马兜铃酸对斑马鱼胚胎肾毒性作用

Nephrotoxic Effects of Aristolochic Acid in Zebrafish(Danio rerio) Embryos

选用肾发育完成（受精后3天）的斑马鱼（Danio rerio）胚胎，用马兜铃酸进行染毒处理，观察胚胎的表型变化及死亡情况，分析马兜铃酸对胚胎的毒性作用及规律；利用肾荧光观察及肾组织切片，观察马兜铃酸处理后胚胎肾形态和肾组织结构的改变情况；利用qPCR检测马兜铃酸处理前后nephrin的表达变化，初步探讨足细胞在马兜铃酸毒性作用中的功能状态。20μmol／L马兜铃酸处理24h后，胚胎出现明显眼周水肿表现；在马兜铃酸高浓度组（40～80μmol／L），除眼周水肿外，胚胎血循环系统功能出现异常，表现为心率降低、血流缓慢甚至停滞；荧光显微镜下观察发现，马兜铃酸处理组胚胎肾出现肾小球囊性膨胀、前。肾管囊性扩张和形态异常；切片显示马兜铃酸处理组胚胎肾组织结构受到损害，表现为肾小球结构疏松、囊性扩张，前肾管上皮细胞细胞排列松散、紊乱及管腔扩张样改变；qPCR结果，马兜铃酸处理组斑马鱼胚胎nephrin的表达水平比对照组显著降低（P〈0．01）。研究表明，马兜铃酸能损害斑马鱼胚胎。肾结构和功能，其毒性作用与肾小球足细胞的功能改变有关。

The nephrotoxicity of aristolochic acid was studied using zebrafish （Danio rerio） embryos at 3 days post-fertilization were treated with various acid for 24 h. Morphological changes of embryos and kidney were observed as a model animal. Zebrafish concentrations of aristolochic under the microscope and the percentage of abnormal embryos was counted. Histological structure of kidney was further examined by tissue sections. The expression level of nephrin was tested by qPCR to explore the podocyte function in embryonic kidney treated with aristolochic acid （AA）. Embryos treated with 20 pmol/L aristolochic acid for 24 h exhibited periocular edema. As the exposure dosages of aristolochic acid increased to 40- 80 μmol/L, besides periocular edema, a large number of embryos also displayed cardiovascular dysfunction characterized by decreased heart rate, slow blood flow and even blood stasis. Expansion of glomeruli and distortion of pronephric tubules were observed in embryos treated with aristolochic acid under a fluorescence microscope. Paraffin sections from AA-treated embryos displayed malformed histological structure of kidney, such as cystic glomeruli and disorganized epithelial cells of pronephric tubules, qPCR showed that aristolochic acid treatment down-regulated the expression of nephrin in half in comparison with mock-control siblings （P 〈 0.01）. The results indicated that aristolochic acid caused profound nephrotoxicity in histological structure and biological function of zebrafish embryos. Dysfunction of podocyte may involve in the renal impairment.