小鼠早期肾脏纤维化加重缺血再灌注诱导的急性肾损伤

Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice

目的研究小鼠早期肾脏纤维化病变对缺血再灌注(IR)诱导的急性肾损伤(AKI)的影响。方法8~12周龄雄性C57BL/6小鼠被随机分为4组:(1)假手术(Sham)组(n=3);(2)单侧输尿管结扎(UUO)组(n=6):分为UUO术后3d组(UU03d,n=3)和UUO术后5d组(UU05d,n=3);(3)肾脏缺血再灌注(IR)组(n=7),双侧肾脏缺血40min再灌注24h;(4)UU03d+IR组(n=6):UUO术后2d双侧肾脏缺血40min再灌注24h(实际UUO时间为3d)。HE及Masson染色观察各组小鼠急、慢性肾组织病理改变;Western印迹及免疫组织化学法(IHC)检测凋亡级联蛋白caspase-3表达;IHC法检测细胞增殖标志蛋白ki67的表达。结果HE及Masson染色结果显示,UUO组小鼠出现随时间延长而加重的肾脏慢性病变,UU03d组和UU05d组小鼠肾间质基质纤维沉积面积明显高于Sham组,UU05d组基质纤维沉积面积明显大于UU03d组(均P<0.05)。UU03d+IR组小鼠肾脏间质出现大量炎性细胞浸润、管腔扩张、刷状缘消失、小管上皮细胞空泡变性、坏死脱落等急性小管损伤,同时合并肾小管萎缩、间质蓝染基质(纤维)增多等慢性损伤;急性小管损伤评分较IR组明显增加(P<0.05)。UU03d+IR组血肌酐水平较Sham组明显增高(P<0.05)。免疫组化结果显示,相对于IR组UU03d+IR组肾小管caspase-3表达明显增加,ki67阳性表达的肾小管细胞占比明显减少(均P<0.05)。结论早期肾脏纤维化病变加重IR诱导的AKI,增加肾小管上皮细胞凋亡,减少肾小管上皮细胞损伤后的增殖修复。

Objective To investigate the influence of earlier renal fibrosis on ischemia and reperfusion induced acute kidney injury.Methods Male C57BL/6mice at eight to twelve weeks old age were divided into 4 groups randomly:(1)Sham (n=3);(2)Unilateral ureter obstruction (UUO,n=6): UUO for 3 days (UUO3d,n=3)and UUO for 5days (UUO5d,n=3);(3)Ischemia and reperfusion (IR,n=7):bilateral kidney ischemia for 40 minutes followed by 24 hours of reperfusion;(4)UUO for 3 days plus IR (UUO3d+IR,n=6):bilateral kidney ischemia after UUO 2days for 40minutes followed by 24 hours of reperfusion,and the real time for UUO was 3days.Pathologic analysis for acute or chronic injury was performed on paraffin embedded kidney sections with hematoxylin and eosin (HE)or Masson staining.Apoptosis was detected by immunohistochemistry(IHC)and Western blotting with anti-caspase-3 antibody,and proliferation was observed by IHC with anti-ki67antibody.Results on kidney sections with HE or Masson staining,it showed that the chronic kidney lesions and fibrosis got more severe as time of UUO prolonged from 3 days to 5 days;the area of matrix deposition increased in UUO5d and .UUO3d mice significantly compared to Sham mice (P<0.05)and was smaller in UUO3d mice compared with UUO5d mice obviously (P<0.05).Acute kidney injury could be observed in UUO3d+IR mice,such as massive inflammatory ceils infiltration,tubules dilation,brush border disappearance,tubular epithelial cells vacuolar degeneration,necrosis,casting formation,coexisting with chronic lesions:thinner cortex,broadened interstitial space,and increased blue stained matrix. Acute kidney injury score in UUO3d+IR mice was higher than that in IR mice significantly (P <0.05), and serum creatinine level increased significantly in UUO3d+IR mice compared to Sham mice (P<0.05).Caspase-3 expression increased and ki67 positive tubular cells decreased in UUO3d+IR mice than those in IR mice obviously (P<0.05).Conclusion Earlier renal fibrosis aggravates acute kidney injury induced by ischemia reperfusion in mice through increasing apoptosis and decreasing proliferation of tubular epithelial cells.