Brambilla, Luca

Abstract Through its classic ATP‐dependent ion‐pumping function, basolateral Na/K‐ATPase (NKA) generates the Na + gradient that drives apical Na + reabsorption in the renal proximal tubule (RPT), primarily through the Na + /H + exchanger (NHE3). Accordingly, activation of NKA‐mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na + reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na + reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na + reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na + /HCO 3 − cotransporter (NBCe1A). Urine output and absolute Na + excretion decreased by 65%, driven by increased RPT Na + reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3 −/− mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na + reabsorption by regulating key apical and basolateral Na + transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP‐driven function of basolateral Na + reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

In the renal proximal tubule (PT), Na + /K + ‐ATPase (NKA) is exclusively located in the basolateral domain. Through its classic ATP‐dependent ion‐pumping function, NKA generates the Na + gradient that drives apical Na + reabsorption, mostly through Na + /H + exchanger (NHE3). Accordingly, activation of NKA‐mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical Na + reabsorption (NHE3). In contrast, pharmacological evidence suggests that activation of the more recently discovered NKA signaling function triggers a cellular redistribution of NKA and NHE3 that decreases transcellular Na + flux in cultured PT cells. To obtain genetic evidence of this NKA/Src mechanism in the PT and asses its physiological importance, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1). Additionally, we genetically targeted NKA α1 in the mouse PT by crossing mice expressing a sodium glucose co‐transporter 2 promoter driven Cre transgene with Floxed NKA α1 mice (PTα1 ‐/‐ ). Knockdown of 90% of NKA α1 in PT LLC‐PK1 cells increased transepithelial 22 Na flux by 2‐fold, activated NHE3 (50% decrease in inhibitory phosphorylation), and increased basolateral Na + /HCO 3 ‐ cotransporter (NBCe1A) protein content. In the PTα1 ‐/‐ mouse (4‐month males and females in a 1:1 ratio), 70% decrease in PT NKA α1 expression decreased urine output (0.51±0.14 vs 1.57±0.21 mL/24h in PTα1 +/+ , p<0.001, n=16) and absolute Na + excretion (0.14±0.05 vs 0.36±0.05 mmol/24h in PTα1 +/+ , p<0.05, n=8) by 65%, without histological or functional evidence of renal injury. Those changes were driven by increased PT Na + reabsorption, as indicated by a 65% decrease in lithium clearance (4‐month males, 1344±220 vs 3932±697 mL/24h in PTα1 +/+ , p<0.001, n=12) with unchanged GFR. This hyper‐reabsorptive phenotype of PTα1 ‐/‐ mice was coupled to increased membrane abundance of NHE3 and NBCe1A, and rescued upon crossing with floxed NHE3 mice, consistent a NKA/NHE3‐dependent mechanism. A dismantlement of caveolar NKA/Src receptor complex and intracellular redistribution of pY418Src occurred in knockdown NKA α1 PT cells, and was also observed in the PTα1 ‐/‐ hypomorphic mouse. Rescue of PT cells with wild‐type but not Src signaling‐null NKA α1 restored NHE3 and NBCe1A to basal levels, indicative of a role for NKA/Src receptor function in the tonic inhibition of Na + transporters in the PT. Hence, NKA signaling exerts a tonic inhibition on Na + reabsorption by regulating key apical and basolateral Na + transporters. This action, which is lifted upon NKA genetic suppression in cells and in vivo , tonically counteracts NKA's ATP‐driven function of basolateral Na + reabsorption. Strikingly, NKA/Src signaling is not only physiologically relevant, it is functionally dominant over NKA ion‐pumping in the control of PT reabsorption. NKA signaling therefore provides a long sought‐after mechanism for the natriuretic action of endogenous NKA ligands such as cardiotonic steroids.