This application describes a proposed investigation of structure-function relationships in isolated perfused segments of the rabbit renal tubule. Our work in the first three years of this project has involved the perfection of optical techniques that would permit both high resolution differential interference-contrast and fluorescence microscopy on isolated tubules in a mode that permitted simultaneous monitoring of physiologic parameters. Coupling this approach with computer-assisted image analysis, investigative studies have centered on a quantitative description of the structural events accompanying antidiuretic hormone-induced osmotic water flow across the cortical collecting tubule. Success with these first goals of the project leads us to propose here a somewhat broader investigation of renal cell biology. Data from studies now completed suggest that transcytoplasmic water flow in the collecting tubule may follow preferential pathways and details of this specialization will be investigated further with a study of cellular vacuolation, fluorescent probe labeling of the water pathway and study of cytoskeletal involvement with both immunofluorescent and electron microscopic techniques. The distribution of function between principal and intercalated cells of this nephron segment will be assessed largely via fluorescent probe analysis with respect to involvement in osmotic water flow and urinary acidification. Studies of the proximal nephron will center on the mechanisms of isotonic fluid absorption with particular emphasis on the behavior of cellular volume and the involvement of volume regulatory mechanisms with variation in the rates of transport. An important corrolary to the proximal tubule study will be an analysis of the process of protein absorption. The topics selected for investigation are central issues in the areas of renal physiology and membrane transport. Health-related significance of these studies will derive from the expansion of our information about basic biological mechanisms for the regulation of salt and water.
