Fig 1: Identification of ISG20 and CORO1A as key biomarkers of RIF. (A) Venn diagrams of key biomarkers shared by LASSO, RF, and SVM-RFE. PROM1, CORO1A, ISG20, and TIMP1 were identified as hub signatures in RIF selected by LASSO, RF, and SVM-RFE. (B-E) Evaluation of the diagnostic value of the hub signatures through the ROC. The AUCs of CORO1A, ISG20, PROM1, and TIMP1 were 0.816, 0.832, 0.726, and 0.763, respectively. (F,G) Validation of the expression of diagnostic biomarkers in GSE22459 and GSE76882. The expression levels of CORO1A (F) and ISG20 (G) were markedly elevated in RIF samples compared with normal samples. LASSO, least absolute shrinkage and selection operator; RF, random forest; SVM-RFE, support vector machine-recursive feature elimination; RIF, renal interstitial fibrosis; ROC, receiver operating curve; AUC, areas under the curve.
Fig 2: Distribution of immune cells in RIF. (A) The infiltration levels in RIF and normal samples. The ssGSEA algorithm in “GSVA” R package was employed to calculate the infiltration levels of 28 immune cell types in 66 samples with RIF and 124 normal samples from the GSE22459 and GSE76882 datasets. (B) Correlation heatmap of 28 types of immune cells. The numbers in the lower left quarter represent the correlation coefficient between row-defining immune cells and column-defining immune cells, while the statistical significance is highlighted in the upper right quarter. Pearson rank correlation test, *, P<0.05; **, P<0.01; ***, P<0.00. (C) Comparisons of immune cell infiltration between RIF and normal samples. Wilcoxon test, *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. (D) Distribution of the cohort differentially infiltrated immune cells (DIICs). t-SNE method was applied to cluster and visualize the distribution of DIICs in the GEO cohort. (E) Correlations between DIICs and diagnostic biomarkers. The numbers in the frame represent the Spearman correlation coefficient between each DIIC and ISG20 or CORO1A. RIF, renal interstitial fibrosis; DIIC, differentially infiltrated immune cell; GEO, Gene Expression Omnibus.
Fig 3: Identification of potential drugs for the treatment of RIF. A chemical-diagnostic biomarker network was constructed based on potential chemicals targeting ISG20 and CORO1A, and their interactions. The red and blue lines represent the respective increasing or decreasing effects of the chemicals (with chemical IDs in the ellipse) on the expression of target genes (with gene names in the ellipse). RIF, renal interstitial fibrosis.
Fig 4: Validation of the expression of CORO1A and ISG20 in fibrotic tubular cells and renal tissue. (A) CORO1A and ISG20 mRNA expression in TGF-ß-stimulated HK-2 cells. HK-2 cells were stimulated with TGF-ß (0 and 2.5 ng/mL) for 48 h. Relative mRNA level of a-SMA, ISG20, and CORO1A in the above cells was measured by qPCR. a-SMA was used as a fibrosis marker induced by TGF-ß. Mean ± SDs were obtained from three technical replicates. Student’s t-test (two-sided), *, P<0.05. (B) CORO1A and ISG20 protein expression in TGF-ß-stimulated HK-2 cells. HK-2 cells were stimulated with TGF-ß (0 and 2.5 ng/mL) for 48 h. Cell lysates from the above cells were immunoblotted against Vimentin, a-SMA, ISG20, CORO1A, and GAPDH. Vimentin and a-SMA were used as fibrosis markers induced by TGF-ß, and GAPDH was used as a loading control. Mean ± SDs are depicted and P value was calculated using Student’s t-test (two-sided) with data from four biological replicates. *, P<0.05; **, P<0.01. (C,D) CORO1A and ISG20 protein expression in fibrotic renal tissue. Renal tissues from CKD patients with different degree of fibrosis were stained for CORO1A and ISG20 by immunohistochemistry. Representative images of Masson staining and immunohistochemistry staining for CORO1A and ISG20 in renal tissues (C). Scale bar: 50 mm. The expression level of CORO1A and ISG20 (D) in renal tissues with different degrees of fibrosis. Student’s t-test (two-sided), *, P<0.05; **, P<0.01. TGF-ß, transforming growth factor-ß; a-SMA, a-smooth muscle actin; CKD, chronic kidney disease.
Fig 5: Fibrosis progression in bleomycin-treated mice: mild, moderate, and severe. (a) Cytoscape network of KEGG Pathways obtained including in the analysis all the 53 proteins statistically significant in the one-way ANOVA; (b) summary of one-way ANOVA statistically significant proteins and with a statistically significant correlation pattern; (c) Cd36 boxplot as an example of the correlation pattern 3-2-1 and a list of all the other ANOVA statistically significant proteins with a statistically significant correlation pattern; (d) Coro1a boxplot as an example of the correlation pattern 1-2-3 and a list of all the other ANOVA statistically significant proteins with a statistically significant correlation pattern; (e) boxplots of the proteins Lyz2 and Trf, characterized by the correlation pattern 1-2-1; and (f) representative immunofluorescence microphotographs of lung parenchyma categorized as mild, moderate, and severe (40X magnification; scale bar 50 µm). Nuclei were visualized using DAPI (blue) and Coro1a was stained in TRITC (red); (g) histogram shows the percentage of Coro1a-positive cells detected after the immunofluorescence reaction. The data were expressed as the percentage of Coro1a-positive cells and were normalized to the total number of cells segmented and classified as mild, moderate, and severe. Asterisks inside the bars indicate statistical significance versus the corresponding control groups (*** p < 0.001), while asterisks above the bars indicate statistical significance between categories (*** p < 0.001) (one-way ANOVA). In all boxplots, mild/saline boxes are colored in light green, moderate BLM are colored in light orange, and severe BLM are colored in dark orange. In all boxplots, black dots represent the abundance of the selected features in all samples, yellow diamond represents the mean abundance.KEGG: Kyoto Encyclopedia of Genes and Genomes; ANOVA: Analysis of Variance; Cd36: Platelet glycoprotein 4; Coro1a: Coronin-1A; Lyz2: Lysozyme C2; Trf: Serotransferrin; BLM: bleomycin.
Supplier Page from Abcam for Anti-Coronin 1a/TACO antibody [EPR19467-36]