Panel A: Immunofluorescence images showing the expression of IFN-lambda2/3 (red), E-cadherin (white), vimentin (cyan), and CD45 (yellow) in kidney sections from sham and UUO mice. The right panel shows quantification of IFN-lambda2/3 positive cells. Panel B-D: Flow cytometry plots showing the purity of isolated E-cadherin positive TECs, CD45 positive immune cells, and vimentin positive fibroblasts from mouse kidneys. Panel E: Bar graph showing RT-qPCR analysis of Ifn-lambda2 and Ifn-lambda3 messenger RNA levels in different renal cell populations from sham and UUO mice. Panel F: Western blot analysis of pathway proteins in renal TECs of sham and UUO mice. Panel G-K: Western blot and RT-qPCR analysis of IFN-lambda2/3 and pathway proteins in renal TECs from various knockout mice subjected to sham or UUO surgery. Panel L: Western blot and RT-qPCR analysis of IFN-lambda2/3, phosphorylated IRF3, and IRF3 in renal TECs treated with PBS or RIG-I inhibitor. Panel M: Immunofluorescence images and quantification of IFN-lambda2/3 or phosphorylated SMAD2/3 in renal TECs from sham and UUO mice treated with PBS or RIG-I inhibitor. Panel N: Immunofluorescence images and quantitative analysis of IFN-lambda2/3 or phosphorylated SMAD2/3 in renal TECs from WT and Mavs minus/minus UUO mice. Panel O: Immunofluorescence images and quantitative analysis of double-stranded RNA in renal TECs from sham and UUO mice. Panel P: Bar graph showing ELISA analysis of double-stranded RNA levels in isolated renal TECs. Panel Q-R: Western blot analysis and RT-qPCR and ELISA evaluation of IFN-lambda2/3 in primary renal TECs treated with RIG-I inhibitor and poly(I:C). Panel S-T: Western blot analysis and RT-qPCR and ELISA evaluation of IFN-lambda2/3 in renal TECs from WT and Mavs minus/minus mice transfected with poly(I:C).
Renal TECs generate IFN-λ upon RIG-I/MAVS signaling activation. (A) Multiplex immunofluorescence staining for IFN-λ2/3 (red), TECs (E-cadherin, white), fibroblasts (vimentin, cyan), and immune cells (CD45, yellow) in kidney sections at day 7 from sham-operated mice (sham) and UUO mice. Scale bars = 50 μm. Quantification of IFN-λ2/3–positive cells are shown on the right panel (n = 6). (B–D) Flow cytometry plots showing the purity of isolated E-cadherin+ TECs (B), CD45+ immune cells (C), and vimentin+ fibroblasts (D) from mouse kidneys (n = 3). (E) RT-qPCR analysis of Ifn-λ2 and Ifn-λ3 mRNA levels in the indicated purified renal cell populations from sham and UUO mice at day 7 (n = 4). (F) Western blot analysis of indicated pathway proteins in renal TECs of sham and UUO mice on day 7 (n = 5 per group). (G–K) WT, Sting–/– (G), Myd88−/− (H), Trl3−/− (I), Mda5−/− (J), and Mavs–/– (K) mice were subjected to sham or UUO surgery (n = 5 per group). Renal TECs were isolated on day 7 after surgery and analyzed for IFN-λ2/3 and pathway protein expression by western blot (left panels) and Ifn-λ2 and Ifn-λ3 mRNA by RT-qPCR (right panels). (L) Renal TECs from sham and UUO treated with PBS or 50 mg/kg of cFP were analyzed for IFN-λ2/3, p-IRF3, and IRF3 protein levels by western blot (left panel) and Ifn-λ2 and Ifn-λ3 mRNA by RT-qPCR (right panel) (n = 5 per group). (M) Immunofluorescence staining for IFN-λ2/3 or p-SMAD2/3 in renal TECs (E-cadherin) in kidneys of sham and UUO mice treated with PBS or 50 mg/kg of cFP on day 7 after surgery. Nuclei were counterstained with DAPI. Quantification is shown on the right. n = 4 per group, scale bars = 50 μm. (N) Representative images and quantitative analysis of IFN-λ2/3 or p-SMAD2/3 in renal TECs (E-cadherin) in kidneys of WT and Mavs–/– UUO mice at day 7. n = 4 per group, scale bar = 50 μm. (O) Representative images and quantitative analysis of dsRNA (J2) in renal TECs (E-cadherin) in kidneys of sham and UUO mice at day 7. n = 4 per group, scale bars = 50 μm. (P) ELISA analysis of dsRNA levels in isolated renal TECs. (Q and R) Primary renal TECs were treated with or without 2.5 mM cFP for 6 h, followed by 1 μg/ml of poly(I:C) transfection for 12 h (n = 3). (Q) Western blot analysis of RIG-I, MAVS, p-IRF3, and IRF3 protein levels. (R) RT-qPCR and ELISA were used to evaluate IFN-λ2/3 mRNA and protein levels, respectively. (S and T) Renal TECs isolated from WT and Mavs–/– mice were transfected with 1 μg/ml of poly(I:C) for 12 h (n = 3). (S) Western blot analysis of MAVS, p-IRF3, and IRF3 protein levels. (T) IFN-λ2/3 mRNA and protein levels were determined by RT-qPCR and ELISA. Data in A, E, and G–P are pooled from two independent experiments. Data in B–D are pooled from three independent experiments. Data in F and Q–T are representative of three independent experiments. Data are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001, by unpaired two-tailed Student’s t test (A–F and O–P), two-way ANOVA with Tukey’s multiple-comparison test (G–K, N, R, and T), and one-way ANOVA with Tukey’s multiple-comparison test (L and M). ns, no significant difference. Source data are available for this figure: SourceData F6.