Renal Function and Transporters in the Na/K-ATPase α1S/S Mouse Model of High Affnity for Cardiotonic Steroids

Abstract

Genetic ablation of ATP1A1 in the mouse renal proximal tubule (RPT) revealed that Na/K-ATPase (NKA) α1 tonically inhibits sodium and water reabsorption. Mechanistically, findings in KO mouse RPT and renal epithelial cells were consistent with a prevalent role of a NKA/Src-dependent downregulation of Na+/H+ exchanger 3 (NHE3). Pharmacologically, functional activation of this pathway has been obtained in vitro and ex vivo using low concentrations of NKA archetypal ligands cardiotonic steroids (CTS). However, the physiological implication of an activation of the endogenous CTS (eCTS)/NKA/Src on renal transporters including NHE3 has not been explored. We assessed the renal impact of CTS/NKA α1 receptor activation by comparing mice expressing the naturally resistant NKA α1 and age-matched mice genetically engineered to express a mutated NKA α1 with high affnity for CTS (R111Q/D122N double substitution). Mice obtained from established colonies at the University of Cincinnati were a gift from Dr. Jerry Lingrel. CTS-sensitive (α1S/S) male mice aged 3-6 months on a C57Bl6 background and age-matched controls (α1R/R) were used. Mice were born with a normal Mendelian ratio, with no observable difference between α1S/S and α1R/R. Renal Na/K-ATPase inhibitory dose-response curve to the CTS ouabain was shifted to the left, as expected, and occurred without detectable change of total renal NKA activity, NKA α1 or β1 protein contents. Metabolic cage studies revealed that increased α1 sensitivity to CTS resulted in a significant increase in urine output in α1S/S mice compared to age-matched α1R/R controls (3.9±0.3 vs. 2.4±0.2 ml/24h; n=6, p<0.01). Consistent with an activation of the NKA/Src receptor by eCTS, western blot analyses of renal cortical preparation revealed an increased phosphorylation of NKA α1 at Y260 (Src target residue) and decreased NHE3 content (43.6 %; n=5, p<0.01). RNA-seq analysis of whole kidneys from α1S/S and α1R/R littermate mice using the Illumina NovaSeq 6000 system (Novogene) and Volcano Plot (DEG data using Python) further showed differential expression of 296 genes (147 upregulated and 149 downregulated). Gene Set Enrichment Analysis (GSEA) using the WebGestalt toolkit showed that the altered pathways were mostly involved in solute transport. Taken together, these results provide genetic evidence of the physiological role of eCTS/NKAα1 on renal function in the intact mouse. Increased affnity to eCTS is suffcient to lead to an activation of NKAα1/Src, a downregulation of NHE3, and transcriptional regulation of renal transporters associated with increased urine output. This work was supported by AHA, Predoctoral fellowship #19PRE34450095, NIH DK129937, and NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence. 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.