The overall goal of this Program Project is to understand the mechanisms underlying renal fluid and electrolyte homeostasis and renal epithelial function in health and the processes that modulate these mechanisms in disease. A broad spectrum of techniques will be used 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 five individual Projects and the four research Cores;joint use of the research core facilities and single administrative core. The research projects comprise a broad range of experimental preparations including ion/solute transport proteins, transport regulatory proteins, transfected mammalian cells in tissue culture, isolated cell membrane vesicles, Xenopus oocyte expression system, isolated kidney cells and tubules, and whole kidney in vivo. We shall use a wide range of methods including molecular cloning and mutagenesis, functional cDNA expression, generation and use of transgenic mice, immune-cytochemistry, phosphopeptide enrichment coupled to mass spectral identification of phosphorylation sites, confocal microscopy, fluorometric assays of cell ion activities, whole cell clamp and patch- and giant patch-clamp techniques, in vivo and in vitro perfusion of defined tubule segments, and clearance studies. Each of the projects and cores is concerned with the central themes of the Program: to provide important new insights into individual transport proteins that play a role in renal electrolyte homeostasis, to elucidate the regulation of these transport-related proteins, to elucidate the cellular pathways involved in epithelial polarity that is an absolute prerequisite for vectorial solute and fluid transport, and to assess the relative contributions of these proteins to tubule and organ function under conditions of normal and deranged electrolyte and energy metabolism. Our strategy to pursue these themes successfully will include close collaboration on interrelated research projects;sharing of expertise, concepts and techniques by Program investigators;and joint use of core facilities.
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