The mTORC2‐SGK1 Signaling Module Mediates Local Sensing of Potassium in Kidney Tubule Principal Cells to Activate ENaC and Promote Potassium Secretion

Abstract

ObjectivePotassium (K+) homeostasis requires precise regulation of K+ secretion by the kidney tubules. The serine‐threonine kinase, SGK1, is essential for normal responses to changes in extracellular K+ across the physiological range. SGK1 stimulates K+ secretion predominantly by activating the epithelial sodium channel (ENaC), which enhances electrogenic Na+ transport into K+ secretory cells and increases the electrical driving force for K+ secretion. Although it is well established that SGK1 activity is regulated through phosphorylation by the kinase, mTOR complex 2 (mTORC2), the physiologically relevant feedback systems that control this phosphorylation remain poorly characterized. In this study, we identified a direct effect of K+ in cortical collecting duct principal cells to stimulate ENaC and enhance K+ secretion, and set out to characterize the underlying mechanism.MethodsmpkCCD cortical collecting duct (CCD) cells were grown on Transwell filters and electrical properties were measured by Evometer following changes in extracellular [K+]. Following electrophysiological measurements, cells were harvested and processed for immunoblot analysis. Additional experiments were performed using patch clamp to detect ENaC currents in intact CCD isolated from mice and subjected to acute changes in bath [K+]. SGK1 phosphorylation state in response to changes in [K+] were also assessed in wild type and WNK1 deficient HEK‐293 cells.ResultsUsing whole cell patch clamp ex vivo, we found that in intact, isolated mouse collecting duct, a change in extracellular [K+] rapidly alters ENaC activity. Further, in cultured collecting duct cells an increase in basolateral but not apical [K+] led to enhanced ENaC‐dependent apical Na+ transport which paralleled an increase in the activated form of SGK1 (phosphorylated at S422). Pharmacologic inhibition of SGK1 or mTOR blocked the K+‐dependent activation of ENaC. Interestingly, the K+‐induced increase in SGK1 phosphorylation was not accompanied by an increase in phosphorylation of Akt2, a close relative of SGK1, which is also phosphorylated by mTORC2. We further explored the underlying mechanism for specific SGK1 activation by testing SGK1 and Akt2 phosphorylation in HEK‐293 cells lacking WNK1 (through CRISPR‐mediated gene deletion). Loss of WNK1 markedly impaired K+‐stimulated phosphorylation of SGK1 but not of Akt2. Additionally, we found that mutation of the chloride binding site of WNK1 led to the loss of K regulation of SGK1 phosphorylation. Further, in cells incubated in low Cl‐medium, increased extracellular K+ failed to increase SGK1 phosphorylation.ConclusionThese data strongly suggest that extracellular K+ can modulate mTORC2‐dependent activation of SGK1 through effects on Cl‐ binding to WNK1, leading to activation of ENaC and increased K+ secretion. We propose a model in which this signaling system supports a cell‐autonomous feedback loop within principal cells, which plays a key role in K+ homeostasis.Support or Funding InformationR01‐DK56695 to DP, R01‐DK54983 to WHW, the James Hilton Manning and Emma Austin Manning Foundation to DP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.