Abstract
Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.
Highlights
Preclinical studies on drug discovery, drug-induced developmental toxicity and safety of chemical compounds require convenient animal models in combination with accurate technologies and reliable test assays
24-hpf-old Tg(wt1b:eGFP) zebrafish embryos were exposed over a period of 24 h to a previously investigated set of FDA-approved drugs affecting kidney development in humans and zebrafish (Figure 1A) [15]
Following automated dorsal imaging (Figure 1E) [15], and consistent dorsal and lateral positioning of 3 dpf larvae into agarose molds. Due to this zebrafish larvae were repositioned to a lateral position (Figure 1D,F) for fluorescein isothiocyanate (FITC)‐inulin injection for
Summary
Preclinical studies on drug discovery, drug-induced developmental toxicity and safety of chemical compounds require convenient animal models in combination with accurate technologies and reliable test assays. Cells 2020, 9, 1269 ex utero development and rapid organogenesis, zebrafish embryos and larvae have been established as a valuable model for large-scale in vivo chemical screening studies, both for toxicological and for phenotypic drug screening approaches [5,6,7]. Despite simpler morphology and tremendous differences in nephron number between humans and zebrafish embryos/larvae, there is homology concerning genetic, structural and functional aspects [10]. On the single-nephron level, the zebrafish pronephros is composed of highly differentiated and segmentally organized glomerular and tubular cells that largely resemble human kidney cells with respect to genetics, metabolism, physiology and morphology, indicating evolutionary conservation [9,10,11]. To fully exploit the in vivo context, newly developed assay platforms for pronephric evaluation should combine scoring of both morphological and functional phenotypes
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