Bioluminescent vdbp-NanoLuc reporter to detect drug nephrotoxicity. (A) Experimental protocol used in gentamicin nephrotoxicity assessment and its rescue by taurine co-treatment using transgenic zebrafish larvae. TEM, transmission electron microscopy. (B) Microscopic examination of 4 dpf gentamicin-treated Tg(PiT1::ctns-EGFP) zebrafish larvae expressing lysosomal marker Ctns-EGFP in PT cells using multiphoton fluorescence microscopy (top). Accumulation of Ctns-EGFP-positive vesicles is observed in PT cells of gentamicin-treated transgenic zebrafish larvae. Dashed lines outline the proximal tubule. PT, proximal tubule. Relative EGFP fluorescence intensity (bottom left) and EGFP-positive vesicle number (bottom right) are quantified for each proximal tubule. n=13 (vehicle), n=11 (gentamicin). Scale bars: 30 µm. (C) Ultrastructure of pronephros in 4 dpf larvae treated with gentamicin or vehicle analyzed by transmission electron microscopy. Accumulation of electron-dense vesicles in PT epithelial cells reflects lysosomal phospholipidosis. n=5. Yellow dashed line squares contain images at higher magnification. BB, brush border. Scale bars: 5 µm (left column), 1 µm (right column). (D) Urine samples were obtained at 2 days post-injection from ½vdbp-NanoLuc larvae treated with vehicle, gentamicin alone or gentamicin/taurine and analyzed by luminometry. The variation observed in the gentamicin-injected group may be due to technical limitations of intravenous injection. n=28 (vehicle), n=26 (gentamicin) and n=28 (gentamicin/taurine). (E) Larval lethality in 4 dpf larvae treated with vehicle, cisplatin (0.5 mM or 1 mM) alone or co-incubated with cisplatin (0.5 mM or 1 mM)+10 mM sodium thiosulfate (STS) for 24 h. Treatment with 1 mM cisplatin for 24 h is lethal for zebrafish larvae but rescued by co-treatment with 10 mM STS. n=40. (F) Morphological examination of 4 dpf larvae treated with vehicle, 0.5 mM cisplatin alone or co-treatment of cisplatin (0.5 mM or 1 mM)+10 mM STS (left) and semi-quantitative scoring of inflation defects of swim bladder (right). n=40. Scale bars: 0.5 mm. (G) Behavioral analysis of 5 dpf zebrafish larvae incubated with vehicle, 0.5 mM cisplatin alone, or co-incubated with cisplatin (0.5 mM or 1 mM)+10 mM STS by movement tracking using the Zebrabox. n=40 (vehicle), n=31 (cisplatin alone), n=40 (0.5 mM cisplatin+10 mM STS), n=38 (1 mM cisplatin+10 mM STS). (H) Overnight urine was collected from 5 dpf ½vdbp-NanoLuc zebrafish larvae treated with vehicle, 0.5 mM cisplatin alone or co-incubated with cisplatin (0.5 mM or 1 mM)+10 mM STS for 24 h and analyzed by luminometry. n=39 (vehicle), n=31 (0.5 mM cisplatin alone), n=39 (0.5 mM cisplatin+10 mM STS), n=37 (1 mM cisplatin+10 mM STS). Plotted data represent mean±s.e.m. Nonparametric Mann–Whitney test, **P<0.01 ***P<0.001. NS, non-significant.
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