Molecular Dissection of Target Antigens and Nephritogenic Antibodies in Membranous Nephropathy: Towards Epitope-Driven Therapies
Pierre Ronco, and
Hanna Debiec*
* INSERM Unité Mixte de Recherche S 702; Université Pierre et Marie Curie-Paris; and Assistance Publique–Hôpitaux de Paris; Hôpital Tenon, Paris, France
Membranous nephropathy (MN) is the most common cause of idiopathicnephrotic syndrome in white adults, accounting for about 20%of cases. The disease is characterized by an accumulation ofimmune deposits on the outer aspect of the glomerular basementmembrane (GBM). The immune deposits consist of IgG (often IgG4),thus far unidentified antigens, and the membrane attack complexof complement C5b-9. Although spontaneous remission of nephroticsyndrome occurs in about a third of patients, MN ends for about40% of patients in end-stage renal failure after 10 yr (1).Treatment of MN is often disappointing (2,3). This is due inpart to heterogeneity of the disease and lack of reliable biomarkersbecause of ignorance of the target antigen(s) and nephritogenicantibodies. Strategies to target B-lymphocytes with anti-CD20antibody (4) and to inhibit complement (5) are steps in theright direction, but more specific, concept-driven therapiesare urgently needed.
We have learned a great deal about idiopathic MN from experimentalHeymann nephritis (HN) (6). The active model of MN is inducedby immunization of Lewis rats with preparations of brush-borderantigens (7), while the passive model is caused by injectingrats with rabbit anti-rat brush-border antibodies. The autoantigenictarget in the rat disease was identified in the early 1980sas the podocyte membrane protein now called megalin (8,9). Thisantigen is expressed with clathrin at the sole of podocyte footprocesses (where immune complexes are formed). This findingprovided the first evidence that podocytes actively contributeto the formation of glomerular immune deposits in MN. Sincethen, attempts have been made to dissect the megalin systemon a molecular level. The article by Tramontano et al. in thisissue of JASN (10) is the latest section of a long road towardidentification of the pathogenic epitope(s) in HN, a prerequisitefor specific immunointervention.
Although a first pathogenic epitope had been identified in theearly 1990s by partial cloning of megalin (11,12), the travelactually 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 molecularweight of approximately 600 kD (14,15) (Figure 1). Megalin isthe endocytotic receptor for which the most ligands have beendescribed, 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 passiveHN could be induced by antibodies specific for a synthetic peptidederived from RAP (16). However, the rats did not develop proteinuria.It seems that RAP by itself cannot induce active HN (17), whichpoints to an essential role for megalin.
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.
Given the large size of megalin, the pinpointing by Raychowdhuryet al. (18) of a 137-aa fragment (aa 1114 to 1250) in the secondligand-binding domain (LBD) as a pathogenic epitope, recognizedby antibodies eluted from the glomeruli of rats with activeHN, represented a major breakthrough (Figure 1). Saito et al.(19) narrowed the epitope to the fifth ligand-binding repeatconsisting of 46 aa (aa 1160 to 1205). In fact, all four putativemegalin LBD actually contain pathogenic epitopes capable ofinducing passive HN (i.e., granular subepithelial immune deposits)(20). However, proteinuria was not reported in either of thesemodels. Therefore, the finding by Makker’s group (21)that a 60-kD N-terminal fragment (nM60) encompassing aa 1 to563 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 157to 236 in the first LBD (Figure 1). Three additional findingsare of interest. First, full immunogenic activity required expressionof the fragments in insect cells, suggesting that posttranslationalmodifications and/or conformational determinants are essentialfor the pathogenic potential (10,22). Second, lymph node cellproliferation assays indicated that the pathogenic epitopescould elicit T cell responses. Third, levels of B cell responsesin rats immunized with different fragments did not correlatewith severity of disease, which suggests that qualitative differencesin the immune response, including epitope specificity and isotypedistribution, are of paramount importance.
However, megalin cannot be held responsible for human MN becauseit has not been found in human glomeruli or podocytes, nor hasit been detected in subepithelial immune deposits in patientswith MN. We have identified neutral endopeptidase (NEP) as thehuman counterpart to the rat antigen megalin in infants bornwith antenatal MN (23,24). Pathogenic antibodies directed againstNEP were transplacentally transferred from the mother to herchild. NEP, a membrane-bound enzyme that can digest biologicallyactive peptides, is expressed on human podocytes, syncytiotrophoblasticcells, polymorphonuclear leukocytes, lymphoid progenitor cells,and epithelial cells of nonlymphoid organs (25). The anti-NEPantibodies were produced against the placental NEP (26) by motherswho are NEP-deficient. Two truncating mutations were identifiedin the MME gene coding for NEP in the three affected families,but truncated proteins could not be detected in the NEP-deficientmothers’ granulocytes or urine, indicating functionalknockout of the mutated mutated metallo-endopeptidase (MME)gene (23,24).
The NEP system bears close similarities with the rat megalinsystem. First, like anti-megalin antibodies, anti-NEP antibodiescause formation of immune complexes at podocyte membranes. Second,complement activation is essential for full-blown expressionof the disease. By confocal microscopy, the membrane attackcomplex of complement (C5b-9) was colocalized, with NEP in immunedeposits, both in the infant and in the rabbits that receivedan injection with the mother’s IgG (27). Third, Ig isotypespecificity is also of crucial importance. Although both IgG1and IgG2a can activate complement through the classic pathwayin the rat (28), complement deposition and proteinuria correlatebest 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, activatecomplement) 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. Pathogenicanti-megalin antibodies inhibit the binding of apolipoproteinsapoE and apoB by podocytes, thereby favoring accumulation ofapoE and apoB in immune deposits (29). These lipids may undergoperoxidation, causing GBM damage and proteinuria. Similarly,anti-NEP antibodies blocked NEP enzymatic activity, therebypotentially increasing the local concentration of vasoactivepeptides (27).
The design of specific therapies for autoimmune diseases isprimarily based on induction of specific immune tolerance. Ideally,this requires identification of the pathogenic epitopes carriedby the antigen. One way to induce tolerance is mucosal administrationof the antigen/immunodominant epitopes (30,31). The inhibitoryeffect of orally or nasally administrated autoantigens or immunodominantpeptides has been widely reported in several experimental modelsof autoimmune disease in rodents. Recently, nasal administrationof recombinant NC1 domain of the a3 chain of type IV collagenwas shown to induce tolerance in a model of anti-GBM glomerulonephritis(32). This resulted in a marked reduction in circulating anddeposited antibodies, albuminuria, severity of glomerular abnormalities,and numbers of glomerular CD8+ T cells and macrophages. We haverecently identified two immunodominant epitopes in the NEP antigenthat are specifically recognized by the mothers’ antibodies(Debiec, unpublished observations, 2006). Because future pregnanciesin 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 emphasizethe need for identification of further podocyte antigens, andfor subsequent molecular dissection of the immune response incommon forms of "idiopathic" MN. Anti-NEP antibodies do notcause common MN, but the experimental and human data stronglysuggest that most antigenic targets sit at the podocyte membrane,where they should be searched for. Translational research inthis area should soon lead to assays of circulating pathogenicantibodies and to better targeted therapies aimed at decreasingspecifically their production.
Acknowledgments
The research of the authors is funded by grants from Institutdes Maladies Rares, INSERM, AURA (Association pour l’Utilizationdu Rein Artificiel), Programme Hospitalier de Recherche Clinique(Vincent Guigonis), and Genzyme Renal Innovations Program (GRIP).We are indebted to Béatrice Mougenot (Tenon Hospital,Paris) and Vincent Guigonis (Limoges University Hospital) forprecious collaboration. We thank Christine Vial for assistancein editing the manuscript.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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