Resistance to loop diuretics in patients is associated with significantly higher mortality in patients. The tubular mechanisms involved in loop diuretic resistance remain unclear. Recent data in patients indicate that upregulation of NCC in distal convoluted tubule (DCT) is associated to loop diuretic resistance. However, whether NCC phosphorylation and SPAK (the kinase upstream) are activated by loop diuretics is unclear. Here, we tested whether a daily single-dose treatment with the loop diuretic bumetanide for 6 days induced loop diuretic resistance. We hypothesize that enhanced NCC and SPAK activation in DCT attenuate the response to loop diuretics after chronic intake of the loop diuretic. Methods: 12-week-old male C57BL/6J mice were divided into bumetanide treatment (chronic bumetanide) and control groups. Mice in the chronic bumetanide group received a single daily dose of a loop diuretic in a small amount of chocolate (0.4g), bumetanide (20mg/kg), for 6 consecutive days whereas control mice received regular chow without bumetanide except on the day of experiment. Urine collection was performed in metabolic cages with subsequent measurement of electrolytes (Na, K, Cl). Protein expression of NKCC2 was performed in medullary thick ascending limb suspensions and NCC and SPAK quantified in cortical tubule suspensions by Western Blot. After 6 days, baseline urine volume was similar between the groups. However, urine osmolality was decreased by 60% in the chronic bumetanide group (Control: 1109 ± 113, bumetanide: 452 ± 21 mOsm/l, p< 0.01, n=7). After 6 days, acute (4h) bumetanide-induced diuresis was decreased in the chronic bumetanide group (Δ4hr Uv: Control: 1.9 ± 0.1ml, chronic bumetanide: 1.6 ± 0.1ml, p< 0.1, n=7). Bumetanide-induced Na and chloride (Cl) excretion were also decreased in the chronic bumetanide group(4hr ΔUNa: Control: 216 ± 31mmol, chronic bumetanide 135 ± 8 mmol from baseline; 4hr ΔUCl: Control: 136 ± 14 mmol, chronic bumetanide: 34 ± 4 mmol from baseline; p< 0.05, n=7) indicating the development of loop diuretic resistance. Next, we determined the response of mice to hydrochlorothiazide (HCTZ), an inhibitor of NCC. Mice underwent chronic bumetanide protocol but received hydrochlorothiazide (HCTZ, 100mg/kg) on day 6. There was no significant difference in HCTZ-induced diuresis between control and chronic bumetanide mice (Δ4hr Uv: Ctrl: 0.59 ± 0.10, chronic bumetanide: 0.68 ± 0.10 ml, p< 0.05, n=7). Chronic bumetanide mice had higher HCTZ-induced natriuresis and Cl excretion (4hr ΔUNa+: Ctrl: 163 ± 17, chronic bumetanide: 201 ± 14 mmol from baseline; 4hr ΔUCl: Ctrl: 71 ± 6, chronic bumetanide: 93 ± 8 mmol from baseline; p< 0.05, n=7). Mice were then placed on regular diet for 1 week, and then half of the group treated with chronic bumetanide for 6 days. We then obtained medullary and cortical tubule suspensions. In medullary TALs, mice in the chronic bumetanide group showed decreased total NKCC2 expression by 40±15%, but no change in the ratio of phosphorylated Thr96,101/total NKCC2. Although total SPAK was increased (118±43%), phosphorylated SPAK was decreased (76±12%). In cortical tubule suspensions of chronic bumetanide mice, total and phosphorylated NCC (Thr53) were increased by 126±20%. Also, chronic bumetanide mice had increased total SPAK (120±25%) and phosphorylated SPAK(242±43%). We conclude that chronic loop diuretic use induces SPAK and NCC activation in the DCT which likely contributes to decreased responsiveness to loop diuretic as seen in patients with loop diuretic resistance. It is not known why chronic loop diuretic use decreases total NKCC2 and SPAK expression in medullary TALs. This model will be useful in studying Na load dependent upregulation of SPAK and NCC. 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.
Baiden et al. (Fri,) studied this question.