MTORc2 in the distal convoluted tubule (DCT) stimulates renal K+ excretion by inhibiting basolateral Kir4.1/Kir5.1 and Na-Cl−cotransporter (NCC) during increasing dietary K+ intake

Abstract

Background: The mTORc2 is a multiprotein complex and the core components of mTORc2 contain mTOR (a serine/threonine protein kinase) and RICTOR (rapamycin insensitive companion of mTOR). Previous studies have strongly suggested the role of mTORc2 in the regulation of renal K+ excretion by stimulating epithelial Na+ channels (ENaC) and ROMK (Kir1.1) in the aldosterone-sensitive distal nephron (ASDN). The mice with deletion of RICTOR in the distal nephron or in the renal tubules are hyperkalemic during increasing dietary K+ intake. A large body of evidence has demonstrated that HK-induced inhibition of NCC is essential for stimulating renal K+ excretion and this is achieved by inhibiting the basolateral Kir4.1/Kir5.1 heterotetramer in the DCT. However, the role of mTORc2 in the regulation of basolateral Kir4.1/Kir5.1 heterotetramer in the DCT and NCC is not explored. Thus, the aim of the present study is to explore whether mTORc2 in the DCT regulates the basolateral Kir4.1/Kir5.1 of the DCT, NCC activity and renal K+ excretion. Method: We have generated DCT-specific RICTOR KO mice (DCT-RICTOR-KO) with C57BL6/J background by crossing the mice expressing Slc12a3-Cre-ERT2 with Rictorflox/flox mice. We used patch-clamp-technique to examine basolateral Kir4.1/Kir5.1 in early-DCT, immunoblotting and immunofluorescence to examine NCC expression and in vivo measurement of urinary K+-excretion to determine baseline renal K+-excretion (EK). We also used metabolic cage to examine the body weight (BW), food/water intakes, urinary Na+ (UNa) /K+ (UK) excretion rate, and plasma Na+ and K+ concentrations in male(m)/female(f) DCT-RICTOR KO mice and Slc12a3-Cre-ERT2 -Rictorflox/flox mice (control). Results: Overnight-HK intake stimulates RICTOR expression in the kidney and increases Rictor mRNA in the DCT. The deletion of RICTOR in the DCT was confirmed by immunofluorescence staining showing that RICTOR is expressed in parvalbumin-positive tubule (DCT) of control mice but is almost absent in DCT-RICTOR-KO mice. Deletion of RICTOR in the DCT increased the Kir4.1/Kir5.1-mediated K+-currents, hyperpolarized DCT membrane and increased the expression of pWNK4 and pNCC. While the two genotypes did not have significant difference regarding BW, food/water intakes, UNa and plasma Na+ concentrations, m/f DCT-RICTOR KO mice had a significantly lower UK and higher plasma K+ concentrations than their corresponding control. In vivo measurement of baseline renal K+ excretion (EK) also shows that renal-EK was lower and plasma K+ was higher in DCT-RICTOR-KO mice than corresponding control mice. Also, overnight-HK did not inhibit Kir4.1/Kir5.1 activity in the DCT and failed to inhibit the expression of pNCC in DCT-RICTOR-KO mice. Overnight-HK stimulated renal-EK in control mice, but this effect was attenuated in DCT-RICTOR-KO mice. Thus, overnight-HK induced hyperkalemia in DCT-RICTOR-KO mice but not in control mice. Conclusion: The mTORc2 of the DCT plays a significant role in mediating the effect of HK intake on Kir4.1/Kir5.1 activity and NCC expression. Since mTORC2 also stimulates ENaC and ROMK in the ASDN, an increase in Na+-delivery is expected to stimulate ENaC-ROMK-dependent K+-excretion and flow-stimulated renal K+-excretion. The work is supported by NIH Grants DK136491 (LDH) and DK133220 (WHW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.