Expression Chang Of Prx6 In Brain Of Renal Ischemia-Reperfusion Injury Rats

The kidney is not only involved in the metabolic wastes, but also participate inhormone secretion and maintain water-electrolyte and acid-base balance. So the dysfunction of kidney will inevitably lead to metabolism and function change of other vital organs. Studies have shown that neighbors and the remote organs of kidney such as heart, lung, liver, intestine and brain function will be severely damaged when kidney function is damaged.Renal ischemia-reperfusion injury(RIRI) is a common pathological physiological phenomena. RIRI often happens in high blood pressure, kidney surgery and so on. RIRI is also an important factor that leads to delayed function recovery after kidney transplantation, poor outcome even acute renal failure. Studies have shown that oxidative stress is one of main mechanisms of kidney ischemia-reperfusion injury. The kidney is in high degree oxidative stress status during ischemia-reperfusion process and kidney will produce large amounts of reactive oxygen species(ROS). ROS include superoxide anion(O2-), hydroxyl radical(OH), hydrogen peroxide and so on. ROS are very reactive and can peroxide reactive with the intracellular biological macromolecules such as protein, DNA and lipid, which can affect cell function, even cause cell and tissue death.Peroxiredoxin is one kind of peroxidases which was found in recent years. It widely exists in various kinds of prokaryotic and eukaryotic cells. Prx6 is one of the members of the family, it expressed in multiple tissues, the maximum expression level is in lung and brain. Studies have shown that Prx6 has the biological activity of glutathione peroxidase. So its main function is responsible for the reduction of H2O2 and phospholipid peroxides.Brain is highly sensitive organ for the oxygen content in the body. Whether the brain tissue will be subject to oxidative stress injury when Renal ischemia-reperfusion injury occurs, the gene and protein level of antioxidant enzymes Prx6 how to change. These questions were not reported.We first 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 MDA content in brain, m RNA and protein expression of Prx6, to explorethe peroxide damage degree of brain, the antioxidant effect of Prx6 during Renal ischemia-reperfusion injury, Which will provide a new way for prevention and treatmemt of brain damage induced by Renal Ischemia- Reperfusion Injury.Objective: Observing the oxidative stress state and expression changes of Prx6 m RNA and protein in brain of renal ischemia-reperfusion injury model, to investigate the role of Prx6 in peroxidation damage induced by RIRI.Methods:1 Animals and preparation of Renal Ischemia-Reperfusion Injury models Male Wister 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 IschemiaReperfusion 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 nondamage 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), Blood Urea Nitrogen(BUN). The rats were killed and harvested the kidney and brain. The kidney was fixed with 4% paraformaldehyde for HE staining and observing the morphological changes. brain was placed in liquid nitrogen for the determination of m RNA expression level, protein level of Prx6, to detecte the MDA content in brain. 2 The index and methods 2.1 The determination of serum SCr and BUN The serum SCr level 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 staining The kidney sample was 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 brain The iced brain tissue were quickly homogenized with 10mg/100μl homogenate buffer(50mmol/LKPB, p H7.4, 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%brain homogenate. The MDA content in 10%brain homogenate was determined by Nanjing Jiancheng assay kit. 2.4 The determination of m RNA level of Prx6 in brain The total RNA were extracted with Trizol, About 3μg total RNA was reverse transcribed into c DNA then RT-PCR. The ratio of amplification products of Prx6 to GAPDH represents the relative m RNA expression levels. 2.5The determination of protein level of Prx6 in brain The protein level of Prx6 was estimated by Western Blot. The rat brain 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 60 ug. The Prx6 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 Ig G 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 microscope The 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 serum The 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). 3 The level of BUN in serum 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 brain homogenate The MDA content in brain of control group was 7.67±1.77mmol/g, The MDA content in brain of RIRI group was 11.67±2.33 mmol/g, The MDA content in brain of RIRI group was significantly higher than that of control group(P<0.01). 5 The relative expression of Prx6 m RNA in brain The relative expression of Prx6 m RNA in brain were determined by RTPCR. The expression level of Prx6 m RNA of control group were 0.69±0.16, the expression level of Prx6 m RNA of RIRI group were 1.08±0.19, The expression level of Prx6 m RNA of RIRI group was significantly higher than that of control group(P<0.01).The result showed that the gene expression ofPrx6 in brain tissue of RIRI group were enhanced. 6 The protein level of Prx6 in brain The protein level of Prx6 in control group was 0.73±0.13, the protein level of Prx6 in RIRI group was 1.12±0.19. The protein level of Prx6 of RIRI group was significantly higher than that of control group(P<0.01). The result showed that the protein level of Prx6 in brain tissue of RIRI group was increased.Conclusion:1The 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 Renal ischemia-reperfusion could lead to oxidative stress injury of brain and the gene and protein expression of Prx6 were significantly enhanced, which showed that Prx6 may participate oxidative stress response in brain induced by kidney ischemia-reperfusion and have protective function for brain cells.