Effect of Apolipoprotein E Gene Polymorphism on Serum Lipid Level Before and After Renal Transplantation

Effect of Apolipoprotein E Gene Polymorphism on Serum Lipid Level Before and After Renal Transplantation

Effect of Apolipoprotein E Gene Polymorphism on Serum Lipid Level Before and After Renal Transplantation H.F. Li, C.F. Han, Y.X. Wang, Y.S. Lu, H.Q. Z...

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Effect of Apolipoprotein E Gene Polymorphism on Serum Lipid Level Before and After Renal Transplantation H.F. Li, C.F. Han, Y.X. Wang, Y.S. Lu, H.Q. Zou, and Q.Q. Xu

ABSTRACT Objective. To investigate the effect of apolipoprotein E (ApoE) gene polymorphism on lipid metabolism among renal transplant recipients before and after transplantation. No prisoners or organs from prisoners were used in this study. Methods. ApoE gene polymorphism was detected with polymerase chain reactionrestriction fragment length polymorphism; serum lipid levels were measured with biochemical methods. Results. Serum lipid levels in the recipients were increased significantly at 3 months after renal transplantation, and further elevated at 6 months and 1 year. The recipients with higher total serum cholesterol (TC) and triglyceride (TG) levels only accounted for 2.9% and 7.6%, respectively, before renal transplantation; but for 28.6% and 46.7%, respectively, at 3 months (P ⬍ .01); 40.0% and 59.0% at 6 months; and 42.9% and 62.9% at 12 months. ApoE gene polymorphism showed no statistical difference in ApoE allele or ApoE genotype between the control and the study groups. The effect of ApoE genotype on serum lipid levels was different between controls and recipients either before or after renal transplantation. The levels of serum TC, TG, low-density lipoprotein cholesterol, ApoB, ApoE were: ␧2/2⫹␧2/3; ␧3/3; ␧3/4⫹␧4/4 from low to high in controls and recipients before transplantation, but the levels of TG and ApoE reversed among recipients after renal transplantation. Conclusion. Renal transplant recipients are liable to develop hyperlipidemia, particularly hypertriglyceridemia among recipients with ApoE genotypes ␧2/2 or ␧2/3. YPERLIPIDEMIA is frequently encountered after renal transplantation, worsening the recipient’s prognosis. However, the mechanisms have not been completely elucidated. Kidney transplant recipients often display hyperlipidemia, which is an important reasons for the arteriosclerosis syndrome and chronic functional loss of renal allografts.1–3 Apolipoprotein (Apo) E is an component of very low-density lipoprotein (VLDL), highdensity lipoprotein (HDL), and chylomicrons.4 –7 It plays an important role in lipid metabolism. The gene polymorphism of ApoE affects lipid metabolism accounting for 14%–17% of the variations in plasma cholesterol concentration,8,9 This study investigated ApoE gene polymorphism among kidney transplant recipients by means of a polymerase chain reaction-restricted fragment length program (PCR-RFLP) seeking to determine its effects on lipid metabolism in renal

H

transplant recipients. No prisoners or organs from prisoners were used in this study. METHODS Patients There were 2 groups of patients in our study: transplanted group and control group. From the Department of Urology (H.F.L., H.Q.Z.), the Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China; the Department of Nephrology (C.F.H.), the Affiliated Xuzhou Hospital, Dongnan University, Xuzhou, Jiangsu Province; the Department of Nephrology (Y.X.W.), the 2nd Hospital of Xiamen, Xiamen, China; and the Department of Nephrology (Y.S.L., Q.Q.X.), the No 1 Hospital of Shanghai, Shanghai, China. Address reprint requests to Yuxin Wang, MD, Department of Nephrology, the 2nd Hospital of Xiamen, Shengguang Road # 566, Xiamen, 361021, China. E-mail: [email protected]

© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/–see front matter doi:10.1016/j.transproceed.2010.04.023

Transplantation Proceedings, 42, 2513–2517 (2010)

2513

Lp(a) (mg/L)

P⬍0.01; compared with 3 months after transplantation: ŒP⬍.05, Compared with control: *P⬍.05, compared with before transplantation:P⬍.05,

1.56 ⫾ 0.65* 1.92 ⫾ 1.15* 1.98 ⫾ 1.24* 2.04 ⫾ 1.51* 1.88 ⫾ 1.17* 4.01 ⫾ 1.11* 5.28 ⫾ 1.18* 5.88 ⫾ 1.30*‘ 5.64 ⫾ 0.94*‘‘ 5.64 ⫾ 0.95* 105 105 105 105 105



1.03 ⫾ 0.51* 1.52 ⫾ 0.52* 1.61 ⫾ 0.57* 1.50 ⫾ 0.47* 1.52 ⫾ 0.39*

2.02 ⫾ 0.61* 2.80 ⫾ 0.89* 3.24 ⫾ 1.20*‘ 2.87 ⫾ 1.02* 3.23 ⫾ 0.87*‘

1.72 ⫾ 0.23* 1.52 ⫾ 0.33* 1.51 ⫾ 0.29* 1.57 ⫾ 0.28* 1.64 ⫾ 0.26*‘

ŒŒ

P⬍.01.

44 ⫾ 12 55 ⫾ 17* 58 ⫾ 14* 58 ⫾ 17* 57 ⫾ 13* 1.14 ⫾ 0.93 1.03 ⫾ 0.31 1.11 ⫾ 0.28*‘ 1.04 ⫾ 0.34 1.10 ⫾ 0.33*

50 ⫾ 10

ApoE (mg/L) ApoB (g/L)

0.98 ⫾ 0.27 1.20 ⫾ 0.17

ApoA1 (g/L) LDLC (mmol/L) HDLC (mmol/L) TC (mmol/L)

TG (mmol/L)

1.23 ⫾ 0.82 4.95 ⫾ 0.89

n

Fig 1. Genotyping of apoE using PCR-RFLP. Lane 1, PCR products; lanes 3 and 8, marker ladder; lane 2, genotypes ␧3/4; lane 4, ␧2/4; lane 5, ␧2/3; lane 6, ␧4/4; lane 7, ␧3/3; lane 9, ␧2/2.

314

Serum from a venous blood sample after an overnight fast was used to measure serum total cholesterol (TC) and triglyceride (TG)

Group

Serum Lipid Analysis

Control Transplant group Before 3 Months after 6 Months after 1 Year after 1.5 Years after

We included 314 healthy people (171 males and 143 females) of overall average age of 41 ⫾ 8 years, with no documented familial hyperlipidemia, cardiovascular, or cerebrovascular diseases, diabetes, or liver or thyroid dysfunction.

Table 1. Comparison of Serum Lipid Levels Between the Control and Transplanted Group

Control Group

2.42 ⫾ 0.66

We included the 105 patients undergoing first kidney transplantations: 59 males and 46 females with an overall average age 43 ⫾ 12 years. All patients had undergone hemodialysis as replacement therapy before transplantation. The immunosuppressive regimens consisted of cyclosporine (CsA), prednisone (Pred), and mycophenolate mofetil (MMF). No acute rejection episodes occurred. They had normal urine output and negative routine urine examinations with serum creatinine levels ⬍170 ␮mol/L. Drugs that affect lipid metabolism, such as ␤-blockers, diuretics, and hypolipidemics, were not used within the first 3 months posttransplantation. Serum lipid levels were routinely measured before as well as 3, 6, 12, and 18 months after transplantation.

1.37 ⫾ 0.29

Transplant Group

189 ⫾ 69 187 ⫾ 74* 196 ⫾ 76* 231 ⫾ 48*‘ 230 ⫾ 85*‘

LI, HAN, WANG ET AL

331 ⫾ 221

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APOE GENE POLYMORPHISM

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concentrations enzymatically with the Cholesterol-E test kit and the triglyceride-E test kit (Wako Pure Chemical Co., Osaka, Japan); HDL cholesterol, with the HDL-cholesterol-E test kit (Wako); low-density lipoprotein cholesterol (LDLC), by the Friede Wald formula (LDLC ⫽ TC ⫺ HDLC ⫺ TG ⫼ 2.2 [mmol/L]). When it was ⬎4 mmol/L, we used a direct method. ApoA1, -B, and -E were tested by an immunoturbidity method. Lipid protein(a) [Lp(a)] was tested by enzyme-linked immunosorbent assay method (Boehringer Mannhein, Germany).

Routine Tests We examined electrocardiographs (ECG), chest x-ray, hemoglobin, alkaline phosphatase, electrolytes, liver function tests, serum albumin, serum uric acid, creatinine, and urea nitrogen levels.

ApoE Gene Polymorphism Test We adopted the PCR-RFLP analytical method using the specific steps of primer design. The upstream sequence was 5=- AACAACTGACCCCGGTGGGG-3= and the downstream sequence was 5=- ATAAATATAAAATATAAATAATGGCGCTGAGGCCGCGCTC-3=; yielding an expanded product of 312 bp with fragments digested by restricted endonuclease Hha I of 91, 83, 72, 61, 48, and 35 bps. For genomic DNA extraction we used a technique of rapid genomic DNA extraction and purification from a small amount of blood with Huashun company kits. PCR was performed with 2 ␮L magnesium chloride 2.5 ␮L of 2 mmol/L 10 ⫻ buffer, 2 ␮l of 1 mmol/L dNTP, 1 U Taq DNA polymerase, 1 ␮L primer, 1 ␮g template DNA mixed, before water was added to bring the mixture to 25 ␮L. After an initial denaturation at 95 °C for 4 minutes, the mixture was put into the following sequence profile: denaturation at 94 °C for 40 seconds, annealing at 60 °C for 1 minutes, and extension at 70 °C for 3 minutes. After 35 cycles, the reaction was ended with a final extension at 70 °C for 7 minutes and cooling to 4 °C. An agarose gel electrophoresis bank of 12 g/L concentration was used to identify the expanded product. ApoE gene polymorphism analysis used 2 ␮L of 10 ⫻ buffer, 0.2 ␮L calf serum, and 5 U endonuclease Hha I added to 20 ␮L of successfully expanded product in a 65 °C bath for 4 hours. Digested product (3 ␮L) was put into polypropylene amide gel electrophoresis for 3 hours (voltage 150 V). Silver nitrate staining was used to observe the results. The length of the expanded ApoE gene product was 312 bps. After digestion of genotype ␧3/4, there were 4 fragments: 91, 72, 61, and 48 bps. After digestion of genotype ␧2/4, there were 5 fragments: 91, 83, 72, 61, and 48 bps. After digestion of genotype ␧2/3, there were 4 fragments: 91, 83, 61, and 48 bps. After digestion by genotype ␧4/4, there were 3 fragments: 72, 61, and 48 bps. After digestion of genotype ␧3/3, there were 3 fragments: 91, 61, and 48. After digestion of genotype ␧2/2, there were 3 fragments: 91, 83, and 61 bps (Fig 1).

Statistical Analysis Statistical analysis was performed using SPSS13.0 software (SPSS Inc., Chicago, Ill). Data were represented as mean values ⫾ standard deviations univariate analysis was made for measurement data. The chi- square test was used to compare categorical data. P ⬍ .05 was considered significant.

RESULTS Comparison of Serum Lipid Levels Between Recipients and Controls

The serum levels of TG and Lp(a) before transplantation were significantly higher than the controls, whereas those of TC, LDLC, and HDLC were lower. However, most recipients showed gradually increasing levels of TC and LDLC within the first year, which shows a downward trend at 1.5 years (Table 1). The proportion of recipients with a serum TC or TG level higher than normal was 2.9% and 7.6% before, increasing to 42.9% and 62.9% at 1 year, but reducing to 35.3% and 48.5% at 1.5 years, respectively. ApoE Genotype and the Frequency of Allele

The most frequent ApoE genotype and allele were ␧3/3 and ␧ 3. Genotypes ␧2/2, ␧2/3, ␧2/4, ␧3/3, ␧3/4, or ␧4/4 were observed in 2, 15, 0, 77, 9, and 2 recipients, respectively, with allele frequencies of ␧2, ␧3 or ␧4 of 9.0%, 84.8%, and 6.2% respectively. Genotypes ␧2/2, ␧2/3, ␧2/4, ␧3/3, ␧3/4, or ␧ 4/4 were observed respectively in 4, 45, 6, 235, 29, and 5 cases of controls with allele frequencies of ␧2, ␧3, or ␧4 of 9.1%, 84.0%, and 6.9%, respectively. There was no significant difference between recipients and controls in the distribution of ApoE genotypes and the frequency of ApoE alleles. Relationship Between ApoE Gene Polymorphism and Serum Lipid Levels

To analyze the pure impact of allele ␧2 and ␧4 on serum lipids, we excluded recipients and controls with genotype ␧ ␧ 2/ 4. The other 5 genotypes were divided into 3 groups: ␧ 2/2 ⫹ ␧2/3, ␧33, and ␧3/4 ⫹ ␧4/4. As a result in controls, the highest serum level of TC, TG, or LDLC was observed in cases with genotype ␧3/4 ⫹ ␧4/4 and the lowest lipid level, in cases with genotype ␧2/2 ⫹ ␧2/3. An opposite result was observed for ApoA1 and HDLC (Table 2). A similar situation was noted in recipients before transplantation. But after transplantation, the serum level of TG or ApoE was the highest among cases with genotype ␧2/2 ⫹ ␧2/3, and lowest level in genotype ␧3/4 ⫹ ␧4/4, which was different

Table 2. Comparison of Serum Lipid Levels of Different ApoE Genotypes in the Control Group Genotype

n

TC (mmol/L)

TG (mmol/L)

HDLC (mmol/L)

LDLC (mmol/L)

ApoA1 (g/L)

ApoB (g/L)

ApoE (mg/L)

Lp(a) (mg/L)

␧2/2⫹␧2/3 ␧3/3 ␧3/4⫹␧4/4 P

49 253 34

4.60 ⫾ 0.73 4.89 ⫾ 0.91 4.99 ⫾ 0.69 .067

1.07 ⫾ 0.76 1.29 ⫾ 0.90 1.58 ⫾ 0.26 .047

1.60 ⫾ 0.30 1.51 ⫾ 0.33 1.40 ⫾ 0.24 .021

2.28 ⫾ 0.68 2.51 ⫾ 0.71 2.58 ⫾ 0.66 .071

1.36 ⫾ 0.24 1.28 ⫾ 0.20 1.24 ⫾ 0.19 .025

0.81 ⫾ 0.21 0.94 ⫾ 0.28 0.99 ⫾ 0.22 .004

52 ⫾ 13 50 ⫾ 11 50 ⫾ 12 .520

193 ⫾ 135 262 ⫾ 235 221 ⫾ 169 .104

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LI, HAN, WANG ET AL Table 3. Comparison of Serum Lipid Levels of Different ApoE Genotypes Before and After Transplantation Group

Before transplantation ␧2/2⫹␧2/3 ␧3/3 ␧3/4⫹␧4/4 P After transplantation ␧2/2⫹␧2/3 ␧3/3 ␧3/4⫹␧4/4 P

n

TC (mmol/L)

TG (mmol/L)

HDLC (mmol/L)

LDLC (mmol/L)

ApoA1 (g/L)

ApoB (g/L)

ApoE (mg/L)

Lp(a) (mg/L)

17 77 11

3.81 ⫾ 0.60 3.84 ⫾ 0.54 4.33 ⫾ 0.68 .046

1.33 ⫾ 0.68 1.64 ⫾ 0.52 2.12 ⫾ 0.80 .008

1.08 ⫾ 0.13 1.00 ⫾ 0.19 1.26 ⫾ 0.22 .051

1.87 ⫾ 0.70 2.16 ⫾ 0.68 2.34 ⫾ 0.74 .022

1.11 ⫾ 0.16 1.09 ⫾ 0.24 1.39 ⫾ 0.32 .090

0.85 ⫾ 0.34 0.93 ⫾ 0.24 0.92 ⫾ 0.24 .662

49 ⫾ 23 38 ⫾ 10 46 ⫾ 7 .018

337 ⫾ 165 207 ⫾ 144 266 ⫾ 5 .070

17 77 11

5.87 ⫾ 1.88 5.50 ⫾ 1.42 5.45 ⫾ 1.30 .662

2.56 ⫾ 0.76 2.11 ⫾ 0.56 1.45 ⫾ 0.68 .033

1.64 ⫾ 0.50 1.56 ⫾ 0.49 1.51 ⫾ 0.46 .778

3.15 ⫾ 0.64 2.81 ⫾ 1.01 3.19 ⫾ 1.08 .279

1.51 ⫾ 0.26 1.55 ⫾ 0.29 1.44 ⫾ 0.16 .387

1.07 ⫾ 0.36 1.05 ⫾ 0.31 1.04 ⫾ 0.26 .968

76 ⫾ 22 57 ⫾ 16 52 ⫾ 11 .008

133 ⫾ 147 191 ⫾ 139 139 ⫾ 112 .279

from the results in the controls (Table 3). In both recipients with ApoE genotype ␧2/2, serum lipid levels were normal before transplantation but significantly increased for TC and TG, with a 4.3-fold increase in TG at 6 months posttransplantation. DISCUSSION

ApoE binds to its receptor as a ligand, thus regulating lipid metabolism. After binding to the LDL receptor-associated protein on the surface of liver cells through ApoE,10 chylomicrons are removed. LDL and VLDL residues are removed by identifying the LDL receptor binding to ApoB or ApoE. Among all ApoE genotypes, ␧3/3 was the most common and ␧3/4 second most frequent.11 ApoE has 3 isomers (E2, E3, E4). The ability to bind E2 receptor is lower than those of E3 or E4. In vitro studies have shown that the low ability of binding to E2 receptor delays catabolism of serum chylomicrons and VLDL residuals. The transition from intermediate-density lipoprotein (IDL) to LDL lessens, the concentration of IDL increases, and the concentration of LDL decreases. The role of E4 is the opposite. Therefore, theoretically, the pure effect of E2 is to increase serum triglyceride and decrease cholesterol levels, but that of E4 is precisely opposite.9,12 At present, many studies have shown that E4 is a risk factor for the pathogenesis of atherosclerosis and E2 is a protective factor. However, in fact, the mechanisms of the effect of ApoE on lipid metabolism and in atherosclerosis are complicated; clinical and animal studies have obtained inconsistent results.13–16 In this study, we showed that TC and LDLC levels increased after the sequence of genotypes ␧2/2⫹␧2/3, ␧3/3, and ␧3/4⫹␧4/4 among either recipients preoperatively or in controls. Thus, TG and LDLC levels of allele ␧4 carriers in both healthy controls and preoperative recipients were higher and those of ␧2 carriers lower. But there was no such change among the transplantation group. The results are consistent with previous reports.16 –21 TC and LDLC levels in recipients increase significantly after transplantation, but the increase did not correlated with ApoE genotypes. Big discrepancies have been reported about the effects of ApoE gene polymorphism on TG levels. It is more accepted that

TG levels in normal people and coronary heart patients with allele ␧2 are higher than those among ␧3 carriers, and TG levels in ␧4 carriers may be higher or lower than those of ␧3 carriers.7 Our present study indicates that ApoE gene polymorphism significantly affects serum lipid levels of renal transplant recipients before and after transplantation in a different way. Before transplantation, TG levels of allele ␧4 carriers may be higher and that of ␧2 carriers lower, but after transplantation, TG and ApoE levels of allele ␧2 carriers may be higher, and those of ␧4 carriers lower. Our present study suggests that ApoE gene polymorphism may play a different role on serum lipid levels in different crowds or in same crowd under different circumstances. ApoE allele ␧4 carriers among controls and preoperative recipients may be prone to develop hypertriglyceridemia, but allele ␧2 carriers in post-operative recipients may be prone to develop hypertriglyceridemia. In this study, there were 2 patients with ApoE genotype ␧2/2. They had normal serum lipid levels before transplantation and their TC and TG levels increased, more significantly for triglyceride levels, 6 months after transplantation. These results suggested that renal transplant recipients with ApoE ␧2/2 genotype should be more alert to the development of hyperlipidemia. However, the mechanism of the effect of ApoE gene polymorphism on serum lipid levels is unclear and further studies are needed. ACKNOWLEDGMENTS C.H. and H.L. contributed equally to this work and should be considered co-first authors.

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2517 14. Kahraman S, Kiykim AA, Altun B, et al: Apolipoprotein E gene polymorphism in renal transplant recipients: effects on lipid metabolism, atherosclerosis and allograft function. Clin Transplant 18:288, 2004 15. Willems Van Dijk K, Hofker MH, et al: Use of transgenic mice to study the role of apolipoprotein E in lipid metabolism and atherosclerosis. Int J Tissue React 22:49, 2000 16. Saito H, Dhanasekaran P, Baldwin F, et al: Effects of polymorphism on the lipid interaction of human apolipoprotein E. J Biol Chem 278:40723, 2003 17. Roussos L, Flor NC, Carlson J, et al: Increased prevalence of apolipoprotein E3/E4 genotype among Swedish renal transplant recipients. Nephron 83:25, 1999 18. Maluf DG, Mas VR, Archer KJ, et al: Apolipoprotein E genotypes as predictors of high-risk groups for developing hyperlipidemia in kidney transplant recipients undergoing sirolimus treatment. Transplantation 80:1705, 2005 19. Rai TS, Khullar M, Sehrawat BS, et al: Synergistic effect between apolipoprotein E and apolipoprotein A1 gene polymorphisms in the risk for coronary artery disease. Mol Cell Biochem 313:139, 2008 20. Liew G, Shankar A, Wang JJ, et al: Apolipoprotein E gene polymorphisms and retinal vascular signs: the atherosclerosis risk in communities (ARIC) study. Arch Ophthalmol 125:813, 2007 21. Koch W, Hoppmann P, Schömig A, et al: Apolipoprotein E gene epsilon2/epsilon3/epsilon4 polymorphism and myocardial infarction: case-control study in a large population sample. Int J Cardiol 125:116, 2008