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.(ABSTRACT TRUNCATED AT 250 WORDS)