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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2017  |  Volume : 28  |  Issue : 5  |  Page : 997-1002
Association between apolipoprotein E polymorphism and nephropathy in Iranian diabetic patients

1 Department of Medical Biochemistry, Tehran University of Medical Sciences, Tehran, Iran
2 Endocrine and Metabolic Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
3 Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Biology, Payam Noor University of Mashhad, Mashhad, Iran
5 Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

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Date of Web Publication21-Sep-2017


Approximately one-third of diabetic patients develop evidence of nephropathy. Pathogenesis of diabetic nephropathy (DN) remains unclear; however, some genetic and metabolic risk factors have been determined for the development and progression of DN. In the recent genetic studies, polymorphism of apolipoprotein E (ApoE) gene has been reported as a risk factor for the development of DN; however, the results are inconsistent. The aim of the present study was to evaluate the association between ApoE polymorphism and nephropathy in Iranian patient with type 2 diabetes. A total of 197 patients with type 2 diabetes in two groups with and without nephropathy (n = 99 and n = 98, respectively) participated in this case–control study. ApoE genotype was determined by restriction fragment length polymorphism analysis. Biochemical factors of all patients were measured. The frequency of Apo ε4 allele was significantly (P <0.05) lower in DN patients (10.6%) than in diabetic patients without nephropathy (20.4%). No significant difference was observed between the groups regarding Apo ε2 and Apo ε3 allele frequencies. Serum level of total and low-density lipoprotein cholesterol in Apo ε2 carriers was lower than Apo ε3 and Apo ε4 carriers, but this difference was not statistically significant. Frequency of Apo ε4 allele is higher in diabetic patients without nephropathy than DN participants. Given to the result, it seems that Apo ε4 has a protective effect in diabetic patients against nephropathy.

How to cite this article:
Karimoei M, Pasalar P, Mehrabzadeh M, Daneshpour M, Shojaee M, Forouzanfar K, Razi F. Association between apolipoprotein E polymorphism and nephropathy in Iranian diabetic patients. Saudi J Kidney Dis Transpl 2017;28:997-1002

How to cite this URL:
Karimoei M, Pasalar P, Mehrabzadeh M, Daneshpour M, Shojaee M, Forouzanfar K, Razi F. Association between apolipoprotein E polymorphism and nephropathy in Iranian diabetic patients. Saudi J Kidney Dis Transpl [serial online] 2017 [cited 2018 Nov 15];28:997-1002. Available from: http://www.sjkdt.org/text.asp?2017/28/5/997/215137

   Introduction Top

Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus and occurs in approximately 40% of diabetic patients.[1] DN is the main cause of end-stage renal disease (ESRD) in the world.[2] Different risk factors are associated with DN development such as long-term hypertension and hyperglycemia which are important but not sufficient for the development of nephro-pathy.[3],[4] Familial clustering and epidemiolo-gical evidences have suggested that genetic susceptibility is required for the development of DN. Despite extensive research, responsible genes and underlying molecular mechanisms remain unclear yet.[5],[6] Identification of genetic determinants would be essential for primary intervention to prevent ESRD and provide clinical and economic benefits.[7],[8]

Abnormal lipid metabolism is proposed as an effective factor for the development and progression of DN.[9],[10] Apolipoprotein E (ApoE) present in the triglyceride-rich lipoprotein such as chylomicron and very low-density lipoprotein (LDL) has a central role in lipid metabolism. There are three alleles for ApoE (ε2, ε3, and ε4) that code ApoE2, ApoE3, and ApoE4 isoforms. Three alleles form six different genotypes as E2/2, E3/2, E3/3, E4/2, E4/3, and E4/4. ApoE3 is the most common form (arginine at residue 158 and cysteine at residue 112). A substitution of arginine to cysteine at residue 158 forms Apo ε2 allele with lower affinity for its receptor while Apo ε4 allele with a substitution of cysteine to arginine at residue 112 shows a higher affinity for its receptor.[11] A reasonable number of studies have investigated the association of the genetic polymorphism of ApoE and DN; however, the results are unconvincing. To the best of our knowledge, there is no report about the possible effect of ApoE gene on DN in Iranians. The aim of this study was to investigate the association of genetic polymorphism of ApoE with DN in Iranian type 2 diabetic patients.

   Patients and Methods Top


In this case–control study which has been conducted between January and December 2015, a total of 197 Iranian diabetic type 2 patients with more than five-year history of diabetes and aged 35–75 years were recruited. The study was carried out in Diabetes and Metabolic Diseases Research Center affiliated to Endocrinology and Metabolism Research Institute (Tehran University of Medical Sciences). Diabetic patients with albumin-to-creatinine ratio (ACR) more than 30 mg/g in an early morning urine sample were considered as DN (n = 99). A group of diabetic patients (n = 98) without nephropathy (ACR less than 30 mg/g) were matched with DN group by sex. Exclusion criteria were hemoglobin A1C (HbA1C) >9%, smoking, uncontrolled hypertension, pregnancy, urinary tract infection, and receiving antihyperlipidemic treatment.

Body height and weight were measured and body mass index was calculated. Blood pressure was measured in a sitting position with standard mercury sphygmomanometers twice, and mean of measurements was recorded. Other information such as medication and diabetes duration were obtained through questionnaire.

Each individual provided a venous blood and a urine sample for biochemical factors measurement and genetic analysis. Protocol was approved by the Ethics Committee of the Endocrinology and Metabolism Research Institute, and each patient provided informed consents.

Genetic analysis

DNA was extracted from 0.3 mL of peripheral blood leukocytes using DNA extraction kit (Bio Basic, Canada) and amplified for the exon 4 region of the APOE gene by poly-merase chain reaction (PCR) as described pre-viously.[12] The primers for this reaction were: forward primer 5’ ACA GAA TTC GCC CCG GCC TGG TAC AC 3’ and reverse primer 5’ TAA GCT TGG CAC GGC TGT CCA AGG A 3’. PCR product (244bp) digested by incubation for 3 h at 37°C with HhaI restriction endonuclease (10 units) (TAKARA, Japan). Then, 8 μL of digested PCR product was electrophoresed on polyacrylamide gel and visualized by silver staining method.

Biochemical analysis

For laboratory measurements, blood samples were collected after an overnight fasting (10–12 h). Fasting blood glucose (FBS), lipid profile [total cholesterol, high-density lipoprotein (HDL), LDL cholesterol, HDL cholesterol, and triglycerides], urea, uric acid, creatinine, and urine albumin/creatinine were determined by commercial kits (Pars Azmun, Iran). HbA1c was measured by means of high-performance liquid chromatography (Tosoh G8, Japan). Estimated glomerular filtration rate (eGFR) was calculated by Cockcroft-Gault equation.

   Statistical Analysis Top

Continuous data were described as mean and standard deviation. Results were compared using Student’s t-test (or Mann–Whitney U-test for nonparametric variables). Allele frequencies were compared between the groups using Chi-square test or Fisher’s exact test. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 21.0 for Windows (SPSS Inc., Chicago, IL, USA), and P <0.05 was considered statistically significant.

   Results Top

Demographic and biochemical parameters of patients are given in [Table 1]. Sex ratio, systolic and diastolic blood pressure, and also lipid profile did not show statistically significant difference between the two groups. Significant differences were observed in age, ACR, eGFR, creatinine, and duration of diabetes, between diabetic and DN groups.
Table 1: Demographic and biochemical parameters of diabetic and DN patients.

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Distribution of the ApoE alleles and genotypes is shown in [Table 2]. The frequency of ε4 allele was significantly higher in diabetic patients (P <0.05) than DN patients. ε2 and ε3 alleles were more frequent in DN group, but the differences were not statistically significant. The frequency of E3/4 genotype was higher in diabetic group (P <0.05). No significant differences were observed in the distribution of E2/4, E4/4, E2/2, and E3/2 genotypes between diabetic and DN groups. Logistic regression analysis with an odd ratio (OR) of 0.5 (95% confidence interval = 0.26–0.97, P <0.05) showed the protective effect of ε4 allele against DN. We did not find significant association between ε2 allele and DN (OR: 1.67 (0.59–4.41), P >0.05). Allele distribution was in Hardy–Weinberg equilibrium.
Table 2: Distribution of ApoE alleles and genotype among diabetic and DN patients.

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To evaluate the relationship between ApoE alleles and serum lipid levels, patients in each group (DN and diabetic without nephropathy) were divided into ε3/ε3 carriers (E3/3), ε4 carriers (E3/4, E4/4), and ε2 carriers (E2/3, E2/2). Three participants who had E2/4 genotype were excluded from this analysis because of the opposing effect of these alleles on lipid metabolism. We did not observe a significant difference in lipid profile among three ApoE allelic groups. Although ε2 carriers had higher levels of HDL cholesterol and lower levels of LDL and total cholesterol than ε3/ε3 and ε4 carriers, the differences were not statistically significant. Other biochemical values did not show any significant relationship with geno-typic or allelic groups.

   Discussion Top

In the present study, we observed a significantly higher frequency of Apo ε4 allele in diabetic patients without nephropathy (20.4%) than patient with DN (10.6%). On the other hand, frequency of ε2 allele tended to be higher in DN patients, but this difference was not statistically significant. These results suggest that Apo ε4 has a protective effect against DN.

Association of ApoE gene polymorphism and DN has been subject of some studies. Leiva et al[13] reported that Apo ε4 allele has a protective role against the development and progression of DN. In two separate studies, Eto et al[11] and Satirapoj et al[14] suggested that ApoE4 is a protective factor and ApoE2 is a prognostic factor for DN. Kimura[15] studied Apo ε allele polymorphisms in Type 2 DM and observed that Apo ε4 allele is a protective factor against diabetic nephropathy and other alleles are risk alleles. The results of these studies are in the agreement with our observations. However, conflicting results were obtained by Tien et al[16] which introduced APO ε4 as a predictor for DN and also Joss study which showed that patients with APO E3/4 are more prone to DN development.[17] There are also some studies which have not found any association between APO E alleles and DN.[18],[19] Araki et al[20] performed a prospective observational study in Japanese population and reported that Apo ε2 could be a prognostic factor for DN. The same results were obtained in Egyptian patients.[21] In our study, although the frequency of ε2 allele was higher in DN group, the difference was not significant. The results in different populations are inconsistent which may be related to the multifactorial nature of DN.

The results of this study showed insignificant differences in total, LDL, and HDL cholesterol in three allelic groups. ε2 carriers had lower serum level of total and LDL cholesterol than ε3 and ε4 carriers but not statistically significant. Results of two population-based studies showed that APO ε4 can be protective for all causes of chronic renal disease regardless of dyslipidemia.[22],[23] Both of mentioned studies are in line of our results and suggested that protective effect of Apo ε4 against DN might be conducted by different mechanisms. The mesangial expansion is an important factor in the pathogenesis of almost all of renal diseases including DN. Chen et al[24] studied ApoE effects on renal function in mice and showed an antiproliferative effect of ApoE on mesan-gial matrix. Furthermore, they previously showed that ApoE induces matrix heparan sulfate proteoglycan, which inhibits proliferation of mesangial cells. A study by Guan demonstrated association of ε2 allele with increased severity of glomerulopathy and expression of ApoE in the nodular lesion. However, more studies are required to clarify the exact mechanisms of effect of the ApoE alleles on renal function.

In conclusion, it seems that Apo ε4 allele reduces the risk of the development of DN. However, a study with a larger population of DN is required to confirm the relationship of ApoE polymorphism and development of DN.

   Acknowledgment Top

The authors would like to thank the patients and staff of the Diabetes and Metabolic Disease Center.

Conflict of interest: None declared.

   Source of Support Top

This study was financially supported by Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

   References Top

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Satirapoj B. Nephropathy in diabetes. Adv Exp Med Biol 2012;771:107-22.  Back to cited text no. 2
Marshall SM. Natural history and clinical characteristics of CKD in type 1 and type 2 diabetes mellitus. Adv Chronic Kidney Dis 2014; 21:267-72.  Back to cited text no. 3
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Pezzolesi MG, Krolewski AS. The genetic risk of kidney disease in type 2 diabetes. Med Clin North Am 2013;97:91-107.  Back to cited text no. 5
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Baumeister SE, Böger CA, Krämer BK, et al. Effect of chronic kidney disease and comorbid conditions on health care costs: A 10-year observational study in a general population. Am J Nephrol 2010;31:222-9.  Back to cited text no. 7
Nasli-Esfahani E, Peimani M, Rambod C, Omidvar M, Larijani B. Developing a Clinical Diabetes Guideline in Diabetes Research Network in Iran. Iran J Public Health 2014; 43:713-21.  Back to cited text no. 8
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Hirano T. Abnormal lipoprotein metabolism in diabetic nephropathy. Clin Exp Nephrol 2014;18:206-9.  Back to cited text no. 10
Eto M, Saito M, Okada M, et al. Apolipo-protein E genetic polymorphism, remnant lipo-proteins, and nephropathy in type 2 diabetic patients. Am J Kidney Dis 2002;40:243-51.  Back to cited text no. 11
Wenham PR, Price WH, Blandell G. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991;337:1158-9.  Back to cited text no. 12
Leiva E, Mujica V, Elematore I, et al. Relationship between Apolipoprotein E polymorphism and nephropathy in type-2 diabetic patients. Diabetes Res Clin Pract 2007;78:196-201.  Back to cited text no. 13
Satirapoj B, Supasyndh O, Dispan R, Punpanich D, Tribanyatkul S, Choovichian P. Apolipo-protein E genetic polymorphisms and the development of nephropathy in type 2 diabetes. J Med Assoc Thai 2013;96:1119-26.  Back to cited text no. 14
Kimura H, Suzuki Y, Gejyo F, et al. Apolipoprotein E4 reduces risk of diabetic nephropathy in patients with NIDDM. Am J Kidney Dis 1998;31:666-73.  Back to cited text no. 15
Tien KJ, Tu ST, Chou CW, et al. Apolipo-protein E polymorphism and the progression of diabetic nephropathy in type 2 diabetes. Am J Nephrol 2011;33:231-8.  Back to cited text no. 16
Joss N, Jardine A, Gaffney D, Boulton-Jones JM. Influence of Apolipoprotein E genotype on progression of diabetic nephropathy. Nephron Exp Nephrol 2005;101:e127-33.  Back to cited text no. 17
Erdogan M, Eroglu Z, Biray C, et al. The relationship of the Apolipoprotein E gene polymorphism Turkish type 2 diabetic patients with and without nephropathy. J Endocrinol Invest 2009;32:219-22.  Back to cited text no. 18
Wen CP, Matsushita K, Coresh J, et al. Relative risks of chronic kidney disease for mortality and end-stage renal disease across races are similar. Kidney Int 2014;86:819-27.  Back to cited text no. 19
Araki S, Koya D, Makiishi T, et al. APOE polymorphism and the progression of diabetic nephropathy in Japanese subjects with type 2 diabetes: Results of a prospective observational follow-up study. Diabetes Care 2003;26: 2416-20.  Back to cited text no. 20
Atta MI, Abo Gabal K, El-Hadidi K, Swellam M, Genina A, Zaher NF. Apolipoprotein E genotyping in Egyptian diabetic nephropathy patients. IUBMB Life 2016;68:58-64.  Back to cited text no. 21
Hsu CC, Kao WH, Coresh J, et al. Apolipo-protein E and progression of chronic kidney disease. JAMA 2005;293:2892-9.  Back to cited text no. 22
Seshasai RK, Katz R, de Boer IH, et al. Apolipoprotein E and kidney function in older adults. Clin Nephrol 2012;78:174-80.  Back to cited text no. 23
Chen G, Paka L, Kako Y, Singhal P, Duan W, Pillarisetti S. A protective role for kidney apolipoprotein E. Regulation of mesangial cell proliferation and matrix expansion. J Biol Chem 2001;276:49142-7.  Back to cited text no. 24

Correspondence Address:
Farideh Razi
Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran
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