Benzamil-mediated urine alkalization is caused by the inhibition of H⁺-K⁺-ATPases

Epithelial Na⁺ channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H⁺ secretion via renal H⁺-K⁺-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K⁺ intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH₄⁺ and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K⁺ diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K⁺ diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α₁-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function.NEW & NOTEWORTHY Bolus application of epithelial Na⁺ channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H⁺-K⁺ pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.