| Background：Ischaemic injury to vital organs such as the heart, brain and kidneyscontributes significantly to morbidity and mortality throughout the world.Renal ischaemia as a consequence of arterial occlusion, shock and organtransplantation is a common cause of renal cell death, renal failure,delayed graft function and renal graft rejection.After an acute renal ischaemic event, early reperfusion remains themost effective strategy to limit organ damage. However, reperfusion ofthe kidney has the potential to cause lethal cell death similar to thatobserved in the heart. Novel protective strategies applied at the time of reperfusion are required to target this injury. Preconditioning maneuver isthe possibility of rendering an organ resistant to subsequent injury by aprior ischaemic insult. Ischaemic preconditioning of the kidney confersprotection against a subsequent ischaemic attack.Nevertheless, the protective potential of ischaemic preconditioninghas not been realized in clinical practice because it necessitates anintervention applied before the onset of ischaemia, which is often difficultto predict in non-surgical situation. A more amenable approach is tointervene at the onset of reperfusion, the timing of which is under thecontrol of the operator. Recently, Zhao et al. showed that repetitive cyclesof short ischaemia during reperfusion significantly reduce heart infarctsize in a canine model. Postconditioning is a mechanical maneuverimposed during the early moments of reperfusion that attenuatesendothelial activation and dysfunction and apoptosis. Formation of reactive oxygen species (ROS) and oxidant stress arethe most invoked disease mechanisms in ischemia–reperfusion injuryincluding hepatic ischemia–reperfusion. In order to successfully targetROS, it is necessary to consider the specific ROS involved, the sourcesgenerating ROS in what particular location, at what time in thepathogenesis, and how much oxidant stress is generated. In addition, it iscritical to understand by what mechanism ROS are actually causing celldeath and organ injury and how relevant the experimental models are forthe human conditions. The current review focuses on these differentaspects of the pathophysiology and the attempts targeting ROS as strategyto limit reperfusion injury.Methods：Male Sprague-Dawley rats were randomly assigned tosham-operation (Sham), ischemia-reperfusion (I/R), postconditioning(POC) and POC with5-HD (5-HD) groups with eightrats per group:group Sham, the abdominal cavity was opened and bilateral kidneyswere separated and exposed for45minutes, no renal ischemia.group I/R, rats were subjected to right nephrectomy and45-min leftkidney occlusion, then the the blood flow was restored withoutintervention.group POC, rats were subjected to right nephrectomy and45-minleft kidney occlusion, complete reperfusion was preceded by7min,30seconds of reperfusion, and30seconds of ischemia, repeated three times.group5-HD,5-HD (10mg/kg) was injected intraperitoneally intoPOC rats10minutes before reperfusion.5-HD（5-hydroxydecanoate）isthe inhibitor of mitoKATP.The roles of mitoKATP were investigated by analyzing fluorescence intensities of mitochondria, mitochondrial membrane potential andintracellular reactive oxygen species (ROS) using appropriate fluorescentmarkers. The relationship between apoptosis and ischemia reperfusioninjury was assessed by TUNEL of kidney tissue.Results：The I/R model was shown to be successful. Significantly higherlevels of creatinine, and abnormal pathology of histologic sections, wereobserved in group I/R, compared with group Sham.5-HD eliminated therenoprotective effects of POC. ROS fluorescence intensities decreased ingroup POC, compared with group I/R and group5-HD. This experimentprepared by renal ischemia reperfusion injury in the rats model to observethe effect of POC on the protection of ischemia-reperfusion injury andoptimization of POC conditions. Learn to compare structure and kidneyfunction from the kidney, and draw the survival curves of each model. The results found that the severe damage occurred in the kidney of I/Rrats, renal tubular epithelial cell swelling, vacuolar degeneration, nuclearsloughed off into the lumen, protein cast and granular casts.The rate of mitochondrial peroxide production was higher in thecontrol group than in the protected group at the end of postconditioningreperfusion. Moreover, mitochondrial protein oxidation was significantlyhigher in control rats than in the postconditioning group at the end ofreperfusion. The experiment found that POC can reduce ROS in thekidney of I/R rats, maintain the mitochondrial membrane potential, helpmitochondria against oxidative stress injury, to explore the relationshipbetween mitochondrial DNA oxidative damage and apoptosis. Detectionof the apoptosis pathway protein Caspase3, results in the Sham group didnot express, group I/R3positive expression of Caspase3, POC group ofweak expression,5-HD positive expression. Expression of renal tissue cell ROS detection, Sham group did not express ROS, there is a largeamount of ROS in I/R group and5-HD group, renal tubular epithelialcells in group POC, ROS production is very small. Detection of DNAoxidative damage marker protein8-OhDG, Sham group and POC groupwas not expressed, whereas I/R group and5-HD positive expression, andthe expression in cytoplasm.The fluorescence intensities of Kir6.2increased in group POC,compared with grouo Sham, group I/R and group5-HD. Through theTUNEL method comparison model the degree of apoptosis. The resultsshowed that the I/R group and the Sham group only a handful of cellapoptosis, a small amount of cell apoptosis in POC group, while a largenumber of cells of the kidney tissue in group5-HD apoptosis. Preparationof I/R group model with multiple time points, by doubleimmunofluorescence staining technique to investigate the relationship between DNA oxidative damage and apoptosis mitochondria. The resultsfound after ischemia reperfusion with expression of8-OhDG graduallyincreased, while in the3hours before the TUNEL results showed no cellapoptosis,6hours a few8-OhDG positive cells apoptosis, apoptotic cellsincreased gradually along with the time. By immunofluorescence andWestern Blot detection of Kir6.2, Sham in group Kir6.2showed generallylow level expression, the positive expression of POC in group Kir6.2, theexpression of I/R and5-HD group, Kir6.2levels were lower in the POCgroup.Conclusions：In the present study, excessive mitochondiral ROS production wasparamount in reperfusion injury which might impair mitochondrialmembrane potential and renal tubular cell apoptosis. Blocking ROSproduction by POC strategy would ameliorate all parameters of renal injury.Kir6.2and mitoKATP participated in POC-induced renoprotectivemechanisms in rat kidneys subjected to ischemia reperfusion injury,possibly through decreased renal tubular epithelial cell ROS production. |
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