Effects of Lipoprotein-Associated Phospholipase A2 on Arginase/Nitric Oxide Pathway in Hemodialysis Patients

Abstract

Lipoprotein-associated phospholipase A2 (Lp-PLA2) and arginase are recently described inflammatory biomarkers associated with cardiovascular disease. In this study, we aimed to investigate the possible effects of serum Lp-PLA2 mass levels on arginase/nitric oxide (NO) pathway as a cardiovascular risk marker in hemodialysis (HD) patients. Forty-three HD patients and 15 healthy subjects were included in this study. Lipid profile, high sensitivity C-reactive protein (hs-CRP), albumin, creatinine, body mass index (BMI), Lp-PLA2 and total nitrite levels, and arginase activity were determined in serum samples from patients and control subjects. Lp-PLA2 levels were found to be positively correlated with arginase, triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and age and negatively correlated with high-density lipoprotein-cholesterol and total nitrite levels, while there was no correlation with BMI and hs-CRP, albumin, and creatinine levels in HD patients. We conclude that elevated Lp-PLA2 mass levels may contribute to impaired arginase/NO pathway in HD patients and that increased the arginase activity and Lp-PLA2 mass levels with decreased total nitrite levels seem to be useful biochemical markers in terms of reflecting endothelial dysfunction and associated cardiovascular risks in HD patients.

• arginase
• hemodialysis
• Lp-PLA2
• nitric oxide

INTRODUCTION

Patients undergoing peritoneal dialysis and hemodialysis (HD) have a high incidence of accelerated atherosclerosis and coronary artery disease.¹ The major cause of death in patients with end-stage renal failure receiving renal replacement therapy is cardiovascular disease.²

A number of factors contribute to atherosclerosis and cardiovascular disease in end-stage renal disease (ESRD) population. These events are driven by oxidative stress, inflammation, endothelial dysfunction, insulin resistance, vascular calcification, and lipid disorders.³ Traditional risk factors cannot explain the high prevalence and incidence of cardiovascular disease; therefore, other nontraditional risk factors have increasingly been studied in chronic kidney disease.⁴

Inflammation plays a key role in the development of atherosclerosis and coronary heart disease (CHD).⁵ Recently, lipoprotein-associated phospholipase A2 (Lp-PLA2) was proposed as a novel inflammatory and endothelial dysfunction marker in HD patients.^6,7 Lp-PLA2 is a potentially useful plasma biomarker associated with cardiovascular disease.^8,9 The enzyme, also known as plasma platelet-activating factor acetylhydrolase (PAF-AH), is a Ca⁺² independent enzyme and has two prominent biological activities. First, it inactivates the major proinflammatory mediator, platelet-activating factor (PAF). Second, Lp-PLA2 hydrolyzes oxidatively modified polyunsaturated fatty acids, producing lysophosphatidylcholine (LysoPC) and oxidized nonesterified fatty acids.^8,10,11

In human plasma, Lp-PLA2 is mainly associated with low-density lipoprotein (LDL) and with high-density lipoprotein (HDL). Lp-PLA2 also bound to very low-density lipoprotein and to intermediate-density lipoprotein, as well as to lipoprotein (a) [Lp(a)].¹²

Arginase is a nitric oxide synthase (NOS)-alternative pathway for l-arginine breakdown, leading to biosynthesis of urea and l-ornithine. Arginase pathway can be induced by inflammatory molecules in macrophages and smooth muscle cells. In addition, it is likely to play a key role in the proliferation of endothelial cells.¹³

In animal vascular tissues, constitutive arginase activity and immunoreactivity were observed in both endothelial and vascular smooth muscle cells (VSMCs). In these cells, arginase pathway is involved in the regulation of cell proliferation. In addition, arginase has been shown to counteract NO-mediated vasodilatation in microvascular endothelial cells, because NOS, the enzyme responsible for NO synthesis, and arginase share l-arginine as the common substrate.¹⁴ Arginase is known to contribute directly to the progression of atherosclerosis and counteract the beneficial effects of NO. On the other hand, inflammation also contributes to the progression of atherosclerosis and influences both arginase and NOS expressions.¹⁵

In this study, we investigated whether levels of Lp-PLA2 on the arginase/NO pathway have an efficacy on the risk prediction of future cardiovascular events in HD patients. We also evaluated the relationship of Lp-PLA2 mass levels and arginase activity with other traditional cardiovascular risk markers.

MATERIALS AND METHODS

This study was conducted in cooperation with two departments: Department of Medical Biochemistry and Department of Nephrology in Eskisehir Osmangazi University Faculty of Medicine in Turkey.

Forty-three patients with ESRD (23 men and 20 women; mean age, 52.91 ± 11.80 years) on HD maintenance therapy for at least 3 months were recruited. The cause of ESRD was hypertensive nephropathy in 12, drug toxicity in 5, chronic glomerulonephritis in 6, polycystic kidney in 5, stone disease in 3, and unknown etiology in 12 patients. All of the patients were routinely dialyzed for 4–5 hours for 3 times weekly using low-flux polysulfone membranes with no dialyzer reuse. Dialysates were bicarbonate based. Pure water used in dialysates was compatible with the standards of European Pharmacopoeia. All patients on HD were anticoagulated with heparin. Fifteen age- and sex-matched healthy volunteers (8 men and 7 women; mean age, 53.67 ± 6.29 years) were used as control group.

Exclusion criteria were the presence of active infection, diabetes mellitus, Hepatitis B and C, active liver disease, familial Mediterranean fever, acute infection, and malignancy in both patients and controls.

Patients and control subjects were all nonsmokers. They were not using vitamin supplementations, antioxidants, angiotensin-converting enzyme (ACE) inhibitors, or statins. Body mass indices (BMIs) of all subjects were calculated as weight in kilograms divided by height in meters squared (kg/m²).

Blood samples were withdrawn after a 12-h overnight fasting just before the breakfast (7.00–9.00 am), and after the patients had been sedentary in sitting or supine position for at least 15 min. Blood samples were collected in tubes without anticoagulants just before commencing the dialysis. Serum was immediately separated and removed by centrifugation. Total cholesterol, triglyceride, LDL-cholesterol, HDL-cholesterol, creatinine, albumin, and high sensitivity C-reactive protein (hs-CRP) were measured with Roche diagnostics kits on Roche/Hitachi modular autoanalyzer at the same day. The separated serum was divided into two polystyrene tubes, and stored for subsequent Lp-PLA2, arginase, and total nitrite analysis (−80°C, and −18°C, respectively).

Lp-PLA2 mass levels were measured with the PLAC test (dia Dexus, Inc., South San Francisco, CA, USA). The assay is a microplate-based enzyme-linked immunoadsorbent assay, employing two monoclonal antibodies specific for Lp-PLA2, and calibrated to recombinant enzyme standard. This procedure was performed with Roche/Hitachi Modular Analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The assay range is 7–500 ng/mL, and intra-assay coefficienct (n = 80) is 1.6–2.4, with a total coefficienct (n = 80) of 1.8–3.2. Arginase activity was estimated by tiyosemikarbazid-diasetil monoksim urea assay.¹⁶ Total nitrite levels were measured according to the method of Cortas et al.¹⁷

This study was approved by Ethical Committee of Eskisehir Osmangazi University Medical School (Eskisehir/Turkey).

Statistical Analysis

The statistical analyses were performed using SPSS for Windows (Statistical Package for the Social Sciences, version 15.0, SSPS Inc., Chicago, IL, USA). The normal distribution of the variables was evaluated using Kolmogorov–Smirnov test. The Mann–Whitney U test was used for the comparison of variables which were not normally distributed and independent samples T test was used for the other comparisons. The Pearson and Spearman tests according to variable distribution were used for evaluation of correlations among the variables. p < 0.05 was accepted as statistically significant. The results of variables with a normal distribution were expressed as means ± SD and those with a non-Gaussian distribution were expressed as median (25th–75th percentile).

RESULTS

The main characteristics and biochemical data of HD patients and control subjects were summarized in Table 1.

Table 1.  Demographic and laboratory data of HD patients and control subjects.

Variables	Controls	HD patients	p-Values
Number	15	43
Gender (M/F)	8/7	23/20
Age (years)	53.67 ± 6.29	52.90 ± 11.80	ns
BMI (kg/m^2)	27.32 ± 4.10	24.86 ± 3.92	<0.05
Albumin (mg/dL)	4.50 (4.40–4.50)	4.10 (3.82–4.30)	<0.001
Creatinine (mg/dL)	0.80 (0.60–0.88)	8.50 (6.77–9.38)	<0.001
Total cholesterol (mg/dL)	163.00 (150.00–178.75)	176.00 (155.00–221.00)	ns
hs-CRP (mg/dL)	1.20 (0.80–1.95)	4.30 (2.32–9.45)	<0.001
HDL-cholesterol (mg/dL)	60.13 ± 8.16	36.77 ± 8.03	<0.001
LDL-cholesterol (mg/dL)	95.00 (80.25–98.00)	105 (85.00–118.25)	<0.05
Triglycerides (mg/dL)	114.00 (71.50–125.75)	220.00 (162.75–298.75)	<0.001

Notes: Values are expressed as means ± SD or medians (25th–75th percentile). BMI, body mass index; hs-CRP, high-sensitivity C-reactive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ns, nonsignificant; HD, hemodialysis.

There were no significant differences in the age of the two groups (p > 0.05). However, BMI was significantly lower in HD patients compared with control subjects (p < 0.05).

The levels of HDL-cholesterol and albumin were significantly lower in HD patients than in control subjects (p < 0.001). On the other hand, levels of triglycerides, hs-CRP, and creatinine were significantly higher in HD patients than that in control subjects (p < 0.001). LDL-cholesterol levels were also significantly higher in HD patients compared with control subjects (p < 0.05). Mean total cholesterol level was not statistically different between HD patients and control groups (p > 0.05).

Levels of Lp-PLA2, arginase, and NO in HD patients and control subjects are summarized in Table 2. Serum Lp-PLA2 mass levels and arginase activity were increased in HD patients compared with control subjects (p < 0.001). On the other hand, total nitrite levels were significantly lower in HD patients than in control subjects (p < 0.001).

Table 2.  Lp-PLA2, arginase, and Nitrite levels of the study population.

Variables	Controls	HD patients	p-Values
Number	15	43
Lp-PLA2 (ng/mL)	160.00 (152.00–191.00)	233.00 (189.25–271.75)	<0.001
Arginase (U/L)	2.18 (1.78–2.59)	4.12 (3.21–7.34)	<0.001
Nitrite (μmol/L)	0.59 ± 0.10	0.40 ± 0.16	<0.01

Note: Values are expressed as means ± SD or medians (25th–75th percentile).

The results of Spearman and Pearson correlations between Lp-PLA2, arginase, and total nitrite levels and other variables are shown in Table 3. In HD patients, Lp-PLA2 mass levels were positively correlated with age (p < 0.05), total cholesterol (p < 0.05), LDL-cholesterol (p < 0.001), triglyceride (p < 0.001), and arginase (p < 0.001) and negatively correlated with HDL-cholesterol (p < 0.05) and total nitrite (p < 0.001) levels. On the other hand, Lp-PLA2 mass levels were not correlated with BMI, albumin, creatinine, and hs-CRP levels (p > 0.05). Arginase activity was positively correlated with albumin (p < 0.05), total cholesterol (p < 0.05), and LDL-cholesterol (p < 0.001) and negatively correlated with total nitrite levels (p < 0.001). Total nitrite levels were correlated positively with total cholesterol (p < 0.05), LDL-cholesterol (p < 0.001), triglyceride levels (p < 0.05), and arginase activity (p < 0.001).

Table 3.  Correlation coefficients of Lp-PLA2, arginase, and nitrite levels of HD patients (n = 43) with other variables.

	Age (years)	BMI (kg/m^2)	Albumin (mg/dL)	Creatinine (mg/dL)	TC (mg/dL)	HDL-C (mg/dL)	LDL-C (mg/dL)	TG (mg/dL)	hs-CRP (mg/dL)	Arginase (U/L)	Nitrite (μmol/L)
Lp-PLA2 (ng/mL)	r = 0.317	r = 0.146	r = 0.015	r = 0.060	r = 0.351	r = −0.303	r = 0.479	r = 0.540	r = 0.232	r = 0.554	r = −0.532
	p = 0.038	p = 0.351	p = 0.926	p = 0.700	p = 0.021	p = 0.049	p = 0.001**	p = 0.001**	p = 0.135	p = 0.001**	p = 0.000**
Arginase (U/L)	r = 0.224	r = 0.086	r = 0.322	r = 0.039	r = 0.352	r = −0.247	r = 0.588	r = 0.295	r = 0.139	–	r = −0.600
	p = 0.149	p = 0.584	p = 0.035	p = 0.806	p = 0.020	p = 0.110	p = 0.000**	p = 0.055	p = 0.373		p = 0.000**
Nitrite (μmol/L)	r = −0.273	r = −0.130	r = −0.094	r = −0.148	r = −0.369	r = 0.194	r = −0.374	r = −0.350	r = −0.151	–	–
	p = 0.077	p = 0.136	p = 0.550	p = 0.345	p = 0.015	p = 0.214	p = 0.014	p = 0.021	p = 0.334	–	–

Notes: Lp-PLA2, lipoprotein-associated phospholipase A2; BMI, body mass index; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TG, trigycerides; hs-CRP, high-sensitivity C-reactive protein.

*p < 0.05, **p < 0.001.

DISCUSSION

In this study, we found elevated Lp-PLA2 mass levels in HD patients compared with control subjects. To the best of our knowledge, this study is the first to investigate the Lp-PLA2 mass levels in HD patients, except one study which showed the presence of higher levels of circulating Lp-PLA2 mass levels in patients with mild renal insufficiency.¹⁸ Two previous studies in uremic patients undergoing various dialysis procedures demonstrated that PAF-AH activity in the plasma of patients with stage 3–4 chronic kidney disease was significantly higher than control subjects.^19,20 In a recent study, Bitla et al.⁶ reported that Lp-PLA2 activity is increased at the end of dialysis session and suggested that HD therapy produces an increase in Lp-PLA2 activity. In contrast, Milionis et al.¹⁹ demonstrated that PAF-AH activity was not different before and after the dialysis procedure.

In this study, Lp-PLA2 mass levels were not correlated with creatinine and albumin levels in HD patients. This finding may exclude the possibility that the increased Lp-PLA2 mass levels are due to a decreased catabolism/clearance of the Lp-PLA2 by the kidneys (Table 3). It is known that the major sources of the plasma Lp-PLA2 are monocyte-derived macrophages and liver cells. Therefore, increased Lp-PLA2 activity in the plasma of HD patients is likely due to enhanced secretion from macrophages. It has been reported that PAF levels increase in patients with renal failure and stimulate the synthesis and secretion of PAF-AH from the liver.^19,21 In our study, HD patients exhibited significantly higher LDL-cholesterol levels than controls; thus, the increased LDL-cholesterol levels could represent the major mechanism for the increased Lp-PLA2 mass levels observed in HD patients in this study. On the other hand, Lp-PLA2 mass levels were found to correlate with LDL-cholesterol and these results may be another mechanism for the increased Lp-PLA2 mass levels in these patients. Not surprisingly, Lp-PLA2 was strongly associated with LDL-cholesterol because LDL is the major carrier of Lp-PLA2.²²

Lp-PLA2 mass levels were strongly correlated positively with triglycerides, age, and total cholesterol and moderately negatively correlated with HDL-cholesterol, as reported in previous studies.^23,24

The prevalence of chronic inflammation is high in dialysis patients and C-reactive protein (CRP) is a widely used inflammatory marker. Korevaar et al.²⁵ suggested that increased CRP levels during a HD session were independently associated with a higher mortality risk. Lp-PLA2 is an independent risk predictor for cardiovascular disease and has been shown to participate in regional vascular inflammation, endothelial dysfunction, and early atherosclerosis in humans.²⁶ Both Lp-PLA2 and hs-CRP levels were increased in HD patients than in control subjects, although there was no correlation between Lp-PLA2 and hs-CRP levels. CRP is a marker of systemic inflammation, while Lp-PLA2 represents a specific marker for local vascular inflammation and endothelial dysfunction, suggesting that both have different pathophysiological mechanisms and providing a plausible explanation for not finding a correlation between these two markers.⁶ Dialysis-induced local inflammation might be able to increase the Lp-PLA2 production as well as CRP production. CRP might not be the ideal marker to study inflammatory changes as a result of dialysis; however, Lp-PLA2 serves as a better biochemical marker to determine the effects of dialysis on inflammatory markers.^6,7

In this study, serum arginase activity was evaluated as another marker of CHD in HD patients. We found increased arginase activity in HD patients compared with control subjects. In a recent study, Eleftheriadis et al.¹⁵ suggested that arginase type 1 may contribute to the pathogenesis of CHD in HD patients and its serum levels could be used as a marker of CHD in this population. Unfortunately, we did not determine activities of arginase isoenzymes separately.

The increased activity of arginase can be attributed to several factors, such as vascular aging,²⁷ wall shear stress,²⁸ hemolysis,²⁹ LysoPC,¹⁴ and OxLDL.³⁴ As mentioned above, LysoPC plays an important role in the effect of Lp-PLA2 on endothelial dysfunction, which impairs endothelial function mainly via oxidative stress.¹⁴ Lyso-PC is known to cause an increase in arginase activity,¹⁴ which results in decreased total nitrite levels as observed in our study. Since increased arginase activity contributes to OxLDL-dependent impairment in NO production,³⁰ it must be noted that both the substrate and the product of Lp-PLA2 increase the activity of arginase.

Arginase activity in endothelium, as well as in VSMC increases under inflammatory conditions. Arginase hydrolyzes l-arginine to urea and l-ornithine, and inhibits NO synthesis by endothelial NOS (eNOS) via competition for the substrate. Other mechanisms incriminated for reduced NO synthesis by arginase are the suppression of the translation of iNOS or inhibition of inducible NOS (iNOS) activity by the product urea and the sensitization of NOS to its endogenous inhibitor asymmetric dimethylarginine. Additionally, the uncoupling of NOS by limited l-arginine availability results in the formation of NO scavenger, superoxide, and finally peroxynitrite, leading to cell damage.¹⁵

Most evidence in the literature shows that total NO production is decreased in renal disease and this is likely due to, in part, impaired NO production by several mechanisms, such as substrate limitation due to decreased renal synthesis of l-arginine and competition with other metabolic pathways that involve arginase activity.³¹ We found that arginase activity was correlated positively with Lp-PLA2 and negatively with total nitrite levels in HD patients. Therefore, it can be suggested that arginase and Lp-PLA2 may be novel inflammatory factors in HD-induced endothelium dysfunction and inflammatory status.

Major limitation of our pilot study is that we could not provide data on other specific factors that could have contributed to the alterations observed in the Lp-PLA2 levels in HD patients such as OxLDL and LysoPC levels, and the activity of NOS. However, we are now planning to study these lacking parameters in addition to the available parameters and to address treatment protocols in a more homogenous patient population in a further study.

Durante et al.¹⁴ suggested that the structure and function of kidney are partly maintained by the balance between the competing-metabolizing enzymes, arginase, and NOS. Increased serum levels of Lp-PLA2 and arginase activity in HD patients are emerging as major cardiovascular risk factors, and are probably primary causes of the endothelial dysfunction associated with renal disease. Therefore, reducing the Lp-PLA2 levels and arginase activity may be an important therapeutic target in patients with renal disease.^32–35

In summary, arginase/NO pathway is impaired during the HD sessions due to several factors. Increased Lp-PLA2 mass levels contribute to impaired arginase/NO pathway via both its substrate (OxLDL) and its product (LysoPC). Therefore, increased arginase activity and Lp-PLA2 levels with decreased total nitrite levels seem to be useful biochemical markers in terms of reflecting endothelial dysfunction and associated cardiovascular risks in HD patients.

ACKNOWLEDGMENTS

The authors acknowledge no financial help in conducting this study. This manuscript has been seen and approved by all authors and it is not under consideration for publication elsewhere.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.