Kynurenine pathway – a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients

Vol. 55(2) · 2010 · pp 196-203 · DOI: 10.2478/v10039-010-0015-6 · Advances in Medical Sciences© ·Medical University of Bialystok, Poland

Kynurenine pathway – a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients Pawlak K1, Myśliwiec M2, Pawlak D1,3* 1 Department of Monitored Pharmacotherapy, Medical University, Bialystok, Poland 2 Department of Nephrology and Clinical Transplantation, Medical University, Bialystok, Poland 3 Department of Pharmacology and Toxicology, University of Warmia and Mazury, Olsztyn, Poland

* CORRESPONDING AUTHOR: Department of Monitored Pharmacotherapy, Mickiewicza 2C str, 15-089 Bialystok, Poland, telephone: 0048 85 7485622, fax: 0048 85 7485622 e-mail: [email protected] (Dariusz Pawlak)

Received 23.07.2009 Accepted 16.03.2010 Advances in Medical Sciences Vol. 55(2) · 2010 · pp 196-203 DOI: 10.2478/v10039-010-0015-6 © Medical University of Bialystok, Poland

ABSTRACT Purpose: The endothelium dysfunction is an important component of atherosclertic cardiovascular disease. It has been also suggested that kynurenine pathway activation may be involved in the pathogenesis of this disease. Material/Methods: This is a cross-sectional study in chronic kidney disease (CKD) patients (n=106; 60 Males). The plasma markers of endothelial dysfunction and kynurenine (KYN), 3-hydroxykynurenine (3-HKYN), kynurenic acid (KYNA), anthranilic acid (AA) and quinolinic acid (QA) were measured in relation to an early indicator of the systemic atherosclerosis - intima-media thickness (IMT). Results: Kynurenines, von Willebrand factor (vWF), thrombomodulin (TM), soluble adhesion molecules (sICAM-1, sVCAM-1) and IMT in each uraemic group were significantly higher than in healthy people. In contrast, no significant differences in sE-selectin and sP-selectin concentrations were observed between CKD patients and controls. Kynurenines were positively associated with vWF, TM, sICAM-1 and sVCAM-1, whereas sP-selectin was inversely associated with the most of kynurenines. IMT was positively correlated both with kynurenines: KYN, 3-HKYN, QA as well as with endothelial markers: TM, vWF, sICAM-1 and sVCAM-1 (all p<0.01). Finally, multiple regression analysis identified age, vWF, sVCAM-1 and QA levels as the independent variables significantly associated with increased IMT in this population (adjusted r2 = 0.51). Conclusions: This study suggests a relationship between kynurenine pathway activation, endothelial dysfunction and the progression of atherosclerosis in CKD patients. It opens a new idea that the inhibition of kynurenine pathway may provide an effective strategy to slow down endothelial dysfunction and thereby the prevalence of atherosclerosis in this population. Key words: atherosclerosis, chronic kidney disease, endothelial dysfunction, kynurenines

INTRODUCTION Atherosclerotic cardiovascular disease (CVD) remains the leading cause of increased morbidity and mortality observed in chronic kidney disease (CKD) patients [1] and there is consistent evidence of a particular association between an endothelial dysfunction and the atherosclerotic process in uraemia [2-4]. Endothelial dysfunction, which is one of the earliest steps of atherogenesis, results in increased adhesiveness and permeability of endothelium for leukocytes [5]. Binding and recruitment of circulating leukocytes to the

vascular endothelium and their further migration into the subendothelial spaces are mediated through a diverse family of cellular adhesion molecules (CAM) [5]. E-selectin and P-selectin mediate initial rolling of leukocytes along the endothelium and intercellular adhesion molecule -1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) play important roles in the firm attachment and transendothelial migration of leukocytes [6]. Moreover, the endothelium is pivotal in the control of hemostasis and thrombosis because it is primary source of many of the major hemostatic regulatory molecules, such as von Willebrand factor (vWF) or thrombomodulin

Pawlak K, Myśliwiec M, Pawlak D

Table 1. Demographic and biochemical characteristics of healthy controls and chronic kidney disease patients. Controls

Non-dialysed CKD

CAPD

HD

Sex, M/F

11/9

20/12

25/24

15/10

Age, years

50.94±6.50

53.19±14.52

51.92±12.56

59.28±13.28

BMI, kg/m2

26.05±3.73

25.21±3.55

25.18±4.16

24.04±3.43

Hematocrit, %

42.07±3.04

32.46±6.32***

36.71±4.73***††

32.98±4.46***^

White blood cells, x109/L

5.86±1.01

6.59±2.27

6.54±2.05

5.43±1.28

Total cholesterol, mmol/L

4.79±7.04

5.49±1.41

5.48±1.16

4.63±1.23

HDL-cholesterol, mmol/L

1.44±0.32

1.39±0.38

1.41±0.45

1.25±0.31

LDL-cholesterol, mmol/L

2.93±0.66

2.76±0.58

3.37±0.98

2.74±1.14

0.81 (0.43-1.68)

2.38 (0.68-6.80)***

1.77 (0.69-3.3)***†

1.15 (0.40-2.40)###

Triglycerides, mmol/L Total protein, g/l

70.2±4.9

62.4±11.4

65.0±5.9

68.2±4.6###^

Albumin, µmol/l

644.8±136.2

454.9±84.0***

498.5±71.0***

546.3±50.7*##

95.5± 17.7

402.2± 209.5***

516.3± 342.1***†

752.3±205.9 ***###^^

Creatinine, µmol/L Hs CRP, µg/ml

0.75 (0.1-10.89)

4.49 (0.1-47.0)***

3.28 (0.1-46.0)**

7.56 (0.1-68.0)***^

SBP, mmHg

126.22±10.18

135.53±11.51

130.27±20.28

134.80±24.29

DBP, mmHg

80.83±6.24

85.86±5.86

82.27±13.05

82.80±12.08

Smokers,%

12

28

25

32

Cardiovascular disease, %

38

49

52

Angiotensin-converting enzyme inhibitors, %

54

49

44

Calcium channel antagonists, %

63

43

66

β-blockers, %

55

41

42

a-blockers, %

9

8

16

EPO treatment, %

-

59

80

EPO, U/kg body weight/week

-

61.81±38.67

96.54±37.08^^

*p<0.05, **p<0.01, ***p<0.001 controls versus patients; ^p<0.05, ^^p<0.01 CAPD versus HD; †p<0.05, ††p<0.01 CAPD versus non-dialysed CKD; #p<0.05, ###p<0.001 HD versus non-dialysed CKD Data are shown as mean±SD or median (range) depending on their normal or skewed distribution. BMI = Body mass index, SBP = systolic blood pressure, DBP = diastolic blood pressure, EPO = erythropoietin, Hs CRP = high sensitivity C-reactive protein

(TM) [7]. The plasma levels of these factors are increased in ESRD patients [4,8,9] and are associated with a marker of preclinical atherosclerosis – carotid intima-media thickness (IMT) in hemodialysed (HD) patients [4]. Recently, it has been postulated that proinflammatory cytokine – interferon γ may be crucially involved in the pathogenesis of atherosclerosis and CVD in general population. Interferon-γ, which is released by activated T-lymphocytes, can stimulate the activity of indoleamine 2.3-dioxygenase (IDO) [10]. This enzyme, by degradation of tryptophan to kynurenine (KYN) [11], induces the kynurenine pathway activation and the production of different metabolites (Fig. 1). Our previous studies demonstrated the accumulation of plasma KYN pathway metabolites both in experimental chronic renal failure [12,13] and in uraemic patients [14]. However, their contribution to vascular physiology and pathology both in general population and in uraemia was still not recognized. More recently, we demonstrated for the first time that some KYN metabolites, particularly 3-hydroxykynurenine (3-HKYN) and quinolinic acid (QA), are associated with increased oxidative stress, inflammation, cardiovascular disease (CVD) prevalence and atherosclerosis in end-stage renal disease patients [15,16].

However, to our knowledge, there are no data concerning KYN pathway activation, endothelial dysfunction and the carotid atherosclerosis in CKD patients. The aim of the present study was therefore to examine the plasma levels of kynurenines; circulating forms of CAM, vWF and TM concentrations which have been implicated as markers of endothelial cell dysfunction and intima-media thickness (IMT), an early reflection of the systemic atherosclerosis in the population of 32 patients with CKD on conservative treatment (non-dialysed CKD), 49 patients on continuous ambulatory peritoneal dialysis (CAPD) and 25 on maintenance hemodialysis (HD).

MATERIAL AND METHODS Subjects

One hundred six patients were enrolled in the study (Tab. 1). All patients were clinically stable and free of active infections and autoimmune diseases. None of the patients received immunosuppressive treatment, lipid-lowering agents, non-

Kynurenine pathway – a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients

Figure 1. Scheme of kynurenine pathway activation.

study volunteered as control subjects. All were on a regular diet and did not have any history of hypertension, DM or renal disease. The study protocol was approved by our institutional ethical board, and informed consent was obtained from all patients and controls.

Blood sampling and laboratory measurements

steroidal anti-inflammatory drugs or antioxidants such as vitamin E, C or allopurinol at the time of the study. Body mass index (BMI) was calculated by dividing the dry weight in kilograms by the square of the height in meters. CVD was defined as documentation in the medical record of a history of myocardial infarction, ischaemic stroke, coronary revascularization procedures, angina pectoris, typical changes on coronary angiograms, typical ischaemic changes on electrocardiogram, peripheral artery surgery (not including the arterio-venous fistula), intermittent claudication or pain at rest. In group of non-dialysed CKD 14 cases had glomerulonephritis, 4 polycystic kidney disease, 6 diabetes mellitus (DM), 2 interstitial nephritis, 1 secondary amyloidosis, 2 hypertensive nephropathy and 3 had unknown etiology. The median estimated glomerular filtration rate (eGFR) in these patients was 13.95 (5.7-121.1 ml/min). The causes of renal failure among HD patients included chronic glomerulonephritis in 8 cases, interstitial nephritis in 6, polycystic kidney disease in 3, secondary amyloidosis in 2, hypertensive nephropathy in 2 and was unknown in 4 cases. The patients received conventional 4-h HD, three times weekly, with modified cellulose membranes, bicarbonate dialysate and low molecular weight heparin - enoxaparin as anticoagulation. The dialysate was endotoxin-free (Coatest Kabi Vitrum). The mean time of HD duration was 40.44 (3-158) months. Dialysis prescription was guided by the goal of achieving a value of Kt/V≥1.2. In CAPD patients, renal failure was due to DM in 6 cases, glomerulonephritis in 17 cases, interstitial nephritis in 4, polycystic kidney disease in 11, hypertensive nephropaty in 4, secondary amyloidosis in 2 and was unknown in 5 cases. All the CAPD patients were performing four 2-liter exchanges a day using either the Baxter TwinBag system or the Fresenius Andy Plus system. Dwell times were generally 4-6 h during the day and 8 h overnight. The glucose concentration ranged from 1.36-3.86%. Dialysis adequacy was assessed by measuring the Kt/V (mean Kt/V = 2.22±0.45). The mean time of CAPD duration was 27.89 (5-125) months. Twenty sex- and age-matched healthy subjects who were receiving no drugs or vitamin supplements at the time of the

Investigations were performed in the morning under fasting conditions. Blood samples were taken directly from the arteriovenous fistula immediately before the beginning of a routine 4-h HD session. In the controls, CAPD and nondialysed CKD group blood was drawn from the antecubital vein to 3.8% sodium citrate in a proportion of 9:1 (v/v). Platelet poor plasma samples were prepared conventionally, aliquoted and stored at -70ºC until the assay. Biochemical parameters were determined by routine laboratory techniques using an automated analyzers. Plasma C-reactive protein levels were measured by high sensitivity ELISA (Imuclone CRP (hs) ELISA, American Diagnostica Inc, Greenwich, USA).

Kynurenine and its metabolites

KYN and its metabolites were determined by high-performance liquid chromatography (HPLC). The chromatographic system was composed of HP 1050 series pump Agillent, and Waters Spherisorb S3 ODS2 150x2.1 mm column (USA). Kynurenine concentration measured using a HP 1050 series UV detector the column effluent was monitored 365 nm. The mobile phase consisted of 0.1 M acetic acid, 0.1 M ammonium acetate (pH 4.65) containing 2% of acetonitrile and was pumped at a flow– rate of 0.25 ml/min. 3-HKYN was measured using a electrochemical detector HP 1049A. Potential of the working electrode was 0.6 V. The mobile phase consisted of 0.1 M triethylamine, 0.1 M phosphoric acid, 0.3 mM EDTA, 8.2 mM heptane-1-sulfonic acid sodium salt, containing 2% of acetonitrile and was pumped at a flow-rate of 0.25 ml/min. KYNA and AA concentrations were determined using fluorescense detector HP 1046A . Excitation and emission wavelengths were set at 254/404 nm for kynurenic acid, 320/420 nm for anthranilic acid. The mobile phase consisted of 50 mM acetic acid, 0.25 M zinc acetate (pH-4.9), containing 1.2% of acetonitrile was pumped at a flow-rate of 0.25 ml/ min. QA was measured using a HP 1050 series UV detector the column effluent was monitored (272 nm). A Phase Separations Partisil 10 SAX 250x4.6 mm (USA) column was eluted with 50 mM potassium phosphate (pH-2.0) containing 12 % methanol at a flow rate of 1.8 ml/min. 2 ml of plasma was concentrated on Sep-Pack cartridges (Accell Plus QMA) washed 2 ml water and eluted with 0.2 ml 4 M H3PO4.

Endothelial dysfunction markers

Thrombomodulin (TM) and von Willebrand factor antigen

Pawlak K, Myśliwiec M, Pawlak D

(vWF) plasma levels were studied using commercially available kits (Thrombomodulin ELISA Kit, American Diagnostica and Asserachrom vWF, Diagnostica Stago; respectively). Circulating forms of ICAM-1 (sICAM-1), VCAM-1 (sVCAM-1), E-selectin (sE-selectin) and P-selectin (sP-selectin) were determined by commercially available ELISA kits and standards (R&D Systems Europe Ltd, Abingdon, UK).

Blood pressure measurements

In dialysis patients, the average blood pressure (BP) was calculated on the basis of a series of 5 measurements taken during the month preceding the study. In controls and in nondialysed CKD group, BP was measured after 15 min of rest and 3 measurements 2 min apart were averaged.

Intima media thickness (IMT)

All subjects underwent measurements of carotid artery IMT by high-resolution real-time B mode ultrasonography with a 7.5-MHz linear transducer (SSH 140A Toshiba, Japan), as previously described [4]. Briefly, the carotid arteries were investigated bilaterally in longitudinal projections. The examination included sections of approximately 2-3 cm of common carotid artery just below the carotid bulb. IMT was defined as the distance between the leading edge of the first echogenic line (lumen-intima interface) and the second echogenic line (media-adventitia interface) of the far wall. Four measurements from both sites were averaged to give the mean IMT. All IMT ultrasound studies were performed by the same investigator, who was blinded to the patients´ clinical and laboratory data.

Statistical analysis

Shapiro-Wilk’s W test of normality was used for data distribution analysis. The normally distributed data were expressed as mean ± SD. The non-Gaussian data were presented as median (full range), depending on their distribution. Nonnormally distributed variables were log-transformed before entering regression analysis. Multiple group comparisons were performed by one-way analysis of variance (ANOVA), with post hock Tukey-Kramer multiple comparison test or Kruskall-Wallis nonparametric ANOVA with post hock Dunn’s multiple comparison test. Univariate correlations were determined by Pearson’s or quasi-Newton and Rosenbrock´s logistic regression analysis. Multiple regression analysis with a forward elimination procedure was used to assess the combined influence of variables on IMT values. A two-tailed p value <0.05 was considered statistically significant. Data given were analyzed using Statistica 5.1 computer software (StatSoft Inc., Tulsa, OK).

RESULTS The demographic and clinical characteristics of the healthy controls and CKD patients are summarized in Tab. 1. Hematocrit and albumin were significantly decreased, whereas creatinine and hs CRP levels were significantly increased in all groups of patients relative to the controls. Triglycerides were significantly elevated in both non-dialysed CKD and CAPD group as compared to the healthy people. Creatinine was significantly higher in dialyzed patients than in non-dialysed CKD patients, whereas hematocrit was significantly lower in non-dialysed CKD and HD patients than in CAPD group. Triglycerides were lower and total protein were higher in HD relative to CAPD and non-dialysed CKD patients. Concentrations of kynurenines, endothelial dysfunction markers and IMT values in controls and CKD patients are presented in Tab. 2. Kynurenines, vWF, TM, sICAM-1, sVCAM-1 and IMT in each uraemic group were significantly higher than in healthy people, particularly in the dialyzed groups. In contrast, no significant differences in sEselectin and sP-selectin concentrations were observed between CKD patients and controls.

The correlations between carotid atherosclerosis marker – IMT and both kynurenines and endothelial dysfunction markers in CKD patients

The main correlations between IMT and both kynurenines and endothelial dysfunction markers levels were shown in Tab. 3. IMT was positively correlated with KYN, 3-HKYN, QA, vWF, and both soluble CAM concentrations. Moreover, IMT was also positively correlated with age (r = 0.602, p<0.0001), TM (r = 0.298, p<0.001), hs CRP levels (r = 0.288, p<0.01), the presence of diabetes mellitus (χ2 = 5.667, p<0.05), and IMT was inversely associated with HDL-cholesterol levels (r = -0.329, p<0.01) in the whole CKD group. Kynurenines were positively associated with vWF, TM, sICAM-1 and sVCAM-1 levels, whereas sP-selectin was inversely associated with 3-HKYN, QA, AA and KYNA concentrations (r = -0.247, r = -0.270, r = -0.282, all p<0.01, and r = -0.220, p<0.05; respectively). The positive correlations were observed between separate endothelial dysfunction markers as well as between kynurenines (Tab.3). The positive association was between inflammatory marker - hs CRP levels and sICAM-1, KYN (r = 0.302 and r = 0.309, both p<0.001), and the weak correlations were also between hs CRP and TM, vWF, 3-HKYN (r = 0.205, r = 0.199, and r = 0.208, all p<0.05; respectively). One subject from each uraemic group showed very high inflammatory marker (hs CRP 47, 46 and 68 µg/ ml in non-dialysed CKD, CAPD and HD group; respectively) without signs or symptoms of disease. When these patients were omitted from the analyses, hs CRP was still correlated with KYN, sICAM-1 and IMT values (r = 0.325, p<0.001, r = 0.296, p<0.01 and r = 0.254, p<0.05; respectively).

Kynurenine pathway – a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients

Table 2. Kynurenines, endothelial dysfunction markers and intima-media thickness (IMT) values in the healthy controls and chronic kidney disease patients. Controls KYN, µM

Non-dialysed CKD

CAPD

HD

1.55 (1.15-2.47)

3.00 (0.79-4.55) ***

2.19 (1.34-4.36) **

3.07 (0.95-6.52) ***

45.0 (22.0-119.0)

140.0 (22.0-313.0) ***

182.0 (36.0-598.0) ***

203.0 (40.0-674.0) ***

KYNA, nM

20.5 (11.8-85.0)

52.11 (18.4-185.5) *

115.00 (18.0-392.0) ***††

120.3 (52.0-282.0) ***##

AA, nM

52.0 (31.0-162.5)

138.6 (15.0-820.0) **

273.0 (54.0-776.0) ***

226.0 (43.5-554.0) ***

QA, µM

0.15 (0.07-0.63)

0.69 (0.15-2.67) **

1.49 (0.36-3.37) ***†

2.05 (0.55-8.00) ***##

TM, ng/ml

2.55 (1.26-5.04)

10.86 (3.76-19.85) ***

13.40 (6.78-25.93) ***†

12.55 (7.15-22.48) ***

3-HKYN, nM

69.8 (59.8-84.6)

106.2 (73.0-132.5) *

123.2 (68.0-245.2) ***††

195.2 (89.0-256.8) ***###^

sICAM-1, ng/ml

vWF, %

223.4 (132.1-283.2)

260.8 (139.0-753.2) *

286.6 (158.0-721.4) ***

324.8 (165.2-958.8) ***#

sVCAM-1, ng/ml

627.0 (295.0-1490.0)

1096.0 (278.0-3416.0) *

1756.0 (586.0-4549.0) ***††

2460.0 (1195.0-4310.0) ***###

sE-selectin, ng/ml

42.8 (16.0-93.5)

35.3 (15.0-68.2)

36.4 (18.4-128.6)

41.0 (16.8-235.0)

sP-selectin, ng/ml

85.5 (32.0-210.6)

73.4 (45.6-160.8)

68.8 (10.6-145.8)

63.8 (17.2-119.2)

0.63±0.05

0.76±0.14 **

0.79±0.15 ***

0.83±0.12 ***

IMT, mm

*p<0.05, **p<0.01, ***p<0.001 controls versus patients; ^p<0.05 CAPD versus HD; †p<0.05 ††p<0.01 CAPD versus non-dialysed CKD; #p<0.05, ## p<0.01, ###p<0.001 HD versus non-dialysed CKD. Data are shown as mean±SD or median (range) depending on their normal or skewed distribution. KYN = kynurenine; 3-HKYN = 3-hydroxykynurenine; KYNA = kynurenic acid; AA = anthranilic acid; QA = quinolinic acid; TM = thrombomodulin; vWF = von Willebrand factor; sICAM-1 = soluble intercellular adhesion molecule -1; sVCAM-1 = soluble vascular adhesion molecule-1

Independent factors associated with IMT values in CKD patients To examine the combined effect of factors affecting increased IMT values in CKD patients, multiple regression analysis were performed based on results of Pearson´s linear or quasi-Newton and Rosenbrock´s logistic regression analysis. Multiple stepwise regression analysis (Table 4) identified age, vWF, sVCAM-1 and QA levels as the independent variables significantly associated with increased IMT in this population.

DISCUSSION Endothelial cell damage can be quantitatively related to the concentrations of plasma proteins involved in intravascular coagulation: TM and vWF factor, but also to endothelial – derived soluble adhesion molecules and selectins. In the present study we found, for the first time, that kynurenine pathway metabolites are associated both with endothelial dysfunction markers and IMT values in the patients with chronic kidney disease. Moreover, we have demonstrated that IMT, reflecting of the systemic atherosclerosis in uraemia, is independently related to increased endothelial dysfunction markers - vWF, sVCAM-1 as well as to QA levels in CKD population. Increased levels of endothelial dysfunction markers were observed by us [4] and by others [2,3] in dialysis patients. Most of all, the raised concentrations of sICAM-1 and sVCAM-1 were found in patients with different stage of CKD, and the accumulation of these molecules were

associated with impaired renal function [8,17- 20]. Moreover, the elevated levels of these adhesion molecules were the independent predictors of mortality in this population [19, 20]. In this study we demonstrated, similarly to the observation of Jacobson et al. [8], the strong positive correlations between the concentrations of vWF, TM, sICAM-1 and sVCAM-1 in CKD patients, suggesting a common pathway for their production. The novel finding is the existence of strong correlations between endothelial dysfunction markers and kynurenines (Tab. 3). This suggests that the disturbances of kynurenine pathway of tryptophan degradation seem to be related to the endothelial dysfunction in CKD patients. The cellular mechanisms responsible for this phenomenon are unknown now. Until now, only separate findings of the relationship between endothelium and kynurenine system were existed in the literature. Hansen et al. [21] demonstrated that vascular endothelial cells were the primary sites of IDO expression in murine malaria infection, and that this response was systemic and INF-γ-dependent. Moreover, it has been demonstrated that vascular endothelium is able to produce and liberate of KYNA [22], and that homocysteine, a risk factor for atherosclerosis, can change its endothelial production [23,24]. Much less is known about the function of KYNA in the periphery. A positive correlation between KYNA serum concentration and homocysteine has been demonstrated in stroke patients [25]. Moreover, in an in vitro vascular flow model, KYNA could be an important early mediator of leukocyte recruitment to vascular endothelium [26]. On the other hand, in vitro experiments suggest that KYNA may have a protective influence on the endothelium during hyperhomocysteinemia

Pawlak K, Myśliwiec M, Pawlak D

Table 3. The associations between kynurenines, endothelial dysfunction markers and intima-media thickness (IMT) in chronic kidney disease patients. KYN

3-HKYN

QA

KYNA

AA

vWF

sICAM-1

sVCAM-1

TM

r = 0.353 p<0.001

r = 0.640 p<0.0001

r = 0.641 p<0.0001

r = 0.466 p<0.0001

r = 0.519 p<0.0001

r = 0.575 p<0.0001

r = 0.519 p<0.0001

r = 0.613 p< 0.0001

vWF

r = 0.253 p<0.01

r = 0.458 p<0.0001

r = 0.586 p<0.0001

r = 0.427 p<0.0001

r = 0.262 p<0.01

r = 0.361 p<0.001

r = 0.608 p<0.0001

sICAM-1

r = 0.402 p<0.0001

r = 0.323 p<0.001

r = 0.356 p<0.001

r = 0.229 p<0.05

r = 0.231 p<0.05

r = 0.361 p<0.001

sVCAM-1

r = 0.216 p<0.05

r = 0.486 p<0.0001

r = 0.537 p<0.0001

r = 0.512 p<0.0001

r = 0.488 p<0.0001

r = 0.608 p<0.0001

r = 0.548 p<0.0001

r = 0.256 p<0.01

3-HKYN

r = 0.339 p<0.001

r = 0.544 p<0.0001

r = 0.471 p<0.0001

r = 0.559 p<0.0001

r = 0.458 p<0.0001

r = 0.323 p<0.001

r = 0.486 p<0.0001

QA

r = 0.314 p<0.001

r = 0.544 p<0.0001

r = 0.393 p<0.0001

r = 0.397 p<0.0001

r = 0.586 p<0.0001

r = 0.356 p<0.001

r = 0.537 p<0.0001

IMT

r = 0.291 p<0.01

r = 0.250 p<0.01

r = 0.017 NS

r = -0.021 NS

r = 0.408 p<0.0001

r = 0.329 p<0.001

r = 0.256 p<0.01

r = 0.369 p<0.001

r = 0.548 p<0.0001

KYN = kynurenine; 3-HKYN = 3-hydroxykynurenine; KYNA = kynurenic acid; AA = anthranilic acid; QA = quinolinic acid; TM = thrombomodulin; vWF = von Willebrand factor; sICAM-1 = soluble intercellular adhesion molecule -1; sVCAM-1 = soluble vascular adhesion molecule-1; IMT = intima-media thickness Table 4. Variables predicting IMT in multiple regression analysis in ESRD patients. Independent variables

Regressioncoefficient

Standarderror

p value

Age

0.356

0.111

0.0022

vWF

0.368

0.155

0.0201

sVCAM-1

0.261

0.122

0.0367

QA

0.223

0.125

0.0421

Multiple r for variables in the model = 0.713, multiple r2 = 0.508, adjusted r2 = 0.469, p<0.0001, standard error of estimate: 0.100.

[27]. In the present study we have demonstrated that both endothelial injury markers (except sP-selectin) as well as KYN and 3-HKYN were positively associated with increased hs CRP levels, reflecting the inflammatory state in these patients. This association suggests that inflammation may be partially responsible for elevated levels of these proteins, and it is in agreement with previous observations demonstrated the relationship between sICAM-1, sVCAM-1 and CRP levels in dialysed patients [2, 3]. Moreover, pro-inflammatory cytokine – interferon-γ, can induce the activity of IDO [10], and by this way the production of different KYN metabolites [11]. This is in the line with observation of Schefold et al. [28] demonstrated that induction of IDO activity in CKD patients may primarily be a consequence of chronic inflammation. On the other hand, it has been demonstrated that KYN metabolites, particularly 3-HKYN and QA, can generate the oxidative stress and cause the neurotoxicity in the central nervous system [29, 30]. More recently, we also observed that kynurenine pathway activation is associated with increased oxidative stress in end-stage renal disease patients [15, 16], and we previously demonstrated [4] that both vWF and TM levels were strongly correlated with oxidative status in hemodialysed patients. Based on these findings it could be

speculated that oxidative stress, generated by kynurenines, may be also partially responsible for increased endothelial dysfunction markers in these patients. This is in agreement with recent evidence that oxidative stress is one of the most potent inductors of endothelial dysfunction and is involved at all stages of atherosclerotic plaque evolution [31]. In the present study, we observed a significant increase in IMT values of all uraemic groups compared with the agematched control subjects. In simple regression analysis, IMT values were significantly correlated with some typical risk factors, such as: age, presence of diabetes mellitus, hs CRP, or HDL-cholesterol. Moreover, IMT was correlated with vWF and TM concentrations, confirming our previous results [4], and it was associated with sICAM-1 and sVCAM-1, which is in agreement with the results presented by Papagianni et al [2, 3]. In the present study increased levels of KYN, 3-HKYN and QA were associated with IMT in uraemic patients. Moreover, multiple stepwise regression analysis (Tab. 4) confirmed age, vWF, sVCAM-1 and QA levels as the parameters independently and significantly predicted elevated IMT values in this population. Thus, we propose that progression of atherosclerosis was independently related to the endothelial dysfunction and accumulation of QA in the plasma of uraemic patients. The previous prospective studies have shown an association between high concentrations of vWF and increased risk for coronary heart disease, myocardial infarction and stroke [32-34]. The concentration of sVCAM-1, which has been shown to be correlated to the expression of VCAM-1 mRNA in human atherosclerotic aorta, has been suggested to be a potential marker for atherosclerosis [35]. In contrast to above mentioned observations, until now there were no data concerning QA and atherosclerosis both in general as well as in uraemic population. There are only separate findings

Kynurenine pathway – a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients

concerning KYN pathway activation in atherosclerosis [36-38]. Recently, it has been demonstrated that IDO activity is associated with IMT values in young female adults [39]. Our results, showing that QA together with endothelial dysfunction markers: vWF and sVCAM-1, independently and significantly predicted elevated IMT in CKD patients, are in line with above mentioned observations. The present study has some limitations that should be considered. First, because of its cross-sectional nature, this study does not demonstrate cause and effect relationships; however, results of this study are hypothesis generating and provide insights into the relationship between KYN pathway activation, endothelial dysfunction and atherosclerosis in uraemic patients. Secondly, the levels of studied parameters are probably not an exact reflection of their vascular tissue levels, and may be affected by many factors. Thirdly, we cannot take into account any variation that may have occurred over time as we did not have repetitive analyses of studied parameters concentrations for all patients.

CONCLUSIONS The present study shows that kynurenine pathway metabolites are associated with endothelial dysfunction markers: vWF, TM, sICAM-1, sVCAM-1 and IMT values in the patients with CKD. The significant increase in these parameters observed in all groups of uraemic patients suggests that atherosclerosis starts at a very early stage of the disease. Finally, carotid IMT is independently related to increased endothelial dysfunction markers- vWF, sVCAM-1 as well as to QA levels in this population. This study opens a new idea that the inhibition of IFN-γ- mediated pathways and in consequence, also KYN metabolites production may provide an effective strategy to slow down endothelial dysfunction and thereby progress of atherosclerosis in this population.

ACKNOWLEDGEMENTS This work is supported by a grant (No. 0754/P01/2007/32) from the National Research Committee, Warsaw, Poland. The authors declare no conflict of interest.

REFERENCES

1. Foley RN, Parfrey PS, Sarnak MJ. Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis. 1998 Nov;32(5 Suppl 3):S112-9. 2. Papagianni A, Kokolina E, Kalovoulos M, Vainas A, Dimitriadis C, Memmos D. Carotid atherosclerosis is associated with inflammation, malnutrition and intercellular adhesion molecule-1 in patients on continuous ambulatory peritoneal dialysis. Nephrol Dial Transplant. 2004 May;19(5):1258-63. 3. Papagianni A, Kalovoulos M, Kirmizis D, Vainas

A, Belechri AM, Alexopoulos E, Memmos D. Carotid atherosclerosis is associated with inflammation and endothelial cell adhesion molecules in chronic haemodialysis patients. Nephrol Dial Transplant. 2003 Jan;18(1):113-9. 4. Pawlak K, Naumnik B, Brzósko S, Pawlak D, Myśliwiec M. Oxidative stress-a link between endothelial injury, coagulation activation and atherosclerosis in haemodialysis patients. Am J Nephrol. 2004 Jan-Feb;24(1):154-61. 5. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999 Jan 14;340(2):115-26. 6. Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis. 2003 Oct;170(2):191-203. 7. Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM. Endothelial cells in physiology and in the pathology of vascular disorders. Blood. 1998 May 15;91(10):3527-61. 8. Jacobson SH, Egberg N, Hylander B, Lundahl J.Correlation between soluble markers of endothelial dysfunction in patients with renal failure. Am J Nephrol. 2002 Jan-Feb;22(1):42-7. 9. Malyszko J, Malyszko JS, Mysliwiec M. Endothelial cell injury markers in chronic renal failure on conservative treatment and continuous ambulatory peritoneal dialysis. Kidney Blood Press Res. 2004;27(2):71-7. 10. Schroecksnadel K, Frick B, Winkler C, Fusch D. Crucial role of interferon-gamma and stimulated macrophages in cardiovascular disease. Curr Vasc Pharmacol. 2006 Jul;4(3):205-13. 11. Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev. 1993 Sep;45(3):309-79. 12. Pawlak D, Tankiewicz A, Buczko W. Kynurenine and its metabolism in the rat with experimental renal insufficiency. J Physiol Pharmacol. 2001 Dec;52(4 Pt 2):755-66. 13. Pawlak D, Tankiewicz A, Matys T, Buczko W. Peripheral distribution of kynurenine metabolism and activity of kynurenine pathway enzymes in renal failure. J Physiol Pharmacol. 2003 Jun;54(2):175-89. 14. Pawlak D, Pawlak K, Malyszko J, Mysliwiec M, Buczko W. Accumulation of toxic products degradation of kynurenine in hemodialyzed patients. Int Urol Nephrol. 2001;33(2):399-404. 15. Pawlak K, Domaniewski T, Mysliwiec M, Pawlak D. The kynurenines are associated with oxidative stress, inflammation and the prevalence of cardiovascular disease in patients with end-stage renal disease. Atherosclerosis. 2009 May;204(1):309-14. 16. Pawlak K, Brzosko S, Mysliwiec M, Pawlak D. Kynurenine, quinolinic acid--the new factors linked to carotid atherosclerosis in patients with end-stage renal disease. Atherosclerosis. 2009 Jun;204(2):561-6. 17. Stam F, van Guldener C, Schalkwijk CG, ter Wee PM, Donker AJ, Stehouwer CD. Impaired renal function

Pawlak K, Myśliwiec M, Pawlak D

is associated with markers of endothelial dysfunction and increased inflammatory activity. Nephrol Dial Transplant. 2003 May;18(5):892-8. 18. Stinghen AE, Goncalves SM, Martines EG, Nakao LS, Riella MC, Aita CA, Pecoits-Filho R. Increased plasma and endothelial cell expression of chemokines and adhesion molecules in chronic kidney disease. Nephron Clin Pract. 2009;111(2):c117-26. 19. Stenvinkel P, Lindholm B, Heimbürger M, Heimbürger O. Elevated serum levels of soluble adhesion molecules predict death in pre-dialysis patients: association with malnutrition, inflammation, and cardiovascular disease. Nephrol Dial Transplant. 2000 Oct;15(10):1624-30. 20. Suliman ME, Qureshi AR, Heimbürger O, Lindholm B, Stenvinkel P. Soluble adhesion molecules in end-stage renal disease: a predictor of outcome. Nephrol Dial Transplant. 2006 Jun;21(6):1603-10. 21. Hansen AM, Ball HJ, Mitchell AJ, Miu J, Takikawa O, Hunt NH. Increased expression of indoleamine 2,3-dioxygenase in murine malaria infection is predominantly localised to the vascular endothelium. Int J Parasitol. 2004 Nov;34(12):1309-19. 22. Stazka J, Luchowski P, Wielosz M, Kleinrok Z, Urbańska EM. Endothelium-dependent production and liberation of kynurenic acid by rat aortic rings expose to L-kynurenine. Eur J Pharmacol. 2002 Jul 19;448(2-3):133-7. 23. Wejksza K, Rzeski W, Parada-Turska J, Zdzisinska B, Rejdak R, Kocki T, Okuno E, Kandefer-Szerszen M, Zrenner E, Turski WA. Kynurenic acid production in cultured bovine aortic endothelial cells. Homocysteine is a potent inhibitor. Naunyn Schmiedebergs Arch Pharmacol. 2004 Mar;369(3):300-4. 24. Stazka J, Luchowski P, Urbanska EM. Homocysteine, a risk factor for atherosclerosis, biophasically changes the endothelial production of kynurenic acid. Eur J Pharmacol. 2005 Jul 11;517(3):217-23. 25. Urbanska EM, Luchowski P, Luchowska E, Pniewski J, Wozniak R, Chodakowska-Zebrowska M, Lazarewicz J. Serum kynurenic acid positively correlates with cardiovascular disease risk factor, homocysteine: a study in stroke patients. Pharmacol Rep. 2006 Jul-Aug;58(4):507-11. 26. Barth MC, Ahluwalia N, Anderson TJ, Hardy GJ, Sinha S, Alvarez-Cardona JA, Pruitt IE, Rhee EP, Colvin RA, Gerszten RE. Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions. J Biol Chem. 2009 Jul 17;284(29):19189-95. 27. Wejksza K, Rzeski W, Turski WA. Kynurenic acid protects against the homocysteine-induced impairment of endothelial cells. Pharmacol Rep. 2009 Jul-Aug;61(4):751-6. 28. Schefold JC, Zeden JP, Fotopoulou C, von Haehling S, Pschowski R, Hasper D, Volk HD, Schuett C, Reinke P. Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms. Nephrol Dial Transplant.

2009 Jun;24(6):1901-8. 29. Okuda S, Nishiyama N, Saito H, Katsuki H. 3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity. J Neurochem. 1998 Jan;70(1):299-307. 30. Santamaria A, Flores-Escartin A, Martinez JC, Osorio L, Galvan-Arzate S, Chaverri JP, Maldonado PD, Medina-Campos ON, Jimenez-Capdeville ME, Manjarrez J, Rios C. Copper blocks quinolinic acid neurotoxicity in rats: contribution of antioxidant systems. Free Radic Biol Med. 2003 Aug 15;35(4):418-27. 31. Schnabel R, Blankenberg S. Oxidative stress in cardiovascular disease. Successful translation from bench to bedside? Circulation. 2007 Sep 18;116(12):1338-40. 32. Morange PE, Simon C, Alessi MC, Luc G, Arveiler D, Ferrieres J, Amouyel P, Evans A, Ducimetiere P, JuhanVague I; PRIME Study Group. Endothelial cell markers and the risk of coronary heart disease: the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study. Circulation. 2004 Mar 23;109(11):1343-8. 33. Thompson SG, Kienast J, Pyke SD, Haverkate F, van de Loo JC. Hemostatic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. N Engl J Med. 1995 Mar 9;332(10):635-41. 34. Folsom AR, Rosamond WD, Shahar E, Cooper LS, Aleksic N, Nieto FJ. Prospective study of markers of hemostatic function with risk of ischemic stroke. The Atherosclerosis Risk in Communities (ARIC) Study Investigators. Circulation. 1999 Aug 17;100(7):736-42. 35. Peter K, Nawroth P, Conradt C, Nordt T, Weiss T, Boehme M, Wunsch A, Allenberg J, Kubler W, Bode C. Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin and thrombomodulin. Arterioscler Thromb Vasc Biol. 1997 Mar;17(3):505-12. 36. Whitman SC, Ravisankar P, Elam H, Daugherty A. Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- moce. Am J Pathol. 2000 Dec;157(6):1819-24. 37. Rudzite V, Sileniece G, Liepina D, Dalmane A, Zirne R. Impairment of kynurenine metabolism in cardiovascular disease. Adv Exp Med Biol. 1991;294:663-7. 38. Wirleitner B, Rudzite V, Neurauter G, Murr C, Kalnins U, Erglis A, Trusinskis K, Fuchs D. Immune activation and degradation of tryptophan in coronary heart disease. Eur J Clin Invest. 2003 Jul;33(7):550-4. 39. Pertovaara M, Raitala A, Juonala M, Lehtimäki T, Huhtala H, Oja SS, Jokinen E, Viikari JS, Raitakari OT, Hurme M. Indoleamine 2,3-dioxygenase enzyme activity correlates with risk factors for atherosclerosis: the Cardiovascular Risk in Young Finns Study. Clin Exp Immunol. 2007 Apr;148(1):106-11.