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

Membranous nephropathy (MN) is one of the most common causes of nephritic syndrome in adults and is the second most common glomerulopathy to progress to end-stage renal disease (ESRD). Most cases are idiopathic (IMN), but ~ 20% are secondary to various causes, including cancers, infections, autoimmune diseases, and medications. The M-type phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain-containing 7A (TSHD7A), and neural epidermal growth factor-like 1 protein (NELL-1) are major autoantigens against podocyte antigens in idiopathic membranous nephropathy (IMN) [1]. PLA2R-associated IMN is a primary membranous nephropathy with positive serum anti-PLA2R antibody, which covers 70% of IMN [2]. Circulating serum autoantibodies against the M-type phospholipase A2 receptor (PLA2R-AB) are a key biomarker in the diagnosis and monitoring of IMN [3]. Moreover, circulating PLA2R-AB is detectable months to years before documented non-nephrotic range proteinuria and biopsy-proven diagnosis in patients with MN [4]. Thus, the immune complex (IC) formed by PLA2R and serum autoantibody deposition on the sub-epithelial side of the glomerular basement membrane resulting in podocyte injury is the characteristic feature of IMN, in which membrane attack complex C5b-9 plays an essential role by inducing a variety of downstream pathways, including protein kinases, lipid metabolism, reactive oxygen species, growth factors/gene transcription, endoplasmic reticulum stress, and the ubiquitin–proteasome system [5, 6].

Vascular endothelial growth factor A (VEGFA), a homodimeric vasoactive glycoprotein, is the key mediator of angiogenesis. Angiogenesis, the formation of new blood vessels, is responsible for a wide variety of physio/pathological processes in kidney diseases, such as diabetic kidney disease and polycystic kidney disease [7]. In the renal glomeruli, VEGFA is mainly expressed in and secreted from podocytes and tubular epithelial cells, which induces renal injury through binding to the receptor VEGFR, mainly VEGFR2, which is expressed on the surface of endothelial cells [8]. In recent decades, studies that investigated dysregulation of VEGFA expression during kidney diseases have led to the identification of a crucial role of this proangiogenic factor in the renal capillary network [9, 10]. Ayumi Matsumoto et al. reported that VEGFA may be important for TSHD7A-associated IMN pathogenesis [11]. However, the role of VEGFA in PLA2R-associated IMN is poorly studied.

In the present study, we attempted to identify crucial genes and pathways that are involved in the pathogenesis of PLA2R-associated IMN. These results may provide new targets for the treatment of IMN.

First, we used a CCK-8 kit to evaluate cell viability after incubation with different concentrations (2 mg/ml, 4 mg/ml, 6 mg/ml and 8 mg/ml) of BV for different times (24 h, 48 h, and 72 h), and saline was used as a control. We found that the higher the concentration of BV added was, the lower the cell viability; the longer the high BV incubation time was, the lower the cell viability (Fig. 5). Thus, we chose 2 mg/kg BV as stimuli in PHN rats. In vitro experiments demonstrated that the VEGFA inhibitor BV decreased proteinuria excretion compared with the model group (p < 0.05) (Fig. 6). Moreover, 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 (Fig. 7A). Immunohistochemistry data showed that along with the decreased expression of VEGFA in the VEGFA inhibitor group, the expression of PI3K and AKT was obviously decreased compared with that in the model group (p < 0.05) (Fig. 7B, C, and D). Furthermore, the glomerular basement membrane and the morphology of glomerular endothelial cells were alleviated in the VEGFA inhibitor group compared with the model group, as shown by electron microscopy (Fig. 8). All data suggested that a VEGFA inhibitor may alleviate MN by decreasing the expression of PI3K and AKT.

In recent years, with the use of vascular endothelial growth factor A (VEGFA) inhibitors in oncology, the number of patients with kidney disease has increased, mainly manifesting as thrombotic microangiopathy, minimal change nephropathy, and collapsing focal segmental glomerulosclerosis [21, 22]. Moreover, rituximab, a B cell-targeted monoclonal antibody that has become the first-line therapy in IMN according to KDIGO CLINICAL PRACTICE GUIDELINE ON GLOMERULAR DISEASES 2021 [23], showed its ability to decrease the VEGFA level in plasma in patients with recurrent mantle cell lymphoma [24]. Additionally, Ayumi Matsumoto et al. reported that VEGFA may be important for TSHD7A-associated IMN pathogenesis [11]. These data suggest that VEGFA may be involved in the development of MN. In this study, we identified 1422 genes as DEGs in kidneys from IMN patients compared to living donors, 3 hub genes, one up-regulated gene VEGFA, two down-regulated genes JUN and FOS, and six significant modules selected from a PPI network, which partially revealed the molecular mechanism in IMN and may be used to develop novel targets for IMN treatment. We validated that the expression of VEGFA, PI3K and AKT was upregulated in vivo. Furthermore, we revealed that the VEGFA inhibitor bevacizumab plays a significant role in decreasing proteinuria and maintaining the integrity of glomerular endothelial cells via PI3K/AKT signalling in vitro. Thus, we concluded that VEGFA promotes the occurrence of PLA2R-associated IMN by stimulating angiogenesis via PI3K/AKT signalling. Moreover, we found that soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) interactions in vesicular transport may be involved in the occurrence or progression of PLA2R-associated IMN.

Angiogenesis is a process of new blood vessel formation from existing vessels, which are essential for the formation of normal kidney structures and for the functions of glomeruli [25]. Abnormal capillary formation in the kidney may cause morphological changes in glomeruli, as well as infiltration of inflammatory cells [21, 26]. Angiogenesis is tightly regulated by a balance of proangiogenic and antiangiogenic factors, in which the most potent proangiogenic factor, vascular endothelial growth factor A (VEGFA), has been intensively investigated [21]. VEGFA is the predominant isoform of VEGF in humans and one of the most potent proangiogenic factors, which is mainly expressed in and secreted from podocytes [9] and renal tubular epithelial cells in glomeruli [7]. Expression of VEGFA promotes endothelial cell proliferation, migration, and survival but can also be associated with vascular hyperpermeability in inflammatory conditions, including inflammatory kidney diseases, by binding to the receptor VEGFR2 [27]. Neuropilin 1 (NRP1) functions as a coreceptor for VEGFA and is required for complete activation of VEGFR2 [28]. As VEGFR2 is expressed on the surface of endothelial cells, VEGFA-mediated epithelial–endothelial crosstalk in the glomeruli plays an essential role in numerous renal injuries [29]. Moreover, Gebran Khneizer et al. reported a case that describes self-limited biopsy-proven membranous nephropathy after intravitreal bevacizumab injections [30]. In the present study, we found that VEGFA and NRP1 are highly expressed in kidney tissue from IMN patients. GSEA confirmed that VEGFA up-regulation promotes angiogenesis in the IMN group. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. Moreover, we validated that the expression of VEGFA, PI3K and AKT was upregulated in vivo. Furthermore, we revealed that the VEGFA inhibitor bevacizumab plays a significant role in decreasing proteinuria and maintaining the integrity of glomerular endothelial cells via PI3K/AKT signalling in vitro. In addition, the biological process of module 3 was clustered in cell migration involved in sprouting angiogenesis, which is a main type of angiogenesis. Thus, VEGFA activates PI3K/Akt signalling by binding to VEGFR2 to accelerate angiogenesis and leads to vascular hyperpermeability, which results in increased filtration of inflammatory factors, complement, and cytokines, eventually leading to immune complex (IC) and MAC formation. Similarly, a study reported that VEGFA may increase vascular permeability and promote inflammatory infiltrations causing glomerular disease in diabetes [31]. These results suggest that regulation of the VEGFA/PI3K/Akt signalling pathway may be a treatment strategy for IMN.

Complement-mediated cell injury has been demonstrated to contribute to IMN [5]. With complement activation, the formation of membrane attack complex C5b-9 in plasma membranes on the soles of the foot processes of visceral glomerular epithelial cells, which has been shown to induce kidney cell injury through activating a variety of downstream pathways, including focal adhesion kinases [32], lipid metabolism, reactive oxygen species, growth factors/gene transcription, endoplasmic reticulum stress, the ubiquitin–proteasome system [5] and DNA damage [33], impacts the integrity of the cytoskeleton and slit diaphragm proteins and contributes to the pathogenesis of mesangial-proliferative glomerulonephritis, thrombotic microangiopathy, and acute kidney injury [5]. In our study, six modules were found from the PPI network. Our finding remains consistent with previous studies. The biological process of genes from six modules were clustered in positive regulation of mitotic metaphase/anaphase transition, regulation of glomerular filtration, cell adhesion mediated by integrin, pentose-phosphate shunt, oxidative branch, intrinsic apoptotic signalling pathway in response to endoplasmic reticulum stress, nucleotide-excision repair, DNA incision, and fatty acid metabolism. The KEGG pathway enrichment was clustered in ubiquitin-mediated proteolysis and ECM-receptor interaction. Thus, it seems that the vascular hyperpermeability induced by VEGFA/PI3K/Akt signalling plays an initial role in the development of IMN (Fig. 9).

Notably, we found that SNARE interactions in vesicular transport were involved in the development of IMN, which was the first report in this paper. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins are essential for exocytosis, mediating the fusion of vesicles with their target membrane. A study reported that SNARE proteins mediate the fusion of autophagosomes with endolysosomal vesicles, which mediates autophagosome maturation, resulting in autophagy [34]. In addition, inhibiting autophagosome-induced excessive autophagy ameliorates proteinuria and protects against glomerular and podocyte injury in nephrotic syndrome rats [35]. Moreover, with the accumulation of autophagosomes, cell viability is altered, which directly induces cellular toxicity [36]. In our report, we found that genes encoding SNARE proteins, including STX6, STX3, VAMP1, VAMP3, VAMP4, STX1B, and SEC22B, are highly expressed in kidney biopsies from IMN patients, which means that inhibitors of the SNARE interaction may provide a new therapeutic strategy in treating IMN; however, this needs to be verified experimentally.

It should be noted that there were some limitations to our research. First, a recent study demonstrated that there are three non-HLA loci, namely, NFKB1, IRF4 and PLA2R1, revealed in IMN, which means that the canonical NF-κB pathway plays an active role in IMN [37]; however, there was no significant difference in the expression of NFKB1 and IRF4 in this study. The shortage may be that the samples from these two groups are too small. Second, focusing on validated target genes may exclude potential targets that have not been validated by the experiment. Third, the antibodies used in this study may have a certain impact on the experimental results, since the results from immunohistochemistry would be better to use antibodies that recognize the phosphorylated forms of PI3K and AKT and not the total form. Fourth, this study explored the relationship between VEGFA and PLA2R-associated IMN, but did not explore the relationship between VEGFA and secondary membranous nephropathy. It is difficult to show that VEGFA is specifically expressed in IMN, which needs more clinical samples to verify. Last, no knock-down or overexpression of core gene was used to confirm the changes.

The present study suggests that VEGFA might be the origin of the process leading to IMN, and its inhibition could be a possible therapeutic approach. VEGFA is overexpressed in IMN and is an important therapeutic target for IMN, which may lead to vascular hyperpermeability via the PI3K-Akt signalling pathway, resulting in increased filtration of inflammatory factors, complements, and cytokines, eventually contributing to immune complex (IC) and MAC formation in IMN. Moreover, SNARE interactions in vesicular transport are involved in the development of IMN, and further basic studies are needed to validate our results and to illuminate the molecular mechanism in IMN. These results may provide new targets for the treatment of IMN.