Mechanism of transforming growth factor- β 1 induce renal fibrosis based on transcriptome sequencing analysis.

OBJECTIVES: To explore the mechanism of transforming growth factor-β1 (TGF-β1) induce renal fibrosis.

METHODS: Renal fibroblast NRK-49F cells treated with and without TGF-β1 were subjected to RNA-seq analysis. DESeq2 was used for analysis. Differentially expressed genes were screened with the criteria of false discovery rate<0.05 and l o g 2 F C >1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for differentially expressed genes. Genes encoding transcription factors were further screened for differential expression genes. Then, the expression of these genes during renal fibrosis was verified using unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model and a public gene expression dataset (GSE104954).

RESULTS: After TGF-β1 treatment for 6, 12 and 24 h, 552, 1209 and 1028 differentially expressed genes were identified, respectively. GO analysis indicated that these genes were significantly enriched in development, cell death, and cell migration. KEGG pathway analysis showed that in the early stage of TGF-β1 induction (TGF-β1 treatment for 6 h), the changes in Hippo, TGF-β and Wnt signaling pathways were observed, while in the late stage of TGF-β1 induction (TGF-β1 treatment for 24 h), the changes of extracellular matrix-receptor interaction, focal adhesion and adherens junction were mainly enriched. Among the 291 up-regulated differentially expressed genes treated with TGF-β1 for 6 h, 13 genes ( Snai1 , Irf8 , Bhlhe40 , Junb , Arid5a , Vdr , Lef1 , Ahr , Foxo1 , Myc , Tcf7 , Foxc2 , Glis1 ) encoded transcription factors. Validation in a cell model showed that TGF-β1 induced expression of 9 transcription factors (encoded by Snai1, Irf8, Bhlhe40, Junb, Arid5a, Vdr, Lef1, Myc, Tcf7 ), while the expression levels of the other 4 genes did not significantly change after TGF-β1 treatment. Validation results in UUO-induced mouse renal fibrosis model showed that Snai1 , Irf8 , Bhlhe40 , Junb , Arid5a , Myc and Tcf7 were up-regulated after UUO, Vdr was down-regulated and there was no significant change in Lef1 . Validation based on the GSE104954 dataset showed that IRF8 was significantly overexpressed in the renal tubulointerstitium of patients with diabetic nephropathy or IgA nephropathy, MYC was highly expressed in diabetic nephropathy, and the expressions of the other 7 genes were not significantly different compared with the control group.

CONCLUSIONS: TGF-β1 induces differentially expressed genes in renal fibroblasts, among which Irf8 and Myc were identified as potential targets of chronic kidney disease and renal fibrosis.