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.
The kidneys play an essential role in adjusting fluids and electrolytes and controlling blood pressure (BP). Hormonal systems are essential for this process, and when functioning normally permit adaptation to enormous variations in environmental water and salt, however, defects can result in severe abnormalities in BP, and cause edema. Our work will shed new light on how these systems are controlled, and suggest avenues toward intervention to correct abnormalities.
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