The overall goal of this Program Project is to understand the mechanisms underlying renal fluid and electrolyte homeostasis. To this end, we will use a broad spectrum of techniques to address a continuum of problems ranging from the molecular characterization of individual transport-related proteins to the contribution of these proteins to integrated renal function at the level of the intact tubule, the organ, and the whole animal. Our strategy to pursue these themes successfully will include close collaboration on interrelated research projects; sharing of expertise, concepts and techniques by Directors of the individual Projects and Cores; and joint use of core facilities. The proposed research projects comprise a broad range of experimental preparations including transport proteins, isolated membrane vesicles, tissue culture cells, Xenopus oocytes, isolated individual kidney cells, single tubules, and whole kidney in vivo. We shall use a wide range of methods including molecular cloning and mutagenesis, functional cDNA expression, generation of transgenic mice, immunocytochemistry, confocal microscopy, fluorometric assays of cell ion activities, whole cell and patch-clamp techniques, in vivo and in vitro perfusion of defined tubule segments, and clearance studies. The proposed research projects comprise a broad range of experimental preparations including transporter and channel proteins, proteins involved in the regulation of transporters and channels, isolated membrane vesicles, cultured cells, Xenopus oocytes, single tubules, the whole kidney in vivo, and the whole rat or mouse, including transgenic mice. We also shall use a wide range of methods, including genetic engineering and mutagenesis, heterologous expression in oocytes and cultured mammalian cells, identification of cellular proteins by mass-spectroscopy approaches, immunocytochemistry, confocal microscopy, fluorometric assays of cell ion activities, patch-clamp techniques, in vivo and in vitro perfusion of defined tubule segments, out-of-equilibrium CO2/HCO-3 solutions, measurement of electrolyte and acid-base parameters in the blood plasma and urine of mice, and clearance studies. The Program is characterized by increased emphasis on transgenic mice and use of the core on Laboratory of Integrated Kidney Function.
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