G蛋白偶联受体激酶4调控小鼠肾脏急性缺血再灌注损伤的机制探讨  

Role of G protein-coupled receptor kinase 4 in regulation of acute renal ischemia-reperfusion injury in mice

作　　者：杨东海 韩愈[1] 巩正藩 周中淑[1] 吴连判 傅春江[1] 曾春雨[1] 周林[1] YANG Donghai;HAN Yu;GONG Zhengfan;ZHOU Zhongshu;WU Lianpan;FU Chunjiang;ZENG Chunyu;ZHOU Lin(Department of Cardiology, Chongqing Institute of Cardiology, Institute of Surgery Research, Third Affiliated Hospital, Army Medical University （Third Military Medical University）, Chongqing, 400042, Chin)

机构地区：[1]陆军军医大学(第三军医大学)第三附属医院野战外科研究所心血管内科,重庆市心血管病研究所,重庆400042

出　　处：《第三军医大学学报》2018年第8期666-672,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(31771281)

摘　　要：目的探讨G蛋白偶联受体激酶4(G protein-coupled receptor kinase 4,GRK4)对小鼠肾脏急性缺血再灌注损伤的影响及其作用机制。方法取SPF级健康野生型[8周龄、体质量(21.34±0.42)g]和GRK4转基因型[8周龄、体质量(21.87±0.68)g]C57BL/6小鼠,各12只。各型分别按随机数字表法分为4组(n=6):野生型假手术对照组、野生型肾脏缺血再灌注损伤组、GRK4转基因型假手术对照组、GRK4转基因型肾脏缺血再灌注损伤组。假手术对照组均采用开腹后不阻断肾动脉血流;缺血再灌注损伤组均采用夹闭肾动脉缺血45 min再灌注24 h,建立小鼠肾脏I/R模型。各组处死小鼠后,取血标本进行肾功能检测(血肌酐、血尿素氮);HE染色观察肾脏病理形态改变,并行肾小管损伤半定量评分;测定肾脏组织中过氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)等氧化应激水平改变;TUNEL染色检测肾脏组织中细胞凋亡情况;蛋白质免疫印记方法检测各组小鼠肾脏组织中GRK4和AT1受体的蛋白表达变化。结果野生型小鼠肾脏I/R模型后肾功能受损,血肌酐、血尿素氮升高,肾脏小管上皮细胞脱落、死亡(P<0.05);肾脏组织中GRK4蛋白表达含量增加,差异有统计学意义(P<0.05)。在GRK4过表达小鼠上研究结果发现,过表达GRK4的肾脏在缺血再灌注损伤后,肾功能损害进一步加重;肾脏病理损伤评分明显增加(P<0.05)。肾脏氧化应激水平明显上升,总SOD下降和MDA升高(P<0.05);肾脏凋亡细胞数目显著增多(P<0.05)。肾脏组织中AT1受体表达量增加(P<0.05),AT1受体含量的升高可以加重小管细胞氧化应激和凋亡的发生。结论 GRK4可以通过上调肾脏AT1受体表达,增加肾脏氧化应激水平和肾小管细胞凋亡,加重肾脏缺血再灌注损伤。Objective To investigate the regulatory role of G protein-coupled receptor kinase 4（ GRK4） in acute renal ischemia-reperfusion（ I/R） injury in mice and explore its mechanism. Methods Twelve8-week-old wild-type C57 BL/6 mice（ body weight 21. 34 ± 0. 42 g） and 12 age-matched GRK4 transgenic C57 BL/6 mice（ body weight 21. 87 ± 0. 68 g） were both randomized into sham-operated group and renal I/R group（ n = 6）. In the 2 I/R groups,the mice underwent laparotomy and the renal artery ischemia was clipped for 45 min followed by reperfusion for 24 h; For sham operation,the renal artery was exposed without occlusion. After the operation,blood samples were collected to measure serum creatinine and blood urea nitrogen levels. The pathological changes of the kidney were observed with HE staining and graded,and TUNEL staining was used to detect cell apoptosis in the renal tissue. Superoxide dismutase（ SOD） activity and malondialdehyde（ MDA） content in the renal tissue were measured to evaluate the changes of oxidative stress. Renal expressions of GRK4 and AT1 R were measured with Western blotting. Results The wild-type mice had significantly increased serum creatinine and blood urea nitrogen levels after renal I/R injury with obvious renal pathologies shown by renal tubular epithelial cell loss and death（ P〈0. 05）; the expression of GRK4 in the kidneys was significantly increased after renal I/R injury（ P〈0. 05）. Compared with wild-type mice,GRK4 transgenic mice exhibited significant impairment of renal function after I/R injury with worsened renal pathologies,significantly increased renal cell apoptosis（ P〈0. 05）,lowered SOD activity（ P〈0. 05）,increased MDA content（ P〈0. 05）,and up-regulated expression of AT1 R in the renal tissue（ P〈0. 05）.Conclusion GRK4 aggravates acute renal I/R injury and increases apoptosis and oxidative stress in renal tubular cells by up-regulating AT1 R expression.

关 键 词：G蛋白偶联受体激酶4 肾脏缺血再灌注损伤 血管紧张素Ⅱ1型受体 凋亡 氧化应激 

分 类 号：R364.12[医药卫生—病理学] R692[医药卫生—基础医学]