| Objective:To establish Non-invasive limb ischemic preconditioning(LIP)model based on acute ischemia-reperfusion (IR) injury in animal model, inorder to explore if the repeated short limb ischemic preconditioning plays a role on renal ischemic reperfusion injury and its possible protective effect.Method:48 healthy Sprague Dawley rats weighing between 260-280g were subjected to right nephrectomy, then randomly divided into four groups:sham operation(Sham,n=12):only dissociate the left renal artery; simple hind limb ischemic preconditioning group(LIP,n=12):5min of the hind limbs of rats clamping with noninvasive hemostat, which shows no flow signal under Color Doppler ultrasound,then 5min relaxation, repeate three cycles;simple renal ischemia reperfusion group(IR,n=12):the left renal artery was clamped for 45min followed by reflow 24h; hind limb ischemic preconditioning-renal ischemia reperfusion group(LIP-IR,n=12):before clamping the left renal artery,give three cycles of hind limb ischemic preconditioning. Exsanguinate from abdominal aortic and to remove the left renal with PBS lavage after 24h reperfusion. Then Serum creatinine(Scr) and blood urea nitrogen (BUN) were tested, pathological changes of renal tissues were observed, the left renal tissues were collected for the determination of NO and the NOS activity, the expression of ICAM-1 was detected by immunohistochemistry,and the gene expression of ICAM-1, iNOS by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Results:1. Renal function:the renal function of rats in IR group and LIP-IR group decreased obviously, the level of Scr and BUN were significantly increased as compared with the sham group (P<0.01); but the level of Scr and BUN in LIP group were significantly decreased as compared with the IR group (P< 0.05).These indicators were no statistically significant differences between LIP group and the sham group (P>0.05)。2. The observation of renal histopathological and the tubular injury scoreThe HE staining of renal tissues showed:the histological changes lighter in the sham group and the LIP group, endothelial cells were normal.The histological changes in kidney tissues of the IR group were most obvious, mainly for the dissolution and shedding of renal tubular epithelial cells,the loss or partial loss of renal tubular brush border, a lot of tube type, the local interstitial edema obvious, the injure were most obvious in the proximal tubule of the outer medulla as well as the junction of the renal cortex and medulla.Compared with the IR group,the injure in the LIP-IR group were。The tubular injury score:Compared with the sham group,The tubular injury score in IR group and LIP-IR group were significantly increased (P<0.01);the score in LIP group were significantly decreased as compared with the IR group (P< 0.05).These indicator was no statistically significant differences between LIP group and the sham group (P>0.05)。3.The expression of ICAM-1 detected by immunohistochemistry and the gene expression of ICAM-1 by RT-PCR.The immunohistochemical detection show that there were only a small amount expression of ICAM-1 in the kidney tissues of the sham group and LIP group, and there was no significant difference between the two groups (P> 0.05); in IR group and LP-IR group, the expression of ICAM-1 were obvious, which mainly locate in the renal tubular epithelial cells,but the expression in LIP-IR group was significantly lower than that of IR group (P< 0.05).The gene expression of ICAM-1 by RT-PCR:The gene expression of ICAM-1 in IR group and LIP-IR group were significantly increased (P<0.01) as compared with sham group. Compared with the IR group, the gene expression of ICAM-1 in LIP-IR group were significantly decreased (P<0.05). Compared with the sham group, The gene expression of ICAM-1 in LIP group have no significant difference (P>0.05).4.The level of NO and the activity of NOS in the kidney tissues and the gene expression of iNOS by RT-PCR.Compared with the sham group,the level of NO and the activity of TNOS and iNOS and cNOS in IR group and LIP-IR group were significantly increased (P< 0.01).The activity of TNOS and iNOS in the kidney tissues were significantly decreased in LIP-IR group as compared with IR group(P<0.05),but the level of NO and the activity of cNOS(which mainly is eNOS) in the kidney tissues were significantly increased (P<0.05). Compared with the sham group, the above indicators in LIP group have no significant difference (P> 0.05).The gene expression of iNOS by RT-PCR:The gene expression of iNOS in IR group and LIP-IR group were significantly increased (P<0.01) as compared with sham group. Compared with the IR group, the gene expression of iNOS in LIP-IR group were significantly decreased (P<0.05). Compared with the sham group, The gene expression of iNOS in LIP group have no significant difference (P> 0.05).Conclusion:1.Eestablished Non-invasive limb ischemic preconditioning model based on acute ischemia-reperfusion injury in animal model.2. Limb ischemia preconditioning can significantly reduce serum creatinine, blood urea nitrogen,reduce the renal tissue damage and the tubular injury score which caused by acute Ischemia-reperfusion injury.These suggest that LIP to a certain extent plays a protective role on the renal.3.The protective effect of limb ischemic preconditioning (LIP) on the renal, which may be related to the inhibition of the expression of ICAM-1 and other inflammatory factors; may also be related to the increased expression of eNOS as well as the reduced expression of iNOS. |
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