Intracellular pH (pH_i) measurements in descending and ascending thin loops of henle in rat inner medulla

In previous in vivo studies, pH in vasa recta blood in rat inner medulla was found to be acidic and pH in ascending (∼6.57) was lower than pH in descending (∼671) vasa recta blood under normal conditions. To determine if low pH was found in loop of Henle cells, we measured pH_i in isolated nonperfused descending thin limb (DTL) and ascending thin limb (ATL) of Henle's loops from rat papilla using pH-sensitive fluorescent dye BCECF. Under control conditions (25 mM HEPES or 20 mM HEPES/5 mM bicarbonate buffer; pH 7.4), pH_i in DTL (∼6.74) was lower than pH_i in ATL (∼6.99) Addition of 20 mM NH₄Cl to bath increased pH_i to ∼7.37 and ∼7.49 in DTL and ATL, respectively. Removal of NH₄Cl from bath reduced pH_i to ∼6.66 and ∼6.77 in DTL and ATL, respectively. Rate of pH change (dpH_i/df, pH U/s), intrinsic buffering capacity (β_i, mM H⁺/pH U), base-lateral NH₃ flux (J_NH3, nmol/cm²/s), and basolateral NH₃ permeability (P_NH3, cm/s X 10^-3) for DTL and ATL segments, respectively, were about: 0.25, 0.29; 28.0, 30.2; 4.1, 3.2; and 8.0, 6.5. Control recovery rate (dpH_i/dt) from decreased pH_i after NH₄Cl removal was approximately 6.6 × 10^-3 and 6.7 × 10^-3 pH U/s in DTL and ATL segments, respectively. This dpH_i/dt was decreased in DTL but not ATL by removal of Na⁺, Cl^-, or Na⁺ and Cl^- simultaneously. The acidic values for pH_i and the differences in pH_i between DTL and ATL are similar to pH values in what would be the neighboring ascending and descending vasa recta in vivo despite bath pH of 7.4 in vitro. These data raise questions about the regulation of pH_i in Henle's loops in the rat inner medulla.