CiteULike: jyuh's Kato

MOCSphaser: a haplotype inference tool from a mixture of copy number variation and single nucleotide polymorphism data.

M Kato — 2008-07-25T06:47:04-00:00

Bioinformatics (Oxford, England), Vol. 24, No. 14. (15 July 2008), pp. 1645-1646.

Detailed analyses of the population-genetic nature of copy number variations (CNVs) and the linkage disequilibrium between CNV and single nucleotide polymorphism (SNP) loci from high-throughput experimental data require a computational tool to accurately infer alleles of CNVs and haplotypes composed of both CNV alleles and SNP alleles. Here we developed a new tool to infer population frequencies of such alleles and haplotypes from observed copy numbers and SNP genotypes, using the expectation-maximization algorithm. This tool can also handle copy numbers ambiguously determined, such as 2 or 3 copies, due to experimental noise. AVAILABILITY: http://emu.src.riken.jp/MOCSphaser/MOCSphaser.zip.

An Algorithm for Inferring Complex Haplotypes in a Region of Copy-Number Variation.

Mamoru Kato — 2008-07-25T05:35:49-00:00

American journal of human genetics (16 July 2008)

Recent studies have extensively examined the large-scale genetic variants in the human genome known as copy-number variations (CNVs), and the universality of CNVs in normal individuals, along with their functional importance, has been increasingly recognized. However, the absence of a method to accurately infer alleles or haplotypes within a CNV region from high-throughput experimental data hampers the finer analyses of CNV properties and applications to disease-association studies. Here we developed an algorithm to infer complex haplotypes within a CNV region by using data obtained from high-throughput experimental platforms. We applied this algorithm to experimental data and estimated the population frequencies of haplotypes that can yield information on both sequences and numbers of DNA copies. These results suggested that the analysis of such complex haplotypes is essential for accurately detecting genetic differences within a CNV region between population groups.

Mapping quantitative trait loci for proteinuria-induced renal collagen deposition

N Kato — 2008-07-05T03:29:42-00:00

Kidney Int, Vol. 73, No. 9. (27 February 2008), pp. 1017-1023.

Attainment of the Japanese Society for Dialysis Therapy guidelines for the management of secondary hyperparathyroidism in chronic hemodialysis patients in our clinic.

Y Sato — 2008-06-30T01:54:17-00:00

Therapeutic apheresis and dialysis : official peer-reviewed journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy, Vol. 11 Suppl 1 (October 2007)

The treatment according to the Japanese Society for Dialysis Therapy guidelines was performed in 189 patients on maintenance dialysis in our clinic. The mean age of the patients was 64.9 years and the mean dialysis period was 6.3 years. The underlying disease was diabetic nephropathy in 40.7% of the patients, chronic glomerulonephritis in 30.2%, and nephrosclerosis in 13.8%. In May 2006 before the use of JSDT guidelines, patients with phosphorus and calcium concentrations in the control goal range were most frequently observed (69.8%), followed in order by those with a high concentration of phosphorus alone (13.8%), those with a low concentration of phosphorus alone (2.6%), those with a high concentration of calcium alone (10.1%), those with high concentration of both phosphorus and calcium (3.7%). Treatment according to JSDT guidelines was performed for 6 months in these patients. In January 2007, the group with both phosphorus and calcium concentrations in the goal range accounted for 82.2%, showing improvement. The intact PTH concentration in patients with normal phosphorus and calcium concentration was in the reference range (60-180 pg/ml) in about 50% of the patients, high (>180 pg/ml) in 35%, low (<60 pg/ml) in 10% during the study periods. The intact PTH concentration was often about 40 pg/ml in patients with a concentration <60 pg/ml, 120 pg/ml in those with a concentration of 60-180 pg/ml, and 200-250 pg/ml in those with a concentration >180 pg/ml. The concentration of NTx was significantly higher in the patients with an intact PTH concentration >180 pg/ml than in those with a concentration of <60 pg/ml or those with a concentration of 60-180 pg/ml and significantly increased with time.

Smad1 as a biomarker for diabetic nephropathy.

H Kato — 2008-06-29T06:25:18-00:00

Diabetes, Vol. 57, No. 6. (June 2008), pp. 1459-1460.

Association of serum adiponectin levels with all-cause mortality in hemodialysis patients.

N Ohashi — 2008-06-29T05:04:42-00:00

Internal medicine (Tokyo, Japan), Vol. 47, No. 6. (2008), pp. 485-491.

OBJECTIVE: Adiponectin (ADPN) has been shown to protect against cardiovascular disease for the general population with problematic metabolic syndrome. However, it remains unclear whether ADPN is associated with mortality in patients on maintenance hemodialysis (HD). METHODS, PATIENTS OR MATERIALS: We selected 85 HD patients [51 men/34 women; mean age, 64+/-2 years; underlying kidney diseases, diabetic nephropathy in 36 patients (42.3%), chronic glomerulonephritis in 29 (34.1%), hypertensive nephrosclerosis in 10 (11.8%), and others in 10 (11.8%)] who survived for more than 3 months after the start of HD. We first measured serum ADPN levels and prospectively followed patients for the next 3 years. RESULTS: We were able to follow 74 of 85 patients; 59 survived, and 15 died. Serum log-transformed ADPN levels were negatively correlated with BMI (r=-0.43, p<0.01). Despite a similar BMI (20.7+/-0.8 vs. 20.3+/-0.4 kg/m(2)), the expired patients had significantly higher ADPN compared with the surviving patients (20.5 microg/ml [14.0-23.5] vs. 14.2 microg/ml [9.7-21.3], p<0.05). Cox-hazards multivariate regression analysis adjusted for conventional case-mix features (age, sex, and underlying kidney disease) revealed that serum ADPN became a significant determinant of all-cause mortality. There was a 10.3% risk increment for each 1-microg/ml increase in ADPN during the follow-up. Kaplan-Meier analysis revealed that patients with higher ADPN levels (> or =15 microg/ml) had a significantly lower survival rate compared with those with lower ADPN levels (<15 microg/ml) (76 vs. 92%, p<0.05). CONCLUSION: These results indicated that high rather than low ADPN independently predict total mortality in HD patients.

Suppressive effect of oral administration of branched-chain amino acid granules on oxidative stress and inflammation in HCV-positive patients with liver cirrhosis.

T Ohno — 2008-06-28T10:01:03-00:00

Hepatology research : the official journal of the Japan Society of Hepatology, Vol. 38, No. 7. (July 2008), pp. 683-688.

Aim: In chronic hepatitis C virus (HCV) infection, it is thought that both chronic persistent inflammation and oxidative stress contribute to the development of hepatocellular carcinoma (HCC), and it has been reported that long-term oral supplementation with branched-chain amino acid (BCAA) granules could inhibit liver carcinogenesis. However, the extent of the involvement of these factors remains obscure. Methods: To clarify the involvement of inflammation and oxidative stress in the inhibition of liver carcinogenesis, we evaluated the effect of oral administration of BCAA granules on oxidative stress and inflammation in HCV-positive patients with liver cirrhosis. Results: Twenty-seven patients were enrolled in the study: 18 of the patients were treated with BCAA granules (administered group) and nine were observed without BCAA granules (non-administered group). In the non-administered group, the production of oxidative stress, as indicated by urine 8-hydroxydeoxyguanosine (8-OHdG) and 15-F2t-Isoprostane (8-IsoPs), significantly increased with time, while in the administered group the levels of ferritin and 8-OHdG decreased significantly. Comparison of the two groups demonstrated that highly sensitive CRP, ferritin, 8-OHdG and 8-IsoPs were significantly reduced by taking BCAA granules. The time-course analysis showed that ferritin and highly sensitive CRP seemed to decrease first, followed by a decrease of 8-OHdG and 8-IsoPs. Conclusion: These findings indicated that the administration of BCAA granules influenced microinflammation and the metabolism of iron in HCV-positive patients with liver cirrhosis, and subsequently seemed to reduce the production of oxidative stress, possibly leading to a decrease in the occurrence of HCC.

Comparison of nutritional and inflammatory markers in dialysis patients with reduced appetite

Juan Carrero — 2008-06-23T13:19:34-00:00

Am J Clin Nutr, Vol. 85, No. 3. (1 March 2007), pp. 695-701.

Background: Anorexia is common in chronic kidney disease and worsens as the disease progresses. Sex hormones and inflammatory cytokines may be related to feeding behavior. Objective: We hypothesized that appetite would be related to inflammation and outcome in hemodialysis patients but that sex may account for differences in the symptoms associated with poor appetite. Design: A cross-sectional study was conducted in patients undergoing prevalent hemodialysis (n = 223; 127 M;[IMG] f1.gif" ALT="x" BORDER="0"> +/- SD age: 66 +/- 14 y). Anthropometric markers of body composition, handgrip strength, and nutritional and inflammatory status were measured, and 3 groups according to their self-reported appetite were established. Overall mortality was assessed after 19 mo (range: 2-29 mo) of follow-up. Results: Poor appetite was associated with a longer vintage time, increased inflammation (higher serum concentrations of interleukin 6 and C-reactive protein), and a worse nutritional status (lower serum concentrations of insulin-like growth factor I, albumin, urea, and creatinine). However, across worsening appetite scale, handgrip strength was incrementally lower in men but not in women (multivariate analysis of variance). In a multivariate logistic regression analysis (pseudo r2 = 0.19), appetite loss was associated with sex [odds ratio (OR): 0.41; 95% CI: 0.24, 0.72], insulin-like growth factor I (3.58; 2.10, 6.32), and C-reactive protein > 10 mg/L (2.39; 1.34, 4.11). Finally, appetite loss was associated with worse clinical outcome even after adjustment for age, sex, inflammation, dialysis vintage, and comorbidity (likelihood ratio = 44.3; P < 0.0001). Conclusions: These results show a close association among appetite, malnutrition, inflammation, and outcome in patients undergoing prevalent hemodialysis. Moreover, our data suggest that uremic men may be more susceptible than are women to inflammation-induced anorexia.

High-density association study and nomination of susceptibility genes for hypertension in the Japanese National Project.

N Kato — 2008-05-23T03:12:59-00:00

Human molecular genetics, Vol. 17, No. 4. (15 February 2008), pp. 617-627.

Essential hypertension is one of the most common, complex diseases, of which considerable efforts have been made to unravel the pathophysiological mechanisms. Over the last decade, multiple genome-wide linkage analyses have been conducted using 300-900 microsatellite markers but no single study has yielded definitive evidence for 'principal' hypertension susceptibility gene(s). Here, we performed a three-tiered, high-density association study of hypertension, which has been recently made possible. For tier 1, we genotyped 80 795 SNPs distributed throughout the genome in 188 male hypertensive subjects and two general population control groups (752 subjects per group). For tier 2 (752 hypertensive and 752 normotensive subjects), we genotyped a panel of 2676 SNPs selected (odds ratio >or= 1.4 and P <or= 0.015 in tier 1) and identified 75 SNPs that showed similar tendency of association in tier 1 and tier 2 samples (P <or= 0.05 for allele frequency and P <or= 0.01 for genotype distribution tests). For tier 3 (619 hypertensive and 1406 normotensive subjects), we genotyped the 75 SNPs and found nine SNPs from seven genomic loci to be associated with hypertension (P <or= 0.05). In three of these loci, the lowest P-values were observed for rs3755351 (P = 1.7 x 10(-5)) in ADD2, rs3794260 (P = 0.0001) in KIAA0789 and rs1805762 (P = 0.0003) in M6PR when case-control comparison was made in the combined data. An SNP (rs3755351) within ADD2 had the lowest P-value and its experiment-wide significance level is 0.13. Thus, these results have nominated several susceptibility genes for hypertension, and independent replication will clarify their etiological relevance.

Hematopoietic origin of hepatic stellate cells in the adult liver.

E Miyata — 2008-05-14T15:38:57-00:00

Blood, Vol. 111, No. 4. (15 February 2008), pp. 2427-2435.

Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. Several studies have reported that bone marrow cells can give rise to hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of clonal populations of cells derived from single enhanced green fluorescent protein (EGFP)-marked Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and examined the histology of liver tissues obtained from the chimeric mice with carbon tetrachloride (CCl(4))-induced injury. After 12 weeks of CCl(4) treatment, we detected EGFP(+) cells in the liver, and some cells contained intracytoplasmic lipid droplets. Immunofluorescence analysis demonstrated that 50% to 60% of the EGFP(+) cells were negative for CD45 and positive for vimentin, glial fibrillary acidic protein, ADAMTS13, and alpha-smooth muscle actin. Moreover, EGFP(+) cells isolated from the liver synthesized collagen I in culture. These phenotypes were consistent with those of hepatic stellate cells. The hematopoietic stem cell-derived hepatic stellate cells seen in male-to-male transplants revealed only one Y chromosome. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells after liver injury and that the process does not involve cell fusion.

Bone mineral density, osteoporosis, and osteoporotic fractures: a genome-wide association study

JB Richards — 2008-05-12T04:50:23-00:00

The Lancet, Vol. 371, No. 9623., pp. 1505-1512.

SummaryBackground Osteoporosis is diagnosed by the measurement of bone mineral density, which is a highly heritable and multifactorial trait. We aimed to identify genetic loci that are associated with bone mineral density.Methods In this genome-wide association study, we identified the most promising of 314[punctuation space]075 single nucleotide polymorphisms (SNPs) in 2094 women in a UK study. We then tested these SNPs for replication in 6463 people from three other cohorts in western Europe. We also investigated allelic expression in lymphoblast cell lines. We tested the association between the replicated SNPs and osteoporotic fractures with data from two studies.Findings We identified genome-wide evidence for an association between bone mineral density and two SNPs (p<5×10-8). The SNPs were rs4355801, on chromosome 8, near to the TNFRSF11B (osteoprotegerin) gene, and rs3736228, on chromosome 11 in the LRP5 (lipoprotein-receptor-related protein) gene. A non-synonymous SNP in the LRP5 gene was associated with decreased bone mineral density (rs3736228, p=6·3×10-12 for lumbar spine and p=1·9×10-4 for femoral neck) and an increased risk of both osteoporotic fractures (odds ratio [OR] 1·3, 95% CI 1·09-1·52, p=0·002) and osteoporosis (OR 1·3, 1·08-1·63, p=0·008). Three SNPs near the TNFRSF11B gene were associated with decreased bone mineral density (top SNP, rs4355801: p=7·6×10-10 for lumbar spine and p=3·3×10-8 for femoral neck) and increased risk of osteoporosis (OR 1·2, 95% CI 1·01-1·42, p=0·038). For carriers of the risk allele at rs4355801, expression of TNFRSF11B in lymphoblast cell lines was halved (p=3·0×10-6). 1883 (22%) of 8557 people were at least heterozygous for these risk alleles, and these alleles had a cumulative association with bone mineral density (trend p=2·3×10-17). The presence of both risk alleles increased the risk of osteoporotic fractures (OR 1·3, 1·08-1·63, p=0·006) and this effect was independent of bone mineral density.Interpretation Two gene variants of key biological proteins increase the risk of osteoporosis and osteoporotic fracture. The combined effect of these risk alleles on fractures is similar to that of most well-replicated environmental risk factors, and they are present in more than one in five white people, suggesting a potential role in screening.Funding Wellcome Trust, European Commission, NWO Investments, Arthritis Research Campaign, Chronic Disease Research Foundation, Canadian Institutes of Health Research, European Society for Clinical and Economic Aspects of Osteoporosis, Genome Canada, Genome Quebéc, Canada Research Chairs, National Health and Medical Research Council of Australia, and European Union.

Practical guide of live imaging for developmental biologists.

Kagayaki Kato — 2008-05-10T01:19:58-00:00

Development, growth & differentiation (16 April 2008)

Time-lapse imaging of fluorescent proteins in living cells has become an indispensable tool in biological sciences. However, its application at the organismal level still faces a number of obstacles, such as large specimen sizes preventing illumination of internal tissues, high background fluorescence and uncontrollable movement of target tissues or embryos. Here we describe our solutions for these issues to obtain 4-D fluorescent images from living Drosophila embryos using confocal microscopes. A computational procedure that detects and corrects the shift of moving objects to virtually stabilize them in time-lapse movies (iSEMS) is presented. We discuss the importance of postimaging treatment of raw image stacks for the discovery of novel phenotypes that have previously escaped attention from the analyses of fixed specimens.

Resolution of liver cirrhosis using vitamin A–coupled liposomes to deliver siRNA against a collagen-specific chaperone

Yasushi Sato — 2008-04-08T04:45:35-00:00

Nature Biotechnology, Vol. 26, No. 4. (30 March 2008), pp. 431-442.

Brain natriuretic peptide in hemodialysis patients: predictive value for hemodynamic change during hemodialysis and cardiac function.

T Matayoshi — 2008-04-17T02:00:00-00:00

American journal of nephrology, Vol. 28, No. 1. (2008), pp. 122-127.

BACKGROUND: Though brain natriuretic peptide (BNP) is widely used as a clinical marker of cardiac function, there is considerable confusion in the interpretation of its value in hemodialysis (HD) patients whose BNPs are often elevated without cardiac diseases. The aim of the present study is to examine the predictive value of BNP for blood pressure (BP) fall during HD and cardiac function. METHODS: Subjects consisted of 205 (160 males, 45 females; age 66.5 +/- 10.5 years) consecutive uremic patients requiring maintenance HD who were admitted to our hospital during 2001-2004. One hundred and eleven cases had a history of ischemic heart disease. We measured BNP in all cases and collected clinical data including age, sex, duration of HD, blood examination and echocardiography. RESULTS: BNP of all 205 cases ranged from 6 to 16,097 pg/ml (median 831). During HD, the average BP change was -24.5 +/- 20.5 mm Hg, and 111 cases showed a systolic BP reduction >20 mm Hg. BNP did not predict the degree of BP fall. After adjusting confounding factors, the presence of ischemic heart disease, ultrafiltration rate, systolic BP before HD and serum sodium concentration showed a significant correlation with BP change (t = -2.84, -2.76, -4.68 and 2.90; p = 0.005, <0.01, <0.0001 and <0.005, respectively). In relation to echocardiographic indices, BNP >785 pg/ml could predict left ventricular dysfunction (fractional shortening of the left ventricle <30%, sensitivity 73%, specificity 65%). CONCLUSION: The level of BNP could not predict BP fall during HD. However, BNP is a good indicator of cardiac function even in uremic patients.

Effect of hyperinsulinemia on renal function in a general Japanese population: the Hisayama study.

M Kubo — 2008-04-17T09:11:32-00:00

Kidney international, Vol. 55, No. 6. (June 1999), pp. 2450-2456.

BACKGROUND: Insulin resistance and hyperinsulinemia induce glomerular hypertension and hyperfiltration, which may result in glomerulosclerosis. However, the relationship between hyperinsulinemia and renal function is uncertain. METHODS: To elucidate whether hyperinsulinemia plays a significant part in the initiation and development of renal dysfunction, we examined in 1988 the relationship between serum insulin and renal function on data from a cross-sectional community survey conducted among residents from Hisayama Town, Japan, who were aged 40 to 79 years old. A total of 1065 men (72.0% of the total population in the same age range) and 1381 women (79.0%) without renal failure (creatinine clearance of more than 30 ml/min) underwent a comprehensive examination, including a 75 g oral glucose tolerance test. RESULTS: The correlation analysis showed that serum insulin, blood pressure, total cholesterol, low-density lipoprotein cholesterol, triglycerides, and body mass index were all negatively correlated with the reciprocal of serum creatinine level (P < 0.01), and alcohol intake was positively correlated (P < 0.05) in both sexes. High-density lipoprotein cholesterol and smoking habits were positively correlated (P < 0.05) in men. When the subjects were divided into quartiles based on the sum of fasting and two-hour postloading insulin levels, the averages of the reciprocal of serum creatinine were significantly lower in the fourth quartile (0.90 +/- 0.10 for men and 1.10 +/- 0.14 for women) compared with the lowest quartile (0.95 +/- 0.12 and 1.13 +/- 0.13, respectively) in both sexes (P < 0.05). In multiple regression analysis, the correlation between the sum of insulin levels and the reciprocal of serum creatinine remained significant even after controlling for age, sex, body mass index, blood pressure, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, alcohol intake, and smoking habits. We could not find appropriate correlations of creatinine clearance calculated by the Cockcroft-Gault formula with the covariates including serum insulin. CONCLUSIONS: The findings of this study suggest that hyperinsulinemia is a significant relevant factor of renal function in the general population.

Robust hepatitis C virus infection in vitro

Jin Zhong — 2008-03-28T04:52:19-00:00

Proceedings of the National Academy of Sciences, Vol. 102, No. 26. (28 June 2005), pp. 9294-9299.

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen. 10.1073/pnas.0503596102

Erythropoietin induces heme oxygenase-1 expression and attenuates oxidative stress.

P Katavetin — 2008-03-12T06:53:29-00:00

Biochem Biophys Res Commun, Vol. 359, No. 4. (10 August 2007), pp. 928-934.

Recent studies have established that erythropoietin (EPO) is a pleiotropic cytokine. In this study we investigated whether pleiotropic effects of EPO may involve regulation of heme oxygenase (HO)-1, an anti-oxidative stress protein. A stimulatory effect of EPO on HO-1 expression was demonstrated in cultured renal endothelial cells, in which EPO decreased intracellular oxidative stress and provided cytoprotection against H(2)O(2). These beneficial effects were partially reversed by a HO-1 inhibitor. We then evaluated whether EPO induces HO-1 and ameliorates renal injury in vivo. Administration of EPO to Dahl salt-sensitive (DS) rats with low salt diet, a model of chronic tubulointerstitial injury, reduced proteinuria, and renal injury including peritubular capillaries rarefaction as compared to vehicle-treated DS rats. This renoprotection was associated with up-regulation of HO-1 in the kidney. In conclusion, EPO-induced HO-1 expression is likely to provide cytoprotection against oxidative stress.

Products of 12/15-Lipoxygenase Upregulate the Angiotensin II Receptor

Zhong-Gao Xu — 2008-03-03T09:35:25-00:00

J Am Soc Nephrol, Vol. 19, No. 3. (1 March 2008), pp. 559-569.

Angiotensin II and its type 1 receptor (AT1R) play important roles in the pathogenesis of renal disease and diabetic nephropathy. The 12/15-lipoxygenase pathway of arachidonate metabolism and its lipid products have also been implicated in diabetic nephropathy. However, it is unclear whether 12/15-lipoxygenase regulates expression of AT1R. In cultured rat mesangial cells, we found that the 12/15-lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) increased AT1R mRNA and protein expression, primarily by stabilizing AT1R mRNA. Pretreatment with 12(S)-HETE also amplified the signaling effects of angiotensin II, likely due to the increased AT1R expression. Levels of AT1R protein expression decreased when 12/15-lipoxygenase was knocked down with specific short hairpin RNA (shRNA) compared with control cells. Similarly, levels of the AT1 receptor, but not the AT2 receptor, were significantly lower in mesangial cells and glomeruli derived from 12/15-lipoxygenase knockout mice compared with control mice. Reciprocally, stable overexpression of 12/15-lipoxygenase increased AT1R expression in cultured mesangial cells. In vivo, modified siRNA targeting 12/15-lipoxygenase reduced glomerular AT1R expression in a diabetic mouse model. Interestingly, angiotensin II induced greater levels of 12/15-lipoxygenase, TGF-1, and fibronectin (FN) in AT1R-overexpressing mesangial cells compared with control cells. Therefore, oxidized lipids generated by the 12/15-lipoxygenase-mediated metabolism of arachidonic acid can enhance AT1R expression in mesangial cells and augment the profibrotic effects of angiotensin II. 10.1681/ASN.2007080939

Reverse white-coat effect as an independent risk for microalbuminuria in treated hypertensive patients.

T Kato — 2008-02-14T05:46:57-00:00

Nephrol Dial Transplant, Vol. 22, No. 3. (March 2007), pp. 911-916.

BACKGROUND: The influence of the converse phenomenon of white-coat hypertension called 'reverse white-coat hypertension' or 'masked hypertension' on hypertensive target organ damage has not been fully elucidated. The present study assessed the hypothesis that this phenomenon may specifically associate with microalbuminuria, a marker of early renal damage, in treated hypertension. METHODS: A total of 267 treated essential hypertensive patients (133 men and 134 women; mean age, 66 years) without renal insufficiency or macroalbuminuria were enrolled in this study. Patients were classified into three groups by the difference between office and day-time ambulatory systolic blood pressure (BP) levels; i.e. subjects with white-coat effect (W group: office--day-time systolic BP > or =20 mmHg, n = 48), with reverse white-coat effect (R group: office - day-time systolic BP < - 10 mmHg, n = 43) and without white-coat or reverse white-coat effect (N group: -10 mmHg < or = office--day-time systolic BP <20 mmHg, n = 176). The urinary albumin (U-Alb) level was measured as the albumin to creatinine excretion ratio in the urine. Microalbuminuria was defined as U-Alb of > or =30 and <300 mg/g Cr. RESULTS: R group had a well-controlled office BP (130/77 mmHg), but their day-time BP (148/87 mmHg) was elevated compared with the other two groups. The levels of U-Alb excretion in N group, W group and R group were 12.3 (8.4, 25.6), 16.0 (10.5, 31.7) and 24.3 (10.2, 79.7) mg/g Cr [median (interquartile range)], respectively. Both U-Alb level and prevalence of microalbuminuria were significantly greater in R group than in N group. Multivariate analyses revealed that the presence of reverse white-coat effect, but not white-coat effect, was a significant predictor for microalbuminuria, independent of various clinical variables including ambulatory BP levels (odds ratio 2.63 vs N group, P = 0.02). CONCLUSION: These findings suggest that the presence of reverse white-coat effect may be an independent risk for early renal damage in treated hypertensive patients.

Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy

Yasuyuki Tsujita — 2008-02-04T05:01:05-00:00

Proceedings of the National Academy of Sciences, Vol. 103, No. 32. (8 August 2006), pp. 11946-11951.

The serine/threonine kinase Akt regulates cellular survival, proliferation, gene transcription, protein translation, metabolism, and differentiation. Although Akt substrates are found throughout the cell, activated Akt normally accumulates in the nucleus, suggesting that biologically relevant targets are located there. Consequences of nuclear Akt signaling in cardiomyocytes were explored by using nuclear-targeted Akt (Akt-nuc). Accumulation of Akt-nuc did not provoke hypertrophy, unlike constitutively activated Akt. Instead, Akt-nuc inhibited hypertrophy concurrent with increased atrial natriuretic peptide (ANP) expression that depended upon phosphatidylinositol-3 kinase activity. Akt-nuc antihypertrophic effects were blocked by inhibition of either guanylyl cyclase A receptor or cyclic guanosine monophosphate-dependent protein kinase in cultured cardiomyocytes. Corroborating evidence showed blunted acute hypertrophic remodeling in Akt-nuc transgenic mice after transverse aortic constriction coincident with higher ANP expression and smaller myocyte volume. In addition, Akt-nuc expression improved systolic function and survival in the chronic phase of transverse aortic constriction-induced hypertrophy. Thus, Akt-nuc antagonizes certain aspects of hypertrophy through autocrine/paracrine stimulation of a phosphatidylinositol-3 kinase-dependent signaling cascade that promotes ANP expression, resulting in a unique combination of prosurvival coupled with antihypertrophic signaling. 10.1073/pnas.0510138103

Establishment of the anti-Klotho monoclonal antibodies and detection of Klotho protein in kidneys.

Y Kato — 2008-01-28T03:41:48-00:00

Biochem Biophys Res Commun, Vol. 267, No. 2. (19 January 2000), pp. 597-602.

A novel gene, klotho (kl), which is involved in the development of a syndrome resembling human aging in mice, was recently identified. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. There also exists a splice variant of kl mRNA which encodes a putative secreted protein in both human and mouse. In this study, to characterize the physiological roles of Klotho protein, we established three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG(2)a), KM2119 (rat IgG(2)b), and KM2365 (mouse IgG(1)). In Western blots, KM2076 and KM2119 specifically recognized a 130 kDa Klotho protein in the mouse and human kidney membrane fractions. To detect the human Klotho protein, the sandwich-type ELISA system with KM2076 and KM2365 was established. Using the ELISA system, we detected the human Klotho protein as low as 20 ng/ml in the supernatant of Chinese hamster ovary cells (CHO cells), introduced the human klotho gene. KM2076 and KM2119 specifically gave a positive staining by immunohistochemical staining in paraffin or frozen sections of the kidneys from wild-type mice but not in those from kl mice. Strong staining was observed especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction pattern in the paraffin sections of rat and human kidneys. The mAbs established in this paper will serve as useful analytical, pathological, and diagnostic tools to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging.

Regional differences in end-stage renal disease and amount of protein intake in Japan.

M Motokawa — 2008-01-24T04:10:00-00:00

J Ren Nutr, Vol. 17, No. 2. (March 2007), pp. 118-125.

OBJECTIVE: We recently showed regional differences in the incidence of end-stage renal disease (ESRD) within Japan, which is generally ethnically homogenous, suggesting that factors other than genetic may contribute to the difference. We examined regional differences in the amounts of dietary nutrient intake, especially protein in our search for an explanation. DESIGN AND SETTING: Annually, the Japanese Society for Dialysis Therapy reports the numbers of patients entering maintenance dialysis in each prefecture of Japan. We used these numbers from 1984 to 2002 to calculate the annual ESRD incidence in each of 12 regions of Japan. The regional differences were analyzed in relation to the amounts of nutrient intake reported annually by National Nutrition Survey in corresponding regions for these 19 years. Each year, approximately 15,000 subjects from 5000 households in randomly selected 300 districts were included to obtain a representative sample of the entire population of Japanese in a manner of age, sex, and body mass matched. RESULTS: There were marked regional differences in the annual ESRD incidence and small regional differences in dietary intake of each nutrient. Multiple regression analysis showed that the annual ESRD incidence was negatively correlated with energy intake (r = -0.65, F = 240, n = 228) and positively correlated with animal protein intake (r = 0.25, F = 30). Across 12 regions in the values averaged for 19 years in each region, however, the incidence of ESRD was negatively correlated only with the amounts of energy intake (r = -0.74, F = 12, n = 12), but not with animal protein (r = 0.07, F = 0.04). CONCLUSION: The present study, relating regional differences between ESRD dynamics and the amounts of nutrient intake in a nationwide population of Japan, revealed that the renal protective effects of dietary restriction of protein, suggested by animal models of progressive nephropathies but yet unproved by large-scale clinical trials, remained unestablished even on a macro level of whole Japan through mapping approaches.

Use of microarrays in transfection of mammalian cells with dicer-digested small interfering RNAs.

Hiroyuki Fujimoto — 2008-01-22T12:26:37-00:00

Anal Biochem (31 December 2007)

Transfection cell arrays provide promising methods for the high-throughput analysis of gene functions. Such analysis is performed efficiently by using small interfering RNA (siRNA). To extend the usefulness of microarrays, this study was devoted to implementing siRNA prepared from complementary DNA (cDNA). The preparation of siRNA involves the transcription of cDNA to generate double-stranded RNA (dsRNA) followed by digestion with endoribonuclease, dicer, by which one can obtain the library of siRNAs without target sequence optimization. In this study, endoribonuclease-digested siRNA (d-siRNA) was prepared using cDNA encoding enhanced green fluorescent protein (EGFP) and loaded onto micropatterned substrates through electrostatic interactions of siRNA complex with substrates. We observed that d-siRNA loading was comparable to the case with siRNA prepared by chemical synthesis. When cotransfected with EGFP plasmid into human embryonic kidney 293 (HEK293) cells on a microarray, d-siRNA suppressed EGFP expression in a loading-dependent manner. In addition, d-siRNA triggered gene silencing at a level similar to that of chemically synthesized siRNA. The similarity between the two types of siRNA regarding silencing efficiency suggests that heterogeneity in nucleotide sequences of d-siRNA has minor effects. From these results, we conclude that the combination of d-siRNA with the array technology provides useful tools for high-throughput screening of gene functions.

Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Attenuates Renal Injury Induced by Protein-Overload Proteinuria

Hideaki Shimizu — 2008-01-15T05:00:17-00:00

J Am Soc Nephrol, Vol. 14, No. 6. (1 June 2003), pp. 1496-1505.

ABSTRACT. It has been postulated that protein filtered through glomeruli activates tubular epithelial cells, which secrete vasoactive and inflammatory substances including chemokines, leading to tubulointerstitial renal injury. The present study was designed to investigate the role of monocyte chemoattractant protein-1 (MCP-1) in this process and to evaluate the effectiveness of a kidney-targeted gene transfer technique using hydrodynamic pressure. Naked plasmid encoding 7ND (an MCP-1 antagonist) or a control plasmid was introduced into the left kidney of rats. Three days after gene transfer (day 0), intraperitoneal administration of bovine serum albumin (10 mg/g body wt per day) was started and continued for 14 or 21 d. RT-PCR showed that 7ND mRNA was expressed only in the gene-transfected kidney. Immunostaining showed that 7ND protein was localized in the interstitial cells. Macrophage infiltration was significantly reduced in the left kidney of rats treated with 7ND on days 14 and 21. In the right kidney, such effects were not observed. 7ND also attenuated tubular damage and decreased the number of apoptotic cells. Computer-assisted analysis revealed that the areas positively stained for alpha-smooth muscle actin (alphaSMA), fibronectin-EDA, type I collagen, and collagen fibrils were significantly reduced in the 7ND-treated kidney on day 21. Furthermore, 7ND gene therapy significantly reduced MCP-1 and TGF-beta1 mRNA expression. These results demonstrate that MCP-1 plays an important role in the development of tubulointerstitial inflammation, tubular damage, and fibrosis induced by proteinuria. The fact that 7ND gene therapy had little effect on the contralateral kidney indicates that 7ND acted locally. This strategy may have a potential usefulness as a gene therapy against tubulointerstitial renal injury. E-mail: marus@med.nagoya-u.ac.jp 10.1097/01.ASN.0000069223.98703.8E

Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells.

K Ota — 2008-01-09T14:38:11-00:00

Biochem Biophys Res Commun, Vol. 357, No. 1. (25 May 2007), pp. 270-275.

Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS.

Long-term phlebotomy with low-iron diet therapy lowers risk of development of hepatocellular carcinoma from chronic hepatitis C.

J Kato — 2007-11-14T08:03:22-00:00

J Gastroenterol, Vol. 42, No. 10. (October 2007), pp. 830-836.

BACKGROUND: We have previously demonstrated that in patients with chronic hepatitis C (CHC), iron depletion improves serum alanine aminotransferase (ALT) levels as well as hepatic oxidative DNA damage. However, it has not been determined whether continuation of iron depletion therapy for CHC favorably influences its progression to hepatocellular carcinoma (HCC). METHODS: We conducted a cohort study on biopsy-proven CHC patients with moderate or severe liver fibrosis who failed to respond to previous interferon (IFN) therapy or had conditions for which IFN is contradicted. Patients were divided into two groups: subjects in group A (n = 35) underwent weekly phlebotomy (200 g) until they reached a state of mild iron deficiency, followed by monthly maintenance phlebotomy for 44-144 months (median, 107 months), and they were advised to consume a low-iron diet (5-7 mg iron/day); group B (n = 40) comprised CHC patients who declined to receive iron depletion therapy. RESULTS: In group A, during the maintenance phase, serum ALT levels decreased to less than 60 IU/l in all patients and normalized (<40 IU/l) in 24 patients (69%), whereas in group B no spontaneous decrease in serum ALT occurred. Hepatocarcinogenesis rates in groups A and B were 5.7% and 17.5% at the end of the fifth year, and 8.6% and 39% in the tenth year, respectively. Multivariate analysis revealed that iron depletion therapy significantly lowered the risk of HCC (odds ratio, 0.57) compared with that of untreated patients (P = 0.0337). CONCLUSIONS: Long-term iron depletion for CHC patients is a promising modality for lowering the risk of progression to HCC.

Investigation of the Molecular Mechanism of ICAN, a Novel Gene Amplification Method.

T Uemori — 2007-11-02T02:48:31-00:00

J Biochem (Tokyo), Vol. 142, No. 2. (August 2007), pp. 283-292.

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, thermostable RNaseH and a DNA polymerase with strand-displacing activity (H. Mukai et al. J. Biochemistry, in the preceding paper in this issue). Here we elucidated the mechanism of ICAN by analysing the nicking site of RNaseH, behaviour of chimeric primers and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN.

Highly Efficient Isothermal DNA Amplification System Using Three Elements of 5'-DNA-RNA-3' Chimeric Primers, RNaseH and Strand-displacing DNA Polymerase.

H Mukai — 2007-11-02T02:42:16-00:00

J Biochem (Tokyo), Vol. 142, No. 2. (August 2007), pp. 273-281.

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe.

Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance

Takashi Matsuzaka — 2007-10-07T05:08:59-00:00

Nat Med, Vol. 13, No. 10. (October 2007), pp. 1193-1202.

Hypovitaminosis D in type 2 diabetes mellitus: Association with microvascular complications and type of treatment.

A Suzuki — 2007-10-02T03:06:43-00:00

Endocr J, Vol. 53, No. 4. (August 2006), pp. 503-510.

The prevalence of hypovitaminosis D has been recently reevaluated, and diabetes is considered as a risk factor for osteoporosis. We studied the association of the prevalence of hypovitaminosis D with the clinical features of diabetes. We conducted the observational study in 581 Japanese patients with type 2 diabetes mellitus and 51 normal subjects, and analyzed the relationship between serum 25-hydroxyvitamin D (25-OHD) concentration and the clinical features associated with type 2 diabetes. Mean serum 25-OHD concentration in type 2 diabetes patients was 17.0 +/- 7.1 ng/ml (Mean +/- SD) in winter, and was not statistically different from normal population (17.5 +/- 3.6 ng/ml). The prevalence of hypovitaminosis D (<20 ng/ml) was 70.6%. Serum concentrations of 25-OHD were associated with HbA1c (P = 0.013), age (P = 0.070) and serum albumin (P < 0.001), but were not related to BMI or the duration of diabetes. The levels of 25-OHD were significantly lower in the population with apparent microvascular complications, although serum creatinine levels were below 2.0 mg/dl. Serum 25-OHD concentrations in the group treated with insulin (15.4 +/- 6.5 ng/ml) was lower than those in the patients treated with diet alone (20.8 +/- 7.6 ng/ml) and with oral hypoglycemic agents (17.3 +/- 7.0 ng/ml). Furthermore, the highest incidence of osteoporotic fracture and/or back deformity was observed in insulin-treated patients with hypovitaminosis D. In conclusion, these results suggest that microvascular complications and insulin treatment in type 2 diabetes patients are associated with the co-existence of hypovitaminosis D, and that hypovitaminosis D in insulin-treated patients is possibly related to the risk of osteoporotic fracture.

Identification of genes up-regulated by histone deacetylase inhibition with cDNA microarray and exploration of epigenetic alterations on hepatoma cells.

T Chiba — 2007-09-26T08:13:30-00:00

J Hepatol, Vol. 41, No. 3. (September 2004), pp. 436-445.

BACKGROUND/AIMS: Epigenetics is the key factor in the regulation of gene expression. We conducted cDNA microarray analysis to screen for genes induced by histone deacetylase (HDAC) inhibition and examined epigenetic alterations. METHODS: Microarray analysis was performed in six hepatoma cell lines and primary hepatocytes treated with trichostatin A (TSA). mRNA expression of several genes was examined by reverse transcription-polymerase chain reaction in TSA-treated cells and hepatoma samples. Acetylated histones and methylation status in 5'CpG islands was assessed by chromatin immunoprecipitation (ChIP) assay and bisulfite genomic sequencing, respectively. RESULTS: Fifty-seven genes showed greater than 2-fold change after TSA treatment in multiple cell lines. Among them, four genes including p21(WAF1) exhibited substantial induction (greater than 5-fold changes). Decreased mRNA levels of these genes in hepatoma tissues were observed in more than half of patients. ChIP assay, in general, demonstrated a good correlation between mRNA expression and histone acetylation, but only a limited correlation with the methylated DNA in the promoter region. CONCLUSIONS: We identified 57 up-regulated genes by TSA treatment in hepatoma cells and some of them appeared to be cancer-related genes in hepatomas. The alterations in acetylated histones are likely closely associated with gene expression.

High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-kappaB activation in bovine aortic endothelial cells.

Hitoshi Iwasaki — 2007-09-19T08:30:29-00:00

Atherosclerosis (31 January 2007)

Recently, it has become evident that elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The molecular mechanisms involved in hyperglycemia-induced PAI-1 expression in bovine aortic endothelial cells (BAEC) were investigated. PAI-1 expression in BAEC was significantly increased in accordance with the concentration of glucose in media from 5.7mM to 23mM. Stimulation with high glucose (23mM) significantly increased small GTPase RhoA activation. Pretreatment with a Rho-kinase inhibitor, Y-27632 (1-10muM), significantly blocked high glucose-induced PAI-1 expression. NF-kappaB activity determined using the luciferase reporter gene assay was significantly enhanced by high glucose, and pretreatment with Y-27632 inhibited high glucose-induced PAI-1 expression at the basal level. An inhibitor of NF-kappaB action, namely parthenolide (0.1muM), BAY 11-7082 (5muM) and SN50 (1muM), significantly blocked high glucose-mediated PAI-1 expression to a level with low glucose (5.7mM). These data suggested that high glucose-induced PAI-1 expression in endothelial cells is mediated by NF-kappaB activation through the Rho/Rho-kinase pathway. Inhibition of Rho/Rho-kinase signaling might be a novel target for diabetes and metabolic syndrome.

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.

M Murphy — 2007-09-13T09:51:22-00:00

J Biol Chem, Vol. 274, No. 9. (26 February 1999), pp. 5830-5834.

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.

Mechanisms of transcriptional repression by 1,25(OH)2 vitamin D.

S Kato — 2007-08-30T09:27:45-00:00

Curr Opin Nephrol Hypertens, Vol. 16, No. 4. (July 2007), pp. 297-304.

PURPOSE OF REVIEW: Vitamin D has diverse biological actions, and consequently the mechanisms behind how it regulates gene transcription are diverse. Unlike its well described positive effects on gene transcription, little is known about how vitamin D induces transcriptional repression. RECENT FINDINGS: Vitamin D-induced transcriptional repression of several negative vitamin D receptor target genes has been studied on a molecular level. A new class of negative vitamin D response elements, which are E-box-type motifs, bind the bHLH-type transcriptional activator (VDIR) together with a histone acetyltransferase coactivator. The vitamin D receptor, activated by vitamin D, does not directly bind to the negative vitamin D response elements, but instead associates with VDIR. This leads to the dissociation of the histone acetyltransferase coactivator and recruitment of a histone deacetylase corepressor to transrepress transcription of the target gene promoter. SUMMARY: Histone inactivation induced by histone deacetylase co-repressors appears to facilitate vitamin D-induced transcriptional repression via the vitamin D receptor. Following vitamin D binding, structural alteration of the DNA-unbound vitamin D receptor triggers transcriptional repression. Given this, the mechanisms behind vitamin D-induced transcriptional repression are probably more complex than those of vitamin D-induced transactivation.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Y Ma — 2007-08-30T09:08:01-00:00

J Clin Invest, Vol. 116, No. 4. (April 2006), pp. 892-904.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Role of the Akt/FoxO3a pathway in TGF-beta1-mediated mesangial cell dysfunction: a novel mechanism related to diabetic kidney disease.

M Kato — 2007-08-29T09:29:41-00:00

J Am Soc Nephrol, Vol. 17, No. 12. (December 2006), pp. 3325-3335.

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrix proteins. TGF-beta is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-beta can mediate MC survival and oxidative stress. TGF-beta treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-beta treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-beta effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-beta and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-beta can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.

Diverse effects of cyclosporine on hepatitis C virus strain replication.

N Ishii — 2007-08-26T13:50:05-00:00

J Virol, Vol. 80, No. 9. (May 2006), pp. 4510-4520.

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.

Different anti-HCV profiles of statins and their potential for combination therapy with interferon.

M Ikeda — 2007-08-26T13:48:46-00:00

Hepatology, Vol. 44, No. 1. (July 2006), pp. 117-125.

We recently developed a genome-length hepatitis C virus (HCV) RNA replication system (OR6) with luciferase as a reporter. The OR6 assay system has enabled prompt and precise quantification of HCV RNA replication. Pegylated interferon (IFN) and ribavirin combination therapy is the world standard for chronic hepatitis C, but its effectiveness is limited to about 55% of patients. Newer therapeutic approaches are needed. In the present study, we used the OR6 assay system to evaluate the anti-HCV activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, called statins, and their effects in combination with IFN-alpha. Five types of statins (atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin) were examined for their anti-HCV activities. Fluvastatin exhibited the strongest anti-HCV activity (IC50: 0.9 micromol/L), whereas atorvastatin and simvastatin showed moderate inhibitory effects. However, lovastatin, reported recently as an inhibitor of HCV replication, was shown to exhibit the weakest anti-HCV activity. The anti-HCV activities of statins were reversed by the addition of mevalonate or geranylgeraniol. Surprisingly, however, pravastatin exhibited no anti-HCV activity, although it worked as an inhibitor for HMG-CoA reductase. The combination of IFN and the statins (except for pravastatin) exhibited strong inhibitory effects on HCV RNA replication. In combination with IFN, fluvastatin also exhibited a synergistic inhibitory effect. In conclusion, statins, especially fluvastatin, could be potentially useful as new anti-HCV reagents in combination with IFN.

Production of infectious hepatitis C virus of various genotypes in cell cultures.

T Kato — 2007-08-26T13:46:51-00:00

J Virol, Vol. 81, No. 9. (May 2007), pp. 4405-4411.

A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.

The association between microalbuminuria and metabolic syndrome in the general population in Japan: the Takahata study.

Z Hao — 2007-08-02T07:54:39-00:00

Intern Med, Vol. 46, No. 7. (2007), pp. 341-346.

OBJECTIVE: The metabolic syndrome is associated with an increased risk of chronic kidney disease, cardiovascular disease and mortality. However, the association between microalbuminuria and the metabolic syndrome has not yet been reported in the general population in Japan. Therefore, we undertook a population-based study to examine the association between microalbuminuria and the metabolic syndrome in Takahata, Japan. METHODS: Subjects of this cross-sectional study were individuals aged from 40 to 87 years old. The metabolic syndrome was defined according to the criteria of the Adult Treatment Panel III. Microalbuminuria was defined as a urine albumin-creatinine ratio of 30 to 300 mg/g. RESULTS: A total of 2,321 subjects (mean age 64 years old) were entered into the final analysis. Among them, the prevalence of the metabolic syndrome and microalbuminuria was 16.5% and 13.7%, respectively. There was a significantly positive correlation between the number of components of the metabolic syndrome and the corresponding prevalence of microalbuminuria (p<0.001). In the subjects with metabolic syndrome compared with those without metabolic syndrome, the age- and gender-adjusted odds ratio of microalbuminuria was 1.99 (95% CI, 1.49-2.66). Multiple logistic regression analysis revealed that high glucose, high blood pressure and obesity were independently associated with microalbuminuria. CONCLUSIONS: Our study revealed a strong relationship between microalbuminuria and the metabolic syndrome in the general population in Japan. More comprehensive and intensive management of the metabolic syndrome at its early stage is important to prevent the progression of renal injury and cardiovascular complications.

Clinical utility of trace proteinuria for microalbuminuria screening in the general population

Konta — 2007-04-29T06:10:30-00:00

Clinical and Experimental Nephrology, Vol. 11, No. 1. (March 2007), pp. 51-55.

Synergistic expression of angiotensin-converting enzyme (ACE) and ACE2 in human renal tissue and confounding effects of hypertension on the ACE to ACE2 ratio.

S Wakahara — 2007-07-25T04:20:49-00:00

Endocrinology, Vol. 148, No. 5. (May 2007), pp. 2453-2457.

Angiotensin-converting enzyme (ACE) 2, a newly emerging component of the renin-angiotensin system, is presumed to be a counterregulator against ACE in generating and degrading angiotensin II. It remains to be elucidated how mRNA levels of these two genes are quantitatively regulated in the kidney and also what kind of clinicopathological characteristics could influence the gene expressions in humans. Seventy-eight cases of biopsy-proven renal conditions were examined in detail. Total RNA from a small part of each renal cortical biopsy specimen was reverse transcribed, and the resultant cDNA was amplified for ACE, ACE2, and glyceraldehyde-3-phosphate dehydrogenase with a real-time PCR system. Then we investigated the relationship between clinicopathological variables and mRNA levels adjusted for glyceraldehyde-3-phosphate dehydrogenase. Statistically significant correlation was not observed between any clinicopathological variables and either of the gene expressions by pairwise comparison. However, a strong correlation was observed between the gene expressions of ACE and those of ACE2. Moreover, the ACE to ACE2 ratio was significantly higher in subjects with hypertension (HT) than that in subjects without HT. Whereas parameters of renal function, e.g. urinary protein excretion (UPE) and creatinine clearance (Ccr), are not significantly related to the ACE to ACE2 ratio as a whole, the HT status may reflect disease-induced deterioration of renal function. That is, UPE and Ccr of subjects with HT are significantly different from those without HT, in which a significant correlation is also observed between UPE and Ccr. Finally, stepwise regression analysis further revealed that only the HT status is an independent confounding determinant of the ACE to ACE2 ratio among the variables tested. Our data suggest that ACE2 might play an important role in maintaining a balanced status of local renin-angiotensin system synergistically with ACE by counterregulatory effects confounded by the presence of hypertension. Thus, ACE2 may exert pivotal effects on cardiovascular and renal conditions.

AGEs activate mesangial TGF-beta-Smad signaling via an angiotensin II type I receptor interaction.

K Fukami — 2007-07-25T01:11:50-00:00

Kidney Int, Vol. 66, No. 6. (December 2004), pp. 2137-2147.

BACKGROUND: The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells. METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [3H]leucine incorporation. RESULTS: AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT1R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta. CONCLUSION: The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.