Physiological pH. Effects on posthypoxic proximal tubular injury.

Abstract

After O2 deprivation, tissue acidosis rapidly self-corrects. This study assessed the effect of this pH correction on the induction, and pathways, of posthypoxic proximal tubular injury. In addition, ways to prevent the resultant injury were explored. Isolated rat proximal tubular segments (PTSs) were subjected to hypoxia/reoxygenation (50/30 or 30/50 minutes) under the following incubation conditions: 1) continuous pH 7.4, 2) continuous pH 6.8, or 3) hypoxia at pH 6.8 and reoxygenation at pH 7.4 (NaHCO3 or Tris base addition). Continuously oxygenated PTSs maintained under these same pH conditions served as controls. Lethal cell injury was assessed by lactate dehydrogenase (LDH) release. pH effects on several purported pathways of hypoxia/reoxygenation injury were also assessed (ATP depletion, lipid peroxidation, and membrane deacylation). Acidosis blocked hypoxic LDH release (pH 7.4, 50 +/- 2%; pH 6.8, 6 +/- 1%) without mitigating membrane deacylation or ATP depletion. During reoxygenation, minimal LDH was released (3-5%) if pH was held constant. However, if posthypoxic pH was corrected, immediate (< or = 5 minutes) and marked cell death (e.g., 55 +/- 3% with Tris) occurred. This was dissociated from lipid peroxidation or new deacylation, and it was preceded by a depressed ATP/ADP ratio (suggesting an acidosis-associated defect in hypoxic/posthypoxic cell energetics). Realkalinization injury was not inevitable, since it could be substantially blocked by 1) posthypoxic glycine addition, 2) transient posthypoxic hypothermia, or 3) allowing a 10-minute reoxygenation (cell recovery) period before base addition. Neither mannitol nor graded buffer Ca2+ deletion conferred protection. Acute pH correction caused no injury to continuously oxygenated PTSs. Conclusions are as follows: 1) Posthypoxic "pH shock" causes virtually immediate cell death, not by causing de novo injury but, rather, by removing the cytoprotective effect of acidosis. 2) This injury can be prevented by a variety of methods, indicating a great potential for salvaging severely damaged posthypoxic PTSs.

Phospholipase A2 activity can protect renal tubules from oxygen deprivation injury.

“…The PP-PLA2 additions were as follows: 20 Al of PP-PLA2 (P-6534, Sigma) was added to 230 Al of the incubation buffer, and then 25 Al of this dilution was added to the appropriate 2.5-ml PTS aliquots (final dilution, 1:1250, equivalent to 0.4 activity unit/ml; PLA2 activity as per Sigma). At completion of the 45-min incubations, cell injury was assessed by recalculating the percentage of LDH release, a well-recognized marker of hypoxic tubular cell death (9,(12)(13)(14)(15).…”

“…Male Sprague-Dawley rats (150-200 g; Charles River Breeding Laboratories), maintained under standard vivarium conditions, were used for all PTS isolations, done as described (13). In brief, after inducing anesthesia with pentobarbital (30-40 mg/kg), the kidneys were resected, minced, and partially digested with collagenase.…”

“…In brief, after inducing anesthesia with pentobarbital (30-40 mg/kg), the kidneys were resected, minced, and partially digested with collagenase. The released, viable PTS were recovered from the digested tissues by centrifugation through Percoll (13) (9,(12)(13)(14)(15) cytoprotectant activity is not a byproduct of PLA2 isolation/ purification processes. To this end, an acetone extract of PP (P-4251, Sigma) was suspended in the PTS incubation buffer (50 mg of powder per ml), and after sonication, the suspension was centrifuged, and the supernatant was passed through a 45-,m filter and retained.…”

“…The PP-PLA2 additions were as follows: 20 Al of PP-PLA2 (P-6534, Sigma) was added to 230 Al of the incubation buffer, and then 25 Al of this dilution was added to the appropriate 2.5-ml PTS aliquots (final dilution, 1:1250, equivalent to 0.4 activity unit/ml; PLA2 activity as per Sigma). At completion of the 45-min incubations, cell injury was assessed by recalculating the percentage of LDH release, a well-recognized marker of hypoxic tubular cell death (9,(12)(13)(14)(15).Because the above PP-PLA2 preparation contains 3.2 M (NH4)2S04, addition ofit to the PTS suspensions created a 2.6 mM (NH4)2SO4 concentration. To exclude the possibility that the (NH4)2SO4 alters hypoxic-reoxygenation injury independent of a PLA2 effect, four pairs of PTS aliquots were Abbreviations: PTS, proximal tubular segments; LDH, lactic dehydrogenase; PLA2, phospholipase A2; PP, porcine pancreas; BP, bovine pancreas; BV, bee venom; SV, snake venom.…”

Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells

“…During 1 h of anoxia, cell viability of freshly isolated PT cells de creased significantly to 54% ± 2% (P < 0.05), while no loss in viability was observed in cultured PT cells. Clamping the pH; during anoxia at 6.7 and 6.1 signifi-shown that acidification of hypoxic tissue, resulting from glycolytic lactate production, ATP hydrolysis and C 0 2 accumulation, can enhance the resistance to the damaging effects of O, deprivation in the kidney [7,24,[26][27][28][29], cardiomyocytes [3,16], and hepatocytes [10,11,13,18]. However, the mechanism behind the protec tion offered by lowering the pH is unknown.…”