| Objective Ischemia reperfusion injury(IRI) inevasible in the course of kidney transplantation is one of the key factors that influence the early function of transplanted organ. IRI is associated with many factors and the mechanism has not been thoroughly clear. Some experimental studies have proved that IRI is related to apoptosis. The recent major aim is to search for the anti-injury substance to prevent and alleviate the renal damage of IRI. The aim of this study is (1)to establish a cold ischemia reperfusion model with rats, (2)to evaluate the effects of Chinese Herb-Eirgeron breviscapus (vant) hand-mazz on cold ischemia and reperfusion injury in rats, and apoptosis of renal tubular epithelial cells, and their mechanisms.Methods Thirty six SD rats were anesthetized with sodium pentobarbital (30mg/kg) and randomly assigned to three groups: sham-operation(A), control (B), and experimental (C)group. In group C, Eirgeron breviscapus (vant) hand-mazz was injected (1.2ml/100g) 15 minutes before operation, meanwhile, in group A and B rats were injected NaCl(1.2ml/100g). Group A was only removed the right kidney. Cold ischemia reperfusion was performed as the follow-listed model in Group B and C. The main process of the model: (1)Perfusing left kidney: after resected the right kidney of the rat, a pipe was put in the remainder right renal artery to perfuse the left kidney. The perfusion flowed out of cava inferior through remainder right renal vein. The blood vessels of left kidney were clipped after cold perfusion.(2)Cold ischemic conservancy: the left kidney was taken out of abdominal cavity then stored in a coldbag which was full of ice and water, but the vessels of that were intact. (3) Reperfusing left kidney: after 60 minutes, the clip was removed. 24 hours after ischemia reperfusion(IR), left kidneys of all rats in three groups were removed to be detected . Structure of the kidney was evaluated by light microscopy and electronic microscopy. Expression of Bcl-2 and Bax was detected by immunohistochemistry. Apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The renal function was also assessed by determining the levels of blood urea nitrogen(BUN) and serum creatinine(Cr) before and 24 hours after IR, and superoxide dismutase(SOD) activity and malondialdehyde(MDA) content in the renal tissues was examined.Results ?Morphologic change (hematoxylin-eosin staining) : A normal morphology was observed by light microscopy in group A. Red blood cells in glomeruli increased and swelling cells of renal tubules appeared with brush border loss in group B. There was only more red blood cells in glomeruli in group C. (D Ultrastructure: A normal ultrastructure was observed by electon microscopy in group A. Swelling and vacuolation of mitochondrion with rare microvillus and cytoplasmic vacuolation appeared in group B, and showing of apoptosis appeared in some nucleuses. The degree of tubular epithelium injury in the group C is milder than that in group B. ?Immunohistochemistry staining: the expression of Bcl-2 in renal tubules in group A were significantly fewer than those in group B and C (P<0.05), meanwhile, there were fewer Bcl-2 positive cells in group B compare with group C (P<0.05). the expression of Bax in renal tubules in group A were significantly fewer than those in group B and C (P<0.05), meanwhile, there were more Bax positive cells in group B compare with group C (P<0.05). The ratio of Bcl-2 I/Bax I in group A was higher than those in group B and C (P<0.05), meanwhile, the ratio in group C was higher than those in group B (P<0.05). ?There were more TUNEL positive cells in group B and C compared with group A in tubular epithelium (P<0.05). TUNEL positive cells in group B were significantly more than those in group C (P<0.05). (§) blood urea nitrogen(BUN) and serum creatinine(Cr): in group A There was not significant difference about BUN and CR between before and after operation (P>0.05), but in Group B and C, those increased significantly at 24 hour after operation (P<0.05). Meanwhile, BUN and CR in group B were significantly higher than those in group C after operation (P<0.05). ?Activity of SOD and content of MDA in renal tissues: Activity of SOD in group A was higher than those in group B and C (P<0.05),meanwhile, Activity of SOD in group C was significantly higher than those in group B (P<0.05). Content of MDA in group A was lower than those in group B and C(P<0.05), meanwhile, content of MDA in group C was significantly lower than those in group B (P<0.05).Conclusions ? The rat renal cold ischemia reperfusion model we established is feasible regardless of experimental conditions with high successful rate and can be studied as the events following IRI. (2) increase of apoptotis in tubular epithelium after cold ischemia-reperfusion show that apoptosis in tubular epithelium may be one of the important factors influencing the cold ischemia reperfusion injury. ?Eirgeron breviscapus (vant) hand-mazz protects against renal cold ischemia-reperfusion injury and decreases apoptotis in tubular epithelium.
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