Mechanistic target of rapamycin complex-2 mediates the effect of overnight high K ⁺ on Kir4.1/Kir5.1 in the DCT and ENaC/ROMK in ASDN

We examine the role of mTORc2 in mediating overnight-high-K⁺-(HK)-intake-induced stimulation of renal outer medullary K⁺ channels (ROMK) in late distal-convoluted-tubule (DCT2)/early connecting-tubule (early-CNT). Also, we explore whether mTORc2 simultaneously inhibits Kir4.1/Kir5.1 and stimulates ROMK/epithelial Na⁺ channel (ENaC) during overnight-HK. We performed patch-clamp experiments, immunoblotting, metabolic cage, and in vivo measurement of urinary K⁺ excretion in kidney tubule-specific RICTOR (rapamycin insensitive companion of mTOR) knockout mice (Ks-RICTOR-KO) and <i>Rictor</i>^flox/flox mice (wild type). Ks-RICTOR-KO mice had a lower urinary K⁺-excretion and higher plasma K⁺ concentration than <i>Rictor</i>^flox/flox mice. Moreover, overnight HK intake further increased plasma K⁺ level in Ks-RICTOR-KO mice but not in <i>Rictor</i>^flox/flox mice. Ks-RICTOR-KO mice had higher basolateral Kir4.1/Kir5.1 activity in the DCT than in <i>Rictor</i>^flox/flox mice. In contrast, tertiapin-Q (TPNQ)-sensitive K⁺ currents (ROMK) were lower in both DCT2/early-CNT and cortical-collecting-duct (CCD) of Ks-RICTOR-KO mice than in <i>Rictor</i>^flox/flox mice. Amiloride-sensitive Na⁺ currents (ENaC) were significantly lower in DCT2/CNT in Ks-RICTOR-KO mice than in wild type. Overnight HK intake decreased Kir4.1/Kir5.1 activity of DCT and increased amiloride-sensitive Na⁺ currents and TPNQ-sensitive K⁺ currents in DCT2/early-CNT and in the CCD in <i>Rictor</i>^flox/flox mice. However, these effects of overnight-HK were absent in Ks-RICTOR-KO mice. Finally, in vivo measurement of urinary K⁺ excretion showed that urinary K⁺ excretion in Ks-RICTOR-KO mice on overnight-HK was lower than in <i>Rictor</i>^flox/flox mice. In summary, mTORc2 plays a role in maintaining baseline activity of Kir4.1/Kir5.1, ROMK, and ENaC and it mediates the effect of overnight-HK on ENaC and ROMK in aldosterone-sensitive distal nephron (ASDN). We conclude that mTORc2 plays a key role in stimulating renal K⁺ excretion during high-K⁺-loading by inhibiting Kir4.1/Kir5.1 in the DCT and stimulating ENaC and ROMK in ASDN.<b>NEW & NOTEWORTHY</b> Our study has two novel findings. First, we demonstrate that mTORc2 plays a key role in mediating high-K⁺-intake-induced stimulation of ENaC/ROMK activity in late DCT. Second, we demonstrate that overnight-HK-induced stimulation of kidney K⁺ excretion is achieved by simultaneously stimulating ENaC and ROMK in the late DCT, CNT, and CCD, and inhibiting Kir4.1/Kir5.1 in the DCT. Thus, we have illustrated an integrated mechanism by which mTORc2 regulates kidney K⁺ excretion.