Fig 2: Relative mRNA expression levels of ECE1-3 and ET1-3 in PT epithelial cells (LLC-PK1) and whole kidneys of neonatal pigs. P < 0.05 (one-way ANOVA, with Holm-Sidak post hoc test).

Fig 3: rUUO did not alter protein expression levels of ETR and TRPC3 but caused early AKI via activation of ECE1/ETR/TRPC3. A-C, representative Western blot showing ETRA, ETRB, and TRPC3 protein expression in renal vessels of neonatal pigs subjected to sham and rUUO operations. ETRA and ETRB blots were stripped and re-probed for ß-actin. D-F, urinary NGAL, L-FABP, and IL-18 in sham-, rUUO-, and CGS (1 µg/kg/min; 30 min) + rUUO-, bosentan (1 µg/kg/min; 30 min) + rUUO-, and Pyr3 (1 µg/kg/min; 30 min) + rUUO-operated neonatal pigs. Urinary NGAL, L-FABP, and IL-18 levels were normalized to urinary creatinine (crea). *P < 0.05 vs. sham; #P < 0.05 vs. rUUO; [unpaired t-test (A–C); one-way ANOVA, with Holm-Sidak post hoc test (D–F)].

Fig 4: Acute rUUO stimulated H2O2 generation and ECE1-dependent ET1-3 production in neonatal pig kidneys. A and B, kidney sections and morphological injury score in contralateral and obstructed kidneys. C and D, urinary isoprostane and H2O2 levels in sham- and rUUO- operated pigs. E and F: catalase immunostaining of sham and rUUO-operated kidney sections. G-J, kidney IHC (n = 4), mRNA expression levels, enzymatic activity, and urinary levels of ECE1 in sham and rUUO pigs. K–N, kidney IHC (n = 4) and urinary levels of ET in sham, rUUO, and CGS + rUUO pigs. *P < 0.05 vs. sham; #P < 0.05 vs. rUUO; [unpaired t-test (B-D, F, H-J); one-way ANOVA, with Holm-Sidak post hoc test (L–N)]. Scale bar = 50 µm.

Fig 5: H2O2 generation stimulated ECE1-dependent ET1-3 production by LLC-PK1 cells. A and B, cellular H2O2 and ECE1 levels in control, GO (1 U/ml)- and EUK (50 µM) + GO-treated cells. C and D, ECE1 immunostaining in control and GO (1 U/ml)-, and EUK (50 µM) + GO-treated cells. E-G, cellular ET1-3 in control, and GO (1 U/ml)-, EUK (50 µM) + GO-, and CGS (10 µM) + GO-treated cells. H and I, panET immunostaining in control and GO (1 U/ml)-, EUK (50 µM) + GO- and CGS (10 µM) + GO-treated cells. *P < 0.05 vs. control; #P < 0.05 vs. GO; (one-way ANOVA, with Holm-Sidak post hoc test). Scale bar = 50 µm.