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Genetic polymorphisms of paraoxonase-1 are associated with chronic kidney disease in Japanese women

2009; Elsevier BV; Volume: 76; Issue: 2 Linguagem: Inglês

10.1038/ki.2009.97

1523-1755

Kazunobu Ichikawa, Tsuneo Konta, Mitsuru Emi, Sayumi Toriyama, Satoshi Takasaki, Ami Ikeda, Yoko Shibata, Noriaki Takabatake, Yasuchika Takeishi, Takeo Kato, Sumio Kawata, Isao Kubota,

Apelin-related biomedical research

Paraoxonase-1 (PON1) is an HDL cholesterol–associated antioxidant enzyme, and some of its polymorphisms are linked with systemic oxidative stress and cardiovascular events. In this study, we genotyped seven single nucleotide polymorphisms (SNPs) within the PON1 gene and determined their association with chronic kidney disease in 2,968 individuals from the general Japanese population. We found that a missense SNP (rs662) with a G-to-A substitution leading to an amino acid substitution (G[Arg]/A[Gln]), was significantly associated with albuminuria and estimated glomerular filtration rate (eGFR), especially in women. The A/A genotype in women had the highest prevalence of albuminuria and the lowest values of adjusted eGFR. In contrast, such relationships were not detected in men. Multivariate regression analysis found that the A/A genotype was an independent and significant factor for albuminuria and renal insufficiency (eGFR less than 60 ml/min/1.73 m2). The serum PON1 activity was lowest in subjects with the A/A genotype. In biopsy specimens, immunohistochemical analysis found increased PON1 expression on the endothelial surface of sclerotic renal arterioles and glomerular capillaries in patients with hypertension or diabetes. Our study shows that this PON1 G-to-A substitution may be a key player in a common pathway to chronic kidney and cardiovascular diseases in women. Paraoxonase-1 (PON1) is an HDL cholesterol–associated antioxidant enzyme, and some of its polymorphisms are linked with systemic oxidative stress and cardiovascular events. In this study, we genotyped seven single nucleotide polymorphisms (SNPs) within the PON1 gene and determined their association with chronic kidney disease in 2,968 individuals from the general Japanese population. We found that a missense SNP (rs662) with a G-to-A substitution leading to an amino acid substitution (G[Arg]/A[Gln]), was significantly associated with albuminuria and estimated glomerular filtration rate (eGFR), especially in women. The A/A genotype in women had the highest prevalence of albuminuria and the lowest values of adjusted eGFR. In contrast, such relationships were not detected in men. Multivariate regression analysis found that the A/A genotype was an independent and significant factor for albuminuria and renal insufficiency (eGFR less than 60 ml/min/1.73 m2). The serum PON1 activity was lowest in subjects with the A/A genotype. In biopsy specimens, immunohistochemical analysis found increased PON1 expression on the endothelial surface of sclerotic renal arterioles and glomerular capillaries in patients with hypertension or diabetes. Our study shows that this PON1 G-to-A substitution may be a key player in a common pathway to chronic kidney and cardiovascular diseases in women. Chronic kidney disease (CKD) is recently attracting the attention of clinicians because of high prevalence in the general population. Especially, its relation with cardiovascular diseases (CVD) observed in several epidemiological studies has a wide influence on daily practice.1Irie F. Iso H. Sairenchi T. et al.The relationships of proteinuria, serum creatinine, glomerular filtration rate with cardiovascular disease mortality in Japanese general population.Kidney Int. 2006; 69: 1264-1271Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar,2Go A.S. Chertow G.M. Fan D. et al.Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization.N Engl J Med. 2004; 351: 1296-1305Crossref PubMed Scopus (8250) Google Scholar The common diseases including CVD and CKD are multifactorial disorders affected by various environmental and genetic factors. The risk factor analyses for the development of renal insufficiency showed that the environmental risk factors such as hypertension, hyperglycemia, dyslipidemia, smoking, and oxidative stress are involved.3Yamagata K. Ishida K. Sairenchi T. et al.Risk factors for chronic kidney disease in a community-based population: a 10-year follow-up study.Kidney Int. 2007; 71: 159-166Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar,4Oberg B.P. McMenamin E. Lucas F.L. et al.Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease.Kidney Int. 2004; 65: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar Conversely, little is known about genetic factors. Oxidative stress is induced by various metabolic disorders and plays an important role in the pathogenesis of atherosclerosis. An antioxidative agent is considered to attenuate the progression of vascular injury. Paraoxonase-1 (PON1), one of the enzymes with antioxidative properties, is a high-density lipoprotein–associated enzyme that promotes the function of high-density lipoprotein (that is, antioxidation, anticoagulation, and anti-inflammation). It is reported that serum PON1 activity was decreased in patients with atherosclerosis, Alzheimer's disease, and chronic renal failure.5Shih D.M. Gu L. Xia Y.R. et al.Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis.Nature. 1998; 394: 284-287Crossref PubMed Scopus (923) Google Scholar, 6Tward A. Xia Y.R. Wang X.P. et al.Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice.Circulation. 2002; 106: 484-490Crossref PubMed Scopus (367) Google Scholar, 7Dantoine T.F. Paraoxonase 1 192/55 gene polymorphisms in Alzheimer's disease.Ann NY Acad Sci. 2002; 977: 239-244Crossref PubMed Scopus (47) Google Scholar, 8Dantoine T.F. Debord J. Charmes J.P. et al.Decrease of serum paraoxonase activity in chronic renal failure.J Am Soc Nephrol. 1998; 9: 2082-2088PubMed Google Scholar Recently, Bhattacharyya et al.9Bhattacharyya T. Nicholls S.J. Topol E.J. et al.Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk.JAMA. 2008; 299: 1265-1276Crossref PubMed Scopus (418) Google Scholar reported that the genotype of PON1 is associated with serum PON1 activity, systemic oxidative levels and cardiovascular events. These observations suggest a possibility that PON1 might be involved in a wide range of diseases induced by oxidative stress. However, it is unknown whether the PON1 genotype is related to the development of chronic kidney disease. In this study, we examined the relation between renal function, urine albumin excretion, and PON1 in the Japanese general population. Baseline characteristics of 2968 subjects who entered into a final analysis were as follows; mean age was 63.0±10.5 years old, 1343 men (45.2%), 1629 subjects (54.9%) with hypertension, 234 subjects (7.8%) with diabetes, 895 subjects (30.2%) with obesity, 988 subjects (33.3%) with hypercholesterolemia, and 653 subjects (22.0%) with albuminuria. PON1 gene is located on chromosome 7q21–q22. We have selected seven single nucleotide polymorphisms (SNPs) regarding PON1 genes that displayed frequent minor allele frequencies in Japanese from dbSNP database of the NCBI and International HapMap Project as described in Materials and Methods section. The chromosomal locations of these seven SNPs are shown in Figure 1 and their characters are summarized in Table 1.Table 1Polymorphisms in PON1 genes examined in the studySNP IDNCBI SNP referenceSNP typePublic location position (B36.2)AlleleAllele frequencyGenotypeHeterozygosity12Allele 1Allele 2111222SNP1rs3917527intron 594778194AG0.880.122274649440.22SNP2rs2057681intron 594776193CT0.660.34129013503260.45SNP3rs3917538intron 594775829TC0.530.4782214926530.50SNP4rs3917541intron 594775560CT0.880.122264647440.22SNP5rs662missense94775382G(Arg)A(Gln)0.660.34128713513270.45SNP6rs2269829intron 694774065CT0.650.35124913693450.45SNP7rs854555intron 894778327TG0.650.35122213353600.46NCBI, National Center for Biotechnology Information; PON1, paraoxonase-1.allele 1, major allele; allele 2, minor allele.Typing call rates were over 99% The Hardy–Weinberg equilibrium P-values did not deviate in all SNPs (P>0.1). Open table in a new tab NCBI, National Center for Biotechnology Information; PON1, paraoxonase-1. allele 1, major allele; allele 2, minor allele. Typing call rates were over 99% The Hardy–Weinberg equilibrium P-values did not deviate in all SNPs (P>0.1). To examine linkage disequilibrium (LD) of the PON1 gene, we performed LD analysis using seven SNP typing data. We observed a consistency of genotypes between SNP5 and SNP7, rs622G/A, rs2269829C/T and rs854555T/G, by detecting a linkage disequilibrium (D′>0.85 and r2>0.70) (Figure 2). It is well known that the missense SNP5 rs662 with amino acid substitution is related to paraoxonase activity and the LD analysis demonstrated that the surrounding SNP6 and SNP7 were in LD block with SNP5 in this population. Therefore, we hereafter focused this SNP5 rs662 and examined its relation with albuminuria and renal function. The comparison of basal characters in subjects with rs662 genotypes A/A, A/G, and G/G was shown in Table 2. There was no significant difference in the basal characters among subjects with these genotypes except uric acid levels.Table 2Genotype-based comparisons of basal characteristics in rs662rs662 GenotypesA/AA/GG/GP-valueNumber32613501287Men (%)50.044.045.3NSaχ2 test.Age (years)62.9±9.963.0±10.363.0±10.3NSbAnalysis of variance, Mean±s.d.Drinker (%)45.440.041.9NSaχ2 test.Current smoker (%)20.217.419.1NSaχ2 test.Hypertension (%)55.253.955.8NSaχ2 test.Diabetes (%)7.77.78.2NSaχ2 test.Hypercholesterolemia (%)32.533.832.9NSaχ2 test.Obesity (%)30.729.630.5NSaχ2 test.Systolic BP (mm Hg)134.7±15.1133.6±16.1134.7±15.7NSbAnalysis of variance, Mean±s.d.Diastolic BP (mm Hg)79.9±9.778.9±10.279.6±9.9NSbAnalysis of variance, Mean±s.d.Serum creatinine (mg/100ml)0.70±0.160.67±0.160.68±0.29NSbAnalysis of variance, Mean±s.d.Hemoglobin A1c (%)5.25±0.705.24±0.655.25±0.69NSbAnalysis of variance, Mean±s.d.HDL-C (mg/100ml)59.4±14.959.2±14.858.9±14.1NSbAnalysis of variance, Mean±s.d.LDL-C (mg/100ml)123.5±29.0125.0±30.3123.5±29.3NSbAnalysis of variance, Mean±s.d.Triglyceride (mg/100ml)108.0±77.0106.4±63.5106.4±60.1NSbAnalysis of variance, Mean±s.d.Uric acid (mg/100ml)5.2±1.45.1±1.35.0±1.40.015bAnalysis of variance, Mean±s.d.Hemoglobin (g/100ml)13.8±1.613.7±1.413.7±1.4NSbAnalysis of variance, Mean±s.d.Past history of CVD (%)12.311.113.7NSaχ2 test.BP, blood pressure; CVD, cardiovascular disease; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein cholesterol; NS, not significant.a χ2 test.b Analysis of variance, Mean±s.d. Open table in a new tab BP, blood pressure; CVD, cardiovascular disease; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein cholesterol; NS, not significant. First we examined the relationship between rs662 genotypes and urine albumin excretion. The prevalence of albuminuria was significantly related to rs662 genotypes with highest value in A/A genotype (A/A: 27.9, A/G: 22.2, and G/G: 21.1%, respectively, P=0.0327) in total subjects. Further analysis revealed the gender difference in the relation. In women A/A genotype showed the strikingly high prevalence as compared with other genotypes (A/A: 32.5, A/G: 21.8 and G/G: 19.5%, P=0.0017). In contrast, there was no significant difference between genotypes in men (Figure 3a). The levels of urine albumin–creatinine ratio showed a similar tendency that the significant relation was observed in women, but not in men (Figure 3b). Next, we examined the relationship between genotype of rs662 and renal function. To exclude the effects of covariates we adjusted estimated glomerular filtration rate (eGFR) for age, gender, body mass index, systolic blood pressure, total protein, total cholesterol, triglyceride, uric acid, HbA1c, hemoglobin, smoking, and drinking, using a general linear model. Then, we performed quantitative trait locus analysis of population having this SNP and adjusted eGFR by an analysis of variance (ANOVA). This analysis showed that adjusted eGFR levels were significantly associated with rs662 genotypes in women. The eGFR values of population having SNP rs662 A/A, A/G, and G/G were 78.7±0.5, 79.5±0.2, and 80.2±0.2 ml/min per 1.73 m2 (mean±s.e.), respectively (P=0.0139). Again, such a relation was not observed in men (Figure 4). These relationships between renal function, albuminuria, and rs662 genotype were preserved in non-diabetic population after excluding diabetic subjects (data not shown). Further, to examine the independent relation of rs662 genotype with albuminuria and renal insufficiency (eGFR <60 ml/min per 1.73 m2), we performed multivariate logistic regression analysis including age, gender, hypertension, diabetes, obesity, smoking, drinking, and hypercholesterolemia. It demonstrated that the rs662 A/A genotype was an independent factor for albuminuria (odds ratio 1.432 (95%CI 1.091–1.879), P=0.0096) and renal insufficiency (odds ratio 1.503 (95%CI 1.016–2.224), P=0.0412) (Table 3).Table 3Odds ratio of rs662 genotype for albuminuria and renal insufficiency (eGFR <60 ml/min per 1.73 m2)UnadjustedAdjustedaAdjusted for gender, age, drinking, smoking, obesity, diabetes, hypertension, and hypercholesterolemia.OR (95% CI)P-valueOR (95% CI)P-valueAlbuminuriars662 A/A1.397 (1.076–1.813)0.01191.432 (1.091–1.879)0.0096Renal insufficiency (eGFR 99%) and were used in this study. Genotypes for these seven SNPs were determined by an Invader assay (Third Wave Technologies, Madison, WI, USA)28Lyamichev V. Mast A.L. Hall J.G. et al.Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.Nat Biotechnol. 1997; 17: 292-296Google Scholar,29Mein C.A. Barratt B.J. Dunn M.G. et al.Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.Genome Res. 2000; 10: 330-343Crossref PubMed Scopus (179) Google Scholar and TaqMan allelic discrimination assay.30Livak K.J. Allelic discrimination using fluorogenic probes and the 5′ nuclease assay.Genet Anal. 1999; 14: 143-149Crossref PubMed Scopus (1180) Google Scholar Reagents were purchased from Applied Biosystems (Foster City, CA, USA). TaqMan probes were designed and synthesized by Applied Biosystems, and distinguish the SNPs at the end of a polymerase chain reaction. One allelic probe was labeled with fluorescent FAM dye and the other with the fluorescent VIC dye. Polymerase chain reaction was performed by TaqMan Universal Master Mix without UNG (Applied Biosystems) with polymerase chain reaction primers at a concentration of 900 nM and TaqMan MGB probes at a concentration of 200 nM. Reactions were performed in 384-well formats in a total reaction volume of 3 μl using 3.0 ng of genomic DNA. The plates were then placed in a GeneAmp PCR System 9700 (Applied Biosystems) and heated at 95°C for 10 min, followed by 40 cycles at 92°C for 15 s and 60°C for 1 min, with a final soak at 25°C. The plates were read by the Prism 7900HT instrument (Applied Biosystems) where the fluorescence intensity in each well of the plate was read.28Lyamichev V. Mast A.L. Hall J.G. et al.Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.Nat Biotechnol. 1997; 17: 292-296Google Scholar Fluorescence data files from each plate were analyzed by the SDS 2.0 allele calling software (Applied Biosystems). Several data (signal intensity) were eliminated to preserve the reliability of the assay system (missing data are guaranteed to be less than 1%). We used a χ2 test to evaluate differences in proportions and an ANOVA with the Bonferroni test as a post hoc test to evaluate differences in means. For some of the clinical and biochemical traits that did not distribute normally, we applied a non-parametric Kruskal–Wallis test. To confirm the Hardy–Weinberg equilibrium among genotypes, a χ2 test was used (P≥0.05). LD for the combination of variations was tested by D' and r2 by using Haploview. Data are expressed as mean±s.d. except as otherwise indicated. A significant difference was defined as P<0.05. All statistical analysis was performed using SPSS version 15.0.1J (SPSS Inc., Chicago, IL, USA). Anti-PON1 goat antibody and peroxidase-conjugated donkey anti-goat immunoglobulin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Immunohistochemical staining was performed on frozen sections of human biopsy specimen using enzyme-labeled antibody method. Frozen sections were air-dried and fixed in acetone for 5 min. Endogenous peroxide activity was quenched by incubating sections in 0.3% H2O2/methanol for 20 min. Sections were incubated with an antibody against PON1 (dilution 1:100) at 4°C overnight. After incubating with secondary antibody at a concentration of 1:100 for 1 h, immunoreaction products were developed using 3,3′-diaminobenzidine as the chromogen, with standardized development times. Sections were then counterstained with hematoxylin acetate. Negative controls were prepared by using irrelevant normal goat IgG as a primary antibody. All the authors declared no competing interests. This study was supported by a grant-in-aid from the 21st Century Center of Excellence (COE) program and the global COE of the Japan Society for the Promotion of Science.