Cellular and Molecular Studies of Renal Transport

总体描述(由申请人提供)： 该计划项目的总体目标是了解肾液和尿液的机制。 电解质动态平衡与健康人肾上皮细胞功能及其调节过程 疾病中的机制。将使用广泛的技术来解决一系列问题，从单个运输相关蛋白的分子特征到这些蛋白在完整小管、器官和整个动物水平上对完整肾脏功能的贡献。我们成功推行这些主题的策略将包括在相互关联的研究项目上密切合作；五个单独项目和四个研究核心的主任分享专业知识、概念和技术；联合使用研究核心设施和单一的行政核心。研究项目包括广泛的实验准备，包括离子/溶质运输蛋白、运输调节蛋白、组织培养中的转基因哺乳动物细胞、分离的细胞膜小泡、非洲爪哇卵母细胞表达系统、分离的肾细胞和肾小管，以及体内的全肾。我们将使用广泛的方法，包括分子克隆和突变，功能基因表达，转基因小鼠的产生和使用，免疫细胞化学，磷酸化位点的质谱学鉴定，共聚焦显微镜，细胞离子活性的荧光分析，全细胞钳和膜片和巨型膜片钳技术，体内和体外灌流确定的小管片段，以及清除研究。每个项目和核心都与该计划的中心主题有关：对在肾脏电解质动态平衡中发挥作用的单个运输蛋白提供重要的新见解，阐明这些运输相关蛋白的调节，阐明作为载体溶质和液体运输的绝对先决条件的上皮极性所涉及的细胞通路，以及评估这些蛋白在正常和错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。我们成功追求这些主题的战略将包括在相互关联的研究项目上密切合作；项目调查人员分享专业知识、概念和技术；以及联合使用核心设施。

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.