Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

Figures

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  Figure 1.. Porcine UII reduces renal blood flow and increases vascular resistance in neonatal pigs. Line graphs showing percent changes from baseline in (A) mean arterial pressure (MAP), (B) cortical perfusion, (C) total renal blood flow (RBF), and (D) renal vascular resistance (RVR) in neonatal pigs infused with lactated ringer’s solution (control; n = 5) or urantide (URTD; 1 µg/kg/min; n = 6). Panels (E–H) show changes in MAP, cortical perfusion, RBF, and RVR, respectively, in pigs infused with lactated ringer’s (n = 3), porcine UII (1 µg/kg/min for 60 min; n = 4), or URTD (1 µg/kg/min for 30 min) followed by porcine UII (1 µg/kg/min for 60 min; n = 4). *p < 0.05 control vs. porcine UII (MAP: 10–30 and 60 min; cortical perfusion: 10–40 min; RBF: 10–40 min; RVR: 10–60 min). #p < 0.05 porcine UII vs. URTD + porcine UII (MAP: 10–40 and 60 min; cortical perfusion: 10–40 min; RBF: 10–50 min; RVR: 10–60 min). Two-way ANOVA and Holm-Šídák’s multiple comparisons tests.

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  Figure 2.. Upregulation of the UII system in the kidneys of neonatal pigs following IR injury. Quantitative RT-PCR analysis showing relative mRNA expression levels of (A) furin, (B) UII, (C) URP, and (D) UT in whole kidney tissue, and (E) UT in intrarenal arteries from sham and IR piglets (n = 4 per group). (F–G) Representative Western blot and densitometric analysis of UT protein expression in intrarenal arteries (n = 4 per group). (H–I) Western blot and quantification of furin protein expression in kidney tissues (n = 4 per group). (J–K) representative immunohistochemistry (IHC) images and quantification of UII immunostaining intensity in renal tubules from sham and IR piglets (4 kidney sections per group). *p < 0.05, unpaired t-test.

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  Figure 3.. UT inhibition mitigates renal IR-induced renal hemodynamic impairment, AKI biomarkers, and inflammatory cytokine expression in neonatal pigs. Cortical perfusion (A), total renal blood flow (RBF; B), and renal vascular resistance (RVR; C) in sham-operated piglets and those subjected to renal IR, with or without UT inhibition using urantide (URTD; 1 µg/kg/min during the first 60 min of reperfusion; n = 5 per group). (D–I) Plasma biomarkers of kidney function and inflammation: (D) creatinine (crea), (E) blood urea nitrogen (BUN), (F) neutrophil gelatinase-associated lipocalin (NGAL), (G) glomerular filtration rate (GFR; n = 4 per group), (H) interleukin-1β (IL-1β), and (I) interleukin-10 (IL-10) in sham, IR, and URTD + IR groups (n = 5 per group for D–F, H–I). *p < 0.05 sham vs. IR; #p < 0.05 IR vs. URTD + IR. One-way ANOVA and Holm-Šídák’s multiple comparisons tests.

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  Figure 4.. UT inhibition attenuates renal IR-induced histological kidney injury in neonatal pigs. (A) Representative hematoxylin and eosin–stained kidney sections and (B) Quantification of combined histological injury scores in sham-operated piglets and those subjected to renal IR, with or without UT inhibition using urantide (URTD; 1 µg/kg/min during the first 60 min of reperfusion; n = 5 per group). *p < 0.05 sham vs. IR; #p < 0.05 IR vs. URTD + IR. One-way ANOVA and Holm-Šídák’s multiple comparisons tests. Scale bar = 50 µm. Black arrowheads indicate areas of tubular degeneration characterized by cytoplasmic vacuolization. Red arrowheads highlight tubular dilatation and intraluminal cast formation.

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  Figure 5.. Furin inhibition reduces cIR-driven UII production, and UT and furin blockade attenuate cIR-induced cytotoxicity in primary neonatal pig proximal tubule epithelial cells. (A) Representative fluorescence microscopy images showing furin and UII immunostaining and their colocalization in primary neonatal pig proximal tubule epithelial cells (PTECs; 3 biological replicates). (B) Quantification of intracellular furin levels in control (ctrl) and chemical ischemia-reperfusion (cIR)–treated cells (n = 4). (C) Secreted UII levels in cIR-treated cells in the absence or presence of the furin inhibitor SSM3 trifluoroacetate (SSM3; n = 4). (D) cIR-induced cytotoxicity was significantly reduced by pharmacological inhibition of furin (SSM3) and by UT inhibition with urantide (URTD). *p < 0.05 ctrl vs. cIR; p < 0.05 cIR vs. SSM3 + cIR; $p < 0.05 cIR vs. both SSM3 + cIR and URTD + cIR. Unpaired t-test (B); One-way ANOVA and Holm-Šídák’s multiple comparisons tests (C–D); n = 4, each. Scale bar = 50 µm.