Renal Proximal Tubule‐Specific Ablation of Atp1a1Reveals a Novel Tonic Inhibitory Mechanism of Sodium Reabsorption

Abstract

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...