Useful Knowledge Of Tubulointerstitial Injury in Diabetic Kidney Disease--Part II
Mar 18, 2022
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Identification and verification of vascular cell adhesion protein 1 as an immune-related hub gene associated with the tubulointerstitial injury in diabetic kidney disease
Yan Jia, et al
ABSTRACT
Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD), but the pathogenesis is not completely understood. Tubulointerstitial injury plays a critical role in the development and progression of DKD (Diabetic kidney disease). The present study aimed to investigate the profile of tubulointerstitial immune cell infiltration and reveal the underlying mechanisms between tubular cell injury and interstitial inflammation in DKD (Diabetic kidney disease) using bioinformatics strategies. First, xCell analysis identified immune cells displaying significant changes in the DKD (Diabetic kidney disease) tubulointerstitium, including upregulated CD4+ T cells, Th2 cells, CD8+ T cells, M1 macrophages, activated dendritic cells (DCs), and conventional DCs, as well as downregulated Tregs. Second, pyroptosis was identified as the main form of cell death compared with other forms of programmed cell death. Vascular cell adhesion protein 1 (VCAM1) was identified as the top-ranked hub gene. The correlation analysis showed that VCAM1 was significantly positively correlated with pyroptosis and infiltrated immune cells in the tubulointerstitium. Upregulation of VCAM1 in the DKD tubulointerstitium was further verified in the European Renal cDNA Bank cohort and was observed to negatively correlate with the glomerular filtration rate (GFR). Our in vitro study validated increased VCAM1 expression in HK-2 cells under diabetic conditions, and pyroptosis inhibition by disulfiram decreased VCAM1 expression, inflammatory cytokine release, and fibrosis. In conclusion, our study identified upregulated VCAM1 expression in renal tubular cells, which might interact with infiltrated immune cells, thus promoting fibrosis. The FDA-approved drug disulfiram might improve fibrosis in DKD (Diabetic kidney disease) by targeting tubular pyroptosis and VCAM1 expression.
KEYWORDS: DKD; tubulointerstitium; immune cells; pyroptosis; VCAM1; disulfiram
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Discussion
Currently available clinical approaches to treating DKD (Diabetic kidney disease) remain limited and mainly involve strict control of hyperglycemia, proteinuria, and blockade of the renin-angiotensin system. Considering the high incidence of DKD-related ESRD, novel and satisfactory strategies are urgently needed. Accumulating evidence indicates a paramount role for immunity and inflammation in the pathogenesis of DKD (Diabetic kidney disease). To date, several studies have screened genes related to glomerular immune cell infiltration in individuals with DKD [20]. Relevant tubulointerstitial studies are rare. In the present study, we analyzed immune cell infiltration and relevant cellular processes in the tubulointerstitium using bioinformatics analysis. Pyroptosis was identified as the main form of cell death among other forms of programmed cell death using GSEA and GSVA. VCAM1 was identified as the top immune-related hub gene and was correlated with renal function in patients with DKD. An in vitro study validated increased VCAM1 expression in HK-2 cells cultured under diabetic conditions, and pyroptosis inhibition by disulfiram decreased VCAM1 expression, inflammatory cytokine release, and fibrosis. Our study identified a close relationship between renal proximal tubular pyroptosis, VCAM1 expression, and the interstitial immune response in patients with DKD (Diabetic kidney disease). This study provided new insights into the tubulointerstitial pathogenesis of DKD (Diabetic kidney disease) and helped identify new therapeutic targets.
xCell was used to conduct a comprehensive evaluation of immune cell infiltration in individuals with DKD (Diabetic kidney disease). The results revealed that both innate immunity and adaptive immunity were involved in tubulointerstitial injury in patients with DKD (Diabetic kidney disease). Macrophages and DCs constitute the mononuclear phagocyte (MNP) system [21], which is particularly complex in the kidney [22]. We found that macrophages, especially M1 macrophages, were significantly enriched in the tubulointerstitium. Interstitial macrophage M1 infiltration was reported to be correlated with interstitial fibrosis, tubular atrophy, renal function, and proteinuria in both animal models and human kidneys [23–25]. M1 macrophages produce cytokines and proteins (e.g., Nitric oxide, platelet-derived growth factor, IL-1, and TNF-α), causing damage to vascular endothelial cells and the proliferation of fibroblasts and mesangial cells, thus aggravating interstitial fibrosis [26]. DCs were reported to be involved in tubulointerstitial inflammation in various progressive kidney diseases, such as lupus nephritis [27,28] and crescentic glomerulonephritis [29,30]. Nevertheless, the role of DCs in many prevalent kidney diseases, such as DKD and hypertensive nephropathy, remains poorly understood. Only one animal study showed that CD11+ dendritic cells infiltrated the glomeruli in NOD mice and correlated with albuminuria [31], and no dendritic cell infiltration in the interstitium has been reported until now. The vast majority of kidney DCs are conventional DCs (cDCs) expressing CD11b and C-X3-C motif receptor 1 [32,33]. We observed increased infiltration of DCs, especially activated DCs and conventional DCs, indicating a role for these cells in the tubulointerstitial pathogenesis of DKD. Previous studies have reported that DCs were identified morphologically within the tubulointerstitium and might be involved in the pathogenesis of interstitial inflammation in drug-induced acute interstitial nephritis [34] and light chainassociated tubulointerstitial nephritis [35]. The specific mechanism requires additional experimental evidence. Helper (CD4+ ) T cells, cytotoxic (CD8+ ) cells, and B cells are important components of the adaptive immune system. In the present study, we observed a significant enrichment of CD4+ and CD8+ T cells in the interstitium, suggesting a role of adaptive immunity in DKD (Diabetic kidney disease). An animal study also detected CD4+ and CD8+ T cell infiltration in the kidney interstitium in streptozotocin-treated rats [36]. In people with diabetes, the number of CD4+ T cells and CD20 cells positively correlates with proteinuria [37]. However, not all CD4+ T cells promote DKD (Diabetic kidney disease). In our study, Tregs were downregulated in DKD and almost negatively correlated with pyroptosis (r = −0.4191, P = 0.0522). Tregs appear to play a protective role in DKD.
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Renal tubular epithelial cells are critical for both tubular and glomerular function, and injured renal tubular cells are an important source of proinflammatory cytokines and chemokines [38]. Multiple studies have shown that regulated cell death (RCD), such as pyroptosis, necroptosis, apoptosis, and ferroptosis, of tubular cells contributes to the pathophysiology of kidney diseases [39–42]. Various forms of cell death were evaluated using GSEA and GSVA in the dataset to explore the relationship between tubular injury and interstitial immune cell infiltration. The results revealed that pyroptosis was the main form of cell death compared with necroptosis, apoptosis, ferroptosis, and autophagy. The accumulation of reactive oxygen species (ROS) in individuals with DKD (Diabetic kidney disease) caused by hyperglycemia is an important activator of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome. Then, NLRP3 activates gasdermin D (GSDMD) and rapidly causes cell membrane rupture and the release of cell contents, leading to the inflammatory response [43]. Pyroptosis is involved in many chronic progressive diseases, such as Alzheimer’s disease [44], Parkinson’s disease [45], atherosclerosis [46], and rheumatoid arthritis [47]. The correlation analysis indicated that pyroptosis was positively correlated with increased interstitial infiltration of CD4+ T cells, CD4+ T memory cells, Th2 cells, CD8+ T cells, CD8+ Tcm cells, CD8+ Tem cells, Tγδ cells, NK cells, M1 macrophages, activated dendritic cells, conventional dendritic cells, and neutrophils. Thus, our study showed that tubular pyroptosis-related interstitial inflammation might be an important contributor to DKD (Diabetic kidney disease). This finding was confirmed in studies of patients with diabetes and db/db mice showing that tubular epithelial cell pyroptosis was accompanied by increases in the levels of the NLPR3 inflammasome, IL-1β, and TGF-β. Treatments targeting renal tubular epithelial cell pyroptosis may be critical to interfere with the progression of DKD (Diabetic kidney disease).
The highest MCC score for VCAM1 obtained from cytoHubba suggested that it is an important hub gene in DKD (Diabetic kidney disease) tubulointerstitial injury. VCAM1 has an important role in leukocyte adhesion to the endothelium. According to an animal study, MRL/lpl (a murine model of lupus nephritis) kidneys display increased VCAM1 expression in the endothelium, cortical tubules, and glomeruli [48]. Patients with type 2 diabetes present significantly elevated serum levels of VCAM1, and VCAM1 levels correlate well with the extent of albuminuria [49]. Previous studies on VCAM1 in DKD (Diabetic kidney disease) mainly focused on glomerular endothelial cells [50]. However, researchers have not completely elucidated whether renal tubular expression of VCAM1 is elevated in patients with DKD and whether it is involved in the interstitial pathogenesis of DKD. In the present study, the upregulated VCAM1 mRNA levels in the DKD (Diabetic kidney disease) tubulointerstitium in the public GEO dataset and markedly increased tubular VCAM1 expression in HK-2 cells confirmed the upregulation of tubular VCAM1 expression in DKD. As VCAM1 is an adhesion molecule for lymphocytes and monocytes, the positive correlation between VCAM1 expression and interstitial immune cell infiltration in our study indicated that elevated tubular VCAM1 expression might allow the protein to interact with mononuclear cells, thus promoting interstitial fibrosis. Consistent with our results, an increase in tubular VCAM1 expression in episodes of acute renal allograft rejection is associated with T cell and monocyte infiltration [50]. In addition, we found a positive correlation between VCAM1 expression and pyroptosis, indicating that pyroptotic tubular cells might release VCAM1. The in vitro study validated increased VCAM1 expression in HK-2 cells cultured under diabetic conditions, and pyroptosis inhibition by disulfiram decreased VCAM1 expression, inflammatory cytokine release, and fibrosis. Our study highlighted renal tubular VCAM1 as an attractive target for DKD (Diabetic kidney disease) therapy (Figure 10). However, the molecular mechanisms underlying the interaction between tubular VCAM1 and immune cell infiltration in the interstitium are essentially unknown, and further experimental studies are urgently needed [51].
Figure 10. A proposed model of VCAM1-expressing tubular epithelial cell promoting interstitial inflammation and fibrosis in DKD (Diabetic kidney disease), and their inhibition by disulfiram.
The current work investigated the profile of tubulointerstitial immune cell infiltration and identified pyroptosis as the main form of programmed cell death in patients with DKD (Diabetic kidney disease) using bioinformatics analysis. The analytical methods were reliable and novel. The correlation between of immune cells, pyroptosis, and VCAM1 provides new information for future research. Furthermore, our in vitro study suggested that the FDA-approved drug disulfiram might alleviate interstitial inflammation and fibrosis by inhibiting tubular pyroptosis and VCAM1 expression in individuals with DKD (Diabetic kidney disease). However, this study has several limitations. First, the sample size applied in the analysis was small.
Second, the results were not confirmed by conducting in-depth experiments. Thus, more combined samples will be needed, and the results will be verified by experiments in DKD (Diabetic kidney disease) animal models and DKD (Diabetic kidney disease) cohorts.
Conclusion
In conclusion, using bioinformatics analysis, we investigated the profile of tubulointerstitial immune cell infiltration and evaluated the role of pyroptosis in the tubulointerstitium of DKD (Diabetic kidney disease). VCAM1 was identified as the hub gene and was positively correlated with pyroptosis and infiltrated immune cells. In addition, VCAM1 expression was validated to be elevated in renal tubular cells. In vitro study revealed that the FDA-approved drug disulfiram inhibited renal tubular cell pyroptosis and decreased VCAM1 expression, inflammatory cytokine levels, and fibrosis. Further experimental analyses of pyroptosis and VCAM1 in the tubulointerstitium of patients with DKD (Diabetic kidney disease) might identify targets for immunotherapy.
Availability of Data and Materials
The datasets used during the study are available from the corresponding author upon reasonable request
Author contributions
Y.J. analyzed the data, interpreted the results, and drafted the article. Y.J. and Z.X. conceived, designed and organized the study. H.X. and L.T. analyzed the data. Y.J. and Q.Y. conducted the in vitro experiments and VCAM1 immunostaining. Z. X. interpreted the results and revised the manuscript. All the authors read and approved the final manuscript.
Funding
is research was supported by grants from the China Postdoctoral Science Foundation (No.2018M640808) and the ‘San-ming’ Project of Medicine in Shenzhen (No.SZSM201812097).
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