The single insult of hypoxic preconditioning induces an antiapoptotic response in human proximal tubular cells, in vitro, across cold storage.

OBJECTIVE

To examine whether hypoxia (one of the many components of ischaemic preconditioning) can induce a protective response in culture renal tubular cells, and thus determine if non-lethal periods of hypoxia could confer protection against apoptotic injury to human proximal tubular cells during cold storage and subsequent cytotoxic insult, and establish the cellular mechanisms by which this protection is induced.

MATERIALS AND METHODS

Human proximal tubular cells (HK-2) were pre-incubated for 24 h in normoxic or hypoxic conditions and then incubated at 4 degrees C for 6 h to mimic cold storage, before being returned to normal conditions and exposed to varying concentrations of cyclosporine A (CSA). Cell viability and apoptosis were measured using propidium iodide staining and flow cytometry. The expression of heat-shock protein (HSP)-70 was determined by Western blotting.

RESULTS

Hypoxia had no effect on cell viability or apoptosis. Pre-exposure of cells to hypoxia significantly protected against CSA-induced damage even after a period of cold storage. Western blotting analysis showed that hypoxia up-regulated the anti-apoptotic protein HSP-70. HK-2 cells over-expressing HSP-70 mimicked hypoxia preconditioning, in that they were protected during cold storage and CSA-induced apoptosis.

CONCLUSION

Exposure of renal tubular cells to a sequential model of cold storage, reperfusion and incubation with CSA resulted in apoptotic cell death. Preconditioning these cells with hypoxia induced a protective response and up-regulation of the anti-apoptotic protein HSP-70. There was a similar response in non-preconditioned cells over-expressing HSP-70. Further understanding of the cellular changes occurring during this period of preconditioning will allow the development of more targeted, clinically relevant methods of preconditioning in renal transplantation.