Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

Abstract

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

mTOR signaling in renal ion transport

“…In agreement with these studies, Chen et al showed that deletion of mTOR from the PCs in mTOR fl/fl *Aqp2Cre mice CD resulted in reduction of all three ENaC subunits at the protein level and attenuated the ability to maintain Na + homeostasis in response to low NaCl diet and benzamil injection. 94 Although deletion of mTOR would influence both mTORC1 and mTORC2 activity, and the current study did not investigate which protein complex mainly played a role in their mice model, SGK1 phosphorylation was decreased in the cortex of KO mice along with increased ubiquitination of α-ENaC, in line with the current mTORC2-SGK1 signaling pathway consensus. 94 Aside from SGK1, SGK3 also modulates Na + transport in the CD through ENaC activity indirectly in a similar manner as SGK1.…”

“…94 Although deletion of mTOR would influence both mTORC1 and mTORC2 activity, and the current study did not investigate which protein complex mainly played a role in their mice model, SGK1 phosphorylation was decreased in the cortex of KO mice along with increased ubiquitination of α-ENaC, in line with the current mTORC2-SGK1 signaling pathway consensus. 94 Aside from SGK1, SGK3 also modulates Na + transport in the CD through ENaC activity indirectly in a similar manner as SGK1. 95 It is currently not known if mTOR signaling is responsible for SGK3-mediated activation of these transporters.…”

Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate Epithelial Sodium Channel: Role in Rapid Renal Responses to Dietary Potassium

“…Another recent study in which AQP2-Cre was used to KO connecting tubule/collecting duct mTOR also showed defects in SGK1 and ENaC. 22 These reports support an important role of mTORC2 in regulating renal tubular K + secretion, however, key aspects remain unknown or controversial: (1) Is mTORC2 a permissive factor, or an acute mediator of aldosterone-independent regulation of K + secretion in vivo? (2) Are ENaC and ROMK both targets of acute and/or longer term effects of K + ?…”

Regulation of distal tubule sodium transport: mechanisms and roles in homeostasis and pathophysiology

“…Hyperkalemia became lethal when animals were treated with the ENaC inhibitor triamterene suggesting a more profound defect in ROMK than in ENaC [ 60 ]. It is notable that in contrast with Rictor KO mice [ 60 ], either acute treatment of mice with an mTOR inhibitor [ 56 ] or mTOR gene deletion [ 27 ] markedly reduces ENaC activity. Although these apparently conflicting findings require additional study to reconcile, they are consistent with the possibility that in vivo mTORC1 and mTORC2 are both able to stimulate ENaC (via SGK1), while only mTORC2 regulates ROMK.…”