免疫球蛋白样结构域受体2缺失加重缺血再灌注诱导的肾纤维化  

Immunoglobulin-like domain-containing receptor 2 deficiency aggravates ischemia-reperfusion-induced renal fibrosis

作　　者：徐梦园 王鑫 巩永凤[1] Xu Mengyuan;Wang Xin;Gong Yongfeng(Department of Physiology,Binzhou Medical University,Yantai 264000,China;Department of Pathology,Affiliated Central Hospital of Jiangnan University,Wuxi 214000,China)

机构地区：[1]滨州医学院生理学教研室,烟台264000 [2]江南大学附属中心医院病理科,无锡214000

出　　处：《中华肾脏病杂志》2023年第4期281-290,共10页Chinese Journal of Nephrology

基　　金：国家自然科学基金(81670620、81870485)。

摘　　要：目的探究免疫球蛋白样结构域受体2(immunoglobulin-like domain-containing receptor 2,Ildr2)在缺血再灌注诱导肾纤维化中的作用。方法利用CRISPR/Cas9技术构建Ildr2敲除小鼠(KO组),其对照组为野生型小鼠(WT组)。通过夹闭左侧肾蒂构建单侧肾脏缺血再灌注(unilateral renal ischemia reperfusion,UIR)模型(UIR组),造模后分为KO-UIR组和WT-UIR组;假手术小鼠(Sham组)不进行缺血处理。采用肌氨酸氧化酶法检测血清肌酐水平;二乙酰肟比色法检测血尿素氮水平;酶联免疫吸附测定法检测尿白蛋白水平,计算尿白蛋白/肌酐比值;HE、PAS及MASSON染色检测肾组织炎性细胞浸润情况和纤维化程度;实时荧光定量PCR检测Ildr2,肾损伤相关分子中性粒细胞明胶酶相关载脂蛋白(neutrophil gelatinase-associated lipocalin,NGAL)和肾损伤分子1(kidney injury molecule-1,KIM-1),纤维化标志分子Ⅰ型胶原α1(type 1 collagenα-1,Col1α1)、纤维连接蛋白1(fibronectin,Fn1)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和结缔组织生长因子(connective tissue growth factor,CTGF),以及炎症相关分子巨噬细胞标志物F4/80、单核细胞趋化因子1(monocyte chemoattractant protein-1,MCP-1)的mRNA表达水平。免疫荧光和Western印迹法检测Ildr2、Col1α1及α-SMA的蛋白表达水平。结果(1)实时荧光定量PCR和Western印迹结果显示UIR组Ildr2 mRNA和蛋白表达水平均显著低于Sham组(均P<0.05)。(2)KO组和WT组小鼠体重、血清肌酐、血尿素氮、总胆固醇、低密度脂蛋白、高密度脂蛋白及三酰甘油等的差异均无统计学意义(均P>0.05)。实时荧光定量PCR结果显示,KO组和WT组NGAL、KIM-1、α-SMA、Col1α1、CTGF、Fn1、MCP-1及F4/80 mRNA表达水平的差异均无统计学意义(均P>0.05)。组织学染色结果显示KO组和WT组均无炎性细胞浸润异常及间质纤维化。(3)与WT-UIR组相比,KO-UIR组血清肌酐和血尿素氮水平均较高(均P<0.05)。实时荧光定量PCR结果Objective To investigate the role of immunoglobulin-like domain-containing receptor 2(Ildr2)in renal fibrosis induced by ischemia-reperfusion.Methods Ildr2 knockout mice(KO group)were constructed using CRISPR/Cas9 technology,and wild-type mice were as the control group(WT group).The unilateral renal ischemia-reperfusion(UIR)model(UIR group)was constructed by clamping the left renal pedicle,and was divided into KO-UIR group and WT-UIR group after modeling.Sham operation mice(sham group)were not treated with ischemia.Serum creatinine was measured by creatinine oxidase method.Blood urea nitrogen was detected by the diacetyloxime colorimetric method.The urinary albumin level was measured by enzyme-linked immunosorbent assay,and urinary albumin/creatinine ratio was calculated.HE,PAS and MASSON staining were used to detect the infiltration of inflammatory cells and the degree of fibrosis in renal tissues.The mRNA expression levels of Ildr2,kidney injury-associated molecules neutrophil gelatinase-associated lipocalin(NGAL)and kidney injury molecule-1(KIM-1),fibrosis markers typeⅠcollagenα1(Col1α1),fibronectin 1(Fn1),α-smooth muscle actin(α-SMA)and connective tissue growth factor(CTGF),as well as inflammation-related molecules macrophage marker F4/80 and monocyte chemoattractant protein-1(MCP-1)were detected by real time quantitative PCR(qRT-PCR).The protein levels of Ildr2,α-SMA and Col1α1 were detected by immunofluorescence and Western blotting.Results(1)qRT-PCR and Western blotting showed that the expression levels of Ildr2 mRNA and protein in UIR group were significantly lower than those in sham group(both P<0.05).(2)There were no significant differences in body weight,serum creatinine,blood urea nitrogen,total cholesterol,low density lipoprotein,high density lipoprotein and triglyceride between KO group and WT group(all P>0.05).qRT-PCR results showed that there were no significant differences in the mRNA expression levels of NGAL,KIM-1,α-SMA,Col1α1,CTGF,Fn1,MCP-1 and F4/80 between KO group and WT group(all

关 键 词：再灌注损伤 肾疾病 炎症 肾纤维化 免疫球蛋白样结构域受体2 

分 类 号：R692[医药卫生—泌尿科学]