SGK Regulation of Epithelial Sodium Transport

摘要 SGK1是调节肾小管离子转运的信号机制的关键组成部分。它在下面 由醛固酮和使特定丝氨酸磷酸化的主激酶mTOR的双重调节(S422) 在SGK1内。控制mTOR依赖的SGK1磷酸化的重要生理信号是 不清楚，监管机制也不清楚。我们的初步数据支持这样的观点，即 血管紧张素II(Ang II)和局部胞外K+浓度([K+])是SGK1的重要激活剂。 通过多亚单位mTOR复合体之一，mTORC2，刺激SGK1 S422磷酸化 从而调节离子转运体，特别是ENaC。本项目的主要目标是阐明 这一调节背后的分子机制，并了解其生理意义。我们将：1： 我们将首先研究血管紧张素II刺激的mTORC2对SGK1的选择性调节。我们会确定残留物 在培养细胞中对Ang II有反应的mTORC2组分SIN1和Rictor被磷酸化 使用质谱学和免疫印迹方法。然后我们将在这些地点测试突变体的能力 支持依赖mTORC2的SGK1磷酸化。我们将研究Ang II对SGK1亚细胞的影响 本地化及其与mTORC2的相互作用。2：表征K+对依赖mTORC2的SGK1的影响 培养细胞中磷酸化及其在调节ENaC和ROMK中的作用。我们的初步数据在培养中 CCD细胞和完整的集合管支持K+调节SGK1的mTORC2磷酸化以 规范ENaC。我们将探索这些发现的机制和生理意义。 细胞，并延伸至ROMK。我们将研究在生理范围内改变[K+]对 MpkCD集合管细胞依赖mTORC2的SGK1磷酸化及ENaC和ROMK电流 生长在Transwell过滤器上。我们还将对这些细胞进行膜片钳，以直接观察通道功能 在根尖膜上。我们将对SGK1中K+调控的信号机制进行研究。 3.体内研究mTORC2在调节Na+、K+排泄中的作用。为了测试关键概念 根据我们的体外实验，并解决最近的出版物之间的差异，我们将执行一系列 在WT和Rictor KO小鼠中使用药物抑制剂的体内实验。我们会比较它们的效果 MTOR对WT与远端肾单位Rictor KO小鼠Na+和K+处理的药理抑制，以及 使用电解质平衡研究和膜片钳评估ENaC和ROMK来协调不同的结果 洋流。最后，我们将使用可诱导的KO模型来检验急性和慢性mTORC2丢失的影响。 比较急性和慢性Rictor缺失。这些研究将为荷尔蒙的调节提供新的线索 并阐明了一种新的K+通过直接作用控制自身排泄的机制。 对醛固酮敏感的远端肾单位/皮质集合管。

ABSTRACT SGK1 is a key component of the signaling machinery that regulates kidney tubule ion transport. It is under dual regulation by aldosterone, and the master kinase mTOR, which phosphorylates a specific serine (S422) within SGK1. The physiologically important signals that control mTOR-dependent SGK1 phosphorylation are not clear, and the regulatory mechanisms are unknown. Our preliminary data support the idea that both angiotensin II (Ang II) and local extracellular K+ concentration ([K+]) are important activators of SGK1, which act through one of the multi-subunit mTOR complexes, mTORC2, to stimulate SGK1 S422 phosphorylation and hence regulate ion transporters, particularly ENaC. The major goal of the present project is to elucidate the molecular mechanisms underlying this regulation, and to understand its physiological implications. We will: 1: We will first examine Ang II-stimulated selective regulation of SGK1 by mTORC2. We will identify residues within mTORC2 components SIN1 and Rictor that are phosphorylated in response to Ang II in cultured cells using mass spectrometry and immunoblot methods. We will then test mutants at these sites for their ability to support mTORC2-dependent SGK1 phosphorylation. We will examine the effect of Ang II on SGK1 subcellular localization and its interaction with mTORC2. 2: Characterize the effects of K+ on mTORC2-dependent SGK1 phosphorylation and its role in modulating ENaC and ROMK in cultured cells. Our preliminary data in cultured CCD cells and intact collecting duct support the idea that K+ modulates mTORC2 phosphorylation of SGK1 to regulate ENaC. We will explore both the mechanism and physiological implications of these findings in cultured cells, and extend to ROMK. We will examine the effect of altering [K+] within the physiologic range on mTORC2-dependent SGK1 phosphorylation and ENaC and ROMK currents in mpkCCD collecting duct cells grown on Transwell filters. We will also perform patch clamp on these cells to look directly at channel function in the apical membrane. We will characterize the signaling mechanisms implicated in K+ regulation of SGK1. 3: Characterize in vivo the role of mTORC2 in regulating Na+ and K+ excretion. In order to test key concepts from our in vitro experiments, and resolve discrepancies between recent publications, we will perform a series of in vivo experiments using pharmacologic inhibitors in WT and Rictor KO mice. We will compare the effects of pharmacologic inhibition of mTOR on Na+ and K+ handling in WT vs. distal nephron Rictor KO mice, and reconcile divergent results using electrolyte balance studies and patch clamp to assess ENaC and ROMK currents. Finally, we will examine the effects of acute vs. chronic loss of mTORC2 using an inducible KO model to compare acute and chronic Rictor deletion. These studies will shed new light on hormonal regulation of renal ion handling, and elucidate a novel mechanism for K+ to control its own excretion through direct effects in the aldosterone sensitive distal nephron/cortical collecting duct.