Background: We have previously shown that preconditioning of human proximal tubular epithelial cells with a sub-lethal dose of hypoxia(Combined Oxygen/Glucose Deprivation (COGD)) increases HSP70 mRNA and protein, and induces tolerance to subsequent hypoxic injury as evidenced by decreased efflux of LDH. We hypothesized that HSP70 may be responsible for this protection.

Methods: To directly determine if overexpression of HSP70 protects renal tubular cells from hypoxic injury, LLC-PK1 cells were stably transfected with the human HSP70 gene and tested for resistance to COGD. In addition, to achieve higher intracellular levels of HSP70, cells were transiently cotransfected with the genes for HSP70 and the marker enzyme luciferase (luc). Loss of luc activity during cellular stress can be used(like LDH efflux) as a marker for cellular injury. Cells transfected with luc alone, and cells cotransfected with luc/HSP70 were exposed to COGD and retained luciferase activity was measured by luminometry at multiple time points. Similar cotransfection experiments were done using heat stress in place of hypoxia.

Results: Six-fold overexpression of HSP70 by stably transfected cells did not reduce LDH efflux during COGD compared to untransfected controls. Transient transfection, which results in much higher levels of HSP70 expression also did not lead to preservation of intracellular protein(luciferase) during COGD or recovery from COGD compared to controls. In contrast, HSP70/luc cotransfected cells had significantly less loss of luc activity during heat stress than did control cells transfected with luc alone.

Conclusion: Overexpression of HSP70 protects LLC-PK1 cells from heat stress but not hypoxia.