| Objectives： We investigated the effects of ischemic preconditioning against renalischemia-reperfusion injury in rats and compared the results with ischemia-reperfusioninjury group at the biochemical, cellular organization and protein levels, providing a newstrategy and experimental basis for the anti-renal ischemia-reperfusion injury.Methods: q36Spargue-Dawley rats (250-300g) were randomly assigned into3groups.Sham operated control (sham, n=12): Laparotomy were carried out in rats under generalanesthesia, the right kidneys were removed for normal control, And sham operation wasdone by mobilization of renal vascular pedicle without clipping, and the kidney wassampled after73min waiting. In I/R group (I/R, n=12), the right kidneys were resected,and after28min waiting, the left kidneys were subjected to45min ischemia. Ischemicpreconditioning group (IPC, n=12), after right nephrectomy, the left kidneys weresubjected to three cycles of2min ischemia separated by5min reperfusion periods before45min of ischemia. At4h,6h,24h time points after reperfusion, blood sampling andkidney harvesting were done in each group.The following two aspects were investgated.1. The protective effects of IPC on renal IRI in rats.The blood urea nitrogen (BUN) and serum creatinine (SCR) were measured withautomatic biochemistry analyzer. The renal pathologic changes were observed byHE-staining.2. The proteins associated with Ischemic preconditioning against renalischemia-reperfusion injury.2.1The antiapoptosis pathways of IPC against renal IRI in ratApoptosis was detected by TUNEL method; The expression of apoptosis relatedproteins：Bcl-2and Bax were assayed by western blot. The renal apoptosis were observedby using transmission electron microscope (TEM). 2.2The autophagy related pathways of IPC against renal IRI in ratThe expression of autophagy related proteins：Beclin-1was assayed by western blot.The renal autophagy were observed by using transmission electron microscope (TEM).Results:1. The protective effects of IPC on renal IRI in rats.①The change of BUN and SCR levels: The BUN levels of sham group were7.72±0.66mmol/L,9.73±1.23mmol/L and9.68±1.50mmol/L at4h,6h and24h afterreperfusion respectively, and the SCR levels were61.13±10.33μmol/L,61.38±9.14μmol/L,87.48±16.50μmol/L respectively; the BUN levels of IPC group were9.80±0.75mmol/L,14.13±2.30mmol/L and20.37±4.23mmol/L respectively, and the SCR levels were86.88±16.94μmol/L,92.55±25.41μmol/L,171.83±33.22μmol/L respectively; the BUNlevels of I/R group were10.47±0.79mmol/L,18.34±2.28mmol/L and33.42±6.41mmol/Lrespectively; and the SCR levels were94.35±16.34μmol/L,127.43±15.28μmol/L,261.88±33.37μmol/L. In IPC group and I/R group SCR and BUN levels weresignificantly higher than that in sham group(P﹤0.05). The BUN and SCR levels of IPCgroup were not significantly different from those in the same point time I/R group, but at6h and24h were significantly higher than that in I/R group (P﹤0.05).②Pathological observation of the renal tissue: The renal tissue of both normalcontrol and sham group were morphologically the same. The renal morphologic score inIPC group were4.20±0.22,4.60±0.42,5.93±0.46repectively at4h,6h and24h afterrenal ischemia-reperfusion injury; The renal morphologic score in I/R group were4.58±0.54,5.45±0.53,7.28±0.95respectively; the morphologic score in6h and24h after renalischemia-reperfusion injury of IPC group were lower than thant in I/R group(P﹤0.05).2. The proteins associated with ischemic preconditioning against renalischemia-reperfusion injury①Tubular epithelial cell apoptosis:the tubular epithelial cell apoptosis was detected inthe kidney at4h after ischemia-reperfusion injury, apoptosis index of I/R group and IPCgroup were higher than that in sham group(P<0.05), while compared with I/R group, theapoptosis index of IPC group was significantly decreased at6h and24h(P<0.05).②The expression of proteins associated with cell apoptosis: There were nosignificant difference between sham group and normal kidney tissue; Compared with sham group, the I/R and IPC group rats resulted in significant up-regulation of Bcl-2, BAX andBcl-2/Bax(P<0.05); while compared with I/R group, IPC group rats resulted in significantup-regulation of Bcl-2and Bcl-2/Bax(P<0.05), and significant down-regulation ofBAX(P<0.05).③The expression of proteins associated with cellAutophagy: There were nosignificant difference between sham group and normal kidney tissue; Compared with shamgroup, the I/R and IPC group rats resulted in significant up-regulation of Beclin-l (P<0.05);while compared with I/R group, IPC group rats resulted in significant up-regulation ofBeclin-l (P<0.05).④The apoptosis and autophagy were observed by using transmission electronmicroscope(TEM): There were few number of autophagosomes and apoptosis in kidney ofsham group. The formation of autophagosomes was detected by TEM at4h afterischemia-reperfusion injury; at6h after ischemia-reperfusion injury, enhanced presence ofautophagosomes and apoptosis were detected by TEM; it was significantly increased at24h.Conclusions:1. The function and structure of renal tissue were damaged by renal ischemicreperfusion in rats with the increase of the level of SCR and BUN,the damage of renaltubula and glomerulum. IPC protected effectively kidney against IRI with the decrease ofthe level of SCR and BUN, and the improvement of the renal morphological features.2. IRI may lead an increase in tubular cell apoptosis, The effects of IPC against renalIRI may be associated with antiapoptosis by regulating the Bcl-2/BAX protein expression.IPC can resulted in significant up-regulation of Bcl-2and Bcl-2/Bax, and significantdown-regulation of BAX. IRI can increased the robust formation of autophagosome whichoccurred earlier than apoptosis. The effects of IPC against renal IRI may be also associatedwith significant up-regulation of Beclin-1. |
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