Mechanism of sgk action in the collecting duct

SGK在集合管中的作用机制

• 批准号: 6624476
• 负责人: GEZA FEJES-TOTH
• 金额: $ 29.23万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6624476
• 关键词: Xenopus oocyte; aldosterone; amiloride; apical membrane; arginine vasopressin; cell line; immunologic assay /test; immunoprecipitation; laboratory rabbit; mass spectrometry; membrane activity; phosphatidylinositol 3 kinase; phosphopeptides; phosphorylation; protein kinase; protein localization; protein purification; protein transport; regulatory gene; renal tubular transport; renal tubule; saluresis; sodium channel; transcription factor

The long-term objective of these studies is to understand the molecular mechanisms through which aldosterone stimulates Na transport and thereby regulates blood pressure. The foundation for the proposed studies is the recent identification of serum- and glucocorticoid-induced kinase (sgk) as an aldosterone-induced early response gene in the cortical collecting duct (CCD). We also demonstrated that in Xenopus oocytes expressing the epithelial Na channel (ENaC) sgk stimulates amiloride-sensitive Na current. These observations make sgk a strong candidate for mediating the early actions of aldosterone on Na transport. We established that in oocytes sgk increases the number of ENaC subunits in the cell membrane and has no effect on open the probability of ENaCs or their rate of their endocytosis, suggesting that in this system sgk increases ENaC exocytosis or recycling. We propose the following specific aims. Aim 1 will test the hypothesis that sgk increases Na absorption in mammalian collecting duct cells by increasing exocytosis or recycling of ENaC, similarly as it does in Xenopus oocytes. The effects of up- or down-regulating sgk in CCD cells on ENaC will be determined by measuring amiloride-sensitive short-circuit current, and analyzing its fluctuations by noise analysis, by determining the amount of ENaC in the apical membrane using biochemical approaches, and by determining the rate of removal of ENaC from the apical membrane. Aim 2 is to elucidate the mechanism by which sgk increases surface expression of ENaC. We will determine if the effect of sgk is mediated by post-translational or transcriptional events by monitoring ENaC current in oocytes following injection of sgk protein and/or RNA synthesis inhibitors. The subcellular localization of sgk will be determined using GFP-sgk chimeras or antibodies against sgk. Finally, in this aim we will identify the downstream targets of sgk action by testing the effects of candidate proteins and by phosphopeptide mapping. Aim 3 is to determine the role of sgk in AVP-stimulated Na transport in CCD cells. The effects of AVP and PI3K inhibitors on the activity and phosphorylation of sgk in mammalian CCD cells will be determined, and these effects will be correlated with changes in amiloride-sensitive current, in normal CCD cells and cells in which endogenous sgk levels are down- regulated. Since the ultimate target of sgk action seems to be a step in ENaC trafficking, identification of the downstream effectors of sgk could lead to insights into the mechanism of ENaC trafficking, which is poorly understood. Unraveling the molecular steps leading to hormone-stimulated Na transport in the CCD could also lead to the identification of genes that might be mutated in some forms of human hypertension, and may eventually lead to therapeutic measures that interrupt this pathway.

这些研究的长期目标是了解醛固酮刺激钠转运从而调节血压的分子机制。这项研究的基础是最近发现的血清和糖皮质激素诱导的激酶(SGK)是一种醛固酮诱导的早期反应基因，位于皮质集合管(CCD)。我们还证明，在非洲爪哇卵母细胞中，表达上皮钠通道(ENaC)的SGK刺激阿米洛利敏感的钠电流。这些观察结果使SGK成为调节醛固酮对钠转运的早期作用的有力候选者。我们证实，在卵母细胞中，SGK增加了细胞膜上ENaC亚基的数量，而对ENaC的开放概率或其内吞速率没有影响，这表明在这个系统中，SGK增加了ENaC的胞吐或再循环。我们提出了以下具体目标。目的1验证SGK通过增加ENaC的胞吐或再循环来增加哺乳动物集合管细胞对Na的吸收的假说，类似于它在非洲爪哇卵母细胞中的作用。通过测量阿米洛利敏感的短路电流，通过噪声分析分析其波动，通过生化方法测定根尖膜中ENaC的量，以及通过测定ENaC从根尖膜上的去除率，来确定上调或下调CCD细胞中SGK对ENaC的影响。目的2阐明SGK促进ENaC表面表达的机制。我们将通过监测注射SGK蛋白和/或RNA合成抑制剂后卵母细胞的ENaC电流来确定SGK的作用是由翻译后事件还是转录后事件介导的。SGK的亚细胞定位将使用GFP-SGK嵌合体或抗SGK抗体来确定。最后，在这个目标中，我们将通过测试候选蛋白的作用和通过磷酸肽定位来确定SGK作用的下游靶点。目的3确定SGK在AVP刺激的细胞钠转运中的作用。AVP和PI3K抑制剂对哺乳动物CCD细胞SGK活性和磷酸化的影响将被确定，这些影响将与正常CCD细胞和内源性SGK水平下调的细胞中阿米洛利敏感电流的变化相关。由于SGK行动的最终目标似乎是ENaC贩运的一步，确定SGK的下游效应者可能有助于深入了解ENaC贩运的机制，这一点尚不清楚。解开导致激素刺激的钠在CCD中运输的分子步骤，也可能导致识别在某些形式的人类高血压中可能发生突变的基因，并最终可能导致中断这一途径的治疗措施。