Role of intracellular pH during cytoprotection of proximal tubule cells by glycine or acidosis

Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca²⁺-induced injury, but there is little information about alterations of intracellular pH (pH_i) in renal tubules during either injury or its modification by decreasing medium pH or other potent protective factors such as glycine. pH_i was assessed with 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein during proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytosolic free Ca₂₊ induced by treatment with the calcium ionophore ionomycin, alone and in combination with antimycin. Freshly isolated rabbit proximal tubules studied under superfusion conditions in the presence of probenecid were suitable for monitoring pH_i during relatively prolonged and severe injury states. Probenecid, used to promote the retention of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pH_i during simple ATP depletion, but pH_i fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence of antimycin, ionomycin induced intracellular alkalinization in Ca²⁺-replete medium, which may have further enhanced the severity of injury. When medium Ca²⁺ was buffered to 100 nM, ionomycin induced intracellular acidification, which likely resulted from a combination of Ca²⁺/H⁺ exchange activity of the ionophore and H⁺ uptake during Ca²⁺-ATPase-mediated extrusion of Ca² released by ionomycin from intracellular pools. Alterations of pH_i did not contribute to glycine cytoprotection because glycine did not affect the behavior of pH_i during treatment with antimycin, ionomycin, or both agents in combination. The lack of effect of glycine on pH_i makes it likely that complementary cytoprotective actions of glycine and reduced pH will be relevant to understanding both In vitro models of injury and in vivo ischemic states.