The relationship of indoxyl sulfate and p-cresyl sulfate with target cardiovascular proteins in hemodialysis patients

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C–C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Proteomic profiling. Serum samples were assessed by the Proseek Multiplex 96 × 96 proximity extension assay using the CV II and III panel (Olink Bioscience, Uppsala, Sweden), as previously described 16 . Each panel simultaneously measures 92 proteins. The detailed methodology was described in Supplementary methods (see Supplementary Materials). During quality control, proteins with more than 15% of the values below the limit of detection (LOD) were removed. After excluding three proteins (natriuretic peptides B, melusin, and chitotriosidase-1), the final data included 181 proteins. The list of all proteins measured is in Supplementary Table 1.
Acute coronary syndrome event definition. Patients were followed up for 2 years or until acute coronary syndrome (ACS) event. The ACS included ST-segment elevation-ACS (STE-ACS) and non-STE-ACS. ACS was diagnosed when any of the following conditions apply: (1) ECG findings met the criteria for a definitive diagnosis during an event; (2) cardiac ischemic chest pain was present, and enzyme levels met the criteria for a definitive diagnosis; (3) cardiac ischemic chest pain was present, enzyme levels met the criteria for suspected myocardial infarction, and ECG findings met the criteria for suspected myocardial infarction; (4) ECG findings, which met the criteria for a definitive diagnosis of myocardial infarction, were newly obtained on current ECG Scientific Reports | (2021) 11:3786 | https://doi.org/10.1038/s41598-021-83383-x www.nature.com/scientificreports/ findings; and (5) elevated level of cardiac enzymes CK, CK-MB, and Troponin-I was measured during an event, and, of these enzymes, elevations greater than twofold the upper limit of normal were found in one or more.
Statistical analysis. Continuous variables of demographic data are demonstrated as mean ± standard deviation (SD), and nominal variables are shown as percentages. In the discovery phase, the two protein-bound uremic toxins (IS and PCS) and 181 proteins were evaluated their association using linear regression models adjusting for age and sex. Associations significant at a false discovery rate (FDR) < 5% were further investigated in the next step. We calculate FDR by Benjamini and Hochberg's method 17 .
In the second analysis phase, we investigated the independent associations using multivariable-adjusted models. We selected the variables based on a causal diagram using the DAGitty software, version 2.2 (www. dagit ty.net; Supplementary Fig. 1). The models included the covariates of age, sex, HD vintage, cause of ESRD, arteriovenous access type, diabetes mellitus, hypertension, dyslipidemia, antiplatelet/warfarin, antihypertensive drugs, diabetes treatment drugs, calcium, phosphate, high sensitivity C-reactive protein (hsCRP), and total Kt/V. Linearity associations between IS or PCS and CV proteins were further tested using restricted cubic splines with three knots. Also, we ranked the CV proteins related to IS or PCS by ascending p-value, with bootstrapped confidence intervals around the ranks. To determine the outcome association, Kaplan-Meier analysis was performed to calculate the cumulative incidence of ACS categorized according to the median value of IS, PCS, and protein biomarkers.
All statistical methods were performed using Stata (version 15, College Station, TX, USA). Results were presented as a beta coefficient (β) with a 95% confidence interval (CI). A two-tailed p < 0.05 was considered statistically significant in this second phase.

Results
Initially, 347 patients were enrolled. Subjects were excluded if the protein-bound uremic toxins measurements (n = 1) or the protein measurements (n = 13) were below the detection limit. Finally, the association between the two protein-bound uremic toxins (free forms of IS and PCS) and 181 proteins were analyzed in 333 patients (Fig. 1).
Demographic and clinical characteristics. The patients' characteristics were listed in Table 1. HD patients with CV comorbidities (coronary artery disease or cerebrovascular disease) were older, had a higher proportion of hypertension, diabetes mellitus, dyslipidemia, and more commonly used antiplatelets/warfarin, antihypertensive drugs, diabetes treatment drugs, and had higher hsCRP levels than patients without CV comorbidities.   Fig. 4). Regarding the associations with PCS, the top 3 hits were protein IL1RL2, CCL15, and complement component C1q ( Supplementary Fig. 5). All the top hits had narrow confidence intervals of the rank.
Cubic spline analysis demonstrated a linear association of IS with CCL15, complement component C1q receptor, perlecan, bleomycin hydrolase, CD166 antigen, SLAM family member 5, and FGF23 (Fig. 3), as well as a linear association of PCS with CCL15, complement component C1q receptor, and IL1RL2 (Fig. 4). assessed by the Kaplan-Meier method and log-rank test. The cumulative incidence of ACS was higher in the high IS group and PCS group than in the low IS group and low PCS group (Log-rank p = 0.016 in IS; Log-rank p = 0.015 in PCS) (Fig. 5). In contrast, there was no significant difference in selected CV protein biomarkers (CCL15, CD93, Perlecan, bleomycin hydrolase, CD166 antigen, CD84, and IL1RL2) between the two groups for the ACS outcomes ( Supplementary Fig. 6). A higher level of FGF23 was found potential increased risks of ACS than a lower level of FGF23 (Log-rank p = 0.05) ( Supplementary Fig. 6).  The possible relationship of protein-bound uremic toxins with inflammation. Uremic toxins could promote cytokine expression and release in a pre-clinical study 18 . In the present study, the free forms of IS and PCS were negatively associated with CCL15, and free form PCS was negatively associated with IL1RL2.    www.nature.com/scientificreports/ CCL15, also known as leukotactin-1, is proinflammatory 19 and involved in monocyte mediated atherosclerotic lesions [19][20][21] . Contrary to a previous report, this study found a negative association between the two protein-bound uremic toxins and CCL15 22 .
IL1RL2, also known as interleukin-36 (IL-36) receptor, is a member of the IL-1 receptor family. IL1RL2 can bind to IL-36, which is involved in several diseases, such as psoriasis 23 or acute kidney injury 24 . Interestingly, genetically manipulated animal models lacking IL1RL2 are protected against the IL-36 triggered an inflammatory response in psoriasis 25,26 and acute kidney injury models 24 . Thus, IL1RL2 could play a role in regulating inflammation. Hence, this negative association between PCS and IL1RL2 may highlight a possible pathway for the uremic toxins associated with CVD.
Protein-bound uremic toxins, endothelial function, and the complement system. In this study, IS was negatively associated with perlecan, which along with β2 microglobulin, are co-localized in HD induced β-amyloidosis 27 . Perlecan is a large multi-domain proteoglycan that binds to extracellular matrix components and cell-surface molecules to maintain the endothelial barrier function 28 . It can stimulate endothelial growth and re-generation, inhibit smooth muscle cell proliferation, and help maintain vascular homeostasis 28,29 . Since perlecan is crucial for vascular repair regulation, the higher IS level associated with low perlecan may be another factor contributing to endothelial dysfunction.
We also demonstrated a negative association between free IS and bleomycin hydrolase in HD patients. Bleomycin hydrolase is an aminohydrolase involved in homocysteine metabolism, and defects in homocysteine metabolism can lead to hyperhomocysteinemia 30 . Pathological manifestations of hyperhomocysteinemia are associated with an increased risk of CV events in HD patients 31 . Furthermore, studies of genetic and nutritional hyperhomocysteinemia in animal models provide additional support for a causal role of homocysteine in atherothrombosis 32 . However, the regulation of IS on bleomycin hydrolase remains unknown.
CD166, also known as activated leukocyte cell adhesion molecule, is expressed on activated monocytes and endothelial cells, involved in cell migration processes 33,34 . The localization of CD166, specifically at cell-cell junctions on endothelial cells, supports its role in transendothelial migration 35 . CD166 can modulate endothelial function and is involved in the response of endothelial cells to inflammation 36 . Therefore, the shedding of CD166 may be impaired in high free IS conditions.
A negative association between uremic toxins (IS and PCS) and complement component C1q receptor was found in the current study. Complement component C1q receptor, known as CD93, plays a role in cell adhesion and phagocytosis in monocytes and macrophages 37 . It is over-expressed during inflammation, and the soluble form (soluble CD93) is increased in various inflammatory conditions. Soluble CD93 is associated with premature myocardial infarction, coronary artery disease, and other inflammatory conditions [38][39][40] , with reduced levels of the soluble CD93 related to metabolic dysregulation 41 . Thus, the release of soluble CD93 has been implicated in response to stressors, such as inflammatory, immune, and angiogenic mediators. However, the mechanism underlying the association between uremic toxins and soluble complement component C1q receptor remains unclear. Our finding proposes a link between protein-bound uremic toxins and complement component C1q receptor related to CVD.
IS could also induce thrombosis and atherosclerosis by enhancing platelet hyperactivity, elevating the response to collagen and thrombin, increasing platelet-derived microparticles and aggregation of platelet-monocytes 42 . The present study showed a positive association between IS and SLAM family member 5 (also known as CD84), which is expressed on the surface of platelets, and may stabilize platelet aggregation and thrombi via homophilic interactions 43 . In a previous clinical study, SLAM family member 5 was highly expressed in inflamed endothelial tissue of the coronary arteries of patients with Kawasaki disease 44 and was also associated with early repeat coronary events in patients with acute coronary syndrome 45 . Thus, SLAM family member 5 could be involved with protein-bound uremic toxins related to platelet aggregation and thrombosis formation. www.nature.com/scientificreports/ Protein-bound uremic toxins, fibroblast growth factor 23/Klotho regulation, and cardiovascular damage. PCS and IS were positively associated with FGF23, but only the association with IS was significant ( Supplementary Fig. 3). Several studies have confirmed the positive association between elevated serum FGF23 levels and mortality or CV events in HD patients 46,47 . Interestingly, the protein-bound uremic toxins and FGF23 association can link to their interference in the Klotho system. Klotho deficiency has been suggested as an upstream step of FGF23 excess, and IS-induced left ventricular hypertrophy (LVH) in Klotho-deficient mice was more severe 48 . By blocking oxidative stress, p38, and extracellular signal-regulated protein kinase 1/2 signaling pathways, exogenous Klotho could overwhelm IS-induced LVH in CKD mice 48 . In vitro and in vivo experiments showed that IS and PCS decreased Klotho expression in renal tubules by epigenetic silencing of the Klotho gene caused by oxidative stress 49 . The decrease in expression of Klotho can lead to a further compensatory increase in levels of FGF-23 50 , causing Klotho-independent adverse effects on off-target organs, such as pathological cardiac remodeling or increased hepatic production of inflammatory cytokines 51 .
Potential biomarkers for ACS in HD patients. We determined an association between protein-bound uremic toxins (IS/PCS) and ACS risk, which was in line with previous studies 6,13,[52][53][54] . Besides, we found a trend association between FGF23 and ACS. FGF23 was a well-established CV biomarker in HD patients 46,47 . However, other selected CV proteins (e.g., CCL15, CD93, Perlecan, bleomycin hydrolase, CD166 antigen, CD84, and IL1RL2) correlated to protein-bound uremic toxins were not found as biomarkers for ACS. Limited sample sizes and event numbers should be acknowledged in our study. More studies should be investigated to confirm our findings.

Strengths and limitations.
The advantages of this study include the comprehensive participant information and use of modern technology that allowed the rapid high-throughput, highly sensitive and specific analyses of limited plasma samples. The study limitations were that the cross-sectional study design precludes any causal inference, potentially introducing prevalence-incidence bias. Secondly, the selection of biomarkers used in this study was pragmatically restricted to CV-specific proteins, and it is possible that other proteins may be involved in the toxicity of IS or PCS. Further in vitro work is necessary to validate the translation of the mechanistic pathways suggested. Thirdly, only HD patients were recruited, so the findings may not be generalizable to other CKD or peritoneal dialysis populations. Fourthly, food intake was not evaluated to assess the intake of tryptophan, tyrosine, and fibers, which are the main sources of IS and PCS. Lastly, although PEA is a promising technique, improvements are warranted to assure clinically valid and reproducible measurements. Studying numerous proteins and protein-bound uremic toxins association may provide new insights on modulators of pathological CVD risk factors in HD patients, but the research remains exploratory. Thus, the proposed mechanisms through which biomarkers may be pathophysiologically related to IS and PCS are hypothesis-generating.
In conclusion, protein-bound uremic toxins (IS and PCS) were important factors for CVD in HD patients. These uremic toxins may contribute to inflammation, endothelial dysfunction, complement system, and FGF23 pathway, thereby providing insight into the mechanisms that lead to higher CVD mortality and death in HD patients. Strategies to reduce the accumulation of uremic toxins could reduce these CVD risk factors, thereby preventing CVD in HD patients.