Impact of Indoxyl Sulfate, a Uremic Toxin, on Non-Culprit Coronary Plaque Composition Assessed on Integrated Backscatter Intravascular Ultrasound

Abstract

Background: Uremic toxin has emerged as an important determinant of cardiovascular risk. The aim of this study was to examine the relationship between serum uremic toxin and coronary plaque composition on integrated backscatter intravascular ultrasound (IB-IVUS).

Methods and Results: IB-IVUS was performed in 47 patients with planned treatment for angina pectoris. Non-culprit intermediate plaque analyzed in this study had to be >5 mm apart from the intervention site. 3-D IB-IVUS analysis was performed to determine percent lipid volume (LV) and fibrous volume (FV). We also measured serum uremic toxins (indoxyl sulfate [IS], asymmetric dimethylarginine [ADMA], and p-cresol [PC]). Glomerular filtration rate correlated with IS (r=–0.329, P=0.04), but did not correlate with ADMA or PC. Percent LV correlated with IS (r=0.365, P=0.02), but did not correlate with ADMA or PC. Percent FV also correlated with IS (r=–0.356, P=0.03), but did not correlate with ADMA or PC. On multivariate regression, only IS was associated with percent LV (r=0.359, P=0.04) and percent FV (r=–0.305, P=0.04) independently of potentially confounding coronary risk factors.

Conclusions: Among the uremic toxins, serum IS might be a novel useful biomarker to detect and monitor lipid-rich coronary plaque on IB imaging. (Circ J 2015; 79: 1773–1779)

Uremic toxin has emerged as an important determinant of cardiovascular risk. Population-based cohort studies have identified chronic kidney disease (CKD) as an important risk factor for cardiovascular disease (CVD).^1,2 In CKD, the rates of CVD increase due to cardiovascular abnormality caused by progression of CKD stage. It is clear that cardio-renal interaction plays an important pathophysiologic role in the development of CVD, but the mechanism of this interaction is not well understood. Recent research has identified indoxyl sulfate (IS), one of the uremic toxins, as a risk factor; IS causes inflammation, as well as oxidative stress on human umbilical cord vein endothelial cells (HUVEC), thereby resulting in endothelial dysfunction (ED).³ Moreover, a correlation between serum IS concentration and arteriosclerosis has been reported.⁴

Editorial p 1691

The aim of this study was to examine the relationship between serum uremic toxins and coronary plaque composition assessed on integrated backscatter intravascular ultrasound (IB-IVUS) during percutaneous coronary intervention (PCI).

Methods

Subjects

We included patients, who had been treated with statin for at least 6 months, with planned treatment for angina pectoris due to coronary stenosis on previous coronary angiography. All patients underwent PCI, and also had IB-IVUS performed in the vessel undergoing the PCI immediately afterwards. Exclusion criteria were acute myocardial infarction (AMI), history of PCI or coronary artery bypass surgery, and dialysis therapy.

Written informed consent was obtained from each patient, and the study protocol of the current report was approved by the Institutional Review Board of Tokushima University Hospital.

IVUS Acquisition

For IVUS, Atlantis (40 MHz; Boston Scientific/Cardiovascular System, San Jose, CA, USA) was used. Data were collected at 0.5 mm/s auto pullback and analyzed using an IVUS console (Galaxy; Boston Scientific/Cardiovascular System). For the prevention of coronary spasm, i.c. optimal dose of isosorbide dinitrate was administered before measurement. A target intermediate stenosis (25%<percent diameter stenosis<50% on quantitative coronary angiography) was selected and plaques analyzed in this study had to be >5 mm from the intervention site. We excluded patients with inadequate IVUS imaging. Target plaques with wide acoustic shadowing (>90°) or with side branch were also excluded. A total of 10 IB-IVUS images (5 mm in length) were captured at an interval of 0.5 mm using a motorized pull-back system in each plaque immediately after PCI (Figures 1A,B).

Figure 1.

Integrated backscatter intravascular ultrasound measurement. (A) Analytical region proximal to the stent placed in the left anterior descending coronary artery (LAD). (B) Analysis was performed at approximately 0.5 mm/s. Target plaque had to be >5 mm from the intervention site. LCx, left circumflex coronary artery; LMT, left main trunk. (C) Color-coded maps of coronary arterial plaque constructed on IB-IVUS in a representative case. Percent volume of the lipid pool component was 43% and that of the fibrous component was 55%.

Conventional IVUS and IB-IVUS Parameters

Each conventional IVUS and IB-IVUS parameter was measured. Cross-sectional lumen area, cross-sectional vessel area within the external elastic membrane, and plaque area (external elastic membrane area minus lumen area) were determined using software provided with the IVUS system. Plaque volume was calculated using integration.

IB values were defined for each histological category by comparing histologic images reported in the previous study.⁵ Plaque properties were diagnosed into 4 types by combining spectral parameters of posterior scattering signals of IVUS: lipid pool, fibrosis, dense fibrosis, or calcification (Figure 1C). The percent area of each component was automatically measured in each plaque. The volume (mm³) of each component in 5 mm was calculated using integration as follows [(area of slice1+area of slice2+ ….. +area of slice10)×0.5] and the percent volume (%) of each component in 5 mm was calculated as follows [(area of slice1+area of slice2+ ….. +area of slice10)×0.5×100/total plaque volume].

Blood Sampling and Uremic Toxin Measurement

Samples for measurement of serum uremic toxins (IS, asymmetric dimethylarginine [ADMA], and p-cresol [PC]) were taken from peripheral vein before heparin or any contrast agents were administered and before any interventional procedures were initiated. Blood samples were obtained by centrifugation at 3,000 rpm for 10 min at 4℃. They were stored at –80℃ until required for analysis. Serum ADMA was measured using an enzyme-linked immunosorbent assay kit (ADMA Xpress ELISA kit; Immundiagnostik AG, Bensheim, Germany). The intra- and inter-assay coefficients of variation (CV) for the measurement of ADMA were from 5.8% (serum ADMA, 0.48 μmol/L) to 7.9% (0.19 μmol/L) and from 7.6% (0.47 μmol/L) to 10.8% (0.19 μmol/L), respectively, which were reported in the instruction manual as typical variation for the assay. Serum IS and PC were measured at SRL (Tokyo, Japan). Serum IS was analyzed on high-performance liquid chromatography (HPLC) using fluorescence detection with a Shiseido Capcell Park MF Ph-1 SC80 column (150×4.6 mm, 5 μm; Japan) and serum PC was analyzed on HPLC using fluorescence detection with Supelco Silica and C18 staturator columns (2.1×150 mm, 5 mm; Sigma-Aldrich, St. Louis, MO, USA). This newly validated method allows for lower limits of detection (as low as 0.1 μmol/L) and has been described in detail by Pretorius et al.⁶ Samples were run in duplicate and the CV for the assays ranged from 1.8% to 2.9%. It was also reported that a sensitive and reproducible HPLC-fluorescence method was developed and validated for the quantitative determination of IS.⁷

Statistical Analysis

All data are expressed as mean±SD. Statistical analysis was performed using Stat View 5.0 (SAS Institute, Cary, NC, USA). Correlations between 2 parameters were assessed using simple linear regression. Multivariate regression analysis was used to determine the effects of clinical atherosclerotic risk factors on the impact of IS on IB-IVUS parameters. Significance was indicated at P<0.05.

Results

Study Sample

We identified 102 consecutive patients who had been treated with statin for at least 6 months, with planned treatment for angina pectoris due to coronary stenosis on previous coronary angiography. We excluded patients with AMI (n=3), history of PCI (n=20), coronary artery bypass surgery (n=6), and dialysis therapy (n=8). Moreover, we excluded patients with inadequate IVUS (n=7) and target plaques with wide acoustic shadowing (n=8) or side branch (n=3). The clinical characteristics and findings of target coronary plaque in the 47 patients are listed in Table 1.

Table 1. Patient Characteristics (n=47)

Clinical characteristics
Age (years)	69±7
Male	29 (62)
Hypertension	39 (83)
Diabetes mellitus	28 (60)
History of smoking	29 (62)
HDL-C (mg/dl)	54±16
LDL-C (mg/dl)	104±31
Triglycerides (mg/dl)	131±65
FBS (mg/dl)	127±50
Hemoglobin A1c (%)	6.1±0.9
GFR (ml/min/1.73 m2)	67.7±15.8
Creatinine (mg/dl)	0.82±0.19
IS (μg/ml)	1.174±0.738
ADMA (μmol/L)	0.565±0.119
PC (μg/ml)	0.769±0.491
Findings of target coronary plaque
Percent diameter stenosis (%)	31.3±12.1
LAD/LCx/RCA	20 (43)/10 (21)/17 (36)
Proximal/Mid	33 (70)/14 (30)
IVUS
Mean vessel area (mm2)	19.8±14.6
Mean lumen area (mm2)	10.7±8.9
Plaque volume (mm3)	20.5±7.7
Plaque burden (%)	46.9±14.1
Lipid volume (mm3)	9.8±4.2
Fibrous volume (mm3)	9.5±4.7
Dense fibrous volume (mm3)	1.0±0.5
Calcified volume (mm3)	0.2±0.2
Relative lipid volume (%)	47.5±14.4
Relative fibrous volume (%)	45.6±14.2
Relative dense fibrous volume (%)	6.2±3.1
Relative calcified volume (%)	1.0±1.2

Data given as mean±SD or n (%). ADMA, asymmetric dimethylarginine; FBS, fasting blood sugar; GFR, glomerular filtration rate; HDL-C, high-density lipoprotein-cholesterol; IS, indoxyl sulfate; IVUS, intravascular ultrasound; LAD, left anterior descending coronary artery; LCx, left circumflex coronary artery; LDL-C, low-density lipoprotein-cholesterol; PC, p-cresol; plaque burden, plaque volume/ vessel volume; RCA, right coronary artery.

Uremic Toxins and Renal Function

Glomerular filtration rate (GFR) correlated with IS (r=−0.329, P=0.04), but did not correlate with ADMA or PC (Figure 2).

Figure 2.

Relationship between glomerular filtration rate (GFR) and indoxyl sulfate (IS), asymmetric dimethylarginine (ADMA) and p-cresol (PC). GFR correlated with IS (r=–0.329, P=0.04), but did not correlate with ADMA or PC.

Coronary Plaque Composition and Uremic Toxins or Lipid

Percent lipid volume (LV) positively correlated with IS and triglycerides (P=0.02, P=0.03, respectively), but did not correlate with ADMA or PC (Figure 3). Percent fibrous volume (FV) also negatively correlated with IS and triglycerides (P=0.03, respectively), but did not correlate with ADMA or PC (Figure 4).

Figure 3.

Relationship between percent lipid volume (%Lip) and indoxyl sulfate (IS), triglycerides (TG), asymmetric dimethylarginine (ADMA) and p-cresol (PC). %Lip correlated with IS and TG (P=0.02, P=0.03, respectively), but not with ADMA or PC.

Figure 4.

Relationship between percent fibrous volume (%Fib) and indoxyl sulfate (IS), triglycerides (TG), asymmetric dimethylarginine (ADMA) and p-cresol (PC). %Fib correlated with IS and TG (P=0.03, respectively), but not with ADMA or PC.

Multivariate Indicators of Lipid-Rich Coronary Plaque on IB Imaging

On multivariate regression analysis, only IS was associated with percent LV (β=0.359, P=0.04) and percent FV (β=−0.305, P=0.04) independently of potentially confounding coronary risk factors (GFR, triglycerides, low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C], fasting blood sugar, smoking, and age; Tables 2,3).

Table 2. Multivariate Indicators of %Fib (R²=0.253)

	β	P value
IS	−0.305	0.04
GFR	−0.070	0.68
Triglycerides	−0.377	0.04
LDL-C	0.138	0.42
HDL-C	−0.042	0.81
FBS	0.166	0.36
History of smoking	0.019	0.92
Age	−0.053	0.78

%Fib, percent fibrous volume. Other abbreviations as in Table 1.

Table 3. Multivariate Indicators of %Lip (R²=0.380)

	β	P value
IS	0.359	0.04
GFR	0.319	0.06
Triglycerides	0.403	0.04
LDL-C	−0.321	0.05
HDL-C	0.015	0.93
FBS	−0.146	0.38
History of smoking	−0.022	0.90
Age	−0.012	0.94

%Lip, percent lipid volume. Other abbreviations as in Table 1.

Discussion

In the present study, we have demonstrated for the first time that only serum IS is positively correlated with percent LV and negatively correlated with percent FV on IB-IVUS among the uremic toxins. In addition, we found that only IS is associated with percent LV and percent FV independently of potentially confounding coronary risk factors (GFR, triglycerides, LDL-C, HDL-C, fasting blood sugar, smoking, and age) on multivariate regression analysis. This suggests that serum IS might be a novel useful biomarker to detect and monitor lipid-rich coronary plaque on IB imaging.

Development of IVUS System

Intravascular ultrasound allows tomographic imaging of coronary arteries and provides a more comprehensive assessment of atherosclerotic plaque. Many techniques for tissue characterization of plaque composition have been developed using IVUS.^8–10 Recently, assessment of plaque composition has become possible using IB-IVUS.^5,11–16 IB-IVUS has recently become clinically applicable, in which plaque properties can be diagnosed by combining spectral parameters of posterior scattering signals of IVUS.¹¹ Previously, we used IB-IVUS of coronary plaque to show that telmisartan had an effect on inflammatory cytokines in hypertensive patients over a 6-month period.¹⁶ In the present study, we examined the relationship between uremic toxins and coronary plaque composition on IB imaging.

Uremic Toxins as Prognostic Markers

In CKD patients, progressive deterioration of renal function might lead to accumulation of uremic toxins, which can induce oxidative stress and favor development of atherosclerosis.^17,18 IS is an organic anion metabolized in the liver from indole, which is produced by intestinal bacteria as a metabolite of tryptophan derived from dietary protein.¹⁹ The concentration of serum IS was shown to have a positive correlation with pulse wave velocity in CKD patients.²⁰ ADMA is produced by methylation of arginine residues in intracellular proteins via arginine N-methyltransferases.^21–23 Plasma ADMA was reported to be related to intima-media thickness of the carotid artery.^23,24 PC is an end-product of protein catabolism produced by intestinal bacteria as a metabolite of tyrosine and phenylalanine.²⁵ PC is known to affect the inflammatory response by decreasing the reaction of activated polymorphonuclear leukocytes and the endothelial cell response to inflammatory cytokines in vitro.^26,27 In the present study, in these uremic toxins, GFR correlated with IS, but did not correlate with ADMA or PC. It was reported that there was a gradual rise in serum IS with the severity of CKD from the very earliest stages of the disease.⁴ Therefore, we consider that the minimal increase in IS reflected early renal dysfunction in the present study.

IS and Atherosclerosis

In the present study, on multivariate regression analysis of several important clinical parameters, we found that IS and triglycerides were associated with percent LV and percent FV independently of potentially confounding coronary risk factors, and that IS, as well as dyslipidemia, were associated with lipid-rich coronary plaque on IB imaging.

Compared with ADMA and PC, it is suspected that IS has a stronger impact on the progression of atherosclerosis. Previous studies showed that IS enhanced reactive oxygen species (ROS) production and induced cellular toxicity in endothelial cells.^28–31 Masai et al reported in their study of endothelial cells that IS caused oxidative stress via nicotinamide adenine dinucleotide phosphate (NADPH) oxidant, activated nuclear factor-κ B (NF-κB), and regulated production of monocyte chemoattractant protein-1 (MCP-1) in HUVEC.³ Tumur et al also found that IS upregulated the expression of intercellular adhesion molecule-1 and MCP-1 by ROS-induced activation of NF-κB in vascular endothelial cells.³⁰ Dou et al found that IS enhanced ROS production, and increased NADPH oxidase activity.³¹

In contrast, there are several reports on the relationship between ADMA and atherosclerosis.^23,24 ADMA inhibits endogenous nitric oxide synthase by competing with l-arginine, causing ED. Zoccali et al reported that ADMA was a strong and independent predictor of cardiovascular outcome and mortality in patients with end-stage renal disease.³² It was reported that ADMA did not correlate with oxidative stress,³³ indicating that ADMA caused atherosclerosis mainly via ED. IS, however, is considered to be associated with oxidative stress in addition to ED,³⁴ therefore, we consider that IS has the strongest impact on the progression of atherosclerosis due to the additional pathways compared with the other uremic toxins, including ADMA (Figure 5).

Figure 5.

Mechanisms for the effects of indoxyl sulfate on cardiovascular disease. CKD, chronic kidney disease; ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α.

Study Limitations

There are several limitations of the current study. First, the sample size was relatively small and a larger number of patients would be required to confirm the impact of IS on coronary plaque composition. Second, all patients had been diagnosed with hyperlipidemia, and statin had been prescribed at the first PCI. Statin therapy has been reported to change plaque component and plaque stabilization in coronary artery,^35,36 therefore we always prescribe statin to patients undergoing PCI if statin has not already been prescribed for them; accordingly we included patients who had been fully treated with statin in order to assess the pure relationship between uremic toxin and coronary plaque composition in the present study. Third, acoustic shadowing by a guidewire or calcification may affect tissue characterization. Therefore, lesions masked by wide acoustic shadowing (>90°) were not included in this study. Fourth, thrombus has several IB values according to its formation. Fresh thrombus and lipid pool have similar IB values, and organized thrombus and fibrosis also have similar IB values. It is possible that fresh thrombus was misclassified as lipid pool (blue), and organized thrombus as fibrosis (green). In the present study, we performed IB-IVUS for non-culprit coronary plaques. Generally, severe stenotic lesions tend to have thrombus or calcifications, therefore, we did not select the culprit lesion, in order to assess the coronary plaque component exactly. Fifth, all patients underwent IB-IVUS in the vessel undergoing the intervention. Ideally, the analytical lesion should be selected in the non-culprit vessels, but analytical plaque in this study had to be >5 mm from the intervention site, therefore, we think that these plaques were less affected by PCI. Sixth, on multivariate regression analysis, LDL negatively correlated with percent LV. We included patients who had been fully treated with statin, but the duration of statin intake, the kind of statin, or endothelial function differed in each patient. We considered that these differences might be associated with the strange relationship between LDL and plaque composition on IB-IVUS. A larger number of patients would be required to confirm the relationship.

Conclusions

Among the uremic toxins, only serum IS was associated with lipid-rich coronary plaque on IB-IVUS in patients with coronary artery disease. Serum IS might therefore be a novel useful biomarker to detect and monitor lipid-rich coronary plaque on IB imaging.

Acknowledgments

We thank the nursing and technical staff of the Cardiac Catheterization Laboratory at Tokushima University Hospital for their expert help in conducting this study. We also thank Dr Etsuko Uematsu of the Department of Cardiovascular Medicine, Institute of Health Biosciences, University of Tokushima Graduate School, for her excellent technical assistance.

Disclosures

None.

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