K+-sparing diuretic actions of trimethoprim: Inhibition of Na+ channels in A6 distal nephron cells

Abstract

Hyperkalemia complicates trimethoprim-sulfamethoxazole (TMP-SMX) therapy in over 20% of HIV-infected patients. TMP is a heterocyclic weak base, similar to amiloride, a 'K+-sparing' diuretic and Na+ channel blocker. Apical TMP is known to inhibit amiloride-sensitive short circuit current in A6 cells, a tissue culture model for mammalian cortical collecting tubule principal cells [1]. We used cell attached patch clamp techniques to investigate the effect of TMP on the 4 pS, highly selective Na+ channel in the apical membrane of A6 cells grown on permeable supports in the presence of 1.5 μM aldosterone. Baseline channel activity at resting membrane potential, measured as NP(o) (N of channels x open probability), was 1.09 ± 0.50 (N = 18). NP(o) (0.92 ± 0.38; N = 9) was unchanged when 10-3 M TMP was added to the basolateral bath for 30 minutes. However, apical exposure with pipettes containing 10-3 or 10-5 M TMP reduced NP(o) ≃ tenfold (0.12 ± 0.08; N = 7 and 0.18 ± 0.14; N = 12, respectively). Kinetic analysis revealed the appearance of a new closed state after apical TMP treatment. Another group of A6 cells were pretreated with 10-3 M apical TMP for 30 minutes prior to patching with pipettes filled with TMP-free saline. NP(o) progressively rose from 0.07 ± 0.09 to 0.87 ± 0.23 (N = 5) as the residual TMP was diluted within the pipette. Apical or basolateral pretreatment (30 min) with 10-3 M SMX did not change Na+ channel activity. In conclusion, in A6 distal nephron cells: (1) TMP reversibly blocks highly selective Na+ channels; (2) direct interaction with the outer channel pore is required since inhibition was observed with apical, but not basolateral TMP; (3) the SMX component of TMP-SMX preparations has no direct effect on Na+ channel activity; (4) This K+-sparing diuretic effect likely contributes to the hyperkalemia associated with TMP therapy in HIV-infected patients.