Correlation analysis of COMP and autophagy in diabetic nephropathy and its functional verification

doi:10.3969/j.issn.1674-8115.2024.07.006

Abstract

Abstract:

Objective ·To further clarify the mechanism of podocyte damage by studying the expression of cartilage oligomeric matrix protein (COMP) in glomerular podocytes and its relationship with podocyte autophagy under high glucose environment. Methods ·The gene expression dataset GSE104948 was downloaded from the GENE EXPRESSION OMNIBUS (GEO) database, and differentially expressed genes (DEGs) were obtained via GEO2R. The molecular functions and signaling pathways related to differential genes were summarized. The most correlated key genes (hub genes) were acquired by Weighted Gene Co-Expression Network Analysis (WGCNA) and the protein-protein interaction network (PPI) of DEGs was constructed with STRING database. The enrichment analysis was performed again. Conditionally immortalized mouse podocyte cells were cultured in vitro. After being fully differentiated, they were stimulated with high glucose, and the expressions of COMP, mammalian target of rapamycin (mTOR), microtubule-associated protein 1 light chain3 (LC3) and other proteins in podocytes were detected by Western blotting. The shRNA constructed by lentiviral vector was further used to infect podocytes to inhibit the expression of COMP, and the stable cell strains were screened by puromycin. The expression of COMP, mTOR, and LC3 of stable strains were detected by Western blotting, in order to observe the effect of COMP on autophagy. Results ·A total of 362 DEGs were filtered for subsequent analysis. Among these DEGs, 284 genes were up-regulated and 78 genes were down-regulated. The results of Gene Onotology (GO) term analysis showed that DEGs in diabetic nephropathy (DN) were mainly enriched in cell surface receptor signaling pathway, receptor binding, etc. The main enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway, extracellular matrix (ECM)-receptor interaction, etc. Sixty-four hub genes were refined through the intersection of WGCNA and PPI hub genes, and the hub genes with significantly increased or decreased expression were sifted. The hub genes were annotated with KEGG again, and it was found that most of the hub genes were enriched in "ECM-receptor interaction" and "PI3K/AKT signaling pathway". The PI3K/AKT/mTOR signaling pathway is a classic autophagy pathway, and COMP was absolutely overexpressed (logFC>2) in the 64 hub genes, suggesting that it may affect autophagy through this pathway. Western blotting showed that compared with the mannitol control group and the low glucose group, the expression of COMP in podocytes was significantly increased under high glucose stimulation. Compared with the control group, the expression of LC3-II in the high glucose group was significantly decreased, indicating that the autophagy initiation of podocytes was inhibited under the high glucose environment. Compared with the negative control, the expression of LC3-II in renal podocytes of mice with knockdown of COMP was significantly increased, and the mTOR decreased with the decrease of the expression of COMP, indicating that inhibiting COMP contributed to the recovery of autophagy in podocytes. Conclusion ·COMP is highly expressed in DN patients and highly enriched in ECM receptor and PI3K/AKT signaling pathway. Autophagy in mouse renal podocytes is inhibited under high glucose conditions, and the high expression of COMP induced by high glucose may be a key factor in autophagy inhibition. Inhibiting COMP helps to restore autophagy in mouse renal podocytes.

Key words: diabetic nephropathy, podocyte, autophagy, bioinformatics, differential gene, cartilage oligomeric matrix protein (COMP)

CLC Number:

WEI Yunxin, JIANG Xushun, CAI Mengyao, WEN Ruizhi, DU Xiaogang. Correlation analysis of COMP and autophagy in diabetic nephropathy and its functional verification[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(7): 847-858.

Figures/Tables 6

Fig 1 Hub genes screened by WGCNA and PPI

Fig 2 Differential expression genes screening

Fig 3 KEGG analysis of differential expression genes

Fig 4 Hub gene enrichment analysis

Fig 5 High glucose-induced COMP overexpressed and inhibited autophagy

Fig 6 Knockdown of COMP levels reduced mTOR expression and increased LC3 expression in podocytes

TrendMD

References 57

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