In vivo High-Content Screening in Zebrafish for Developmental Nephrotoxicity of Approved Drugs

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 3Dprinted 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.

Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis

“…Unfortunately, these model organisms are not easily usable in high‐throughput assays and even in the era of CRISPR/Cas9 genetic manipulation is rather difficult and time‐consuming. In the zebrafish model, the small larval size and conserved morphology of the pronephros offers a general applicability for high‐throughput assays as larvae can easily be treated and screened in the 96‐well format as it has been shown before for modulators of polycystic kidney disease using a morpholino‐based genetic zebrafish model 24 or a developmental drug nephrotoxicity assay 25 which both used pronephric morphometry as the primary readout.…”

Fiji plugins for qualitative image annotations: routine analysis and application to image classification

“…The multi-class (dropdown) plugin (Figure 3) allows to choose keywords from several lists of choices using dropdown menus (Figure 3B). The labels and choices for the dropdown menus are defined by the user in a simple csv file (Extended data, Supplementary Figure 1) (Thomas, 2020). This is convenient if multiple image features should be reported in separate columns (content, quality, etc.)…”

“…For the annotation of ROIs, the presented plugins can be used in combination with our previously published ROI 1-click tools (Thomas & Gehrig, 2020), which facilitate the creation of ROIs of predefined shapes, and the automated execution of custom commands for these ROIs. The generated ROIs can then be described with qualitative features using the hereby presented plugins, either for one ROI at a time, or by simultaneously selecting multiple ROIs.…”

Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases

“…This enables the automated acquisition of consistent views of 48–96 hpf zebrafish larvae in large-scale screening scenarios (Figure 2C ). For instance, we employed that methodology for imaging of embryonic kidneys in automated large-scale microscopy assays to score for morphological alterations of the pronephros upon compound exposure ( 93 , 125 , 128 ) (Westhoff et al unpublished data) or capture phenotypic changes in cystic kidney disease models ( 129 ) (Pandey et al, unpublished data). Other more complex technical solutions employ microfluidic systems that combine automated detection and rotational orientation within glass capillaries followed by microscopic imaging ( 127 , 130 ).…”