Molecular Dissection of Target Antigens and Nephritogenic Antibodies in Membranous Nephropathy
Authors: Pierre Ronco, Hanna Debiec
Journal: Journal of the American Society of Nephrology (2006)

Membranous nephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in white adults, accounting for about 20% of cases. The disease is characterized by an accumulation of immune deposits on the outer aspect of the glomerular basement membrane (GBM). The immune deposits consist of IgG (often IgG4), thus far unidentified antigens, and the membrane attack complex of complement C5b-9. Although spontaneous remission of nephrotic syndrome occurs in about a third of patients, MN ends for about 40% of patients in end-stage renal failure after 10 yr (1). Treatment of MN is often disappointing (2,3). This is due in part to heterogeneity of the disease and lack of reliable biomarkers because of ignorance of the target antigen(s) and nephritogenic antibodies. Strategies to target B-lymphocytes with anti-CD20 antibody (4) and to inhibit complement (5) are steps in the right direction, but more specific, concept-driven therapies are urgently needed. We have learned a great deal about idiopathic MN from experimental Heymann nephritis (HN) (6). The active model of MN is induced by immunization of Lewis rats with preparations of brush-border antigens (7), while the passive model is caused by injecting rats with rabbit anti-rat brush-border antibodies. The autoantigenic target in the rat disease was identified in the early 1980s as the podocyte membrane protein now called megalin (8,9). This antigen is expressed with clathrin at the sole of podocyte foot processes (where immune complexes are formed). This finding provided the first evidence that podocytes actively contribute to the formation of glomerular immune deposits in MN. Since then, attempts have been made to dissect the megalin system on a molecular level. The article by Tramontano et al. in this issue of JASN (10) is the latest section of a long road toward identification of the pathogenic epitope(s) in HN, a prerequisite for specific immunointervention. Although a first pathogenic epitope had been identified in the early 1990s by partial cloning of megalin (11,12), the travel actually started with the complete cloning of the gene in 1994 (13). Megalin is an approximately 4600–amino acid (aa), transmembrane, polyspecific receptor protein with a molecular weight of approximately 600 kD (14,15) (Figure 1). Megalin is the endocytotic receptor for which the most ligands have been described, including the receptor-associated protein (RAP) (15). The latter is a 39-kD protein, which acts as a chaperone (15). Antibodies to RAP were also detected in rats with HN, and passive HN could be induced by antibodies specific for a synthetic peptide derived from RAP (16). However, the rats did not develop proteinuria. It seems that RAP by itself cannot induce active HN (17), which points to an essential role for megalin. Given the large size of megalin, the pinpointing by Raychowdhury et al. (18) of a 137-aa fragment (aa 1114 to 1250) in the second ligand-binding domain (LBD) as a pathogenic epitope, recognized by antibodies eluted from the glomeruli of rats with active HN, represented a major breakthrough (Figure 1). Saito et al. (19) narrowed the epitope to the fifth ligand-binding repeat consisting of 46 aa (aa 1160 to 1205). In fact, all four putative megalin LBD actually contain pathogenic epitopes capable of inducing passive HN (i.e., granular subepithelial immune deposits) (20). However, proteinuria was not reported in either of these models. Therefore, the finding by Makker’s group (21) that a 60-kD N-terminal fragment (nM60) encompassing aa 1 to 563 could induce full-blown active HN was a significant feat. Now, by successive C-terminal truncations, Tramontano et al. (10) have further narrowed the pathogenic epitopes to aa 157 to 236 in the first LBD (Figure 1). Three additional findings are of interest. First, full immunogenic activity required expression of the fragments in insect cells, suggesting that posttranslational modifications and/or conformational determinants are essential for the pathogenic potential (10,22). Second, lymph node cell proliferation assays indicated that the pathogenic epitopes could elicit T cell responses. Third, levels of B cell responses in rats immunized with different fragments did not correlate with severity of disease, which suggests that qualitative differences in the immune response, including epitope specificity and isotype distribution, are of paramount importance. However, megalin cannot be held responsible for human MN because it has not been found in human glomeruli or podocytes, nor has it been detected in subepithelial immune deposits in patients with MN. We have identified neutral endopeptidase (NEP) as the human counterpart to the rat antigen megalin in infants born with antenatal MN (23,24). Pathogenic antibodies directed against NEP were transplacentally transferred from the mother to her child. NEP, a membrane-bound enzyme that can digest biologically active peptides, is expressed on human podocytes, syncytiotrophoblastic cells, polymorphonuclear leukocytes, lymphoid progenitor cells, and epithelial cells of nonlymphoid organs (25). The anti-NEP antibodies were produced against the placental NEP (26) by mothers who are NEP-deficient. Two truncating mutations were identified in the MME gene coding for NEP in the three affected families, but truncated proteins could not be detected in the NEP-deficient mothers’ granulocytes or urine, indicating functional knockout of the mutated mutated metallo-endopeptidase (MME) gene (23,24). The NEP system bears close similarities with the rat megalin system. First, like anti-megalin antibodies, anti-NEP antibodies cause formation of immune complexes at podocyte membranes. Second, complement activation is essential for full-blown expression of the disease. By confocal microscopy, the membrane attack complex of complement (C5b-9) was colocalized, with NEP in immune deposits, both in the infant and in the rabbits that received an injection with the mother’s IgG (27). Third, Ig isotype specificity is also of crucial importance. Although both IgG1 and IgG2a can activate complement through the classic pathway in the rat (28), complement deposition and proteinuria correlate best with glomerular IgG1 deposition (10). In antenatal MN, maternal production of anti-NEP IgG1 seems necessary for disease; if only anti-NEP IgG4 (which do not, or only weakly, activate complement) is produced, then proteinuria does not result (24). Fourth, like megalin, which plays a key role in endocytosis, NEP is endowed with important enzymatic activity. Pathogenic anti-megalin antibodies inhibit the binding of apolipoproteins apoE and apoB by podocytes, thereby favoring accumulation of apoE and apoB in immune deposits (29). These lipids may undergo peroxidation, causing GBM damage and proteinuria. Similarly, anti-NEP antibodies blocked NEP enzymatic activity, thereby potentially increasing the local concentration of vasoactive peptides (27). The design of specific therapies for autoimmune diseases is primarily based on induction of specific immune tolerance. Ideally, this requires identification of the pathogenic epitopes carried by the antigen. One way to induce tolerance is mucosal administration of the antigen/immunodominant epitopes (30,31). The inhibitory effect of orally or nasally administrated autoantigens or immunodominant peptides has been widely reported in several experimental models of autoimmune disease in rodents. Recently, nasal administration of recombinant NC1 domain of the a3 chain of type IV collagen was shown to induce tolerance in a model of anti-GBM glomerulonephritis (32). This resulted in a marked reduction in circulating and deposited antibodies, albuminuria, severity of glomerular abnormalities, and numbers of glomerular CD8+ T cells and macrophages. We have recently identified two immunodominant epitopes in the NEP antigen that are specifically recognized by the mothers’ antibodies (Debiec, unpublished observations, 2006). Because future pregnancies in NEP-immunized mothers are at high risk for the fetus (26), epitope-driven therapies, including induction of mucosal tolerance, are urgently needed. The findings by Makker’s group and our own data emphasize the need for identification of further podocyte antigens, and for subsequent molecular dissection of the immune response in common forms of “idiopathic” MN. Anti-NEP antibodies do not cause common MN, but the experimental and human data strongly suggest that most antigenic targets sit at the podocyte membrane, where they should be searched for. Translational research in this area should soon lead to assays of circulating pathogenic antibodies and to better targeted therapies aimed at decreasing specifically their production.Figure 1: The structure of megalin featuring the regions containing pathogenic epitopes in Heymann nephritis (HN). Megalin is an approximately 4600 amino acid transmembrane protein. The extracellular domain contains four cystein-rich clusters of LDL-receptor type A repeats that constitute the ligand-binding domains, and are separated and followed by 17 EGF-type repeats and eight spacer regions that contain Tyr-Trp-Thr-Asp ( YWTD) repeats. Heterologous antibodies against all four ligand-binding domains (black bars in the sketch) can induce formation of glomerular immune deposits but not proteinuria. Molecular determinants in the region spanning residues 157 to 236 (red bars in the sketch) are critical for expression of the full disease.The research of the authors is funded by grants from Institut des Maladies Rares, INSERM, AURA (Association pour l’Utilization du Rein Artificiel), Programme Hospitalier de Recherche Clinique (Vincent Guigonis), and Genzyme Renal Innovations Program (GRIP). We are indebted to Be[Combining Acute Accent]atrice Mougenot (Tenon Hospital, Paris) and Vincent Guigonis (Limoges University Hospital) for precious collaboration. We thank Christine Vial for assistance in editing the manuscript.