The prevalence of end-stage kidney disease (ESKD) continues to increase globally owing to improved patient survival, higher prevalence of risk factors for ESKD, and increasing availability of kidney replacement therapy [1]. In patients undergoing hemodialysis (HD), vascular access is recognized as a ‘lifeline’ and provides adequate blood flow for HD treatment. According to the current guidelines, arteriovenous (AV) access, including AV fistulas (AVFs) and prosthetic AV grafts (AVGs), is preferred over central venous catheters in prevalent HD patients due to fewer vascular access-related events (e.g., infections, thrombotic and non-thrombotic complications) [2]. Thrombosis is a leading cause of AV access failure, potentially leading to missed dialysis sessions, hospitalizations, the need for endovascular or surgical thrombectomy, and all-cause mortality [3,4].

Several risk factors for vascular access thrombosis have been identified, including older age, female sex, diabetes, obesity, peripheral vascular disease, atherosclerosis, pre-dialysis hypotension, smoking, the use of an AVG, a distal fistula site, hypoalbuminemia, and elevated inflammatory biomarkers [5–8]. Recent studies have highlighted the association between vascular calcification and AV access patency [9,10]. Vascular calcification is highly prevalent in patients with ESKD and is a well-known risk factor for cardiovascular events and all-cause mortality in this population [11,12].

Osteoprotegerin (OPG), a member of the tumor necrosis factor (TNF) receptor superfamily, functions as a soluble decoy receptor for the receptor activator of nuclear factor kappa B ligand (RANKL) and regulates osteoclast activation in bone remodeling [13]. Increasing evidence suggests the involvement of the OPG/receptor activator of nuclear factor kappa B (RANK)/RANKL axis in coronary artery disease, atherosclerotic disorders, and vascular calcification [14–17]. Some studies have demonstrated elevated OPG concentrations in patients with calcified vessels, suggesting an interacting pathway that increases OPG expression during the development of vascular calcification [18,19]. Additionally, many studies have observed a potential link between the OPG/RANK/RANKL axis and unprovoked venous thromboembolic disease and ischemic stroke [20,21]. A previous experimental study also showed that OPG can regulate platelet adhesion to von Willebrand factor secreted from endothelial cells [22]. However, studies exploring the relationship between blood OPG, soluble RANKL, and AV access outcome are limited. Hence, in this study, we prospectively evaluated the association between AV access thrombosis and clinical factors including OPG and RANKL blood levels in a cohort of stable HD patients.

This study suggested that higher blood OPG is associated with an increased risk of AV access thrombosis. Additionally, our results revealed that different types of AV access correspond to distinct risk factors for developing thrombotic events. Specifically, in patients using an AVF, elevated blood levels of OPG and RANKL were strongly associated with AVF thrombosis, whereas increased levels of CRP were associated with thrombosis in patients using an AVG.

A key finding of the study is that the patients with higher levels of OPG and RANKL were at a higher risk of developing AVF thrombosis, while these associations were not observed in the patients using AVG. Furthermore, the highest log OPG tertile was linked to an increased risk of an AVF thrombotic event. Most previous studies highlight that blood levels of OPG are associated with arterial calcification, such as coronary arterial calcification, and mortality in HD patients [16,23,24]. Clinical studies link serum OPG levels with AV access dysfunction or thrombosis, but the results have been inconsistent. Kim et al. [25] reported that serum OPG levels were positively correlated with the degree of AVF stenosis using ultrasonography. On the other hand, Morena et al. [26] found no association between OPG and RANKL levels with AVF thrombosis in a study with 128 HD patients. In a prospective study of 727 patients with HD, Lyu et al. [9] investigated markers of vascular calcification and vascular access complications and suggested that OPG was not significantly associated with the risk of an AVF intervention. Instead, they found that other vascular calcification markers, including fetuin-A, osteopontin, and bone morphogenetic protein 7, were associated with an AVF intervention. There are various possible explanations for the link between OPG and AVF thrombosis. As a marker of vascular calcification, higher OPG levels are more likely to be associated with preexisting AV access calcification, which has been suggested to predict AVF failure in HD patients [10,27]. In addition, prior research has shown that OPG is expressed in normal vasculature and is more predominant in advanced atherosclerotic plaques [19], suggesting that circulating OPG may originate from these vascular plaques. The presence of atherosclerosis has been linked to an increased risk of AVF failure [7,8], establishing OPG as a potential biomarker associated with AVF thrombosis [15]. Despite the strong correlation between OPG/RANKL and vascular calcification, there is currently no literature focusing on the direct relationship between OPG and AVF or venous calcification, possibly because AVF calcification is difficult to detect, thus limiting research on this topic. Noteworthy, OPG has been associated with many risk factors that cause vascular calcification and AVF thrombosis, such as old age, diabetes mellitus, and chronic inflammation [5,8,28]. These risk factors may also contribute to eventual AVF thrombosis. Among these risk factors, inflammation, indicated by elevated inflammatory markers and cytokines such as CRP, interleukin-6, and TNF-α, is particularly relevant to AV access thrombosis [29,30]. OPG, belonging to the TNF receptor family, is stimulated by pro-inflammatory agents such as TNF in vascular smooth muscle and endothelial cells [31,32]. Furthermore, emerging evidence has revealed the complex interplay between inflammation and vascular thrombosis [33]. Therefore, OPG levels may link to AVF thrombotic events under inflammatory conditions. However, the association was not observed in patients using AVGs in the present study, which is consistent with Lyu et al. [9], who reported no significant correlation between calcification markers, including OPG, and AVG intervention. Generally, stenosis induced by neointimal hyperplasia at the venous anastomoses is the primary cause of AVG thrombosis. The histology of neointimal hyperplasia comprises myofibroblasts, extracellular matrices, pro-inflammatory cells, neovasculature, and various growth factors and cytokines [30]. Because of the strong correlation of neointimal hyperplasia-induced stenosis with AVG thrombosis, which is less related to vascular calcification, this may partly explain the insignificant association between OPG and AVG thrombosis. Moreover, the small sample size of AVG users in our study may have resulted in reduced statistical power. Further research is needed to investigate this association in AVG users.

In this study, a positive correlation was found between blood RANKL and thrombosis of the AVF. However, the tertiles of blood RANKL were not significantly associated with AVF survival in the Kaplan-Meier analysis. Prior research has suggested the pathogenic effects of the RANKL/OPG/RANK axis in atherosclerosis [15,34], and vascular calcification has been linked to RANKL activity [35]. Nevertheless, the use of serum RANKL as a biomarker for CVD remains controversial. Lieb et al. [36] observed no association between serum RANKL and coronary artery calcification, incident CVD, or mortality in the general population. Similarly, in another cohort study conducted by Spartalis et al. [16] involving 80 HD patients, RANKL levels did not correlate with the presence or progression of vascular calcification in the abdominal aorta and muscular arteries. However, the Bruneck Study, including 909 participants, found that baseline serum RANKL levels strongly predicted CVD [37]. Generally, OPG levels are significantly higher than RANKL levels in circulation, and previous research suggests that OPG is a more stable and reliable indicator of the RANKL/OPG/RANK axis than soluble RANKL [15].

Another important finding in this study is that the patients with elevated CRP levels had a higher risk of developing AV access thrombosis, and the risk was more pronounced among the patients who used an AVG. In general, patients using AV access experience localized and systemic inflammation due to factors such as oxidative stress, wall shear stress, endothelial dysfunction, needle cannulation, AV access surgery, graft material biocompatibility, and uremic status [30,38,39]. Several clinical studies have reported an association between inflammation in HD patients with AV access thrombosis. Chou et al. [29] found that higher serum CRP levels in chronic HD patients were linked to a higher risk of vascular access thrombosis. Notably, compared to AVF users, those with an AVG have been reported to be more likely to have low-grade inflammation at baseline [40], which may lead to an increased risk of AV access thrombosis. These findings are consistent with our study, as suggested by the association of elevated CRP levels with AVG thrombosis. Although the timing of blood sample collection in the present study may not have aligned with that of AV access thrombosis, potentially limiting its ability to reflect the inflammatory status at the time of the thrombosis, our findings still indicated that the HD patients with elevated baseline CRP levels, suggesting chronic inflammation, were at an increased risk of AV access thrombosis, and particularly those with AVG.

The present study has several limitations. First, the small sample size may have decreased the statistical power, particularly in stratified analyses of the patients using AVGs. Second, an observational study may not adequately adjust for all potential confounders. For example, we didn’t have detailed data on AV access vintage, which may have a potential link with AV access thrombosis. Third, the absence of serial measurements of serum OPG and RANKL meant that we could not analyze associations between their fluctuations and the development of AV access thrombosis. Fourth, while we observed an association between higher OPG levels and AVF thrombosis, it is still unclear whether increased OPG contributes directly to the development of a thrombosed AVF or is simply a compensatory response to atherosclerotic disease. In addition, we didn’t perform other imaging studies, such as sonography, to confirm the presence of AV access calcification, despite the significant correlation between OPG and calcification found in previous research. Further investigations are needed to understand the underlying mechanisms of this observation.

In conclusion, our results identified high blood OPG levels associated with AV access thrombosis, particularly in the AVF, suggesting that OPG may be a potential biomarker in clinical use. Further research may be required to investigate the underlying pathophysiological mechanisms for preventing AV access thrombosis.