未折叠蛋白反应参与小鼠急性肾缺血/再灌注损伤

Unfolded protein response involves in acute renal ischemia/reperfusion injury in mice

目的探讨未折叠蛋白反应(UPR)在小鼠急性肾缺血/再灌注损伤中的作用。方法将C57B6小鼠分为正常、假手术组和0、4、12、24 h手术组(分别于术后0、4、12和24 h处死小鼠),留取各组小鼠的左侧肾脏。苏木精-伊红(H-E)、过碘酸雪夫反应(PAS)和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色观察各组肾脏组织学变化,Western印迹法和实时聚合酶链反应(PCR)检测葡萄糖调节蛋白78(GRP78)和caspase12在各组小鼠肾组织中的表达。结果H-E、PAS染色可见24 h手术组的肾小管上皮细胞坏死明显,病变最重的部位为外髓外带近端小管,肾小管坏死评分为(2.930 0±0.131 1)分,显著高于其他组(P值均<0.01)。TUNEL染色可见24 h手术组的肾小管上皮细胞凋亡指数为(36.17±7.53)%,显著高于其他各组(P值均<0.01)。Western印迹法结果显示,12 h手术组的GRP78和caspase12蛋白水平显著高于其他各组(P值均<0.01)。实时PCR结果显示,4 h手术组的GRP78和caspase12 mRNA水平显著高于其他各组(P值分别<0.01、0.05)。结论在夹闭左侧肾动脉致急性肾缺血/再灌注损伤模型中,小鼠左侧肾脏组织学损伤显示再灌注后24 h最严重。GRP78和caspase12在再灌注后其mRNA和蛋白表达水平明显升高或激活,其变化早于组织学改变。提示UPR可能在急性肾缺血/再灌注损伤中发挥重要作用。

Objective To explore the role of unfolded protein response （UPR） in acute renal ischemia/ reperfusion injury （ARIRI） in mice, Methods C57B6 mice were divided into the normal group, sham operation group and the surgery group： the surgery group was further divided into 0 h, 4 h, 12 h, and 24 h subgroups according to the time they were put into death. The left kidneys of the mice were harvested in each group. H-E, PAS staining and TUNEL staining were employed to observe the histological changes of the kidney. Western-blot and real-time polymerase chain reaction （PCR） were used to examine the changes of glucose-regulated protein 78 （GRP78） and Caspase12 expression. Results H-E and PAS staining showed that there were obvious necroses of renal tubular epithelial cells in the 24 h-operation group, with the severest necrosis seen in the outer medullary proximal tubules and the tubular necrosis score being 2. 930 0±0.131 1, which was significantly higher than those of other groups （P〈0.01）. TUNEL staining showed that the 24 h-operated group had the highest apoptosis index in renal tubular epithelial cells （[36.17±7.53] % ）, followed by the 12 h-operation group. Western-blot showed that the protein levels of caspase12 and GRP78 peaked at 12 hours after renal ischemia/reperfusion （P〈0.01）. Real-time PCR results showed that the mRNA levels of caspase12 and GRP78 peaked at 4 hours after renal ischemia/reperfusion （P〈0.01）. Conclusion In ARIRI model caused by clipping the left renal artery, the worst histology injury of the left kidney occurs at 24 hours after reperfusion. The mRNA and protein expression levels of GRP78 and caspase12 are up-regulated or activated, which is earlier than the changes in the renal histology, suggesting that the UPR may play an important role in acute renal ischemia/reperfusion injury. （Shanghai Med J, 2009, 32, 180-183）