Expression Chang Of EC-SOD In Brain Of Renal Ischemia-Reperfusion Injury Rats

The primary function of the kidney is maintenance of body homeostasis by regulating tubular reabsorption of water,ions,glucose,nutrients and removal of waste metabolic products via glomerular filtration.Therefore,the other organs in body can also be seriously affected when function and structure of kidney is damaged.Studies showed that function and structure of liver,lung,heart,brain and intestine will be severely damaged when kidney function is damaged.Renal ischemia-reperfusion injury?RIRI?is frequently seen in clinical surgery,such as the treatment of hypertension,renal transplantation and renal artery occlusion and so on,Which is one of important factors that leads to acute renal injury,delayed graft function and acute renal failure.RIRI has a high incidence and mortality and can seriously affects the patient's prognosis.So far,the exact pathophysiological mechanism of ischemia reperfusion injury is not unkown.But research shows that: Renal ischemia reperfusion injury is caused by a sudden transient interruption of blood supply to the kidney and then reperfusion.during the process of hypoxia / reperfusion,the kidney will be in a state of high oxidative stress and then seriously affect the renal function.Therefore,oxidative stress is considered to be one of the important factors that cause renal ischemia reperfusion injury.The superoxide dismutases?SOD?are a family of enzymes that play a pivotal role protecting tissues from damage by oxidant stress by scavenging superoxide anion,which prevents the formation of other more potent oxidants such as peroxynitrite and hydroxyl radical.Extracellular SOD?EC-SOD?is found predominantly in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS.EC-SOD has been shown to be protective in several models of interstitial lung disease,including pulmonary fibrosis.In addition,alterations in EC-SOD expression are also present in human idiopathic pulmonary fibrosis.Brain is highly sensitive to ischemia and hypoxia,When renal ischemiareperfusion injury occurs and kidney was in high oxidative stress state,whether brain tissue was also in oxidative stress state and suffered oxidative damage?The expression of EC-SOD how to change and whether EC-SOD play antioxidant role during the process?The first,we established Renal Ischemia-Reperfusion Injury model of rats by clipping the left renal artery.After 24 h of reperfusion,we detected the H₂O₂ content,MDA content,m RNA and protein expression change of EC-SOD,And analyze the correlation between MDA content,H₂O₂ content and EC-SOD expression level.To investigate the effect of renal ischemic reperfusion injury on the oxidative stress in brain and the antioxidant effect plaied by EC-SOD during the process,to provide a new way for the prevention and treatment of brain tissue damage induced by RIRIObjective: Observing MDA content,H₂O₂ content,m RNA and protein expression level of EC-SOD,the correlation between MDA content,H₂O₂ content and EC-SOD expression level.To investigate the effect of renal ischemic reperfusion injury on the oxidative stress in brain and the antioxidant effect plaied by EC-SOD during the process.Methods:1 The preparation of RIRI models,animals group and assay sampleThe male Wistar rat weighting 200±10g were divided randomly into Renal Ischemia-Reperfusion Injury?RIRI?group and control group?Con?.There are 6 rats in each group.Renal Ischemia-Reperfusion Injury model of rats was established according to the method of YU Xiao-Dong et al.First the rats were anesthetized with 6% chloral hydrate?5ml/kg?,We exposed the kidneys of RIRI group rats,first removed the right kidney,then separated the left renal artery and cliped the left renal artery with non-damage vascular clamp near the renal hilum.We could see that the colour of kidney became gradually dark red from bright red.Removed the vascular clamp after 45 mins and restored the blood supply.The colour of kidney quickly became bright red from dark red again which showed that the reperfusion was successful.The control group rats were only removed the right kidney and separated the left renal artery,but not cliped the left renal artery.After 24 hours,the blood of rats was collected 5ml and was centrifuged for 10 mins at 3000 rpm in order to isolate the serum.The serum Creatinine?SCr?and Blood Urea Nitrogen?BUN?were determinated.The rats were harvested the left kidney and brain.The left kidney was fixed with 4% paraformaldehyde for HE staining and observing the morphological of kidney.The brain was placed in liquid nitrogen for the determination the content of MDA,H₂O₂,and the m RNA expression level,protein level of EC-SOD.2 The observation index and its method2.1 The determination of the serum SCr and BUNThe serum SCr level was measured by the picric acid method.The serum BUN level was measured with enzyme-coupled rate method2.2 The morphological and structure of kidney were observed by HE stainingThe left kidney sample was dehydrated with alcohol,transparent with dimethylbenzene.After it was embedded in paraffin,cranked out the 5 micron thick section,HE stained,then observed the morphological and structure of the kidney by Olympus light microscope.2.3 The preparation of rats brain homogenate and the determination of MDA contentThe iced brain tissue was taken out from the-80 refrigerator and was quickly homogenized with 10mg/100?l homogenate buffer?1mmol/L Benzamidine,p H7.4,50mmol/LKPB,1mmol/LPMSF,0.5mol/L Na Cl,0.1% Tween-20,1mmol/L EDTANa3,?-Mercaptoethanol?.The homogenate was centrifuged with 4000rpm?20min,4??,Then collected the supernatant to preparate the 10% brain homogenate.The MDA content in 10% brain homogenate was determined by the Nanjing Jiancheng assay kit.2.4 The preparation of rats brain homogenate and the determination of H₂O₂ contentThe iced brain tissue was taken out from the refrigerator of-80 was quickly homogenized with 10mg/100?l homogenate buffer?1mmol/L Benzamidine,p H7.4,50mmol/LKPB,1mmol/LPMSF,0.5mol/L Na Cl,0.1% Tween-20,1mmol/L EDTANa3,?-Mercaptoethanol?.The homogenate was centrifuged with 4000rpm?20min,4??,Then collected the supernatant to preparate the 10% brain homogenate.The H₂O₂ content in brain homogenate was determined by the Molybdate colorimetric method,and was expressed with H₂O₂ content in the each gram of sample protein?pro mmol/g?.2.5 The determination of the EC-SOD m RNA level in rats brainTotal RNA were extracted from the brain tissue by the trizol method.About 3?g total RNA were reverse transcribed into c DNA.After PCR with GAPDH as the internal reference.The ratio of EC-SOD to GAPDH amplification products was represented the relative m RNA expression levels.2.6 The determination of the EC-SOD protein level in rats brainThe protein level of the EC-SOD in rats brain was estimated by Western Blot.The rats brain tissue were homogenized and collected the supernatant after the centrifugation.The total protein was determined with the modified Lowry method.The amount of loading protein was 62 ug.After electrophoresis,transfering film and sealing treatment,The antibody of anti-rabbit EC-SOD was added to the PVDF membrane.The PVDF membrane was held for overnight with room temperature.Then antibody of anti-rabbit Ig G with the Fluorescence labeled was added again.Then the image was scaned by two-color infrared imaging systems then analyzed images value.2.7 The correlation analysis between MDA content,H₂O₂ content and EC-SOD expression levelThe correlation between MDA content,H₂O₂ content and EC-SOD expression level were analysed with Pearson correlation analysis.Results: 1 The morphology change of kidneyunder light microscope,The structure and shape of renal capsule,glomerulus,proximal tubule,distal convoluted tubule and collecting duct of control group were neat.But the glomerulus of RIRI group was shrinking.The size of glomerular became smaller.Renal capsule cavity was expanded.Lumen of tubular was also expanded obviously.Some epithelial cells of proximal tubule showed edema change and their cytoplasm became loose.Renal stroma also showed edema change.The gap between tubular was expanded.Lumen of collecting duct was expanded and their epithelial cell showed edema change.2 The level of serum SCrThe serum SCr of RIRI group?131.153±17.814?mol/L?was significantly higher than that of control group?103.444±8.465?mol/L?,P<0.05.3 The BUN level in the serumThe serum BUN was 13.685±4.397mmol/L in the RIRI group,was 4.462±0.541mmol/L in the control group,the RIRI group was significantly higher than the control group,P<0.05.4 The MDA content in the brain homogenateThe MDA content of brain homogenate was 11.67±2.33mmol/g in the RIRI group,was 7.67±1.77mmol/g in the control group,RIRI group was significantly higher than control group,P<0.01.5 The H₂O₂ content in the brain homogenateThe H₂O₂ content brain homogenate was 30.68±3.07mmol/g in the RIRI group,was 20.41±2.36mmol/g in the control group,RIRI group was significantly higher than control group,P<0.01.6 The expression level of the EC-SOD m RNA in brainThe expression level of the EC-SOD m RNA in brain were determined by RT-PCR?The expression level of the EC-SOD m RNA was 0.94±0.12 in the RIRI group,was 0.57±0.11 in the control group,RIRI group was significantly higher than control group,P<0.01,The result showed that the gene expression of the EC-SOD in the RIRI group were enhanced.7 The protein level of the EC-SOD in brainThe protein level of the EC-SOD was estimated by Western Blot.The protein level of the EC-SOD was 0.71±0.08 in the RIRI group,was 0.49±0.08 in the control group,RIRI group was significantly higher than control group,P<0.01,The result showed that the protein level of EC-SOD in the RIRI group were increased.8 The correlation between MDA content,H₂O₂ content and EC-SOD expression levelBetween the MDA content and EC-SOD m RNA level?r=0.629,P<0.05?,MDA content and EC-SOD protein level?r=0.651,P<0.05?,H₂O₂ content and EC-SOD m RNA level?r=0.832,P<0.01?,H₂O₂ content and EC-SOD protein level?r=0.791,P<0.01?were positive correlation.Conclusion: 1 Renal ischemia reperfusion injury causes the brain to be in a high degree of oxidative stress and suffer from oxidative damage.2 after Renal ischemia reperfusion injury,the gene and protein expression of EC-SOD were significantly enhanced,and the correlation between MDA content,H₂O₂ content and EC-SOD expression level were positive correlation,which showed that EC-SOD may play the anti-oxidative stress role during the process.