Blood flow restoration next an anoxic or hypoxic period alters the function of numerous organs, such as the heart , brain , liver  and kidney . This event induces abnormal activation of many signal transduction cascades, which lead to cellular dysfunction and initiation of the apoptosis/necrosis. In agreement with previous studies [3, 26], our results confirmed that renal I/R provokes severe glomerular and tubular damage. Excessive production of reactive oxygen species (ROS) plays a principal role leading to structural and functional injury of the ischemic kidneys .
The understanding of renal I/R syndrome is still incomplete although several mechanisms were henceforth defined. Some recent evidences strongly suggest the involvement of ER stress as an initiator of cell death during ischemic glomerular and tubular epithelial injury [28, 29]. The ER provides an optimized environment for formation and maturation of cell proteins, but several insults can interfere with this machinery causing aberrant protein folding. Accumulation of these unfolded or misfolded proteins in the ER lumen induces ER stress and in turn activates a well-conserved adaptive response.
The present study shows that the ER stress is enhanced in kidney following I/R. We observed increased amount of the ER chaperone GRP78, which is a central regulator of ER function. This expression was associated with amplified levels of p-PERK, ATF4 and XBP-1. In addition, apoptosis is induced given that caspase 12 and p-JNK, the hallmarks of ER associated apoptosis, and cytochrome c were found to be over-expressed in ischemic kidney. Other studies have also produced evidence suggesting ER stress following in vivo renal I/R. I/R induces phosphorylation of PERK and eIF2α, indicating ER stress and activation of the UPR [28, 30, 31]. It has been observed that I/R of rat kidneys augmented expression of the ER molecular stress chaperone GRP78, as well as TUNEL labeling and the apoptotic protein caspase 12. These increases are concomitant with oxidative stress . In their study, Wang et al. have established that one of the downstream pathways of apoptotic cell death after ER stress is mediated by JNK . They found that JNK is translocated to the mitochondrial membrane, and in turn it is decisive for cytochrome c release. Furthermore, the increased level of p-PERK after I/R injury seems to play a significant pro-apoptotic role, as it induces the activation of the CCAAT/enhancer-binding protein homologous protein (CHOP) . Gao et al. showed that taurousodeoxycholic acid (TDUCA), an inhibitor of ER stress, could represent effective strategy to improve renal function and to reduce apoptosis of tubular epithelial cells. These beneficial effects were associated with a reduction of GRP78, CHOP and caspase 12 . Taken together, these data and our results suggest that I/R-induced oxidative injury activates ER stress and consequently leads to damage cells of rat kidneys. However, it is unclear how ROS induce ER stress. One possibility is that ROS cause inhibition of Ca2+-ATPase on the ER membrane and thus depletion of Ca2+ stores in this organelle [13, 34, 35]. Another possibility is that ROS may cause generation and accumulation of oxidatively modified and abnormal proteins in the ER .
The detrimental effects of renal I/R injury are now well recognized. Interestingly, IPostC has been shown to protect multiple organs [5–7] including kidney  from I/ R injury. Chen et al. reported that IPostC application attenuated renal dysfunction . In agreement with this our results clearly indicate an improvement of creatinine clearance and Na reabsorption rate in the treated kidneys versus those none treated. Parallely, IPostC enhances cell integrity and decreases lipid peroxydation as compared to I/R group. Eldaif et al. demonstrated that IPostC could stabilize the structure of the tubular epithelial cells . However, the cellular mechanism remains to be defined. Our results show a marked decline in the levels of GRP78, p-PERK, ATF4, and XBP-1 in IPostC group. Concomitantly, we note that IPostC reduces the phosphorylation of the JNK and the caspase 12 level in ischemic kidneys. Yeh et al. observed that renal hypoxic conditioning attenuated oxidative stress, and diminished GRP78 and caspase 12 in the ischemic renal tubules. These effects are correlated with enhanced HSP70 protein expression and hypoxia-inducible transcription factor-1α (HIF-1α) activation . In their study, Liu et al. confirmed that IPostC reduced I/R induced ER stress in the heart via the mobilization of the p38 mitogen-activated protein kinase (p38 MAPK)/JNK signaling pathways . Moreover, Yuan et al. suggested in their study that IPostC protected ischemic brain through suppressing ER stress-induced apoptosis and that PI3K/Akt pathway was involved . Therefore we could suggest that IPostC could protect kidneys against renal I/R insults throughout modulation of ER stress.
There is accumulating evidence that lethal reperfusion injury could be reduced by TMZ preconditioning in animals. It was found that TMZ conserves ATP production and lowers intracellular acidosis, while maintaining cellular homeostasis [19–21, 37]. In addition, it reduces oxidative damage to the mitochondria and protects the organ from I/R-induced damage arising from mitochondrial respiration [22, 38, 39]. When given before ischemia, TMZ attenuates the inflammatory response and the tubulointerstitial development of fibrosis prevalent in ischemic kidney injury and reduces the rate of apoptosis expression [19, 22]. These effects were correlated with an early and great expression of HIF-1α. The same impact was also found in liver . In another study, the protective effect appeared to be through activation of p38 MAPK and Akt signaling . However, there is no data about the impact of TMZ treatment on ER stress processes after I/R. Here, we found that contrary to IPostC treatment, it seems that reduction of ER stress is not involved in the protective mechanism of TMZ of ischemic kidney, at least at the early period of reperfusion. As observed, all levels of the ER stress parameters (GRP78, p-PERK, ATF4, XBP-1 and caspase 12) were decreased but not significantly as compared with those of I/R group. However, we noted a significant decrease of mitochondrial parameters (p-VDAC and cytochrome c) and a significant increase in p-GSK3-β content in TMZ group versus I/R group.
TMZ as mostly described, acts on mitochondria by restoring ATP synthesis and by maintaining the mitochondrial membrane impermeability . Indeed, TMZ might bind to inner mitochondrial membrane  to modulate the mitochondrial permeability transition pore (mPTP) opening . Moreover, TMZ might interact with VDAC which has been also proposed to control the release of cytochrome c without mPTP opening . Parallely, previous studies demonstrated that inhibition of GSK3-β phosphorylation was associated with a decrease in VDAC phosphorylation and a delayed mPTP opening [19, 42, 43].
Overall, the data from the present study suggest that ER might not be a target through which TMZ exerts its cytoprotective effect at early reperfusion and that TMZ beneficial effect attenuated essentially mitochondrial dysfunction after renal I/R. In agreement with this, our results show that TMZ protected mitochondria from deleterious consequences of renal I/R injury, via promotion of GSK3-β phosphorylation and prevention of VDAC phosphorylation.
We also evaluated the effect of the combination of both TMZ and IPostC treatments on renal function. Interestingly, we noted a significant improvement of the renal functional parameters as shown by a rise of GFR and Na reabsorption rate versus TMZ and IPostC separately. Such result may be explicated, at least partially, by the improvement of structural parameters. In fact, we observed a significant decline of MDA level and LDH activity as compared to each treatment alone. Histopathological assessment confirmed our suggestion. Taken together, our results supported the idea of a synergetic outcome of both treatments to ameliorate kidney function following warm I/R. The protective mechanisms of the association TMZ+IPostC implicate distinct organelles. In fact, the beneficial effects of TMZ triggered essentially mitochondrial disturbance, while IPostC reduced ER stress. We noted, after the association of both treatments, a significant decline in the rate of XBP-1 and caspase 12, as compared to the TMZ group and I/R group. However, we noted a paradoxical increase in the levels of the PERK and its downstream factor (ATF4) in the group TMZ+IPostC, as compared to IPostC only. These results may be explained, by the fact that the combination TMZ+IPostC did not implicate the PERK pathway and its downstream factor (ATF4). Probably, the effect of IPostC on the PERK pathway is lost, when it was combined to TMZ treatment. The underlying mechanisms are not well understood and other investigations are necessary to confirm this hypothesis.