Expression Chang Of Prx ? In Cardica Muscle Of Renal Ischemia-Reperfusion Injury Rats

The kidneys is the important organ for excretion and secretion in the body.It play an important role for maintaining acid-base balance and potassium,sodium,calcium and other electrolyte stability.So the kidney is very important for maintaining the internal environment balance of the body.Studies found that the function of remote organs and neighbors organs of kidney such as liver,lung,heart,brain and intestine will be damaged when the function of kidney was damaged.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(O^2-),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.Peroxiredoxin?Prx?discovered in recent yearsis is one kind of peroxidase system,which can remove ROS.Prx? is one member of the Peroxiredoxin family.Prx? is only one enzyme protein in the whole family which was located in mitochondria.The study found that Prx? was produced in Endoplasmic reticulum and then located in the mitochondria by the positioning signal.The mitochondria are the main source of ROS in the cell,are also the primary target for ROS.Recent years,mamy studies found that Prx? has a close relationship with H₂O₂ removal in cellular mitochondria.Prx? may be the important cleaner of ROS in mitochondria.The heart has a great need for oxygen,so it is highly sensitive to ischemia and hypoxia reaction in the body.ATP produced by mitochondrial oxidative phosphorylation is the main energy source of myocardial cells.During the process,there will be a large number of electrons leaked from respiratory chain to form ROS.So,the heart is the most vulnerable to ROS peroxide damage in the body.Whether the heart was peroxidation damaged and oxidative stress caused myocardial damage is also one of the main mechanisms when RIRI happened and the kidney was in highly oxidative stress state.How to change of the Prx? expression in myocardial tissue and whether Prx? participate in oxidative stress process.It is not reported.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 heart,gene and protein expression change of Prx?,to explore the oxidative stress mechanism of myocardial tissue damage induced by RIRI and the antioxidant effect of Prx? during Kidney ischemia-reperfusion injury,Which will provide a new way for prevention and treatmemt of myocardial tissue damage induced by Renal Ischemia-Reperfusion Injury.Objective:Observing the oxidative stress level and expression changes of antioxidant enzyme Prx? mRNA and protein in cardiac muscle tissue after renal ischemia-reperfusion injury,to investigate the oxidative stress mechanism of myocardial injury induced by renal ischemia reperfusion injury,and antioxidant role which may be played by Prx? 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 and heart.the kidney was fixed with 4% paraformaldehyde for HE staining and observing the morphological changes.The heart was placed in liquid nitrogen for the determination of mRNA expression level,protein level of Prx?,to detecte the MDA and H₂O₂ content in heart.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 myocardial tissue.The iced myocardial tissue taken out from-70 refrigerator were quickly homogenized with 10mg/100?l homogenate buffer?50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L NaCl,1mmol/L EDTANa3 ?-Mercaptoethanol?.The homogenate was centrifuged at 4000rpm?20min,4??,Then collected the supernatant to preparate the 10%iced myocardial tissue homogenate.The MDA content in 10 % iced myocardial tissue homogenate was determined by Nanjing Jiancheng assay kit.2.4 The determination of H₂O₂ content in myocardial tissue.The iced myocardial tissue taken out from-70 refrigerator were quickly homogenized with 10mg/100?l homogenate buffer?50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L NaCl,1mmol/L EDTANa3 ?-Mercaptoethanol?.The homogenate was centrifuged at 4000rpm?20min,4??,Then collected the supernatant to preparate the 10%myocardial tissue homogenate.The H₂O₂ content in myocardial 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 Prx? in myocardial tissueThe total RNA were extracted with Trizol,About 3?g total RNA was reverse transcribed into cDNA then RT-PCR,using GAPDH as internal control.The ratio of amplification products of Prx? to GAPDH represents the relative mRNA expression levels.2.6The determination of protein level of Prx? in myocardial tissueThe protein level of Prx? was estimated by Western Blot.The rat myocardial 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 62 ug.The antirabbit Prx? 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.Renal stroma showed edema change.The gap between tubular was expanded.Lumen of collecting duct was expanded.2 The level of SCr in serumThe serum SCr of control group was 103.444±8.465?mol/L,The serum SCr of RIRI group was 131.153±17.814?mol/L.The serum SCr levels of RIRI group was significantly higher than that of control group?P <0.05?.3 The level of BUN in serumThe 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 myocardial tissue homogenateThe MDA content in myocardial tissue of control group was 10.18±1.77mmol/g,The MDA content in myocardial tissue of RIRI group was 14.01±2.29 mmol/g,The MDA content in myocardial tissue of RIRI group was significantly higher than that of control group?P <0.01?.5 The H₂O₂ content in myocardial tissue homogenateThe H₂O₂ content in myocardial tissue of control group was 13.93±1.88mmol/g,The H₂O₂ content in myocardial tissue of RIRI group was 18.56±2.56 mmol/g,The H₂O₂ content in myocardial tissue of RIRI group was significantly higher than that of control group?P <0.01?.6 The relative expression of Prx? mRNA in myocardial tissue.The relative expression of Prx? mRNA in myocardial tissue were determined by RT-PCR.The expression level of Prx? mRNA of control group were 0.95±0.17,the expression level of Prx? mRNA of RIRI group were 1.34±0.18,The expression level of Prx? mRNA of RIRI group was significantly higher than that of control group?P <0.01?.The result showed that the gene expression of Prx? in myocardial tissue of RIRI group were enhanced.7 The protein level of Prx? in myocardial tissueThe protein level of Prx? in control group was 0.58±0.09,the protein level of Prx? in RIRI group was 1.01±0.17.The protein level of Prx ? of RIRI group was significantly higher than that of control group?P <0.01?.The result showed that the protein level of Prx? in myocardial 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.2 Myocardial tissue was also in a high level of oxidative stress after renal ischemia reperfusion injury,which caused the peroxide damage of myocardial tissue.3 The gene and protein expression of Prx ? in myocardial tissue of renal ischemia reperfusion injury modle were significantly enhanced,which showed that Prx? may be involved in the oxidative stress response of myocardial tissue induced by ischemia-reperfusion and play an antioxidative effect in myocardial tissue.