Transcriptome analysis of the transformation of podocytes into mesenchymal cells in high glucose memory model / 解放军医学杂志

ABSTRACT

Objective: To investigate, with transcriptome sequencing, the mechanism of metabolic memory in epithelial-mensenchymal transition (EMT) of podocytes induced by high glucose. Methods: Mouse glomerular podocytes were cultured in four groups normal glucose group (NG, 5 mmol/L D-glucose X48 h); high glucose group (HG, 30 mmol/L D-glucose X48 h); metabolic memory group (MG, 30 mmol/L D-glucose x48 h, +5.5 mmol/L D-glouse x48 h); osmotic pressure group (OSM, 5.5 mmol/L D-glucose + 24.5 mmol/L mannitol X48 h). The expressions of Nephrin and α-SMA in podocyte EMT were detected by Western blotting and immunofluorescence. Illumina Hiseq 2000 was applied to characterize the transcriptome profiles of NG, HG and MG groups, DESeq was used to identify the differentially expressed genes (DEGs), gene ontology (GO) enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were implemented for DEGs. The expression levels of the selected DEGs were detected by real-time fluorescence quantitative PCR. Results: Compared with NG group, both Western blotting and immunofluorescence showed that the expression level of Nephrin decreased, while of ct-SMA increased (P0.05). RNA-Seq showed that 194 genes (119 up-regulation and 75 down-regulation) were differentially expressed between HG and NG groups, while 532 genes (316 up-regulation and 216 down-regulation) were differentially expressed between MG and NG groups. Further comparison of the above DEGs showed 108 genes (35 up-regulation and 73 down-regulation) had similar expression patterns in HG and MG groups. The GO enrichment analysis showed 10 GO terms were significantly enriched, and the KEGG pathway enrichment analysis showed TGF-β, focal adhesion plaque, Rap1 and pluripotent regulation of stem cells signal pathways were significantly enriched. PCR showed that the expression levels of Smad9, Snai1 and Mapkapk3 were higher, while of Igf1, Fgf22, Lama1 and Zfhx3 were lower in HG and MG group than in NG group. Conclusions: High glucose can induce podocyte EMT in vitro, and there is a "memory phenomenon" even after normal glucose culture. The occurrence of this phenomenon may be potentially related to TGF-β, focal adhesion plaque, Rap1 and pluripotent regulation of stem cells signal pathways, and upregulation of Smad9, Snai1 and Mapkapk3, as well as downregulation of Igf1, Fgf22, Lama1 and Zfhx3.