Abstract
Aim: Atherosclerotic cardiovascular disease is one of the major causes of mortality and morbidity in peritoneal dialysis (PD) patients. S100A12 is an endogenous receptor ligand of advanced glycation end-products. It was shown to contribute to the development of atherosclerosis in animal models. The aim of this study was to evaluate the relationship between S100A12 levels and carotid atherosclerosis in PD patients. Methods: A cross-sectional study was performed in 56 PD patients and 20 control subjects. Plasma S100A12 levels were measured from all participants beside routine laboratory evaluation. All subjects underwent high-resolution B-mode ultrasonography to determine carotid intima media thickness (CIMT). S100A12 levels were compared between patient and control groups. Correlation analyses of S100A12 with other laboratory values and CIMT were also performed. Results: Plasma S100A12 levels were higher in PD patients compared with control subjects (129.5 ± 167.2 ng/mL vs. 48.5 ± 30.3 ng/mL, respectively, p < 0.001). In the patient group, CIMT was found to be positively correlated with age (r = 0.354; p = 0.007), CRP level (r = 0.269; p = 0.045), and S100A12 (r = 0.293; p = 0.028) level while it was found to be negatively correlated with hemoglobin concentration (r = −0.264; p = 0.049). In the linear regression analysis, the model, including CRP, S100A12, age, and Hgb, was found to be significant (F: 4.177, p: 0.005). When the parameters are analyzed age and S100A12 were found to be independent determinants of CIMT (β = 0.308, p = 0.018 and β = 0.248, p = 0.049, respectively). Conclusions: This study suggests that an elevated plasma S100A12 level was closely associated with atherosclerosis. With aging elevated plasma S100A12 may show a powerful proatherogenic potential in patients undergoing PD.
Introduction
Atherosclerotic cardiovascular disease is one of the leading causes of mortality and morbidity in patients with end stage renal disease. Multiple traditional and non-traditional cardiovascular risk factors take part in the atherosclerotic process. The final common pathway for development of atherosclerosis (AS) is vascular inflammation causing endothelial dysfunction.Citation1
Advanced glycation end-products (AGEs) are involved in the non-traditional risk factors in peritoneal dialysis (PD) patients. They are the glycoxidation products of proteins and lipids and accumulate in patients with renal failure.Citation2,Citation3 Accelerated AGE formation is observed in PD patients. Heat sterilization produces glucose degradation products which can stimulate AGE formation. Therefore, over-expression of plasma receptors for AGEs (RAGE) is expected in PD patients.Citation4
Recently, biomarkers linked to the AGEs, receptor of AGE (RAGE) system, have attracted increasing attention.Citation5 S100A12, also known as EN-RAGE, is a circulating RAGE ligand. RAGE functions as a multiligand pattern recognition receptor. It mediates pro-inflammatory signals following binding to circulating AGEs, S100A12, and other circulating peptide and protein ligands.Citation6,Citation7 Up-regulation of RAGE is linked to inflammation, obesity, insulin resistance, diabetes, CKD, and CVD.Citation8,Citation9
S100A12 is overexpressed on the cell surface of macrophages, lymphocytes, and endothelium at sites of local inflammation and it acts as co-facilitator/initiator of the AGE-RAGE-mediated inflammatory response.Citation10 Circulating S100A12 correlates with inflammatory markers and may reflect disease activity.Citation11–13 In chronic pro-inflammatory conditions such as AS, plasma S100A12 is increased and it is found to be associated with increased risk of CVD, both in diabetic and in non-diabetic patients.Citation14 The role of S100A12 as biomarker in CKD is not completely understood. Higher S100A12 level has been reported to be associated with inflammation, CVD, and mortality in CKD patients but this is not a consistent finding.Citation10,Citation15–24
In prevalent hemodialysis (HD) patients, plasma concentrations of S100A12 were not only elevated compared with healthy individuals but also were associated with CVD-related mortality.Citation21 Moreover, in a study of Isoyama et al. 200 CKD patients are followed for median 23 months and increased concentration of S100A12 was found to be an independent predictor of mortality risk.Citation25
The aim of this study was to evaluate the association of plasma S100A12 level with carotid AS determined by measurement of carotid intima media thickness (CIMT) in PD patients.
Materials and methods
Study population
This cross-sectional study included 56 PD patients followed in Diskapi Yildirim Beyazit Training and Research Hospital PD unit and 20 healthy age- and gender-matched controls. This study was approved by the local ethics committee and was conducted in accordance with Declaration of Helsinki. All participants provided written informed consent to participate in the study.
All subjects were clinically stable at the time of evaluation. Exclusion criteria were the presence of infection, chronic inflammatory disease, CV disease history, and malignancy. Patients who had PD catheter insertion in last 30 days were also excluded. For all patients’ demographic parameters, co-morbidities, and routine laboratory values at the time of inclusion were recorded from patients’ medical files.
Measurement of S100A12
Blood samples from the subjects were collected in the PD unit from a peripheral vein after an overnight fasting and plasma were separated and kept frozen at −70 °C for a maximum of three months. Plasma concentrations of S100A12 (ng/mL) were measured via commercially available ELISA kit according to instructions of the manufacturer (CircuLex S100A12 ELISA kit; Cyclex Co., Ltd., Nagano, Japan).
Measurement of carotid intima media thickness
Ultrasonographical B-mode imaging of bilateral carotid arteries were performed with a high-resolution real-time ultrasonography with 12 MHz linear-assay transducer (Mindray DC7, China). Evaluations were performed by a single trained radiologist who was blinded to the clinical status and laboratory parameters of the patients. Carotid arteries, carotid bulb, and internal carotid arteries were examined by two different longitudinal projections. At each longitudinal projection CIMT was conducted from the site of the greater thickness. CIMT was defined as the distance between the leading edges of the lumen interface at the far wall in plaque-free arterial segments. The value was expressed as an average of the maximal CIMT.
Statistical analysis
Statistical analysis was performed by using statistical package SPSS version 19.0 (SPSS Inc., Chicago, IL). All variables were expressed as the mean ± SD unless otherwise indicated. The Kolmogorov–Simirnov test was used to analyze the normality of distribution. Pearson’s correlation analysis was used to evaluate the relation between CIMT, inflammatory parameters, and S100A12. Patients were grouped in two according to the median level of S100A12. Parameters shown to correlate with CIMT were analyzed by linear regression analysis. A level of p < 0.05 was accepted as significant.
Results
Baseline characteristics of the patient and healthy control groups are shown in . The two groups were accurately matched for age and gender. For PD patients mean age was 48.1 ± 12.9 years, mean duration of dialysis was 71.8 ± 39.4 months and 55.4% of the patients were female. The prevalence of diabetes and hypertension was 12.5% and 60.7%, respectively. Compared with controls, PD patients showed higher blood pressure, elevated serum urea nitrogen, creatinine, higher CRP, and had lower serum albumin and HDL-cholesterol level (p < 0.05). CIMT was also higher in the PD group and the difference was statistically significant (0.07 ± 0.02 cm vs. 0.06 ± 0.01 cm; p = 0.015). Plasma concentrations of S100A12 were significantly higher in PD patients compared with healthy controls (129.5 ± 167.2 ng/mL vs. 48.5 ± 30.3 ng/mL; p = 0.001).
Table 1. Demographic characteristics and laboratory values of study population.
Patients were grouped in two with regard to the median level of S100A12: group I: low and group II: high (). WBC count and CRP levels were statistically significantly higher in group II when compared with group I (8714.3 ± 2280.1 vs. 6867.9 ± 1458.6; p = 0.001 and 2.3 ± 2.8 vs. 0.9 ± 0.9; p = 0.020, respectively).
Table 2. Demographic and laboratory values of patients according to the S100A12 groups.
Patient group was analyzed in two groups according to the presence of residual renal function (RRF) (). There was no statistically significant difference in respect to S100A12 and CIMT between groups (129.38 ± 180.228 vs. 129.228 ± 140.672; p = 0.522 and 0.7 ± 0.2 vs. 0.7 ± 0.2; p = 0.781). The only statistically significant differences were observed in terms of creatinine level (10.05 ± 1.5 vs. 8.61 ± 2.3; p = 0.02), waist circumference (95.74 ± 6.4 vs. 102.43 ± 8.61; p = 0.017) and BMI (24.55 ± 3.3 vs. 27.33 ± 4.6; p = 0.03). Waist circumference and BMI were statistically significantly higher in the patients who had RRF while creatinine level was significantly higher in the patients who had no RRF.
Table 3. Patients according to the presence of RRF.
In the patient group, CIMT was found to be positively correlated with age (r = 0.354; p = 0.007), CRP level (r = 0.269; p = 0.045), and S100A12 (r = 0.293; p = 0.028) level while it was found to be negatively correlated with hemoglobin concentration (r = −0.264; p = 0.049) (). No significant relation determined between CIMT and albumin, total-cholesterol, LDL-cholesterol, and HDL-cholesterol.
Table 4. Carotid intima media thickness correlations.
The parameters found to be correlated with CIMT were examined by linear regression analysis. In a regression analysis, the model, including CRP, S100A12, age, and Hgb, was found to be statistically significant (F: 4.177, p: 0.005). When the parameters are analyzed age and S100A12 but not Hgb or CRP were found to be independent determinants of CIMT (β = 0.308, p = 0.018 and β = 0.248, p = 0.049, respectively). R2 value explaining the overall biological variability in CIMT explained by age and S100A12 was found to be 0.247 ().
Table 5. Regression analysis.
Discussion
The main finding of this study is that (1) mean plasma S100A12 level is higher in PD patients compared with control subjects, (2) plasma concentrations of S100A12 seem to reflect systemic inflammatory status (3) age and S100A12 seem to be the major determinant of CIMT. Therefore, we could suggest that elevated S100A12 level is associated with systemic AS in PD patients. This may be caused by induction of inflammation.
In non-CKD populations higher plasma S100A12 may show increased disease activity, and, in inflammatory conditions such as Kawasaki’s diseaseCitation26 and in Familial Mediterranean fever, inflammatory bowel disease and rheumatoid arthritis, S100A12 concentrations may be 5–10 times higher than in healthy subjects.Citation27–29 Also in a study of Isoyama et al. s100a12 was four times higher in level 5 CKD 5 patients than in controls and increased with increasing hsCRP.Citation25 Likewise, in the current study, S100A12 and CRP were higher in PD patients when compared with controls.
The effects of decreased renal function on the elevation of S100A12 level have been documented in previous studies with CKD or HD patients.Citation10,Citation14,Citation17,Citation21,Citation30–34 It remains a question if the elevation of S100A12 is caused by decreased renal clearance or a result of increased inflammation. Patients with and without RRF were found to have similar levels of S100A12 so the cause of elevation may be caused by inflammatory stimuli. Increased S100A12 level may be caused by a positive feedback or increased synthesis by activated leucocytes in uremia.Citation21,Citation35
Another finding is the positive correlation between S100A12, WBC, and CRP level. Considering the pro-inflammatory functions of S100A12, it is possible that neutrophil-derived S100A12 might be involved in an inflammatory process in CKD and PD patients. Indeed, S100A12 is secreted from activated neutrophils and exerts its pro-inflammatory effects on the endothelium and leukocytes by binding to RAGE expressed on these cells.Citation36,Citation37 Interaction of S100A12 with RAGE on the endothelium, mononuclear phagocytes and lymphocytes triggers cellular activation with generation of key pro-inflammatory mediators.Citation38 Also, S100A12 concentrations indicate neutrophil activation in various diseases such as IBD,Citation37 RA,Citation39 and MPO-ANCA-associated glomerulonephritisCitation40 and type 2 DM.Citation30
Another key finding of our study is the relation of AS determined by measurement of CIMT with CRP, age, hemoglobin concentration and S100A12 level. Linear regression analysis showed S100A12 and age to be the main determinant of CIMT.
AS has an inflammatory etiology, and elevated CRP in patients with AS not only serves a biomarker of cardiovascular disease risk but it also functions as an active mediator of AS by its direct proatherogenic effects on vasculature.Citation41,Citation42
A number of studies focused on the pivotal role of RAGE signaling in the pathogenesis of AS.Citation10,Citation16 Plasma S100A12 activates the RAGE pathway as a pro-inflammatory ligand. It activates key inflammatory signals such as NF-κβ and MAP-kinase stimulating cell adhesion molecules like VCAM-1 and ICAM-1. Stimulation of adhesion molecules promotes leucocyte recruitment and macrophage infiltration into vessel walls. Therefore, increased levels of S100A12 may be an indicator for accelerated AS caused by induction of inflammation.Citation21
Mori et al.Citation10 in their study of 72 HD patients and 42 controls measured S100A12 levels and CIMT. In this study, S100A12 level was approximately two fold higher in the patient group in accordance with our findings. In the regression analysis, the WBC was found to be the independent determinant of S100A12, which is in turn found to be the determinant of maximum CIMT.
In our study, the relation of CIMT with CRP and WBC did not persist when regression analysis made. The reason for this may be that we could not measure high sensitive CRP or another inflammatory marker. Also study population was small.
In an observational study of Nakashima et al. the relationship between mortality and S100A12 was examined in a group of 184 HD patients. They showed that the S100A12 level was an independent predictor of all cause and CVD-related mortality.Citation21
In a cross-sectional study of Isoyama et al. in 200 CKD stage 5 patients high plasma S100A12 was found to be an independent predictor of increased mortality risk.Citation25
In a study of Kim et al. S100A12 was found to be independent risk factor for development of AS.Citation23 These findings can suggest higher S100A12 levels to be associated with amplified inflammatory response and extensive vascular damage with severe AS.Citation10
Our study has some limitations. First due to cross-sectional design of the study a longitudinal causative relationship cannot be established between the plasma S100A12 levels and advanced AS. Furthermore, we could not analyze the association of S100A12 with long-term cardiovascular or all-cause mortality. Therefore, follow up data are needed to clarify the precise significance of this parameter. Second, the influence of confounding factors on AS and their interference by unrecognized factors cannot be ruled out.
In conclusion, our findings suggest that an elevated plasma S100A12 level was closely associated with systemic inflammation. With aging, elevated plasma S100A12 showed a powerful proatherogenic potential in patients undergoing PD.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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