Objective: To investigate the role of SPP1 gene in acute kidney injury induced by renal ischemia-reperfusion injury (IRI).
Methods: Twelve Sprague-Dawley rats were randomly divided into sham group and IRI group (n=6) and subjected to sham operation and renal ischemia for 30 min induced by penal pedicle clamping using non-traumatic microvascular clamps, respectively.Serum creatinine and blood urea nitrogen levels were detected, and PAS staining was used for pathological examination of the kidneys in the two groups.The renal expressions of SPP1, α-SMA and caspase-3 were detected using immunohistochemistry and immunofluorescent staining.In cultured renal tubular epithelial cells (HK-2 cells), Western blotting was performed to detect the changes in expressions of SPP1, caspase-3, and Kim-1 proteins following hypoxiareoxygenation (H/R) and transfection with si-NC or si-SPP1;flow cytometry was employed to analyze apoptosis of the treated cells.
Results: Renal IRI caused significant elevations of serum creatinine and blood urea nitrogen levels (P<0.05) and induced severe shedding and necrosis of the renal tubular epithelial cells in the rats, resulting also in significantly up-regulated renal expressions of SPP1, α-SMA and caspase-3(P<0.05).In HK-2 cells, H/R significantly increased the protein expression levels of SPP1, caspase-3, and Kim-1(P<0.05), and compared si-NC transfection, transfection with SPP1 obviously reduced caspase-3 and Kim-1 expressions and lowered apoptosis rate of the cells with H/R exposure (P<0.05).
Conclusion: SPP1 is up-regulated in the kidneys of rats with renal IRI, and down-regulation of SPP1 expression can inhibit H/R-induced apoptosis of renal tubular epithelial cells.
目的: 探究SPP1基因在肾脏缺血再灌注损伤(IRI)中的作用及机制。
方法: GSE131454数据集显示了肾I/R组和Sham组基因的差异表达。在GSE131454数据库中搜索, 通过相关文献我们选择了SPP1作为研究对象。12只SD大鼠随机分为缺血再灌注组(IRI组)和假手术组(Sham组), 各6只, IRI组使用无损伤显微血管夹钳夹肾蒂使肾脏缺血30 min, Sham组不做处理。采集肾组织并检测两组大鼠血清肌酐、尿素氮的变化, PAS染色检测两组肾脏组织病理变化, 免疫组化检测SPP1基因和α-SMA基因表达变化。免疫荧光染色检测肾脏SPP1和Caspase-3的表达。肾小管上皮HK-2细胞缺氧复氧(H/R)模型分为Control组和H/R组, Western blot检测各组SPP1、Caspase-3、Kim-1蛋白的相对表达。HK-2细胞分别转染si-NC和si-SPP1, 检测各组干扰效率后分为si-NC+Control组、si-NC+H/R组、si-SPP1+H/R组, Western blot检测各组细胞中Caspase-3、Kim-1蛋白相对表达量, 流式细胞术检测各组细胞凋亡率。
结果: IRI组大鼠血清肌酐、尿素氮相比Sham组含量显著增加(P<0.05), 并观察到严重的肾小管上皮细胞脱落及坏死。在大鼠肾脏缺血再灌注损伤后, 肾脏组织中SPP1和α-SMA含量显著增加(P<0.05)。与Sham组相比, IRI组大鼠组织显示SPP1和Caspase-3的荧光强度显著升高(P<0.05)。肾小管上皮细胞缺氧复氧后SPP1、Caspase-3、Kim-1蛋白表达显著增加(P<0.05)。si-SPP1组细胞SPP1蛋白表达低于si-NC组细胞(P<0.05)。si-NC+H/R组的Caspase-3、Kim-1蛋白相对表达量和凋亡率均高于si-NC+Control组(P<0.05), si-SPP1+H/R组的Caspase-3、Kim-1蛋白相对表达量和调亡率均低于si-NC+H/R组(P<0.05)。
结论: SPP1在肾脏缺血再灌注损伤中的表达上调, 下调SPP1表达可抑制H/R肾小管上皮细胞调亡。
Keywords: HK-2 cells; SPP1; apoptosis; hypoxia-reoxygenation; ischemia-reperfusion injury.