Fig 2: Cisplatin induces renal injury in an administration time-dependent manner.Mice were injected with cisplatin (20 mg/kg) or equivalent volume of saline at ZT1 or ZT13, respectively. In all, 72 h thereafter, mouse kidney and serum samples were collected for the following experiments. n = 5 for each group. a H&E, PAS staining and TUNEL assays of kidney sections. b, c BUN and serum Cr levels. d, e RT-qPCR analyses of renal Kim-1 and Ngal mRNA expression. f Western blot analysis of renal Kim-1 and Ngal protein expression. g, h Quantitative data of panel. f. *P < 0.05 and **P < 0.01 vs. CTL group, #P < 0.05 and ##P < 0.01 vs. ZT1 CTL group. i IHC analyses of Kim-1 and Ngal proteins. All values are presented as the mean ± SD.

Fig 3: Overexpression of Bmal1 accelerates cisplatin-induced renal injury in vivo.Mice were transduced with adenovirus expressing either Bmal1 CDS domain or GFP via tail-vein injection for 4 days, followed by cisplatin treatment at ZT1 or ZT13 for another 72 h. Mouse kidney and serum samples were collected for the following experiments. n = 5 for each group. a H&E, PAS staining and TUNEL assays of kidney sections. b, c BUN and serum Cr levels. d–g RT-qPCR and western blot analyses of renal expression levels of Bmal1, Kim-1 and Ngal. h IHC analyses of Kim-1 and Ngal protein expression from kidney sections. *P < 0.05 and **P < 0.01 vs. Ad-GFP + cisplatin group. All values are presented as the mean ± SD.

Fig 4: Knockdown of Bmal1 alleviates cisplatin-induced renal injury in vivo.Mice were transduced with adenovirus expressing either Bmal1 shRNA or scramble shRNA via tail-vein injection for 4 days, followed by cisplatin treatment at ZT1 or ZT13 for another 72 h. Mouse kidney and serum samples were collected for the following experiments. n = 5 for each group. a H&E, PAS staining and TUNEL assays of kidney sections. b, c BUN and serum Cr levels. d–g RT-qPCR and western blot analyses of renal expression levels of Bmal1, Kim-1 and Ngal. h IHC analyses of Kim-1 and Ngal from kidney sections. *P < 0.05 and **P < 0.01 vs. Ad-Scramble shRNA+ cisplatin group. All values are presented as the mean ± SD.

Fig 5: Ephx2 KO mice abolished the protective effect of alisol B in the apoptosis, inflammation, and oxidative stress. (A-D) Representative images of immunostaining for KIM-1 (A), ICAM-1 (B), 8-OXO (C), and Grp78 (D) in WT and sEH-/- mice with Cis-induced AKI treated with alisol B; (E) Quantitative analysis of immunostaining for KIM-1, ICAM-1, 8-OXO, and Grp78; data were presented as mean ± SEM, n=5, ** p<0.01, ***p<0.001; n.s.= no significance; (F) Ephx2 KO abolished effects of alisol B on expression levels of proteins related to inflammation, apoptosis, and oxidative stress in Cis-induced AKI; (G) Quantitative analysis for the protein levels in (F); data were presented as mean ± SEM, n=3, *p<0.05, ** p<0.01, ***p<0.001, n.s.= no significance.