Journal: Redox Biology

Article Title: Macrophage AMPK activated by oxidative stress drives profibrotic crosstalk with tubular cells to accelerate renal fibrosis after ischemic and reperfusion injury

doi: 10.1016/j.redox.2025.104002

Figure Lengend Snippet: AMPK orchestrated the development of profibrotic Arg1 + MMP12 + macrophages. (A) Sub-clustering analysis of myeloid cells. Neu, neutrophil; DC, dendritic cell; Mono, monocyte; Mac, macrophage. (B) Violin plots and (C) bubble plots showing the expression of specific feature genes in myeloid cells. (D) Barplot and (E) heatmap showing the cell abundance of myeloid cells in sham kidneys from WT mice and IRI day 7 kidneys from WT and mKO mice. (F) GO term enrichment analysis of Arg1 + MMP12 + macrophages. (G) Representative immunofluorescent staining for F4/80 (Cyan), Arg1 (Green), MMP12 (Yellow), and TWEAK (Red) in WT and mKO kidneys at day 28 post-IRI. Scale bars, 40 μm. (H) Representative flow cytometry plots and quantification showing macrophage abundance in IRI kidneys from WT and mKO mice at day 28 post-IRI (n = 6). (I) GSEA analysis showing enrichment of mitochondrial long-chain fatty acid β-oxidation pathway in Arg1 + MMP12 + macrophages according to the upregulated differentially expressed genes from WT and mKO mice. (J) The intracellular levels of acetyl-CoA in WT and AMPKα1 −/− BMDMs (n = 5). The results represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Following fixation and permeabilization with True-Nuclear Transcription Factor Buffer Set (Biolegend, 424401), cells were sequentially incubated with primary antibodies against phospho-AMPKα (CST, 2535T), Arg1 (Proteintech, 16001-1-AP) and MMP12 (Proteintech, 22989-1-AP), followed by corresponding Alexa Fluor-594 conjugated secondary antibodies.

Techniques: Expressing, Staining, Flow Cytometry

Journal: Redox Biology

Article Title: Macrophage AMPK activated by oxidative stress drives profibrotic crosstalk with tubular cells to accelerate renal fibrosis after ischemic and reperfusion injury

doi: 10.1016/j.redox.2025.104002

Figure Lengend Snippet: Arg1 + MMP12 + macrophage-derived TWEAK licenses PDGFB production in fibroTECs via Fn14 signaling. (A) Ligand-receptor communication analysis between different cell types in IRI kidneys. (B) The major incoming signaling of different cell types. (C) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between myeloid cell subsets and TEC subsets. (D) Bubble plots showing the expression of specific ligand-receptor pairs in the interaction between TEC subsets and myeloid cell subsets. (E, F) WT/AMPKα1 −/− BMDMs were treated with IL-4 and IL-13, and the expression levels of Arg 1 , Mmp12 , and Tnfsf12 were analyzed by qPCR (n = 5). (G, H) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs. The expression of Pdgfb , Cxcl1 , and Egfr in TCMK-1 cells was analyzed by qPCR (n = 5). (I, J) TCMK-1 cells were treated with CM from IL-4 and IL-13-stimulated WT/AMPKα1 −/− BMDMs or together with recombinant TWEAK (rTWEAK). The expression of Pdgfb in TCMK-1 cells was analyzed by qPCR (n = 5). (K, L) TCMK-1 cells were treated with rTWEAK or rTWEAK and an Fn14 inhibitor. The expression of Pdgfb in TCMK-1 cells was detected by qPCR (n = 5). (M) Immunoblots and quantification of IKK-β and nuclear NF-κB p65 in TCMK-1 cells treated with PBS or rTWEAK (n = 3). The results represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Following fixation and permeabilization with True-Nuclear Transcription Factor Buffer Set (Biolegend, 424401), cells were sequentially incubated with primary antibodies against phospho-AMPKα (CST, 2535T), Arg1 (Proteintech, 16001-1-AP) and MMP12 (Proteintech, 22989-1-AP), followed by corresponding Alexa Fluor-594 conjugated secondary antibodies.

Techniques: Derivative Assay, Expressing, Recombinant, Western Blot