Abstract
Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the Tg(wt1b:EGFP) zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for in vivo large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.
Highlights
Pharmaceutical drugs and other chemicals can negatively impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants
To score developmental phenotypes of the zebrafish developing kidney in a large scale, we have previously reported the development of an automated imaging pipeline for the analysis of pronephroi in live zebrafish embryos and larvae (Westhoff et al, 2013)
To enable rapid visual assessment of screening results, image data processing was carried out to generate maximum z-projection images followed by automated region of interest detection, cropping of kidney regions and generation of thumbnail overview images (Figure 1B)
Summary
Pharmaceutical drugs and other chemicals can negatively impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants. 65–94% of women get at least one drug prescription (Ramoz and Patel-Shori, 2014) with the use of over-the-counter medications being assumed even higher (Werler et al, 2005). Developmental toxicity testing of promising drug candidates according to current international guidelines includes a series of screening tests (e.g., OECD Prenatal Developmental Toxicity Study, Two-Generation Reproduction Toxicity Study, Reproduction/Developmental Toxicity Screening Test) that are predominantly performed in historically established mammalian models (e.g., rat, rabbit). These in vivo tests require large numbers of animals, raising ethical concerns, and are highly laborious and expensive (Piersma, 2004)
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