COLLECTING DUCT SPECIFIC GENE EXPRESSION IN KIDNEY

关于控制肾小管发育和分化的分子机制的知识是不完整的。集合管的个体发育是特别重要的，因为两种主要的细胞类型，即插层细胞和主细胞，对于维持酸碱、钠和水平衡至关重要;这些细胞的功能障碍可能导致尿酸化、水肿形成和浓缩障碍、肾脏发育障碍和恶性肿瘤。我们计划研究肾主细胞特异性调节的水通道蛋白-2基因，因为它是特定的和必不可少的集合管内的主细胞的功能。我们的具体目标是：1）建立转基因小鼠模型，用于水通道蛋白-2基因内肾主细胞特异性调控区的体内研究。2)建立水通道蛋白2基因主要细胞特异性调控区体外研究的细胞培养模型。3)鉴定水通道蛋白2基因内负责水通道蛋白2的主要细胞特异性表达的核转录因子的结合位点。4)通过使用体外细胞培养模型和体内转基因小鼠模型的组合，鉴定水通道蛋白-2基因内共同赋予主要细胞特异性表达的调控区域。5)分离编码负责水通道蛋白-2基因的肾主细胞特异性表达的转录因子的cDNA。这些研究将利用转基因小鼠和转染或显微注射肾细胞中的报告基因研究、DNA酶I超敏反应分析、体内足迹法和电泳迁移率变化试验的组合，以确定负责肾主细胞特异性表达的水通道蛋白-2基因内的特异性调控区和核蛋白结合位点。编码与这些调控区相互作用的转录因子的cDNA将使用单杂交克隆系统或亲和纯化、微测序和简并RT-PCR克隆技术的组合来分离。参与肾脏和集合管特异性基因表达的现有或新的转录因子的鉴定将为理解肾小管分化的分子基础打开大门，并将提供对肾脏潜在疾病机制的深入了解。

Knowledge about the molecular mechanisms that control development and differentiation of the renal tubule is incomplete. Ontogeny of the collecting duct is of particular importance because the two major cell types, intercalated and principal cells, are critical to maintenance of acid-base, sodium and water balance; and dysfunction of these cells likely accounts for urinary acidification, edema forming and concentrating disorders, developmental disorders of the kidney, and malignancies. We plan to study renal principal cell specific regulation of the aquaporin-2 gene, because it is specific for and essential to the function of principal cells within the collecting duct. Our specific aims are: 1) Develop a transgenic mouse model for the In vivo study of renal principal cell specific regulatory regions within the aquaporin-2 gene. 2) Develop a cell culture model for the in vitro study of the principal cell specific regulatory regions within the aquaporin-2 gene. 3) Identify the binding sites for nuclear transcription factors within the aquaporin-2 gene that are responsible for principal cell specific expression of aquaporin-2. 4) Identify regulatory regions within the aquaporin-2 gene that collectively confer principal cell specific expression by using a combination of the in vitro cell culture model and in vivo transgenic mouse model. 5) Isolate cDNAs encoding transcription factors that are responsible for renal principal cell specific expression of the aquaporin-2 gene. These studies will utilize a combination of reporter gene studies in transgenic mice and either transfected or microinjected kidney cells, Dnase I hypersensitivity analysis, in vivo footprinting, and electrophoretic mobility shift assays to define the specific regulatory regions and nuclear protein binding sites within the aquaporin-2 gene that are responsible for renal principal cell specific expression. cDNAs encoding transcription factors that interact with these regulatory regions will be isolated using one- hybrid cloning system, or a combination of affinity purification, microsequencing and degenerate RT-PCR cloning techniques. The identification of existing or novel transcription factors involved in kidney and collecting duct specific gene expression will open the door to understanding the molecular basis for renal tubule differentiation, and will provide insight into potential disease mechanisms in the kidney.