Panel A: Two sets of immunohistochemical images show Masson's trichrome and Picrosirius red staining of kidney sections from mice at days 0, 7, and 14 post-UUO surgery. The bar graphs quantify the fibrotic area percentage, showing an increase over time. Panel B: Immunohistochemical images display alpha-SMA and fibronectin staining in kidney sections at days 0, 7, and 14 post-UUO. The bar graphs show the relative mean optical density, indicating increased levels over time. Panel C: Western blot images show protein levels of alpha-SMA, fibronectin, and vimentin in kidneys at days 0, 7, and 14 post-UUO. Panel D: Bar graphs display the relative mRNA levels of Acta2, fibronectin, and vimentin, normalized to Gapdh, showing an increase over time. Panel E: Immunohistochemical images show IFN-λ2/3 staining in kidneys of WT and Ifn-2/3-knockout mice at days 0, 7, and 14 post-UUO. The bar graph quantifies the relative mean optical density. Panel F: Western blot images show IFN-λ2/3 protein levels in kidneys of WT and Ifn-2/3-knockout mice at days 0, 7, and 14 post-UUO. Panel G: Bar graphs display the relative mRNA levels of Ifn-λ2 and Ifn-λ3, normalized to Gapdh, showing differences between WT and Ifn-λ2/3-knockout mice. Panel H: Bar graphs show the expression levels of IFN-λ2 and IFN-λ3 in control and CKD patients from the Nephroseq database. Panel I: Immunohistochemical images show Masson's trichrome and Picrosirius red staining of kidney sections from control and CKD patients. The bar graph quantifies the fibrotic area percentage. Panel J: Immunohistochemical images show IFN-λ2/3, alpha-SMA, and fibronectin staining in kidneys of control and CKD patients. The bar graphs quantify the relative mean optical density. Panel K: Bar graphs display the relative mRNA levels of IFN-λ2/3, ACTA2, and FIBRONECTIN, normalized to ACTINB, showing differences between control and CKD patients.
IFN-λ is upregulated in fibrotic murine and human kidneys. (A–D) WT mice underwent UUO surgery, and kidneys were harvested at 0, 7, and 14 days (n = 6 per group). (A) Representative images and quantitative analysis of fibrotic areas with Masson’s trichrome and PSR staining in mouse kidneys. (B) Representative immunohistochemical images and quantitative analysis of α-SMA and fibronectin relative mean OD (MOD) in the kidneys of UUO mice. (C) Western blot analysis showing α-SMA, fibronectin, and vimentin protein levels in kidneys of UUO mice at days 0, 7, and 14. (D) RT-qPCR analysis of Acta2, fibronectin, and vimentin mRNA levels in kidneys of UUO mice. Relative fold changes in target gene mRNA expression were normalized to Gapdh. (E–G) WT and Ifn-λ2/3–/– mice following UUO, and kidneys were harvested at 0, 7, and 14 days (n = 6 per group). (E) Representative immunohistochemical images and quantitative analysis of IFN-λ2/3 relative MOD in the kidneys of UUO-treated WT and Ifn-λ2/3–/– mice. (F and G) Western blot (F) and RT-qPCR (G) analysis showing IFN-λ2/3 protein and mRNA levels in kidneys of UUO mice. Relative fold changes in target gene mRNA expression were normalized to Gapdh. (H) The expression levels of IFN-λ2 and IFN-λ3 in control (Ctrl) and CKD patients from large cohorts by the Nephroseq version 5.0 database. (I) Kidney sections from control (Ctrl) and CKD patients were stained with Masson’s trichrome and PSR. The percentage of fibrotic area was quantified (n = 12 per group). (J) Immunohistochemical images and quantitation of IFN-λ2/3, α-SMA, and fibronectin MOD in Ctrl and CKD patients (n = 12 per group). (K) RT-qPCR analysis of IFN-λ2/3, ACTA2, and fibronectin mRNA levels in kidneys of Ctrl and CKD patients (n = 12 per group). Relative fold changes in target gene mRNA expression were normalized to ACTB. Scale bars = 50 μm (A, B, E, I, and J). Data in A–G and I–K are represented as individual data points pooled from two independent experiments. Data in H were adapted from Nephroseq version 5.0 database. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Tukey’s multiple-comparison test (A–D), two-way ANOVA with Tukey’s multiple-comparison test (E–G), or unpaired two-tailed Student’s t test (H–K). Source data are available for this figure: SourceData F1.