During the last 10 years our research has been directed toward identifying and characterizing glomerular components known to be important in filtration and/or pathology. In the past we have focused our attention on heparan sulfate proteoglycans (HSPG), epithelial polyanion, and gp330, the Heymann nephritis (HN) antigen. Major findings of the previous renewal period were: 1) Evidence was obtained for the existence of a family of closely-related basement membrane (HSPG that differ from one basement membrane to another; 2) Demonstration that podocalyxin contains N- and O- linked oligosaccharides that are both sialylated and sulfated; 3) Generation of a panel of MABs that recognize native glomerular membrane proteins and demonstration that circulating antibodies can bind to their respective antigens in all layers of the glomerular capillary wall; 4) Discovery of a 44 kD protein that associates with gp330 to form the Heymann nephritis antigenic complex (HNAC); 5) Demonstration that the complex is assembled in the ER and expressed at the surface of glomerular and proximal tubule epithelia; 6) Demonstration that the 44 kD protein is identical to RAP; 7) Discovery of a cell line that synthesizes and expresses gp330 and RAP; 8) Demonstration that the filtration slit membrane is a variant of the tight junction; 9) Demonstration that tyrosine phosphorylation is involved in signalling during pathogenic changes in junctional morphology and that the tight junction protein Z)-1 is one of the newly phosphorylated proteins. The studies proposed in this application represent a direct continuation of our ongoing work.
Our specific aims are: 1) To further characterize the gp330/RAP (HNAC) complex. Specific concerns are to determine the nature of the pathogenic epitopes that initiate formation of immune deposits in HN and to map the binding sites on RAP for heparin, gp330 and LRP; 2) To study the trafficking of gp330 and RAP along the exocytic and endocytic pathways and to identify the natural ligands for gp330. These studies will be greatly facilitated by our discovery of a cell line that expresses HNAC; 3) To determine the pattern of expression of cell-cell and cell-matrix junctional proteins in podocytes of normal rats and those with PAN nephrosis. We will determine which of these proteins undergoes phosphorylation under normal and pathologic conditions and investigate the role of ANG II, ANF and integrin receptors in effecting tyrosine phosphorylation; 4) To extend knowledge of both the protein and oligosaccharide moieties of podocalyxin. Specifically we intend to complete the cloning of podocalyxin and to express this protein (by transfection) in cultured cells in order to assess its effect on cell behavior. Detailed analysis will also be made of the nature of the sialic acid and other oligosaccharide moieties on podocalyxin from normal rats and those with PAN nephrosis. It is our hope and expectations that these studies will provide new insights into our understanding of the cellular and molecular mechanisms of glomerular filtration and its alterations in glomerular diseases.
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