Cytoplasmic pH regulation in canine renal proximal tubule cells

Reid, Ian R.; Civitelli, Roberto; Westbrook, Scott L.; Avioli, Louis V.; Hruska, Keith A.
May 1987
Kidney International;May1987, Vol. 31 Issue 5, p1113
Academic Journal
The precise mechanisms by which the mammalian kidney proximal tubule transports H+ and HC03- 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 Ha+. This recovery was blocked by removal of Ha+ or addition of 0.2 mM amiloride to the cell suspension. In the presence of 0.2 mM amiloride and Ha+, 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)2 SO4 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)2 SO4, 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.


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