Ischemic postconditioning inhibits apoptosis in an in vitro proximal tubular cell model

Abstract

Ischemia-reperfusion is a common injury of clinical ischemic disease and surgical lesions. Ischemic postconditioning (IPO) improves the ability of organs subjected to ischemia to tolerate injury. However, renal IPO studies have been based on animal models. In order to gain insights into IPO-induced alterations at the cellular level, an in vitro model for IPO was designed using the rat proximal tubular cell line NRK-52 E. This model was established by placing NRK-52 E cells in ischemic conditions for 3 h, then exposing cells to three cycles of reperfusion for 10 min and finally to ischemic conditions for 10 min (postconditioning). The cells were cultured further in reperfusion conditions for 3, 6, 12 and 24 h. Flow cytometry and Hoechst were used to assess apoptosis. The protein expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, cleaved caspase-3 and caspase-8 were analyzed by western blotting. The results demonstrated that apoptosis occurred in cells subjected to ischemia/reperfusion (I/R) alone or with postconditioning following reperfusion for 24 h. Cells subjected to I/R demonstrated increased expression of Bax, cleaved caspase-3 and caspase-8 at the end of reperfusion. However, the levels of Bax, cleaved caspase-3 and caspase-8 were significantly attenuated in cells, which had undergone IPO. In conclusion, apoptosis was observed in cells subjected to 3 h of ischemia-reperfusion injury and IPO was able to inhibit this apoptosis. IPO inhibited apoptosis by inhibiting the caspase pathway thereby exerting protective effects.

Figure 1

Experimental procedure used to determine the effect of IPO following I/R in an in vitro model. Normal, normal condition culture; control, cells cultured in control medium followed by reperfusion; I/R, cells cultured in ischemic conditions followed by reperfusion; IPO, cells cultured in ischemic conditions for 3 h, then replaced with complete medium and cultured under normal conditions for 10 min, followed by placing cells in ischemic conditions for 10 min. IPO1 group, one cycle of IPO; IPO2, two cycles of IPO; IPO3, three cycles of IPO. IPO+, cells cultured under mimic ischemic conditions for 3 h. The ischemic buffer was not changed and another 0.5 ml fresh complete medium was added. The cells were grown under normal conditions for 10 min. Following this, the cells were exposed to ischemic conditions for 10 min without changing the mixed medium. IPO1+, one cycle of IPO+; IPO2+, two cycles of IPO+; IPO3+, three cycles of IPO+. I, ischemia; R, reperfusion; IPO, ischemic postconditioning.

Figure 2

Apoptotic rate of NRK-52E cells following IPO. (A) The apoptotic rate of NRK-52E cells was significantly affected by IPO. The typical flow cytometry results of nine groups cultured for 24 h culture following simulating ischemia and reperfusion injury are shown. (B) Results were obtained from three independent experiments. ^*P<0.05, versus normal; ^{^}P<0.05, versus I/R; ^#P<0.05, versus IPO1+ (n=3 in each group). IPO, ischemic postconditioning; I/R, ischemia/reperfusion; FITC, fluorescein isothiocyanate.

Figure 3

Effect of postconditioning on ischemia and reperfusion-induced apoptosis of NRK-52E cells. (A) Normal, NRK-52E cells cultured under normal conditions. (B) I/R, cells cultured in ischemic conditions and then reperfused for 24 h. (C) IPO3+, cells cultured in ischemic conditions, followed by three cycles of IPO and then reperfused for 24 h. Arrows indicate the apoptotic cells (Hoechst staining; all fluorescence photomicrographs original magnification, ×200).

Figure 4

Expression of Bcl-2 and Bax following IPO. The effects of IPO on Bcl-2 and Bax expression in the NRK-52E cells, which were subjected to three cycles of 10 min reperfusion/10 min ischemia for postconditioning. I/R injury increased the expression of Bax and decreased the expression of Bcl-2. However, the alterations induced by I/R injury were opposite in the IPO group. IPO, ischemic postconditioning; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; I/R, ischemia/reperfusion.

Figure 5

Expression of apoptotic proteins following IPO. The effects of IPO on caspase-3, cleaved caspase-3 and caspase-8 expression in the NRK-52E cells, which were subjected to three cycles of 10 min reperfusion/10 min ischemia for postconditioning. I/R injury increased the expression of cleaved caspase-3 and caspase-8 after 24 h, and decreased the expression of cas-pase-3. However, the alterations induced by I/R injury were opposite in the IPO group. IPO, ischemic postconditioning; I/R, ischemia/reperfusion.