Schematic representation of experimental protocols performed in this study. Five groups (I-V) were applied in the treatment experiments based on combinations of different exposure onsets: (I) no treatment group, water only; (II) AA group, exposed to AA from 24 to 31 hpf; (III) ZDW group, exposed to ZDW from 12 to 24 hpf; (IV) ZDW, AA group (defined as pretreatment), exposed to ZDW from 12 to 24 hpf, withdrawn ZDW and followed by exposure with AA from 24 to 31 hpf; (V) ZDW+AA group (defined as cotreatment), coexposure with AA and ZDW from 24 to 31 hpf.

ZDW dose titration and survival rate analysis in this study. For ZDW dose titration, wild-type (WT; AB strain) embryos were collected, randomly divided into 50 per experimental group, and exposed to either water (no treatment, control: 0 ppm) or water containing ZDW (1000, 500, and 100 ppm, (a)–(c)) and/or 3 ppm AA. All embryos were cultivated in 6-well cell culture plates, and survival rates were determined at 12, 24, 31, and 48 hpf. The x- and y-axes represent the developmental stages and embryo survival rates, respectively. The cotreatment group (V) was only applied in low-dose ZDW (100 ppm).

Kidney phenotypes of zebrafish embryos after prevention of ZDW. Morphological changes of Tg(wt1b:EGFP) embryos after exposure to water (a) or water containing 1000, 500, and 100 ppm of ZDW (b–d). AA-induced malformed kidney phenotypes which included curved pronephric tube (pt) and swollen, asymmetrical distribution of glomerulus (gl) were observed after being exposed with 3 ppm AA (e). Those phenotypic changes could be attenuated by either pretreatment (3 ppm AA; 100 ppm ZDW) (f) or cotreatment (3 ppm AA+100 ppm ZDW) with ZDW (g). Representative figures are shown here. All photos were taken from a dorsal view at the developmental stage of 48 hpf.

Malformation rates of kidney-defective phenotypes of zebrafish embryos after prevention of ZDW. Malformation rates of Tg(wt1b:EGFP) embryos after exposure to different doses of ZDW ((a): 1000 ppm; (b): 500 ppm; (c): 100 ppm) in combination with or without 3 ppm of AA were calculated. Malformation rates for each treatment group were calculated at 48 hpf. The cotreatment group (V) was only applied in low-dose ZDW (100 ppm).

ZDW pretreatment attenuated AA-induced defective pronephric ducts. Immunohistochemical staining of the basolateral marker Na⁺/K⁺-ATPase alpha1 subunit (α6F) was performed on transverse sections of the pronephric tubes from 48 hpf zebrafish embryos after exposure to water (no treatment group, (a)), 3 ppm of AA (b), 10 ppm of ZDW (c), and AA combined with ZDW (pretreatment (ZDW; AA group) (d) or cotreatment (ZDW+AA) (e)). The sections were stained with a monoclonal antibody (α6F) and counterstained with hematoxylin. (a′)-(a^″), (b′)-(b^″), (c′)-(c^″), (d′)-(d^″), and (e′)-(e^″) were different positions of the embryos indicated by red lines shown in (a), (b), (c), (d), and (e), respectively.

Quantitative gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The total RNA was extracted at the 48 hpf, and the relative expression levels of the indicated genes were determined by qRT-PCR. The gene expression results of the indicated genes between no treatment control (NT) and different experimental groups. The data in triplicate were normalized for zebrafish β-actin expression.