Thick Ascending Limb-Specific Kir4.1 Deletion in Mice Increases Urine Output and Induces Metabolic Alkalosis Independent of Sodium-Chloride Cotransporter (NCC)

Background: Loss-of-function mutations in KCNJ10, encoding the inward rectifier K+ channel Kir4.1, cause EAST syndrome—characterized by salt wasting, hypokalemia, and metabolic alkalosis. While Kir4.1 is well studied in the distal convoluted tubule (DCT), its role in the thick ascending limb (TAL) remains less defined. We recently identified Kir4.1 expression in discrete TAL cell types conserved across species, suggesting a shared mechanism for cell-specific K+ transport (PMID: 39896580). We hypothesized that Kir4.1-expressing cells maintain K+ homeostasis by modulating TAL Na+ transport in response to circulating K+, analogous to its function in the DCT. Methods: Inducible TAL-specific Kir4.1 knockout mice (Slc12a1-CreERT2; Kir4.1^flx/flx) were treated with tamoxifen (1 mg/day × 5 days) or vehicle (5% ethanol in sunflower oil) IP. Control groups included vehicle-treated Cre+ and tamoxifen-treated Cre- littermates. Two weeks post-induction, male mice were placed in metabolic cages for 24-hour urine collection on standard Na+ (0.33%) and K+ (1.16%) gel diet (n=7-10). Urine electrolytes were measured via flame photometry (n=3-4); blood chemistry via handheld autoanalyzer (n=7-10). Kir4.1 expression was assessed by immunofluorescence (n=3); NKCC2 and NCC by immunoblotting (n=3–4). Results: Kir4.1 staining in the TAL was mosaic in controls and reduced in knockouts, with preserved distal expression. Compared to controls, knockout mice had increased urine volume (0.23 vs. 0.17 mL/gbw/day, p=0.0087) and trended toward greater urinary Na+ (0.019 vs. 0.014 mEq/gbw/day, p=0.13) and K+ (0.039 vs. 0.029 mEq/gbw/day, p=0.18) excretion. Blood bicarbonate was elevated (22.1 vs. 17.1 mmol/L, p<0.0001) and K+ trended lower (3.6 vs. 4.1 mEq/L, p=0.078). Immunoblots demonstrated lower pNKCC2 (p=0.0024) with no change in total NKCC2 (p=0.57) , and increased total NCC (p=0.033) and pNCC (p=0.016). Conclusion: TAL-specific deletion of Kir4.1 leads to increased urine volume and metabolic alkalosis, with trends toward urinary salt and K+ loss. These changes are accompanied by reduced pNKCC2 and compensatory upregulation of NCC, supporting a Kir4.1-dependent role in TAL Na+ transport. Future work will include female mice and dietary K+ interventions to define cell-type–specific Kir4.1 responses in the TAL. Funding: NIDDK Support - NIDDK Support, NIDDK Support