Title: Membranous nephropathy: recent travels and new roads ahead
Abstract: Insights from experimental studies have been recently translated into substantial advances in understanding the pathogenesis of human membranous nephropathy (MN). These include identification of neutral endopeptidase (NEP) as the target antigen in alloimmune MN resulting from fetomaternal immunization in NEP-deficient mothers, and our demonstration that a high proportion of patients with idiopathic MN (IMN) have circulating antibodies to the M-type phospholipase A2 receptor (PLA2R), a transmembrane protein located on podocytes. Here we highlight the studies that led to these discoveries and our current knowledge about the possible role of anti-PLA2R autoantibodies in the pathogenesis of IMN. Given that the sensitivity and specificity of anti-PLA2R for IMN are >75 and 100%, respectively, we foresee that a widely available assay for anti-PLA2R will prove to be valuable for diagnosing IMN, distinguishing it from secondary MN, and evaluating response to therapy. We suggest reasons why 25% of patients with IMN have tested negative for anti-PLA2R, and propose possible explanations for the presence of complement deposits in IMN despite the fact that immunoglobulin G4 (IgG4), the predominant anti-PLA2R IgG subclass, is incapable of activating the classical complement pathway. Finally, we point out avenues to be explored, including the events that induce production of anti-PLA2R, their ability to cause podocyte injury, the role of complement, and the nature of the antibodies in secondary forms of MN. Insights from experimental studies have been recently translated into substantial advances in understanding the pathogenesis of human membranous nephropathy (MN). These include identification of neutral endopeptidase (NEP) as the target antigen in alloimmune MN resulting from fetomaternal immunization in NEP-deficient mothers, and our demonstration that a high proportion of patients with idiopathic MN (IMN) have circulating antibodies to the M-type phospholipase A2 receptor (PLA2R), a transmembrane protein located on podocytes. Here we highlight the studies that led to these discoveries and our current knowledge about the possible role of anti-PLA2R autoantibodies in the pathogenesis of IMN. Given that the sensitivity and specificity of anti-PLA2R for IMN are >75 and 100%, respectively, we foresee that a widely available assay for anti-PLA2R will prove to be valuable for diagnosing IMN, distinguishing it from secondary MN, and evaluating response to therapy. We suggest reasons why 25% of patients with IMN have tested negative for anti-PLA2R, and propose possible explanations for the presence of complement deposits in IMN despite the fact that immunoglobulin G4 (IgG4), the predominant anti-PLA2R IgG subclass, is incapable of activating the classical complement pathway. Finally, we point out avenues to be explored, including the events that induce production of anti-PLA2R, their ability to cause podocyte injury, the role of complement, and the nature of the antibodies in secondary forms of MN. Membranous nephropathy (MN), despite its recognition as one of the leading causes of idiopathic nephrotic syndrome, remains a relatively rare disease. Why then, has it garnered so much attention in the nephrology community over the last several decades? The answer, in part, is that idiopathic MN is a conceptually simple organ-specific autoimmune disease with pronounced direct and secondary effects. Through studies detailed below, it has become clear that circulating autoantibodies bind to target antigens on the podocyte foot process to initiate the disease process. There, the deposited antibody–antigen complexes not only give rise to the microscopic features by which the disease is characterized but also activate the complement system, leading to injury to the podocyte and subsequent proteinuria. This review will serve to highlight the most recent additions to this ever-evolving field of research. The features that characterize MN as a histopathological entity result from the immune deposits that form at the base of the foot processes of the glomerular visceral epithelial cell or podocyte. The name membranous derives from the thickened glomerular basement membrane (GBM) that is often evident by light microscopy in later stages of the disease. The immune deposits and, more important, the additional matrix material laid down by the injured podocytes are responsible for thickening of the GBM as the disease progresses. Using a silver methenamine stain, David Jones in 1957 first described argyrophilic ‘spikes’ or ‘clubs’ that represent this new matrix material between and around the unstained immune deposits.1.Jones D.B. Nephrotic glomerulonephritis.Am J Pathol. 1957; 33: 313-329PubMed Google Scholar In contrast, immunofluorescence for total immunoglobulin G (IgG) or IgG4 highlights the deposits themselves, yielding fine granular staining in a capillary loop pattern. Electron microscopy details the amorphous, electron-dense deposits that occur in a subepithelial and intramembranous position, as well as the effacement of the podocyte foot processes and other signs of podocyte injury. Most cases of MN in developed countries such as the United States are idiopathic MN (IMN); however, a number of secondary processes can also cause MN that is clinically and histologically similar to IMN. Worldwide, chronic infections such as hepatitis B, malaria, syphilis, and schistosomiasis are the most important causes of secondary MN. Systemic lupus erythematosus can give rise to a membranous form of glomerular disease, classified as class V lupus nephritis. Other autoimmune diseases such as rheumatoid arthritis, autoimmune thyroid diseases, and Sjögren's syndrome can all be associated with MN. Historically, certain medications used for the treatment of rheumatoid arthritis such as gold salts, penicillamine, and some NSAIDs were causally linked to MN. Solid tumors are associated with secondary MN more often than chance alone would predict,2.Lefaucheur C. Stengel B. Nochy D. et al.Membranous nephropathy and cancer: epidemiologic evidence and determinants of high-risk cancer association.Kidney Int. 2006; 70: 1510-1517Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar and on rare occasions remissions and relapses of the glomerular disease have been noted to occur with removal or relapse of the malignancy. Finally, MN can occur de novo after renal transplantation or allogeneic hematopoietic stem cell transplantation, perhaps reflecting alloimmunization to a minor histocompatibility antigen expressed in the glomerulus.3.Ronco P. Debiec H. Molecular pathomechanisms of membranous nephropathy: from Heymann nephritis to alloimmunization.J Am Soc Nephrol. 2005; 16: 1205-1213Crossref PubMed Scopus (103) Google Scholar,4.Brukamp K. Doyle A.M. Bloom R.D. et al.Nephrotic syndrome after hematopoietic cell transplantation: do glomerular lesions represent renal graft-versus-host disease?.Clin J Am Soc Nephrol. 2006; 1: 685-694Crossref PubMed Scopus (104) Google Scholar Secondary forms of MN often exhibit histopathological clues that distinguish them from IMN, although this is not always the case. As opposed to the exclusively subepithelial and intramembranous deposits seen in IMN, secondary forms, especially membranous lupus nephritis, often have mesangial and subendothelial deposits. Tubuloreticular inclusions may also been seen within the glomerular endothelium on electron microscopy in lupus-associated MN. The IgG subclasses found within the glomerular deposits also differ. In contrast to the predominant IgG4 found in IMN, IgG2 and IgG3 are typically most abundant in secondary (lupus- and malignancy-associated) forms of MN.5.Haas M. IgG subclass deposits in glomeruli of lupus and nonlupus membranous nephropathies.Am J Kidney Dis. 1994; 23: 358-364Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 6.Kuroki A. Shibata T. Honda H. et al.Glomerular and serum IgG subclasses in diffuse proliferative lupus nephritis, membranous lupus nephritis, and idiopathic membranous nephropathy.Intern Med. 2002; 41: 936-942Crossref PubMed Scopus (105) Google Scholar, 7.Ohtani H. Wakui H. Komatsuda A. et al.Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy.Nephrol Dial Transplant. 2004; 19: 574-579Crossref PubMed Scopus (140) Google Scholar Finally, the nature of the electron dense material itself may herald a secondary cause. A form of MN characterized by spherular structures within the subepithelial deposits has been described that appears to be distinct from its idiopathic cousin.8.Kowalewska J. Smith K.D. Hudkins K.L. et al.Membranous glomerulopathy with spherules: an uncommon variant with obscure pathogenesis.Am J Kidney Dis. 2006; 47: 983-992Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar An experimental rat model developed by Walter Heymann in the late 1950s has provided much of our knowledge about the pathophysiological mechanisms of human MN.9.Heymann W. Hackel D.B. Harwood S. et al.Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions.Proc Soc Exp Biol Med. 1959; 100: 660-664Crossref PubMed Scopus (397) Google Scholar Known as Heymann nephritis (HN), the model is induced by the active or passive immunization of rats with fraction 1A (Fx1A), a mixture of antigens derived from rat proximal tubular brush border. These rats develop a proteinuric glomerulopathy with histological features virtually identical to the human disease. The model and its findings have been well reviewed in detail elsewhere;10.Kerjaschki D. Neale T.J. Molecular mechanisms of glomerular injury in rat experimental membranous nephropathy (Heymann nephritis).J Am Soc Nephrol. 1996; 7: 2518-2526Crossref PubMed Google Scholar however, several key findings that have helped to establish the pathogenetic paradigm in MN need special emphasis. There was initial debate as to the source of the subepithelial deposits, and the prevailing early view was that they represented circulating immune complexes that deposited in the glomerulus. Others speculated that freely circulating antigens were initially ‘planted’ in the GBM, followed later by antibody binding. Definitive experiments using in vitro and ex vivo perfused rat kidneys in a single-pass system showed that the antibodies bind in situ to a target antigen intrinsic to the glomerular capillary wall.11.Couser W.G. Salant D.J. In situ immune complex formation and glomerular injury.Kidney Int. 1980; 17: 1-13Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 12.Couser W.G. Steinmuller D.R. Stilmant M.M. et al.Experimental glomerulonephritis in the isolated perfused rat kidney.J Clin Invest. 1978; 62: 1275-1287Crossref PubMed Scopus (256) Google Scholar, 13.Van Damme B.J. Fleuren G.J. Bakker W.W. et al.Experimental glomerulonephritis in the rat induced by antibodies directed against tubular antigens. V. Fixed glomerular antigens in the pathogenesis of heterologous immune complex glomerulonephritis.Lab Invest. 1978; 38: 502-510PubMed Google Scholar The primary antigen targeted in HN was ultimately identified as the transmembrane protein megalin, an endocytic receptor in the low-density lipoprotein receptor family that is located within coated pits on rat podocyte foot processes and proximal tubular brush borders.14.Kerjaschki D. Farquhar M.G. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border.Proc Natl Acad Sci USA. 1982; 79: 5557-5561Crossref PubMed Scopus (384) Google Scholar, 15.Kerjaschki D. Farquhar M.G. Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats.J Exp Med. 1983; 157: 667-686Crossref PubMed Scopus (358) Google Scholar, 16.Makker S.P. Singh A.K. Characterization of the antigen (gp600) of Heymann nephritis.Lab Invest. 1984; 50: 287-293PubMed Google Scholar, 17.Raychowdhury R. Niles J.L. McCluskey R.T. et al.Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor.Science. 1989; 244: 1163-1165Crossref PubMed Scopus (208) Google Scholar, 18.Saito A. Pietromonaco S. Loo A.K. et al.Complete cloning and sequencing of rat gp330/’megalin,’ a distinctive member of the low density lipoprotein receptor gene family.Proc Natl Acad Sci USA. 1994; 91: 9725-9729Crossref PubMed Scopus (470) Google Scholar Another key finding in the HN model is that the development of proteinuria in most strains of rats is critically dependent on complement activation within the glomerular immune deposits. Rats depleted of complement with cobra venom factor do not develop proteinuria when injected with anti-Fx1A,19.Salant D.J. Belok S. Madaio M.P. et al.A new role for complement in experimental membranous nephropathy in rats.J Clin Invest. 1980; 66: 1339-1350Crossref PubMed Scopus (192) Google Scholar,20.Saran A.M. Yuan H. Takeuchi E. et al.Complement mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.Kidney Int. 2003; 64: 2072-2078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar and deficiency or depletion of the terminal complement components C6 and C8 precludes assembly of the membrane attack complex C5b-9 and prevents podocyte injury and proteinuria, despite formation of subepithelial immune deposits.21.Cybulsky A.V. Rennke H.G. Feintzeig I.D. et al.Complement-induced glomerular epithelial cell injury. Role of the membrane attack complex in rat membranous nephropathy.J Clin Invest. 1986; 77: 1096-1107Crossref PubMed Google Scholar, 22.Cybulsky A.V. Quigg R.J. Salant D.J. The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.J Immunol. 1986; 137: 1511-1516PubMed Google Scholar, 23.Baker P.J. Ochi R.F. Schulze M. et al.Depletion of C6 prevents development of proteinuria in experimental membranous nephropathy in rats.Am J Pathol. 1989; 135: 185-194PubMed Google Scholar These and subsequent findings in HN established that complement-fixing anti-megalin antibodies aggregate the antigen and activate complement leading to C5b-9-induced sublethal podocyte injury facilitated by accompanying antibodies that abrogate the effect of local complement-regulatory proteins.24.Schiller B. He C. Salant D.J. et al.Inhibition of complement regulation is key to the pathogenesis of active Heymann nephritis.J Exp Med. 1998; 188: 1353-1358Crossref PubMed Scopus (49) Google Scholar The injured podocytes undergo a number of cytopathological changes, including the generation of reactive oxygen species and eicosanoids, reorganization of the actin cytoskeleton, and dissociation of slit diaphragm complexes.25.Cybulsky A.V. Quigg R.J. Salant D.J. Experimental membranous nephropathy redux.Am J Physiol Renal Physiol. 2005; 289: F660-F671Crossref PubMed Scopus (96) Google Scholar Although the active and passive HN models have been invaluable in defining pathogenic mechanisms in MN, they do have certain limitations, some of which have called into question HN's relevance to the human disease. First and most important is the fact that megalin is not expressed on human podocytes.26.Ronco P. Debiec H. Target antigens and nephritogenic antibodies in membranous nephropathy: of rats and men.Semin Immunopathol. 2007; 29: 445-458Crossref PubMed Scopus (34) Google Scholar Second, attempts by several investigators failed to disclose a similar antigen in human MN. Third, the pathogenic IgG antibodies in HN are clearly able to fix complement, whereas IgG4, the predominant antibody in human MN, is believed to be an anti-inflammatory Ig incapable of activating the classical pathway of complement.27.Van der Zee J.S. Aalberse R.C. The role of IgG in immediate-type hypersensitivity.Eur Respir J Suppl. 1991; 13: 91s-96sPubMed Google Scholar Fourth, although complement factors C3 and C5b-9 are found in the same fine granular pattern as IgG in MN biopsies,28.Hinglais N. Kazatchkine M.D. Bhakdi S. et al.Immunohistochemical study of the C5b-9 complex of complement in human kidneys.Kidney Int. 1986; 30: 399-410Abstract Full Text PDF PubMed Scopus (72) Google Scholar and the C5b-9 complex has been found in the urine of such patients,29.Brenchley P.E. Coupes B. Short C.D. et al.Urinary C3dg and C5b-9 indicate active immune disease in human membranous nephropathy.Kidney Int. 1992; 41: 933-937Abstract Full Text PDF PubMed Scopus (65) Google Scholar,30.Schulze M. Donadio Jr, J.V. Pruchno C.J. et al.Elevated urinary excretion of the C5b-9 complex in membranous nephropathy.Kidney Int. 1991; 40: 533-538Abstract Full Text PDF PubMed Scopus (77) Google Scholar it is important to note that there are several animal models in which antibodies directed at podocyte antigens cause proteinuria in the absence of complement,31.Kawachi H. Kurihara H. Topham P.S. et al.Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes.Am J Physiol. 1997; 273: F984-F993PubMed Google Scholar, 32.Natori Y. Shindo N. Proteinuria induced by anti-dipeptidyl peptidase IV (gp108); role of circulating and glomerular antigen.Clin Exp Immunol. 1994; 95: 327-332Crossref PubMed Scopus (8) Google Scholar, 33.Salant D.J. Natori Y. Kawachi H. Glomerular injury due to antibody alone.in: Neilson E.G. Neilson W.G. Immunologic Renal Diseases. 2nd edn. Lippincott Williams & Wilkins, Philadelphia2001: 347-365Google Scholar which suggests a direct cytopathic effect. The first and best evidence that the paradigm of in situ immune complex formation also applies to human MN was established by Debiec et al.34.Debiec H. Guigonis V. Mougenot B. et al.Antenatal membranous glomerulonephritis due to anti-neutral endopeptidase antibodies.N Engl J Med. 2002; 346: 2053-2060Crossref PubMed Scopus (378) Google Scholar in a rare form of neonatal MN. In a single case report, followed later by several more cases,35.Debiec H. Nauta J. Coulet F. et al.Role of truncating mutations in MME gene in fetomaternal alloimmunisation and antenatal glomerulopathies.Lancet. 2004; 364: 1252-1259Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar they described an infant born with the nephrotic syndrome, the cause of which was found to be MN on renal biopsy. Investigation into the cause revealed that the mother was genetically deficient in neutral endopeptidase (NEP), and was immunized to this protein during a previous miscarried pregnancy from an NEP-positive father. In her subsequent term pregnancy, circulating anti-NEP antibodies crossed the placenta as well as the fetal GBM, bound NEP on the fetal podocytes, and instigated a similar disease process as that described in the experimental model. Two important features should be noted in these cases of alloimmune antenatal MN. First, the disease resolved in the infant once the maternal anti-NEP antibody was cleared from its system. Second, proteinuria occurred only in the children of mothers who had both IgG1 and IgG4 anti-NEP.35.Debiec H. Nauta J. Coulet F. et al.Role of truncating mutations in MME gene in fetomaternal alloimmunisation and antenatal glomerulopathies.Lancet. 2004; 364: 1252-1259Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar A mother who was found to have only the IgG4 subclass of anti-NEP did not give birth to an affected infant. As IgG4 is unable to activate the classical complement system, this suggests the need for complement fixing IgG1 or IgG3 to cause clinically evident disease. Our laboratory has been involved in the search for the human MN antigen, and similar to others, we approached the problem by using sera from individuals with MN to screen human glomerular proteins by immunoblot analysis. We soon realized that a number of these sera contained antibodies that specifically recognized a high-molecular-weight glycoprotein in normal human glomeruli. This protein was detected only in the absence of reducing agents, suggesting the presence of one or more epitopes in the molecule whose conformation was dependent on disulfide bonds and providing a potential explanation as to why other laboratories had not identified this protein using similar techniques. Capitalizing on several properties of this glycoprotein and with mass spectrometric analysis, we ultimately identified the protein reactive with these antibodies as the M-type phospholipase A2 receptor (PLA2R).36.Beck Jr, L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1288) Google Scholar This protein, also expressed in lung and on neutrophils,37.Granata F. Petraroli A. Boilard E. et al.Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor.J Immunol. 2005; 174: 464-474Crossref PubMed Scopus (111) Google Scholar,38.Silliman C.C. Moore E.E. Zallen G. et al.Presence of the M-type sPLA(2) receptor on neutrophils and its role in elastase release and adhesion.Am J Physiol Cell Physiol. 2002; 283: C1102-C1113Crossref PubMed Scopus (57) Google Scholar appears largely restricted in the kidney to the podocyte, in which it is abundantly expressed.36.Beck Jr, L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1288) Google Scholar Autoantibodies to PLA2R are largely IgG4, the least abundant IgG subclass in general, but known to be the predominant subclass in IMN immune deposits. In confirmation of this latter point, IgG4 could be colocalized with the PLA2R antigen within the immune deposits of IMN biopsy samples, and anti-PLA2R IgG could be eluted from biopsy samples of patients with IMN, but not lupus-associated MN or IgA nephropathy. All anti-PLA2R autoantibodies tested to date share the same property of detecting PLA2R only in its non-reduced state (LH Beck and DJ Salant, unpublished data). PLA2R is an intriguing molecule, with properties that may ultimately prove important for its proposed role as target antigen in MN. It is a member of the mannose receptor family of proteins, which includes the mannose receptor, Endo180, DEC205, and the avian IgY receptor.39.East L. Isacke C.M. The mannose receptor family.Biochim Biophys Acta. 2002; 1572: 364-386Crossref PubMed Scopus (456) Google Scholar,40.Lambeau G. Lazdunski M. Receptors for a growing family of secreted phospholipases A2.Trends Pharmacol Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar Three of these members have been shown to undergo a conformational change, forming an N-terminal hairpin bend in response to changes in pH or in association with ligand binding.41.Boskovic J. Arnold J.N. Stilion R. et al.Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor.J Biol Chem. 2006; 281: 8780-8787Crossref PubMed Scopus (61) Google Scholar, 42.Rivera-Calzada A. Robertson D. MacFadyen J.R. et al.Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.EMBO Rep. 2003; 4: 807-812Crossref PubMed Scopus (23) Google Scholar, 43.West Jr, A.P. Herr A.B. Bjorkman P.J. The chicken yolk sac IgY receptor, a functional equivalent of the mammalian MHC-related Fc receptor, is a phospholipase A2 receptor homolog.Immunity. 2004; 20: 601-610Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Furthermore, all undergo constitutive endocytic recycling at the plasma membrane,44.Zvaritch E. Lambeau G. Lazdunski M. Endocytic properties of the M-type 180-kDa receptor for secretory phospholipases A2.J Biol Chem. 1996; 271: 250-257Crossref PubMed Scopus (106) Google Scholar which could provide a constant source of PLA2R at the podocyte foot process. We do not yet know whether the anti-PLA2R antibodies interfere with the normal function of PLA2R in podocytes, and the biological role of the protein in the kidney is unknown. PLA2R has recently been reported to promote replicative senescence in human fibroblasts, in part, by causing reactive oxygen species production and DNA damage.45.Augert A. Payre C. de Launoit Y. et al.The M-type receptor PLA2R regulates senescence through the p53 pathway.EMBO Rep. 2009; 10: 271-277Crossref PubMed Scopus (91) Google Scholar It has also been shown to activate cytosolic PLA2 leading to membrane phospholipid hydrolysis and eicosanoid production.46.Lambeau G. Gelb M.H. Biochemistry and physiology of mammalian secreted phospholipases A2.Annu Rev Biochem. 2008; 77: 495-520Crossref PubMed Scopus (388) Google Scholar Similar processes have been observed as downstream processes in the HN model,25.Cybulsky A.V. Quigg R.J. Salant D.J. Experimental membranous nephropathy redux.Am J Physiol Renal Physiol. 2005; 289: F660-F671Crossref PubMed Scopus (96) Google Scholar and it will be of interest to see if such effects occur in human disease, mediated through PLA2R. A transcript encoding a form of human PLA2R that lacks the transmembrane and intracellular domains has been described,47.Ancian P. Lambeau G. Mattei M.G. et al.The human 180-kDa receptor for secretory phospholipases A2. Molecular cloning, identification of a secreted soluble form, expression, and chromosomal localization.J Biol Chem. 1995; 270: 8963-8970Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar but we have not detected this putative soluble form in normal or MN serum. Current sensitivity and specificity analyses show that anti-PLA2R autoantibodies are present in greater than 75% of individuals with IMN, but never in those with secondary causes of MN, other glomerular or autoimmune diseases or normal controls. There may be several reasons that the sensitivity is not 100%, including potential technical issues with the immunoblot assay, additional antigens targeted in the disease, or misclassification of patients with IMN (who may have an unrecognized secondary cause). However, our leading hypothesis as to why there remain 25% of IMN patients who do not have anti-PLA2R antibodies is that there was an absence of immunologic disease activity at the time when their serum was sampled, despite proteinuria. We have shown presence of anti-PLA2R in patients initially nephrotic from IMN, an absence in many of those that undergo complete remission, and a recurrence of the autoantibodies in cases of relapse. However, for reasons explained below, the waters become muddier in those patients with subnephrotic proteinuria who have achieved a partial remission. It is important to make this distinction between proteinuria, as a clinical marker of disease, and the presence of anti-PLA2R, a marker of immunologic activity in IMN. The two are closely, but not perfectly, related due to the time it takes to form and resorb the subepithelial deposits (see Figure 1). Protocol biopsies of renal allografts demonstrate that the earliest deposits that occur in recurrent MN can be associated with minimal proteinuria.48.El-Zoghby Z.M. Grande J.P. Fraile M.G. et al.Recurrent idiopathic membranous nephropathy: early diagnosis by protocol biopsies and treatment with anti-CD20 monoclonal antibodies.Am J Transplant. 2009; 9: 2800-2807Crossref PubMed Scopus (73) Google Scholar Conversely, proteinuria can remain after there is no further evidence of circulating autoantibodies. We have shown a linear correlation between anti-PLA2R and amount of proteinuria (LH Beck and DJ Salant, unpublished data), and note that at the point where autoantibody level reaches zero, there is on average still residual proteinuria, in the 2–3 g/day range. In patients who achieve complete remission in response to immunosuppressive therapy, anti-PLA2R levels become undetectable months before the proteinuria resolves completely. Although these findings could alternatively be explained by the continued circulation of additional unidentified pathogenic antibodies, they are perhaps more compatible with residual structural deficits in the absence of immunologic activity. In the experimental HN model, transplantation of an affected kidney to a naive host results in a significant decrease in proteinuria, but not its complete elimination.49.Makker S.P. Kanalas J.J. Course of transplanted Heymann nephritis kidney in normal host. Implications for mechanism of proteinuria in membranous glomerulonephropathy.J Immunol. 1989; 142: 3406-3410PubMed Google Scholar In addition, GBM remodeling and sclerotic changes that occur in the glomerulus and interstitium in the face of longstanding MN can also cause persistent proteinuria unrelated to an ongoing immunologic process. Given the strong association of anti-PLA2R with disease activity, it appears likely that these autoantibodies have inherent pathogenicity. Proof, however, must await suitable transgenic animal models, as all small animals tested thus far do not express PLA2R on their podocytes, at least not in a form recognized by human autoantibodies. Further proof of pathogenicity might be forthcoming from studies of recurrent MN post-renal transplantation if it is found that persistence or reappearance of anti-PLA2R predates clinical or histological evidence of MN. Regarding potential mechanisms by which anti-PLA2R might cause podocyte damage, both complement-dependent and independent mechanisms are possible. As noted above, there is substantial evidence that complement is activated in the glomerular immune deposits in idiopathic MN; whereas, IgG4, the major IgG subclass present in the glomeruli as well as the predominant anti-PLA2R subclass, is generally regarded as being incapable of activating complement through the classical pathway. While it is possible that the smaller amo