Abstract
Background: Cardiovascular diseases (CVDs) are the leading cause of death of patients with chronic renal failure. Apolipoprotein E (apoE) plays an important role in the homeostasis of cholesterol and triglycerides. Objective: We aimed to investigate the possible link(s) between apoE gene polymorphism, inflammation and lipoproteins in hemodialysis patients. Methods: We studied 109 end-stage renal disease (ESRD) patients and 97 controls. The serum lipids, apolipoproteins, lipoprotein particles, high-sensitivity C-reactive protein (hs-CRP) and total homocysteine (t-Hcy) levels and paraoxonase (PON) activity were determined in our patients. We also analyzed apoE gene polymorphism in the patients and controls. Results: The analysis of the apoE gene demonstrated a predominance of the e3 allele in both the patients and controls, followed by the e4 and then the e2 alleles. The analysis of the apoE genotype and allele frequencies showed significantly higher e4 allele and E3E4 genotype frequencies and decreased e3 allele and E3E3 genotype frequencies in the patients compared with the controls. The e2, e4 and E3E4 carriers within the ESRD patient population presented an atherogenic lipid profile. However, there were no significant variations in the serum PON activity and the hs-CRP and t-Hcy levels between individuals with different apoE polymorphisms. Conclusions: Our findings suggest an association between the e4 allele, E3E4 genotype and ESRD. The apoE polymorphism affects the serum lipoprotein levels, and the ESRD patients who are e4 and e2 allele carriers are more likely to present an atherogenic lipoprotein profile that may be a major factor associated with increased risk of CVD.
Introduction
Atherosclerotic vascular diseases are the leading cause of death among dialysis patients. Chronic renal insufficiency (CRI) is classified as a risk factor of vascular events. Dyslipidemia, malnutrition, oxidant stress and inflammation have been reported as specific vascular risk factors that appear in CRI. Genetic factors are not excluded. Several studies have demonstrated the existence of genetic abnormalities among dialysis patients that lead to lipid metabolism disturbances, such as familial hypercholesterolemia,Citation1 polymorphism of the apolipoprotein (apo) AI-CII-AIV gene clusterCitation2 and paraoxonase (PON) polymorphism.Citation3 However, it is not yet well established how these factors contribute to the development of accelerated atherosclerosis in dialysis patients. Thus, the interactions between these factors need to be investigated. The genetic polymorphism of apolipoprotein E (apoE) has been reported by several studies to be involved in lipid metabolism abnormalities and in several metabolic diseases, such as Alzheimer disease and cataracts, in the general population.Citation4 ApoE is a plasma protein that serves as a ligand for low-density lipoprotein (LDL) receptors and is synthesized in various organs, including the liver, brain, spleen and kidney. This protein is present at high concentrations in interstitial fluid, where it appears to participate in the cholesterol distribution among cells by transferring excess cholesterol to those cells that require cholesterol. ApoE also appears to be involved in the repair response to tissue injury; for example, markedly increased levels of apoE are found at sites of peripheral nerve injury and regeneration. Other functions of apoE that are unrelated to lipid transport have been investigated, and these include immunoregulation and the modulation of cell growth and differentiation. The gene encoding this apo has been found to exhibit a polymorphism that results in six phenotypes, namely E2/E2, E2/E3, E2/E4, E3/E3, E3/E4 and E4/E4 from the three co-dominant alleles e2, e3 and e4.Citation5 Several studies have demonstrated a correlation between the frequency of the e4 allele compared with other alleles and the lipid disturbances that are responsible for cardiovascular events. Thus, the aim of this study was to investigate the lipid profile in end-stage renal disease (ESRD) patients on long-term hemodialysis and to determine the distribution of apoE allele and genotype frequencies and their effects on lipid metabolism.
Patients and methods
Study subjects
One-hundred nine ESRD patients on long-term hemodialysis whose clinical data were described in detail previously were enrolled in this study.Citation6 The exclusion criteria included antioxidant and/or hypolipidemic medication and clinical signs of infectious diseases. Briefly, the patients were aged 44.92 ± 14.4 years. The length of dialysis treatment was 9.44 ± 4.42 years. All of the patients were under maintenance of dialysis for 4 h during three sessions per week. The dialysis membrane was composed of polysulfone, and bicarbonate was used as a dialysis solution for all the patients. The mean levels of albumin were 40.26 ± 4.16 g/dL. Twenty percent of the patients had hypertension, and 3% suffered from anemia. All the patients were administered calcium. Sixteen percent of the patients were given various vitamin D supplements, and 20% received antihypertensive drugs. Ninety-seven healthy subjects matched for age and gender were used as the controls. The mean age of the controls was 46.81 ± 11.75 years. All of the patients and controls provided informed consent, and the study was approved by the local ethics committee.
Biochemical tests
Blood samples were collected after 12 h of overnight fasting prior to the hemodialysis session. The sera were stored at −20 °C until analysis. Fresh sera were used for the determination of lipids using enzymatic methods. Specimens with an anti-coagulant were stored at −20 °C until analysis of the apoE gene polymorphism. LDL-cholesterol and pure LDL-cholesterol (LDL-p) were calculated according to the Friedewald formula.Citation7 Apo AI, B and E were determined by nephelometry (Dade Behring Nephelometer BN100; Marburg GmbH, Marburg, Germany). Lipoprotein particles (Lp), namely LpAI and LpAI:AII, were determined by immunoelectrophoresis on agarose gels, as previously described.Citation8 Lp(a) was determined through the Dade Behring N Latex agglutination assay. The serum levels of high-sensitivity C-reactive protein (hs-CRP) were measured through the Dade Behring Cardio phase hs-CRP assay. We evaluated the total L-homocysteine using the AxSYM homocysteine assay based on the fluorescence polarization immunoassay method with the Abbott AxSYM system (Abbott Diagnostics, Abbott Park, IL). The PON activity was determined using a paraoxon-like substrate, as previously described.Citation9
Molecular analysis of the apoE gene
The genetic polymorphism of apoE was determined as described by Hixson and VernierCitation10 after the leukocyte DNA (deoxyribonucleic acid) was extracted through the phenol–chloroform method from the frozen specimens. Briefly, the DNA was amplified by polymerase chain reaction (PCR) using the oligonucleotide primers F4 (5′-ACAGAATTCGCCCCGGCCTGGTACAC-3′) and F6 (5′-TAAGCTTGGCACGGCTGTCCAAGGA-3′). After PCR amplification, five units of the HhaI restriction endonuclease were added directly to each reaction mixture for digestion of the apoE sequences (> 3 h at 37 °C). Each reaction mixture was loaded onto an 8% polyacrylamide nondenaturing gel and electrophoresed for 3 h under a constant current (45 mA). After electrophoresis, the gel was treated with ethidium bromide (0.2 mg/L) for 10 min, and the DNA fragments were visualized by ultraviolet illumination. The sizes of the HhaI restriction endonuclease fragments were estimated by comparisons with markers of known size.
Statistical analysis
The allele and genotype frequencies were estimated by the gene-counting method. Chi-square test was used to investigate the differences between the observed and expected genotype frequencies assuming Hardy–Weinberg equilibrium and to study the differences in the allele frequencies between the ESRD patients and controls. The odds ratios (OR) with 95% confidence intervals (CI) and p values were calculated to examine the association of apoE polymorphism and ESRD in patients on hemodialysis. The results of all of the studied parameters are summarized by their means ± standard deviation (SD). Student t-test and ANOVA were performed to compare the lipids, lipoproteins and apos between the groups with different apoE genotypes and alleles, which were grouped according to the following form: the group of apoe3-containing genotypes, which is considered the reference group, includes E3/E3 and E2/E3, and the group of apoe4-containing genotypes includes E3/E4 and E2/E4. The statistical analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL). p Values less than 0.05 were considered statistically significant.
Results
ApoE allele and genotype frequencies in ESRD patients and healthy subjects
As shown in , the E3/E3 genotype and the e3 allele were the most common in ESRD patients on hemodialysis and in the controls. In addition, the E2/E2 and E4/E4 homozygous genotypes were absent from both groups. The chi-square test showed that the e3 and e4 allele frequency distribution was significantly different between ESRD patients on hemodialysis and control subjects. The E3/E3 and E3/E4 genotype frequency distribution was also significantly different between both groups. In fact, the ESRD patients exhibited lower frequencies of the e3 allele and the E3/E3 genotype (p < 0.001) and higher frequencies of the e4 allele and the E3/E4 genotype compared with the controls (p < 0.001). The distribution of the e2 allele and the frequencies of the apoe2-containing genotypes (E2/E3 and E2/E4) presented no significant differences between both groups. The ESRD association analysis () of ESRD patients compared with the controls revealed that carriers of the e4 allele and the E3E4 genotype exhibit higher risks of ESRD (OR = 0.491, p = 0.009 and OR = 0.316, p < 0.001, respectively).
Table 1. Distribution of apoE alleles and genotypes in ESRD patients on hemodialysis and controls.
Table 2. Disease association analyses according to apoE polymorphism in ESRD patients.
Lipoprotein disturbance and inflammation in ESRD patients with different apoE alleles
As indicated in , we separated the ESRD patients on hemodialysis into three allele groups. The e2 allele carriers showed an increase in the serum levels of total cholesterol (TC), LDL-C and LDL-p and the TC/high-density lipoprotein-cholesterol (HDL-C) and LDL-C/HDL-C ratios compared with the e4 and e3 carriers. The ESRD patients with the e2 allele also exhibited increased apoE serum levels and decreased PON activity compared with the e3 carriers, although these differences were not significant. In contrast, the e4 allele carriers showed significantly increased serum levels of LDL-C compared with the e3 allele carriers and lower HDL-C, apoAI, and LpAI levels than the e2 carriers. However, there were no significant differences in the serum levels of total homocysteine (t-Hcy) and hs-CRP between the three groups.
Table 3. Lipids, apolipoproteins, lipoprotein particles and inflammation in ESRD patients on hemodialysis according to the apoE alleles.
Lipoprotein disturbance and inflammation in ESRD patients with different apoE genotypes
We separated our ESRD patients on hemodialysis into two groups based on their apoE genotypes: apoe4-containing genotypes (E3/E4 and E2/E4) and apoe3-containing genotypes (E3/E3 and E2/E3). We then observed that the ESRD patients with apoe4-containing genotypes showed a significant increase in their serum concentrations of TC, LDL-C and LDL-p, the TC/HDL-C and LDL-C/HDL-C ratios, and the apoB levels compared with ESRD patients with apoe3-containing genotypes (). However, there were no significant differences in the serum levels of t-Hcy and hs-CRP and in the PON activity between the two groups.
Table 4. Lipids, apolipoproteins, lipoprotein particles and inflammation in ESRD patients on hemodialysis according to the apoE genotypes.
Discussion
In this study, we analyzed the lipoprotein profile, inflammation, serum PON activity and t-Hcy levels with respect to apoE gene polymorphism. ESRD patients on long-term hemodialysis showed disturbed lipid and lipoprotein profiles that were characterized by hypertriglyceridemia, increased serum levels of LDL-C, apoB, apoE and Lp(a), and decreased HDL-C, apoAI, LpAI and LpAI:AII serum levels, as reported previously.Citation6 These patients presented enhanced inflammation associated with the atherogenic lipoprotein profile and decreased serum PON activity.Citation6 As this work is the first study enrolled in Moroccan ESRD patients on hemodialysis, it should provide new information about the relationship between the apoE gene polymorphism, lipoproteins and ESRD in Arab population, especially Moroccan patients. The apoE gene analysis performed in this study indicates that the E3/E3 genotype and the e3 allele were the most common genotype and allele in ESRD patients and control subjects. These results agree with the findings reported by Tascilar et al.Citation11 The E2/E2 and E4/E4 homozygous genotypes were not found in the ESRD patients and controls. The frequency distribution of the e2 allele and the e2-containing genotypes (E2/E3 and E2/E4) showed no significant differences between both groups. In contrast, in Japanese ESRD patients, the e2 allele is more frequent and the e4 allele is less common compared with the controls, and the e2 allele may be associated with ESRD.Citation12,Citation13 Hubacek et al. reported in a large population of Caucasians ESRD patients an association between apoe2 allele and all-cause ESRD especially in one-year non-survivors.Citation14 However, most studies in renal disease patients showed no significant difference in both allele and genotype frequencies between the patients and healthy subjects.Citation15–18
It was recently suggested that the e4 allele has an independent negative impact on renal patient and graft survival in the long term, particularly in older patients.Citation19 In contrast, we observed that the e3, e4, E3/E3 and E3/E4 frequencies distributions are significantly different between ESRD patients and controls. In fact, ESRD patients presented a lower frequency distributions of the e3 allele and the E3/E3 genotype and higher frequency distributions of the e4 allele and E3/E4 genotype than the controls. These results are in agreement with the findings in Caucasians and Swedish patientsCitation20,Citation21 and suggested that the e4 allele and E3E4 genotype are associated with ESRD. A weak association between the e4 allele and a low glomerular filtration rate was recently demonstrated.Citation22 However, the role of apoE in human renal diseases remains controversial. Roussos et al. showed decreased frequency of e4 allele in patients with glomerulonephritis and increased frequency of this allele in patients with autosomal dominant polycystic kidney disease.Citation23 In our study, the most cause of ESRD was chronic glomerulonephritis (n = 33) and nephrosclerosis (n = 15). Unfortunately, in 43.12% of patients, chronic kidney disease was unspecified. Thus, we were unable to analyze the relationship between apoE gene polymorphism and ESRD with regard to the primary disease.
Compared with other healthy Moroccan populations,Citation24–26 the healthy subjects in this study showed a similar apoE allele distribution (): the most common allele was the e3 allele followed by the e4 allele, and a small proportion of the e2 allele was observed. However, compared to other Arab populations,Citation27–29 the control subjects exhibited different allele frequencies, particularly higher frequencies of the e4 allele (). In contrast, our ESRD patients on hemodialysis also showed different allele distributions compared with Arab and non-Arab hemodialysis populationsCitation13,Citation27,Citation30,Citation31 () that was characterized by decreased e2 allele and increased e4 allele frequencies. This difference is likely due to the different number of patients in the different populations examined, their ethnicity, and the rarity of studies that have enrolled Arab patients.
Table 5. ApoE allele frequencies in Moroccan healthy subjects.
Table 6. ApoE allele frequencies in Morrocan healthy subjects and Arab populations.
Table 7. ApoE allele frequencies in ESRD patients, Arab and no-Arab ESRD populations.
The analysis of the lipid parameters in ESRD patients with different apoE polymorphisms revealed that the serum levels of lipids and the atherogenicity ratios are increased in e4 and e2 allele carriers compared with e3 carriers (reference allele). The e2 allele carriers also showed increased apoE serum levels and decreased PON activity compared with the e3 carriers, although these differences were not significant. The e4 allele carriers presented significantly lower HDL-C, apoAI and LpAI levels than the e2 allele carriers. In addition, the ESRD patients on hemodialysis with apoe4-containing genotypes exhibited a significant increase in the serum concentrations of TC, LDL-C and LDL-p, the TC/HDL-C and LDL-C/HDL-C ratios, and the level of apoB compared with the ESRD patients with apoe3-containing genotypes. These results indicate that the e2 and e4 alleles and the E3/E4 genotype are associated with an atherogenic lipid profile that may be a major factor associated with increased risk for coronary artery diseases (CAD). These findings are supported by several studies in ESRD patients. Liberopoulos et al. demonstrated increases in the TC, LDL-C, TG and apoB levels in e4 carriers.Citation13
Arikan et al. showed increased levels of TC, LDL-C and apoB in HD patients with E3E4 genotype.Citation32 Furthermore, Akanji et al. studied an Arab population and demonstrated that the apoe2 allele exhibits some important associations with the lipoprotein levels, and the LDL sub-fractions that may variably influence the risk for coronary heart diseases (CHD) in both CHD patients and healthy controls.Citation33 It was recently demonstrated that patients with the apoe4 isoforms present increased carotid intima-media thickness and more severe and extensive CAD than patients with the apoe3 isoforms.Citation34 It has been well evidenced that the apoE polymorphism affects cholesterol metabolism. However, no patient-based study has demonstrated the relationship between this polymorphism and cardiovascular disease (CVD) incidence.
To our knowledge, this work is the first study to investigate the PON activity and inflammation with regard to the apoE gene polymorphism in ESRD patients on hemodialysis. However, the hs-CRP and t-Hcy levels and the PON activity showed no significant differences between different apoE alleles and genotypes in this report. These results are inconsistent with the findings reported by Ravaglia et al., who demonstrated that elderly patients who are apoe4 carriers exhibit a lower risk of hyperhomocysteinemia and higher CRP than those who are not carriers of the apoe4 genotype.Citation35 Thus, because of the few number of studies on the apoE gene that enrolled ESRD patients on hemodialysis, the examination of inflammation with respect to the apoE gene polymorphism requires a large cohort of patients, and several investigations are necessary to confirm these findings.
Conclusion
In conclusion, this study suggests an association between the e4 allele, the E3E4 genotype and ESRD. The ApoE polymorphism affects the serum lipoprotein profile in ESRD patients on hemodialysis, and the e4 and e2 allele carriers exhibited an atherogenic lipoprotein profile that may be a major factor associated with increasing risk for CVDs. Further investigations of ESRD patients on hemodialysis and Arab populations may be required to confirm these findings.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Van Tits L, De Graf J, Hak-Lemmers H, et al. Increased levels of low-density lipoprotein oxidation in patients with familial hypercholesterolemia and in end-stage renal disease patients on hemodialysis. Lab Invest. 2003;83:13–21
- Atman PO, Samuelsson OG, Moberly J, et al. Apolipoprotein B-containing lipoproteins in renal failure: The relation to mode of dialysis. Kidney Int. 1999;55:1536–1542
- Schiavon R, Battaglia P, De Fanti E, et al. HDL3-related decreased serum paraoxonase (PON) activity in uremic patients. comparison with the PON1 allele polymorphism. Clin Chim Acta. 2002;324:39–44
- Cedazo-Mínguez A. Apolipoprotein E and Alzheimer’s disease: Molecular mechanisms and therapeutic opportunities. J Cell Mol Med. 2007;11:1227–1238
- Mahley RW. Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science. 1988;240:622–630
- Lahrach H, Ghalim N, Taki H, et al. Serum paraoxonase activity, high-sensitivity C-reactive protein, and lipoprotein disturbances in end-stage renal disease patients on long-term. J Clin Lipid. 2008;2:43–50
- Friedwald WT, Levy RI, Frederickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499–502
- Parra HJ, Mezdour H, Ghalim N, Bard JM, Fruchart JC. Differential electro-immunoassay of human LpAI lipoprotein particles on ready to-use-plates. Clin Chem. 1990;36:1431–1435
- Paragh G, Asztalos L, Seres I, et al. Serum paraoxonase activity changes in uremic and kidney-transplanted patients. Nephron. 1999;83:126–131
- Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545–548
- Tascilar N, Dursun A, Ankarali H, et al. Relationship of apoE polymorphism with lipoprotein(a), apoA, apoB and lipid levels in atherosclerotic infarct. J Neurol Sci. 2009;277:17–21
- Oda H, Yorioka N, Ueda C, Kushihata S, Yamakido M. Apolipoprotein E polymorphism and renal disease. Kidney Int Suppl. 1999;71:25–27
- Liberopoulos EN, Miltiadous GA, Cariolou M, Tselepis AD, Siamopoulos KC, Elisaf MS. The influence of serum apolipoprotein E concentration and polymorphism on serum lipid parameters in patients. Am J Kidney Dis. 2004;44:300–308
- Hubacek JA, Bloudickova S, Kubinova R, Pikhart H, Viklicky O, Bobak M; and for the MIA group. Apolipoprotein E polymorphism in hemodialysed patients and healthy controls. Biochem Genet. 2009;47:688–693
- Güz G, Nurhan Ozdemir F, Sezer S, et al. Effect of apolipoprotein E polymorphism on serum lipid, lipoproteins, and atherosclerosis in patients. Am J Kidney Dis. 2000;36:826–836
- Feussner G, Wey S, Bommer J, Deppermann D, Grutzmacher P, Ziegler R. Apolipoprotein E phenotypes and hyperlipidemia in patients under maintenance of hemodialysis. Hum Genet. 1992;88:307–312
- Eggertsen G, Heimburger O, Stenvinkel P, Berglund L. Influence of variation at the apolipoprotein E locus on lipid and lipoprotein levels in CAPD patients. Nephrol Dial Transplant. 1997;12:141–144
- Imura T, Kimura H, Gejyo F. Apolipoprotein E phenotypes in patients on hemodialysis. Kidney Int Suppl. 1999;71:245–247
- Cofán F, Cofan M, Rosich E, et al. Effect of apolipoprotein E polymorphism on renal transplantation. Transplant Proc. 2007;39:2217–2218
- Lerique B, Moulin B, Delpero C, Purgus R, Olmer M, Boyer J. Apolipoprotein E phenotype and hyperlipoproteinemia in nephrotic syndrome. Clin Chem. 1994;40:849–850
- Roussos L, Floren CH, Carlson J, Svensson PJ, Wallmark A, Ekberg H. Increased prevalence of apolipoprotein E3/E4 genotype among Swedish renal transplant recipients. Nephron. 1990;83:25–30
- Chu AY, Parekh RS, Astor BC, et al. Association of APOE polymorphism with chronic kidney disease in a nationally representative sample: A Third National Health and Nutrition Examination Survey (NHANES III) Genetic Study. BMC Med Genet. 2009;10:108
- Roussos L, Ekström U, Ehle PN, Oqvist B, Floren CH. Apolipoprotein E polymorphism in 385 patients on renal replacement therapy in Sweden. Scand J Urol Nephrol. 2004;38(6):504–510
- Valveny N, Esteban E, Kandil M, Moral P. Apo E polymorphism in Spanish and Moroccan populations. Clin Genet. 1997;51:354–356
- Kacemi LN, Bamou Y, Guedira A, et al. Polymorphisme de l'apolipoprotéine E dans une population marocaine: Fréquence allélique et relation avec les paramètres lipidiques plasmatiques. Ann Biol Clin. 2002;60:73–78
- Bennouar N, Allami A, Laraqui A, et al. Implication du polymorphisme génétique de l’apolipoprotéine E et de l’enzyme de conversion de l’angiotensine dans l’athérosclérose coronarienne. Ann Biol Clin. 2004;62:295–304
- Al-Muhanna F, El-Mueilo S, Al-Ali A, et al. Polymorphism in methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and apolipoprotein E in patients. Saudi J Kidney Dis Transplant. 2008;19:937–941
- Al-Yahyaee SA, Al-Kindi MN, Al-Bahrani AH. Distribution of apolipoprotein E alleles in the Omani population. Med Princ Pract. 2005;14:73–78
- Al-Shammari S, Fatania H, Al-Radwan R, Akanji AO. Apolipoprotein E polymorphism and lipoprotein levels in a Gulf Arab population in Kuwait: A pilot study. Ann Saudi Med. 2004;24:361–364
- Zahálková J, Vaverková H, Novotný D, Kosatíková Z. Impaired triglyceride tolerance in patients with different apolipoprotein E (Apo E) isoforms. Biomed Papers. 2002;146:73–76
- Oda H, Yorioka N, Ueda C, Kushihata S, Yamakido M. Apolipoprotein E polymorphism and renal disease. Kidney Int Suppl. 1999;71:25–27
- Arikan H, Koc M, Sari H, Tuglular S, Ozener C, Akoglu E. Associations between apolipoprotein E gene polymorphism and plasminogen activator inhibitor-1 and atherogenic lipid profile in dialysis patients. Ren Fail. 2007;29(6):713–719
- Akanji AO, Suresh CG, Fatania HR, Al-Radwan R, Zubaid M. Associations of apolipoprotein E polymorphism with low-density lipoprotein size and subfraction profiles in Arab patients with coronary heart disease. Metabolism. 2007;56:484–490
- Granér M, Kahri J, Varpula M, et al. Apolipoprotein E polymorphism is associated with both carotid and coronary atherosclerosis in patients with coronary artery disease. Nutr Metab Cardiovasc Dis. 2008;18:271–277
- Ravaglia G, Forti P, Maioli F, et al. Apolipoprotein E e4 allele affects risk of hyperhomocysteinemia in the elderly. Am J Clin Nutr. 2006;84:1473–1480