Expression Chang Of EC-SOD In Kidney Of Renal Ischemia-reperfusion Injury Rats

Renal Ischemia-Reperfusion Injury（RIRI）is a common clinical pathological physiological reaction.it is often occurred during the clinical treatment such as kidney transplantation or acute renal artery occlusion and so on.RIRI is also an important factor that leads to delayed function recovery after kidney transplantation,even acute renal failure and increased mortality.The kidney will produce a lot of reactive oxygen species（ROS）when the blood flow to kidneys was temporaryly blockd,then restored its blood and oxygen supply,which lead to the high oxidative stress status of kidney.This is one of important mechanisms for ischemia-reperfusion injury.Members of ROS include superoxide anion（O2-）,hydroxyl radical（OH）,hydrogen peroxide（H₂O₂）and so on.ROS are very reactive and can peroxide react with function proteins,DNA in the cell and lipids on cell membrane,which can promote cell apoptosis and death,aggravate kidney tissue damage.Superoxide dismutase（SOD）is the one of important antioxidant enzymes which is responsible for the removal of O2-,It can convert O2-to oxygen,but at the same time,the byproduct H₂O₂ was produced.SOD plays an important role in clearing ROS and antioxidant reaction in body.There are three subtypes of SOD in mammals.Manganese-SOD（Mn-SOD）was located in mitochondria,the Copper / zinc-SOD（Cu/Zn-SOD）was mainly located in cytoplasm and nucleus.Extracellular superoxide dismutase（extracellular,SOD,EC-SOD）is the only one subtype of SOD located outside the cell.It is binded with the cell surface and extracellular matrix through the heparinbinding domain（HBD）.Secondly,EC-SOD is also present in the extracellular fluid,such as serum,cerebrospinal fluid,synovial fluid,ascites and so on.The previous studies about SOD functions were mainly focused on the antioxidant effect of Mn-SOD and Cu / Zn-SOD.But recent studies show that EC-SOD may have a stronger anti-oxidative stress and anti-inflammatory effects.How to change of EC-SOD in RIRI model and if EC-SOD has antioxidant function,which need to be researched.We established Renal Ischemia-Reperfusion Injury model of rats by clipping the renal artery with non-damage vascular clamp.After 24 h of reperfusion,we observed the H₂O₂ content and MDA content in kidney,gene and protein expression change of EC-SOD,to confirm the oxidative stress mechanism of RIRI,to explore the antioxidant effect of EC-SOD during Kidney ischemia-reperfusion injury,Which will provide a new way for prevention and treatmemt of Renal Ischemia-Reperfusion Injury.Objective: Observing the oxidative stress level of RIRI model rat after Renal Ischemia-Reperfusion Injury occurrence and expression changes of antioxidant enzyme EC-SOD mRNA and protein,to confirm the oxidative stress mechanism of RIRI and antioxidant role which may be played by EC-SOD during this process.Methods: 1 Animals and preparation of Renal Ischemia-Reperfusion Injury modelsMale Wistar rat weighting 200±10g were purchased from the Experimental Animal Center of Hebei Medical University.The rats were divided randomly into control group（Con）and Renal Ischemia-Reperfusion Injury（RIRI）group.There are 6 rats in each group.First the rats were anesthetized with 6% chloral hydrate,then we established Renal Ischemia-Reperfusion Injury model of rats according to the method of YU Xiao-Dong et al.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 was collected and centrifuged for 10 mins at 3000 rpm to isolate the serum for determination of Serum Creatinine（SCr）and Blood Urea Nitrogen（BUN）.The rats were killed and harvested the kidney.One part of kidney was fixed with 4% paraformaldehyde for HE staining and observing the morphological changes.The other part of kidney was placed in liquid nitrogen for the determination of mRNA expression level,protein level of EC-SOD,to detecte the MDA and H₂O₂ content in kidney.2 The index and methods 2.1 The determination of serum SCr and BUNThe SCr level in serum was measured using the picric acid method.The BUN level in serum was measured by enzyme-coupled rate method.2.2 The morphological observation of the kidney by HE stainingThe kidney sample were dehydrated,transparent.embedded in paraffin,cranked out 5 micron thick common section,HE stained,then observed the morphological change of kidney by light microscope 2.3 The determination of MDA content in kidneyThe iced kidney tissue taken out from-70 refrigerator were quickly homogenized with 10mg/100μl homogenate buffer（50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L Na Cl,1mmol/L EDTANa3 β-Mercaptoethanol）.The homogenate was centrifuged at 4000rpm（20min,4℃）,Then collected the supernatant to preparate the 10%iced kidney tissue homogenate.The MDA content in 10% iced kidney tissue homogenate was determined by Nanjing Jiancheng assay kit.2.4 The determination of H₂O₂ content in kidney tissueThe iced kidney tissue taken out from-70 refrigerator were quickly homogenized with 10mg/100μl homogenate buffer（50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L NaCl,1mmol/L EDTANa3 β-Mercaptoethanol）.The homogenate was centrifuged at 4000rpm（20min,4℃）,Then collected the supernatant to preparate the 10%kidney tissue homogenate.The H₂O₂ content in kidney tissue homogenate was determined by Molybdate colorimetric method and expressed in amount of hydrogen peroxide in per gram sample（mmol/g pro）2.5 The determination of mRNA level of EC-SOD in kidney tissueThe total RNA were extracted with Trizol,About 3μg total RNA was reverse transcribed into cDNA then RT-PCR,using GAPDH as internal control.The ratio of amplification products of EC-SOD to GAPDH represents the relative mRNA expression levels.2.6 The determination of protein level of EC-SOD in kidney tissueThe protein level of EC-SOD was estimated by Western Blot.The rat kidney tissue was homogenized and collected the supernatant after centrifugation.The total protein was determined with the modified Lowry method.The amount of loading protein in electrophoresis was 63 ug.The antirabbit EC-SOD antibody was added to the PVDF membrane after transfer film and closed process.The PVDF membrane was stood for overnight at room temperature.Then the anti-rabbit IgG antibody labeled by Fluorescence was added again.Then the image was scaned with two-color infrared imaging systems to analyzed images value.Results:1 The morphology change of kidney under light microscopeThe structure and shape of glomerulus,renal capsule,proximal tubule,distal convoluted tubule and collecting duct of control group were neat under light microscope.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 SCr in serumThe serum SCr of control group was 103.444±8.465μmol/L,The serum SCr of RIRI group was 131.153±17.814μmol/L.The serum SCr levels of RIRI group was significantly higher than that of control group（P<0.05）.The serum BUN of control group was 4.462±0.541 mmol/L,The serum BUN of RIRI group was 13.685±4.397 mmol/L.The serum BUN levels of RIRI group was significantly higher than that of control group（P<0.05）.4 The MDA content in kidney tissue homogenateThe MDA content in kidney tissue of control group was 9.15± 1.53 mmol /g,The MDA content in kidney tissue of RIRI group was 12.92± 2.43 mmol/g,The MDA content in kidney tissue of RIRI group was significantly higher than that of control group（P<0.01）.5 The H₂O₂ content in kidney tissue homogenateThe H₂O₂ content in kidney tissue of control group was 11.92±1.9mmol/g,The H₂O₂ content in kidney tissue of RIRI group was 16.62±2.35 mmol/g,The H₂O₂ content in kidney tissue of RIRI group was significantly higher than that of control group（P<0.01）.6 The relative expression of EC-SOD mRNA in kidney tissue.The relative expression of EC-SOD mRNA in kidney tissue were determined by RT-PCR.The expression level of EC-SOD m RNA of control group were 0.67±0.12,the expression level of EC-SOD mRNA of RIRI group were 0.94±0.17,The expression level of EC-SOD mRNA of RIRI group was significantly higher than that of control group（P<0.01）.The result showed that the gene expression of EC-SOD in kidney tissue of RIRI group were enhanced.7 The protein level of EC-SOD in kidney tissueThe protein level of EC-SOD in control group was 0.51±0.09,the protein level of EC-SOD in RIRI group was 0.81±0.15.The protein level of EC-SOD of RIRI group was significantly higher than that of control group（P<0.01）.The result showed that the protein level of EC-SOD in kidney tissue of RIRI group were increased.Conclusion:1 The Renal Ischemia-Reperfusion Injury model of rats can be successfully established by clipping left renal artery with non-damage vascular clamp near the renal hilum.3 The level of BUN in serum2 kidney tissue was also in a high level of oxidative stress after renal ischemia reperfusion injury,which caused the peroxide damage of kidney tissue3 The gene and protein expression of EC-SOD in kidney tissue of renal ischemia reperfusion injury modle were significantly enhanced,which showed that EC-SOD may be involved in the oxidative stress response of kidney tissue induced by ischemia-reperfusion and play an antioxidative effect in kidney tissue...