| Preface: Ischemic injury to renal tubular cells can cause acute renal failure(ARF). 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. It 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 apoptosis pathways in in vivo and in vitro models of acute renal failure. Bcl-2 encodes a 26KD protein, which localizes to outer mitochondrial membranes by a hydrophobic C terminus, and affects the function of mitochondria and blocks apoptosis in the early phase. HSP 70 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 andmarked increase in intracellular calcium. To preserve the organ function and morphologic integrity after I/R, many tactics, such as induction of heat shock protein or surpress cell apoptosis by drug administration have been tried. Ulinastatin is a potent protease inhibitor and has protective effect on many organs. Whether it has protective effect against renal ischemia/reperfusion injury or not, and its mechanism of induction is not known to us. To explore the protective effects and the beneficial mechanism of induction after ulinastatin administration, a model of bilateral post-ischemic renal injury by clamping renal pedicles for 45 minutes was set up, and we examined the renal function, histology, and the expression of HSP 70 and bcl-2 by immunohistochemical method, the ultrastructure of kidney. Methods:1.Animals and chemicalsMale Sprague-Dawley rats(n=75) weighing 210-250g(Experiment Animal Center of ZheJiang Medical Institute)were used. The bcl-2 and HSP 70 monoclonal antibodies were purchased from MaiXin Biological Technology Company, Fuzhou. Ulinastatin were purchased from Guangdong Techpool Biological Chemical Company.2.Treatment of rats1)Animal group 75 Sprague-Dawley rats were divided randomly into 3 groups: sham-operate group (group C, n=25), ischemic control group(group I, n=25), ulinastatin administration group (group U, n=25), each group were divided into 5 parts according to reperfusion duration: Oh, 2h,6h,12h,24h(n=5 in each part).2) Animal model Rat were anesthetized with ketamine50mg · kg-1ip.The vein in the tail were cannulated for fluid and drug administration. Group U received ulinastatin 12.5 thousands units intravenous each whereas group I and group C received normal saline 1ml. Then each rat received continuous intravenous normal saline 1ml/100g/h with a pump.Half an hour after the drug or saline intravenous injection, a midline incision was made, the renal pedicles was occluded with nontraumatic clamps for 45 minutes. Sham-operated group was not occluded. Left nephrectomy were performed after 0 hour, 2 hours, 6 hours, 12 hours, 24hours of reperfusion, and blood samples were collected from the heart. All renal specimens were fixed by formalin and paraffin-embedded and cut into 4 um slices to HE stain and bcl-2, HSP 70 immunostaining. After the left nephrectomy, the left kidney of the rats reperfusion for 24h was 1/3 cut out for the examination of ultrastructure. 3. Laboratory measurements.1) Renal function Measurements of serum creatinine and BUN values after Oh, 2h, 6h,12h,24h of reperfusion.2) HE stain and immunohistochemical studies All kidney samples were fixed by formalin and paraffin-embedded, and cut into 4um sections which were studied by imrnunohistochemical staining and HE stain. Cell counts were performed at high power (x400) of Olympus-HB2. Histologic studies were scored with semiquantitative scale evaluating changes by... |
