
51. Rajagopal K, Whalen EJ, Violin JD, Stiber 60. Jackson TR, Blair LA, Marshall J, Goedert
JA, Rosenberg PB, Premont RT, Coffman M, Hanley MR (1988) The mas oncogene
TM, Rockman HA, Lefkowitz RJ (2006) encodes an angiotensin receptor. Nature
Beta-arrestin2-mediated inotropic effects of 335(6189):437–440. doi:10.1038/335437a0
the angiotensin II type 1A receptor in iso- 61. Santos RA, Simoes e Silva AC, Maric C, Silva
lated cardiac myocytes. Proc Natl Acad Sci U DM, Machado RP, de Buhr I, Heringer-
S A 103(44):16284–16289. doi:10.1073/ Walther S, Pinheiro SV, Lopes MT, Bader M,
pnas.0607583103 Mendes EP, Lemos VS, Campagnole-Santos
52. Rakesh K, Yoo B, Kim IM, Salazar N, Kim MJ, Schultheiss HP, Speth R, Walther T
KS, Rockman HA (2010) Beta-arrestin-biased (2003) Angiotensin-(1-7) is an endogenous
agonism of the angiotensin receptor induced ligand for the G protein-coupled receptor
mas. Proc Natl Acad Sci U S A 100(14):8258– 71. Sampaio WO, Henrique de Castro C,
8263. doi:10.1073/pnas.1432869100 Santos RA, Schiffrin EL, Touyz RM (2007)
62. Lemos VS, Silva DM, Walther T, Alenina Angiotensin-(1-7) counterregulates angio-
N, Bader M, Santos RA (2005) The tensin II signaling in human endothelial cells.
endothelium-dependent vasodilator effect of Hypertension 50(6):1093–1098. doi:10.1161/
the nonpeptide Ang(1-7) mimic AVE 0991 is HYPERTENSIONAHA.106.084848.
abolished in the aorta of mas-knockout mice. HYPERTENSIONAHA.106.084848 [pii]
J Cardiovasc Pharmacol 46(3):274–279 72. Hayashi N, Yamamoto K, Ohishi M, Tatara
63. Reis AB, Araujo FC, Pereira VM, Dos Reis Y, Takeya Y, Shiota A, Oguro R, Iwamoto Y,
AM, Santos RA, Reis FM (2010) Angiotensin Takeda M, Rakugi H (2010) The counter-
(1-7) and its receptor mas are expressed in the regulating role of ACE2 and ACE2-mediated
human testis: implications for male infertil- angiotensin 1-7 signaling against angiotensin
ity. J Mol Histol 41(1):75–80. doi:10.1007/ II stimulation in vascular cells. Hypertens Res
s10735-010-9264-8 33(11):1182–1185. doi:10.1038/hr.2010.
64. Leal MC, Pinheiro SV, Ferreira AJ, Santos 147. hr2010147 [pii]
RA, Bordoni LS, Alenina N, Bader M, Franca 73. Thatcher SE, Zhang X, Howatt DA, Lu H,
LR (2009) The role of angiotensin-(1-7) Gurley SB, Daugherty A, Cassis LA (2011)
receptor mas in spermatogenesis in mice and Angiotensin-converting enzyme 2 deficiency
rats. J Anat 214(5):736–743. doi:10.1111/ in whole body or bone marrow-derived cells
j.1469-7580.2009.01058.x increases atherosclerosis in low-density lipo-
65. Santos RA, Castro CH, Gava E, Pinheiro SV, protein receptor-/- mice. Arterioscler Thromb
Almeida AP, Paula RD, Cruz JS, Ramos AS, Vasc Biol 31(4):758–765. doi:10.1161/
Rosa KT, Irigoyen MC, Bader M, Alenina N, atvbaha.110.221614
Kitten GT, Ferreira AJ (2006) Impairment of 74. Zhang F, Hu Y, Xu Q, Ye S (2010) Different
in vitro and in vivo heart function in angio- effects of angiotensin II and angiotensin-(1-7)
tensin-(1-7) receptor MAS knockout mice. on vascular smooth muscle cell prolifera-
Hypertension 47(5):996–1002. doi:10.1161/ tion and migration. PLoS One 5(8):e12323.
01.HYP.0000215289.51180.5c doi:10.1371/journal.pone.0012323
66. Santos SH, Fernandes LR, Mario EG, Ferreira 75. Lautner RQ, Villela DC, Fraga-Silva RA, Silva
AV, Porto LC, Alvarez-Leite JI, Botion LM, N, Verano-Braga T, Costa-Fraga F, Jankowski
Bader M, Alenina N, Santos RA (2008) Mas J, Jankowski V, Sousa F, Alzamora A, Soares
deficiency in FVB/N mice produces marked E, Barbosa C, Kjeldsen F, Oliveira A, Braga J,
changes in lipid and glycemic metabolism. Savergnini S, Maia G, Peluso AB, Passos-Silva
Diabetes 57(2):340–347. doi:10.2337/ D, Ferreira A, Alves F, Martins A, Raizada
db07-0953 M, Paula R, Motta-Santos D, Klempin F,
67. Silva AR, Aguilar EC, Alvarez-Leite JI, da Pimenta A, Alenina N, Sinisterra R, Bader M,
Silva RF, Arantes RM, Bader M, Alenina N, Campagnole-Santos MJ, Santos RA (2013)
Pelli G, Lenglet S, Galan K, Montecucco F, Discovery and characterization of alamandine:
Mach F, Santos SH, Santos RA (2013) Mas a novel component of the renin-angiotensin
receptor deficiency is associated with wors- system. Circ Res 112(8):1104–1111.
ening of lipid profile and severe hepatic ste- doi:10.1161/CIRCRESAHA.113.301077
atosis in ApoE-knockout mice. Am J Physiol 76. Chen Z, Tan F, Erdos EG, Deddish PA (2005)
Regul Integr Comp Physiol 305(11):R1323– Hydrolysis of angiotensin peptides by human
R1330. doi:10.1152/ajpregu.00249.2013 angiotensin I-converting enzyme and the resen-
68. Page IH, Helmer OM (1940) A crystalline sitization of B2 kinin receptors. Hypertension
pressor substance (Angiotonin) resulting 46(6):1368–1373. doi:10.1161/01.HYP.
from the reaction between renin and renin- 0000188905.20884.63
activator. J Exp Med 71(1):29–42 77. Coutinho DC, Foureaux G, Rodrigues KD,
69. Menendez EB, Fasciolo JC, Houssay BA, Leloir Salles RL, Moraes PL, Murca TM, De Maria
LF, Munoz JM, Taquini AC (1943) Angiotonin ML, Gomes ER, Santos RA, Guatimosim S,
or hypertensin. Science 98(2553):495. Ferreira AJ (2014) Cardiovascular effects of
doi:10.1126/science.98.2553.495 angiotensin A: a novel peptide of the renin-
angiotensin system. J Renin-Angiotensin-
70. Zhang F, Ren X, Zhao M, Zhou B, Han Y Aldosterone Syst 15(4):480–486. doi:10.1177/
(2016) Angiotensin-(1-7) abrogates angio- 1470320312474856
tensin II-induced proliferation, migration and
inflammation in VSMCs through inactivation 78. Grobe N, Weir NM, Leiva O, Ong FS,
of ROS-mediated PI3K/Akt and MAPK/ Bernstein KE, Schmaier AH, Morris M,
ERK signaling pathways. Sci Rep 6:34621. Elased KM (2013) Identification of prolyl
doi:10.1038/srep34621 carboxypeptidase as an alternative enzyme
14 Sean E. Thatcher
for processing of renal angiotensin II using 89. Zennaro MC, Le Menuet D, Viengchareun
mass spectrometry. Am J Physiol Cell S, Walker F, Ricquier D, Lombes M (1998)
Physiol 304(10):C945–C953. doi:10.1152/ Hibernoma development in transgenic mice
ajpcell.00346.2012 identifies brown adipose tissue as a novel
79. Grobe N, Elased KM, Cool DR, Morris M target of aldosterone action. J Clin Invest
(2012) Mass spectrometry for the molecular 101(6):1254–1260. doi:10.1172/JCI1915
imaging of angiotensin metabolism in kidney. 90. Liu G, Grifman M, Keily B, Chatterton JE,
Am J Physiol Endocrinol Metab 302(8):E1016– Staal FW, Li QX (2006) Mineralocorticoid
E1024. doi:10.1152/ajpendo.00515.2011 receptor is involved in the regulation of
80. Carey RM, Levens NR, Peach MJ (1980) genes responsible for hepatic glucose pro-
Studies of the functional role of the intrare- duction. Biochem Biophys Res Commun
nal renin-angiotensin system. Prog Biochem 342(4):1291–1296. doi:10.1016/j.
Pharmacol 17:6–13 bbrc.2006.02.065
81. Cassis LA, Saye J, Peach MJ (1988) Location 91. Carruthers BM, Ledray RD, Seraglia M,
and regulation of rat angiotensinogen messen- McIntosh HW, Walsh GC (1963) Effect of an
ger RNA. Hypertension 11(6 Pt 2):591–596 aldosterone antagonist (spironolactone) on
82. Schelling P, Ganten U, Sponer G, Unger T, patients with severe congestive heart failure.
Ganten D (1980) Components of the renin- Can Med Assoc J 89:633–641
angiotensin system in the cerebrospinal fluid 92. Lazar G, Pagano M, Agarwal MK (1990)
of rats and dogs with special consideration Purification and characterization of the
of the origin and the fate of angiotensin activated mineralocorticoid receptor from
II. Neuroendocrinology 31(5):297–308 rat myocardium. Biochim Biophys Acta
83. Liu S, Xie Z, Daugherty A, Cassis LA, 1033(1):41–48
Pearson KJ, Gong MC, Guo Z (2013) 93. Kornel L, Kanamarlapudi N, Travers T, Taff
Mineralocorticoid receptor agonists induce DJ, Patel N, Chen C, Baum RM, Raynor WJ
mouse aortic aneurysm formation and rup- (1982) Studies on high affinity binding of
ture in the presence of high salt. Arterioscler mineralo- and glucocorticoids in rabbit aorta
Thromb Vasc Biol 33(7):1568–1579. cytosol. J Steroid Biochem 16(2):245–264
doi:10.1161/ATVBAHA.112.300820 94. Hilzendeger AM, Morgan DA, Brooks L,
84. Schwartz F, Hadas E, Harnik M, Solomon Dellsperger D, Liu X, Grobe JL, Rahmouni
B (1990) Enzyme-linked immunosorbent K, Sigmund CD, Mark AL (2012) A brain
assays for determination of plasma aldo- leptin-renin angiotensin system interac-
sterone using highly specific polyclonal tion in the regulation of sympathetic nerve
antibodies. J Immunoass 11(2):215–234. activity. Am J Physiol Heart Circ Physiol
doi:10.1080/01971529008053270 303(2):H197–H206. doi:10.1152/ajpheart.
85. Prome D, Viger A, Marquet A (1988) Use 00974.2011
of tandem mass spectrometry (MS-MS) for 95. Young CN, Morgan DA, Butler SD, Rahmouni
aldosterone assay at the nanogram level in K, Gurley SB, Coffman TM, Mark AL, Davisson
complex biological mixtures. Anal Biochem RL (2015) Angiotensin type 1a receptors in the
172(1):264–269 forebrain subfornical organ facilitate leptin-
86. Lombes M, Farman N, Oblin ME, Baulieu induced weight loss through brown adipose tis-
EE, Bonvalet JP, Erlanger BF, Gasc JM sue thermogenesis. Mol Metab 4(4):337–343.
(1990) Immunohistochemical localization doi:10.1016/j.molmet.2015.01.007
of renal mineralocorticoid receptor by using 96. Grobe JL, Grobe CL, Beltz TG, Westphal SG,
an anti-idiotypic antibody that is an internal Morgan DA, Xu D, de Lange WJ, Li H, Sakai
image of aldosterone. Proc Natl Acad Sci U S K, Thedens DR, Cassis LA, Rahmouni K, Mark
A 87(3):1086–1088 AL, Johnson AK, Sigmund CD (2010) The
87. Van Eekelen JA, Jiang W, De Kloet ER, Bohn brain renin-angiotensin system controls diver-
MC (1988) Distribution of the mineralocorti- gent efferent mechanisms to regulate fluid and
coid and the glucocorticoid receptor mRNAs energy balance. Cell Metab 12(5):431–442.
in the rat hippocampus. J Neurosci Res doi:10.1016/j.cmet.2010.09.011
21(1):88–94. doi:10.1002/jnr.490210113 97. Contreras C, Nogueiras R, Dieguez C,
88. Fukushima K, Sasano H, Sasaki I, Nagura H, Medina-Gomez G, Lopez M (2016)
Funayama Y, Matsuno S (1994) Increased Hypothalamus and thermogenesis: heat-
expression of mineralocorticoid receptor in ing the BAT, browning the WAT. Mol Cell
human ileum after total colectomy: immuno- Endocrinol. doi:10.1016/j.mce.2016.08.002
histochemical and immunoblotting studies. 98. Littlejohn NK, Keen HL, Weidemann BJ,
Tohoku J Exp Med 173(4):383–390 Claflin KE, Tobin KV, Markan KR, Park S,
Naber MC, Gourronc FA, Pearson NA, Liu signaling pathways. Regul Pept 185:44–51.
X, Morgan DA, Klingelhutz AJ, Potthoff MJ, doi:10.1016/j.regpep.2013.06.007
Rahmouni K, Sigmund CD, Grobe JL (2016) 108. Campbell JH, Fennessy P, Campbell GR

Suppression of resting metabolism by the angio- (1992) Effect of perindopril on the develop-
tensin AT2 receptor. Cell Rep 16(6):1548– ment of atherosclerosis in the cholesterol-fed
1560. doi:10.1016/j.celrep.2016.07.003 rabbit. Clin Exp Pharmacol Physiol Suppl
99. Laursen JB, Rajagopalan S, Galis Z, Tarpey 19:13–17
M, Freeman BA, Harrison DG (1997) Role 109. Ambrosioni E, Bacchelli S, Degli Esposti D,
of superoxide in angiotensin II-induced but Borghi C (1992) ACE-inhibitors and athero-
not catecholamine-induced hypertension. sclerosis. Eur J Epidemiol 8(Suppl 1):129–133
Circulation 95(3):588–593 110. Chobanian AV, Haudenschild CC, Nickerson
100. Fukai T, Siegfried MR, Ushio-Fukai M,
C, Hope S (1992) Trandolapril inhibits ath-
Griendling KK, Harrison DG (1999) erosclerosis in the Watanabe heritable hyper-
Modulation of extracellular superoxide dis- lipidemic rabbit. Hypertension 20(4):473–477
mutase expression by angiotensin II and 111. Nickenig G, Jung O, Strehlow K, Zolk O,
hypertension. Circ Res 85(1):23–28 Linz W, Scholkens BA, Bohm M (1997)
101. Ushio-Fukai M, Alexander RW, Akers M,
Hypercholesterolemia is associated with
Griendling KK (1998) p38 mitogen-acti- enhanced angiotensin AT1-receptor expres-
vated protein kinase is a critical component sion. Am J Phys 272(6 Pt 2):H2701–H2707
of the redox-sensitive signaling pathways 112. Daugherty A, Rateri DL, Lu H, Inagami T,
activated by angiotensin II. Role in vascular Cassis LA (2004) Hypercholesterolemia stim-
smooth muscle cell hypertrophy. J Biol Chem ulates angiotensin peptide synthesis and con-
273(24):15022–15029 tributes to atherosclerosis through the AT1A
102.
Touyz RM, Schiffrin EL (1999) Ang receptor. Circulation 110(25):3849–3857.
II-stimulated superoxide production is medi- doi:10.1161/01.CIR.0000150540.54220.
ated via phospholipase D in human vascular C4. 01.CIR.0000150540.54220.C4 [pii]
smooth muscle cells. Hypertension 34(4 Pt 113. Hirano T, Ran J, Adachi M (2006) Opposing
2):976–982 actions of angiotensin II type 1 and 2 recep-
103. Schieffer B, Luchtefeld M, Braun S, Hilfiker tors on plasma cholesterol levels in rats.
A, Hilfiker-Kleiner D, Drexler H (2000) J Hypertens 24(1):103–108
Role of NAD(P)H oxidase in angiotensin 114. Lu H, Balakrishnan A, Howatt DA, Wu C,
II-induced JAK/STAT signaling and cytokine Charnigo R, Liau G, Cassis LA, Daugherty
induction. Circ Res 87(12):1195–1201 A (2012) Comparative effects of different
104. Du J, Peng T, Scheidegger KJ, Delafontaine modes of renin angiotensin system inhibition
P (1999) Angiotensin II activation of insulin- on hypercholesterolaemia-induced athero-
like growth factor 1 receptor transcription sclerosis. Br J Pharmacol 165(6):2000–2008.
is mediated by a tyrosine kinase-dependent doi:10.1111/j.1476-5381.2011.01712.x
redox-sensitive mechanism. Arterioscler 115. Lu H, Rateri DL, Feldman DL, Charnigo

Thromb Vasc Biol 19(9):2119–2126 RJ Jr, Fukamizu A, Ishida J, Oesterling
105.
Modesti A, Bertolozzi I, Gamberi T, EG, Cassis LA, Daugherty A (2008) Renin
Marchetta M, Lumachi C, Coppo M, Moroni inhibition reduces hypercholesterolemia-
F, Toscano T, Lucchese G, Gensini GF, induced atherosclerosis in mice. J Clin Invest
Modesti PA (2005) Hyperglycemia activates 118(3):984–993. doi:10.1172/JCI32970
JAK2 signaling pathway in human failing 116. Investigators O, Yusuf S, Teo KK, Pogue J,
myocytes via angiotensin II-mediated oxida- Dyal L, Copland I, Schumacher H, Dagenais
tive stress. Diabetes 54(2):394–401 G, Sleight P, Anderson C (2008) Telmisartan,

106. Griendling KK, Sorescu D, Lassegue B, ramipril, or both in patients at high risk for
Ushio-Fukai M (2000) Modulation of protein vascular events. N Engl J Med 358(15):1547–
kinase activity and gene expression by reac- 1559. doi:10.1056/NEJMoa0801317
tive oxygen species and their role in vascular 117. Zulli A, Burrell LM, Widdop RE, Black

physiology and pathophysiology. Arterioscler MJ, Buxton BF, Hare DL (2006)
Thromb Vasc Biol 20(10):2175–2183 Immunolocalization of ACE2 and AT2
107. Song B, Jin H, Yu X, Zhang Z, Yu H, Ye J, receptors in rabbit atherosclerotic plaques.
Xu Y, Zhou T, Oudit GY, Ye JY, Chen C, Gao J Histochem Cytochem 54(2):147–150.
P, Zhu D, Penninger JM, Zhong JC (2013) doi:10.1369/jhc.5C6782.2005
Angiotensin-converting enzyme 2 attenuates 118. Thomas MC, Pickering RJ, Tsorotes D, Koitka
oxidative stress and VSMC proliferation via the A, Sheehy K, Bernardi S, Toffoli B, Nguyen-
JAK2/STAT3/SOCS3 and profilin-1/MAPK Huu TP, Head GA, Fu Y, Chin- Dusting J,
16 Sean E. Thatcher
Cooper ME, Tikellis C (2010) Genetic Ace2 126. Clancy P, Koblar S, Golledge J (2016)

deficiency accentuates vascular inflammation and Involvement of angiotensin II type 1 and 2
atherosclerosis in the ApoE knockout mouse. receptors in gelatinase regulation in human
Circ Res 107(7):888–897. doi:10.1161/ carotid atheroma in vitro. J Atheroscler
CIRCRESAHA.110.219279 Thromb 23(7):773–791. doi:10.5551/
119. Zhang C, Zhao YX, Zhang YH, Zhu L,
jat.31401
Deng BP, Zhou ZL, Li SY, Lu XT, Song 127.
Daugherty A, Whitman SC (2003)
LL, Lei XM, Tang WB, Wang N, Pan CM, Quantification of atherosclerosis in mice.
Song HD, Liu CX, Dong B, Zhang Y, Cao Methods Mol Biol 209:293–309
Y (2010) Angiotensin-converting enzyme 128. Azizi M, Rousseau A, Ezan E, Guyene TT,
2 attenuates atherosclerotic lesions by tar- Michelet S, Grognet JM, Lenfant M, Corvol P,
geting vascular cells. Proc Natl Acad Sci U Menard J (1996) Acute angiotensin-converting
S A 107(36):15886–15891. doi:10.1073/ enzyme inhibition increases the plasma level of
pnas.1001253107 the natural stem cell regulator N-acetyl-seryl-
120. Tesanovic S, Vinh A, Gaspari TA, Casley D, aspartyl-lysyl-proline. J Clin Invest 97(3):839–
Widdop RE (2010) Vasoprotective and ath- 844. doi:10.1172/JCI118484
eroprotective effects of angiotensin (1-7) in 129. Rousseau A, Michaud A, Chauvet MT,

apolipoprotein E-deficient mice. Arterioscler Lenfant M, Corvol P (1995) The hemoregu-
Thromb Vasc Biol 30(8):1606–1613. latory peptide N-acetyl-Ser-Asp-Lys-Pro is a
doi:10.1161/ATVBAHA.110.204453. natural and specific substrate of the N-terminal
ATVBAHA.110.204453 [pii] active site of human angiotensin-converting
121. Pernomian L, do Prado AF, Gomes MS,
enzyme. J Biol Chem 270(8):3656–3661
Pernomian L, da Silva CH, Gerlach RF, de 130. Haznedaroglu IC, Tuncer S, Gursoy M (1996)
Oliveira AM (2015) MAS receptors medi- A local renin-angiotensin system in the bone
ate vasoprotective and atheroprotective marrow. Med Hypotheses 46(6):507–510
effects of candesartan upon the recovery 131. Mrug M, Stopka T, Julian BA, Prchal JF,

of vascular angiotensin-converting enzyme Prchal JT (1997) Angiotensin II stimulates
2-angiotensin-(1-7)-MAS axis function- proliferation of normal early erythroid pro-
ality. Eur J Pharmacol 764:173–188. genitors. J Clin Invest 100(9):2310–2314.
doi:10.1016/j.ejphar.2015.07.007 doi:10.1172/JCI119769
122. Habiyakare B, Alsaadon H, Mathai ML, Hayes 132. Haznedaroglu IC, Savas MC, Benekli M

A, Zulli A (2014) Reduction of angiotensin (1997) Renin-like activity in leukemic blast
A and alamandine vasoactivity in the rabbit cells: an initial clue to a local renin-angiotensin
model of atherogenesis: differential effects of system in the bone marrow. Ann Hematol
alamandine and Ang(1-7). Int J Exp Pathol 75(1–2):69–70
95(4):290–295. doi:10.1111/iep.12087
133. Joshi S, Balasubramanian N, Vasam G,

123. Nehme A, Marcelo P, Nasser R, Kobeissy
Jarajapu YP (2016) Angiotensin convert-
F, Bricca G, Zibara K (2016) The kinet- ing enzyme versus angiotensin convert-
ics of angiotensin-I metabolism in human ing enzyme-2 selectivity of MLN-4760 and
carotid atheroma: an emerging role for angio- DX600 in human and murine bone marrow-
tensin (1-7). Vasc Pharmacol 85:50–56. derived cells. Eur J Pharmacol 774:25–33.
doi:10.1016/j.vph.2016.08.001 doi:10.1016/j.ejphar.2016.01.007
124. Clancy P, Seto SW, Koblar SA, Golledge
134. Thatcher SE, Gupte M, Hatch N, Cassis

J (2013) Role of the angiotensin con- LA (2012) Deficiency of ACE2 in bone-
verting enzyme 1/angiotensin II/ marrow-derived cells increases expression of
angiotensin receptor 1 axis in interstitial TNF-alpha in adipose stromal cells and aug-
collagenase expression in human carotid ments glucose intolerance in obese C57BL/6
atheroma. Atherosclerosis 229(2):331–337. mice. Int J Hypertens 2012:762094. doi:
doi:10.1016/j.atherosclerosis.2013.05.022 10.1155/2012/762094
125. Clancy P, Koblar SA, Golledge J (2014) 135. Strawn WB, Richmond RS, Ann Tallant

Angiotensin receptor 1 blockade reduces E, Gallagher PE, Ferrario CM (2004)
secretion of inflammation associated cytokines Renin- angiotensin system expression in
from cultured human carotid atheroma and rat bone marrow haematopoietic and stro-
vascular cells in association with reduced extra- mal cells. Br J Haematol 126(1):120–126.
cellular signal regulated kinase expression and doi:10.1111/j.1365-2141.2004.04998.x
activation. Atherosclerosis 236(1):108–115.
doi:10.1016/j.atherosclerosis.2014.06.011 136. Gomez RA, Norling LL, Wilfong N, Isakson
P, Lynch KR, Hock R, Quesenberry P (1993)
Leukocytes synthesize angiotensinogen. in acute glucocorticoid-induced hyperten-

Hypertension 21(4):470–475 sion. J Am Soc Nephrol 19(7):1291–1299.
137. Rodgers K, Xiong S, DiZerega GS (2003) doi:10.1681/ASN.2007080911
Effect of angiotensin II and angiotensin(1-7) 148. Marmot MG (1985) Psychosocial factors and
on hematopoietic recovery after intrave- blood pressure. Prev Med 14(4):451–465
nous chemotherapy. Cancer Chemother 149. Morrison JF, Pickford M (1971) Sex differ-
Pharmacol 51(2):97–106. doi:10.1007/ ences in the changes in sympathetic nerve
s00280-002-0509-4 activity when arterial pressure is raised by
138. Rodgers KE, Xiong S, Steer R, diZerega GS infusion of angiotensin and noradrenaline.
(2000) Effect of angiotensin II on hema- J Physiol 216(1):69–85
topoietic progenitor cell proliferation. 150. Keys JR, Zhou RH, Harris DM, Druckman
Stem Cells 18(4):287–294. doi:10.1634/ CA, Eckhart AD (2005) Vascular smooth
stemcells.18-4-287 muscle overexpression of G protein-coupled
139. Gollan F, Richardson E, Goldblatt H (1948) receptor kinase 5 elevates blood pressure,
Hypertension in the systemic blood of ani- which segregates with sex and is depen-
mals with experimental renal hypertension. dent on Gi-mediated signaling. Circulation
J Exp Med 88(4):389–400 112(8):1145–1153. doi:10.1161/
140. Goldblatt H (1938) Studies on experimen- CIRCULATIONAHA.104.531657
tal hypertension: vii. The production of the 151. Xue B, Pamidimukkala J, Hay M (2005) Sex
malignant phase of hypertension. J Exp Med differences in the development of angioten-
67(5):809–826 sin II-induced hypertension in conscious
141. Hicks JD, Giltinan P, Pye J (1965) A new mice. Am J Physiol Heart Circ Physiol
method of measuring blood-pressure in mice. 288(5):H2177–H2184. doi:10.1152/
Lancet 2(7419):930–932 ajpheart.00969.2004
142. Mills PA, Huetteman DA, Brockway BP,
152. Venegas-Pont M, Sartori-Valinotti JC, Glover
Zwiers LM, Gelsema AJ, Schwartz RS, PH, Reckelhoff JF, Ryan MJ (2010) Sexual
Kramer K (2000) A new method for measure- dimorphism in the blood pressure response
ment of blood pressure, heart rate, and activ- to angiotensin II in mice after angiotensin-
ity in the mouse by radiotelemetry. J Appl converting enzyme blockade. Am J Hypertens
Physiol (1985) 88(5):1537–1544 23(1):92–96. doi:10.1038/ajh.2009.203
143. Oliverio MI, Kim HS, Ito M, Le T, Audoly 153. Mirabito KM, Hilliard LM, Head GA,

L, Best CF, Hiller S, Kluckman K, Maeda N, Widdop RE, Denton KM (2014) Pressor
Smithies O, Coffman TM (1998) Reduced responsiveness to angiotensin II in female
growth, abnormal kidney structure, and type mice is enhanced with age: role of the angio-
2 (AT2) angiotensin receptor-mediated blood tensin type 2 receptor. Biol Sex Differ 5:13.
pressure regulation in mice lacking both AT1A doi:10.1186/s13293-014-0013-7
and AT1B receptors for angiotensin II. Proc 154. Gupte M, Thatcher SE, Boustany-Kari CM,
Natl Acad Sci U S A 95(26):15496–15501 Shoemaker R, Yiannikouris F, Zhang X,
144. Gurley SB, Allred A, Le TH, Griffiths R,
Karounos M, Cassis LA (2012) Angiotensin
Mao L, Philip N, Haystead TA, Donoghue converting enzyme 2 contributes to sex differ-
M, Breitbart RE, Acton SL, Rockman HA, ences in the development of obesity hyperten-
Coffman TM (2006) Altered blood pressure sion in C57BL/6 mice. Arterioscler Thromb
responses and normal cardiac phenotype in Vasc Biol 32(6):1392–1399. doi:10.1161/
ACE2-null mice. J Clin Invest 116(8):2218– atvbaha.112.248559
2225. doi:10.1172/JCI16980 155.
Schneider MP, Wach PF, Durley MK,
145. Krege JH, John SW, Langenbach LL, Hodgin Pollock JS, Pollock DM (2010) Sex differ-
JB, Hagaman JR, Bachman ES, Jennette JC, ences in acute ANG II-mediated hemody-
O'Brien DA, Smithies O (1995) Male-female namic responses in mice. Am J Physiol Regul
differences in fertility and blood pressure in Integr Comp Physiol 299(3):R899–R906.
ACE-deficient mice. Nature 375(6527):146– doi:10.1152/ajpregu.00638.2009
148. doi:10.1038/375146a0 156. Ji H, Zheng W, Wu X, Liu J, Ecelbarger
146. Makhanova N, Lee G, Takahashi N, Sequeira CM, Watkins R, Arnold AP, Sandberg K
Lopez ML, Gomez RA, Kim HS, Smithies O (2010) Sex chromosome effects unmasked
(2006) Kidney function in mice lacking aldoste- in angiotensin II-induced hypertension.
rone. Am J Physiol Renal Physiol 290(1):F61– Hypertension 55(5):1275–1282. doi:10.1161/
F69. doi:10.1152/ajprenal.00257.2005 HYPERTENSIONAHA.109.144949
147. Goodwin JE, Zhang J, Geller DS (2008)
157. Alsiraj Y, Thatcher SE, Charnigo R, Kuey
A critical role for vascular smooth muscle C, Blalock E, Daugherty A, Cassis LA
18 Sean E. Thatcher
(2016) Female mice with an XY sex chro- by angiotensin II-induced hypertension.

mosome complement develop severe Circ Res 107(2):263–270. doi:10.1161/
angiotensin II-induced abdominal aor- CIRCRESAHA.110.217299
tic aneurysms. Circulation. doi:10.1161/ 167. Kintscher U, Hartge M, Hess K, Foryst-

CIRCULATIONAHA.116.023789 Ludwig A, Clemenz M, Wabitsch M,
158. Simon MR, Kamlay MT, Khan M, Melmon Fischer-Posovszky P, Barth TF, Dragun
K (1989) Angiotensin II binding to human D, Skurk T, Hauner H, Bluher M, Unger
mononuclear cells. Immunopharmacol T, Wolf AM, Knippschild U, Hombach V,
Immunotoxicol 11(1):63–80. doi:10.3109/ Marx N (2008) T-lymphocyte infiltration
08923978909082143 in visceral adipose tissue: a primary event in
159. Luft FC (2001) Angiotensin, inflammation, adipose tissue inflammation and the develop-
hypertension, and cardiovascular disease. ment of obesity-mediated insulin resistance.
Curr Hypertens Rep 3(1):61–67 Arterioscler Thromb Vasc Biol 28(7):1304–
160. Wassmann S, Stumpf M, Strehlow K, Schmid 1310. doi:10.1161/ATVBAHA.108.165100
A, Schieffer B, Bohm M, Nickenig G (2004) 168.
Itani HA, Xiao L, Saleh MA, Wu J,
Interleukin-6 induces oxidative stress and Pilkinton MA, Dale BL, Barbaro NR, Foss
endothelial dysfunction by overexpres- JD, Kirabo A, Montaniel KR, Norlander
sion of the angiotensin II type 1 receptor. AE, Chen W, Sato R, Navar LG, Mallal SA,
Circ Res 94(4):534–541. doi:10.1161/01. Madhur MS, Bernstein KE, Harrison DG
RES.0000115557.25127.8D (2016) CD70 exacerbates blood pressure
161. Touyz RM (2005) Intracellular mechanisms elevation and renal damage in response
involved in vascular remodelling of resistance to repeated hypertensive stimuli. Circ
arteries in hypertension: role of angiotensin Res 118(8):1233–1243. doi:10.1161/
II. Exp Physiol 90(4):449–455. doi:10.1113/ CIRCRESAHA.115.308111
expphysiol.2005.030080 169. Feau S, Garcia Z, Arens R, Yagita H, Borst
162. Guzik TJ, Hoch NE, Brown KA, McCann J, Schoenberger SP (2012) The CD4(+)
LA, Rahman A, Dikalov S, Goronzy J, T-cell help signal is transmitted from APC
Weyand C, Harrison DG (2007) Role of the to CD8(+) T-cells via CD27-CD70 interac-
T cell in the genesis of angiotensin II induced tions. Nat Commun 3:948. doi:10.1038/
hypertension and vascular dysfunction. J Exp ncomms1948
Med 204(10):2449–2460. doi:10.1084/ 170. Kirabo A, Fontana V, de Faria AP, Loperena
jem.20070657 R, Galindo CL, Wu J, Bikineyeva AT, Dikalov
163. Yusof M, Kamada K, Gaskin FS, Korthuis RJ S, Xiao L, Chen W, Saleh MA, Trott DW, Itani
(2007) Angiotensin II mediates postischemic HA, Vinh A, Amarnath V, Amarnath K, Guzik
leukocyte-endothelial interactions: role of cal- TJ, Bernstein KE, Shen XZ, Shyr Y, Chen SC,
citonin gene-related peptide. Am J Physiol Mernaugh RL, Laffer CL, Elijovich F, Davies
Heart Circ Physiol 292(6):H3032–H3037. SS, Moreno H, Madhur MS, Roberts J 2nd,
doi:10.1152/ajpheart.01210.2006 Harrison DG (2014) DC isoketal- modified
proteins activate T cells and promote hyper-
164. Crowley SD, Frey CW, Gould SK, Griffiths tension. J Clin Invest 124(10):4642–4656.
R, Ruiz P, Burchette JL, Howell DN, doi:10.1172/JCI74084
Makhanova N, Yan M, Kim HS, Tharaux
PL, Coffman TM (2008) Stimulation of 171. Saleh MA, McMaster WG, Wu J, Norlander
lymphocyte responses by angiotensin II pro- AE, Funt SA, Thabet SR, Kirabo A, Xiao
motes kidney injury in hypertension. Am L, Chen W, Itani HA, Michell D, Huan T,
J Physiol Renal Physiol 295(2):F515–F524. Zhang Y, Takaki S, Titze J, Levy D, Harrison
doi:10.1152/ajprenal.00527.2007 DG, Madhur MS (2015) Lymphocyte
adaptor protein LNK deficiency exacer-
165. Shirakawa K, Yan X, Shinmura K, Endo J,
bates hypertension and end-organ inflam-
Kataoka M, Katsumata Y, Yamamoto T, Anzai mation. J Clin Invest 125(3):1189–1202.
A, Isobe S, Yoshida N, Itoh H, Manabe I, doi:10.1172/JCI76327
Sekai M, Hamazaki Y, Fukuda K, Minato
N, Sano M (2016) Obesity accelerates T cell 172. Briones AM, Nguyen Dinh Cat A, Callera GE,
senescence in murine visceral adipose tissue. Yogi A, Burger D, He Y, Correa JW, Gagnon
J Clin Invest. doi:10.1172/JCI88606 AM, Gomez-Sanchez CE, Gomez- Sanchez
EP, Sorisky A, Ooi TC, Ruzicka M, Burns KD,
166. Marvar PJ, Thabet SR, Guzik TJ, Lob
Touyz RM (2012) Adipocytes produce aldoste-
HE, McCann LA, Weyand C, Gordon FJ, rone through calcineurin- dependent signaling
Harrison DG (2010) Central and periph- pathways: implications in diabetes mellitus-
eral mechanisms of T-lymphocyte activa- associated obesity and vascular dysfunction.
tion and vascular inflammation produced
Hypertension 59(5):1069–1078. doi:10.1161/ Blocking angiotensin-converting enzyme

HYPERTENSIONAHA.111.190223 induces potent regulatory T cells and modulates
173. Platten M, Youssef S, Hur EM, Ho PP, Han TH1- and TH17-mediated autoimmunity. Proc
MH, Lanz TV, Phillips LK, Goldstein MJ, Bhat Natl Acad Sci U S A 106(35):14948–14953.
R, Raine CS, Sobel RA, Steinman L (2009) doi:10.1073/pnas.0903958106
Chapter 2
A Color Segmentation-Based Method to Quantify

Atherosclerotic Lesion Compositions with Immunostaining
Congqing Wu, Alan Daugherty, and Hong Lu
Abstract
There is an increasing recognition that atherosclerotic lesion composition, rather than size, is the determi-
nant of acute events. Immunostaining is a commonly used method to characterize atherosclerotic lesion
compositions. Here, we describe a color segmentation-based approach in HSI (hue, saturation, and inten-
sity) color mode, which minimizes subjectivity and produces accurate and consistent quantifications of
atherosclerotic lesion compositions.
Key words Immunostaining, Atherosclerosis, Antibody, Imaging, Color
1 Introduction
Mouse models are commonly used to study pathologies and mecha-

nisms of atherosclerosis attributed to the ease of genetic manipula-
tions, availability of large numbers, and cost benefits relative to large
species. The two commonly used mouse models, low-density lipo-
protein (LDL) receptor −/− and apolipoprotein E (apoE) −/−
mice, rapidly develop atherosclerosis when fed a saturated
fat-enriched diet (generally referred to as “Western diet”) [1–4].
There is also consistent evidence that activation of the renin angio-
tensin system contributes to hypercholesterolemia-induced athero-
sclerosis [5–17]. One direct evidence is that infusion of angiotensin
II, the major bioactive peptide of the renin angiotensin system,
accelerates formation and progression of atherosclerosis in these two
hypercholesterolemic mouse models [5, 13, 14]. Conversely, inhib-
iting key components of the renin angiotensin system reduces ath-
erosclerosis, as demonstrated in both animal models and human
trials (a few examples from thousands of publications [8, 10, 12,
15–17]. Therefore, determination of the renin angiotensin compo-
nents in atherosclerotic lesions may provide mechanistic insights
into the development of atherosclerosis.
21
22 Congqing Wu et al.
Fig. 1 Normalization of image background. Overexposed or underexposed images can be normalized to same
background by the “Best Fit” mode
Immunostaining detects cell types and molecular markers of

the pathologic process at different stages of atherosclerotic lesions.
This provides insights into the complexity and dynamic nature of
lesion development.
Aortic root is a commonly studied region for atherosclerotic
lesions in mouse models. Atherosclerotic lesions in mice are char-
acterized by subendothelial accumulation of leukocytes, of which
macrophages are the most abundant cell type with lipid content. A
cap develops over these macrophages, which contains smooth
muscle cells, is also detected in mouse atherosclerotic lesions.
Antibodies that specifically target macrophages (e.g., CD68) and
the major resident cell type (smooth muscle cells) of the aortic wall
have been well established [18]. Therefore, in Subheading 3
method for quantification of positive immunostaining we will use
CD68 (Figs. 1, 2, 3, 4, and 5) and smooth muscle α-actin (Fig. 5)
positive staining as examples.
Here, we also provide insights into chicken antibodies (Table 1)
we developed that target mouse angiotensinogen (AGT), renin, or
angiotensin-converting enzyme (ACE), as well as antibodies tar-
geting mouse AngII type 1 (AT1) receptors. Chicken antibodies
we developed consist of pre-immune IgY (prior to the introduc-
tion of antigen from the same hen for the specific antibody), affin-
ity purified chicken anti mouse antibody, and IgY after eluting the
specific antibody (depleted IgY). Therefore, this provides two
appropriate negative controls for the antibody, pre-immune IgY
and depleted IgY. For renin and ACE immunostaining, we have
also used tissues from renin and ACE whole body-deficient mice,
respectively, as negative controls to validate the specificity of the
antibodies to these two respective targets.
To determine whether these renin angiotensin components are
present in mouse atherosclerotic lesions, it is important to first vali-
date these antibodies in their major expressing organs. AGT is ubiq-
uitously expressed in many organs. Hepatocytes are the systemic
source for AGT [12, 19, 20]. After synthesis, AGT is released to
circulating blood, and also redistributed to other organs such as kid-
ney [12, 21, 22]. Therefore, liver, despite being the major source of
systemic AGT, is not optimal to validate an AGT antibody. Instead,
HSI Method for IHC Quantification 23
Fig. 2 Defining area of interest. Atherosclerotic lesions in an aortic root section

were visualized with CD68 and hematoxylin staining. Individual lesion areas
(within green lines) are manually traced using the “Area” tool bar. Three areas of
interest are defined as shown by green outlines
Fig. 3 Determination of color histogram. HUE channels of the color histogram for CD68 positive immunostain-
ing (red) are defined by comparing to the negative control (non-immune rat IgG2a). Two color segments of HUE
(0–30) and (211–255) appearing on CD68 positive staining, but not on the negative control, represent CD68
positive immunostaining
AGT accumulates in proximal convoluted tubules of kidney, which

provides an optimal tissue for immunostaining of AGT [22]. In con-
trast to dispersed expression of AGT in many organs, juxtaglomeru-
lar cells (JG) of the kidney are the primary source for renin expression.
We have demonstrated that chicken anti mouse renin antibody listed
Fig. 4 Quantification of positive staining. An example for setting up the color segment (A), selecting measure-
ments (B), and automatic calculation of positive staining (C)
in Table 1 only detects renin in kidney JG cells of wild-type, but not

renin −/− mice. ACE is predominantly expressed in vasculature,
which is abundant in both endothelial and smooth muscle cells [6,
11, 23]. Therefore, vessels such as the aorta are appropriate tissues
for ACE immunostaining. We have demonstrated that chicken anti
mouse ACE antibody listed in Table 1 only develops positive stain-
ing in wild-type, but not ACE-deficient tissues.
Angiotensin II contributes to atherosclerosis through its inter-
action with AT1a receptors [6, 7]. There are many commercially
available antibodies against mouse AT1 or AT1a receptors. We have
A Distance (mm) from the Transition Section
-300 -200 -100 0 100 200
CD 68
Oil Red O
a-actin
Transition Section
B C
Lesion Area
CD68
1.0 CD68 100 SM a-Actin
SM a-Actin
Percent Positive Staining Area Oil Red O
Oil Red O
0.8 80
Area (mm2)
0.6 60
0.4 40
0.2 20
0.0 0
-300 -200 -100 0 100 200 -300 -200 -100 0 100 200
Distance (mm) from Transition Section Distance (mm) from Transition Section
Fig. 5 Analysis of positive staining in atherosclerotic lesions. A male LDL receptor −/− mouse was fed “Western diet”
for 7 months. (A) Images of CD68, oil red O, and smooth muscle α-actin staining (red denotes positive staining)
throughout the aortic root. “Transition” denotes the ending of the aortic sinus and the beginning of the ascending aorta.
(B) Positive area of oil red O, CD68, and smooth muscle α-actin. (C) Percent positive area is calculated by comparing
positive staining area of oil red O, CD68, and smooth muscle α-actin to lesional area (namely, total area of interest)
Table 1
Chicken anti-mouse IgY antibodies for immunostaining of angiotensinogen, renin, and angiotensin-
converting enzyme
Immunogenic peptide Recommended positive Recommended working

Target sequence control tissue concentration References
AGT CZEEEQPTTSVQQPGSPE Kidney (proximal 10 μg/ml [6]
convoluted tubules)
Renin CZRKFYTEFDRHNNR Kidney (present in JG 10 μg/ml [6, 8]
cells under normal
condition)
ACE CZDLE TDE Vasculature 3 μg/ml [6, 11]
AKADRFVEEYD RT
AGT angiotensinogen, ACE angiotensin-converting enzyme, JG juxtaglomerular
also developed multiple chicken anti-mouse antibodies targeting

different sequences of the AT1a receptor. Consistent with reports
from Dr. Saavedra and Dr. Coffman’s laboratories [24, 25], we
have failed to demonstrate that AT1 receptor can be stained specifi-
cally with any antibody in mouse tissues. Therefore, we are not
aware of any antibodies that have validated for AT1a receptor
immunostaining of mouse atherosclerosis.
Many image analysis programs provide tools to quantify pos-
itive staining with RGB color mode (Red, Green, Blue) as their
defaulted setting. While it is optimal to select single homogeneous
color [26, 27], RGB mode has shortcomings to quantify composi-
tions on complex immunostaining images. It lacks reproducibility
since a range of each color channel is arbitrarily selected by the
observer. Therefore, it is difficult to define heterogeneous positive
staining colors with RGB color mode, which are commonly seen in
atherosclerotic lesions. Here, we describe the use of HSI model for
color selection. HSI stands for hue (H), saturation (S), and inten-
sity (I) triplet, each component of which can vary from 0 to 255 in
8-bit computing. Hue defines the color itself. For example, red is
distinct from blue and yellow. In theory, the values for the hue axis
run from 0 to 360°, beginning and ending with red and running
through green, blue, and all intermediary colors such as greenish-
blue, orange, purple, and other colors. In an image analysis pro-
gram, 0–360° is converted to 0–255. Saturation denotes the degree
to which the hue differs from a neutral gray. Saturation of 0–100%
is converted to 0–255. Intensity means the level of illumination. 0%
appears black, whereas 100% is full illumination, which washes out
color. The 0–100% mode of intensity is also converted to 0–255.
HSI matches the manner how we perceive complex color ranges,
while remaining computationally simple. Endpoints of color seg-
ments representing positive staining are readily determined without
personal bias by comparing histograms between a positive staining
image and a negative control image in HSI mode. Appropriately
applied, this color segmentation-based method is a reliable tech-
nique to provide accurate and consistent quantification of positive
immunostaining, a useful tool to look into the development of ath-
erosclerosis as well as many other disease processes.
2 Materials
1. Immunostained cross-sections of aortic root samples.

2. A bright-field microscope with a digital camera for image
acquisition.
3. Imaging software that has HSI color mode (Hue, Saturation,
Intensity) for quantification of positive immunostaining area
and atherosclerotic lesion area.
3 Methods
Macrophages are the most abundant cell type in atherosclerotic

lesions of hypercholesterolemic mice, whereas smooth muscle cells
are the predominant resident cell type of the aortic wall, which also
frequently form a boundary (the cap) of atherosclerotic lesions.
Lesions are stained with oil red O to determine the amount of
neutral lipid. In this Section, we use immunostaining of CD68
(Clone FA-11) and smooth muscle α-actin as an example to illus-
trate the Method. Immunostaining of the renin angiotensin com-
ponents can also be analyzed similarly.
Tissue sections cut on a cryostat were derived from the aortic
root of a male LDL receptor−/− mouse fed “Western diet” for
7 months. Sections were stained for neutral lipids with oil red O and
immunostained for CD68 (macrophage marker) and smooth muscle
α-actin (smooth muscle cell marker). Images of aortic root sections
that were 100 μm apart were analyzed using the Image-Pro Plus
software. We follow the steps listed below to quantify each image:
1. Image quality control: Make sure all images are taken at same
camera settings. Use the “Best Fit” function of the imaging
software to fix overexposed or underexposed images (Fig. 1)
so that every image has similar background color distribution.
2. Defining area of interest (AOI): Manually trace the whole
lesion area as shown in Fig. 2 that green color isolates AOI
from the rest of the image. This is to ensure that positive stain-
ing is quantified within the defined AOI. Multiple AOIs are
allowed. As shown in Fig. 2, three AOIs are defined.
3. Determination of positive immunostaining color range using
histogram: Color histogram is a representation of the frequency
distribution of colors in an image, determined by counting the
number of pixels of each given set of color ranges. This func-
tion is under “Measure” in the Image-Pro Plus software.
Figure 3 shows the HUE channel of the color histogram of
negative control and CD68 positive immunostaining, respec-
tively. Two color segments of HUE (0–30) and (211–255)
only appear on CD68 staining but not on its negative control,
representing CD68 positive immunostaining. Obtaining these
endpoints of color segments using color histogram of the HSI
mode is the key to this method (see Note 1).
4. Quantification of positive immunostaining: After setting color seg-
ments with HUE value (Fig. 4a), select “Measurements” as shown
in Fig. 4b. Positive staining area is instantly selected and calculated
automatically. Selection of positive immunostaining can be modi-
fied by adjusting INTENSITY value. The results are shown as pre-
sented in Fig. 4c. Use the same HUE and INTENSITY values for
every image of same immunostaining (see Note 2).
5. Analysis of lesional composition: There are two methods to

analyze data obtained from step 4. One is to determine abso-
lute area of lesional composition as shown in Fig. 5b, and the
other is to normalize lesional composition to total lesion area
as shown in Fig. 5c. There is no standard which method is bet-
ter. The selection can depend on what content to state. For
example, if the user wants to compare whether there are more
macrophages accumulated in lesions in one group than the
other, the former method is feasible. If the user wants to
emphasize whether the ratio of macrophage accumulation in
lesions is more in one group than in the other, the latter
method is optimal (see Note 3).
4 Notes
1. Specificity of positive immunostaining is a premise for accurate

quantification [18]. This is determined by appropriate negative
controls including antibody-equivalent immunoglobulin from
non-immune or pre-immune same host, exclusion of primary
antibody, omission of both primary and secondary antibodies.
Availability of tissue sections as a negative control would
enhance the reliability of the specificity for immunostaining.
2. Quality of images is the key for accurate measurements using
this HSI color segmentation-based method. It is also impor-
tant to acquire all images using same microscope, same camera
setting, and same light conditions.
3. Measuring multiple sections (> 5 serial sections), rather than a
single section, enhances the accuracy of quantification. Same as
atherosclerotic lesion measurements in a specific region [28,
29], this also requires to use a defined landmark to ensure posi-
tive immunostaining in same region is compared.
Acknowledgments
Congqing Wu is supported by an American Heart Association

Postdoctoral fellow award (16POST31140008). The authors’
research work is supported by an Institutional Development Award
from the National Institute of General Medical Sciences of the
National Institutes of Health under grant number P20 GM103527
and R01 under grant numbers HL107319 and HL133723 from
the National Institutes of Health of the United States of America.
The content in this manuscript is solely the responsibility of the
authors and does not necessarily represent the official views of the
National Institutes of Health.
References
1. Breslow JL (1996) Mouse models of athero- Angiotensinogen exerts effects independent of
sclerosis. Science 272:685–688 angiotensin II. Arterioscler Thromb Vasc Biol
2. Ishibashi S, Goldstein JL, Brown MS, Herz J, 36:256–265
Burns DK (1994) Massive xanthomatosis and 13. Daugherty A, Cassis L (1999) Chronic angio-
atherosclerosis in cholesterol-fed low density tensin II infusion promotes atherogenesis in
lipoprotein receptor-negative mice. J Clin low density lipoprotein receptor -/- mice. Ann
Invest 93:1885–1893 N Y Acad Sci 892:108–118
3. Plump AS, Smith JD, Hayek T, Aaltosetala K, 14. Weiss D, Kools JJ, Taylor WR (2001)
Walsh A, Verstuyft JG et al (1992) Severe Angiotensin II-induced hypertension acceler-
hypercholesterolemia and atherosclerosis in ates the development of atherosclerosis in apoE-
apolipoprotein-E-deficient mice created by deficient mice. Circulation 103:448–454
homologous recombination in ES cells. Cell 15. Imanishi T, Tsujioka H, Ikejima H, Kuroi A,
71:343–353 Takarada S, Kitabata H et al (2008) Renin
4. Zhang SH, Reddick RL, Piedrahita JA, Maeda inhibitor aliskiren improves impaired nitric
N (1992) Spontaneous hypercholesterolemia oxide bioavailability and protects against athero-
and arterial lesions in mice lacking apolipopro- sclerotic changes. Hypertension 52:563–572
tein E. Science 258:468–471 16. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R,
5. Daugherty A, Manning MW, Cassis LA (2000) Dagenais G (2000) Effects of an angiotensin-
Angiotensin II promotes atherosclerotic lesions converting-enzyme inhibitor, ramipril, on car-
and aneurysms in apolipoprotein E-deficient diovascular events in high-risk patients. The
mice. J Clin Invest 105:1605–1612 heart outcomes prevention evaluation study
6. Daugherty A, Rateri DL, Lu H, Inagami T, investigators. N Engl J Med 342:145–153
Cassis LA (2004) Hypercholesterolemia stimu- 17. ONTARGET Investigators, Yusuf S, Teo KK,
lates angiotensin peptide synthesis and contrib- Pogue J, Dyal L, Copland I et al (2008)
utes to atherosclerosis through the AT1A Telmisartan, ramipril, or both in patients at
receptor. Circulation 110:3849–3857 high risk for vascular events. N Engl J Med
7. Wassmann S, Czech T, van Eickels M, Fleming 358:1547–1559
I, Bohm M, Nickenig G (2004) Inhibition of 18. Lu H, Rateri DL, Daugherty A (2007)
diet-induced atherosclerosis and endothelial Immunostaining of mouse atherosclerosis
dysfunction in apolipoprotein E/angiotensin lesions. Methods Mol Med 139:77–94
II type 1A receptor double-knockout mice. 19. Wu C, Xu Y, Lu H, Howatt DA, Balakrishnan
Circulation 110:3062–3067 A, Moorleghen JJ et al (2015) Cys18-Cys137
8. Lu H, Rateri DL, Feldman DL, Charnigo RJ disulfide bond in mouse angiotensinogen does
Jr, Fukamizu A, Ishida J et al (2008) Renin not affect AngII-dependent functions in vivo.
inhibition reduces hypercholesterolemia- Hypertension 65:800–805
induced atherosclerosis in mice. J Clin Invest 20. Yiannikouris F, Wang Y, Shoemaker R, Larian
118:984–993 N, Thompson J, English VL et al (2015)
9. Daugherty A, Lu H, Rateri DL, Cassis LA Deficiency of angiotensinogen in hepatocytes
(2008) Augmentation of the renin-angiotensin markedly decreases blood pressure in lean and
system by hypercholesterolemia promotes vas- obese male mice. Hypertension 66:836–842
cular diseases. Future Lipidol 3:625–636 21. Wu C, Lu H, Cassis LA, Daugherty A (2011)
10. Lu H, Balakrishnan A, Howatt DA, Wu C, Molecular and pathophysiological features of
Charnigo R, Liau G et al (2012) Comparative angiotensinogen: a mini review. N Am J Med
effects of different modes of renin angiotensin Sci (Boston) 4:183–190
system inhibition on hypercholesterolaemia- 22. Matsusaka T, Niimura F, Shimizu A, Pastan I,
induced atherosclerosis. Br J Pharmacol Saito A, Kobori H et al (2012) Liver angioten-
165:2000–2008 sinogen is the primary source of renal angio-
11. Chen XC, Lu H, Zhao M, Tashiro K, Cassis tensin II. J Am Soc Nephrol 23:1181–1189
LA, Daugherty A (2013) Angiotensin- 23. Chen XC, Howatt DA, Balakrishnan A,
converting enzyme promotes atherosclerosis Moorleghen JJ, Wu CQ, Cassis LA et al (2016)
through an angiotensin I to angiotensin II Angiotensin-converting enzyme in smooth mus-
pathway involving leukocytes. Arterioscler cle cells promotes atherosclerosis. Arterioscler
Thromb Vasc Biol 33:2075–2080 Thromb Vasc Biol 36:1085–1089
12. Lu H, Wu C, Howatt DA, Balakrishnan A, 24. Benicky J, Hafko R, Sanchez-Lemus E, Aguilera
Moorleghen JJ, Chen X et al (2016) G, Saavedra JM (2012) Six commercially available
angiotensin II AT(1) receptor antibodies are non- 27. Vrekoussis T, Chaniotis V, Navrozoglou I,
specific. Cell Mol Neurobiol 32:1353–1365 Dousias V, Pavlakis K, Stathopoulos EN et al
25. Herrera M, Sparks MA, Alfonso-Pecchio AR, (2009) Image analysis of breast cancer
Harrison-Bernard LM, Coffman TM (2013) immunohistochemistry- stained sections using
Response to lack of specificity of commercial ImageJ: an RGB-based model. Anticancer Res
antibodies leads to misidentification of angio- 29:4995–4998
tensin type-1 receptor protein. Hypertension 28. Daugherty A, Whitman SC (2003)
61:e32 Quantification of atherosclerosis in mice.
26. Zhang G, Chen Y, BilalWaqar A, Han L, Jia M, Methods Mol Biol 209:293–309
Xu C et al (2015) Quantitative analysis of rab- 29. Daugherty A, Lu H, Howatt DA, Rateri DL
bit coronary atherosclerosis. Practical tech- (2009) Modes of defining atherosclerosis in
niques utilizing open-source software. Anal mouse models: relative merits and evolving
Quant Cytol Histol 37:115–122 standards. Methods Mol Biol 573:1–15
Chapter 3
Assessment of Protein Carbonylation and Protein Tyrosine

Phosphatase (PTP) Oxidation in Vascular Smooth Muscle
Cells (VSMCs) Using Immunoblotting Approaches
Sofia Tsiropoulou and Rhian M. Touyz
Abstract
Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in
cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing
phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the
cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irre-
versible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome
of vascular smooth muscle cells (VSMC).
Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of
protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in
the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-
PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible
protein carbonylation in the sample by immunoblotting.
Oxidation of PTPs can be evaluated using a monoclonal antibody against the “hyperoxidized” (SO3H)
catalytic site of these enzymes. The described methodology offers the ability to discriminate between irre-
versible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S−)
are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other
part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both
untreated and PV-treated samples are analyzed by SDS-PAGE and “hyperoxidized” PTPs are detected by
immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference
between the signals in untreated and the PV-treated samples.
The above immunoassays provide general approaches to detect and quantify global levels of irrevers-
ible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context.
Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms
underlying chronic diseases associated with oxidative stress. This is particularly important in systems influ-
enced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox
signaling.
Key words Protein oxidation, Carbonylation, Oxyblot, PTP oxidation, Cysteine thiol, Irreversible
modifications, Reversible modifications
31
32 Sofia Tsiropoulou and Rhian M. Touyz
1 Introduction
1.1 Protein Oxidation Reactive oxygen species (ROS), such as superoxide anion (O2.−)
and hydrogen peroxide (H2O2), are natural by-products of enzy-
matic activity, which at physiological low levels play a key role in
cell homeostasis by participating in redox signaling and antioxidant
defence. However, excess ROS generation in the cellular environ-
ment leads to alterations in cellular function and even cell death,
through lipid peroxidation, protein oxidation, and DNA damage.
Sustained imbalance in ROS concentration in the system is associ-
ated with pathological conditions such as hypertension [1].
Angiotensin II (Ang II), a vasoactive peptide and a major pro-
hypertensive hormone, has been implicated in processes associated
with endothelial dysfunction, arterial remodeling, vascular fibrosis,
and inflammation by stimulating generation of ROS [2].
Interaction of protein macromolecules with highly reactive
oxygen and nitrogen species (ROS/RNS) or electrophiles leads to
oxidative post-translational protein modification. Such modifica-
tions alter the biochemical properties of proteins, including its
function and activity, structure, localization, and interactions with
other macromolecules, resulting in aberrant cell signaling and
function [3]. Depending on the degree of oxidation, protein mod-
ifications are classified into two categories: (1) irreversible oxida-
tion that eventually leads to loss of function and protein degradation
and (2) reversible oxidation, which is emerging as important cel-
lular regulatory mechanism [4, 5].
The different types of oxidative modifications can be assessed
by employing different methodologies. Here, we describe immu-
noblotting approaches for comprehensive detection of irreversible
protein carbonylation and oxidation of protein tyrosine phospha-
tases (PTP), as crucial cell signaling molecules, highly susceptible
to Ang II-induced oxidation.
1.2 Immunoblot Carbonylation is the most frequent form of irreversible protein

Detection of Protein oxidation as it occurs on a number of amino acids including argi-
Carbonylation nine, lysine, proline, threonine, histidine, and cysteine. As such,
carbonylation is used as a marker of increased oxidative stress [6].
Metal-catalyzed oxidation of proteins leads to formation of car-
bonyl groups (C = O; aldehydes and ketones) on side chains of
amino acids, with consequent loss of function, aggregation, and
ultimately degradation of proteins.
The most commonly used methodology to capture carbonylation
is the OxyBlot™ assay, which was initially described by Levine et al.
(1994) [7]. Protein carbonyl groups (C = O) are indirectly detected
by immunoblotting after reaction with 2,4-dinitrophenylhydrazine
(DNPH). VSMCs lysate is initially treated with a reducing agent, such
as dithiothreitol (DTT), for reduction of reversibly oxidized proteins
and for prevention of artifactual oxidation during sample processing.
Immunodetection of Oxidatively Modified Proteins in VSMCs 33
Subsequent treatment with DNPH derivatizes carbonyl groups on

oxidized proteins, forming a stable dinitrophenyl (DNP) hydrazone
product. Samples are analyzed by SDS-PAGE and transferred on an
immunoblotting membrane, which is then blocked for nonspecific
binding. DNP is detected by the use of a specific primary antibody.
Use of horseradish peroxidase (HRP)-conjugated secondary antibody
against the primary antibody, along with chemiluminescent substrates,
allows for visualization and quantification of total protein carbonyl
formation. The intensity of the detected bands is proportional to the
degree of protein carbonylation in the sample. Along with the deriva-
tized sample, a negative control (sample treated with derivatization
control solution) is analyzed to account for any artifactual carbonyl-
ation occurring during the sample processing.
The main advantage of the OxyBlot™ methodology is that no
expensive equipment is required, and the main limitation is that no
information can be gained regarding the specific site of the
modification.
1.3 Immunoblot Protein tyrosine phosphatases (PTPs) are important regulatory

Assessment of Protein molecules, which influence numerous cellular signaling pathways.
Tyrosine Phosphatases Under normal conditions, a highly conserved free cysteine thiol at
(PTPs) their active center is essential for their catalytic activity on protein
Oxidation States dephosphorylation. High levels of ROS react readily with the
nucleophilic cysteine thiol, to cause PTP oxidation, which renders
the enzyme inactive. The reversibility of the oxidative modification
is a crucial mechanism for downstream signaling regulation. Cys
oxidation to sulfenic acid (PTP-SOH) can be reversed by antioxi-
dants, whereas higher oxidation to sulfinic (PTP-SO2H) or sul-
fonic (PTP-SO3H) acid states is normally irreversible [8]. A number
of approaches have been developed for direct or indirect monitor-
ing of PTP oxidation [9].
The approach described in this chapter, for accessing PTP oxi-
dation, is a modified version of the methodology originally pro-
posed by Persson et al. (2005) [10]. Initially, the method exploits
the ability of unmodified PTP-thiols (S−) to be irreversibly blocked
by an alkylating agent such as N-ethylmaleimide (NEM). This
results in the elimination of those PTPs that remain in a reduced
state after a stimulus, from the sample. Subsequently, the cell lysate
is split into two. One half of the sample is stored away to be used
for directly capturing irreversible PTP-SO3H hyperoxidation levels.
In the second half, reversibly modified PTP-SOH levels are indi-
rectly estimated. -SOH is first reduced into -S− by dithiothreitol
(DTT) and then hyperoxidized into −SO3H using pervanadate
(PV). Both the untreated and PV-treated parts of the same sample
are analyzed on the same polyacrylamide gel by SDS-PAGE and
transferred on an immunoblotting membrane, which is blocked for
nonspecific binding. The hyperoxidized state is captured using a
mouse-monoclonal antibody specific to the SO3H-modified cata-

lytic center of PTPs. Subsequent incubation with a horseradish
peroxidase (HRP)-conjugated secondary antibody against the
primary antibody and use of chemiluminescent substrates facilitate
detection of any PTPs in the −SO3H state. Calculation of PTP-
SOH levels is based on the subtraction of the untreated-sample
signal from the PV-treated-sample signal.
The advantage of the strategy described in this chapter is that
analysis of the two differentially treated parts of the cell lysate on
the same gel enables measurement and direct comparison of the
irreversibly and reversibly modified oxidation states of PTPs. Such
an approach allows for a more comprehensive assessment of the
PTP redox status under different (patho)physiological settings.
The main limitation is that this methodology does not capture the
irreversible PTP-SO2H oxidative modification, since it cannot be
hyperoxidized by pervanadate [11]. Moreover, the ox-PTP anti-
body is highly specific only for the classical PTP signature motif
and does not detect dual specificity PTPs (DUSPs) [12]. Finally,
sulfenic acid (−SOH) being the unstable oxidized intermediate, it
can rapidly transform into the more stable irreversible sulfinic or
sulfonic acids after cell lysis, leading to false-positive results if sam-
ples are not processed fresh and fast enough.
In general, to minimize variability in immunodetection assays,
use of multiple replicates and appropriate controls is essential for
testing the reproducibility and producing high-quality results.
2 Materials
All aqueous solutions are prepared in Milli-Q grade water (ultra-

pure; ~18 MΩ.cm), unless otherwise stated.
2.1 Angiotensin 1. Cell culture petri dishes 100 mm.

II-Stimulation of VSMC 2.
Complete growth media: Dulbecco’s Modified Eagle’s
Medium (DMEM) with 4 mM L-glutamine, 1 g/l D-glucose
and 110 mg/l sodium pyruvate, supplemented with 10% (v/v)
fetal calf serum (FCS) and 5% (v/v) penicillin (100 IU/ml)—
streptomycin (100 μg/ml) solution.
3. Starvation media: DMEM with 4 mM L-glutamine, 1 g/L
D-glucose and 110 mg/L sodium pyruvate, supplemented
with 0.5% (v/v) FCS and 5% (v/v) penicillin (100 IU/ml)—
streptomycin (100 μg/ml) solution.
4. Angiotensin II peptide solution: 10−5 M.
5. 1× Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4.
2.2 OxyBlot 1. Lysis buffer: 50 mM Na4P2O7, 50 mM NaF, 50 mM NaCl,
5 mM Na2EDTA, 10 mM HEPES, 0.5% (v/v) triton X-100,
pH 7.4, supplemented with 2 mM Na3VO4, 1 mM PMSF and
1 μg/ml of aprotinin, leupeptin and pepstatin.
2. Cell scrapers.
3. OxyBlot™ Protein Oxidation Detection Kit (S7150) from
Merck Millipore (Billerica, Massachusetts, USA) (see Note 1).
4. 1.5 ml Eppendorf tubes: two pre-labeled sets for collection of
lysate and sample for Western blotting.
5. 96-well plates: to be used for protein quantification assay and
OxyBlot assay.
6. Dithiothreitol (DTT): 1 M stock solution (see Note 2).
7. Sodium dodecyl sulfate (SDS): 12% w/v.
8. Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories,
Hercules, CA).
9. Microplate spectrophotometer.
2.3 PTP Oxidation 1. 1× Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM

KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4.
2. Lysis buffer: 50 mM HEPES pH 6.5, 150 mM NaCl, 1%
(v/v) Nonidet P-40, and 10% (v/v) glycerol supplemented
with 40 μg/ml PMSF, 2 μg/ml pepstatin, 20 μg/ml leu-
peptin, 20 μg/ml aprotinin, 20 mM NaF and 10 mM
β-glycerophosphate (see Note 3).
3. Vacuum pump and vacuum flask (see Note 4).
4. Cell scrapers.
5. 1.5 ml Eppendorf tubes: four pre-labeled sets for collection of
lysate, sample without DTT/PV treatment, sample treated
with DTT, sample treated with PV.
6. N-ethylmaleimide (NEM): added to lysis buffer at a final con-
centration of 10 mM (see Note 5).
7. Catalase: added to lysis buffer at a final concentration of
100 μg/ml (see Note 6).
8. Micro Bio-Spin 6 desalt column pre-packed with specially-
sized Bio-Gel®polyacrylamide size exclusion gel and pre-
hydrated with equilibration buffer 10 mM Tris pH 7.4
(Bio-Rad) (see Note 7).
9. Dithiothreitol (DTT): 1 M stock solution (see Note 8).
10. Tube rotating wheel.

11. Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories,
Hercules, CA).
12. Spectrophotometer.
13. Sodium orthovanadate (Na3VO4): 100 mM stock solution

(see Note 9).
14. HEPES buffer: 20 mM, pH 7.3.
15. H2O2: 30% stock solution.
2.4 Western Blotting 1. Electrophoresis and transfer apparatus.

2. SDS-PAGE resolving buffer: 10% (v/v) acrylamide/bis-
acrylamide (40%, 37.5:1), 375 mM Tris–HCl pH 8.8, 0.1%
(w/v) SDS, 1% (v/v) TEMED, 10% (v/v) ammonium persul-
fate (APS).
3.
SDS-PAGE stacking buffer: 4% (v/v) acrylamide/bis-
acrylamide (40%, 37.5:1), 125 mM Tris–HCl pH 6.8, 0.1%
(w/v) SDS, 1% (v/v) TEMED, 10% (v/v) APS.
4. Water saturated isopropanol: 80% isopropanol (v/v).
5. Protein Standard Molecular Weight Marker.
6. 6× Laemmli buffer: 260 mM Tris–HCl pH 6.8, 10% (w/v)
SDS, 30% (v/v) glycerol, 0.012% (w/v) bromophenol blue,
6% (v/v) β-mercaptoethanol (see Note 10).
7. Running buffer: 0.1% (w/v) SDS, 25 mM Tris, 192 mM
glycine.
8. Transfer buffer: 25 mM Tris, 192 mM glycine, 20% (v/v)
methanol.
9.
Polyvinylidene difluoride (PVDF) or nitrocellulose
membrane.
10. Ponceau S: 0.5% (w/v) Ponceau S in 1% (v/v) acetic acid.
11. Tris-buffered saline/Tween-20 (TBS-T): 140 mM NaCl,
20 mM Tris pH 7.6, 0.05% (v/v) Tween-20.
12. Blocking buffer: 3% (w/v) BSA/TBS-T or nonfat milk
(see Note 11).
13. Antibody-dilution buffer: 1% (w/v) BSA/TBS-T (see Note 12).
14. Primary Antibody: Rabbit Anti-DNP antibody (provided in
OxyBlot kit); Mouse anti-oxidized-PTP (#MAB2844, R&D
Systems, Michigan, USA).
15. Secondary Antibody: Goat Anti-Rabbit IgG (HRP-conjugated)
antibody (provided in OxyBlot kit); Rabbit Anti-mouse IgG
(HRP-conjugated).
16. Enhanced chemiluminescence reagent (ECL).
3 Methods
3.1 Angiotensin 1.
VSMCs are maintained in complete growth media
II-Stimulation of VSMC (DMEM/10% FBS), in 100 mm cell culture dishes at 37 °C in
a 5% CO2 humidified incubator, until 70–80% confluent
(see Note 13).
2. Growth media is aspirated and replaced by starvation media
(DMEM/0.5% FBS) for ~16 h. (see Note 14).
3. Starvation media is refreshed 30 min prior to Ang II-stimulation
and dishes are returned to the incubator.
4. VSMCs are stimulated with Ang II, 10−7 M, for the desirable
timepoint(s) (see Note 15).
5. Stimulation is terminated by washing off the media three times
with ice-cold PBS (see Note 16).
6. Excess PBS is removed completely and VSMCs are processed
immediately or dishes are stored at −80 °C until use
(see Note 17).
3.2 OxyBlot Assay 1. VSMCs are scraped in lysis buffer on ice, using a cell scrapper
(see Note 18).
3.2.1 Derivatization
of Carbonyl Groups 2. The lysate is transferred into labeled eppendorf tubes and incu-
bated on ice for 30 min (see Note 19).
3. The sample is centrifuged for 10 min at 12,000 × g and at
4 °C.
4. The supernatant is transferred to a new tube and the pellet is
discarded.
5. The supernatant is then split:
(a) 5 μl into a 96-well plate to be used for the protein assay
(see Note 20).
(b) Half of the sample into an eppendorf tube to be used for
the OxyBlot assay.
(c) The remaining material is saved for western blotting and
stored at −80 °C.
6. Based on the protein quantification, all samples designated for
OxyBlot are diluted down to the same protein concentration,
using lysis buffer.
7. DTT is added to a final concentration of 50 mM and the sam-
ples are vortexed briefly (see Note 21).
8. All the following steps are performed at room temperature
(Fig. 1).
9. Equal “x” amounts of sample, containing 15–20 μg of protein,
are transferred into two separate wells; one will be subjected to
the derivatization reaction (DR) and the second will serve as
the negative control (NC) (see Note 22).
Fig. 1 Oxyblot flowchart. Derivatization of protein carbonyl groups (C = O) by 2,4-dinitrophenylhydra-

zine (DNPH)
10. An equal volume “x” of 12% SDS is then added to the sample
for protein denaturation.
11. 2 “x” volumes of 1× DNPH or 1× derivatization control solu-
tion are added into the wells designated for the DR reaction or
NC, respectively (see Note 23).
12. The samples are mixed by shaking the plate and incubated for
15 min at room temperature (see Note 24).
13. Derivatization reaction is terminated by addition of 3/2 “x”
volumes of neutralization solution to all samples, with suffi-
cient force to mix the sample.
14. Samples can be stored at 4 °C and run on a gel within 7 days.
For longer periods of time samples should be aliquoted and
stored at −20 °C.
3.2.2 Western Blotting 1. OxyBlot samples must be allowed to reach room temperature
prior to loading on a gel (see Note 10).
2. 10% polyacrylamide gels are casted using the appropriate
apparatus and by following the manufacturer’s instructions.
3. The samples are loaded in the wells starting with the molecu-
lar weight protein standards. For Oxyblot samples equal
volumes of each sample are loaded. It is recommended that
each DR and the respective NC samples are loaded side by
side to allow more efficient comparison during the analysis
(see Note 25).
4. SDS-PAGE is run following standard procedures [13].
5. The proteins are transferred onto a nitrocellulose or PVDF
membrane using an appropriate electroblotting apparatus and
by following the manufacturer’s instructions (see Note 26).
6. The membrane is soaked into Ponceau S for 5 min and then
rinsed with water to visualize the bands and ensure equal load-
ing of the samples.
7. The membrane is rinsed in TBS-T until Ponceau S is washed
off.
8. Nonspecific primary antibody binding is blocked by incubat-
ing the membrane in blocking/dilution buffer for 1 h at room
temperature, under gentle shaking.
9. The membrane is incubated in 15 ml of the primary antibody
diluted 1:150 in blocking/dilution buffer, under gentle shak-
ing, overnight, at 4 °C (see Note 27).
10. Excess, unbound primary antibody is washed off five times for
5 min with TBS-T.
11. The membrane is incubated in 15 ml of the secondary anti-
body diluted 1:300 in blocking/dilution buffer, for 1 h at
room temperature, under gentle shaking.
12. Excess, unbound secondary antibody is washed off three times
for 5 min with TBS-T.
13. The membrane is incubated in enhanced chemiluminescence
solution according to manufacturer’s specifications.
14. The bands are developed either by exposing the membrane to
film or to a chemiluminescence western blot imager.
3.2.3 Data Interpretation 1. Signal intensity is directly proportional to the amount of pro-
tein carbonyl groups in the samples.
2. Biological samples exposed to stress conditions will have higher
carbonylation content.
3. Development of the OxyBlot membrane will detect multiple
bands in the derivatization reaction samples and theoretically
no bands in the negative controls. Bands appearing in the neg-
ative control represent proteins that have undergone oxidation
during the sample processing (artifacts) and their signal should
be subtracted from the derivatization signal.
Fig. 2 Flowchart for assessment of PTP oxidation in VSMCs stimulated with Ang II. Bottom right panel, example
of oxPTP-immunoblot including samples before (basal −SO3H) and after (total −SO3H) treatment with PV
3.3 PTP Oxidation The following protocol describes a three-step method (S− alkyla-
tion, −SOH reduction, and S− hyperoxidation) designed to cap-
3.3.1 Sample
ture both irreversible (−SO3H) and reversible (−SOH) PTP
Preparation
oxidation states (see Note 28) (Fig. 2).
1. PBS and lysis buffer are degassed for 20 min prior to use, using
a vaccum pump (see Notes 4 and 29).
2. Lysis buffer is supplemented with fresh NEM 10 mM and cata-
lase 100 μg/ml, and degassed for a further 5 min (see Note 5).
3. VSMCs are rinsed with degassed, ice-cold 1× PBS.
4. Cells are scraped in degassed lysis buffer on ice, using a cell
scrapper (see Note 18).
5. The lysate is transferred into labeled eppendorf tubes and incu-
bated for 1 h at 4 °C, protected from light (see Note 30).
6. The sample is centrifuged for 15 min at 16,000 × g and at
4 °C.
7. The alkylated supernatant is directly transferred onto a Micro

Bio-Spin 6 chromatography column (see Note 7).
8. Excess NEM is removed by centrifugation for 4 min at 1000 × g
and at 4 °C.
9. The purified flow-through is split into two eppendorf tubes
(see Note 31): One tube will be used for protein quantification
and for assessing the irreversible PTP-SO3H levels and can be
stored at −80 °C until use.
10. The flow-through in the other tube is treated with 10 mM
DTT for 30 min at room temperature, under rotation, pro-
tected from light (see Note 32).
11. The reduced sample is passed through a Micro Bio-Spin 6
chromatography column.
12. Excess DTT is removed by centrifugation for 4 min at 1000 × g
and at room temperature.
13. 5 μl of the flow-through is kept for protein quantification.
14. The rest of the purified flow-through is treated with 100 μM
PV for 1 h at 4 °C, under rotation, protected from light. For
the preparation of 1 mM PV stock solution (see Note 33):
(a) 30% H2O2 is diluted ten times with HEPES buffer and
mixed gently.
(b) 3% H2O2 is further diluted ten times with HEPES buffer
and mixed gently.
(c) 50 μl of 0.3% H2O2 is added to 10 μl of 100 mM Na3VO4
and 940 μl of H2O and is mixed gently by reversing the
eppendorf tube, to make up 1 mM PV stock.
(d) After 5 min, a small amount of catalase is scooped using a
pipette tip and is mixed into the PV stock, producing a
burst of O2 bubbles. The lid of the tube is left open to
release the air pressure (see Note 34).
(e) The PV stock solution is good for several hours (<2 h) (see
Note 35).
15. Hyperoxidized samples can be further processed to be ana-
lyzed by WB or stored at −80 °C until use.
3.3.2 Western Blotting 1. Equal amounts of protein (20–30 μg) from both DTT/PV
untreated and treated samples are diluted to an equal final vol-
ume, using lysis buffer free of NEM and catalase.
2. Protein samples are mixed with 6× Laemmli buffer (to 1× final
concentration) for denaturation of proteins by β-mercaptoethanol
and are boiled at 80 °C, for 10 min.
3. 10% polyacrylamide gels are casted using the appropriate appa-

ratus and by following the manufacturer’s instructions.
4. The samples are loaded in the wells starting with the molecular
weight protein standards (see Note 36).
5. Protein electrophoresis, transfer, and membrane blocking are
carried out as described in Subheading 3.2.2, steps 4–8.
6. The membrane is incubated in 5 ml of the anti-ox-PTP pri-
mary antibody at 1 μg/ml blocking/dilution buffer, under
gentle shaking, overnight, at 4 °C.
7. Excess, unbound primary antibody is washed off five times for
5 min with TBS-T.
8. The membrane is incubated in 10 ml of the secondary anti-
body diluted 1:2000 in blocking/dilution buffer, for 1 h at
room temperature, under gentle shaking (see Note 37).
9. Membrane rinsing of the secondary antibody and development
is carried out as described in Subheading 3.2.2, steps 12–14.
3.3.3 Data Interpretation 1. Signal intensity is directly proportional to the degree of PTP-
oxidation in the samples.
2. The cumulative signal of PTP-SOH and PTP-SO3H oxidation
is provided by the samples treated with PV.
3. Levels of irreversible PTP-SO3H oxidation are provided by
those samples not treated with PV.
4. Levels or reversible PTP-SOH oxidation is calculated as the
difference between the cumulative and the irreversible PTP-
SO3H oxidation levels.
5. Biological samples exposed to stress-inducing stimuli will
exhibit higher PTP-SOH content, whereas PTP-SO3H con-
tent seems to only be affected by exposure to extremely high
levels of oxidative stress [12].
4 Notes
1. The mixture of standard proteins with attached DNP residues,

provided with the kit, should be aliquoted and stored at
−20 °C after the first use. All other reagents are stored at 4 °C.
2. The working concentration of DTT is 50 mM and should not
be stored for more than 6 h at room temperature. Prepare just
prior to use. Discard any unused reagent.
3. The protease and phosphatase inhibitors are added immedi-
ately before use. Sodium orthovanadate (Na3VO4) should not
be included in the lysis buffer, since it is a known competitive
inhibitor of PTPs by occupying their catalytic site, mimicking
phospho-Tyrosine, and thus compromising the efficiency of
the following steps in this technique [11].
4. Oxygen in the air is dissolved in PBS and lysis buffer and can
induce spontaneous PTP oxidation. Therefore, degassing of
these solutions prior to use is critical to minimize spontaneous
PTP oxidation during cell lysate preparation.
5. NEM is used for free thiol (S−) alkylation. It is a relatively
unstable compound in solution and should be always prepared
fresh prior to use. Protect from light by preparing in an amber
1.5 ml tube. Dissolve in a minimum amount of ethanol and
then add to lysis buffer.
6. Always add to the lysis buffer fresh, immediately before use.
7. Follow manufacturer’s instructions for the preparation of the
Micro Bio-Spin 6 chromatography columns before use.
Briefly, drain the original storage buffer off the column by
short spinning and equilibrate the column by applying
degassed lysis buffer three times, before applying the sample.
The maximum volume of the sample loaded on the column
should not exceed 100 μl.
8. DTT is used for reduction of reversibly oxidized thiols. It is a
relatively unstable compound due to air oxidation and solu-
tions should be preferably prepared fresh. Alternatively, stock
solution of 1 M can be stored at −20 °C in small aliquots to
avoid multiple freezing-thawing cycles.
9. Prepare orthovanadate fresh before use and discard any unused
material. The powder must be white/colorless. Yellow coloration
means that vanadate (VO4) forms 12-mer and cannot be used.
Alternatively, stock solution of 100 mM can be stored at −20 °C
in small aliquots to avoid multiple freezing-thawing cycles.
10. Oxyblot samples do not require mixing with Laemmli buffer
and boiling. The neutralization buffer makes the sample dense,
so it sinks to the bottom of the well.
11. Both BSA and nonfat milk seem to work equally well with pri-
mary antibodies for oxyblot and ox-PTP.
12. Secondary antibodies are normally diluted in 1% (w/v) nonfat
milk.
13. In this protocol, primary VSMC were isolated from rat mesen-
teric bed and used at low-passage (5–6), as healthy cells work
better for measuring inducible protein oxidation and respond
better to Ang II. Higher passage VSMCs may demonstrate
loss of expression of Ang II receptors AT1R and AT2R. Growth
media is refreshed every 48 h. Cells are split when they reach
~70–80% confluence using trypsin-EDTA for cell detach-
ment and ensuring they were not subject to stress by over or
under-confluence. If the experimental design does not require
treatment/stimulation of the cells, the complete growth
media is replaced by starvation media for approximately 16 h
before harvesting.
14. Serum deprivation is used as a technique to induce growth

arrest and synchronize all cell cycles to the G0 quiescent stage
before any treatment, so all cells respond in a similar fashion.
15. For VSMCs isolated from rat mesenteric artery the optimal
effect of Ang II is achieved at a concentration of 10−7 M,
according to response curves performed previously in our lab-
oratory. The timepoints used to observe an effect of Ang II on
protein/PTP oxidation are relatively short and range between
5 and 60 min. It is important to use negative controls stimu-
lated with just the vehicle, i.e. the solvent that Ang II is dis-
solved/diluted in, to account for any false-positive effects of
the vehicle on the parameters tested. If additional treatment
with Ang II-receptor antagonists or antioxidant agents is
required, then VSMCs are treated with these agents for a
period of 30 min prior to the addition of Ang II. Normally, the
final concentration of the antagonists/antioxidants in the cell
culture should be at least 10–100 times higher than the stimuli
concentration.
16. It is recommended that dishes are kept on ice during the
washes to more effectively and acutely stop/slow down any
cellular signaling and metabolic reactions, especially if one is
interested in capturing very transient changes such as PTP
reversible sulfenylation (PTP-SOH).
17. Storage period at −80 °C should not exceed 2–3 months.
18. Harvesting of cells must proceed rapidly to minimize post-lysis
spontaneous protein/PTP oxidation due to oxidizing air con-
ditions. The amount of cell lysis buffer used can be adjusted
according to the desirable protein concentration of the sample
to perform the assays.
19. The samples must be maintained on ice at all times to avoid
proteolysis.
20. DTT is not compatible with most protein assays, at the con-
centration used in this protocol. Therefore, an aliquot of the
sample needs to be kept aside for protein quantification, before
treatment with DTT.
21. Ideally, OxyBlot should be performed on fresh VSMC lysates.
Alternatively, samples treated with DTT can be stored at
−20 °C for up to 1 month. DTT is a reducing agent that pre-
vents spontaneous protein oxidation after cell lysis.
22. Larger volumes of sample can be processed to allow running
more than one Western blot for carbonylation per sample.
Avoid protein concentrations >10 μg/μl, as sample solubility
during the derivatization reaction can be affected.
23. The sample for the DR reaction will take an orange color upon
addition of DNPH.
24. Samples should not stand in derivatization solution for more

than 15–20 min, as this could cause artifact increase in the pro-
tein carbonyl content.
25. All samples should have equal protein concentrations, based
on the protein quantification assay and the dilutions performed
in Subheading 3.2, step 6. To further ensure equal loading,
probe membranes for GAPDH or any other commonly used
loading control.
26. If a PVDF membrane is used, activation of the membrane by
soaking it in ethanol prior to use is required.
27. It is recommended to prepare fresh primary antibody each time.
28. If lots of samples/sets need to be analyzed, it is recommended
to process fewer samples through the full protocol within 1
day, rather than processing all sets simultaneously but breaking
the protocol at more than one step.
29. Lysis buffer may foam due to NP-40. Let the foam sit
before use.
30. NEM is light sensitive and is used to alkylate free thiols. It is
recommended that a positive control of a sample not treated
with NEM is included, to validate the efficiency of the free
S− blocking.
31. The columns can be thoroughly washed with 10 ml of ultra-
pure H2O, followed by 10 ml of 20% (v/v) ethanol or PBS
supplemented with 0.1% (v/v) sodium azide. Columns can be
stored in PBS/sodium azide buffer and can be reused for
another three to four times.
32. DTT is light sensitive and is used for reduction/reactivation of the
reversibly oxidized PTPs. Omitting this step prevents PV from
accessing the active site of reversibly oxidized PTPs and limits
hyperoxidation. It is recommended that a negative control of a
sample not treated with DTT is included, to evaluate the efficiency
of the NEM-mediated alkylation in the first step of the cell lysis.
33. Pervanadate is rapidly converted to vanadate, in the presence
of thiol reductants such as DTT [11]. Therefore, complete
removal of excess DTT in the previous step is critical for effi-
cient PTP hyperoxidation.
34. Catalase is used to remove excess H2O2 that has not reacted
with Na3VO4 in the solution, seen as a burst of O2 bubbles.
35. It is recommended to use PV within 5 min to minimize decom-
position of the vanadate-H2O2 complex.
36. Untreated and PV-treated samples should be loaded on the
same gel to allow for direct comparisons between the –SO3H
and –SOH oxidation states of PTPs.
37. Antibody dilutions can be adjusted empirically.
References
1. Montezano AC, Dulak-Lis M, Tsiropoulou S, oxidatively modified proteins. Methods

Harvey A, Briones AM, Touyz RM (2015) Enzymol 233:346–357
Oxidative stress and human hypertension: vas- 8. Ostman A, Frijhoff J, Sandin A, Böhmer FD
cular mechanisms, biomarkers, and novel ther- (2011) Regulation of protein tyrosine phos-
apies. Can J Cardiol 31(5):631–641. phatases by reversible oxidation. J Biochem
doi:10.1016/j.cjca.2015.02.008 150(4):345–356. doi:10.1093/jb/mvr104
2. Nguyen Dinh Cat A, Montezano AC, Burger 9. Karisch R, Neel BG (2013) Methods to moni-
D, Touyz RM (2013) Angiotensin II, NADPH tor classical protein-tyrosine phosphatase oxi-
oxidase, and redox signaling in the vasculature. dation. FEBS J 280(2):459–475.
Antioxid Redox Signal 19(10):1110–1120. doi:10.1111/j.1742-4658.2012.08626.x
doi:10.1089/ars.2012.4641 10. Persson C, Kappert K, Engström U, Ostman
3. Dean RT, Fu S, Stocker R, Davies MJ (1997) A, Sjöblom T (2005) An antibody-based
Biochemistry and pathology of radical- method for monitoring in vivo oxidation of
mediated protein oxidation. Biochem protein tyrosine phosphatases. Methods
J 324:1–18 35(1):37–43
4. Cai Z, Yan LJ (2013) Protein oxidative modifi- 11. Huyer G, Liu S, Kelly J, Moffat J, Payette P,
cations: beneficial roles in disease and health. Kennedy B, Tsaprailis G, Gresser MJ,
J Biochem Pharmacol Res 1(1):15–26 Ramachandran C (1997) Mechanism of inhibi-
5. Tabet F, Schiffrin EL, Callera GE, He Y, Yao G, tion of protein-tyrosine phosphatases by vana-
Ostman A, Kappert K, Tonks NK, Touyz RM date and pervanadate. J Biol Chem
(2008) Redox-sensitive signaling by angioten- 272(2):843–851
sin II involves oxidative inactivation and 12. Karisch R, Fernandez M, Taylor P, Virtanen C,
blunted phosphorylation of protein tyrosine St-Germain JR, Jin LL, Harris IS, Mori J, Mak
phosphatase SHP-2 in vascular smooth muscle TW, Senis YA, Östman A, Moran MF, Neel BG
cells from SHR. Circ Res 103(2):149–158 (2011) Global proteomic assessment of the
6. Dalle-Donne I, Giustarini D, Colombo R, classical protein-tyrosine phosphatome and
Rossi R, Milzani A (2003) Protein carbonyl- "Redoxome". Cell 146(5):826–840.
ation in human diseases. Trends Mol Med doi:10.1016/j.cell.2011.07.020
9:169–176 13. Laemmli UK (1970) Cleavage of structural
7. Levine RL, Williams JA, Stadtman ER, Shacter proteins during the assembly of the head of
E (1994) Carbonyl assays for determination of bacteriophge T4. Nature 227:680
Chapter 4
Methods for Studying the Role of RAAS in the Modulation

of Vascular Repair-Relevant Functions of Stem/Progenitor
Cells
Yagna P.R. Jarajapu
Abstract
In recent years, previously unknown functions have been conferred to the RAAS and have been explored
in mechanistic studies and disease models. Implication of bone marrow stem/progenitor cells in the car-
diovascular protective or detrimental effects of RAAS is a prominent advancement because of the transla-
tional significance. Selected members of RAAS are now known to modulate migration, proliferation, and
mobilization of bone marrow cells in response to ischemic insult, which are sensitive indicators of vascular
repair-relevant functions. In this Chapter, protocols for most frequently used, in vitro, ex vivo, and in vivo
assays to explore the potential of RAAS members to stimulate vascular repair-relevant functions of bone
marrow stem/progenitor cells of human and murine origin.
Key words CD34+ cells, LSK cells, Bone marrow, Mobilization, Flow cytometry, Migration,
Proliferation
1 Introduction
The existence of stem/progenitor cells that accelerate ischemic

vascular repair by re-endothelialization has now been well docu-
mented in experimental studies. These cells proliferate in response
to ischemia-regulated factors, and have the ability to mobilize from
the bone marrow niches into systemic circulation and to migrate to
the areas of ischemia [1]. Importantly, clinical studies have con-
firmed the therapeutic potential of progenitor cells for the treat-
ment of ischemic cardiovascular disease.
Evidence has now been accumulated that implicates the renin-
angiotensin-aldosterone system (RAAS) in cardiovascular repair
and regeneration by modifying the vasoreparative functions of bone
marrow stem/progenitor cells [2, 3]. Impaired proliferation or
migration in response to stromal-derived factor-1α (SDF) or vascu-
lar endothelial growth factor (VEGF) predicts the defective mobili-
47
48 Yagna P.R. Jarajapu
zation in response to injury and impaired reparative propensity of

stem/progenitor cells, all of which are apparent in clinical condi-
tions such as diabetes [4, 5]. We have been consistently using pro-
liferation, migration, and ischemia-induced mobilization as sensitive
indicators of vascular repair-relevant functions of human and murine
cells for evaluating the potential of ACE2/Angiotensin-(1-7)/Mas
receptor pathway [6–10]. Here, we describe preparation of cells
from human and mouse samples, and protocols for carrying out
these functional assays.
2 Materials
2.1 Cell Culture 1. C57Bl/6J mice (Jackson laboratories) or C57Bl/6 NHsd

Reagents (Harlan Laboratories, Inc.).
2. Human peripheral blood, preferably in the form of leukopaks
or leucocyte reduction systems (LRS) following apheresis.
3. Buffers/media: PBS (Ca2+ and Mg2+ free), HBSS, StemSpan®,
and RPMI.
4. Stain buffer: PBS with 2% FBS, 0.09% sodium azide.
5. Sorting buffer: PBS with 0.5% FBS.
6. 0.4% Trypan blue solution in PBS, particle-free.
7. 10× RBC lysis buffer.
8. Ficoll® Paque.
9. 96-well plates, U-bottom.
10. 40-μm mesh filters.
11. 5 mL round bottom, and 15 mL and 50 mL conical polypro-
pylene tubes.
2.2 Fluorescence- 1. FACSAria cell sorter or FACS jazz for cell sorting and flow
Activated Cell Sorting cytometry. Accuri C6 flow cytometer.
(FACS) 2. Mouse antibodies: Rat anti-mouse lineage cocktail, anti-Sca-1-
and Immunomagnetic APC, and anti-cKit-PE. Rat isotype controls. Mouse anti-
Enrichment human antibodies: CD34-Pacific blue, CD45-PE-Cy7,
CD34-PE, CD45-PE-Cy7, and mouse isotype controls.
3. Cell enrichment kits for murine hematopoietic cells, murine
cKit+ cells, human hematopoietic cells and human CD34+ cells.
2.3 Assay Kits 1. Colony-forming unit (CFU) assay: Methocult® GF M3434

complete methylcellulose medium with species-specific recom-
binant cytokines for colony assay.
2. Migration assay: QCM-Chemotaxis-Cell Migration assay,
5 μm, 96-well format, for non-adherent cells.
3. Proliferation assay: Cell proliferation ELISA, BrdU (colorimetric).
RAAS and Stem/Progenitor Cells 49
3 Methods
In this section, protocols for the isolation of human CD34+ cells or

mouse lineage−, c-Kit+ and Sca-1+ cells from peripheral blood or
bone marrow by flow-assisted cell sorting or by immunomagnetic
enrichment are described followed by functional assays, by which
the potential of RAAS components in modulating these functions
can be evaluated.
3.1 Low-Density 1. Dilute human blood with PBS if the samples were obtained as
Gradient Separation a leukopak or in LRS cones with PBS containing 2% FBS in
of Total Mononuclear 50 mL tubes and mix gently by inverting tightly capped tubes
Cells (MNCs) up and down (see Note 1).
from Human 2. Aliquot 15 mL of Ficoll into 50 mL polypropylene tubes.
Peripheral Blood 3. 35 mL of blood sample is layered over Ficoll (see Note 2).
Mark the cells/Ficoll-paque interface with a felt tip pen.
4. Tubes are centrifuged for 30 min at 800 × g at room
temperature.
5. The suspension of MNCs is aspirated to the edge of the inter-
face into another 50 mL tube.
6. Cells are slow-washed with PBS to remove platelets and resid-
ual plasma and Ficoll by centrifuging at 120 × g for 10 min at
room temperature.
7. Remove supernatant and resuspend the cell pellet in 45 mL of
PBS and repeat step 7.
8. Repeat step 8 for a third wash. Remove supernatant and resus-
pend pellet in 2 mL PBS.
9. Determine the cell number by diluting an aliquot with Trypan
blue solution (1:1) and by using hemocytometer. Trypan blue
stains dead cells.
3.2 Enrichment This is carried out by using commercially available kits for example
of Lin- Human from StemCell Technologies.
Progenitor Cells
1. Cell suspension containing 1 × 108 cells/mL, not more than
with CD41 Depletion 2 mL, is aliquoted into 5 mL polypropylene tubes.
2. Enrichment cocktail, 50 μL/mL of cells, is added and the cell
suspension is gently mixed and incubated at room temperature
for 10 min.
3. Nanoparticle suspension is mixed vigorously by using a pipette
at least five times to a uniform suspension (see Note 3).
4. 50 μL of nanoparticle suspension is added to 1 mL of cell sus-
pension and mixed well, and incubated at room temperature
for 15 min.
5. Suspension is remixed thoroughly and volume is made up to
5 mL with PBS.
6. Tube is placed in the EasySep magnet and incubated for 10 min

at room temperature.
7. Magnet holding the tube is inverted in one continuous motion
to decant the desired fraction into another tube. This is the
unlabeled fraction or negative selection, enriched for
lineage− cells.
8. Total number of cells is determined as described above. This
fraction is used for isolation of CD34+ cells by FACS or by
immunomagnetic positive selection.
3.3 Enrichment 1. Lin− cell suspension (1 × 106 cells/mL stain buffer) is treated
of CD34+ Cells by FACS with human FcR blocking reagent.
2. Cells are stained with pacific blue-labeled mouse anti-human
CD34 and PE-Cy7-labeled mouse anti-human CD45 antibod-
ies will resolve CD34 and CD45 populations with no fluores-
cence overlap. Cells are incubated for 45 min at 4 °C.
3. Total volume is made up to 5 mL with PBS and centrifuged at
200 × g for 10 min.
4. Supernatant is discarded and step 3 is repeated.
5. Cell pellet is resuspended in 1 mL of sort buffer.
6. Cells are stained with 7-aminoactinomycin D (7-AAD) to
exclude dead cells before sorting (see Note 4).
7. A tube with cells stained with isotype control antibodies is pre-
pared as per steps above and used for setting the gates for
sorting.
8. CD45lowCD34high population is selected for sorting.
9. Cells are collected in a sterile tube with medium for culture.
10. Collected cells are enumerated, and spun down at 200 × g and
supernatant is discarded.
11. Cell pellet is resuspended in the medium, with or without
cytokines, in 96-well plate U-bottom, at a density of 2 × 104
cells/well in 150 μL.
3.4 Alternatively, 1. The Lin− cell suspension is aliquoted at a density of 2 × 108

Lin- Cells Can cells/mL in PBS with 2% FBS and 1 mM EDTA in 5 mL poly-
Be Enriched for CD34 propylene tubes.
Cells by 2. Positive selection cocktail is added to the cell suspension,
Immunomagnetic 100 μL/mL, and mixed thoroughly. Cells are incubated at
Selection by Using room temperature for 15 min.
a Kit. With Practice, 3. Magnetic nanoparticle suspension is mixed vigorously with a
this Approach Can pipette to uniform suspension.
Obtain 95% Purity
4. Nanoparticle suspension, 50 μL/mL, is added to cells and
of Isolated Cells
mixed well. Cell suspension is incubated for 10 min at room
temperature.
5. Volume is made to 2.5 mL and mixed well.

6. Tube with diluted cell suspension is placed in the EasySep
magnet for 5 min at room temperature.
7. Magnet is inverted in one continuous motion to decant the
buffer with unwanted cells.
8. Step 7 is repeated three more times by adding 2.5 mL of PBS
to the tube with retained cells, mixing well before placing in
the magnet.
9. CD34+ cells that are retained in the tube are resuspended in
PBS and enumerated.
10. Cells are plated in StemSpan as described in Subheading 3.3.
Shown in Fig. 1 are representative dot plots enumerating the
CD34+ cell population in MNCs, Lin- cells or in the prepara-
tion enriched as per the protocol in Subheading 3.4 (Fig. 1).
3.5 Low-Density Bone marrow cells are obtained by either flushing the bone mar-
Gradient Separation row or by crunching femorae and tibiae.
of Total Mononuclear 1. Total bone marrow obtained by crushed bones derived from
Cells from Mouse one mouse is passed through 40 μm mess filters and suspended
Bone Marrow in PBS with 2% FBS at 3 × 107 cells/mL.
2. Cell suspension is layered over 3 mL of Ficoll® Paque in 15 mL
conical tubes.
3. The tubes are spun down at 500 × g for 30 min at room
temperature.
4. The interphase cell layer is aspirated into 50 mL of PBS, and
slow-washed by centrifugation at 200 × g for 10 min at room
temperature.
5. Cells are counted in a hemocytometer as described above.
6. The cells are resuspended in PBS.
Fig. 1 Representative dot plots showing the purity of CD34+ cells that were enriched by immunomagnetic selec-
tion from human peripheral blood. Shown in a was an isotype control. Density of CD34+ cells was checked (b)
in the total mononuclear cells, and (c) after enrichment of Lineage negative (Lin−) cells and (d) CD34+ cells
3.6 FACS of Lin-/ 1. An aliquot of cells in stain buffer with labeled rat isotype anti-
Sca-1+/c-Kit+ Cells bodies as no-stain control to set the gates for FACS.
2. The rest of cells from Subheading 3.5 are treated with mouse
FcR blocking antibody in stain buffer, and stained with lineage
cocktail-FITC, c-Kit-PE, and Sca-1-APC.
3. Cell suspensions from steps 1 and 2 are incubated for
45 min at 4 °C. The tubes are covered with aluminum foil
to prevent exposure to light. Concentration of each anti-
body for mouse cells must be predetermined as per stan-
dard protocols.
4. 7-AAD is added before proceeding for sorting to exclude dead
cells.
5. Lin- Sca-1+c-Kit+ cells appear as a well-defined population in a
dot plot (Fig. 2).
6. Cells are collected in sterile RPMI and enumerated as described
above.
7. Cell suspension is centrifuged at 200 × g and the supernatant
is discarded.
8. Cells are resuspended in RPMI and plated in a 96-well plate,
U-bottom, at a density of 2 × 104 cells/well/150 μL.
Fig. 2 Representative dot plots showing the protocol for flow cytometric enumeration of lineage-negative (Lin-),
Sca-1+ and C-Kit+ cells from mouse peripheral blood. Shown in the top panel is a representative of a sample
stained with isotype control antibodies, and shown in the bottom panel resolution of LS, LK, and LSK popula-
tions. Note the exclusion of dead cells that are stained by 7-AAD, and the exclusion of doublets or cell clumps
by plotting FSC-A against FSC-H
3.7 For Flow This is frequently used for evaluating the effect of treatments on
Cytometric mobilization of bone marrow LSK cells by collecting peripheral
Enumeration of LSK blood samples at different time intervals.
Cells from Mouse Shown in Fig. 2 are representative dot plots from the flow
Peripheral Blood or cytometric enumeration of LSK cells from peripheral blood.
Bone Marrow,
the Same Protocol
as above Is Followed
(Steps 1–5), and Can
Be Carried
Out in Simple Flow
Cytometer with four
Different Channels
(e.g., Accuri C6)
3.8 Immuno This is accomplished by using commercially available isolations

magnetic Enrichment kits.
of Mouse Lin- Cells
1. A suspension of cells obtained in 2.5 is prepared at a density of
1 × 108 cells/mL in Ca2+ and Mg2+ free PBS with 2% FBS and
1 mM EDTA in a 5 mL polypropylene tube.
2. The cell suspension is treated with naive rat serum at a concen-
tration of 50 μL/mL and mixed thoroughly.
3. Mouse hematopoietic progenitor cell enrichment cocktail is
added at a concentration of 50 μL/mL. Suspension is mixed
thoroughly and incubated at 2–8 °C for 15 min.
4. Total volume is made up to 2 mL and centrifuged at 200 × g
for 5 min.
5. Cell pellet is resuspended in the medium as suggested in step 1.
6. Biotin selection cocktail is added at a concentration of
100 μL/mL of cell suspension. Suspension is mixed well and
incubated at 2–8 °C for 15 min.
7. Magnetic particle suspension is vigorously mixed with a pipette
to a uniform suspension.
8. Magnetic particle suspension is added at a concentration of
50 μL/mL. Cell suspension is mixed thoroughly and incu-
bated at 2–8 °C for 10 min.
9. The cell suspension is mixed thoroughly again and volume is
made up to 2.5 mL with PBS with 2% FBS and 1 mM EDTA.
10. Cell suspension is then placed in the EasySep magnet and set
aside for 3 min.
desired fraction of the cell suspension into a new tube.
12. The number of Lin- cells collected in step 11 is determined
and are ready for further enrichment.
3.9 Immuno Using this approach we can obtain Lin−c-Kit+ cells not LSK cells.
magnetic Enrichment These cells are preferred over LSK cells for functional assays because
of Mouse Lin-c-Kit+ of their higher number from bone marrow and these cells possess
Cells (LK Cells) vascular-repair relevant functions similar to LSK cells. Importantly,
a majority of these cells are positive for Sca-1. Along similar lines,
LS cells could be isolated in even higher number than LK cells;
however, a majority of LS cells are not positive for c-Kit.
1. Lin- cells obtained in Subheading 3.8 are resuspended in
100 μL of PBS with 2% FBS and 1 mM EDTA in a 5 mL poly-
propylene tube.
2. Mouse FcR (CD16/32) blocking antibody is added at 1 μg/
mL, mixed thoroughly and left aside for 5 min at room
temperature.
3. CD117-PE labeling reagent is added 5 μL/100 μL/mL of cell
suspension, mixed well and incubated at room temperature for
15 min.
4. PE selection cocktail is then added, 7 μL/100 μL suspension,
mixed well and incubated at room temperature for 15 min.
5. Magnetic nanoparticle suspension is vigorously mixed with a
pipette to a uniform suspension.
6. Magnetic nanoparticles were added, 5 μL/100 μL suspension,
and incubated for 10 min.
7. Cell suspension is made up to 2.5 mL and the tube is kept in
the magnet for 5 min.
buffer with unwanted cells.
9. Step 8 is repeated three more times by adding 2.5 mL of PBS,
mixing well before placing in the magnet.
10. LK cells that are retained in the tube are resuspended in PBS
and cell count is determined.
3.10 Quantification This is accomplished by using species-specific medium, Methocult®.

of Colony Forming
1. Peripheral blood or bone marrow MNCs are mixed with
Units (CFUs)
Methocult medium in a 5 mL round-bottom tube.
2. Tubes are vortexed and allowed to rest for 10 min at room
temperature.
3. Medium with cells is dispensed into 35 mm culture dishes.
Final cell density is 1 × 104 cells/mL for bone marrow cells, or
1 × 105 cells/mL for peripheral blood cells.
4. Dishes are placed in a 100 mm petri dish with an open 35 mm
dish with sterile water.
5. The 100 mm dish containing the 35 mm dishes is placed in an
incubator at 37 °C, 5% CO2 for 10 days.
6. CFUs are identified as clusters with more than 50 cells and

counted. All colonies, including BFU-E, CFU-GM, and CFU-
Mix are counted. Our routine practice is to count the total
number of CFUs as a measure of the number of functional
progenitor cells present in the given sample.
3.11 Protocol This protocol is used for human CD34+ or mouse LK or LSK cells,
for Migration Assay and is carried out by using QCM-Chemotaxis cell migration assay
kit (5 μm) (see Note 5).
1. Cells are counted and suspended in Hank’s buffered saline
solution (HBSS). Make sure 2 × 104 cells are available for each
well/treatment.
2. 150 μL of HBSS is added in the 96-well feeder tray, either
control or containing factors such as SDF or VEGF that are
species-specific.
3. Cell migration chamber plate, 96-well, is carefully placed on
feeder tray and cell suspension containing 2 × 104 cells in
100 μL is added to each well.
4. Chemotaxis plate is incubated at 37 °C for 6 h (see Note 6).
5. The upper migration chamber plate is removed and the cells are
discarded by gently flipping off the plate onto blotting paper.
6. Lower chamber with migrated cells is kept at room tempera-
ture for use in step 11.
7. In another 96-well plate, 150 μL of cell detachment buffer is
added, and the migration chamber plate is placed on it and
incubated for 30 min at 37 °C.
8. A mixture of cell lysis buffer and a dye solution, provided in
the kit, is prepared at 4:1 ratio and dispensed into a black well
plate appropriate for fluorescence plate reader.
9. Cell suspension, 75 μL from lower chamber in step 6 and
75 μL from step 7 are added to black well plate.
10. Fluorescence is read at 480/520 nm in a plate reader.
Alternatively, migration assay can be carried out by using CFU
assay at step 4 by using species-specific medium as described in
Subheading 3.10.
3.12 Proliferation Proliferation of cells can be evaluated in several ways, ranging from
Assay Protocol simple cell counting or by sequential dilution of intracellular fluo-
rescent dye [9]. We find the assay involving BrdU incorporation is
more reproducible, accurate, and efficient as it requires less num-
ber of cells per individual treatment. This is carried out by using a
colorimetric, cell proliferation BrdU ELISA.
1. Cells are counted and resuspended in appropriate medium,
StemSpan for human cells or RPMI for murine cells, with no
addition of FBS or supporting factors.
2. Dispense 1 × 104 cells per each well/or treatment in 100 μL of

medium. Cells with no treatment, control, and cells with
10 μM mitomycin treatment, a negative control, relative to
which fold-proliferation will be expressed, are included.
3. Proliferation can be monitored for 24, 48, and 72 h, therefore
plan accordingly with required number of cells for the time
intervals.
4. Plate with cells and treatments is incubated at 37 °C.
5. Label cells with BrdU for about 18 h with the reagent pro-
vided in the kit.
6. Transfer labeled cells from different wells to 1.5 mL eppendorf
tubes.
7. Cell suspension is made up to a volume of 1 mL with PBS.
8. Tubes are centrifuged at 120 × g for 10 min at room
temperature.
9. Supernatant is discarded and dried at 60 °C for 1 h.
10. Fixation-DNAdenat buffer, 200 μL, is added to each tube and
tubes are incubated at room temperature for 30 min.
11. Tubes are centrifuged at 120 × g for 10 min and the superna-
tant is discarded.
12. Anti-BrdU-POD, 100 μL/well, is added and then incubated
at room temperature for 90 min.
13. Tubes are centrifuged at 120 × g for 10 min and the superna-
tant is discarded.
14. 1 mL of PBS is added and mixed with gentle tapping the bot-
tom of the tube. Tubes are centrifuged at 300 × g for 10 min.
15. The supernatant is discarded and the substrate solution is
added.
16. Tubes are incubated at room temperature for 15 min.
17. Cells are mixed by pipetting gently for two to three times and
then transferred into a 96-well plate (flat-bottom).
18. The absorbance is measured at ODs 405 and 490 nm in a plate
reader.
19. A405-A490 is an indicator of BrdU incorporation or
proliferation.
3.13 Mobilization We have standardized this protocol in the model of hind-limb isch-
of Cells in Response emic (HLI) injury in mice [11] and this can be applied to any
to Ischemic Injury other models of ischemic insults.
1. Number of mice per group is determined according to the
principles of experimental design and power analysis.
2. All mice undergoing ischemic injury are tested for the basal
levels of LSK cells in the circulation prior to the ischemic insult.
Peripheral blood samples, 70–80 μL, are obtained under iso-
flurane anesthesia (see Notes 7–9).
3. Blood samples are treated with RBC lysis buffer, 2–5 mL, in
15 mL tubes, and are incubated at 4–8 °C for 10 min. Volume
of RBC lysis is required to be optimized by an individual
researcher (see Note 10).
4. Total volume is made up to10 mL with PBS and centrifuged at
200 × g for 10 min.
5. The supernatant is discarded and step 4 is repeated.
6. If RBCs are still left in the cell pellet, repeat steps 3–5.
7. Enumerate the total number of cells as described above.
8. Cells are now ready for enumeration of LSK cells as described
in Subheading 3.7.
9. Following ischemic insult, blood samples are collected on days,
1, 2, 3, 5, 7, and 10, and blood samples are processed as
described for the numeration of LSK cells (see Note 11).
4 Notes
1. All centrifugations in the methods described above are prefer-

ably carried out in a break-of mode, which would prevent likely
disturbance of the cell pellet.
2. Overlaying blood over Ficoll layer is a crucial step that deter-
mines clarity of separation of MNC-rich layer from other layers
in the gradient. Tilt tube with Ficoll at a 45° angle and overlay
blood very slowly by using a 10 mL pipette. An automated
pipetting system can be used to overlay blood only if operated
in “g” (gravity) mode.
3. Thorough mixing of magnetic particles has to be done at every
time of use as the particles tend to settle down to the bottom
of the vial and form loose clumps relatively quickly.
4. 7-AAD can be conveniently added 5–10 min before flow cyto-
metric enumeration or sorting for staining dead cells at a con-
centration of 0.25 μg/mL.
5. Migration and proliferation assays can be carried out in human
CD34 cells or LK cells obtained from mice that have received
pharmacological treatments, or mice that were genetically
modified.
6. In our experience with migration assay for non-adherent cells,
5–6 h of incubation is optimal for observing maximum migra-
tory response with standard treatments such as SDF or VEGF
as well as Angiotensin-(1-7). With prolonged periods of time,

increased migration is observed; however, the untreated cells
also show higher mobilization.
7. Blood sampling for enumeration of circulating bone marrow
cells can be done either from orbital sinus or from subman-
dibular vein. The latter is preferred if frequent sampling is
needed for example post-HLI for 2–3 weeks at regular time
intervals.
8. It is highly recommended that blood sampling for the enu-
meration of circulating bone marrow cells is performed under
light anesthesia. This is necessary to prevent stress-induced
mobilization of bone marrow cells, which occurs
instantaneously.
9. Blood volume that is required for enumeration of circulating
LSK cells requires optimization by an individual researcher and
this varies with the treatments or a disease model. For example,
following treatment with a strong mobilizer such as granulo-
cyte colony stimulating factor, G-CSF, as low as 25 μL of blood
would be more than enough. In contrary, in mice with long-
term diabetes, very few cells would be identified in a sample of
100 μL of blood.
10. To lyse RBCs, the recommended volume is in the ratio of 1:4,
one volume of cell suspension with four volumes of the lysis
buffer, followed by incubation for 10 min at 4 °C. This dura-
tion of incubation can be reduced for low volumes of blood;
on the other hand for larger volumes of blood this step requires
to be repeated. Longer incubation times for larger volumes is
not recommended as it would result in damage of cells and
poor enrichment. Therefore, this step requires optimization by
the individual researcher.
11. In our experience with HLI model in C57Bl/6J mice, peak
mobilization of LSK cells is observed in 2–3 days and returns
back to basal levels by day 7 following HLI. This pattern, and
the number of cells in the circulation at the peak of mobiliza-
tion, vary with disease, strain, age or following pharmacologi-
cal treatments that manipulate RAAS. Use of appropriate
controls is necessary to avoid false interpretation of results.
Acknowledgments
The author wishes to acknowledge Mr. Shrinidh Joshi, and Mr.

Goutham Vasam, for their contributions in refining some of the
protocols described in this chapter.
References
1. Asahara T, Kawamoto A, Masuda H (2011) 7. Jarajapu YP, Bhatwadekar AD, Caballero S
Concise review: circulating endothelial pro- et al (2013) Activation of the ACE2/angio-
genitor cells for vascular medicine. Stem Cells tensin-(1-7)/mas receptor axis enhances the
29(11):1650–1655 reparative function of dysfunctional diabetic
2. Jarajapu YP, Grant MB, Raizada MK (2012) endothelial progenitor cells. Diabetes
ACE2/angiotensin-(1-7)/mas axis and cardio- 62(4):1258–1269
vascular regeneration. Curr Hypertens Rev 8. Singh N, Vasam G, Pawar R et al (2014)
8(1):35–46 Angiotensin-(1-7) reverses angiogenic dys-
3. Roks AJM, Rodgers K, Walther T (2011) function in corpus cavernosum by acting on
Effects of the renin angiotensin system on the microvasculature and bone marrow-
vasculogenesis-related progenitor cells. Curr derived cells in diabetes. J Sex Med
Opin Pharmacol 11:162–174 11(9):2153–2163
4. Fadini GP, Derraro F, Quaini F et al (2014) 9. Jarajapu YP, Hazra S, Segal M et al (2014)
Concise review: diabetes, the bone marrow Vasoreparative dysfunction of CD34+ cells in
niche, and impaired vascular regeneration. diabetic individuals involves hypoxic desensiti-
Stem Cells Transl Med 3(8):949–957 zation and impaired autocrine/paracrine
5. Jarajapu YP, Grant MB (2010) The promise of mechanisms. PLoS One 9(4):e93965
cell based therapies for diabetic complica- 10. Singh N, Joshi S, Guo L et al (2015) ACE2/
tions: challenges and solutions. Circ Res Ang-(1-7)/mas axis stimulates vascular repair-
106(5):854–869 relevant functions of CD34+ cells. Am J Physiol
6. Jarajapu YP, Caballero S, Verma A et al (2011) Heart Circ Physiol 309(10):H1687–H1707
Blockade of NADPH oxidase restores vasore- 11. Niiyama H, Huang NF, Rollins MD et al
parative function in diabetic CD34+ cells. (2009) Murine model of hindlimb ischemia.
Invest Ophthalmol Vis Sci 52(8):5093–5104 J Vis Exp 23:1035. doi:10.3791/1035
Chapter 5
Use of a Fluorescent Substrate to Measure ACE2 Activity

in the Mouse Abdominal Aorta
Yu Wang, Lisa A. Cassis, and Sean E. Thatcher
Abstract
The use of fluorogenic substrates to measure enzymatic activity is widely used to understand function
within different experimental models. ACE2 is important in understanding the balance between AngII
and Ang-(1-7) and how this balance could then in turn influence hypertension or other disease outcomes.
Here, we describe a method to measure ACE2 activity in abdominal aorta of hyperlipidemic mice under
both saline and AngII infusion.
Key words Angiotensin converting enzyme 2, Aorta, Activity, Enzyme, Fluorescence
1 Introduction
Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypepti-

dase that cleaves angiotensin II (AngII) to angiotensin-(1-7)(Ang-
(1-7)) [1]. ACE2 is known to have other substrates; however, it
has been best characterized in the catabolism of AngII [2–4]. Our
lab uses iodinated AngII (125I-AngII) and separates out the angio-
tensin peptides by high-performance liquid chromatography
(HPLC) to measure Ang-(1-7) levels in tissues [5, 6]. This method
has the benefit of using AngII as the substrate; however, this
method requires dedicated lab equipment and the use of radiola-
bels which may not be practical for all labs. Recently, more labs are
using fluorescently quenched compounds to measure enzymatic
activity in cells and tissues [7, 8]. This principle uses a quencher
molecule, in this case dinitrophenol (Dnp), to block the fluores-
cence of methoxycoumarin (Mca). This interaction is abolished
however when the enzyme cleaves the proline-lysine residue and
allows for the Mca to then emit light at a given wavelength. These
fluorogenic compounds offer more flexibility since more samples
can be screened in a relatively shorter amount of time (e.g., higher
throughput). ACE2 is a metalloproteinase; therefore, it requires a
61
62 Yu Wang et al.
divalent cation positioned at the active site to perform catalysis.

Optimal pH balance is also important for ACE2 and inhibition of
angiotensin-converting enzyme 1 (ACE1) and other endopepti-
dases (e.g., neprilysin and thimet oligopeptidase) are necessary to
prevent the influence of these enzymes on Ang-(1-7) formation.
Within this protocol, we will use the abdominal aorta as an exam-
ple of how to measure ACE2 activity in hyperlipidemic mice that
have been infused for 28 days with either saline or AngII.
2 Materials
Prepare all solutions using ultrapure water (18 MΩ at 25 °C).

Store all reagents at room temperature (unless otherwise indicated)
and protect fluorescent substrates from light when working with
them at the lab bench. Also, dispose of all wastes properly and
according to hazardous waste regulations.
2.1 Stock Solutions 1. 1 M Tris–HCl, pH 7.5: Measure out 75 mL of water and place
in beaker with a stir bar. Weigh out 15.76 g of Tris–HCl and
place in a beaker and mix to dissolve. Adjust the pH with 1 M
NaOH to get to a pH of 7.5. Make up to 100 mL with water.
2. 5 M NaCl solution: Measure out 100 mL of water and place in
a beaker with a stir bar. Weigh out 29.2 g of NaCl and place in
a beaker and mix to dissolve.
3. 1 M ZnCl2 solution: Measure out 75 mL of water and place in
a beaker with a stir bar. Weigh out 13.6 g of ZnCl2 and place
in a beaker and mix. Then add 3 mL of 1 N HCl (see Note 1)
to solution and then add water to make up 100 mL.
2.2 ACE1, ACE2, 1. 100 mM stock of captopril (ACE1 inhibitor): Weigh out a
and Prolyl small amount (10–20 milligrams (mg) or 0.01–0.02 g) and
Endopeptidase divide this amount by 0.02173 g (Molecular weight (MW) of
Inhibitors captopril is 217.3) to determine the amount of water needed
(see Note 2). Next, do a tenfold serial dilution twice, to get a
final concentration of 1 mM.
2. 10 mM solution of MLN-4760 (ACE2 inhibitor, MW 472.3):
Weigh out 4.7 mg and place in 1 mL of water to get this stock
solution. Aliquot stocks into eppendorf tubes at 100 μL and
store at −20 °C. MLN-4760 is used to determine any activity
that is not ACE2 dependent (see Note 3). You may also use
ACE2-deficient cells or tissues as a negative control.
3. 50 mM stock of Z-Pro-Prolinal (Prolyl endopeptidase inhibi-
tor, MW 376.45): Weigh 18.8 mg and dissolve in 1 mL of 50%
methanol. Aliquot stocks into eppendorf tubes at 25 μL and
store at −20 °C.
Fluorescent ACE2 Activity in Mouse Abdominal Aorta 63
4. 1 mg of Mca-APK (Dnp)(ACE2 substrate, MW 696.7): Dilute

1 mg into 143 μL of DMSO to get a 10 mM stock solution (see
Note 4). Store solution at −20 °C and protect from light at all
times.
5. Make 10 mL of a 1× ACE2 buffer with the following compo-
nents: 750 μL of 1 M Tris–HCl, pH 7.5, 2 mL of 5 M NaCl,
5 μL of 1 M ZnCl2, 100 μL of 1 mM captopril, 20 μL of
50 mM Z-Pro-Prolinal, and 7 mL of ultrapure H2O. Make
1 mL of a 10× ACE2 buffer by using 750 μL of 1 M Tris–HCl,
pH 7.5, 5 μL of 1 M ZnCl2, and 245 μL of ultrapure H2O.
6. In order to make up the fluorescent substrate, make the following
master mix solution: 265 μL of 1× ACE2 buffer, 10 μL of 10×
ACE2 buffer, 20 μL of 5 M NaCl, and 5 μL of fluorescent sub-
strate (10 mM stock, final concentration is 50 μM) (see Note 5).
3 Methods
Carry out all procedures on ice unless otherwise specified.

1. Add a Complete “Mini” protease inhibitor tablet (EDTA-free)
into the 10 mL of 1× ACE2 buffer and vortex until dissolved
(see Note 6).
2. Take 5 mL of the 1× ACE2 buffer and 25 μL of Triton-X to
get a 0.5% solution. The other 5 mL of 1× ACE2 buffer will be
used for the enzymatic assay.
3. Homogenize the tissue using a Dounce homogenizer (glass-
on-glass) in the 0.5% Triton-X 1× ACE2 buffer. For abdominal
aorta, we weigh the tissue and use the weight to figure out
how much buffer to add (see Note 7).
4. Centrifuge samples at 12,000 × g’s for 15 min at 4 °C. Take
clarified supernatant to measure the protein concentration
using the Bicinchoninic acid (BCA) assay (see Note 8). If you
have enough protein, perform duplicates for all samples.
5. Take 100 μg of abdominal aortic protein from each sample for
loading into the assay (see Note 9). From the given volume
amount subtract it from 75 μL to figure out how much 1×
ACE2 buffer to use as a diluent. Load 70 μL of this solution
into each well and allow the inhibitors to incubate with the
samples for at least 10 min before loading any substrate.
Include a “blank” that contains only 1× ACE2 buffer and no
protein in your 96-well plate.
6. Before adding the master mix, make sure to turn on spectro-
photometer and allow it to warm up for at least 10 min. During
this time, you can edit your plate, and set your excitation and
emission filters (see Note 10) (Fig. 1).
64 Yu Wang et al.
4
Saline (Ace2 +/y)
Saline (Ace2 -/y)
Ang II (Ace2 +/y)
Ang II (Ace2 -/y)
Relative Fluorescent Units/100 mg protein

3
0
0 50 100 150
Time (minutes)
Fig. 1 Abdominal aortic ACE2 activity in saline and angiotensin II (AngII) infused
mice (28-day infusion protocol). Note that the ACE2 −/y LDLr−/− mice have slopes
that are close to zero, whereas the ACE2 +/y LDLr−/− mice have a slope of 4.8 and
a slope of 13.7 (10−3 RFU/100 μg protein/5 min) under saline and AngII infusion,
respectively. This represents a 2.9-fold increase in ACE2 activity under AngII infu-
sion. Each data point represents an average of 3–5 mice per group
7. Once everything is ready, add 30 μL of master mix using a

multi-channel pipettor to all wells and take readings at 5 min
for 2.5 h. This will give you a total of 31 readings.
4 Notes
1. A slight amount of hydrochloric acid is needed in order to get

ZnCl2 into solution. Heating the solution will not help with
solubility of ZnCl2.
2. In order to not waste these compounds, we typically weigh out
the smallest amount possible and then use the formula weight
to divide to get a certain molarity of the compound. If we
weighed out 0.01 g of captopril, then we would need 0.46 mL
of water to get a 100 mM stock. Captopril is very stable, so
you can keep at room temperature or at 4 °C for 3–6 months.
3. We use MLN-4760 and we incubate tissues or cells with this

compound for 30 min at 37 °C before the addition of any
ACE2 substrate. There are other ACE2 inhibitors, such as
DX-600; however, this inhibitor may have different affinities
to human versus mouse ACE2 [7, 8].
4. In our hands, Mca-APK(Dnp) works the best in both ACE2-
proficient and ACE2-deficient tissues. There is another fluo-
rescent substrate, Mca-YVADAPK (Dnp); however, this
substrate shows a higher background level in ACE2-deficient
tissues (unpublished data) and is also reactive to other enzymes,
such as caspase-1 and ACE1.
5. You may need to test the amount of substrate needed for your
given reactions. We have found that 50 μM works best, how-
ever other labs have used less (1–30 μM) [7, 9, 10]. It is impor-
tant to make sure that the substrate is in excess during the
entire incubation time. This master mix is enough for nine
reactions. If there are more samples, then you can recalculate
for your given number of samples.
6. There are numerous methods for inhibiting proteases and it is
important to recognize that the literature has not conformed
to one single method. Some labs, including ours, use numer-
ous inhibitors such as pepstatin (renin inhibitor), bestatin
(aminopeptidase inhibitor), benzyl succinate (carboxypepti-
dase inhibitor), and thiorpan/phosphoramadon (neprilysin
inhibitors) to inhibit these enzymes. Depending on the tissue
type, you may need to test different combinations of inhibitors
to find the one that best fits your needs.
7. For abdominal aorta, it is good to use the least amount of buf-
fer necessary to homogenize the tissue. For example, if the
abdominal weighs 10 mg, then we will use 100 μL of the
homogenization buffer (1 mg per 10 μL of buffer).
8. It should be noted that ACE2 can reside on the cell membrane
and within the cell [11]. If cell membrane ACE2 is what is
preferred, we would recommend using a high centrifugation
rate of 25,000–30,000 × g’s at 4 °C for 20 min with 1× ACE2
buffer only without Triton-X. Once you have the pellet, then
you resuspend the pellet in 0.5% Triton-X ACE2 buffer. Allow
this to incubate overnight and then perform a 5000 × g’s spin
at 4 °C for 5–10 min to get membrane ACE2. This assay is to
look at total ACE2 from abdominal aorta.
9. Abdominal aorta and fat tissue [5, 12] have much lower ACE2
activity than kidney, heart, or lungs; therefore, it is necessary to
load a higher amount of protein. It is best to keep this volume
as low as possible. The protein lysate should not be any more
than 20–25% of the total reaction volume as other proteins
could interfere with the assay. It is also wise to try different
66 Yu Wang et al.
amounts of protein lysate to determine what protein concen-

tration will work best for this assay.
10. Make sure that the spectrophotometer can incubate the plate
at 37 °C. Also, we do not use the well mixing function before
each reading. This can influence the absorbance reading and so
the incubation time of 2.5 h may need to be shortened if this
function is performed. Also, we do use the auto-cutoff func-
tion for performing all spectrophotometry readings at 420 nm.
It is best to use a confined emission wavelength as other pro-
teins or substrates could produce background fluorescence
within the samples.
References
1. Tipnis SR, Hooper NM, Hyde R, Karran E, bone marrow-derived cells increases atherosclero-
Christie G, Turner AJ (2000) A human homo- sis in low-density lipoprotein receptor −/− mice.
log of angiotensin-converting enzyme. Cloning Arterioscler Thromb Vasc Biol 31(4):758–765.
and functional expression as a captopril- doi:10.1161/atvbaha.110.221614
insensitive carboxypeptidase. J Biol Chem 7. Pedersen KB, Sriramula S, Chhabra KH, Xia
275(43):33238–33243. doi:10.1074/jbc. H, Lazartigues E (2011) Species-specific inhib-
M002615200. M002615200 [pii] itor sensitivity of angiotensin-converting
2. Crackower MA, Sarao R, Oudit GY, Yagil C, enzyme 2 (ACE2) and its implication for ACE2
Kozieradzki I, Scanga SE, Oliveira-dos- activity assays. Am J Physiol Regul Integr
Santos AJ, da Costa J, Zhang L, Pei Y, Comp Physiol 301(5):R1293–R1299.
Scholey J, Ferrario CM, Manoukian AS, doi:10.1152/ajpregu.00339.2011
Chappell MC, Backx PH, Yagil Y, Penninger 8. Ye M, Wysocki J, Gonzalez-Pacheco FR, Salem
JM (2002) Angiotensin-converting enzyme 2 M, Evora K, Garcia-Halpin L, Poglitsch M,
is an essential regulator of heart function. Schuster M, Batlle D (2012) Murine recombi-
Nature 417(6891):822–828. doi:10.1038/ nant angiotensin-converting enzyme 2: effect
nature00786. nature00786 [pii] on angiotensin II-dependent hypertension
3. Donoghue M, Hsieh F, Baronas E, Godbout and distinctive angiotensin-converting enzyme
K, Gosselin M, Stagliano N, Donovan M, 2 inhibitor characteristics on rodent and
Woolf B, Robison K, Jeyaseelan R, Breitbart human angiotensin-converting enzyme 2.
RE, Acton S (2000) A novel angiotensin- Hypertension 60(3):730–740. doi:10.1161/
converting enzyme-related carboxypeptidase HYPERTENSIONAHA.112.198622
(ACE2) converts angiotensin I to angiotensin 9. Liu J, Ji H, Zheng W, Wu X, Zhu JJ, Arnold
1-9. Circ Res 87(5):E1–E9 AP, Sandberg K (2010) Sex differences in renal
4. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, angiotensin converting enzyme 2 (ACE2)
Tang J, Godbout K, Parsons T, Baronas E, activity are 17beta-oestradiol-dependent and
Hsieh F, Acton S, Patane M, Nichols A, sex chromosome-independent. Biol Sex Differ
Tummino P (2002) Hydrolysis of biological 1(1):6. doi:10.1186/2042-6410-1-6
peptides by human angiotensin-converting 10. Wysocki J, Ye M, Soler MJ, Gurley SB, Xiao
enzyme-related carboxypeptidase. J Biol Chem HD, Bernstein KE, Coffman TM, Chen S,
277(17):14838–14843. doi:10.1074/jbc. Batlle D (2006) ACE and ACE2 activity in dia-
M200581200. M200581200 [pii] betic mice. Diabetes 55(7):2132–2139.
5. Gupte M, Boustany-Kari CM, Bharadwaj K, doi:10.2337/db06-0033
Police S, Thatcher S, Gong MC, English VL, 11. Lambert DW, Yarski M, Warner FJ, Thornhill P,
Cassis LA (2008) ACE2 is expressed in Parkin ET, Smith AI, Hooper NM, Turner AJ
mouse adipocytes and regulated by a high-fat (2005) Tumor necrosis factor-alpha convertase
diet. Am J Physiol Regul Integr Comp (ADAM17) mediates regulated ectodomain
Physiol 295(3):R781–R788. doi:10.1152/ shedding of the severe-acute respiratory syn-
ajpregu.00183.2008. 00183.2008 [pii] drome-coronavirus (SARS-CoV) receptor,
6. Thatcher SE, Zhang X, Howatt DA, Lu H, Gurley angiotensin-converting enzyme-2 (ACE2). J Biol
SB, Daugherty A, Cassis LA (2011) Angiotensin- Chem 280(34):30113–30119. doi:10.1074/
converting enzyme 2 deficiency in whole body or jbc.M505111200. M505111200 [pii]

12.
Wang Y, Shoemaker R, Thatcher SE, obesity-hypertension through an ACE2-
Batifoulier-Yiannikouris F, English VL, Cassis dependent mechanism. Am J Physiol
LA (2015) Administration of 17beta-estradiol Endocrinol Metab 308(12):E1066–E1075.
to ovariectomized obese female mice reverses doi:10.1152/ajpendo.00030.2015
Chapter 6
Measuring Blood Pressure Using a Noninvasive Tail Cuff

Method in Mice
Abstract
The renin angiotensin system (RAS) is well known for its role in regulating blood pressure (BP). An acti-
vated RAS contributes to elevated blood pressure and is evident in both human and animal models of
hypertension. Drugs that target the classic vasoconstrictive arm of the RAS (angiotensin II/AT1 receptor
signaling) are potent anti-hypertensive agents in clinical setting. However, the newly discovered
angiotensin-converting enzyme 2 (ACE2)/angiotensin—(1–7)/Mas receptor axis added new vitality to
the hypertension field. Advances in genetic manipulation and the relative low cost made the mouse model
as one of the most popular animal models to study hypertension. Since a reliable and accurate method for
BP assessment is the key for such experiments, here we provide a protocol for BP measurement in mice
using a noninvasive BP system. The CODA noninvasive BP system (a tail-cuff Method, Kent Scientific
Corporation) enables blood pressure (BP) measurements in mice. This method uses a specialized volume
pressure recording (VPR) sensor, and measures blood volume changes that are placed over the animal’s
tail. Mice do need to be restrained in specific holders and artificially heated to maintain normal BP.
Key words Blood pressure, Blood volume, Tail cuff, Noninvasive, Mice
1 Introduction
The use of small animal models of hypertension has led to a sub-

stantial improvement in understanding the etiology of human
hypertension. The ability to accurately assess BP in small animals is
the key point to elucidate the underlying determinants of hyper-
tension. Although a direct intra-arterial assessment of BP in unre-
strained, conscious animals is generally considered the most
translationally relevant, issues associated with this method such as
the invasive nature, the technical difficulty of the surgery, and the
expensive price limit its usage in a general laboratory setting [1, 2].
Within this protocol, we demonstrate the use of a noninvasive tail-
cuff method to measure BP in mice. This tail cuff method is very
user friendly and is well correlated with other methods, such as
assessment of intra-arterial BP.
69
70 Yu Wang et al.
2 Materials
CODA High Throughput Noninvasive Blood Pressure system

(Product # CODA-HT8, Kent Scientific corporation), neutral
cleaning reagent and kimwipes.
3 Methods
3.1 Setting 1. The area for setting up the Coda system and performing exper-
Up Equipment iments should be in a room where the temperature is stable
and Software (20–23 °C). Avoid locations near air conditioning vents or fans
(see Note 1).
2. Turn on the Coda controller and then use the warming plat-
form (set at appropriate temperature levels accordingly, for
room temperature, use level 1 or 2, see Note 2).
3. Perform the controller diagnostic test.
●● Open Coda software, select coda device by click on it, select
(test selected Device), select the cuffs and the channels to
test and click (test).
●● After the test’s complete, close the device test window. Select
the Coda controller to use then click (use these devices).
4. Setting up software (Coda noninvasive blood pressure system).
●● Select (Tools) >> (Manage personnel).
●● Under (Researchers), enter performer’s name and save data.
●● Under (Specimens), name animals and animal type and save
data.
5. Begin a new experiment.
●● Open (File) >> (New) >> (Experiment).
●● In experiment wizard window, enter experiment name,
select key researcher, and begin date and then click (Next)
to enter session name information (example: session 1).
●● Set Acclimation Cycles at 5; Number of sets at 1; Time
between Sets at 30 s; Cycles per set at 20;
●● Time between Cycles at 5 s, click (Next) to (Specimen
selection).
●● Select appropriate specimens from the specimen pool (on
left) and assign to available channels (on right) and then
click (Next) to (Session Parameters).
●● Set Maximum Occlusion pressure at 250 mmHg; Deflation
time at 20 s for mice; Minimum volume at 15 μL, and
Display style “One Channel per Graph”; Click (Next) to
(Review Session Script) and Click (Next) to (Experiment
Blood Pressure by Tail Cuff 71
Configured). Click (Finish) only after completion of the

“Preparing the Animals” Subheading 3.2.
3.2 Preparing 1. Select appropriate holders for mice based on their size
the Animals (see Note 3).
2. Place the animal into a holder (mouse should be able to freely
enter the holder) (Fig. 1) [3].
3. Picking up a mouse by the tail and gently place it into the rear
of the holder which faces the open end of the nose cone.
4. Carefully secure the rear hatch to the holder by turning a screw
on the rear hatch. Be careful not to pinch the tail or any other
parts of the body which secure the rear hatch.
5. Next, slide the nose cone toward the rear hatch limiting the
movement of the animal. The nose cone should be in a position
to limit the animal from turning around while inside the holder.
6. Place the holder onto the warming platform in the designated
position. Allow the animal at least 5 min to acclimate to the
holder (see Note 4). Do not touch the animals in the holder,
unnecessary contact could irritate the animal. Never leave the
animals unattended.
7. Place the occlusion tail cuff through the tail and to the base of
tail without force. After that, thread the tail through the VPR
sensor cuff and place it within 2 mm of the occlusion cuff
(Fig. 2, see Note 5) [3].
8. Secure the air tubing in the notch on the top rear of the holder.
9. After all the animals have been placed in the holder and their
tails cuffed, allow them at least 5 min to acclimate to surround-
ings. Record the temperature of the animal with an infrared
thermometer if needed. The temperature of animals should
between 32 and 35 °C.
Fig. 1 Schematic of mouse holder for the Kent CODA system. Note the position of
the nose cone and rear hatch for proper restraint of the mouse
72 Yu Wang et al.
Fig. 2 Proper placement of the occlusion and VPR cuffs on the mouse tail. Note
the air tubing is not twisted and placed in open notch of the mouse holder
3.3 Data Collection 1. Click (Finish) on the (Experiment configured) window to

and Processing begin the experiment and data collection (see Note 6).
2. When the experiment has ended, remove the animals immedi-
ately from the cuffs and the holder and place back to their own
cages.
3. A simple session summary report should be displayed. The
data collected in the experiment is saved as an excel file.
4. There are several ways to process the data within the excel file.
A common practice is to obtain the average and standard
deviation.
5. Delete measurements if the standard deviation is greater than
30. Additionally, rejected cycles may be viewed and the entire
data base can be easily exported to excel (see Notes 7 and 8).
4 Notes
1. Exposing animals to loud noise or extraneous odors may irri-

tate and stress the animal. Be quite and do not wear perfume
when performing experiment.
2. It is critical to maintain the mouse’s body temperature at
32–35 °C, use higher heating levels if the room temperature is
too low. Also obese mice may take a longer time to reach target
temperature.
3. Since BPs were measured by directly sensing changes in vol-
ume pressure in the tail with VPR sensor instead of sensing
light penetration with a photo-resistor, BP can be measured in
dark-skinned mice (C57BL/6J) without difficulty. However,
in case of obese mice (usually with thick tail due to excess fat
Blood Pressure by Tail Cuff 73
accumulation underneath the skin), BP measurement using tail

cuff method could be challenging and the BP values are usually
low.
4. Two to three days of acclimation measurements are usually
recommended followed by another two to three days of data
collection measurements to assure accurate measurements.
5. The occlusion tail cuff is inflated to inhibit the blood flow to
the tail. The occlusion cuff is slowly deflated and the VPR cuff
incorporating the specially designed sensor measures the physi-
ological characteristics of the returning blood flow. As the
blood returns to the tail, the VPR sensor cuff measures the tail
swelling as a result of the arterial pulsations from blood flow.
Systolic blood pressure (SBP) is automatically measured at the
first appearance of tail swelling and diastolic blood pressure
(DBP) is automatically calculated with the increasing rate of
swelling in the tail.
6. In the real-time BP graph, the red line represents the deflation
of the occlusion cuff and the VPR sensor cuff, the blue line
represents blood volume changes in the tail. The first inflection
of the blue line, minimum rate of change identifies the SBP;
the second inflection of the blue line, maximum rate of change
identifies the DBP.
7. Do not schedule the BP measurement in male and female mice
at the same time. The presence of an opposite sex may irritate
the mice. Always do the male mice first if your experiments hap-
pen to have both sexes in the same study design. Clean devices
(platforms, holders, etc.) after each round of measurement.
8. Appropriate training for software operating and animal han-
dling is required prior to perform this method for BP measure-
ment. It is most important to reduce animal stress by following
this procedure in order to get accurate and consistent
measurements.
References
1. Krege JH, Hodgin JB, Hagaman JR, Smithies by radiotelemetry and tail-cuff methods. Am
O (1995) A noninvasive computerized tail-cuff J Physiol Heart Circ Physiol 286(6):H2408–
system for measuring blood pressure in mice. H2415. doi:10.1152/ajpheart.01089.2003
Hypertension 25(5):1111–1115 3. Daugherty A, Rateri D, Hong L, Balakrishnan
2. Whitesall SE, Hoff JB, Vollmer AP, D'Alecy LG A (2009) Measuring blood pressure in mice
(2004) Comparison of simultaneous measure- using volume pressure recording, a tail-cuff
ment of mouse systolic arterial blood pressure method. J Vis Exp 27. doi:10.3791/1291
Chapter 7
Blood Pressure Monitoring Using Radio Telemetry

Method in Mice
Abstract
The TA11PA-C10 implantable transmitter (Data Sciences International, DSI) is designed to measure
blood pressure (BP) and activity in freely moving laboratory mice. The fluid filled catheter is placed in the
free flowing blood of the systemic artery (inserted into the left carotid artery and extended into the aorta),
and the transmitter body is placed in a benign location for long-term biocompatibility. The transmitter can
be used to monitor BP in mice (as small as 17 g) under normal physiological and unrestricted conditions
24 h a day while remaining free from stress associated with human interaction. Thus, telemetry is consid-
ered the gold standard for BP monitoring in small animals such as mice. However, this methodology does
require a good understanding of the system as well as appropriate training to perform the delicate transmit-
ter implantation surgery.
Key words Blood pressure, Radio telemetry, Catheter, Transmitter, Left carotid artery
1 Introduction
A direct intra-arterial assessment of BP in freely moving small

laboratory animals such as mice is generally considered the most
translationally relevant and the gold standard [1–4]. With the
implantable radio telemetry, mice are unrestrained and can be
freely moving in their own environment while their BP and activity
are being remotely monitored 24 h a day. It is by far the only
method which allows the BP to be continuously monitored while
remaining free from stress associated with human interaction [5].
Also a diurnal pattern of BP is usually present and can be analyzed
in normal mice housed in regular day and light cycle. This is par-
ticularly useful if the oscillation of the BP is studied [6]. The
method is feasible for measuring blood pressure in both lean and
obese mice [7]. However, carrying a transmitter inside the body
may negatively influence the development of obesity in mice in a
chronic setting (over 6 weeks). Benefit from its high accuracy and
low variability, less animals are usually needed for a given statistical
75
76 Yu Wang et al.
power as compared to other BP monitoring methods (e.g., tail-

cuff method). However, this radio telemetry method requires a
delicate surgical procedure for catheter insertion and transmitter
implantation. Within this protocol, we demonstrate how to per-
form transmitter implantation and measure BP using a radio telem-
etry system in mice.
2 Materials
DSI radio telemetry blood pressure monitoring system for data

acquisition and analysis (including platform receivers, matrix and
linked computer with installed software), DSI TA11PA-C10 trans-
mitter, equipment and tools for surgical implantation are listed in
surgical preparation section.
3 Methods
3.1 Device Operation 1. On and off switch.

The implantable transmitter has two operational modes and
can be switched “on” and “off” by passing a strong magnet.
Place a working FM radio in close proximity while turning on
the transmitter using a strong magnet. An audible tone from the
FM radio indicates a successful “on” operation (see Note 1).
2. Offset determination.
Activate the transmitter well advance of the implantation
surgery and determine the pressure offset value before placing
the device into the animal. Place the transmitter on an empty
telemetry receiver, and use the Acquisition system software to
determine the BP reading. Normal values are expected to fall
between ±3 mmHg. Record the BP offset value to a designated
file for later references. If the offset values exceed ±3 mmHg
range, send the transmitter back to DSI for exchange or
refurbishment.
3. Calibration and configuration
Each individual transmitter unit has a unique set of calibra-
tion values that must be manually configured into the data
acquisition system to allow the computer system interpreting
signals received from the telemetry device. Those calibration
values are associated with the transmitter serial number. Thus,
it is important to record the serial number of the transmitter
that each mouse received during implantation, and display the
number on animal’s cage card after surgery (see Note 2).
3.2 Software 1. Acquisition (this is for offset determination as well as data col-
Operation (Acquisition lection in real experimental setting).
and Analysis)
Blood Pressure by Radio Telemetry 77
Open Acquisition software, click {Configuration} >>

{Hardware}, select an empty receiver. Right click to add {new
transmitter}, enter all required information of the transmitter
(type, name, serial number, and BP calibration data set). Close
configuration wizard and save changes, the newly added trans-
mitter should appear in the main window. Select all target
transmitters and right click, select {start sampling} >> {trace
and save}. Now the computer system is receiving signals from
the transmitter. Record the start and stop date and time. For
offset determination, stop sampling after 1 h by selecting all
target transmitters and right click, select {stop sampling}. For
experimental data collection, stop sampling accordingly (e.g.,
We typically collect data for 5 consecutive days for each study
endpoint).
2. Analysis (this is for data extraction).
Open Analysis software, click {File} >> {Load data}, click
select “Parameters.” Select target transmitters from {Form
list}. Select time or duration accordingly (refer to the records
of starting and stopping time) for data extraction. For offset
determination, a time period of half an hour before stop sam-
pling is chosen for data extraction. Record BP values at
Mean ± SEM. For experimental data extraction, pulse pressure,
systolic BP, mean arterial pressure, diastolic BP, heart rate, and
physical activity data can be extracted separately. Also a circa-
dian pattern should be existed in all of these parameters if the
animals were housed in normal light dark circle.
3.3 Surgical 1. As with any small animal survival surgery, sterile instruments
Preparation and strict aseptic procedure are recommended. Any individual
anesthetic protocol should be tested for each animal strain and
variation before tempting survival surgery (see Note 3).
2. Equipment and tools:
Sterile dissecting microscope.
Heating pad.
Isoflurane machine for inhalation anesthesia (Induction 3–5%
in O2, maintenance 1.5–2% in O2).
Small needle holder.
Small, blunt-tipped dissecting scissors.
Sterile cotton swabs.
6-0 braided silk suture for skin closure.
25-gauge syringe needles.
Curved thumb forceps.
Vessel dilation forceps (curved and straight, fine tips and
smooth finish).
78 Yu Wang et al.
Vessel cannulation forceps (for proper manipulating catheter

for insertion into the vessel without causing damage to the
catheter).
Veterinary tissue adhesive.
Lint-free gauze pads.
7-0 braided silk for aorta occlusion.
Magnet (switch the transmitter on and off).
Working FM radio.
3.4 Implantation 1. Flood the catheter channel with sterile saline at room tempera-
Procedure ture prior to the surgery to dimensionally stabilize the catheter
(see Note 4).
2. After the animals are properly anesthetized, place mouse on a
heating pad (see Note 5), shave and clean the throat area,
secure front legs with tape, and allow great access to the vessels
along the trachea (Fig. 1).
3. To expose the submandibular throat area, using a small dissect-
ing scissors to make a 3 cm long incision through the skin
along the midline (from Lower mandible posteriorly to the
sternum). Using two moist sterile surgical swabs gently retract
glands and muscles along the tracheal.
4. The left carotid artery can typically be found next to the tracheal
and just beneath a thin layer of muscle (Fig. 2). Since the vagus
nerve is usually in close proximity to the left carotid artery, great
care should be taken to avoid injuring this vital nerve.
Fig. 1 Proper positioning of the mouse

Fig. 2 Isolating left carotid artery
Fig. 3 Ligation (anterior end) and occlusion (posterior end) of the left carotid
artery with sutures
5. Carefully isolate the left carotid artery using two vessel dilation
forceps and then thread two pieces of 7-0 silk suture beneath
the vessel.
6. The more anterior suture will be used to permanently ligate the
artery. It is ideally placed just posterior to the bifurcation of the
interior and exterior carotid arteries. (This anatomic feature is a
useful landmark for determining the proper insertion length for
80 Yu Wang et al.
the implanted catheter). Locate the bifurcation and ligate the

artery with a secure surgical node. The suture at the more pos-
terior end will be used temporarily to occlude blood flow in the
vessel and allow insertion of the catheter (Fig. 3, see Note 6).
7. Cannulation of the carotid artery. Bend the tip of a small nee-
dle (25 gauge), and use it to insert the catheter (Fig. 4).
8. Remove the protective tip cover from the end of the catheter
(if it is new out of box). Holding the non-compliant stem sec-
tion with a catheter forceps, using thumb forceps grasp the tip
cover and remove the tip cover gently. Avoid contact of the
catheter to any other surface.
9. Elevate the vessel by applying gentle tension to the ligation
and occlusion sutures.
10. Grasp the end of the catheter at the overlap section with vessel
cannulation forceps, use the bend-tip needle to insert the cath-
eter, puncture the vessel near the ligation suture, and slip the
catheter tip into the carotid artery (Fig. 5, see Note 7).
11. Carefully release the tension on the occlusion suture and
advance the catheter further into the vessel (Fig. 6).
12. Once the catheter is in an ideal position, tie the occlusion
suture on the vessel and allow it to seal around the catheter.
13. Take the loose ends of the ligation suture and securely tie them
around the notch of the catheter (Fig. 7); this will prevent the
catheter from moving once the animals resume normal
movement.
Fig. 4 Bending the needle tip to roughly 80–90°

Fig. 5 Puncture the vessel with the bended needed near the ligation suture and
slip the catheter tip into the carotid artery
Fig. 6 Slowly loosen the occlusion suture and advance the catheter further into
the artery
14. Irrigate the incision area with sterile saline and release the sur-
gical retraction (Fig. 8).
15. Make a pouch underneath the skin along the mouse flank using
sterile hemostatic forceps to place the transmitter body (see
Note 8).
16. Once the pouch is formed, irrigate the tunnel using sterile
saline and slid in the transmitter (see Note 8). Use a small drop
82 Yu Wang et al.
Fig. 7 Secure the catheter in place. Tie the loose occlusion suture on the vessel
and seal around the catheter. Tie the loose end of the ligation suture around the
notch of the catheter
Fig. 8 Clean the loose end of the sutures and irrigate the surgical area
of tissue adhesive to secure the transmitter in place until the

connective tissue has a chance to form.
17. To close the skin, irrigate the opening with sterile saline and
pull together the tissue edges with 6-0 silk suture and then seal
the incision with tissue adhesive.
3.5 Post- Place the animals back to their own cages on a heating pad after
Surgical Care transmitter implantation. Monitor their recover for an hour or two
before transfer back to housing room. Check on mice every day and
fill out surgery records accordingly. Administer analgesic to surgical
mice for the first couple days following implantation according to
individual anesthetic protocol. Here, we administer Flunixin
(2.5 mg/kg) via subcutaneous injection every 12 h for 2 day fol-
lowing implantation. Typically, for mice that weigh 25 g or more,
expect 7–10 days for the mice to resume normal circadian blood
pressure rhythm and return to their pre-implantation body weight.
4 Notes
1. Transmitters are shipped in the off mode; only turn on the

transmitter prior to its use. Transmitters should be in off mode
after each use to save battery life. When switching on or off by
passing over a strong magnet, hold both the transmitter and
magnet tightly to avoid direct impact.
2. The BP calibration values for each individual transmitter appear
on the backside of the sterile case. Record those values together
with transmitter serial numbers. Loss of such information will
prohibit the use of the transmitter.
3. We usually use isoflurane inhalation as our anesthesia strategy
for mice. Different anesthetic methods can be used for this
surgical procedure as well. However, each anesthetic protocol
should be tested prior to experimentation since it will have a
substantial impact on BP recording. Appropriate doses should
be given to maintain unconsciousness throughout the whole
surgical process (takes 30–45 min per mouse). Over dosing of
anesthesia may have negative effects on body temperature;
results could delay recovery or even induce death.
4. The catheter (especially the tip area) needs to be clean and
straight for a smooth insertion into the vessel. Usually, this is
not a problem for new transmitters, but be careful if using a
reused transmitter. For reused transmitters, we carefully clean
the transmitter (e.g., soaked in Tergazyme enzyme-active pow-
dered detergent to digest any tissue attached to the catheter)
under a dissecting microscope with vessel dilation and cannula-
tion forceps without force. The transmitters are then soaked in
CIDEX OPA solution for disinfection followed by rinsing with
ultrapure water. Thread the whole length of catheter into thin
plastic tubing (1 mm in diameter, 6–7 cm in length) and incu-
bate in 37 °C overnight to straighten the catheter.
5. According to our experience, properly maintaining the ani-
mal’s body temperature has been found to be critical for
increasing survival and improving post-surgical recovery.
84 Yu Wang et al.
Fig. 9 A close demonstration of the sections of the catheter.
Fig. 10 Position of the tip of the catheter into the free-flowing blood of the aorta
6. The terminal 10 mm of the catheter is composed a thin-wall

section of tubing. At the transition point there is a small notch
in the profile of the catheter, this notch will be a useful land-
mark for optimal positioning of the catheter. The tip section of
the tubing (treated with anti-thrombogenic film) is designed
to be inserted to the animal’s blood stream (Fig. 9). It is the
distal 4 mm of the catheter that actually transmits the animal’s
BP. Therefore, it is essential that a significant portion of this
thin-wall tip section is placed in the free flow blood of the
aorta. Ideally at least 2 mm of the catheter tip should be
extended out of the left carotid artery into the free-flowing
blood of the aorta. In most mouse models, this can be accom-
plished by advancing the catheter into the carotid artery until
the catheter notch reaches the level of carotid bifurcation
(Fig. 10). Minor adjustment may be required based on the size
of the mouse. Also, the left carotid artery is chosen to avoid
disruption of blood flow to the right branchial subclavian
artery which branches off the right carotid artery.
7. The occlusion suture needs to be semi-tied and elevated at the

time of puncture and catheter insertion to occlude blood flow.
Failure to do so will cause blood to flow out from the punc-
ture site and cause animal death due to excess blood loss. It is
normal if only a small amount of blood comes out from the
insertion site. Use sterile cotton swabs to clean the blood to
retain a clear view.
8. Normally, the transmitter will be placed on the opposite side of
the body from the catheter; however for animals that were
expected to grow to a size greater than 30 g, place the trans-
mitter on the same side of body as the catheter insertion to
reduce stress applied to the catheter as the animal moves about
in its cage.
References
1. Brockway BP, Mills P, Kramer K (1998) Fully 5. Whitesall SE, Hoff JB, Vollmer AP et al (2004)
implanted radio-telemetry for monitoring labo- Comparison of simultaneous measurement of
ratory animals. Lab Anim 27:40–45 mouse systolic arterial blood pressure by radio-
2. Mattson DL (1998) Long-term measurement telemetry and tail-cuff methods. Am J Physiol
of arterial blood pressure in conscious mice. Am Heart Circ Physiol 286(6):H2408–H2415.
J Physiol 274(2 Pt 2):R564–R570 Epub 2004 February 12
3. Van Vliet BN, Chafe LL, Antie V et al (2000) 6. Xie Z, Su W et al (2015 Jan) Smooth-muscle
Direct and indirect methods used to study arte- BMAL1 participates in blood pressure circadian
rial blood pressure. J Pharmacol Toxicol rhythm regulation. J Clin Invest 125(1):324–336
Methods 44(2):361–373 7. Gupte M et al (2012) Angiotensin converting
4. Huetteman DA, Boqie H (2009) Direct blood enzyme 2 contributes to sex differences in the
pressure monitoring in laboratory rodents via development of obesity hypertension in
implantable radio telemetry. Methods Mol Biol C57BL/6 mice. Arterioscler Thromb Vasc Biol
573:57–73 32(6):1392–1399
Chapter 8
Characterization and Functional Phenotyping of Renal

Immune Cells via Flow Cytometry
Abstract
A variety of immune cell subsets contribute to the pathogenesis of hypertension and associated kidney
damage following inappropriate activation of the renin–angiotensin system (RAS). These immune cell
subsets often express common surface markers, which complicates their separation and characterization
in vivo. Accordingly, flow cytometry has become an invaluable tool for parsing immune cell populations
because this technique permits the simultaneous detection of up to 18 markers on a single cell. Below we
describe a process by which one can determine the immune cell subsets in the kidney via flow cytometry.
Key words Kidney diseases, Inflammation, Flow cytometry, Hypertension, Cytokines
1 Introduction
Dysregulation of the renin–angiotensin system (RAS) contributes

to the pathogenesis of hypertension and chronic kidney disease, as
evidenced by the efficacy of RAS inhibitors in lowering blood pres-
sure and slowing the progression of kidney diseases in a wide range
of patients [1, 2]. Moreover the immune system has been impli-
cated in RAS-mediated renal pathologies [3, 4]. Experimentally
both myeloid and lymphoid cells infiltrate the kidney in models of
RAS-dependent hypertension and kidney disease and mediate
sodium retention, tissue injury, and fibrosis [3, 5, 6]. In order to
understand the inflammatory mechanisms that drive kidney dys-
function and injury, it is imperative to characterize and study the
specific immune cell subsets that accumulate in the kidney.
Myeloid and lymphoid cells are divided into many functionally
distinct subsets. For example, CD4+ T lymphocytes are further
divided into four subsets with disparate inflammatory capacities:
Th1, Th2, Th17, and T regulatory cells (Treg). Unfortunately
The original version of this chapter was revised. The erratum to this chapter is available at:
DOI 10.1007/978-1-4939-7030-8_16
87
88 Nathan P. Rudemiller and Steven D. Crowley
these subsets cannot be differentiated by single surface markers,

but they can be distinguished by multiple surface markers in com-
bination with unique expression profiles of cytokines and/or
transcription factors [7–10]. Available immunohistochemical tech-
niques can illuminate the distribution of immune cells in the kid-
ney, but fail to provide the granularity necessary to distinguish
specific myeloid and lymphoid subsets. In contrast, flow cytometry
is a laser-based technique that permits the simultaneous staining
and analysis of up to 18 extracellular or intracellular epitopes on a
single cell [11], allowing intricate parsing of immune cell subsets
based on phenotypic and functional characteristics. Furthermore
fluorescent cell sorting can be employed to separate and collect
highly purified cell populations for protein or RNA analysis. Flow
cytometry requires that the starting biological sample be a single
cell suspension of live cells. This can prove difficult if the cells of
interest (e.g., immune cells) are integrated into a solid organ such
as the kidney. Therefore, below we outline a methodology to dis-
sociate kidneys—drawing from our experience and the experience
of others in the literature—in such a way that will enable detailed
analysis of intra-renal immune cells via flow cytometry to further
develop our understanding of the immune mechanisms that
regulate RAS-dependent renal disease.
2 Materials
Confirm that all tools, media, and work surfaces are sterile.
1. Surgical instruments: Forceps, standard surgical scissors,
spring-action micro scissors.
2. Animal clippers.
3. Cotton tip applicators.
4. Gauze.
5. 25 G 5/8 needle.
6. 3 ml syringe.
7. 1 ml syringe plunger.
8. Petri dish (60 × 15 mm).
9. 100, 70, and 40 μm cell strainers.
10. 15 ml conical tubes.
11. Centrifuge with 15 ml conical tube adaptor.
12. Phosphate buffered solution (PBS; 137 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4).
13. Deoxyribonuclease I (DNase): reconstitute in PBS at 1 mg/ml,
aliquot, and store at −20 °C.
14. Collagenase type IV: reconstitute in PBS at 100 mg/ml, aliquot,
and store at −20 °C (see Note 1).
Flow Cytometric Analysis of Renal Immune Cells 89
15. RPMI-10: RPMI 1640 containing 10% fetal bovine serum

(FBS).
16. Digestion solution: RPMI 1640 containing 3% FBS, 10 μg/ml
DNase, and 1 mg/ml collagenase type IV. Allow digestion
buffer to reach room temperature (RT). Add DNase and col-
lagenase immediately before use.
17. Incubator with agitator (37 °C).
18. Wash buffer: PBS containing 2% FBS and 2 mM EDTA. Store
at 4 °C.
19. Red blood cell (RBC) lysis buffer (155 mM NH4Cl, 12 mM
NaCHO3, 0.1 mM EDTA).
20. Trypan blue solution, 0.4%.
21. Appropriate fluorochrome-conjugated primary antibodies to
stain the cells of interest.
22. Access to a flow cytometer and data analysis software such as
FACSDiva or FlowJo.
Optional materials if intracellular staining of inducible factors
(e.g., cytokines) is required:
23. (Optional) Atmosphere-controlled incubator (37 °C with
5% CO2).
24. (Optional) Monensin: reconstitute in DMSO at 10 mg/ml.
25. (Optional) Brefeldin A: reconstitute in DMSO at 10 mg/ml.
26. (Optional) Cell dissociation media (trypsin, EDTA, etc.).
3 Methods
3.1 Collection 1. Deeply anesthetize the mouse and shave the abdomen (see
of Kidney Samples Note 2).
2. Place the anesthetized mouse supine on a surgical table and
secure the limbs, thus immobilizing the mouse.
3. Make a longitudinal incision through the skin and abdominal
wall spanning from the lower abdomen to the sternum, making
sure not to puncture the diaphragm. Make two transverse
incisions, bisecting either side of the abdominal wall.
4. Using sterile cotton-tip applicators, gently shift the stomach,
intestines, liver, spleen, and any excess fascia laterally to expose
the left kidney and vena cava. Upon lifting the sternum with for-
ceps, the heart should be visible through the intact diaphragm.
5. Clasp the sternum with forceps, cut the vena cava below the
left renal vein with micro scissors (see Note 3) and pierce the
left ventricle through the intact diaphragm with a 25 G needle
attached to a 3 ml syringe filled with ice-cold PBS (see Note 4).
6. Infuse the PBS through the left ventricle at an approximate

rate of 200 μl/s until all 3 ml have been exhausted; absorb the
fluid exiting from the lacerated vena cava with gauze. The left
kidney can provide a proxy of the efficacy of the PBS flush. The
kidney should become pale, indicating that it has been cleared
of blood (see Note 5). Flushing the kidneys ensures that only
those immune cells that have infiltrated the renal parenchyma
will be included in downstream analysis.
7. Excise and decapsulate the left kidney (see Note 6). Place the
kidney in RPMI-10 on ice. Collect all subsequent kidney sam-
ples according to steps 1–7 (see Note 7).
3.2 Mechanical 1. Place a 100 μm cell strainer in a petri dish. Aliquot 5 ml of RT
and Enzymatic digestion buffer into the filter.
Digestion of Kidneys 2. Place a kidney in the filter and mince it with a pair of scissors
into several small pieces. With the blunt end of a 1 ml syringe
plunger, grind the kidney pieces into the filter until the tissue
is homogenized.
3. Overturn the filter and hold it over the petri dish. Scoop the
kidney homogenate into the petri dish with the syringe
plunger. Rinse the overturned filter with 1–2 ml digestion
buffer while holding it over the petri dish to collect residual
sample (see Note 8).
4. Mix the contents of the petri dish and transfer the contents to
a 15 ml conical tube using a transfer pipette. Incubate the kidney
homogenate under constant agitation for 30 min at 37 °C.
5. Remove the sample from 37 °C and place it on ice. Add 3 ml
ice-cold wash buffer to the 15 ml conical tube containing the
sample and invert the tube several times to mix the solution.
6. Centrifuge the sample for 1 min at 10 × g to pellet heavy debris.
7. Rinse a 70 μm cell strainer with wash buffer. Pour the kidney
homogenate through the rinsed filter into a new 15 ml conical
tube and discard the pellet of heavy debris. Centrifuge the
sample for 7 min at 300 × g.
8. Discard the supernatant and suspend the cell pellet in 3 ml RBC
lysis buffer to eliminate residual RBCs. Incubate cells for 5 min
on ice. Add 5 ml wash buffer and invert the tube to mix.
9. Rinse a 40 μm cell strainer with wash buffer. Pour the sample
through the cell strainer into a new 15 ml conical tube.
Centrifuge the cells for 7 min at 300 × g (see Note 9).
10. Suspend the cell pellet in 5 ml wash buffer. Add 10 μl of the
cell suspension to a microcentrifuge tube containing 10 μl
Trypan blue solution and pipette to mix. Determine the cell
concentration by counting live (Trypan blue-negative) cells on
a hemocytometer (see Notes 10 and 11), and use this concen-
tration to determine the total number of live cells in your sam-

ple—a value that can be utilized in the data analysis to quantify
immune cell subsets.
3.3 (Optional) 1. Wash cells once with PBS to remove EDTA that may carry
Activation of Renal over from the wash buffer. Suspend cells at 2 × 106 cells/ml in
Immune Cells to Elicit RPMI-10 containing the protein transport inhibitors monen-
Measureable Cytokine sin and brefeldin A (5 μg/ml each) (see Note 13). Aliquot cells
Production and Other into a 24-well culture dish at 1 ml/well and add an appropriate
Inducible Factors for cell stimulant. For example, the combination of PMA (10 ng/ml)
Antibody Staining and ionomycin (1 μg/ml) has been used to induce the produc-
(See Note 12) tion of TNF, IFNG, and IL-17a in renal T cells [12–14],
while LPS can be used to stimulate iNOS production in renal
monocytes/macrophages [6]. Prepare duplicate wells for each
sample as well as control wells that receive protein transport
inhibitor but no stimulation. Place the samples in a cell culture
incubator for 4–6 h (see Note 14).
2. Following incubation, combine the desired cells from the
duplicate wells into 12 × 75 mm round-bottom test tubes. For
suspension cells, such as T cells, transfer the supernatant. To
collect adherent cells, such as myeloid cells, aspirate the super-
natant, wash the wells with PBS, and remove the adherent cells
from the wells with cell dissociation media.
3.4 Staining Cell Once the kidney has been dissociated into a single-cell suspension,
Surface Markers and/ the method for staining the cells for flow cytometry is no different
or Intracellular from staining any other single-cell population. This methodology,
Antigens for Flow utilizing fluorochrome-conjugated antibodies that recognize
Cytometry extracellular or intracellular epitopes, has been thoroughly
described elsewhere, and may vary slightly depending on the anti-
body manufacturer. We recommend consulting the manufacturer
website for the appropriate staining protocol. Therefore, instead of
describing the staining procedure, below we suggest an epitope
panel to measure the capacity of renal T cells to produce TNF and
IFNG (Table 1). This epitope panel requires that the flow cytom-
eter is equipped with a violet laser (roughly 405 nm), a blue laser
(roughly 488 nm), a red laser (roughly 633 nm), and all of the
appropriate filters to detect the listed fluorochromes. The number
and types of fluorochromes that one can use will depend on the
spectral overlap of the fluorochromes and the cytometer’s capabil-
ity to excite the fluorochromes and detect their emittances. Consult
with the personnel of the flow cytometry core regarding the equip-
ment capability and read further about the construction of an anti-
body panel [15–18]. Additionally fluorescence-minus-one (FMO)
controls should be prepared for each antibody in the panel. FMO
controls are experimental cells that have been stained with all but
one of the fluorochromes in the panel. These controls are essential
Table 1
Seven-color antibody panel to measure T cell cytokine production
Fluorochrome conjugate Primary antibody (clone)

BV421 CD4 (RM4-5)
BV510 CD45 (30-F11)
FITC CD3ε (145-2C11)
PE IFNG (XMG1.2)
PE-cy7 CD8α (53-6.7)
APC TNF (MP6-XT22)
Near IR dead cell stain –
Fig. 1 Using FMO controls to determine positive staining. The FMO control (a) is
stained with all but one fluorochrome-conjugated antibody in the staining panel.
In this example, the FMO for the antibody conjugated to BV711 allows the appro-
priate determination of positive staining in experimental samples (b) with no
clear distinction between the positive and negative populations
in determining positive staining, especially for sweeping epitope

expression without a clear distinction between the negative and
positive populations (Fig. 1). A completely unstained sample will
not suffice in determining positive staining since the presence of
other fluorochromes in the FMO may shift the negative population
due to emission spillover.
3.5 Analysis of Flow The purpose of this section is not to explain how to acquire data on
Cytometry Data a flow cytometer, as that process will vary depending on the cytom-
from Renal Immune eter and software used to collect the data and has been explained
Cells elsewhere [16, 18]. We would, however, like to suggest a gating
strategy to analyze the cytokine expression of renal T cells from data

generated using the epitope panel in Table 1. This gating strategy is
illustrated in Figs. 2 and 3. Gates 1A-1D will be common among
most staining panels of renal immune cells, not just the panel pre-
sented in this protocol. The subsequent gates will vary depending
upon the additional antibodies in the panel. The data in Fig. 2 were
generated with renal cells from a 129/SvEv mouse after 14 days of
chronic angiotensin II infusion, collected on a BD LSRII cytome-
ter, and analyzed using FlowJo software. Each dot on the scatter
plots represents a recorded event. These events are not necessarily
cells, as the cytometer does not discriminate between debris, cells,
etc. Therefore, the data will be referred to as events until we reach
a point in the analysis where debris has been excluded.
1. Place side scatter area (SSC-A), a measure of granularity or
internal complexity, on the Y-axis and near-IR dead cell stain
on the X-axis to gate the events that do not have positive fluo-
rescence (Fig. 2a). The intensity of the fluorescence of the
A B C
Debris Live
/dead cells
Singlets cells
80.3
SSC-A
FSC-H
SSC-A
38.0
8.21
Near -IR dead cells stain FSC-A FSC-A
D E F
38.0 14.2 0.22
CD8-PE/Cy7
SSC-A
SSC-A
15.5
5.03 80.5
CD45-BV510 CD3-FITC CD4-BV421
Fig. 2 Gating strategy to analyze renal T cells. All events collected on the flow cytometer are first assessed for
expression of dead cell stain (a), and those events with positive expression are excluded from downstream
analysis. Live events are further narrowed to single events (b). Debris and residual dead cells are excluded
within the single events (c). Live cells are then selected for expression of CD45 (d), followed by CD3 (e), and
then parsed by expression of CD4 and CD8 (f)
CD4+ T cells CD8+ T cells

A B
IFNG+ IFNG+TNF+ IFNG+ IFNG+TNF+
0.49 0.11 0.62 0
IFNG-PE
IFNG-PE
Control
negative TNF+ negative TNF+

98.9 0.54 98.2 1.23
TNF-APC TNF-APC
C D
IFNG+ IFNG+TNF+ IFNG+ IFNG+TNF+
12.6 6.89 3.05 0.38
IFNG-PE
IFNG-PE
PMA +
ionomycin
negative TNF+ negative TNF+

63.4 17.1 78.6 17.9
TNF-APC TNF-APC
Fig. 3 Cytokine profile of renal T cells. Renal immune cells were cultured for 6 h in the presence or absence of
PMA/ionomycin and subsequently stained for flow cytometry. Control T cells (a, b) showed little expression of TNF
and IFNG whereas T cells stimulated with PMA + ionomycin (c, d) displayed robust expression of TNF and IFNG
dead cell stain is dependent on its ability to permeate the cell

membrane. As such, cells with compromised membranes will
(e.g., dead cells) display high fluorescence.
2. Within the “live” gate, graph forward scatter height (FSC-H)
against forward scatter area (FSC-A) to select single events
(Fig. 2b). These two parameters indicate event signal and event
size, respectively, and should report the same value. When
events fall outside of the linear range, this indicates a dispro-
portionate size to signal ratio, which often represents cellular
aggregates (doublets, triplets, etc.). Those events should be
excluded from analysis since they might display markers from
multiple cell types and thus obscure the data.
3. In order to set the singlet gate, which may appear unclear at
first, one can utilize “back-gating” (Shown in Fig. 4). By gat-
ing a large, labeled cell population, such as CD45+ events, and
displaying that population in the FSC-H vs. FSC-A plot
(Fig. 4a), one can clearly set a singlet gate that can then be
applied to the upstream population, e.g., live events (Fig. 4b).
A B
Singlets Singlets
FSC-H
FSC-H
FSC-A
C D FSC-A
Debris Live Debris Live
/dead cells /dead cells
cells cells
SSC-A
SSC-A
FSC-A FSC-A
Fig. 4 Performing back-gating. CD45+ events were gated for single events (a),
which was then be applied to the upstream “live event” population (b). Likewise,
CD45+ events were gated to exclude debris/dead cells (c), and this gate was
applied to the upstream singlet population (d)
4. Within the singlet gate, graph SSC-A against FSC-A to exclude

debris and residual dead cells (Fig. 2c) (see Note 15). Debris/
dead cells can be discriminated by their low FSC-A. Again this
gate can be determined by back-gating using the CD45+ popu-
lation (Fig. 4c and d).
5. Within the live cell gate, graph SSC-A against CD45-BV510
to select all CD45+ cells, or leukocytes (Fig. 2d).
6. Within the CD45+ gate, graph SSC-A against CD3-FITC to
select CD3+ cells, or T cells (Fig. 2e). The FMO control was
essential in setting this gate.
7.
Within the T cell gate, graph CD8-PE/Cy7 against
CD4-BV421 to differentiate CD8+, CD4+, and double nega-
tive T cells (Fig. 2f).
8. Within the CD4+ gate and CD8+ gate, graph TNF-APC against
IFNG-PE to determine the percentage of these populations
that can produce TNF and/or IFNG (Fig. 3). Both unstimu-
lated (Fig. 3a and b) and stimulated (Fig. 3c and d) samples
from the same kidney are shown to illustrate the necessity of a
stimulant to elicit the generation of these cytokines for
measurement.
4 Notes
1. Collagenase Type IV is comparable to Collagenase D and

Collagenase CLS-4.
2. It is essential that the mouse be anesthetized during, not euth-
anized before, the kidney harvest in order to reduce blood
clotting and cell death in the kidney.
3. The vena cava must be punctured to allow fluid to exit the
vascular network during the infusion.
4. The left ventricle is sometimes difficult to visualize and pierce
through an intact diaphragm. Alternatively incisions can be
made through the ribs on both sides of and parallel to the ster-
num, followed by cutting the diaphragm away, thus completely
exposing the heart. The left ventricle can then be accessed with
ease and pierced with the 25 G needle. This process must be
done quickly, as deoxygenated blood will begin to circulate
and could cause clotting issues in peripheral tissue.
5. In some forms of kidney disease, microvascular damage may
occur. In this case, the kidney may appear splotchy. This may
not be due to inefficient flushing, but the inability to clear
blood due to injury in the vascular architecture.
6. The right kidney will be cleared of blood as well and can be
utilized in this protocol if the left kidney is required for other
forms of analysis (histology, RNA isolation, etc.).
7. Keep in mind that the number of samples collected and the
pace of collection may affect cell viability, as the precedent kid-
ney samples will be incubating on ice during all subsequent
sample collections. Intersperse the collection of experimental
and control kidneys to account for this temporal factor.
8. Alternatively automatic tissue dissociators such as the gen-
tleMACS Dissociator may be used to streamline the dissection
process and reduce user variability during kidney dissociaton.
9. Following this step, many groups subject the cells to a percoll
gradient, which separates particles based on density [13, 14].
The utility of this gradient is to further enrich for leukocytes by
exploiting their known density range. However, in our hands,
we are able to record a sufficient number of renal immune cells
via flow cytometry without percoll enrichment, and others
have reported decreased cell recovery and reduced viability
when using density enrichment [19]. Therefore, we suggest to
perform this density enrichment only if necessary, for example,
to capture a rare cell population.
10. An aliquot of the cell solution can be diluted further if the
sample is too concentrated to accurately count.
11. Much debris may be present in your sample. This is not unex-
pected, though the parsing of cell from debris may be difficult
while counting on a hemocytometer. Look for cellular charac-
teristics such as a circular shape or the presence of a nucleus to
identify cells.
12. Ex vivo activation of the renal immune cells is necessary only if
the measurement of inducible factors, such as cytokines, is
required.
13. The addition of protein transport inhibitors is necessary only if
secreted factors such as cytokines are being measured via intra-
cellular staining. Furthermore, monenesin and brefedin A have
each been shown to effect the expression of particular surface
makers or intracellular cytokines [20, 21]. This should be
taken into consideration during experimental design.
14. The user must determine the appropriate cell stimulant and
time of incubation to elicit the inducible factor of interest from
the relevant cell type.
15. The live cell population represented in Fig. 2c is analogous to
the initial cell count performed on the hemocytometer at the
end of the mechanical digestion protocol. Because live, single
cells were counted on the hemocytometer, one can assume
that the total count of cells is represented in the live cell gate in
Fig. 2c. Therefore, the total number of any following gates can
be determined by simple mathematics using the gate percent-
ages afforded by the data analysis program. This type of quan-
tification is appropriate only when performing flow cytometry
staining immediately after counting, as manipulations, such as
activation in culture, skew the population due to cell adher-
ence to the plate, increased apoptosis, etc.
References
1. Dahlof B, Devereux RB, Kjeldsen SE, Julius S, 3. Zhang JD, Patel MB, Griffiths R, Dolber PC,
Beevers G, de Faire U, Fyhrquist F, Ibsen H, Ruiz P, Sparks MA, Stegbauer J, Jin H, Gomez
Kristiansson K, Lederballe-Pedersen O, JA, Buckley AF, Lefler WS, Chen D, Crowley
Lindholm LH, Nieminen MS, Omvik P, Oparil SD (2014) Type 1 angiotensin receptors on
S, Wedel H, Group LS (2002) Cardiovascular macrophages ameliorate IL-1 receptor-
morbidity and mortality in the losartan interven- mediated kidney fibrosis. J Clin Invest
tion for endpoint reduction in hypertension 124(5):2198–2203. doi:10.1172/JCI61368
study (LIFE): a randomised trial against ateno- 4. Zhang JD, Patel MB, Song YS, Griffiths R,
lol. Lancet 359(9311):995–1003. doi:10.1016/ Burchette J, Ruiz P, Sparks MA, Yan M, Howell
S0140-6736(02)08089-3 DN, Gomez JA, Spurney RF, Coffman TM,
2. Brenner BM, Cooper ME, de Zeeuw D, Keane Crowley SD (2012) A novel role for type 1
WF, Mitch WE, Parving HH, Remuzzi G, angiotensin receptors on T lymphocytes to
Snapinn SM, Zhang Z, Shahinfar S, limit target organ damage in hypertension.
Investigators RS (2001) Effects of losartan on Circ Res 110(12):1604–1617. doi:10.1161/
renal and cardiovascular outcomes in patients CIRCRESAHA.111.261768
with type 2 diabetes and nephropathy. N Engl 5. Guzik TJ, Hoch NE, Brown KA, McCann LA,
J Med 345(12):861–869. doi:10.1056/ Rahman A, Dikalov S, Goronzy J, Weyand C,
NEJMoa011161 Harrison DG (2007) Role of the T cell in
the genesis of angiotensin II induced hyper- Y, Takaki S, Titze J, Levy D, Harrison DG,
tension and vascular dysfunction. J Exp Madhur MS (2015) Lymphocyte adaptor pro-
Med 204(10):2449–2460. doi:10.1084/jem. tein LNK deficiency exacerbates hypertension
20070657 and end-organ inflammation. J Clin Invest
6. Zhang J, Rudemiller NP, Patel MB, Karlovich 125(3):1189–1202. doi:10.1172/JCI76327
NS, Wu M, McDonough AA, Griffiths R, 14. Wei Z, Spizzo I, Diep H, Drummond GR,
Sparks MA, Jeffs AD, Crowley SD (2016) Widdop RE, Vinh A (2014) Differential phe-
Interleukin-1 receptor activation potentiates notypes of tissue-infiltrating T cells during
salt reabsorption in angiotensin II-induced angiotensin II-induced hypertension in mice.
hypertension via the NKCC2 Co-transporter PLoS One 9(12):e114895. doi:10.1371/jour-
in the nephron. Cell Metab 23(2):360–368. nal.pone.0114895
doi:10.1016/j.cmet.2015.11.013 15. Radcliff G, Jaroszeski MJ (1998) Basics of flow
7. Korn T, Bettelli E, Oukka M, Kuchroo VK cytometry. Methods Mol Biol 91:1–24
(2009) IL-17 and Th17 cells. Annu Rev 16. Maecker HT, Trotter J (2006) Flow cytometry
Immunol 27:485–517. doi:10.1146/annurev. controls, instrument setup, and the determina-
immunol.021908.132710 tion of positivity. Cytometry A 69(9):1037–
8. Szabo SJ, Kim ST, Costa GL, Zhang X, 1042. doi:10.1002/cyto.a.20333
Fathman CG, Glimcher LH (2000) A novel 17. Shapiro HM (2004) Excitation and emission
transcription factor, T-bet, directs Th1 lineage spectra of common dyes. Curr Protoc Cytom
commitment. Cell 100(6):655–669 Chapter 1:Unit 1.19. doi:10.1002/04711
9. Hori S, Nomura T, Sakaguchi S (2003) Control 42956.cy0119s26
of regulatory T cell development by the tran- 18. Roederer M (2001) Spectral compensation for
scription factor Foxp3. Science 299(5609):1057– flow cytometry: visualization artifacts, limita-
1061. doi:10.1126/science.1079490 tions, and caveats. Cytometry 45(3):194–205
10. Zheng W, Flavell RA (1997) The transcription 19. Yu YR, O'Koren EG, Hotten DF, Kan MJ,
factor GATA-3 is necessary and sufficient for Kopin D, Nelson ER, Que L, Gunn MD
Th2 cytokine gene expression in CD4 T cells. (2016) A protocol for the comprehensive flow
Cell 89(4):587–596 cytometric analysis of immune cells in normal
11. Chattopadhyay PK, Roederer M (2012) and inflamed murine non-lymphoid tissues.
Cytometry: today’s technology and tomor- PLoS One 11(3):e0150606. doi:10.1371/
row’s horizons. Methods 57(3):251–258. journal.pone.0150606
doi:10.1016/j.ymeth.2012.02.009 20. Schuerwegh AJ, Stevens WJ, Bridts CH, De
12. Itani HA, Xiao L, Saleh MA, Wu J, Pilkinton Clerck LS (2001) Evaluation of monensin and
MA, Dale BL, Barbaro NR, Foss JD, Kirabo A, brefeldin A for flow cytometric determination
Montaniel KR, Norlander AE, Chen W, Sato R, of interleukin-1 beta, interleukin-6, and tumor
Navar LG, Mallal SA, Madhur MS, Bernstein necrosis factor-alpha in monocytes. Cytometry
KE, Harrison DG (2016) CD70 exacerbates 46(3):172–176
blood pressure elevation and renal damage in 21. Nylander S, Kalies I (1999) Brefeldin A, but
response to repeated hypertensive stimuli. Circ not monensin, completely blocks CD69
Res. doi:10.1161/CIRCRESAHA.115.308111 expression on mouse lymphocytes: efficacy of
13. Saleh MA, McMaster WG, Wu J, Norlander inhibitors of protein secretion in protocols for
AE, Funt SA, Thabet SR, Kirabo A, Xiao L, intracellular cytokine staining by flow cytome-
Chen W, Itani HA, Michell D, Huan T, Zhang try. J Immunol Methods 224(1–2):69–76
Chapter 9
Assessment of the Renin–Angiotensin System in Cellular

Organelle: New Arenas for Study in the Mitochondria
Bryan A. Wilson and Mark C. Chappell
Abstract
The renin–angiotensin system (RAS) is an important hormonal system composed of various protein and
peptide components that contribute to blood pressure regulation. Although originally characterized as a
circulating system, there is increasing evidence for the intracellular expression of RAS elements on the nucleus
and mitochondria that may function in concert with or independent of the circulating system. The present
chapter describes several experimental approaches to quantify the expression of RAS components in isolated
mitochondria from the kidney. These approaches are intended to provide a framework to understand the
mitochondrial RAS within a cell-free environment.
Key words Mitochondria, Renin–angiotensin system, Subcellular fractionation, Peptide metabolism,

Ang II, Ang-(1–7), Renin, Mas protein, Neprilysin, Thimet oligopeptidase
1 Introduction
The renin–angiotensin system (RAS) is a key hormonal system that

regulates blood pressure through both central and peripheral
mechanisms [1]. It is also quite clear that the RAS constitutes a
multifunctional set of bioactive peptides, processing enzymes, and
distinct receptor subtypes [2]. Figure 1 outlines these pathways of
the RAS that ultimately begins with conversion of the precursor
protein angiotensinogen to Ang I by the aspartyl protease renin
and the subsequent conversion to Ang II by the metallo-
dipeptidylcarboxpeptidase angiotensin I converting enzyme
(ACE). Ang II binds to and activates the AT1 receptor (AT1R)
subtype to elicit a number of actions that include an increase in
blood pressure, vasoconstriction, tissue fibrosis, cellular prolifera-
tion, and inflammation (Fig. 1). Ang II also recognizes the AT2R
subtype that generally opposes actions of the AT1R including
vasodilation mediated through the release of nitric oxide (NO).
Ang II undergoes further processing by the mono-aminopeptidases
to form Ang-(2–8) [Ang III] and Ang-(3–8) [Ang IV]. Ang III
99
100 Bryan A. Wilson and Mark C. Chappell
Fig. 1 Processing pathways within the renin-angiotensin system. The protease

renin converts angiotensinogen to Ang I. In the classical pathway, ACE converts
Ang I to Ang II which binds to both AT1R and AT2R. Ang II is further converted by
aminopeptidase A (APA) to Ang III that may interact with AT1R and AT2R. Ang III is
converted by aminopeptidase N to Ang-(3–8) [Ang IV] that interacts with the
insulin regulated aminopeptidase (IRAP). In the alternative pathway, Ang I is pro-
cessed to Ang-(1–7) by the endopeptidases neprilysin (NEP) and thimet oligo-
peptidase (TOP). Ang-(1–7) is also derived from processing of Ang II by ACE2 and
prolyl oligopeptidase (POP). Ang-(1–7) recognizes the AT7R/Mas protein. Ang-
(1–7) is metabolized by ACE to Ang-(1–5). Ang-(1–7) can be modified by an
aspartyl decarboxylase (DC) to form [Ala1]-Ang-(1–7) that recognizes the Mas-
related receptor mRG-D. Adapted from Chappell, American Journal of
Physiology—Heart and Circulatory Physiology 310: H137–H152, 2016
recognizes both AT1R and AT2R, while the hexapeptide Ang IV

apparently interacts with insulin regulated aminopeptidase (IRAP)
(Fig. 1). Ang I is also directly processed to Ang-(1–7) by several
endopeptidases including neprilysin (NEP) and thimet oligopepti-
dase (TOP) through hydrolysis of the Pro7-Phe8 bond; the mono-
carboxypeptidases ACE2 and prolyl oligopeptidase (POP) generate
Ang-(1–7) from Ang II (Fig. 1). The heptapeptide Ang-(1–7) pri-
marily recognizes the AT7R/Mas protein to elicit vasodilation
through NO release, as well as attenuate fibrosis, inflammation,
proliferation oxidative stress, and epithelia to myofibroblast transi-
tion [2, 3]. Ang-(1–7) may undergo further modification through
decarboxylation of the aspartyl residue to form Ala1-Ang-(1–7)
[almandine] that recognizes the Mas-related receptor mRG-D to
induce vasorelaxation (Fig. 1) [3]. The importance of these “alter-
native pathways” is most likely evident during therapeutic block-
ade of the RAS as ACE inhibitors markedly increase circulating
levels of Ang-(1–7) while AT1R antagonists (ARBs) increase both
Mitochondrial Renin-Angiotensin System 101
Ang II and Ang-(1–7) to stimulate AT2R and AT7R, respectively.

Although the activation of the RAS is typically considered to reflect
the binding of Ang II to the extracellular AT1R, substantial evi-
dence suggests an intracellular pool of functional receptors local-
ized to the nucleus, endoplasmic reticulum and mitochondria
[4–15]. Indeed, the intracellular administration of Ang II to car-
diac myocytes, vascular smooth muscle cells and proximal tubule
epithelial cells results in an immediate increase in the intracellular
levels of calcium [9, 16, 17]. Moreover, Ang II elicits a rapid
increase in reactive oxygen species (ROS) that is blocked by the
ARBs losartan and candesartan, as well as the NAD(P)H oxidase
inhibitor diphenyliodium in nuclei isolated from the rat and sheep
renal cortex [6, 10]. The direct application of Ang II to isolated
nuclei also elicits an increase in the mRNA levels of angiotensino-
gen, renin, MCP-1, and NHE-3 [18]. Intracellular angiotensin
receptors do not solely comprise the AT1R subtype as both func-
tional AT2R and AT7R/Mas linked to NO stimulation were iden-
tified on the nucleus and mitochondria [4, 5, 7, 10, 11]. Abadir
and colleagues presented evidence for an intra-mitochondrial RAS
by identifying AT1 and AT2 receptors within isolated liver mito-
chondria of mice; Ang II activation of mitochondrial AT2R stimu-
lated local levels of NO [4, 8]. The expression of the AT1 to AT2
receptor ratio was higher in the mitochondria of aged animals, and
the chronic treatment of the aged mice with the ARB losartan mit-
igated the enhanced expression of the AT1R [19].
We expanded the characterization of an intra-mitochondrial
RAS through the demonstration of an Ang-(1–7)-AT7R axis in
mitochondria isolated from the sheep renal cortex [7]. Renal mito-
chondria expressed angiotensinogen, active renin, and the peptides
Ang-(1–7), Ang II, and Ang I [29]. Additionally, these studies
revealed the processing of Ang I to Ang-(1–7) in isolated mito-
chondria that reflected the contribution of the endopeptidases
NEP and TOP [7].
Lastly, we demonstrated immunoreactive evidence for expres-
sion of the AT7R/Mas in cortical mitochondria. These studies col-
lectively support the existence of an Ang-(1–7) pathway within
mitochondria and the possibility for the peptide to elicit direct
actions on mitochondrial function. Indeed, the beneficial aspects
of the Ang-(1–7) axis may counteract the deleterious actions of the
Ang II-AT1R pathway within the mitochondria [7].
Due to emerging evidence for the complex interplay between
mitochondrial function and the RAS, we review various method-
ologies to assess the RAS components in isolated mitochondria.
The intent of the chapter is to provide a biochemical approach to
elaborate the mitochondrial components and functions in a cell-
free context. We detail mitochondrial isolation from the kidney, as
well as describe methodologies to assess mitochondrial purity,
renin activity, and angiotensin peptide content and metabolism.
Mitochondria are isolated utilizing a combination of differen-

tial centrifugation and densitometric separation by a discontinuous
Percoll gradient. The separation of mitochondria and other organ-
elles on Percoll is a widely utilized method that typically retains the
functional integrity of the organelle [4, 7, 20, 21]. Moreover, iso-
lation of mitochondrial on Percoll gradients is rapidly obtained
from intact tissues (<2 h) and can be achieved with a high-speed
centrifuge obviating the necessity of an ultracentrifuge. We rou-
tinely utilize a Percoll density gradient consisting of a 24% and 40%
layered suspensions (Fig. 6). The crude mitochondria pellet derived
from the differential centrifugation separation is reconstituted in
12% Percoll/buffer A and gently layered on top of the 24% inter-
face. Following centrifugation at 15,000 × g for 5 min, the mito-
chondria sediments in a visible band between the 24% and 40%
Percoll layers. The purity and enrichment of the isolated mito-
chondria are assessed by immunoblot analysis using antibodies
against the following mitochondrial proteins: voltage-dependent
anion channel (VDAC), an outer mitochondrial membrane marker,
and the inner mitochondrial markers of the electron transport
chain: ATP synthase complex V (CV-ATP5A); ubiquinolcyto-
chrome c reductase core protein II complex III (CIII-UCRC2);
and succinate dehydrogenase complex subunit B complex II (CII-
SDHB) (Fig. 2a–c). Additionally, a cathepsin B antibody is utilized
as a marker for lysosomes and renin granules, as well a β-actin anti-
body for cytosolic contamination [22].
Renin constitutes the rate-limiting enzyme catalyzing the pro-
cessing of angiotensinogen to Ang I (Fig. 1). Peters and colleagues
originally demonstrated an alternative cytosolic transcript of renin
that was predicted to be retained within the cell and not enter the
secretory pathway [23]. This truncated form of renin was active
and preferentially localized to the mitochondria. Interestingly, the
renin isoform was actively taken up and internalized by isolated
mitochondria, but full-length prorenin did not undergo internal-
ization [23, 24]. Ishigami et al. recently identified a constitutively
active form of renin that lacks a portion of the pro-segment in the
proximal tubules of the mouse kidney [25]. In transgenic mice, the
forced overexpression of this renin isoform increased blood pres-
sure, but did not elevate circulating renin [25].
Based on these earlier studies, we assessed renin activity in iso-
lated mitochondria from male and female sheep by quantifying the
disappearance of intact angiotensinogen (Fig. 3). In contrast to
typical renin assays that quantify the formation of Ang I, our assay
was facilitated by the specificity of the antibody to recognize the
N-terminal Ang I sequence of angiotensinogen. As shown in the
immunoblot in Fig. 3a–d, renin activity in isolated mitochondria
was evident by the time-dependent disappearance of intact angio-
tensinogen at 37 °C; angiotensinogen processing was abolished by
pretreatment of the mitochondria with aliskiren (Fig. 3g and h).
Fig. 2 Assessment of mitochondria purity from isolated fractions of the sheep

renal cortex. Whole renal cortex homogenate (lane 1), crude mitochondrial frac-
tion from a 10,000 × g differential centrifugation step (lane 2), and the Percoll-
purified mitochondrial fraction (lane 3) were subjected to 10% SDS gel and
immunoblotting with anti-VDAC (a—50 μg total protein) as well as ATP5A,
UQCRC2, SDHB (b—5 μg total protein) and Cathepsin B (c—50 μg total protein),
as well as β-actin. Purified mitochondria appear to exhibit greater expression of
both the outer mitochondria marker (VDAC) and the inner mitochondrial markers
(ATP5A, UQCRC2, and SDHB), while Cathepsin B and β-actin expression were
diminished or absent. Data adapted from Wilson et al., Am J Physiol Renal Physiol
310: F637–F645, 2016
To determine prorenin levels, mitochondria were pretreated with

trypsin to convert prorenin to active renin, and angiotensinogen
processing was determined in the presence of the trypsin inhibitor
SBTI. Trypsin exposure did not appear to markedly increase renin
activity as the disappearance rate of Ang I-angiotensinogen was
comparable for both the trypsin and non-trypsin treated mito-
chondrial samples (Fig. 3c–f).
Fig. 3 Renin activity in the kidney mitochondria. Renin activity in the mitochondria of male (a, c, e, g) and
female (b, d, f, h) sheep. Non-trypsin (a, b) or trypsin (c, d) treated mitochondria with (+) or without (−) the
renin inhibitor aliskiren. Immunoblots were probed with an antibody directed to the Ang I sequence of angio-
tensinogen (AI-Aogen). The disappearance or half-life (t½) of AI-Aogen without (e, f) or with (g, h) aliskerin was
determined by Prism 6 statistical program. Data adapted from Wilson et al., Am J Physiol Renal Physiol 310:
F637–F645, 2016
The expression of renin in isolated mitochondria was confirmed

by immunoblot analysis using two different renin antibodies. As
shown in Fig. 4a and b, both antibodies detected two predominant
bands at 37 and 55 kDa. The 37 kDa immunoreactive band corre-
sponds to renin and the higher 55 kDa may represent “big renin,”
Fig. 4 Full-length immunoblot gels for renin, pro-renin receptor, and the AT7R/Mas
in isolated mitochondria. Renin protein detected by the Aviva (a) and Inagami (b)
antibodies reveals two prominent bands at 37 and 55 kDa. A 35-kDa band is evi-
dent for the pro-renin receptor (PRR) in the renal cortex, but not in the mitochon-
drial fractions (c). The AT7R/Mas protein visualized with the Alomone antibody
reveals a single band at 37 kDa (d). Total cortical homogenate (lane 1) and three
separate mitochondrial preparations (lanes 2–4). MW: molecular weight markers.
Data adapted from Wilson et al., Am J Physiol Renal Physiol 310: F637–F645, 2016
an inactive form of the enzyme that is complexed to the renin-bind-

ing protein acetyl-glucosamine-2-epimerase [26]. We did not detect
a protein band for the pro-renin receptor (PRR) in the isolated
mitochondria, although a 35 kDa band was evident in the total cor-
tical homogenate (Fig. 4c). The absence of the PRR in the mito-
chondria would appear to be consistent with lack of prorenin in this
organelle. Finally, the immunoblot analysis revealed the Mas protein
at ~37 kDa in three purified mitochondria preparations, as well as
the total cortical homogenate (Fig. 4d). Using the same Alomone
antibody, we previously demonstrated immunoreactive localization
of the Mas protein to the proximal tubules, thick ascending limb,
collecting ducts, and vasa recta of the sheep kidney [10].
Angiotensin peptides were detected within isolated mitochon-
dria from the sheep renal cortex by distinct radioimmunoassays
(RIAs) directed to the C-terminus of Ang I, Ang II and Ang-(1–7).
Mitochondrial content of Ang II and Ang-(1–7) were both signifi-
cantly higher than Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg
protein; respectively] [7]. Identification of angiotensin peptides

within the mitochondria may portend for Ang-(1–7) and Ang II to
influence mitochondrial function and integrity. Indeed Kim et al.
found that Ang II induced oxidative stress, depolarization of the
mitochondrial membrane potential, and cytosolic secretion of cyto-
chrome C and apoptosis-inducing factor (AIF) in cultured rat prox-
imal tubule cells [27]. Moreover, treatment with exogenous
Ang-(1–7) attenuated the Ang II-induced mitochondrial produc-
tion of ROS via reduced NOX4 expression and apoptosis [27].
Renal mitochondrial content of Ang I was markedly lower than
Ang II and Ang-(1–7) and may reflect active processing within the
mitochondria, therefore, we examined the potential processing
pathways of Ang I in isolated mitochondrial homogenates using the
125
I-labeled peptide as a substrate coupled to HPLC-based separa-
tion and in-line γ-detection [2, 7, 28]. As shown in the chromato-
graphs for Fig. 5a, 125I-Ang I was converted primarily to
125
I-Ang-(1–7). Addition of the NEP inhibitor SCH39370 (SCH)
reduced but did not abolish the 125I-Ang-(1–7) peak (Fig. 5b).
Co-addition of SCH and a TOP inhibitor (CPP) further reduced
the 125I-Ang-(1–7) peak and preserved the 125I-Ang I peak (Fig. 5c).
Immunoblot analysis of the isolated mitochondria confirmed both
NEP and TOP expression (Fig. 5d). Quantification of Ang I metab-
olism revealed that the NEP inhibitor significantly increased Ang I
and reduced Ang-(1–7) (Fig. 5e and f, respectively). The addition
of the TOP inhibitor further reduced Ang-(1–7), but this did not
attain significance compared with the effect of the NEP inhibitor
alone. Importantly, we could not demonstrate the processing of
Ang I to Ang II in the mitochondrial preparation either with or
without the endopeptidase inhibitors (Fig. 5a–c). The failure to
detect Ang II generation from Ang I may reflect the absence of
ACE or other Ang II-generating enzymes in the isolated renal
mitochondria. These data are consistent with the recent study by
Astin et al. that did not detect ACE immunoreactivity within iso-
lated liver mitochondria of the rat [29].
2 Materials
2.1 Tissue Collection 1. Renal Cortex: Sheep (Ovis aries): male and female, adult mixed
breed (10–212 months of age).
2. Anesthesia: ketamine and isoflurane.
3. Ice-cold saline solution.
2.2 Preparation 1. Freshly dissected sheep renal cortex tissue: 5–10 g.

of Isolated 2. Percoll.
Mitochondria
3. Buffer A: 10 mM HEPES (Na+ Free), 0.1 mM EDTA (Na+),
1 L of MilliQ H2O, pH = 7.4 (adjust with KOH only).
Fig. 5 Mitochondrial processing of 125I-Ang I. Chromatograph reveals that 125I-Ang I [125I-AI] was converted to
125
I-Ang-(1–7) [125I-A7] in renal mitochondrial homogenate fractions (a). 125I-AI metabolism to 125I-A7 was
reduced by 10 μM SCH (b), and 125I-AI metabolism to 125I-A7 was reduced by 10 μM SCH in combination with
10 μM CPP (c). Immunoblot analysis revealed evidence for protein expression of neprilysin (NEP) and thimet
oligopeptidase (TOP) in cortical homogenate (lane 1) and mitochondrial homogenates (lanes 2–4, n = 3) (d).
125
I-labeled products were separated by HPLC under gradient conditions. Statistical analysis of 125I-AI metabo-
lism demonstrated Scheme (10 μM) and SCH in combination with CPP (10 μM) preserved 125I-Ang I, while
Scheme (10 μM) and CPP (10 μM) reduced 125I-Ang-(1–7) formation (e, f). The addition of SCH and CPP tended
to further reduce 125I-Ang-(1–7) and increase 125I-Ang I (e, f). Data are mean ± SEM, n = 3; *p < 0.05 vs. control
(CON). Data adapted from Wilson et al., Am J Physiol Renal Physiol 310: F637–F645, 2016
4. Homogenization buffer: 225 mM mannitol, 75 mM sucrose;

dissolve the mannitol and sucrose in 200 mL of buffer A.
5. Percoll Buffer: 250 mM sucrose, 0.4 mM EDTA; dissolve the
sucrose and EDTA in 200 mL of buffer A.
6. Polytron Ultra-Turrax T25 basic tissue homogenizer

7. 50 mL all-glass Dounce Homogenizer.
8. Nalgene round centrifuge tube, PC 16 mL.
9. 70-μm metal strainer.
10. Stir-Pak laboratory stirrer.
2.3 Immunoblot 1. Isolated sheep renal cortex mitochondria: 100–500 μg.

Analysis 2. 2× Laemmli Sample Buffer.
of Mitochondria Purity
3. Heating block.
4. 10% SDS-PAGE gels.
5. Mini-electrophoresis cell.
6. Mini-electrophoresis transfer cell.
7. Polyvinylidene difluoride (PVDF) membrane.
8. Methanol.
9. Running Buffer: 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS.
10. Transfer Buffer: 25 mM Tris, 192 mM glycine, 20% (v/v)
methanol, pH 8.3.
11. Tris buffered saline (TBS): 20 mM Tris, 500 mM sodium
chloride, pH 7.5.
12. TBS-Tween 20 (TBST): 20 mM Tris, 500 mM sodium chloride,
pH 7.5, 0.05% (v/v) Tween 20.
13. Blotting Grade Blocker (Nonfat dry milk): This is added to
TBST to make 5% (w/v) blocking solution.
14. ECL anti-mouse horseradish peroxidase (HRP) linked whole
antibody, ECL anti-rabbit HRP linked whole antibody.
15. ECL detection kit.
16. X-ray/autoradiography film.
17. Antibodies:
●● Angiotensinogen, 1:1000, (Ang I directed, residues #25–34;
Dr. Mark Chappell, WFUSM).
●● Angiotensinogen 1:1000, (Distal to Ang I sequence, residues
#42–57; Dr. Mark Chappell, WFUSM).
●● ATP5A, UQCRC2, SDHB, 1:1000.
●● β-actin, 1:500 (#AC-15).
●● Cathepsin B, 1:400 (#CA10).
●● Mas receptor, 1:250 (#AAR-013).
●● Neprilysin, 1:2000 (#NEP11-S).
●● Prorenin receptor (ATP6IP2), 1:500 (#ab63957).
●● Renin.
–– 1:3000 (#826; Dr. Tad Inagami, Vanderbilt University,
Nashville TN).
–– 1:100 (#T100).
●● Thimet oligopeptidase, 1:800 (#EPR7651).
●● VDAC, 1:1000 (#4866).
2.4 Renin Assay 1. Isolated sheep renal cortex mitochondria: ~400 μg.

2. Nephrectomized sheep plasma.
3. Metabolism Buffer: 25 mM HEPES, 125 mM NaCl, 10 μM
ZnCl2 at pH 7.4 in MilliQ H2O.
4. Trypsin, 5 mg/mL.
5. Soybean trypsin inhibitor (SBTI): 1 mg/mL.
6. EDTA.
7. o-Phenanthroline.
2.5 Angiotensin 1. Isolated sheep renal cortex mitochondria.

Peptide Quantification 2. Milli-Q Ultrapore H2O [≥18 MΩ].
3. Heptafluorobutyric acid (Sequanal Grade HFBA).
4. Tissue sonicator.
5. High-speed centrifuge up to 15,000 × g.
6. Methanol.
7. Sep-Pak C18 extraction columns.
8. Extraction manifold.
9. Radioimmunoassay (RIA) kits.
●● Angiotensin I.
●● Angiotensin II.
●● Angiotensin-(1–7)—in-house [29].
2.6 Angiotensin 1. Isolated sheep renal cortex mitochondria: Mitochondrial

Peptide Metabolism lysate.
2. Metabolism buffer: 25 mM HEPES, 125 mM NaCl, 10 μM
ZnCl2 at pH 7.4 in MilliQ H2O.
3. Iodine-125 (125I) radionuclide (5 mCuries).
4. 125I-Angiotensin I,
5. N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl]-
(S)-isoserine (SCH;10 μM): neprilysin inhibitor.
6. N-[1-(R,S)-carboxy-3-phenylpr opyl]-AlaAla-Phe-p-
aminobenzoate (CPP; 10 μM); thimet oligopeptidase inhibitor.
7. Shimadzu Prominence HPLC binary system.
2.7 HPLC Equipment 1. Aeris Peptide XB-C18 narrow bore column (2.1 × 100 mm).
2. Bioscan Inline γ-detector.
3. Phosphoric acid (HPLC grade).
4. Filter vials—0.45 μm PTFE, 450 μL capacity
3 Methods
3.1 Mitochondria Procedure requires all steps performed on ice or at 4 °C

Isolation from 1. Dissect and weigh out 10 g of kidney cortex tissue, and mince
Renal Cortex very finely using surgical scissors and tissue grossing and trim-
ming blade tools.
2. Split the 10 g of kidney tissue into two separate 15 mL conical
tubes (5 g each tube).
3. Add homogenization buffer to each tube up to a total volume
of 15 mL.
Homogenize on ice
1. Initially homogenize minced kidney utilizing a power blade
metal tissue homogenizer. Then utilize the Teflon Dounce
homogenizer (mortar and pestle) with 5 up and down strokes
at setting of 5.
2. Filter homogenate through a 70 μm metal strainer.
3. Centrifuge tubes at 1500 × g for 5 min at 4 °C.
4. Remove supernatant and recentrifuge at 10,000 × g for 10 min
at 4 °C to pellet the crude mitochondria.
5. Remove the supernatant and the pellet consists of the crude
mitochondrial fraction.
Mitochondria crude pellet is resuspended in 12% Percoll media
1. Gently resuspend crude mitochondrial pellet in 12% Percoll
media by diluting the 24% Percoll 1:1 (i.e., 3 mL of Percoll
Buffer + 3 mL of 24% Percoll).
Layering the Percoll gradient
While a variety of tubes may be used for layering the Percoll
gradient, we recommend using Nalgene Round Centrifuge Tube,
PC 16 mL.
Percoll Gradient Calculations
24% Percoll media = 4.8 mL (100% Percoll) + 15.2 mL (Percoll
Buffer) = 20 mL.
For a lesser volume of 24% Percoll media if preferred.
2.4 mL (100% Percoll) + 7.6 mL (Percoll Buffer) = 10 mL.
40% Percoll media = 6.0 mL (100% Percoll) + 9.0 mL (Percoll

Buffer) = 15 mL.
1. Add 4 mL of 40% media to each tube. Then gently layer 5 mL
of 24% Percoll media on top. For ease, even layering may be
achieved by pipetting the 24% Percoll media in such a way that
it sleeks slowly down the side of the tube. Finally, layer the
3 mL mitochondrial crude pellet in 12% Percoll media on top
of 24% Percoll media layer.
2. Centrifuge at 15,000 × g for 5 min at 4 °C
3. Pure mitochondria will sediment with a prominent band
between the 24% and 40% Percoll layers. Gently pipet off the
mitochondrial band between the 24% and 40% interface from
each tube and place into a clean Nalgene tube.
4. To dilute out the Percoll solution, add 8–10 mL of buffer A to
each tube. Note: To further dilute the Percoll, divide the two
tubes in half a second time. This will result in four tubes, and
add ~4–5 mL of buffer A to each tube to further dilute the
Percoll. Failure to efficiently dilute the Percoll media will pre-
vent the sufficient formation of the mitochondrial pellet.
5. Centrifuge at 11,000 × g for 10 min at 4 °C.
6. Gently remove the supernatant so as not to disrupt the pure
mitochondrial pellet.
7. Resuspend each mitochondrial pellet in preferred volume of
metabolism buffer and transfer into 1.5 mL microfuge tubes
and store at 4 °C or −80 °C for later use (Fig. 6).
3.2 Western Blot 1. Utilize purified mitochondrial preparations as described in

Analysis Subheading 3.1.
2. Protein concentration of samples is determined by the Bradford
assay (Bio-Rad).
3. A 2× Bio-Rad Laemmli buffer is added and mitochondrial
samples are boiled in screw cap 1.5 mL tubes for 5 min. Equal
volumes of samples are loaded onto a Bio-Rad MiniPROTEAN
TGX StainFree 10% Gel.
4. Gels are run at 120 V until the dye front reaches the bottom of
the gel.
5. Gels are removed from their plastic casts and are assembled on
the semidry transfer cell. Two pieces of transfer buffer-soaked:
filter paper and filter pad, methanol-activated PVDF transfer
membrane are placed between the gel.
6. Ensure there are no air pockets trapped between the sandwich
layers.
7. Transfer takes place for 60 min at 100 V in transfer buffer.
Transfer cassette contains an ice pack to maintain temperature
at 4 °C.
Fig. 6 Mitochondrial purification from renal cortex tissue. Mitochondria were isolated from fresh sheep renal
cortex by a discontinuous Percoll gradient. Tissue was homogenized in mitochondrial homogenization buffer
and centrifuged for 5 min at 1000 × g, and the resultant supernatant filtered through a 70 μm metal strainer
and centrifuged for 10 min at 10,000 × g. This pellet, representing the crude mitochondrial fraction was resus-
pended in 12% Percoll media and gently layered onto a discontinuous Percoll gradient (24% and 40% in
Percoll buffer). The gradient was centrifuged for 5 min at 15,000 × g and the band at the 24% and 40% Percoll
layers was collected
8. The membrane is removed from the sandwich and is incubated

with agitation at room temperature for 1 h in TBST supple-
mented with 5% Bio-Rad nonfat dry milk (w/v).
9. The buffer is removed and replaced by the same solution sup-
plemented with the primary antibody. The membrane and
antibody in buffer are agitated overnight at 4 °C.
10. The membrane is washed once for 10 min at room tempera-
ture in TBS and twice for 10 min at room temperature in
TBST.
11. The membrane is incubated with agitation in TBST supple-
mented with 5% (w/v) Bio-Rad nonfat dry milk powder and
the appropriate secondary antibody for 1–2 h at room
temperature.
12. Wash the membrane as in step 10.
13. The membrane is finally incubated with ECL detection reagent,

covered in a transparent plastic sheet protector, and exposed to
X-ray film for an appropriate time. Films are subsequently
developed in an automated developer.
3.3 Renin Assay Mitochondria Reaction with Nephrectomized Sheep Plasma

1. We recommend utilizing ~400 μg of mitochondrial protein for
the total reaction.
2. Trypsin activation (see Note 1).
3. Set up trypsin activation reaction mixture in a 1.5 mL tube by
adding the reaction components listed sequentially in the table
below and incubate for 1 h at 37 °C in a water bath (Table 1).
4. Stop the trypsin activation by adding the trypsin inhibitor
SBTI (25 μL of 10 mg/mL stock solution and incubate at
37 °C for 15 min.
5. Divide the reaction volume by aliquoting 200 μL of total reac-
tion volumes in separate 1.5 mL tubes. Label tubes by specify-
ing “+” or “−” Aliskiren (Table 2).
6. Add aliskiren or metabolism buffer, mix and incubate for
10 min at 37 °C (see Note 2–4).
7. Add 10 μL of nephrectomized sheep plasma to each tube and
vortex.
Table 1
Components of trypsin activation reaction mix for mitochondrial protein
Total reaction volume 400 μL

Metabolism buffer 335 μL
o-Phenanthroline (1 mM) 5 μL
EDTA (1 mM) 5 μL
Sheep mitochondria 50 μL
Trypsin (5 mg/mL) 5 μL
Table 2
Components of reaction mixture with and without the renin inhibitor,
Aliskiren
(+) Aliskiren (−) Aliskiren

200 μL—Total reaction mixture 200 μL—total reaction
mixture
2 μL of (100 μM Aliskiren stock) = Final 2 μL of metabolism buffer
concentration 1 μM
8. Remove 30 μL aliquots of (+) and (−) aliskiren at 30 min, 1 h,

2 h, and 4 h time points. When removing the aliquots at the
specified time points, add the contents to a 1.5 mL screw cap
tube and add 30 μL of 2× Bio-Rad Laemmli buffer.
9. Only 20 μL of the 60 μL total (30 μL Laemmli + 30 μL sample)
will be loaded onto the gel for Western blot analysis (Fig. 7).
Western Blot Gel Map (Table 3).
10. 20 μL of sample is loaded in each cell well [2–10]. Control
sample (lane 2) was prepared by taking 10 μL of nephrecto-
mized sheep plasma and diluting it in 200 μL of metabolism
buffer. A 30 μL aliquot from the 210 μL (total volume) was
removed and added to 30 μL of Laemmli buffer.
11. Immunoblot analysis is conducted with methods described in
Subheading 3.2 utilizing the Ang I sequence of angiotensino-
gen antibody (Ang I-angiotensinogen, residues 25–34,
1:1000).
3.4 Angiotensin 1. After isolating mitochondria as described in Subheading 3.1,

Peptide Quantification pellet mitochondria at 16,000 × g for 1 min.
2. Rapidly dilute the sonicated mitochondria in 10 mL of MilliQ
H2O in a 15 mL plastic conical tube. Puncture the cap of the tube.
Fig. 7 Renin assay utilizing isolated sheep renal cortex mitochondrial homogenates. Mitochondria were iso-
lated as described in Fig. 6. The mitochondrial pellet was reconstituted in 1 mL of metabolism buffer and
stored on ice. For basal renin activity assay, 400 μg of mitochondrial homogenate was added to 10 μL of
nephrectomized sheep plasma as the source of exogenous angiotensinogen (Aogen) substrate. The assay was
performed at 37 °C for 4 h in the presence or absence of the renin inhibitor aliskiren (1 μM, final concentra-
tion). Aliquots were removed at 30 min, 1, 2 and 4 h and analyzed by Western blot using the Ang I-angiotensinogen
(AI-Aogen) antibody. Activation of prorenin was performed by addition of 5 mg/mL trypsin to the mitochondrial
sample for 60 min. Trypsin was inactivated by incubation with an excess of soybean trypsin inhibitor (1 mg/
mL) for 15 min at room temperature
Table 3
Gel map for Western blotting mitochondrial angiotensinogen and other proteins of the RAS
1 2 3 4 5 6 7 8 9 10
Molecular Control 30 min 30 min 1 h 1 h 2 h 2 h 4 h 4 h
weight Neph Sheep + − + − + − + −
marker Plasma Aliskiren Aliskiren Aliskiren Aliskiren Aliskiren Aliskiren Aliskiren Aliskiren
3. Immerse tube in boiling water for 15 min.

4. Sonicate the mitochondrial homogenate ~30 s at a setting of 5.
5. Remove an aliquot for protein analysis by Bradford (100 μL).
6. Add 10 μL of HFBA to the mixture to acidify the solution
(0.1% HFBA final concentration).
7. Centrifuge at 10,000 × g for 20 min at 4 °C.
8. Collect supernatant and transfer to a clean 15 mL conical tube.
Discard pellet.
9. Place supernatant sample on ice until SepPak extraction.
Angiotensin Peptide Extraction
10. Peptide extraction is performed on Sep-Pak C18 columns cou-
pled to 10 mL BD plastic syringes. The syringe fits snugly on
the column and is ideal as a 10 mL reservoir. The acidified
supernatant is applied to the syringe and is gravity fed through
the Sep-Pak column (see Fig. 8).
11. Activate Sep Pak (300 mg cartridges; Millipore, MA):
●● 5 mL 100% methanol–0.1% HFBA.
●● 5 mL 0.1% HFBA in MilliQ H2O.
12. Apply samples (in 0.1% HFBA/MilliQ H2O), gravity feed
through the syringe reservoir.
13. Wash:
●● 10 mL 0.1% HFBA in MilliQ H2O.
●● 10 mL MilliQ H2O.
14. Elute in 3 mL 100% methanol–0.1% HFBA gravity feed.
15. Aliquot specified volume for RIA analysis. It is important to
completely evaporate samples in a vacuum centrifuge as any
residual solvent may drastically interfere with the RIA analysis
(see Notes 8–11).
3.5 Angiotensin Reaction Vessel—Example in 1.5 mL tube (Table 4)

Peptide Metabolism
1. In a 1.5 mL reaction tube, combine reaction contents in the
following order.
Fig. 8 Angiotensin peptide extraction from isolated renal cortex mitochondria. Sheep renal cortex mitochondria
were isolated as described in Fig. 6. The mitochondrial pellet was reconstituted in Milli-Q water and placed in a
boiling water bath for 15 min. The mitochondrial fraction was acidified with heptafluorobutyric acid (HFBA) to a
final concentration of 0.1%, sonicated and centrifuged at 10,000 × g for 20 min at 4 °C. The resultant superna-
tant was applied to an activated Sep-Pak C18 extraction column, washed with 0.1% HFBA, and the peptide
fraction eluted with 3 mL of 100% methanol in 0.1% HFBA. Measurement of immunoreactive Ang I, Ang II, and
Ang-(1–7) in the extracted mitochondria was performed using three separate radioimmunoassays (RIAs)
Table 4
Reaction mix for measuring mitochondrial angiotensin peptides
Total reaction volume 500 μL

Metabolism buffer—Volume will vary based on protein 390 μL
concentration of sample
Inhibitor of choice prepared at a 10× higher concentration 50 μL
Mito sample (~50 μg)—Volume will vary based on protein 10 μL
concentration of sample
125
I-Ang peptide (2 × 106 cpm/100 μL) in 0.1% Triton— 50 μL
Volume may vary based on the cpm of radioactivity
2. Add precalculated volume of metabolism buffer.

3. Add inhibitors at desired concentration.
4. Add mitochondria sample in desired protein concentration
and pre-incubate for ~5 min.
5. Add 125I-labeled peptide substrate and vortex.
6. Incubate reaction at 37 °C for the predetermined time period
(i.e., 15, 30, or 60 min).
7. At every time point, vortex each tube and immediately pipette

220 μL of suspension into a tube containing 220 μL of 1%
phosphoric acid to terminate the metabolism reaction.
8. Repeat for each reaction sample sequentially.
9. Vortex aliquots and centrifuge at 16,500 × g for 1 min.
10. Filter samples using HPLC grade Filter Vials (0.45 μm PTFE,
450 μL capacity).

11. Refrigerate samples until submission for HPLC analysis
(Fig. 9).
12. HPLC conditions for separation of 125I-peptides are described
below (see Notes 11–14):
●● Solvent A: 0.1% phosphoric acid in Milli-Q ultrapure water.
●● Solvent B: 80% acetonitrile–0.1% phosphoric acid.
●● Flow rate: 0.35 mil/min.
●● Gradient: Isocratic 5 min at 10% B; Liner 10–30% B;
Isocratic 30% B 10 min.
Fig. 9 Scheme for HPLC-based detection of 125I-Ang I metabolism in isolated renal cortex mitochondria. 125I-
Ang I metabolism is measured at 37 °C in the mitochondrial lysate. The metabolism reaction contains a final
concentration of 0.5 nM 125I-Ang I and 100 nM unlabeled Ang I with or without peptidase inhibitors. The reac-
tion is stopped with ice-cold 1.0% phosphoric acid and centrifuged at 16,000 × g. The supernatants are fil-
tered by filter vials, and the products are separated by C18 reverse phase chromatography. The individual
radioactive peaks separated by the HPLC column are detected by an inline γ detector
4 Notes
1. The rationale for trypsin activation is that renin is initially syn-

thesized in a pro-form that is inactive towards the angioten-
sinogen substrate. Upon enzymatic cleavage of the pro-segment
by serine proteases such as trypsin, renin is fully active and
hydrolyzes the Ang I segment from angiotensinogen.
2. Nephrectomized sheep plasma is used as an exogenous source
of angiotensinogen. The removal of the kidneys abolishes the
major source of circulating pro-renin and renin. As a result of
the essentially complete depletion of renin, circulating levels of
intact angiotensinogen rise markedly due to the inhibition of
negative feedback by the lower levels of Ang II. Therefore, the
plasma isolated from nephrectomized sheep blood constitutes
an enriched source of intact angiotensinogen for experimental
studies.
3. Renin is a highly specific protease and the sole endogenous
substrate for the enzyme is angiotensinogen. Sheep renin will
readiy hydrolyze angiotensinogen from other species [30].
4. To distinguish renin activity in the mitochondria from that of
other aspartyl proteases, we pretreat the sample with the spe-
cific renin inhibitor aliskiren. The difference between the
control sample and the renin-added sample is considered renin
activity.
5. The classical renin assay is to quantify the generation of Ang I
from angiotensinogen using a specific RIA or ELISA. Precautions
must be taken to prevent the degradation of Ang I in the assay
and various inhibitors are typically added; however, these
inhibitors should not interfere with Ang I assay or attenuate
renin activity [2, 30].
6. Alternatively, ELISAs are available to quantify immunoreactive
protein levels of active renin. To detect pro-renin, aliskerin is
added to induce a conformational change in pro-renin and
allow the ELISA antibody to detect renin. The difference
between the renin values with and without aliskerin is pro-
renin [2, 30].
7. Samples can be placed in a desiccator under vacuum overnight
to ensure the extraction solvents are completely removed.
8. RIA analysis requires an appropriate “blank sample” that lacks
the mitochondria but is extracted in the exact same manner
and assayed under identical conditions.
9. The recovery of angiotensins during the extraction process can
be determined by adding a small amount of the radiolabeled
peptide to each sample homogenate. The % recovery is calcu-

lated by dividing the recovered cpm by the initial cpm × 100;
typical recovery values vary from 60% to 90%. Peptide content
can be corrected for each sample by normalizing to the recov-
ery value. Alternatively, an excess of unlabeled peptide is added
to a duplicate set of homogenate samples and the recovery is
based on this value.
10. The RIA or ELISA peptides values can be further validated by
prior separation of the sample on HPLC and the collected frac-
tions assayed by the RIA or ELISA. Alternatively, samples can
be fractionated by HPLC and the peptides quantified by
mass-spectroscopy.
11. The metabolites are identified by comparison of their retention
times to the 125I-standard peptides, the response to the addi-
tion of peptidase inhibitors and immunoprecipitation by selec-
tive antibodies. 125I-standards are developed using the
chloramine T method to iodinate the tyrosine residue of angio-
tensin peptides and purify the radiolabeled peptides by HPLC
to a specific activity of 2200 Curies/mmol [2, 28].
12. Radioactive peaks are quantified based on the specific activity
of the 125I-peptide substrate [~2200 Curies/mmol]. In brief, 1
Curie = 2.22 × 1012 dpm; the sample peak cpm is converted to
dpm based on the efficiency of the γ-gamma counter (~70%)
and divided by the specific activity to yield the mole content.
13. The Shimadzu auto-sampler employs an extensive wash cycle
between sample injections to eliminate the radioactive carry-
over from the previous samples.
14. This method detects only the 125I-tyrosine signature and
metabolites that do not contain the 125I-tyrosine remain
unidentified [2, 28].
Acknowledgment
The authors gratefully acknowledge Nancy Pirro, Eric LeSaine,

and Pamela Dean for technical and surgical support. During the
completion of this work, B.A. Wilson was supported by an
American Heart Association (AHA) predoctoral fellowship grant
(15PRE25120007). Additional support for these studies was pro-
vided by National Institutes of Health Grants HD-047584,
HD-017644, HD-084227, HL-51952, T32 grant (HL091797);
the Groskert Heart Fund, the Wake Forest Venture Fund, and the
Farley-Hudson Foundation (Jacksonville, NC).
References
1. Campbell DJ (2014) Clinical relevance ally coupled to the formation of nitric oxide.
of local renin angiotensin systems. Front Am J Physiol Renal Physiol 299(5):F983–
Endocrinol (Lausanne) 5:113. doi:10.3389/ F990. doi:10.1152/ajprenal.00371.2010
fendo.2014.00113 12. Kumar R, Thomas CM, Yong QC, Chen
2. Chappell MC (2016) Biochemical evaluation W, Baker KM (2012) The intracrine
of the renin-angiotensin system: the good, renin-angiotensin system. Clin Sci (Lond)
bad, and absolute? Am J Physiol Heart Circ 123(5):273–284. doi:10.1042/CS20120089
Physiol 310(2):H137–H152. doi:10.1152/ 13. Kurdi M, De Mello WC, Booz GW (2005)
ajpheart.00618.2015 Working outside the system: an update on
3. Santos RA (2014) Angiotensin-(1–7). the unconventional behavior of the renin-
Hypertension 63(6):1138–1147. doi:10.1161/ angiotensin system components. Int J Biochem
HYPERTENSIONAHA.113.01274 Cell Biol 37(7):1357–1367. doi:10.1016/j.
4. Abadir PM, Foster DB, Crow M, Cooke CA, biocel.2005.01.012
Rucker JJ, Jain A, Smith BJ, Burks TN, Cohn 14. Re RN, Cook JL (2011) Noncanonical intra-
RD, Fedarko NS, Carey RM, O'Rourke B, crine action. J Am Soc Hypertens 5(6):435–
Walston JD (2011) Identification and char- 448. doi:10.1016/j.jash.2011.07.001
acterization of a functional mitochondrial 15. de Cavanagh EM, Inserra F, Ferder M, Ferder
angiotensin system. Proc Natl Acad Sci U L (2007) From mitochondria to disease: role
S A 108(36):14849–14854. doi:10.1073/ of the renin-angiotensin system. Am J Nephrol
pnas.1101507108 27(6):545–553. doi:10.1159/000107757
5. Gwathmey TM, Shaltout HA, Pendergrass 16. Haller H, Lindschau C, Erdmann B, Quass P,
KD, Pirro NT, Figueroa JP, Rose JC, Diz DI, Luft FC (1996) Effects of intracellular angio-
Chappell MC (2009) Nuclear angiotensin II tensin II in vascular smooth muscle cells. Circ
type 2 (AT2) receptors are functionally linked Res 79(4):765–772
to nitric oxide production. Am J Physiol Renal 17. Zhuo JL, Li XC, Garvin JL, Navar LG,
Physiol 296(6):F1484–F1493. doi:10.1152/ Carretero OA (2006) Intracellular ANG
ajprenal.90766.2008 II induces cytosolic Ca2+ mobilization by
6. Pendergrass KD, Gwathmey TM, Michalek stimulating intracellular AT1 receptors in
RD, Grayson JM, Chappell MC (2009) The proximal tubule cells. Am J Physiol Renal
angiotensin II-AT1 receptor stimulates reac- Physiol 290(6):F1382–F1390. doi:10.1152/
tive oxygen species within the cell nucleus. ajprenal.00269.2005
Biochem Biophys Res Commun 384(2):149– 18. Li XC, Zhuo JL (2008) Intracellular ANG
154. doi:10.1016/j.bbrc.2009.04.126 II directly induces in vitro transcription of
7. Wilson BA, Nautiyal M, Gwathmey TM, Rose TGF-beta1, MCP-1, and NHE-3 mRNAs in
JC, Chappell MC (2015) Evidence for a mito- isolated rat renal cortical nuclei via activation
chondrial angiotensin-(1–7) system in the kid- of nuclear AT1a receptors. Am J Physiol Cell
ney. Am J Physiol Renal Physiol 00479:02015. Physiol 294(4):C1034–C1045. doi:10.1152/
doi:10.1152/ajprenal.00479.2015 ajpcell.00432.2007
8. Abadir PM, Walston JD, Carey RM (2012) 19. de Cavanagh EM, Piotrkowski B, Basso N,
Subcellular characteristics of functional intracellu- Stella I, Inserra F, Ferder L, Fraga CG (2003)
lar renin-angiotensin systems. Peptides 38(2):437– Enalapril and losartan attenuate mitochondrial
445. doi:10.1016/j.peptides.2012.09.016 dysfunction in aged rats. FASEB J 17(9):1096–
9. De Mello WC (1998) Intracellular angiotensin 1098. doi:10.1096/fj.02-0063fje
II regulates the inward calcium current in car- 20. Nautiyal M, Katakam PV, Busija DW, Gallagher
diac myocytes. Hypertension 32(6):976–982 PE, Tallant EA, Chappell MC, Diz DI (2012)
10. Gwathmey TM, Pendergrass KD, Reid SD, Rose Differences in oxidative stress status and expres-
JC, Diz DI, Chappell MC (2010) Angiotensin- sion of MKP-1 in dorsal medulla of trans-
(1–7)-angiotensin-converting enzyme 2 genic rats with altered brain renin-angiotensin
attenuates reactive oxygen species forma- system. Am J Physiol Regul Integr Comp
tion to angiotensin II within the cell nucleus. Physiol 303(8):R799–R806. doi:10.1152/
Hypertension 55(1):166–171. doi:10.1161/ ajpregu.00566.2011
HYPERTENSIONAHA.109.141622 21. Rajapakse N, Shimizu K, Payne M, Busija D
11. Gwathmey TM, Westwood BM, Pirro NT, (2001) Isolation and characterization of intact
Tang L, Rose JC, Diz DI, Chappell MC (2010) mitochondria from neonatal rat brain. Brain
Nuclear angiotensin-(1–7) receptor is function- Res Brain Res Protoc 8(3):176–183
22. Taugner R, Buhrle CP, Nobiling R, Kirschke 2 7. Kim SM, Kim YG, Jeong KH, Lee SH,

H (1985) Coexistence of renin and cathep- Lee TW, Ihm CG, Moon JY (2012)
sin B in epithelioid cell secretory granules. Angiotensin II-induced mitochondrial
Histochemistry 83(2):103–108 Nox4 is a major endogenous source of oxi-
23. Clausmeyer S, Sturzebecher R, Peters J (1999) dative stress in kidney tubular cells. PLoS
An alternative transcript of the rat renin gene One 7(7):e39739. doi:10.1371/journal.
can result in a truncated prorenin that is trans- pone.0039739
ported into adrenal mitochondria. Circ Res 28. Shaltout HA, Westwood BM, Averill DB,
84(3):337–344 Ferrario CM, Figueroa JP, Diz DI, Rose JC,
24. Wanka H, Kessler N, Ellmer J, Endlich N, Chappell MC (2007) Angiotensin metabolism
Peters BS, Clausmeyer S, Peters J (2009) in renal proximal tubules, urine, and serum
Cytosolic renin is targeted to mitochondria of sheep: evidence for ACE2-dependent pro-
and induces apoptosis in H9c2 rat cardiomyo- cessing of angiotensin II. Am J Physiol Renal
blasts. J Cell Mol Med 13(9A):2926–2937. Physiol 292(1):F82–F91. doi:10.1152/
doi:10.1111/j.1582-4934.2008.00448.x ajprenal.00139.2006
25. Ishigami T, Kino T, Chen L, Minegishi S, Araki 29. Astin R, Bentham R, Djafarzadeh S, Horscroft
N, Umemura M, Abe K, Sasaki R, Yamana H, JA, Kuc RE, Leung PS, Skipworth JR, Vicencio
Umemura S (2014) Identification of bona JM, Davenport AP, Murray AJ, Takala J, Jakob
fide alternative renin transcripts expressed SM, Montgomery H, Szabadkai G (2013)
along cortical tubules and potential roles in No evidence for a local renin-angiotensin sys-
promoting insulin resistance in vivo without tem in liver mitochondria. Sci Rep 3:2467.
significant plasma renin activity elevation. doi:10.1038/srep02467
Hypertension 64(1):125–133. doi:10.1161/ 30. Campbell DJ, Nussberger J, Stowasser M,
HYPERTENSIONAHA.114.03394 Danser AH, Morganti A, Frandsen E, Menard
26. Takahashi S, Hori K, Ogasawara H, Hiwatashi J (2009) Activity assays and immunoassays for
K, Sugiyama T (2006) Effects of nucleotides on plasma renin and prorenin: information pro-
the interaction of renin with GlcNAc 2-epimer- vided and precautions necessary for accurate
ase (renin binding protein, RnBP). J Biochem measurement. Clin Chem 55(5):867–877.
140(5):725–730. doi:10.1093/jb/mvj201 doi:10.1373/clinchem.2008.118000
Chapter 10
Comprehensive Assessments of Energy Balance in Mice

Justin L. Grobe
Abstract
Increasing evidence supports a major role for the renin‐angiotensin system (RAS) in energy balance
physiology. The RAS exists as a circulating system but also as a local paracrine/autocrine signaling mecha-
nism in target tissues including the gastrointestinal tract, the brain, the kidney, and distinct adipose beds.
Through activation of various receptors in these target tissues, the RAS contributes to the control of food
intake behavior, digestive efficiency, spontaneous physical activity, and aerobic and anaerobic resting
metabolism. Although the assortment of methodologies available to assess the various aspects of energy
balance can be daunting for an investigator new to this area, a relatively straightforward array of entry-level
and advanced methodologies can be employed to comprehensively and quantitatively dissect the effects of
experimental manipulations on energy homeostasis. Such methodologies and a simple initial workflow for
the use of these methods are described in this chapter, including the use of metabolic caging systems,
bomb calorimetry, body composition analyzers, respirometry systems, and direct calorimetry systems.
Finally, a brief discussion of the statistical analyses of metabolic data is included.
Key words Energy balance, Caloric balance, Obesity, Calorimetry, Metabolism, Digestive efficiency,
Energy efficiency, Resting metabolic rate, Aerobic, Anaerobic
1 Introduction
Genetic, dietary, behavioral, and pharmacological manipulations

often result in changes in energy balance. Such changes are primar-
ily mediated through altered food intake behavior, digestive effi-
ciency, spontaneous physical activity, and aerobic or anaerobic
resting metabolism (Table 1). As we recently reviewed, the renin‐
angiotensin system (RAS) has been implicated in the physiologi-
cal/pathophysiological control of each of these endpoints [1].
Intriguingly, the RAS can simultaneously activate opposing effects
(depending upon the tissue site of action, receptors activated, and
endpoint of interest) which can be reflected in large and opposing
changes in individual phenotypes (e.g., food intake and resting
metabolic rate) that ultimately result in no net change in body
mass. Thus, investigators studying the effects of various
manipulations of the RAS upon metabolic physiology may benefit
123
124 Justin L. Grobe
Table 1
The four major mechanisms of energy flux, and common methodologies to assess these components
Energy intake mechanisms Energy output mechanisms
Resting
Food intake Digestive efficiency Physical activity metabolism
Type of Behavioral Biological Behavioral Biological
mechanism
Contribution to Maximally sets the Scales energy Typically contributes Typically
energy balance amount of energy (calories) actually 0–40% of total contributes
to be balanced by absorbed from 0 energy 60–100% of
expenditure to 100% of those expenditure total energy
consumed expenditure
Common Food weights Bomb calorimetry Photoelectric beam Direct calorimetry
methods (quantitative), (quantitative), break, wheel (for total
consumption fecal acid rotations, metabolic rate),
patterning steatocrit or radiotelemetric respirometry
(qualitative) metabolomics implant (all (aerobic
(qualitative) qualitative) metabolism) (all
quantitative)
from a simple rubric for dissecting the relative effects of the RAS
upon individual mechanisms that contribute to energy balance in
addition to the complex, integrated endpoint of total body mass.
It is important to recognize that at the population level, human
obesity is the result of very small net imbalances of caloric intake
versus output spread over long periods of time. One highly publi-
cized estimate suggests that roughly 7.2 kcal/day imbalance
between input and output can explain the obesity epidemic in the
USA [2]. Compared to a normal 2000 kcal/day diet, 7.2 kcal/day
reflects a 0.35% imbalance. While the exact accuracy of this esti-
mate has been contested by various groups, there is essentially uni-
versal agreement that obesity is the result of very small differences
between caloric input and output. By extension, any physiological
or behavioral mechanisms that contribute to energy balance must
be very tightly controlled. Therefore, all mechanisms contributing
to energy balance represent potential therapeutic targets for obe-
sity and stopping one’s investigation of the effects of any manipula-
tion (of the RAS or any system) after only measuring body mass
and food intake behaviors is inadequate and inappropriate. At min-
imum, investigators should consider calculating the fraction of an
observed change in body mass that can be attributed to a specific
endpoint such as altered food intake behavior.
In this chapter, we outline a simple workflow (Fig. 1) which
uses a combination of classic and emerging technologies to allow
for straightforward, comprehensive, and quantitative assessments
Assessing Energy Balance in Mice 125
Fig. 1 Proposed workflow to assess energy balance in laboratory mice. To comprehensively identify the
mechanism(s) that contribute to an observed body mass phenotype, a quantitative approach is required. One
simple workflow is to use body composition analysis to clarify the total amount of energy change that is
required to produce an observed metabolic phenotype, then sequentially—and quantitatively—determine the
relative contributions of the four major mechanisms of energy balance to this total. Herein we propose that the
use of metabolic caging systems and bomb calorimetry can quantify the contributions of food intake behavior
and digestive efficiency (and in combination, caloric intake); if these mechanisms account for essentially all of
the total change in energy flux, then the investigator can conclude that caloric intake changes explain the
overall phenotype. If these mechanisms do not quantitatively account for the total energy change, then subse-
quent studies to explore resting metabolic rate (RMR) are required. Only after an investigator exhausts meth-
ods to assess RMR can changes in physical activity be quantitatively determined. Qualitative methods to
assess physical activity are then recommended to complement the preceding quantitative methods
126 Justin L. Grobe
of energy balance in laboratory mice. Similar workflows could be

adapted for any species of interest, but equipment descriptions
herein are tailored for use with mice.
2 Materials
2.1 Metabolic One common type of metabolic caging system for use with mice is the
Caging Systems Nalgene-style single-mouse sized metabolic cage. These cages are
designed to be used with a single mouse per cage, and are treated with
special coatings to maximize urine collection efficiency. Great care
must be taken when washing the cages to prevent loss of the coatings
by using specific soaps as recommended by the manufacturer.
Our group has frequently utilized Techniplast USA model
3600M021 to study the effect of manipulations of the RAS upon
metabolic and fluid balance physiology [3–8] (Fig. 2). These cages
have been modified to replace the standard water bottle with two
50 mL burets, to allow for two-bottle choice testing.
Fig. 2 Illustration of one type of mouse-sized metabolic cage. A single mouse-

sized metabolic cage (Techniplast USA model 3600 M021 shown) typically
includes a detachable food hopper, water bottle, urine and fecal collection tubes.
Cages can be modified (as shown) to provide multiple drink tubes, to permit two-
bottle choice tests
2.2 Bomb A bomb calorimeter of appropriate size must be selected. One

Calorimeter commonly used design is a 25–200 mg bomb (e.g., Parr Instrument
Company, model 6725). Importantly the investigator will need a
pellet press to uniformly prepare samples for use in the bomb (e.g.,
Parr Instrument Company, model 2817). We have used these
models of equipment to examine digestive efficiency and energy
efficiency in animals with manipulations of mitochondrial function
and the gut microbiome [9, 10] (Fig. 3).
2.3 Body Body composition analyzers are typically far beyond the budget of
Composition Analyzer an individual laboratory, and are more typically offered through an
institutional core facility. It is important to know what equipment is
available in the facility, however, as occasionally the resolution of a
given scanner (e.g., a rat-sized scanner) is insufficient to accurately
assess body composition of smaller species (e.g., mice). We have
used NMR and MRI scanners within core facilities at the University
of Iowa in a wide array of studies of energy balance [3, 8, 10–12].
Protocols for using such equipment will be provided by core facility
staff and are beyond the scope of this chapter. These analyzers typi-
cally provide bodily composition of fat mass, lean mass, and water.
Fig. 3 Illustration of a semi-micro bomb calorimetry system. A 25–200 mg bomb

calorimeter system (Parr model 6725 shown) consists of a bomb that is loaded
with the sample pellets (prepared by a press as shown), which is then submerged
in a specific volume of water within a vacuum-lined flask. The flask is then placed
into the mixing chamber that includes a high-resolution thermometer in the water
bath. Systems such as the Parr model 6725 perform scripted analysis runs
(roughly 20 min per sample) and also log and analyze data for the user
128 Justin L. Grobe
2.4 Respirometry Gas analyzer equipment is available from a long list of manufactur-
Equipment ers, including AEI Technologies, Sable Systems, and Columbus
Instruments. It is again important to evaluate the specifications of
the equipment under comparison based on the resolution of the
analyzers for the species of animal that will be studied. Equipment to
correct air flow rates to standard temperature and pressure is avail-
able from many commercial sources as well. Some manufacturers
offer equipment to record data, while others require that you inter-
face these systems with third-party chart recorder equipment (e.g.,
ADInstruments). Finally, housing chambers can be obtained from
some manufacturers, or custom-built. Investigators who wish to be
able to expose animals to a range of ambient temperatures may con-
sider the use of custom-modified water-jacketed chambers coupled
to circulating heating/cooling water baths to provide such options.
We have described a push-pull respirometry array using AEI
gas analyzers and custom thermally controlled chambers (ACE
Glass, Vineyard, NJ) in multiple recent publications [3, 5, 7, 8, 13,
14] (Fig. 4).
2.5 Comprehensive Several companies offer “comprehensive” phenotyping equipment

Phenotyping Systems (e.g., “OxyMax/CLAMS” from Columbus Instruments,
“Promethion” from Sable Systems, “PhenoMaster” from TSE).
These combinatorial systems allow simultaneous assessment of food
intake, respiratory gas exchange, physical activity by beam break,
and core temperature via implanted telemeters. Such systems offer
repeated, longitudinal assessments of various phenotypes through-
out the circadian cycle but generally fail to include mechanisms to
assess all aspects of energy balance by ignoring digestive efficiency
and anaerobic metabolism. Using an OxyMax/CLAMS system
available through core facilities at the University of Iowa, we have
published studies using this system to investigate the effects of
dietary sodium and the RAS upon energy balance [8].
2.6 Direct There are currently no commercial sources of direct calorimetry

Calorimetry Equipment equipment for use with rodents for biomedical research. Various
groups have described the custom fabrication of direct calorimetry
chambers and coupling these chambers to respirometry equipment
to generate “total” or “combined” calorimeter equipment [11, 12,
15–19]. While the adoption of such equipment by the field is very
strongly recommended, the fabrication, optimization, validation
and use of such equipment is well beyond the current article. Any
laboratory planning to develop such equipment is strongly advised
to employ a professional electrical and biomedical engineer.
The combined calorimeter system currently operational in our
laboratory (Fig. 5), which was custom-fabricated by Colin
M.L. Burnett, MD, MS, is thoroughly described in several recent
publications [8, 10–12]. Briefly, conditioned air (see Note 1) is
analyzed for flow, temperature, humidity, and pressure to calculate
the enthalpy of air entering the chamber. Heat dissipated by the
Fig. 4 Illustration of a simple respirometry system. (a) A custom-fabricated push-pull continuous respirometry
system such as currently in use in our laboratory is illustrated. Pressurized air from a standard laboratory sup-
ply line is passed through a humidity and temperature regulator system (explained more fully in Fig. 5), and
conditioned air is then passed at ~300 mL/min STP(Standard Temperature and Pressure) into a water-jacketed
animal chamber. The animal chamber (a 2-L custom-modified chamber from ACE Glass, Vineland, NJ) is main-
tained at 30 °C by constant water jacket perfusion via a heated circulating water bath (not shown). Animals
are placed into the chamber atop a plastic pedestal (to negate behavioral consequences of placing the ani-
mal’s thermally sensitive paws upon the heated glass). Air exits the chamber via a port drilled through a tight-
fitting rubber stopper that is seated in the mouth of the beaker. Effluent air is then subsampled by a respirometry
system, including a desiccant column (anhydrous calcium sulfate/Drierite), a CO2 analyzer, O2 analyzer, and
finally the air pump that pulls subsampled air through the system. Data from the analyzers is then logged on
a chart recorder. (b) A photograph of a four-chamber continuous respirometry system currently in use in our
laboratory
subject passes through thermopiles that line the interior walls of a

water-jacketed chamber, which generates a proportional voltage
that is recorded. Flow, temperature, humidity, and pressure are
then again measured in effluent air to determine its enthalpy. In
addition, core body temperature is recorded using surgically
implanted telemeters and a custom miniaturized receiver antenna
inside the chamber (Data Sciences International). Effluent air is
then subsampled (i.e., flow is less than through the chamber) by a
standard respirometry system as above.
130 Justin L. Grobe
Fig. 5 Illustration of a combined/total calorimeter. (a) A custom-fabricated combined calorimeter system is

illustrated. First, pressurized air (of uncontrolled humidity and temperature) from a standard laboratory supply
line is passed through a humidity and temperature regulator system to supply a constant stream of 30 °C/~35%
RH/300 mL/min STP (Standard Temperature and Pressure) air to the chamber (see Note 1). Enthalpy of the
influent and effluent air streams is calculated by measuring flow, temperature, humidity, and pressure. Heat
radiated into the walls of the chamber is determined from the voltage generated by thermopiles that line the
interior walls of the water-jacketed chamber. Core body temperature is recorded continuously by radiotelemet-
ric probe. Effluent air is then passed to a respirometry system, as above. (b) A photograph of the humidity and
temperature regulator system currently in use in our laboratory. (c) A photograph of the gradient-layer direct
calorimetry chamber currently in use in our laboratory
3 Methods
3.1 Method 1: When an experimental manipulation results in a change in body

Metabolic Caging mass, one simple and superior method to investigate energy bal-
Systems ance is to use metabolic caging systems to quantitatively assess
food intake. While some investigators prefer to examine food
intake behavior in home cages to minimize behavioral interrup-
tions due to neophobia, it is virtually impossible to quantify food
intake with high resolution in this setting due to the loss of spilled
food dust into bedding materials. Subsequently, food intake in
home cages is often reported at higher rates than in metabolic cag-
ing systems, but the relative contribution of neophobia-induced
anorexia versus food spillage to this difference is impossible to dis-
tinguish. Further, mice often consume bedding materials and
therefore the caloric content of this additional “food” source is
impossible to quantify. (Also, regardless of the caloric value of the
bedding material, consumption of that material may also have
unexpected consequences on the gut microbiome and thereby
energy balance control.) Therefore, we prefer to consistently use
metabolic caging systems, but acknowledge the need for explicit
training of the animals to minimize the influence of neophobia
upon food intake behavior.
Metabolic caging systems (whether commercial or custom-
fabricated) typically include a food hopper and fluid delivery system
that minimizes spillage of food and drink solutions, and a wire mesh
or metal grate floor which allows for efficient and quantitative col-
lection of both urine and fecal samples. Animals should be placed
into caging systems for several days to acclimate (see Note 2) before
measurements are performed. Following acclimation, the investiga-
tor can measure food intake (mass change of food hopper) and fecal
output (mass change of fecal collection tube) daily for several con-
secutive days. In our laboratory, we typically use at least 48–72 h of
acclimation, as our previous studies have demonstrated the instabil-
ity of body mass, food intake, and feces production when animals
are acclimated for shorter periods (Fig. 6).
On a daily basis the investigator should record the animal’s
body mass, the end masses/volumes for food, water, urine, and
feces. Then after resetting the cage, tare masses/volumes for food,
water, urine, and feces should again be recorded. Date, time of
cage servicing, room temperature and humidity, and operator are
also critical information to record for future reference (see Table 2
for an example data recording sheet; notably on day 0 there will be
no “end” masses/volumes, and on the last day of study there will
be no “tare” values). Seasonal variations in ambient temperature
and humidity can have major effects on fluid intake behavior and
urine production in addition to the rates of evaporation of urine
from the collection tube. Evaporation rates can either be
132 Justin L. Grobe
Fig. 6 Example empirical determination of required acclimation time for metabolic caging systems. It is neces-
sary for investigators to empirically determine the requisite acclimation phase for animals in metabolic cages,
as acclimation needs can wildly vary based on an array of environmental and animal-specific factors. In this
example, double-transgenic “sRA” mice, which exhibit brain-specific hyperactivity of the RAS [3, 6, 34] and their
phenotypically normal single- and non-transgenic littermates, were placed into Nalgene-style single-mouse
metabolic cages for seven consecutive days and allowed ad libitum access to chow and water. *P < 0.05 vs
control at indicated timepoint by ANOVA followed by Bonferroni multiple-comparisons procedure
Table 2
Example daily log sheet for metabolic cage studies
Date: Technician: Room Temperature:
Time: Room Humidity:
End End Water End End Tare Tare Water Tare Tare
Cage # Animal Body (g) Food (g) (g or mL) Urine (g) Feces (g) Food (g) (g or mL) Urine (g) Feces (g)
determined by daily recordings of the mass of water lost from a

water-filled tube kept in the animal room, or controlled by placing
a small amount of oil in the urine collection tube to “trap” the
aqueous urine layer and prevent its evaporation.
Using metabolic caging systems or home cage systems, the
investigator can immediately calculate food intake behavior from
these data. Using the caloric density of the food (which can be
found on the dietary information specifications supplied by the
manufacturer), the investigator can then easily calculate the total

calories consumed daily by the animal per day (Eq. 1):
[Total calories consumed daily ] = [ Mass of food consumed ] ´ [Caloric density of food ] (1)
Using bomb calorimetry methods detailed below, we have

determined that the batch-to-batch variability of caloric density of
standard chow and high-fat diets from various manufacturers is
appreciable. Thus, while the use of a published caloric density is a
good starting point, we recommend the routine analysis of the
caloric density of individual batches of food for quantitative studies
of energy balance using bomb calorimetric methods.
3.2 Method 2: Bomb By carefully and analytically collecting the feces produced by an
Calorimetry animal per unit time, the investigator can then perform a series of
tests to qualitatively or quantitatively assess digestive efficiency and
caloric absorption. Various methods including fecal acid steatocrit
(see Note 3) or other metabolomics methods can be employed to
qualitatively determine whether animals are exhibiting changes in
the absorbance rates of fatty acids or other metabolites. To quanti-
tatively assess digestive efficiency and caloric absorption, however,
bomb calorimetry is the gold standard. Its less-than-universal use
in animal physiology laboratories may stem from the perceived
complexity of this physical/analytical chemistry method, or simply
from unpalatable but necessary handling and use of feces.
Bomb calorimetry is a methodology that is easily adaptable for
use in physiological studies. In essence, this method allows for the
determination of the caloric density of a sample material (see Note 4).
After an investigator quantitatively collects feces from study animals
via metabolic caging systems (this is extremely difficult to do if the
investigator uses home cages), the fecal samples are desiccated. It is
critical to calculate the “wet” mass of the feces before desiccation and
the “dry” mass after drying. This allows the investigator to calculate
the water content of the feces, which may be an endpoint of interest
in its own right. Further, it is necessary to know the contribution of
“dry” mass to “wet” mass to ultimately calculate digestive efficiency.
Desiccated fecal samples are then pressed into pellets, and the pellet
mass is carefully determined. It is important to use high-resolution
balances for all steps, as significant digit and error propagation issues
can become unwieldy after sequential mathematical calculations.
Further, the use of an appropriately sized bomb calorimeter is
required (see Note 5).
Fecal samples are then burned to completion in the bomb
calorimeter according to the protocol of the individual calorim-
eter. Typically, the sample is loaded into the bomb along with
fuse wire, the bomb is pressurized with pure oxygen, and the
bomb is submerged into a known volume of water within a well-
insulated container. A very high-resolution thermometer is placed
134 Justin L. Grobe
into the water bath, and a water circulating system (such as a

paddle wheel or turbine) is submerged and activated. After a suit-
able thermal stabilization phase, which is subject to ambient con-
ditions and the material composition of various equipment
components involved, an electrical current is passed through the
bomb’s fuse wire and the fecal sample is rapidly combusted to
completion. The energy from the combustion is transferred into
the water bath, and the resulting temperature change in the water
is noted by the thermometer. Having previously calibrated the
thermometer system by combusting known masses of pure fuels
(e.g., benzoic acid) according to bomb manufacturer’s protocol,
the user can then calculate the caloric release from the feces.
Using that value and the known mass of the fecal pellet that was
burned, the investigator can determine the caloric density of the
desiccated fecal sample (Eq. 2):

[Energy released from fecal pellet ]
[Caloric density of desiccated feces ] = (2)
[Mass of fecal pellet ]
Once the investigator has determined the caloric density of a
desiccated fecal sample, he or she can then calculate the caloric
density of the original fecal sample (Eq. 3):

[Caloric density of original sample ] = [Caloric density of desiccated feces ]
é [ Mass of dry feces ] ù
´ê ú (3)
êë [ Mass of wet feces ] úû
Once the caloric density of the original sample has been deter-
mined, it is simple to calculate the total daily caloric loss to feces
(Eq. 4):
[Total daily caloric loss to feces ] = [ Mass of wet feces ] ´ [Caloric density of original sample ] (4)
Next, the investigator can then calculate total daily caloric

absorption using the total calories consumed daily (Eq. 1) and
the total daily caloric loss to feces (Eq. 4) by simple subtraction
(Eq. 5):
[Total daily caloric absorption ] = [Total calories consumed daily ]

- [Total daily caloric loss to feces ] (5)

Total daily caloric absorption is an important value to consider
by itself, and the investigator should consider this value as more
important to total energy balance than the calories consumed, as
ultimately the net energy balance is subject to the balance between

calories absorbed versus expended.
In addition to total caloric absorption, bomb calorimetry data
can be used to calculate digestive efficiency. Digestive efficiency is
subject to many different biological mechanisms (mechanics of the
gastrointestinal tract, hepatic and pancreatic function, disease
states, etc.), but from an energetics perspective these individual
mechanisms are only of interest if there is a change in net absorp-
tion efficiency. Digestive efficiency simply reflects the fraction of
calories absorbed from total calories consumed (Eq. 6):
[Total daily caloric absorption ]
[Digestive efficiency ] = (6)
[Total calories consumed daily ]

Finally, the results of bomb calorimetry can also be used to
estimate the relative importance of changes in energy intake versus
output in observed changes in body mass. Energy efficiency is a
term for the amount of weight gain exhibited by an animal per
calorie absorbed over a given period of time (Eq. 7):
[Change in body mass per unit time ]
[Energy efficiency ] = (7)
[Total calories absorbed per unit time ]
Energy efficiency is typically calculated for each of several treat-
ment groups that were matched under baseline conditions (such as
before initiation of an experimental diet). For example, after treat-
ing two groups of mice with chow or a 45% high-fat diet for
28 days, bomb calorimetry is used to calculate the daily caloric
absorption of each animal. For each mouse, energy efficiency is
calculated as the weight gained over the 28 days, divided by the
total calories absorbed over the 28 days (i.e., the daily absorption
× 28 days). A positive deflection is expected in the mice fed the
high-fat diet, which would be interpreted as an increase in weight
gained for any individual calorie that was absorbed. If the two
groups of animals exhibited similar total daily caloric absorption
but differences in energy efficiency (e.g., increased with high-fat
feeding), then the investigator can conclude that the increased
weight gain is due primarily to a reduction in energy expenditure.
While the conclusion may seem obvious (e.g., the same calories
were absorbed, but one group got larger), the use of this method-
ology can quantify the effect over multiple time points, which can
be used to illustrate the effect of serial treatments or compensatory
changes in the animal groups. Further, this methodology allows
the investigator to mathematically describe the relationship.
3.3 Method 3: Body Many laboratories now have access to core facilities that are
Composition equipped with sophisticated body composition analyzers. These
analyzers come in multiple forms, including dual-energy X-ray
136 Justin L. Grobe
absorptiometry (DEXA), nuclear magnetic resonance (NMR), or

magnetic resonance imaging (MRI), and each type of technology
sports relative benefits and trade-offs. DEXA scanners use X-ray
technology to assess lean, fat, and bone masses, and can be used
with animals that have metal implants such as implanted mini-
pumps for drug delivery. NMR equipment provides rapid and
highly quantitative assessments of lean, fat, and fluid masses,
though animals instrumented with metal implants cannot be ana-
lyzed in these systems. MRI equipment uses essentially the same
methodology as NMR equipment, but is more commonly used for
providing images to illustrate body composition rather than simply
quantifying body composition. Regardless of method, animals
must be either anesthetized or restrained to prevent movement
during scanning.
Use of these various methods can provide substantial insight
into the effects of treatments upon the subject, as serial, longitudi-
nal measures within an individual can be performed (i.e., baseline
and post-treatment values within subject can be easily captured and
compared). In addition, data from these types of analyses can be
used in conjunction with bomb calorimetry methods to further
estimate the contribution of various energy-balance mechanisms to
weight gain. For example, lean tissue is generally estimated to have
a caloric value of approximately 4 kcal/g, while fat tissue is esti-
mated to have a caloric value of 9 kcal/g. If an animal gains 1 g
each of fat and lean tissues, then the investigator is left to account
for 13 kcal of increased energy intake/absorption or reduced
energy expenditure. If the animal absorbed an additional 10 kcal
during the experimental period, then the investigator can roughly
estimate that 77% of the extra weight gain was a result of increased
caloric absorption (i.e., 10 kcal absorbed, divided by 13 kcal total).
By extension, 23% of the extra weight gain (3 of the 13 kcal) likely
resulted from reduced energy expenditure.
3.4 Method 4: One of the mechanisms of energy expenditure available to any ani-
Physical Activity mal is spontaneous physical activity, though physical activity for a
Methods typical human accounts for only 0–40% of total daily energy expen-
diture. Various methods have been developed to estimate physical
activity in rodents, including systems which quantify distance trav-
elled by photoelectric beam interruption (“beam breaks”), running
wheel revolutions, and systems that track proximity between radio-
telemetric transmitter implants and stationary receiver antennas.
It is critical to appreciate that all methods used to assess the
caloric value of physical activity are qualitative. This is due to the
wide array of variables that contribute to the energy equivalency of
any given physical motion. As a result, the relationship between
recorded motion by an animal and the caloric expenditure is of a
multivariate form. For example, if a human weighing 70 kg walks
up two flights of stairs and a 140 kg human walks up one flight of
stairs, it is essentially impossible to calculate how much energy was

spent by each subject, and which subject spent more energy. Body
composition, exercise conditioning and capacity, muscle efficiency,
blood chemistry, feeding status, disease state, height, altitude,
ambient temperature, relative humidity, etc. all contribute to the
final energy expenditure required by each subject to perform a
given motion. Thus, while the above systems can provide critical
qualitative insights into circadian rhythms of activity or gross
changes in total activity between treatment groups, it remains
impossible to calculate energetic equivalents of these motions.
Instead, caloric equivalents of any given amount of physical activity
are instead typically calculated as the remaining balance between
total calories expended (calculated by bomb calorimetry) and calories
expended via resting metabolism (Fig. 1).
3.5 Method 5: Resting metabolic rate (RMR) refers to the energy expended by a
Aerobic Resting body when resting. Typically this refers to an animal that is station-
Metabolism ary, awake, and calm but not sleeping, though it is difficult to dis-
Method— sociate “resting” from “sleeping” bouts in rodent species. RMR
Respirometry typically accounts for 60–100% of total energy expenditure, and it
is often assessed using respirometric methods due to the availability
of high-quality “turn-key” commercial systems [20–22].
Respirometry is one form of “indirect calorimetry,” which is a
catch-all term describing a collection of methods that estimate met-
abolic rate by measuring some simple surrogate. For example,
“indirect calorimetry” methods also include methods such as iso-
tope dilution. In the case of respirometry, metabolic rate is esti-
mated by measuring respiratory gas exchange. As early as 1928,
Lusk published a table of coefficients that related the amount of
heat generated per volume oxygen consumed by a combustion
reaction of mixtures of fats and carbohydrates [23]. (Popularized by
Lusk, this table represented a corrected version of such a table pub-
lished by Zuntz and Schumburgin [24].) Relatively simple algebraic
regressions of these interpolated data lead to an equation to relate
rates of oxygen consumption (VO2) and carbon dioxide production
(VCO2) to heat production [20, 21], which are calculated from the
changes in air O2 and CO2 content as it passes a live animal (with
flow rates measured and corrected for standard temperature and
pressure, STP), and many investigators and commercial equipment
use this equation to estimate metabolic rate (Eqs. 8–10):
[VO 2 ] = [ DO 2 %] ´ [STP - corrected chamber air flow ]

(8)
[ VCO 2 ] = [ DCO 2 %] ´ [STP - corrected chamber air flow ] (9)

[Metabolic rate ] = 3.815 ´ [ VO2 ] + 1.232 ´ [VCO2 ] (10)

138 Justin L. Grobe
This equation and other similar equations are used by virtually

every commercial respirometry-based system, be it a stand-alone
system or part of a comprehensive monitoring system.
Respirometry systems can be in many forms [20] (see Note 6),
but many investigators utilize “push-pull” design systems, where
air of known composition is “pushed” through an air-tight testing
chamber, and effluent air is “pulled” into a subsampling system
including a desiccant system to dry the air, then through oxygen
and carbon dioxide analyzers. Testing chambers can be maintained
at any desired temperature, though assessments of resting metabo-
lism are typically performed at thermoneutral temperatures (e.g.,
~30 °C for mice [25]) both to promote “resting” behavior and
also to minimize the activation of adaptive thermoregulatary
mechanisms (e.g., sweating, licking, panting, shivering, or non-
shivering thermogenesis). Various publications have demonstrated
that the thermoneutral zone for wild-type C57BL/6 mice is
between 26 and 34 °C [25], and therefore most investigators use
an ambient/chamber temperature of 30 °C for measurements of
RMR. Although perhaps not immediately obvious, it is also impor-
tant that air flow through such systems must be corrected to
Standard Temperature and Pressure (STP) for calculations of met-
abolic rate, and any custom-fabricated systems must be designed to
make this correction. This is necessary because an air flow of, say,
100 mL per hour will not represent the same mass of air if it were
at a different temperature or pressure.
RMR measures must be taken while animals are “resting.”
Investigators can ensure that animals are resting via visual inspection
(either in person or via video camera so as to not disturb the resting
animal), or more simply by tracking oxygen consumption and
carbon dioxide production rates in real-time. Whereas awake and
moving animals exhibit highly variable second-to-second rates,
resting/sleeping animals will achieve a low, steady plateau of rates
that are easily noted in strip charts or electronic equivalents (Fig. 7).
Such assessments are only possible if investigators utilize ‘continuous’
measurement methods; systems with discontinuous measures are not
favorable for precise assesments of RMR.
3.6 Method 6: Total It is very important to note that by definition, respirometry is lim-
Resting Metabolism ited to assessments of oxygen-dependent (i.e., aerobic) metabo-
Method—Direct lism. In fact, the use of respirometry relies upon several untested/
Calorimetry untestable assumptions which most users ignore [22]. Growing
evidence now supports the conclusion that the tacit assumption
that anaerobic processes are both negligible and constant is simply
wrong. Our group and others have demonstrated that anaerobic
processes appear to contribute large, variable fractions of total
RMR in various species using “combined calorimetry” or “total
calorimetry” methods, which are described below. Walsberg and
Hoffman [17, 18] demonstrated that anaerobic processes contribute
0–40% of total RMR in snakes, birds, and kangaroo rats. Pittet et al.
Fig. 7 Example real-time tracing of oxygen consumption and carbon dioxide production by a mouse. A typical
recording session of a mouse placed into a (continuous) push-pull respirometer system, with chamber tempera-
ture maintained at 30 °C and with air flowing through the chamber at ~300 mL/min STP (Standard Temperature
and Pressure). The oxygen consumption rate (ΔO2%; reflected in a downward deflection in effluent O2 content)
and carbon dioxide production rate (ΔCO2%; reflected in an upward deflection in effluent CO2 content) can be
determined both during bouts of wakefulness (Awake) and during periods of rest (Resting). Periods of rest are
easily identified in tracings of effluent air composition as periods of steady, minimum-value plateaus in both
ΔO2% and ΔCO2% endpoints. Aerobic metabolic rate is then calculated using Eqs. 8–10, and aerobic RMR
simply reflects the output of these calculations when inputting ΔO2% and ΔCO2% values recorded during peri-
ods of rest
[15, 16] demonstrated that in lean humans, anaerobic processes

contribute nearly 10% of total RMR, while they contribute essen-
tially 0% in obese humans. Van Breukelen’s group has also started
investigating these processes in desert and aquatic species and in
the context of torpor [19, 26–29]. Our group has determined that
in laboratory mice, anaerobic RMR contributes around 10% of
total RMR, and that this contribution can be suppressed or stimu-
lated by various genetic (e.g., disruption of the angiotensin AT2
receptor), pharmacological (anesthesia), dietary (high-fat diet), and
microbial (risperidone) interventions [8, 10–12]. Thus, there is a
major need for the field to adopt methods that can measure the
140 Justin L. Grobe
entire range of processes (aerobic and anaerobic) contributing to

RMR [22, 30].
At the chemical level, metabolic rate is the rate of heat produc-
tion. In steady-state, the rate of heat production by an animal is
equivalent to the rate of heat release from the body. Thus, “direct”
calorimetry describes methods that measure heat dissipation to cal-
culate metabolic rate. There are currently no commercial sources
for direct calorimetry systems for rodents, though several groups
have described simplified [19] and more elaborate [11, 22] sys-
tems with a range of capabilities and resolutions which can be used
for rodents of various sizes. Critically, the investigator must insert/
implant a core temperature probe within the animal to either
ensure that measurements are performed while the animal is at
steady state (indicated by a stable core temperature) or to correct
direct calorimeter data for the rate of core temperature change,
using complicated equations that depend upon knowledge of the
subject’s exact body composition [31].
As air must be fluxed through the chamber of a direct calorim-
eter for the animal to survive, it is straightforward to simply sub-
sample effluent air from a direct calorimeter and perform
respirometric measurements to simultaneously measure aerobic
metabolic rate. Using such a “combined calorimetry” or “total
calorimetry” setup, the investigator can therefore measure total
metabolic rate (via direct calorimetry) and aerobic metabolic rate
(via respirometry). The difference between these methods, there-
fore, represents an index of the anaerobic metabolic rate.
Use of a combined calorimeter is essentially identical to the use
of a respirometer system. The testing chamber is maintained at an
experimentally dictated temperature (typically, thermoneutrality),
and the heat production/release within the box and the composition
and flow rate of the air fluxing through the box are determined
while the animal is in the box and at rest, compared to when the
animal is not in the box.
3.7 Method 7: As described in the above sections, quantitative evaluations of

Putting It All Together energy flux in a mouse (or any animal subject) can be efficiently
performed using metabolic caging systems and a bomb calorime-
ter. Additional dissection of the contribution of resting metabolic
processes versus physical activity requires far more sophisticated
methodologies that are not as commonly (or inexpensively) avail-
able. By multiplexing these various methods, however, it is possible
to comprehensively evaluate all aspects of energy balance in the
animal, and to specifically attribute the relative importance of indi-
vidual mechanisms to the net changes in body mass (Fig. 1).
Metabolic caging and bomb calorimetry allows the investiga-
tor to evaluate caloric intake, absorption, digestive efficiency, and
energy efficiency. These methods directly address the contributions
of food intake and gastrointestinal function to energy balance. By
additionally utilizing body composition analysis methods, investi-

gators can illustrate the relative importance of observed processes
to weight gain/loss, compared to what is expected based on
changes in body composition. By using quantitative measures of
resting metabolism (both aerobic and anaerobic), investigators can
then determine the contributions of these processes to total energy
balance, and finally by simple subtraction, remaining calories are
most easily ascribed to physical activity. Such designations can be
complemented/confirmed by qualitative methods of assessing
physical activity behavior.
3.8 Method 8: Substantial discussion has raged in the literature over the last
Statistical decade with regard to the most appropriate way to compare meta-
Considerations bolic phenotyping data across subjects of differing body size/body
mass/body composition. Of course a larger animal would be
expected to eat more food, but documenting a larger food intake
in a larger animal does not necessarily indicate that the greater food
intake preceded or caused the additional weight gain. As explained
in other recent review articles, investigators often normalize data
by simply dividing food intake or RMR data by body mass, but
from both mathematical and biological perspectives this approach
is untenable. Instead, more reasonable solutions to this problem
have been proposed to include either simply reporting all of the
data (such that the reader is left to draw their own conclusions), or
to account for differences in subject body mass using the ANCOVA
statistical correction method [32].
As an oversimplification, ANCOVA analyses involve performing
linear regressions of metabolic rate (or food intake etc.) versus body
mass data pairs. One assumes (and tests) that the slopes of the lines
which best describe data from each treatment group are indistin-
guishable, and then a comparison of the two groups at a single aver-
age value for body mass is made (Fig. 8). Statistical packages are
available commercially which can perform one-way ANCOVA, or
various online calculators for free (see Note 7). Univariate regression
modeling must be used if more than one variable is considered.
4 Notes
1. To control ambient air temperature and humidity for subject

animals in calorimeter systems throughout various seasons, a
simple system that controls the dew point of the air stream can
be used. Water is bubbled through room-temperature water
to saturate the air (i.e., 100% relative humidity, RH). This air
is then passed through condenser columns maintained by a
chilled circulating water bath, thereby “setting” the dew point
of the air stream. Warming this air maintains the quantity of
142 Justin L. Grobe
Fig. 8 Illustration of an ANCOVA analysis. (a) A simple comparison between two different groups indicates no
difference in mean aerobic RMR between groups. (b) Comparison of body masses between groups uncovers
a difference between groups, necessitating the reanalysis of RMR data to account for body size. (c) Metabolic
rate values are plotted against the body mass of individual animals within each group, and linear regressions
are performed for each group. Tests of homogeneity of regression are performed to confirm that the slopes of
lines through each group are indistinguishable. (In this case the lines are indistinguishable, P = 0.96, so we
can proceed with ANCOVA analyses). (d) The interpolated average RMR value (intercept) at the average body
mass value for all animals in the study (21.97 g) is then reported as the “ANCOVA-corrected” RMR value
water vapor and reduces the relative humidity. For example,

setting the chilled water to 10 °C and warming to 30 °C
results in a relative humidity of about 35%. Air flow through
this conditioner system can be controlled by modulating the
pressure supplied to the system.
2. Because of the neophobia exhibited by rodents, animals will
often exhibit abnormal behaviors (and by extension, energy
balance) for between 1 and 7 days when placed into metabolic
caging systems. Users should empirically determine the accli-
mation period required for animals in their laboratory, as accli-
mation needs can differ depending upon the animal strain and
the complexity of the caging system. For example, in our labo-
ratory we have repeatedly determined that food and water
intake, urine and fecal production all drop over the first
24–96 h when C57BL/6J mice and transgenic mice on this
background are placed into Nalgene-style single-mouse sized
metabolic cages when housed at 22–25 °C with 30–70% rela-
tive humidity, but that intake and output behaviors rebound
and achieve a steady plateau after 72–96 h (Fig. 5). In contrast,
other laboratories insist upon (based on various levels and
types of evidence) waiting up to 7 days of acclimation. We
advocate empirical determinations in each laboratory to gain
confidence in one’s own data.
3. Fatty acid content of feces can be assessed using a very simple
biochemical method [8, 33]. Briefly, a known mass of desiccated
feces (e.g., 50 mg) is reconstituted in freshly prepared perchloric

acid (200 μL of 1 N). After reconstitution, a small amount of
oil-red-O stain (100 μL of 0.5%) is then added to the slurry. The
slurry is then loaded in a capillary tube and centrifuged. The
layer stained with oil-red-O is then compared (as a percentage)
against the entire column in a very similar method to determin-
ing the hematocrit of blood. Relative increases or decreases in
the contribution of this layer to the total column height reflect
decreases or increases, respectively, in fatty acid absorption effi-
ciency. That is, an increase in steatocrit means a decrease in
absorption. This qualitative method provides some insight into
digestive efficiency, but because it is impossible to determine the
caloric value of the fatty acid species present and the value of the
other, nonfat components in the feces, this method often serves
as only a screening tool or qualitative complement to bomb
calorimetric methods. Notably, we have found that when ani-
mals are maintained on a chow diet the fatty acid content of the
stool is very low and it is difficult to discern changes by this
method. In contrast, when animals are maintained on high-fat
diets (45 or 60% kcal from fat), changes in fatty acid absorption
are easy to detect by this method.
4. It is worth noting that analyses of caloric density using a bomb
calorimeter ignore the bioavailability of calories in a sample. For
example, a chow-based diet typically contains some amount of
undigestible fiber, yet combustion of that food sample in a bomb
calorimeter will yield a caloric density including calories from
both the digestible and undigestible components. Ultimately if
calculations are performed correctly this will not matter as the
same undigestible materials are represented in feces, and the
same constant value is present in both the consumed and fecal
masses. Subtraction of fecal calories from consumed calories
(Eq. 5) therefore yields the same value for total daily caloric
absorption as this constant is subtracted from itself.
5. A single mouse can be expected to reliably produce ~100 mg of
desiccated fecal sample per day, so use of an appropriately sized
bomb is necessary. For example, use of a bomb which requires
1 g of sample would necessitate combining fecal samples from
10 individual mice or serially collecting fecal samples from a
single mouse for 10 consecutive days. In contrast, a bomb
which requires 25–200 mg of sample is suitable to analyze
samples from single-day collections from individual mice.
6. Many multichamber metabolic monitoring systems are avail-
able commercially through an array of companies including
Columbus Instruments, Sable Systems, and TSE. Some of
these systems make use of continuous-recording respirometer
arrays in which each cage has a dedicated pair of oxygen and
carbon dioxide analyzers continuously analyzing effluent air;
such systems are analogous to the system described herein.
144 Justin L. Grobe
In contrast, some systems keep costs down by using an auto-

mated sampling manifold such that one single analyzer pair is
used for a number of chambers. This second, discontinuous-
type system samples effluent air from each of several cages
for a few seconds every few minutes to provide a survey of
metabolic rate values across time. As a result, the addition of
more chambers to such a discontinuous system reduces the
sampling frequency. As sampling time points are separated, it
becomes increasingly difficult to interpret bouts of rest.
Therefore for assessments specifically of RMR (as opposed to
simply metabolic rate in general), we have had much greater
success using a continuous-sampling system as illustrated in
Fig. 5.
7. One free source for one-way ANCOVA analyses can be found
at: http://vassarstats.net/. It is important to note that if more
than one independent variable is in play, that univariate regres-
sion methods must be used instead of the simple ANCOVA
method.
Acknowledgments
The Grobe laboratory is supported by grants from the NIH

(HL134850, HL084207), the American Diabetes Association (1-14-
BS-079), the American Heart Association (15SFRN23730000), the
University of Iowa Office of the Vice President for Research and
Economic Development, the Fraternal Order of Eagles’ Diabetes
Research Center, and the UIHC Center for Hypertension Research.
Some of the equipment illustrated was purchased with support from
the Roy J. Carver Trust. Connie C. Grobe, PhD, Jeremy A. Sandgren,
John R. Kirby, PhD, and Colin M.L. Burnett, MD, MS provided criti-
cal revisions of the chapter text.
References
1. Littlejohn NK, Grobe JL (2015) Opposing AL, Johnson AK, Sigmund CD (2010) The
tissue-specific roles of angiotensin in the patho- brain renin-angiotensin system controls diver-
genesis of obesity, and implications for obesity- gent efferent mechanisms to regulate fluid and
related hypertension. Am J Physiol Regul energy balance. Cell Metab 12(5):431–442.
Integr Comp Physiol 309(12):R1463–R1473. doi:10.1016/j.cmet.2010.09.011
doi:10.1152/ajpregu.00224.2015 4. Grobe JL, Dickson ME, Park S, Davis
2. Hall KD, Sacks G, Chandramohan D, DR, Born EJ, Sigmund CD (2010)
Chow CC, Wang YC, Gortmaker SL, Cardiovascular consequences of genetic varia-
Swinburn BA (2011) Quantification of the tion at −6/235 in human angiotensinogen
effect of energy imbalance on bodyweight. using "humanized" gene-targeted mice.
Lancet 378(9793):826–837. doi:10.1016/ Hypertension 56(5):981–987. doi:10.1161/
s0140-6736(11)60812-x hypertensionaha.110.157354
3. Grobe JL, Grobe CL, Beltz TG, Westphal SG, 5. Grobe JL, Buehrer BA, Hilzendeger AM,
Morgan DA, Xu D, de Lange WJ, Li H, Sakai Liu X, Davis DR, Xu D, Sigmund CD
K, Thedens DR, Cassis LA, Rahmouni K, Mark (2011) Angiotensinergic signaling in the
brain mediates metabolic effects of deoxy- metabolism. Physiol Genomics 43(6):286–294.

corticosterone (DOCA)-salt in C57 mice. doi:10.1152/physiolgenomics.00208.2010
Hypertension 57(3):600–607. d oi:10.1161/ 14. Hilzendeger AM, Cassell MD, Davis DR,
hypertensionaha.110.165829 Stauss HM, Mark AL, Grobe JL, Sigmund
6. Littlejohn NK, Siel RB Jr, Ketsawatsomkron CD (2013) Angiotensin type 1a receptors in
P, Pelham CJ, Pearson NA, Hilzendeger AM, the subfornical organ are required for deoxy-
Buehrer BA, Weidemann BJ, Li H, Davis DR, corticosterone acetate-salt hypertension.
Thompson AP, Liu X, Cassell MD, Sigmund Hypertension 61(3):716–722. doi:10.1161/
CD, Grobe JL (2013) Hypertension in mice hypertensionaha.111.00356
with transgenic activation of the brain renin- 15. Pittet P, Gygax PH, Jequier E (1974) Thermic
angiotensin system is vasopressin depen- effect of glucose and amino acids in man stud-
dent. Am J Physiol Regul Integr Comp ied by direct and indirect calorimetry. Br J Nutr
Physiol 304(10):R818–R828. doi:10.1152/ 31(3):343–349
ajpregu.00082.2013 16. Pittet P, Chappuis P, Acheson K, De Techtermann
7. Grobe JL, Rahmouni K, Liu X, Sigmund F, Jequier E (1976) Thermic effect of glucose
CD (2013) Metabolic rate regulation by the in obese subjects studied by direct and indirect
renin-angiotensin system: brain vs. body. calorimetry. Br J Nutr 35(2):281–292
Pflugers Arch 465(1):167–175. doi:10.1007/ 17. Walsberg GE, Hoffman TC (2005) Direct
s00424-012-1096-9 calorimetry reveals large errors in respiro-
8. Weidemann BJ, Voong S, Morales-Santiago metric estimates of energy expenditure. J Exp
FI, Kahn MZ, Ni J, Littlejohn NK, Claflin KE, Biol 208(Pt 6):1035–1043. doi:10.1242/
Burnett CM, Pearson NA, Lutter ML, Grobe jeb.01477
JL (2015) Dietary sodium suppresses digestive 18. Walsberg GE, Hoffman TC (2006) Using
efficiency via the renin-angiotensin system. Sci direct calorimetry to test the accuracy of indirect
Rep 5:11123. doi:10.1038/srep11123 calorimetry in an ectotherm. Physiol Biochem
9. Fink BD, Herlein JA, Guo DF, Kulkarni C, Zool 79(4):830–835. doi:10.1086/505514
Weidemann BJ, Yu L, Grobe JL, Rahmouni K, 19. Burger M, van Breukelen F (2013) Construction
Kerns RJ, Sivitz WI (2014) A mitochondrial- of a low cost and highly sensitive direct heat
targeted coenzyme q analog prevents calorimeter suitable for estimating metabolic rate
weight gain and ameliorates hepatic dys- in small animals. J Therm Biol 38(8):508–512.
function in high-fat-fed mice. J Pharmacol doi:10.1016/j.jtherbio.2013.09.002
Exp Ther 351(3):699–708. doi:10.1124/
jpet.114.219329 20. Lighton JRB (2008) Measuring metabolic
rates: a manual for scientists. Oxford University
10. Bahr SM, Weidemann BJ, Castro AN, Walsh JW, Press, Oxford. https://global.oup.com/aca-
deLeon O, Burnett CM, Pearson NA, Murry demic/product/measuring-metabolic-rates-
DJ, Grobe JL, Kirby JR (2015) Risperidone- 9780195310610?cc=us&lang=en& DOI:
induced weight gain is mediated through shifts 10.1093/acprof:oso/9780195310610.001.0001
in the gut microbiome and suppression of energy
expenditure. EBioMedicine 2(11):1725–1734. 21. McLean JA, Tobin G (2007) Animal and
doi:10.1016/j.ebiom.2015.10.018 human calorimetry. Cambridge University Press,
Cambridge
11. Burnett CM, Grobe JL (2013) Direct calorim-
etry identifies deficiencies in respirometry for 22. Kaiyala KJ, Ramsay DS (2011) Direct animal
the determination of resting metabolic rate in calorimetry, the underused gold standard for
C57Bl/6 and FVB mice. Am J Phys Endocrinol quantifying the fire of life. Comp Biochem
Metab 305(7):E916–E924. doi:10.1152/ Physiol A Mol Integr Physiol 158(3):252–264.
ajpendo.00387.2013 doi:10.1016/j.cbpa.2010.04.013
12. Burnett CM, Grobe JL (2014) Dietary effects 23. Lusk G (1928) The elements of the science of
on resting metabolic rate in C57BL/6 mice nutrition, 4th edn. W.B. Saunders Company,
are differentially detected by indirect (O2/ Philadelphia, PA
CO2 respirometry) and direct calorimetry. 24. Zuntz, N. 1847-1920. (1901). Studien zu
Mol Metab 3(4):460–464. doi:10.1016/j. einer Physiologie des Marsches: von Zuntz und
molmet.2014.03.003 Schumburg. Berlin: Hirschwald. pp 1847–1920
13. Xu D, Borges GR, Davis DR, Agassandian K, 25. Maloney SK, Fuller A, Mitchell D, Gordon C,
Sequeira Lopez ML, Gomez RA, Cassell MD, Overton JM (2014) Translating animal model
Grobe JL, Sigmund CD (2011) Neuron- or research: does it matter that our rodents are
glial-specific ablation of secreted renin does not cold? Physiology (Bethesda) 29(6):413–420.
affect renal renin, baseline arterial pressure, or doi:10.1152/physiol.00029.2014
146 Justin L. Grobe
26. Utz JC, Velickovska V, Shmereva A, van

31. Blaxter K (1989) Energy metabolism in ani-
Breukelen F (2007) Temporal and tem- mals and man. Cambridge University Press,
perature effects on the maximum rate of Cambridge
rewarming from hibernation. J Therm 32. Tschop MH, Speakman JR, Arch JR, Auwerx
Biol 32(5):276–281. doi:10.1016/j. J, Bruning JC, Chan L, Eckel RH, Farese
jtherbio.2007.02.002 RV Jr, Galgani JE, Hambly C, Herman MA,
27. Utz JC, van Breukelen F (2013) Prematurely Horvath TL, Kahn BB, Kozma SC, Maratos-
induced arousal from hibernation alters Flier E, Muller TD, Munzberg H, Pfluger PT,
key aspects of warming in golden-mantled Plum L, Reitman ML, Rahmouni K, Shulman
ground squirrels, Callospermophilus lateralis. GI, Thomas G, Kahn CR, Ravussin E (2012)
J Therm Biol 38(8):570–575. doi:10.1016/j. A guide to analysis of mouse energy metabo-
jtherbio.2013.10.001 lism. Nat Methods 9(1):57–63. doi:10.1038/
28. Heuton M, Ayala L, Burg C, Dayton K, nmeth.1806
McKenna K, Morante A, Puentedura G, Urbina 33. Takahashi N, Li F, Hua K, Deng J, Wang
N, Hillyard S, Steinberg S, van Breukelen CH, Bowers RR, Bartness TJ, Kim HS,
F (2015) Paradoxical anaerobism in desert Harp JB (2007) Increased energy expendi-
pupfish. J Exp Biol 218(Pt 23):3739–3745. ture, dietary fat wasting, and resistance to
doi:10.1242/jeb.130633 diet-induced obesity in mice lacking renin.
29. van Breukelen F, Martin SL (2015) The hiber- Cell Metab 6(6):506–512. doi:10.1016/j.
nation continuum: physiological and molecu- cmet.2007.10.011
lar aspects of metabolic plasticity in mammals. 34. Sakai K, Agassandian K, Morimoto S, Sinnayah
Physiology (Bethesda) 30(4):273–281. P, Cassell MD, Davisson RL, Sigmund CD
doi:10.1152/physiol.00010.2015 (2007) Local production of angiotensin II in
30. Kaiyala KJ (2014) What does indirect calorim- the subfornical organ causes elevated drinking.
etry really tell us? Mol Metab 3(4):340–341. J Clin Invest 117(4):1088–1095. d oi:10.1172/
doi:10.1016/j.molmet.2014.03.005 jci31242
Chapter 11
In Vitro Assays to Determine Smooth Muscle Cell

Hypertrophy, Protein Content, and Fibrosis
Katherine J. Elliott and Satoru Eguchi
Abstract
This chapter provides information on how to culture primary rat vascular smooth muscle cells and how to
induce cellular changes similar to those associated with angiotensin II activation in vivo. We describe how
to assess the cellular changes by determining cell size with an automated coulter cell counter to measure
cell volume. In addition, we describe a method to assess total protein content. Finally, we describe a stan-
dard technique to quantify angiotensin II-induced pro-fibrotic response using the Chondrex Sirius Red
Total Collagen Detection Kit.
Key words Explant primary cell culture, VSMC, Angiotensin II, Hypertrophy, Fibrosis
1 Introduction
This chapter provides the reader with instructions on how to cul-

ture rat vascular smooth muscle cells (VSMC) and how to stimulate
them with angiotensin II (Ang II) to induce a cellular hypertrophic
phenotype similar to that observed in hypertension. Primary cell
cultures, derived from dissection of living tissue and maintained in
culture for a limited number of passages, provide a great tool to
simplify the study of complex biologic disease processes such as
hypertension and associated organ damage. This explant method
allows VSMC to migrate from the rat aorta onto a cell culture dish
forming a monolayer of adherent cells, which will continue to pro-
liferate for up to 12 passages [1, 2]. These cells can be stimulated by
various agonists, and downstream physiologic changes such as
hypertrophy [3] and pro-fibrotic responses can be assessed [4–6].
This is a relatively easy to use and inexpensive model for the study
of pathophysiological changes occurring in cells with Ang II stimu-
lation. We do not include descriptions of cell proliferation assays
because Ang II does not stimulate proliferation in VSMC that have
been isolated via the explant method [3].
147
148 Katherine J. Elliott and Satoru Eguchi
2 Materials
2.1 Explant Method This technique was adapted from Ross et al. [2, 4].
for VSMC Primary Cell
1. 12-week-old Male Sprague Dawley rat.
Culture
2. Surgical scissors and tweezers.
3. VSMC Growth Medium: Dulbecco’s Modification of Eagle’s
Medium (DMEM: 4.5 g/l glucose), 10% (v/v) fetal bovine
serum, 1% (v/v) penicillin–streptomycin solution.
4. 0.05% trypsin–0.53 mM EDTA.
5. Hank’s buffered saline solution (HBSS).
6. Tissue culture incubator (37 °C, 5% CO2).
2.2 VSMC 1. Serum-free medium: DMEM, 1% (v/v) penicillin–streptomy-

Stimulation cin solution.
with Angiotensin II 2. Angiotensin II.
2.3 VSMC 1. 0.5% SDS.

Hypertrophy 2. BCA protein assay kit.
3. Plate reader or spectrophotometer.
4. Trypsin.
5. HBSS.
6. Phosphate buffered saline (PBS).
7. Scepter Handheld Automated Cell Counter with 60 μm
Scepter sensors.
2.4 VSMC Fibrosis 1. Chondrex Sirius Red Total Collagen Detection Kit (Catalog
#9062).
2. Pepsin (porcine gastric mucosa) 1 mg/ml in 0.05 M acetic
acid.
3. Cell scraper.
3 Methods
3.1 Explant Method 1. Excise the thoracic aorta of a male Sprague Dawley rat (200–
for VSMC Primary Cell 250 g) directly below the left subclavian artery and above the
Culture diaphragm. Rapidly immerse in VSMC growth medium.
Carefully remove connective tissue and adherent fat.
2. Longitudinally cut open isolated aorta and remove endothe-
lium by gently rubbing the intimal surface with a dull scissor
(see Note 1).
3. Cut denuded aorta into approximately 3-mm square sections.
Place intimal side down into 6-well culture dish.
SMC Hypertrophy, Protein and Fibrosis 149
4. Gently add enough VSMC Growth medium to minimally

cover the tissue, about 100–200 μl, without disturbing the ori-
entation of the explant and without causing the explant to
float. Culture at 37 °C in 5% CO2 incubator. Avoid handling
plate for 48 h after explant so as not to dislodge the tissue (see
Note 2).
5. Beginning on day 2, monitor plate for cell growth and add
additional medium as necessary to avoid drying of tissue and
migrating cells.
6. Allow VSMCs to grow out from the explant for 7–14 days (Fig. 1).
7. Once cells have established a monolayer culture surrounding
the tissue, remove tissue and culture medium, rinse with PBS,
and harvest cells by trypsinization. Transfer collected cells to a
100-mm flask.
8. The cells obtained by this method should express the “hills and
valleys” growth characteristics of cultured VSMC. The expres-
sion of myosin light chain and smooth muscle α-actin can be
confirmed by immunocytochemistry.
Cells should be maintained by passaging once a week with
trypsin and seeding in 75-cm2 flasks at approximately 0.5–1.0 × 106
cells. Cells should not be passaged more than 12 times and should
never be frozen for storage (see Note 3).
3.2 VSMC 1. Seed VSMC on 6- or 12-well plate and allow to grow to

Stimulation confluence.
with Angiotensin II 2. Serum-starve confluent VSMC for 48–72 h by removing
(Ang II) growth medium, wash cells with serum-free medium and incu-
bate with 1 ml of serum-free medium (Table 1).
3. On the day of stimulation, replace serum-free medium and let
cells rest for 1 h in incubator.
Fig. 1 VSMC explant culture, 14 days post-explant

Table 1
Guidelines for seeding VSMC in different sized culture wells
Approximate cell
number at
Plate size Seeding density Days to confluence confluence
6 well 2 × 105 3 5 × 105
12 well 1 × 105 2 2 × 105
24 well 5 × 104 2 1 × 105
96 well 3 × 103 2 2 × 104
4. Add Ang II at a final concentration of 100 nM. Stimulation

time should be 24–72 h in order to observe maximal hypertro-
phy (see Note 4).
3.3 VSMC Cellular protein measurement/BCA Assay

Hypertrophy
1. Seed VSMC in 24-well plates with n = 6 for each sample. Allow
cells to grow to confluence. Stimulate cells with agonist as
needed for experiment.
2. Wash cells with PBS.
3. Lyse cells in well using 150 μl of 0.5% SDS. Let stand for 5 min.
4. Add 25 μl cell lysate to 96-well plate and add BCA reagent
(mix according to package instructions).
5. Prepare standard curve.
6. Measure protein amount with plate reader at 570 nm (Fig. 2).
Cell volume
1. Seed VSMC in 6-well plates with n = 3 for each sample. Allow
2. Wash cells twice with HBSS.
3. Remove cells from plate with 250 μl of trypsin. Add 750 μl
PBS to each well. Pipet up and down to disrupt cell clumps
and transfer cell suspension to 1.5 ml tube.
4. Centrifuge tube at 250 × g for 3 min. Carefully remove
supernatant.
5. Resuspend cells in 100 μl PBS.
6. Dilute cells in PBS to an acceptable concentration (see Note
5). The Coulter Cell Counter can measure 10,000–500,000
cells/ml (Fig. 3).
30
*
Protein (µg/cm2)
20
10
0
control Ang II
Fig. 2 Representative graph illustrating hypertrophy via increased total protein in

AngII-stimulated VSMC cultures. VSMC were plated in a 12-well dish, grown to
confluence, and serum-starved for 48 h prior to AngII-stimulation for 72 h.
*p < 0.05 (n = 6)
4
*
Cell Volume (pL)
2
control Ang II
Fig. 3 Representative graph illustrating hypertrophy via increased cell volume in

*p < 0.05 (n = 6)
3.4 VSMC Fibrosis 1. Seed VSMC in 12-well plates with n = 6 for each sample. Allow
2. Wash cells with cold, distilled water.
3. Add 50 μl of 0.05 M acetic acid. Scrape cell layer with cell
scraper and transfer to 1.5 ml Eppendorf tubes.
4. Rinse wells with 50 μl 0.05 M acetic acid and transfer to respec-
tive Eppendorf tube.
5. Add 10 μl of pepsin solution sample. Incubate at 4 °C for 48 h
on a rotator.
6. Add 250 μl of Sirius Red solution to each tube. Vortex and
incubate at room temperature for 20 min followed by centrifu-
gation at 10,000 × g for 3 min.
7. Carefully remove the supernatant without disturbing the pellet
(see Note 6).
8. Resuspend pellet in 250 μl of Washing Solution. Centrifuge at
10,000 × g for 3 min.
4
*
Collagen (µg/cm2)
3
0
control Ang II
Fig. 4 Representative graph illustrating fibrosis via increased collagen levels in

*p < 0.05 (n = 6)
9. Carefully remove the supernatant without disturbing the

pellet.
10. Resuspend pellet in 125 μl Extraction Buffer. Vortex briefly.
11. Transfer 100 μl to clear bottom 96-well plate. Measure colla-
gen amount with plate reader at 570 nm (Fig. 4).
12. Prepare standard curve by same method as samples.
4 Notes
1. From this point, an aseptic technique should be performed

inside a tissue culture hood.
2. It is important that the tissue remain fixed with the intimal side
facing the plate so that the VSMC can attach and migrate onto
the floor of the culture well.
3. Seeding density may need to be adjusted according to how
quickly the cells are growing. Unpublished observations indi-
cate that freezing VSMC results in a phenotypic change in
their response to Ang II stimulation. Freezing the cells will
select for a subset of highly proliferative cells.
4. Ang II is prepared in H2O and stored in small aliquots at
−20 °C. Store 5 μl of 1 mM (10,000×) stock solution in small
tube. Dilute 1:10 with water (1000×) solution.
5. In our hands, we have found that a dilution of 1:100 works
well.
6. Using a p200 pipet to remove the supernatant is safer than try-
ing to pour off or aspirate the supernatant because the pellet
can be small and soft.
References
1. Geisterfer AA, Peach MJ, Owens GK (1988) formation of elastic fibers. J Cell Biol
Angiotensin ii induces hypertrophy, not hyper- 50:172–186
plasia, of cultured rat aortic smooth muscle 5. Nakashima H, Frank GD, Shirai H, Hinoki A,
cells. Circ Res 62:749–756 Higuchi S, Ohtsu H, Eguchi K, Sanjay A,
2. Eguchi S, Hirata Y, Imai T, Kanno K, Marumo Reyland ME, Dempsey PJ, Inagami T, Eguchi S
F (1994) Phenotypic change of endothelin (2008) Novel role of protein kinase C delta
receptor subtype in cultured rat vascular smooth Tyr311 phosphorylation in vascular smooth
muscle cells. Endocrinology 134:222–228 muscle cell hypertrophy by angiotensin
3. Ohtsu H, Higuchi S, Shirai H, Eguchi K, II. Hypertension 51:232–238
Suzuki H, Hinoki A, Brailoiu E, Eckhart AD, 6. Takayanagi T, Forrester FJ, Kawai T, Obama T,
Frank GD, Eguchi S (2008) Central role of gq Tsuji T, Elliott KJ, Nuti E, Rossello A, Kwok
in the hypertrophic signal transduction of HF, Scalia R, Rizzo V, Eguchi S (2016) Vascular
angiotensin ii in vascular smooth muscle cells. ADAM17 as a novel therapeutic target in medi-
Endocrinology 149:3569–3575 ating cardiovascular hypertrophy and perivascu-
4. Ross R (1971) The smooth muscle cell. lar fibrosis induced by angiotensin
II. Growth of smooth muscle in culture and II. Hypertension 68(4):949–955
Chapter 12
A New Mouse Model for Introduction of Aortic Aneurysm

by Implantation of Deoxycorticosterone Acetate Pellets
or Aldosterone Infusion in the Presence of High Salt
Shu Liu, Ming C. Gong, and Zhenheng Guo
Abstract
Dysfunction of the renin-angiotensin-aldosterone system (RAAS) has been implicated in the etiologies of
many cardiovascular diseases, including aortic aneurysm. In particular, the infusion of angiotensin II (Ang
II) in the apolipoprotein E-deficient mice (apoE−/−) and low density lipoprotein receptor knockout mice
(LDLR−/−) to induce aortic aneurysm has been extensively used in the field. In contrast, whether aldoste-
rone (Aldo), an essential component of RAAS and a downstream effector of Ang II, is involved in aortic
aneurysm is largely unknown. Here, we describe a new animal model for induction of aortic aneurysm in
mice in which administration of deoxycorticosterone acetate (DOCA) and high salt or aldosterone and high
salt, but not DOCA or high salt alone, to C57BL/6 male mice can potently induce aortic aneurysm forma-
tion and rupture in an age-dependent manner. This new aortic aneurysm mouse model is different from
Ang II infusion mouse model and exhibits several unique features that mimic human aortic aneurysm.
Key words Aneurysm, Angiotensin, Aldosterone, Sodium, Deoxycorticosterone
1 Introduction
Aortic aneurysm is defined as a permanent localized dilation of the

aorta with at least 50% increase in diameter compared with the
normal aortic diameter [1]. Aortic aneurysm can be classified
according to location as thoracic aortic aneurysm (TAA) and
abdominal aortic aneurysm (AAA). AAA is the most common form
of aortic aneurysm [2], affecting 4–8% of men and 0.5–1.5% of
women over the age of 60, and accounting for nearly 2% of all
deaths in Western countries [3, 4]. Due to an incomplete under-
standing of molecular mechanisms and pathophysiologic processes,
no drug has been approved for treatment of progression and rup-
ture of either type of this devastating disease.
Several aortic aneurysm animal models have been developed
over the last few decades [3]. Among them, the infusion of Ang II
in the apoE−/− and LDLR−/− mice to induce aortic aneurysm is
155
156 Shu Liu et al.
mostly used [5–10]. In contrast to overwhelming evidence for a

significant role of Ang II in aortic aneurysm [3, 4], little is known
whether aldosterone (Aldo), an essential component of the RAAS
and a downstream effector of Ang II, is involved in aortic aneu-
rysm. We recently developed a new mouse model for aortic aneu-
rysm in which administration of mineralocorticoid receptor (MR)
agonists, DOCA or Aldo plus high salt, but not DOCA or high salt
alone, to C57BL/6 male mice can potently induce aortic aneu-
rysm formation and rupture in an age-dependent manner [11].
Both AAA and TAA could be observed in aortic aneurysm induced
by DOCA or Aldo plus high salt but AAA was mostly evident and
TAA appeared as a result of AAA progression [11]. Compared to
the Ang II infusion mouse model, DOCA or Aldo plus high salt
mouse model described here exhibits several unique features,
including: (1) using WT mice, not the apoE−/− and LDLR−/−
mice, avoids the potential confounding effects of hyperlipidemia
on AAA; (2) infusing the mice with 200 μg/kg/day Aldo (which
is sufficient to induce aortic aneurysm) increases plasma Aldo con-
centration to 10.58 nM [11], which is below that in patients with
heart failure [12, 13]. This indicates that the Aldo-salt model is a
pathological model that mimics human diseases rather than a phar-
macological model that would cause concerns due to use of high
doses of the molecule of interest that never happens in human
patients; (3) using 10-month-old mice rather than 10-week-old
mice (mostly used by the Ang II and other models) resembles
human AAA; (4) dependent on MR but independent on Ang II
receptor since eplerenone (a specific MR antagonist), but neither
losartan (an Ang II receptor blocker) nor enalapril (an angiotensin
converting enzyme inhibitor), effectively blocks Aldo and high salt
induced AAA [11]; (5) requiring high salt for DOCA or Aldo to
induce aortic aneurysm implies that high salt intake may be a new
risk factor for aortic aneurysm.
Given that there are no effective drug therapies for aortic
aneurysms, DOCA or Aldo plus high salt mouse model described
here not only provides a new aortic aneurysm animal model
induced by MR agonist and high salt but reveals a previously
unrecognized but potentially significant role of aldosterone and
high salt in the pathogenesis of aortic aneurysm.
2 Materials
2.1 Animals, Ten-month-old male C57BL/6J or C57BL/6N mice are either

Chemicals, Osmotic purchased from colonies of the National Institute on Aging (Charles
Minipumps, River, Raleigh, NC, USA; see Note 1) or from the Jackson Laboratory
and Equipment (Bar Harbor, ME, USA) as retired breeders (see Note 2). All proto-
cols are approved by the University of Kentucky Institutional Animal
Care and Use Committee (IACUC).
Aortic Aneurysms in DOCA and Salt Mouse Model 157
1. DOCA pellets (50 mg, 21-day release, see Note 3).

2. d-Aldosterone (Aldo)(200 μg/kg/day) and solubilized in 50%
dimethyl sulfoxide (DMSO, see Note 4).
3. Isoflurane and/or ketamine–xylazine.
4. Depilatory cream (e.g., Nair®).
5.
Surgical instruments (forceps, hemostat, scissors, and
auto-stapler).
6. Osmotic minipumps (Alzet model 2004, 28-day release, see
Note 4).
7. High salt: 0.9% NaCl plus 0.2% KCl.
8. Noninvasive CODA® tail-cuff blood pressure system.
9. High-resolution ultrasound imaging Vevo® 2100 System.
10. Nikon SMZ800 Stereo microscope with digital imaging soft-
ware NIS-Elements (Nikon Instruments Inc.).
3 Methods
3.1 Preparation Mice are anesthetized by intraperitoneal injection of ketamine

for DOCA Pellet (100 mg/kg) and xylazine (10 mg/kg; see Note 5).
Implantation, Surgical
1. Shave mice and then use a depilatory cream to remove all hair
Procedure, from the surgical site.
and Administration
of High Salt
2. Lift the skin on the lateral side of the neck of the animal.
in Drinking Water 3. Make an incision equal in diameter to that of the pellet.
4. Make a pocket horizontally with a pair of forceps about 2 cm
beyond the incision site.
5. Put DOCA pellets into the pocket with forceps.
6. Close the wound with wound clips. Two clips will normally
suffice.
7. Mice are given with the drinking water containing high salt
(0.9% NaCl plus 0.2% KCl) immediately after DOCA implan-
tation for 3 weeks (see Note 6).
3.2 Measurement Systolic blood pressure (SBP) is measured using a noninvasive tail
of Mouse Blood cuff system. Measurements are performed for 5 consecutive days
Pressure by Tail-Cuff, for determination of weekly average measures as described [11].
Quantification SBP is measured 1 week before DOCA administration (basal) and
of Abdominal Aortic again during the third week after DOCA and salt administration
Dilation by Ultrasound (see Note 7).
Imaging, and External A high-resolution ultrasound imaging system is used to image
Abdominal Aortic and quantify intraluminal diameters of mouse suprarenal abdominal
Diameter aortas [11, 14]. Briefly, mice are anesthetized by inhalation of isoflu-
Measurements rane mixed with O2 (3–5% isoflurane/97% O2) and maintained by
158 Shu Liu et al.
inhalation of isoflurane mixed with O2 (1–3% isoflurane/97% O2)

throughout the procedure. Mouse hair is removed from the abdo-
men by using a depilatory cream. Mice are laid supine on a heated
table at 37 °C. Warmed ultrasound transmission gel is placed on the
abdomen. Cine loops of 300 frames are acquired throughout the
renal region of the abdominal aorta and used to determine the maxi-
mal diameters of the abdominal aorta in the suprarenal region.
“Portal Triad” (hepatic artery, hepatic vein, and bile duct) is used as
anatomic marker to assess transverse images in the same location in
each mouse. Ultrasound images are acquired 1 week before and
weekly after DOCA or aldosterone administration, and are indepen-
dently validated by two different operators.
Based on the definition of human aortic aneurysm, AAA and
TAA are defined in the current study as having at least a 50%
increase in maximal intraluminal and external diameters compared
with the same region of aorta in control mice. Maximal intralumi-
nal diameters of suprarenal abdominal aortas are quantified in vivo
by ultrasound imaging as described above. Maximal external diam-
eters of thoracic and abdominal aortas are photographed by Nikon
SMZ800 stereomicroscope with digital imaging software NIS-
Elements and then quantified as described [6, 11, 14].
3.3 Induction 1. Weigh study mice before calculating the amount of Aldo
of Aortic Aneurysm needed for infusion (see Note 8).
by Aldo and High Salt 2. Use the template (Table 1) to calculate the Aldo mass needed
for the experiment. Calculate a 300 μL total volume of Aldo
solution or each mouse since each pump requires approxi-
mately 250 μL.
3. Weigh the calculated Aldo mass (4.6 mg as shown in Table 1)
into a sterile plastic tube.
4. Add a half calculated volume of DMSO (1800 μL) into the
plastic tube containing the Aldo, cap, and mix thoroughly by
vortexing until the solution is clear.
5. Add another half calculated volume of room temperature
dH2O (1800 μL) into the Aldo solution, cap, and mix thor-
oughly by vortexing until the solution is clear.
6. Label mouse numbers #1, #2, #3, etc. on individual sterile
plastic tubes with caps. Prepare Aldo solution for each mouse
based on body weight as shown in Table 1.
3.4 Osmotic Pump 1. Weigh each pump (including both the main pump body and flow
Filling moderator). This weight, termed “Pump Weight empty” in the
template (Table 1), will be used to calculate the filled ratio.
2. Attach the pump filling needle to a 1 mL sterile syringe and care-
fully fill the syringe with Aldo solution from the appropriately
Table 1
Calculation of Aldo for osmotic pump infusion
Experimental characteristics
Dose required 139 ng/kg/min = 200 μg/kg/day
Start body weight (largest mouse) 37.9 g
Total estimated body weight gain 1.0 g
Pump rate 0.25 μL/h
Start body weight (largest mouse) 37.9 g
Number of mice 12
Calculations (see Note 8)
Does per hour for animals 320.256 nga
Concentration needed 1281. 024 ng/μLb
For 300 μ1 solution 0.384 mg/300 μLc
Solution needed
Total aldosterone (mg) 4.608 mgd
Dissolved in (DMSO + H20) 3600 μLe
Mouse # BW (g) Dilution Factor f Aldo (μL)g H2O (μL)h
#1 30.5 0.805 241 59
#2 31.8 0.839 252 48
#3 31.2 0.823 247 53
#4 34.2 0.902 271 29
#5 33.5 0.884 265 35
#6 35.9 0.947 284 16
#7 36.8 0.971 291 9
#8 37.2 0.982 294 6
#9 37.9 1.000 300 0
#10 33.3 0.879 264 36
#11 34.1 0.900 270 30
#12 33.2 0.876 263 37
a −3
139 × 10 × (37.9 + ½ × 1) × 60 = 320.56 ng
b
320.56/0.25 = 1281.024 ng
c
1281.024 ng × 300 = 0.384 mg/300 μL
d
0.384 mg × 12 = 4.608 mg
e
300 × 12 = 3600 μL
f
BW/37.9 = Dilution factor
g
300 × dilution factor = Aldo (μL)
h
300 – Aldo = H2O (μL)
160 Shu Liu et al.
numbered plastic tube. It is important to avoid drawing air into

the syringe.
3. Gently insert the filling needle into the pump body. Push the
syringe plunger slowly to fill the pump with Aldo solution.
Stop filling the pump and carefully remove the needle as soon
as a bead of fluid rises out of the pump.
4. Insert flow moderator into pump through the hole and weigh
the filled pump.

5.
Calculate Filling Ratio (%) = (Pump Weight
“filled” − “empty”) × 1000/mean fill volume × 100.
6. Place filled pump into the labeled 15 mL tube with the mod-
erator head facing upward. Add 5 mL of sterile saline or PBS
to cover the pump.
7. Place tubes in a 37 °C incubator overnight to allow the pump
to prime.
3.5 Preparation 1. Mice are anesthetized by intraperitoneal injection of ketamine

for Pump Implantation (100 mg/kg) and xylazine (10 mg/kg; see Note 5).
and Surgical 2. An about 1 cm incision is made just behind the neck of animals
Procedures (mid-scapular incision). The incision is perpendicular to the
long axis of the implant (see Note 9).
3. A hemostat is inserted into the incision to create a subcutane-
ous pocket for the pump. Open the jaws to bluntly dissect the
subcutaneous tissue. Withdraw the hemostat slightly, close
jaws, and repeat the blunt dissection steps until a suitable
implant pocket has been created (see Note 10).
4. The pump is inserted into the pocket, delivery portal first,
which minimizes interaction between the compound delivered
and the healing of the incision (see Note 11).
5. The wound is closed with wound clips (two clips will normally
suffice).
6. Perform blood pressure measurement, ultrasound imaging,
and quantification of aortic aneurysms as described in
Subheading 3.1.
4 Notes
1. C57BL/6 mice have at least two different strains: C57BL/6J

(the Jackson Laboratory) and C57BL/6N (the NIA aged
rodent colonies), which have different genetic backgrounds
[15]. C57BL/6J and C57BL/6N mice may potentially
respond to DOCA or Aldo plus high salt with different sensi-
tivity, which we have not extensively investigated. However,
we found that there is no difference regarding induction of
aortic aneurysm by DOCA or Aldo plus high salt. In addition,

the NIA aged rodent colonies are only available for projects
that are directly related to aging and that are funded by grants
from the NIH, other US government agencies, and US-based
private foundations that fund aging research. Contract-funded
projects are only eligible if they are funded by the NIA.
2. The age of C57BL/6J retired breeders from the Jackson
Laboratory may be variable. Since DOCA or Aldo plus high
salt model described here is age dependent [11], the retired
breeders that are less than 10 months old are required for sin-
gly cage housing due to their aggression until 10 months old.
Alternatively, 6-month-old C57BL/6J mice are available from
the Jackson Laboratory. But again these mice are also required
for singly cage housing up to 10 months of age prior to
experiments.
3. Different dose (0.001–200 mg/pellet) and time release (21–
90 days) of DOCA pellets are available. We use DOCA pellets
at a dose of 50 mg/pellet for a 21-day release and demon-
strated that it is sufficient to potently induce hypertension and
aortic aneurysm in 10-month-old male C57BL/6J mice.
However, it should be pointed out that the low dose of DOCA
pellets may be also effective.
4. There are many different way to solubilize Aldo to use Alzet®
osmotic pumps to deliver Aldo to mice (http://www.alzet.
com/research_applications/ALDO.html). We used 50%
DMSO and demonstrated that 50% DMSO alone did not have
any effect on aortic aneurysm formation [11].
5. Mice may need to be redosed with 25–35% of the original dose
of ketamine alone to achieve desirable anesthetic effect.
Alternatively, mice could be anesthetized by inhalation of iso-
flurane mixed with O2 (3–5% isoflurane/97% O2) at a flow rate
of 600 mL/min and anesthesia is then maintained by inhala-
tion of isoflurane mixed with O2 (1–3% isoflurane/97% O2) at
a flow rate of 600 mL/min throughout the surgical procedure.
During anesthesia, the animal’s eyes are protected with petro-
leum ophthalmic ointment.
6. Upon administration of DOCA and high salt, mice usually
drink considerable amounts of water and urinate frequently,
which indicates the effectiveness of DOCA and high salt.
Personnel need to monitor and refill drinking water if neces-
sary and change cage bedding at least every other day.
7. Increases in blood pressure by DOCA and high salt compared
to basal blood pressure are as expected and indicate the effec-
tiveness of DOCA and high salt.
162 Shu Liu et al.
8. The protocol described here uses the example of infusion of

Aldo (200 μg/kg/day or 139 ng/kg/min) for 4 weeks to
twelve 10-month-old male mice (Table 1).
9. Implants are best tolerated in the dorsal region of the body
slightly caudal (posterior) to the shoulders. An appropriately
inserted implant in this site will not interfere with vital organs,
or restrict normal activities of the animal. This site is also rela-
tively inaccessible to manipulation by the animal. Care must be
exercised to cut only the skin, and not the underlying tissues.
10. The pocket will be slightly wider than the implant diameter
and about 1 cm longer than the pump. Avoid making the
pocket too large, as this will allow the pump to turn around or
slip down on the flank of the animal.
11. The pump location is checked to assure that it does not rest
immediately beneath the incision, which could interfere with
the healing of the incision.
Acknowledgment
This work was supported by NIH grants HL106843 and

HL125228 (to M.C. Gong and Z. Guo) and VA Merit Award
BX002141 (to Z. Guo) as well as a National Institute of General.
Medical Sciences grant (P20 GM103527-05 to L. Cassis).
References
1. Hiratzka LF, Bakris GL, Beckman JA et al (2010) 5. Cassis LA, Gupte M, Thayer S et al (2009) ANG
2010 ACCF/AHA/AATS/ACR/ASA/SCA/ II infusion promotes abdominal aortic aneu-
SCAI/SIR/STS/SVM guidelines for the diag- rysms independent of increased blood pressure
nosis and management of patients with thoracic in hypercholesterolemic mice. Am J Physiol
aortic disease. A report of the American College Heart Circ Physiol 296:H1660–H1665
of Cardiology Foundation/American Heart 6. Cassis LA, Helton MJ, Howatt DA et al
Association task force on practice guidelines, (2005) Aldosterone does not mediate angio-
American Association for Thoracic Surgery, tensin II-induced atherosclerosis and abdomi-
American College of Radiology, American Stroke nal aortic aneurysms. Br J Pharmacol
Association, Society of Cardiovascular 144:443–448
Anesthesiologists, Society for Cardiovascular 7. Daugherty A, Manning MW, Cassis LA (2000)
Angiography and Interventions, Society of Angiotensin II promotes atherosclerotic lesions
Interventional Radiology, Society of Thoracic and aneurysms in apolipoprotein E-deficient
Surgeons, and Society for Vascular Medicine. mice. J Clin Invest 105:1605–1612
J Am Coll Cardiol 55:e27–e129
8. Rateri DL, Moorleghen JJ, Balakrishnan A
2. Isselbacher EM (2005) Thoracic and abdominal et al (2011) Endothelial cell-specific deficiency
aortic aneurysms. Circulation 111:816–828 of Ang II type 1a receptors attenuates Ang
3. Lindsay ME, Dietz HC (2011) Lessons on the II-induced ascending aortic aneurysms in LDL
pathogenesis of aneurysm from heritable con- receptor−/− mice. Circ Res 108:574–581
ditions. Nature 473:308–316 9. Thomas M, Gavrila D, Mccormick ML et al
4. Golledge J, Muller J, Daugherty A et al (2006) (2006) Deletion of p47phox attenuates angio-
Abdominal aortic aneurysm: pathogenesis and tensin II-induced abdominal aortic aneurysm
implications for management. Arterioscler formation in apolipoprotein E-deficient mice.
Thromb Vasc Biol 26:2605–2613 Circulation 114:404–413
10. Zhang X, Thatcher SE, Rateri DL et al (2012) 13. He BJ, Joiner ML, Singh MV et al (2011)
Transient exposure of neonatal female mice to Oxidation of CaMKII determines the cardio-
testosterone abrogates the sexual dimorphism toxic effects of aldosterone. Nat Med
of abdominal aortic aneurysms. Circ Res 17:1610–1618
110:e73–e85 14. Barisione C, Charnigo R, Howatt DA et al
11. Liu S, Xie Z, Daugherty A et al (2013) (2006) Rapid dilation of the abdominal aorta
Mineralocorticoid receptor agonists induce during infusion of angiotensin II detected by
mouse aortic aneurysm formation and rupture noninvasive high-frequency ultrasonography.
in the presence of high salt. Arterioscler J Vasc Surg 44:372–376
Thromb Vasc Biol 33:1568–1579 15. Mekada K, Abe K, Murakami A et al (2009)
12. Weber KT (2001) Aldosterone in congestive Genetic differences among C57BL/6 sub-
heart failure. N Engl J Med 345:1689–1697 strains. Exp Anim 58:141–149
Chapter 13
Fluorescence-Based Binding Assay for Screening

Ligands of Angiotensin Receptors
Maiia E. Bragina, Nikolaos Stergiopulos, and Rodrigo A. Fraga-Silva
Abstract
Binding assay is a common technique used to characterize ability of a ligand to interact with a specific
biological target. A number of parameters, such as binding affinity, receptor density, and association/dis-
sociation rate constants, can be measured by means of this technique. In most cases, implementation of the
binding assay requires specific infrastructure for labeling and detecting the ligand, which impedes realiza-
tion of this technique in a standard laboratory. Here we describe a simple fluorescence-based binding assay
for angiotensin peptides and receptors, which does not require complex equipment and can be used for
initial screening of the novel ligands or mutational studies.
Key words Binding assay, Ligand, Receptor, Fluorescently labeled, Screening, Renin–angiotensin
system
1 Introduction
The relevance of the renin angiotensin system (RAS) is demon-

strated by the remarkable clinical success of the angiotensin type I
receptor (AT1) blockers and angiotensin converting enzyme
(ACE) inhibitors [1–3]. Nowadays, drugs targeting RAS represent
one of the most effective class of medications used for treatment of
cardiovascular diseases [1, 2]. Recent discovery of new RAS com-
ponents and their essential role in various pathophysiological pro-
cesses pointed out novel pharmacological targets. For instance,
newly identified G-protein-coupled receptors (GPCRs), AT2 and
Mas, activated by angiotensin II and angiotensin-(1–7), respec-
tively, have been shown to evoke a number of effects opposite to
ones associated with AT1 signaling [4–6]. Thus compounds, which
are able to activate these receptors may become potential therapeu-
tic tools against cardiovascular disorders [5].
Binding of ligand to the receptor is a fundamental step, respon-
sible for modulation of the receptor activity and downstream signal-
ing, which in turn results in development of various physiological,
165
166 Maiia E. Bragina et al.
pathological, or pharmacological effects [7, 8]. Binding assay is a

prominent technique used to detect and characterize ligand–recep-
tor interactions for elucidation of molecular regulatory mechanisms
[4, 9] as well as development of novel drugs [10, 11] or diagnostic
tools [12, 13]. Although a wide variety of methods for realization
of binding assays have been established, in most cases this assay
consists of incubation of labeled ligand with the corresponding
receptor preparation and following quantification of the ligand
bound to the receptor [8].
Specific parameter to be determined by means of binding assay
defines the experimental design. In this sense three types of bind-
ing assay can be highlighted: saturation binding assay, kinetic stud-
ies, and competition binding. In particular, saturation experiments
are used to assess receptor density (Bmax) and affinity of the ligand
to the receptor (Kd) [14, 15]. Generally, it involves incubation of
receptor preparation with increasing concentrations of labeled
ligand [15]. Ligand bound to the receptor is further quantified
and plotted against its concentration in the assay mixture [14, 15].
On its turn, kinetic studies are generally employed to measure the
association/dissociation (kon/koff) rate constants of the binding
reaction and dissociation half-life (t½) [16]. In order to evaluate
the association kinetics, receptor preparation is incubated with
labeled ligand at one or more fixed concentrations. At the same
time, dissociation studies are performed by pre-incubation of
receptors with labeled ligand until equilibrium is reached and sub-
sequent initiation of the dissociation process by addition of unla-
beled competitor or immersion in the buffer solution [15]. Both
reactions are allowed to run for different time periods and conse-
quently intensity of the signal, created by the label, is plotted with
respect to the time point [15].
Among all types of binding assay, competition binding is the
most common one used to characterize binding features of angio-
tensin peptides. Competition experiment tests the ability of unla-
beled ligand to compete for the binding site of the receptor with the
labeled ligand of fixed concentration [15]. Competing ligand can be
identical to the labeled compound (labeled vs. non-labeled) or be
structurally different (e.g., synthetic agonist/antagonist vs. endog-
enous ligand) [14]. In case of the identical ligands used, competitive
binding assay can be employed as an alternative to saturation studies
to assess Kd and Bmax [14]. Experiment with structurally different
ligands gives rise to the concentration of the unlabeled compound,
which displaces 50% of the labeled ligand bound to the target recep-
tor (IC50) [8]. Final result of the competition binding assay is the
curve of displacement, where binding of labeled ligand is plotted
with respect to log concentration of the non-labeled ligand of inter-
est [14]. Binding of labeled compound is expressed as a fraction of
total binding, which arises from incubation of the receptor prepara-
tion with the labeled ligand only [14].
Fluorescence-Based Binding Assay 167
Ligand labeling strategy is the most important parameter of

the binding assay as it impacts the whole experimental procedure.
Due to its sensitivity and specificity, radioligand binding assay,
which implies incorporation of the radioactive moiety into the
ligand molecule, has become a conventional technique used to
characterize ligand–receptor interactions. However, employment
of this method is associated with a number of drawbacks like neces-
sity to handle radioactive substances as well as production of haz-
ardous waste [8].
In the current chapter we describe a simple binding assay tech-
nique, which is based on usage of fluorescently labeled ligands.
While being less accurate as the radioligand methods, it is safer, has
lower costs and requires standard laboratory equipment. Although
not accurate enough to determine Kd or Bmax, the technique intro-
duced in this chapter can serve as a valuable tool to rapidly test
large number of compounds for their ability to bind the receptor.
Another useful application of the current method is mutational
studies to identify specific amino acids within the binding site of
the receptor.
2 Materials
1. Cells naturally expressing the target receptor or transfected

cells. A number of approaches are currently available for deliv-
ery of foreign genetic material into the cell: biological (virus-
mediated), chemical (cationic polymers and lipids, calcium
phosphate), and physical (direct injection, electroporation,
etc.) [17]. One should select the method, which will ensure
high transfection efficiency, reproducibility, and protein yield
(see Note 1).
2. Assay solution: Hanks’ Balanced Salt solution or standard Tris
buffer supplemented with 0.2% bovine serum albumin (BSA),
0.01% bacitracin, 0.002% phenylmethylsulfonyl fluoride (PMSF),
0.01% 1,10-phenanthroline (see Notes 2 and 3). To reduce
background fluorescence Hanks’ solution without phenol red is
preferred. Since 1,10-phenanthroline and PMSF have only mod-
erate solubility in water, it is recommended to pre-solubilize
them in methanol and isopropanol, respectively. For 100 mL of
assay solution, dilute 2 mg of PSMF in 150 μL of isopropanol
and 10 mg of 1,10-phenanthroline in 50 μL of methanol. Final
formulation of the assay solution must be done just before the
experiment, since the supplementary components, PMSF in par-
ticular, have limited stability in aqueous solutions.
3. Angiotensin peptides or testing compounds in unlabeled form as
a competing ligand, for example: (a) Angiotensin II-Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe; (b) Angiotensin-(1–7)-Asp-Arg-Val-Tyr-
Ile-His-Pro; (c) Angiotensin-A-Ala-Arg-Val-Tyr-Ile-His-Pro-Phe;
(d) Alamandine- Ala-Arg-Val-Tyr-Ile-His-Pro; (e) Angiotensin-

(1–9)-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His; and (f) Angiotensin
IV-Val-Tyr-Ile-His-Pro-Phe. In case binding properties of the
competing ligand are not known, we recommend to test several
concentrations of it in order to cover the condition of its possible
interaction with the target receptor. Assay solution containing dif-
ferent amount of the competing ligand should be prepared right
before the experiment and kept on ice.
4. Fluorescently labeled angiotensin peptide (labeled with Mono-
5-(and-6)-carboxyfluorescein (FAM) or Mono-Rhodamine B
(Rhod), e.g.: FAM-Angiotensin II; Rhod-Angiotensin II;
FAM-Angiotensin-(1–7); Rhod-Angiotensin-(1–7)). Dilute
labeled peptide in the assay solution right before the experi-
ment initiation and keep it protected from light and on ice (see
Note 4).
5. Receptor antagonists can also be used in certain experiment
design (e.g.: losartan, AT1 antagonist; A-779, Mas antagonist;
PD123319, AT2 antagonist) (see Note 5). Similar to the pep-
tide preparation, dilute the antagonist in the assay buffer, right
before the experiment initiation and keep it on ice.
6. Twelve-well plate with coverslips fitting into the well (e.g.,
15 mm circular coverslip) or any other support/flask, which
allows for cell cultivation and consequent microscopy of the
samples.
7. Glass slides (e.g., 25 × 75 × 1.0 mm), DAPI containing mount-
ing medium (see Note 6) and nail polish to prepare coverslips
with cells for microscopy.
8. Fine tweezers to manipulate the coverslips and mount them on
the glass slides.
9. Fluorescent or confocal microscope for image acquisition.
10. Ice to keep assay components and cell preparation at low
temperature.
Diligently follow all waste disposal regulations when discard-
ing waste materials.
3 Method
The current chapter describes a method to test the ability of the

compound of interest to bind to angiotensin-related GPCRs
[18–20].
Cells constitutively expressing the GPCR of interest (target
receptor) should be seeded on coverslips, placed in the 12-well
plate, 1–2 days in advance of the binding assay (see Note 7). In case
transiently transfected cells are used in the experiment, duration of
the cell cultivation should correspond to the maximum level of the

receptor expression (see Note 1).
When conducting binding experiments one should distinguish
between specific and nonspecific binding of the ligand [7]. While
specific binding (SB) implies association of the ligand exclusively
with the target receptor, nonspecific binding (NSB) results from
the labeled ligand binding to the other components present in the
assay mixture. Since NSB may introduce a significant disturbance
in the binding measurements, it is important to quantify it in every
binding experiment. NSB is determined in the presence of excess
of the unlabeled ligand or receptor antagonist, which leads to the
occupation of the target receptors by non-detectable molecule and
labeled ligand binding mostly to the nonspecific sites. Although it
is advisable to have NSB no more than 20% [14], significant results
can be still obtained at NSB level up to 30–60% [9, 20, 21].
All the procedures should be performed on ice to minimize the
protease activity and inhibit a range of the processes such as inter-
nalization of the receptor [7].
We recommend to run triplicates for each assay condition.
1. Once the monolayer of cells expressing the target receptor is
formed on the coverslips, aspirate the medium and wash twice
with 1–2 mL of the ice-cold assay solution. This step should be
done gently to avoid damage of the cell monolayer.
2. Incubate cells for 5–10 min with prepared dilutions of the test-
ing compound or unlabeled angiotensin (at designated con-
centrations) to occupy the binding sites of the target receptor.
For total binding measurements, the pre-incubation must be
performed using assay buffer without testing compound or
unlabeled angiotensin peptide, while nonspecific binding mea-
surements should include pre-incubation with high concentra-
tion of unlabeled angiotensin peptide (e.g., 10−5 M). We
recommend to add 1 mL of the solution per well in case of
12-well plate (see Note 8).
3. Gently wash the cells with the assay solution.
4. Add the solution containing unlabeled peptide or testing com-
pound at same concentrations as for pre-incubation step plus
labeled angiotensin peptide into the wells. The concentration
of labeled ligand must be the same in every experimental con-
dition. Let the incubation last for 60 min on ice with gentle
shaking every 10–15 min. From this step on, the preparation is
photosensitive and must be protected from light exposure.
5. After incubation, wash gently 2–3 times with ice-cold assay
solution (see Note 9).
6. Place a small drop of the mounting medium on the microscopy
slide. Using the fine tweezers, take the coverslip from the well,
carefully remove the excess of liquid without touching the cell
Fig. 1 Representative images corresponding to conditions of (a) total binding and (b) nonspecific binding of
FAM-angiotensin-(1–7) to CHO cells overexpressing Mas receptor
monolayer and subsequently mount the coverslip with cells

down on the microscope slide. Seal the edges of coverslip
using, for example, nail polish. Do not forget to properly label
the microscope slide.
Of note, it could be problematic to remove the coverslip
from the well in wet environment. The selection of a suitable
tweezer with pointed tips can facilitate the coverslip handling.
7. Directly proceed with imaging by means of fluorescent or con-
focal microscope (see Note 10). As images are taken for the
following signal quantification, all acquiring parameters must
be kept unchanged during imaging. It is recommended to cap-
ture several pictures (at least 5) from every slide for better
overview of each condition (Fig. 1).
8. Quantification can be done with different image processing
software, such as Image J (NIH, Bethesda, MD, USA). The
main principle of the image analysis is to quantify the mean
grey value (signal intensity) on every image and normalize it to
the number of cells present in the field of view. The number of
cells can be obtained by counting number of nuclei stained by
DAPI. Artefacts exhibiting amorphous strong fluorescent sig-
nal should be excluded from the area of quantification.
4 Notes
1. Since the introduced fluorescence-based method has low sensi-

tivity, we highly recommend to use cells, which overexpress the
receptor of interest. In case transfection is done to achieve the
receptor overexpression, it is advisable to carry out preliminary
optimization of the transfection conditions as well as duration of

cell cultivation to achieve the highest possible receptor density.
Although radioligand-based methods allow usage of tissue sec-
tions for binding studies, we do not recommend to employ
them with the current technique. Tissues often present high
level of background fluorescence, which can produce consider-
able noise, impairing the sensitivity of the experiment. Exceptions
can be made in case tissue has high expression of the target
receptor, as shown in previous publications assessing binding of
Angiotensin-(1–7) to Mas receptor in mouse testis [22] and
heart [23] sections.
2. Angiotensin peptides have a very short half-life and enzymes,
constitutively expressed by the cultured cells, can cause their
rapid degradation during the experiment. In order to increase
the stability of the peptides, protease inhibitors (bacitracin,
PMSF, and 1,10-phenanthroline) must be added to the assay
solution. Additionally, BSA should be added in the assay solu-
tion to prevent nonspecific adsorption of the peptide onto
surfaces.
3. pH of the solution is a critical parameter, which can signifi-
cantly change the ligand–receptor binding affinities through
disturbance of electrostatic/hydrophobic interactions as well
as receptor conformation. The pH of the assay mixture must
correspond to the physiological range and stay constant during
the experiment, and therefore, buffer should be added into the
composition of assay solution. We recommend to use standard
Tris buffer (e.g., 150 mM NaCl, 50 mM Tris–HCl, 20 mM
EDTA) with pH adjusted to 7.4, or Hanks’ solution, which
does not contain sodium bicarbonate, but instead buffered by
20 mM HEPES. While the bicarbonate-buffered solutions
tend to alkalinize at low CO2 content in the ambient atmo-
sphere, HEPES keeps the pH constant. In some cases, calcium
and/or magnesium salts could be added to promote the bind-
ing events [14].
4. Competition binding assays require labeled ligand concentra-
tions equal or less than Kd for the given receptor–ligand pair
[14] since higher concentrations may be not displaced by the
competitive ligand. As an example, for FAM-labeled angioten-
sin-(1–7) the optimal concentration is 10−8–10−9 M [20, 21].
In case of limited information about binding properties of the
labeled ligand, additional optimization of its concentration
may be required.
5. Antagonist of high affinity and selectivity (e.g., losartan, AT1
antagonist; A-779, Mas antagonist; PD123319, AT2 antago-
nist) may be employed at high concentration to additionally
confirm nonspecific binding [21].
6. We recommend using DAPI-containing mounting medium to

facilitate imaging of the samples and cell quantification during
the image analysis.
7. It is advisable that cells have the confluence of no more than
60–70% by the day of the experiment since monolayer of
higher density may complicate cell quantification and overall
image analysis.
8. In order to use the current technique for the mutational stud-
ies, conditions with (a) fluorescently labeled angiotensin (total
binding) and (b) excess of unlabeled form co-incubated with
fluorescently labeled angiotensin (nonspecific binding) are suf-
ficient to drive the conclusion whether the angiotensin peptide
is able to bind to the mutated receptor. Difference between
total binding and nonspecific binding will indicate the binding
event.
9. The final washing step is critical to guarantee the removal of
the unbound labeled ligands, which can significantly increase
the background fluorescence. However, taking into account
that removal of the ligand-containing solution disturbs the
equilibrium and triggers the dissociation of the ligand, we
advise that final washing step and subsequent preparation of
the microscope slide are done as fast as possible. Please refer to
Fig. 2 for processing microscope slides.
10. For image acquisition, it is recommended to use high resolu-
tion fluorescent microscope, equipped with appropriated fil-
ters, or confocal microscope. However, when using confocal,
an adequate opening of the pinhole must be considered.
Steps 1 2 3 4 5 6 7 8
Conditions Washing Pre-Incubation Washing Incubation Washing Mounting Imaging Analysis
Image acquisition (fluorescent or confocal
Image analysis (intensity / cell number)
Assay Solution
Wash the cells with assay solution
Total Binding FLP

Mount the coverslip on the slide
(without ligand)
microscopy)
FLP
Non-Specific Binding UAP +
UAP
FLP
Binding of Testing Compound
TC +
(at different concentration)
TC
UAP - unlabeled angiotensin peptide (diluted in assay buffer)

FLP - fluorescently labeled peptide (diluted in assay buffer)
TC - testing compound (diluted in assay buffer)
Fig. 2 Schematic process-flow

References
1. Schiffrin EL (2002) Vascular and cardiac ben- 10. Violin JD, DeWire SM, Yamashita D,
efits of angiotensin receptor blockers. Am Rominger DH, Nguyen L, Schiller K, Whalen
J Med 113(5):409–418 EJ, Gowen M, Lark MW (2010) Selectively
2. Ma TK, Kam KK, Yan BP, Lam YY (2010) engaging beta-arrestins at the angiotensin II
Renin-angiotensin-aldosterone system block- type 1 receptor reduces blood pressure and
ade for cardiovascular diseases: current status. increases cardiac performance. J Pharmacol
Br J Pharmacol 160(6):1273–1292. Exp Ther 335(3):572–579. doi:10.1124/
doi:10.1111/j.1476-5381.2010.00750.x jpet.110.173005
3. Bader M (2010) Tissue renin-angiotensin- 11. Wan Y, Wallinder C, Plouffe B, Beaudry H,
aldosterone systems: targets for pharmaco- Mahalingam AK, Wu X, Johansson B, Holm
logical therapy. Annu Rev Pharmacol M, Botoros M, Karlen A, Pettersson A, Nyberg
Toxicol 50:439–465. doi:10.1146/annurev. F, Fandriks L, Gallo-Payet N, Hallberg A,
pharmtox.010909.105610 Alterman M (2004) Design, synthesis, and
4. Santos RA, Simoes e Silva AC, Maric C, Silva biological evaluation of the first selective non-
DM, Machado RP, de Buhr I, Heringer- peptide AT2 receptor agonist. J Med Chem
Walther S, Pinheiro SV, Lopes MT, Bader M, 47(24):5995–6008. doi:10.1021/jm049715t
Mendes EP, Lemos VS, Campagnole-Santos 12. Slavik R, Herde AM, Bieri D, Weber M, Schibli
MJ, Schultheiss HP, Speth R, Walther T R, Kramer SD, Ametamey SM, Mu L (2015)
(2003) Angiotensin-(1–7) is an endogenous Synthesis, radiolabeling and evaluation of
ligand for the G protein-coupled receptor mas. novel 4-oxo-quinoline derivatives as PET trac-
Proc Natl Acad Sci U S A 100(14):8258– ers for imaging cannabinoid type 2 receptor.
8263. doi:10.1073/pnas.1432869100 Eur J Med Chem 92:554–564. doi:10.1016/j.
5. Fraga-Silva RA, Ferreira AJ, Dos Santos RA ejmech.2015.01.028
(2013) Opportunities for targeting the 13. Ni R, Gillberg PG, Bergfors A, Marutle A,
angiotensin-converting enzyme 2/angioten- Nordberg A (2013) Amyloid tracers detect
sin-(1–7)/mas receptor pathway in hyperten- multiple binding sites in Alzheimer’s disease
sion. Curr Hypertens Rep 15(1):31–38. brain tissue. Brain 136(Pt 7):2217–2227.
doi:10.1007/s11906-012-0324-1 doi:10.1093/brain/awt142
6. Steckelings UM, Paulis L, Namsolleck P, 14. Auld DS, Farmen MW, Kahl SD, Kriauciunas A,
Unger T (2012) AT2 receptor agonists: hyper- McKnight KL, Montrose C, Weidner JR (2012)
tension and beyond. Curr Opin Nephrol Receptor binding assays for HTS and drug dis-
Hypertens 21(2):142–146. doi:10.1097/ covery. In: Sittampalam GS, Coussens NP et al
MNH.0b013e328350261b (eds) Assay guidance manual. Eli Lilly &
7. Sridharan R, Zuber J, Connelly SM, Mathew Company and the National Center for Advancing
E, Dumont ME (2014) Fluorescent approaches Translational Sciences, Bethesda, MD
for understanding interactions of ligands with 15. Maguire JJ, Kuc RE, Davenport AP (2012)
G protein coupled receptors. Biochim Biophys Radioligand binding assays and their analysis. In:
Acta 1838(1 Pt A):15–33. doi:10.1016/j. Davenport AP (ed) Receptor binding techniques,
bbamem.2013.09.005 vol 897, 3rd edn. Springer, New York, NY
8. de Jong LA, Uges DR, Franke JP, Bischoff R 16. Hulme EC, Trevethick MA (2010) Ligand
(2005) Receptor-ligand binding assays: tech- binding assays at equilibrium: validation and
nologies and applications. J Chromatogr B interpretation. Br J Pharmacol 161(6):1219–
Anal Technol Biomed Life Sci 829(1–2):1–25. 1237. doi:10.1111/j.1476-5381.2009.00604.x
doi:10.1016/j.jchromb.2005.10.002 17. Kim TK, Eberwine JH (2010) Mammalian cell
9. Lautner RQ, Villela DC, Fraga-Silva RA, Silva transfection: the present and the future. Anal
N, Verano-Braga T, Costa-Fraga F, Jankowski Bioanal Chem 397(8):3173–3178. doi:10.1007/
J, Jankowski V, Sousa F, Alzamora A, Soares E, s00216-010-3821-6
Barbosa C, Kjeldsen F, Oliveira A, Braga J, 18. Jankowski V, Tolle M, Santos RA, Gunthner
Savergnini S, Maia G, Peluso AB, Passos-Silva T, Krause E, Beyermann M, Welker P, Bader
D, Ferreira A, Alves F, Martins A, Raizada M, M, Pinheiro SV, Sampaio WO, Lautner R,
Paula R, Motta-Santos D, Klempin F, Pimenta Kretschmer A, van der Giet M, Zidek W,
A, Alenina N, Sinisterra R, Bader M, Jankowski J (2011) Angioprotectin: an angio-
Campagnole-Santos MJ, Santos RA (2013) tensin II-like peptide causing vasodilatory
Discovery and characterization of alamandine: effects. FASEB J 25(9):2987–2995.
a novel component of the renin-angiotensin doi:10.1096/fj.11-185470
system. Circ Res 112(8):1104–1111. 19. Pinheiro SV, Simoes e Silva AC, Sampaio WO, de
doi:10.1161/CIRCRESAHA.113.301077 Paula RD, Mendes EP, Bontempo ED, Pesquero
JB, Walther T, Alenina N, Bader M, Bleich M, involves mas-mediated NO release from plate-
Santos RA (2004) Nonpeptide AVE 0991 is an lets. Mol Med 14(1–2):28–35. doi:10.2119/
angiotensin-(1–7) receptor mas agonist in the 2007-00073.Fraga-Silva
mouse kidney. Hypertension 44(4):490–496. 22. Leal MC, Pinheiro SV, Ferreira AJ, Santos RA,
doi:10.1161/01.HYP.0000141438.64887.42 Bordoni LS, Alenina N, Bader M, Franca LR
20. Savergnini SQ, Beiman M, Lautner RQ, de (2009) The role of angiotensin-(1–7) receptor
Paula-Carvalho V, Allahdadi K, Pessoa DC, mas in spermatogenesis in mice and rats.
Costa-Fraga FP, Fraga-Silva RA, Cojocaru G, J Anat 214(5):736–743. doi:10.1111/
Cohen Y, Bader M, de Almeida AP, Rotman j.1469-7580.2009.01058.x
G, Santos RA (2010) Vascular relaxation, anti- 23. Santos RA, Castro CH, Gava E, Pinheiro SV,
hypertensive effect, and cardioprotection of a Almeida AP, Paula RD, Cruz JS, Ramos AS,
novel peptide agonist of the MAS receptor. Rosa KT, Irigoyen MC, Bader M, Alenina N,
Hypertension 56(1):112–120. doi:10.1161/ Kitten GT, Ferreira AJ (2006) Impairment of
HYPERTENSIONAHA.110.152942 in vitro and in vivo heart function in angioten-
21. Fraga-Silva RA, Pinheiro SV, Goncalves AC, sin-(1–7) receptor MAS knockout mice.
Alenina N, Bader M, Santos RA (2008) The Hypertension 47(5):996–1002. doi:10.1161/
antithrombotic effect of angiotensin-(1–7) 01.HYP.0000215289.51180.5c
Chapter 14
A Primer to Angiotensin Peptide Isolation, Stability,

and Analysis by Nano-Liquid Chromatography
with Mass Detection
Mariola Olkowicz, Stefan Chlopicki, and Ryszard T. Smolenski
Abstract
The renin-angiotensin system (RAS) is an important element of cardiovascular and renal physiology and
targeting the RAS by renin inhibitors, angiotensin (Ang) converting enzyme (ACE) inhibitors and Ang II
type 1 receptor antagonists is effective in the treatment of hypertension, heart failure, and atherosclerosis.
Quantification of Ang peptides is critical to establish the status of the RAS, but it is challenging due to low
Ang peptides concentrations (fmol/mL or fmol/g), abundance of interfering substances, post sampling
conversions, and difficulties with the specificity of the assay.
In this chapter, we describe a new nano-LC/MS-based methodology for comprehensive, specific,
sensitive, and accurate quantification of Ang peptides profile in plasma and tissue. We optimized sample
pretreatment method (protein removal (acetonitrile precipitation) followed by solid-phase extraction (C18
silica bonded phase)), chromatographic conditions (reversed-phase nanochromatography with preconcen-
tration), and mass detection (multiple reaction monitoring) of nine peptides: Ang-(1–12), Ang I (1–10),
Ang-(1–9), Ang II (1–8), [Ala1]-Ang II, Ang III (2–8), Ang IV (3–8), Ang-(1–7), and [Ala1]-Ang-(1–7).
Assessment of plasma and cardiac concentrations of Ang peptides in genetically modified atherosclerotic
apolipoprotein E/LDL receptor double knockout (ApoE−/−/LDLR−/−) mice vs. wild types revealed
changes in renin-angiotensin system consistent with an overactivation of ACE and impairment of ACE2.
The method could be easily adopted for high-throughput analysis and for use in clinical applications such
as diagnosis of the RAS abnormalities or monitoring of the RAS inhibition-based therapies.
Key words Renin-angiotensin system, Angiotensin peptides, Sample handling and extraction,
Nanoflow liquid chromatography, Multiple reaction monitoring, Angiotensin concentrations in
plasma and tissues
1 Introduction
The renin-angiotensin system (RAS) has been originally character-

ized as a set of circulating factors that play a critical role in cardio-
vascular and renal physiology [1, 2]. Further research revealed that
disturbances of this system lead to hypertension, atherosclerosis,
cardiac hypertrophy, heart failure, and renovascular disorders [3–5].
175
176 Mariola Olkowicz et al.
Angiotensin II (Ang II), the primary effector of the RAS is involved

not only in control of blood pressure but is also affecting the func-
tion of most of the organs (including heart, blood vessels, kidney,
and brain) causing mostly deleterious effects. On the other hand,
the counterregulatory axis of the RAS, centered on the actions of
angiotensin-converting enzyme 2 (ACE2), and the resultant Ang-
(1–7) and Ang-(1-9) might oppose some harmful effects of Ang II
[6–9]. Moreover, newly identified RAS family peptides: Ang A and
alamandine further extend the complexities in understanding the
cardiovascular/renal physiopathology of the RAS [10–12].
Altogether, the RAS could be described as a complex array of com-
ponents with diverse biological actions that can be functionally
partitioned into distinct peptidases, peptide signaling molecules,
and receptors that are downstream from the initial processing of
angiotensinogen to form Ang I and Ang-(1–12) precursor pep-
tides [13] (Fig. 1).
In view of the importance of the RAS and increased concerns
on reliable quantification of endogenous angiotensin peptides in
various biological compartments, a new approach is needed to
accurately characterize the status of this system. The immunoassay-
based methods such as radioimmunoassay (RIA) or enzyme-linked
immunosorbent assay (ELISA) are valuable tools and currently
remain the most commonly used procedure to quantitate individ-
ual angiotensins [14–17]. However, limitations of these assays
? Classical RAS Pathway Protective Arm of the RAS

Ang-(1–12) Aogen
Chymase
Renin
ACE2
Ang II (1–8) Ang I (1–10) Ang -(1–9)
DC ACE ACE2 ACE

DC
[Ala1]-Ang II Ang II (1–8) Ang -(1–7)
APA
DC
APN
IRAP Ang IV (3–8) Ang III (2–8) [Ala1]-Ang -(1–7)
AT1 AT2 MAS MRGD
Brain RAS Pathway

Vasoconstriction, inflammation, Vasodilation, anti-inflammatory,
growth, fibrosis antihypertrophic, and antifibrotic effects
Fig. 1 Simplified view of the renin–angiotensin system (RAS) cascade. Aogen angiotensinogen, Ang angiotensin,
ACE angiotensin-converting enzyme, ACE2 angiotensin-converting enzyme 2, APA aminopeptidase A, APN
aminopeptidase N, DC decarboxylase, AT1 angiotensin type 1 receptor, AT2 angiotensin type 2 receptor, IRAP
insulin-regulated aminopeptidase, Mas Ang-(1–7) receptor Mas, MrgD Mas related G-protein coupled receptor D
Angiotensin Quantification by NanoLC/MS 177
such as variable specificity that dependent on the characteristics for

each antibody, laborious and time-consuming procedures as well as
problems with recovery assessment made search for new technolo-
gies a high priority. An alternative approach is based on application
of liquid chromatography/mass spectrometry (LC/MS) that
enables detecting peptide fragmentation signatures/mass transi-
tions that are highly specific for individual angiotensins. This in
combination with high sensitivity and high-throughput capability
for sample processing and analytical step make this method ideal
for such analysis [18–20].
This chapter provides protocols from the initial sample pro-
cessing to angiotensin peptides analysis by nano-LC-MS/MS as
well as their application in the study of the systemic and tissue RAS
abnormalities. The method proposed was used for simultaneous
detection and quantification of nine angiotensins in plasma and
heart of wild type (C57BL/6J) and atherosclerotic (ApoE−/−/
LDLR−/−) mice, indicating substantial dysregulation of the RAS in
these atherosclerosis-prone mice.
2 Materials
Prepare all solutions for mass spectrometry analysis using ultrapure

18 MΩ water (obtained by reverse osmosis/deionization) and ana-
lytical/MS grade reagents. MS analysis can be performed with any
mass spectrometer with MS/MS capabilities. In this report con-
centrations and amounts of reagents and solutions were optimized
for a nanoflow liquid chromatography system coupled online to a
TSQ Vantage triple quadrupole mass spectrometer.
2.1 Equipment 1. Water purification system.

2. Ultrasonic bath.
3. Digital vortex mixer.
4. Eppendorf microcentrifuge.
5. SpeedVac concentrator.
6. Vac elut 20 manifold/Solid phase extraction system.
7. UltiMate 3000 Rapid Separation nano-LC system.
8. ChipMate nanoelectrospray ion source.
9. TSQ Vantage EMR triple quadrupole mass spectrometer.
10. Xcalibur version 2.1 software.
2.2 Reagents 1. Peptide standards: Ang-(1–12), Ang I (1–10), Ang-(1–9),

Ang II (1–8), [Ala1]-Ang II, Ang III (2–8), Ang IV (3–8),
Ang-(1–7), [Ala1]-Ang-(1–7) and internal standard: [Asn1,
Val5]-Ang II (Phoenix Europe GmbH, Karlsruhe, Germany
and Sigma-Aldrich, Poznan, Poland).
2. Protease Inhibitor Cocktail: 10 mM ethylenediaminetetraacetic

acid (EDTA) disodium salt, 5 mM 1,10-phenanthroline, 20
mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 400
μM amastatin, 150 μM pepstatin A, 100 μM thiorphan, and
500 μM aliskiren.
3. 0.9% solution of sodium chloride.
4. Protein Extraction Solution: 0.9% saline, 0.1 M HCl, 10% pro-
tease inhibitor cocktail (v/v).
5. BCA (bicinchoninic acid) protein assay kit.
2.3 Chromatogra- 1. Blood collection tubes containing EDTA.

phic Accessories and 2. Low-binding 0.5/1.5-mL microcentrifuge tubes and low-
Other Materials retention pipette tips.
3. C18 silica bonded SPE cartridges (100 mg/1 mL), Sep-Pak
type.
4. Trapping column for the nanoflow LC system: Acclaim
PepMap100 C18, 2 cm × 75 μm, 3 μm, 100 Å.
5. Separation column: Acclaim PepMap100 RSLC C18, 150 mm
× 75 μm I.D., 2 μm, 100 Å.
3 Methods
3.1 Determination The whole blood was collected via cardiac puncture into chilled
of Plasma Angiotensin tubes (containing EDTA as anticoagulant) from 8-month-old male
Content ApoE/LDLR double knockout (n = 7) and wild type C57BL/6J
mice (n = 5) under isoflurane anesthesia (see Notes 1 and 2).
Afterwards, the samples were immediately mixed with protease
inhibitor cocktail at a ratio of 9:1 (v/v) and centrifuged at 2000 ×
g, 4 °C for 20 min to isolate plasma (see Notes 3–8). After the
centrifugation, the resulting plasma was transferred into the Protein
LoBind tubes, split into aliquots and if not used stored at −80 °C
(see Note 9).
3.1.1 Preparation 1. Prepare the stock solution of each standard at the concentra-
of the Standard Solutions, tion of 1 mg/mL in the amber glass vials, 50% acetoni-
Calibration Standards (CS), trile (ACN)/1% acetic acid and keep frozen at −80 °C until
Internal Standards (IS), used (see Notes 10 and 11).
and Quality Control (QC) 2. Prepare the working standard solutions by dilution of the stock
Samples solutions with deionized water to obtain concentration in the
range of 50–2500 pg/mL.
3. Mix and dilute of IS stock solution with water to obtain the IS
working solution at 2000 pg/mL.
4. Prepare the calibration standards/or quality control samples
just before use by spiking 70 μL (pooled blank) plasma with 20
μL appropriate working standard solution and 10 μL IS – [Asn1,

Val5]-Ang II (2000 pg/mL).
5. Calibration curve standards should be made in at least six levels
covering the expected range. In our protocol the standards
were prepared at: 5; 10; 50; 100; 250; 400; 500 pg/mL
(IS – 200 pg/mL) concentrations, respectively.
6. Pretreat each of the samples in accordance with the protocol
outlined below.
3.2 Sample Sample cleanup procedure consisted of two stages: (1) protein pre-
Extraction cipitation and (2) solid-phase extraction that were performed as
outlined below (see Note 12). Samples should be kept on ice unless
otherwise specified.
3.2.1 Protein 1. Mix 70 μL aliquot of each plasma sample (except for the cali-
Precipitation bration standards/or quality control samples) with 20 μL of
deionized water in order to make the sample volume and
condition identical with calibration standards/quality control
samples.
2. Add 10 μL of the IS working solution, mix rapidly and extract
with 400 μL of pure, HPLC-grade ACN.
3. Vortex the mixture and allow to stand at −20 °C for 45 min.
4. Remove the precipitated proteins by centrifugation at 20,000 × g
for 20 min and evaporate the collected supernatant to dryness.
3.2.2 Solid-Phase 1. Dissolve the residues obtained in 50 μL 1% (v/v) acetic acid in

Extraction water, vortex and sonicate two times for 2 min to facilitate pep-
tides dissolution and then centrifuge (20,000 × g, 10 min, 4
°C) to separate any insoluble particles.
2. Precondition each cartridge with 2 mL ACN followed by 2 mL
of water (both containing 1% (v/v) acetic acid) at a suction
pressure of 5 mmHg.
3. Apply the thawed sample (~50 μL) to the cartridge and wash
with 2 mL of 1% (v/v) acetic acid in water.
4. Elute the angiotensin peptides retained at the columns with
0.5 mL of H2O/ACN, 20:80 (v/v) with 1% (v/v) acetic acid
(suction pressure less than 5 mmHg) into the Protein LoBind
polypropylene tubes.
5. Concentrate the eluates using a vacuum centrifuge, freeze ali-
quots at −80 °C and lyophilize them for l5–18 h until com-
pletely dry.
6. Reconstitute dried peptide samples with 35 μL of 1% (v/v)
acetic acid in water and analyze using LC/MS system.
3.3 Analysis 1. Perform two replicate injections (3 μL) per sample in the
by Nano-LC-MS/MS mobile phase – ACN/H2O, 2:98 (v/v) containing 1% (v/v)
acetic acid, at a flow rate of 5 μL/min for 5 min.
2. Load peptides onto a trapping column to preconcentrate and
desalt samples.
3. Perform a reversed-phase liquid chromatography (RPLC)
using UltiMate nano-LC system on a C18 column in a gradi-
ent of phase A (acetic acid (1%, v/v) in water) and phase B
(acetic acid (1%, v/v) in acetonitrile).
4. Elute the peptides at a flow rate of 300 nL/min in a continu-
ous acetonitrile gradient: 2% B for 5 min, 2–98% B for 15 min,
and 98% B for 5 min.
5. Set the mass spectrometer to operate in positive ion mode and
adjust other parameters depending on the type/model used.
Measure two different mass transitions for each peptide includ-
ing internal standard. Analytical method details as well as the
MS/MS transitions monitored in our protocol are presented
in Table 1 (see Notes 13 and 14).
6. Analyze the peptides quantitatively by means of the standard
addition calibration.
7. In our case, Xcalibur v. 2.1 software was used for control of
both, the Ultimate nano-LC and TSQ Vantage system.
Representative LC/MS chromatograms of selected angioten-
sin peptides and internal standard from the plasma sample of
ApoE−/−/LDLR−/−mouse are presented on Fig. 2.
3.4 Determination As in the case of determinations in plasma described above, heart speci-
of Cardiac Angiotensin mens were obtained from 8-month-old male ApoE/LDLR double
Content knock-out and wild type C57BL/6J mice. Hearts were rapidly removed,
immediately briefly rinsed with isotonic saline by retrograde perfusion.
Then, left ventricles were dissected, weighed, and homogenized on ice
in protein extraction solution. The protocol has been optimized for
mice cardiac ventricles weighing approximately 50 mg (see Note 15).
3.4.1 Sample Extraction 1. Homogenize the tissues in extraction solution keeping the

and Nano-LC/MS Analysis ratio: 1:4 (w/v), tissue:solution.
2. Centrifuge the homogenate at 4 °C, 20,000 × g for 20 min.
3. Determine the total protein content of an aliquot of the homog-
enate using the BCA protein assay with bovine serum albumin
as a standard.
4. Perform peptide extraction and nano-LC-MS/MS analysis as
described previously in Subheadings 3.2–3.3.
Representative plasma angiotensin profile and cardiac angio-
tensin profile determined in 8-month-old male atherosclerotic
ApoE−/−/LDLR−/− and wild type C57BL/6J mice are presented
on Figs. 3 and 4, respectively (see Notes 16 and 17).
Table 1
Molecular description and multiple reaction monitoring (MRM) settings for detection of angiotensin
peptides
Precursor Charge Product Collision

Peptide Amino acid sequence MW (Da) ion (m/z) state iona (m/z) energy (eV)
Ang-(1–12) DRVYIHPFHLLY 1572.8 524.92 3+ 583.58 18
(b92+) 33
109.65
Ang I (1–10) DRVYIHPFHL 1296.5 432.91 3+ 647.19 17
(b5+) 32
109.79
Ang-(1–9) DRVYIHPFH 1183.3 395.10 3+ 156.03 20
(y+) 33
109.85
Ang II (1–8) DRVYIHPF 1046.2 523.85 2+ 784.26 19
(b6+) 20
262.74
Ang A/ ARVYIHPF 1002.2 501.62 2+ 740.36 18
[Ala1]-Ang II (b6+) 19
263.02
Ang-(1–7) DRVYIHP 899.0 450.18 2+ 647.41 19
(b5+) 28
109.97
Alamandine/ ARVYIHP 855.0 428.09 2+ 603.25 17
[Ala1]- (b5+) 23
Ang-(1–7) 109.77
Ang III (2–8) RVYIHPF 931.1 466.19 2+ 669.46 17
(b5+) 18
262.73
Ang IV (3–8) VYIHPF 774.9 388.17 2+ 262.85 12
(b2+) 20
136.07
[Asn1, Val5]- NRVYVHPF 1031.2 516.19 2+ 769.34 19
Ang II (IS) (b6+) 20
263.25
Transition used for quantification of each peptide in biological sample refers to the transition in bold
a
4 Notes
1. Appropriate handling and subsequent extraction of plasma

and tissue samples are critical to obtain reliable data on angio-
tensin levels, independent of the analytical method that is used
for detection.
2. Regardless of the detection method used for angiotensin
determination, such as RIA, RP-HPLC-RIA, or HPLC/MS,
RT: 0.00 - 25.00 SM: 9G

RT: 11.12
100
[Ala1]-Ang-(1–7)
50
RT: 11.79
0
100 RT: 11.49
50 Ang-(1–7)
0
RT: 11.84
100
50
[Asn1, Val5]-Ang II
0
100 RT: 11.88
Ang-(1–9)
50
0
100 RT: 12.29
50 Ang III
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
RT: 0.00 - 25.00 SM: 9G

RT: 12.36
100
50 [Ala1]-Ang II
0
100 RT: 12.87
50
Ang II
0
100 RT: 12.88
50 Ang-(1–12)
0
100 RT: 13.05
Ang I
50
0
100 RT: 13.17
Ang IV
50
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
Fig. 2 Representative extract ion multiple reaction monitoring (MRM) chromatograms of nine angiotensin pep-
tides and internal standard ([Asn1, Val5]-Ang II) obtained by analysis of a selected (ApoE−/−/LDLR−/−) unspiked
plasma sample using nano-LC-MS/MS
improper sample handling and extraction may yield errone-

ously higher or lower concentrations.
3. Blood or tissue samples for angiotensin quantification should
be immediately spiked with a potent protease inhibitor cock-
tail that enables efficient stabilization of angiotensin
metabolites.
4. Because of above, the sampling time has to be kept as short as

possible which could be challenging in some animal studies.
5. Optimal protease inhibitor cocktail blocking all angiotensin
conversion enzymes has not been identified and still evolves
with our expanding knowledge on peptide generation/degra-
dation pathways.
6. Specific targets of each component of the Protease Inhibitor
Cocktail are: EDTA and 1,10-phenanthroline act against
metalloproteases; AEBSF acts to inhibit serine proteases,
including trypsin, chymotrypsin-like enzymes, and plasmin
among others; amastatin inhibits aminopeptidases, including
aminopeptidase A; pepstatin A is an inhibitor of acid proteases
(aspartyl peptidases) such as pepsin, renin, cathepsin D; thior-
phan acts against neutral endopeptidase (NEP); and aliskiren
is an inhibitor of renin activity.
7. Metalloenzymes such as ACE, ACE2, neprilysin (NEP),
aminopeptidase A are the dominant class of peptidases and
occurrence of divalent cations is essential for the preservation
***
500
WT
300 ApoE-/-/LDLR-/-
300
***
200
[fmol/mL]
*** *
** *** ***
100
0
)
)
2)
)
)
ne
10
-8
-7
-9
-8
-8
-1
di
ng
(2
-(1
-(1
(1
(3
1-
-(1
an
I(
III
II
IV
ng
ng
m
ng
ng
ng
A
A
la
ng
ng
A
A
A
A
Fig. 3 Plasma angiotensin profile in 8-month-old WT (n = 5) and 8-month-old ApoE−/−/LDLR−/− (n = 7) mice.

Values were presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT (age-matched) group
350
WT
*
ApoE-/-/LDLR-/-
300
***
250
[fmol/g wet tissue]
200
**
150
**
***
** *
100
***
50
0
ne
)
)
2)
)
A
)
10
-8
-7
-9
-8
-8
-1
di
ng
(2
-(1
-(1
(1
(3
1-
-(1
an
I(
III
II
IV
ng
ng
m
ng
ng
ng
A
A
la
ng
ng
A
A
A
A
Fig. 4 Cardiac angiotensin profile in 8-month-old WT (n = 5) and 8-month-old ApoE−/−/LDLR−/− (n = 7) mice.

Values were presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT (age-matched) group
of biological activity of these enzymes. However, strong che-

lating agents such as EDTA or phenanthroline, usually used to
abolish peptide metabolism, are not sufficient to completely
suppress the metalloenzyme activity and more specific inhibi-
tors should be used.
8. When angiotensin–converting enzyme is inhibited, it leads to
high renin and Ang I levels; speed of sample processing, quick
freezing, and the use of a typical renin inhibitor – pepstatin A
may not be sufficient to completely block renin activity, and a
more potent and specific renin inhibitor such as enalkiren,
aliskiren should be considered.
9. Before sample collection, animals should be maintained in a
calm environment and anesthetic agents such as pentobarbital
that significantly activate the RAS should be avoided.
10. Many earlier studies highlighted a significant loss of angioten-
sin peptides during storage, that was also noticed in the stan-
dard stock solutions [16, 18]. The reasons for this loss are not
clear but could be due to binding to storage vial walls. To

avoid/reduce sample depletion by nonspecific peptide adsorp-
tion, only low-binding 0.5/1.5-mL vials and low-retention
pipette tips were used. Moreover, fresh standards for each
analysis series were used. Also, the samples were extracted and
analyzed immediately to shorten the sample preparation time.
11. To optimize angiotensin recoveries and prevent irreversible
adsorption of the peptides to the polypropylene tubes, the
Tris-albumin-buffer coated tubes are often used [16, 21] but
this procedure is more compatible with RIA-based assays.
12. So far, various extraction methods that block peptide metabo-
lism, deplete the sample from contaminating proteins and
other compounds that may interfere with the assay and enrich
peptide content in a manageable volume have been developed
[22]. They usually include protein precipitation (MeOH,
HCl–EtOH, guanidine thiocyanate) and subsequent solid-
phase extraction (C18 or phenyl). In our procedure, we have
used acetonitrile precipitation followed by extraction on the
C18 silica bonded SPE cartridges. Peptide recovery with our
protocol for plasma samples typically ranges from 75 to 90%,
whereas in tissues recoveries are generally lower in the 60–80%
range.
13. RIA and ELISA are most common methods used to character-
ize angiotensins [22, 23]. Specificity of these methods is highly
dependent on the characteristics for each antibody, as well as
the proper sample extraction and assay conditions. The anti-
bodies used in RIAs or ELISAs are typically directed against
the unique COOH-terminus of the peptide, and distinction of
the NH2-terminal metabolites without additional chromato-
graphic separation step can be problematic. LC/MS is there-
fore a more specific tool.
14. Our nano-LC-MS/MS approach enables to quantify compre-
hensively nine angiotensins in plasma and tissue samples with
a sensitivity comparable to RIA and superior accuracy, high-
throughput potential, and robustness.
15. Lower limits of quantification for angiotensin metabolites esti-
mated by our nano-LC-MS/MS method are: 5 fmol/mL in
plasma and 5 fmol/g in tissue samples.
16. The optimized nano-LC-MS/MS method allows the measure-
ment of Ang metabolites in the same sample, providing a highly
reliable estimate of their ratios. A specific metabolic pattern,
called “RAS-fingerprint” could be drawn (Fig. 5) that provides
visual information on the RAS alterations and possible changes
in the angiotensin processing enzymes in the sample. This type
of analysis may be well used for the monitoring of the efficacy of
PLASMA WT ApoE-/-/LDLR-/-
ACE2 Ang I Ang I
ACE2
Ang-(1–9) Ang-(1–9)
Ang-(1–12) ACE
ACE Ang-(1–12)
Chymase ACE
ACE Chymase
Ang II Ang II
ACE2 ACE2
APA APA
Ang-(1–7) DC Ang III Ang-(1–7) Ang III

DC
1
ACE2 [Ala ]- APN DC APN
DC Ang II ACE2 [Ala1]-
Ang IV Ang II
1 1
[Ala ]-Ang-(1–7) [Ala ]-Ang-(1–7) Ang IV
HEART WT ApoE-/-/LDLR-/-
Ang I
Ang I ACE2
ACE2
Ang-(1–9) Ang-(1–12)
Ang-(1–9) ACE ACE
Ang-(1–12)
ACE
ACE Chymase
Chymase ACE2
Ang II Ang II
ACE2 APA
APA
Ang-(1–7)
Ang-(1–7) DC Ang III Ang III
DC
ACE2 DC ACE2 [Ala1]-

[Ala1]- APN APN
DC Ang II Ang II
1
[Ala1]-Ang-(1–7) Ang IV [Ala ]-Ang-(1–7) Ang IV
Fig. 5 Angiotensin peptide levels in WT and ApoE−/−/LDLR−/− mice depicted using the diameter of the circles to
reflect their concentration in plasma (upper panel) and heart (lower panel). The peptides formed by ACE2
cleavage are in very low concentrations (green) after the RAS dysregulation in atherosclerotic mice as opposed
to those formed by ACE/chymase cleavage (red). Ang angiotensin, ACE angiotensin-converting enzyme, ACE2
angiotensin-converting enzyme 2, APA aminopeptidase A, APN aminopeptidase N, DC decarboxylase
the RAS-targeted therapies that constitute a major treatment

modality in cardiovascular diseases [22, 23].
17. The accurate and highly sensitive procedure presented here
combined with appropriate sampling conditions may bring
our understanding of the RAS to the next level, although the
results of nano-LC/MS and classical (RIA, ELISA) quantifica-
tion methods still should be rigorously compared.
Acknowledgments
This work was supported by the European Union from the resources
of the European Regional Development Fund under the Innovative
Economy Programme (a grant coordinated by JCET-UJ, No.
POIG.01.01.02-00-069/09) and TEAM programme of the
Foundation for Polish Science (TEAM/2011-8/7).
References
1. Mehta PK, Griendling KK (2007) Angiotensin 13. Ferrario CM (2010) New physiological con-
II cell signaling: physiological and pathological cepts of the renin-angiotensin system from the
effects in the cardiovascular system. Am investigation of precursors and products of
J Physiol Cell Physiol 292:C82–C97 angiotensin I metabolism. Hypertension
2. Balakumar P, Jagadeesh G (2014) A century 55:445–452
old renin-angiotensin system still grows with 14. Allred AJ, Chappell MC, Ferrario CM, Diz DI
endless possibilities: AT1 receptor signaling (2000) Differential actions of renal ischemic
cascades in cardiovascular physiopathology. injury on the intrarenal angiotensin system.
Cell Signal 26:2147–2160 Am J Physiol Renal Physiol 279:F636–F645
3. Te Riet L, van Esch JH, Roks AJ, van den 15. Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani
Meiracker AH, Danser AH (2015) G, Brunner HR, Nussberger J (2000) Increased
Hypertension: renin-angiotensin-aldosterone cardiac angiotensin II levels induce right and
system alterations. Circ Res 116:960–975 left ventricular hypertrophy in normotensive
4. Oparil S, Schmieder RE (2015) New mice. Hypertension 35:985–991
approaches in the treatment of hypertension. 16. Nussberger J, Brunner DB, Nyfeler JA, Linder
Circ Res 116:1074–1095 L, Brunner HR (2001) Measurement of immu-
5. Passos-Silva DG, Brandan E, Santos RA (2015) noreactive angiotensin-(1–7) heptapeptide in
Angiotensins as therapeutic targets beyond human blood. Clin Chem 47:726–729
heart disease. Trends Pharmacol Sci 17. Yamamoto K, Ohishi M, Katsuya T et al (2006)
36:310–320 Deletion of angiotensin-converting enzyme 2
6. Donoghue M, Hsieh F, Baronas E et al (2000) accelerates pressure overload-induced cardiac
A novel angiotensin-converting enzyme- dysfunction by increasing local angiotensin
related carboxypeptidase (ACE2) converts II. Hypertension 47:718–726
angiotensin I to angiotensin 1-9. Circ Res 18. Lortie M, Bark S, Blantz R, Hook V (2009)
87:E1–E9 Detecting low-abundance vasoactive peptides
7. Keidar S, Kaplan M, Gamliel-Lazarovich A in plasma: progress toward absolute quantita-
(2007) ACE2 of the heart: from angiotensin I tion using nano liquid chromatography-mass
to angiotensin (1–7). Cardiovasc Res spectrometry. Anal Biochem 394:164–170
73:463–469 19. Olkowicz M, Radulska A, Suraj J et al (2015)
8. McKinney CA, Fattah C, Loughrey CM, Development of a sensitive, accurate and robust
Milligan G, Nicklin SA (2014) liquid chromatography/mass spectrometric
Angiotensin-(1–7) and angiotensin-(1–9): method for profiling of angiotensin peptides in
function in cardiac and vascular remodelling. plasma and its application for atherosclerotic
Clin Sci (Lond) 126:815–827 mice. J Chromatogr A 1393:37–46
9. Jiang F, Yang J, Zhang Y et al (2014) 2 0. Olkowicz M, Chlopicki S, Smolenski RT
Angiotensin-converting enzyme 2 and angio- (2015) Perspectives for angiotensin profiling
tensin 1–7: novel therapeutic targets. Nat Rev with liquid chromatography/mass spectrome-
Cardiol 11:413–426 try to evaluate ACE/ACE2 balance in endo-
10. Jankowski V, Vanholder R, van der Giet M thelial dysfunction and vascular pathologies.
et al (2007) Mass-spectrometric identifica- Pharmacol Rep 67:778–785
tion of a novel angiotensin peptide in human 21. Nussberger J, Brunner DB, Waeber B, Brunner
plasma. Arterioscler Thromb Vasc Biol HR (1985) True versus immunoreactive angio-
27:297–302 tensin II in human plasma. Hypertension
11. Lautner RQ, Villela DC, Fraga-Silva RA et al 7:I1–I7
(2013) Discovery and characterization of ala- 2 2. Chappell MC (2016) Biochemical evaluation
mandine: a novel component of the renin- of the renin-angiotensin system: the good, bad,
angiotensin system. Circ Res 112:1104–1111 and absolute? Am J Physiol Heart Circ Physiol
12. Etelvino GM, Peluso AA, Santos RA (2014) 310:H137–H152
New components of the renin-angiotensin sys- 23. Poglitsch M, Sturrock ED, Danser AH (2016)
tem: alamandine and the MAS-related G Letter to the editor: angiotensin quantification
protein-coupled receptor D. Curr Hypertens by mass spectrometry. Am J Physiol Heart Circ
Rep 16:433 Physiol 310(33):H452–H453
Chapter 15
Analysis of Angiotensin Metabolism in the Kidney

Using Mass Spectrometry
Nadja Grobe and Khalid M. Elased
Abstract
The renin angiotensin system (RAS) is a highly complex enzymatic system consisting of multiple peptide
hormones, enzymes, and receptors. A thorough characterization of angiotensin peptide metabolism is
crucial for understanding pathological states associated with an imbalanced RAS. Here, we describe two
matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) approaches for the assess-
ment of in vitro and in situ RAS enzymatic activities in the kidney using the natural angiotensin peptide
substrates. These MS techniques demonstrate high specificity and are superior over conventional spectro-
photometric or colorimetric assays since multiple proteolytic cleavage sites can be detected, thus unravel-
ing the complexity of the RAS.
Key words Mass spectrometry, MALDI, Imaging, Renin-angiotensin system, Kidney, Enzyme
activity, Angiotensin metabolism, ACE, ACE2, NEP
1 Introduction
The renin-angiotensin system (RAS) plays a fundamental role in

the pathophysiology of hypertension and the onset and progres-
sion of renal injury. However, the intrarenal RAS network and the
role of specific peptidases in mediating renal and cardiovascular
health are incompletely understood partly due to limited analytical
techniques that are able to unravel its complexity. Mass spectrom-
etry (MS) is a fundamental technique that has been widely used to
characterize cell systems by assessing metabolites, peptides, and
proteins. Over the last 15 years, several MS approaches have been
developed to measure the RAS, replacing traditional fluorogenic
substrate, colorimetric, ultraviolet, or radioimmunoassay (RIA)
based techniques. Since molecules are ionized and analyzed accord-
ing to their mass-to-charge (m/z) ratios, the natural angiotensin
(Ang) peptide substrates and products resulting from RAS pepti-
dase cleavage are more selectively and specifically detected using
MS. Indeed, Fredline et al. showed for the first time a higher
189
190 Nadja Grobe and Khalid M. Elased
specificity for assessing plasma renin activity using MS as compared

to RIA [1]. MS was also used to characterize various substrates and
multiple proteolytic cleavage sites for the newly discovered angio-
tensin converting enzyme 2 (ACE2), a novel component of the
protective arm of the RAS [2]. We and others developed several
MS-based assays to detect and quantify metabolism of the RAS
peptides in cells, plasma, tissue homogenates, and tissue sections
[3–8]. Although a local RAS exists in many organ systems, we have
extensively focused our studies on the in vitro and in situ metabo-
lism of the intrarenal RAS due to its pivotal role in blood pressure
regulation, salt and acid–base homeostasis, and pathogenesis of
kidney diseases. Compared to other ionization techniques and
liquid chromatography-based approaches, matrix-assisted laser
desorption/ionization (MALDI) MS is uniquely suited for the
characterization of RAS peptidase activities because of a softer
ionization technique, easy-to-follow sample preparation, short
analysis time, and high-throughput capability. Here, we demon-
strate that the intrarenal RAS peptidases and metabolism of several
Fig. 1 Angiotensin metabolism in the kidney detected by mass spectrometry. ACE Ang converting enzyme, NEP
neprilysin, ACE2 Ang converting enzyme 2, APA aminopeptidase A, APN aminopeptidase N, PRCP prolyl
carboxypeptidase, PREP prolyl endopeptidase, TOP thimet oligopeptidase
MS Analysis of Renal Angiotensin Metabolism 191
Ang peptides can be efficiently measured using MALDI MS. Our

current RAS test platform is capable of localizing the multistep
enzymatic cascade of the RAS, starting from the decapeptide Ang
I down to smaller Ang peptides such as Ang-(1-4) (Fig. 1).
2 Materials
Prepare all solutions using ultrapure, LC-MS grade water and

LC-MS grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations when disposing of waste materials.
2.1 MS Analysis 1. Phenylmethanesulfonyl flouride (PMSF): 100 mM PMSF in

of In Vitro Renal dimethyl sulfoxide (DMSO). Store at −20 °C.
Angiotensin 2. Lysis buffer: Dissolve one tablet protease inhibitor in 10 ml
Metabolism Complete Lysis-M EDTA-free lysis buffer and add 250 μl
100 mM PMSF. Store at 4 °C.
3. Kimble Kontes Duall Tissue Grinder, size 22.
4. Bovine serum albumin (BSA) standard for protein assay:
Prepare 1 mg BSA in 1 ml water. Store at −20 °C.
5. 2-(N-morpholino)ethanesulfonic acid (MES) buffer: 0.4 M
MES buffer, pH 6.75. Weigh 3.9 g MES hydrate and add
about 30 ml of water to a 100-ml beaker. Adjust pH using 1 N
NaOH. Make up to 50 ml with water.
6. Bestatin: 500 μM in 100% isopropyl alcohol. Store at −20 °C.
7. Ang peptides: Prepare 1 mg Ang peptide in 1 ml water. Store
at −20 °C.
8. RAS inhibitors: Prepare 10 mM in water (see Note 1). Store at
−20 °C.
9. Stop solution: 10% trifluoroacetic acid (TFA) in water.
10. Diluent: 90% acetonitrile–0.3% TFA (v/v). Mix 900 μl aceto-
nitrile with 30 μl 10% TFA and 70 μl water.

11. Ang internal standards: stable-isotope, [13C5, 15N]-Valine
labeled Ang peptide standards aliquoted as 1000 pmol/μl in
30% acetonitrile with 0.1% formic acid and stored at
−80 °C. Dilute 1:10 with water (see Note 2).
12. MALDI matrix: 10 mg/ml α-cyano-4-hydroxycinnamic acid
(CHCA), 60% methanol, 10% acetone, and 0.3% TFA (see
Note 3). Dissolve 10 mg α-cyano-4-hydroxycinnamic acid in
0.6 ml methanol, 0.1 ml acetone, 0.03 ml 10% TFA, and
0.27 ml water.
13. Peptide calibration standard II: Peptide mixture for calibration
of matrix-assisted laser desorption and ionization time-of-flight
(MALDI-TOF) mass spectrometer (MS) consisting of the

peptides Bradykinin 1–7, Angiotensin II, Angiotensin I,
Substance P, Bombesin, ACTH clip 1–17, ACTH clip 18–39,
and Somatostatin 28. Dissolve one tube in 125 μl 0.1% TFA
and store at −20 °C. Spot 0.5 μl peptide calibration standard
II and mix with 0.5 μl MALDI matrix. Spot peptide calibration
standard II onto each corner of the 384 ground steel MALDI
target plate (see Note 4).
2.2 MS Analysis 1. Ang peptides and RAS inhibitors.

of In Situ Renal 2. Peptide calibration standard II. Spot onto indium-tin-oxide
Angiotensin coated glass slides.
Metabolism
3. MALDI matrix: prepare 20 ml of 10 mg/ml α-cyano-4-
hydroxycinnamic acid, 60% methanol, 10% acetone, and 0.3%
TFA. Dissolve 200 mg CHCA in 12 ml methanol, 2 ml ace-
tone, 0.6 ml 10% TFA, and 5.4 ml of water (see Note 3).
4. Slide moisture chamber.
5. Laboratory incubator set to 37 °C.
6. Vacuum desiccator connected to a vacuum pump.
7. Scanner.
8. 10-ml thin layer chromatography sprayer connected to a
Drierite gas drying unit and nitrogen gas cylinder.
9. Light microscope with 10× magnification.
3 Methods
3.1 MS Analysis 1. Kidney collection: Quickly remove the kidney, transfer into a
of In Vitro Renal 1.5-ml Eppendorf tube, and immediately freeze using liquid
Angiotensin nitrogen or dry ice. Store at −80 °C.
Metabolism 2. Kidney homogenates: Rinse mouse kidney twice in 1 ml ice-
cold Roche EDTA-free protease inhibitor buffer containing
2.5 mM PMSF. Transfer washed kidney to a chilled glass
homogenizer and homogenize on ice using 30 strokes. Transfer
first homogenate to a chilled 5-ml tube. Add 1 ml to the chilled
homogenizer and homogenize remaining kidney pieces with
five strokes. Combine second homogenate with first homoge-
nate in 5-ml tube. Divide combined homogenate into two
chilled 1.5-ml tubes and centrifuge at 9279 × g for 10 min and
4 °C. Combine supernatants in a fresh, chilled 5-ml tube and
prepare 100-μl aliquots to be stored at −80 °C.
3. Protein assay: Determine protein content of kidney homoge-
nate using BSA standard and Bradford reagent according to
the manufacturer’s instructions.
4. Incubation: Mix 20 μg kidney homogenate protein with

12.5 μl 0.4 M MES buffer, pH 6.75, and 2 μl 100 mM PMSF
in a 1.5 ml tube (see Note 5). Make up to 100 μl water. Start
reaction by adding 5 μl 1 mg/ml Ang peptide (see Note 6).
Incubate for 30–120 min at 37 °C in a thermomixer under
shaking at 350 rotations per minute (rpm).
5. Inhibitor studies: Prepare the same reaction mixture as in
Subheading 3.1, step 4, and add 1 μl 10 mM inhibitor (see
Note 7). Make volume up to 100 μl water and pre-incubate
test tubes for 10 min at 37 °C at 350 rpm. Start reaction by
adding Ang peptide. Incubate for 30–120 min at 37 °C in a
thermomixer under shaking at 350 rpm.
6. MS sample preparation: Add 10 μl 10% TFA and 10 μl 1:10
diluted stable isotope labeled Ang internal standards to reac-
tion mixture. Take out 2 μl and mix with 18 μl of diluent con-
sisting of 90% acetonitrile containing 0.3% TFA, vortex,
centrifuge, spot 0.5 μl onto a 384 MALDI target plate and mix
with 0.5 μl MALDI matrix.
7. MS acquisition: Mass calibrate by collecting 200 laser shots of
Bruker peptide calibration standard II using an Autoflex III
smartbeam MALDI TOF/TOF instrument (Bruker Daltonics,
MA, USA). Operate mass spectrometer with positive polarity
in reflectron mode and analyze spots using the method “RP-
pepmix.” Acquire a total of 2000–3000 laser shots (2–3× 1000
shots) randomly for each spot in the range of m/z 500–3000
at a laser frequency of 100 Hz. Identify and confirm detected
molecules using the Bruker Lift method upon comparison to
standard compounds (Fig. 2).
8. Data analysis: Analyze data with proprietary Bruker Flex
Analysis software using baseline subtraction, smoothing, and
normalization. Quantitate MALDI signals for Ang peptides by
comparing to signal intensities obtained for the stable-isotope
labeled Ang internal standards (Fig. 3).
3.2 MS Analysis 1. Kidneys.

of In Situ Renal 2. Kidney sections: Let tissue adjust to −20 °C for 30 min. Cut
Angiotensin kidney sagittally in half using a chilled blade. Cut 12 μm-sized
Metabolism tissue sections at −20 °C using a cryostat (see Note 8). Move
tissue sections carefully on indium-tin-oxide coated glass slides
at −20 °C using chilled brushes (see Note 9). Mount tissue
section onto glass slide by holding finger underneath the glass
slide containing the tissue section for 1 min at −20 °C. Desiccate
tissue sections for 5 min at room temperature.
3. Incubation: Apply 5 μl of 1 mg/ml Ang peptide to tissue sec-
tions using a 20-μl pipet at room temperature. For inhibitor
studies, add 1 μl 10 mM inhibitor to 100 μl 1 mg/ml Ang
Fig. 2 Confirmation of detected Ang III and Ang-(1-7) peptides upon comparison to standard compounds using
the Bruker Lift method. (a) Shows the MS/MS of Ang III (m/z 931) produced by kidney homogenate after incu-
bation with Ang II. (b) Shows the MS/MS of the peptide standard Ang III (m/z 931). (c) Shows the MS/MS of
Ang-(1-7) (m/z 899) produced by kidney homogenate after incubation with Ang II. (d) Shows the MS/MS of the
peptide standard Ang-(1-7) (m/z 899)
peptide solution (see Note 7). Incubate tissue sections with

test solution in humidity chamber placed inside an incubator at
37 °C for 5 min (see Note 10).
4. Preparing tissue sections and glass slides for matrix coating:
Desiccate tissue sections at room temperature for at least
30 min. When dry, spot immediately 0.5 μl peptide calibration
standard II next to the tissue sections in a corner of the indium-
tin-oxide coated glass slide and mix with 0.5 μl MALDI matrix.
Let spot dry at room temperature for 5 min. Add teaching
points to each corner of the indium-tin-oxide coated glass slide
using a permanent marker and white-out. Scan glass slide con-
taining tissue sections, peptide calibration standard, and teach-
ing points.
5. Matrix coating of tissue sections: Spray 20 ml MALDI matrix
per glass slide using a thin layer chromatography nebulizer
with nitrogen gas (10 psi) from a distance of 20 cm. Repeat
spray coating 10–15× allowing for 30 s of drying time between
3 and 5 passes until a uniform matrix coating is achieved.
Desiccate tissue sections at room temperature for at least
10 min. Use light microscopic visualization at 10× magnifica-
tion to confirm even matrix coating.
6. MS acquisition: Mass calibrate spectra by collecting 200 laser
shots of Bruker peptide calibration standard II. Start
automated analysis of tissue sections using an Autoflex III
Fig. 3 MS analysis of in vitro renal angiotensin metabolism. (a) Shows the MS signals obtained after incubation
of Ang II with 20 μg kidney homogenate protein in 50 mM phosphate buffer pH 7.0 for 60 min. (b) Shows the
MS signals obtained after incubation of Ang II with 20 μg kidney homogenate protein in 50 mM citrate buffer
pH 5.0 for 60 min. (c, d) Show the MS signals obtained after 60-min incubation of Ang I with 20 μg kidney
homogenate protein in 50 mM MES buffer, pH 6.75, in absence or presence of 100 μM ACE inhibitor captopril,
respectively. (e, f) Show the MS signals obtained after a 60-min incubation of Ang I with 20 μg kidney homog-
enate protein in 50 mM MES buffer pH 6.75 in absence or presence of 100 μM NEP inhibitor thiorphan, respec-
tively. The asterisk indicates stable-isotope, [13C5, 15N]-Valine labeled Ang peptide standards
smartbeam MALDI TOF/TOF instrument (Bruker Daltonics,

MA, USA) in positive polarity and reflectron mode acquiring a
total of 200 spectra for each spot of a 100 or 200 μm raster in
the range of m/z 500–3000 and at a laser frequency of 100 Hz.
Identify and confirm detected molecules using the Bruker Lift
method upon comparison to standard compounds.
Fig. 4 MS analysis of in situ renal angiotensin metabolism. Incubation of mouse kidney sections with Ang II
shows distinct spatial distribution patterns for the two main products Ang-(1-7) and Ang III
7. Data analysis: Analyze date with proprietary Bruker Flex

Analysis and Imaging software using baseline subtraction,
smoothing, and normalization. Quantitate MALDI imaging
signals represented in color on pseudocolor images (Fig. 4) as
integrated intensity and normalized to the area of the whole
kidney section using MetaMorph image analysis software.
4 Notes
1. Some inhibitors have a low solubility in water and need sonica-

tion to be dissolved. Some inhibitors need a few initial micro-
liters of ethanol or DMSO. Check with manufacturer for
detailed instructions. For instance, we found the following
solubilities: 10 mM ACE inhibitor captopril in water, 10 mM
ACE2 inhibitor MLN-4760 (MLN) in water, 10 mM NEP
inhibitor thiorphan in DMSO, 10 mM PRCP/PREP inhibitor
Z-pro-prolinal (ZPP) in water, 10 mM APA inhibitor
4-aminophosphobutyric acid in water.
2. Prepare the dilution of Ang internal standards freshly right
before use.
3. Prepare MALDI matrix freshly right before use. Vortex
thoroughly.
4. Clean target plate before first use with isopropanol followed by
water. Wet a tissue and wipe surface. Sonicate the target plate
for 10 min covered with isopropanol and dry the target plate
using a stream of nitrogen or let it air dry in a dust-free envi-
ronment. The target plate can be reused following the cleaning
procedure.
5. Buffers other than MES buffer, pH 6.75, may be used to char-
acterize the RAS. We have used 50 mM phosphate buffer,
pH 7, or 50 mM citrate buffer, pH 5 (Fig. 3).
6. If Ang II is tested as substrate, add 4 μl 500 μM Bestatin before
making up to 100 μl water to stabilize Ang II.
7. Different inhibitors can be used to test RAS enzyme activities,
including ACE inhibitor captopril, ACE2 inhibitor MLN-
4760, NEP inhibitor thiorphan, PRCP/PREP inhibitor

Z-pro-prolinal, or APA inhibitor 4-aminophosphobutyric acid.
Figure 3 shows an example of kidney homogenate incubated
with Ang I in absence and presence of ACE inhibitor captopril
or NEP inhibitor thiorphan.
8. To obtain intact kidney sections, adjust temperature of the
cryostat to −20 ± 3 °C. Cutting performance depends on
room temperature and humidity. Cryostat temperature has to
be adjusted accordingly.
9. One indium-tin-oxide coated glass slide can hold up to 12
mouse kidney sections.
10. Subheading 3.2, steps 1–5 are key features unique to this
technique. These steps are highly temperature- and time-
sensitive. Proper processing is crucial for visualization of tissue
enzyme activities, including (a) quick removal and rapid freez-
ing of organs after collection, (b) avoidance of localized tissue
warming and contamination of tissue cutting surface with
freezing medium, (c) care in applying the peptide solution
such that the tissue is not damaged, and (d) minimal move-
ment of slides during incubation and transfer to desiccator.
Acknowledgment
This work was supported by National Institutes of Health Grants

F32 DK-093226 and the Carl W. Gottschalk Research Scholar
Grant of the ASN Foundation for Kidney Research to N.G. and
R01 HL-093567 to K.M.E.
References
1. Fredline VF, Kovacs EM, Taylor PJ, Johnson imaging of angiotensin metabolism in kidney.
AG (1999) Measurement of plasma renin activ- Am J Physiol Endocrinol Metab 302:
ity with use of HPLC-electrospray-tandem E1016–E1024
mass spectrometry. Clin Chem 45:659–664 6. Grobe N, Di FM, Kashkari N, Chodavarapu H,
2. Donoghue M, Hsieh F, Baronas E, Godbout K, Somineni HK, Singh R, Elased KM (2015)
Gosselin M, Stagliano N, Donovan M, Woolf B, Functional and molecular evidence for
Robison K, Jeyaseelan R, Breitbart RE, Acton S expression of the renin angiotensin system and
(2000) A novel angiotensin-converting enzyme- ADAM17-mediated ACE2 shedding in COS7
related carboxypeptidase (ACE2) converts angio- cells. Am J Physiol Cell Physiol 308:
tensin I to angiotensin 1-9. Circ Res 87:E1–E9 C767–C777
3. Elased KM, Cool DR, Morris M (2005) Novel 7. Stettner D, Bujak-Gizycka B, Olszanecki R,
mass spectrometric methods for evaluation of Rytlewski K, Huras H, Korbut R (2013)
plasma angiotensin converting enzyme 1 and Assessment of angiotensin I metabolism in the
renin activity. Hypertension 46:953–959 human placenta using an LC/MS method.
4. Elased KM, Cunha TS, Gurley SB, Coffman Folia Med Cracov 53:31–39
TM, Morris M (2006) New mass spectrometric 8. Velez JC, Ierardi JL, Bland AM, Morinelli TA,
assay for angiotensin-converting enzyme 2 Arthur JM, Raymond JR, Janech MG (2012)
activity. Hypertension 47:1010–1017 Enzymatic processing of angiotensin peptides
5. Grobe N, Elased KM, Cool DR, Morris M by human glomerular endothelial cells. Am
(2012) Mass spectrometry for the molecular J Physiol Renal Physiol 302:F1583–F1594
ERRATUM TO
Chapter 8
Characterization and Functional Phenotyping of Renal
Immune Cells via Flow Cytometry
DOI 10.1007/978-1-4939-7030-8_16
This chapter was originally published with two errors in the Materials section found on
pages 88 and 89.
14. should read “…reconstitute in PBS at 100 mg/ml…” not 0.1 mg/ml as is written
16. should read “…containing 3% FBS, 10 ug/ml DNase…” not 10 mg/ml as is written
The updated original online version for this chapter can be found at
http://dx.doi.org/10.1007/978-1-4939-7030-8_8
E1
Index
A Apoptosis �� 4, 97, 106

Abdominal aorta ��3, 61, 64, 157, 158 Aseptic �� 77, 152
Abdominal aortic aneurysm (AAA) �� 6, 155, 156, 158 Atherosclerosis �� 5, 7–8, 21, 24, 26, 175, 177
Acclimation ��70, 73, 131, 132, 142 Atherosclerosis mouse models
Activity �� 1–4, 6, 8, 9, 32, 33, 61, 62, 75, 77, 101–104, apolipoprotein E-deficient mice (ApoE-/-)�� 21, 155,
114, 118, 119, 123, 125, 128, 136–137, 140, 141, 156, 177, 178, 180, 182–184, 186
165, 169, 183, 184, 190 low density lipoprotein-deficient mice (Ldlr-/-)��64,
Aerobic �� 123, 124, 137–142 155, 156, 177, 178, 180, 182–184, 186
Aldosterone (Aldo) �� 6, 155–162
Aliskiren ��2, 7, 102, 104, 113–115, 118, 178, 183, 184
Amastatin �� 178, 183 B
Anaerobic ��123, 128, 138, 140, 141 Beta-actin (β-actin)�� 102, 103, 108
Aneurysm ��155, 156, 158, 160, 161 Bicinchoninic acid (BCA)��63, 148, 150, 178, 180
Angiotensin Binding assay �� 5, 165, 170, 172
alamandine �� 5, 7, 168, 176 Blood pressure ��1, 5, 6, 8, 9, 69–73, 75–85, 87, 99,
angiotensin A��5, 167 102, 157–158, 160, 161, 176, 190
angiotensin I/angiotensin-(1-10) �� 2, 99, 109, Blood volume ��73
177, 192 Body composition ��125, 127, 135–137, 140, 141
angiotensin II/angiotensin-(1-8) �� 2, 21, 24, 32, Bone marrow �� 47, 49, 51–54, 58
34, 37, 40, 43, 44, 61, 62, 64, 93, 99–101, Bradford reagent ��192
105, 106, 109, 116, 118, 147–150, 152, Bromodeoxyuridine (BrdU) �� 48, 55, 56
155, 165, 167, 176, 177, 179, 181, 182, 192
angiotensin III/angiotensin-(2-8) �� 99, 100, 177,
181, 194, 196 C
angiotensin IV/angiotensin-(3-8) �� 99, 100, 168, 177 C57Bl/6J�� 6, 48, 58, 72, 142, 156,
angiotensin-(1-7) �� 3–5, 7, 8, 48, 58, 61, 62, 100, 160, 161, 177, 178, 180
101, 105, 106, 109, 116, 165, 167, 170, 171, 176, Caloric balance ��123
177, 181, 194, 196 Calorimetry ��124, 125, 127–131, 133–140
angiotensin-(1-9) �� 5, 168, 176, 177 Captopril �� 3, 62–64, 195, 196
angiotensin-(1-12) �� 176, 177, 181 Carbonylation �� 7, 32–45
Angiotensin-converting enzyme 1 (ACE1) �� 3, 8, Carotid artery �� 78–80, 84
62–63, 65 Cathepsin B �� 102, 103, 108
Angiotensin-converting enzyme 2 Catheter �� 76, 78, 80–85
(ACE2) ��3–4, CD34 �� 8, 48–51, 55, 57
61–66, 176, 183, 186, 190, 196 CD45 �� 48, 50, 92–95
Angiotensin receptor Cell culture
angiotensin receptor 1a (AT1aR) �� 4, 6, 8 D-glucose ��34
angiotensin receptor 1b (AT1bR) ��4 Dulbecco’s Modified Eagle’s Medium
angiotensin receptor 2 (AT2R) �� 4–5, 99–101 (DMEM) �� 34, 37, 148
angiotensin-(1-7) receptor/mas receptor (AT7R/MasR), fetal bovine serum (FBS)�� 37, 48–51,
4–5, 7, 8, 48, 100, 101, 105, 108, 170, 171 53–55, 89, 148
Angiotensinogen (AGTN) ��2, 4, 6, 22, 23, 25, L-glutamine ��34
99–102, 104, 108, 114, 115, 118, 176 penicillin-streptomycin �� 34, 148
Antibody Roswell Park Memorial Institute
IgG��36 Medium (RPMI) �� 48, 52, 55, 89
IgY��22, 25 sodium pyruvate ��34
DOI 10.1007/978-1-4939-7030-8, © Springer Science+Business Media LLC 2017
199
The Renin-Angiotensin-Aldosterone System: Methods and Protocols
200 Index

Cell volume�� 150, 151 H
Chinese Hamster Ovary cells (CHO cells)��170 Hank’s buffered saline solution (HBSS)�� 48, 55, 148, 150
Circadian��8, 77, 83, 128, 137 Hematopoiesis �� 8, 48, 53
c-Kit (CD117)((Stem cell growth High-pressure liquid chromatography (HPLC)�� 3, 4,
factor receptor) �� 49, 52–54 61, 106, 107, 109, 110, 117, 119, 179, 181
Collagen ��152 Horseradish peroxidase (HRP) �� 33, 34, 108
Collagenase �� 88, 89, 96 Hue Saturation Intensity method (HSI)��26–28
Color imaging �� 21–28, 44, 196 Hypertension �� 1, 5, 6, 8–9, 32, 69, 87, 147,
Competition �� 166, 171 161, 175, 189
Confocal �� 168, 170, 172 Hypertrophy �� 147, 149–152, 175
Cytokines �� 9, 48, 50, 87, 89, 91–95, 97
I
D Imaging �� 26, 27, 157–158, 160, 170, 172, 196
Deoxycorticosterone (DOCA) ��155 Immune �� 9, 22, 87, 92–95
Deoxyribonucleic acid (DNA) ��32 Immunoblotting ��2, 32, 38, 40, 103
Diabetes ��48, 58 Immunohistochemical ��88
Diamidino-2-phenylindole (DAPI)�� 168, 170, 172 Immunomagnetic �� 48–51, 53, 54
Diastolic �� 8, 73, 77 Immunostaining ��21–28
Digestive efficiency �� 123, 125, 127, 128, 133, 135, Inflammation �� 9, 32, 99, 100
140, 143 Infusion �� 7, 9, 21, 64, 93, 96, 155
Diurnal ��75 Insulin regulated aminopeptidase (IRAP) �� 100, 176
Drug screening ��165 Interferon-gamma (INF-γ) ��9
Dual energy x-ray absorptiometry (DEXA)��136 Interleukin ��7
Isoflurane �� 57, 77, 83, 106, 157, 161, 178
E
Enalapril ��156 K
Energy balance �� 6, 123–144 Kidney disease �� 87, 96, 190
Enzyme �� 4, 5, 33, 61, 62, 65, 83, 99,
102, 105, 106, 171, 176, 183, 184, 186,
190, 196, 197 L
Eplerenone ��156 Labeling �� 54, 167
Ethylenediaminetetraacetic acid (EDTA)�� 2, 3, 43, Ligand �� 5, 165, 170, 172
50, 53, 54, 63, 89, 91, 106, 107, 109, 113, Lineage-negative (Lin-) ��49–54
148, 171, 178, 183, 184, 191, 192 Lipopolysaccharide (LPS) ��91
Explant ��147–149 Liquid chromatography (LC) �� 5, 177, 179–182,
185, 186, 190
Losartan �� 101, 156, 168, 171
F LSK cells �� 53–55, 57, 58
Fibrosis �� 32, 87, 99, 100, 148, 151–152 Lymphocytes ��9, 87
Flow cytometry ��8, 9, 48, 87, 92–95 Lymphoid ��87, 88
Fluorescein isothiocyanate (FITC)�� 52, 92, 95
Fluorescence ��5, 50, 55, 61, 66, 91, 93, 165, 170, 172
Fluorescence-activated cell sorting (FACS)�� 48, 50, M
52–53 Mass spectrometry (MS)�� 5, 6, 177, 189, 190, 194–196
Forward scatter area (FSC-A)�� 52, 94, 95 Matrix-assisted laser desorption/ionization
Forward scatter height (FSC-H)��52, 94 (MALDI)��190–196
Fractionation ��3, 50, 53, 103, 105, 107, 110, 112, Metabolism �� 101, 106, 107, 109, 111, 113–118,
116, 119, 124, 135, 138, 166 123, 124, 128, 137–141, 184, 185, 189,
190, 194–196
Metalloenzymes �� 183, 184
G Metalloproteinase (MMP)��61
G-protein-coupled receptors (GPCRs), �� 4–5, 165, 168, 176 Microscope �� 26, 28, 77, 83, 157, 170, 172, 192
Gut microbiome �� 127, 131 Migration ��47, 48, 55, 57, 58
The Renin-Angiotensin-Aldosterone System: Methods and Protocols
201
Index

Mineralocorticoid receptor (MR) ��6, 156 Red blood cell (RBC)�� 48, 57, 89, 90
Mitochondria ��4, 99, 100, 103–105, 107, Redox ��32, 34
112, 114, 116, 117 Renin �� 1, 2, 4, 6, 21–23, 25, 27, 65, 99–105, 109,
MLN-4760�� 3, 62, 65, 196–197 113–114, 118, 183, 184, 190
Mono-5-(and-6)-carboxyfluorescein (FAM)��168 Renin-angiotensin system (RAS)�� 4, 21, 87, 88,
Mouse �� 4, 6, 8, 21–27, 34, 36, 48–55, 99, 100, 103–105, 107, 112, 114, 116, 117, 123,
61, 64, 71, 72, 76, 78, 81, 83, 84, 89, 93, 96, 102, 124, 126, 128, 132, 165, 175–177, 184–186,
126, 132, 135, 139, 140, 142, 143, 155, 171, 180, 189–192, 196
192, 196, 197 Renin-angiotensin-aldosterone system
Myeloid �� 87, 88, 91 (RAAS)�� 1–9, 47–58, 156
Resting metabolic rate (RMR)��123, 125, 137–142, 144
Rhodamine (Rhod)��168
N Ribonucleic acid (RNA)��88, 96
Nanoflow �� 177, 178
Neprilysin (NEP)�� 4, 62, 65, 100, 101, 106–109,
183, 190, 195–197 S
Non-invasive �� 69, 71, 72 Salt �� 2, 6, 8, 155, 167, 171, 178, 190
Nucleus �� 4, 97, 101 SDS-PAGE �� 33, 36, 39, 108
Side scatter area (SSC-A) ��93, 95
Sodium �� 1, 3, 6, 34–36, 42, 45, 48, 87,
O 108, 128, 171, 178
Obesity ��5, 6, 9, 75, 124 Stem cell antigen-1 (Sca-1)�� 49, 52–54
Osmotic ��156–161 Stem cells ��8
Oxidative stress �� 4, 7, 9, 32, 42, 100, 106 Stromal-derived factor-1 alpha (SDF-1α) �� 47, 55, 57
Oxyblot ��32, 33, 35–39, 43, 44 Survival surgery ��77
Systolic ��5, 8, 73, 77, 157
P
PD123319 �� 168, 171 T
Pepstatin ��35, 65, 178, 183, 184 Tail-cuff ��8, 69, 71, 72, 76, 157–158
Petri dish(es) �� 34, 54, 88, 90 Temperature �� 2, 37–39, 41, 42, 49–51, 54–56,
Phosphate-buffered saline (PBS)��34, 35, 37, 40, 62, 64, 70–72, 78, 83, 89, 111, 112, 128–131, 134,
43, 45, 48–51, 53, 54, 56, 57, 148–150, 160 137–141, 151, 158, 168, 191, 193, 194, 197
Phosphoramadon ��65 Thimet oligopeptidase (TOP) ��62, 100, 101, 106,
Progenitor cells �� 8, 47, 51, 52 107, 109, 190
Proliferation �� 4, 8, 47, 48, 55–57, 99, 100, 147 Thiorpan ��65
Prolyl endopeptidase �� 62–63, 190 Time-of-flight (TOF) �� 191, 193, 195
Pro-renin (PR)��102, 103, 105, 114, 118 Tris �� 35, 36, 108
Pro-renin receptor (PRR)�� 105, 108 Trypan blue solution �� 48, 49, 89, 90
Protein Trypsin �� 89, 103, 104, 109, 113, 114, 118,
oxidation�� 32, 35, 43, 44 148–150, 183
reduction��32 Tumor necrosis factor-alpha (TNF-α) ��9
Protein tyrosine phosphatase (PTP) �� 7, 32, 38, 40 Tween-20 �� 36, 108
Protein tyrosine phosphatase (PTP)
oxidation �� 7, 32, 38, 40
Pump ��35, 40, 129, 158–162, 192 V
Vascular endothelial growth factor (VEGF) �� 47, 55, 57
Vascular smooth muscle cells (VSMCs) �� 7, 32, 38,
R 40, 101, 147–152
Radioimmunoassay (RIA)�� 2, 105, 109,
115, 116, 118, 119, 176, 181, 185, 186, 189, 190
Radio telemetry �� 75, 78–82, 84 W
Reactive nitrogen species (RNS)��32 Western ��4, 5, 9, 21, 25, 27, 35–39, 41–42, 44,
Reactive oxygen species (ROS) �� 32, 33, 101, 106 111–114, 155

The Renin-Angiotensin-Aldosterone System - Methods and Protocols (PDFDrive)

Uploaded by

Document Informationclick to expand document information

Document Informationclick to expand document information

Copyright:

Available Formats

The Renin-Angiotensin-Aldosterone System - Methods and Protocols (PDFDrive)

Uploaded by

Document Information

Original Title

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

The Renin-Angiotensin-Aldosterone System - Methods and Protocols (PDFDrive)

Uploaded by

Copyright:

Available Formats

Methods in

Molecular Biology 1614

Sean E. Thatcher Editor

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2017937363

© Springer Science+Business Media LLC 2017

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

Lexington, KY, USA Sean E. Thatcher

1 A Brief Introduction into the Renin-Angiotensin-­Aldosterone System:

13 Fluorescence-Based Binding Assay for Screening Ligands

Maiia E. Bragina • Laboratory of Hemodynamics and Cardiovascular Technology,

Mariola Olkowicz • Department of Biochemistry, Medical University of Gdansk, Gdansk,

A Brief Introduction into the Renin-Angiotensin-­

Key words Angiotensin, Historical, RAAS, Techniques, Methods

The renin-angiotensin-aldosterone system (RAAS) is an important

helped to discern the overall activation of the RAAS. Renin is an

1.2 ACE Angiotensin-converting enzyme 1 (ACE1, 150–180 kDa) is a

1.3 ACE2 Angiotensin-converting enzyme 2 (ACE2, 90–120 kDa) is also a

1.4 Neprilysin Neprilysin (90–100 kDa) can process AngI directly to Ang-(1-7)

1.5 Angiotensinogen Angiotensinogen is roughly a 450 amino acid protein (55–60 kDa)

blood pressure regulation [46–48]. Unfortunately, antibodies tar-

(Figure 4 of Chapter 15). These advanced techniques can help to

performed in the aortic arch by en face analysis or in the aortic

and infusion of AngII, can be used to increase blood pressure and

enzyme-related carboxypeptidase (ACE2) con- Gong M, Cassis LA (2015) Deficiency of angio-

Leukocytes synthesize angiotensinogen. in acute glucocorticoid-induced hyperten-

(2016) Female mice with an XY sex chro- by angiotensin II-induced hypertension.

Hypertension 59(5):1069–1078. doi:10.1161/ Blocking angiotensin-converting enzyme

A Color Segmentation-Based Method to Quantify

Key words Immunostaining, Atherosclerosis, Antibody, Imaging, Color

Mouse models are commonly used to study pathologies and mecha-

Immunostaining detects cell types and molecular markers of

Fig. 2 Defining area of interest. Atherosclerotic lesions in an aortic root section

AGT accumulates in proximal convoluted tubules of kidney, which

in Table 1 only detects renin in kidney JG cells of wild-type, but not

Immunogenic peptide Recommended positive Recommended working

also developed multiple chicken anti-mouse antibodies targeting

1. Immunostained cross-sections of aortic root samples.

Macrophages are the most abundant cell type in atherosclerotic

5. Analysis of lesional composition: There are two methods to

1. Specificity of positive immunostaining is a premise for accurate

Congqing Wu is supported by an American Heart Association

Assessment of Protein Carbonylation and Protein Tyrosine

1.2 Immunoblot Carbonylation is the most frequent form of irreversible protein

Subsequent treatment with DNPH derivatizes carbonyl groups on

1.3 Immunoblot Protein tyrosine phosphatases (PTPs) are important regulatory

mouse-monoclonal antibody specific to the SO3H-modified cata-

All aqueous solutions are prepared in Milli-Q grade water (ultra-

2.1 Angiotensin 1. Cell culture petri dishes 100 mm.

2.3 PTP Oxidation 1. 1× Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM

13. Sodium orthovanadate (Na3VO4): 100 mM stock solution

2.4 Western Blotting 1. Electrophoresis and transfer apparatus.

Fig. 1 Oxyblot flowchart. Derivatization of protein carbonyl groups (C = O) by 2,4-dinitrophenylhydra-

7. The alkylated supernatant is directly transferred onto a Micro

Lexington, KY, USA Sean E. Thatcher

1 A Brief Introduction into the Renin-Angiotensin-Aldosterone System:

A Brief Introduction into the Renin-Angiotensin-

3.8 Immuno This is accomplished by using commercially available isolations

power as compared to other BP monitoring methods (e.g., tail-