MATERIAL F BUN DESPRE SISTEMUL COMPLEMENT

LINK: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272665/

Complement in Kidney Disease: Core Curriculum 2015

Joshua M. Thurman, MD
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The publisher's final edited version of this article is available at Am J Kidney
Dis
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The complement cascade is part of the innate immune system and provides an important line
of defense against invasive pathogens. However, the complement system also causes kidney
injury in a variety of different diseases, and clinical evaluation of the complement system is
an important part of the diagnostic workup of patients with glomerulonephritis. Complement
activation is particularly important in the pathogenesis of atypical hemolytic uremic syndrome
(aHUS) and C3 glomerulopathy. Complement inhibition is effective for treatment of aHUS,
and complement inhibitors will likely be tested in other kidney diseases in the future. While
the role of the complement system in the pathogenesis of many kidney diseases is well
established, however, there is not a simple algorithm for identifying which patients should be
treated with complement inhibitors or for how long complement inhibition should be
continued.
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OVERVIEW OF THE COMPLEMENT SYSTEM
Complement proteins provide an important line of defense against bacteria, fungi, and
viruses. The complement system also facilitates the efficient removal of damaged cells and
immune complexes. Inactive complement proteins (zymogens) circulate in plasma, and are
activated through three distinct pathways: the classical pathway, the alternative pathway, and
the mannose binding lectin pathway (Figure 1). Once activated, the complement system
generates several different activation fragments that have potent pro-inflammatory or
cytolytic effects. Some of these fragments are soluble and can be measured in plasma (e.g.
C3a and C5a), and some become covalently bound to target cells (e.g. C4d and C3d). A key
feature of this system is that it is rapidly activated on pathogens and damaged cells, but it is
not activated on host surfaces. The ability of the complement system to discriminate between
different surfaces is accomplished by a finely tuned balance between activator proteins and
regulatory proteins.
Figure 1

Overview of the complement system

The complement system is activated through three distinct pathways: the classical
pathway, the mannose binding lectin pathway, and the alternative pathway. Activation
through the classical and mannose binding lectin pathways causes cleavage of the
protein C4, and fixation of C4d (a fragment of C4) to nearby tissues. Activation
through all pathways leads to cleavage of C3. The cleavage of C3 generates a soluble
fragment (C3a) and a tissue bound fragment (C3b). Further proteolysis of C3b
generates iC3b and finally C3d. Full activation of the complement system also
generates C5a and C5b-9, important mediators of tissue inflammation and injury.
Role of Complement in Fighting Infection
The importance of the complement system for protecting against pathogens is demonstrated
by the susceptibility of patients with congenital complement deficiencies to opportunistic
infections. Patients deficient in C3 are predisposed to bacterial infections. Levels of C3 can
become depleted in patients deficient in the complement regulatory proteins factor I and
factor H, and this acquired deficiency of C3 is also associated with recurrent pyogenic
infections. Patients with deficiencies of classical pathway proteins (C1q, C1r, C1s, C2, and
C4) are at increased risk of infections with encapsulated bacteria. Those with deficiencies of
terminal complement proteins (C5, C6, C7, C8, or C9) and those with deficiencies of
alternative pathway proteins (factor B, factor D, and properdin) are at increased risk of
infections with Neisseria species. Similarly, patients treated with eculizumab are susceptible
to Neisseria infections and should be immunized with the meningococcal vaccine prior to
treatment.

Additional Readings


 Skattum L, van Deuren M, van der Poll T, Truedsson L. Complement deficiency
states and associated infections. Mol Immunol. 2011;48(14):1643–1655.




Walport MJ. Complement. First of two parts. N Engl J Med. 2001;344(14):1058–

1066.

Role of Complement in Tissue Inflammation

Complement activation causes tissue injury in a wide range of autoimmune and inflammatory
diseases. Complement activation can damage host tissues if the response to infection is
sufficiently strong or widespread, when activated by autoantibodies and immune complexes,
in patients deficient in complement regulatory proteins, and in patients with gain of function
mutations. Systemic complement activation, as occurs during sepsis or dialysis with
incompatible membranes, causes vascular leak and sequestration of leukocytes in the
pulmonary circulation. At the local level, complement activation within the glomerular
capillary walls causes cell activation, glomerular inflammation, and injury.

Additional Readings

Quigg RJ. Complement and the kidney. J Immunol. 2003;171(7):3319–3324.




Trouw LA, Seelen MA, Daha MR. Complement and renal disease. Mol Immunol.
2003;40(2–4):125–134.




Walport MJ. Complement. Second of two parts. N Engl J Med. 2001;344(15):1140–

1144.

Key Role of Complement Regulatory Proteins

As highlighted above, the complement system must adequately respond to pathogens, but
activation must be controlled on host tissues. This balance is maintained by a group of
complement regulatory proteins. Some of these proteins are expressed on cell membranes
[such as membrane cofactor protein, decay accelerating factor, and CD59], whereas others
(such as factor H and factor I) are soluble plasma proteins that are synthesized in the liver.
Several inflammatory diseases are directly caused by congenital or acquired deficiencies of
regulatory proteins, permitting uncontrolled complement activation on host cells. Patients
with paroxysmal nocturnal hemoglobinuria, for example, have a clonal defect that prevents
the expression of two complement regulatory proteins (decay accelerating factor and CD59)
on the surface of erythrocytes. As a result, complement activation causes lysis of erythrocytes
and hemolytic anemia.
aHUS and C3 glomerulopathy are also strongly associated with defects in the proteins that
regulate alternative pathway activation. These defects in complement regulation are generally
systemic, and it is not clear why the kidney is vulnerable to complement-mediated injury in
patients with these mutations. Factor H is a soluble protein, for example, and mutations
should increase alternative pathway activation throughout the body. Injury of other organs
does occur in patients with factor H mutations, but the kidney is the most frequent and the
most severely affected organ.

Additional Readings

de Cordoba SR, de Jorge EG. Translational mini-review series on complement factor

H: genetics and disease associations of human complement factor H. Clin Exp
Immunol. 2008;151(1):1–13.




Naik A, Sharma S, Quigg RJ. Complement regulation in renal disease models. Semin

Nephrol. 2013;33(6):575–585.




Noris M, Caprioli J, Bresin E, et al. Relative role of genetic complement

abnormalities in sporadic and familial aHUS and their impact on clinical
phenotype. Clin J Am Soc Nephrol. 2010;5(10):1844–1859.




Zipfel PF, Skerka C. Complement regulators and inhibitory proteins. Nat Rev

Immunol. 2009;9(10):729–740.

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THE SPECTRUM OF COMPLEMENT-MEDIATED KIDNEY DISEASES
Complement proteins are seen in biopsies from patients with virtually all forms of
glomerulonephritis, and each of the three activation pathways have been linked with various
kidney diseases (Figure 2). Although complement activation occurs downstream of immune
complex deposition or antibody-mediated injury in many of these diseases, complement
activation is also observed in kidney diseases that are not antibody-mediated. The wide
variety of kidney diseases associated with complement activation suggests there is something
about the structure or function of the kidney that makes it particularly susceptible to
complement-mediated injury.
Figure 2

Mechanisms of complement activation in kidney disease

Immune complex deposition within glomeruli activates the classical pathway of
complement in some forms of glomerulonephritis, and antibodies specific to renal
antigens also activate the classical pathway. Uncontrolled activation of the alternative
pathway of complement is associated with aHUS and C3 glomerulopathy, and the
alternative pathway has also been implicated in ANCA-associated vasculitis and post-
streptococcal glomerulonephritis. Mannose binding lectins have been detected in the
kidneys of some patients with IgA nephropathy, membranous nephropathy, and post-
streptococcal glomerulonephritis. Abbreviations: HUS, hemolytic uremic syndrome;
ANCA, anti-neutrophil cytoplasmic antibody; MBL, mannose binding lectin; IgA,
immunoglobulin A; MPGN, membranoproliferative glomerulonephritis; GBM,
glomerular basement membrane
aHUS (Atypical Hemolytic Uremic Syndrome)
HUS is a clinical syndrome of hemolysis, thrombocytopenia, and acute kidney injury. In
approximately 90% of patients, HUS is triggered by enteric infections by Shiga-toxin
producing bacteria. The remaining cases are referred to as “atypical HUS”. Over the past 15
years, work from laboratories around the world has shown that 40–60% of patients with
aHUS have mutations in complement proteins, and approximately 10% of patients have
autoantibodies to factor H that impair its function. The 30–40% of patients with aHUS who
do not have identified mutations in complement-related genes may have mutations in genes
for other proteins, as yet unidentified, that also help control alternative pathway activation. It
is also possible that even normal complement regulation can be overwhelmed during some
illnesses. Thrombi form in the glomeruli and capillaries of patients with aHUS, similar to
what occurs in Shiga toxin-associated HUS and thrombotic thrombocytopenic purpura
(Figure 3), and studies have revealed multiple links between the complement system, the
coagulation system, and platelet activation.

Figure 3

Histology of atypical hemolytic uremic syndrome

A kidney biopsy from a patient with atypical HUS and dysregulated alternative
pathway activity demonstrates typical findings of thrombotic microangiopathy. (A)
Thrombi are seen within the glomerular capillaries (labeled with asterisks). (B) A
thrombus is also seen in an arteriole (labeled with a crosshatch). Tissue was stained
with Masson’s trichrome. Reproduced by permission from Macmillan Publishers Ltd:
Fakhouri F and Fremeaux-Bacchi V. Does hemolytic uremic syndrome differ from
thrombotic thrombocytopenic purpura? Nat Clin Pract Nephrol 2007;3:679–687.
All of the complement defects associated with aHUS have similar functional effects and
enhance alternative pathway activation (Table 1). The mutations in factor B and C3 are gain
of function mutations. The mutations in the complement regulatory proteins, on the other
hand, decrease the function of these proteins. Interestingly, most factor H mutations cluster in
the region of the protein that mediates binding to endothelial cells. The mutant proteins can
still control alternative pathway activation, but binding to endothelial cells and other surfaces
is impaired. Autoantibodies to factor H bind this same region of the protein and likely have a
similar functional effect. For patients with these defects, cells or tissues that require bound
factor H for regulating the complement system are vulnerable to complement-mediated
inflammation.
Table 1

Kidney diseases associated with mutations or variations in the genes for complement
regulatory proteins, categorized by syndrome.

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CFHR, complement factor h related protein; HELLP, a syndrome of hemolysis,
elevated liver enzymes, low platelets; MPGN, membranoproliferative
glomerulonephritis; Ig, immunoglobulin.
Although patients with congenital complement mutations usually present in childhood, some
patients present as adults. Furthermore, the mutations have incomplete penetrance. Most of
the mutations are heterozygous, but 3% of patients carry compound mutations in more than
one complement related gene. Patients carrying these mutations have impaired ability to
control alternative pathway activation, and complement regulation is overwhelmed by events
that promote intravascular complement activation, including infections, pregnancy, and
medications.
Plasma exchange is beneficial in some patients with aHUS. This treatment removes
autoantibodies or dysfunctional complement proteins, and replacement of patient plasma with
fresh frozen plasma can restore deficient proteins (factor H and factor I). Thus, it addresses
many of the possible underlying defects. Unfortunately, many patients with aHUS do not
respond to plasma, and in some patients the administration of plasma simply provides
additional substrate for complement activation. Case reports describe patients who did not
respond to plasma exchange but quickly responded to eculizumab. Eculizumab was approved
by the US Food and Drug Administration (FDA) for treatment of aHUS, primarily based on
two trials showing its efficacy. One of the trials included patients considered to have stable
disease controlled by regular plasma exchange, and the kidney function in this group of
patients improved after starting treatment with eculizumab. Because eculizumab blocks the
complement cascade at the level of C5, activation through C3 is not directly blocked by the
drug, and it is not known to what degree C3 activation fragments contribute to tissue injury in
this disease.
It can be challenging to distinguish aHUS from thrombotic thrombocytopenic purpura (TTP).
Although there is probably some overlap in the underlying mechanisms of tissue injury in
aHUS and TTP, aHUS is regarded as a disease of uncontrolled complement activation and
TTP as a disease of abnormal ADAMTS13 activity. ADAMTS13 is a metalloprotease that
cleaves von Willebrand factor multimers, and up to 90% of patients with TTP have deficient
ADAMTS13 activity (<10%). Clinical findings suggestive of deficient ADAMTS13 activity
and TTP are a platelet count <30,000/μL and a serum creatinine level less than 1.7 mg/dL.
Distinguishing aHUS and TTP is clinically important because eculizumab may be more
effective than plasma exchange in patients with aHUS, whereas plasma exchange is the
treatment of choice for TTP. However, there are case reports of patients with TTP who have
been successfully treated with eculizumab, and it is possible that a single approach to these
diseases will be possible in the future.
Shiga-Toxin aHUS
There is evidence of complement activation in patients with HUS triggered by Shiga toxin. A
few patients with particularly severe courses have been identified as having mutations in
genes for complement regulatory proteins. Several case reports have described patients who
were successfully treated with eculizumab. Eculizumab was also used in an open label, multi-
center trial of patients in France and Germany during a severe outbreak of HUS in
2011caused by Escherichia coli O104:H4. Outcomes of patients treated with plasma exchange
and eculizumab were not better than those treated with plasma exchange alone, although the
treatments were not randomized and some clinical parameters were worse in the group that
received eculizumab. Thus, the role of eculizumab in the treatment of non-aHUS forms of
thrombotic microangiopathy is unclear at this point.

Additional Readings

Cataland SR, Yang S, Wu HM. The use of ADAMTS13 activity, platelet count, and
serum creatinine to differentiate acquired thrombotic thrombocytopenic purpura from
other thrombotic microangiopathies. Br J Haematol. 2012;157(4):501–503.



 Lapeyraque AL, Malina M, Fremeaux-Bacchi V, et al. Eculizumab in severe Shiga-
toxin-associated HUS. N Engl J Med. 2011;364(26):2561–2563.




Legendre CM, Licht C, Muus P, et al. Terminal complement inhibitor eculizumab in

atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368(23):2169–2181.




Noris M, Mescia F, Remuzzi G. STEC-HUS, atypical HUS and TTP are all diseases
of complement activation. Nat Rev Nephrol. 2012.




Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med.

2009;361(17):1676–1687.




Noris M, Remuzzi G. Thrombotic microangiopathy after kidney transplantation. Am

J Transplant. 2010;10(7):1517–1523.




Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fremeaux-Bacchi V. Use of

eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat
Rev Nephrol. 2012;8(11):643–657.

C3 Glomerulopathy
C3 glomerulopathy is a recently described disease diagnosed by the detection of glomerular
C3 deposits in the absence of concomitant immunoglobulin (Figure 4). Alternative pathway
mutations have been identified in many patients with C3 glomerulopathy, indicating that this
is a disease of dysregulated alternative pathway activation. Dense deposit disease (DDD;
formerly referred to as membranoproliferative glomerulonephritis type 2) is considered a
subset of C3 glomerulopathy, but it is distinguished by the presence of electron dense deposits
within the glomerular basement membrane by electron microscopy, and it may have a worse
prognosis than C3 glomerulopathy in the absence of dense deposits.
Figure 4

Biopsy characteristics of C3 glomerulopathy

The diagnosis of C3 glomerulopathy is based upon the detection of C3 in the relative
absence of immunoglobulin or classical pathway proteins. (A) C3 fragments are seen
in the mesangium and capillary loops of a patient with C3 glomerulopathy. C3 (B)
and IgG (C) from the same patient with C3 glomerulopathy, demonstrating that some
immunoglobulin deposition is occasionally seen. (D) Electron microscopy from a
patient with dense deposit disease demonstrates dense intramembranous deposits. (E)
Electron dense deposits are seen in the glomerular basement membrane of patients
with C3 glomerulopathy, although they are not as dense or well defined as those in
patients with dense deposit disease. (F) Large, humped subepithelial deposits are
sometimes seen in patients with C3 glomerulopathy, resembling those seen in patients
with post-infectious glomerulonephritis. Reproduced with permission from Macmillan
Publishers Ltd: Pickering MC, D’Agati VD, Nester CM et al. C3 glomerulopathy:
consensus report. Kidney Int. 2013;84(6):1079–1089 and with permission from BMJ
Publishing Group Ltd: Servais A, Frémeaux-Bacchi V, Lequintrec M, et al. Primary
glomerulonephritis with isolated C3 deposits: a new entity which shares common
genetic risk factors with haemolytic uraemic syndrome. J Med
Genet. 2007;44(3):193–199.
Similar to aHUS, C3 glomerulopathy (and DDD) is associated with mutations in the genes for
C3, factor H, factor B, and for complement factor H related proteins 1, 2 and 5 (CFHR1,
CFHR2, and CFHR5). More than 70% of patients with DDD and more than 40% of patients
with other forms of C3 glomerulopathy have a circulating auto-antibody, referred to as C3
nephritic factor (C3Nef) that stabilizes the alternative pathway C3-convertase and protects it
from inactivation by factor H. It is still not known whether C3Nef is a pathogenic factor, but
its functional effect is similar to the described mutations in factor H, C3, and factor B that are
associated with the disease. Investigators have also discovered autoantibodies to factor H, C3,
and factor B in some patients.
Several therapies have theoretical ability to block the complement defects associated with C3
glomerulopathy. Rituximab could potentially ameliorate disease in those with autoantibodies,
and some case reports describe successful use of rituximab in patients with C3Nef or
autoantibodies to complement proteins. Standard immunosuppressive drugs do not directly
reduce complement activation. There are anecdotal reports of some patients improving with
standard immunosuppressive drugs, but a benefit was not detected in other reports. Plasma
exchange could theoretically benefit those patients with autoantibodies or mutations in
complement proteins. There are case reports of this therapy helping some patients, but the
evidence to support this treatment is also quite limited. Eculizumab has had mixed results in
C3 glomerulopathy. Treatment with the drug was associated with clinical improvement in at
least four reported patients, but has not been of benefit in all cases. In a recent series of 6
patients with C3 glomerulopathy (three of whom had DDD) who were treated with
eculizumab, three of the patients seemed to respond to therapy with the drug. Thus, although
a great deal has been learned about the pathogenesis of C3 glomerulopathy, at this time there
are no validated therapeutic strategies.

Additional Readings

Bomback AS, Smith RJ, Barile GR, et al. Eculizumab for dense deposit disease and
C3 glomerulonephritis. Clin J Am Soc Nephrol. 2012;7(5):748–756.




Lu DF, Moon M, Lanning LD, McCarthy AM, Smith RJ. Clinical features and
outcomes of 98 children and adults with dense deposit disease. Pediatr Nephrol.
2012;27(5):773–781.




Nasr SH, Valeri AM, Appel GB, et al. Dense deposit disease: clinicopathologic study
of 32 pediatric and adult patients. Clin J Am Soc Nephrol. 2009;4(1):22–32.




Pickering MC, D’Agati VD, Nester CM, et al. C3 glomerulopathy: consensus

report. Kidney Int. 2013;84(6):1079–1089.




Servais A, Fremeaux-Bacchi V, Lequintrec M, et al. Primary glomerulonephritis with

isolated C3 deposits: a new entity which shares common genetic risk factors with
haemolytic uraemic syndrome. J Med Genet. 2007;44(3):193–199.



 Smith RJ, Alexander J, Barlow PN, et al. New approaches to the treatment of dense
deposit disease. J Am Soc Nephrol. 2007;18(9):2447–2456.




Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fremeaux-Bacchi V. Use of

eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat
Rev Nephrol. 2012;8(11):643–657.

C3 Glomerulopathy Versus aHUS: Flip Sides of the Same Coin?

C3 glomerulopathy and aHUS are both associated with defective control of the alternative
pathway. In particular, many patients with these diseases have defective function of the factor
H protein. What, then, determines whether a patient develops one of these diseases or the
other? Factor H has several functional domains, including a complement regulatory domain at
the amino terminus of the protein and a binding region at the carboxyl terminus of the protein
(Figure 5). It controls alternative pathway activation in the fluid phase (i.e. in plasma) and on
tissue surfaces (i.e. on glomerular basement membrane and endothelial cells). The factor H
mutations associated with aHUS cluster in the carboxyl-terminal region of the protein that is
responsible for binding tissue surfaces. Factor H protein with these mutations can regulate the
alternative pathway in the circulation but has an impaired ability to regulate activation on
surfaces. Similarly, autoantibodies to the carboxy-terminus of factor H impair alternative
pathway regulation on tissue surfaces.
Figure 5

Complement regulation in the glomerulus by factor H

Factor H inhibits alternative pathway activation in the fluid phase and on the surface
of cells and the basement membrane. It is a string-like protein; the complement
regulatory region is at the amino terminus, and a region that mediates binding to
surfaces is at the carboxy terminus. Factor H defects in patients with aHUS are
predominantly in the binding region of the protein, suggesting that the disease is
caused by impaired complement regulation by factor H on endothelial cells and/or the
glomerular basement membrane (GBM). Some patients with C3 glomerulopathy have
an absolute deficiency in complement regulation by factor H. Disease in these patients
may be caused by uncontrolled fluid phase activation of the alternative pathway or
insufficient regulation on the GBM.
In C3 glomerulopathy, the defects (mutations and autoantibodies) tend to decrease all
alternative pathway regulation by factor H. This presumably affects both fluid phase and
surface regulation. Animal models support the concept that C3 glomerulopathy is a
consequence of overactive fluid phase complement activation, whereas aHUS is caused by
activation specifically on the capillary walls. An elegant series of experiments by Pickering’s
group showed that an absolute deficiency of factor H in mice causes fluid phase complement
activation and kidney disease similar to C3 glomerulopathy, whereas the absence of the
binding region of factor H predisposes mice to thrombotic microangiopathy. Excessive fluid
phase complement activation could even prevent thrombotic microangiopathy by reducing the
amount of C3 available for surface activation.
In reality, this distinction between alternative pathway regulation in the fluid phase and on
glomerular surfaces is probably not absolute. There are patients with complement mutations
or autoantibodies who have developed membranoproliferative glomerulonephritis (MPGN)
and thrombotic microangiopathy at different times, indicating a shared pathophysiology. The
analysis of glomeruli from patients with DDD also demonstrates that complement activation
occurs within the glomeruli of some patients. Patients with C3 glomerulopathy also develop
macular drusen that are similar to those seen in macular degeneration. This has been
attributed to similarities in the fenestrated capillaries of these two tissues, and may also
suggest alternative pathway activation on tissue surfaces. Finally, mutations in the
complement factor H related proteins have been associated with C3 glomerulopathy, and this
appears to be due to the ability of the mutant complement factor H related proteins to displace
factor H from surfaces.

Immune-Complex Glomerular Diseases

Immune complexes deposit within the glomeruli in a number of different kidney and systemic
autoimmune diseases. Immune complexes directly activate the classical pathway of
complement, and the alternative pathway amplifies this process.
Membranoproliferative Glomerulonephritis
MPGN is a histologic pattern of injury caused by a number of different pathologic processes.
Immunofluorescence microscopy and electron microscopy of MPGN kidneys reveals
different patterns, and subcategories of the disease have been developed. In MPGN type 1,
immune-complexes in the mesangium and subendothelial space activate the classical pathway
of complement. Clinical evidence of complement activation includes deposits of complement
proteins in the glomeruli and consumption of plasma C3 and C4. Some patients have
profound complement abnormalities, although levels are normal in approximately 30% of
patients. About 40% of patients have circulating C3nef. Heterozygous mutations in factor H
or factor I have been identified in some affected patients, suggesting that dysregulated
alternative pathway activation is also a risk factor for this disease.
MPGN, as a disease entity, has been modified several times. Ultrastructural examination led
to the development of the MPGN type 2 and MPGN type 3 sub-classifications. The electron
dense deposits that are pathognomonic for MPGN type 2 can also occur with histologic
patterns of injury other than MPGN, so the name MPGN type 2 was subsequently supplanted
by the term “dense deposit disease”. As discussed above, patients with DDD also frequently
have defects in their control of alternative pathway activation and fulfill the diagnostic criteria
for C3 glomerulopathy. This evolution of disease classifications – MPGN type 2 to DDD to
C3 glomerulopathy – represents the transition from a classification based on light microscopy
to one based on the underlying pathophysiologic process. As our understanding of the
molecular causes of this disease improves, the nomenclature and classification will likely
change further.
Cryoglobulinemia
Cryoglobulinemic kidney disease is usually associated with a MPGN pattern of injury.
Immune-complexes are seen within capillary loops and in the subendothelial space, and C1q,
C3, and C4 are usually detected. The involvement of classical pathway proteins (C1q and C4)
is not surprising since this is an immune-complex disease. Cryoglobulinemic kidney disease
is associated with a drop in C4 greater than what is typically seen in other forms of immune-
complex glomerulonephritis.
Lupus nephritis
The complement system has a paradoxical role in the development of lupus nephritis.
Complement deficiencies, particularly of components of the classical pathway, are strong risk
factors for the development of lupus due to defective clearance of nuclear antigens released
by injured and apoptotic cells. On the other hand, complement activation mediates glomerular
injury in lupus nephritis. C3 and C4 levels are depressed in greater than 90% of patients with
diffuse proliferative lupus nephritis, and a fall in these proteins often reflects an increase in
disease activity. In patients with congenital deficiency of C4, the levels of this protein do not
rise during remission, and serial measurements of C4 must be performed to determine
whether the level changes with disease activity or whether it is always low or absent in an
individual patient.
Membranous nephropathy
Membranous nephropathy is caused by immune-complexes in the subepithelial space of the
glomerular capillary wall. Extensive work in animal models demonstrates that complement
activation is central to the pathogenesis of the disease. An exciting breakthrough was the
identification of M-type phospholipase A2 receptor (PLA2R) as the target antigen in
idiopathic membranous nephropathy. Most of the IgG in the glomeruli and most of the anti-
PLA2R antibody in the serum of patients with idiopathic membranous nephropathy is of the
IgG4 subclass, and IgG4 is a poor activator of the classical pathway of complement.
Nevertheless, C3 deposits are seen in 60–80% of the biopsies of patients with membranous
nephropathy. The mechanisms by which the IgG4 deposits engage the complement system are
not yet understood, although it is possible that the class of antibody changes during the course
of the disease. Mannose binding lectin is also often detected in the glomeruli of patients with
membranous nephropathy, suggesting that complement activation proceeds through the
mannose binding lectin pathway. In secondary membranous nephropathy, the glomerular
immune deposits usually contain IgG1 and IgG3, which are effective activators of the
classical pathway.

Additional Readings

Beck LH, Jr., Salant DJ. Membranous nephropathy: recent travels and new roads
ahead. Kidney Int. 2010;77(9):765–770.




Huang CC, Lehman A, Albawardi A, et al. IgG subclass staining in renal biopsies
with membranous glomerulonephritis indicates subclass switch during disease
progression. Mod Pathol. 2013;26(6):799–805.




Manderson AP, Botto M, Walport MJ. The Role of Complement in the Development
of Systemic Lupus Erythematosus. Annu Rev Immunol. 2004;22:431–456.




Ramos-Casals M, Stone JH, Cid MC, Bosch X. The cryoglobulinaemias. Lancet.

2012;379(9813):348–360.




Sethi S, Fervenza FC. Membranoproliferative glomerulonephritis--a new look at an

old entity. N Engl J Med. 2012;366(12):1119–1131.


 

Walker PD, Ferrario F, Joh K, Bonsib SM. Dense deposit disease is not a
membranoproliferative glomerulonephritis. Mod Pathol. 2007;20(6):605–616.

Other Antibody-Mediated Kidney Diseases

Antibody-Mediated Rejection
Acute antibody-mediated rejection of kidney allografts carries a poor prognosis and is
resistant to most standard immunosuppressive therapies. Detection of C4d deposition in the
peri-tubular capillaries is one of the diagnostic criteria for the condition, and probably reflects
classical pathway activation by donor specific antibodies against HLA. It is not clear why C3
fragments and immunoglobulin are less reliable markers of antibody-mediated rejection than
C4d, but these other proteins are frequently absent in C4d positive biopsies. The association
of positive C4d staining with the titer of donor specific antibodies and graft failure suggests a
pathogenic role of complement. Treatment of patients with acute antibody-mediated rejection
often includes therapies aimed at reducing the levels of donor specific antibodies, such as
plasmapheresis, IV Ig, and rituximab. Eculizumab has been used to prevent antibody-
mediated rejection in sensitized patients and as salvage treatment for patients with refractory
disease. A randomized controlled open-label trial of eculizumab in sensitized allograft
recipients is currently underway.
Antiphospholipid Antibody Syndrome
Antiphospholipid antibody syndrome can cause thrombotic microangiopathy or kidney failure
due to thrombosis in renal capillaries, arterioles, and arteries. Animal studies indicate that
complement activation by the antibodies is an important mechanism of disease pathogenesis.
Serum C3 and C4 are low in some patients with primary antiphospholipid antibody syndrome,
and levels of C3a and C4a (generated during activation) are frequently elevated. Cases of C3
glomerulopathy in patients with antiphospholipid antibody syndrome have also been reported,
suggesting that complement activation within glomeruli by antiphospholipid antibodies
triggers C3 glomerulopathy in susceptible patients. “Catastrophic” antiphospholipid antibody
syndrome is a rare but serious form of antiphospholipid antibody syndrome involving three or
more organ systems, and kidney involvement is reported in 78% of these patients.
Eculizumab has been successfully used as salvage therapy in patients with catastrophic
antiphospholipid antibody syndrome. It has also been used prophylactically in patients with
catastrophic antiphospholipid antibody syndrome undergoing kidney transplantation and in
patients with antiphospholipid antibody syndrome who have developed recurrent thrombotic
microangiopathy post-transplant. A randomized trial of eculizumab is currently underway for
patients with catastrophic antiphospholipid antibody syndrome undergoing kidney
transplantation. Complement inhibition may also be beneficial in patients with catastrophic
antiphospholipid antibody syndrome who have failed conventional therapy.
Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis
Studies in animal models of ANCA-associated vasculitis have shown that complement
activation contributes to the pathogenesis of this disease. Activation is through the alternative
complement pathway, and agents that block C5 cleavage or C5a signaling are protective in
these models. In humans, active disease is associated with elevations in plasma levels of C3a,
Bb, C5a, and sC5b-9, but not with elevations in plasma C4d, consistent with alternative
pathway activation. Although ANCA-associated glomerulonephritis is often called “pauci-
immune”, most biopsies contain some immunoglobulin and/or complement proteins. One
report identified deposits of alternative pathway proteins (factor B and properdin), but C4d
was absent. Complement activation in humans with the disease therefore mirrors that
described in the animal models. There are not, at this time, any reports of patients with
ANCA-associated vasculitis who have been treated with eculizumab.
IgA Nephropathy
IgA nephropathy is associated with aberrant glycosylation of IgA1 molecules, and the
development of autoantibodies specific for the altered IgA1. IgA1-containing immune
complexes deposit within the mesangium, and likely initiate glomerular injury. Although
plasma C3 levels are usually normal, plasma C3a is elevated in some patients and glomerular
C3 deposits are detected in approximately 85% of biopsies. IgA activates the complement
system through either the alternative or mannose binding lectin pathway. Glomerular
mannose binding lectin is seen in a subset of patients with IgA nephropathy, and mannose
binding lectin deposition correlates with greater disease severity and a worse prognosis. C4d
deposition, which may be the result of mannose binding lectin pathway activation, has also
been linked with worse outcomes. A genome wide association study in patients with IgA
nephropathy found that an allele with deletion of CFHR1 and CFHR3 was protective. Recent
work indicates that the complement factor H related proteins can compete with factor H,
positively activating the alternative pathway. Thus, the deficiency of the complement factor H
related proteins may make factor H more effective and reduce complement activation within
affected glomeruli, explaining the association of this allele with protection from disease.

Additional Readings

Colvin RB. Antibody-mediated renal allograft rejection: diagnosis and

pathogenesis. J Am Soc Nephrol. 2007;18(4):1046–1056.




Gou SJ, Yuan J, Chen M, Yu F, Zhao MH. Circulating complement activation in

patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Kidney Int.
2013;83(1):129–137.




Haas M, Eustace JA. Immune complex deposits in ANCA-associated crescentic

glomerulonephritis: a study of 126 cases. Kidney Int. 2004;65(6):2145–2152.




Liu LL, Liu N, Chen Y, et al. Glomerular mannose-binding lectin deposition is a

useful prognostic predictor in immunoglobulin A nephropathy. Clin Exp Immunol.
2013;174(1):152–160.




Oku K, Atsumi T, Bohgaki M, et al. Complement activation in patients with primary

antiphospholipid syndrome. Ann Rheum Dis. 2009;68(6):1030–1035.

Complement and Tubular Injury

Tubulointerstitial Injury in Chronic Proteinuria Diseases
Complement regulatory proteins are not expressed on the apical (urinary) surface of tubular
epithelial cells. Ordinarily the complement proteins are restricted from passing through the
glomerular filtration barrier and accessing this surface, so complement regulation is
unnecessary. In proteinuric diseases, however, complement proteins enter the urinary space
and the absence of regulatory proteins on the apical surface of tubular cells permits
complement activation. Complement activation fragments are detectable in the urine of
patients with many forms of nephrotic syndrome. In general, a higher degree of proteinuria is
associated with a worse renal prognosis, and complement activation on the renal tubules may
be a mechanism linking the glomerular process with progressive tubulointerstitial injury.
Acute Kidney Injury
The alternative pathway is activated in the tubulointerstitium of rodents with ischemic acute
kidney injury, and complement activation in the tubulointerstitium directly contributes to
kidney injury. C3d is also seen in kidney biopsies with histologic evidence of acute tubular
injury. Cross-talk between the complement system and the adaptive immune system may
affect the immune response to foreign antigens. In the transplant setting, this may link kidney
injury and complement activation at the time of transplantation with increased risk of
rejection and worse long-term outcomes. Therapeutic complement inhibitors could, in that
context, reduce delayed graft function and also improve long-term graft survival. APT070 is
an experimental complement inhibitor that binds to cell membranes, and a Phase II clinical
trial of this agent is currently underway in the United Kingdom to test whether it protects
kidney allografts from delayed graft function.

Additional Readings

McCullough JW, Renner B, Thurman JM. The role of the complement system in
acute kidney injury. Semin Nephrol. 2013;33(6):543–556.




Nangaku M, Pippin J, Couser WG. Complement membrane attack complex (C5b-9)

mediates interstitial disease in experimental nephrotic syndrome. J Am Soc Nephrol.
1999;10(11):2323–2331.




Pratt JR, Jones ME, Dong J, et al. Nontransgenic hyperexpression of a complement

regulator in donor kidney modulates transplant ischemia/reperfusion damage, acute
rejection, and chronic nephropathy. Am J Pathol. 2003;163(4):1457–1465.




Thurman JM, Lucia MS, Ljubanovic D, Holers VM. Acute tubular necrosis is
characterized by activation of the alternative pathway of complement. Kidney Int.
2005;67(2):524–530.

Go to:
CLINICAL COMPLEMENT TESTS
The most common complement labs used by nephrologists are measurement of complement
protein levels (C3 and C4), immunostaining of biopsies for complement proteins (C3 and C4
fragments), and measurement of the hemolytic potential in a patient’s serum (CH50 and
AH50). These tests have long been used to focus the differential diagnosis in patients with
glomerular disease and to monitor disease activity. More recently, screening for genetic
mutations has been used for diagnostic and prognostic evaluation of aHUS and C3
glomerulopathy.
Genetic testing of disease-associated complement genes is not widely available, but several
laboratories can help get this testing done. A list of complement laboratories in Europe is
available from the European Complement Network website (www.ecomplement.org). Genetic
testing is under development at several labs in North America, including the Molecular
Otolaryngology and Renal Research Laboratories at the University of Iowa
(www.healthcare.uiowa.edu/labs/morl/) and the Molecular Genetics Laboratory at The
Hospital for Sick Children (SickKids) at the University of Toronto
(www.sickkids.ca/molecular). The Complement Laboratory at the National Jewish Medical
and Research Center (www.nationaljewish.org/professionals/clinical-
services/diagnostics/adx/about-us/lab-expertise/complement) can quantitatively measure a
broad range of complement proteins and activation fragments, and also performs functional
tests of the complement system.
The rapid and accurate assessment of the complement system is particularly important for
patients with aHUS and C3 glomerulopathy. Biomarkers of complement activation would
help with the diagnosis of these diseases and be useful for monitoring disease activity. Given
the limitations of the available tests, however, there is no strict diagnostic algorithm for these
diseases. Nevertheless, the available tests provide valuable clinical information (Tables 2 and 
and33).

Table 2

Complement testing in patients with C3 glomerulopathy

I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
C3 andCN
C4 3o
levels n
f-
rs
ep
qe
uc
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
ei
nf
ti
lc
y

d
e
p
r
e
s
s
e
d

a
n
d

s
u
p
p
o
r
t
s

d
i
a
g
n
o
s
i
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
s
;

n
o
r
m
a
l

C
4

s
u
g
g
e
s
t
s

a
n

a
l
t
e
r
n
a
t
i
v
e
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
p
a
t
h
w
a
y

p
r
o
c
e
s
s
SolublMT
e C5b-ae
9 ys
t
b
en
o
it
n
dw
i
cd
ae
tl
oy
r
a
ov
fa
i
al
ca
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
tb
il
ve
e

d
i
s
e
a
s
e
;

m
a
y

i
d
e
n
t
i
f
y

p
a
t
i
e
n
t
s

w
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
h
o

w
i
l
l

b
e
n
e
f
i
t

f
r
o
m

C
5

b
l
o
c
k
a
d
e
C3 AL
nephrise
tic sv
factoroe
cl
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
is
a
td
eo
d
n
wo
it
t
hc
o
Cr
3r
e
gl
la
ot
me
e
rw
ui
lt
oh
p
ad
ti
hs
ye
;a
s
me
a
ya
c
it
di
ev
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
ni
t
iy
f;
y
a
pl
as
to
i
es
ne
te
sn

wi
hn
o
M
wP
iG
lN
l
t
by
ep
ne
e
f1
i
t

f
r
o
m
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
B

c
e
l
l

t
a
r
g
e
t
e
d

t
h
e
r
a
p
i
e
s
FactorM
H a
proteiy
n
levelsi
d
e
n
t
i
f
y
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns

u
n
d
e
r
l
y
i
n
g

m
e
c
h
a
n
i
s
m

o
f

a
l
t
e
r
n
a
t
i
v
e
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
p
a
t
h
w
a
y

a
c
t
i
v
i
t
y
;

m
a
y

i
d
e
n
t
i
f
y

p
a
t
i
e
n
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
t
s

w
h
o

w
i
l
l

b
e
n
e
f
i
t

f
r
o
m

p
l
a
s
m
a

i
n
f
u
s
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
i
o
n
/
e
x
c
h
a
n
g
e
AutoanMT
tibodiae
es to ys
factor t
H and i
factordn
B eo
nt
t
iw
fi
yd
e
ul
ny
d
ea
rv
la
yi
il
na
gb
l
me
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
e
c
h
a
n
i
s
m

o
f

a
l
t
e
r
n
a
t
i
v
e

p
a
t
h
w
a
y

a
c
t
i
v
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
i
t
y
;

m
a
y

i
d
e
n
t
i
f
y

p
a
t
i
e
n
t
s

w
h
o

w
i
l
l

b
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
e
n
e
f
i
t

f
r
o
m

c
e
l
l

t
a
r
g
e
t
e
d

t
h
e
r
a
p
i
e
s
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
GenetiMN
c ao
mutatiyt
on
screeniw
ing: di
ed
ne
tl
Fiy
af
cya
tv
oua
rni
dl
Hea
rb
l
ye
i;
Cn
Fgc
H l
Rmi
1en
,ci
hc
2a
,nl
i
asi
nm
dp
ol
5fi
c
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
a
lt
ti
eo
Frn
ans
ca
ttu
oin
rvk
en
Io
pw
an
t
h
Cw
3a
y

a
c
Ft
ai
cv
ti
ot
ry

Abbreviations: MPGN, membranoproliferative glomerulonephritis.

Table 3
Complement testing in patients with aHUS

I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
C3 andLA
C4 on
w
i
Cn
3s
e
sn
us
pi
pt
oi
rv
te
s
i
tn
hd
ai
tc
a
dt
io
sr
e
ao
sf
e
c
io
nm
vp
ol
le
vm
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
e
sn
t
t
ha
ec
t
ci
ov
ma
pt
li
eo
mn
e
ni
tn

sa
yH
sU
tS
e
m
;

n
o
r
m
a
l

C
4

s
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
u
g
g
e
s
t
s

a
n

a
l
t
e
r
n
a
t
i
v
e

p
a
t
h
w
a
y

p
r
o
c
e
s
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
s
SolublAT
e C5b- e
9 s
et
n
sn
io
t
i
vw
ei
d
ie
nl
dy
i
ca
av
ta
oi
rl
a
ob
fl
e
c
o
m
p
l
e
m
e
n
t
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
a
c
t
i
v
a
t
i
o
n

a
n
d

m
a
y

a
l
s
o

r
e
f
l
e
c
t

a
c
t
i
v
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
e

d
i
s
e
a
s
e
LevelsML
of ae
factoryv
H, e
factoril
I, MCPds
e
nm
ta
iy
f
yb
e
u
n
do
er
rm
la
yl
i
ni
gn

mp
ea
ct
hi
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
ae
n
it
s
m
w
oi
ft
h
a
ld
ty
es
rf
nu
an
tc
it
vi
eo
n
p
ap
tr
ho
wt
ae
yi
n
a
c
t
i
v
i
t
y
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
AutoanMT
tibodiae
es to ys
factor t
H i
dn
eo
nt
t
iw
fi
yd
e
ul
ny
d
ea
rv
la
yi
il
na
gb
l
me
e
c
h
a
n
i
s
m

o
f
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
a
l
t
e
r
n
a
t
i
v
e

p
a
t
h
w
a
y

a
c
t
i
v
i
t
y
GenetiMT
c ae
mutatiys
on t
i
screen s
ing: d
en
no
t
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
Fi
afw
cyi
td
oue
rnl
dy
He
ra
lv
ya
i
Mnl
Cga
Pb
ml
e
c;
h
Fat
ane
cis
tst
oms
r
ot
Ifa
k
ae
l
t
Ceo
3ro
n
al
to
in
 I
n
t
eL
ri
pm
ri
et
ta
at
ti
io
on
Test ns
Fvg
ae
ct
tpo
oa
rth
he
Bwl
ap
y
w
ai
Tct
hth
ri
ova
mic
btu
oyt
m e
o
c
d
a
u
r
l
e
i
n

Abbreviations: aHUS, atypical hemolytic uremic syndrome; MCP, membrane

cofactor protein.
Immunofluorescence Microscopy
Most native kidney biopsies are immunostained for C3 and C4 fragments, and kidney
allograft biopsies are now routinely immunostained for C4d. Although one generally refers to
“C3” deposits, in truth the immunostaining is performed to detect C3 fragments that are
covalently bound to kidney surfaces during complement activation (C3b, iC3b, C3d;
see Figure 1). C3 is the central component to complement activation by all pathways, so the
presence of C3 fragments reflects complement activation by any of the three activation
pathways.
Immune Complex Glomerulonephritis
Glomerular immune complexes activate the classical pathway of complement, and classical
pathway proteins (C1q, C4, and C3) are present in the deposits. C3 fragments are detected in
nearly 100% of kidney biopsies from patients with membranoproliferative glomerulonephritis
(MPGN) type 1, lupus nephritis, and post-infectious glomerulonephritis.
C3 Glomerulopathy
C3 glomerulopathy is a recently described disease that is diagnosed by detection of prominent
glomerular C3 in the relative absence of immunoglobulin, C1q, or C4d. This
immunofluorescence pattern indicates alternative pathway activation, and the absence of
immunoglobulin distinguishes C3 glomerulopathy from immune complex diseases such as
MPGN type 1 and lupus nephritis. The histologic pattern of injury varies and can resemble
other forms of proliferative or crescentic glomerulonephritis. The diagnosis of this disease
therefore depends upon the pattern of immune deposits seen on immunofluorescence
microscopy.
Antibody-Mediated Rejection of the Kidney Allograft
In patients with antibody-mediated allograft rejection, C4d is seen in the peritubular
capillaries. This finding is now incorporated into the diagnosis of acute and chronic antibody-
mediated kidney allograft rejection. C4d is deposited due to classical pathway activation on
endothelial cells by donor specific antibodies to human leukocyte antigens (HLA) class I and
class II antigens. C4d, which becomes covalently bound to target endothelial cells, persists
longer than the donor specific antibodies that triggered complement activation.

Additional Readings

Cohen D, Colvin RB, Daha MR, et al. Pros and cons for C4d as a biomarker. Kidney
Int. 2012;81(7):628–639.




Pickering MC, D’Agati VD, Nester CM, et al. C3 glomerulopathy: consensus

report. Kidney Int. 2013;84(6):1079–1089.




West C. Complement and Glomerular Disease. In, The Human Complement System
in Health and Disease. John E. Volanakis and Michael M. Frank, Eds. Marcel
Dekker, Inc. New York. 1998.

Complement Levels
Measurement of C3 and C4 can narrow the differential diagnosis in patients with the nephritic
syndrome (Box 1), and serial measurement of these proteins may detect disease remissions or
flares. The concentration of complement proteins in plasma is influenced by the rate of
production in the liver and consumption of the proteins throughout the body. Complement
activation within the kidney is probably only responsible for a small component of the overall
consumption of complement proteins. Consequently, intra-renal complement activation can
cause tissue injury without causing a decrease in the plasma level of complement proteins. In
membranous nephropathy, for example, complement fragments are seen in the majority of
biopsies and complement activation probably causes podocyte injury, yet the levels of
circulating C3 and C4 are usually normal. The level of C3 is thus a poor indicator of whether
the complement system is “on” or “off”, although in some diseases (such as lupus) the
magnitude of the decrease in these proteins does reflect the overall disease activity.

Box 1

Serum C3 Levels in Patients with Various Kidney Diseases

Normal
Serum
Low Serum Comple
Complement ment
Level Level
Systemic System
diseases ic
diseas
 es

SLE 

 P
o
 l
y
C a
l r
a t
s e
s r
i
I t
I i
I s

( n
7 o
5 d
% o
) s
a

 

C
l H
a y
s p
s e
r
 Normal
Serum
Low Serum Comple
Complement ment
Level Level
I s
V e
n
( s
9 i
0 t
% i
) v
i
 t
 y

C v
l a
a s
s c
s u
l
V i
t
( i
6 s
0
% 
) 

 G
r
 a
n
Subacu u
te l
bacteri o
al m
endoca a
rditis t
(90%) o
s
 i
 s
“Shunt w
” i
nephrit t
is h
 Normal
Serum
Low Serum Comple
Complement ment
Level Level

 p
 o
l
Cryogl y
obulin a
emia n
(90%) g
i
 i

t
i
aHUS s
(50%)

 

M
i
c
r
o
s
c
o
p
i
c

p
o
l
y
a
n
g
i
i
t
i
s




H
 Normal
Serum
Low Serum Comple
Complement ment
Level Level
e
n
o
c
h
-
S
c
h
ö
n
l
e
i
n

p
u
r
p
u
r
a




G
o
o
d
p
a
s
t
u
r
e

s
y
n
d
r
o
 Normal
Serum
Low Serum Comple
Complement ment
Level Level
m
e




V
i
s
c
e
r
a
l

a
b
s
c
e
s
s

Primary Primar
kidney y
diseases kidney
diseas
 es

Poststr 
eptoco
ccal I
glomer g
ulonep A
hritis
(90%) n
e
 p
 h
r
MPGN o
type I p
(70%) a
t
 Normal
Serum
Low Serum Comple
Complement ment
Level Level
h
 y


C3 
glomer
ulonep A
hritis n
(75%) t
i
 g
 l
o
Dense m
deposit e
disease r
(80%) u
l
 a

r
MPGN b
type III a
(80%) s
e

m
e
n
t

m
e
m
b
r
a
n
e

d
i
s
e
a
s
 Normal
Serum
Low Serum Comple
Complement ment
Level Level
e




R
e
n
a
l

l
i
m
i
t
e
d

A
N
C
A
-
a
s
s
o
c
i
a
t
e
d

v
a
s
c
u
l
i
t
i
s
 Normal
Serum
Low Serum Comple
Complement ment
Level Level

Note: The percentage of patients with low C3 levels is shown in parentheses

Abbreviations: SLE, systemic lupus erythematosus; MPGN, Membranoproliferative
glomerulonephritis; ANCA, anti-neutrophil cytoplasmic antibody; aHUS, atypical
hemolytic uremic syndrome; Ig, immunoglobulin.
In glomerular diseases that are associated with decreased C3 levels, the sensitivity of this test
varies from 50–90% (Box 1). Immune-complex-mediated diseases, such as lupus nephritis,
tend to decrease the levels of both C3 and C4, consistent with complement activation through
the classical pathway (Figure 1). Alternative pathway driven diseases such as aHUS and C3
glomerulopathy, on the other hand, are sometimes associated with low levels of C3 and
normal C4 levels. Low complement levels can be caused by decreased production of the
proteins by the liver or consumption of the proteins in tissues other than the kidneys, so low
C3 or C4 levels are not specific markers of glomerulonephritis. Other diseases can also cause
C3 consumption, including sepsis, atheroembolic disease, pancreatitis, and HIV infection.
Production of complement proteins may be decreased in patients with malnutrition or liver
disease, and production can increase during pregnancy or the acute phase response. A
pregnant patient with active lupus nephritis might therefore have normal C3 and C4 levels in
spite of high consumption of these proteins.

Additional Readings

Birmingham DJ, Irshaid F, Nagaraja HN, et al. The complex nature of serum C3 and
C4 as biomarkers of lupus renal flare. Lupus. 2010;19(11):1272–1280.




Hebert LA, Cosio FG, Neff JC. Diagnostic significance of

hypocomplementemia. Kidney Int. 1991;39(5):811–821.




Noris M, Caprioli J, Bresin E, et al. Relative role of genetic complement

abnormalities in sporadic and familial aHUS and their impact on clinical
phenotype. Clin J Am Soc Nephrol. 2010;5(10):1844–1859.




Sethi S, Fervenza FC, Zhang Y, et al. C3 glomerulonephritis: clinicopathological

findings, complement abnormalities, glomerular proteomic profile, treatment, and
follow-up. Kidney Int. 2012;82(4):465–473.
 


Varade WS, Forristal J, West CD. Patterns of complement activation in idiopathic

membranoproliferative glomerulonephritis, types I, II, and III. Am J Kidney Dis.
1990;16(3):196–206.

Hemolytic Assays: CH50 and AH50

The CH50 assay measures the overall hemolytic capacity of a patient’s plasma, and is used as
a screening test for complement deficiencies. In this test, antibody coated sheep erythrocytes
are incubated with patient serum, and the degree of lysis reflects classical pathway activity of
the serum. Consequently, deficiency or consumption of classical pathway components, C3, or
terminal pathway components causes a decrease in the CH50. In the AH50 assay, the lysis of
rabbit or guinea pig erythrocytes is measured in a reaction that only permits activation via the
alternative pathway. A patient with low C3 should have low hemolytic activity by both the
CH50 and AH50 assays since C3 is central to complement activation through either pathway.
Enzyme-linked immunosorbent assays (ELISAs) have also been developed to measure the
activation potential of the classical, alternative, or mannose binding lectin pathway, and are
used in a growing number of clinical laboratories.

Genetic Analysis
A number of genetic defects in complement regulation have been identified in patients with
kidney disease. The most striking example is aHUS, in which disease-associated mutations
have been identified in the genes for factor H, factor I, C3, factor B, membrane cofactor
protein, thrombomodulin, and genes for the complement factor H related proteins (Table 1).
Mutations in complement regulatory genes have also been identified in patients with C3
glomerulopathy, other forms of thrombotic microangiopathy, and MPGN type 1. The
functional consequence of these different mutations is, in most cases, over-activity of the
alternative pathway.
Because these kidney diseases are associated with so many different mutations, identifying
the specific mutation in an individual patient is complicated. The genetic studies can be very
helpful, however. Patients with aHUS do not always present with all of the typical clinical
findings, and the identification of an associated complement mutation provides support for
this diagnosis. The underlying complement defect also influences the prognosis, although
genetic analysis takes too long to guide therapy during acute flares. For patients with aHUS
who develop end stage kidney disease, detection of an underlying mutation is also important
for transplant planning. Membrane cofactor protein is a transmembrane protein, and a kidney
allograft from a healthy donor corrects the defect. On the other hand, kidney donation from a
relative is contraindicated if the relative carries the same complement mutation as the patient,
even if the potential donor is disease free. It is worth noting, however, that the recurrence of
aHUS after transplantation in patients with membrane cofactor protein mutations is higher
than 10% in some series. This may be due to co-existing mutations in other complement-
related genes in some patients. Because factor H, factor I, C3, and factor B are primarily
synthesized in the liver, transplant recipients with mutations in the genes for these proteins
have limited ability to control complement activation within the allograft and are at high risk
of disease recurrence during the peri-transplant period.

Additional Readings

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Caprioli J, Noris M, Brioschi S, et al. Genetics of HUS: the impact of MCP, CFH,
and IF mutations on clinical presentation, response to treatment, and outcome. Blood.
2006;108(4):1267–1279.

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

Fang CJ, Fremeaux-Bacchi V, Liszewski MK, et al. Membrane cofactor protein

mutations in atypical hemolytic uremic syndrome (aHUS), fatal Stx-HUS, C3
glomerulonephritis, and the HELLP syndrome. Blood. 2008;111(2):624–632.

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

Jodele S, Licht C, Goebel J, et al. Abnormalities in the alternative pathway of

complement in children with hematopoietic stem cell transplant-associated
thrombotic microangiopathy. Blood. 2013.

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

Le Quintrec M, Zuber J, Moulin B, et al. Complement Genes Strongly Predict

Recurrence and Graft Outcome in Adult Renal Transplant Recipients with Atypical
Hemolytic and Uremic Syndrome. Am J Transplant. 2013.




Noris M, Caprioli J, Bresin E, et al. Relative role of genetic complement

abnormalities in sporadic and familial aHUS and their impact on clinical
phenotype. Clin J Am Soc Nephrol. 2010;5(10):1844–1859.




Servais A, Fremeaux-Bacchi V, Lequintrec M, et al. Primary glomerulonephritis with

isolated C3 deposits: a new entity which shares common genetic risk factors with
haemolytic uraemic syndrome. J Med Genet. 2007;44(3):193–199.




Servais A, Noel LH, Roumenina LT, et al. Acquired and genetic complement
abnormalities play a critical role in dense deposit disease and other C3
glomerulopathies. Kidney Int. 2012;82(4):454–464.




Sethi S, Fervenza FC. Membranoproliferative glomerulonephritis--a new look at an

old entity. N Engl J Med. 2012;366(12):1119–1131.

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THERAPEUTIC COMPLEMENT BLOCKADE

Complement Inhibitory Drugs

Eculizumab has been approved by the FDA for the treatment of aHUS, and has been tested in
several other kidney diseases. It is a humanized murine monoclonal antibody to complement
C5 that prevents the formation of C5a and C5b-9, but it does not prevent the generation of
C3a and C3b. Leaving the early complement system intact may reduce the risk of infection,
but C3a and C3b may also contribute to the pathogenesis of some inflammatory diseases.
Because eculizumab prevents formation of the membrane attack complex, the CH50 in treated
patients should be close to 0. This functional readout aids in monitoring the dosing of the
drug. C5 blockade does not directly affect the levels of circulating C3 and C4, nor does it
block C3 deposition within glomeruli. The primary risk of complement inhibition is that of
infection, and all patients who receive eculizumab should be either immunized for
meningococcus or prophylactically treated for this infection with antibiotics.
Eculizumab is an expensive drug, limiting its use for diseases in which its benefit is uncertain
or that have other effective treatments. Even in patients with aHUS, the optimal duration of
treatment is unknown. Patients with complement mutations are at lifelong risk of recurrence,
and it is not clear whether patients should be treated indefinitely or only during periods of
active disease. Disease flares are often triggered by illness (particularly diarrheal illnesses),
pregnancy, and certain drugs. In some cases disease recurrence has been attributed to very
minor stressors, such as vaccination. A dilemma, then, is that flares may be triggered by
minor events and may rapidly lead to irreversible kidney injury, yet prevention of these flares
could require life-long therapy.
Other complement inhibitory drugs are in development, some of which block specific
activation pathways or activation fragments. One difficulty in developing new drugs for the
treatment of aHUS and C3 glomerulopathy is the large number of underlying genetic
mutations and autoantibodies associated with these diseases. Depending upon their
mechanisms of action, many of the new drugs will likely not work for patients with particular
underlying complement defects. For example, gain of function mutations in C3 that resist
inactivation by endogenous complement regulatory proteins might also be resistant to
inactivation by some anti-complement drugs. Although complete genetic and molecular
testing of each patient may take weeks or months to complete, functional assays of
complement inhibition may provide a rapid means of testing complement inhibition by a
specific drug in an individual patient.

Additional Readings

Ricklin D, Lambris JD. Progress and Trends in Complement Therapeutics. Adv Exp

Med Biol. 2013;735:1–22.

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

Rother RP, Rollins SA, Mojcik CF, Brodsky RA, Bell L. Discovery and development
of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal
hemoglobinuria. Nat Biotechnol. 2007;25(11):1256–1264.



 Zhang Y, Nester CM, Holanda DG, et al. Soluble CR1 therapy improves complement
regulation in C3 glomerulopathy. J Am Soc Nephrol. 2013;24(11):1820–1829.




Zuber J, Quintrec ML, Krid S, et al. Eculizumab for Atypical Hemolytic Uremic
Syndrome Recurrence in Renal Transplantation. Am J Transplant. 2012.

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FUTURE DIRECTIONS
The complement system plays a central role in the pathogenesis of a wide range of kidney
diseases. Mutations and auto-antibodies that impair control of the alternative pathway are
associated with the development of aHUS, C3 glomerulopathy, and several other kidney
diseases. Eculizumab has been approved for the treatment of aHUS, and a variety of new anti-
complement drugs are in development. Additional therapeutic options may lead to greater
complexity regarding which drugs to use, but improved biomarkers of complement activation
may improve our ability to identify appropriate patients for treatment. Nevertheless, it is clear
that the complement system is an important mediator of kidney injury and the role of anti-
complement therapies in nephrology will expand in the coming years.
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Acknowledgments
Support: Dr. Thurman is supported by National Institutes of Health grants DK076690
and HD070511.
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Footnotes
Financial disclosure: Dr. Thurman receives royalties from Alexion
Pharmaceuticals, Inc.
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