Klotho蛋白对脓毒症急性肾损伤小鼠的肾脏保护作用及其机制研究  

Protective effects of Klotho protein on acute kidney injury in septic mice and its mechanism

作　　者：孙敏[1] 樊恒[1] 乐健伟[1] 陈国栋[1] 陈红[1] 李洁[1] 朱建华[1] Sun Min;Fan Heng;Le Jianwei;Chen Guodong;Chen Hong;Li Jie;Zhu Jianhua(Department of Intensive Care Unit, Xingbo First Hospital, Ningbo 315010, Zhejiang, China)

机构地区：[1]宁波市第一医院重症医学科,浙江宁波315010

出　　处：《中华危重病急救医学》2019年第2期160-164,共5页Chinese Critical Care Medicine

基　　金：浙江省自然科学基金(LQ18H150001);浙江省医药卫生科技计划项目(2017KY134,2017KY585,2018KY669);浙江省宁波市自然科学基金(2018A610296,2017A610188).

摘　　要：目的探讨Klotho蛋白(一种单向跨膜蛋白)对脓毒症急性肾损伤(AKI)小鼠的保护作用及其作用机制。方法选取6~8周龄、SPF级健康雄性C57BL/6小鼠60只,按随机数字表法分为假手术组(Sham组)、脓毒症模型组(CLP组)和Klotho蛋白干预组(CLP+KL组),每组20只。采用盲肠结扎穿孔术(CLP)制备脓毒症AKI小鼠模型;Sham组除不结扎、穿刺盲肠外,其余手术步骤相同。CLP+KL组术后连续4 d腹腔注射Klotho蛋白0.02 mg/kg;Sham组和CLP组注射等量生理盐水。术后24 h取各组小鼠眼眶血,采用肌氨酸氧化酶法和脲酶法分别检测血肌酐(SCr)、尿素氮(BUN)水平。术后5 d于麻醉后取小鼠肾组织,采用苏木素-伊红(HE)染色评估肾脏病理损伤;透射电镜下观察肾小管上皮细胞线粒体超微结构;用微量酶标法测定线粒体中还原型谷胱甘肽(GSH)水平,用硫代巴比妥酸法测定线粒体中丙二醛(MDA)含量,用比色法测定线粒体中一氧化氮合酶(NOS)活性;用蛋白质免疫印迹试验(Western Blot)检测肾组织Klotho、Bcl-2和细胞色素C(Cyt C)蛋白表达。结果 Sham组肾组织结构清晰、完整。与Sham组相比,CLP组肾小球和肾小管上皮细胞明显水肿,小管腔内可见透明管型,间质有大量炎性细胞浸润;血清SCr和BUN水平均显著高于Sham组〔SCr(μmol/L):182.60±6.97比47.20±5.37,BUN(mmol/L):53.70±5.12比18.70±2.62,均P<0.01〕;透射电镜下可见线粒体嵴肿胀、变形,嵴结构破坏,基质密度降低,外膜丧失;线粒体氧化应激因子水平显著升高〔MDA(μmol/g):1.172±0.046比0.746±0.094,GSH(μmol/g):5.765±0.059比4.223±0.072,NOS(kU/g):0.91±0.05比0.68±0.03,均P<0.01〕;肾组织Klotho蛋白、Bcl-2蛋白表达水平明显下降,Cyt C蛋白表达水平显著增加(Klotho/-?actin:0.188±0.020比0.538±0.024,Bcl-2/-?actin:0.311±0.010比0.391±0.015,Cyt C/-?actin:0.226±0.010比0.135±0.006,均P<0.01)。与CLP组比较,CLP+KL组肾小球和小管上皮细胞水肿减轻,小管腔内透明�Objective To investigate the protective effects of Klotho protein, a kind of single-pass transmembrane protein, on acute kidney injury (AKI) in septic mice and its mechanism. Methods Sixty SPF healthy male C57BL/6 mice (6-8 weeks) were randomly divided into sham operation group (Sham group), sepsis model group (CLP group) and Klotho protein injection group (CLP+KL group), with 20 in each group. The septic AKI mice model was established by cecal ligation and puncture (CLP);Sham group had the same procedure except that the cecal was not ligated. The CLP+KL group was received Klotho protein (0.02 mg/kg) by intraperitoneal consecutive injection for 4 days after operation;Sham group and CLP group were injected with the same amount of saline. Blood samples were obtained at 24 hours after operation, the levels of serum creatinine (SCr) and urea nitrogen (BUN) were measured by sarcosine oxidase and urease method. The mice were sacrificed under anesthesia at 5 days after operation to harvest renal tissues, and the pathological damage of the kidney was evaluated by hematoxylin-eosin (HE) staining. The ultrastructure of mitochondria in mouse renal tubular epithelial cells was observed under transmission electron microscope. The levels of reduced glutathione hormone (GSH), malondialdehyde (MDA) and nitric oxide synthase (NOS) in mitochondrion were determined by micro-enzyme method, thiobarbituric acid method, colorimetry method, respectively. The protein expressions of Klotho, Bcl-2 and cytochrome C (Cyt C) were detected by Western Blot. Results The pathological structure of the kidneys in the Sham group was clear and intact. Compared with the Sham group, the renal tissue edema of the mice in the CLP group was significant, and the transparent tube type was observed in the small lumen, and the interstitial inflammatory cells infiltrated;the levels of SCr and BUN were significantly increased [SCr (μmol/L): 182.60±6.97 vs. 47.20±5.37, BUN (mmol/L): 53.70±5.12 vs. 18.70±2.62, both P < 0.01];the mitochondria were swollen and

关 键 词：KLOTHO蛋白 脓毒症 肾损伤 急性 线粒体 

分 类 号：R459.7[医药卫生—急诊医学]