Cellular and Molecular Studies of Renal Transport

总体描述（由申请人提供）： 本计划项目的总体目标是了解肾液的潜在机制， 电解质稳态和肾上皮功能的健康和过程，调节这些 疾病的机制。一个广泛的技术将被用来解决一系列的问题，从个别运输相关蛋白质的分子特征，这些蛋白质的贡献，在完整的肾小管，器官和整个动物的水平上的综合肾功能。我们成功推行这些课题的策略，包括在互相关连的研究项目上紧密合作;五个个别项目和四个研究中心的主任分享专业知识、概念和技术;共同使用研究中心设施和单一行政中心。研究项目包括广泛的实验制剂，包括离子/溶质转运蛋白、转运调节蛋白、组织培养中的转染哺乳动物细胞、分离的细胞膜囊泡、爪蟾卵母细胞表达系统、分离的肾细胞和肾小管以及体内的整个肾。我们将使用广泛的方法，包括分子克隆和诱变，功能性cDNA表达，转基因小鼠的产生和使用，免疫细胞化学，磷酸肽富集与磷酸化位点的质谱鉴定，共聚焦显微镜，细胞离子活性的荧光测定，全细胞钳和膜片钳和巨膜片钳技术，体内和体外特定小管段的灌注，和清除研究。每个项目和核心都与方案的中心主题有关：为在肾电解质稳态中起作用的单个转运蛋白提供重要的新见解，阐明这些转运相关蛋白的调节，阐明上皮极性中涉及的细胞途径，上皮极性是载体溶质和液体转运的绝对先决条件，并评估这些蛋白质在正常和紊乱的电解质和能量代谢条件下对小管和器官功能的相对贡献。我们的战略，以追求这些主题的成功将包括密切合作，相互关联的研究项目;分享专业知识，概念和技术的计划调查;和核心设施的共同使用。

DESCRIPTION, OVERALL (provided by applicant): 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.