Deletion of thick ascending limb Kir4.1 blunts diuretic response to furosemide

Background: We recently reported that thick ascending limb (TAL)–specific deletion of Kir4.1 produces polyuria, relative metabolic alkalosis, and sex-dependent alterations in potassium homeostasis, with males exhibiting higher urinary K + excretion. These physiological changes were accompanied by reduced expression of total and phosphorylated forms of the furosemide-sensitive Na + -K + -2Cl - cotransporter (NKCC2) and trends toward increased expression of the amiloride-sensitive epithelial sodium channel (ENaC). Based on these findings, we hypothesized that the observed molecular adaptations would manifest functionally as a blunted natriuretic response to furosemide and an exaggerated response to amiloride. Methods: Male (n=5 per group) and female (n=3 per group) mice with TAL-specific Kir4.1 deletion were generated by treating Slc12a1-CreERT2; Kir4.1flx/flx with tamoxifen (TAL-Kir4.1-/-, 1 mg/day × 5 days) or vehicle (control, 5% ethanol in sunflower oil) IP. All mice underwent a 2-week tamoxifen washout period prior to functional testing and were maintained on standard Na + (0.33%) and K + (1.16%) diet. A series of diuretic challenge protocols were performed sequentially, with ≥4-week washout periods between each test to avoid carry-over effects. For the furosemide response test, mice received consecutive daily injections of vehicle (Day 1; 1.2% ethanolamine in 0.9% NaCl) followed by furosemide (Day 2; 25 mg/kg body weight), with 4-hour urine collections obtained in metabolic cages after each injection. Following a washout period, an amiloride response test was performed by consecutive injections of vehicle (Day 1; 0.9% NaCl) and amiloride (Day 2; 40 µg per 25 g body weight), followed by 6-hour urine collections. Urine electrolytes were measured by flame photometry or colorimetric assays. Results: Furosemide-sensitive urinary Na + excretion was significantly lower in TAL-Kir4.1-/- compared with controls (delta Na + : 0.005 vs 0.007 mmol Na + /g.b.w./4 hours, p=0.0196 by unpaired t-test). In contrast, urinary Ca++ excretion trended higher in response to furosemide (delta Ca++: 0.0020 vs. 0.0010 mg/g.b.w./4 hours, p=0.1119 by unpaired t-test). Similarly, while sex-dependent differences in baseline urinary Mg++ excretion were apparent, furosemide-sensitive urinary Mg++ excretion trended higher in both males (p=0.0980) and females (p=0.1033). No difference was detected in amiloride response between TAL-Kir4.1-/- and controls (0.0035 vs. 0.0045 mmol Na + /g.b.w./6 hours, p=0.4104 by unpaired t-test ). Conclusion: Deletion of Kir4.1 specifically in the TAL significantly blunts the natriuretic response to furosemide, consistent with reduced NKCC2 activity in this segment. In contrast, ENaC–mediated sodium handling, assessed by the amiloride response, remains preserved. These findings identify Kir4.1 as a critical regulator of NKCC2 function in the TAL. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.