Key Points

Rapamycin inhibited Kir4.1/Kir5.1, decreased Na-Cl-cotransporter (NCC), and increased renal potassium (K+) excretion.Inhibition of mechanistic target of rapamycin complex 1 in the distal convoluted tubule suppressed Kir4.1/Kir5.1, inhibited NCC, and increased renal K+ excretion.The mechanistic target of rapamycin complex 1 in the distal convoluted tubule plays a role in maintaining K+ homeostasis by controlling baseline activity of Kir4.1/Kir5.1 and NCC.

Background

Mechanistic target of rapamycin complex 1 (mTORc1) plays a role in maintaining potassium (K+) homeostasis. We now examine whether mTORc1 of distal convoluted tubule (DCT) regulates Kir4.1/Kir5.1 channels and thiazide-sensitive Na-Cl cotransporter (NCC), which plays a role in regulating renal K+ excretion.

Methods

We used patch clamp technique to examine basolateral Kir4.1/Kir5.1 in early DCT, immunoblotting to examine NCC expression, and in vivo measurement of urinary K+ excretion to determine baseline renal K+ excretion (EK) in the mice treated with rapamycin and in DCT-specific regulatory-associated protein of mechanistic target of rapamycin knockout mice (DCT-RAPTOR-KO).

Results

Application of rapamycin decreased Kir4.1/Kir5.1-mediated K+ currents and depolarized DCT membrane potential in Fkbp1aflox/flox mice. However, the effect of rapamycin on Kir4.1/Kir5.1 was absent in kidney-specific 12kDa FK506-binding protein knockout mice. Rapamycin decreased basolateral 40-pS K+ channel activity (Kir4.1/Kir5.1 heterotetramer) of the DCT. This effect was absent in the DCT treated with hydrogen peroxide, which stimulated the 40-pS K+ channel activity, suggesting the role of reactive oxygen species in mediating the effect of mTORc1 on Kir4.1/Kir5.1. Rapamycin treatment significantly decreased the abundance of both phosphorylated NCC and total NCC in Fkbp1aflox/flox mice but not in kidney-specific 12kDa FK506-binding protein knockout mice. Moreover, in vivo measurement of urinary Na+ excretion and urinary K+ excretion demonstrated that rapamycin treatment decreased hydrochlorothiazide-induced natriuresis but increased renal K+ excretion in Fkbp1aflox/flox mice. Moreover, Kir4.1/Kir5.1-mediated K+ currents of the DCT were lower, and DCT membrane potential was less negative in DCT-RAPTOR-KO than those of Ncc-Cre-Raptorflox/flox mice. In addition, the abundance of phosphorylated NCC was lower in DCT-RAPTOR-KO mice than Ncc-Cre-Raptorflox/flox mice. By contrast, the abundance of phosphorylated type II Na-Cl-K cotransporter was the same between two genotypes, while cleaved alpha subunit of epithelial sodium channel abundance was higher in DCT-RAPTOR-KO mice than Ncc-Cre-Raptorflox/flox mice. Consequently, DCT-RAPTOR-KO mice had a higher urinary K+ excretion and lower plasma K+ concentrations than Ncc-Cre-Raptorflox/flox.

Conclusions

mTORc1 in the DCT plays a significant role in maintaining K+ homeostasis by controlling the basolateral Kir4.1/Kir5.1 of the DCT and NCC.