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Original Report: Laboratory Investigation

Am J Nephrol 2011;33:510–514 DOI: 10.1159/000327822

Haptoglobin Polymorphism as a Risk Factor for Chronic Kidney Disease: A Case-Control Study

Yi-Chun Chen   a, e Ching-Chih Lee   c, e Chih-Yuan Huang   a Hsien-Bin Huang   d Chi-Chia Yu   b Yu-Chen Ho   c Yu-Chieh Su   b, e  

Divisions of a   Nephrology and b   Hematology-Oncology, Department of Internal Medicine, and c   Department of Otolaryngology, Buddhist Dalin Tzu Chi General Hospital, and d   Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chiayi , and e   School of Medicine, Tzu Chi University, Hualien , Taiwan, ROC

Introduction

Chronic kidney disease (CKD) is a significant world-wide public health problem and financial burden [1] . Tra-ditional risk factors for CKD include advanced age, dia-betes, hypertension, dyslipidemia and analgesic use [2] . However, susceptibility to CKD varies considerably in different geographic regions and individuals, suggesting a genetic basis for CKD.

Haptoglobin (Hp) is mainly synthesized in the liver and secreted into the plasma. At the physiological level, Hp functions as an important antioxidant, anti-inflam-matory agent and angiogenic factor [3] . The Hp protein is the major hemoglobin (Hb)-binding plasma protein and protects against heme- and iron-driven oxidative damage to the vascular system and kidney. In addition, inflam-matory cytokines can induce the Hp gene in the liver and extrahepatic tissues, including the kidney of mice [4] . The level of Hp increases dramatically upon acute stress and inflammation, so it is considered an acute-phase plasma protein. Furthermore, Hp promotes differentiation and proliferation of vascular endothelium and the formation of new blood vessels [4] .

In humans, Hp is characterized by a genetic polymor-phism and can be expressed as 3 major genotypes (Hp1-1, Hp2-1 and Hp2-2) that are determined by 2 alleles (Hp 1 and Hp 2 ) of the Hp gene, which is located on chromosome 16q22 [5] . Hp1-1 has more potent antioxidative and anti-

Key Words

Haptoglobin polymorphism � Chronic kidney disease

Abstract

Aims: Taiwan has the highest incidence and prevalence of end-stage renal disease worldwide. Haptoglobin (Hp) has a role in renal protection, and there are known differences in the function of different Hp alleles. We aim to study the as-sociation between Hp genotype and chronic kidney disease (CKD) in Taiwan. Methods: We performed one hospital-based, age-matched case-control study of 213 patients with CKD and 213 controls to evaluate the association between Hp polymorphism and CKD. Three major Hp genotypes were determined using polymerase chain reaction and electro-phoresis. An unconditional logistic regression model was used to identify the associated risk factors for the develop-ment of CKD. Results: The frequency of Hp2-2 genotype and Hp 2 allele was significantly higher in the CKD group than in controls (p = 0.032 and 0.024, respectively). After adjustment for covariates, the Hp2-2 genotype (vs. Hp1-1; OR 3.841) re-mained significantly associated with the development of CKD, together with diabetes (OR 3.131), hypertension (OR 1.748) and dyslipidemia (OR 1.646). Conclusion: This present study shows that Hp2-2 genotype is an independent risk fac-tor for CKD. Determination of the Hp genotype may be of potential value to the prediction of genetic risk for CKD.

Copyright © 2011 S. Karger AG, Basel

Received: February 10, 2011 Accepted: March 28, 2011 Published online: May 5, 2011

NephrologyAmerican Journal of

Yu-Chieh Su, MD Division of Hematology-Oncology, Department of Internal MedicineBuddhist Dalin Tzu Chi General Hospital No. 2, Minsheng Rd., Dalin Township, Chiayi County 622, Taiwan (ROC) Tel. +886 5 264 8000, ext. 5665, E-Mail hepatoma   @   ms3.hinet.net

© 2011 S. Karger AG, Basel0250–8095/11/0336–0510$38.00/0

Accessible online at:www.karger.com/ajn

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inflammatory activity than Hp2-2; however, Hp2-2 has more angiogenic activity than Hp1-1, while Hp2-1 is moderately active [5] . It has been reported that the Hp2-2 genotype is linked to susceptibility to diabetic vascular complications and certain neoplasms [5–8] . However, the association between Hp polymorphism and diabetic ne-phropathy remains conflicting among different ethnic populations [7, 9–13] .

Taiwan has a high prevalence of CKD and the highest incidence and prevalence of end-stage renal disease (ESRD) in the world [1] . Large-scale epidemiological studies of CKD in Taiwan have identified several major risk factors, including advanced age, diabetes mellitus (DM), hypertension, hyperlipidemia, and use of Chinese herbs and analgesics [14, 15] . However, there have been limited genetic association studies of CKD. Furthermore, the frequency distribution of the Hp allele varies geo-graphically, and there is a higher incidence of the Hp 2 al-lele in Asia than in the West [16] . It is uncertain whether Hp polymorphism has a role in the development of CKD in Taiwan. In the present study, we aimed to investigate the association between different Hp genotypes and CKD.

Subjects and Methods

Participants and Design This was a hospital-based case-control study conducted at our

institution located in the center of the Chiyi-Yunlin-Tainan area, the region with the highest incidence of dialysis in Taiwan. The study participants comprised 213 CKD patients (men/women 110/103; mean age 66.9 8 12.5 years) randomly 10-year age fre-quency-matched with control subjects at a 1: 1 ratio, all of whom visited our outpatient departments between December 2008 and November 2009 for renal consultations or for annual health check-ups. CKD was defined as the presence of persistent protein-uria or decreased kidney function (estimated glomerular filtra-tion rate, eGFR ! 60 ml/min/1.73 m 2 ) for at least 3 months based on National Kidney Foundation Kidney Disease Outcome Qual-ity Initiative guidelines [15] and no need for renal replacement therapy. For each patient, eGFR was calculated by the simplified Modification of Diet in Renal Disease equation [17] and then clas-sified as having CKD stage 1–5. There were 9 patients (4.2%) with stage 1 disease, 8 patients (3.8%) with stage 2, 85 patients (39.9%) with stage 3, 52 patients (24.4%) with stage 4, and 59 patients (27.7%) with stage 5. Underlying causes for the CKD group in-cluded diabetic nephropathy (n = 113; 53.1%), chronic glomerulo-nephritis with small size of both kidneys (n = 71; 33.3%), biopsy-proved immunoglobulin A nephropathy (n = 4; 1.9%), lupus ne-phritis (n = 6; 2.8%), hypertensive nephrosclerosis (n = 3; 1.4%), gout nephropathy (n = 5; 2.3%), obstructive uropathy (n = 3; 1.4%), and unknown causes (n = 8; 3.8%). The control group included 213 subjects (men/women 108/105; mean age 66.3 8 11.4 years), each with an eGFR 6 60 ml/min/1.73 m 2 and no proteinuria. The study protocol and consent document were approved by our in-

stitutional review board, and the study adhered to the principles of the Declaration of Helsinki.

Definitions Hypertension was defined by blood pressure 6 140/90 mm

Hg, a previous diagnosis of hypertension or treatment with anti-hypertensive medication(s). Diabetes was defined by fasting glu-cose 6 126 mg/dl, a previous diagnosis of diabetes or treatment by hypoglycemic agent(s). Dyslipidemia was defined by the presence of serum triglycerides of 6 150 mg/dl or total serum cholesterol of 6 240 mg/dl [18] .

Hp Genotyping Genomic DNA was extracted from peripheral blood mono-

nuclear cells using a DNA extraction kit (Qiagen, Valencia, Calif., USA). PCR was used to identify the Hp 1 and Hp 2 alleles, as de-scribed previously [8] . The oligonucleotide primers A (5 � -GAG-GGGAGCTTGCCTTTCCATTG-3 � ) and B (5 � -GAGATTTTTG-AGCCCTGGCTGGT-3 � ) were used to amplify the Hp 1 allele-spe-cific sequence of 1,757 bp and the Hp 2 allele-specific sequenceof 3,481 bp. The primers C (5 � -CCTGCCTCGTATTAACTGC-ACCAT-3 � ) and D (5 � -CCGAGTGCTCCACATAGCCATGT-3 � ) were used to amplify the Hp 2 allele-specific sequence of 349 bp. PCR was performed in a final volume of 50 � l with 10–20 ng of genomic DNA. For protocol 1, which used primers A and B, PCR was performed in 1 ! PCR buffer (Qiagen) mixed with 1.5 m M MgCl 2 , 0.2 m M dNTPs, 0.4 � M of each primer, and 2 units of Qia-gen tag DNA polymerase. A thermal cycler (MyCycler, BioRad, Calif., USA) was used for initial denaturation at 95   °   C for 5 min, followed by 35 cycles of 95   °   C for 1 min, 66   °   C for 40 s, 72   °   C for90 s, and a final extension at 72   °   C for 10 min. For protocol 2, which used primers C and D, PCR was performed in 1 ! PCRbuffer mixed with 1.5 m M MgCl 2 , 0.05 m M dNTPs, 0.2 � M of each primer, and 2 units of ABgene tag DNA polymerase. Initial dena-turation was at 95   °   C for 5 min, followed by 35 cycles at 95   °   C for 1 min, 69   °   C for 1 min, and a final extension at 72   °   C for 10 min. The resulting PCR products were subjected to electrophoresis in a 1% agarose gel, then stained with ethidium bromide. All samples were analyzed using protocol 1 with primers A and B. If the 1,757-bp product was detected, samples were further analyzed accord-ing to protocol 2 with primers C and D.

Hp1-1 appeared as a single band of 1,757 bp, Hp2-2 as a single band of 3,481 bp, and Hp2-1 by both of these bands. For Hp2-1, the Hp2 band was generally more faint than the Hp1 band, lead-ing to possible misinterpretation. Thus, for participants with the 1,757-bp band, all the results were confirmed using protocol 2 with primers C and D to show the presence of the Hp2 band.

Collection of Demographic and Laboratory Data We collected demographic data including age, gender and

comorbid conditions including diabetes and hypertension. Bio-chemical data were measured from fasting blood samples using an autoanalyzer (Hitachi 7170, Hitachi Ltd., Tokyo, Japan).

Statistical Analyses All data were analyzed using SPSS version 13.0 (SPSS Inc., Chi-

cago, Ill., USA). All data are expressed as percentages and num-bers, or as mean 8 SD. The differences between groups were com-pared by Pearson’s � 2 test for categorical variables or by an inde-pendent t test for continuous variables. The Hardy-Weinberg

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equilibrium for Hp polymorphism was analyzed with 1 degree of freedom. Unconditional logistic regression analysis was used to estimate multivariate-adjusted odds ratio (OR) and 95% confi-dence interval (CI). A 2-sided p value ! 0.05 was considered sta-tistically significant.

Results

CKD patients and matched controls were similar with respect to age and gender ( table 1 ). CKD patients were found to be significantly associated with a higher per-centage of DM, hypertension and dyslipidemia. The dis-

tribution of Hp genotypes in CKD patients was not in Hardy-Weinberg equilibrium (p = 0.032). The frequency of the Hp2-2 genotype was significantly higher in the CKD group than in the control group (p = 0.032). Table 2 shows that the allele frequency of controls ( Hp 1 vs. Hp 2 allele, 0.33 vs. 0.67) in our study was in accordance with the Taiwanese population [19] and our previous reports [8] . The Hp 2 allele was significantly overexpressed in CKD patients compared with controls (0.74 vs. 0.67; OR 1.409, 95% CI 1.046–1.896; p = 0.024).

Next, multiple unconditional regression analysis after adjustment for covariates shows that the Hp2-2 genotype was independently associated with the development of CKD in an OR of 3.841 versus Hp1-1 (95% CI 1.394–10.583; p for trend = 0.003), in addition to diabetes (OR 3.131, 95% CI 1.926–5.088; p ! 0.001), hypertension (OR 1.748, 95% CI 1.107–2.759; p = 0.017) and dyslipidemia (OR 1.646, 95% CI 1.048–2.587; p = 0.031) ( table 3 ). Mul-tiple conditional regression analysis (table not listed) also shows that the Hp2-2 genotype was an independent risk factor for CKD (adjusted OR 3.359; p for trend = 0.004).

Discussion

This is the first age-matched case-control study to identify a relationship between Hp genotype and CKD by use of multivariate analysis. Compared with traditional risk factors for CKD, our results further indicate that the Hp2-2 genotype is an independent risk factor for the de-velopment of CKD, and that the Hp 2 allele is overex-pressed among CKD patients. These findings have im-portant predictive implications for Taiwan, which has the highest incidence and prevalence of ESRD.

Oxidative stress is a well-known feature of CKD and progresses as CKD advances [20] . The Hp gene is also ex-pressed in the kidney and inducible through cytokines such as interleukin-6 [4] . The pathogenic mechanism by which the Hp2-2 genotype adversely affects CKD may be related in part to the weaker antioxidative activity of Hp2-2 relative to Hp1-1, as indicated by mounting evi-dence. Hp functions as an antioxidant through the abil-ity to bind free Hb at the extravascular site, followed by the clearance of the Hp-Hb complex by CD163 macro-phage scavenger receptor [21] . Hp genotype and serum Hp level determine the Hb-binding capacity and the re-moval of the Hp-Hb complex [5] . Hp2-2 has less efficient Hb binding because of its lowest serum level and higher molecular weight (170–900 kDa) so as to limit its penetra-tion into the extravascular space. The clearance of Hp2-

Table 1. C haracteristics and Hp genotype distribution of study participants (n = 426)

Variable Controls(n = 213)

CKD group(n = 213)

p

Gender 0.846Male 108 (50.7) 110 (51.6)Female 105 (49.3) 103 (48.4)

Age, years 66.3811.4 66.9812.5 0.568Hypertension, % 48.4 66.7 <0.001Diabetes, % 24.4 53.1 <0.001Dyslipidemia, % 28.1 39.1 0.021Laboratory parameters

Creatinine, mg/dl 0.880.2 3.282.5 <0.001eGFR, ml/min/1.73 m2 103.6829.6 33.1831.6 <0.001

Genotype 0.032Hp1-1 20 (9.4) 8 (3.8)Hp2-1 99 (46.5) 93 (43.7)Hp2-2 94 (44.1) 112 (52.6)

�2 testa 0.7 4.58

D ata are expressed as the mean 8 SD or number of subjects with percentages in parentheses, unless otherwise indicated.

a �2 test for deviation from Hardy-Weinberg equilibrium.p values from goodness-of-fit �2 testing in Hardy-Weinberg

equilibrium were 0.403 in the control group and 0.032 in the CKD group.

Table 2. Hp allele frequency distribution in CKD patients and controls

Hp allele Controls(n = 213)

CKD group(n = 213)

Hp1 0.33 0.26Hp2 0.67 0.741

1 OR 1.409, 95% CI 1.046–1.896; p = 0.024.

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2-Hb by CD163 is also less effective than that of Hp1-1-Hb. In addition, some experimental studies have sup-ported the importance of Hp against oxidative damage and the superiority of Hp1-1 over Hp2-2. For example, Hp knockout mice were more liable to renal oxidative dam-age following hemolysis [22] . Hp2-2 DM mice had greater histologic features of diabetic nephropathy, which im-proved after vitamin E supply [23] . In an apolipoprotein E knockout background, Hp2 mice had greater inflam-mation and lipid peroxidation in the plaques compared to Hp1 mice [24] . Collectively, Hp2-2 serves as an inferior antioxidant to Hp1-1 or Hp2-1.

Previous studies of Hp have investigated its impact on diabetic nephropathy [7, 9–13] , one of the common pre-cursors to CKD; however, the relationship varies among different ethnic populations. Studies of Israeli, Irish and Egyptian subjects [7, 9, 10] indicated that the Hp2-2 gen-otype may confer susceptibility to diabetic nephropathy. However, there was no association between Hp genotype and diabetic nephropathy in studies of Japanese, North American and Brazilian subjects [11–13] . These contra-dictory results may be due to differences in the distribu-tion of Hp alleles in different populations, linkage dis-equilibrium near the Hp locus [11] or study design and sample size, as well as baseline characteristics of enrolled patients. Our study was a case-control design, while the studies of Japanese, North American and Brazilian sub-jects [11–13] were not. Moreover, our study was compa-rable in size to the studies of Irish and North American subjects [9, 12] and larger than the studies of Israeli and Japanese subjects [7, 11] . The study of North American subjects [12] enrolled patients with CKD and ESRD, but did not consider CKD stage, and the study of Japanese subjects [11] did not report renal function. Our study only enrolled patients with CKD stage 1–5 and excluded ESRD patients to reduce possible bias. In addition, our study focused on the impact of Hp on the development of CKD, regardless of cause. Our results indicate that subjects car-rying the Hp2-2 genotype are at higher risk of developing CKD than those with traditional risk factors such as dia-betes, hypertension and dyslipidemia ( table 3 ), although there are no comparable studies in the literature.

The present study has some limitations. First, although all study participants were from a single hospital, the al-lele frequency of controls ( Hp 1 vs. Hp 2 allele 0.3 vs. 0.7) in our study was the same as that of the Taiwanese popula-tion [19] . In addition, CKD staging, Hp genotyping and laboratory measurements were all performed in the same hospital, thus minimizing information bias. Second, the CKD patients in this study may or may not have had acute

renal insufficiency, leading to possible overidentification of CKD stage. However, all enrolled CKD patients had regular follow-ups for at least 3 months and were only from the outpatient departments, reducing this possible cause of bias. Third, we could not adjust for some CKD risk factors, such as smoking or use of herbal remedies and analgesics because of inevitable recall bias. Fourth, we did not measure serum Hp concentration because the serum Hp level is significantly higher in patients with early-stage renal injury than in those with normal kid-neys and does not increase as CKD progresses to more advanced stages [25] . Fifth, the case number in this case-control study was relatively small, and a large population-based cohort should be conducted to further validate the finding in the near future.

Despite these limitations, our findings are clinically significant. First, this is the first study to assess the influ-ence of Hp genotype on CKD risk by use of multivariate analysis. Second, this hospital-based study was per-formed in an area with the greatest number of dialysis patients in Taiwan. Third, previous population-based ep-idemiological studies of CKD risk factors in Taiwan [14, 15, 18] did not identify genetic markers for CKD risk, but we identified the Hp2-2 genotype as a risk factor for CKD.

Table 3. Multiple unconditional logistic regression analysis of risk factors for the development of CKD (n = 426)

Risk factors Adjusted OR1 p

Age (per 1 year) 0.987 (0.968–1.006) 0.182Gender 0.822

Female 1Male 0.953 (0.624–1.454)

Hp genotypeHp1-1 1 0.0032

Hp2-1 2.758 (1.000–7.606)Hp2-2 3.841 (1.394–10.583)

Diabetes <0.001No 1Yes 3.131 (1.926–5.088)

Hypertension 0.017No 1Yes 1.748 (1.107–2.759)

Dyslipidemia 0.031No 1Yes 1.646 (1.048–2.587)

Figures in parentheses are 95% CIs.1 Adjusted for age, gender, Hp genotype, diabetes, hyperten-

sion and dyslipidemia. 2 p value for trend.

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Conclusions

With an endemically representative sample, our re-sults document that the Hp2-2 genotype is linked with susceptibility to CKD. At this time, it would be premature to recommend clinical Hp genotyping in CKD preven-tion care programs. Nevertheless, this study highlights the potential for analyzing genetic risk factors of CKD that, in addition to traditional risk factors, may be useful in future CKD prevention programs.

Acknowledgements

The authors would like to thank Professor Shang-Jyh Hwang, Division of Nephrology, Department of Internal Medicine, Kaoh-siung Medical University, Kaohsiung, Taiwan, who encouraged us to pioneer in early CKD prevention in rural areas in Taiwan. The study was supported by grant No. DTCR 99(1)-14 from the Buddhist Dalin Tzu Chi General Hospital.

Disclosure Statement

The authors declare that they have no conflicts of interest.

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