Regulation of ENaC by sgk1

描述(由申请人提供)：肾脏远端小管的钠重吸收对于维持细胞外容量和血压是必不可少的。醛固酮、加压素和胰岛素通过上调ENaC的活性来刺激钠重吸收。越来越多的证据表明，sgk1(血清和糖皮质激素诱导的激酶)介导了这些激素的作用，然而，sgk1所采用的信号通路和改变ENaC活性的分子机制尚未阐明。我们的工作假说源于一个模型，在该模型中，SGK1主要定位于上皮细胞的基底膜，与输入的激动剂如胰岛素或AVP密切相关。这些和/或其他未知的刺激使sgk1磷酸化，触发到达顶膜的信号级联，增加ENaC的丰度和/或活性。这项拨款提案的具体目的是：1)确定SGK1对ENaC影响的潜在机制，特别是研究通道流量的变化，从根尖膜插入和/或取出，以及通道动力学的变化。2)确定SGK1在醛固酮、加压素和胰岛素反应中的作用，以及3)识别通过与SGK1结合将其拴在膜上、调节其活性或构成SGK1信号级联的底物的分子。我们已经开发了工具，并设计了生化(ENaC亚基周转、磷酸化分析、改进的RAS招募系统)和电生理学(膜片钳研究和阻滞剂诱导的噪声分析)实验，以便在体外(在四环素控制下修饰表达各种形式的sgk1和siRNAs的A6细胞株)和大鼠组织中共同阐明拟议的目标。SGK1在许多信号通路整合中的中心作用使其成为调节与高血压疾病相关的钠滞留的候选分子。因此，了解这一信号通路的分子机制对高血压的诊断和新的治疗策略的开发具有潜在的意义。

DESCRIPTION (provided by applicant): Sodium reabsorption in the distal tubule of the kidney is essential for maintenance of extracellular volume and blood pressure. Aldosterone, vasopressin and insulin stimulate sodium reabsorption by upregulating the activity of ENaC. There is growing evidence that sgkl (serum- and glucocorticoid-induced kinase) mediates the effects of these hormones; however, the signaling pathways employed by sgkl and the molecular mechanisms that modify ENaC activity have not been elucidated. Our working hypothesis stems from a model in which sgkl localizes predominantly at the basolateral membrane of epithelial cells in close proximity with incoming agonists such as insulin or AVP. Phosphorylation of sgkl by these and/or other stimuli to date unknown triggers a signal cascade that reaches the apical membrane to increase the abundance and/or activity of ENaC. The specific aims of this grant proposal are to: 1) Identify the mechanism underlying the effects of sgkl on ENaC in particular, to investigate changes in the traffic of channels, insertion and/or retrieval from the apical membrane, and changes in channel kinetics. 2) Determine the contribution of sgkl to aldosterone, vasopressin and insulin responses, and 3) To identify molecules that by associating with sgkl tether it to membranes, modulate its activity or constitute substrates of the sgkl-signaling cascade. We have developed tools and designed biochemical (turnover of ENaC subunits, phosphorylation assays, modified Ras recruitment system) and electrophysiological (patch-clamp studies and blocker-induced noise analysis) experiments to be conducted in vitro (A6 cell lines modified to express various forms of sgkl and siRNAs under the control of tetracycline) and in rat tissues that together will elucidate the proposed aims. The central role of sgkl in the integration of many signaling pathways makes it a candidate to mediate sodium retention associated with hypertensive disorders. Therefore, understanding the molecular mechanisms of this signaling pathway has potential implications for the diagnosis and development of new treatment strategies for the correction of high blood pressure.