摘要
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肾小管上皮细胞凋亡。
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 hypoxia reoxygenation(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.
作者
虞亘明
王鑫玮
骆金光
苏萧
陶怀祥
闻志远
关翰
YU Genming;WANG Xinwei;LUO Jinguang;SU Xiao;TAO Huaixiang;WEN Zhiyuan;GUAN Han(Department of Urology,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Anhui Provincial Key Laboratory of Immunology in Chronic Disease,Bengbu Medical College,Bengbu 233030,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2023年第11期1947-1954,共8页
Journal of Southern Medical University
基金
安徽省自然科学基金重点项目(2008085QH358)
慢性疾病免疫学基础与临床安徽省重点实验室开放课题基金(AHIAI2022K01)。
