During the last 5 years our research has been directed toward identifying and partially characterizing glomerular components known to be important in filtration and/or pathology. We have focused our attention on heparin sulfate proteoglycans (HSPG), epithelial polyanion, and the Heymann nephritis antigen, gp330. Major findings obtained were: 1) The glomerulus contains two types of antigenically-distinct HSPG associated with basement membranes and cell membranes, respectively; 2) Basement membrane HSPG are concentrated in the laminae rarae of the GBM; 3) Antibodies against these HSPG produce an anti-GBM nephritis when given in vivo; 4) Podocalyxin, a 140 kD glycoprotein, represents the biochemical equivalent of epithelial polyanion; 5) The sialic acid content of podocalyxin is reduced in PAN nephrosis; 6) Podocalyxin represents a differentiation marker for the polocyte; 7) The membranes of glomerular and proximal tubule epithelia contain microdomains of distinctive protein composition; 8) In Heymann nephritis immune deposits, formed as a result of binding of anti-gp330 to the gp330 antigen, are rapidly shed, and become cross-linked to the GBM. The studies proposed in this application represent a direct continuation of our ongoing work. At this juncture information on the structure of the molecules we have identified is sorely needed in order to gain insights into their nature, functions and pathology. Specifically, we wish to further characterize basement membrane HSPG, podocalyxin and gp330, determine their molecular structure (by protein and DNA sequencing) and study their biosynthesis in normal and diseased rats. We also intend to investigate further the cellular and molecular mechanisms involved in the formation of subepithelial immune deposits in membranous nephropathy.
Our Specific Aims are: 1) To characterize the core protein of the HSPG from the rat GBM; 2) To study the intracellular transport, processing and assembly into the G8M of these HSPG in normal vs. nephrotic rats; 3) To investigate the mechanisms of formation of subepithelial immune deposits by injecting antibodies that recognize endogenous glomerular proteins: 4) To characterize the antigens recognized by a family of monoclonal antibodies generated against glomerular components; 5) To determine the kinetics and steps in the biosynthesis of podocalyxin and the nature of its postranslational modifications in controls and PAN rats. It is hoped that these studies will provide new insights into the cellular and molecular aspects of glomerular filtration and its alterations in various glomerular diseases.
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