Complement activation plays an important role in many human glomerular diseases and in the animal models that mimic these conditions.Although the deleterious effects of glomerular complement activation have been characterized in detail, the role of complement regulation in glomerular disorders is not well understood. During the tenure of the current R29 award, the Principal Investigator has isolated three complement regulatory substances produced by cultured rat glomerular epithelial cells (GEC). These are: 1) a chondroitin sulfate B proteoglycan termed glomerular complement regulatory factor (GCRF); 2) complement receptor type 1 (CR1); and, 3) Crry. These substances are present in vivo, and, therefore, are likely to have complement regulatory actions in that rat glomerulus. Previous studies in humans, and in models of human disease, suggest a role for abnormal complement regulation in several glomerulophathies. By defining the roles of glomerular CR1, Crry, and GCRF under normal and pathological conditions, the proposed research will test the hypothesis that impaired glomerular complement regulation results in disease.
Specific aims 1 and 2 are planned in cultured GEC. The capacity of CR1, Crry, and GCRF to limit complement activation on GEC under disease conditions will be evaluated. The likelihood that the pathogenic antibody of experimental rat membranous nephropathy binds and inhibits CR1 and Crry to account for its nephritogenicity will be assessed. The role for CR1 in processing immune complexes formed with both extrinsic and intrinsic antigens will also be evaluated. Expression of CR1, Crry, and GCRF will be determined in cultured GEC by measuring immunoreactive protein and mRNA quantities. GEC will be studied to discover which inflammatory stimuli account for absent CR1 protein but elevated mRNA expression in human lupus nephritis. The possibility that Crry and GCRF are affected similarly to CR1 under disease conditions will also be determined.
Specific aims 3 and 4 will be accomplished in rats. Alterations of Cr1, Crry, and GCRF proteins in different experimental glomerular diseases will be evaluated by immunoassay and histochemistry, while in situ and northern hybridizations will be performed to assess the localization and quantities of their mRNA. Rat models of immune complex glomerulonephritis, anti-glomerular basement membrane (GBM) nephritis, and membranous nephropathy will be studied. In human pathology specimens, CR1 has been shown to be reduced in each of these disorders. The functions of CR1, Crry, and GCRF under basal conditions in rats will be evaluated by the passive administration of F(ab')2 antibodies that neutralize the activity of the complement regulators. In addition, the role for inhibition of complement regulation by nephritogenic antibodies in rat models of anti-GBM nephritis and membranous nephropathy will be assessed. The part GEC CR1 plays in clearing immune complexes will be evaluated in a model of chronic serum sickness, in which circulating immune complexes traverse the glomerulus. The results of these studies in cultured cells and in animals will help define the role for complement regulation in human glomerular diseases.
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