MINERALOCORTICOID-REGULATED EPITHELIAL SODIUM TRANSPORT

脊椎动物的细胞外液容量主要由矿化皮质激素--醛固酮调节，它刺激紧密的上皮细胞，特别是肾脏集合管对Na+的吸收。这一过程在血压控制中起着举足轻重的作用，而由于激素过多或靶细胞缺陷导致的血压调节失调是高血压的重要原因。虽然已知醛固酮通过细胞内受体调节基因转录，但其影响Na+转运的机制仍不清楚。醛固酮最早的作用是在不改变ENaC亚单位基因转录的情况下刺激顶膜ENaC插入和/或增加ENaC开放概率。近年来，随着对血清和糖皮质激素调节的蛋白激酶(SGK)是一种醛固酮诱导的ENaC调节剂的发现，对醛固酮调节Na+转运的分子基础的研究取得了重大进展。肾脏集合管中的SGK mRNA和蛋白在醛固酮作用下迅速增加，野生型SGK可使非洲爪哇卵母细胞ENaC介导的Na+电流增加7倍以上。有趣的是，一个不能与ATP结合的SGK突变体(“激酶死亡突变体”)不仅不能增加Na+电流，而且实际上减少了它。野生型和突变型SGK的这些相反的功能作用与ENaC在卵母细胞中质膜定位的显著相反作用相关。此外，磷脂酰肌醇-3-激酶(PI3K)信号通路的拮抗对SGK活性和ENaC介导的Na+转运的影响类似于激酶死亡突变体。这些发现强烈暗示SGK是一种由醛固酮诱导的ENaC活性调节因子。然而，SGK如何调节CD细胞的ENaC活性以及P13K在这一过程中扮演什么角色仍不清楚。考虑到这些观察结果，这项工作的主要目标是：(1)确定SGK是否必要和/或足以介导盐皮质激素对CD细胞Na+转运的影响。我们将在外源启动子的控制下，在培养的CD细胞中表达野生型SGK和激酶死亡突变体，以使它们的水平不受盐皮质激素的控制，并检测它们对Na+转运的选择性作用。我们还将确定糖皮质激素刺激的集合管细胞的Na+转运是否因SGK表达或活性的中断而延迟或受阻。(2)研究ENaC磷酸化和顶膜重定位在SGK刺激的Na+转运中的作用。我们将确定SGK是否直接与ENaC亚基相互作用和/或磷酸化。我们将确定SGK是否刺激ENaC定位到培养细胞的顶膜。我们将使用突变技术来检测卵母细胞和培养细胞中SGK结构与功能的关系。(3)研究PI3K在激活SGK中的作用以及在盐皮质激素对Na+转运的调节中的作用。我们将研究阻断PI3K活性对ENaC介导的Na+转运的影响。我们将确定SGK的结构性活性突变体或PI3K的下游产物是否克服了PI3K抑制Na+转运的影响。总之，这些研究有望使人们更好地理解血压调节的机制基础，并有可能找到治疗盐敏感型高血压的新途径。

Extracellular fluid volume in vertebrates is regulated primarily by the mineralocorticoid hormone, aldosterone, which stimulates Na+ absorption in tight epithelia, particularly the kidney collecting duct. This process plays a pivotal role in the control of blood pressure, and its dysregulation, either due to excess hormone or to target cell defects, is an important cause of hypertension. Although aldosterone is known to act through intracellular receptors to regulate gene transcription, the mechanistic basis of its effects on Na+ transport remains poorly understood. The earliest effect of aldosterone is to stimulate apical membrane ENaC insertion and/or increase ENaC open probability without altering ENaC subunit gene transcription. Recently, significant progress was made in understanding the molecular basis of aldosterone-regulated Na+ transport with the discovery that serum and glucocorticoid-regulated kinase (SGK) is an aldosterone-induced ENaC modulator. SGK mRNA and protein are rapidly increased by aldosterone in kidney collecting duct and wild type SGK stimulates ENaC-mediated Na+ current more than seven-fold in Xenopus oocytes. Interestingly, an SGK mutant that cannot bind ATP ("kinase-dead mutant") not only fails to increase Na+ current but actually decreases it. These opposite functional effects of wild type and mutant SGK are correlated with striking opposite effects on ENaC plasma membrane localization in oocytes. Moreover, antagonism of the phosphatidylinositol-3-kinase (PI3K) signaling pathway has an effect on SGK activity and ENaC-mediated Na+ transport similar to the kinase-dead mutant. These findings strongly implicate SGK as an aldosterone-induced regulator of ENaC activity. However, it remains unknown how SGK modulates ENaC activity in CD cells and what role P13K plays in this process. With these observations in mind, the major goals of the work described in this proposal are to: (1) Determine if SGK is necessary and/or sufficient to mediate the effects of mineralocorticoids on Na+ transport in cultured CD cells. We will express wild type SGK and the kinase-dead mutant in cultured CD cells under the control of a heterologous promoter so that their levels can be controlled independently of mineralocorticoids and their selective effects on Na+ transport examined. We will also determine if mineralocorticoid-stimulated Na+ transport in collecting duct cells is delayed or blocked by disruption of SGK expression or activity. (2) Examine the role of ENaC phosphorylation and apical membrane relocalization in SGK- stimulated Na+ transport. We will determine if SGK directly interacts with and/or phosphorylates ENaC subunits. We will determine if SGK stimulates ENaC localization to the apical membrane of cultured cells. We will use mutagenesis to examine SGK structure-function relationships in oocytes and cultured cells. (3) Examine the role of PI3K in activating SGK and in mediating the effects of mineralocorticoids on Na+ transport. We will examine the effect on ENaC-mediated Na+ transport of blocking PI3K activity. We will determine if constitutively active mutants of SGK, or downstream products of PI3K, overcome the effect of PI3K inhibition on Na+ transport. Together, these studies promise to lead to a greater understanding of the mechanistic basis of blood pressure regulation and potentially to new avenues of treatment for salt sensitive hypertension.