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 micro perfused hamster ATL. Basal [Na⁺](i) was 19.0 ± 1.2 nM (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⁺], 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.