Panel A shows Western blot images of phosphorylated and total ERK, JNK, p38, PI3K, and mTOR proteins in renal fibroblasts treated with IFN-lambda2 for 0, 30, 60, and 120 minutes. Panel B shows Western blot images comparing phosphorylated and total ERK and JNK proteins in WT and Ifnlr1 minus/minus renal fibroblasts treated with IFN-lambda2 or PBS. Panel C shows Western blot images of phosphorylated ERK, total ERK, TGF-beta, and GAPDH proteins in renal fibroblasts treated with IFN-lambda2 with or without ERK inhibitor. Panel D shows vertical bar graphs with the x-axis representing treatment groups and the y-axis representing Tgf-beta messenger RNA levels relative to GAPDH and TGF-beta concentration in picograms per milliliter. Panel E shows Western blot images of phosphorylated JNK, total JNK, TGF-beta, and GAPDH proteins in renal fibroblasts treated with IFN-lambda2 with or without JNK inhibitor. Panel F shows vertical bar graphs with the x-axis representing treatment groups and the y-axis representing Tgf-beta messenger RNA levels relative to GAPDH and TGF-beta concentration in picograms per milliliter. Panel G shows vertical bar graphs with the x-axis representing treatment groups and the y-axis representing relative messenger RNA levels of Acta2, fibronectin, and vimentin. Panel H shows Western blot images of phosphorylated and total ERK and JNK proteins in renal fibroblasts isolated from sham and UUO mice. Panel I shows a Western blot image of TGF-beta and GAPDH proteins and a vertical bar graph with the x-axis representing treatment groups and the y-axis representing Tgf-beta messenger RNA levels relative to GAPDH. Panel J shows a Western blot image of TGF-beta and GAPDH proteins and a vertical bar graph with the x-axis representing treatment groups and the y-axis representing Tgf-beta messenger RNA levels relative to GAPDH. Panel K shows Masson's trichrome, PSR, and phosphorylated SMAD2/3 stained kidney images, alongside vertical bar graphs with the x-axis representing treatment groups and the y-axis representing fibrotic area percentage, PSR fibrotic area percentage, and phosphorylated SMAD2/3 relative mean optical density. Panel L shows vertical bar graphs with the x-axis representing treatment groups and the y-axis representing relative messenger RNA levels of Acta2, fibronectin, and vimentin.
IFN-λ induces TGF-β synthesis in renal fibroblasts through activation of the ERK and JNK signaling pathways. (A) Primary renal fibroblasts from WT mice were treated IFN-λ2 (100 ng/ml) for 0, 30, 60, and 120 min. Phosphorylated and total ERK, JNK, p38, PI3K, and mTOR were assessed by western blot. (B) Primary renal fibroblasts from WT and Ifnlr1−/− mice were treated with IFN-λ2 (100 ng/ml) or PBS for 120 min p-ERK, ERK, p-JNK, and JNK were analyzed by western blot. (C and D) Primary renal fibroblasts were pretreated with ERK inhibitor SCH772984 (ERKi, 1 μM) for 1 h, then stimulated with IFN-λ2 (100 ng/ml) for 2 h (to assess ERK activation) or 24 h (to evaluate TGF-β expression) (n = 6). (C) p-ERK and ERK were assessed by western blot; TGF-β expression were analyzed by western blot (C), RT-qPCR (D, left panel), and ELISA (D, right panel). (E and F) Primary renal fibroblasts were pretreated with JNK inhibitor SP600125 (JNKi, 10 μM) for 1 h, then stimulated with IFN-λ2 (100 ng/ml) for 2 h (to assess JNK activation) or 24 h (to evaluate TGF-β expression) (n = 6). (E) p-JNK and JNK were assessed by western blot; TGF-β expression were analyzed by western blot (E), RT-qPCR (F, left panel), and ELISA (F, right panel). (G) Primary renal fibroblasts were treated with IFN-λ2 (100 ng/ml) for 24 h in the presence or absence of ERKi or JNKi. Acta2, fibronectin, and vimentin mRNA levels were measured by RT-qPCR (n = 6). (H) WT mice underwent sham or UUO surgery for 7 days. Renal fibroblasts were isolated and analyzed for p-ERK, ERK, p-JNK, and JNK by western blot. (I and J) WT UUO mice were subcutaneously injected with 1 μg of IFN-λ2 on days −1, 1, 3, and 5, combined with intraperitoneal administration of ERKi (50 mg/kg) (I) or JNKi (50 mg/kg) (J). Kidneys were collected at day 7 after UUO. TGF-β protein and mRNA levels in renal fibroblasts were assessed by western blot and RT-qPCR (n = 5). (K) Representative images and quantitative analysis of fibrotic area and p-SMAD2/3 MOD in kidneys using Masson’s trichrome, PSR, and IHC (scale bars = 50 μm) (n = 5). (L) Renal Acta2, fibronectin, and vimentin mRNA levels were measured by RT-qPCR (n = 5). Data are representative of three (A–C, E, and H–J) independent experiments. Data in D, F–G, and K–L 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 F5.