This application is the continuation of a proposal to determine cellular mechanisms of water and solute transport in specific segments of mammalian renal tubule. We will use in vitro isolated perfused and nonperfused tubules, cell culture, brightfield and fluorescence microscopy of intact tubules and cultured cells, and recombinant DNA methods to address in rabbit tubfule segments and MDCK cells two major research areas that have emerged from our previous work. I. The following specific aims will test the hypothesis that renal epithelial cells utilize short and long term mechanisms to restore intracellular volume and chemical composition to normal in chronic anisotonic states: a) determine if sustained alterations in cell volume in acute and chronic anisotonic states perturb tubule cell function (PAH transport in proximal tubules and ADH-mediated hydrosmotic water flow in CCT). b) determine the cellular mechanisms of intracellular solute redistribution in anisotonic states in tubules and MDCK cells, and if MDCK cells, whose state of differentiation and function can be varied in vitro, express volume regulation mechanisms found in terminally differentiated tubule cells; c) determine the effect of acute and chronic anisotonic states on the cell proliferation response of cells to mitogens. II. The following specific aims will test the hypothesis that the transcellular organic anion transport mechanisms for urate and hippurate in proximal tubules have several important biologic functions, and are regulated by peritubular plasma proteins: a) develop methods to monitor the fluorescence of aryl anions in isolated S2 segments; b) develop methods to monitor fluid-phrase and receptor-mediated endocytosis of fluorescence-labelled plasma proteins (albumin, globulin); c) determine the meachanisms by which short chain fatty acids interfere with hippurate transport and potently support isovolumetric regulation of cells in hyperosmotic media; d) determine the relations among cell volume regulation, anion transport, fluid secretion and oncogene expression. The studies outlined in this application will establish the importance of intracellular volume regulation is the expression of highly differentiated structure and function in renal cortical cells, clarify the biologic roles of basolateral organic anion transport systems, and reveal the cellular mechanisms by which hippurate and urate secretion are regulated in proximal tubule cells.
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