Characterization of Na+ transport across the cell membranes of the ascending thin limb of Henle's loop

Characterization of Na+ transport across the cell membranes of the ascending thin limb of Henle's loop. In the ascending thin limb of Henle's loop (ATL), intracellular Na+ is extruded by Na+/K+ ATPase in the basolateral membrane. To further characterize Na+ transport across the cell membranes of the ATL, the intracellular sodium concentration ([Na+]i) was monitored using a sodium-sensitive fluorescent probe, SBFI, in the in vitro microperfused hamster ATL. Basal [Na+]i was 19.0 ± 1.2 mM (N = 24). Removal and replacement of luminal Na+ did not change [Na+]i in the presence of Na+ in the bathing fluid. In contrast, luminal Na+ removal reduced [Na+]i from 11.6 ± 0.9 to 6.3 ± 0.8 mM in the absence of peritubular Na+ (P < 0.0005, N = 21). Replacement of luminal Na+ increased [Na+]i to 12.6 ± 0.9 mM. In the absence of Na+ in the bath, the addition of 1 µM benzamil, 0.1 mM 5-(N,N-dimethyl)-amiloride (DMA), 0.1 mM furosemide, or 0.1 mM trichlormethiazide to the lumen did not change [Na+]i or the rate of change in [Na+]i (d[Na+i/dt) after removal and replacement of luminal Na+. Decreases in luminal pH in a Hepes-buffered solution and luminal HCO3- did not affect [Na+]i. In the absence of peritubular Na+, DMA in the bathing fluid decreased [Na+]i from 11.4 ± 1.3 to 6.4 ± 1.2 mM (P < 0.01, N = 5) and completely inhibited the changes in [Na+]i after removal and replacement of luminal Na+. Removal of peritubular Na+ reduced [Na+]i from 18.8 ± 1.2 to 11.3 ± 0.7 mM (P < 0.0001, N = 23). Addition of DMA in the bathing fluid reduced [Na+]i and inhibited the changes in [Na+]i after removal and replacement of peritubular Na+. Addition of benzamil, furosemide or trichlormethiazide to the bathing fluid did not alter [Na+]i or the changes in [Na+]i after removal and replacement of peritubular Na+. Decreases in peritubular pH in a Hepes-buffered solution and peritubular HCO3- did not affect [Na+]i. These results indicate that Na+ transport across the cell membranes of the ATL is mediated by the Na+/H+ antiporter in the basolateral membrane together with Na+/K+ATPase in the basolateral membrane and demonstrate the absence of Na+ permeability in the luminal membrane of the ATL