VEGFA promotes the occurrence of PLA2R-associated idiopathic membranous nephropathy by angiogenesis via the PI3K/AKT signalling pathway

Abstract

Background: The M-type phospholipase A2 receptor (PLA2R)-associated idiopathic membranous nephropathy (IMN) is a common immune-related disease in adults. Vascular endothelial growth factor A (VEGFA) is the key mediator of angiogenesis, which leads to numerous kidney diseases. However, the role of VEGFA in IMN is poorly understood.

Methods: In the present study, we downloaded the microarray data GSE115857 from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified with R software. The cytoHubba plug-in were used to identify hub genes from the protein-protein interaction network. Gene set enrichment analysis (GSEA) was used to identify signalling pathway in IMN. CCK8 was performed to assess the cell viability in human vascular endothelial cells (HVECs). Then, passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Animals treated with VEGFA inhibitor bevacizumab (BV), with saline as a positive control. Proteinuria was evaluated by biochemical measurements. Immunohistochemistry and immunofluorescence was used to evaluate relative proteins expression. Electron microscopy was performed to observe the thickness of the glomerular basement membrane (GBM).

Results: We revealed 3 hub genes, including one up-regulated gene VEGFA and two down-regulated genes JUN and FOS, which are closely related to the development of PLA2R-associated IMN. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. GSEA showed that the signalling pathway of DEGs in GSE115857 was focused on angiogenesis, in which VEGFA acts as a core gene. We confirmed the high expression of VEGFA, PI3K, and AKT in IMN renal biopsy samples with immunohistochemistry. In HVECs, we found that BV suppresses cell viability in a time and dose dependent manner. In vivo, we found low dose of BV attenuates proteinuria via inhibiting VEGFA/PI3K/AKT signalling. Meanwhile, low dose of BV alleviates the thickening of the GBM.

Conclusion: VEGFA/PI3K/AKT signalling may play significant roles in the pathogenesis of IMN, which may provide new targets for the treatment of IMN.

Keywords: Bioinformatics; PI3K/AKT signalling; PLA2R-associated idiopathic membranous nephropathy; VEGFA.

Fig.1

A Volcano plot of DEGs in IMN. The cut-off criteria were |log2Fc|> 1 and P < 0.05. The red dots represent the up-regulated genes, and the blue dots denote the down-regulated genes. The grey dots indicate the genes with |log2Fc|< 1 and/or P > 0.05. B Heatmap of the top 50 up-regulated genes and the top 50 down-regulated genes. Grey indicates relatively low expression, and red indicates relatively high expression. C GO enrichment and KEGG pathway analysis of DEGs: (a) Biological process. (b) Cellular component. (c) Molecular function. (d) KEGG pathway

Fig. 2

Six modules identified by MCODE: 6 modules from the PPI network were selected. The red nodes represent the up-regulated genes, and the green nodes represent the down-regulated genes: (a) module 1; (b) module 2; (c) module 3; (d) module 4; (e) module 5; (f) module 6

Fig. 3

A The hub genes in the PPI network screened out via intersected by Betweenness, Bottleneck, Eccentricity, EPC, and MNC methods. B Signalling pathway activated in kidney biopsies from IMN patients using GSEA

Fig. 4

The relative expression of VEGFA, PI3K, and AKT. A Immunohistochemistry; B The relative expression of VEGFA, PI3K, and AKT. ^*p < 0.05, compared with control group

Fig. 5

Bevacizumab (BV) downregulates the viability of human vascular endothelial cells (HVECs) in a dose-dependent manner

Fig. 6

Bevacizumab (BV) decreased proteinuria excretion compared with the model group (p < 0.05)

Fig. 7

A Immunofluorescence and immunohistochemistry showed that the expression of VEGFA in the BV group was alleviated and accompanied by suppression of PI3K and AKT compared with the model group. B Immunohistochemistry data showed that the expression of VEGFA in the BV group was alleviated compared with that in the model group (p < 0.05). C Immunohistochemistry data showed that the expression of PI3K in the BV group was decreased compared with that in the model group (p < 0.05). D Immunohistochemistry data showed that the expression of AKT in the BV group was alleviated compared with that in the model group (p < 0.05)

Fig. 8

Electron microscopy showed that the glomerular basement membrane and the morphology of glomerular endothelial cells were alleviated in the VEGFA inhibitor group compared with the model group

Fig. 9

The possible mechanisms by which VEGFA/PI3K/Akt is involved in IMN