Mechanism of sgk action in the collecting duct

SGK在集合管中的作用机制

基本信息

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
项目类别：
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

项目摘要

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。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）作为皮质集合管（CCD）中醛固酮诱导的早期反应基因。我们还证明，在非洲爪蟾卵母细胞中，表达上皮钠通道（ENaC）的 sgk 会刺激阿米洛利敏感的钠电流。这些观察结果使 sgk 成为介导醛固酮对 Na 转运的早期作用的有力候选者。我们确定，在卵母细胞中，sgk 增加细胞膜中 ENaC 亚基的数量，并且对 ENaC 的开放概率或其内吞作用速率没有影响，表明在该系统中 sgk 增加 ENaC 胞吐作用或再循环。我们提出以下具体目标。目标 1 将检验以下假设：sgk 通过增加 ENaC 的胞吐作用或再循环来增加哺乳动物集合管细胞中 Na 的吸收，类似于非洲爪蟾卵母细胞中的作用。上调或下调 CCD 细胞中 sgk 对 ENaC 的影响将通过测量阿米洛利敏感短路电流并通过噪声分析分析其波动、使用生化方法确定顶膜中 ENaC 的量来确定，并确定 ENaC 从顶膜去除的速率。目标 2 是阐明 sgk 增加 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中的 ENaC 电流来确定 sgk 的作用是否是由翻译后或转录事件介导的。 sgk 的亚细胞定位将使用 GFP-sgk 嵌合体或针对 sgk 的抗体来确定。最后，为了这个目标，我们将通过测试候选蛋白的作用和磷酸肽图谱来确定 sgk 作用的下游靶标。目标 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抑制剂对哺乳动物CCD细胞中sgk的活性和磷酸化的影响，并且这些影响将与正常CCD细胞和内源性sgk水平下降的细胞中阿米洛利敏感电流的变化相关。受监管。由于 sgk 作用的最终目标似乎是 ENaC 贩运的一个步骤，因此识别 sgk 的下游效应器可能有助于深入了解 ENaC 贩运的机制，而目前人们对此知之甚少。解开CCD中激素刺激的Na转运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，并可能最终导致中断该途径的治疗措施。