PATHOGENESIS OF MEMBRANOUS GLOMERULONEPHROPATHY

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date9/1/8311/30/04

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $11,710.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $225,819.00
  • National Institutes of Health

ASJC

  • Medicine(all)

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