Regulation Of PI3K/Akt Signaling Pathway In The Protective Effect Of Remifentanil On Renal Ischemia-reperfusion Injury In Rats

Objective: Renal ischemia-reperfusion injury(ischemia/reperfusion, I/R) injury is a relatively common clinical pathological phenomenon,and is a major cause of acute renal failure and renal transplant failure. The cause of ischemia-reperfusion injury is complex, not only related to the inflammatory response, but also involving apoptosis. PI3 K / Akt signaling pathway is composed by PI3 K and its downstream molecules Akt, which widely exists in cells,regulating cell proliferation, differentiation, apoptosis and migration. Kwon et al reported that PI3 K / Akt signaling pathway is activated after renal ischemia-reperfusion injury, and through its anti-apoptotic effect to promote cell survival, thus involved in renal ischemic injury and repair. Remifentanil is a ultra short-acting higher choice μ-opioid receptor agonist, our previous study found that remifentanil alleviate renal I/R injury by inhibiting apoptosis,but the exact mechanism is imperfect.In the study, a model of renal I/R injury of rat was adopted. By observing the apoptotic rate of renal tissue, PI3 K m RNA and Akt m RNA expression,and the expression of Akt protein and Akt phosphorylation changes at different time points of renal I/R,to explore remifentanil alleviate renal I/R injury in cell apoptosis effect whether is realated to the PI3 K / Akt signaling pathway.Methods: sixty male Sprague-Dawley rats, weighing 220-230 g were randomly divided into 5 groups(n=12 each): sham operation group(group S),I/R group, remifentanil group(group R), remifentanil + Wortmannin group(group RW), Wortmannin group(group W). The renal arteries were exposed, but were not clamped in group S. The renal I/R model was established by clamping the bilateral renal arteries for 45 min using an atraumatic clamp followed by reperfusion in other four groups. Remifentanil was infused at 1.0 μg· kg-1· min-1 via the caudal vein starting from 15 min before ischemia until 30 min of reperfusion in group R and group RW, while the equal volume of normal saline substitution was received in group S, group I/R and group W. In group RW and group W, the rats were injected wortmannin 15 μg/kg 20 min before ischemia, the same volume of saline was injected in group S, group I/R, group R. At 24 h and 48 h of reperfusion, a 1-ml blood sample was cllected via the femoral vein, respectively from 6 rats, then centrifuged and obtained the blood serum used for the measurements of serum creatinine(s Cr) and blood urea nitrogen(BUN). The animals were sacrificed at 24 h and 48 h of reperfusion, and the renal specimens were obtained to observe the renal pathological changes with light microscope and to detect the expression of PI3 K m RNA and Akt m RNA by RT-PCR and to detect the expression of Akt and phosphorylated Akt with the method of Western Blot. The apoptotic rate was determined by flow cytometry double staining method.Results:1 s Cr and BUN concentrationss Cr and BUN concentrations at 24 h and 48 h of reperfusion in other four groups were higher as compared with group S(P<0.01 or 0.05). s Cr and BUN concentrations at 24 h and 48 h of reperfusion in group R were lower as compared with group I/R(P<0.01 or 0.05).s Cr and BUN concentrations at 24 h and 48 h of reperfusion in group RW and group W were higher as compared with group R(P<0.01 or 0.05). No significant differences were seen in s Cr and BUN concentrations among the three groups: goup I/R、group RW and group W(P>0.05).2 Observed under light microscopeNormal tissue was observed, without apparently histopathologic changes in all time points in group S.In group I/R, glomerular pycnosisthe, renal interstitials edema, tubular dilatation were predominant and the renal tubular epithelial cells were edema, degeneration and necrosis at 24 h after reperfusion. The injuries above were aggravated at 48 h after reperfusion and a large number of cell debris were observated in the lumen of renal tubulars. In group R, The renal interstitial can be seen mild edema and inflammatory cell infiltration 24 h after reperfusion, and the renal interstitials hyperemia, the tubular lumen mild expansion, exfoliated tubular cells and cast were observed accidental in the tubular lumen 48 h after reperfusion. In group RW and group W, the renal histopathologic changes were similar with those in group I/R.3 The apoptosis ratio of renal tissueCompared with group S, the apoptosis ratio at 24 h and 48 h of reperfusion were increased in other four groups(P<0.01). Compared with group I/R, the apoptosis ratio at 24 h and 48 h of reperfusion were decreased in group R(P<0.01),no significant differences were seen in the apoptosis ratio between group RW and group W(P>0.01). Compared with group R, the apoptosis ratio at 24 h and 48 h of reperfusion were increased in group RW and group W(P<0.01).4 The expression of PI3 K m RNA and Akt m RNACompared with group S, the PI3 K m RNA expression was significantly upregulated in group I/R and group R and there were no significant differences in PI3 K m RNA expression between group RW and group W(P>0.05). Compared with group I/R, the PI3 K m RNA expression was upregulated in group R and the PI3 K m RNA expression was significantly downregulated in group RW and group W(P<0.01). There were no significant differences in Akt m RNA expression among the 5 goups(P>0.05).5 The expression of Akt and phosphorylated AktThere were no significant differences in total Akt among the 5 goups(P>0.05). Compared with group S, the phosphorylated Akt and p-Akt/Akt expression was significantly upregulated in group I/R and group R and there were no significant differences in phosphorylated Akt and p-Akt/Akt expression between group RW and group W(P>0.05). Compared with group I/R, the phosphorylated Akt and p-Akt/Akt expression was upregulated in group R, the phosphorylated Akt and p-Akt/Akt expression was significantly downregulated in group RW and group W(P<0.01).Conclusion: The PI3K/Akt signialing pathaway activation is involved in the protective effect of remifentanil on renal tissue cellular apoptosis induced by renal ischemia/reperfusion.