NDT Advance Access originally published online on March 22, 2006
Nephrology Dialysis Transplantation 2006 21(7):1921-1926; doi:10.1093/ndt/gfl115
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Dialysis and Transplantation
The E-selectin gene polymorphism and carotid atherosclerosis in end-stage renal disease
1 CNR-IBIM, National Research Council-Institute of Biomedicine, Clinical Epidemiology and Physiopathology of Renal Diseases and Hypertension, Reggio Calabria, 2 Cardiology Unit, Morelli Hospital, Reggio Cal and 3 Department of Internal Medicine, Catania University, Catania, Italy
Correspondence and offprint requests to: Prof. Carmine Zoccali, CNR-IBIM, Istituto di Biomedicina, Epidemiologia Clinica e Fisiopatologia delle Malattie Renali e dell’Ipertensione Arteriosa, c/o Ki Point – Gransial Srl, Via Filippini, 85, 89125 Reggio Calabria, Italy. Email: carmine.zoccali{at}tin.it
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Abstract |
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Background. E-selectin is a cell surface glycoprotein that mediates the adhesion of leucocytes to vessels endothelium, an important early step in the atherosclerotic process. End-stage renal disease (ESRD) is a highly atherogenic disease but it is unknown whether genetic polymorphism(s) in the E-selectin gene plays a role in the severity of arterial damage in this condition.
Method. In this study, we tested whether the Leu554Phe variant in the E-selectin gene is linked to carotid atherosclerosis in 134 well-characterized ESRD patients. The frequency of this polymorphism was also measured in a population sample of the same geographical area.
Results. A total of 84% patients had the CC genotype, 13% had the CT genotype, 3% had the TT genotype and this distribution did not differ from that in the control population. Intima-media thickness (IMT) (P = 0.01) and cross-sectional area (P = 0.02) were significantly higher in patients with the T-allele than in those without this allele. Furthermore, the degree of carotid stenosis was significantly higher (P = 0.02) in patients with T-allele than in CC patients. On multivariate analyses including the traditional and non-traditional risk factors, the Leu554Phe polymorphism was confirmed as an independent correlate of IMT (P = 0.02), cross-sectional area (P = 0.03) and carotid stenosis (P = 0.02).
Conclusion. In ESRD, the Leu554Phe polymorphism of E-selectin gene is associated with the severity of carotid atherosclerosis, suggesting that genetically-determined alterations in the E-selectin molecule may render ESRD patients with this gene variant particularly susceptible to the detrimental effects of inflammation on the arterial wall.
Keywords: atherosclerosis; dialysis; E-selectin gene; polymorphism
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Introduction |
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In the last decade, inflammation has emerged as a risk factor of paramount importance in patients with end-stage renal disease (ESRD) [1–3]. Inflammation markers such as C-reactive protein (CRP) [3], interleukin-6 (IL-6) [4,5] and serum fibrinogen [6] represent strong predictors of death and incident cardiovascular (CV) events in this population. Inflammation is a complex, multifactorial and multistage process, and the adhesion of leucocytes to an activated endothelium is a fundamental early event in this process. The selectin family of adhesion molecules shares a structure consisting of an extracellular domain with a variable number of repeats, a transmembrane domain and a short cytoplasmic domain. E-selectin, one of the three members of this family, has been shown to mediate the recruitment of circulating leucocytes by physically supporting adhesive interactions, participating in cell signalling and rolling, inturn that lead to firm adhesion through the activation and binding of ß2-integrin [7–10].
Various environmental factors may trigger inflammation in the arterial wall such as low-density lipoprotein cholesterol, infection, smoking and other factors [11]. On the other hand, several lines of evidence indicate that besides environmental factors, the genetic background has an important influence an inflammation-induced arterial damage [12]. In this regard, it is of particular interest that a polymorphism in the E-selectin gene (Leu554Phe) altering the E-selectin shedding from the endothelium was associated with severe coronary atherosclerosis [13]. Inflammation in the arterial system in ESRD [14] may be incited by disparate mechanisms. Whether the genetic background plays a role in inflammation and atherosclerosis in this population is unknown. The question is relevant because risk factors for atherosclerosis in uraemia do not coincide with those in the general population. Given the crucial role of adhesion molecules in atherosclerosis, we focused our attention on the Leu554Phe polymorphism because it increases the E-selectin anchoring to the endothelium [15], an alteration that may amplify the inflammatory effect of leucocytes on the arterial wall. In this study, we report the association between this gene variant and carotid atherosclerosis in a sizable group of ESRD patients.
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Methods |
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The protocol was in conformity with the local ethical guidelines of our institution and informed consent was obtained from each participant.
Patients
A total of 134 dialysis patients (80 males and 54 females), all residents in the urban area of Reggio Calabria, 88 on haemodialysis (HD) and 46 on chronic ambulatory peritoneal dialysis (CAPD) who had been on regular dialysis treatment (RDT) for at least 6 months and who were free of overt infections (fever, infected vascular access or peritonitis or exit-site infection) were recruited for the study. HD patients were being treated thrice a week with standard bicarbonate dialysis (Na, 138 mmol/l; HCO3, 5 mmol/l; K, 1.5 mmol/l; Ca, 1.25 mmol/l; Mg, 0.75 mmol/l) either with Cuprophan or semi-synthetic membranes. The average urea Kt/V in these patients was 1.29±0.29. The remaining 46 patients were on CAPD (weekly Kt/V 1.66±0.33). All HD patients were virtually anuric (24 h urine volume <200 ml/day), while a minority of CAPD patients (n = 6) had a 24 h diuresis >200 ml/day. Nineteen patients were diabetic and 67 were habitual smokers (22±18 cigarettes/day). Seventy-seven patients were treated with various antihypertensive drugs (51 on mono-therapy with ACE inhibitors, AT-1 antagonists, calcium-channel blockers and alpha- and beta-blockers, and the remaining 26 on double or triple therapy with various combinations of these drugs). Sixty-six patients were on treatment with erythropoietin.
Control subjects
For genetic data analysis, we considered as control group a series of 105 healthy blood donors from the same geographical area of patients. Controls were Caucasians, and at the time of the recruitment all the participants were apparently healthy and free from acute or serious chronic diseases such as cancer, hypertension, diabetes and coronary heart disease. All participants were CV event-free. These subjects were accurately matched to the study population as for age (56±7 years) and sex (males: 71, females: 34). The matching procedure for age was done by 5 year categories.
Carotid ultrasonography
In all patients, intima media thickness (IMT) was measured as previously reported [16]. Cross-sectional area was calculated by the standard formula [17]. In stenotic arteries, the degree of stenosis was estimated by the method proposed by Blackshear et al. [18]. Repeated studies of 105 dialysis patients by a blinded observer in our laboratory showed that IMT represents a reliable measurement in dialysis patients because its coefficient of variation was 5.5%.
Laboratory measurements
For HD patients, fasting blood sampling was performed during the mid-week non-dialysis day, and for CAPD patients on an empty stomach. Serum cholesterol, albumin, calcium and phosphate and haemoglobin measurements were made using standard methods in the routine clinical laboratory. CRP and plasma total homocysteine were determined as previously reported [19]. The immuno-enzymometric technique (ELISA) for serum E-selectin assay was supplied from R&D (Abingdon, UK). The intra-assay and inter-assay coefficients of variation were 4.9 and 6.8%, respectively. The sensitivity of the assay was <0.1 µg/l. No cross-reactivity was found with natural human IgG, recombinant human VCAM-1, ICAM-1 or natural P-selectin. In our laboratory, the reference range in sex- and age-matched healthy subjects was 27–55 µg/l.
Genetic analysis of Leu554Phe polymorphism of E-selectin gene, restriction fragment length polymorphism (RFLP) validation
Genomic DNA was extracted from peripheral blood leucocytes using the salting-out technique [20]. The C1839T polymorphism in exon 10 leads to the substitution of leucine (Leu) by phenylalanine (Phe) at amino acid 554 (Leu = allele-C; Phe = allele-T). This alteration was identified by polymerase chain reaction (PCR) using the following primer pair: F1, 5'-TCTCCCTCCTGACATTAGCA-3' and R1, 5'-CTGAAACACTTCCCACACTGAG-3'. PCR was carried out in 25 µl final volume containing 100 ng of genomic DNA, 0.2 µmol/l of each primer, 200 µmol/l of each deoxyribonucleoside triphosphate (dNTP), 2.5 µl of 10x buffer [100 mmol/l of Tris–HCl (pH 8.3), 500 mmol/l of KCl and 15 mmol/l of MgCl2], and 0.2 U of Taq DNA polymerase. The reaction mixture was subjected to 5 min of denaturation at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 53°C and 30 s at 72°C, and a final extension of 7 min at 72°C. The mutation abolishes a Bsa XI restriction site; the PCR resulting 239 bp fragment was digested in the wild allele in three fragments of 199, 30 and 10 bp while the mutated allele remained unmodified. After the restriction analysis, the resulting fragments were separated on 2% agarose gel. To validate the results of RFLP analysis, a 10% random sample of the study population was re-analysed by gene sequencing which fully confirmed the results of RFLP.
Statistical analysis, study power
Data are expressed as mean and SD, median (inter-quartile range) or as percent frequency, and comparisons between two groups were made by t-test, Mann–Whitney test or chi-square test, as appropriate. Comparisons among ordered groups were made by P for trend [21]. Variables showing a positively skewed distribution (Kolmogorow–Smirnow test) were log transformed (log10).
The relationship between the Leu554Phe polymorphism of the E-selectin gene (defined according to a dominant model) and the severity of carotid atherosclerosis (as estimated by IMT, cross-sectional area and carotid stenosis) was analysed by univariate and multivariate linear regression analysis. Tested covariates included the Leu554Phe polymorphism as well as a series of traditional risk factors [age, sex, body mass index (BMI), smoking, previous CV events, diabetes, parathormone (PTH), cholesterol, arterial pressure, heart rate and anti-hypertensive treatment], factors peculiar to ESRD (duration of RDT, albumin, haemoglobin, calcium phosphate product, treatment modality and anti-hypertensive treatment) and emerging risk factors (like CRP and homocysteine). To obtain parsimonious models, significant independent variables were identified by a backward elimination strategy. By this approach, we constructed models of adequate statistical power (at least 16 patients for each variable in the final model). Furthermore, to assess differences in multiple dependent variables (IMT, cross-sectional area and carotid stenosis considered simultaneously) in patients with and without the T-allele (dominant model), we performed a multiple analysis of variance (MANOVA).
To calculate the study power, we considered as relevant a difference in IMT of 0.1 mm between TT + CT and CC patients. Testing for such a difference (study power 80%, 
-error = 0.05 two-tailed) demands that the number of patients in the study population should be 176 patients.
Data are expressed as correlation coefficients (R), standardized regression coefficient (ß), partial correlation coefficients (partial R) and P-value. All calculations were done using a standard statistical package (SPSS for Windows Version 9.0.1, 11 March 1999).
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Results |
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The genotype distribution of the Leu554Phe polymorphism of the E-selectin gene did not deviate from Hardy–Weinberg equilibrium either in patients (CC genotype, 84%; CT genotype, 13%; TT genotype, 3%) or in control healthy subjects (CC genotype, 83%; CT genotype, 11%; TT genotype, 6%), and the allele frequency was almost identical in these two populations. When patients were categorized on the basis of the presence/absence of the risk allele (i.e. TT and CT vs CC patients), no difference emerged between the two groups as for demographic and somatometric data or for clinical and biochemical parameters (Table 1). Likewise, plasma E-selectin did not differ between the two groups.
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E-selectin gene and carotid atherosclerosis
IMT (P = 0.01), cross-sectional area (P = 0.02) and the degree of carotid stenosis (P = 0.02) were significantly higher in patients with the T-allele (i.e. in CT and TT patients) than in those without this allele (i.e. CC patients) (Figure 1). MANOVA analysis showed that there was a significant global difference (P = 0.03) in the three indicators of carotid atherosclerosis (IMT, cross-sectional area and carotid stenosis), all being significantly higher in patients with the T-allele than in those without such an allele (dominant model). Collectively, plasma E-selectin was unrelated to indicators of carotid atherosclerosis (R ranging from 0.009 to –0.10, P = NS). However, separate analysis according to the genotype revealed that plasma E-selectin was inversely related to IMT (R = –0.48, P = 0.03) (Figure 2) in patients with the T-allele, while no such relationship existed in those without the T-allele (R = 0.06, P = 0.55). It is notable that IMT was related in a dose–response fashion with the number of T-alleles, being 1.03±0.23 mm in patients without such an allele, 1.13±0.22 mm in those with one T-allele and 1.26±0.22 mm in those with two T-alleles (P for trend = 0.008).
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E-selectin gene and carotid atherosclerosis: multiple regression analyses
Since the association between E-selectin genotypes and carotid atherosclerosis could be confounded by other risk factors, we performed multiple regression analyses adjusting for all variables which were associated with the three indicators of carotid atherosclerosis. In these analyses, the Leu554Phe polymorphism of E-selectin gene was confirmed as a correlate of IMT, cross-sectional area and the severity of carotid stenosis (Table 2).
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Discussion |
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In patients with ESRD, the Leu554Phe polymorphism of E-selectin gene is coherently associated to three indicators of carotid atherosclerosis. These associations suggest that genetically determined alterations in this adhesion molecule participate in the pathogenesis of atherosclerosis in ESRD.
Carotid atherosclerosis and inflammation in ESRD
Atherosclerosis is the main cause of death and CV complications in ESRD. Traditional risk factors (i.e. hypertension, smoking, diabetes and dyslipidaemia) only in part explain the increased risk of death and CV disease in this population [22]. Attention has therefore been focused on disease-specific factors like hyperparathyroidism, hypoalbuminaemia and anaemia and on emerging factors like hyperhomocysteinaemia and inflammation. Inflammation in ESRD is a complex, multifactorial phenomenon because it may be triggered and/or amplified by a variety of patient-related and dialysis treatment-related risk factors [2,4,14]. CRP [3] and IL-6 [4,5] are considered the most reliable indicators of systemic inflammation in ESRD because these substances are coherently associated with the severity of atherosclerosis and predict death and incident CV complications in this population.
E-selectin polymorphism
Environmental factors apart, a substantial body of evidence has been accrued indicating that genetic factors are of relevance in atherosclerosis. Association studies between genetic factors and indicators of arterial damage are therefore important in order to understand the unique severity of CV disease in ESRD [23]. In the general population, various polymorphisms in genes regulating the inflammatory response have been associated with atherosclerosis. Among these gene variants, a point mutation in the gene producing E-selectin (Leu554Phe) appears to be of particular interest. Indeed, this polymorphism alters the release of the gene product and may, therefore, result in a tighter anchoring of leucocytes into the endothelium [15]. It is notable that this mutation was associated with the early onset atherosclerosis [13] and with hypertension in overweight and obese individuals [24], an observation which fits with the notion that obesity represents a pro-inflammatory condition and that endothelial adhesion molecules are up-regulated in this condition [25]. Ours is the first study associating the Leu554Phe polymorphism of E-selectin gene to objective measures of atherosclerosis. The dialysis population is an interesting natural model of atherosclerosis because uraemia is a strong amplifier of arterial damage [26]. In the present study, we considered three well-established indicators of carotid atherosclerosis, namely IMT [27], cross-sectional area [18] and arterial stenosis [19]. These indicators reflect different aspects of the atherosclerotic process [28]. In fact, on multivariate analyses we found that the independent correlates of these indicators did not coincide (Table 2). Yet, inflammation emerged as a coherent determinant of carotid atherosclerosis in the ESRD population because CRP, an inflammatory marker sensitive to environmental factors such as dialysis treatment [29], and a genetic marker of the same process like the E-selectin polymorphism were the sole factors which maintained an independent association with all indicators of carotid atherosclerosis. Notably, the association of the Leu554Phe with atherosclerosis was largely independent of CRP. This phenomenon indicates that this polymorphism reflects a component of the inflammatory process different from that of CRP. This contention is coherent with biological knowledge indicating that a mechanism(s) whereby CRP induces arterial damage does not coincide with that of E-selectin. In this respect, our finding that an inverse link exists between circulating E-selectin and IMT in patients with the T-allele but not in those without such an allele, would support the biological interpretation that this gene variant affects the inflammatory process by increasing the anchoring of E-selectin to the vascular endothelium. However, this speculation needs to be tested in a larger database and also independently of E-selectin genotype.
In the present study, the frequency of the T-allele was higher than that reported in the French population [24] and in the German population [13]. To test whether the higher prevalence of the T-allele is due to a co-segregation of the Leu554Phe polymorphism with ESRD, we measured the frequency of the same polymorphism in a carefully matched sample of healthy subjects of the same geographic area. In this reference group, the frequency of the T-allele (11%) resulted in being almost identical to that of dialysis patients (9%), indicating that the higher frequency of this allele in our patients depends on genetic background rather than on co-segregation of the T variant with ESRD.
A limitation of this study is that we failed to enroll the number of patients required by power analysis (n = 176 vs 134 actually enrolled into the study). Patients included in this study are part of a larger cohort of dialysis patients enrolled in 1997 and prospectively followed for about 8 years (CREED study). Carotid ultrasonography was performed only in patients of a single centre (i.e. in all patients of Reggio Calabria) to avoid problems due to different operators and equipment. This was a prevalent-incident cohort. The number of incident patients was less than expected, and for this reason we failed to reach the study target.
Formally, the power of the present study for detecting as significant (-error = 0.05 two-tailed) a 0.1 mm difference of IMT between TT and CT + CC patients was less (71%) than the conventional threshold of 80%.
In conclusion, in ESRD patients the Leu554Phe polymorphism of E-selectin gene is associated with severe atherosclerosis. Genetically determined alterations in the E-selectin molecule may render dialysis patients with this polymorphism particularly sensitive to the detrimental effect of inflammation on the arterial system.
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Acknowledgments |
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We thank Rosa Maria Parlongo for her technical contribution. This study was supported by grants of the National Research Council of Italy (CNR).
Conflict of interest statement. None declared.
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Accepted in revised form: 22. 2.06
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