马兜铃酸I致急性肾损伤的分子机制研究

Study on mechanism of aristolochic acid I induced acute kidney injury

本研究通过开展马兜铃酸I(aristolochic acid I,AAI)致急性肾损伤的转录组学分析,以探讨AAI肾毒性的分子机制。雄性C57BL/6小鼠15只,随机分为正常组(n=6)和模型组(n=9)。模型组小鼠腹腔注射AAI 20 mg/kg,每天1次,连续5天,第6天处死。检测血清肌酐(Scr)、尿素氮(BUN)水平,苏木精-伊红染色(HE)观察肾组织病理变化;两组各随机选取3例肾组织提取RNA进行高通量转录组测序,将两组间差异倍数(fold change,FC)≥2.0且P值<0.01定义为差异基因,筛选正常肾与模型肾组织差异基因,应用GO、KEGG数据库进行功能分析;并选择两组间差异倍数变化最为明显的前5个基因进行qRT-PCR验证。最终发现,与正常组相比,模型组小鼠血清Scr、BUN含量明显升高;HE染色显示,正常肾组织结构正常,肾小球、肾小管、肾间质均未见到明显改变;模型组肾组织结构紊乱,肾小球水肿、固缩,近曲小管上皮细胞脱落。采用高通量转录组测序,按照筛选条件最终获得差异基因共计4975个,其中上调基因有2511个,下调基因有2464个。聚类分析发现差异表达基因中前5位为kap、Spp1、Aldh1a2、Serpine1、Tnc。GO与KEGG富集分析发现,差异基因主要在小分子代谢过程、胞外间质组织、中性粒细胞参与的免疫应答、颗粒腔、细胞质囊泡腔等相关的GO分类中显著富集;在炎症信号通路、过氧化反应、糖代谢等途径中富集明显。qRT-PCR检测提示,模型组kap表达减少,Spp1、Aldh1a2、Serpine1与Tnc表达增加,与转录组结果一致。这表明AAI具有明显的肾毒性,其毒性机制可能与炎症反应、氧化应激、糖代谢等途径相关。

In this study,transcriptomics analysis of aristolochic acid I(AAI)induced acute kidney injury was carried out to explore the molecular mechanism of AAI nephrotoxicity.Fifteen male C57BL/6 mice were randomly divided into normal group(n=6)and model group(n=9).The mice in the model group were intraperitoneally injected with 20 mg/kg AAI once a day for consecutive 5 days and sacrificed on the 6th day.Serum and renal tissue were collected for later detection,Scr and BUN values were detected,and HE staining of renal tissue was performed to observe pathological changes.High-throughput transcriptome sequencing was performed on RNA extracted from kidney tissue samples,which randomly selected from each group.Fold change≥2.0 as well as P-value<0.01 were defined as differentially expressed genes,which screened separately.Meanwhile,functional analysis was performed using GO and KEGG databases.The top 5 genes with the most obvious difference multiple changes between the two groups were selected for qRT-PCR verification.Compared with the normal group,Scr and BUN of the model group were significantly increased,HE staining showed that the kidney tissue in normal group had a normal structure,and the glomeruli,tubules,interstitium were not obviously changed.While in model group,the kidney tissue turned out globular edema,glomerular pyknosis,and loss of proximal convoluted tubule epithelial cells.Through high-throughput transcriptome sequencing,a total of 4975 differential mRNAs were obtained,of which 2511 were up-regulated genes and 2464 were down-regulated genes.Cluster analysis identified Kap,Spp1,Aldh1a2,Serpine1,Tnc as the top 5 differentially expressed genes.GO and KEGG enrichment analysis found that these differential genes were mainly significantly enriched in GO categories related to small molecule metabolism,extracellular matrix tissue,immune response involving neutrophils,granular cavity and cytoplasmic vesicle cavity.It is also significantly enriched in pathways such as inflammatory signaling,peroxidation,and glucose metabolism.qRT-PCR indicated that the expression of Kap decreased,while the expression of Spp1,Aldh1a2,Serpine1 and Tnc increased in model group,which were consistent with the transcriptome results.The results suggest that AAI has obvious nephrotoxicity,and its toxic mechanism may be related to inflammatory response,oxidative stress,glucose metabolism and other pathways.