During the last 15 years, our research has been directed toward understanding the cellular and molecular mechanisms of glomerular permeability and protein absorption and in characterizing the derangements in these processes that occur in glomerular diseases. Major findings of the previous renewal period were: 1) Validation of the multiple epitope model of Heymann nephritis; 2) Obtaining the complete sequence of megalin; 3) Mapping the main pathogenic epitope in megalin to 45 aa in the second set of ligand binding repeats; 4) Mapping the pathogenic epitope of RAP; 5) Demonstration that the ligand binding site in megalin and the main pathogenic epitope coincide; 6) Demonstration that pathogenic antibodies inhibit the binding of apolipoproteins to megalin; 7) Demonstration that megalin is the main endocytic receptor for insulin and other peptides in the proximal tubule; 8) Establishing the trafficking itinerary of megalin and RAP along the exocytic and endocytic pathways; and 9) Demonstration of increased phosphorylation of the tight junction protein ZO-1 in PAN nephrosis. The studies proposed in this application represent a direct continuation of our ongoing work.
Our specific aims are: 1) To determine the nature of the recognition mechanisms by which megalin binds and clears multiple ligands in the glomerular and proximal tubule epithelium in normal and disease states. 2) To characterize the signaling pathways for megalin in the podocyte and proximal tubule epithelium by identifying proteins that interact with the extracellular domain and cytoplasmic tail of megalin. Our hypothesis, based on preliminary work to date is that novel signal transduction pathways are involved and that these pathways are altered in PAN nephrosis and other diseases affecting protein absorption. 3) To extend knowledge of the functions and interactions of podocalyxin. We will attempt to identify proteins that interact with podocalyxin by carrying out yeast two hybrid screening and protein cross-linking studies and assess the effects of expression of podocalyxin on cultured kidney cells and CHO cell glycosylation mutants deficient in sialic acid. We will also characterize the oligosaccharide moieties of podocalyxin purified from normal glomeruli and those from rats with PAN nephrosis. Advantage will be taken of the expertise of the UCSD Glycobiology core. It is our hope and expectation that these studies will provide new insights into our understanding of the cellular and molecular mechanisms of glomerular filtration and protein absorption as well as alterations in these processes that occur in glomerular diseases.
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