Panel A: Two bar graphs show the relative Tgf-beta messenger ribonucleic acid levels and TGF-beta protein levels in primary renal fibroblasts from WT and Ifnlr1 minus/minus mice treated with IFN-lambda2 or phosphate-buffered saline. The x-axis represents different treatment groups, and the y-axis represents relative messenger ribonucleic acid levels and TGF-beta concentration. Panel B: A Western blot image shows TGF-beta protein expression in cell lysates from WT and Ifnlr1 minus/minus mice treated with IFN-lambda2 or phosphate-buffered saline. Panel C: Bar graph and a Western blot image show TGF-beta messenger ribonucleic acid and protein levels in renal fibroblasts from WT and Ifnlr1 minus/minus mice subjected to UUO surgery and treated with IFN-lambda2 or phosphate-buffered saline. Panel D: A Western blot image shows the levels of phosphorylated or total SMAD2/3, SMAD4, and SMAD7 in primary kidney fibroblasts from WT and Ifnlr1 minus/minus mice treated with IFN-lambda2 or phosphate-buffered saline. Panel E: A Western blot image shows the levels of phosphorylated and total SMAD2/3 in primary renal fibroblasts from WT mice treated with IFN-lambda2 or phosphate-buffered saline in the presence of anti-TGF-beta or IgG1 isotype control. Panel F: Three bar graphs show the messenger ribonucleic acid levels of Acta2, fibronectin, and vimentin in primary renal fibroblasts from WT mice treated with IFN-lambda2 or phosphate-buffered saline in the presence of anti-TGF-beta or IgG1 isotype control. Panel G: Three bar graphs show the messenger ribonucleic acid levels of Acta2, fibronectin, and vimentin in primary renal fibroblasts from WT mice treated with IFN-lambda2 or phosphate-buffered saline in the presence or absence of SMAD2/3 inhibitor. Panel H: Representative images of kidney sections stained with Masson's trichrome, PSR, and phosphorylated SMAD2/3 staining from WT mice subjected to UUO surgery and treated with IFN-lambda2 or phosphate-buffered saline in the presence of anti-TGF-beta or IgG1 isotype control. Panel I: Bar graphs show the quantitative analysis of fibrotic area and phosphorylated SMAD2/3 mean optical density from the stained kidney sections. Panel J: Three bar graphs show the messenger ribonucleic acid levels of Acta2, fibronectin, and vimentin in kidney tissues from WT mice subjected to UUO surgery and treated with IFN-lambda2 or phosphate-buffered saline in the presence of anti-TGF-beta or IgG1 isotype control.
IFN-λ promotes TGF-β–Smad2/3 signaling in renal fibroblasts that drives fibrogenesis. (A and B) Primary renal fibroblasts from WT and Ifnlr1−/− mice were treated with 100 ng/ml of IFN-λ2 or PBS for 24 h (n = 4). (A)Tgf-β mRNA levels were assessed by RT-qPCR, while the culture supernatants were evaluated for TGF-β by ELISA. (B) TGF-β protein expression in cell lysates was examined by western blot. GAPDH was employed as a loading control. (C) WT and Ifnlr1−/− mice (n = 6 per group) underwent UUO surgery and were subcutaneously injected with IFN-λ2 (1 μg) or PBS on days −1, 1, 3, and 5. Renal fibroblasts were isolated on day 7 after surgery to measure TGF-β mRNA and protein levels using RT-qPCR and western blot. (D) Primary kidney fibroblasts from WT and Ifnlr1−/− mice were treated with 100 ng/ml of IFN-λ2 or PBS for 24 h. Western blot was used to assess the phosphorylated or total SMAD2/3, SMAD4, and SMAD7 levels. (E and F) Primary renal fibroblasts from WT mice were treated with 100 ng/ml IFN-λ2 or PBS in the presence of 2 μg/ml anti-TGF-β–neutralizing antibody (anti-TGF-β) or IgG1 isotype control (anti-IgG1) for 24 h (n = 4). (E) The phosphorylated and total SMAD2/3 levels were analyzed by western blot. (F)Acta2, fibronectin, and vimentin mRNA levels were measured by RT-qPCR. (G) Primary renal fibroblasts from WT mice were treated with 100 ng/ml of IFN-λ2 or PBS in the presence or absence of 20 μM SMAD2/3 inhibitor (LY2109761) for 24 h (n = 4). Acta2, fibronectin, and vimentin mRNA levels were determined by RT-qPCR. (H–J) WT mice were subjected to UUO surgery and received subcutaneous injections of 1 μg of IFN-λ2 or PBS, combined with intraperitoneal injections of 25 μg of TGF-β–neutralizing antibody (anti–TGF-β) or IgG isotype control (anti-IgG1) on days −1, 1, 3, and 5. The kidneys were collected on day 7 after surgery (n = 6). (H and I) Representative images and quantitative analysis of fibrotic area and p-SMAD2/3 MOD using Masson’s trichrome and PSR staining and p-SMAD2/3 staining (scale bars = 50 μm). (J)Acta2, fibronectin, and vimentin mRNA levels were determined by RT-qPCR. Data are representative of two (H) or three (A–G) independent experiments. Data in I and J are pooled from two independent experiments. Data are shown as mean ± SEM. ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test. MOD, mean OD. Source data are available for this figure: SourceData F4.