The detrimental effects of renal I/R injury are now well recognized. Interestingly, IPC has been shown to protect multiple organs [13, 14, 26] including kidney  from I/R injury in animals. In humans, some investigations have demonstrated the usefulness of IPC in cardiac , and liver surgery and transplantation [28, 29], but no studies on human kidneys have been carried out . The molecular bases of IPC involve the liberation of several mediators that afterwards take place on a multiple and complex intracellular signal pathways [31, 32]. There is a biphasic temporal relationship between IPC-induced protection and the duration of reflow . It offers an initial protection during 2-3 hours after reperfusion and a remote protection at the 12-24 hours that lasts for 2 to 3 days. In fact, this acute window was defined as the strongest form of in vivo protection against myocardial I/R [30, 34]. This early window activates various signal transduction pathways and second messengers which then, serve as signals responsible for activation of the molecular and genetic responses of the delayed IPC . In the current study, we were interested to explore the protective effect of IPC during early reperfusion.
Among the numerous signaling pathways involved in the protective mechanism of IPC, Akt signaling pathway plays a crucial role in defending various organs against I/R injury. Joo et al showed that acute renal IPC is associated with rapid phosphorylation of the Akt and that the inhibition of Akt pathway blocked the protective effects of renal IPC . In agreement with this, our results revealed a marked increase of the phosphorylated form of Akt after IPC in comparison to I/R group. Strong evidences showed that eNOS is an important target of p-Akt [36, 37]. Taken together, our results showed that IPC increased Akt phosphorylation, which in turn activated eNOS and NO generation to alleviate the I/R injury of ischemic kidneys.
It has emerged that NO plays a key role in triggering IPC phenomenon in different organs via its antioxidant , anti-apoptotic  and anti inflammatory properties . It is known that inducible NOS (iNOS) activity was not detected before and immediately after ischemia, but it gradually increased after reperfusion . In kidney, a previous investigation has showed that the expression and the activity of iNOS increase 24 h after IPC application . Besides, it has been observed that pharmacological inhibition of NO synthesis or genetic deletion of the iNOS gene augments mouse kidney susceptibility and mitigates the protection afforded by IPC . From these studies, the increase of iNOS expression could be considered as an important component of long-term protection induced by the IPC in the kidney. Nevertheless, eNOS-mediated NO production plays a crucial role in the acute preconditioned kidney . Interestingly, our study showed that IPC resulted in the increase of nitrite/nitrate level which was markedly higher than this of I/R group. However, pharmacological inhibition of NOS with L-NAME abolished these beneficial effects of IPC against renal I/R injury. These results may reflect the critical role of NO and eNOS, at least partially, to trigger mechanisms responsible for inducing the protective effect of the early IPC.
Several regulatory molecules have been described to activate various signaling pathways able to reduce renal I/R injury. For instance, pharmacological up-regulation of HIF-1α represents a novel strategy in the prevention of acute kidney injury . HIF-1α, a master regulator of gene transcription, mediates many processes of adaptation to low oxygen tension during hypoxia and ischemia . Nevertheless under normoxic conditions, degradation of HIF-1α is mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative alpha-subunits by HIF prolyl hydroxylases (PHD). It was demonstrated that inhibition of HIF-1α degradation by pharmacologic inhibition of PHD would confer protection against subsequent ischemic injury . Recently, it was proved that renal hypoxic conditioning increased the expression of HIF-1α which correlated with the decrease of oxidative stress [1, 38]. In line with this, our results clearly indicate that IPC application increased the level of HIF-1α as compared to I/R. We found that the IPC enhanced the stabilization of HIF-1α to protect ischemic kidneys against reperfusion injury. However, L-NAME treatment abolished this effect. Such results may reflect the involvement of NO in the preservation of HIF-1α stability in ischemic kidney. Zaouali et al. demonstrated that NO could favor the stabilization of HIF-1α in the liver . Based on these findings and our results, it thus seems that IPC ameliorates kidney tolerance against I/R injury throughout enhancement of NO induced HIF-1α stabilization.
The involvement of ER stress in renal cells has been shown to be critical in acute kidney injury in humans and in various animal models of I/R injury [39, 40]. Several factors including oxidative stress, disturbance of calcium homeostasis, and over expression of normal and/or incorrectly folded proteins, interfered with ER function and induced ER stress . The stressed ER triggered the UPR which induces signal transduction events to increase ER resident chaperones, to inhibit protein translation, and to accelerate the degradation of unfolded proteins . Our results showed a marked decline in the levels of p-PERK, ATF4, and TRAF2 and an increase of GRP78 level in IPC group. Studies performed during the last decade identified GRP78 as a ubiquitous luminal resident protein of the ER that plays a key role in assisting the corrected folding and secretion of protein . This protein can protect cells from ER stress and its induction is crucial for maintaining the viability of cells subjected to stress [43, 44]. Hayashi et al. found that induction of GRP78 by IPC reduced ER stress and prevented delayed neuronal cell death . Moreover, Hung et al. observed that blocking the induction of GRP78 sensitized the renal epithelial cells to oxidative stress. Therefore, we could suggest that IPC protected kidneys against renal I/R insults throughout modulation of ER stress and that up regulation of GRP78 made kidneys more resistant to the stressful conditions. Nevertheless, this beneficial effect was abolished when L-NAME had been administered before IPC treatment. In line with this, Bachar et al. proved in pancreatic beta cells subjected to glucolipotoxic conditions, that chronic inhibition of NO production exacerbates ER stress . Taken together, our results revealed that IPC modulated ER stress through the activation of the eNOS pathway, since NOS inhibitor counteracted the protective effect of IPC to reduce ER stress. In addition, Xu et al. strongly suggest in their study that the NO increased the GRP78 expression. They proposed that NO regulates the flux of Ca2+ between the mitochondria, the Golgi and the ER. This results in activation of the ER-stress response transcription factor ATF6 which then translocates to the nucleus and activates ER stress-responsive genes, such as GRP78 .