ADH increases apical Na⁺,K⁺,2Cl^- entry in mouse medullary thick ascending limbs of Henle

These studies were designed to evaluate the mechanism for the ADH-dependent increase in transcellular conductance (G(c), mS·cm^-2), which accompanied hormone-dependent increases in the spontaneous transepithelial voltage (V(e), mV) and in the net rate of Cl^- absorption in single medullary thick ascending limbs of Henle (mTALH) isolated from mouse kidney. The total transepithelial conductance (G(e), mS·cm^-2) was measured with perfusing solutions containing 5 mM K⁺, zero Ba²⁺; G(c) was that component of G(e) blocked by luminal 20 mM Ba²⁺, zero K⁺. In paired experiments, antidiuretic hormone (ADH) increased G(c) from 44.5 ± 5.6 to 58.9 ± 8.9 mS·cm^-2 (Δ = 14.3 ± 5.5; P < 0.02); however, in the presence of 10^-4 M luminal furosemide, ADH had no significant effect on G(c) (Δ = 5.0 ± 4.3; NS). A set of similarly paried measurements together with paired observations on the effects of bath Cl^- deletion, permitted an assessment of the effect of ADH on the magnitude of the fall in G(c) on bath Cl^- removal (ΔG(c)(Cl), mS·cm^-2). ΔG(c)(Cl) was clearly larger with ADH, 29.6 ± 4.3, than without ADH, 19.2 ± 1.0 (Δ = 10.4 ± 4.9; P < 0.05). However, with luminal furosemide, ADH had no significant effect on ΔG(c)(Cl) (Δ = 1.7 ± 4.5; NS). These results indicate that the ADH-dependent increase in G(c) is secondary to increased salt entry across the apical membrane. We computed apical (g(a), mS·cm^-2) and basolateral (g(b), mS·cm^-2) membrane conductances from the G(c) measurements and apical-to-basolateral membrane resistance ratios (R(a)/R(b)) obtained from cell impalement: the ADH-dependent G(c) increase was due to an increase in g(b), which was blocked entirely by luminal furosemide. We propose that ADH increases the number of functioning apical membrane Na⁺,K⁺,2Cl^- transport units, and that g(b) increases because cell Cl^- activity rises and depolarizes the basolateral membrane. Thus the calculated cellular Cl^- activity was 16.3 mM without ADH, and 25 mM with ADH.