Klotho对慢性肾脏病相关的动脉粥样硬化斑块不稳定性的保护作用  

Klotho protects against chronic kidney disease-associated atherosclerotic plaque vulnerability in mice

作　　者：毕先金 杨江信 熊加川 杨可 赵景宏 BI Xianjin;YANG Jiangxin;XIONG Jiachuan;YANG Ke;ZHAO Jinghong(Department of Nephrology,Kidney Center of PLA,Chongqing Key Laboratory for Prevention and Treatment of Chronic Kidney Diseases,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院肾内科,全军肾脏病中心,重庆市慢性肾脏病防治重点实验室,重庆400037

出　　处：《第三军医大学学报》2020年第5期460-465,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81873605);国家自然科学基金青年科学基金(81700379)。

摘　　要：目的探讨慢性肾脏病(chronic kidney disease,CKD)对动脉粥样硬化斑块稳定性的影响和Klotho蛋白在此病理过程中的作用。方法采用8周龄雄性Apoe^(-/-)小鼠制备5/6肾毁损CKD动物模型,高脂饮食12周,并通过腹腔注射Klotho蛋白(0.01 mg/kg,每2天注射1次)进行治疗。实验分为假手术组(sham组)、CKD组和Klotho+CKD组,每组8只小鼠。对各组小鼠主动脉根部层面切片,行HE和α-SMA免疫荧光染色,观察CKD对动脉粥样硬化斑块大小和纤维帽厚度的影响。体外培养人血管平滑肌(vascular smooth muscle cells,VSMCs)细胞,实验分4组,分别使用CKD 5期患者血清(CKD组)或者健康人血清(对照组)单独及联合Klotho蛋白进行孵育,观察各组细胞ROS水平和衰老细胞所占比例。结果与sham组比较,CKD组动脉粥样硬化斑块面积显著增加[(3.53±0.34)×10^5vs(2.21±0.21)×10^5μm^2,P<0.01],纤维帽明显变薄[(8.43±1.74)vs(21.21±2.27)μm,P<0.01],腹腔注射Klotho蛋白后可以显著抑制以上病理变化(P<0.01)。与对照组比较,CKD组半乳糖苷酶阳性VSMCs细胞比例明显增加(P<0.01),VSMCs细胞ROS水平显著增加(P<0.01),Klotho蛋白预孵育后能显著抑制CKD导致的半乳糖苷酶阳性VSMCs细胞增加(P<0.01)和ROS水平增加(P<0.01)。结论VSMCs细胞衰老是CKD导致动脉粥样硬化斑块不稳定性发生的重要原因,Klotho蛋白在此过程中发挥保护作用。Objective To explore the effects of chronic kidney disease(CKD)on the instability of atherosclerotic plaques and investigate the role of Klotho in this pathological process.Methods A total of 24 ApoE-/-mice(8 weeks old)were randomly divided into sham group,CKD group and CKD+Klotho group(n=8 in each group).Mouse model of CKD was established by 5/6 nephrectomy in the corresponding mice.Then,these mice were fed with high-fat diet for 12 weeks,and Klotho was supplemented intraperitoneally at 0.01 mg/kg,every other day to the mice from the CKD+Klotho group.HE staining andα-SMA immunofluorescence staining were performed to evaluate the effects of CKD and Klotho on the atherosclerotic plaque size and fibrous cap thickness.Cultured human vascular smooth muscle cells(VSMCs)were treated with the serum from CKD stage 5 patients or health individuals,in presence of Klotho protein.β-Gal staining and DCFH-DA probe were conducted to detect the proportion of senescent VSMCs and production of reactive oxygen species(ROS).Results The CKD group had significantly enlarged atherosclerotic plaque size[(3.53±0.34)×10^5 vs(2.21±0.21)×10^5μm^2,P<0.01],and decreased fibrous cap thickness(8.43±1.74 vs 21.21±2.27μm,P<0.01)when compared with the sham group.Supplement of Klotho could reverse these effects(P<0.01).In vitro experiment showed that there were larger proportion of galactosidase positive cells and higher ROS level in the VSMCs treated with the serum form CKD patients(P<0.01).However,pre-incubation of Klotho could alleviate theses effects(P<0.01).Conclusion Senescence of VSMCs is an important reason for instability of atherosclerotic plaques caused by CKD,and Klotho plays a protective role in the process.

关 键 词：KLOTHO蛋白 慢性肾脏病 斑块稳定性 血管平滑肌细胞 细胞衰老 

分 类 号：R341[医药卫生—基础医学] R543.5