MCP-1 and soluble TWEAK levels are independently associated with coronary artery disease severity in patients with chronic kidney disease

Abstract

Purpose: Patients diagnosed with chronic kidney disease (CKD) have a greater rate of cardiovascular mortality when compared with the general population. The soluble form of TNF-like weak inducer of apoptosis (TWEAK) and monocyte chemoattractan protein 1 (MCP-1) play important roles in cellular proliferation, migration and apoptosis. The current study aimed to analyze whether soluble TWEAK (sTWEAK) and MCP-1 levels are associated with the severity of coronary arterial disease (CAD) in CKD patients. Methods: Ninety-seven patients diagnosed with CKD stages 2–3 according to their estimated glomerular filtration rate and the presence of kidney injury were included in the study. Plasma sTWEAK and MCP-1 concentrations were determined using commercially available ELISA kits. Coronary angiographies were performed through femoral artery access using the Judkins technique. Results: Correlation analysis of sTWEAK and Gensini scores showed significant association (p < 0.01, r² = 0.287). Also significant correlation has been found in MCP-1 levels and Gensini scores (p < 0.01, r² = 0.414). When patients were divided into two groups with a limit of 17 according to their Gensini score, sTWEAK levels indicated a statistically significant difference (p < 0.01). Conclusions: Our findings support a relationship between sTWEAK and MCP-1 levels and CAD in CKD stages 2–3 patients.

• Chronic kidney disease
• coronary arterial disease
• Gensini score
• MCP-1
• TWEAK

Introduction

Chronic kidney disease (CKD) patients have higher mortality rates when compared with the general population based on cardiovascular diseases (CVD).1 Progression in CKD increases the risk of CVD. Many patients die before initiation of renal replacement therapy.2 Alterations in heart and vascular structures seem to be the main factors involved.3 Various pathways have been hypothesized to play a role in the pathological process.

TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) is a type II trans-membrane glycoprotein of the TNF super-family that is found in plasma as a soluble form (sTWEAK) with a molecular mass of 18 kDa.4 The binding of TWEAK to its receptor, Fn14, mediates multiple biologic effects, such as cellular growth, proliferation and migration, osteoclastogenesis, angiogenesis and apoptosis.5 It has been reported that sTWEAK plasma levels in non-diabetic, pre-dialysis CKD patients decrease with the progression of CKD, which is associated with the aggravation of endothelial function.6 It is not known whether sTWEAK levels are associated with the severity of coronary arterial disease (CAD).

The monocyte chemoattractant protein-1 (MCP-1/CCL2) is a member of the C-C chemokine family.7 MCP-1 is a proinflammatory cytokine that has been found to be associated with insulin resistance, metabolic syndrome and lipid abnormalities.8

The aim of this study was to clarify any relationship between sTWEAK and circulating MCP-1 levels with the CAD severity in patients diagnosed with CKD stages 2–3 who underwent coronary arterial imaging using Gensini scoring.

Materials and methods

Patients

Patients who were indicated to receive coronary arterial imaging due to stable or unstable angina pectoris at the Department of Cardiology of Dr. Siyami Ersek Thoracic and Cardiovascular Surgery Center, Istanbul, and who had CKD were included in the study. Ethical approval has been granted by Local Ethics Committee. The study included 97 patients diagnosed with CKD stages 2–3 according to their estimated glomerular filtration rate (eGFR) and the presence of kidney injury as defined by National Kidney Foundation Kidney Disease Outcomes Quality Initiative Guidelines.6 CKD etiologies of the patients are listed in Table 1. Mean age of the study population was 65.53 ± 8.6 years.

Table 1. Demographic and clinical characteristics of the entire study population.

eGFR mL/dk/1.73 m^2	60–89	30–59	p-Value
Age (years)	64.82(41–78)	68.38 (49–82)	0.08
Sex (M/F)	44/7	37/2	0.65
Body mass index (kg/m^2)	29.35	26.47	0.54
History of CVD	3	4	0.66
CKD etiology
Diabetes mellitus	19	20	0.72
Hypertension	23	8	0.021
Glomerulonephritis	6	9	0.15
Unknown	3	2	0.53
Currently smoking	16	4	0.017

Seven percent of the patients had a previous history of CAD. Forty-eight patients were treated with angiotensin converting enzyme inhibitor or angiotensinogen receptor blocker, eight patients were treated with calcium channel antagonists, four patients were treated with beta blocker agents, two patients were treated with alpha-blockers and six patients were treated with loop diuretics. Forty-four patients were receiving anti-diabetic treatment (23 patients were treated with oral anti-diabetics and 21 patients were treated with insulin).

Stratification of patients was performed using eGFR levels as determined by Kidney Disease Outcomes Quality Initiative Guidelines, according to the simplified version of the Modification of Diet in Renal Disease formula.9

Plasma sTWEAK and MCP-1 measurements

Plasma sTWEAK concentrations were determined using commercially available ELISA kits (Bender MedSystems, Vienna, Austria). The minimum detectable level of sTWEAK was set at 10 pg/mL. Serum MCP-1 levels were measured with the enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, Minneapolis, MN).

Coronary arterial imaging

Coronary angiographies were performed through femoral artery access using the Judkins technique. Angiograms were visually examined by two interventional cardiologists who were blind to both the study plan and each other. Each coronary angiographic index was established based on lesion location and percentage of stenosis among all coronary lesions. CAD was defined as >50% stenosis in the luminal diameter in at least one major epicardial coronary artery.10 The Gensini scoring system was used to describe the severity of the CAD, as previously described.11 Scoring chart is shown in Figure 1.

Figure 1. Gensini scoring system.

Statistical analyses

Statistical analyses were performed using SPSS 11.0 statistical software (SPSS, Chicago, IL). Abnormally distributed variables are expressed as median (range), and normally distributed variables are expressed as mean ± SD. Intra-group comparisons were assessed for nominal variables using the t-test and the Kruskal–Wallis test (ANOVA). Spearman’s rank correlation was used to determine correlations between variables. A p-value of <0.05 was considered statistically significant.

Results

The demographic and clinical characteristics of the study population are shown in Table 1. There were no statistically significant differences found between the different CKD stages with regard to age, gender, body mass index or etiology of CKD. There were more patients in the CKD stage 2 group than CKD stage 3 group who were diagnosed with hypertensive nephropathy and were current smokers; a statistically significant observation (p < 0.05).

Biochemical evaluation and clinical results of the study groups and the entire study population are shown in Table 2. Hemoglobin levels were significantly higher in the CKD stage 2 group compared with the CKD stage 3 group. The mean Gensini score was calculated as 46.14 ± 35.4 from the entire study population. In CKD stages 2 and 3, Gensini scores were calculated as 41.94 ± 36.8 and 53.75 ± 32.4, respectively. The difference between the groups was statistically significant (p = 0.047).

Table 2. Biochemical evaluation of the study population according to CKD stages.

	All patients	CKD stage 2	CKD stage 3	p-Value
eGFR (mL/min)	64.18 ± 16.5	71.9 ± 8.86	50.11 ± 7	<0.01
SBP (mmHg)	133.76 ± 5.1	134.21 ± 5.13	133.84 ± 4.79	0.857
DBP	84.79 ± 6.41	86.07 ± 6.58	83.2 ± 5.9	0.31
Albumin (g/dL)	4.12 ± 0.42	3.9 ± 0.72	4.2 ± 0.54	0.43
Total cholesterol (mg/dL)	188.38 ± 48.88	193.68 ± 50.26	178.05 ± 45.56	0.111
Triglyceride (mg/dL)	157.8 ± 78.14	171.29 ± 90.05	135.32 ± 55.71	0.29
Low denstiy lipoprotein (mg/dL)	116.29 ± 43.54	118.41 ± 45.01	113.23 ± 41.81	0.597
High density lipoprotein (mg/dL)	40.03 ± 11.27	41.64 ± 12.4	37.6 ± 8.8	0.112
Calcium (mg/dL)	8.52 ± 0.63	9.09 ± 0.43	8.73 ± 0.32	0.09
Phosphate (mg/dL)	5.35 ± 1.82	4.64 ± 0.83	4.84 ± 0.69	0.102
Hemoglobin (g/dL)	14.09 ± 1.75	14.55 ± 1.73	13.6 ± 1.7	<0.01
iPTH (pg/mL)	132 ± 76	72 ± 28	162 ± 53	0.086
CRP (mg/L)	6.25 ± 11.8	3.8 ± 4.5	7.99 ± 11.38	0.062
MCP-1 (pg/mL)	67.41 ± 28.11	62.56 ± 31.17	72.9 ± 22.3	0.107
sTWEAK (pg/mL)	439.29 ± 223.18	453.72 ± 240.19	435.71 ± 215.39	0.542
Gensini score	46.14 ± 35.4	41.94 ± 36.8	53.75 ± 32.4	0.047

Notes: eGFR: estimated glomerular filtration rate, SBP: systolic blood pressure, DBP: diastolic blood pressure, iPTH: intact parathyroid hormone, CRP: c-reactive protein, sTWEAK: soluble TNF-like weak inducer of apoptosis, MCP-1: monocyte chemoattractant protein-1.

In the study population, 23 individuals were active smokers and 27 were receiving statin treatment. When these patients were analyzed, neither smoking nor statin treatment were found to have any impact on the severity of CAD.

In patients stages 2 and 3 CKD, soluble TWEAK measurements were 453.72 ± 240.19 and 435.71 ± 215.39 pg/mL, respectively. Circulating MCP-1 levels were measured as 62.56 ± 31.17 and 72.9 ± 22.3 There were no statistically significant differences between groups.

Correlation analysis of sTWEAK and Gensini scores showed significant associations (p < 0.01, r² = 0.287) (Figure 2). Correlation analysis of MCP-1 and Gensini scores showed significant importance (p < 0.01, r² = 0.171) (Figure 3). Patients were divided into two groups with the cut-off limit set at 46.14 according to their Gensini score mean. Multivariate analysis was performed. eGFR, sTWEAK and MCP-1 levels indicated an independently statistical significance (p < 0.01). Data are shown in Table 3.

Figure 2. Correlation analysis of sTWEAK and Gensini scores.

Figure 3. Correlation analysis of MCP-1 and Gensini scores.

Table 3. Demographic and laboratory values of the low- and high-Gensini groups.

	Gensini group
	Group 1	Group 2	p-Value
Age (years)	65.02 ± 9.42	66.06 ± 7.91	0.557
BMI (kg/m^2)	28.72 ± 3.26	28.21 ± 2.60	0.398
Systolic BP	133.27 ± 5.82	134.27 ± 4.25	0.335
Diastolic BP	85.61 ± 6.01	83.96 ± 6.76	0.206
eGFR	67.97 ± 17.28	60.33 ± 14.87	0.022
Hemoglobin	13.92 ± 1.86	14.28 ± 1.62	0.303
HsCRP	7.26 ± 12.95	5.22 ± 7.49	0.345
Total cholesterol	195.52 ± 42.88	181.09 ± 47.27	0.119
Triglyceride	162.97 ± 75.52	152.55 ± 70.01	0.483
HDL cholesterol	40.84 ± 9.89	39.21 ± 11.10	0.449
LDL cholesterol	121.99 ± 40.25	110.47 ± 39.82	0.16
VLDL cholesterol	32.62 ± 15.06	30.57 ± 14.03	0.49
sTWEAK	322.44 ± 139.30	558.59 ± 230.55	<0.01
MCP-1	55.80 ± 27.56	79.21 ± 23.59	<0.01

Notes: Values are shown as mean ± standard deviation.

BMI: body mass index, BP: Blood pressure, eGFR: estimated glomerular filtration rate, HDL: high density lipoprotein, LDL: low density lipoprotein, VLDL: very low density lipoprotein, sTWEAK: soluble TWEAK, MCP-1: monocyte chemoattractant protein-1.

Discussion

CVD are still the leading cause of mortality in patients diagnosed with CKD. Healthy population interventions or imaging studies using radio-contrast agents show a greater risk in CKD patients with regard to CKD progression.12 The best approach to prevent contrast-induced nephropathy still seems to be the avoidance of unnecessary imaging or using lesser amounts of contrast agents.

Our results also suggest that the severity of CAD is associated with eGFR. In a recently reported study, higher serum cystatin levels were found to be associated with increased severity of CAD;13 our results align with these findings. Also our results support that sTWEAK and MCP-1 levels are associated with the severity of CAD in CKD patients.

sTWEAK was first identified as a soluble protein that is released in low concentrations from injured vessels rather than from healthy vessels.14 Decline in eGFR has been reported to be associated with gradual reductions in sTWEAK plasma levels previously, as we also observed.6 A progressive reduction in sTWEAK levels has been observed in association with the decrease in eGFR. sTWEAK is also reported to be low in hemodialysis patients.15 In such patients, it was reported that increased sTWEAK was associated with a higher probability of death.13 Our results indicated that CAD severity is associated with higher levels of sTWEAK. It is not clear whether sTWEAK levels may be used as a predictive biomarker.

In an experimental model, it has been shown that the pathological effects of TWEAK are mediated by binding sTWEAK with its receptor, Fn14.16–18 In normal physiology, Fn14 is not expressed in healthy human aorta, but its expression increases in pro-inflammatory conditions and also allows CD163 to sequester and degrade sTWEAK by acting as a scavenger receptor.18–21

In addition to conventional risk factors, our results support that the possibility of sTWEAK being a non-traditional risk for CVD. In animal models, it has been reported that atorvastatin prevents the pro-atherogenic effects induced by TWEAK, our results did not find any significant association between MCP-1 and TWEAK levels and statin or angiotensin converting enzyme inhibitor treatments.

It has been recently reported that in type I diabetes mellitus patients who are at greater risk for CVD, sCD163 (soluble sCD163) and sTWEAK levels increase.21 However, conflicting data in the literature still exist. In one study, it was reported that sTWEAK levels are negatively correlated with IL-6 levels in patients with ischemic heart disease who had been diagnosed with CKD.22

It is probable that sTWEAK and sCD163 levels and CVD are associated in patients who have pro-inflammatory diseases such as diabetes mellitus or CKD.

Monocytes, representatives of the innate immune system, play a pivotal role in the initiation, propagation and progression of atherosclerosis from a stable to an unstable state. Therapies, such as acetyl salicylic acid and the statins, are not approved medicines currently for management of stable or complicated atherosclerosis has inflammation as a primary target.23

MCP-1 has been known as one of the key chemokines that regulate migration and infiltration of monocytes/macrophages from the blood stream across the vascular endothelium for routine immunological surveillance of tissues, as well as in response to inflammation.24 It has been found that MCP-1 is produced by a variety of cells including endothelial, fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, monocytic and microglial cells after induction by oxidative stress, cytokines or growth factors.25

MCP-1 acts through CCR2 receptor. In a study performed on CCR2 lack out mice showed regression in the atherosclerosis.26

Polymorphism in the CCL2 promoter has been found to be associated increased risk of an individual suffering from coronary artery disease.27

Our data show that CAD severity is associated with the proinflammatory markers such as STWEAK and MCP-1. Our findings may help to explain the high rate for cardiovascular mortality rates in CKD patients.

Prospective studies with greater sample sizes may clarify this association.

Declaration of interest

The authors have no conflicts of interest to declare.