| The kidney is the main secretion organs in body. kidney is not only involved in the metabolic wastes, hormone secretion, but also can maintain water-electrolyte and acid-base balance. 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 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 damage proteins, DNA and lipids in the cell, 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. So far, six members of the family were found in mammals. Prx I is one member of the Peroxiredoxin family, the maximum expression level is in liver. Prx I is mainly responsible for the reduction of peroxide and superoxide, protects cells and tissues against oxidative stress injury. Prx I plays an important role in the removal of ROS.The liver is also one of the important organs in the body. It is not only involved in a variety of nutrient metabolic reactions, but also has secretion, detoxification, drainage and other important function. Whether the adjacent organs liver also suffered from oxidative stress injury, the gene and protein expression level of antioxidant enzymes Prx I how to change, when kidney ischemia-reperfusion injury occurs and a large number of ROS were produced, which is 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 serum ALT, morphological changes of liver, MDA content in liver, m RNA and protein expression of Prx I, to explore function change, morphology change, peroxide damage degree of liver, the antioxidant effect of Prx I during Kidney ischemia-reperfusion injury, Which will provide a new way for prevention and treatmemt of liver damage induced by Renal Ischemia- Reperfusion Injury.Objective: Observing the oxidative stress state and expression changes of Prx I m RNA and protein in liver of renal ischemia-reperfusion injury model, to investigate the role of Prx I in peroxidation damage induced by RIRI.Methods: 1 Animals and preparation of Renal Ischemia-Reperfusion Injury modelsMale 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 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), Blood Urea Nitrogen(BUN) and Alanine Aminotransferase(ALT). The rats were killed and harvested the kidney and liver. One part of kidney and liver were fixed with 4% paraformaldehyde for HE staining and observing the morphological changes. One part of liver was placed in liquid nitrogen for the determination of m RNA expression level, protein level of Prx I, to detecte the MDA content in liver. 2 The index and methods 2.1 The determination of serum ALT, SCr and BUNThe serum ALT levels was determined by automatic biochemical analyzer. The 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 liver and kidney by HE stainingThe liver and kidney sample were dehydrated, transparent. embedded in paraffin, cranked out 5 micron thick common section, HE stained, then observed the morphological change of liver and kidney by light microscope. 2.3 The determination of MDA content in liver.The iced liver tissue were quickly homogenized with 10mg/100 μ l 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%liver homogenate. The MDA content in 10%liver homogenate was determined by Nanjing Jiancheng assay kit. 2.4 The determination of m RNA level of Prx I in liverThe 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 Prx I to GAPDH represents the relative m RNA expression levels. 2.5The determination of protein level of Prx I in liverThe protein level of Prx I was estimated by Western Blot. The rat liver 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 58 ug. The Prx I 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 and liver 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. The hepatocyte cords of control group were arranged neatly, dyeing uniformity, no abnormal. The hepatocyte cords of RIRI group were not arranged neatly, dyeing was lighter than that of control group, the liver blood sinus showed a slightly expansion.2 The level of ALT in serumThe serum ALT of control group was 19.8±3.76U/L, The serum ALT of RIRI group was 64.3±7.92 U/L. The serum ALT levels of RIRI group was significantly higher than that of control group(P<0.01).3 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).4 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).5 The MDA content in liver homogenateThe MDA content in liver of control group was 9.03±2.11mmol/g, The MDA content in liver of RIRI group was 16.12±1.89 mmol/g, The MDA content in liver of RIRI group was significantly higher than that of control group(P<0.01).6 The relative expression of Prx I m RNA in liverThe relative expression of Prx I m RNA in liver were determined by RT-PCR. The expression level of Prx I m RNA of control group were 0.75±0.18, the expression level of Prx I m RNA of RIRI group were 1.23±0.21, The expression level of Prx I m RNA of RIRI group was significantly higher than that of control group(P<0.01).The result showed that the gene expression of Prx I in liver tissue of RIRI group were enhanced.7 The protein level of Prx I in liverThe protein level of Prx I in control group was 0.69±0.13, the protein level of Prx I in RIRI group was 1.04±0.16. The protein level of Prx I of RIRI group was significantly higher than that of control group(P<0.01). The result showed that the protein level of Prx I in liver 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 Renal ischemia-reperfusion could lead to oxidative stress injury of liver and the morphological structure and function was damaged.3 The gene and protein expression of Prx I in liver of renal ischemia reperfusion injury modle were significantly enhanced, which showed that Prx I may play an important role in oxidative stress process and have protective function for liver cells. |
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