Mechanistic Target of Rapamycin Complex 1 in Distal Convoluted Tubule and Renal Potassium Handling

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 (E K ) 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 Fkbp1a flox/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 Fkbp1a flox/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 Fkbp1a flox/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-Raptor flox/flox mice. In addition, the abundance of phosphorylated NCC was lower in DCT-RAPTOR-KO mice than Ncc-Cre-Raptor flox/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-Raptor flox/flox mice. Consequently, DCT-RAPTOR-KO mice had a higher urinary K + excretion and lower plasma K + concentrations than Ncc-Cre-Raptor flox/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.