TY - JOUR
AU1 - Kato,, Yuki
AU2 - Tonomura,, Yutaka
AU3 - Hanafusa,, Hiroyuki
AU4 - Nishimura,, Kyohei
AU5 - Fukushima,, Tamio
AU6 - Ueno,, Motonobu
AB - Abstract Drug-induced kidney injury is a serious safety issue in drug development. In this study, we evaluated the usefulness of adult zebrafish as a small in vivo system for detecting drug-induced kidney injury. We first investigated the effects of typical nephrotoxicants, gentamicin and doxorubicin, on adult zebrafish. We found that gentamicin induced renal tubular necrosis with increased lysosome and myeloid bodies, and doxorubicin caused foot process fusion of glomerular podocytes. These findings were similar to those seen in mammals, suggesting a common pathogenesis. Second, to further evaluate the performance of the model in detecting drug-induced kidney injury, adult zebrafish were treated with 28 nephrotoxicants or 14 nonnephrotoxicants for up to 4 days, euthanized 24 h after the final treatment, and examined histopathologically. Sixteen of the 28 nephrotoxicants and none of the 14 nonnephrotoxicants caused drug-induced kidney injury in zebrafish (sensitivity, 57%; specificity, 100%; positive predictive value, 100%; negative predictive value, 54%). Finally, we explored genomic biomarker candidates using kidneys isolated from gentamicin- and cisplatin-treated zebrafish using microarray analysis and identified 3 candidate genes, egr1, atf3, and fos based on increased expression levels and biological implications. The expression of these genes was upregulated dose dependently in cisplatin-treated groups and was > 25-fold higher in gentamicin-treated than in the control group. In conclusion, these results suggest that the adult zebrafish has (1) similar nephrotoxic response to those of mammals, (2) considerable feasibility as an experimental model for toxicity studies, and (3) applicability to pathological examination and genomic biomarker evaluation in drug-induced kidney injury. atf3, egr1, fos, microarray, pathology, small in vivo system, genomic biomarkers Drug-induced kidney injury (DIKI) is a common toxicity observed in pharmaceutical development (Troth et al., 2019). This is on account of the kidney being one of the target organs for drug-induced toxicity because a large amount of blood filtered through the glomerulus and high activity of numerous transporters, which are largely responsible for the elimination of various drugs and their metabolites (Benjamin et al., 2015; International Transporter Consortium et al., 2010; Loghman-Adham et al., 2012). Therefore, it is important to efficiently select drug candidates with a low risk of DIKI at the preclinical toxicological screening stage. However, although various efforts make in vitro systems for investigating DIKI promising, these systems are not always supported by corresponding in vivo outcome. Soo et al. (2018) reported that current in vitro cellular models do not replicate both the morphology and function of renal tubules, and thus, fail to demonstrate injury responses to drug candidates that would be nephrotoxic under in vivo conditions. Furthermore, the use of animal models such as rats and mice for DIKI screening requires large amounts of test substance, leading to low throughput and productivity. In recent years, zebrafish, a small freshwater fish, has been used as an alternative experimental animal model for mammals. Importantly, the nephrons of the adult zebrafish kidney have a similar histological structure and physiological function to those of the mammalian kidney (McCampbell et al., 2014; McKee and Wingert, 2015). For example, zebrafish pronephros and mesonephros have a segmental organization similar to that of mammals with glomeruli and tubules that contain proximal and distal segments (Kroeger and Wingert, 2014). Moreover, a few studies have demonstrated the usefulness of zebrafish as a model for DIKI assessment although only a few nephrotoxicants were used in these experiment studies (Gorgulho et al., 2018; Kamei et al., 2015; McKee and Wingert, 2016). Compared with mammalian models of DIKI, teleosts such as the zebrafish are cost effective and promote animal welfare. Therefore, we focused on adult zebrafish as a small in vivo system. In the current study, we examined the feasibility of zebrafish as an alternative animal model to mammals for evaluating DIKI. For this purpose, we conducted the following 3 experiments. First, to investigate whether zebrafish exhibits the same pathological response as mammals, we administered the representative nephrotoxicants gentamicin and doxorubicin and comprehensively examined the morphological changes of the kidney. Second, to determine the accuracy of DIKI diagnosis, adult zebrafish were treated with 28 nephrotoxicants or 14 nonnephrotoxicants for up to 4 days and were evaluated histopathologically. Finally, to explore genomic biomarkers as alternative tools for pathological determination of DIKI, gene expression was analyzed in the kidney of adult zebrafish-administered cisplatin or gentamicin. MATERIALS AND METHODS Animal care and maintenance Adult golden zebrafish (Danio rerio, 3–6-month old; body length, 2–3 cm) were purchased from a local aquarium farm (Meito Suien, Aichi, Japan) and allowed to acclimate for 2 weeks. Fish were maintained in a noncirculating water tank maintained at a temperature of 27°C to 29°C under a 12-h light/dark cycle. Fish were fed twice daily with live hatched brine shrimp and dry food (Japan SLC, Inc, Shizuoka, Japan). Fish were used without distinction between males and females. During the test substance treatment, zebrafish in each administration group were bred in a small noncirculating water tank. Zebrafish were grossly observed for mortality daily during the administration period at 3 timepoints (before and after dosing in the morning, and in the afternoon). Zebrafish that died or were moribund during the treatment period were immediately fixed with modified Davidson’s solution for pathological examination after euthanasia, if necessary. All study experimental procedures were conducted with the approval of the Institutional Animal Care and Use Committee of Shionogi Pharmaceutical Research Center. Administration of test substances Summarized information pertaining to the list of tested substances, dosage, route of administration, length of study, number of animals tested, and pathological findings is presented in Tables 1 and 2 and Supplementary Table 1. Adult zebrafish were intraperitoneally (i.p.) or orally (p.o.) treated with nephrotoxicants or nonnephrotoxicants for up to 4 days repeatedly (once daily). Body weight was regarded as 400 mg in the experiments in this study, and drugs were administered using 0.5-ml syringes attached to 34-gauge needles (Reactsystem, Osaka, Japan) at 10 µl/zebrafish or 1-ml syringes attached to plastic tubes at 5 μl/zebrafish under anesthesia with 0.6 mM tricaine (FujifilmWako Pure Chemical Corporation, Osaka, Japan) (Drummond and Davidson, 2010; Zang et al., 2011). In addition, 4% (v/v) dimethyl sulfoxide (Fujifilm Wako Pure Chemical Corporation) in saline (Otsuka Pharmaceutical Co, Ltd) and 0.5% (w/v) methylcellulose (Fujifilm Wako Pure Chemical Corporation) solution were used as i.p. and p.o. vehicles respectively (Zang et al., 2011). Either of these vehicles was used for dosing the control animals. The i.p. administration route was first selected because the i.p. bioavailability was expected to be higher than that of p.o., whereas poorly soluble compounds were administered p.o. The highest dose level of each test substance was set at 2000 mg/kg as the limit dose. Where the zebrafish did not tolerate a 2000 mg/kg dose, the maximal tolerated dose was selected as the highest dose. Furthermore, if the maximum concentration of the test substance prepared solutions was lower than maximal tolerated dose, then that maximum concentration was set as the highest dose used. Nephrotoxicants were defined as compounds previously reported to induce renal injury in mice, rats, dogs, monkeys, or rabbits (Table 1). Table 1. Summary of Toxicological Studies in Adult Zebrafish Using Nephrotoxic Compounds Nephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . Main Target Cells for Mammals . Reference (for Mammalian DIKI) . Allopurinola 250 i.p. Days 2, 5 8 (1) No noteworthy findings Positive RT, I Ansari and Rajaraman (1992) and NIBIOHN (2008) 500 i.p. Days 2, 5 8 (1) Necrosis of renal tubule Aristolochic acid Naa 5, 10, 30 i.p. Days 2, 4, 5 8 (2), 8 (3), 5 Necrosis and regeneration of renal tubule Positive RT Hu et al. (2017) Cefepimeb 160, 800 i.p. Days 2, 5 5, 5 (3) Hyaline droplet accumulation of renal tubule Positive RT Elsayed et al. (2014) Cefotaximea 500, 2000 i.p. Day 5 6 (1), 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Ceftazidimec 500, 2000 i.p. Days 3, 5 6, 6 (5) Hyaline droplet accumulation of renal tubule Positive RT Abe et al. (1988); Hayashi et al. (1988) Ceftriaxonec 500, 2000 i.p. Day 5 6, 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Vomiero et al. (2002) Cisplatind 10, 60 i.p. Days 2, 5 8, 8 (6) Necrosis and regeneration of renal tubule Positive RT Jesse et al. (2014) Colistinb 6.25, 12.5 i.p. Day 4 5 (2), 5 (2) Necrosis and hyaline droplet accumulation of renal tubule Positive RT Keirstead et al. (2014; Wallace et al. (2008) Dicloxacillina 500, 1000 i.p. Days 2, 5 6 (1), 6 (4) Necrosis and regeneration of renal tubule Positive RT Nordbring (1984) Doxorubicina 10 i.p. Days 2, 3, 5 16 (7) No noteworthy findings Positive G, RT NIBIOHN (2008) 40, 150 i.p. Days 2, 3, 5 16 (2), 15 (13) Necrosis and regeneration of renal tubule Ethioninec 150, 300 i.p. Days 2, 5 8, 8 (2) Necrosis of renal tubule Positive RT NIBIOHN (2008) Gentamicine 100, 250 i.p. Days 2–5 19 (2), 6 (5) Necrosis, cellular cast, and regeneration of renal tubule Positive RT NIBIOHN (2008) Imipenemf 100, 200 i.p. Day 2 6 (3), 6 (4) Necrosis and cellular cast of renal tubule Positive RT Topham et al. (1989) Meropenemg 250, 500 i.p. Day 5 6 (1), 6 (1) Necrosis, regeneration, and hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Streptozotocine 150, 300, 500 i.p. Day 4 5, 5, 5 Necrosis and regeneration of renal tubule Positive RT Moss et al. (2009); Olsen et al. (2010) Tigecyclineb 100 i.p. Day 5 6 Necrosis and regeneration of renal tubule Positive RT Pfizer (2012) BEAa 125, 250 i.p. Days 2, 3, 5 8, 8 (1) No noteworthy findings Negative P, I Gregg et al. (1990); Price et al. (2010) Amphotericin Ba 15, 30 i.p. Days 2, 5 10 (6), 8 No noteworthy findings Negative RT McDuffie et al. (2016) Cefamandolea 500, 2000 i.p. Days 3, 5 6, 6 (3) No noteworthy findings Negative RT Beck et al. (1980) Cefazolinc 100, 250 i.p. Day 5 6, 6 No noteworthy findings Negative RT Hayashi et al. (1988) Cyclophosphamidea 150, 300 i.p. Days 2, 5 8 (2), 8 (3) No noteworthy findings Negative G, P Abraham and Rabi (2011); Okamura et al. (1992) 150, 600 p.o. Day 5 8, 8 (1) No noteworthy findings Cyclosporine Ac 250, 500 i.p. Days 2, 5 8 (3), 8 (4) No noteworthy findings Negative G, RT Hill 1986; Ryffel and Mihatsch (1986) Ethionamidec 500, 1000 p.o. Day 5 5, 5 No noteworthy findings Negative RT NIBIOHN (2008) Indomethacina 25, 50 p.o. Day 5 5 (1), 5 No noteworthy findings Negative P Brix (2002); Nagappan et al. (2014) Monocrotalinea 25, 50 i.p. Days 2, 5 8, 8 No noteworthy findings Negative G Kurozumi et al. (1983) 25, 500 p.o. Day 5 4, 4 No noteworthy findings Piperacillinc 500, 2000 i.p. Day 5 6, 6 (1) No noteworthy findings Negative RT Hori et al. (2000) PANa 100, 200 i.p. Days 2, 3, 5 8, 8 (2) No noteworthy findings Negative G, RT Herman-Edelstein et al. (2016); Hill (1986) Thioacetamidee 50, 100 i.p. Days 2, 5 8 (1), 8 (1) No noteworthy findings Negative RT Hafez et al. (2019) 50, 500 p.o. Day 5 4, 4 No noteworthy findings Nephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . Main Target Cells for Mammals . Reference (for Mammalian DIKI) . Allopurinola 250 i.p. Days 2, 5 8 (1) No noteworthy findings Positive RT, I Ansari and Rajaraman (1992) and NIBIOHN (2008) 500 i.p. Days 2, 5 8 (1) Necrosis of renal tubule Aristolochic acid Naa 5, 10, 30 i.p. Days 2, 4, 5 8 (2), 8 (3), 5 Necrosis and regeneration of renal tubule Positive RT Hu et al. (2017) Cefepimeb 160, 800 i.p. Days 2, 5 5, 5 (3) Hyaline droplet accumulation of renal tubule Positive RT Elsayed et al. (2014) Cefotaximea 500, 2000 i.p. Day 5 6 (1), 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Ceftazidimec 500, 2000 i.p. Days 3, 5 6, 6 (5) Hyaline droplet accumulation of renal tubule Positive RT Abe et al. (1988); Hayashi et al. (1988) Ceftriaxonec 500, 2000 i.p. Day 5 6, 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Vomiero et al. (2002) Cisplatind 10, 60 i.p. Days 2, 5 8, 8 (6) Necrosis and regeneration of renal tubule Positive RT Jesse et al. (2014) Colistinb 6.25, 12.5 i.p. Day 4 5 (2), 5 (2) Necrosis and hyaline droplet accumulation of renal tubule Positive RT Keirstead et al. (2014; Wallace et al. (2008) Dicloxacillina 500, 1000 i.p. Days 2, 5 6 (1), 6 (4) Necrosis and regeneration of renal tubule Positive RT Nordbring (1984) Doxorubicina 10 i.p. Days 2, 3, 5 16 (7) No noteworthy findings Positive G, RT NIBIOHN (2008) 40, 150 i.p. Days 2, 3, 5 16 (2), 15 (13) Necrosis and regeneration of renal tubule Ethioninec 150, 300 i.p. Days 2, 5 8, 8 (2) Necrosis of renal tubule Positive RT NIBIOHN (2008) Gentamicine 100, 250 i.p. Days 2–5 19 (2), 6 (5) Necrosis, cellular cast, and regeneration of renal tubule Positive RT NIBIOHN (2008) Imipenemf 100, 200 i.p. Day 2 6 (3), 6 (4) Necrosis and cellular cast of renal tubule Positive RT Topham et al. (1989) Meropenemg 250, 500 i.p. Day 5 6 (1), 6 (1) Necrosis, regeneration, and hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Streptozotocine 150, 300, 500 i.p. Day 4 5, 5, 5 Necrosis and regeneration of renal tubule Positive RT Moss et al. (2009); Olsen et al. (2010) Tigecyclineb 100 i.p. Day 5 6 Necrosis and regeneration of renal tubule Positive RT Pfizer (2012) BEAa 125, 250 i.p. Days 2, 3, 5 8, 8 (1) No noteworthy findings Negative P, I Gregg et al. (1990); Price et al. (2010) Amphotericin Ba 15, 30 i.p. Days 2, 5 10 (6), 8 No noteworthy findings Negative RT McDuffie et al. (2016) Cefamandolea 500, 2000 i.p. Days 3, 5 6, 6 (3) No noteworthy findings Negative RT Beck et al. (1980) Cefazolinc 100, 250 i.p. Day 5 6, 6 No noteworthy findings Negative RT Hayashi et al. (1988) Cyclophosphamidea 150, 300 i.p. Days 2, 5 8 (2), 8 (3) No noteworthy findings Negative G, P Abraham and Rabi (2011); Okamura et al. (1992) 150, 600 p.o. Day 5 8, 8 (1) No noteworthy findings Cyclosporine Ac 250, 500 i.p. Days 2, 5 8 (3), 8 (4) No noteworthy findings Negative G, RT Hill 1986; Ryffel and Mihatsch (1986) Ethionamidec 500, 1000 p.o. Day 5 5, 5 No noteworthy findings Negative RT NIBIOHN (2008) Indomethacina 25, 50 p.o. Day 5 5 (1), 5 No noteworthy findings Negative P Brix (2002); Nagappan et al. (2014) Monocrotalinea 25, 50 i.p. Days 2, 5 8, 8 No noteworthy findings Negative G Kurozumi et al. (1983) 25, 500 p.o. Day 5 4, 4 No noteworthy findings Piperacillinc 500, 2000 i.p. Day 5 6, 6 (1) No noteworthy findings Negative RT Hori et al. (2000) PANa 100, 200 i.p. Days 2, 3, 5 8, 8 (2) No noteworthy findings Negative G, RT Herman-Edelstein et al. (2016); Hill (1986) Thioacetamidee 50, 100 i.p. Days 2, 5 8 (1), 8 (1) No noteworthy findings Negative RT Hafez et al. (2019) 50, 500 p.o. Day 5 4, 4 No noteworthy findings The starting day of administration is designated as day 1. Abbreviations: RT, renal tubule; I, interstitium; G, glomerular; P, papilla; BEA, 2-bromoethylamine hydromide; PAN, puromycin aminonucleoside; DIKI, drug-induced kidney injury; i.p., intraperitoneal(ly); p.o., per os (oral[ly]). a Sigma Aldrich Chemical Co (St Louis, Missouri). b Synthesized for research at Shionogi Pharmaceutical Research Center. c Tokyo Chemical Industry (Tokyo, Japan). d Sequoia Research Products Ltd (Pangbourne, United Kingdom). e Wako Pure Chemical Industries, Ltd (Osaka, Japan). f Cayman Chemical (Ann Arbor, Michigan). g Pharmaceutical grade. Open in new tab Table 1. Summary of Toxicological Studies in Adult Zebrafish Using Nephrotoxic Compounds Nephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . Main Target Cells for Mammals . Reference (for Mammalian DIKI) . Allopurinola 250 i.p. Days 2, 5 8 (1) No noteworthy findings Positive RT, I Ansari and Rajaraman (1992) and NIBIOHN (2008) 500 i.p. Days 2, 5 8 (1) Necrosis of renal tubule Aristolochic acid Naa 5, 10, 30 i.p. Days 2, 4, 5 8 (2), 8 (3), 5 Necrosis and regeneration of renal tubule Positive RT Hu et al. (2017) Cefepimeb 160, 800 i.p. Days 2, 5 5, 5 (3) Hyaline droplet accumulation of renal tubule Positive RT Elsayed et al. (2014) Cefotaximea 500, 2000 i.p. Day 5 6 (1), 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Ceftazidimec 500, 2000 i.p. Days 3, 5 6, 6 (5) Hyaline droplet accumulation of renal tubule Positive RT Abe et al. (1988); Hayashi et al. (1988) Ceftriaxonec 500, 2000 i.p. Day 5 6, 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Vomiero et al. (2002) Cisplatind 10, 60 i.p. Days 2, 5 8, 8 (6) Necrosis and regeneration of renal tubule Positive RT Jesse et al. (2014) Colistinb 6.25, 12.5 i.p. Day 4 5 (2), 5 (2) Necrosis and hyaline droplet accumulation of renal tubule Positive RT Keirstead et al. (2014; Wallace et al. (2008) Dicloxacillina 500, 1000 i.p. Days 2, 5 6 (1), 6 (4) Necrosis and regeneration of renal tubule Positive RT Nordbring (1984) Doxorubicina 10 i.p. Days 2, 3, 5 16 (7) No noteworthy findings Positive G, RT NIBIOHN (2008) 40, 150 i.p. Days 2, 3, 5 16 (2), 15 (13) Necrosis and regeneration of renal tubule Ethioninec 150, 300 i.p. Days 2, 5 8, 8 (2) Necrosis of renal tubule Positive RT NIBIOHN (2008) Gentamicine 100, 250 i.p. Days 2–5 19 (2), 6 (5) Necrosis, cellular cast, and regeneration of renal tubule Positive RT NIBIOHN (2008) Imipenemf 100, 200 i.p. Day 2 6 (3), 6 (4) Necrosis and cellular cast of renal tubule Positive RT Topham et al. (1989) Meropenemg 250, 500 i.p. Day 5 6 (1), 6 (1) Necrosis, regeneration, and hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Streptozotocine 150, 300, 500 i.p. Day 4 5, 5, 5 Necrosis and regeneration of renal tubule Positive RT Moss et al. (2009); Olsen et al. (2010) Tigecyclineb 100 i.p. Day 5 6 Necrosis and regeneration of renal tubule Positive RT Pfizer (2012) BEAa 125, 250 i.p. Days 2, 3, 5 8, 8 (1) No noteworthy findings Negative P, I Gregg et al. (1990); Price et al. (2010) Amphotericin Ba 15, 30 i.p. Days 2, 5 10 (6), 8 No noteworthy findings Negative RT McDuffie et al. (2016) Cefamandolea 500, 2000 i.p. Days 3, 5 6, 6 (3) No noteworthy findings Negative RT Beck et al. (1980) Cefazolinc 100, 250 i.p. Day 5 6, 6 No noteworthy findings Negative RT Hayashi et al. (1988) Cyclophosphamidea 150, 300 i.p. Days 2, 5 8 (2), 8 (3) No noteworthy findings Negative G, P Abraham and Rabi (2011); Okamura et al. (1992) 150, 600 p.o. Day 5 8, 8 (1) No noteworthy findings Cyclosporine Ac 250, 500 i.p. Days 2, 5 8 (3), 8 (4) No noteworthy findings Negative G, RT Hill 1986; Ryffel and Mihatsch (1986) Ethionamidec 500, 1000 p.o. Day 5 5, 5 No noteworthy findings Negative RT NIBIOHN (2008) Indomethacina 25, 50 p.o. Day 5 5 (1), 5 No noteworthy findings Negative P Brix (2002); Nagappan et al. (2014) Monocrotalinea 25, 50 i.p. Days 2, 5 8, 8 No noteworthy findings Negative G Kurozumi et al. (1983) 25, 500 p.o. Day 5 4, 4 No noteworthy findings Piperacillinc 500, 2000 i.p. Day 5 6, 6 (1) No noteworthy findings Negative RT Hori et al. (2000) PANa 100, 200 i.p. Days 2, 3, 5 8, 8 (2) No noteworthy findings Negative G, RT Herman-Edelstein et al. (2016); Hill (1986) Thioacetamidee 50, 100 i.p. Days 2, 5 8 (1), 8 (1) No noteworthy findings Negative RT Hafez et al. (2019) 50, 500 p.o. Day 5 4, 4 No noteworthy findings Nephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . Main Target Cells for Mammals . Reference (for Mammalian DIKI) . Allopurinola 250 i.p. Days 2, 5 8 (1) No noteworthy findings Positive RT, I Ansari and Rajaraman (1992) and NIBIOHN (2008) 500 i.p. Days 2, 5 8 (1) Necrosis of renal tubule Aristolochic acid Naa 5, 10, 30 i.p. Days 2, 4, 5 8 (2), 8 (3), 5 Necrosis and regeneration of renal tubule Positive RT Hu et al. (2017) Cefepimeb 160, 800 i.p. Days 2, 5 5, 5 (3) Hyaline droplet accumulation of renal tubule Positive RT Elsayed et al. (2014) Cefotaximea 500, 2000 i.p. Day 5 6 (1), 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Ceftazidimec 500, 2000 i.p. Days 3, 5 6, 6 (5) Hyaline droplet accumulation of renal tubule Positive RT Abe et al. (1988); Hayashi et al. (1988) Ceftriaxonec 500, 2000 i.p. Day 5 6, 6 (1) Hyaline droplet accumulation of renal tubule Positive RT Vomiero et al. (2002) Cisplatind 10, 60 i.p. Days 2, 5 8, 8 (6) Necrosis and regeneration of renal tubule Positive RT Jesse et al. (2014) Colistinb 6.25, 12.5 i.p. Day 4 5 (2), 5 (2) Necrosis and hyaline droplet accumulation of renal tubule Positive RT Keirstead et al. (2014; Wallace et al. (2008) Dicloxacillina 500, 1000 i.p. Days 2, 5 6 (1), 6 (4) Necrosis and regeneration of renal tubule Positive RT Nordbring (1984) Doxorubicina 10 i.p. Days 2, 3, 5 16 (7) No noteworthy findings Positive G, RT NIBIOHN (2008) 40, 150 i.p. Days 2, 3, 5 16 (2), 15 (13) Necrosis and regeneration of renal tubule Ethioninec 150, 300 i.p. Days 2, 5 8, 8 (2) Necrosis of renal tubule Positive RT NIBIOHN (2008) Gentamicine 100, 250 i.p. Days 2–5 19 (2), 6 (5) Necrosis, cellular cast, and regeneration of renal tubule Positive RT NIBIOHN (2008) Imipenemf 100, 200 i.p. Day 2 6 (3), 6 (4) Necrosis and cellular cast of renal tubule Positive RT Topham et al. (1989) Meropenemg 250, 500 i.p. Day 5 6 (1), 6 (1) Necrosis, regeneration, and hyaline droplet accumulation of renal tubule Positive RT Topham et al. (1989) Streptozotocine 150, 300, 500 i.p. Day 4 5, 5, 5 Necrosis and regeneration of renal tubule Positive RT Moss et al. (2009); Olsen et al. (2010) Tigecyclineb 100 i.p. Day 5 6 Necrosis and regeneration of renal tubule Positive RT Pfizer (2012) BEAa 125, 250 i.p. Days 2, 3, 5 8, 8 (1) No noteworthy findings Negative P, I Gregg et al. (1990); Price et al. (2010) Amphotericin Ba 15, 30 i.p. Days 2, 5 10 (6), 8 No noteworthy findings Negative RT McDuffie et al. (2016) Cefamandolea 500, 2000 i.p. Days 3, 5 6, 6 (3) No noteworthy findings Negative RT Beck et al. (1980) Cefazolinc 100, 250 i.p. Day 5 6, 6 No noteworthy findings Negative RT Hayashi et al. (1988) Cyclophosphamidea 150, 300 i.p. Days 2, 5 8 (2), 8 (3) No noteworthy findings Negative G, P Abraham and Rabi (2011); Okamura et al. (1992) 150, 600 p.o. Day 5 8, 8 (1) No noteworthy findings Cyclosporine Ac 250, 500 i.p. Days 2, 5 8 (3), 8 (4) No noteworthy findings Negative G, RT Hill 1986; Ryffel and Mihatsch (1986) Ethionamidec 500, 1000 p.o. Day 5 5, 5 No noteworthy findings Negative RT NIBIOHN (2008) Indomethacina 25, 50 p.o. Day 5 5 (1), 5 No noteworthy findings Negative P Brix (2002); Nagappan et al. (2014) Monocrotalinea 25, 50 i.p. Days 2, 5 8, 8 No noteworthy findings Negative G Kurozumi et al. (1983) 25, 500 p.o. Day 5 4, 4 No noteworthy findings Piperacillinc 500, 2000 i.p. Day 5 6, 6 (1) No noteworthy findings Negative RT Hori et al. (2000) PANa 100, 200 i.p. Days 2, 3, 5 8, 8 (2) No noteworthy findings Negative G, RT Herman-Edelstein et al. (2016); Hill (1986) Thioacetamidee 50, 100 i.p. Days 2, 5 8 (1), 8 (1) No noteworthy findings Negative RT Hafez et al. (2019) 50, 500 p.o. Day 5 4, 4 No noteworthy findings The starting day of administration is designated as day 1. Abbreviations: RT, renal tubule; I, interstitium; G, glomerular; P, papilla; BEA, 2-bromoethylamine hydromide; PAN, puromycin aminonucleoside; DIKI, drug-induced kidney injury; i.p., intraperitoneal(ly); p.o., per os (oral[ly]). a Sigma Aldrich Chemical Co (St Louis, Missouri). b Synthesized for research at Shionogi Pharmaceutical Research Center. c Tokyo Chemical Industry (Tokyo, Japan). d Sequoia Research Products Ltd (Pangbourne, United Kingdom). e Wako Pure Chemical Industries, Ltd (Osaka, Japan). f Cayman Chemical (Ann Arbor, Michigan). g Pharmaceutical grade. Open in new tab Table 2. Summary of Toxicological Studies in Adult Zebrafish Using Nonnephrotoxic Compounds Nonnephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Dosing Frequency . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . 1,3-Dinitrobenzenea 10, 40 p.o. Repeat Day 5 8, 8 No noteworthy findings Negative 2-Acetamidofluoreneb 250, 500 p.o. Repeat Day 5 10 (1), 10 No noteworthy findings Negative 6-Mercaptopurinec 100, 400 p.o. Repeat Day 5 8, 8 (2) No noteworthy findings Negative Allyl alcoholc 50, 100 i.p. Repeat Day 4 8 (2), 8 (3) No noteworthy findings Negative Allylamined 10, 50 i.p. Repeat Day 5 8 (2), 8 (6) No noteworthy findings Negative 50, 200 p.o. Repeat Day 5 8, 8 (5) No noteworthy findings Negative Ampicillinb 500, 2000 i.p. Repeat Day 5 6 (1), 6 (1) No noteworthy findings Negative Chlorpromazineb 75, 150, 300 i.p. Single/repeat Days 2, 3 6 (3), 8 (4), 8 (4) No noteworthy findings Negative 25, 50 p.o. Repeat Day 5 4, 4 No noteworthy findings Negative Colchicineb 15, 30 i.p. Single/repeat Days 2, 3 8 (4), 8 (5) No noteworthy findings Negative Ethambutolb 375, 750 p.o. Repeat Day 5 5 (1), 5 (2) No noteworthy findings Negative EGMEe 100, 500 i.p. Repeat Day 5 8 (1), 8 No noteworthy findings Negative 500, 2000 p.o. Repeat Day 5 8 (2), 8 No noteworthy findings Negative Iodate Naf 30 i.p. Single/repeat Day 5 8 (5) No noteworthy findings Negative Methapyrileneb 50, 250 i.p. Repeat Day 5 14 (2), 8 No noteworthy findings Negative Phalloidinb 0.25, 0.5 i.p. Single/repeat Days 2, 5 8, 8 No noteworthy findings Negative Voriconazoleb 75, 150 i.p. Single/repeat Days 2, 5 8 No noteworthy findings Negative Nonnephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Dosing Frequency . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . 1,3-Dinitrobenzenea 10, 40 p.o. Repeat Day 5 8, 8 No noteworthy findings Negative 2-Acetamidofluoreneb 250, 500 p.o. Repeat Day 5 10 (1), 10 No noteworthy findings Negative 6-Mercaptopurinec 100, 400 p.o. Repeat Day 5 8, 8 (2) No noteworthy findings Negative Allyl alcoholc 50, 100 i.p. Repeat Day 4 8 (2), 8 (3) No noteworthy findings Negative Allylamined 10, 50 i.p. Repeat Day 5 8 (2), 8 (6) No noteworthy findings Negative 50, 200 p.o. Repeat Day 5 8, 8 (5) No noteworthy findings Negative Ampicillinb 500, 2000 i.p. Repeat Day 5 6 (1), 6 (1) No noteworthy findings Negative Chlorpromazineb 75, 150, 300 i.p. Single/repeat Days 2, 3 6 (3), 8 (4), 8 (4) No noteworthy findings Negative 25, 50 p.o. Repeat Day 5 4, 4 No noteworthy findings Negative Colchicineb 15, 30 i.p. Single/repeat Days 2, 3 8 (4), 8 (5) No noteworthy findings Negative Ethambutolb 375, 750 p.o. Repeat Day 5 5 (1), 5 (2) No noteworthy findings Negative EGMEe 100, 500 i.p. Repeat Day 5 8 (1), 8 No noteworthy findings Negative 500, 2000 p.o. Repeat Day 5 8 (2), 8 No noteworthy findings Negative Iodate Naf 30 i.p. Single/repeat Day 5 8 (5) No noteworthy findings Negative Methapyrileneb 50, 250 i.p. Repeat Day 5 14 (2), 8 No noteworthy findings Negative Phalloidinb 0.25, 0.5 i.p. Single/repeat Days 2, 5 8, 8 No noteworthy findings Negative Voriconazoleb 75, 150 i.p. Single/repeat Days 2, 5 8 No noteworthy findings Negative The starting day of administration is designated as day 1. Abbreviations: DIKI, drug-induced kidney injury; i.p., intraperitoneal(ly); p.o., per os (oral[ly]). a Alfa Aesar (Heysham, United Kingdom). b Sigma Aldrich Chemical Co. c Wako Pure Chemical Industries, Ltd. d abcr GmbH & Co (Karlsruhe, Germany). e MP Biomedicals (Santa Ana, California). f Nacalai Tesque, Inc (Kyoto, Japan). Open in new tab Table 2. Summary of Toxicological Studies in Adult Zebrafish Using Nonnephrotoxic Compounds Nonnephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Dosing Frequency . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . 1,3-Dinitrobenzenea 10, 40 p.o. Repeat Day 5 8, 8 No noteworthy findings Negative 2-Acetamidofluoreneb 250, 500 p.o. Repeat Day 5 10 (1), 10 No noteworthy findings Negative 6-Mercaptopurinec 100, 400 p.o. Repeat Day 5 8, 8 (2) No noteworthy findings Negative Allyl alcoholc 50, 100 i.p. Repeat Day 4 8 (2), 8 (3) No noteworthy findings Negative Allylamined 10, 50 i.p. Repeat Day 5 8 (2), 8 (6) No noteworthy findings Negative 50, 200 p.o. Repeat Day 5 8, 8 (5) No noteworthy findings Negative Ampicillinb 500, 2000 i.p. Repeat Day 5 6 (1), 6 (1) No noteworthy findings Negative Chlorpromazineb 75, 150, 300 i.p. Single/repeat Days 2, 3 6 (3), 8 (4), 8 (4) No noteworthy findings Negative 25, 50 p.o. Repeat Day 5 4, 4 No noteworthy findings Negative Colchicineb 15, 30 i.p. Single/repeat Days 2, 3 8 (4), 8 (5) No noteworthy findings Negative Ethambutolb 375, 750 p.o. Repeat Day 5 5 (1), 5 (2) No noteworthy findings Negative EGMEe 100, 500 i.p. Repeat Day 5 8 (1), 8 No noteworthy findings Negative 500, 2000 p.o. Repeat Day 5 8 (2), 8 No noteworthy findings Negative Iodate Naf 30 i.p. Single/repeat Day 5 8 (5) No noteworthy findings Negative Methapyrileneb 50, 250 i.p. Repeat Day 5 14 (2), 8 No noteworthy findings Negative Phalloidinb 0.25, 0.5 i.p. Single/repeat Days 2, 5 8, 8 No noteworthy findings Negative Voriconazoleb 75, 150 i.p. Single/repeat Days 2, 5 8 No noteworthy findings Negative Nonnephrotoxic Compounds . Dosage (mg/kg/day) . Dosing Route . Dosing Frequency . Sacrifice Timing . Number of Animals (Number of Dead Animals) . Pathological Findings . DIKI for Zebrafish . 1,3-Dinitrobenzenea 10, 40 p.o. Repeat Day 5 8, 8 No noteworthy findings Negative 2-Acetamidofluoreneb 250, 500 p.o. Repeat Day 5 10 (1), 10 No noteworthy findings Negative 6-Mercaptopurinec 100, 400 p.o. Repeat Day 5 8, 8 (2) No noteworthy findings Negative Allyl alcoholc 50, 100 i.p. Repeat Day 4 8 (2), 8 (3) No noteworthy findings Negative Allylamined 10, 50 i.p. Repeat Day 5 8 (2), 8 (6) No noteworthy findings Negative 50, 200 p.o. Repeat Day 5 8, 8 (5) No noteworthy findings Negative Ampicillinb 500, 2000 i.p. Repeat Day 5 6 (1), 6 (1) No noteworthy findings Negative Chlorpromazineb 75, 150, 300 i.p. Single/repeat Days 2, 3 6 (3), 8 (4), 8 (4) No noteworthy findings Negative 25, 50 p.o. Repeat Day 5 4, 4 No noteworthy findings Negative Colchicineb 15, 30 i.p. Single/repeat Days 2, 3 8 (4), 8 (5) No noteworthy findings Negative Ethambutolb 375, 750 p.o. Repeat Day 5 5 (1), 5 (2) No noteworthy findings Negative EGMEe 100, 500 i.p. Repeat Day 5 8 (1), 8 No noteworthy findings Negative 500, 2000 p.o. Repeat Day 5 8 (2), 8 No noteworthy findings Negative Iodate Naf 30 i.p. Single/repeat Day 5 8 (5) No noteworthy findings Negative Methapyrileneb 50, 250 i.p. Repeat Day 5 14 (2), 8 No noteworthy findings Negative Phalloidinb 0.25, 0.5 i.p. Single/repeat Days 2, 5 8, 8 No noteworthy findings Negative Voriconazoleb 75, 150 i.p. Single/repeat Days 2, 5 8 No noteworthy findings Negative The starting day of administration is designated as day 1. Abbreviations: DIKI, drug-induced kidney injury; i.p., intraperitoneal(ly); p.o., per os (oral[ly]). a Alfa Aesar (Heysham, United Kingdom). b Sigma Aldrich Chemical Co. c Wako Pure Chemical Industries, Ltd. d abcr GmbH & Co (Karlsruhe, Germany). e MP Biomedicals (Santa Ana, California). f Nacalai Tesque, Inc (Kyoto, Japan). Open in new tab Pathological examination At 24 h after the final drug treatment, zebrafish were euthanized by exsanguination, in which the tails were cut under 0.6 mM tricaine anesthesia. Then, the zebrafish were fixed with modified Davidson’s solution for approximately 48 h and 10% buffered formalin for more than 24 h (Wolf et al., 2015). Four vertical longitudinal sections (3 µm) of whole body paraffin-embedded specimens were prepared per animals as follows. The first section was cut at the center of the eyeball and the fourth at the midline, whereas the second and third sections were cut between them. The kidney was observed primarily in the third and fourth sections, which were stained with hematoxylin and eosin using a routine method. Tissues for each individual fish were scored semiquantitatively for degree of pathology using a scale graded into the following 5 categories: none (0), minimal (1), mild (2), moderate (3), and marked (4). Electron microscopy Zebrafish were euthanized by exsanguination by cutting the tails under 0.6 mM tricaine anesthesia. Using scissors, an incision was made from the anus to the lower jaw along the ventral midline, and the zebrafish were then laid in the dorsal position to fix the abdominal wall on a rubber board using a pin. Abdominal organs such as the gut and associated organs (including the liver and pancreas), gonads (testes or ovaries), and the swim bladder were removed from the abdominal cavity. The kidney, which was attached to the dorsal abdominal wall (Figure 1A), was soaked in 0.1 M phosphate buffer (pH 7.4) containing 3% glutaraldehyde for several minutes and then the trunk kidney was excised using a scalpel. The isolated kidney was fixed in 0.1 M phosphate buffer (pH 7.4) containing 3% glutaraldehyde, postfixed in 2% osmium tetroxide in 0.5 M sucrose buffer, and then embedded in Epon. After staining with platinum blue followed by lead citrate, ultrathin sections were observed using a JEM-1400 plus electron microscope (JEOL, Tokyo, Japan). Figure 1. Open in new tabDownload slide Morphological features of control adult zebrafish kidney. A, Image of a normal kidney seen from the ventral side after organs inside the thoracoabdominal cavity were removed. B, Image of hematoxylin and eosin (H&E) staining of a normal kidney. C, Images of platinum blue and lead citrate staining of the glomerulus (C), proximal tubule (D), and distal tubule (E). a, Glomerulus; b, proximal tubule; c, distal tubule; d, hematopoietic cells. Arrow, kidney border; arrowhead, podocyte foot process; asterisk, basement membrane. Original magnification, 400× (B). Scale bars: (C) 1 µm and (D, E), 5 µm. Abbreviations: N, nuclei; Ly, lysosome; Mv, microvilli; Er, erythrocyte; Pd, podocyte; Bs, Bowman’s space; L, lumen. Figure 1. Open in new tabDownload slide Morphological features of control adult zebrafish kidney. A, Image of a normal kidney seen from the ventral side after organs inside the thoracoabdominal cavity were removed. B, Image of hematoxylin and eosin (H&E) staining of a normal kidney. C, Images of platinum blue and lead citrate staining of the glomerulus (C), proximal tubule (D), and distal tubule (E). a, Glomerulus; b, proximal tubule; c, distal tubule; d, hematopoietic cells. Arrow, kidney border; arrowhead, podocyte foot process; asterisk, basement membrane. Original magnification, 400× (B). Scale bars: (C) 1 µm and (D, E), 5 µm. Abbreviations: N, nuclei; Ly, lysosome; Mv, microvilli; Er, erythrocyte; Pd, podocyte; Bs, Bowman’s space; L, lumen. Microarray analysis For microarray analysis, gentamicin (100 mg/kg) or cisplatin (10 mg/kg) was administered once, i.p. to adult zebrafish, whereas vehicle-treated controls received 4% (v/v) dimethyl sulfoxide/saline. At 48-h postdosing (hpd), fresh kidneys were isolated as described in the electron microscopy section, immersed in RLT lysis buffer (RNeasy Mini Kit, Qiagen, Valencia, California) containing β-mercaptoethanol, and stored in a freezer until the analysis. Total RNA was prepared from the isolated kidney using the miRNeasy mini kit (Qiagen, Valencia, California) according to the manufacturer’s instructions. The concentration and purity of the RNA were assessed using a Multiskan GO (Thermo Fisher Scientific, Yokohama, Japan) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California). RNA extraction and amplification yielded rRNA ratios with an 18S:28S of 0.7–1.2. RNA integrity number values were 8.2–9.2 for the samples. RNA concentrations were 29–111 ng/µl, and the RNA integrity number values tended to be good in the samples with high RNA concentrations. The procedure was conducted according to the manufacturer’s instructions using a 3′ IVT Express kit (Affymetrix, Santa Clara, California). In brief, 150 ng total RNA was used as the template for each reaction. Fragmented biotin-labeled cRNA was hybridized to the Zebrafish Genome Array (Affymetrix) for 17 h at 45°C and 60 rpm. The array was then washed and stained with streptavidin-phycoerythrin using a Fluidics Station 450 (Affymetrix) and scanned using a Gene Array Scanner GCS3000 7G (Affymetrix). Microarray Analysis Suite 5.0 (MAS, Affymetrix) was used to quantify the microarray signals. The microarray data were first imported into Spotfire DecisionSite for functional genomics (Spotfire, Göteborg, Sweden), and all signal intensities for each chip were normalized to the global mean. After excluding absent probes, expressed sequence tags, and probe annotation grades E or R, probes that were upregulated > 3-fold that of the vehicle control kidneys were selected as significantly changed genes. The Affymetrix Detection Call algorithm was also used for this step, and upregulated probes showing all present calls in treated zebrafish were selected. PCR analysis The following gene expression assays (Thermo Fisher Scientific) were used for quantitative real-time PCR (qRT-PCR): early growth response protein 1 (egr1), activating transcription factor (atf3), and Fos proto-oncogene (fos), and hepatitis A virus cellular receptor 1 (HAVCR1, kidney injury molecule 1 [kim1]). Total RNA (40 ng) was used as the template and TaqMan gene expression assay kits (for egr1, assay ID: Dr03074044_m1; for atf3, assay ID: Dr03100567_m1; for fos, assay ID: Dr03100811_g1; for havcr1 (kim1), assay ID: Dr03133931_mH) or TaqMan endogenous controls (rpl13a, ribosomal protein L13a, Dr03101114_g1) were used as gene-specific probes and primer sets. The qRT-PCR was performed using a QuantiTect probe RT-PCR kit (Qiagen), and transcript levels were quantified using an ABI PRISM 7500 Fast System (Applied Biosystems) according to the manufacturer’s instructions. RT and amplification conditions were set as follows: 50°C for 30 min, 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The resulting cycle threshold (Ct) value was processed using the comparative Ct method. The ΔCt values for all genes were determined relative to the control gene rpl13a. The ΔΔCt values were calculated using treated group means relative to control group means and fold change data were calculated from the ΔΔCt values. Statistical significance was evaluated using a one-way ANOVA and Dunnett’s multiple comparison post hoc test for the vehicle control group versus cisplatin-treated groups, or Welch’s t test for the vehicle control group versus gentamicin-treated group. Statistical significance was determined using GraphPad Prism 7 (San Diego, California). RESULTS Exp. 1: Pathological Features of DIKI in Adult Zebrafish Adult zebrafish kidney showed elongated and flattened tissue that occupied the space between the vertebral column and swim bladder (Figure 1A). The zebrafish nephron was also composed of the glomerulus, proximal and distal tubules, and collecting ducts, but no thin limb segment was observed (Figure 1B). A major difference between the mammalian and zebrafish kidney is that many hematopoietic cells were seen in the interstitium of the zebrafish kidney. The glomerulus was composed of podocytes, vascular endothelial cells, and mesangial cells, similar to that of mammals, and podocyte foot processes regularly connected by slit-diaphragms covered the glomerular basement membrane of the Bowman’s space side (Figure 1C). The proximal tubule was continuous from the glomerulus and was lined by columnar cells. Ultrastructurally, epithelial cells of the proximal tubule near the glomerulus (segment 1: S1) contained large lysosomes, a nucleus located in the center to basal aspect, and many large mitochondria located in the basal aspect that were covered with extensive basal cytoplasmic invaginations and prominent brush-border microvilli on the apical surface (Figure 1D). The proximal tubule near the distal tubule (segment 2: S2) consisted of numerous vacuoles and mitochondria-poor/lysosome-poor cytoplasm, less brush-boarder microvilli than S1, and the round nucleus located in the basal aspect. The distal and connecting tubules showed similar epithelial structures with minimal or absent microvilli and an extensive system of basolateral membrane infoldings associated with mitochondria, whereas the collecting duct had a dilated lumen (Figure 1E). Gentamicin was administered once i.p. to zebrafish at the doses of 0 (control) and 100 mg/kg to sequentially assess pathological changes in the kidney. At 24 hpd, mild to moderate necrosis and sloughing of affected epithelium into the tubular lumina were observed in the renal tubule (Figure 2A). From 48 to 72 hpd, in addition to tubular necrosis, minimal to moderate tubular regeneration was seen in the tubular epithelium (Figures 2B and 2C). At 96 hpd, a few regenerative tubules and nephron neogenesis defined by dark basophilic nephron segments were also observed adjacent to the original nephrons (Figure 2D). Figure 2. Open in new tabDownload slide Chronological histopathological changes in gentamicin-treated adult zebrafish kidney. Images of hematoxylin and eosin (H&E) staining of gentamicin-treated zebrafish at (A) 24 hpd (B), 48 hpd, (C) 72 hpd, and (D) 96 hpd. Black arrowhead, tubular necrosis; white arrowhead, nephron neogenesis; arrow, tubular regeneration. Original magnification, 400×. Abbreviation: hpd, hour postdosing. Figure 2. Open in new tabDownload slide Chronological histopathological changes in gentamicin-treated adult zebrafish kidney. Images of hematoxylin and eosin (H&E) staining of gentamicin-treated zebrafish at (A) 24 hpd (B), 48 hpd, (C) 72 hpd, and (D) 96 hpd. Black arrowhead, tubular necrosis; white arrowhead, nephron neogenesis; arrow, tubular regeneration. Original magnification, 400×. Abbreviation: hpd, hour postdosing. To investigate the pathogenesis of gentamicin-induced kidney injury, ultrastructural changes of the adult zebrafish kidney were examined, following administration of a single i.p. injection of 100 mg/kg gentamicin. Following this treatment, irregular lysosomes of various sizes increased, and dilatation and degeneration of the mitochondria were also observed in the proximal tubule at 2 hpd (Figure 3A). These changes were exacerbated, and lamellar bodies, swelling of the cytoplasm, and detachment of microvilli were seen at 6 hpd (Figure 3B). Many organelles were denatured while cell membrane rupture, nuclear fragmentation, and inflammatory cell infiltration were also observed at 24 hpd (Figure 3C). Figure 3. Open in new tabDownload slide Chronological ultrastructural changes of the proximal tubules of gentamicin-treated adult zebrafish. Images of platinum blue and lead citrate staining of gentamicin-treated zebrafish at (A) 2 hpd, (B) 6 hpd, and (C) 24 hpd. Arrowhead, lysosomal degeneration; white arrow, myeloid body; black arrow, mitochondrial degeneration; white asterisk, degenerative mitochondria-containing secondary lysosome; black asterisk, swelling in cytoplasm. Scale bars: (A) 10 µm and (B, C) 5 µm. Abbreviations: N, nuclei; Ly, lysosome; Mv, microvilli; hpd, hour postdosing. Figure 3. Open in new tabDownload slide Chronological ultrastructural changes of the proximal tubules of gentamicin-treated adult zebrafish. Images of platinum blue and lead citrate staining of gentamicin-treated zebrafish at (A) 2 hpd, (B) 6 hpd, and (C) 24 hpd. Arrowhead, lysosomal degeneration; white arrow, myeloid body; black arrow, mitochondrial degeneration; white asterisk, degenerative mitochondria-containing secondary lysosome; black asterisk, swelling in cytoplasm. Scale bars: (A) 10 µm and (B, C) 5 µm. Abbreviations: N, nuclei; Ly, lysosome; Mv, microvilli; hpd, hour postdosing. To determine whether, in addition to tubular injury, glomerular injury was also induced, ultrastructural changes of the glomeruli of zebrafish kidneys were examined following administration of 4-day repeated i.p. injections of 40 mg/kg doxorubicin. The results showed mild fusion of epithelial cell foot processes observed in the glomerulus (Figure 4). Figure 4. Open in new tabDownload slide Ultrastructural changes of the glomerulus of doxorubicin-treated adult zebrafish. Images of platinum blue and lead citrate. Arrow, podocyte foot process; asterisk, basement membrane. Scale bar: 1 µm. Abbreviations: Bs, Bowman’s space; Endo, endothelial cells. Figure 4. Open in new tabDownload slide Ultrastructural changes of the glomerulus of doxorubicin-treated adult zebrafish. Images of platinum blue and lead citrate. Arrow, podocyte foot process; asterisk, basement membrane. Scale bar: 1 µm. Abbreviations: Bs, Bowman’s space; Endo, endothelial cells. Exp. 2: Accuracy of Nephrotoxin Detection Using Adult Zebrafish To determine the performance of the adult zebrafish model in diagnosing DIKI, the zebrafish were treated i.p. or orally with 28 nephrotoxicants or 14 nonnephrotoxicants for up to 4 days (once daily) and examined histopathologically. Tables 1 and 2 show the presence and absence of nephrotoxicity in adult zebrafish caused by nephrotoxic and nonnephrotoxic compounds, respectively. In this study, the reference data of adult zebrafish in the vehicle control groups (Table 3) defined DIKI as the presence of necrosis, cell debris, and regeneration in the renal tubule in one or more individual specimens, and hyaline droplet accumulation in the renal tubule in 2 or more individuals in the test substance-treated group. Of the 28 nephrotoxicants, the zebrafish model detected the nephrotoxicity of 16 compounds. Table 3 Pathological Reference Data of Adult Zebrafish Kidneys of Vehicle Control Group Findings . Sex . Male . Female . Total . Number of Animals Examined . 110 . 110 . 220 . Accumulation, brown pigment: renal tubule 2 3 5 Accumulation, hyaline droplets: renal tubule 0 1 1 Cell debris: renal tubule 0 0 0 Dilatation: renal tubule 1 2 3 Granuloma: focal 0 1 1 Necrosis: renal tubule 0 0 0 Nephron neogenesis 1 1 2 Regeneration/nephrogenesis: renal tubule 0 0 0 Vacuolation, cytoplasmic: renal tubule 4 0 0 Findings . Sex . Male . Female . Total . Number of Animals Examined . 110 . 110 . 220 . Accumulation, brown pigment: renal tubule 2 3 5 Accumulation, hyaline droplets: renal tubule 0 1 1 Cell debris: renal tubule 0 0 0 Dilatation: renal tubule 1 2 3 Granuloma: focal 0 1 1 Necrosis: renal tubule 0 0 0 Nephron neogenesis 1 1 2 Regeneration/nephrogenesis: renal tubule 0 0 0 Vacuolation, cytoplasmic: renal tubule 4 0 0 The incidence of the histopathological lesions in controls is shown. Data show no necrosis or regeneration of the renal tubule in controls. Open in new tab Table 3 Pathological Reference Data of Adult Zebrafish Kidneys of Vehicle Control Group Findings . Sex . Male . Female . Total . Number of Animals Examined . 110 . 110 . 220 . Accumulation, brown pigment: renal tubule 2 3 5 Accumulation, hyaline droplets: renal tubule 0 1 1 Cell debris: renal tubule 0 0 0 Dilatation: renal tubule 1 2 3 Granuloma: focal 0 1 1 Necrosis: renal tubule 0 0 0 Nephron neogenesis 1 1 2 Regeneration/nephrogenesis: renal tubule 0 0 0 Vacuolation, cytoplasmic: renal tubule 4 0 0 Findings . Sex . Male . Female . Total . Number of Animals Examined . 110 . 110 . 220 . Accumulation, brown pigment: renal tubule 2 3 5 Accumulation, hyaline droplets: renal tubule 0 1 1 Cell debris: renal tubule 0 0 0 Dilatation: renal tubule 1 2 3 Granuloma: focal 0 1 1 Necrosis: renal tubule 0 0 0 Nephron neogenesis 1 1 2 Regeneration/nephrogenesis: renal tubule 0 0 0 Vacuolation, cytoplasmic: renal tubule 4 0 0 The incidence of the histopathological lesions in controls is shown. Data show no necrosis or regeneration of the renal tubule in controls. Open in new tab Histological changes such as tubular necrosis and regeneration in the kidney were observed in adult zebrafish–administered compounds that directly cause tubular damage such as cisplatin, gentamicin, and streptozotocin (Figure 5). In adult zebrafish treated with cephem antibiotics such as cefepime, cefotaxime, ceftazidime, and ceftriaxone, hyaline droplet accumulation with characteristics such as single or multiple, round or irregular, and brown or pink heterogeneous droplets was observed in the cytoplasm of the proximal tubules in 2 or more individuals in each antibiotic-treated group. Under these experimental conditions, histopathological lesions determined to be DIKI were observed only in the renal tubules, whereas no glomerular injury due to doxorubicin, cyclophosphamide, puromycin aminonucleoside, and monocrotaline was observed under a light microscopy. Papillary necrosis and interstitial nephritis due to indomethacin (a nonsteroidal anti-inflammatory drug) and 2-bromoethylamine hydrobromide were also not observed in the zebrafish. In these experiments, there was no obvious sex difference in the frequency and extent of DIKI. Figure 5. Open in new tabDownload slide Representative histopathological changes in drug-induced nephrotoxicity. Images of hematoxylin and eosin (H&E) staining of zebrafish treated with (A) cisplatin at day 2, (B) streptozotocin at day 3, (C) cefotaxime at day 5, and (D) meropenem at day 5. Arrowhead (black), tubular necrosis; arrowhead (white), hyaline droplet accumulation in the cytoplasm of the proximal tubule; arrow, tubular regeneration. Original magnification, 400×. Abbreviations: H&E, hematoxylin and eosin; day 1, starting day of administration. Figure 5. Open in new tabDownload slide Representative histopathological changes in drug-induced nephrotoxicity. Images of hematoxylin and eosin (H&E) staining of zebrafish treated with (A) cisplatin at day 2, (B) streptozotocin at day 3, (C) cefotaxime at day 5, and (D) meropenem at day 5. Arrowhead (black), tubular necrosis; arrowhead (white), hyaline droplet accumulation in the cytoplasm of the proximal tubule; arrow, tubular regeneration. Original magnification, 400×. Abbreviations: H&E, hematoxylin and eosin; day 1, starting day of administration. None of the 14 nonnephrotoxic compounds induced nephrotoxicity in adult zebrafish and included hepatotoxic compounds such as 2-acetamidofluorene and allyl alcohol and testicular toxic compounds such as 6-mercaptopurine and ethylene glycol monomethyl ether. The nephrotoxicity detection performance of the adult zebrafish model was determined based on the following measurement results: accuracy, 68% (30/42 compounds); sensitivity, 57% (16/28 compounds); specificity, 100% (14/14 compounds); positive predictive value, 100% (16/16 compounds); and negative predictive value, 54% (14/26 compounds). Exp. 3: Exploration of Nephrotoxic Genomic Biomarkers in Adult Zebrafish To explore genomic biomarkers as an alternative tool for the pathological examination of nephrotoxicity in adult zebrafish, we first analyzed gene expression using microarray analysis of isolated kidneys from gentamicin- and cisplatin-treated adult zebrafish at 48 hpd. Table 4 shows the representative upregulated probes in the nephrotoxicant-treated group. These genes showed a > 3-fold upregulation in the gentamicin- and cisplatin-treated group compared with the control group. Genes with unknown functions or less associated with cell injury were excluded even when they were upregulated > 3-fold. We first focused on the signal intensity variation of the microarray analysis of candidate genes in the control zebrafish (ie, coefficient of variance < 30%) and the increase in expression level (ie, the higher the expression level, the more promising the genomic biomarker candidate is). Furthermore, we considered it preferable for the genomic biomarker candidates to be related to cell viability (see Discussion section). According to these criteria, egr1, atf3, and fos showed low expression levels and few variations in the control kidney. In addition, these 3 genes were more significantly upregulated in the gentamicin-treated group than other genes were. Therefore, we selected egr1, atf3, and fos as candidate DIKI markers in adult zebrafish on the basis of the microarray analysis results. Table 4 Representative Upregulated Probes of Cisplatin- and Gentamicin-Treated Kidneys of Adult Zebrafish. Probe Set ID . Gene Title . Gene Symbol . Signal Intensity of Controlsa . CV of Controls (%) . Cisplatin Groupb . Gentamicin Groupb . Dr.10183.1.S2_at Early growth response 1 egr1 0.07– 0.08 5 4.0 24.2 Dr.14282.1.S1_at Activating transcription factor 3 atf3 0.22–0.30 13 6.7 35.0 Dr.13050.1.S1_at Pleckstrin homology-like domain, family A, member 2 phlda2 0.34–0.58 22 3.8 7.2 Dr.994.1.S1_at Claudin b cldnb 0.42–0.73 25 4.0 6.5 Dr.12986.1.A1_a_at v-fos FBJ murine osteosarcoma viral oncogene homolog fos 0.06–0.10 29 5.3 44.7 Dr.8723.1.S1_at Thioredoxin txn 0.40–0.74 31 12.4 9.6 Dr.5853.1.A1_at Lectin, galactoside-binding, soluble, 3 binding protein b lgals3bpb 0.35–0.71 33 6.3 20.5 Dr.12153.1.A1_at Radial spoke head 9 homolog rsph9 0.12–0.23 34 3.7 6.6 Dr.17659.1.S1_at ISG15 ubiquitin-like modifier isg15 0.32–0.92 54 6.1 27.0 Dr.20106.1.S1_at Apoptosis inhibitor 5 api5 0.01–0.02 64 4.8 4.9 Dr.1246.1.S1_at Apolipoprotein Eb apoeb 0.22–0.89 66 3.2 7.3 Dr.6391.3.S1_at EF-hand calcium binding domain 1 efcab1 0.01–0.09 92 4.8 5.3 Dr.19239.1.S1_at Myxovirus (influenza) resistance A mxa 0.00–0.05 113 7.5 30.8 Dr.10914.1.A1_at Immunoresponsive gene 1, like irg1l 0.02–0.49 150 7.4 4.8 Probe Set ID . Gene Title . Gene Symbol . Signal Intensity of Controlsa . CV of Controls (%) . Cisplatin Groupb . Gentamicin Groupb . Dr.10183.1.S2_at Early growth response 1 egr1 0.07– 0.08 5 4.0 24.2 Dr.14282.1.S1_at Activating transcription factor 3 atf3 0.22–0.30 13 6.7 35.0 Dr.13050.1.S1_at Pleckstrin homology-like domain, family A, member 2 phlda2 0.34–0.58 22 3.8 7.2 Dr.994.1.S1_at Claudin b cldnb 0.42–0.73 25 4.0 6.5 Dr.12986.1.A1_a_at v-fos FBJ murine osteosarcoma viral oncogene homolog fos 0.06–0.10 29 5.3 44.7 Dr.8723.1.S1_at Thioredoxin txn 0.40–0.74 31 12.4 9.6 Dr.5853.1.A1_at Lectin, galactoside-binding, soluble, 3 binding protein b lgals3bpb 0.35–0.71 33 6.3 20.5 Dr.12153.1.A1_at Radial spoke head 9 homolog rsph9 0.12–0.23 34 3.7 6.6 Dr.17659.1.S1_at ISG15 ubiquitin-like modifier isg15 0.32–0.92 54 6.1 27.0 Dr.20106.1.S1_at Apoptosis inhibitor 5 api5 0.01–0.02 64 4.8 4.9 Dr.1246.1.S1_at Apolipoprotein Eb apoeb 0.22–0.89 66 3.2 7.3 Dr.6391.3.S1_at EF-hand calcium binding domain 1 efcab1 0.01–0.09 92 4.8 5.3 Dr.19239.1.S1_at Myxovirus (influenza) resistance A mxa 0.00–0.05 113 7.5 30.8 Dr.10914.1.A1_at Immunoresponsive gene 1, like irg1l 0.02–0.49 150 7.4 4.8 Abbreviation: CV, coefficient of variation. a The minimum to maximum signal intensity of controls are shown. b Fold change to controls (mean) are shown, n = 4 animals/group. Open in new tab Table 4 Representative Upregulated Probes of Cisplatin- and Gentamicin-Treated Kidneys of Adult Zebrafish. Probe Set ID . Gene Title . Gene Symbol . Signal Intensity of Controlsa . CV of Controls (%) . Cisplatin Groupb . Gentamicin Groupb . Dr.10183.1.S2_at Early growth response 1 egr1 0.07– 0.08 5 4.0 24.2 Dr.14282.1.S1_at Activating transcription factor 3 atf3 0.22–0.30 13 6.7 35.0 Dr.13050.1.S1_at Pleckstrin homology-like domain, family A, member 2 phlda2 0.34–0.58 22 3.8 7.2 Dr.994.1.S1_at Claudin b cldnb 0.42–0.73 25 4.0 6.5 Dr.12986.1.A1_a_at v-fos FBJ murine osteosarcoma viral oncogene homolog fos 0.06–0.10 29 5.3 44.7 Dr.8723.1.S1_at Thioredoxin txn 0.40–0.74 31 12.4 9.6 Dr.5853.1.A1_at Lectin, galactoside-binding, soluble, 3 binding protein b lgals3bpb 0.35–0.71 33 6.3 20.5 Dr.12153.1.A1_at Radial spoke head 9 homolog rsph9 0.12–0.23 34 3.7 6.6 Dr.17659.1.S1_at ISG15 ubiquitin-like modifier isg15 0.32–0.92 54 6.1 27.0 Dr.20106.1.S1_at Apoptosis inhibitor 5 api5 0.01–0.02 64 4.8 4.9 Dr.1246.1.S1_at Apolipoprotein Eb apoeb 0.22–0.89 66 3.2 7.3 Dr.6391.3.S1_at EF-hand calcium binding domain 1 efcab1 0.01–0.09 92 4.8 5.3 Dr.19239.1.S1_at Myxovirus (influenza) resistance A mxa 0.00–0.05 113 7.5 30.8 Dr.10914.1.A1_at Immunoresponsive gene 1, like irg1l 0.02–0.49 150 7.4 4.8 Probe Set ID . Gene Title . Gene Symbol . Signal Intensity of Controlsa . CV of Controls (%) . Cisplatin Groupb . Gentamicin Groupb . Dr.10183.1.S2_at Early growth response 1 egr1 0.07– 0.08 5 4.0 24.2 Dr.14282.1.S1_at Activating transcription factor 3 atf3 0.22–0.30 13 6.7 35.0 Dr.13050.1.S1_at Pleckstrin homology-like domain, family A, member 2 phlda2 0.34–0.58 22 3.8 7.2 Dr.994.1.S1_at Claudin b cldnb 0.42–0.73 25 4.0 6.5 Dr.12986.1.A1_a_at v-fos FBJ murine osteosarcoma viral oncogene homolog fos 0.06–0.10 29 5.3 44.7 Dr.8723.1.S1_at Thioredoxin txn 0.40–0.74 31 12.4 9.6 Dr.5853.1.A1_at Lectin, galactoside-binding, soluble, 3 binding protein b lgals3bpb 0.35–0.71 33 6.3 20.5 Dr.12153.1.A1_at Radial spoke head 9 homolog rsph9 0.12–0.23 34 3.7 6.6 Dr.17659.1.S1_at ISG15 ubiquitin-like modifier isg15 0.32–0.92 54 6.1 27.0 Dr.20106.1.S1_at Apoptosis inhibitor 5 api5 0.01–0.02 64 4.8 4.9 Dr.1246.1.S1_at Apolipoprotein Eb apoeb 0.22–0.89 66 3.2 7.3 Dr.6391.3.S1_at EF-hand calcium binding domain 1 efcab1 0.01–0.09 92 4.8 5.3 Dr.19239.1.S1_at Myxovirus (influenza) resistance A mxa 0.00–0.05 113 7.5 30.8 Dr.10914.1.A1_at Immunoresponsive gene 1, like irg1l 0.02–0.49 150 7.4 4.8 Abbreviation: CV, coefficient of variation. a The minimum to maximum signal intensity of controls are shown. b Fold change to controls (mean) are shown, n = 4 animals/group. Open in new tab The expression of the 3 selected genes was further confirmed using real-time qRT-PCR analysis of kidney samples following i.p. treatment with 10 and 60 mg/kg cisplatin and 100 mg/kg gentamicin at 48 hpd (Figure 6). The expression of these genes was upregulated in a dose-dependent manner in the cisplatin-treated groups and was upregulated > 25-fold in the gentamicin-treated group than in the control group (Figs. 6A–C). These genes were more significantly upregulated than kim1 was (Figs. 6A–D). The kim1 gene is a well-known nephrotoxic biomarker in mammals (Vaidya et al., 2010). Furthermore, the chronological changes of these genes during renal injury were investigated using gentamicin-treated adult zebrafish. The expression of egr1, fos, and atf3 genes was upregulated by > 75-fold and > 14-fold at 24 and 48 hpd than in the control group, which was apparently higher than that of kim1 (Figs. 7A–D). The expression levels of the 3 genes had returned to baseline by 96 hpd (Figs. 7A–C). Finally, to investigate whether these candidate genes could be used as reporter genes in creating a transgenic zebrafish DIKI screening model, we measured their expression levels in whole body tissues. The genes were globally expressed, but the signal intensity in all tissues examined in this study was similar to that in the vehicle control kidneys (Supplementary Table 2 and Figure 8). Figure 6. Open in new tabDownload slide Expression of nephrotoxicity markers in cisplatin- or gentamicin-treated adult zebrafish. Mean ± SD values are shown, n = 4 animals/group. Inset, higher magnification showing each gene upregulation was dose dependent in the cisplatin-treated group. Statistical significance was evaluated using ANOVA and Dunnett’s multiple comparison post hoc test for cisplatin group and Welch’s t test for gentamicin group. *p < .05 and**p < .01 versus vehicle controls. Abbreviations: CONT, control; CSP, cisplatin; GMC, gentamicin. Figure 6. Open in new tabDownload slide Expression of nephrotoxicity markers in cisplatin- or gentamicin-treated adult zebrafish. Mean ± SD values are shown, n = 4 animals/group. Inset, higher magnification showing each gene upregulation was dose dependent in the cisplatin-treated group. Statistical significance was evaluated using ANOVA and Dunnett’s multiple comparison post hoc test for cisplatin group and Welch’s t test for gentamicin group. *p < .05 and**p < .01 versus vehicle controls. Abbreviations: CONT, control; CSP, cisplatin; GMC, gentamicin. Figure 7. Open in new tabDownload slide Chronological expression changes of nephrotoxicity markers in gentamicin-treated adult zebrafish. Mean ± SD values are shown, n = 4 animals/group. Inset, higher magnification showing expression of each gene decreases over time. Statistical significance was evaluated using Welch’s t test for each gentamicin group versus vehicle controls. *p < .05 versus vehicle controls. Abbreviations: CONT, control; GMC, gentamicin. Figure 7. Open in new tabDownload slide Chronological expression changes of nephrotoxicity markers in gentamicin-treated adult zebrafish. Mean ± SD values are shown, n = 4 animals/group. Inset, higher magnification showing expression of each gene decreases over time. Statistical significance was evaluated using Welch’s t test for each gentamicin group versus vehicle controls. *p < .05 versus vehicle controls. Abbreviations: CONT, control; GMC, gentamicin. Figure 8. Open in new tabDownload slide Tissue distribution of genomic biomarker candidate and housekeeping genes in normal adult zebrafish. Tissue distribution of selected genomic biomarker candidates was analyzed in liver, kidneys, heart (n = 9 each), spleen (n = 4), intestine (n = 9), swim bladder (n = 9), testes (n = 6), and ovaries (n = 3). Quantitative PCR conducted using 40 ng total RNA. Red: 4.0 × 10−7 higher; white: 1.0 × 10−8 lower expression. Figure 8. Open in new tabDownload slide Tissue distribution of genomic biomarker candidate and housekeeping genes in normal adult zebrafish. Tissue distribution of selected genomic biomarker candidates was analyzed in liver, kidneys, heart (n = 9 each), spleen (n = 4), intestine (n = 9), swim bladder (n = 9), testes (n = 6), and ovaries (n = 3). Quantitative PCR conducted using 40 ng total RNA. Red: 4.0 × 10−7 higher; white: 1.0 × 10−8 lower expression. DISCUSSION We examined the usefulness of adult zebrafish as a DIKI screening model. Zebrafish larvae have been reported as a potential alternative model to mammals. However, larvae have only one pair of nephrons, whereas adult zebrafish have several hundred (Diep et al., 2011; Kroeger and Wingert, 2014; Zhou et al., 2010). Histopathological examination is a definitive method for evaluating toxicity, and the adult zebrafish was considered suitable for histopathological evaluation. The results of Exp. 1 showed that the nephron structures of adult zebrafish such as the glomeruli, proximal tubules, and distal tubules were similar to those of mammals, supporting our hypothesis of the models suitability. We did not administer test substances in the water bath but chose the i.p. or p.o. dosing route to reduce the test substance amount. Exp. 1 showed numerous similarities in responses of mammals and zebrafish to nephrotoxicants. In rats, gentamicin filtered through the glomeruli undergoes proximal tubular reabsorption by binding to anionic phospholipids in the brush border, followed by endocytosis and sequestration in lysosomes of the S1 and S2 segments of proximal tubules (Schnellmann, 2019). The first morphological changes after gentamicin administration in rats are an increase in lysosome size and number, with the detection of electron-dense lamellar structures (myeloid bodies) containing undegraded phospholipids (Beauchamp et al., 1991; Schnellmann, 2019). Subsequently, the brush border, endoplasmic reticulum, and mitochondria were damaged, which led to tubular cell necrosis. In our experiment, similar ultramorphological changes were observed in kidneys of gentamicin-treated adult zebrafish, including increased irregular lysosomes of various size at 2 hpd and myeloid bodies at 6 hpd. In adult zebrafish, the tubular regeneration induced after necrosis was also similar to that in mammals. A study assessing the ultrastructure of doxorubicin-induced glomerular injury in rats showed that typical changes to glomerular visceral epithelial cells, including fusions of foot processes, cytoplasmic blebs, protein droplets, and villous transformation induced 21 days after a single intravenous administration (Remuzzi et al., 1985). We also observed doxorubicin-induced glomerular lesions such as slight fusion of epithelial cell foot processes in zebrafish kidneys. These results indicate that pathophysiological features of glomerular podocyte injury and proximal tubule injury and the repair process in adult zebrafish are consistent with those of mammals. However, some differences occurred between mammals and zebrafish. In whole nephron neogenesis in teleosts including zebrafish, de novo nephron formation occurs throughout the lifetime of the fish, likely due to the continued growth of the organism and the subsequent need to meet a higher demand for waste excretion (Davidson, 2011; Diep et al., 2011; McCampbell and Wingert, 2014; Zhou et al., 2010). In contrast, whole nephron neogenesis does not occur in mammals, where partial regeneration was observed in tubule epithelial cells (Li et al., 2014). Histologically, it is not rare to observe low numbers of small, dark immature (embryonic) nephrons in the kidneys of healthy adult fish (nephron neogenesis). Immature nephrons may be found at a high frequency in DIKI, and McKee and Wingert (2015) reported that nephron neogenesis was observed from 5 to 14 days after gentamicin treatment. We also observed nephron neogenesis around original nephrons in gentamicin-treated zebrafish at 96 hpd. Another difference between zebrafish and mammals is that zebrafish lack a comparable structure to the loop of Henle found in mammals, because freshwater fish do not need to concentrate urine (Nishimura and Imai, 1982). Furthermore, histopathological examinations differ in difficulty between zebrafish and mammals. Particularly, the identification and characterization of renal inflammation can be challenging in zebrafish because of the high resident cellularity of hematopoietic cells in the normal renal interstitium. The histopathological evaluation of several nephrotoxicants with various target tissues and toxicity mechanisms in Exp. 2 showed that the sensitivity and negative predictive value were not particularly high at 57% and 54%, respectively. In contrast, the specificity and positive predictive value were both 100%. These indicators suggest that the adult zebrafish DIKI screening system did not detect half of the compounds with possible DIKI effects. However, the compounds, determined to be positive using this system, were assessed as absolutely positive. Therefore, we considered the adult zebrafish a useful model for confirming drug candidates determined as positive that should not be further evaluated in subsequent stages. Many compounds that mainly target the renal tubules in mammals such as cisplatin, gentamicin, doxorubicin, and streptozotocin frequently induce degenerative changes in zebrafish kidney (Table 1). In addition, cephem antibiotics cause the accumulation of hyaline droplets in the cytoplasm of renal tubules in zebrafish. Abe et al. (1988) demonstrated that in the kidneys of rats treated with cephem antibiotics, brown granules accumulated in the cytoplasm of the renal tubule, which are observed as expanded lysosomes using electron microscopy. In contrast, most compound that damage the glomerulus, interstitial tissue, and renal papilla were negative for nephrotoxicity in adult zebrafish, and the reason for this was unclear in the current study. However, it may be related to the anatomical species difference described above. With respect to glomerular injury, it is difficult to detect slight changes to the glomeruli such as foot process fusion using a light microscope as shown in Figure 3. However, electron microscopy assessment is too cumbersome to be routinely conducted. Additionally, compared with mammals, glomerular morphology is less consistent in fish; the same kidney can have marked variability in mesangial and vascular loop thickness, cellularity, and the size of glomeruli and Bowman’s spaces (Wolf et al., 2015). Some of this variability may be attributable to nephron neogenesis, which produces glomeruli at different stages of development. This may explain why subtle damage to fish glomeruli may be difficult to detect at the light microscopic level. Therefore, assessing DIKI for longer dosing periods using the adult zebrafish model may allow glomerular injury to be detected using light microscopy. In the future, the usefulness of adult zebrafish should be further verified by examining the similarity of the molecular mechanism of toxicity of each compound in mammals and zebrafish. In addition, differences in the anatomy and physiological functions described above should also be elucidated. Exp. 1 and 2 revealed that adult zebrafish showed some usefulness as an evaluation system for nephrotoxicity. However, the screening system would be considerably taxing if pathological examinations were performed each time. Therefore, we explored genomic biomarkers as alternatives to pathological examination. For this purpose, we conducted microarray analysis using the kidneys isolated from nephrotoxicant-treated zebrafish and identified biomarkers candidates based on expression level and biological significance. Consequently, erg1, atf3, and fos were selected as candidate DIKI markers in this study. These genes are also functionally related to cell injury such as necrosis. Specifically, Egr-1 is a zinc-finger transcriptional factor that belongs to a group of early response genes together with the Egr family and the tumor suppressor, Wilms’ tumor gene product. Egr-1 has been implicated in the regulation of cell growth, survival, and transformation (Ahmed, 2004; Mahalingam et al., 2010; Thiel and Cibelli, 2002). Ho et al. (2016) reported that Egr-1 is of clinical significance because its activity in renal tubular cells was upregulated in patients with renal failure. ATF3 is a member of the ATF/cyclic AMP response element binding family of transcription factors and is induced by DNA damage and other oncogenic stimuli (Hai and Hartman, 2001; Hai et al., 1999; Thompson et al., 2009). Sato et al. (2014) reported that the model of 5-fluorodeoxyuridine (FUdR)-induced cell death indicates that ATF3 expression was induced strongly during necrosis but not apoptosis. ATF3 was suggested to be involved in doxorubicin-induced cytotoxicity in the human renal proximal tubular cell line HK-2 (Park et al., 2012). ATF3 increases in the urine of early acute kidney injury (AKI) patients and is considered an early diagnostic marker for AKI (Zhou et al., 2008). c-Fos is a proto-oncogene that is the human homolog of the retroviral oncogene v-fos and plays an important role in many cellular functions. c-Fos forms a heterodimer with c-jun, and an activator protein-1 (AP-1) complex in the promoter and enhancer region of the target gene to convert extracellular signals into changes in gene expression (Chiu et al., 1988). c-fos is believed to be associated with kidney injury because administration of a selective inhibitor of c-fos/AP-1 in an endotoxin-induced AKI mouse model suppressed renal inflammation (Miyazaki et al., 2012). These genomic biomarker candidates showed characteristic high expression levels accompanied by histopathological lesions in the kidneys of zebrafish, and low expression in the absence of nephrotoxicity in whole body (Figures 6–8). Therefore, these genomic biomarkers have the potential usage in combination with reporter genes such as the green fluorescent protein in transgenic zebrafish. This notion is corroborated by the use of fabp10 promoter for the detection of hepatotoxicity in zebrafish (Zhang et al., 2014). In addition, as a new animal model of nephrotoxicity, the possibility of gene editing in zebrafish is highly anticipated (Barnett and Cummings, 2018). However, in this study, only gene expression of these biomarker candidates in the kidneys of zebrafish treated with gentamicin and cisplatin was examined. To demonstrate the influence of these biomarker candidate genes, further verification is necessary such as examining changes in the expression of these genes in the kidneys of zebrafish treated with other nephrotoxic substances and nonnephrotoxic substances. In conclusion, we investigated the usefulness of adult zebrafish as a DIKI screening model and clarified the presence of certain similarity to mammals in the histopathological properties of the kidney. Histopathological examination of the reactivity of various nephrotoxic substances revealed that adult zebrafish can be used to detect renal injury with high specificity, especially with nephrotoxicants targeting renal tubules. Furthermore, egr1, atf3, and fos were identified as candidate genes for DIKI in adult zebrafish, which may be applied as an efficient screening system in combination with transgenic methods. It may be possible to efficiently screen for nephrotoxicity by complementing small in vivo systems using adult zebrafish with various high throughput in vitro systems. DECLARATION OF CONFLICTING INTERESTS The authors declare that they have no conflicts of interest to disclose in connection with this study. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Yuki Kato and Yutaka Tonomura contributed equally to this study. © The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Adult Zebrafish Model for Screening Drug-Induced Kidney Injury
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfaa009
DA - 2020-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/adult-zebrafish-model-for-screening-drug-induced-kidney-injury-3rykNTo9iz
DP - DeepDyve
ER -