The development of SLE is closely associated with the alteration in molecular functions, biological processes and signaling pathways. However, the molecular characteristics and the associated molecular functions, pathways, and interactions in different stage of SLE are not well understood. To explore whether distinct molecular and biological functions were enriched in active JSLE, in the currently study, multiple molecular and biological function as well as pathways were observed in the common DEGs, implying that these factors were involved the development of JSLE. Since the non-invasive urine samples can be easily collected and diagnose, in this study, we proposed to reflect specific and sensitive of renal activity by using JSLE urine samples, and revealed that 105 different proteins in the comparison between JSLE patients and healthy controls based on proteomic assay. The results showed that these specific DEGs had different molecular functions and associated with different biological pathways. In addition, these common and unique DEGs formed complex interactome networks. The most important discovery of our study is that we have found both urine EPHA4 and VTN levels are reduced in inactive and active JSLE patients when compared to healthy controls. Further bioinformatics analysis implied us the EPHA4 and VTN in urine might have a potential possibility to be biomarkers of JSLE patients. Furthermore, VTN in the urine of JSLE patients, including inactive and active JSLE, was inversely correlated with the serum dsDNA and ANA. And VTN appeared to be selectively distinguished in inactive JSLE from active JSLE, suggesting that it might serve as a useful biomarker for clinical prognosis.
Coincidentally, in some previous studies, the presence of soluble membrane attack complex (MAC) has been well documented in the renal pathological process along with immune deposits containing circulating immune complexes (CIC) and C3 . In addition, it is reported that in SLE patients, the elevated urinary MAC has been used as a biomarker for disease flare . Furthermore, in another study, the high levels of VTN are associated with the CIC-MAC in SLE patients with active nephritis . Further expanded studies are required to establish the value of VTN binds to CIC as a biomarker of active nephritis. Interestingly, in our study, we found a significant decreased level of urinary VTN and lower expression of serum C3 and C4 in inactive JSLE, and also a positive association between VTN and C3/C4, which may derive from the metabolic dissociation of CIC-MAC in JSLE glomerulus. These findings of identifying a novel mechanism may contribute to the JSLE nephritis.
Besides, it has been demonstrated that EPHA4 performs an important role in a lot of cellular processes, like promoting cell proliferation and cell adhesion-mediated drug resistance via the Akt signaling pathway . While COL18A1 interacted with VTN and EPHA4, plays a crucial role in response of acute liver injury through binding α1β1 integrin on hepatocytes . In addition, the PI3K/Akt and Wnt/β-catenin signaling pathways as well as ERK1/2, the downstream of EPHA4 receptor activation, play an important role in the regulation of events related with the epithelial-mesenchymal transition development . These findings in line with the analysis of these pathways are shown in Fig. 4D-E, including PI3K-Akt, cell adhesion molecules and focal adhesion. Specifically, EPHA4 is the most abundant ephrin receptor, which interacted with almost all ephrin ligands, ranging from effects on inflammatory responses to axonal degeneration and regeneration in the central nervous system, especially in neurological functional recovery by regulating various processes, such as neuroinflammation, angiogenesis, neurogenesis, axonal reorganization and synaptic plasticity [20, 30]. Although update to now, there is no available evidence proved the function of EPHA4 in the pathogenesis of SLE, EPHA4 as a possible inflammatory mediator may contribute to the immunopathological disease like multiple sclerosis and neuropsychiatric disorder in SLE [31–36]. Occasionally, in our study, we have found a significant decreased protein level of EPHA4 in the urine of JSLE patients, which may imply a potential role of EPHA4 in neurodevelopment of JSLE patients via PI3K-Akt signaling pathway. However, because the expressions of EPHA4 in some JSLE patients are below the detection limit, we cannot obtain the valid comparison of urinary EPHA4 between JSLE patients and healthy controls. The correlation between EPHA4 and clinical parameters will be further addressed in future study with a large number of JSLE patients.
In addition to the EPHA4, we found that dsDNA and ANA had a negative correlation with urine VTN level, which was similar with many other studies like circulating S100 and serum triggering receptor expressed on myeloid cell-1 [37–38]. Otherwise, VTN was reported to involve in promoting the expression of inflammatory factors, such as IL-6 and leukemia inhibitory factor via integrin-focal adhesion kinase and uPAR signaling pathways , and to promote neurogenesis . Another study also found an increased co-localization of MAC, VTN and VTN receptor (avβ3 integrin), both of which are within and around the subepithelial deposits in membranous nephropathy . What's more, prevention of autoimmune diseases depends on immune homeostasis, which results from the balances of different T-lymphocyte subsets. Therefore, in this study, we have found that urine VTN level was significantly negative associated with %CD19 + B cells in PBMC and %CD3 + CD4-CD8- T cells in the whole blood of inactive JSLE (Fig. 6A-B). The similar relationship was found in an analysis for the correlations of VTN with %CD3 + CD8 + suppressor T cells in PBMC of active JSLE (Fig. 6F). These results imply that due to the higher expression of VTN in active JSLE and lower expression of VTN in inactive JSLE, the corresponding increased %CD3 + CD8 + suppressor T cells in active JSLE and higher %CD19 + B cells in inactive JSLE may involve into the auto-inflammation and accumulation of MAC. This reminds us the urine VTN level may become a novel biomarker for the diagnostics of JSLE progression. However, due to the limited number of JSLE patients and healthy controls in this investigation, several concerns may be not clarified precisely. For example, what kind of relationship between EPHA4 and VTN, and whether EPHA4 is a novel receptor of VTN still need to be further justified.