CELLULAR AND MOLECULAR STUDIES OF RENAL TRANSPORT

本计划项目的总体目标是了解机制 潜在的肾液和电解质动态平衡。为此，我们将 使用广泛的技术来解决一系列问题 从单个转运蛋白的克隆和特征 这些转运蛋白对综合肾功能的贡献 完整的小管和器官的水平。 我们成功追求这些主题的战略将包括关闭 在相互关联的研究项目上进行合作；分享专业知识， 方案调查人员的概念和技术；以及联合使用CORE 设施。 拟议的研究项目包括广泛的实验项目 制剂包括转运蛋白、分离的膜泡、 组织培养细胞，非洲爪哇卵母细胞，分离的单个肾脏细胞， 单肾小管，活体全肾。我们将使用广泛的 方法包括分子克隆和诱变，功能基因 表达、转基因小鼠的生成、免疫细胞化学、共聚焦 细胞离子活度的显微镜、荧光分析、全细胞和 膜片钳技术、体内和体外对明确小管的灌流 节段和净空研究。 在课程中引入新方法和新技术 这也是本提案的一个重要特点。中的一个关键要素 这方面是适应清除、微穿孔和 微灌流技术(近端和远端小管)在研究近端和远端小管中的应用 转基因和基因敲除小鼠的肾功能。

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 cloning and characterization of individual transporters to the contribution of these transporters to integrated renal function at the level of the intact tubule and organ. 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. 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 introduction into the program of novel approaches and new techniques is also an important feature of the present proposal. A key element in this regard is the adaptation of clearance, micropuncture and microperfusion techniques (proximal and distal tubules) to the study of renal function in transgenic and knockout mice.