| Background and objectiveKidney is easily subjected to ischemic reperfusion injury due to its abundance of blood supply. The temporary discontinuation of renal blood supply is encountered in many clinical situations, such as kidney transplantation, partial nephrectomy, renal artery angioplasty, cardiopulmonary bypass, aortic bypass surgery, accidental or iatrogenic trauma, sepsis, hydronephrosis and elective urological operations. Acute renal failure (ARF) resulting from renal ischemia reperfusion injury (IRI) remains a major clinical problem because of its frequent occurrence and high mortality. How to treat the renal IRI is always keeping in attention.Currently, ischemic preconditioning (IPreC) is a well-known method in treating organ IRI. Toosy demonstrated that several short cycles of ischemia and reperfusion before renal ischemia could effectively render the renal resistant to a subsequent ischemic insult, although extensive researches have demonstrated that IPreC reduced renal IRI. However, IPreC is clinically feasible only when the occurrence of ischemia is predictable. It is fail to treat unpredictable accident events. It is need to find out other useful method to amelioration renal IRI.Compared to ischemia, the onset of reperfusion is more predictable. In 2003, Vinten-Johnsen and his colleagues reported that brief episodes of myocardial ischemia and reperfusion employed during reperfusion after a prolonged ischemic insult had been demonstrated to play a cardioprotective role. This phenomenon was termed "Ischemic postconditioning (IPostC)". In a dog model, IPostC limited infarct size, reduced tissue edema and poly morphonuclear neutrophil accumulation in the area at risk myocardium, and improved endothelial function. It has been suggested that IPostC is as effective as IPreC in limiting infarct size and preserving postischemic endothelial function. Numerous studies demonstrated that IPostC was a simple and harmless method which provided a new tool to protect organ from IRI.Therefore, we investigated the protective role and mechanism of ischemic postconditioning against renal IRI in rats and compared the results with IPreC. Our study may not only provide a useful theoretical and practical evidence for reducing renal IRI, but also provide a novel approach for clinic application.MethodsAdult male Sprague-Dawley rats (180～220 g) were randomly separated into four groups: a) Sham group: the right kidneys were removed only; b) IR group: after right nephrectomy, kidneys were subjected to 45 min of ischemia; c) IPreC group: kidneys were subjected to three cycles of 2 min ischemia separated by 5 min reperfusion periods before 45 min of ischemia; d) IPostC group: kidneys were subjected to 45 min of ischemia followed by six cycles of 10 sec reperfusion separated by 10 sec ischemia. Rats were sacrificed and kidneys were harvested after reperfusion. Each group comprised eight rats. The following two aspects were investgated.1. The protective effects of IPostC on renal IRI in rats.Serum Cr and BUN were evaluated by Cr assay kits and BUN assay kits at 24 h after IRI, respectively. The renal pathologic changes were observed by HE-staining at 24 h after IRI.2. The mechanism associated with renal IRIA The antioxidative mechanism of IPostC against renal IRI in ratSOD, CAT, and GSH-Px activity were investigated. GSH and MDA at 1 h, 3 h, 6 h, 12 h, and 24 h were measured. The mRNA and protein expression of SODat 1 h, 6 h and 24 h were evaluated by Real Time RT-PCR and Western blotting.B The antiinflammatory mechanism of IPostC against renal IRI in ratMPO activity at 1 h, 3 h, 6 h, 12 h and 24 h was investigated.The mRNA andprotein expression of SOD at 1 h, 3 h, 6 h, 12 h and 24 h were evaluated by RealTime RT-PCR and Western blotting.C The antiapoptosis mechanism of IPostC against renal IRI in ratApoptosis was compared by TUNEL method; Real time RT-PCR assay for theBcl-2 and Bax mRNA at 6 h was evaluated; Western blotting assay for the proteinexpression of Bcl-2 and Bax at 6 h was evaluated.Results1. The protective effects of IPostC on renal IRI in ratsThe renal function of rats 24 h after IRI was significantly different between groups. Rats subjected to IRI showed significant increase in serum Cr and BUN compared with Sham group (P < 0.05). The renal function changes caused by IRI were significantly improved by IPreC and IPostC treatments (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).Renal IRI resulted in significant renal injury as evidenced by tubular necrosis, medullary hemorrhage, congestion, and development of proteinaceous casts. In contrast, treatments of IPreC and IPostC ameliorated these severe renal damages. According to Jablonski scores, 45 min renal ischemia followed by 24 h reperfusion resulted in severe acute tubular necrosis. Quantitative analysis showed a dramatically decreased score in both IPreC group and IPostC group compared with IRI group (P < 0.05). There were no significant difference between IPreC and IPostC (P> 0.05).2. The antioxidative mechanism of IPostC against renal IRI in ratCompared with Sham group, the I/R group resulted in significant down-regulation of the activities of SOD, CAT, and GSH-Px in renal at 1.5 h, 3 h, 6 h, 12 h and 24 h (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant up-regulation of their activities at each time (P < 0.05); compared with Sham group, the IRI rats resulted in significant increases of the levels of MDA, and decreases of GSH in renal tissue at 1 h, 3 h, 6 h, 12 h and 24 h. While compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the levels of MDA, and increases of GSH at each time in renal tissue (P < 0.05). There were no significant difference between IPreC and IPostC (P> 0.05).The mRNA and protein expression of SOD was evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant down-regulation of SOD mRNA and protein expression in renal (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant up-regulation of SOD mRNA and protein expression at 1 h, 6 h, and 24 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).3. The antiinflammatory mechanism of IPostC on renal IRI in ratscompared with Sham group, the IRI rats resulted in significant increases ofthe MPO activity in renal tissue at 1 h, 3 h, 6 h, 12 h and 24 h (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the MPO activitiy at each time (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).The mRNA and protein expression of COX-1 and COX-2 were evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant up-regulation of COX-2 expression in renal (P < 0.05); while Compared with I/R group IPreC and IPostC treatments resulted in significant down-regulation of COX-2 expression at 1.5 h, 3 h, 6 h, 12 h and 24 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).4. The antiapoptosis mechanism of IPostC against renal IRI in ratcompared with Sham group, the IRI rats resulted in significant increases of the apoptosis cell in renal tubular at 6 h and apoptosis index (AI) was significantly increased (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the apoptosis cell and AI was significantly decreased (P < 0.05) in renal tubular at 6 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).The mRNA and protein expressiones of Bcl-2 and Bax were evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant down-regulation of Bcl-2 mRNA and protein expression, and up-regulation of Bax mRNA and protein expression in renal and increase of Bcl-2/Bax (P < 0.05); while compared with I/R group IPreC and IPostC treatments resulted in significant up-regulation of Bcl-2 and down-regulation of Bax expression in renal (P < 0.05) and increase of Bcl-2/Bax. There were no significant difference between IPreC and IPostC (P > 0.05).Conclusions1. The function and structure of renal tissue were damaged by renal ischemic reperfusion in rats with the increase of the level of Cr and BUN, the damage of renal tubula and glomerulum, the elevation of MDA and MPO, the activity decreasing of GSH,SOD,CAT and GSH-Px, the decreasing of SGD mRNA and protein expression, the elevation of COX-2 mRNA and protein expression, the increasing of cell apoptosis, the decreasing of Bcl-2 mRNA and protein expression, and the increasing of Bax mRNA and protein expression.2. Postconditioning was effectively protected kidney against IRI with the decrease of the level of Cr and BUN, and the improvement of the renal morphological features.3. The mechanism of ischemic postconditioning against renal IRI may be related to antioxidative effect on the reducing the free radical, inhibiting lipid peroxidation, increasing the activity of antioxidative enzyme and SOD mRNA and protein expression, which improve antioxidative ability of kidney. 4. The mechanism of ischemic postconditioning against renal IRI may be related to anti-inflammatory effect on the inhibiting of PMN aggregation, the mRNA and protein expression of COX-2, and the producing of inflammatory mediator.5. The mechanism of ischemic postconditioning against renal IRI may be associated to antiapoptosis effect by up-regulating Bcl-2 mRNA and protein expression and down-regulating Bax mRNA and protein expression.6. The protective effect and mechanism of postconditoning against renal IRI were similar to that of ischemic preconditioning.7. This study may not only provide a novel approach for clinic application, but also a useful theoretical and practival evidence for treating renal IRI.
|
PDF Full Text Request