Cellular and Molecular Studies of Renal Transport

本计划的总体目标是了解肾脏液体和电解质稳态的机制。为此，我们将使用广泛的技术来解决一系列问题，从单个转运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综合肾功能的贡献。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;个别项目和核心项目的主任分享专业知识、概念和技术;以及共同使用核心设施。 拟议的研究项目包括广泛的实验制剂，包括运输蛋白，分离的膜囊泡，组织培养细胞，非洲爪蟾卵母细胞，分离的单个肾细胞，单个肾小管和整个肾脏在体内。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，免疫细胞化学，共聚焦显微镜，细胞离子活性的荧光测定，全细胞和膜片钳技术，体内和体外灌注， 确定的小管节段和清除研究。 拟议的研究项目包括广泛的实验制剂，包括转运蛋白和通道蛋白，参与转运蛋白和通道调节的蛋白质，分离的膜囊泡，培养细胞，非洲爪蟾卵母细胞，单个肾小管，体内整个肾脏，以及整个大鼠或小鼠，包括转基因小鼠。我们还将使用广泛的方法，包括基因工程和诱变，在卵母细胞和培养的哺乳动物细胞中的异源表达， 质谱法、免疫细胞化学、共聚焦显微镜、细胞离子活性的荧光测定、膜片钳技术、特定小管节段的体内和体外灌注、失衡CO2/HCO-3溶液、小鼠血浆和尿液中电解质和酸碱参数的测量以及清除研究。该计划的特点是更加重视转基因小鼠和使用综合肾功能实验室的核心。

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.