Length Polymorphism in Heme Oxygenase-1 and Risk of CKD Among Patients with Coronary Artery Disease

CLINICAL RESEARCH www.jasn.org

Length Polymorphism in Heme Oxygenase-1 and Risk of CKD among Patients with Coronary Artery Disease

† ‡ ‡ | Yu-Hsin Chen,* Ko-Lin Kuo, Szu-Chun Hung, Chih-Cheng Hsu,§ Ying-Hwa Chen,* and †† Der-Cherng Tarng*¶**

*Faculty of Medicine, ¶Institute of Clinical Medicine, and **Department and Institute of Physiology, National Yang- Ming University, Taipei, Taiwan; †Division of Nephrology, Department of Internal Medicine, Taipei City Hospital, Yang-Ming Branch, Taipei, Taiwan; ‡Division of Nephrology, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, Taipei, Taiwan; §Division of Geriatrics and Gerontology, Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Taiwan; and Divisions of |Cardiology and ††Nephrology, Department of Medicine and Immunology Center, Taipei Veterans General Hospital, Taipei, Taiwan

ABSTRACT The length polymorphism of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter is associated with cardiovascular events and mortality in high-risk populations. Experimental data suggest that heme oxygenase-1 protects against kidney disease. However, the association between this polymorphism and long-term risk of CKD in high-risk patients is unknown. We analyzed the allelic frequencies of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter in 386 patients with coronary artery disease recruited from January 1999 to July 2001 and followed until August 31, 2012. The S allele represents short repeats (,27), and the L allele represents long repeats ($27). The primary renal end points consisted of sustained serum creatinine doubling and/or ESRD requiring long-term RRT. The secondary end points were major adverse cardiovascular events and mortality. At the end of study, the adjusted hazard ratios (95% conﬁdence intervals) for each L allele in the additive model were 1.99 (1.27 to 3.14; P=0.003) for the renal end points, 1.70 (1.27 to 2.27; P,0.001) for major adverse cardiovascular events, and 1.36 (1.04 to 1.79; P=0.03) for mortality. With cardiac events as time-dependent covariates, the adjusted hazard ratio for each L allele in the additive model was 1.91 (1.20 to 3.06; P=0.01) for the renal end points. In conclusion, a greater number of guanosine thymidine dinucleotide repeats in the heme oxygenase-1 gene promoter is associated with higher risk for CKD, cardiovascular events, and mortality among patients with coronary artery disease.

J Am Soc Nephrol 25: 2669–2677, 2014. doi: 10.1681/ASN.2013111205

Heme oxygenase (HO) is the rate-limiting enzyme in thymidine dinucleotide [(GT)n] repeats in the HO- heme degradation. The enzyme generates free iron, 1 gene microsatellite promoter is inversely associated biliverdin, and carbon monoxide. Biliverdin is sub- with HO-1 mRNA levels and enzyme activity.7,8 sequently converted to bilirubin by biliverdin re- ductase, and free iron is rapidly sequestered by Received November 19, 2013. Accepted March 16, 2014. ferritin.1 HO is a cytoprotective enzyme that poten- ﬂ Published online ahead of print. Publication date available at tially exerts antioxidant, anti-in ammatory, and www.jasn.org. antiapoptotic functions.2 HO-1 is the inducible iso- form, whereas HO-2 is constitutively expressed. Correspondence: Dr. Ying-Hwa Chen, Faculty of Medicine, Na- tional Yang-Ming University, and Division of Cardiology, Department HO-1 is expressed in various tissues and is upregula- of Medicine, Taipei Veterans General Hospital, 201, Section 2, ted by cellular stress. HO-1 is known to be protective Shih-Pai Road, Taipei 11217, Taiwan, or Dr. Der-Cherng Tarng, against atherosclerosis, hepatic ischemia-reperfusion Department and Institute of Physiology, National Yang-Ming University, and Division of Nephrology, Department of Medicine injury, hyperoxia-induced lung injury, and corneal and Immunology Center, Taipei Veterans General Hospital, 201, inﬂammation in experimental studies.3–6 Section 2, Shih-Pai Road, Taipei 11217, Taiwan. Email: yhchen@ The human HO-1 gene has been mapped to chro- vghtpe.gov.tw or [email protected] mosome 22q12 and the number of guanosine Copyright © 2014 by the American Society of Nephrology

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Previous studies have demonstrated an increased susceptibility polymorphism in patients with CAD were comparable with to cardiovascular events and increased mortality of longer those in healthy controls and Hardy–Weinberg equilibrium (GT)n repeats in the HO-1 gene promoter in high-risk popu- was met. In this study, all participants were Taiwanese and lations such as patients with diabetes mellitus, had similar ethnic backgrounds. Therefore, statistical artifacts hypercholesterolemia, a history of smoking, peripheral artery caused by population stratification could be ruled out as de- disease, or arsenic exposure.8–16 Our recent study also showed scribed by Pritchard and Rosenberg.28 that longer (GT)n repeats in the HO-1 gene promoter were associated with a higher risk of long-term cardiovascular events Baseline Demographic and Laboratory Data and mortality in hemodialysis patients.17 Until now, the inves- Patients with CAD among the three genotypes of HO-1 pro- tigations for this polymorphism were mostly restricted to graft moter polymorphism had no significant differences in age at survival in kidney transplant recipients.18 The effect of the entry, sex, comorbidity, LDL and HDL cholesterol levels, and length polymorphism in the HO-1 gene promoter on the risk smoking status (Table 2). No significant differences across of CKD is a largely undefined body of knowledge. genotype groups were found in the baseline eGFR, protein- Coronary artery disease (CAD) per se is a major indepen- uria, use of statins or renin-angiotensin system blockades, dent risk factor for subsequent development and progression serum albumin, calcium, phosphorus, and hemoglobin levels. of CKD.19–21 Kiyosue et al. found that the deterioration of Intriguingly, the serum bilirubin level was lower in L allele renal function was correlated with the severity of CAD in Jap- carriers (L/L and S/L genotypes) and higher in patients with anese patients.20 We recently revealed that the GFR declined the S/S genotype (Figure 2). Serum ferritin and malondialde- fasterinCADthanthatinnormalcoronaryarteryofTai- hyde levels were highest in the L/L genotype and lower in the 21 wanese patients. Longer (GT)n repeats in the HO-1 gene S/L and S/S genotypes (Figure 2). promoter associated with long-term outcomes of CAD, such as major adverse cardiovascular events, target vessel revascu- larization, and overall mortality8–12; however, the relevance of this polymorphism in native kidney function of patients with CAD is unknown. The protective effect of HO-1 against kidney injury is well known in a variety of animal models.2,18,22–26 We therefore performed an association study in patients with CAD to investigate whether the length polymorphism of the HO-1 gene promoter is associated with the risk of CKD.

RESULTS

Initially, 402 patients with CAD were enrolled from a tertiary hospital. During a median follow-up period of 10.2 years (interquartile range, 6.31211.6), 16 patients were excluded from the study because of a follow-up time of ,6 months Figure 1. Frequency distribution of the number of (GT)n repeats of (n=3) and insufﬁcient creatinine follow-up data (n=13). Fi- the heme oxygenase-1 microsatellite promoter. There are 386 pa- nally, 386 patients with CAD with a mean age of 70 years were tients with CAD and 361 healthy controls. The (GT)n repeats range analyzed. from 16 to 38. There is a bimodal distribution with one peak located at 23 repeats and the other located at 30 repeats in both groups. ﬁ HO-1 The frequency distributions of (GT)n repeats are not signi cantly Length Polymorphism of (GT)n Repeats in the different between patients with CAD and healthy controls. Gene Promoter Figure 1 shows the frequency distribution of (GT)n repeats of HO-1 the microsatellite promoter in the 386 patients with Table 1. Genotype and allelic frequency of HO-1 CAD and 361 healthy controls. The allelic distribution ranged microsatellite promoter polymorphism in healthy controls from 16 to 38 GTrepeats, with 23 and 30 GTrepeats being the and patients with CAD two most common alleles. We chose 27 GT repeats as a cutoff Healthy Controls Patients with CAD to classify the participants for allele typing, and the cutoff (n=361) (n=386) value was consistent with the previously published litera- Genotype, n (%) 10,14,17,27 , ture. Thus, short repeats with 27 GT repeats were S/S 78 (21.6) 94 (24.4) designated as S alleles, and long repeats with at least 27 GT S/L 194 (53.7) 187 (48.4) repeats were designated as L alleles. Accordingly, the partici- L/L 89 (24.7) 105 (27.2) pants were categorized into S/S, S/L, or L/L genotypes. In Table L-allelic frequency 0.52 0.51 HO-1 , $ 1, the genotype proportions and L-allelic frequencies of S allele, number of (GT)n repeats 27; L allele, number of (GT)n repeats 27.

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Risk of CKD three types of cumulative events (Table 3). This implies that a At the end of study, 47 patients (12.2%) reached the renal end recessive model would best fit the observed data. In the re- points, which included sustained serum creatinine doubling cessive model of a Cox regression analysis, individuals with the and ESRD necessitating long-term RRT. In this study, we used L/L genotype had approximately 2.4-fold the risk for the renal the composite renal end points to evaluate the risk of CKD. To end points as the S allele carriers, which led to an adjusted ensure the events were CKD but not AKI, the patients followed- hazard ratio (HR) of 2.43 (95% confidence interval [95% CI], up for fewer than 6 months were ruled out. Data on at least two 1.28 to 2.04; P=0.01) (Table 3). In the additive model, each L follow-up creatinine measurements were obtained. The events allele had an adjusted HR of 1.99 (95% CI, 1.27 to 3.14; of creatinine doubling were restricted to sustained high P=0.003) for the renal end points (Table 3). We further vali- creatinine levels; this means that if the creatinine level regressed dated that the risk of CKD is independent to the risk of cardiac to lower than the doubling value during follow-up, the renal outcomes that developed during follow-up using the cardiac end point was not reached. ESRD is defined as necessitating events as time-dependent covariates in the Cox regression long-term RRT but not for a short temporary period. analysis (Table 4). The L/L genotype led to an adjusted HR Moreover, acute elevation of creatinine and dialysis starting of 2.41 (95% CI, 1.31 to 4.55; P=0.01) for the renal end points before death were assumed to be AKI and were not included in as the S allele carriers, and each L allele had an adjusted HR of the renal end points. Kaplan–Meier analysis curves among 386 1.91 (95% CI, 1.20 to 3.06; P=0.01) for the renal end points patients with CAD showed that the risk of the renal end points (Table 4). was significantly higher in the L/L genotype than in the S/S genotype (P=0.01) and S allele carriers (S/L and S/S geno- Major Adverse Cardiovascular Events and All-Cause types) (P=0.02) (Figure 3A). When we assessed the association Mortality of this polymorphism with each outcome, there were no major Of the study patients, 101 (26.4%) developed major adverse differences between the S/S and S/L genotype groups for all cardiovascular events and 117 (30.3%) died. Of the deceased

Table 2. Baseline demographic characteristics and laboratory data of patients with CAD stratiﬁed by genotypes of HO-1 microsatellite promoter polymorphism HO-1 Promoter Genotype Characteristic All Patients (n=386) S/S (n=94) S/L (n=187) L/L (n=105) P Value Age, yr 70687168706871680.76 Men, % 92.7 93.6 92.5 92.4 0.93 Current smoker, % 33.1 36.7 30.3 34.7 0.53 Comorbidities, % Hypertension 73.8 80.9 73.3 68.6 0.14 Diabetes mellitus 33.7 30.9 34.8 34.3 0.80 Congestive heart failure 12.4 11.7 11.8 14.3 0.80 Cerebrovascular disease 6.5 4.3 5.3 10.5 0.14 Peripheral artery disease 3.4 5.3 1.1 5.7 0.05 Total no. of antihypertensive medications, % 2.761.6 2.761.4 2.561.5 3.161.7 0.75 Renin-angiotensin system blockades, % 45.6 45.7 44.9 46.6 0.96 Statins, % 23.8 24.5 23.5 23.8 0.99 Laboratory parameters Creatinine, mg/dl 1.360.4 1.360.4 1.260.3 1.360.6 0.22 eGFR, ml/min per 1.73 m2 64617 63617 65615 63619 0.44 Proteinuria, % 12.2 12.1 13.4 10.1 0.71 Albumin, g/dl 4.160.4 4.060.4 4.160.4 4.160.4 0.29 Total cholesterol, mg/dl 187637 193641 183632 188640 0.08 LDL cholesterol, mg/dl 122627 125631 121626 121627 0.46 HDL cholesterol, mg/dl 38683868376839680.12 Triglyceride, mg/dl 1516118 1576103 143687 1596168 0.46 Fasting serum glucose, mg/dl 116646 114638 115646 119651 0.64 Uric acid, mg/dl 7.361.9 7.461.7 7.162.0 7.361.8 0.51 Calcium, mg/dl 9.060.5 9.060.6 8.960.4 9.060.5 0.77 Phosphorus, mg/dl 3.460.6 3.560.5 3.460.6 3.460.7 0.55 Hemoglobin, g/dl 13.261.5 13.361.7 13.161.5 13.361.5 0.43 Iron, mg/dl 82698 69672 83690 916129 0.27 Transferrin saturation, % 26614 25613 26613 28615 0.26

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(P=0.001) (Figure 3B). In the recessive model, patients with the L/L genotype had approximately 2-fold the risk for major adverse cardiovascular events and 1.5-fold the risk for death as the S allele carriers, which resulted in adjusted HRs of 2.05 (95% CI, 1.36 to 3.11; P=0.001) and 1.47 (95% CI, 1.02 to 2.18; P=0.01), respectively (Table 3). In the additive model, each L allele had adjusted HRs of 1.70 (95% CI, 1.27 to 2.27; P,0.001) for major adverse cardiovascular events and 1.36 (95% CI, 1.04 to 1.79; P=0.03) for death, respectively (Table 3).

DISCUSSION

This study, with a median follow-up of 10.2 years among 386 patients with CAD, is the first to demonstrate that there is a significantly increased risk of CKD for patients with the L/L genotype in HO-1 gene promoter polymorphism. Individuals with this genotype had approximately 2.4-fold the risk for sustained serum creatinine doubling and/or ESRD necessitating long-term RRT of those with S/S or S/L genotypes. The significant association of this polymorphism with cardiovascular outcome and mortality in patients with CAD also corroborated previous studies in these high-risk populations.8–16 Ample evidence has shown the beneficial effect of HO-1 in experimental kidney disease, including ischemia-reperfusion kidney injury, nephrotoxin or angiotensin II–induced renal injury, kidney transplantation, and diabetic models.2,18,22 Kim et al.23 demonstrated the renoprotective effect of HO-1 using its inducer and inhibitor through an antiapoptotic pathway in the unilateral ureteral obstruction (UUO) model. Kie et al.24 showed that HO-1 deficiency promoted epithelial- mesenchymal transition and renal fibrosis in the UUO model in HO-1 knockout mice. Desbuards et al.25 used hemin, an inducer of HO-1, to attenuate interstitial fibrosis in 5/6 neph- rectomized rats via inhibiting TGF-b and caspase-3 expression and increasing bone morphogenetic protein-7 expression. Correa-Costa et al.26 further revealed that pretreatment or delayed treatment with hemin both reduced renal dysfunction and decreased the expression of proinflammatory molecules, fibrosis-related molecules, and collagen deposition in rats subjected to UUO. In addition, HO-2, which contrib- Figure 2. Levels of serum bilirubin, ferritin, and malondialdehyde by genotypes. Baseline serum bilirubin (A), ferritin (B), and ma- utes the bulk of HO activity in the unstressed kidney, was londialdehyde (C) in 386 patients with CAD stratified by the ge- also suspected to play a protective role in the animal kidney notype of the HO-1 microsatellite promoter polymorphism. models.29,30 However, the association between the HO-2 Whisker plots show the 10th, 25th, 50th, 75th, and 90th per- gene polymorphism and kidney disease in humans is still centiles of distribution. aP,0.05 versus S/S genotype; bP,0.05 obscure. versus S/L genotype. The mechanism of HO-1 protection in the kidney is related to heme degradation because a large amount of free heme is pro-oxidant, proinflammatory, and proapoptotic and HO-1 is patients, 42 (35.9%) died from cardiovascular causes. In the responsible for regulating its cellular levels. HO-1 also exerts Kaplan–Meier curve analyses, the risk of all-cause mortality protective effects through its reactive products, bilirubin and was significantly higher in the L/L genotype than in the S/S carbon monoxide.2,18,22 Bilirubin has both antioxidant and genotype (P=0.001) and the S/L genotype (P=0.01) (Figure anti-inflammatory properties.31,32 In humans, the length of 3B). The S allele noncarriers had a significantly higher risk of (GT)n repeats in the HO-1 gene promoter is inversely associ- mortality than the S allele carriers (S/L and S/S genotypes) ated with HO-1 mRNA levels and enzyme activity.8 Because

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using a Mendelian randomization ap- proach.35 For example, S alleles result in higher HO-1 expressions of life-long per- sistence. Therefore, a higher protective effect against renal function decline from these alleles can be expected. The association between the polymorphism with clinical outcomes is less likely to be influenced by reverse causation or confounding. Human studies assessing the length polymorphism of the HO-1 gene promoter and renal outcome are mostly restricted to graft survival in kidney transplant recipients and the results are conflicting.18 Only two cross-sectional studies examined the relationship of this polymorphism in CKD. Courtney et al.36 found that the HO-1 genotype conferred no significant influence on the mean age at commence- ment of RRT among patients with autosomal dominant polycystic kidney disease or IgA nephropathy. Chin et al.37 found that the longer length of the HO-1 gene pro- – moter was related to an eGFR,60 ml/ Figure 3. Survival curves for renal end points and all-cause mortality by genotypes. Kaplan 2 Meier cumulative survival curves for renal end points (A) and all-cause mortality (B) among min per 1.73 m at diagnosis of IgA ne- the 386 patients with CAD in relation to the genotype of the HO-1 microsatellite promoter phropathy. The discrepancy between our polymorphism. The upper panels are the three genotypes (S/S, S/L, and L/L), and the lower study and that by Courtney et al. may be panels are the genotypes in a recessive model (S/L+S/S and L/L). mainly because the milieu of factors linked to the progression of CKD were not adjusted in the study by Courtney et al. In HO-1 is a rate-limiting enzyme for bilirubin production, the addition, differences in study populations (autosomal domi- serum bilirubin concentration is determined in part by this nant polycystic kidney disease or IgA nephropathy versus length polymorphism. Our study confirmed previous findings CAD), genotype distribution, sample size, and study design that the L allele was significantly associated with lower serum (cross-sectional analysis versus longitudinal follow-up) are bilirubin,33 and the gene explained a large proportion of the also noted. To our knowledge, our study is the first longitudi- variation in bilirubin levels. We further found that patients nal long-term study to investigate HO-1 promoter polymor- with the L/L genotype had less ability against oxidative stress phism and risk of CKD. and inflammation, which was exhibited by higher levels of Given the estimated allele frequency and its effect on serum serum malondialdehyde and ferritin. The results corroborated bilirubin, the L allele of the HO-1 gene promoter is a likely previous studies.9,11,33 Although ferritin increases in response candidate for the reported trait. If approximately 27% of the to oxidative damage and inflammation and is considered an CAD population has a strong adverse effect in renal function inflammation marker, ferritin has been shown to have antiox- decline because of the HO-1 length polymorphism, it would idant properties through iron sequestering and the potential have significant implications in future CKD prevention for the protective effects in the kidney.34 Being a potential cytopro- high-risk population. tective protein and an inflammation marker at the same time, Some limitations in this study should be acknowledged. the mechanisms by which HO-1 polymorphism confers the First, this is a single-center study in patients with CAD in the variance in ferritin values remain to be elucidated. In this Taiwanese population and confirmatory replication studies study, the Cox regression with time-varying covariate analyses in other centers and other populations are needed. Second, (Supplemental Table 1) revealed that lower total bilirubin was the creatinine follow-up interval was not regular in usual significantly associated with the risk of CKD progression. Our clinical care instead of regular study visits, and the creatinine additional data suggest that the association of the L/L genotype doubling time might be underdiagnosed. Finally, our study or L allele with the risk of CKD is at least in part mediated by was observational in nature, so it cannot prove causality. the intermediate phenotype. However, to ensure adequate statistical power (a=0.05; Evidence for causality between inflammation-oxidative 12b=0.8; no loss of follow-up), at least 364 patients with stress and renal function progression may be possible CAD should be enrolled to examine a relative risk increment

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of 40%. Apart from impracticability to conduct a randomized controlled trial, emerging evidence38 suggests that well 0.001 Value , P designed observational studies could yield comparable out- Adjusted comes. In conclusion, this prospective cohort study shows that homozygote L allele carriers exhibited a strong associationwith higher risk of CKD, major adverse cardiovascular events, and

(95% CI) overall mortality among patients with CAD. Further studies are Adjusted HR required to validate this association. 0.001 1.70 (1.27 to 2.27) Value CONCISE METHODS Crude , P microsatellite promoter polymorphism Research Participants Study participants were recruited from January 1999 to July 2001 at a HO-1 a tertiary hospital in Taipei, a metropolitan city that is not the arseniasis-endemic area in Taiwan. Atotal of 419 patients undergoing (95% CI) Crude HR

ripheral arterial disease, total cholesterol, HDL cholesterol, serum coronary angiography agreed to participate in this study. Seventeen patients were excluded from the study because of non-CAD (n=4), insigniﬁcant CAD (n=11), history of receiving a renal transplant (n=1), and ESRD undergoing dialysis at entry (n=1). CAD was docu- Value $ P mented by angiographic evidence of at least 75% stenosis of at least Adjusted

Cox Regression Models one major coronary artery or a history of prior angioplasty, coronary artery bypass surgery, or myocardial infarction by history validated by electrocardiographic changes. During follow-up, patients who had only one or no follow-up creatinine data and who were followed-up

(95% CI) for fewer than 6 months were also excluded from the study. A total Adjusted HR of 386 patients with CAD were enrolled. A group of 361 healthy controls were also recruited from volunteers who were receiving health checkups. The control patients were enrolled for genotyping of the 0.001 2.05 (1.36 to 3.11 0.001 1.77 (1.33 to 2.37) Value length polymorphism of (GT)n repeats in the HO-1 gene promoter. Crude , P The healthy volunteers had no CAD or cardiovascular or kidney diseases. The study protocol was approved by the institutional review board of Taipei Tzuchi Hospital. Informed consent was obtained Recessive Model for L/L versus S/L+S/S Additive Model for Each L Allele from all participants, and our study complies with the Declaration of Helsinki. (95% CI) Crude HR

Clinical Data Collection Baseline demographic data were recorded at the time of recruitment. Hypertension was deﬁned as a measured systolic BP.140 mmHg, a diastolic BP.90 mmHg, and/or the use of antihypertensive medications. Diabetes was diagnosed on the basis of the World Health Or- ganization criteria. Congestive heart failure was diagnosed as a left ventricular ejection fraction ,40% by echocardiography and symptoms/signs of clinical criteria. Cerebrovascular disease was diagnosed as a brain infarction or hemorrhage by brain imaging and clinical symptoms/signs. Peripheral artery disease was diagnosed as an ankle- 7.4 13.1 22.1 2.02 (1.13 to 3.62) 0.02 2.43 (1.28 to 2.04) 0.01 1.74 (1.14 to 2.66) 0.01 1.99 (1.27 to 3.14) 0.003 Person-Years by Genotype S/S S/L L/L 20.2 25.8 56.2 2.33 (1.57 to 3.46) Cumulative Events per 1000 brachial index #0.9 and clinical symptoms/signs.

Laboratory Measurements Venous blood samples were drawn from the patients who had fasted Cumulative renal, major adverse cardiovascular and mortality events, and HRs (95% CIs) of genotypes of the overnight. Albumin was measured using the bromocresol green method. Iron, total cholesterol, triglyceride, HDL cholesterol, LDL

End Point cholesterol, glucose, calcium, phosphorus, and creatinine levels in cardiovascular events doubling and/ or ESRD A multivariate Cox regression model was adjusted for age, sex, smoking status, diabetes, hypertension, prior congestive heart failure, stroke or pe albumin, hemoglobin, eGFR, presence of proteinuria at baseline, and use of renin-angiotensin system blockades or statins. Table 3. a in patients with CAD with a median follow-up of 10.2 years Major adverse Serum creatinine All-cause mortality 22.0 30.7theserumweredeterminedusingc 53.4 1.87 (1.29 to 2.71) 0.001 1.47 (1.02 to 2.18) 0.01 1.58 (1.22 to 2.06)ommercialkitsandaHitachi7600 0.001 1.36 (1.04 to 1.79) 0.03

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Table 4. Adjusted HRs (95% CIs) of genotypes of the HO-1 microsatellite promoter polymorphism for renal end pointsa in patients with CAD with a median follow-up of 10.2 years Recessive Model for L/L versus S/L+S/S Additive Model for Each L Allele Model Adjusted HR (95% CI) P Value Adjusted HR (95% CI) P Value Cox regressionb 2.43 (1.28 to 2.04) 0.01 1.99 (1.27 to 3.14) 0.003 Cox regression with time-varying covariatec 2.41 (1.31 to 4.55) 0.01 1.91 (1.20 to 3.06) 0.01 aRenal end points are serum creatinine doubling and/or ESRD necessitating long-term RRT. bA Cox regression model was adjusted for age, sex, smoking status, diabetes, hypertension, prior congestive heart failure, stroke or peripheral arterial disease, total cholesterol, HDL cholesterol, serum albumin, hemoglobin, eGFR, presence of proteinuria at baseline, and use of renin-angiotensin system blockadesorstatins. cThe multivariate Cox regression model was further analyzed using the cardiac events as the time-dependent covariates. autoanalyzer (Roche Modular; Hitachi, Ltd., Tokyo, Japan). The gastrointestinal bleeding, chronic obstructive lung disease, and total iron binding capacity (TIBC) was measured using the TIBC chronic liver disease. Microtest (Daiichi, Tokyo, Japan), and serum ferritin was determined using an RIA (Incstar, Stillwater, MN). Transferrin saturation Statistical Analyses wascalculatedastheratioofserumirontoTIBCandwaspresentedas A comparison of the genotypes and allelic frequencies of the HO-1 percentages. Proteinuriawas defined as a value of trace or higher on a microsatellite promoter polymorphism between patients with CAD urine dipstick assay (Clinitek Atlas 10; Bayer HealthCare, Misha- and healthy individuals was performed using a chi-squared test. Baseline waka, IN). Plasma malondialdehyde was determined with a thio- descriptive variables were expressed as percentages for categorical data, barbituric acid test. The adducts consisting of two molecules of means6SDs for continuous data with a normal distribution, and me- thiobarbituric acid were separated by an HPLC method described by dians (interquartile ranges) for continuous data without a normal dis- Nielsen et al.39 Total serum bilirubin was measured using the meta- tribution. Potential differences among the three patient groups of the vanadate oxidation method (Wako Pure Chemical Industries, Ltd., HO-1 promoter genotype were assessed with ANOVA for normally Osaka, Japan). distributed data, the Kruskal–Wallis test for non-normally distributed data, or the Pearson chi-squared test for categorical variables. Cumula- tive survival curves for the renal end points and all-cause mortality were Length Polymorphism of (GT) Repeats in the HO-1 n generated using the Kaplan–Meier method. Between-group survival Gene Promoter HO-1 Genomic DNAs were extracted from leukocytes with conventional rates among the genotypes of the promoter polymorphism procedures. The 59-flanking region containing (GT) repeats of the were compared using a log-rank test. The assumption of proportional n fi HO-1 gene was amplified by the PCR with a 5-carboxyfluorescein– hazards was con rmed by a log minus log plot and was met in the Cox labeled sense primer (59-AGAGCCTGCAGCTTCTCAGA-39)andan models. A multivariate Cox regression model was used to estimate the antisense primer (59-ACAAAGTCTGGCCATAGGAC-39), according HRs of the renal end points, major adverse cardiovascular events, and HO-1 to a previously published procedure.40 The PCR products were mixed all-cause mortality in relation to the genotypes of the microsat- together with the GenoType TAMRA DNA ladder (size range, 50–500 ellite promoter polymorphism. The analysis was adjusted for age, sex, bp; GibcoBRL) and analyzed with an automated DNA sequencer smoking status, diabetes, hypertension, prior congestive heart failure, stroke, and peripheral artery disease, total cholesterol, HDL cholesterol, (ABI Prism 377). Each size of the (GT)n repeats was calculated with GeneScan analysis software (PE Applied Biosystems). serum albumin, hemoglobin, eGFR, presence of proteinuria at baseline, and use of renin-angiotensin system blockades and statins. Because the length polymorphism in the HO-1 promoter was significantly associ- Outcome Data Collection ated with bilirubin, ferritin, and malondialdehyde, these three variables The primary renal end points included sustained serum creatinine were not offered simultaneously in a Cox regression model to avoid doubling and/or ESRD necessitating long-term RRT from the time of multicollinearity. The HRs of the renal end points were further exam- inclusion in the study. The secondary end points were major adverse ined using cardiac events as time-dependent covariates in the Cox re- cardiovascular events and all-cause mortality. The cohort was gression model in relation to the genotypes of the HO-1 microsatellite followed until August 31, 2012. The patients were followed-up for promoter polymorphism, total bilirubin, serum ferritin, and malondial- at least 6 months. A trained physician who was blinded to the length dehyde, respectively. Statistical analysis was performed using SPSS soft- polymorphism of (GT) repeats in the HO-1 gene promoter inde- n ware (version 16.0; SPSS Inc., Chicago, IL). All P values were two-tailed. pendently obtained information about the occurrence of interim P values,0.05 were considered statistically significant. ESRD, major adverse cardiovascular events, and cause of death by reviewing hospital records and making phone calls to the study patients. ESRD means the patients undergo long-term RRT, including hemodialysis, peritoneal dialysis, or renal transplantation. Major ad- ACKNOWLEDGMENTS verse cardiovascular events consist of myocardial infarction and is- chemic stroke. All-cause mortality included cardiovascular death and We thank P.C. Lee for her expert secretarial assistance and graphic noncardiovascular causes comprising infection, sepsis, malignancy, design.

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This study was supported by grants from the National Science 14. Wu MM, Chiou HY, Chen CL, Wang YH, Hsieh YC, Lien LM, Lee TC, Council (NSC 99-2314-B-010-002-MY3 and NSC 102-2314-B-010- Chen CJ: GT-repeat polymorphism in the heme oxygenase-1 gene 004-MY3), the Taipei Veterans General Hospital (V96ER2-012 and promoter is associated with cardiovascular mortality risk in an arsenic- ’ exposed population in northeastern Taiwan. Toxicol Appl Pharmacol V99C1-121), and the Ministry of Education s Aim for the Top Uni- 248: 226–233, 2010 versity Plan. 15. Wu MM, Chiou HY, Lee TC, Chen CL, Hsu LI, Wang YH, Huang WL, Hsieh YC, Yang TY, Lee CY, Yip PK, Wang CH, Hsueh YM, Chen CJ: GT- repeat polymorphism in the heme oxygenase-1 gene promoter and the DISCLOSURES risk of carotid atherosclerosis related to arsenic exposure. J Biomed Sci 17: 70, 2010 None. 16. Wu MM, Chiou HY, Chen CL, Hsu LI, Lien LM, Wang CH, Hsieh YC, Wang YH, Hsueh YM, Lee TC, Cheng WF, Chen CJ: Association of heme oxygenase-1 GT-repeat polymorphism with blood pressure REFERENCES phenotypes and its relevance to future cardiovascular mortality risk: An observation based on arsenic-exposed individuals. Atherosclerosis 219: 704–708, 2011 1. Maines MD: Heme oxygenase: Function, multiplicity, regulatory 17. Chen YH, Hung SC, Tarng DC: Length polymorphism in heme oxygenase-1 – mechanisms, and clinical applications. FASEB J 2: 2557 2568, 1988 and cardiovascular events and mortality in hemodialysis patients. Clin 2. Nath KA: Heme oxygenase-1: A provenance for cytoprotective path- JAmSocNephrol8: 1756–1763, 2013 – ways in the kidney and other tissues. Kidney Int 70: 432 443, 2006 18. Courtney AE, Maxwell AP: Heme oxygenase 1: Does it have a role in 3. Juan SH, Lee TS, Tseng KW, Liou JY, Shyue SK, Wu KK, Chau LY: renal cytoprotection? Am J Kidney Dis 51: 678–690, 2008 Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the 19. Elsayed EF, Tighiouart H, Griffith J, Kurth T, Levey AS, Salem D, Sarnak fi development of atherosclerosis in apolipoprotein E-de cient mice. MJ, Weiner DE: Cardiovascular disease and subsequent kidney dis- – Circulation 104: 1519 1525, 2001 ease. Arch Intern Med 167: 1130–1136, 2007 4. Tamura T, Kondo T, Ogawa K, Fukunaga K, Ohkohchi N: Protective 20. Kiyosue A, Hirata Y, Ando J, Fujita H, Morita T, Takahashi M, Nagata D, effect of heme oxygenase-1 on hepatic ischemia-reperfusion injury Kohro T, Imai Y, Nagai R: Relationship between renal dysfunction and through inhibition of platelet adhesion to the sinusoids. J Gastroenterol severity of coronary artery disease in Japanese patients. Circ J 74: 786– – Hepatol 28: 700 706, 2013 791, 2010 5. Otterbein LE, Kolls JK, Mantell LL, Cook JL, Alam J, Choi AM: Exoge- 21. Hsu TW, Kuo KL, Hung SC, Huang PH, Chen JW, Tarng DC: Progression nous administration of heme oxygenase-1 by gene transfer provides of kidney disease in non-diabetic patients with coronary artery disease: protection against hyperoxia-induced lung injury. J Clin Invest 103: Predictive role of circulating matrix metalloproteinase-2, -3, and -9. 1047–1054, 1999 PLoS ONE 8: e70132, 2013 6. Laniado-Schwartzman M, Abraham NG, Conners M, Dunn MW, Levere 22. Agarwal A, Nick HS: Renal response to tissue injury: Lessons from heme RD, Kappas A: Heme oxygenase induction with attenuation of experi- oxygenase-1 GeneAblation and expression. JAmSocNephrol11: mentally induced corneal inflammation. Biochem Pharmacol 53: 1069– 965–973, 2000 1075, 1997 23. Kim JH, Yang JI, Jung MH, Hwa JS, Kang KR, Park DJ, Roh GS, Cho GJ, 7. Kutty RK, Kutty G, Rodriguez IR, Chader GJ, Wiggert B: Chromosomal Choi WS, Chang SH: Heme oxygenase-1 protects rat kidney from localization of the human heme oxygenase genes: Heme oxygenase-1 ureteral obstruction via an antiapoptotic pathway. J Am Soc Nephrol (HMOX1) maps to chromosome 22q12 and heme oxygenase-2 17: 1373–1381, 2006 (HMOX2) maps to chromosome 16p13.3. Genomics 20: 513–516, 1994 24. Kie JH, Kapturczak MH, Traylor A, Agarwal A, Hill-Kapturczak N: Heme 8. Exner M, Schillinger M, Minar E, Mlekusch W, Schlerka G, Haumer M, oxygenase-1 deficiency promotes epithelial-mesenchymal transition Mannhalter C, Wagner O: Heme oxygenase-1 gene promoter micro- and renal fibrosis. J Am Soc Nephrol 19: 1681–1691, 2008 satellite polymorphism is associated with restenosis after percutaneous 25. Desbuards N, Hyvelin JM, Machet MC, Eder V, Garrigue MA, Halimi transluminal angioplasty. J Endovasc Ther 8: 433–440, 2001 JM, Antier D: Heme oxygenase-1 inducer hemin attenuates the pro- 9. Chen YH, Lin SJ, Lin MW, Tsai HL, Kuo SS, Chen JW, Charng MJ, Wu TC, gression of remnant kidney model. Nephron, Exp Nephrol 113: e35– Chen LC, Ding YA, Pan WH, Jou YS, Chau LY: Microsatellite poly- e44, 2009 morphism in promoter of heme oxygenase-1 gene is associated with 26. Correa-Costa M, Semedo P, Monteiro AP, Silva RC, Pereira RL, susceptibility to coronary artery disease in type 2 diabetic patients. Gonçalves GM, Marques GD, Cenedeze MA, Faleiros AC, Keller AC, Hum Genet 111: 1–8, 2002 Shimizu MH, Seguro AC, Reis MA, Pacheco-Silva A, Câmara NO: In- 10. Kaneda H, Ohno M, Taguchi J, Togo M, Hashimoto H, Ogasawara K, duction of heme oxygenase-1 can halt and even reverse renal tubule- Aizawa T, Ishizaka N, Nagai R: Heme oxygenase-1 gene promoter interstitial fibrosis. PLoS ONE 5: e14298, 2010 polymorphism is associated with coronary artery disease in Japanese 27. Lüblinghoff N, Winkler K, Winkelmann BR, Seelhorst U, Wellnitz B, patients with coronary risk factors. Arterioscler Thromb Vasc Biol 22: Boehm BO, März W, Hoffmann MM: Genetic variants of the promoter of 1680–1685, 2002 the heme oxygenase-1 gene and their influence on cardiovascular 11. Schillinger M, Exner M, Minar E, Mlekusch W, Müllner M, Mannhalter C, disease (the Ludwigshafen Risk and Cardiovascular Health study). BMC Bach FH, Wagner O: Heme oxygenase-1 genotype and restenosis after Med Genet 10: 36, 2009 balloon angioplasty: A novel vascular protective factor. JAmColl 28. Pritchard JK, Rosenberg NA: Use of unlinked genetic markers to detect Cardiol 43: 950–957, 2004 population stratification in association studies. Am J Hum Genet 65: 12. Chen YH, Chau LY, Lin MW, Chen LC, Yo MH, Chen JW, Lin SJ: Heme 220–228, 1999 oxygenase-1 gene promotor microsatellite polymorphism is associated 29. Goodman AI, Chander PN, Rezzani R, Schwartzman ML, Regan RF, with angiographic restenosis after coronary stenting. Eur Heart J 25: Rodella L, Turkseven S, Lianos EA, Dennery PA, Abraham NG: Heme 39–47, 2004 oxygenase-2 deficiency contributes to diabetes-mediated increase in 13. Dick P, Schillinger M, Minar E, Mlekusch W, Amighi J, Sabeti S, Schlager superoxide anion and renal dysfunction. J Am Soc Nephrol 17: 1073– O,RaithM,EndlerG,MannhalterC,WagnerO,ExnerM:Haemoxygenase-1 1081, 2006 genotype and cardiovascular adverse events in patients with peripheral 30. Nath KA, Grande JP, Farrugia G, Croatt AJ, Belcher JD, Hebbel RP, artery disease. Eur J Clin Invest 35: 731–737, 2005 Vercellotti GM, Katusic ZS: Age sensitizes the kidney to heme

2676 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2669–2677, 2014 www.jasn.org CLINICAL RESEARCH

protein-induced acute kidney injury. Am J Physiol Renal Physiol 304: 37. Chin HJ, Cho HJ, Lee TW, Na KY, Yoon HJ, Chae DW, Kim S, Jeon US, Do F317–F325, 2013 JY, Park JW, Yoon KW, Shin YT, Lee KW, Na KR, Cha DR, Kang YS; Pro- 31. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN: Bilirubin gressive REnal disease and Medical Informatics and gEnomics Research is an antioxidant of possible physiological importance. Science 235: (PREMIER) members: The heme oxygenase-1 genotype is a risk factor to 1043–1046, 1987 renal impairment of IgA nephropathy at diagnosis, which is a strong 32. Willis D, Moore AR, Frederick R, Willoughby DA: Heme oxygenase: A predictor of mortality. JKoreanMedSci24[Suppl]: S30–S37, 2009 novel target for the modulation of the inﬂammatory response. Nat Med 38. Concato J, Shah N, Horwitz RI: Randomized, controlled trials, obser- 2: 87–90, 1996 vational studies, and the hierarchy of research designs. NEnglJMed 33. Chen YH, Chau LY, Chen JW, Lin SJ: Serum bilirubin and ferritin levels 342: 1887–1892, 2000 link heme oxygenase-1 gene promoter polymorphism and suscepti- 39. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean P: bility to coronary artery disease in diabetic patients. Diabetes Care 31: Plasma malondialdehyde as biomarker for oxidative stress: Reference 1615–1620, 2008 interval and effects of life-style factors. Clin Chem 43: 1209–1214, 1997 34. Zarjou A, Bolisetty S, Joseph R, Traylor A, Apostolov EO, Arosio P, Balla 40. Kimpara T, Takeda A, Watanabe K, Itoyama Y, Ikawa S, Watanabe M, J, Verlander J, Darshan D, Kuhn LC, Agarwal A: Proximal tubule H- Arai H, Sasaki H, Higuchi S, Okita N, Takase S, Saito H, Takahashi K, ferritin mediates iron trafﬁcking in acute kidney injury. JClinInvest123: Shibahara S: Microsatellite polymorphism in the human heme oxygenase-1 4423–4434, 2013 gene promoter and its application in association studies with Alzheimer 35. Katan MB: Apolipoprotein E isoforms, serum cholesterol, and cancer. and Parkinson disease. Hum Genet 100: 145–147, 1997 Lancet 1: 507–508, 1986 36. Courtney AE, McNamee PT, Heggarty S, Middleton D, Maxwell AP: Association of functional haem oxygenase-1 gene promoter polymorphism with polycystic kidney disease and IgA nephropathy. This article contains supplemental material online at http://jasn.asnjournals. Nephrol Dial Transplant 23: 608–611, 2008 org/lookup/suppl/doi:10.1681/ASN.2013111205/-/DCSupplemental.

J Am Soc Nephrol 25: 2669–2677, 2014 HO-1 Promoter Polymorphism and CKD 2677 Supplementary data

Length Polymorphism in Heme Oxygenase-1 and Risk of Chronic Kidney Disease among Coronary Artery Disease Patients

Yu-Hsin Chen,§* Ko-Lin Kuo,** Szu-Chun Hung,** Chih-Cheng Hsu,ψ Ying-Hwa

Chen,§† and Der-Cherng Tarng§ǁ¶‡

§Faculty of Medicine, ǁInstitute of Clinical Medicine and ¶Department and Institute of

Physiology, National Yang-Ming University, Taipei; *Division of Nephrology,

Department of Internal Medicine, Taipei City Hospital Yang-Ming Branch, Taipei;

**Division of Nephrology, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical

Foundation, Taipei; ψDivision of Geriatrics and Gerontology, Institute of Population

Health Sciences, National Health Research Institutes; Divisions of †Cardiology and

‡Nephrology, Department of Medicine and Immunology Center, Taipei Veterans

General Hospital, Taipei, Taiwan

1 Supplementary Table 1. Adjusted hazard ratio (95% confidence interval) of

total bilirubin, serum ferritin and malondialdehyde for renal endpointsa

in CAD patients with a median follow-up of 10.2 years

Adjusted Hazard Ratio (95% Confidence Interval)

Bilirubin Ferritin Malondialdehyde

For each 1 For each 100 For each 1

mg/dL increase μg/L increase μmol/L increase

Cox regression modelb 0.91 (0.860.95) 1.20 (1.031.32) 1.23 (1.011.42) P = 0.022 P = 0.012 P = 0.010

Cox with time-varying 0.90 (0.850.99) 1.16 (0.991.35) 1.12 (0.901.45) covariate modelc P = 0.048 P = 0.058 P = 0.072 aRenal endpoints are serum creatinine doubling and/or end-stage renal disease

necessitating long-term renal replacement therapy. bA Cox regression model was adjusted for age, sex, smoking status, diabetes,

hypertension, prior congestive heart failure, stroke or peripheral arterial disease, total

cholesterol, high-density lipoprotein-cholesterol, serum albumin, hemoglobin,

estimated glomerular filtration rate, the presence of proteinuria at baseline, and the

use of renin-angiotensin system blockades or statins. cThe multivariate Cox regression model was further analyzed using the cardiac events as the time-dependent covariates. dTo avoid multicollinearity, the genotypes of length polymorphism in HO-1 promoter were not offered in the Cox regression model and Cox with time-varying covariate model.