MINERALOCORTICOID-REGULATED EPITHELIAL SODIUM TRANSPORT

脊椎动物的细胞外液量主要由盐皮质激素醛固酮调节，醛固酮刺激致密上皮细胞（特别是肾集合管）中的Na+吸收。 这一过程在血压控制中起着关键作用，其失调，无论是由于过量的激素还是靶细胞缺陷，都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，但其对Na+转运作用的机制基础仍然知之甚少。 醛固酮的最早作用是刺激顶膜ENaC插入和/或增加ENaC开放概率而不改变ENaC亚基基因转录。近年来，随着血清和糖皮质激素调节激酶（serum and glucocorticoid-regulated kinase，SGK）是醛固酮诱导的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。 肾集合管中的醛固酮迅速增加SGK mRNA和蛋白，野生型SGK刺激爪蟾卵母细胞中ENaC介导的Na+电流超过7倍。 有趣的是，不能结合ATP的SGK突变体（“激酶死亡突变体”）不仅不能增加Na+电流，而且实际上降低了它。野生型和突变体SGK的这些相反的功能效应与卵母细胞中ENaC质膜定位的显著相反效应相关。此外，磷脂酰肌醇-3-激酶（PI 3 K）信号通路的拮抗作用对SGK活性和ENaC介导的Na+转运具有与激酶死亡突变体相似的影响。 这些发现强烈暗示SGK作为醛固酮诱导的ENaC活性调节剂。 然而，SGK如何调节CD细胞中的ENaC活性以及P13 K在此过程中发挥什么作用仍然未知。 考虑到这些观察结果，本提案中描述的工作的主要目标是：（1）确定SGK是否是必需的和/或足以介导盐皮质激素对培养CD细胞中Na+转运的影响。 我们将表达野生型SGK和激酶死亡的突变体在培养的CD细胞的控制下的异源启动子，使其水平可以控制独立的盐皮质激素和他们的选择性影响Na+运输检查。 我们还将确定盐皮质激素刺激的Na+转运在集合管细胞被延迟或阻断SGK的表达或活性的破坏。(2)检查ENaC磷酸化和顶端膜再定位在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ENaC亚基。 我们将确定SGK是否刺激ENaC定位到培养细胞的顶膜。 我们将使用诱变来研究SGK在卵母细胞和培养细胞中的结构-功能关系。(3)检查PI 3 K在激活SGK和介导盐皮质激素对Na+转运的作用中的作用。 我们将研究阻断PI 3 K活性对ENaC介导的Na+转运的影响。 我们将确定SGK的组成型活性突变体或PI 3 K的下游产物是否克服了PI 3 K抑制对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，并可能为盐敏感性高血压的治疗开辟新的途径。

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.