Cytoplasmic pH regulation in canine renal proximal tubule cells

The precise mechanisms by which the mammalian kidney proximal tubule transports H⁺ and HCO₃^- and regulates cytosolic pH (pHi) remain in doubt, though both a H⁺-ATPase pump and Na⁺/H⁺ exchange at the luminal membrane are known to function in the export of protons. The mechanisms of HCO₃^- transport are less clear though recent reports suggest an important role for an electrogenic Na⁺/HCO₃^- symport in the basolateral membrane. The importance of chloride-dependent bicarbonate transport is unknown. In the present studies, the pH-sensitive fluorescent dye, bis-(carboxyethyl)-carboxyfluorescein (BCECF) has been used to study pHi changes in suspensions of canine proximal tubule cells following acidification or alkalinization of the cytosol. Cells were acid-loaded to pH 6.5 by exposure to the H⁺/K⁺ ionophore, nigericin. Following removal of nigericin, pHi returned to basal levels (pHi = 7.1) when the cells were resuspended in a buffer containing 100 mM Na⁺. This recovery was blocked by removal of Na⁺ or addition of 0.2 mM amiloride to the cell suspension. In the presence of 0.2 mM amiloride and Na⁺, partial excretion of the acid load occurred if the buffer also contained HCO₃^-/CO₂, but this effect was blocked by the removal of Na⁺ or the addition of 1 mM 4-acetomido-4'-isothiocyano-2,2'-stilbene disulfonic acid (SITS). When cell membrane potential was monitored in these experiments using the potential-sensitive fluorescent dye, bis-(1,3-dibutylbarbiturate)trimethine oxonol, the increase in pHi seen in the presence of Na⁺ was found to be electroneutral, whereas when that occurred in the presence of Na⁺, amiloride and HCO₃^-/CO₂ was associated with membrane hyperpolarization. In a further series of experiments, the chloride dependence of bicarbonate transport was studied. In the absence of chloride, basal pHi was increased (7.31 ± 0.02 vs. 7.15 ± 0.03, P < 0.005), but the rise in pHi produced by 10 mM (NH₄)₂SO₄ was blunted (0.12 ± 0.01 vs. 0.16 ± 0.01, P < 0.02). Cells suspended in chloride-containing bicarbonate buffer showed a fall in pHi towards basal values after cytoplasmic alkalinization by (NH₄)₂SO₄, but no such recovery was detectable in chloride free conditions. These data are consistent with pHi regulation of these cells by means of an amiloride-inhibitable Na⁺/H⁺ exchanger and a SITS-inhibitable, electrogenic, Na⁺/HCO₃^- symport. Chloride-dependent bicarbonate transfer also occurs and is important in the maintenance of basal pHi and the excretion of a base load.