The kidney is a major target organ of toxicity. Numerous structurally diverse drugs, natural compounds and chemicals can cause kidney injury. Presently, nephrotoxic effects of drugs and chemicals are identified primarily through regulatory toxicity tests in animals. With the aim of more efficient safety assessment and reduction of animal testing, toxicology is currently undergoing a paradigm shift in the way toxicity testing and risk assessment is carried out - away from tests in animals towards mechanism-based in vitro methods. While cell-based in vitro assays are particularly attractive for high-throughput screening, they fail to adequately reflect the complex processes that occur in vivo and thus do not allow prediction of potential health risks of drugs and chemicals with high confidence. Embryonic zebrafish (Danio rerio), which according to animal welfare legislation are considered as an in vitro model, are recognized as an alternative model for toxicity screening as they are easily accessible, releatively cheap and suitable for high-throuput methods, and - more importantly - better reflect the physiological situation of an intact organism (e.g. with respect to toxicokinetics, biotransformation) than cell-based in vitro assays.
Fig. 1: Three day old embryo of the transgenic wt1b:GFP zebrafish line in brightfield and fluorescence illumination.
The GFP-tagged transcription factor wt1b (Wilm´s tumor gene) marks the embryonic zebrafish pronephros. The GFP tag allows isolation and subsequent analysis of kidney cells to determine expression of transporters and drug-metabolizing enzymes and molecular changes induced by nephrotoxins.
Despite its relative simple anatomical structure composed of only two nephrons, the anatomical organization (glomerulus, proximal tubule, distal tubule, collecting duct) and function of the pronephros in embryonic zebrafish closely resemble the human kidney (metanephros). Cell types, differentiation pathways and molecular signaling pathways are conserved between kidney of zebrafish and human. Key transporters (e.g. slc20a1a, sglt, slc13a3, slc4a4) and the endocytic receptor megalin are expressed in the respective nephron segments. Glomerular filtration starts within 48h post fertilization. Thus, embryonic zebrafish fulfil fundamental requirements for an alternative model for nephrotoxicity testing. Preliminary data demonstrate toxic effects of selected model nephrotoxins on the pronephros of embryonic zebrafish. While these data suggest a huge potential of the model as a screening tool for the identification of compounds with nephrotoxic potential, several data gaps need to be filled to establish embryonic zebrafish as an alternative model suitable for safety assessment that may close the gap between cell-based high-throughput assays and toxicity tests in animals. Within the project, the following key questions will be addressed.
- Are there major species-differences in the expression of drug-transporters and drug-metabolizing enzymes recognized as being critical for the organ-specific toxicity of chemicals in humans/rodents between pronephros of embryonic zebrafish and metanephros of higher vertebrates?
- Can organ-specific toxic effects known to occur in kidney of higher vertebrates be adequately reflected in adult and ebryonic zebrafish with regard to type, localization and underlying molecular alterations?
- Which molecular markers are most suitable for identification of nephrotoxic effects in the embryonic zebrafish model?
- Besides identification of chemicals with nephrotoxic potential, can embryonic zebrafish deliver data relevant for quantitative risk assessment?
Group Prof. Dr. Angela Mally:
Institut für Pharmakologie und Toxikologie
Versbacher Straße 9, 97078 Würzburg
Dr. Daniel Liedtke:
Institut für Humangenetik
06/2018 - 05/2020