Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells

Background. This study tested the following hypotheses: (a) renal tubular e pithelial cells subjected to transient adenosine triphosphate (ATP) depleti on undergo apoptosis, and (b) induction of heat stress proteins (HSPs) inhi bits cell death following ATP depletion, possibly by interacting with anti- apoptotic signal proteins. Methods. To simulate ischemia in vivo, cells derived from opossum kidney pr oximal tubule (OK) were subjected to ATP depletion (5 mM cyanide, 5 mM 2-de oxy-D-glucose, and 0 mM glucose) for 1 to 1.5 hours, followed by recovery ( 10 mM glucose without cyanide). The presence of apoptosis was assessed by m orphological and biochemical criteria. The effect of prior heat stress or c aspase inhibition on apoptosis and cell survival were assessed. Results. In the ATP-depleted cell, both Hoechst dye and electron microscopy revealed morphological features that are typical of apoptosis. On an agaro se gel, a "ladder pattern" typical of endonucleosomal DNA degradation was o bserved. Prior heat stress reduced the number of apoptotic-appearing cells, significantly decreased DNA fragmentation, and improved cell survival comp ared with controls (73.0 +/- 1% vs. 53.0 +/- 1.5%; P < 0.05). Two different caspase inhibitors also improved survival, suggesting that apoptosis is a cause of cell death in this model. Compared with ATP-depleted controls, pri or heat stress inhibited the pro-apoptotic changes in the ratio of Eel, to BAX, proteins known to regulate the apoptotic set point in renal cells. HSP 72, a known cytoprotectant, coimmunoprecipitated with Bcl(2), an anti-apop totic protein. Prior heat stress markedly increased the interaction between HSP 72 and Bcl(2). Conclusions. Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. N ovel interactions between HSP 72 and Bcl(2) may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depleti on injury.