Nephrotoxicity in Drug Development: Detecting Drug-Induced Kidney Toxicity with Zebrafish

What Is Nephrotoxicity and Why Drug-Induced Kidney Damage Goes Undetected

Nephrotoxicity refers to kidney damage caused by exposure to chemical substances, including therapeutic drugs. The kidneys are particularly vulnerable because their role in filtering, concentrating, and excreting xenobiotics exposes tubular cells to high local concentrations of potentially toxic substances. Drug-induced nephrotoxicity causes approximately 20% of community- and hospital-acquired episodes of acute renal failure, and drug-induced acute kidney injury (AKI) accounts for 19-26% of all hospitalized cases. 

Despite these data, the nephrotoxic potential remains unknown for most compounds entering development. The number of compounds requiring evaluation far exceeds the capacity of available screening tools. Standard in vitro methods lack the physiological complexity of a functioning kidney, while most in vivo methods involve rodent models that are too resource-intensive for early-stage screening, such as the repeated-dose 28-day oral toxicity study in rodents according to OECD TG 407. 

There is a screening gap. Compounds advance through the pipeline without reliable renal liability data, and nephrotoxicity only surfaces when it is already expensive, or too late to act on it.

Mechanisms of Renal Toxicity: Target Sites, Pathways and Vulnerable Kidney Structures

Drug-induced nephrotoxicity develops through three primary mechanisms:

• Proximal tubular injury and acute tubular necrosis (ATN), a dose-dependent process driven by direct apical contact with drugs or their metabolites, intracellular drug accumulation via uptake from the tubular lumen, and the secretion of compounds from the basolateral surface into the tubular lumen. 
• Tubular obstruction by crystals or casts formed by drugs and their metabolites that are insoluble in urine, a process that activates NLRP3 inflammasome-mediated inflammation and leads to AKI. 
• Acute tubulointerstitial nephritis (ATIN), a dose-independent immune-mediated response predominantly driven by T-cell type IV hypersensitivity, triggered by drugs such as antibiotics, Non-steroidal anti-inflammatory drugs (NSAIDs), and proton pump inhibitors.

At the molecular level, the most studied mechanisms of chemical-induced nephrotoxicity include:

• Oxidative stress, through the overproduction of reactive oxygen species (ROS), triggers mitochondrial dysfunction and pro-apoptotic pathways.
• Inflammation mediated by pro-inflammatory markers such as TNF-α, IL-1β, cox2, and myeloperoxidase (MPO).
• Apoptosis via caspase activation and disruption of the Bcl-2/Bax balance.
• DNA damage.
• Cell death.
• Fibrosis.

These pathways are interconnected, and kidney injury is rarely the product of a single mechanism.

Nephrotoxic Drug Classes: From Aminoglycosides to Contrast Agents

Antibiotics, particularly aminoglycosides such as gentamicin and amikacin, are among the most well-documented nephrotoxins, with nephrotoxicity occurring in 10-25% of therapeutic courses. They cause direct tubular cell toxicity by accumulating in proximal tubular cells, disrupting mitochondrial function, and triggering apoptosis. Antineoplastic agents, most notably cisplatin, cause nephrotoxicity in 30-40% of treated patients through a similar tubular toxicity mechanism, driving oxidative stress and inflammatory cascades that lead to tubular cell death and fibrosis. Vancomycin, amphotericin B, tenofovir, and other antiretrovirals are additional antimicrobial and antiviral agents with well-established renal liability.

Contrast agents used in diagnostic imaging represent a distinct and clinically significant category. Contrast-induced nephropathy (CIN) is the third most common cause of acute renal failure in hospitalized patients. The injury combines direct tubular cytotoxicity with renal vasoconstriction that reduces medullary blood flow and generates oxidative stress. Risk is highest in patients with pre-existing renal insufficiency, diabetes, heart failure, or volume depletion. NSAIDs, proton pump inhibitors, and certain antibiotics round out the picture, acting primarily through interstitial nephritis rather than direct tubular damage.

Zebrafish for Nephrotoxicity Detection: Anatomy, Assays and Endpoints

Zebrafish (Danio rerio) have emerged as a powerful tool for early nephrotoxicity screening. 

The zebrafish pronephros, the embryonic kidney, develops rapidly and is complete by approximately 48 hours post-fertilization (hpf). Despite its simplicity (composed of just two nephrons), it contains all the key structural components of the mammalian nephron: glomerulus, neck, proximal tubules, distal tubules, and collecting duct. Because zebrafish embryos are optically transparent, the pronephros can be directly observed for structural and functional changes without dissection, a key practical advantage for screening. 

Brightfield dorsal view of a 2-day post-fertilization (dpf) zebrafish embryo (upper panel). The rectangle in the anterior trunk indicates the location of the proximal pronephric structures with a fused glomerulus at the midline that connects to the segmented pronephric tubules as labeled in the Tg(wt1b:egfp) zebrafish line by GFP expression (lower panel). Source: Gehrig, Pandey, and Westhoff, 2018.

A critical aspect of the zebrafish model's translational value is that nephrotoxic compounds known to induce AKI in humans produce equivalent damage in zebrafish in a dose-dependent manner that recapitulates mammalian AKI. Gentamicin exposure, for example, causes flattening of the proximal tubule brush border, tubular and glomerular distension, lysosomal phospholipidosis, formation of debris in the nephron lumen, and accumulation of leukocytes.

Nephron composition and segment pattern is conserved between zebrafish and mammals. Nephrons in the mature mammalian metanephric kidney are composed of a renal corpuscle followed by proximal, intermediate, and distal tubule segments. This anatomical pattern is conserved in the zebrafish pronephros, which form a renal corpuscle at the midline that sends filtrate to the pair of nephrons that contain a series of proximal and distal segments. The podocytes and tubule cells in zebrafish express a shared suite of conserved genetic markers in common with mammals. Source: Drummond, Ercanbrack, and Wingert, 2023.

This translational fidelity, combined with the small size and high fecundity of zebrafish, makes them compatible with high-throughput 96-well plate formats. Compounds that would require weeks to evaluate in rodents can be assessed in days at a fraction of the cost and with significantly reduced animal use. Embryo and larval zebrafish are considered a fast screening model for renal toxicity assessment, while adult zebrafish, whose mesonephros more closely resembles the complexity of mammalian kidneys, are preferred for mechanistic studies. 

Zebrafish are used to determine glomerular filtration using either direct or indirect assays. In the direct method, fluorescence-labeled dextran or inulin is microinjected into the zebrafish heart. Renal clearance is then quantified over time by fluorescence microscopy, and decreased clearance indicates impaired filtration function. For an indirect measure of glomerular damage, proteinuria can be tracked by analyzing protein concentration in the water or using transgenic zebrafish lines expressing GFP-linked reporter proteins such as vitamin D-binding protein (VDBP), the zebrafish homolog of mammalian albumin.

Morphological and histological endpoints capture structural changes in the glomerulus, pronephric tubule, and duct, including cyst formation, podocyte disruption, tubular distension, necrosis, collagen deposition, and renal edema. Transgenic lines with GFP-labeled kidney tissues enable direct visualization of these changes even at early developmental stages. At the cellular and molecular level, apoptosis markers (TUNEL staining, caspase activity, Bax/Bcl-2 ratio), oxidative stress indicators (ROS levels, antioxidant enzyme activity, TBARS), inflammation markers (mpo, cox2, il-1β, tnfα), and DNA damage markers (γH2AX) provide mechanistic resolution that in vitro assays cannot replicate.

The regenerative capacity of zebrafish is an added advantage for their use in nephrotoxicity studies. Contrary to mammals, zebrafish kidneys can regenerate after injury and generate new functional nephrons. This feature makes the adult zebrafish a uniquely powerful model for studying the determinants of renal repair.

In summary, zebrafish in nephrotoxicity screening offer whole-organism, in vivo data at in vitro-compatible throughput, with the mechanistic resolution to differentiate compounds and predict mammalian outcomes, all while supporting 3Rs compliance.

Sources

Drummond BE, Ercanbrack WS, Wingert RA. Modeling Podocyte Ontogeny and Podocytopathies with the Zebrafish. J Dev Biol. 2023 Feb 20;11(1):9. doi: 10.3390/jdb11010009. 

Gehrig J, Pandey G, Westhoff JH. Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases. Front Pediatr. 2018 Jun 28;6:183. doi: 10.3389/fped.2018.00183. 

Kwiatkowska E, Domański L, Dziedziejko V, Kajdy A, Stefańska K, Kwiatkowski S. The Mechanism of Drug Nephrotoxicity and the Methods for Preventing Kidney Damage. Int J Mol Sci. 2021 Jun 6;22(11):6109. doi: 10.3390/ijms22116109.

Lim S, Kang H, Kwon B, Lee JP, Lee J, Choi K. Zebrafish (Danio rerio) as a model organism for screening nephrotoxic chemicals and related mechanisms. Ecotoxicol Environ Saf. 2022 Sep 1;242:113842. doi: 10.1016/j.ecoenv.2022.113842.

Poureetezadi SJ, Wingert RA. Little fish, big catch: zebrafish as a model for kidney disease. Kidney Int. 2016 Jun;89(6):1204-10. doi: 10.1016/j.kint.2016.01.031.

Header image: Pronephric areas of the Tg(wt1b:egfp) zebrafish transgenic line. Credit: Gehrig J, Pandey G, Westhoff JH. Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases. Front Pediatr. 2018 Jun 28;6:183. doi: 10.3389/fped.2018.00183.

By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.