COLLECTING DUCT SPECIFIC GENE EXPRESSION IN KIDNEY

对控制肾小管发育和分化的分子机制的了解还不完全。集合管的个体发育具有特别重要的意义，因为两种主要的细胞类型，即间质细胞和主细胞，对维持酸碱、钠和水的平衡至关重要；而这些细胞的功能障碍可能是尿酸、水肿形成和集中障碍、肾脏发育障碍和恶性肿瘤的原因。我们计划研究水通道蛋白-2基因对肾脏主细胞的特异性调控，因为它是集合管中主细胞功能所特有的，也是必不可少的。我们的具体目标是：1)建立转基因小鼠模型，用于体内研究水通道蛋白-2基因内肾主细胞特异性调控区。2)建立细胞培养模型，用于体外研究水通道蛋白-2基因的主要细胞特异性调控区。3)确定水通道蛋白-2基因中负责水通道蛋白-2主细胞特异性表达的核转录因子的结合位点。4)利用体外细胞培养模型和体内转基因小鼠模型相结合的方法，确定水通道蛋白-2基因中共同赋予主细胞特异性表达的调节区。5)分离肾主细胞水通道蛋白-2基因转录因子的编码基因。这些研究将利用转基因小鼠和转基因或显微注射肾细胞的报告基因研究、DNase I超敏分析、体内足迹和凝胶迁移率改变分析相结合的方法来确定水通道蛋白-2基因中负责肾主细胞特异性表达的特定调节区和核蛋白结合部位。编码与这些调控区域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。鉴定现有的或新的参与肾脏和集合管特异基因表达的转录因子将为理解肾小管分化的分子基础打开大门，并将为深入了解肾脏潜在的疾病机制提供线索。

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.