pH-stat experiments in proximal renal tubules

Abstract

The pH-stat technique has been used to measure H+ fluxes in gastric mucosa and urinary bladder "in vitro" while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1 N HC1-filled microelectrodes to inject OH- or H+ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H(+)-ion conductance). These ratios were reduced after luminal 10(-4) M acetazolamide, during injection of OH-, but they increased during injection of H+. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H+ fluxes per cm2 epithelium. The relation between JH+ and pH gradient showed saturation kinetics of H fluxes, both during OH- and H+ injection. This kinetic behavior is compatible with inhibition of JH by luminal H+. It is also compatible with dependence on Na+ and H+ gradients of a saturable Na/H exchanger. H(+)-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H+ flow is mediated by a membrane component, most likely the Na(+)-H+ exchanger.