mTORc1 in Distal Convoluted Tubule (DCT) and Renal Potassium (K+) Handling

Background: Mechanistic-target-of-rapamycin-complex-1 (mTORc1) plays a role in maintaining K + homeostasis. We now examine whether mTORc1 of distal-convoluted-tubule (DCT) regulates Kir4.1/Kir5.1 channels and thiazide-sensitive-Na-Cl cotransporter, 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 r egulatory- a ssociated- p rotein-of-mechanisticc- t arget- o f- r apamycin 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-FKBP12-knockout mice (Ks-FKBP12-KO). 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 H 2 O 2 which stimulated the 40-pS K + -channel activity, suggesting the role of reactive-oxygen-species (ROS) 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 Ks-FKBP12-KO 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. Also, the abundance of phosphorylated-NCC was lower in DCT-RAPTOR-KO mice than Ncc-Cre-Raptor flox/flox mice. In contrast, the abundance of phosphorylated-NKCC2 was the same between two genotypes while cleaved αENaC 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.