The goals of this proposal are to study the biochemical differences of the plasma membrane in different regions of the kidney tubular epithelium with special emphasis being given to the establishment epithelial cell polarity and its maintenance by tight junctions. Our approach will be to raise monoclonal antibodies against the plasma membranes of the MDCK kidney epithelial cell line and kidney plasma membranes. These antibodies will be used as highly specific probes to study the distribution of antigens on the cell surface of kidney tubules by both immunoflourescence microscopy and immunoelectron microscopy. Antigens recognized by monoclonal antibodies will be characterized by immunoprecipitation and SDS gel electrophoresis. Our goal will be to acquire a library of monoclonal antibodies that recognize cell surface proteins on both the apical and basal-lateral plasma membrane domains in different regions of the nephron. Using purified kidney epithelial primary cultures isolated by immunoabsorption and a high resistance MDCK clonal cell line, we plan to study the cellular mechanisms involved in the development of the biochemically unique apical and basal-lateral membrane domains. These studies should provide further insights into the mechanisms of transepithelial ion, solute and fluid transport. The knowledge gained from these studies can then be applied to the investigation of transport related diseases such as cyctic fibrosis and diabetes insipidus.
