Contrasting Roles of Complement Activation and Its Regulation in Membranous Nephropathy

Patrick N. Cunningham and Richard J. Quigg Section of Nephrology, The University of Chicago, Chicago, Illinois

The complement system is involved in defense against microorganisms, the processing of immune complexes and apoptotic debris, and the development of an appropriate immune response. Along with these physiologic effects, complement activation has the potential to result in tissue pathology. To limit this, various complement regulatory proteins (CRP) are present on host cells, including the glomerular podocyte. Experimental data from the Heymann nephritis (HN) rat model of human membranous nephropathy (MN) have shown that IgG antibodies in subepithelial immune deposits initiate complement activation and C5b-9–mediated damage of the overlying podocyte. Although IgG can activate the classical pathway, there also is evidence that alternative pathway activation occurs in MN, which could occur because of absent, dysfunctional, or inhibited podocyte CRP. Related to this are experimental data in HN showing the presence of antibodies that bind and inhibit podocyte CRP; although such antibodies have not been documented in human MN, a decrease in CR1 quantity on the podocyte has been observed. A s a result of a relative lack of CRP and the exposure of activating complement proteins to tubular cells, alternative complement pathway activation and C5b-9–mediated tubular injury can occur in MN and other proteinuric diseases. Overall, in a disease such as MN, the balance between complement regulation and activation is tipped toward its being activated. Therefore, a number of therapeutic approaches have been developed to counteract this, including recombinant forms of endogenous CRP and complement-inhibitory monoclonal antibodies. There is good reason to be optimistic that approaches to block complement activation will become viable therapy for human MN in the future. J Am Soc Nephrol 16: 1214–1222, 2005. doi: 10.1681/ASN.2005010096

embranous nephropathy (MN) is a common cause Activation and Regulation in the of nephrotic syndrome in adults, with as many as Complement System M half of affected individuals progressing to renal The complement system is an important component of both failure over a period of years to decades (1). Although some innate and adaptive immunity, consisting of the classical, alter- cases are associated with malignancy, infections, or drugs, most native, and lectin pathways, altogether containing approxi- are of a primary “idiopathic” nature and are due to underlying mately 35 proteins involved in its activation and regulation autoimmunity (2). Pathologically, MN is characterized by thick- (Figure 1) (reviewed in references 3–6). Under normal circum- ening of the glomerular capillary wall, which is visible by light stances, complement activation occurs on pathogens and un- microscopy, but little in the way of increased cellularity. As wanted host material (e.g., apoptotic debris) but not on normal seen by electron microscopy, there are subepithelial immune host cells; distinction between the two is based on the molec- deposits and effacement of the overlying visceral glomerular ular repertoire presented, such as antigens to which antibodies epithelial cell (podocyte). By immunohistologic techniques, are directed or pathogen-associated molecular patterns (PAMP) these immune deposits contain IgG as well as complement (5). The classical pathway is initiated on biologic surfaces by the binding of complement component C1q to complement-fixing components. This review details the evidence from human and isotypes of IgG, as well as IgM. The activated C1 engages and animal data that the complement system plays a key role in the activates C4 and C2 in sequence, thereby forming the C3 con- pathogenesis of MN and speculates on ways in which comple- vertase, which catalyzes the ongoing cleavage of C3 to the C3a ment inhibitory strategies could be used as therapy. anaphylatoxin and C3b, which becomes transiently reactive, allowing it to become bound to the growing immune complex. The C5 convertase, C4b2a3b, in turn cleaves circulating C5 into C5a and C5b; C5a is similar to C3a in its activity as an anaphylatoxin, whereas C5b is like C3b and remains associated with Published online ahead of print. Publication date available at www.jasn.org the immune complex, where it recruits C6, C7, C8, and C9 to Address correspondence to: Dr. Richard Quigg, Chief of the Section of Nephrol- form the C5b-9 complex. When complement activation occurs ogy, The University of Chicago, AMB-S523, MC 5100, 5841 South Maryland Avenue, Chicago, IL 60637. Phone: 773-702-0757; Fax: 773-702-4816; E-mail: in the vicinity of a lipid membrane such as on bacteria, eryth- [email protected] rocytes, and nucleated cells, the newly expressed hydrophobic

tor H and the cell membrane proteins decay accelerating factor (DAF; CD55), membrane co-factor protein (CD46), and complement receptor 1 (CR1; CD35). CR2 (CD21) is also a member of this family, whereas CR3 (CD11b/CD18) is a ␤2 integrin; both have affinity for C3 proteins but do not limit complement activation. Confusing to all but the aficionados is CR1-related gene/protein y (Crry) (9), which is a rodent member of the regulators of complement activation family with fairly broad activity (10). Relevant to MN and this discussion are the CRP located on the podocyte. DAF and CD59 are linked to the rat and human podocyte membrane via a glycosylphosphatidyl- inositol (GPI) anchor (11–15). As its transmembrane CRP, human podocytes use CR1 (16), whereas rat podocytes have Crry (15). Overall, it seems that human podocytes use DAF and CR1 to limit C3 and C5 activation and CD59 to restrict C5b-9 formation. Figure 1. The complement system. Activation can occur through Besides inflammation, complement activation clearly is in- the classical, lectin, or alternative pathways. Proinflammatory volved in the immune response to foreign antigens or lack of it mediators include anaphylatoxins C3a and C5a, C3b (and its in tolerance to self and in immune complex processing (17–19). cleavage fragments), which can interact with complement re- Simplistically, for a productive immune response to be propa- ceptors, and the C5b-9 membrane attack complex (in boldface). gated when the B lymphocyte antigen receptor is engaged by Regulation occurs throughout the pathways by complement antigen, there must be a second signal that can be delivered by regulatory proteins (CRP), which are depicted in boxes adjacent the interaction of immune complex-bearing C3d with B lym- to and matched in black type for the complement proteins that phocyte CR2 (20). Complement activation is also important for they inhibit. Factor I (fI) can cleave and inactivate C4b and C3b the processing of circulating immune complexes, both by lim- by using C4-binding protein (C4bp), complement receptor 1 iting their size and through interactions with erythrocyte CR1 (CR1), membrane co-factor protein (MCP), and factor H (fH) as (or platelet factor H in rodents [21]), which shuttles immune co-factors. CR1-related protein y (Crry) is found exclusively in ␥ rodents. Ag, antigen; Ab, antibody; C1inh, C1-inhibitor; MBL, complexes to Fc R- and CR3-bearing cells of the mononuclear mannose-binding lectin; MASP-2, MBL-associated serine pro- phagocyte system. Complement activation also can profoundly tease-1; DAF, decay-accelerating factor. affect the fate of immune complexes in glomeruli and may well be responsible for the movement of certain immune complexes from subendothelial to subepithelial sites (22). Thus, in addi- domains of C7, C8, and C9 can insert into the cell membrane. In tion to the pathophysiologic effects from complement activa- some circumstances, this can lead to osmotic cellular death. In tion products, the complement system has the potential to be other instances in which so-called sublytic quantities of C5b-9 involved in earlier immunologic events that occur in MN. have formed, a variety of cellular events can occur, including activation of signaling pathways and induction of the active process of endocytosis and shedding of C5b-9, likely as a pro- Heymann Nephritis as a Model of MN tective mechanism. In contrast to the classical pathway, the We are fortunate to have an excellent animal model of MN, alternative pathway of complement is independent of antibody Heymann nephritis (HN) (23). In the active model of HN and has a low-level constitutive activity, as a result of the (AHN), rats are immunized with Fx1A, an extract of rat kidney spontaneous hydrolysis of C3 in plasma. The alternative path- that contains components of the proximal tubule brush border. way C3 and C5 convertases (C3bBb and C3bBb3b) are homol- Animals respond to this immunization by developing autoan- ogous to those of the classical pathway and similarly generate tibodies against various brush border proteins, some of which C3a, C3b, C5a, and C5b-9. are also expressed on the podocyte. Within 8 to 10 wk, most rats Besides the capacity of complement proteins to be discrimi- develop clinical aspects seen in humans with MN, namely natory in their binding, such as to PAMP or immune complexes heavy proteinuria (consisting of albumin and higher molecular that bear IgG, an additional level of security is provided self- weight proteins), features of the nephrotic syndrome, and mild tissue by circulating and cell-bound complement regulatory renal insufficiency (24,25). Although initially few changes are proteins (CRP) (7,8). The various CRP act throughout the com- visible by light microscopy, immunofluorescence microscopy plement activation cascades, from prevention of C1 activation demonstrates the typical granular peripheral capillary wall by C1 inhibitor to blocking formation of C5b-9 by CD59 (Figure deposition of IgG and C3, visible ultrastructurally as electron- 1). A number of proteins are contained within the regulators of dense deposits beneath effaced podocytes. With time, the on- complement activation gene family and are concentrated at the going generation of immune complexes decreases, but protein- C3/C5 step, which is where all three pathways converge and uria remains for the life of the animal. The passive model of HN generate C3a, C3b, and C5a and begin the formation of C5b-9. (PHN) has similar features but is induced by the passive ad- These include the plasma proteins C4-binding protein and fac- ministration into rats of antiserum from sheep or rabbits that 1216 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 1214–1222, 2005 were immunized with Fx1A. This leads to rapid induction of mone receptor in Graves disease (41) and potentially to podo- ␤ these changes within 5 to 6 d after injection. cyte 1 integrins in PHN (42). Along with its clinical manifestations, AHN has other simi- Studies in HN have taught us much about the disease process larities to human MN; both are autoimmune diseases charac- in human MN. Depletion of serum complement with cobra terized by the production of pathogenic autoantibodies (26–28), venom factor immediately before induction of PHN prevents and both have a genetic susceptibility involving genes both foot process effacement and proteinuria almost completely inside and outside the MHC with incomplete penetrance (i.e., without affecting the quantity of subepithelial immune com- not all animals of the same background in any given experi- plexes (43). Complement activation in the subepithelial space ment develop disease) (2,29). Just as must occur in humans, produces the C3a and C5a anaphylatoxins and the C3 opsonins. tolerance to self-antigens is lost in AHN; in this model, a fairly However, as elegantly dissected by Salant et al. (33), these specific immunization scheme must be followed, and variations proinflammatory components of complement are not of conse- on this can prevent autoimmunity from occurring, including quence when generated in the subepithelial space because of its through the induction of antigen-specific T suppressor cells being separated from the blood by the glomerular capillary (30). The primary antigenic target in AHN is megalin, which is wall. Thus, MN is a noninflammatory disease. Using an iso- shared by podocytes and proximal tubular epithelial cells (26– lated perfused kidney model of PHN, Cybulsky et al. (44) 28). Unfortunately, AHN with its anti-megalin autoantibodies showed that C8 was required in the plasma perfusate for podo- is unlikely truly to reflect human MN, as megalin is absent from cyte injury and proteinuria to occur. In further studies, Baker et the human podocyte. In fact, human MN is likely to be a al. (45) showed that C6-depleted rats did not develop protein- heterogeneous disease, with multiple possible antibody-anti- uria in PHN. Overall, because C6 and C8 have no known role gen specificities and routes of subepithelial immune complex other than contributing to the formation of C5b-9 (Figure 1), accumulation (see also Ronco and Debiec in this Frontiers in these provided strong evidence for the role of C5b-9 to result in Nephrology). Antibodies can bind directly to intrinsic glomer- podocyte injury in HN. This topic is covered in detail by ular antigens such as megalin in HN or neutral endopeptidase Nangaku, Shankland, and Couser in this Frontiers in Nephrol- (NEP) in congenital MN (31); antibodies can bind to antigens ogy. extrinsic to the glomerulus that have accumulated in this re- Consistent with complement activation occurring in human gion, including a variety of cationic proteins (32,33), and im- and experimental MN is the finding of C3 and C5b-9 in sub- mune complexes originally formed in the circulation can ulti- epithelial immune deposits and in the urine (46–49). In AHN, mately end up in this area, such as occurs in serum sickness even if there are large amounts of IgG1 and IgG2c antibodies in (34). Related to the latter mechanism, the experimental evi- glomerular immune deposits, complement is not activated un- dence is overwhelming that circulating immune complexes less IgG2b antibodies are present (25). This complement acti- cannot directly deposit in subepithelial sites but rather must go vation (as evidenced by C3 deposition) is associated with podo- through intermediate steps, such as binding in subendothelial cyte ultrastructural abnormalities and proteinuria. In keeping locations and then dissociating and reforming in the subepithe- with these observations is that rat IgG2b is very effective at lial space (35) (see also Nangaku, Shankland, and Couser in this binding C1q and leading to subsequent complement activation, Frontiers in Nephrology section). As noted previously, the whereas IgG1 and IgG2c are not (50). In PHN, injection of the complement system can profoundly affect the disposition of complement-activating sheep ␥1 anti-Fx1A leads to disease, immune complexes and surely plays a role in this localization whereas the non–complement-activating ␥2 anti-Fx1A does not and movement of glomerular immune complexes (22,36,37). (43). A similar requirement for a classical pathway-activating Given the broad autoantibody repertoire in systemic lupus human IgG isotype is seen in congenital MN. Maternal produc- erythematosus, including to cellular and matrix proteins, pos- tion of anti-NEP IgG1 seems necessary for disease; if only itively charged histones, and antigens contained in circulating anti-NEP IgG4 is produced, then proteinuria does not result immune complexes, it is conceivable that lupus MN could (51). Of the human IgG isotypes, IgG1 and IgG3 are capable of involve any one or more of these three possibilities (38,39). binding C1q and leading to C4 activation (52).

Role of CRP and the Alternative Pathway in Complement Activation in Human and Human and Experimental MN Experimental MN These data in humans and experimental animals would lead Relevant to the complement system and the pathogenesis of one to believe that MN should have a uniform pathogenesis: MN is the presence of IgG contained in the subepithelial im- Irrespective of exact antigen specificity, the presence of subep- mune deposits. Although most of the pathogenicity of IgG is ithelial C1q-fixing IgG leads to classical pathway complement the result of the ␥ heavy chain constant domains (which form activation all of the way to C5b-9, which exerts its effects on the the Fc) interacting with the C1q complement protein and Fc␥R podocyte. Unfortunately for this simple paradigm, most cases on inflammatory cells, other effects can be the direct conse- of idiopathic human MN have a predominance of IgG4, with quence of IgG binding to particular antigens or even its shear less IgG3 and no IgG1 in subepithelial immune deposits (53,54). bulk when accumulated in large latticed immune complexes as In addition, there is little to no demonstrable C1q and C4 in occurs in MN (40). Examples of antigen reactivities that can be these deposits (55), consistent with the lack of classical path- of direct consequence include those to thyroid stimulating hor- way-activating capacity of IgG4 (56) and that the large hetero- J Am Soc Nephrol 16: 1214–1222, 2005 Complement Activation and Its Regulation in Membranous Nephropathy 1217

ligomeric C1 complex (C1qr2s2, Mr approximately 800 kD) undoubtedly has limited access across the glomerular basement membrane to the subepithelial space. Although hereditary complete C4 deficiency is rare and has been associated with lupus-like and proliferative glomerulonephritis, MN has also been reported in a C4-deficient patient (57). Thus, the absence of classical pathway-activating IgG isotypes, or C1q and C4 in glomeruli, is evidence against classical pathway activation as predominating in idiopathic MN and that alternative pathway activation may be occurring instead. The alternative pathway is spontaneously active; the presence of CRP on host but not foreign tissue confers the specificity of activation. The podocyte primarily relies on membrane CR1 (Crry in rodents) and GPI-linked DAF; whether circulating factor H has access to the podocyte is not clear but may not occur substantially, given the asymmetry of factor H (58). Per- Figure 2. Effects of CRP inhibition in models of membranous haps because of this, the podocyte does have the capability to nephropathy (MN). Injection of anti-megalin antibodies alone into rats led to subepithelial immune deposit formation but no make its own factor H (59) and related proteins (60,61). apparent complement activation or proteinuria (A). This can be Alternative pathway activation and C5b-9 generation on the attributed to functional complement regulation on podocytes, podocyte in MN could occur because of absent, dysfunctional, as injection of inhibitory antibodies to Crry and CD59 resulted or inhibited CRP. Related to the last mechanism, we have found in development of abnormal proteinuria (Ͼ5 mg/d) in all an- that the crude Fx1A preparation that is used to induce AHN in imals (B), which was not apparent in rats that received anti- rats contains Crry and CD59 and that within anti-Fx1A gener- Crry and anti-CD59 only (data not shown). Anti-Fx1A antibod- ated in rats (or in sheep as used in PHN), there are antibodies ies are reactive with a variety of proteins, including Crry and to both Crry and CD59 that can neutralize their complement CD59, and can neutralize their functions in vitro. However, regulatory activity in podocytes in vitro (15,62,63). In a study by their removal from anti-Fx1A did not affect complement acti- Schiller et al. (63), rats that were immunized with Fx1A lacking vation or proteinuria in passive Heymann nephritis (C) com- Crry generated anti-Fx1A autoantibodies that accumulated in pared with native anti-Fx1A (D), indicating that the tempo of complement activation by anti-Fx1A can overwhelm intrinsic subepithelial immune deposits, but there was no evidence for podocyte complement regulation. The dashed line represents complement activation in glomeruli and abnormal proteinuria the limit of daily protein excretion in normal rats (Ͻ5 mg/d). did not result. Disease could be reconstituted through the in- Data are mean Ϯ SEM (n ϭ 5 to 6 per group) and are from clusion of recombinant Crry in the immunogen or by the pas- reference (64). sive transfer of anti-Crry antibodies. Thus, generation of autoantibodies to functional podocyte CRP could render these cells susceptible to complement activation and C5b-9 formation. CR1 has long been associated with glomerular diseases, in Further support for this concept is our finding that injection of particular lupus nephritis (68,69). The possibility that CR1 is function-neutralizing anti-Crry and anti-CD59 antibodies to cleaved at its membrane proximal site such as by inflammatory rats with preexisting immune deposits that consisted only of cell proteases (leaving a “stump”) or that it is absent altogether anti-megalin antibodies led to development of abnormal pro- has been investigated by Schifferli et al. (70), who have shown teinuria (64) (Figure 2). The activation of complement through that the entire CR1 protein is absent in glomerular diseases, any pathway represents a balance between activating and in- including MN. As the authors have postulated, the absence of hibiting influences; even with a full repertoire of CRP, comple- CR1 on podocytes could render these cells “highly sensitive to ment activation can overwhelm its regulation (65). This seems complement attack.” Whether absence of CR1 in glomerular to occur in the PHN model in which immune complex forma- diseases such as MN represents an intrinsic or acquired defect tion occurs rapidly and with a sufficient amount of antibody and, importantly, whether this contributes to disease pathogen- (66) to overwhelm any protective effect of the CRP, thereby esis remain to be established. resulting in complement activation and disease (64) (Figure 2). This guides the underlying premise of providing extrinsic CRP Role of Complement in the to podocytes in therapy (discussed below). Tubulointerstitial Injury in MN Despite the data in AHN discussed above, there has been no In addition to the pivotal role of complement in mediating evidence that podocyte CRP are the target of autoantibodies in injury to the podocyte in MN, complement activation seems to human MN. As an alternative possibility, a reduction in CRP be involved in the tubular injury that culminates in the histo- could enhance susceptibility to complement activation; such a logic picture of tubulointerstitial atrophy and fibrosis (71–73). It mechanism underlies paroxysmal nocturnal hemoglobinuria, a has long been known that progression in MN and other glo- clonal abnormality in hematopoietic cells in which there is an merulopathies correlates closely with the degree of proteinuria inability to anchor GPI-linked proteins, including DAF and (74,75). Increasingly, evidence suggests that proteinuria per se CD59, to the plasma membrane (67). An alteration in podocyte may actually be responsible for this tubulointerstitial injury, 1218 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 1214–1222, 2005 through exposure of the luminal surface of tubules to albumin, highest, which is difficult to predict in a disease such as MN, transferrin, lipoproteins, and proteins of the complement sys- particularly given our ignorance of target antigens. One in- tem (76,77). Because of their large size, key complement com- triguing possibility is to identify periods of disease activity as ponents such as C3 and C5 are not normally present in the reflected by ongoing complement activation on the podocyte by glomerular ultrafiltrate; therefore, nature has limited expres- measuring the appearance of C5b-9 in the urine (48,49,90,91). sion of CRP on the apical membrane of tubular cells to only Given these limited options, strategies to inhibit complement, weak expression of CD59 (12). The relative imbalance between such as through the use of recombinant CRP or anti-comple- CRP and proactivating complement proteins results in the al- ment antibodies, could potentially offer a significant advance in ternative pathway’s being activated on proximal tubular cells, the therapy of MN. There is growing evidence in animal mod- which can be modeled in vitro (78,79) and also detected in vivo els that such an approach would be effective and feasible. A (71,80). Compelling evidence that C5b-9 activation occurs and recombinant soluble form of human CR1 was used successfully is injurious to tubular epithelia comes from the studies of by Couser et al. (92) to decrease the extent of glomerular com- Nangaku and colleagues (81–83) in which rats deficient in C6 plement deposition and proteinuria in PHN. Similarly, the were protected from tubulointerstitial injury in the puromycin, rodent molecule Crry, which inhibits the C3 and C5 convertases adriamycin, and remnant kidney proteinuric models. Thus, of the classical and alternative pathways in a manner similar to strategies to inhibit the complement system in MN may protect human CR1, has been used successfully to prevent injury in not only against glomerular damage but also directly against various mouse models of glomerular diseases. Specifically, tubular injury. daily administration of the fusion protein Crry-Ig, comprising two molecules of Crry linked to the constant region of mouse CRP as Therapy for MN IgG1, decreased multiple markers of renal injury in mouse Current therapy for MN consists primarily of immunosup- models of nephrotoxic and lupus nephritis (93,94). In addition, pressive drugs such as corticosteroids, cytotoxic agents, and a monoclonal antibody that blocked C5 activation had a signif- calcineurin inhibitors, aimed mainly at decreasing the produc- icant effect on renal disease and survival in murine lupus (95). tion of the autoantibodies presumed to be present in MN As with the latter studies in murine lupus models in which (84,85). These immunosuppressive therapies come at the cost of homologous proteins were administered chronically, use of serious side effects, such as bone marrow suppression and an endogenous CRP or antibodies in which all but antigen-binding increase in the risk for infection. Furthermore, despite intensive variable regions are “humanized” would avoid any concern of effort and study, available treatment has limited efficacy (86); immunogenicity. Given the multiple beneficial roles of the com- the topic of conventional management of MN is covered by plement system, including its activity against infectious agents, Cattran in this Frontiers in Nephrology section. One potential its ability to enhance a normal humoral immune response while clue to why rational (albeit, nonspecific) therapy targeting the maintaining tolerance, and its processing of immune com- immune system for an autoimmune disease such as MN has not plexes, alteration of any one of these theoretically could occur consistently shown a benefit again comes from the AHN model when inhibition of the complement system is used in therapy. and studies by Noble et al. (87) of its natural course. As time Clues to which of these may occur in human therapy comes passes after immunization with Fx1A, autoantibodies that are from our relatively long-term treatment with Crry-Ig in a detectable in blood wane as does glomerular complement acti- mouse model of lupus nephritis, in which there was no change vation, yet basement thickening and disorganization occur, in autoantibody levels or apparent infectious complications, yet incorporating these immune deposits, and proteinuria persists. there was markedly altered immune complex handling, in this Upon reimmunization with Fx1A, there is the prompt reap- case, protecting the glomerulus (94). Because it does not affect pearance of anti-Fx1A antibodies forming fresh immune depos- the generation of C3, anti-C5 is unlikely to affect immune its and another wave of glomerular complement activation. complex metabolism, although this was not specifically ad- Related to this concept are the findings of Schulze et al. (88) that dressed in its study in lupus mice (95). Unfortunately, because the presence of C3c reflects ongoing complement activation and there is no universally accepted mouse model of MN, compa- tracks with disease activity, whereas the presence of C3d is a rable studies for MN in the mouse have not been performed. marker for past complement activation and those of Makker Potentially even more promising is the strategy of targeting and Kanalas (89) in which kidneys that were from rats with CRP specifically to the site where complement is being acti- AHN and transplanted into normal hosts had prompt disap- vated, which can decrease systemic toxicity of complement pearance of glomerular C3 but persistent glomerular IgG and inhibitors while at the same time increasing their potency. Such proteinuria up to 28 wk posttransplantation. Similar findings of an approach has been used to target CR1 to cell membranes apparent disease activity and inactivity are seen in human MN, with addressins (96) and to selectins with sialyl Lexis x (sLex) as C3d is present in nearly all cases of MN, whereas C3c is glycosylation (97). More recently, the Tomlinson group devel- found much less frequently in biopsy specimens and is associ- oped a chimeric protein that consists of DAF or CD59 fused to ated with higher levels of proteinuria (55). The presence of C3d CR2, the latter targeting the protein to C3d (and iC3b) present in subepithelial immune deposits presumably reflects the ac- at the site of complement activation (98). This targeted inhibitor tion of podocyte CR1 to act as a factor I co-factor for the is approximately 20 times more potent at inhibiting comple- cleavage of iC3b. It seems logical that targeting immunologic ment-mediated lysis of cells compared with soluble DAF or aspects with therapy should be timed when disease activity is CD59 alone, and when injected into lupus-prone MRL/lpr J Am Soc Nephrol 16: 1214–1222, 2005 Complement Activation and Its Regulation in Membranous Nephropathy 1219

into complete remission), providing optimism that eculizumab will be effective therapy for MN. Thus, despite all of the evidence favoring that complement mediates MN in humans, whether eculizumab or any other complement inhibitory strategy such as those presented here will be used as therapy in human MN remains a question for the future.

Acknowledgments Much of the authors’ work presented here has been supported over the years by National Institutes of Health Grant R01DK041873. P.N.C. is also supported by K08DK61375.

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