During the last 25 years our research has been directed toward understanding the cellular and molecular mechanisms of glomerular permeability and protein absorption as well as understanding the derangements in these processes that occur in glomerular diseases. Major findings of the previous renewal period were: 1) Demonstration that megalin is the main endocytic receptor for insulin and other peptides in the proximal tubule; 2) Identification of two novel proteins--ANKRA and ARH--that bind to the cytoplasmic tail of megalin; 3) Demonstration that ARH facilitates protein uptake by megalin in the proximal tubule; 4) Identification of the targeting signal in the megalin tail that directs it to the brush border region of the proximal tubule cell; 5) Demonstration that podocalyxin is connected to the actin cytoskeleton via two linker proteins--NHERF and ezrin; 6) demonstration that the association of podocalyxin with the cytoskeleton is lost in PAN nephrosis and in other conditions in which there is proteinuria and loss of foot processes; and 7) Preliminary indications that nephrin is associated with adherens junction proteins. The studies proposed in this application represent a direct continuation of our ongoing work.
Our specific aims are: 1) To characterize interacting partners of the cytoplasmic tail of megalin with emphasis on ARH and two Ring finger proteins that are involved in the regulation of megalin's functions in the uptake of filtered proteins by podocytes and proximal tubules of normal and PAN nephrotic rats; 2) To investigate the functional interactions of nephrin with other components of the slit membranes in normal and nephrotic glomeruli; and 3) To investigate the molecular mechanisms by which podocalyxin is involved in maintenance of the foot process organization of the podocyte and the flattening of the foot processes that occurs in proteinuric states. We will focus on the interactions of podocalyxin's tail with NHERF and ezrin and their involvement in signaling from both outside and inside the cell. In this work we will use a combination of morphological, biochemical, molecular biological and proteomics approaches. It is our hope and expectation that these studies will continue to provide new insights into our understanding of the cellular and molecular mechanisms of glomerular filtration, protein absorption, and their alterations in glomerular diseases associated with proteinuria.
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