Fig 1.
The cellular landscape of the kidney 7 days after uIRI-induced AKI and in Sham control mice.
(a) Single-cell transcriptome atlas of mouse kidneys identifies 11 cell types, including proximal tubular cells, endothelial cells, epithelial cells, adipocytes, Fibroblasts and six types of immune cells (monocytes, neutrophils, T cells, B cells, NK cells, and macrophages). (b) Marker gene expression for each cell type, with dot size indicating the proportion of expressing cells and color representing average expression level. (c) Pie charts showing decreased proximal tubular and epithelial cells and increased immune cells, particularly macrophages (26.7%), in AKI kidneys compared to controls. (d) Bar plots of cell type proportions across datasets confirm increased macrophage infiltration in all AKI samples relative to controls.
Fig 2.
Ccl6/Ccr2 axis mediates macrophage communication in day 7 post-AKI.
(a) Analysis of cell-cell communication shows differences in interaction number and weight among cell subtypes in AKI and sham control groups. In the AKI group, macrophages exhibit increased interactions with themselves, neutrophils, and lymphocytes. (b) Ligand-receptor interaction weights indicate enhanced signal reception and outgoing signaling by macrophages in AKI kidneys compared to controls. (c) Intercellular signaling pathway analysis reveals intensified communication among immune cells, particularly within macrophage subtypes, in the AKI group. (d) Chemokine CCL signaling pathways are significantly upregulated in AKI macrophages, indicating activation of this pathway. (e) Ccl6/Ccr2 as the key driver of macrophage-macrophage interactions, promoting the recruitment of bone marrow-derived macrophages to the injured kidney and potentially influencing renal repair. (f) Validation of CCL signaling pathway activation in AKI. CellChat analyses across all AKI datasets at 7 days post-injury revealed consistent activation of the CCL signaling pathway, with the Ccl6–Ccr2 demonstrating strong interactions and emerging as a significantly enriched communication axis.
Fig 3.
Trajectory analysis reveals temporal expression consistency of Ccl6 and Ccr2 in macrophages during AKI.
(a) UMAP visualization of 5,009 macrophages from Control and AKI mouse kidney tissues, reclustered into six distinct subpopulations. (b-c) Heatmap and FeaturePlot showing the marker genes for each macrophage subpopulation. (d) Pseudotime analysis using Monocle 3 reveals a differentiation trajectory from Stage I to Stage II. (e) Heatmap of pseudotime-ordered gene expression, highlighting differentially expressed genes along the trajectory. Ccl6 and Ccr2 showed significant expression at the terminal stage of the trajectory. (f) FeaturePlot showing the expression patterns of Ccl6 and Ccr2 across macrophage clusters. A subset of Cluster 0 co-expresses both genes, suggesting a potential autocrine loop, while Cluster 3 predominantly expresses Ccl6 alone, supporting a paracrine model.
Fig 4.
Ccl6-Ccr2 interaction drives macrophage migration and M2 polarization during AKI.
(a) Differential expression analysis of macrophages in the AKI group compared to the Control group. A total of 132 genes were upregulated, including Ccl6 and Ccr2, while 295 genes were downregulated. (b) The FeaturePlot displays the top 10 upregulated genes in macrophages from AKI kidneys. (c) UMAP visualization and violin plots showing the expression distribution of Ccl6 and Ccr2 across cell clusters in the AKI group. (d) Correlation analysis reveals positive relationships between Ccl6, Ccr2, and key genes such as Retnla, Fn1, and Arg1. (e) GO enrichment analysis of 132 upregulated genes in macrophages from the AKI group. The genes are enriched in pathways such as cytokine-mediated signaling, leukocyte migration, and myeloid leukocyte chemotaxis. (f) Heatmap of co-expressed genes involved in the enriched pathways. (g) Gene relationship network showing the co-expression of Ccl6 and Ccr2 in pathways related to cytokine-mediated signaling, chemokine activity, and leukocyte migration. (h) Protein-Protein Interaction (PPI) network of upregulated genes in the AKI group. Ccl6 and Ccr2 demonstrate strong interactions within three core pathways: chemokine receptor-ligand binding, chemokine-mediated signaling, and cellular responses to chemokines. (i) Ccl6+Ccr2+Arg1+ macrophages exhibit high Spp1, Lgals3, and Mmp12 expression, suggesting pro-fibrotic remodeling potential.
Fig 5.
Ccl6+Ccr2+Arg1+ cell infiltration correlates with the progression of renal interstitial fibrosis following AKI.
(a) Schematic representation of the experimental workflow using the uIRI model to induce AKI. Kidney samples were harvested 7 days post-injury (n = 6). (b) Transcriptional analysis of Ccl6, Ccr2, Retnla, and Arg1 expression levels in kidney tissue, showing significant upregulation in the AKI group compared to sham controls (n = 6). (c) Histological analysis at day 7 post-AKI. H&E staining reveals tubular necrosis, epithelial detachment, and brush border loss. PSR and Masson staining demonstrate significant interstitial fibrosis. Quantification of tubular injury scores and fibrosis areas highlights incomplete renal repair and fibrotic remodeling (n = 6). Scale bar: 50 μm. (d, e) Colocalization analysis of Ccl6, Ccr2, and Arg1 in the OSOM region of the kidney at day 7 post-AKI. Immunofluorescence-based quantification showed significantly increased Pearson’s correlation coefficients among the three markers, indicating enhanced co-expression within the same cells (n = 6). Scale bar: 50 μm (f) Quantification of Ccl6+Ccr2+ and Ccl6+Ccr2+Arg1+ cells revealed a significant increase in kidneys 7 days after AKI compared to sham controls (n = 6). (g, h) The infiltration of Ccl6+Ccr2+Arg1+ cells positively correlate with the severity of renal interstitial fibrosis. (i) Western blot analysis of Ccr2 protein levels in kidney tissue shows significant difference between AKI and sham controls. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance.
Fig 6.
The role of the Ccl6/Ccr2 axis in macrophage migration, M2 polarization, and renal fibrosis following AKI.
(a) Co-immunofluorescent staining for Arg1 and F4/80 in kidney tissues 7 days post-AKI showed a significant increase in expression compared to the Sham group, which was reduced following Ccr2 antagonist treatment (n = 3). Scale bar: 50 μm. (b, c) Co-localization analysis of Arg1 and F4/80 signals revealed a significant correlation, with no significant differences in Pearson’s correlation coefficient or Manders’ overlap coefficient among groups. (d) Quantification of Arg1 mean fluorescence intensity, Arg1-positive area percentage, and Arg1+F4/80+ double-positive macrophages showed significant increases in the AKI group, which were reduced following Ccr2 blockade. (e, f) Masson and Sirius Red staining demonstrated a marked reduction in renal fibrosis upon Ccr2 inhibition (n = 3). Scale bar: 50 μm. (g) Confirmation of macrophage identity by Cd68 immunofluorescence staining in isolated bone BMDMs. Scale bar: 10 μm (h) CCK8 assay revealed no significant effect of 24-hour Ccl6 treatment on BMDM proliferation (n = 6). (i, j) Transwell migration assays showed a significant increase in BMDMs migration in the Ccl6-treated group (n = 6). (k, l) Flow cytometry analysis indicated an elevated mean fluorescence intensity of Arg1, confirming that Ccl6 promoted M2 polarization of BMDMs (n = 3). Results are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance.