提取的尿蛋白及糖基化终产物对肾小管上皮细胞自噬通路的影响  

Impact of extracted urinary proteins and advanced glycation end product on autophagy pathway in renal tubular epithelial cells

作　　者：王燕劲 沈婷婷[1] 吴洪銮 陈秋华[1] 胡海滢[1] 潘庆军[1] 刘华锋[1] 刘伟敬[1] 

机构地区：[1]广东医学院附属医院肾病研究所,湛江524001

出　　处：《中华肾脏病杂志》2014年第9期695-701,共7页Chinese Journal of Nephrology

基　　金：广东省自然科学基金（S2012040006276）;湛江市科技攻关计划（2013B01056）;广东医学院及广东医学院附属医院博士启动基金（B2012080及BK201201）资助项目

摘　　要：目的 探讨尿蛋白超负荷及糖基化终产物对肾小管上皮细胞（TECs）自噬活性的影响.方法 在广东医学院附属医院肾脏病理库中选择微小病变肾病综合征（MCNS）及糖尿病肾病（DN）患者的肾组织,以临床表现为血尿而肾组织活检基本正常的标本为对照,免疫染色观察自噬膜蛋白-微管相关蛋白轻链3（LC3-Ⅱ）在肾脏的表达.分别以提取的MCNS患者的尿蛋白（8 g/L）以及晚期糖基化终产物-牛血清白蛋白（AGE-BSA,100 mg/L）刺激人肾小管上皮细胞（HK-2细胞株）,Western印迹检测LC3-Ⅱ蛋白表达,以溶酶体抑制剂（亮肽素200 mg/L或氯喹10 μmol/L）阻断溶酶体对自噬的降解,观察LC3-Ⅱ的转化;转染tfLC3质粒以观察溶酶体与自噬泡融合及降解的情况;分别以自噬诱导剂雷帕霉素（10 μmol/L）以及自噬抑制剂氯喹（10 μmol/L）增强及抑制自噬后,ELISA法检测细胞损伤因子中性粒细胞明胶酶相关性脂质运载蛋白（NGAL）及肾损伤分子-1（KIM-1）.结果 （1）与正常对照组相比,MCNS及DN患者TEC内LC3-Ⅱ蛋白表达明显增加（P＜0.01）.（2）尿蛋白刺激后,HK2细胞LC3-Ⅱ蛋白表达显著增多（P＜0.01）,且自噬抑制剂亮肽素使LC3-Ⅱ表达进一步增加;（3）通过转染tfLC3质粒,观察到尿蛋白刺激使HK2细胞早期自噬体及自噬溶酶体都明显增加（P＜0.01）.（4）AGE-BSA刺激HK2细胞后,LC3-Ⅱ表达也明显增多（P＜0.01）,但自噬抑制剂氯喹并未使LC3-Ⅱ蛋白表达进一步增加;（5）通过转染tfLC3质粒,观察到AGE-BSA刺激可使早期自噬体增多（P＜0.01）,但无晚期自噬体增多（自噬泡不能被降解）.（6）自噬诱导剂雷帕霉素可降低尿蛋白诱导的细胞损伤因子NGAL和KIM-1的上调,自噬抑制剂氯喹则作用相反.结论 尿蛋白可激活TEC自噬活性,但AGE-BSA则在下游阻断自噬通路而降低自噬活性.自噬通路的激活可能在原发和继发性肾病所致TEC损伤�Objective To investigate the effect of urinary proteins extracted from minimal change nephritic syndrome （MCNS） and advanced glycation end products （AGEs） on autophagy activity in renal tubular epithelial cells （TECs）.Methods Kidney tissue specimens of patients with MCNS and DN were obtained from the kidney pathology library of the Affiliated Hospital of Guangdong Medical College.The kidney tissue from patients with hematuria and proven to be minimal change by pathology examination were used as control.The expression of LC3-Ⅱ in kidney was examined by immune histochemistry in vivo.Expression of LC3-Ⅱ was also studied after exposing HK-2 cells to 8 g/L urinary proteins and 100 mg/L AGE-BSA respectively.LC3-Ⅱ turnover was examined after exposure to urinary proteins in presence of Lysosomal inhibitors leupeptin （200 mg/L） or chloroquine（10μmol/L） by western blot assay.In addition,the autophagosome or autolysosome formation was assessed after transfecting a tandem mRFP-GFP tagged LC3 （tfLC3） plasmid into HK-2 cells.Finally,the production of neutrophil gelatinase-associated lipocalin （NGAL） and kidney injury molecule-1 （KIM-1） were measured by ELISA after exposure of cells to autophagy enhancer rapamycin （10 μmol/L） and autophagy inhibitor chloroquine （10 μmol/L） in addition to urinary proteins.Results （1）In comparison with the control group,the expression of LC3-Ⅱ was significantly increased in TECs from patients with MCNS and DN（P ＜ 0.01）.（2）The expression of LC3-Ⅱ was enlarged after exposed to urinary proteins（P ＜ 0.01）,and further increased after leupeptin （autophagy inhibitor） addition.（3） Exposure to urinary proteins increased the autophogosomes and autolysosomes when observed by transfection of tfLC3 plasmid（P ＜ 0.01）.（4）The expression of LC3-Ⅱ was also elevated after treatment with AGE-BSA（P ＜ 0.01）,but no further increase after chloroquine （autophagy inhibitor） addition.（5） Only the autophogosome formati

关 键 词：自噬通路 糖尿病肾病 糖基化终末产物 肾小管上皮细胞 

分 类 号：R692[医药卫生—泌尿科学]