We have investigated the potential cytoprotective role of endoplasmic reticulum (ER) molecular chaperones in a cultured cell model of renal ischemia. Madin-Darby canine kidney (MDCK) cells were pretreated with tunicamycin (an inducer of ER but not cytosolic molecular chaperones) for 12-16 h, followed by 6 h of ATP depletion. A rapid and severe depletion of cellular ATP was noted in both control and tunicamycin-treated cells. Trypan blue exclusion assays indicated that pretreatment of MDCK cells with tunicamycin reduced ATP depletion-induced cell damage by ~80% compared with nonpretreated controls. This apparent cytoprotective effect was also found following pretreatment with another inducer of ER molecular chaperones (i.e., A23187). For example, A23187 was found to reduce lactate dehydrogenase release by ~50% compared with untreated controls, whereas E-64, a cysteine protease inhibitor which may affect degradation of some proteins in the ER, had little or no effect on cell injury. Moreover, a fluorescent assay confirmed the marked reduction in cell damage following ATP depletion (up to 80% reduction in tunicamycin-pretreated cells). Together, these findings are consistent with the notion that induction of ER molecular chaperones leads to the acquisition of cytoprotection in the face of ATP depletion. However, inhibition of protein translation by cycloheximide was found to only partially attenuate the observed cytoprotective effect, raising the possibility that other, as yet to be identified, nonprotein synthesis-dependent mechanisms may also play a role in the observed cytoprotection.