肾小管上皮细胞有机阴离子转运蛋白1在马兜铃酸Ⅰ跨细胞转运及其在细胞毒性中的作用  

Role of rat organic anion transporter 1 in renal cellular uptake of aristolochic acid Ⅰ and the induction of cellular toxicity

作　　者：张锐[1] 阳晓[1] 刘眉[1] 伍军[1] 张云芳[1] 彭文兴[1] 孔庆瑜[1] 董秀清[1] 余学清[1] 

机构地区：[1]中山大学附属第一医院肾内科卫生部重点实验室,广州510080

出　　处：《中华肾脏病杂志》2009年第8期624-629,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30873432）

摘　　要：目的探讨肾小管上皮细胞有机阴离子转运蛋白1（OAT1）在马兜铃酸Ⅰ（AAⅠ）跨细胞转运中的作用及其毒效关系。方法体外构建重组大鼠OAT1（rOAT1）基因的真核表达质粒，将含rOAT1质粒导入人胚肾细胞（HEK-293）。观察rOAT1及被其特异性底物对氨基马尿酸（PAH）阻断后对AAⅠ摄取的作用。并分别观察不同浓度AAⅠ对转染细胞caspase-3mRNA表达和细胞凋亡的影响。Western印迹检测转染细胞rOAT1表达；毛细管电泳法检测细胞内PAH浓度；高效液相色谱法（HPLC）检测细胞内AA浓度。RT—PCR检测细胞caspase-3mRNA表达；流式细胞法（AnnexinV—FITC）检测细胞凋亡水平。结果转染rOAT1质粒的HEK-293细胞特异性表达rOAT1。以40、80、120、160mg／LAAⅠ分别刺激细胞45min，转染rOAT1组细胞内AAⅠ浓度显著高于空载体组（均P〈0．05）。以120mg／LAAⅠ分别刺激细胞30、60、90、120min，转染组细胞AAⅠ浓度显著高于空载体组（均P〈0．05）。加入PAH（5mmol／L）特异性阻断OAT135min后，以上述不同浓度AAⅠ分别刺激转染细胞，细胞内AAⅠ浓度显著低于未阻断组（均P〈0．05）。以上述不同浓度AAⅠ分别刺激转染细胞和空载体细胞，两组细胞caspase-3mRNA表达及细胞凋亡随AAⅠ浓度上升而增加，转染组细胞凋亡率显著高于空载体组（均P〈0．05）。结论转染含rOAT1质粒的HEK-293细胞表达rOAT1。AAⅠ呈浓度依赖性和时间依赖性进入IOAT1转染细胞，并呈剂量依赖性诱导OAT1表达细胞凋亡。提示OAT1介导AAⅠ的肾细胞转运及细胞毒性。Objective To investigate the role of rat organic anion transporter 1 （OAT1, SLC22A6） in the renal cellular uptake of AAⅠ and its impact on cellular toxicity. Methods HEK-293 cells were transfected with rat OAT1 eDNA or empty vectors. The over-expression of rOAT1 was confirmed by Western blot analysis and its activity was validated by using paraaminohippurate （PAH） as a probe. Cellular apoptosis was examined by flow eytometery using propodium iodode （PI） and annexin V-FITC staining. Results Concentration- and timedependent intracellular accumulation of AAⅠ was observed in rOAT1-transfected HEK-293 cells. After treatment with AAⅠ at the concentrations of 40 mg/L, 80 rag/L, 120 mg/L and 160 mg/L,respectively, for 45 min, the intracellular concentrations of AAⅠ in rOAT1-transfected HEK-293 cells were higher than those in controls （P〈0.05）. After treatment with AAⅠ （120 mg/L for 30 min, 60 min, 90 rain and 120 min, respectively, the intracellular concentrations of AAⅠ in rOAT1-transfected HEK-293 cells were higher than those in controls （P〈0.05）. PAH significantly reduced the intracellular accumulations of AAⅠ in rOAT1-transfected HEK-293 cells. After treatment with AAⅠ at the concentrations of 40 mg/L, 80 mg/L, 120 mg/L and 160 mg/L respectively for 35 min, the intracellular accumulations of AAⅠ in rOAT1-transfected HEK-293 cells that treated with PAH were lower than those that were not treated by PAH. Cellular apoptosis and caspase-3 expression in rOAT1-transfeeted HEK-293 cells were significantly up-regulated as compared to controls （P〈0.05）. Conclusion rOAT1 is involved in the cellular uptake of AAⅠ which leads to increased epithelial apoptosis. Further studies are suggested to investigate the role of human OAT in the disposition of AA and its toxicological consequences.

关 键 词：有机阴离子转运蛋白1 马兜铃酸 细胞凋亡 转染 肾毒性 

分 类 号：R692[医药卫生—泌尿科学]