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 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.

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.…”

A Multiparametric Assay Platform for Simultaneous In Vivo Assessment of Pronephric Morphology, Renal Function and Heart Rate in Larval Zebrafish

“…For renal phenotyping, quantitative measurements of kidney alterations were carried out for five morphological pronephric parameters: glomerular separation, glomerular height, glomerular width, tubular diameter and tubular distance. To measure these features, cropped maximum projection data of fluorescently labelled kidney was loaded in Fiji, 16 reference points were set manually on each projection [27], and the geometrical parameters (distances) were calculated automatically using a Fiji macro. Following normalization to quantitative measurements in control larvae that were co-assessed in each 96-well plate, heatmaps were generated using Graphpad Prism (Version 8.3.1, La Jolla, CA, USA).…”

“…While morphological analysis was performed by a combination of quantitative and qualitative parameters that each had to be determined separately in a time-consuming manner in our previous work [15], in the present project, quantitative measurements of renal parameters were carried out by loading fluorescent image data into Fiji and manually setting 16 reference points in the images for automated calculation of the geometrical parameters using a Fiji macro [27]. Representative images of pronephroi of 75-hpf-old drug-exposed larvae are shown in Figures 1E and 2.…”