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MicroRNAs have been shown to play a role in B‐cell differentiation and activation. Here, we found miR‐182 to be highly induced in activated B cells. However, mice lacking miR‐182 have normal B‐cell and T‐cell development. Interestingly, mutant mice exhibited a defective antibody response at early time‐points in the immunization regimen when challenged with a T‐cell‐dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR‐182‐deficient mice. Mutant mice were also not able to respond to a T‐cell‐independent type 2 antigen, which typically elicited an extrafollicular B‐cell response. Taken together, the data indicated that miR‐182 plays a critical role in driving extrafollicular B‐cell antibody responses.

The environmental factors such as aging, smoking, and alcohol consumption have been reported to influence DNA methylation (DNAm). However, the versatility of DNAm measurement by DNAm array systems is low in clinical use. Thus, we developed the MethyLight assay as a simple method to measure DNAm. In the present study, we isolated peripheral blood DNA from 33 healthy volunteers and selected cg25809905, cg02228185, and cg17861230 as aging, cg23576855 as smoking, and cg02583484 as alcohol consumption biomarkers. The predicted age by methylation rates of cg25809905 and cg17861230 significantly correlated with chronological age. In immortalized B-cells, DNAm rates of two sites showed a younger status than the chronological age of donor. On the other hand, the predicted age of the patients with myocardial infarction (MI) was not accelerated. The methylation rate of cg23576855 was able to discriminate the groups based on the smoking status. The DNAm rate of cg02583484 was reduced in subjects with habitual alcohol consumption compared to that of subjects without habitual alcohol consumption. In conclusion, our MethyLight assay system reconfirms that aging, smoking, and alcohol consumption influenced DNAm in peripheral blood in the Japanese. This MethyLight system will facilitate DNAm measurement in epidemiological and clinical studies.

MicroRNAs (miRNAs) are small noncoding RNAs of 18–23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various physiological and pathological conditions. The present study details the conserved miRNA expression profiles of tubular tissues, and discusses whether they could be used to distinguish between proximal tubule injury, diagnose acute kidney injury (AKI), and the early-stage renal tubular dysfunction. miRNA expression was assessed with miRNA array and real-time reverse transcription polymerase chain reaction using the TaqMan system. The expression profiles of miR-200a/b/c, miR-145, miR-192, miR-194, miR-216a/b, miR-217, and miR-449a in human and rat tubular tissues such as the kidneys, lung, small intestine, and various exocrine glands were adequate for discriminating tubular tissues. In the kidney, miR-192 and miR-194 were highly expressed, whereas miR-145 and miR-449a were absent. miR-145 and miR-449a were relatively specifically expressed in small intestine and lung, respectively. Therefore, the combined levels of miR-200a/b/c, miR-192, and miR-194 in plasma were very useful in diagnosing AKI induced by contact freezing in mice. Moreover, urinary miR-200a levels were useful for the diagnosis of renal tubular dysfunction in Dahl salt-sensitive rat with high salt administration. Our results indicate that miRNA expression profiles are useful as biomarkers for identification of various kidney injuries.

Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing nonalcoholic fatty liver (NAFL). Peroxisomes have many important functions in lipid metabolism, including fatty acid β-oxidization. However, the pathogenic link between NAFL and peroxisome biogenesis remains unclear. To examine the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Body weights and hepatic TG concentrations in Pex11α−/−mice were significantly higher than those in wild-type (WT) mice fed a normal or a high-fat diet. Hepatic TG concentrations in fasted Pex11α−/−mice were significantly higher than those in fasted WT mice. Plasma TG levels increased at lower rates in Pex11α−/−mice than in WT mice after treatment with the lipoprotein lipase inhibitor tyloxapol. The number of peroxisomes was lower in the livers of Pex11α−/−mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherically shaped peroxisomes were more prevalent in Pex11α−/−mice fed normal chow supplemented without or with fenofibrate. We observed a significantly higher ratio of empty peroxisomes containing only PMP70, a peroxisome membrane protein, but not catalase, a peroxisome matrix protein, in Pex11α−/−mice. The mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, subfamily D, member 2, and acyl-CoA thioesterase 3) were significantly higher in WT mice than those in Pex11α−/−mice under fed conditions. Our results demonstrate that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, which contributes to increased lipid accumulation in the liver.

MicroRNAs (miRNAs) are endogenous small RNAs (length, 18-ss23 nucleotides) that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored cell- or tissue-specific miRNAs and assessed the applicability of miRNA profiling for identifying biomarkers of tissue injuries. miRNA analyses in various human and rat tissues identified several candidate miRNAs with possible tissue-specific expression, some of which have already been reported. In the present study, we focused on pancreas-specific miRNAs, miR-216a and miR-216b. Laser microdissection revealed that miR-216a and 216b were predominantly expressed in acinar cells of the pancreas as compared to Langerhans' islet. Plasma concentrations of miR-216a and miR-216b considerably increased in a rat model of L-arginineinduced acute pancreatitis. The current results have confirmed that miRNA expression profiling in various cells is useful for providing biomarkers for cell- or tissue-specific injuries.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses.Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively.We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P0.01).Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.

Background: Noncoding RNAs (ncRNAs), including microRNA (miRNA) and long noncoding RNA (lncRNA), are functional and non-protein coding RNA molecules. miRNAs are small ncRNAs of 18-23 nucleotides, whereas LncRNAs are longer than 200 nucleotides and were recently recognized as a class of genomic regulatory molecules and reported in various species. Nonetheless, their physiological functions have not been elucidated. Methods: In order to investigate a possible involvement of ncRNAs in salt-sensitive hypertension, we performed ncRNA sequencing and transcriptome analysis in Dahl salt-sensitive rats and Lewis rats. The pathway analysis were used to understand the biological roles of differently expressed ncRNAs. Results: Following ncRNA sequencing, 591 transcripts were defined as mature miRNAs and 2636 transcripts as lncRNAs. Of these, 9 miRNA were mapped to candidate chromosomal regions (chromosome 1, 10, and 12), previously reported to be involved in salt-sensitive hypertension. Differential expression was detected for 3 of these miRNAs (miR-150-5p, miR-7a-5p, miR-21-5p) (fold up/down ≥ 1.2, q < 0.05). However, only one lncRNA (XLOC_081666) encoded by the candidate region of chromosome 1 showed no difference. In contrast, 10 miRNAs (miR-1b, miR-221-3p, miR-300-3p, miR-411-5p, miR-184, miR-205, miR-129-5p, miR-741-3p, miR-379-5p, miR-138-5p) and 5 lncRNAs (XLOC_102471, XLOC_047839, XLOC_253638, XLOC_778532, XLOC_1029112), located in other regions, showed significant differential expression (fold up/down ≥ 2.0, q < 0.05). The pathway analysis showed that Wnt and MAPK signaling pathways are associated with the targets of detected miRNAs and metabolic pathways are associated with the possible targets of detected lncRNAs. Although expression levels of target mRNAs in these pathways showed no differences between Dahl salt-sensitive and Lewis rats, it is likely that effects of a single ncRNA on mRNA expression are subtle and may exhibit different response to salt intake. Conclusions: Our results demonstrate the probable implication of miRNAs and lncRNAs in salt-sensitive hypertension. This new finding may contribute to the exploration of candidate factors involved in the hypertension mechanism.

The P2X7 receptor is a ligand-gated ion channel, and genetic variations in the P2X7 gene significantly affect blood pressure. P2X7 receptor expression is associated with renal injury and inflammatory diseases. Uninephrectomized wild-type (WT) and P2X7-deficient (P2X7 KO) mice were subcutaneously implanted with deoxycorticosterone acetate (DOCA) pellets and fed an 8% salt diet for 18 days. Their blood pressure was assessed by a telemetry system. The mice were placed in metabolic cages, and urine was collected for 24 h to assess renal function. After 18 days of DOCA-salt treatment, P2X7 mRNA and protein expression increased in WT mice. Blood pressure in P2X7 KO mice was less than that of WT mice (mean systolic blood pressure 133 ± 3 vs. 150 ± 2 mmHg). On day 18, urinary albumin excretion was lower in P2X7 KO mice than in WT mice (0.11 ± 0.07 vs. 0.28 ± 0.07 mg/day). Creatinine clearance was higher in P2X7 KO mice than in WT mice (551.53 ± 65.23 vs. 390.85 ± 32.81 μl·min−1·g renal weight−1). Moreover, renal interstitial fibrosis and infiltration of immune cells (macrophages, T cells, B cells, and leukocytes) were markedly attenuated in P2X7 KO mice compared with WT mice. The levels of IL-1β, released by macrophages, in P2X7 KO mice had decreased dramatically compared with that in WT mice. These results strongly suggest that the P2X7 receptor plays a key role in the development of hypertension and renal disease via increased inflammation, indicating its potential as a novel therapeutic target.

Adiponectin is most abundantly expressed in adipose tissue and well known to play an important role in metabolic regulation. Several studies have attempted to identify the genetic determinants of metabolic syndrome (MetS), though no study has revealed a cis- or trans-single nucleotide polymorphism (SNP) that affects plasma adiponectin levels, except the adiponectin structure gene and genes encoding adiponectin-regulatory proteins. We performed a genome-wide association study in regards to plasma adiponectin concentrations in 3,310 Japanese subjects. We identified the strongest statistically associated SNP (rs4783244) with adiponectin levels (P = 3.8 × 10(-19)) in the first intron of CDH13 (T-cadherin) gene in a 30-kb haplotype block covering the promoter region to first intron. In addition, rs12051272 SNP genotypes in linkage disequilibrium with rs4783244 were found to be more significantly associated with adiponectin levels (P = 9.5×10(-20)) and specifically with the levels of high-molecular weight (HMW) adiponectin, a subtype form associated with parameters related to glucose metabolism. Our results did show more significant association with adiponectin levels than rs12444338 (in CDH13) SNP genotypes reported recently. We suggest that the phenotype-affecting haplotype tagged by rs12051272 SNP would affect the plasma adiponectin levels and that we have to take the CDH13 genotype into account before considering the functional relevance of the adiponectin level.

We conducted a meta-analysis of genome-wide association studies of systolic (SBP) and diastolic (DBP) blood pressure in 19,608 subjects of east Asian ancestry from the AGEN-BP consortium followed up with de novo genotyping (n = 10,518) and further replication (n = 20,247) in east Asian samples. We identified genome-wide significant (P < 5 × 10(-8)) associations with SBP or DBP, which included variants at four new loci (ST7L-CAPZA1, FIGN-GRB14, ENPEP and NPR3) and a newly discovered variant near TBX3. Among the five newly discovered variants, we obtained significant replication in the independent samples for all of these loci except NPR3. We also confirmed seven loci previously identified in populations of European descent. Moreover, at 12q24.13 near ALDH2, we observed strong association signals (P = 7.9 × 10(-31) and P = 1.3 × 10(-35) for SBP and DBP, respectively) with ethnic specificity. These findings provide new insights into blood pressure regulation and potential targets for intervention.

Background: Large-scale genome-wide association studies (GWAS) have been successful in identifying genes that contribute to common diseases and phenotypes. A GWAS of hypertension-related phenotypes in a Japanese population was conducted in the current study. Methods and Results: A total of 936 participants were recruited from the Suita Study and a GWAS with 538,732 single nucleotide polymorphisms (SNP) was performed. The phenotypes included were systolic and diastolic blood pressure (SBP and DBP), body mass index (BMI), waist-to-hip ratio (WHR), plasma renin activity (PRA), plasma aldosterone concentration (PAC), plasma brain natriuretic peptide (BNP) concentration and alcohol consumption (AC). The SNP exceeding the genome-wide significance level were subjected to subsequent association studies using samples available from the Suita Study and Nomura Study. There is no master gene in the Japanese population that profoundly affects SBP, DBP, BMI, WHR, PRA and PAC. AC was influenced by the functional polymorphism in ALDH2, which affected BP levels in men. The BNP concentration was influenced by a polymorphism in the 3' region of the gene encoding for BNP. However, this polymorphism did not influence blood pressure (BP). Six SNP were identified to be associated with hypertension in both the Suita and Nomura studies. Conclusions: Although several candidate SNP relevant to hypertension and those influencing AC and BNP were identified, our middle-sized GWAS indicated that there is no master gene in Japanese people that profoundly affects BP-related phenotypes. (Circ J 2010; 74: 2353-2359)

Background: Recent genome-wide association studies (GWAS) have identified genes or loci affecting lipid levels. Given the difference in allele frequencies and linkage disequilibrium patterns across the populations, a GWAS was conducted using the Illumina 550K in a Japanese population (n=900) in search of population-specific genetic variations associated with high-density lipoprotein (HDL)-cholesterol. Methods and Results: Among the 368,274 single nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 0.1, 43 SNPs exceeded the arbitrary threshold of -log10P >4.0. The most significant SNP was rs3764261, located 5'upstream of CETP, exhibiting a -log10P value of 6.17. Increasing the sample size by genotyping in the additional Suita sample (n=1,810) further improved the level of significance, with each additional copy of the minor allele being associated with an increase in HDL-cholesterol by 6.2 mg/dl (P =3.4×10-12). Interestingly, the minor allele was more prevalent in cases with myocardial infarction than in controls (0.221 vs 0.196, nominal P=0.02). Conclusions: The association between genetic variants at CETP and HDL-cholesterol was replicated in our sample. None of the genetic variants exerted a greater influence on HDL levels than those at CETP. Associations for the top-ranked SNPs need to be tested for further replication in an independent sample. (Circ J 2009; 73: 1119-1126)

The Dahl salt-sensitive (DS) rat is the most prevalently used animal model of salt-sensitive hypertension. The purpose of the present study was to test the hypothesis that mitochondrial DNA (mtDNA) polymorphisms influence blood pressure in DS rats. We produced two strains of female F1 rats, one from female DS and male Lewis rats (DL) and the other from Lewis female and DS male rats (LD). These two strains had the same autosomal genetic background, but their mitochondria had different origins. The DL and LD rats had DS and Lewis mitochondria, respectively. A high-salt diet was started at 4 weeks of age. Radiotelemetry devices were implanted into the lower abdominal aorta of these F1 rats at 9 weeks of age. Blood pressure was monitored for 24 h at 11, 12, 13, 14, and 19 weeks of age. No significant differences were observed in blood pressure levels between the strains. Although more than 100 polymorphisms were detected between DS and Lewis rats, it is unlikely that polymorphisms in mtDNA contribute to hypertension in DS rats. Moreover, we found no difference between DS and Lewis rats in the mtDNA copy number in the kidneys, the liver, and the ventricles of the heart before and after salt loading. Thus, it is unlikely that mitochondrial dysfunction due to high blood pressure exacerbated target organ damage. Intriguingly, the time course of body weight gain differed significantly between DL and LD F1 rats, suggesting the influence of mitochondrial polymorphisms on body composition.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways. J.T. and P.H. are consultants for Servier. J.C. received research grants and honoraria from Servier. K.S. obtained research support from Boehringer Ingelheim. The remaining authors declared no competing financial interests.

Aldosterone plays essential roles in body fluid and electrolyte homeostasis and blood pressure. However, the association between polymorphisms in the CYP11B2 gene and hypertension is controversial. We resequenced CYP11B1 and CYP11B2 and identified 35 polymorphisms in this region. We performed association studies between the plasma aldosterone concentration and 13 polymorphisms in this region in 1443 subjects. The subjects were all obtained from the Suita Cohort Study. Multiple regression analysis indicated that aldosterone levels were determined by renin activity, age, total cholesterol, and hematocrit. Residuals of the aldosterone levels after adjusting for these confounding factors were nominally associated with the T(−344)C ( P =0.0026), C(595)T ( P =0.0180), −(4837)C ( P =0.0310), and G(4936)A ( P =0.0498) polymorphisms. Only the T(−344)C polymorphism was significantly associated with the aldosterone level after a correction for multiple testing (Bonferroni). A significant interaction was observed between the T(−344)C polymorphism and renin activity in determining aldosterone levels. Moreover, a significant interaction was observed in 2063 subjects between urinary sodium excretion, which reflects sodium intake, and the T(−344)C polymorphism in determining systolic blood pressure. Only subjects with the TT genotype showed a positive correlation between urinary sodium excretion and systolic blood pressure. In vitro experiments confirmed the functional significance of this T(−344)C polymorphism in terms of angiotensin II reactivity. Thus, the T(−344)C polymorphism in CYP11B2 appears to affect salt sensitivity in Japanese and to have clinical significance.

Background Subclinical hypothyroidism, defined as high serum thyroid-stimulating hormone (TSH) levels and normal serum free-triiodothyronine (fT3) and serum free-thyroxine (fT4) levels, is a common medical problem among the elderly, but it is unclear whether it should be treated with thyroid hormone replacement therapy. Methods and Results A cross-sectional study of 3,607 participants in a community health survey in Suita, in the northern part of Osaka, was performed. Participants were categorized into 5 groups: normal, hyperthyroidism, hypothyroidism, subclinical hypothyroidism, and subclinical hyperthyroidism. The association between each group and various phenotypes was examined, in relation to cardiovascular disease and metabolic syndromes. Serum TSH levels increased and fT3 and fT4 levels decreased with age. A total of 14.6% of subjects aged 70-80 years and 20.1% of subjects aged older than 80 years were classified as having subclinical hypothyroidism. Subclinical hypothyroidism was not associated with glycol-hemoglobin A1c, body mass index, pulse rate, hypertension, total cholesterol, high-density lipoprotein cholesterol or triglyceride levels or intima - media thickness. It was only associated with higher fasting blood glucose and glycol-hemoglobin A1c levels compared with euthyroidism. Conclusions The present observation does not support the need for treatment of subclinical hypothyroidism or subclinical hyperthyroidism. (Circ J 2007; 71: 191 - 195)

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines. Several studies have suggested that this enzyme may play a role in blood pressure regulation. We previously reported that the expression levels of Comt mRNA in Dahl salt-sensitive (DS) rats were lower than those in Lewis (LEW) rats. However, the physiological significance of this phenomenon has not been investigated. The purpose of the present study was to evaluate the significance of lower expression of Comt in Dahl salt-sensitive hypertension. The Comt gene in DS rats has a palindromic insertion in 3'-untranslated region, which appears to be responsible for reduced mRNA stability. A genome-wide quantitative trait loci (QTL) analysis of blood pressure using 107 F2 rats indicated that a statistically significant QTL for pulse pressure was located at the Comt locus in chromosome 11. Microarray analysis confirmed that Comt was the only gene differentially expressed between DS and LEW rats in this chromosomal region. However, COMT inhibitors had no significant effects on blood pressure in either DS or LEW rats. Thus, Comt was excluded from the candidate genes contributing to salt-sensitive hypertension in DS rats. A true gene responsible for pulse pressure in this chromosome 11 region remains to be determined.

Background Associations between exposure to particulate matter and susceptibility to cardiovascular events have been reported. Although the underlying mechanisms are not fully understood, this association seems to be particularly exaggerated in the presence of atherothrombotic risk factors. The present study was undertaken in low-density lipoprotein receptor knockout (LDLR/KO) mice to test the hypothesis that long-term exposure to a high dose of nano-sized carbon black (CB) exacerbates atherosclerotic lesions. Methods and Results LDLR/KO mice were subjected to a 10-week intratracheal dispersion of CB (1 mg/week) or air under a 0% or 0.51% cholesterol (Chol) diet. Development of aortic lipid-rich lesions was detected in mice under a 0.51% Chol diet with or without CB dispersion, but not in mice fed a 0% Chol diet with or without CB. Quantification of the area stained with oil red O revealed the highest percentage in CB-treated mice on a 0.51% Chol diet among the 4 groups. One-way ANOVA indicated CB-treated mice with 0.51% Chol diet had a significantly higher percentage of positive staining than vehicle-treated mice with 0.51% Chol diet (p

Background Nanomaterials have numerous potential benefits for society, but the potential hazards of nanomaterials on human health are poorly understood. Nanomaterials are known to pass into the circulatory system in humans, causing vascular injuries that might play a role in the development of atherosclerosis. The present study aimed to determine the effects of chronic exposure to nanomaterials on macrophage phenotype and platelet aggregation. Methods and Results Cultured macrophages (RAW264.7) were treated with carbon black (CB) and water-soluble fullerene (C60(OH)24) from 7 to 50 days. Individually, CB had no significant effects on RAW264.7 cell growth, whereas C60(OH)24 alone or CB and C60(OH)24 together with oxidized low-density lipoprotein (Ox-LDL) (100 μg/ml) induced cytotoxic morphological changes, such as Ox-LDL uptake-induced foam cell-like formation and decreased cell growth, in a dose-dependent manner. C60(OH)24 induced LOX-1 protein expression, pro-matrix metalloprotease-9 protein secretion, and tissue factor mRNA expression in lipid-laden macrophages. Although CB or C60(OH) 24 alone did not induce platelet aggregation, C60(OH) 24 facilitated adenosine diphosphate (ADP)-induced platelet aggregation. Furthermore, C60(OH)24 acted as a competitive inhibitor of ADP receptor antagonists in ADP-mediated platelet aggregation. Conclusions The present study confirmed novel effects of nanomaterials in macrophages and platelets. These effects suggest that exposure to nanomaterials might be a risk for atherothrombotic diseases. (Circ J 2007; 71: 437 - 444)

SummaryUnderstanding the fiber-type specification and metabolic switch in skeletal muscle provides insights into energy metabolism in physiology and diseases. Here, we show that miR-182 is highly expressed in fast-twitch muscle and negatively correlates with blood glucose level. miR-182 knockout mice display muscle loss, fast-to-slow fiber-type switching, and impaired glucose metabolism. Mechanistic studies reveal that miR-182 modulates glucose utilization in muscle by targeting FoxO1 and PDK4, which control fuel selection via the pyruvate dehydrogenase complex (PDHC). Short-term high-fat diet (HFD) feeding reduces muscle miR-182 levels by tumor necrosis factor α (TNFα), which contributes to the upregulation of FoxO1/PDK4. Restoration of miR-182 expression in HFD-fed mice induces a faster muscle phenotype, decreases muscle FoxO1/PDK4 levels, and improves glucose metabolism. Together, our work establishes miR-182 as a critical regulator that confers robust and precise controls on fuel usage and glucose homeostasis. Our study suggests that a metabolic shift toward a faster and more glycolytic phenotype is beneficial for glucose control.

Nanomaterials have numerous potential benefits for society, but the effects of nanomaterials on human health are poorly understood. In this study, we aim to determine the genotoxic effects of chronic exposure to nanomaterials in various cell lines.Chinese hamster ovary (CHO) cells, human epidermoid-like carcinoma (Hela) cells and human embryonic kidney 293 (HEK293) cells were treated with the water-soluble fullerence C(60)(OH)(24) for 33-80 days. Cell proliferation, cytotoxic analysis and micronucleus tests were performed.When treated with C(60)(OH)(24) (0, 10, 100, or 1000 pg/ml) for 33 days, both the HEK293 and Hela cells showed increased cell proliferation, but cellular lactate dehydrogenase (LDH) activity was not affected. After long-term exposure (80 days) to C(60)(OH)(24) (0, 10, 100, or 1000 pg/ml), the CHO, Hela and HEK293 cells showed increased genotoxicity on the micronucleus test.This study suggests that nanomaterials, such as C(60)(OH)(24), have genotoxic effects.

To the Editor: Graham et al. (June 15 issue)1 report that serum levels of retinol-binding protein 4 (RBP4) correlated with the magnitude of insulin resistance in various subjects. However, RBP4 and plasma insulin levels were dissociated in subjects who did not have an improvement in insulin sensitivity after exercise.1 Thus, we believe that the importance of RBP4 should be assessed in more diverse groups. We assessed serum RBP4 levels in 473 subjects with normal glucose tolerance (fasting blood glucose level

Nanoscale materials are presently under development for diagnostic (nanomedicine) and electronic purposes. In contrast to the potential benefits of nanotechnology, the effects of nanomaterials on human health are poorly understood. Nanomaterials are known to translocate into the circulation and could thus directly affect vascular endothelial cells (ECs), causing vascular injury that might be responsible for the development of atherosclerosis. To explore the direct effects of nanomaterials on endothelial toxicity, human umbilical vein ECs were treated with 1–100 μg/ml hydroxyl fullerene [C60(OH)24; mean diameter, 7.1 ± 2.4 nm] for 24 h. C60(OH)24 induced cytotoxic morphological changes such as cytosolic vacuole formation and decreased cell density in a dose-dependent manner. Lactate dehydrogenase assay revealed that a maximal dose of C60(OH)24 (100 μg/ml) induced cytotoxic injury. Proliferation assay also showed that a maximal dose of C60(OH)24 inhibited EC growth. C60(OH)24 did not seem to induce apoptosis but caused the accumulation of polyubiquitinated proteins and facilitated autophagic cell death. Formation of autophagosomes was confirmed on the basis of Western blot analysis using a specific marker, light chain 3 antibody, and electron microscopy. Chronic treatment with low-dose C60(OH)24 (10 μg/ml for 8 days) inhibited cell attachment and delayed EC growth. In the present study, we have examined, for the first time, the toxicity of water-soluble fullerenes to ECs. Although fullerenes changed morphology in a dose-dependent manner, only maximal doses of fullerenes caused cytotoxic injury and/or death and inhibited cell growth. EC death seemed to be caused by activation of ubiquitin-autophagy cell death pathways. Although exposure to nanomaterials appears to represent a risk for cardiovascular disorders, further in vivo validations are necessary.

Background Epidemiological studies indicate a significant link between exposure to environmental air pollution and mortality and morbidity from ischemic heart disease. Because nanoparticles can translocate into blood circulation, the present study aimed to clarify their direct effects on human vascular endothelial cells (ECs). Methods and Results Human umbilical vein ECs (HUVECs) were treated with carbon black (CB), a component of diesel exhaust particles, for 24 h. CB induced cytotoxic morphological changes such as cytosolic vacuole formation, cell disorientation and decreased density. Lactate dehydrogenase assay revealed that CB induced cytotoxic injury in both the cells and plasma membranes. Proliferation assay showed that CB inhibited cell growth. Monocyte chemoattractant protein-1 but not vascular cell adhesion molecule-1 was induced by CB. CB reduced the expressions of connexin37 and endothelial nitric oxide (NO) synthase. Microarray analysis revealed the induction of pro-inflammatory molecules by CB. Conclusions The present results demonstrate for the first time that CB directly affects the endothelium, causing cytotoxic injury, inflammatory responses, and inhibition of cell growth. As EC injury/inflammation and membrane disintegration are related to the initiation of atherosclerosis, and NO is anti-atherogenic and anti-thrombogenic, the direct effects of nanoparticles on ECs may represent one mechanism behind environmental air pollution-mediated atherosclerosis and ischemic heart disease. (Circ J 2006; 70: 129 - 140)

MicroRNAs constitute a growing class of non-coding RNAs that are thought to regulate gene expression via translational repression. MicroRNAs are initially transcribed as several hundred-nucleotide pri-miRNAs and are then processed to approximately 60-nucleotide hairpin pre-miRNAs. We hypothesized that polymorphisms in both pre-miRNA and mature microRNA modify various biological processes by influencing the processing and/or target selection of microRNAs. In the present study, we sequenced 173 human pre-miRNA genome regions in 96 subjects and found 10 polymorphisms in the 10 pre-miRNA hairpin regions. Although most of these polymorphisms seem to have no effect on microRNA processing, we identified a C to A polymorphism in the mature miR-30c-2 sequence. This polymorphism may alter target selection and thus exert profound biological effects. To the best of our knowledge, this is the first report of polymorphisms in pre-miRNAs.

Recently, a member of the GATA-binding family of transcription factors was shown to be involved in human hypoparathyroidism, sensorineural deafness, and renal abnormality (HDR) syndrome. We report here a Japanese family in which two of the members are affected with HDR syndrome. Sequence analysis of GATA3 showed a heterozygous novel mutation in this family: an unusual mutation at exon 3 (709insC) resulting in a premature stop at codon 302 with a loss of both of the zinc finger domains.

Background Reproducibility of results is important for the validity of genetic association studies. Recently, 3 functional polymorphisms, G(-930)A in CYBA, T481S in CLCNKB, and E65K in KCNMB1, were reported to be associated with blood pressure (BP) status and the aim of this study was to confirm those findings using a large cohort representing the general Japanese population. Methods and Results The study population consisted of 3,652 subjects recruited from the Suita study as representive of the general population in Japan. The genotypes of the 3 polymorphisms were determined by the TaqMan method. Logistic analysis indicated that the CYBA/G(-930)A polymorphism was associated with hypertension in male subjects. In the male population, the odds ratio of the GG genotype over GA + AA was 1.27 (95% confidence interval 1.01-1.57, p=0.034). Moreover, residuals of systolic and diastolic BP values were significantly higher in subjects with the GG genotype than in those with the GA or AA genotype (p=0.0007). However, such significant effects of the genotype on BP status were not observed in the female population. The significance of the CLCNKB/T481S and KCNMB1/E65K polymorphisms were not replicated in the present study. Conclusion The significance of the G(-930)A polymorphism of CYBA was confirmed in the present study with adequate statistical power, which strengthens the hypothesis that this polymorphism is important in the pathogenesis of hypertension and confers susceptibility. (Circ J 2005; 69: 138 - 142)

Background Recently, the 5-lipoxygenase activating protein gene (ALOX5AP) was reported to confer a risk of myocardial infarction (MI) and stroke, independent of conventional risk factors. The purpose of the present study was to validate those findings in a Japanese population. Methods and Results The study population consisted of 1,875 subjects (males 871, females 1,004) recruited from the Suita study (control group) and 353 subjects (males 306, females 47) with MI. The promoter, all of the exons, and 3'UTR regions of ALOX5AP were sequenced in 96 subjects, and 8 polymorphisms were found. There were significant differences in the frequencies of the haplotypes constructed from the 2 SNPs (A162C and T8733A) between the control and MI groups. Multiple logistic analysis indicated that the homozygous genotype of the (CA) haplotype was significantly associated with a reduced risk for MI. Conclusion The hypothesis that ALOX5AP contributes to susceptibility for MI was validated in a Japanese population. (Circ J 2005; 69: 1029 - 1034)

Background Recently, a mutation in the human MEF2A gene was reported to be responsible for an autosomal dominant form of coronary artery disease, so the purpose of the present study was to assess the significance of MEF2A mutations in Japanese subjects with myocardial infarction (MI). Methods and Results The study population consisted of 589 control subjects recruited from the Suita study and 379 subjects with MI. The promoter, all the exons, and 3'-UTR regions of MEF2A were sequenced in 190 subjects with myocardial infarction. We found 2 amino acid length polymorphisms, a 7-amino acid deletion polymorphism, and a nonsense mutation (R447X) in exon 12. The length and deletion polymorphisms did not confer susceptibility to MI. Although the nonsense mutation was detected in 1 subject with MI, and in none of the control subjects, the impact of this mutation does not appear to be great; the subject had the MI while in his 70 s, had 2 major risk factors, and no family history of ischemic heart disease. Conclusion MEF2A polymorphism does not contribute appreciably to MI in the Japanese population. (Circ J 2005; 69: 1192 - 1195)

Association studies were performed to confirm the effect of polymorphisms in apolipoprotein A1 ( ApoA1) on the high-density lipoprotein cholesterol (HDL-C) level and the incidence of myocardial infarction (MI). A sequence analysis identified nine polymorphisms in ApoA1. After considering linkage disequilibrium, four polymorphisms in ApoA1 and four polymorphisms in the 5'-flanking regions and 3'-flanking regions from the JSNP database were determined in 1,880 subjects recruited from the Suita study, which represents the general population in Japan. Of the eight polymorphisms tested, the ApoA1 T84C polymorphism had the greatest effect on the levels of HDL-C ( P=0.0005, P(c)=0.0040 corrected by the Bonferroni method) and triglyceride ( P0.0001, P(c)=0.0008). The ApoA1 MspI polymorphism was not associated with HDL-C or triglyceride levels. We confirmed that the ApoA1 T84C polymorphism was associated with the HDL-C level but not the triglyceride level in patients with MI ( n=637). Moreover, this polymorphism was associated with the incidence of MI in male subjects ( P=0.0326). A logistic analysis indicated that the frequency of MI in the CC genotype was lower than that in the CT+TT genotype ( P=0.0145, OR=0.4955, 95% CI: 0.2746-0.8525). The ApoA1 T84C polymorphism is an important marker for the HDL-C level and may be a new risk marker for MI in Japanese.

We performed association studies between 118 single-nucleotide polymorphisms (SNPs) of 22 candidate genes (or gene family) and hypertension in a Japanese population.The study population consisted of 1880 subjects representing the general population in Japan, recruited from the Suita study. The candidate genes were selected based on their functions, including insulin resistance (APM1, CD36, HSD11B1), oxidative stress (CYBA, GPX1, GSTMs), steroid hormone (ESR1, ESR2, HSD11B2), renal functions (PTGS2, KLK1, NPHS1, NPHS2, SGK, SLC12A1, PTGES), and others related to cardiovascular physiology (GJA4, NOS1, NTRK3, P2RX4, SPP1, ALDH2).Multiple logistic analyses, with age and body mass index as covariates, indicated that 13 SNPs (eight genes), six SNPs (four genes) and 11 SNPs (four genes) were associated with hypertension (P0.05) in the total, male, and female populations, respectively. PTGS2 seems to be a promising candidate gene for hypertension in men. GSTM3 and SLC12A1 seem to be promising candidate genes for hypertension in women. Especially, a polymorphism in SLC12A1 was significantly associated with hypertension in women even after correction by the Bonferroni method (corrected P = 0.0236). Multiple logistic analyses, with age and body mass index as covariates, indicated that the prevalence of hypertension in females was significantly higher in subjects with the CC genotype than in those with the TT + TC genotypes (P0.0001, odds ratio = 1.967, 95% confidence interval = 1.430-2.712).Although the present results should be replicated in other study populations for confirmation, the present results suggest that SLC12A1 may contribute to hypertension in Japanese women.

To investigate the effects of polymorphisms in the ATP-binding cassette transporter A1 ( ABCA1) gene on the high-density lipoprotein cholesterol (HDL-C) level and the incidence of myocardial infarction (MI), we performed association studies. Sequence analysis identified 14 polymorphisms in the promoter region of ABCA1. After considering linkage disequilibrium, three polymorphisms in the promoter region and 11 polymorphisms from the JSNP database were determined in 1,880 subjects recruited from the Suita Study, representing the general population in Japan. We evaluated the association between the ABCA1 genotype and HDL-C level adjusted not only for standard factors, but also for genetic factors including ApoA1 and ApoE genotypes. Of the 14 polymorphisms tested, the G(-273)C ( P=0.0074), C(-297)T ( P=0.0195), and IMS-JST071749 ( P=0.0093) polymorphisms were significantly associated with the HDL-C level in the Suita population. We could reconfirm that the G(-273)C genotype was influential in another set of subjects ( P=0.0310, n=743). However, the distribution of the ABCA1 G(-273)C genotype in subjects with MI ( n=598) was not different from that in the control population ( n=801). These results indicate that ABCA1 G(-273)C has a significant effect on the HDL-C level in the general Japanese population, but not on the incidence of MI.

To identify candidate genes responsible for hypertension in Dahl salt-sensitive rats (Dahl-S), an oligonucleotide microarray analysis was performed to find differentially expressed genes in kidneys of Dahl-S and Lewis rats. We obtained 101 F2 male rats from Dahl-S and Lewis rats and performed precise measurements of blood pressure (BP) and heart rate by telemetric monitoring at 14 weeks of age after 9 weeks of salt-loading. The correlation analysis between genotypes of differentially expressed genes and BP in F2 rats indicated that pyridoxine 5'-phosphate oxidase (Pnpo) and catecholamine-O-methyltransferease (Comt) showed a highly significant association with BP. However, in the case of Comt, the Dahl-S genotype correlated with low BP. Short/branched chain acyl-CoA dehydrogenase and Sah also showed a significant association with systolic blood pressure. The present study provided evidence that Pnpo is a candidate gene responsible for hypertension in Dahl-S rats.

OBJECTIVE To examine the relationship between the gene for haeme oxygenase (HO)-1 (HMOX-1) and human essential hypertension, because both the acute and systemic induction of HMOX-1 have been suggested to attenuate vascular tone and blood pressure. METHODS We screened for sequence variations in HMOX-1 and conducted an association study, using these polymorphisms, in a large cohort (1998 individuals) representing the general Japanese population. RESULTS We sequenced HMOX-1 and found a T(-413)A polymorphism in the promoter region. The frequency of hypertensive individuals and the use of antihypertensive drugs were significantly greater in the AA genotype than in other genotypes among women: 45.5, 34.2, and 35.0% (P = 0.0099) and 23.4, 17.5, and 15.0% (P = 0.038), respectively, for the AA, AT, and TT genotypes, respectively. However, this association was not observed in men. Multiple logistic analyses indicated that the T(-413)A (AA/TA+TT) polymorphism, age, and body mass index affected the occurrence of hypertension in women. The odds ratio of the AA genotype for hypertension in women was 1.59 (P = 0.0058; 95% confidence interval 1.14 to 2.20). A luciferase reporter assay indicated that the A allele-promoter had eight-fold greater activity than the T allele promoter (P < 0.01). CONCLUSIONS The AA genotype of HMOX-1 is associated with an increased incidence of hypertension in women. Oestrogen attenuates vasoconstriction by increasing the expression of inducible nitric oxide synthase. As carbon monoxide, which is one of the products of HO-1, can attenuate nitric oxide-induced vasodilatation, a high expression of HO-1 may cause hypertension, especially in women.

The gene SAH (chromosome 16p12.3) is of interest in the etiology of human hypertension. In Caucasians a PstI restriction fragment length polymorphism (RFLP) of SAH has been correlated with body weight in individuals with hypertension. To extend this finding we carried out a case-control study of several recently identified polymorphisms in SAH: 1) an insertion/deletion of TTTAA at nucleotide -1037 in the promoter; 2) an insertion/deletion of two Alu like sequences in intron 1; and 3) an A→G variant in intron 12 located 7 bp upstream from exon 13. Subjects were 121 hypertensives with 2 hypertensive parents and 178 normotensives whose parents were both normotensive. All were Anglo-Celtic Caucasians and 51% of the hypertensives were overweight (body mass index (BMI)>25 kg/m2). The SAH promoter and intron 1 variants, but not the intron 12 or PstI RFLP, were in linkage disequilibrium (LD) (D′=100%, p

Selective blockade of the norepinephrine transporter with reboxetine has been reported to induce a slight but significant increase in blood pressure. This study was designed to examine the relation of genetic variants of the norepinephrine transporter gene (solute carrier family 6, member 2; SLC6A2) with hypertension in a Japanese population. We genotyped five genetic variants of SLC6A2, three in the promoter region and two in the intronic sequence, in 1,950 subjects recruited from the Suita study. One of the variants, an A>G polymorphism in the promoter region (Promoter 3 polymorphism), was found to be associated with hypertension. Multiple logistic analysis indicated that sex (p =0.0223), age (p

Spontaneously hypertensive rats (SHR) are a well-known animal model for hypertension. We have previously identified eleven differentially expressed genes in the kidneys between SHR/Hos and Wistar-Kyoto rats (WKY/Hos) using an oligonucleotide microarray and analyzed the correlation between these genes and hypertension. In the present study, we analyzed the differentially expressed genes in the kidneys between SHR/NCrj and WKY/NCrj obtained from an other source to clarify the common and/or specific gene expression between the different sources. Furthermore, expression changes in the representative genes were characterized by Northern blot analysis using samples prepared from a third source, the Izm strain. The comparison revealed quite different changes in the differentially expressed genes among them. Sequence analysis of one of the differentially expressed genes, cytosolic epoxide hydrolase, revealed that two haplotypes could in part explain the expression level. Our study showed the complex nature of the genetic heterogeneity between SHR and WKY from different sources.

The rostral ventrolateral medulla (RVLM) plays a critical role in the tonic and reflexive regulation of arterial blood pressure. Recent studies have demonstrated that injection of kynurenic acid (KYN) into the RVLM of spontaneously hypertensive rats (SHR) decreases arterial blood pressure. We hypothesized that a relative increase in the excitatory amino acid-mediated drive of RVLM vasomotor neurons in SHR may be due to derangement of one of the enzymes that affect the KYN level in the brain. We selected kynureninase, kynureninase hydroxylase, kynurenine aminotransferase type I, and kynurenine aminotransferase type II as candidates that may affect the KYN level in the brainstem. We conducted association studies between polymorphisms of these genes and blood pressure in an F2 population derived from SHR and Wistar-Kyoto rats (WKY). The cosegregation analysis indicated that only the kynureninase gene (KYNU) polymorphism influenced systolic blood pressure (SBP) and residuals of systolic blood pressure after adjusting for heart rate and body weight (RSBP). KYNU was found to be located on rat chromosome 3, and quantitative trait loci analysis at this locus indicated that the logarithms of the odds scores for KYNU in terms of SBP and RSBP were 2.0 and 3.3, respectively. This association with blood pressure decreased in proportion to the distance from KYNU. The expression level of KYNU mRNA in the brainstem was about 3.1 and 2.9 times higher in 10-week-old and 16-week-old SHR than in age-matched WKY, respectively. The increased expression of KYNU in SHR is thought to decrease the KYN level. KYNU seems to be one of the genes that contributes to hypertension in SHR. (Hypertens Res 2002; 25: 135-140)

Background — The SA gene ( SAH ) has been isolated by differential screening from a genetically hypertensive rat strain as a candidate gene that may contribute to hypertension. Recently, the SA protein has been reported to be highly homologous to bovine xenobiotic–metabolizing medium-chain fatty acid:CoA ligase. Methods and Results — To clarify the pathophysiological significance of SAH , we searched for polymorphisms of human SAH and performed association studies using a large cohort (4000 subjects) representing the general population in Japan. We found 2 polymorphisms in the promoter region and single-nucleotide polymorphisms in introns 5, 7, and 12 and exon 8. One of the variants, an A/G polymorphism in intron 12, just 7 bp upstream from exon 13, strongly affected plasma triglyceride, plasma cholesterol, body mass index (BMI), waist-to-hip ratio (W/H), and blood pressure status. The effect of this genotype on blood pressure seems to be conveyed through its effects on BMI and W/H. Transient expression of the SA protein in mammalian cells confirmed that it is expressed in mitochondria and has medium-chain fatty acid:CoA ligase activity. The A/G polymorphism was found to be associated with the expression level of SA mRNA in peripheral mononuclear cells in vivo. Conclusions — The G allele of SAH was found to be associated with multiple risk factors, including hypertriglyceridemia, hypercholesterolemia, obesity, and hypertension. This observation should open a new area for future research in multiple-risk-factor syndromes.

Recent studies have described a linkage between angiotensin converting enzyme (ACE) gene polymorphism and hypertension in a large number of hypertensive sibs. Moreover, among Japanese, the DD genotype of the ACE gene has been reported to be a genetically predisposing factor for hypertension in a large general population. However, there is some controversy regarding the association between the ACE insertion/deletion (I/D) polymorphism and systemic hypertension. Therefore, we examined the influence of the ACE I/D polymorphism in a random Japanese population. The participants were 2, 892 subjects (1, 110 males: mean age, 58.4±15.5; 1, 782 females: mean age, 56.2±15.7) who underwent medical examinations in 1999 in Shigaraki, a suburban town located in an urban area in Shiga prefecture. Among them, 2, 395 subjects (917 males: mean age, 58.0±15.7; 1478 females: mean age, 56.3±15.6) who gave their informed consent for genetic analysis were enrolled in the present study. Every year since 1991, we have repeatedly performed medical examinations in Shigaraki. ACE genotypes were determined by polymerase chain reaction (PCR) methods. Logistic analysis revealed that age (p

The SCNNIA gene, which is located on human chromosome 12p13.3, encodes the alpha subunit of the amiloride-sensitive epithelial sodium channel, and mutations in SCNNIA can result in pseudohypoaldosteronism type I. It was postulated that genetic variations in SCNN1A could lead to an increased risk of hypertension. Sequence variations in SCNN1A were identified, and the association between these polymorphisms and BP was examined in a large cohort (n = 3898) representing the general population in Japan. Four polymorphisms in the promoter region, three polymorphisms in the exonic region, and one polymorphism in the first intron were identified. Because association studies with one-half of the study population indicated that the A(2139)G polymorphism, among others, significantly affected BP, this polymorphism was studied in the entire study population. Multiple logistic analyses indicated that the odds ratio for hypertension with the GA+GG genotype was 1.31 (P = 0.0154) in the total population and 1.77 (P = 0.0035) among subjects

Although proteinuria is known to be associated with the deterioration of chronic kidney disease, the molecular basis of this mechanism is not fully understood. We previously found that Pex11a deficiency was associated with a reduction of functional peroxisomes and impaired fatty acid metabolism in hepatocytes and resulted in steatosis. Proximal tubule cells are rich in peroxisomes. We assessed whether Pex11a deficiency might result in the derangement of peroxisome systems in proximal tubule cells and the aggravation of tubulointerstitial lesions in chronic kidney disease. Histological analyses showed that the number of functional peroxisomes in proximal tubule cells was reduced in Pex11a knockout ( Pex11a −/− ) mice. To clarify whether a decrease in the number of tubular peroxisomes might aggravate interstitial lesions, we assessed 2 models in which proximal tubule cells are overloaded with fatty acids (ie, deoxycorticosterone acetate and salt hypertension and the overload of fatty acid–bound albumin). Deoxycorticosterone acetate -salt–treated Pex11a −/− mice exhibited greater interstitial lesions than deoxycorticosterone acetate-salt–treated wild-type mice in terms of tubular lipid accumulation, blood pressure, urinary albumin, urinary N-acetyl-β- d -glucosaminidase, urinary 8-iso-prostane, and the histological evaluation of fibrosis and inflammation. An overload of fatty acid–bound albumin also resulted in more severe tubulointerstitial lesions in Pex11a −/− mice than in wild-type mice. Fenofibrate, a peroxisome proliferator-activated receptor-α agonist, restored the abundance of peroxisomes and reduced the tubulointerstitial lesions induced by deoxycorticosterone acetate-salt hypertension. In conclusion, our results indicate that proximal tubule peroxisomes play an important role in proteinuria-induced interstitial lesions. The activation of tubular peroxisomes might be an excellent therapeutic strategy against chronic kidney disease.

There has been increasing interest in the use of circulating DNA as biomarkers for various tissue injuries, cancers, and fetal conditions. DNA methylation is a well-characterized mechanism underlying the epigenetic regulation of gene expression, and many diagnostic tests based on DNA methylation patterns have been developed. We developed a novel TaqMan-based assay for the detection of acute kidney injury using a hypomethylated promoter region of Slc22a12, a urate transporter specifically expressed in proximal tubular cells. Bisulfite sequencing analysis confirmed that the CpG islands in the promoter region of mouse Slc22a12 were preferentially hypomethylated in the kidney cortex. TaqMan minor groove binder (MGB) probes reliably discriminated the DNA fragments corresponding to the unmethylated and methylated promoter regions of Slc22a12. Plasma levels of unmethylated DNA corresponding to the Slc22a12 promoter region were undetectable at baseline and were significantly elevated after acute kidney cortex necrosis. This study showed the usefulness of the TaqMan system in discriminating methylated and unmethylated DNA fragments, and the similar strategy can be applied for establishing biomarkers for various cellular injuries or pathological conditions.

OBJECTIVES Endothelium-derived nitric oxide plays a key role in the regulation of vascular tone. Recently, endothelial nitric oxide synthase (eNOS) gene polymorphisms were reported to be associated with hypertension or coronary spasm. We investigated the association between the eNOS gene polymorphisms and hypertension in a large population-based sample of 4055 Japanese. DESIGN AND METHODS We investigated two polymorphisms of the eNOS gene, Glu298Asp polymorphism of exon 7 and T(-786)C polymorphism of the promoter region. The genotype distribution in hypertensive subjects was compared to that in the other subjects. The influence of the genotype on blood pressure values was analyzed in the subjects not taking hypertensive medication. The promoter activities of the eNOS gene with the (-786)T or (-786)C allele were measured by a luciferase reporter gene assay. RESULTS There was significant linkage disequilibrium between the two polymorphisms (P < 0.0001). The genotype distribution of the Glu298Asp or T(-786)C polymorphism did not differ between the hypertensive and the other subjects. No significant differences in the blood pressure of subjects not taking hypertensive medication were observed among the three genotypes of Glu298Asp or T(-786)C polymorphisms. No significant differences in the promoter activity were observed between bovine endothelial cells transfected with the (-786)T and (-786)C alleles. CONCLUSIONS Our data suggested that these polymorphisms of the eNOS gene are unlikely to be major factors in the susceptibility to hypertension in the Japanese population studied.

A common variant at codon 235 of the angiotensinogen gene with methionine to threonine amino acid substitution ( AGT M235T ) has been reported as a genetic risk for essential hypertension. However, the frequency of AGT T235 was heterogeneous among races, and a positive association between AGT M235T and hypertension was not settled. To examine the association in a general population of Japanese (n=4013), we introduced the TaqMan polymerase chain reaction method and examined the relation between hypertension and T+31C polymorphism, which was in absolute linkage disequilibrium with AGT M235T. The C+31 allele of AGT was significantly associated with the positive family history of hypertension (FH) but not with the presence of hypertension or blood pressure. The subjects with CC tended to have hypertensive relatives, especially a hypertensive father or siblings, and its statistical significance was stronger in men. Adjustment of confounding factor did not alter the results of simple association study, suggesting that this positive association with FH is independent and significant. Our findings revealed that the TaqMan polymerase chain reaction method is a powerful tool for genetic association study with a large number of subjects and that AGT T+31C is significantly associated with paternal FH.

Excessive alcohol consumption is a potent risk factor for high blood pressure. About half of Japanese show an extremely high sensitivity to alcohol, which is due to a genetic deficiency in an isoenzyme of aldehyde-dehydrogenase with a low Km (ALDH2). It is possible that the effects of alcohol consumption on blood pressure differ according to the ALDH2 genotype. The purpose of the present study was to assess the influence of the ALDH2 genotype on the pressor effects of alcohol. The influence of the ALDH2 genotype on blood pressure was investigated in a large cohort (4, 000 subjects) representing the general population in Japan. The genotype was determined by the TaqMan method. The genotype was significantly associated with alcohol consumption, gamma-GTP level, and HDL cholesterol level in both males and females. The odds ratio for the presence of hypertension for the Glu/Glu genotype in comparison to other genotypes was 1.67 (p

Background —Prostacyclin (prostaglandin I 2 ) is a strong vasodilator that inhibits the growth of vascular smooth muscle cells and is also the most potent endogenous inhibitor of platelet aggregation. Therefore, it has been considered to play an important roles in cardiovascular disease. On the basis of the hypothesis that variations of the prostacyclin synthase gene may also play an important role in human cardiovascular disease, we performed a screening for variations in the human prostacyclin synthase gene. Methods and Results —We have detected a repeat polymorphism in the promoter region of the human prostacyclin synthase gene. The number of 9-bp (CCGCCAGCC) repeats in the promoter region, which encodes a tandem repeat of Sp1 transcriptional binding sites, varied between 3 and 7 in Japanese subjects. Luciferase reporter analysis indicated that the alleles of 3 and 4 repeats (R3 and R4, respectively) had less promoter activity in cultured human umbilical vein endothelial cells. We then investigated the possible association of this repeat polymorphism with blood pressure in a large population-based sample (the Suita Study), which consisted of 4971 Japanese participants. Multivariate models indicated that participants with the R3R3, R3R4, or R4R4 genotype (SS genotype, n=80) had significantly higher systolic pressure ( P =0.0133) and pulse pressure ( P =0.0005). The odds ratio of hypertension (140/90 mm Hg) for the SS genotype was 1.942 (95% confidence interval 3.20 to 1.19, P =0.0084). Conclusions —Repeat polymorphism of the human prostacyclin synthase gene seems to be a risk factor for higher pulse pressure and is consequently a risk factor for systolic hypertension in the Japanese population.

Background —Impaired renal hemodynamic function has been suspected to be responsible for hypertension in the spontaneously hypertensive rat (SHR). Methods and Results —We measured renal hemodynamic functions, including the glomerular filtration rate, renal plasma flow, and renal vascular resistance, in an F2 rat population derived from spontaneously hypertensive and Wistar-Kyoto (WKY) rats and performed a genome-wide screening to map quantitative trait loci that influence these functions to gain insight into the relationship between renal hemodynamic functions and blood pressure control. The D1 Mit7 locus was identified as a major locus that influenced renal hemodynamic functions, and we transferred the SHR chromosomal segment around the D1 Mit7 locus into the WKY strain. The congenic rats exhibited impaired renal hemodynamic functions. The systolic blood pressure of the congenic rats was significantly higher than that of age-matched WKY rats, but only at nighttime. No significant differences in systolic blood pressure during daytime or diastolic blood pressure were observed between the 2 strains Conclusions —We have identified a chromosome segment that influences renal hemodynamic function. The SHR chromosome segment around the D1 Mit7 locus had significant, but not dramatic, effects in increasing blood pressure in the WKY genetic background. However, further studies will be necessary to determine the significance of this locus in SHR hypertension.