Role of mTORC2/SGK1 Signaling in Rapid Response to Acute K Load to Maintain K+ Homeostasis

Background: The kinase mTORC2 phosphorylates SGK1 and is required for normal K+ secretion in the aldosterone-sensitive distal nephron. Aldosterone is known to play a role in mediating a sustained response through effects on SGK1 gene transcription, however, it is unknown how rapid responses are mediated. Here we have explored the role of mTORC2 and ENaC activity in the early response to an acute K+ load to regulate K secretion. Methods: Inducible tubule-specific Rictor (a core component of mTORC2) knockout mice (TRKO) were generated (Pax8-rtTA/LC-1/Rictorflox/flox). Both WT and TRKO mice received control or 2% KCl via gavage following intraperitoneal vehicle or Benzamil (ENaC inhibitor) injection. Spot urine was collected. ENaC and ROMK activity were measured in split open tubules by apical membrane patch clamp 3h post gavage. Membrane and cytoplasmic proteins were extracted from kidneys for immunoblot analysis. Results: Adult TRKO mice on normal diet displayed no abnormality except significantly elevated aldosterone. K+ administration by gavage triggered markedly greater Na+ excretion and lower K+ excretion in TRKO than WT mice, with differences detectable within 1 h of gavage. Benzamil induced a greater natriuresis in WT than in TRKO mice, and more strongly suppressed kaliuresis, consistent with greater ENaC activation in WT than in TRKO. The response of WT occurred rapidly, before significant change in aldosterone. In benzamil-treated mice, the natriuresis and kaliuresis of WT and TRKO mice were comparable, strongly supporting the idea that KCl induced ENaC-dependent K+ secretion in WT, and that this response is defective in TRKO. Patch clamp measurement demonstrated increased ENaC activity in WT but not in TRKO mice and no change in ROMK activity in WT or TRKO by KCl gavage. Membrane expression of cleaved α- and gENaC were significantly increased in WT but not in TRKO mice receiving KCl gavage. No significant increase in membrane expression of ROMK was observed in WT or TRKO post gavage. Finally, both SGK1 and Nedd4-2 phosphorylation were increased in WT but not TRKO mice receiving KCl gavage. Conclusions: Overall, the data strongly suggest that an acute K+ load acts through mTORC2/SGK1 to rapidly stimulate ENaC but not ROMK to promote K+ secretion. These effects are primarily due to local renal tubular K+ sensing. Funding: NIDDK Support, Private Foundation Support