| It is necessary to block renal circulation temporarily in some complicated renal operations such as nephropyelolithotomy and nephron sparing nephrectomy of renal tumors. How to attenuate renal ischemia-reperfusion injury is a highlight in nephroprotective studies. Some studies indicates that iNOS expression increases in renal ischemia-reperfusion injury. It was reported that protection by glycine against renal hypoxic injury relates to their physicochemical effects, which stabilize kidney tubule membrane protein tertiary structure and protect the mitochondria. The purpose of this study was to investigate the nephropretective effects of glycine on renal ischemia-reperfusion injury and the possible mechanism of nephroprotection by glycine.Materials and MethodsPreparation of glycineGlycine was dissolved in 500ml of 0.45% sodium chloride for injection,adjusted pH of solution to pH6.5,then filtrated and asepticized for preparation.Animal protocolsTwenty-two male Wistar rats weighing 250g 18g were used in this experiment. Rats were randomly divided into 3 groups, Group A: sham-operated group(n=6), Group B: control group(n=8),Group C: glycine-treated group(n=8). Rats were anesthetized with injection (60mg/kg) of pentobarbitone sodium. A femoral vein catheter was placed for the administration of injection. Normal saline(Group A B)or Amino acids(Group C) was infused at a rate of lml*100g-1 *h-1 1 hour before ischemia and whole period of 2-hour reperfusion. A bladderurine catheter was placed for collecting urine in a tube (1.5ml), which had been weighed previously, to calculate the urine flow rate. After one hour infusion, a midlinc incision was made, the both renal pedicles were occluded with nontraumatic clamps for 30 minutes. The sham-operated rats were not occluded. Urine samples were collected at 0.5h, Ih, 1.5h and 2 hours of reperfusion. Blood samples were collected from the end of cut tails after 2 hours of reperfusion, samples were also collected from portal vein and bilateral nephrectomy were performed after 24 hours of reperfusion(n=24). Samples of urine or serum were stored at -20 until measurement. The kidneys were fixed in 4% buffered formaldehyde and embedded in paraffin. General methodsSerum and urinary creatinine values, serum BUN levels, urine sodium concentrations, urine potassium concentrations were measured by Automatic Biochemistry Analyzer (Hitachi). Morphology and immunohistochemistryRenal tissue fixed in formalin was processed by dehydration and embedded in paraffin. Sections were cut at 4 urn and stained with hematoxylin and eosin for histological assessment. Examination and scoring of the sections were performed on a blinded basis. In the assessment of kidneys harvested after 24 h of reperfusion, the severity of renal damage in terms of morphological changes was scored with semiquantitative scale that was established by Paller The ultrastructural changes of the kidney were observed by the transmission electron microscope. Expression of iNOS protein were examined in the kidney by immunohistochemistry (Envision method). Statistic AnalysisThe parameters in this study were expressed as mean plus or minus standard deviation( xs). Differences between groups were evaluated by Student-Newman-keul test. The Wilcoxon rank sum test was used to analyze the histological data. Statistical significance was considered at p< 0.05.ResultsChanges of urine volumeUrine volume increased remarkably after 30 minutes of reperfusion in glycine- treated group, compared with the control group (p<0.05). Changes of serum creatinine and BUN values after 24 hours of reperfusionSerum creatinine( 95.74 4.03umol/L) and serum BUN concentration (10.16 1.59mmol/L) in glycine protected rats after 24 hours of reperfusion were significantly lower(p<0.01) than those in the control rats (serum creatinine 115.51 14.47umoI/L and BUN 17.24 3.09mmol/L). Change of creatinine excretion, creatinine clearance after 2 hours of reperfusionCreatinine excretion in protected rats was s... |
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