Tg(cdh17:Dendra2-NTR) transgenic line construction. (A) Constructs used to generate the cdh17-driven Dendra2-NTR transgenic line is shown. Dendra2, green-to-red photo-switchable fluorescent protein; NTR, nitroreductase. Dendra2 expression in Tg(cdh17:Dendra2-NTR) transgenic fish renal tubules (arrowheads) at 24 hpf (B), 48 hpf (C) and 72 hpf (D), respectively. Scare bar: 200 μm. (E) Detailed display of the laser confocal image in the middle section of the 72 hpf Tg(cdh17:Dendra2-NTR) transgenic fish renal tubule. Scare bar: 100 μm.

MTZ induces renal tubular epithelial cells-specific ablation. (A) Treatment with MTZ resulted in the attenuation of the Dedra2 signal in the renal tubules of Tg(cdh17:Dendra2-NTR) zebrafish larvae (n = 30). The white arrowheads in Con larvae indicated the intact renal tubules (head, body and tail). The white and red arrowhead in MTZ larvae respectively indicated the remaining and the injured parts of the renal tubules. Scare bar: 100 μm. (B) Edema could be seen in MTZ-treated Tg(cdh17:Dendra2-NTR) zebrafish larvae but not in the control at 1 dpi to 4 dpi (n = 30). Scale bar: 200 μm. Con, control. n, the number of larvae per group under each treatment condition. Each experiment was repeated more than three times.

Renal tubular injury leads to loss of renal function. (A) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive apoptotic cells (red) were detected in the renal tubules of Tg(cdh17:Dendra2-NTR) zebrafish larvae treated with MTZ, but not in the control (n = 30). Scale bar: 50 μm. (B,C) Tg(cdh17:Dendra2-NTR) zebrafish larvae treated with MTZ or the control were injected with 2 nL 5% FITC-inulin, and FITC-inulin fluorescence intensity of the caudal artery was detected in three separate regions 15 min and 4 h after injection. White or red arrows respectively indicated the same measuring position of Con larvae or MTZ larvae. (D) Zebrafish larvae treated with MTZ or control zebrafish larvae (30 larvae/Sample) were collected at 1 dpi to 4 dpi for the detection of UA and UN. (E) Tg(cdh17:Dendra2-NTR) zebrafish larvae treated with MTZ and control zebrafish larvae were collected (n = 60, respectively), and their survival rate was calculated from 1 dpi to 6 dpi. The data are expressed as the mean ± SEM; * p < 0.05, ** p < 0.01, and **** p < 0.001 (ANOVA/Dunnett’s test).

ARF induced heart failure in zebrafish larvae. The heart rate (A–C) and heart size (D) of Tg(cdh17:Dendra2-NTR;cmlc2:GFP) zebrafish larvae treated with MTZ or control were detected (n = 5). (E) TUNEL-positive apoptotic cardiomyocytes (red) were detected in the heart of Tg(cdh17:Dendra2-NTR;cmlc2:GFP) zebrafish larvae treated with MTZ, but not in the control (n = 30). Scare bar: 100 μm. Mean ± SEM; ns, not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001, ANOVA/Dunnett’s test compared with control.

Changes of blood flow and blood vessels in ARF larvae. (A) The erythrocytes in the caudal artery (CA) (green arrow), caudal vein (CV) (white arrow), posterior cardinal vein (PCV) (green arrow) and dorsal aorta (DA) (white arrow) of Tg(cdh17:Dendra2-NTR; flk1:GFP;gata1:DsRed) zebrafish larvae treated with MTZ or control are shown. Scale bar: 200 μm. (B) After treatment with MTZ, the caudal artery blood flow velocity of zebrafish larvae (n = 5) was evaluated using ImageJ. (C–E) Confocal images of blood vessels around the heart and tail of Tg(cdh17:Dendra2-NTR; flk1:GFP;gata1:DsRed) zebrafish larvae treated with MTZ or control (n = 5). The Scale bars of (C) and (D,E) are 100 μm and 200 μm, respectively. The red and white line segments represent the vessel lumen of zebrafish larvae. CA, caudal artery; CV, caudal vein. Mean ± SEM; * p < 0.05 and **** p < 0.001, ANOVA/Dunnett’s test compared with control.

Evaluation of the effect of digoxin and enalapril on the CRS-3 model. (A) Heart images of zebrafish larvae (Control, zebrafish larvae treated with MTZ, zebrafish larvae treated with digoxin or enalapril) (n = 5) at 2 dpi are shown. Scale bar: 100 μm. (B,C) Heart rate was detected by the dynamic change pattern in the ventricle of zebrafish larvae. (D) ARF-induced heart enlargement can be decreased by digoxin and enalapril (n = 5). (E) Compared with the control group, treatment of ARF larvae with digoxin and enalapril resulted in significant increases in blood flow velocity (n = 5). (F) After treatment with digoxin or enalapril, the survival rate of ARF zebrafish larvae was increased (n = 60). Mean ± SEM; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, ANOVA/Dunnett’s test compared with untreated ARF zebrafish larvae.

Effect of α-lipoic acid on CRS-3. (A–F) The heart rate, blood flow, heart dilatation and survival rate of ARF larvae treated with α-lipoic acid (4 μM, 8 μM) and the untreated group are presented. The number of larvae in each group was five, except for the survival rate group (n = 60). Scale bar: 100 μm. (G,H) MTZ-induced ROS in zebrafish larvae (n = 5) can be attenuated by α-lipoic acid. The image shows that ROS significantly decreased as the drug concentration increased. The white arrow points to the heart area. The Scale bar of (B,G) are 100 μm and 200 μm, respectively. Mean ± SEM; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, ANOVA/Dunnett’s test compared with untreated ARF larvae.