Ischemic postconditioning inhibits apoptosis of renal cells following reperfusion: a novel in vitro model.

PURPOSE: The majority of renal ischemic/reperfusion (I/R) and ischemic postconditioning (IPO) studies have been based on animal models. To gain mechanistic insights into ischemic postconditioning-induced alterations at the cell level, a novel in vitro model of I/R and IPO is set up by using the rat proximal tubule cell line NRK-52E.

METHODS: Cells are incubated in 1 mL ischemic buffer under hypoxia conditions for 3 h to simulate the clinical condition of a cellular microenvironment representative of ischemia, including oxygen deprivation, carbon dioxide elevation, nutrient depletion, and waste accumulation. IPO model is established by exposing the cells to three cycles of 'mimic reperfusion condition' for 10 min and ischemic condition for 10 min after placing the cells in ischemic condition for 3 h. Flow cytometry and Hoechst are used to assessing apoptosis. The expression spot and protein levels of PDK, Akt, and ERK are also analyzed.

RESULTS: I/R results in severe injury in NRK-52E cells as evidenced by increased LDH leakage in the culture medium, as well as increased apoptotic index, which may be significantly attenuated by IPO treatment applied before the abrupt reperfusion (P < 0.05 vs. I/R group). Meanwhile, IPO, compared with I/R, increases phosphorylation levels of Akt and ERK (P < 0.05 vs. I/R group), which have been identified to play a vital role in the regulation of cell proliferation, survival, and metabolism.

CONCLUSION: A new in vitro model of I/R and IPO is established successfully. These results offer evidence that 3 h of simulating ischemic/reperfusion injury may cause cell apoptosis, and IPO is effective to attenuate renal cell apoptosis and potentially mediate via activation of Akt and ERK signal.