| Ischemic injury to renal tubular cells can cause acute renal failure(ARF).The mortality in patients with ARF has been unchanged since the mid-1960s and remains high at approximately 50%.How to prevent renal injury of ischemia-reperfusion is the major subject of renal protection.lt has long been recognized that the tubule necrosis is a major form of cell death associated with ischemic acute renal failure.However,there is accumulating evidence indicating a role of apoptotic pathways in in vivo and in vitro models of acute renal failure.Bcl-2 is a member of bcl-2 gene family, which plays an important role in programmed cell death.Bcl-2 encodes a 26KD protein,which localizes to outer mitochondrial membranes by a hydrophobic C terminus,and affects the function of mitochrondria and blocks apoptosis in the early phase.HSP70 is a member of the family of proteins known as chaperones.These proteins can function to promote correct protein folding and prevent in appropriate protein interactions because during ischemia/reperfusion tertiary structure of proteins may change sufficiently to alter function by free radicals and marked increase in intracellular calcium.To preserve the organ function and morphologic integrity after I/R, many tactics,such as induction of heat shock protein by thermal stimulation or drug administration have been tried.Recently,Our previous study had revealed that amino acids could protect renal function against ARF induced by cisplatin.To explore the protective effects and the beneficial mechanism of induction after amino acids preconditioning, a model of bilateral post-ischemic renal injury by clamping renal pedicles for 45 minutes was set up, and we examined the urine flow,renal function, histology,and the expression of HSP70 and bcl-2 by immunohistochemical method.Materials and Methods1. Animals and chemicalsMale Sprague-Dawley rats(n-24) weighing 210-250g(Experiment Animal Center of ZheJiang University)were used. The bcl-2 and HSP70 monoclonal antibodies were purchased from Boster Biological Technology Company, Wu han.SL-amino acids were purchased from Shanghai Biological Chemical Company. Preparation of a mixture of 8L-animo acids:glycine,alanine etc. Were dissolved and asepticized for preparation.2.Treatment of rats1) Animal group 24 Sprague-Dawley rats were divided randomly into 3 groups:sham-operated group (group A,n=8),ischemic control group (group' B,n=8),amino acids preconditioning group (group C,iT=8).2) Animal model Rats were anesthetized with intraperitoneal injection of sodium pentobarbitone (60mg/kg).A femoral vein cathether was placed for the admini-stration of drugs (1 ml/100g/h).A bladder urine cathether was placed for collecting urine in a tube(1.5ml),which was weighted previously. After a hour ofinfusing,a midline incision was made,the renal pedicles were occluded with nontraumatic clamps for 45 minutes. Sham-operated group was not occluded. The samples of urine after 30,60,90,120,150, ISOminutes of reperfusion were collected, and the rate of urine flow were calculated.Blood samples were collected from portal vein, and left nephrectomy were performed after 24 hours of reperfusion. All renal specimens were fixed by formalin and paraffin-embedded and cut into 4um slices to HE stain and bcl-2,HSP70 immunostainig. 3.Laboratory measurements1) Rate of urine flow Calculation of the urine flow rate after 3 0,60,90,120,150,1 SOminutes of reperfusion.2) Renal function Measurements of serum creatinine and BUN values after 3 and 24 hours of reperfusion.3) HE stain and immunohistochemical studies All kidney samples were fixed by formalin and paraffin-embedded, and cut into 4um sections which were studied by immunohistochemical staining and HE stain . Cell counts were performed at high power (X400)of Olympus-HB2. By counting the number of positively stained kidney cells in 5 high-power fields per case, we determined positive rate of bcl-2 and HSP70. 4 .Statistical analysesData were analyzed with analysis of variance and H-test... |
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