Indoxyl sulfate – the uremic toxin linking hemostatic system disturbances with the prevalence of cardiovascular disease in patients with chronic kidney disease

Chronic kidney disease (CKD) is an established risk factor for the occurrence of hemostatic disorders and the increased prevalence of cardiovascular disease (CVD) [1, 2]. Traditional cardiovascular risk factors are insufficient to explain the high coincidence of CVD among CKD patients, suggesting the existence of “missing links” connecting cardiovascular system and kidney [3].

Clinical observations provide evidence of the coexistence of two opposite thrombotic and bleeding tendencies that are commonly observed in CKD patients. Disturbances in hemostasis during CKD are manifested in episodes of spontaneously induced prothrombotic states or severe bleeding symptoms and represent an important cause of the morbidity and mortality in patients with impaired renal function [1, 4]. The causes of hemostasis abnormalities in CKD are extremely complicated, depend on many factors and are still not fully understood despite many years of research.

Hypercoagulability is caused by abnormal activity of coagulation regulatory factors and the platelet hyper-reactivity, while excessive bleeding is the result of inadequate function of platelets, the coagulation cascade factors and/or intensified activity of the fibrinolysis [5]. The endothelial cells (ECs), cellular adhesion molecules (CAMs), the vessel wall and its extracellular matrix play some roles in the etiology of hemostatic disturbances [6]. Furthermore, patients with impaired renal function are at enhanced risk of inflammation, oxidative stress (SOX), fluid retention, further impairment of endothelial function, and anemia – conditions specific to renal disease [7, 8]. Above-mentioned components are firmly influenced by uremic toxins and metabolic compounds accumulated in CKD patients serum due to its inadequate renal clearance [9]. Altogether, it results in a mutual intensification of hemostatic disorders and progression of CKD.

Indoxyl sulfate (IS) is an aggressive uremic toxin that is markedly accumulated in the plasma of patients with CKD and its concentration in CKD patients can increase even 50-fold compared to healthy people [10]. IS is the end-stage product of dietary tryptophan metabolism and due to its high-affinity binding to albumins cannot be efficiently removed by hemodialysis. It exerts prooxidative and proinflammatory activity, triggers of the immune response, and stimulates the progression of CKD [11]. The compound exhibits its cellular toxicity in renal tubular cells, glomerular mesangial cells, and vascular smooth muscle cells (VSMCs) via numerously signaling pathways alterations [12]. Besides, IS inhibits ECs proliferation and viability, stimulates endothelium for secretion of chemokines and CAMs, and enhance expression of hemostasis-related molecules on their surface [13]. Recently, uremic toxins have been suggested as a potential “missing link” between CKD and the presence of CVD [3]. Many authors indicate a potential role of IS in the progression of vascular and hemostatic dysfunctions through the induction of SOX and progressive inflammatory process. According to Shivanna et al. [14] and Chitalia et al. [15] IS is a potential CKD-related prothrombotic uremic toxin that induces tissue factor (TF) in VSMCs and increases post-vascular interventional prothrombotic risk by TF-dependent manner. Recent study of Tang et al. indicated that IS exerts modulatory activities towards K⁺ channels leading to the development of arrhythmogenesis in CKD patients [16]. Previous studies conducted using in vitro methods and animal models provided presumptive evidence suggesting that IS may be considered as a molecule responsible for thrombotic events in CKD patients [14, 15]. Despite the existence of background information on the potential IS impact on hemostasis, currently, there are no studies discussing in a comprehensive manner this influence, especially during CKD, when the levels of IS are permanently increased. Taking this into consideration, the aim of our study was to investigate the potential associations between plasma IS levels and the parameters of: coagulation, fibrinolysis and endothelial function in CKD patients on conservative treatment. Because SOX, inflammation, and cellular immune activation are the recognized factors that may affect the hemostatic system in this population the markers of SOX and inflammatory state were also determined [17]. Moreover, we also wanted to determine, if IS-dependent hemostatic system disturbances were associated with the prevalence of CVD in this population.

The aim of our study was to examine the precise impact of IS on the hemostatic system in the aspect of the prevalence of cardiovascular incidents. Existing basis lead us to consideration that IS may be one of the “missing links” between CVD and CKD that are closely interrelated and reinforce each other [19‐23]. The results of our study demonstrated for the first time that: (1) the increased plasma IS concentrations were associated with disturbances of hemostatic system, increased oxidative stress and monocyte activation in patients with CKD; (2) IS levels, oxidative stress, and monocyte activation were independently associated with part of the evaluated parameters of hemostasis; (3) accumulation of IS in the plasma of CKD patients may participate in the risk of CVD prevalence through the mechanism associated with disturbances of hemostatic system.

Our results are in line with other observations proving that IS levels are strongly correlated with stages of CKD [23]. We also found a strong positive association of IS with Cu/Zn SOD – a recognized marker of oxidative stress in CKD population [24] as well as a positive relationship between IS and neopterin, which is synthesized by monocytes and macrophages in response to interferon-γ produced by activated T cells [25], what is more, we demonstrated the strong and independent effect of IS on these biomarkers, which indicated the direct role of IS in the generation of SOX and monocyte activation. Moreover, another uremic toxin – p-cresol but not p-cresol sulfate (PCS) stimulates monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC [26]. Previously, IS was shown to induce SOX in cell cultures [27]. Ito et al. [28] used a nephrectomized mouse model to demonstrate that oral administration of IS activates numbers of proinflammatory functions and reactive oxygen species (ROS) production in monocytes. However, the present study is the first research demonstrating the direct effect of IS on monocyte activation in CKD patients.

In accordance with our previous [29, 30] and other findings [31], we demonstrated the abnormalities of the hemostatic system in CKD. The maintenance of hemostasis, which is a complex mechanism, depends on many factors. Both components of the hemostatic system: coagulation and fibrinolysis, are affected by impaired endothelial function, inflammation, immune response, oxidative stress and effects of accumulated toxins [32]. In the current study, the activation of coagulation cascade was reflected by increased TF level, TF/TFPI ratio, and an increment of prothrombin fragments F1 + 2 – an indicator of in vivo thrombin generation [33]. Simultaneously, the activation of fibrinolysis was observed in these patients. Among the analyzed parameters of coagulation and fibrinolysis, IS was positively associated with TF and suPAR values. However, the results of the multivariate analysis revealed that TM was the only independent factor affecting increased TF levels in CKD patients. In contrast, suPAR concentrations were independently affected by different factors, precisely by uPA, TM, inflammatory status, monocyte activation and oxidative stress (Table 3, section B). Interestingly, TF, suPAR, and TM levels were associated with neopterin and oxidative status, and the strong relationships existed between these molecules, as shown in Table 4. Although ECs are regarded as the main source of circulating TF, suPAR and TM [34], the obtained results suggest that above-mentioned molecules may partially come from the activated monocytes [35, 36]. This hypothesis is supported by observation that plasma levels of vWF, which is a reliable marker of ECs activation, did not correlate with TF or TM concentrations (Table 4). Thus, the significant elevation of circulating TF and TM, combined with their strong relationship with the monocyte activation marker – neopterin, makes activated monocytes probable (apart from endothelium) source of these molecules in CKD patients. Because in the current study IS strongly and independently affects the markers of monocyte activation and oxidative stress, we hypothesized that this uremic toxin can be partially responsible for activation of ECs and monocytes, which in turn can lead to the increased release of TF and TM. This hypothesis is supported by previous observation of Gondouin et al. [37] showing that IS, through a ROS-mediated mechanism, induces TF expression in monocytes and ECs. In the case of suPAR, which strongly correlated with vWF, neopterin and the markers of oxidative stress and inflammation, it seems that the different types of activated cells release this molecule in CKD patients [38].

Endothelial dysfunction is frequently observed in uremic conditions [32]. The present study confirmed that the markers of endothelial dysfunction like vWF, TM, sICAM-1, and sVCAM-1 were markedly elevated in the plasma of CKD group compared to controls. The strong positive associations existed between IS, as well as Cu/Zn SOD and the majority of the endothelial markers, whereas between IS and vWF only tendency to positive correlation was observed. Interestingly, all endothelial markers were affected by inflammation (Table 5 - Appendix). These results indicate that inflammatory state may be mainly responsible for endothelial dysfunction in CKD patients, and this is in line with previous observations [27]. The adhesion of circulating monocytes to endothelium is mediated by cell adhesion molecules, such as ICAM-1 and VCAM-1, which are upregulated on the ECs surface. Furthermore, ROS serves as common intracellular messengers for redox-sensitive pathways, playing a role in the expansion of vascular disease [39]. ROS can induce endothelial injury through activation of transcription factor – NF-kB, a key redox-sensitive regulator of chemokines, cytokines, and CAMs [40]. Moreover, the mechanism linking ROS with vascular inflammation has already been documented [41]. In the condition of the present study, sVCAM-1 was independently affected by plasma IS, neopterin, and sICAM-1 levels. In addition, we noticed the strong relationship between this adhesion molecule and the markers of SOX and inflammation. Although plasma sICAM-1 was independently associated with sVCAM-1 and suPAR, its levels were also related to oxidative stress and inflammatory state. What is more, there was a positive relationship between the markers of oxidative stress and inflammation in our CKD patients. On the basis of above results, we proposed the hypothesis that IS could provoke ROS production and CAMs expression, leading to monocyte-endothelial cell interaction, the initiation of vascular inflammation and endothelial dysfunction. This hypothesis is supported by the study of Ito et al. [28], who demonstrated IS-dependent ROS production in a monocytic cell line, and enhanced adhesion of these cells to vascular endothelium in vitro [28]. They also showed that IS reduction, by the administration of IS absorbent – AST-120, significantly decreased ROS level in monocytes of nephrectomized mice. Besides, Stinghen et al. [42] demonstrated with in vitro and in vivo models that exposure of the endothelium to uremic plasma results in the increase of sVCAM-1 expression, which indicated a link between vascular activation, systemic inflammation, and uremic toxicity. In addition, Tumur et al. [43] showed that IS upregulated the expression of ICAM-1 by ROS-induced activation of NF- κB in vascular ECs, and through this mechanism may play an important role in the development of CVD.

IS is one of the most investigated uremic toxins on account of its negative impact on the cardiovascular system. Clinical studies demonstrated that serum IS level is a predictor of overall and cardiovascular mortality [22, 23, 44]. On the other hand, Lin et al. proved that elevated levels of PCS and IS are associated with increased mortality in patients with CKD, while PCS, but not IS, is associated with an increased risk of cardiovascular events [45]. Although we could not establish the direct correlation between plasma levels of IS and CVD prevalence, we found an association between CVD and some hemostatic factors, such as vWF, suPAR and sICAM-1. These factors were not only associated with CVD prevalence, but also the strong interrelationships existed between them (Table 4). This fact indicates that multiple dysfunctions of the vascular cells were present among patients with CKD [46]. vWF is released into circulation by activated ECs and mediates platelet adhesion to injured endothelium – the first step in thrombus formation [47]. It is established that vWF has independent prognostic value for all-cause mortality and CV events in peritoneal dialyzed and hemodialyzed patients [48]. In accordance with these data, our previous study [30] demonstrated that vWF was independently associated with an early indicator of systemic atherosclerosis – intima-media thickness in CKD patients. An elevated suPAR level is thought to reflect activation of the inflammatory and immune systems, and it predicts cancer, CVD, diabetes and mortality in the general population [49]. The impact of suPAR on the cardiovascular system in CKD is relatively unknown. Previously, we showed that uPA/suPAR system was associated with hyperfibrinolysis, oxidative status and CVD prevalence in pre-dialysis and hemodialysis CKD patients [29, 50]. The study of Meijers et al. [31] confirmed that the higher suPAR level was directly associated with both overall mortality and cardiovascular events in the uremic population. sICAM-1 and sVCAM-1 trigger leukocyte adhesion and migration into the subendothelial space, initiating the formation of atherosclerotic lesions [39]. Data of Stenvinkel et al. [51] suggest that sICAM-1 is an independent predictor of mortality in pre-dialysis patients, who are malnourished, inflamed, and have signs of CVD.

Although it is well established that both oxidative stress, as well as monocyte activation, were associated with CVD development in uremia [52], we showed for the first time in clinical conditions that IS can be a factor linking these abnormalities with the prevalence of CVD by a common mechanism associated with the disorders of hemostatic system.

This study is limited due to its cross-sectional design that makes us unable to establish precise mechanisms underlying observed associations in view of various factors related to processes discussed in this work and characteristic for CKD. Due to relatively small numbers of patients, our results require further prospective cohort studies. Furthermore, our study did not focus on the effect of the neutralization of IS toxicity mechanisms by counteragents.

In conclusion, this study demonstrated for the first time the involvement of IS in the disturbances of the hemostatic system by the mechanism associated with oxidative stress and monocytes activation, which can result in the development of CVD in CKD patients on conservative treatment. Previously, we observed the impact of kynurenines, the other components of tryptophan metabolism pathway, on disturbances of hemostasis and CVD prevalence in CKD patients [24, 29, 31]. In this context, the current study extends the knowledge concerning the impact of tryptophan metabolites on a risk of cardiovascular complications in uremia. The clinical relevance of this work may reside on the novel characterization of CKD patients’ populations at increased risk of cardiovascular events, which are dependent from hemostatic disorders.