Hormone-regulated Na+ transport in the kidney tubules is critical to the control of blood pressure and other vital processes in health and disease. The serine-threonine kinase SGK1 is one of the most intensively studied regulators of epithelial Na+ transport, and has been particularly recognized for its importance in mediating the effects of aldosterone on the epithelial sodium channel (ENaC). SGK1 is under dual control: its expression is controlled by aldosterone through effects on gene transcription, and its activity is controlled by phosphorylation involving a network of kinase cascades. Recent evidence has identified mammalian target of rapamycin (mTOR) as the kinase mediating a key "gateway" phosphorylation event required for SGK1 activation. We hypothesize that SGK1 physically interacts with specific component(s) of a multi-protein complex containing mTOR, which is essential for the gateway phosphorylation at serine 422 (S422). We further hypothesize that SGK1 together with this mTOR-containing complex is recruited to another protein complex, where SGK1 regulates ENaC by phosphorylating targets such as Nedd4-2. With these hypotheses in mind, our specific aims for this competing renewal are to: 1: Determine the mechanistic basis of mTOR physical association with, and phosphorylation of SGK1. There are two mTOR-containing multi-protein complexes, mTORC1 and mTORC2, which have distinct components and substrates, and regulate distinct cellular processes. We will: A) Examine the physical interactions of individual mTORC1 &2 components with SGK1 using the yeast two hybrid assay and in vitro interaction. B) Identify domains and specific amino acids within SGK1 and mSin1, which mediate their interaction. C) Determine if physical interaction between mSin1 and SGK1 is necessary for SGK1 to be phosphorylated by mTORC2. D) Examine mTORC2 regulation of SGK1 relatives, Akt, SGK2, and SGK3. 2: Characterize the functional effects of mTOR and SGK1 on ENaC in cultured cortical collecting duct (CCD) cells.
This aim will establish the functional role of the above characterized physical interactions and SGK1 modifications in controlling ENaC-mediated Na+ transport in cultured cells. We will: A) Assess the functional role of SGK1 as a mediator of mTOR-dependent activation of ENaC in a CCD cell line, using a combination of small molecule inhibitors of mTOR and SGK1, and genetic manipulation. B) Assess the functional implications of SGK1 interaction with mTORC2 by characterizing the effect of mutants on SGK1 phosphorylation and ENaC current. C) Characterize the effect of cellular conditions and hormonal milieu on mTOR-dependent activation of SGK1 and ENaC. 3: Characterize the role of mTOR and SGK1 in Na+ homeostasis in vivo. In order to advance these findings into native tissues, we will: A) Examine the effects of mTOR activators and inhibitors on Na+ balance, blood pressure, and ENaC subcellular localization in wild type and SGK1-null mice. B) Examine mTOR regulation of amiloride-inhibitable transepithelial Na+ current in isolated perfused CCD harvested from aldosterone-treated mice. C) Examine the role of mTOR in regulation of amiloride-inhibitable currents in isolated CCD using patch clamp.

Public Health Relevance

This project is focused on understanding the molecular mechanisms underlying the control of sodium excretion by the kidneys, which is critical to the regulation of blood pressure, body fluid volume (and hence edema formation), and the concentrations of ions in the blood. More than 50 million individuals in the US, and close to a billion world-wide, have high blood pressure, which markedly increases their risk for heart attack, stroke and kidney failure. Edema and ion concentration disorders are common in patients with heart failure and liver disease. Although there are drugs for the treatment of these disorders, in more than 25% of patients, blood pressure is poorly controlled and edema is not effectively treated. We have identified a key regulator of sodium excretion, called SGK1. SGK1 is contained mostly in the cells that control sodium excretion, where it exists in an inactive state, until hormones signal it to become active. Our research focuses on understanding how SGK1 gets activated, and once it is activated, how it regulates sodium excretion. We hypothesize that in response to the appropriate sodium-regulating hormones, SGK1 physically interacts with other cellular proteins, which activate it and then get it to the appropriate locations in kidney cells to alter sodium excretion. With this hypothesis in mind, our specific goals for this grant are to: (1) Identify the precise mechanism by which SGK1 gets activated, and (2) Determine the functional effects of activated SGK1 on sodium handling by the kidney. These studies will shed new light on the molecular mechanisms of sodium excretion, and provide key information for the design of new drugs for the treatment of hypertension, edematous disorders and defects in blood electrolyte concentrations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056695-12
Application #
8546327
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2011-09-20
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
12
Fiscal Year
2013
Total Cost
$324,277
Indirect Cost
$114,389
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Soundararajan, Rama; Pearce, David; Ziera, Tim (2012) The role of the ENaC-regulatory complex in aldosterone-mediated sodium transport. Mol Cell Endocrinol 350:242-7
Soundararajan, Rama; Lu, Ming; Pearce, David (2012) Organization of the ENaC-regulatory machinery. Crit Rev Biochem Mol Biol 47:349-59
Lu, Ming; Wang, Jian; Ives, Harlan E et al. (2011) mSIN1 protein mediates SGK1 protein interaction with mTORC2 protein complex and is required for selective activation of the epithelial sodium channel. J Biol Chem 286:30647-54
Yao, Li-Jun; McCormick, James A; Wang, Jian et al. (2011) Novel role for SGK3 in glucose homeostasis revealed in SGK3/Akt2 double-null mice. Mol Endocrinol 25:2106-18
Pao, Alan C; Bhargava, Aditi; Di Sole, Francesca et al. (2010) Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney. Am J Physiol Renal Physiol 299:F1496-506
Soundararajan, Rama; Pearce, David; Hughey, Rebecca P et al. (2010) Role of epithelial sodium channels and their regulators in hypertension. J Biol Chem 285:30363-9
Hallows, Kenneth R; Bhalla, Vivek; Oyster, Nicholas M et al. (2010) Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel. J Biol Chem 285:21671-8
Zemtsova, Irina M; Heise, Nicole; Frohlich, Henning et al. (2010) Blunted IgE-mediated activation of mast cells in mice lacking the serum- and glucocorticoid-inducible kinase SGK3. Am J Physiol Cell Physiol 299:C1007-14
Soundararajan, Rama; Wang, Jian; Melters, Daniel et al. (2010) Glucocorticoid-induced Leucine zipper 1 stimulates the epithelial sodium channel by regulating serum- and glucocorticoid-induced kinase 1 stability and subcellular localization. J Biol Chem 285:39905-13
Mauro, Theodora M; McCormick, James A; Wang, Jian et al. (2009) Akt2 and SGK3 are both determinants of postnatal hair follicle development. FASEB J 23:3193-202

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