PATHOGENESIS OF MEMBRANOUS GLOMERULONEPHROPATHY

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The long term objective of this project is to understand the pathogenesis of membranous glomerulonephropathy (MGN), a common kidney disease in humans. Heymann Nephritis (HN) of rat, an accepted model of MGN, will be used to gain insight into the mechanisms of this disease. The specific aims are: 1) Test the autoimmunogencity and nephritogenicity of reduced and nonreduced individual subunits of the HN antigen, gp600. Rats immunized with subunits isolated by preparative polyacrylamide gel electrophoresis will be examined for development of HN. Sera will be analyzed for autoantibodies to native, reduced and nonreduced gp600 and individual subunits by indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA) and immunoreactivity on Western blots. Kidneys will be examined by immunofluorescence and immunoelectron microscopy for typical features of HN, and the identity and ultrastructural location of the epitopes reacting with deposited autoantibodies using rat monoclonal antibodies to gp600. Data will provide information on whether the necessary disease producing epitopes are present on all subunits and whether intact disulfide bonds in subunits are essential for nephritogenicity; 2) Test the hypothesis that some nephritogenic epitopes are shared among subunits while others are not. This will be performed with rat monoclonal antibodies to gp600 which show identity for epitope reactivity with the nephritogenic autoantibodies. Methods will include competitive inhibition immunoassays, enzyme immunodetection on Western blots, and autoradiography; 3) Further characterize biochemically and immunologically the peptides of subunits produced by limited proteolytic cleavage for relatedness in peptide composition, nature of nephritogenic epitopes, and autoimmunogenic and nephritogenic potential. This will involve peptide mapping of individual subunits using 2-D gel electrophoresis to determine the relatedness, if any, of the subunits. Partial characterization of the nature of the nephritogenic epitopes will be done using the proteolytic technique in the presence of antibody followed by gel electrophoretic and Western analyses of proteolysed fragments. The fragments which still bind antibody will be further analyzed for nephritogenic and autoimmunogenic potential; 4) Determine the significance of carbohydrate residues in gp600 towards autoantibody binding autoimmunogenicity and nephritogenicity: this will be examined by performing deglycosylation of gp600 with exo- and endoglycosidases and then testing the deglycosylated preparation for-autoantibody binding by ELISA and for nephritogenicity by immunizing Lewis rats. The proposed studies will provide new information regarding the biochemical and immunological structure of gp600 and how that relates to the nephritogenicity of gp600. This knowledge will lead to further progress in the understanding of the mechanism of MGN.