Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. CTS are classically known as specific inhibitors of Na/K‐ATPase (NKA) α1 ion‐pumping function, which is critical for Na+ and water reabsorption in the renal proximal tubule (PT). At much lower concentrations, closer to the range of those reported for endogenous CTS in the blood, they also initiate NKA/Src‐mediated signaling in renal cell cultures. To investigate the respective roles of PT NKA α1/Src signaling and ion‐pumping function in physiological salt handling, we developed a PT‐specific NKA α1 knockout (RPTα1−/−) mouse with PT‐specific rescue by wild‐type NKA α1 (RPT α1WT) or a mutant NKA α1 with intact ion‐pumping function and blunted NKA/Src signaling (RPTα1Y260A).The PT‐specific NKA α1 knockout mouse was generated by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed ATP1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce α1WT or α1Y260A expression at the Rosa26 locus. Mice were born with the expected Mendelian ratio and appeared phenotypically normal, with no detectable change in water and food intake, or body weight. Renal phenotyping was further assessed using metabolic cages in 2–4 month‐old male and female mice.PT‐specific KO and rescue was confirmed by immunohistochemistry in PT cells isolated from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice. Kidney size, morphology and structure appeared overall normal following periodic acid shift (PAS) and trichrome staining. Functionally, in contrast with the predicted outcome based solely on the classical enzymatic function of NKA, a significant decrease in daily urine output (0.55±0.2 vs 1.76±0.3 mL/24h in RPTα1+/+, n=8–10, p<0.01) and absolute Na+ excretion (0.13±0.07 vs 0.35±0.0.5 mmol/24h in RPTα1+/+, n=6, p<0.01) were observed in the absence of PT NKA. The urinary clearance of lithium was significantly decreased in RPTα1−/− (1.42±0.3 vs 4.13±0.5 mL/min in RPTα1+/+, n=3, p<0.05), suggesting that the observed increase in Na+ reabsorption originated in the PT. The urinary output was rescued in the RPTα1WT (2.12±0.3 vs 2.16±0.4 mL/24h in RPTα1WTcontrol, n=7–10, p>0.05) but not in the RPTα1Y260A (0.42±0.1 vs 1.56±0.2 mL/24h in RPTα1Y260Acontrol, n=6–8, p<0.01), which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption.These results support a previously unascertained physiological role of NKA/Src signaling in PT Na+ and water reabsorption, and warrant further investigation to assess the mechanism and possible impact on blood pressure regulation.Support or Funding InformationMarshall Institute of Interdisciplinary Research FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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+/HCO3− 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 22Na flux by 2‐fold, activated NHE3 (50% decrease in inhibitory phosphorylation), and increased basolateral Na+/HCO3‐ 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.

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion, and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. Rather than the classic CTS‐mediated inhibition of Na+/K+‐ATPase (NKA)‐mediated ion transport in the renal proximal tubule (RPT), in vitro pharmacological approaches have suggested that low concentrations of CTS (in the range of those reported in the blood) may initiate NKA/Src‐mediated signaling to reduce apical Na+/H+ Exchanger‐3 (NHE3) and transepithelial Na+ flux in the RPT. To obtain genetic evidence of this putative NKA/Src mechanism in the RPT and asses its physiological impact, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1) and generated a PT‐specific NKA α1 knockout mouse (RPTα1−/−) by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed Atp1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce expression of wild‐type NKA α1 (RPT α1WT) or a Src‐null mutant NKA α1Y260A (RPTα1Y260A) with intact ion‐pumping. In cells with 90% NKA α1 knockdown compared to the parent LLC‐PK1 cell line, we observed a 50% decrease in phosphorylated NHE3 (inactive form) without change in total NHE3, and a 50% increase in total Sodium‐Bicarbonate cotransporter‐1A (NBCe1A) expression. Comparable NHE3 activation with NBCe1A increase was observed when NKA α1 knockdown cells were rescued with a Src‐binding NKA α1 null‐mutant or non‐src binding NKA α2, but not with Src‐binding gain‐of‐function α2 mutant or the WT NKA α1, suggesting a role for NKA/Src receptor function in the tonic inhibition of NHE3. RPT‐specific KO and rescue confirmed by immunohistochemistry in kidney cross‐section from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice did not alter kidney size, morphology or overall structure as assessed by periodic acid shift (PAS) and Masson’s trichrome staining. Western blot analyses of RPTα1−/− cortex indicated a decrease in phosphorylated NHE3 with no change in total NHE3 and an increase in NBCe1A expression comparable to those observed in vitro. Functionally, a 65% decrease in daily urine output and absolute Na+ excretion was observed in RPTα1−/− mice (n=6–10). Consistent with a NKA‐dependent tonic inhibition of NHE3, increased PT Na+ reabsorption was indicated by a 65% decrease in urinary lithium clearance in RPTα1−/−, with no change in glomerular filtration rate measured by FITC‐sinistrin clearance (n=10–12). The absolute Na+ excretion was rescued in the RPTα1WT but not in the RPTα1Y260A mouse, which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption (n=5). These studies reveal a novel mechanism of tonic inhibition of NHE3 and NBCe1A by Atp1a1. In vitro results provide genetic evidence that NKA/Src receptor function is critical to this mechanism, which was corroborated in vivo. Animal studies further indicate a significant physiologically impact of this hitherto unrecognized regulation of Na+ reabsorption in the PT, which may be regulated by endog...