Mechanism of sgk action in the collecting duct

SGK在集合管中的作用机制

• 批准号: 6475280
• 负责人: GEZA FEJES-TOTH
• 金额: $ 30.65万
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6475280
• 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NA转运并从而调节血压的分子机制。拟议研究的基础是最近鉴定出血清和糖皮质激素诱导的激酶（SGK）是醛固酮诱导的皮质收集导管（CCD）中醛固酮诱导的早期反应基因。 我们还证明，在表达上皮Na通道（ENAC）SGK的异武卵母细胞中，SGK刺激了对艾米洛里德敏感的Na电流。 这些观察结果使SGK成为介导醛固酮在NA运输方面的早期作用的有力候选者。 我们确定，在卵母细胞中，SGK增加了细胞膜中的ENAC亚基数，并且对打开ENAC的概率或其内吞作用率没有影响，这表明在该系统中，SGK会增加eNAC外胞胞菌病或再生。 我们提出以下特定目标。 AIM 1将检验以下假设：SGK通过增加胞吐作用或回收ENAC的哺乳动物收集导管细胞的Na吸收，就像在Xenopus卵母细胞中一样。 CCD细胞中向上或下调的SGK对ENAC的影响将通过测量Amiloride敏感的短路电流来确定，并通过使用生物化学方法确定顶膜中的ENAC数量，并通过确定来自Apical eNAC的ENAC的速率来确定噪声分析的波动。 目的2是阐明SGK增加ENAC表面表达的机制。 我们将通过在注射SGK蛋白和/或RNA合成抑制剂后监测卵母细胞中的ENAC电流来确定SGK的作用是否是通过翻译后或转录事件介导的。 SGK的亚细胞定位将使用针对SGK的GFP-SGK嵌合体或抗体确定。最后，在此目标中，我们将通过测试候选蛋白和磷酸肽映射的影响来确定SGK作用的下游靶标。 AIM 3是确定SGK在CCD细胞中AVP刺激的Na转运中的作用。 将确定AVP和PI3K抑制剂对SGK在哺乳动物CCD细胞中SGK活性和磷酸化的影响，并且这些作用将与正常CCD细胞和内源性SGK水平的正常CCD细胞和细胞中的amiloride敏感电流变化相关。由于SGK行动的最终目标似乎是ENAC贩运方面的一步，因此对SGK的下游效应子的识别可能会导致对ENAC贩运机制的见解，而ENAC贩运的机制知之甚少。 阐明导致CCD中激素刺激的Na转运的分子步骤也可能导致鉴定可能在某些形式的人类高血压中突变的基因，并最终可能导致中断这一途径的治疗措施。