In recent years, although the overall incidence and prevalence of acute allograft in kidney transplantation have decreased, it is also a vital reason which leads to graft dysfunction and even death [18]. Presently, as the result of the usage of more effective and advanced immunosuppressive drugs, the incidence of acute rejection in the first year is reduced to approximately 7.9% [19]. Currently, renal biopsy remains the gold standard for the specific injury of AR. However, in consideration of its side effects, performing serial renal biopsies are impractical at the same periodicity as blood or urine examination. Besides, Dharnidharka and his colleagues found that cellular and molecular events of AR occur before the rise of the clinical biomarker like serum creatinine as well as according to serum creatinine elevated, clinical doctors can’t distinguished a superimposed AR from chronic allograft injury progression, which suggests that functional markers like serum creatinine for glomerular filtration are not as specific as injury markers[20]. Therefore, it is be in urgent need of more accurate and less invasive methods to diagnose AR so that potent immunosuppressive can be applied in time. Although peripheral blood biomarkers are not as general as that in cardiopathy and tumors due to the complicated molecular mechanisms, it doesn’t mean that it makes no sense to perform peripheral blood biomarker to diagnose. Roedder et al. [21] used a kidney transplant cohort called the Assessment of Acute Rejection in Renal Transplantation (AART)study to verify a 17-gene signature in the peripheral blood that uncovered an area under curve (AUC) of 0.92 to predict AR. The above studies revealed the potential of peripheral blood biomarkers in diagnosis of AR. In our study, we implemented a synthetic investigation on the expression profiling of PBL from transplant patients with AR which included 6 kidney transplant patients undergoing AR and 9 patients with well-functioning transplant with no clinical evidence of rejection from the GEO database of GSE1563. By the way of defining the absolute value of the logarithm (base 2) fold change (log FC) greater than 2, We identified totally 347 DEGs (account for 2.75% of all genes) where 301 genes were upregulated and 46 were downregulated. In order to have a further detecting and analyzing these DEGs, we performed GO function, KEGG pathway to select 10 sensitive genes and top 18 hub genes were detected via PPI network and connectivity analysis of these DEGs. Additionally, we also applied GSEA to reveal the potential mechanisms of AR through these hub genes.
There were a great many factors related to transplant patients occurring AR, but the primary mechanism to contribute to the AR is still controversial, which is the reason that why it is difficult to diagnose and treat AR. According to our study, we used GO term enrichment and PPI analysis to uncover that the DEGs were closely interrelated with immune response and inflammatory process, especially downregulated DEGs. It is clear that the biological process associated with immunity plays an important role in the development of AR.
On the basis of pathophysiology, AR fall into two categories, which are antibody-mediated rejection (ABMR) and acute T-cell mediated rejection (TCMR) respectively [22]. On the one hand, Inflammation of glomeruli and peritubular capillary are always attributed to ABMR. Patients with ABMR usually verifies evidence of circulating donor-specific alloantibodies and immunological evidence of antibody-mediated injury [23]. It is reported that acute ABMR occur in about 7% of patients and it can be up to 50% in patients with human leukocyte antigen (HLA)-incompatible transplants [24–25]. It is much worse plenty of these patients may develop transplant glomerulopathy or chronic ABMR and have beyond a quadruple risk of graft loss compared to control groups [26]. On the other hand, TCMR is characterized by lymphocytic infiltration of the renal tubules, interstitium and arterial intima [27]. Zoghby et al. proved that TCMR is one of the most important and serious complications occurring in the renal transplant patients and it will cause graft loss and permanent impairment of graft function if not early diagnosis [28]. Therefore, we found that both ABMR and TCMR are equally essential for renal transplant patients which need to early detection and treatment.
In our GO analysis, it is indicated that DEGs, especially down-regulated DEGs, were mainly associated with immune response as well as neutrophil chemotaxis, innate immune response and inflammatory response. To our knowledge, ABMR is linked with antibodies against foreign donor antigens, especially HLA antigen which contributes to impairing the allograft by the way of activating the complement-dependent pathway and independent mechanisms recruiting NK cells, macrophages, polymorphonuclear cells and platelets to attack the allograft [29]. According to this mechanism, we uncovered that there were 3 genes (MAPK8, CCL5 and HMGB1) possibly related to the immune response occurring in ABMR. Haylett et al. discovered that MHC class II molecules can adjust mitogen-activated protein kinase 8 (MAPK8) pathway and the expression of c-Fos in B cells which can induce B cell to release the antibody [30]. In another study, MAPK8 was related to the release of BECN1 form its complex with BCL2 apoptosis regulator (BCL2), which in turn results in macrophages, an important class of antigen-presenting cells that activated adaptive immune responses autophagy [31]. Besides, it is reported that Fingolimod can increase the C-C motif chemokine ligand 5 (CCL5) in peripheral blood to inhibit the egress of lymphocyte involved in antigen presentation, which restrain B cells for humoral immunity [32]. As the same result, Sullivan et al. demonstrated endogenous CCL5 had a neutralization effect and inhibited B cell proliferation and IgM secretion when B cells stimulation [33]. As for high mobility group box 1 (HMGB1), through Sprague-Dawley rat hearts transplanted heterotopically into BALB/c mice, it is indicated HMGB1 regulated B cell activation and IFN-γ and IL-17A production which plays an important role in mediating acute xenograft rejection[34]. Thus, our results suggested the activation and proliferation of B cell and other antigen-presenting cells (APC) attach great importance to the development of ABMR via MAPK8, CCL5 and HMGB1 and further affect the severity of AR.
In TCMR, APC recognize the foreign donor antigens in the transplanted organ via various pathways to activate the recipient’s lymphocytes. As a result, T cells were activated and infiltrated which can damage the allograft. Thus, the most important part in TCMR is the activation of T cell. It is reported that some molecules can shared the important rejection mechanisms between B cells and NK cells in ABMR and effector T cells in TCMR [35]. Therefore. We predicted the mechanisms of T lymphocytes activity and recruitment may also be associated with those 3 hub genes (MAPK8, CCL5 and HMGB1) through GO and KEGG analysis. MAPK8 signaling has been implicated in multiple T cell functions. Similar to MAPK8 activation response in B cells, the MAPK8 is also synergistically activated by co-stimulation of the TCR with antibodies to its CD3 component and the CD28 auxiliary receptor [36]. Meanwhile, the activation of MAPK8 in T cells can inhibit the proliferation and infiltration due to neutralization. Additionally, the misbalanced level of chemokines like CCL5 is associated with the activation and function of cytotoxic T lymphocytes and may constrain CD8 + T cell out of lymphoid organs [32, 37]. Furthermore, it is reported that HMGB1 can recruits cells across endothelial barriers and induced the production of tumor-necrosis (TNF) and interferon, which is an nuclear weapon in the immune arsenal [38]. In short, from our perspective based on the GO and KEGG analyses, the regulation of immune-related genes involving MAPK8, CCL5 and HMGB1 can play an essential role in ABMR and TCMR which equally have an important effect in the occurrence and progression of AR. Thus, the detection of these target genes in peripheral blood may be a potential candidate for early diagnose of AR.
The PPI network could construct an obvious framework to have a better comprehension of the function of the proteome [39]. Through the enriched pathway of top 3 modules, we indicated that the interactions among the proteins in AR are specifically associated with inflammatory response, chemokine-mediated signaling pathway and neutrophil chemotaxis, which is as the same results as above. It emphasizes again that the interaction of immune-related genes can regulate the humoral immunity and cellular immunity associated with AR. Surprisingly, from enriched pathway of Modules 1, we found that some related proteins (NCBP2 and XPO1) in AR are also linked with RNA transport and mRNA surveillance pathway. It implies that RNA transport will be another mechanism of AR development. Coincidentally, we performed GSEA and discovered that non-coding RNA (ncRNA) export from nucleus is one of the key biological processes of AR. We applied DAVID to uncover the function of 18 hub genes and verified the same evidence that nuclear cap binding protein subunit 2 (NCBP2) and exportin 1 (XPO1) have a relationship with RNA transport. Therefore, we speculated that RNA transport can regulate some ncRNA transport which can be detected for diagnosis. Anglicheau et al. demonstrated that the levels of six miRNAs (miR-142–5p, miR-155, miR-223, miR-10b, miR-30a-3p, and let-7c) can become the biomarkers to diagnose AR, among which miR-142-5p, miR-155 and miR-233 can predicted AR with above 90% specificity and sensitivity[40]. Büssing et al. used C. elegans and Drosophila to prove that as a nuclear export receptor, XPO1 interacted with NCBP2 which is a subunit of the nuclear cap-binding complex to promote the pri-miRNA to pre-miRNA processing and induce nuclear export of miRNAs [41]. The above studies further consolidated our analysis, which revealed that NCBP2 and XPO1 were inextricably linked with the process of ncRNA, especially miRNA transport. Hence, monitoring the ncRNA transport genes and even specific miRNA of AR is of great importance for the diagnosis of AR.
Moreover, when we sifted the GSEA carefully, we uncovered that the development of AR may also be attributed to oxidoreductase activity acting on the aldehyde or oxo group of donors associated with amyloid beta precursor protein (APP). A part of oxidoreductase like Xanthine oxidase can increase the oxidative radical production, which may result in the dysregulation of redox homeostasis and excessive generation of reactive oxygen species (ROS), culminating in oxidative stress and the linked oxidative damage of cellular components [42]. Oxidative stress and ROS play an essential role in the pathogenesis of AR. Additionally, oxidative stress can activate autophagy, which can bring about the promotion of inflammatory response indirectly and enhance the progression of AR. APP is an oxidative stress responsive molecule. With APP overexpression, it will damage the structure of mitochondria and increase ROS production, which enhanced oxidative stress [43]. Besides, Sennvik et al. used western blotting to compare the cerebrospinal fluid from Alzheimer’s disease patients with healthy individual and revealed APP underwent posttranslational proteolytic processing by α-, β-, γ-secretases, which can produce soluble amyloid protein or APP components with amyloidogenic characters to induce oxidative stress [44]. It is consistent with our GO and KEGG analyses that protein stabilization is an important process of AR. Therefore, we hypothesized that APP is linked with oxidative stress resulting in protein hydrolysis and inflammatory response. Detecting this gene can early discover the subtle injury of AR in order to deal with it as early as possible.
Conclusively, applying a series of bioinformatics analysis, we provide a comprehensive and novel demonstration of gene expression profiles to recognize DEG expressing in peripheral blood, which may play critical roles in the occurrence and development in renal transplant patients with AR. Genes implicated with immune, inflammation, RNA transport as well as oxidative stress were apparently altered in AR patients, among which MAPK8, CCL5 and HMGB1 play core roles in the immune response of AR including ABMR and TCMR. Additionally, the interaction of NCBP2 and XPO1 will become the new mechanism to speculate the ncRNA transport of AR and may discover more novel miRNA of AR via the mechanism of these two genes. Furthermore, the graft dysfunction and fibrosis were associated with oxidative stress linked with APP in AR, which can be a promising strategy to diagnose and treat AR from another perspective. Taken together, we found that MAPK8, CCL5, HMGB1, NCBP2, XPO1 and APP may be regarded as potential serum biomarkers and targets of therapy. Certainly, we need further experiments and investigations to make sure the correlation of these dysregulated genes in order to allow these biomarkers to be applied more ordinarily in clinic. We expect this kind of analyzing method will provide more valuable and precise information for our future study on the molecular mechanisms of AR and offer more accurate evidences for detection of new diagnosis biomarkers and therapeutic strategies.