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Pathogenesis of membranous nephropathy: recent advances and future challenges

Pierre Ronco and Hanna Debiec
Abstract | Over the past few years, considerable advances have been made in our understanding of the molecular pathomechanisms of human membranous nephropathy, inspired by studies of Heymann nephritis, a faithful experimental model of this disease. This research led to the identification of neutral endopeptidase, the Mtype receptor for secretory phospholipaseA2 (PLA2R1) and cationic bovine serum albumin as target antigens of circulating and deposited antibodies in alloimmune neonatal, adult idiopathic and early-childhood membranous nephropathy, respectively. A genome-wide association study has provided further evidence for a highly significant association between PLA2R1 and HLA-DQA1 loci and idiopathic membranous nephropathy in patients of white ancestry. Additional antibody specificities for cytoplasmic antigens have also been identified, but their pathogenic role is uncertain. The time has come to revisit the spectrum of membranous nephropathies based on the newly identified antigenantibody systems that should be considered as molecular signatures of the disease and that challenge the uniform histological definition. These signatures will soon have a major impact on patient care.
Ronco, P . & Debiec, H. Nat. Rev. Nephrol. 8, 203213 (2012); published online 28 February 2012; doi:10.1038/nrneph.2012.35

Introduction
Membranous nephropathy is one of the most common forms of nephrotic syndrome in adults, accounting for about 20% of cases.1 This condition can be idiopathic without an identified cause (7080% of cases), or secondary to various clinical conditions, including infections (for example, hepatitisB and syphilis), systemic lupus erythematosus, cancer and drug intoxication. 1,2 Membranous nephropathy is an immunologically mediated disease characterized by the deposition of immune complexes in the subepithelial space, which causes a membrane-like thickening of the glomerular basement membrane. The immune deposits consist of several components, including IgG, antigens that have long eluded identification, and the membrane attack complex, which is assembled from complement components to form C5b9. IgG4 is usually the most prominent IgG subclass deposited in idiopathic membranous nephropathy, although variable amounts of IgG1 are also associated with immune deposits; by contrast, deposition of IgG1, IgG2 and IgG3 exceeds deposition of IgG4 in secondary membranous nephropathy.36 The formation of subepithelial immune deposits and complement activation are responsible for functional impairment of the glomerular capillary wall, which causes proteinuria. Idiopathic membranous nephro pathy is usually considered an autoimmune disease, whereas exogenous antigens such as viral and tumoral antigens are thought to be involved in secondary forms of the disease.7
Competing interests The authors declare no competing interests.

Although spontaneous remission is a well-known characteristic of idiopathic membranous nephropathy, occurring in about one-third of patients,8,9 membranous nephropathy leads to end-stage renal failure in about 40% of patients after 10years.10 Treatment for membranous nephropathy remains controversial and challenging because of uncertain benefits or adverse effects of immunosuppressive therapy, and, apart from proteinuria, there is a lack of reliable biomarkers of disease activity owing to the fact that the antigens targeted by nephrito genic antibodies have remained elusive.1114 The key to a substantial improvement of disease monitoring and treatment is the identification of pathogenic mechanisms. In the past 10years, great progress has been made in the understanding of the molecular pathomechanisms of human membranous nephropathy. These advances were inspired by studies of experimental models of membranous nephropathy, such as Heymann nephritis1517 and membranous nephropathy induced by cationic bovine serum albumin (BSA).18,19 Studies of Heymann nephritis led to the concept that a podocyte antigen, megalin, could serve as a target of circulating antibodies leading to the insitu formation of immune complexes,20,21 whereas cationic BSA-related membranous nephropathy first illustrated the case of planted antigen.22 In humans, progress in this area started in 2002 with the identification of neutral endopeptidase (NEP; also known as neprilysin) as the responsible antigen in a rare subset of patients with alloimmune neonatal membranous nephropathy.23,24 This finding provided the proof of concept that a podocyte antigen could be responsible for human membranous nephropathy, as is the case for megalin in the rat, and

INSERM UMR S 702 and Division of Nephrology, Tenon Hospital, 4 Rue de la Chine, Paris 75020, France (P. Ronco, H.Debiec). Correspondence to: P . Ronco pierre.ronco@ tnn.aphp.fr

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Key points
Antibodies to neutral endopeptidase, the first podocyte antigen described in human membranous nephropathy, are responsible for alloimmune neonatal membranous nephropathy, which develops in children of mothers deficient in this enzyme The secretory phospholipaseA2 receptor (PLA2R1) is a major target antigen in idiopathic membranous nephropathy in adults, and antibodies to PLA2R1 are detected in 6080% of these patients before immunosuppressive treatment In addition to their diagnostic value, anti-PLA2R1 antibodies can be used to monitor response to treatment Variants in PLA2R1 and HLA-DQA1 are strongly associated with idiopathic membranous nephropathy in patients of white ancestry Immunization against cationic bovine serum albumin is a cause of earlychildhood membranous nephropathy, which points to a possible role for food and environmental antigens in membranous nephropathy The newly identified antigenantibody systems should be considered as molecular signatures of membranous nephropathy that challenge the uniform histological definition and will have a major impact on patient care in the near future

laid the foundation for the identification of the Mtype receptor for secretory phospholipaseA2 (PLA2R1), which is the first podocyte autoantigen shown to be involved in idiopathic membranous nephropathy in humans. 25 Genome-wide association studies further demonstrated that single nucleotide polymorphisms in the PLA2R1 gene were strongly associated with idiopathic membranous nephropathy,26 which again pointed to the involvement of this antigen using an unbiased genetic approach. Other antigens, such as aldose reductase, superoxide dismutase2 and enolase, have also been identified in humans, although their role remains to be established because they have not been detected on the surface of normal podocytes.27,28 In addition to podocyte antigens, exogenous antigens such as cationic BSA have also been implicated in some patients with early-childhood membranous nephropathy.22 All of these findings have opened up a new era for the diagnosis and monitoring of membranous nephropathy from early infancy to adulthood. In this Review, we focus on the molecular patho mechanisms of membranous nephropathy, with particular emphasis on the antibodies involved because they provide new tools for patient monitoring as well as hope for innovative treatments with fewer adverse effects. We describe experimental models of membranous nephro pathy that have helped to elucidate systems involved in human disease. We also discuss unresolved questions and future challenges in understanding the pathogenesis of membranous nephropathy.

Serum sickness and circulating immune complexes Deposition of circulating immune complexes in tissue is a hallmark of chronic serum sickness, which experimentally is induced by repeated administration of a constant dose of exogenous antigen. In glomeruli, the location of immune deposits is highly dependent on the magnitude of the antibody response, the size of the immune complex (determined by antigen antibody ratio and lattice formation), the characteristics of the antigen (for example, size and charge), as well as hemodynamics and pharmacological factors.29 If the antigen dose is adjusted according to the circulating antibody response in order to maintain a state of chronic antigen excess, deposits are almost exclusively subepithelial irrespective of the level of antibody response.30,31 It is possible that small circulating immune complexes comprised of low-avidity antibody and oligo valent antigen can traverse the capillary wall, but one cannot exclude the possibility that, owing to their properties, the immune complexes dissociate on the endothelial side of the capillary wall and reassociate on the subepithelial side. Heymann nephritis and podocyte antigens In 1959, Heymann etal.15 described a rat model of membranous nephropathy, referred to as active Heymann nephritis, which was induced by the immunization of Lewis rats with crude kidney extracts and was reminiscent of the disease in humans. Because the subepithelial deposits were induced by fractions of renal brush-border membrane rather than by glomerular extracts, the deposits were initially believed to result from the glomerular trapping of circulating immune complexes composed of brush-border-related antigens and the corresponding antibodies. Subsequently, however, the development of passive Heymann nephritis in rats injected with rabbit anti-rat brush-border antibodies argued against a role for circulating immune complexes.16,17 In the passive Heymann nephritis model, immune deposits occur within minutes of antibody injection, which argues against formation of immune compexes in the circulation followed by their deposition in the glomerular capillary wall. With the use of exvivo and isolated perfused kidney systems, Van Damme etal.16 and Couser etal.17 demonstrated that anti-brush-border antibodies bound to an antigenic target located on podocytes, which indicated that the disease was caused by the insitu formation of immune complexes. The principal autoantigen in both active and passive Heymann nephritis was identified in the early 1980s.20,21 This autoantigen is a large podocyte transmembrane protein called megalin (also known as LDL receptor-related protein2), with a molecular weight of ~600kDa. 32,33 This polyspecific receptor, which belongs to the LDL receptor superfamily, is present at the sole of podocyte foot processes in clathrin-coated pits. The formation of immune deposits is fueled by the denovo synthesis of megalin. 34 Epitope mapping showed that although subepithelial immune deposits could be induced by all four megalin ligand-binding domains, 32,33,35 full-blown disease with proteinuria
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Experimental models
Three possible mechanisms can explain the formation of subepithelial deposits in experimental and human membranous nephropathy (Figure1). The first mechanism involves the deposition of circulating immune complexes as described in chronic serum sickness; the second and third mechanisms involve the insitu formation of immune complexes in the glomerular capillary wall, where the antigen is either a native podocyte antigen or a planted exogenous antigen artificially bound to the basement membrane or the podocyte.
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was associated with a specific epitope located in the first ligand-binding domain of megalin, in a 60kDa Nterminal fragment,3638 as well as with intramolecular epitope spreading.39 Heymann nephritis-like membranous nephropathy was also reported in rats and mice,4043 in which the target antigen was dipeptidyl peptidase4. More recently, a model of passive Heymann nephritis was induced in mice injected with antipodocyte serum.44,45 Although studies of Heymann nephritis have greatly contributed to our understanding of the pathomechanisms of membranous nephropathy, there is no evidence that megalin is involved in human membranous nephropathy.
a Deposition of immune complexes from the circulation
Animal model Rabbit Chronic serum sickness Low-avidity antibody and oligovalent antigen Human MN No evidence yet

Circulating immune complexes

b In situ formation of immune deposits

Animal model Heymann nephritis Megalin Human MN Alloimmune MN Neutral endopeptidase Autoimmune MN Phospholipase A2 receptor

Complement-mediated injury Complement activation and formation of the C5b9 attack complex, which is triggered by immune complex deposition, have major roles in sublethal podocyte injury and proteinuria.4648 C5b9, in sublytic quantities that do not kill podocytes, activates various mediators, such as phospholipases, protein kinases, cyclo oxygenases, transcription factors and cytokines. These signals influence podocyte cell metabolic pathways, structure and function of key cytoskeletal proteins, expression and localization of nephrin, the turnover of extracellular matrix, and DNA integrity.4648 Evidence also shows that assembly of the sublytic C5b9 complex on podocytes causes upregulation of NADPH oxidoreductase expression and its translocation to the cell surface. Subsequently, reactive oxygen species are produced locally, which leads to damage of podocyte lipids, membrane proteins and glomerular basement membrane components.2,4648 Cationic BSA and planted antigen Three decades ago, Border etal.18 reported a model of membranous nephropathy in rabbits immunized with cationic BSA. The experiments were driven by the hypothesis that antigen charge could be a key factor for the formation of subepithelial deposits, given the negative charge of the glomerular capillary wall. Only rabbits immunized with cationic BSA were found to have subepithelial deposits of IgG and complementC3, whereas those receiving anionic or native (neutral) BSA featured mostly mesangial deposits. Proteinuria was also more severe in those animals immunized with cationic BSA. Experiments performed in rabbit perfused kidneys confirmed that immune complexes were formed insitu after cationic BSA bound to anionic heparin sulfate proteoglycans of the basement membrane, followed by binding of the antibody.19 Chemical cationization of BSA altered its immunogenicity and resulted in the formation of antibody of low precipitability and low avidity, thus facilitating immune complex dissociation and access of free cationic BSA to the rabbit glomerular capillary wall.49,50 This model was later established in dogs, mice and rats.5155 In addition to cationic BSA, other potential antigens can become implanted in the glomerular capillary wall, such as enzymatically modified nucleosomes that contain cationic histones and might have a role in
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Circulating antibodies

c Exogenous planted antigen targets

Animal model Rabbit or mouse Cationic BSA Human MN Early-childhood MN Cationic BSA

Non-native antigens

Circulating antibodies

Figure 1 | Possible mechanisms of immune complex deposition in experimental and human membranous nephropathy. a | Glomerular IgG deposits might result from an accumulation of circulating immune complexes, as has been established in a rabbit model of chronic serum sickness. Among IgG subtypes, non-complement-fixing IgG4 complexes that contain diverse antigens might have a major role because these complexes cannot be eliminated by complement-mediated pathways. As yet no proof exists that this model operates in human disease. b | The insitu formation of immune complexes is initiated by binding of circulating antibodies to antigens that may be endogenous constituents of the podocyte membrane. Three endogenous antigenic targets, all integral membrane glycoproteins of podocytes, have been identified including the polyspecific receptor megalin in Heymann nephritis, neutral endopeptidase in alloimmune neonatal membranous nephropathy, and the phospholipaseA2 receptor in idiopathic membranous nephropathy. c | Alternatively, insitu immune complexes involving exogenous antigens planted in the glomerular basement membrane are formed in rabbits and mice injected with repeated doses of cationized BSA, which serves as a planted antigen. In the human counterpart of this experimental model, which occurs in young children, cationized BSA from the diet might become implanted in the anionic glomerular capillary wall and contribute to the insitu formation of immune complexes. Abbreviations: BSA, bovine serum albumin; MN, membranous nephropathy.

lupus-related membranous nephropathy, as well as lectin or cationized ferritin.5659

Alloimmune neonatal disease

A human counterpart to passive Heymann nephritis was characterized in a neonate with membranous nephro pathy.23,24 Because of the early occurrence of the disease, we hypothesized that the mother became immunized during pregnancy and the nephritogenic antibodies were transferred to the fetus. Interspecies differences observed when rat and rabbit kidneys were incubated with sera from the mother and infant were suggestive of reactivity with NEP, which was confirmed by immunoprecipitation of rat brush-border extracts.23,24 Transfer experiments, in
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N-terminal domain M292V H300D NH2 Cysteine Fibronectin -rich -like type II region domain 1 C-type lectin domain 2 3 4 5 6 7 8 COOH Negative regulator Internalization Degradation G1106S Transmembrane C-terminal domain domain Positive regulator MAPK activation Lipid mediator production ROS production and activation of DNA-damage pathways

Receptor binding PLA2

Figure 2 | Structural and functional properties of PLA2R1. This receptor is a typeI transmembrane glycoprotein composed of a large extracellular portion that consists of an N-terminal cysteine-rich region, a fibronectin-like typeII domain, a tandem repeat of eight Ctype lectin domains, a transmembrane domain and a short intracellular Cterminal domain. PLA 2R1 can function as a positive regulator of PLA2 by inducing a variety of biological responses, or as a negative regulator through rapid internalization and degradation of PLA2. SNPs associated with the risk of developing membranous nephropathy identified in European (yellow) and Asian (red) cohorts are circled. Abbreviations: MAPK, mitogen-activated protein kinase; PLA 2, secretory phospholipaseA2; PLA2R1, PLA2 receptor; ROS, reactive oxygen species; SNP , single nucleotide polymorphism.

which a rabbit was injected with the IgG fraction from the mother, showed co-localization of NEP, IgG and C5b9 within the immune deposits, which established that the disease was caused by anti-NEP antibodies.23,24,60 Because the mother did not show any renal manifestations even though she had persistently high titers of antiNEP antibody, we thought she might be deficient in NEP. This hypothesis was supported by the lack of reactivity of her granulocyte extracts with a panel of anti-NEP antibodies.23,24 We have identified a further three families with alloimmune neonatal membranous nephro pathy.23,24 All four immunized mothers in this study were NEP deficient as a result of truncating mutations in exon7 and exon15 of the MME gene coding for NEP. Mutations in the MME gene are asymptomatic so one can expect a high prevalence in selected populations, such as the Dutch, Moroccan and Portuguese populations from which the cases originated.

Idiopathic membranous nephropathy

PLA2R1 as a major target antigen Considerable efforts have been devoted to the identification of antigen(s) in autoimmune idiopathic membranous nephropathy. For many years, these efforts failed as it was impossible to detect antipodocyte antibodies in the blood, podocyte antigens in immune deposits, and specific reactivity in eluates from kidneys of patients with membranous nephropathy because of a lack of sensitive methods. The use of glycoprotein-enriched glomerular extracts from kidneys as a source of antigens, and of nonreducing conditions for western blots, enabled the identification of a 185kDa protein band that was detected in 70% of patients with idiopathic membranous nephropathy, but not in those with secondary membranous nephropathy or in controls.25 Reactivity to this protein persisted after Ndeglycosylation, but dis appeared after reduction of disulfide bonds. PLA2R1 was identified by mass spectro metry analysis of the reactive band. Furthermore, sera from patients with membranous nephropathy immunoprecipitated PLA2R1 from glomerular extracts. Although anti-PLA 2 R1 antibodies were mostly IgG4, other subclasses were also involved to a lesser extent.
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Evidence for insitu formation of immune complexes was provided by the detection of PLA2R1 in normal human glomeruli, apparently in podocytes. 25 PLA2R1 and IgG4 were found to be co-localized within the subepithelial immune deposits. IgG that was eluted from biopsy samples of patients with idiopathic membranous nephropathy reacted with PLA2R1, whereas IgG eluted from biopsy specimens from patients with lupus membranous nephropathy or IgA nephropathy did not. These data suggest that, like Heymann nephritis and allo immune neonatal membranous nephropathy, autoimmune idiopathic membranous nephropathy involves insitu formation of subepithelial deposits through binding of circulating anti-PLA2R1 autoantibodies to podocyte PLA2R1. Transfer experiments to confirm the pathogenic effects of anti-PLA2R1 antibody have been hampered by an apparent lack of PLA2R1 expression in rodent glomeruli. The recurrence of membranous nephropathy in kidney transplant recipients with circulating anti-PLA2R1 antibodies supports a pathogenic role for these antibodies,61 although some PLA2R1-positive patients did not recur.62 PLA2R1 is a type1 transmembrane receptor for secretory phospholipaseA2 (Figure2), a member of the mannose receptor family, which also includes the cationdependent mannose6-phosphate receptor, the Ctype mannose receptor2, the dendritic cell receptor DEC205, and the avian yolk sac IgY receptor FcRY.6365 Several common features are shared by members of this family. First, all members are recycled between the plasma membrane and the endosomal machinery leading to the internalization of extracellular ligands. Second, these receptors can present with at least two configurations: an extended conformation with the Nterminal cysteine-rich domain pointing outwards from the cell surface, or a bent conformation where the Nterminal domain folds back to interact with Ctype lectin-like domains at the middle of the structure, thus affecting ligand binding and oligomerization. Because auto antibodies in patients with membranous nephro pathy only recognize a conformation- dependent epitope,25 the target epitope might occur in only one of these two
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configurations. Changes in the molecular architecture of the Goodpasture autoantigen has a key role in eliciting an autoimmune response,66,67 which suggests that, like Goodpasture syndrome, idiopathic membranous nephropathy might be an autoimmune conformational disease, also known as conformeropathy. Further work based on identification and modeling of pathogenic PLA2R1 epitopes is required to confirm this hypothesis.
n = 286 68.5% n = 45 4.4% n = 14 28% n = 18 17% n = 13 15% n = 90 n = 153 0% 0%

75 Positive for anti-PLA2R1 (%)

50

25

Genes linked to membranous nephropathy The influence of genetic factors in membranous nephropathy is well established both in rats and mice as only Lewis strain rats and ICR or BALB/c mice develop the typical clinical and pathological patterns of membranous nephropathy.53,68 European Caucasoids show a strong association of membranous nephropathy with an HLAB8DR3 haplotype.6972 Genome-wide association studies have described significant associations between the 6p21 HLA-DQA1 and 2q24 PLA2R1 loci and idiopathic membranous nephropathy in patients of white ancestry.26 Interestingly, carrying the risk alleles of these two genes had an additive effect. Patients with all four risk alleles had an odds ratio of 78.46 for the disease compared with individuals who had only the protective alleles.26 Genetic variations in both immune response and podocyte genes could lead to the production of autoantibodies. However, this assumption has not yet been confirmed experimentally. A more comprehensive evaluation of rare variants with next-generation sequencing methods and cell and molecular biological analyses will help to establish causative variants. Two studies from Asia that used a candidate-gene approach confirmed that single nucleotide polymorphisms in the PLA2R1 gene that resulted in amino acid changes were associated with a predisposition to membranous nephropathy.73,74 Other non-HLA alleles also predispose indivi duals to the development or progression of membranous nephropathy. For instance, the tumor necrosis factor ( TNF ) allele G308A and TNFd microsatellite poly morphisms located downstream of the TNF gene are associated with increased production of TNF and strongly associated with the occurrence and initiation of membranous nephropathy.75,76 A direct role for TNF in the pathogenesis of membranous nephropathy is supported by its increased serum and urine levels, and increased glomerular expression in patients with membranous nephropathy compared with patients with other renal diseases.77,78 TNF is able to induce discohesion at the slit membrane with disassembly of actin filaments,79 suggesting that TNF might be involved in the induction or maintenance of glomerular barrier dysfunction. Nephrin (NPHS1), a podocyte protein that links the slit diaphragm to modulators of actin dynamics, has a critical role in maintaining glomerular permselectivity.80 Various polymorphisms in NPHS1 have been associated with susceptibility to membranous nephropathy and remission of proteinuria during disease progression.81 Plasminogen activator inhibitor1 (PAI1) activity has an important role in renal fibrosis. A correlation between the 4G allele in PAI1, renal deterioration and increased
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0 Idiopathic MN Lupus Cancer nephritis MN HBV GVHD Controls Healthy with controls disease

Secondary MN

Figure 3 | Prevalence of anti-PLA2R1 antibody in patients with membranous nephropathy. Prevalence of anti-PLA2R1 antibody was calculated on the basis of results presented in several studies25,85,87,88,90,91 and obtained from patients at our institution. Abbreviations: GVHD, graft-versus-host disease; HBV, hepatitisB virus; MN, membranous nephropathy; PLA2R1, secretory phospholipaseA2 receptor.

cardiovascular events has been observed in patients with membranous nephropathy. 82 The presence of the 4G allele is associated with increased transcriptional activity, resulting in higher PAI1 levels and activity than occurs in the presence of the 5G allele.82 Two studies in the Taiwanese population revealed that individuals with polymorphisms in the IL6 and STAT4 genes were more susceptible to developing membranous nephropathy and renal failure. 83,84 Overall, these data suggest that a combination of gene variants initiate disease and that modifier genes controlling glomerular permeability, inflammation and fibrosis might be involved in the pathogenesis of membranous nephropathy.

PLA2R1 test for diagnosis and monitoring Although the exact role of PLA2R1 in the pathogenesis of idiopathic membranous nephropathy is still unknown, the presence of anti-PLA2R1 antibodies is highly specific for idiopathic membranous nephropathy. A low prevalence of PLA2R1 antibodies has been observed in secondary forms of membranous nephropathy,8587 but in these cases, coincidental occurrence of idiopathic membranous nephropathy with the associated disease cannot be excluded. Patients with other causes of the nephrotic syndrome or healthy individuals have no detectable level of PLA 2R1 antibodies (Figure3).25,85,87 Several studies indicate that anti-PLA2R1 antibodies correlate with disease activity and disappear during a spontaneous or treatment-induced remission and reappear during a relapse.8890 Because an immunoassay for circulating antibodies to PLA2R1 is now commercially available, monitoring antibody levels prospectively might help to titrate membranous nephropathy therapy on the basis of immunological activity and clinical outcome.87 However, it should be mentioned that PLA 2 R1 antigen has also been found in immune deposits in some seronegative patients, which indicates rapid clearance of antibodies and deposition in glomeruli.91 Conversely, detection of circulating antibodies is not
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always associated with the presence of the antigen in the immune deposits, which could suggest that not all antibodies to PLA2R1 are pathogenic.91 Assessment of both circulating anti-PLA2R1 antibodies and PLA2R1 in biopsy samples might better select patients for appropriate therapy. diseases without glomerular involvement and in healthy individuals.94,95 No comparison of circulating anti-PLA2R1 antibodies or deposited PLA2R1 antigen with the presence of other antibody and antigen specificities has as yet been carried out in individual patients. Using a proteomic approach and lysates from human podocytes, our group identified several other antigens involved in metabolic pathways, cytoskeleton architecture and cell stress; the reactivity of these antigens with sera from patients was confirmed with the respective commercially available recombinant protein (H. Debiec and P. Ronco, unpublished work). Similarly, more than 20 intracellular proteins were identified as possible antigens to sheep antipodocyte antibodies in a model of passive Heymann nephritis in mice.44,45 How can we explain the multiplicity of candidate target antigens in adult idiopathic membranous nephropathy? First, membranous nephropathy is likely to be a heterogeneous disease. Second, the antibodies identified so far may be involved in the initiation phase of the disease (for example, anti-megalin in the rat, anti-NEP in neonates, and possibly anti-PLA2R1 in adult patients with idiopathic membranous nephropathy), or later in the progression phase. One can hypothesize that following the initial injury induced by complement activation and production of reactive oxygen species, intra cellular proteins, cryptic epitopes and neoepitopes might be exposed and induce a second wave of immunization (Figure4). Whether the antibodies raised against those proteins or epitopes have a pathogenic effect remains to be established.

Recurrent membranous nephropathy Recurrence of membranous nephropathy might occur because of the binding of circulating anti-PLA 2R1 antibodies to PLA2R1 antigen expressed on podocytes in the donor kidney, as first shown by Stahl etal.61 in 2010. However, a recent study performed by our group in 10 patients with recurrent membranous nephro pathy indicates that the picture might be more complex than previously thought. 62 We showed that PLA 2R1 was implicated in recurrent membranous nephropathy in five of 10 patients, but in none of the nine patients with denovo membranous nephropathy. Nevertheless, the kinetics of anti-PLA2R1 antibodies was markedly heterogeneous as tested by disappearance of antibodies months before the diagnosis of recurrent membranous nephropathy or reappearance at the time of diagnosis, suggesting that other antigenantibody systems including allo antibodies might be initially involved at least in some cases. Moreover, donor kidney injury owing to ischemiareperfusion could unmask hidden epitopes or create neoantigens that become target antigens for circulating antibodies.92 The most surprising finding from this study was the absence of recurrence on biopsies performed 2646months after grafting in three patients who had high titers of anti-PLA2R1 antibodies at the time of transplantation.62 PLA2R1 sequence variants in the donor kidney might differ from those in the recipient kidney, thus altering antigen presentation by podocytes or antigen presentation by dendritic cells. Alternatively, cross-presentation of PLA 2R1 epitopes by recipient dendritic cells through donor HLA molecules93 might represent another way of donorrecipient interaction, which could potentially lead to the inhibition of specific memory Bcells. These issues will hopefully be solved by comparative sequencing of the PLA2R1 gene and HLA-DQA1 locus in the donor and the recipient. A role for other autoantigens? Anti-PLA 2R1 autoantibodies or PLA 2R1 antigen in immune deposits are not found in all cases of idiopathic membranous nephropathy.25,85,87,91 Some patients with idiopathic membranous nephropathy have been shown to have circulating antibodies to cytoplasmic antigens such as aldose reductase, superoxide dismutase2 and enolase, together with the corresponding antigen in glomerular immune deposits.27,28 These ubiquitous enzymes are not, or only very weakly, expressed on the membrane of normal podocytes, but as shown for superoxide dismutase2, they can be induced invitro by oxidative stress, such as exposure to H2O2.27 Furthermore, anti-enolase antibodies are not specific for membranous nephropathy as they have been detected in a wide range of autoimmune
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Secondary membranous nephropathies

The term idiopathic is used to dissimulate our lack of knowledge of the causes and pathogenesis of membranous nephropathy in most patients. It has been suggested that one should no longer use this term in PLA2R1related membranous nephropathy. However, we would recommend waiting until the triggers of immunization are identified and the issues of the plurality and timing of involvement of potential candidate antigens have been solved.

BSA-related membranous nephropathy In the wake of studies devoted to childhood membranous nephropathy, our group found high levels of anti-BSA antibodies in four of five children, all aged less than 5years, and seven of 41 adults with idiopathic membranous nephropathy analyzed consecutively. 22 These 11 patients also had elevated levels of circulating BSA; however, isoelectric focusing showed that affinity- purified BSA from the blood turned out to be cationic only in the four children, whereas in the adults, BSA had the same, slightly anionic isoelectric point as native BSA. BSA was detected in subepithelial immune deposits only in children with both anti-BSA antibodies and high levels of circulating cationic BSA, and it co-localized with IgG in the absence of PLA2R1. IgG eluted from kidney biopsy samples of children with BSA deposits belonged to IgG1 and IgG4 subclasses and specifically reacted with BSA, but not with human serum albumin.
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Cows milk is the major source of BSA in young children. Although it is unknown why cationic BSA was formed, differences in food processing and intestinal microbiota might be responsible for modifications of BSA. Whatever the chemical modifications involved, membranous nephropathy related to cationic BSA seems to be the human counterpart of the experimental model.18,19 Our findings strongly support the scenario of planted antigen in the human disease. From a clinical point of view, a diagnosis of BSA-related membranous nephropathy should be considered in young children with membranous nephropathy, which should prompt a search for BSA in immune deposits. If BSA is detected, eliminating this protein from the diet could be beneficial.
Initiation Progression Podocyte

Antigen Cytoplasmic protein Neoantigen Neoepitope

Disease-induced alterations

C5b9

Other candidate antigens HepatitisB, hepatitisC and Helicobacter pylori antigens, tumor antigens, thyroglobulin, and DNA-containing material were detected in subepithelial deposits in patients with secondary membranous nephropathy.9698 These antigens might have been trapped in the glomerular basement membrane as a result of physicochemical properties, as is the case for cationic albumin. Alternatively, small, circulating, nonprecipitating IgG4 complexes containing these antigens could become deposited in the glomerular basement membrane as is seen in the chronic serum sickness model (Figure1a), although there is as yet no experimental evidence to support this hypothesis in humans.

Figure 4 | Mechanisms of immune-mediated podocyte injury and disease progression. Antipodocyte antibodies targeting integral membrane proteins cross the basement membrane, induce clustering of antigens, and shedding of immune complexes in the extracellular space. This process results in complement activation and formation of C5b9 on the membrane of podocytes, which in turn leads to a variety of intracellular events, including production of reactive oxygen species and proteases, cytoskeletal changes and a stress response. In addition to forming immune complexes, antipodocyte antibodies can directly alter podocyte biology by activation of signaling pathways irrespective of complement activation. Under disease conditions, cytoplasmic proteins can be routed to the cell membrane, and a variety of neoantigens or neoepitopes may be induced by injury. Circulating antibodies raised against those antigens could be nephritogenic and implicated in the perpetuation of the disease. The multiple antigenantibody systems that are generated could be involved at different time points and in different subcategories of patients with membranous nephropathy.

Unresolved issues and future challenges

Although substantial advances have been made in the pathophysiology of membranous nephropathy, some important issues remain unsolved, and the objective of antigen-driven therapy has still not been achieved.

Triggering and spreading the immune response How the immune response is triggered and spreads remains unknown, except in alloimmune neonatal membranous nephropathy where mothers become allo immunized against an antigen of the placenta, which they do not express because of genetic deficiency. The conditions that lead to the appearance of the PLA2R1 epitope on the podocyte surface and the events that trigger immunization are unknown, but could involve the following: any kind of podocyte injury associated with the release of the PLA2R1 epitope; conformational change in the PLA2R1 epitope induced by environmental or other factors, as strongly suspected in Goodpasture syndrome;67 stimulation of the relevant autoreactive Bcell clones; or molecular mimicry by a bacterial or another exogenous antigen, as shown by Kain etal.99 in pauci-immune focal necrotizing glomerulonephritis. B-cell epitope spreading, that is the extension of the primary immune response against the immunodominant epitope(s) to other epitopes within the same molecule or in different molecules, might be relevant to the pathogenesis of membranous nephropathy. This phenomenon has been observed in many models of antibody-mediated autoimmune diseases and in Heymann nephritis.39,100,101
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Role of IgG subclasses Membranous nephropathy is characterized by a predominant Thelper2-type immune response with production of IgG4 both in humans and in animals.102,103 IgG4 has unique properties among the IgG subclasses because it does not activate complement and behaves mostly as a monovalent, low-affinity immuno g lobulin. 104,105 However, there is much evidence that complement is activated and that assembly of the membrane attack complex C5b9 leads to injury of podocytes.48 The concomitant presence of IgG1 in immune deposits could possibly be responsible for complement activation through the classical pathway. However, most patients with membranous nephropathy have very low or undetectable levels of complementC1q in deposits in contrast to patients with secondary forms of membranous nephropathy. Together, these observations suggest that the alternative or lectin pathways might be involved in complement activation and formation of the C5b9 complex. Indeed, mannose-binding lectin has been identified in the glomeruli of many patients with idiopathic membranous nephropathy.106 Effects of antibodies beyond complement Despite numerous studies supporting a role for complement in the pathogenesis of experimental membranous nephropathy, proteinuria and podocyte damage may also occur without assembly of C5b9.107,108 In addition to forming immune complexes, antipodocyte anti bodies might directly alter podocyte biology. For example, in
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passive Heymann nephritis, anti-megalin IgG inhibits the binding and uptake of lipoproteins by megalin invivo during the formation of immune deposits.109 Accumulated lipids may undergo peroxidation causing damage to the glomerular basement membrane and proteinuria.109 Similarly, anti-PLA2R1 antibodies can interfere with the normal function of PLA2R1, either as an agonist or an antagonist. The precise function of PLA2R1 in podocytes remains unclear. Binding of PLA2G1B to PLA2R1 is known to induce various effects depending on the cell type, such as activation of the mitogen-activated protein kinase cascade, production of lipid mediators and selective release of arachidonic acid from mast cells derived from bone marrow.110,111 Phospholipid hydrolysis and eicosanoid production have been observed as downstream events in experimental models;112 therefore it will be important to investigate whether these pathways are mediated by PLA2R1 in human disease. In addition to its function as a receptor, PLA2R1 has endocytic properties and rapidly internalizes PLA2 ligand.113 PLA2R1mediated endocytosis is particularly important for the clearance of extracellular PLA2 in order to protect cells from potent enzymatic activities.110 PLA2R1 also regulates cellular senescence through the production of reactive oxygen species and the activation of DNA-damage pathways.114 Because markers of senescence are over expressed in podocytes from patients with membranous nephropathy, a potential role for agonistic activity of anti-PLA2R1 can be considered.115 localized to Bcells, Tcells, antigen-presenting cells, and podocytes.118 In the rat model of passive Heymann nephritis, treatment with MC1R agonists reduced protein uria, improved glomerular morphology and reduced oxidative stress.119 ACTH might reduce the production of nephritogenic antibodies and could also have a direct effect on podocytes, thus stabilizing glomerular architecture and ameliorating proteinuria. Future research is needed to further define the role of ACTH in therapy. The design of specific therapies requires identification of pathogenic epitopes. We have recently identified two mimotopes that mimic the conformational epitope recognized by maternal alloantibodies against NEP. We have also shown that immunoadsorption of maternal sera on a peptide-coupled affinity column removed most of the anti-NEP antibodies (P. Ronco and H.Debiec, unpublished work). Because future pregnancies in NEP-immunized mothers are high risk for the fetus,120 quick and selective elimination of antibodies from the maternal circulation could be achieved by extracorporeal immunoadsorption of specific anti b odies. A similar approach could be of value in patients with idiopathic membranous nephropathy once pathogenic epitopes are firmly established. The pathogenic role of C5b9 provides an opportunity to develop targeted interventions based on complementspecific antibodies.48 New approaches that target active complement regulatory molecules to specific cell surfaces where complement is deposited could also be useful.121,122 Future therapies could combine immunosuppression or antibody inactivation, protection of podocytes from injury and restoration of glomerular barrier disrupted by the disease.

New therapies
The treatment of human membranous nephropathy is based on nonspecific immunosuppressive agents that have their own toxic effects.11,14 A better understanding of disease pathomechanisms will result in more specific concept-driven therapies. Given the key role of IgG antibodies in membranous nephropathy, strategies to target Blymphocytes with anti-CD20 antibody could be a safe and effective way to induce remission of nephrotic syndrome through inhibition of antibody formation. However, responses vary widely from complete to no response87,90 and further research is needed in order to identify those patients who are likely to benefit from rituximab therapy and those who will be resistant. In Europe and the USA, clinical observations indicate that adrenocorticotropic hormone (ACTH) in a synthetic form or as natural highly purified gel can be an effective therapy for idiopathic membranous nephro pathy.116,117 Good results have been achieved in patients who have previously failed other immunosuppressive therapies. ACTH acts via the melanocortin receptors (MC1RMC5R) distributed throughout the body.118 Of note, MC1R has been
1. Glassock, R.J. The pathogenesis of idiopathic membranous nephropathy: a 50-year odyssey. Am. J. Kidney Dis. 56, 157167 (2010). Kerjaschki, D. Pathogenetic concepts of membranous glomerulopathy (MGN). J. Nephrol. 13 (Suppl. 3), S96S100 (2000). Imai, H., Hamai, K., Komatsuda, A., Ohtani, H. & Miura, A.B. IgG subclasses in patients with

Conclusions
Considerable advances have been achieved in unraveling the molecular pathomechanisms of membranous nephropathy. The spectrum of human membranous nephropathies needs to be revisited according to the newly identified antigenantibody systems, which should be considered as molecular signatures of the disease. These signatures will soon have a major impact on patient care, including kidney biopsy policy, diagnosis and monitoring, as well as on choice and titration of therapy.
Review criteria
Articles for this Review were identified by searching PubMed using the terms membranous nephropathy, antigens, antibodies and Heymann nephritis. Original English-language, full-text papers from this search were added to our own collection of papers on these topics.

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Ohtani, H. et al. Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy. Nephrol. Dial. Transplant. 19, 574579 (2004). Segawa, Y. et al. IgG subclasses and complement pathway in segmental and global membranous nephropathy. Pediatr. Nephrol. 25, 10911099 (2010).

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7. 8. Ponticelli, C. Membranous nephropathy. J.Nephrol. 20, 268287 (2007). Polanco, N. et al. Spontaneous remission of nephrotic syndrome in idiopathic membranous nephropathy. J. Am. Soc. Nephrol. 21, 697704 (2010). Polanco, N. et al. Spontaneous remission of nephrotic syndrome in membranous nephropathy with chronic renal impairment. Nephrol. Dial. Transplant. 27, 231234 (2012). Glassock, R.J. Diagnosis and natural course of membranous nephropathy. Semin. Nephrol. 23, 324332 (2003). Cattran, D. Management of membranous nephropathy: when and what for treatment. J.Am. Soc. Nephrol. 16, 11881194 (2005). Glassock, R.J. The treatment of idiopathic membranous nephropathy: a dilemma or a conundrum? Am. J. Kidney Dis. 44, 562566 (2004). Waldman, M. & Austin, H.A. III. Controversies in the treatment of idiopathic membranous nephropathy. Nat. Rev. Nephrol. 5, 469479 (2009). Cattran, D.C., Reich, H.N., Kim, S.J. & Troyanov,S. Have we changed the outcome in membranous nephropathy? 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