Effect of Extracellular Calcium on Survival of Human Proximal Tubular Cells Exposed to Hypoxia

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Abstract
Removal of extracellular calcium has been demonstrated to improve membrane integrity of rodent myocytes, astrocytes, and renal tubular cells injured by hypoxia. In this study, the effect of extracellular calcium on long-term survival of cultured human proximal tubular epithelial cells (PTEC) subjected to hypoxia was evaluated. In addition, the effect of extracellular calcium on release of arachidonic acid metabolites (AAM) was assessed during and after hypoxia. To induce hypoxic injury, PTEC were incubated in an anaerobic chamber in glucose-free buffer (combined oxygen/glucose deprivation, COGD). Long-term survival was assessed by measuring lactate dehydrogenase (LDH) efflux during COGD and after an additional 24-h “recovery'’ period (in routine culture medium in 95% air/5% CO2). To determine if extracellular calcium influenced AAM release from membrane phospholipids, cells were preincubated with [3H]arachidonic acid and the release of AAM was measured during COGD and recovery. With this model system, PTEC exhibited minimal LDH efflux during ≤ 12 h COGD, but LDH efflux increased to 73.9 ± 4.7% by 24 h COGD. With 12–18 h of COGD, the extent of LDH efflux was greater during recovery than during COGD, indicating that, for human PTEC, the extent of membrane damage does not become fully evident by LDH efflux for hours after hypoxia. PTEC exposed to 24 h of COGD in the absence of extracellular calcium exhibited strikingly less LDH efflux during COGD than cells incubated in the presence of extracellular calcium, suggesting that extracellular calcium contributes to membrane damage during COGD. However, upon reexposure of PTEC to extracellular calcium, LDH efflux rapidly increased to control levels. Furthermore, despite allowing cells to recover in oxygen or oxygen and glucose before exposure to calcium-containing medium, a rapid increase in LDH efflux could not be avoided. These results suggest that COGD induces an irreversible injury that ultimately leads to loss of membrane integrity whether or not extracellular calcium is present; however, extracellular calcium accelerates the loss of membrane integrity caused by hypoxia. Extracellular calcium did not alter AAM release, indicating that the effect of extracellular calcium on membrane damage (as indicated by LDH efflux) was not mediated by an increased activity of phospholipases (such as phospholipase A2) that are involved in the release of AAM.