Department of Toxicology, University of Würzburg, Versbacher Strasse 9, 97078, Würzburg, Germany.
Institute of Human Genetics, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.
Toxicol Lett. 2021 Jun 15;344:69-81. doi: 10.1016/j.toxlet.2021.03.005. Epub 2021 Mar 17.
Due to an increasing demand for testing of new and existing chemicals and legal restrictions for the use of animals, there is a strong need for alternative approaches to assess systemic toxicity. Embryonic and larval zebrafish (Danio rerio) are increasingly recognized as a promising alternative whole-animal model that may be able to overcome limitations of cell-based in vitro assays and bridge the gap between high-throughput in vitro screening and low-throughput in vivo tests in animals. Despite the relatively simple anatomical structure of the zebrafish larval kidney (pronephros) - composed of only two nephrons - the pronephros shares major functions and cell types with mammalian nephrons. Glomerular filtration begins at 48 h post fertilization. The aim of the present study was to investigate if early zebrafish larvae might be a suitable model for nephrotoxicity testing. On day 3 post fertilization, larval zebrafish were treated with selected nephrotoxins (aristolochic acid, cadmium chloride, potassium bromate, ochratoxin A, gentamicin) for 48 h. Histological evaluation of zebrafish larvae exposed to model nephrotoxins revealed tubule injury as evidenced by dilated tubules with loss of the brush border, tubule cell necrosis and disorganization of the tubular epithelium. These changes were most severe after treatment with gentamicin, which also impaired pronephros function as evidenced by reduced clearance of FITC-dextran. Whole-mount in situ hybridization showing loss of cdh17 expression revealed site-specific injury to the proximal tubule segment. Analysis of genes previously identified as novel biomarkers of kidney injury in mammals showed upregulation of the kidney injury marker genes heme oxygenase 1 (hmox1), clusterin (clu), secreted phosphoprotein/osteopontin (spp1), connective tissue growth factor (ctgf) and kim-1 (havcr-1) in response to nephrotoxin treatment, although the response of individual genes varied across compounds. Consistent with the severity of lesions and impaired kidney function, the most prominent gene expression changes occurred in larvae exposed to gentamicin. Overall, our results suggest that larval zebrafish may be a suitable alternative model organism for nephrotoxicity screening, yet further improvements and integration with quantitative in vitro to in vivo extrapolation will be needed to predict human toxicity.
由于对新的和现有的化学物质进行测试的需求不断增加，以及对动物使用的法律限制，因此迫切需要替代方法来评估系统毒性。胚胎和幼体斑马鱼（Danio rerio）越来越被认为是一种很有前途的替代整体动物模型，它可能能够克服基于细胞的体外测定的局限性，并在高通量体外筛选和低通量体内试验之间架起桥梁。尽管斑马鱼幼体肾脏（前肾）的解剖结构相对简单——仅由两个肾单位组成——但前肾与哺乳动物肾单位具有主要的功能和细胞类型。肾小球滤过作用在受精后 48 小时开始。本研究的目的是研究早期斑马鱼幼体是否可能成为肾毒性测试的合适模型。受精后第 3 天，用选定的肾毒物（马兜铃酸、氯化镉、溴酸钾、赭曲霉毒素 A、庆大霉素）处理斑马鱼幼体 48 小时。用模型肾毒物处理的斑马鱼幼体的组织学评价显示出肾小管损伤，表现为刷状缘丢失的扩张小管、肾小管细胞坏死和管状上皮组织紊乱。用庆大霉素处理后，这些变化最为严重，这也表明前肾功能受损，因为 FITC-右旋糖酐的清除率降低。全胚胎原位杂交显示 cdh17 表达缺失，表明近端肾小管节段发生了部位特异性损伤。分析先前在哺乳动物中鉴定为肾脏损伤的新型生物标志物的基因显示，血红素加氧酶 1（hmox1）、簇蛋白（clu）、分泌型磷蛋白/骨桥蛋白（spp1）、结缔组织生长因子（ctgf）和 kim-1（havcr-1）等肾脏损伤标记基因的表达上调对肾毒物处理的反应，尽管个别基因的反应在不同化合物之间有所不同。与损伤的严重程度和肾脏功能受损一致，暴露于庆大霉素的幼体发生最显著的基因表达变化。总的来说，我们的研究结果表明，斑马鱼幼体可能是一种合适的替代肾毒性筛选的模型生物，但需要进一步改进并与定量体外到体内外推相结合，以预测人类毒性。
