Adult Zebrafish Model for Screening Drug-Induced Kidney Injury.

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.

Microarray analysis of 6-mercaptopurine-induced-toxicity-related genes and microRNAs in the rat placenta.

MicroRNAs (miRNAs) are small single-stranded RNAs of 19-25 nucleotides and are important in posttranscriptional regulation of genes. Recently, the role of miRNAs in toxicity incidence is reported to be a regulator of key-stopper of gene expression, however the detailed mechanism of miRNAs is not well known yet. 6-Mercaptopurine (6-MP), the anti-leukemic and immunosuppressive drug, produced teratogenicity and pregnancy loss. We focused on the placenta to evaluate toxicity in embryo/fetal development produced by 6-MP treatment. MiRNA expression in the placenta was analyzed by miRNA microarray. Fifteen miRNAs were upregulated on GD13 and 5 miRNAs were downregulated on GD15 in 6-MP treatment rat placentas. Some miRNAs may have functions in apoptosis (miR-195, miR-21, miR-29c and miR-34a), inflammation (miR-146b), and ischemia (miR-144 and miR-451). In the maternal plasma, expression of miR-144 was significantly reduced by 6-MP treatment when examined by real-time RT-PCR. We determined toxicity-related gene expression in the rat placenta. Gene expression analysis was carried out by DNA oligo microarray using rat placenta total RNAs. Compared between predicted targets of miRNAs and microarray data in 6-MP-treated rat placenta, expressions of hormone receptor genes (estrogen receptor 1; Esr1, progesterone receptor; Pgr, and prolactin receptor; Prlr), xanthine oxidase (Xdh), Slc38a5 and Phlda2 genes were changed. The histopathologically found increase in trophoblastic giant cells and reduced placental growth by 6-MP treatment were well correlated to these gene expressions. These data suggest that some miRNAs may link to toxicological reactions in 6-MP-induced placental toxicity.

MicroRNAs expression in the ethylene glycol monomethyl ether-induced testicular lesion.

Ethylene glycol monomethyl ether (EGME) induces testicular lesion in rats and human. To investigate miRNAs expression in EGME testicular lesion, miRNA array assay and real-time RT-PCR analysis were conducted by using testis in rats treated with 50 and 2,000 mg/kg EGME for 6 and 24 hr. The expression of corresponding target gene for miRNAs was also examined. At 50 mg/kg, there were no changes in the gene expression and histopathological examination. At 2,000 mg/kg, slight decrease of phacytene spermatocytes with cell shrinkage and nucleus pyknosis at 6 hr and remarkable decrease (or cell death) of phacytene spermatocytes with Sertoli cell vacuolation at 24 hr were observed. After 24 hr, miR-449a and miR-92a decreased obviously and, miR-320, miR-134 and miR-188 increased, while only miR-760-5p increased after 6 hr. Above these miRNAs are reported to have an important role for spermatogenesis. The gene expression of Bcl-2, target for miR-449a, increased and therefore it is considered anti-apoptotic reaction has started in this stage. The expression of high mobility group AT-hook 2 (target of miR-92a) which regulates histone structure, was increased. Furthermore, histone deacethylase 4, targets for miR-320, was also affected. Above prohibiting apoptosis or activating epigenetic genes might be protective reaction to spermatocytes death under the miRNAs regulation in EGME testicular lesion.