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

Abstract

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. 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. We used patch clamp technique to examine basolateral Kir4.1/Kir5.1 in early DCT, immunoblotting to examine NCC expression, and <i>in vivo</i> 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). Application of rapamycin decreased Kir4.1/Kir5.1-mediated K⁺ currents and depolarized DCT membrane potential in <i>Fkbp1a</i>^{<i>flox/flox</i>} 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 <i>Fkbp1a</i>^{<i>flox/flox</i>} mice but not in kidney-specific 12kDa FK506-binding protein knockout mice. Moreover, <i>in vivo</i> measurement of urinary Na⁺ excretion and urinary K⁺ excretion demonstrated that rapamycin treatment decreased hydrochlorothiazide-induced natriuresis but increased renal K⁺ excretion in <i>Fkbp1a</i>^{<i>flox/flox</i>} 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 <i>Ncc-Cre-Raptor</i>^{<i>flox/flox</i>} mice. In addition, the abundance of phosphorylated NCC was lower in DCT-RAPTOR-KO mice than <i>Ncc-Cre-Raptor</i>^{<i>flox/flox</i>} 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 <i>Ncc-Cre-Raptor</i>^{<i>flox/flox</i>} mice. Consequently, DCT-RAPTOR-KO mice had a higher urinary K⁺ excretion and lower plasma K⁺ concentrations than <i>Ncc-Cre-Raptor</i>^{<i>flox/flox</i>}. 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.