DNA methylation of GJA1, BMP2 and BMP4 in a human cementoblast cell line induced by lipopolysaccharide.

AIM: To examine DNA methylation of GJA1, BMP2 and BMP4 in human cementoblasts (HCEM) induced by lipopolysaccharide (LPS). METHODOLOGY: HCEM were cultured in osteoinduction medium. After 24 h, Escherichia coli LPS (1 µg/mL) was added to the medium, which was changed every 2-3 days. Untreated samples were used as controls. Messenger RNA was extracted after 4 weeks, and quantitative real-time polymerase chain reaction (qRT-PCR) for GJA1, BMP2, BMP4 and DNMT1 was performed. Genomic DNA was extracted after 4 weeks, and quantitative methylation-specific polymerase chain reaction was carried out for GJA1, BMP2 and BMP4. To detect mineralization, alizarin red and alkaline phosphatase staining were performed. The cells were also treated with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza) and examined. The significance of differences amongst groups was assessed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with P < 0.05 being significant. RESULTS: Decreased expression of mRNA was seen in GJA1, BMP2 and BMP4 after 4 weeks (P < 0.05). DNA hypermethylation was detected in GJA1, BMP2 and BMP4 (P < 0.05). Alizarin red staining and alkaline phosphatase staining revealed decreased mineralization levels in HCEM stimulated with LPS. 5Aza abolished the effects of DNA methylation in HCEM stimulated with LPS. CONCLUSIONS: These results suggest that long-term LPS stimulation induces DNA methylation of GJA1, BMP2 and BMP4 in HCEM.

Evaluating dose-limiting toxicities of MDM2 inhibitors in patients with solid organ and hematologic malignancies: A systematic review of the literature.

INTRODUCTION: Mouse double minute 2 protein (MDM2), a negative regulator of the p53 tumour suppressor gene, is frequently amplified in malignancies. MDM2 antagonists have shown efficacy in treating malignancies with MDM2 overexpression and can overcome chemoresistance in acute myeloid leukemia. We systematically evaluated the safety profile of MDM2 inhibitors in the treatment of solid organ and hematologic malignancies. MATERIALS AND METHODS: We searched Medline and EMBASE from January 1947 to November 2018 for prospective clinical studies, in English or French, investigating any MDM2 inhibitor in pediatric or adult cancers, and reporting dose and toxicity outcomes. Primary outcome was dose-limiting toxicity (DLT) and secondary outcome was death. RESULTS: The search yielded 493 non-duplicate citations. Eighteen studies of 10 inhibitors met inclusion criteria (total N = 1005 patients). Two-thirds of included studies did not define DLTs and the reporting of toxicities was highly variable. The most commonly reported DLTs were cytopenias, gastrointestinal toxicity, metabolic disturbances, fatigue and cardiovascular toxicity; there was one death attributed to treatment toxicity. CONCLUSION: MDM2 antagonists have been studied in a variety of malignancies with toxicities similar to other commonly used chemotherapy agents and may represent a safe adjuvant treatment for further study in in acute leukemia.

Silver nanoparticles coated with dodecanethiol used as fillers in non-cytotoxic and antifungal PBAT surface based on nanocomposites.

In the present study, we report the preparation of antifungal and non-cytotoxic polymer nanocomposites with potential application in biomedical materials. Dodecanethiol-protected silver nanoparticles (AgNPs-DDT) were synthesized by a reduction/precipitation method and dispersed in chloroform to obtain stable colloidal dispersions. PBAT-based nanocomposites containing 0.25, 0.5 and 2 wt% AgNPs-DDT were prepared by casting method. The incorporation of AgNPs-DDT in PBAT matrix resulted in nanocomposites which combine improved mechanical performance and antifungal properties with a non-cytotoxic characteristic.

Is the oral fungal pathogen Candida albicans a cariogen?

Pathobiology of dental caries is complex. Data from recent molecular microbiologic studies have further redefined the role of the oral microbiome in the etiology of dental caries. This new information challenges the conventional view on the hegemony of classic cariogenic prokaryotes such as Streptococcus mutans in caries etiology, and raises the intriguing possibility of the participation of the eukaryotic oral fungal pathogen Candida in the caries process. The virulence attributes of Candida species such as their acidogenicity and aciduric nature, the ability to develop profuse biofilms, ferment and assimilate dietary sugars, and produce collagenolytic proteinases are all indicative of their latent cariogenic potential. Based on the above, oral candidal counts have been used by some as a caries risk indicator. On the contrary, other studies suggest that Candida is merely a passenger extant in an acidic cariogenic milieu, and not a true pathogen. In this review, we critically examine the varying roles of Candida, and traditionally accepted cariogens such as the mutans group of streptococci in the pathobiology of dental caries. The weight of available data tends to imply that Candida may play a pivotal role as a secondary agent perpetuating the carious process, especially in dentinal caries.

Impact of glucose tolerance status on the development of coronary artery disease among working-age men.

AIMS: To examine the impact of glucose tolerance status on the development of coronary artery disease (CAD) in working-age men in Japan. METHODS: This population-based retrospective cohort study included 111,621 men aged 31-60 years [63,558 with normal glucose tolerance (NGT); 37,126 with prediabetes; 10,937 with diabetes]. The Cox proportional-hazards regression model was used to identify variables related to the incidence of CAD. RESULTS: Multivariate analysis showed that, compared with NGT, diabetes increased the risk of CAD by 17.3 times (95% CI: 6.36-47.0) at ages 31-40 years, by 2.74 times (95% CI: 1.85-4.05) at ages 41-50 years and by 2.47 times (95% CI: 1.69-3.59) at ages 51-60 years. The HRs for CAD in men with diabetes aged 31-40 equaled that of men with NGT aged 51-60 [18.2 (7.15-46.4) and 19.4 (8.28-45.4), respectively]. CONCLUSION: The impact of diabetes on CAD was markedly greater in men aged 31-40 years compared with those aged 41-60 years.

Use of the Japanese health insurance claims database to assess the risk of acute pancreatitis in patients with diabetes: comparison of DPP-4 inhibitors with other oral antidiabetic drugs.

This study was initiated to evaluate the association of acute pancreatitis (AP) with the use of dipeptidyl peptidase-4 (DPP-4) inhibitors among patients with diabetes in Japan. A retrospective cohort study of a large medical and pharmacy claims database was performed to compare the incidence of AP among those receiving DPP-4 inhibitors and those receiving other oral antidiabetic drugs. The incidence of all AP and hospitalizations for AP was similar between the two groups. Previous exposure to DPP-4 inhibitors did not affect occurrence of AP in patients on other oral antidiabetic drugs. The Kaplan-Meier curve for time to AP was similar between the two groups, and was not affected by previous exposure to DPP-4 inhibitors. The Cox proportional hazard models showed the incidence of AP was not significantly higher in those receiving DPP-4 inhibitors. Despite numerous, important limitations related to claims database-based analyses, our results indicate that there is no increased risk of AP with use of DPP-4 inhibitors among patients with diabetes in Japan.

Systematic screening of plant extracts from the Brazilian Pantanal with antimicrobial activity against bacteria with cariogenic relevance.

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants--(1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens--using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease.

Hydroxyurea therapy in sickle cell anemia patients aids to maintain oral fungal colonization balance.

BACKGROUND: The aim of this study was to evaluate the frequency of Candida species and presence of lesions in the oral cavity of patients with sickle cell anemia (SS). METHODS: The study included 30 patients diagnosed with sickle cell anemia and taking hydroxyurea for at least 90 days (SS/HU+); and 39 patients with sickle cell anemia and without hydroxyurea therapy (SS/HU-). Two control groups were constituted by healthy individuals matched to the test groups in age, gender, and oral conditions (C/HU+ for SS/HU+ and C/HU- for SS/HU-). Oral clinical examination and anamnesis were performed. Yeasts were collected by oral rinses and identified by API system. Antifungal susceptibility evaluation was performed according to the CLSI methodology. Data obtained for microorganisms counts were compared by Student's t test (SS/HU+ vs. C/HU+ and SS/HU- vs. C/HU-) using MINITAB for Windows 1.4. Significance level was set at 5%. RESULTS: No oral candidosis lesions were detected. Significant differences in yeasts counts were observed between SS/HU- group and the respective control, but there were no differences between SS/HU+ and C/HU+. Candida albicans was the most prevalent species in all groups. Candida famata was observed both in SS and control groups. Candida dubliniensis, Candida glabrata, Candida krusei, Candida tropicalis, Candida pelliculosa, and Candida parapsilosis were observed only in SS groups. Most strains were susceptible to all antifungal agents. CONCLUSION: Hydroxyurea therapy seems to decrease candidal counts and resistance rate in sickle cell anemia patients. However, further studies should be conducted in the future to confirm this finding. Hydroxyurea therapy in sickle cell anemia patients maintains fungal species balance in oral cavity.

Unique structures of organelles observed in primary spermatocytes after micro-injection of protein solutions such as immunoglobulin into the lumen of the seminiferous tubules in mice and rats.

Unique membranous structures of intracytoplasmic organelle, sting of a stack of a few flat cisternae about 50 nm in thickness, were found in mouse and rat spermatocytes after micro-injection of immunoglobulin G into the lumina of the seminiferous tubules. Other proteins such as BSA and cytochrome c used in this study also induced the structures. In most cases, the stacks of cisternae were rolled up like cigars or cylinders. The structures varied in length and diameter, the largest one observed in this study being 10.7 µm in length. The structures did not appear when the testes were fixed just after micro-injection and were formed transiently: they were observed in the spermatocytes fixed between 1 and 4 h after injection. Cytochrome c, micro-injected as an inter-cellular tracer, was visualised by a diaminobenzidine reaction. As the reaction product was not contained in the cisternae of the unique structures, the lumen of the cisternae of the organelles was not continuous with the inter-cellular space. A flocculent material of low density was observed in the cisternae of the organelle. Similar material was observed in the lumina of solitary cisternae of the rough endoplasmic reticulum in the spermatocytes, suggesting that the structures derived from endoplasmic reticulum.

Oral microbial colonization in patients with systemic lupus erythematous: correlation with treatment and disease activity.

Treating patients with systemic lupus erythematosus (SLE) with steroids and immunosuppressive drugs may interfere in the presence of potentially opportunistic microorganisms in the oral cavity. The aim of this study was to evaluate the presence of Candida spp., Staphylococcus spp., Enterobacteria and Pseudomonas spp. in the oral cavity of SLE patients, compared with healthy controls. A group of 40 patients who had received therapy for at least 60 days was selected (19-53 years). For the control group, 40 healthy individuals matched for age, gender and use of partial prosthesis were selected. Oral rinse samples were collected and plated on specific culture media. After incubation, the number of colony forming units (CFU) was obtained and the isolates were identified at species level. Microbial counts were compared between SLE and control by analysis of variance (ANOVA) and Mann-Whitney (p < 0.05 significant). Microorganism counts in patients with and without immunosuppressive drugs, as well with active and inactive disease (according to SLEDAI score) were also compared. No significant differences in CFU/mL between SLE and control patients were observed (yeasts, p = 0.55; Staphylococci, p = 0.24; Enterobacteria/Pseudomonas spp., p = 0.26). No differences in microbial counts were observed regarding clinical parameters tested. The most frequent species isolated in the SLE group were Candida albicans, Staphylococcus epidermidis and Klebsiella oxytoca. In conclusion, no differences in frequency and microorganism levels were found between SLE patients and healthy individuals.

Interleukin-10 expression induced by adeno-associated virus vector suppresses proteinuria in Zucker obese rats.

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6 mg per mg·creatinine versus 88.8±30.0 mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.

Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse.

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175(-/-) ) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175(-/-) mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175(-/-) testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175(-/-) testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus.

OBJECTIVE: The goal of the study was to measure the prevalence of Candida spp. in the oral cavity of patients with diabetes types 1 and 2 when compared to healthy individuals and to study antifungal resistance profile of the isolates. DESIGN: There were 162 subjects in the study: diabetes type 1 (n=39); control group 1 (n=50): healthy individuals matched in gender, age, and oral conditions to diabetes type 1 patients; diabetes type 2 (n=37); control group 2 (n=36) who were matched to each patient of the diabetes type 2 group. Stimulated saliva was collected and isolates were identified with phenotypic tests. The presence of C. dubliniensis was determined by multiplex PCR. RESULTS: There were no statistically significant differences in Candida spp. frequency between the diabetes 1 group and its control (p=0.443) nor between the diabetes 2 group and its control (p=0.429). C. albicans was the most frequently isolated yeast in all groups. In the diabetes groups, C. stellatoidea, C. parapsilosis, C. tropicalis, C. lipolytica, C. glabrata, and C. krusei were also identified. Additionally, in control groups, C. kefyr was also detected. None of the isolates were resistant to amphotericin B and flucytosine. A low percentage of the isolates were resistant to ketoconazole. CONCLUSIONS: No differences were detected in colonization of Candida spp. oral isolates from type 1 and type 2 diabetes when compared to matched controls. The antifungal resistance of Candida spp. isolates for ketoconazole from type 1 diabetes patients was significantly higher than that of its matched control.

Antimicrobial analysis of chlorhexidine gel and intracanal medicaments against microorganisms inoculated in root canals.

AIM: The aim of this paper was to evaluate the antimicrobial activity of 2% chlorhexidine gel (CLX) associated with various intracanal medicaments against Candida albicans and Enterococcus faecalis inoculated in root canals. METHODS: Thirty six human single-rooted teeth were contaminated with C.albicans and E.faecalis. The canals were instrumented using 2% CLX gel and were divided into three groups according to the intracanal medicaments (ICM) used. Group 1: calcium hydroxide paste [Ca(OH)2], Group 2: 2% chlorhexidine gel (CLX) and Group 3: 2% CLX gel + Ca(OH)2. The root canal collections were performed after 21 days of contamination (control collection), after instrumentation (1st collection), after 14 days of intracanal medicament (2nd collection) and 7 days after medicament removal (3rd collection). The microbiological samples were plated in culture media and incubated for 48 hours. The results were submitted to Kruskal-Wallis test (P ≤ 0.05). RESULTS: It was verified that the instrumentation with CLX reduced the number of CFU/ml significantly when compared with the confirmation collection (control). However, the use of the ICM was only capable to eliminate completely the microorganisms in the root canals without difference statistics between them. CONCLUSION: Although the use of 2% chlorherixidine gel reduces the number of microorganisms significantly, only the ICM calcium hydroxide and calcium hydroxide associated with chlorhexidine are able to eliminate these microorganisms completely.

Hypertension induced by erythropoietin has a correlation with truncated erythropoietin receptor mRNA in endothelial progenitor cells of hemodialysis patients.

Endothelial nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contribute to erythropoietin (EPO)-induced hypertension, a major adverse reaction associated with EPO therapy. To investigate the mechanism of EPO-induced hypertension, we examined circulating endothelial progenitor cells (EPCs) taken from 56 hemodialysis (HD) patients. Among these EPCs (which reflect the condition of the endothelium), we looked for EPO receptor (EPOR) mRNAs. A truncated form of EPOR acts as a dominant negative regulator of EPO signaling, leading to hypertension. We found that the ratio of truncated EPOR mRNA in EPCs has a correlation with EPO-induced increase in blood pressure (r = 0.36, P = 0.02). The ratio of truncated to total EPOR mRNA in EPCs had an inverse correlation with EPO-induced cGMP production in vitro (r = -0.31, P = 0.02). A similar correlation was observed in cultured human endothelial cells after transfection of the full-length or truncated forms of EPOR (r = -0.92, P < 0.001). It follows, therefore, that evaluation of EPOR isoform mRNA in EPCs can predict EPO-induced hypertension. The termination of the EPO signal by truncated EPORs may decrease NO/cGMP production after EPO exposure, thereby raising blood pressure.

Reduced efficacy of topical corticosteroid in preventing cytarabine-induced kerato-conjunctivitis in patients receiving high-dose cytarabine and total body irradiation for allogeneic hematopoietic stem cell transplantation.

This study aimed to retrospectively evaluate the incidence of kerato-conjunctivitis in patients receiving TBI followed by high-dose cytarabine, and to clarify how effectively topical corticosteroid eye drops prevent kerato-conjunctivitis in these patients. Fifty-three patients who received cytarabine at a dose of 3 g/m2 every 12 h for 4 days after receiving TBI (12 Gy) as a conditioning for allogeneic hematopoietic stem cell transplantation (HSCT) were evaluated. For the prophylaxis of kerato-conjunctivitis, all patients received betamethasone sodium phosphate eye drops every 6 h, starting 1 day before the first dose of cytarabine and continuing until 1 day after the last dose of cytarabine or the complete resolution of ocular symptoms. For grading of kerato-conjuncitivitis, the National Cancer Institute-Common Toxicity Criteria were used. Among the 53 patients, the grades of kerato-conjunctivitis were grade 0 in 13 patients, grade 1 in 6 patients (11.3%), grade 2 in 10 patients (18.9%) and grade 3 in 25 patients (47.2%). These results strongly suggest that topical corticosteroid eye drops could not effectively prevent the development of cytarabine-induced kerato-conjunctivitis in HSCT recipients who receive high-dose cytarabine following TBI. Further investigation into a more effective prophylaxis for cytarabine-induced kerato-conjunctivitis in this setting is required.

Variations in the HHEX gene are associated with increased risk of type 2 diabetes in the Japanese population.

AIMS/HYPOTHESIS: Recently, several groups have carried out whole-genome association studies in European and European-origin populations and found novel type 2 diabetes-susceptibility genes, fat mass and obesity associated (FTO), solute carrier family 30 (zinc transporter), member 8 (SLC30A8), haematopoietically expressed homeobox (HHEX), exostoses (multiple) 2 (EXT2), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), which had not been in the list of functional candidates. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) in these genes and type 2 diabetes in participants from the Japanese population. METHODS: Sixteen previously reported SNPs were genotyped in 864 Japanese type 2 diabetes individuals (535 men and 329 women; age 63.1 +/- 9.5 years (mean+/-SD), BMI 24.3 +/- 3.9 kg/m(2)) and 864 Japanese control individuals (386 men and 478 women; age 69.5 +/- 6.8 years, BMI 23.8 +/- 3.7 kg/m(2)). RESULTS: The SNPs rs5015480 [odds ratio (OR) = 1.46 (95% CI 1.20-1.77), p = 2.0 x 10(-4)], rs7923837 [OR = 1.40 (95% CI 1.17-1.68), p = 2.0 x 10(-4)] and rs1111875 [OR = 1.30 (95% CI 1.11-1.52), p = 0.0013] in HHEX were significantly associated with type 2 diabetes with the same direction as previously reported. SNP rs8050136 in FTO was nominally associated with type 2 diabetes [OR = 1.22 (95% CI 1.03-1.46), p = 0.025]. SNPs in other genes such as rs7756992 in CDKAL1, rs10811661 in CDKN2B and rs13266634 in SLC30A8 showed nominal association with type 2 diabetes. rs7756992 in CDKAL1 and rs10811661 in CDKN2B were correlated with impaired pancreatic beta cell function as estimated by the homeostasis model assessment beta index (p = 0.023, p = 0.0083, respectively). CONCLUSIONS/INTERPRETATION: HHEX is a common type 2 diabetes-susceptibility gene across different ethnic groups.

New approach for chronic renal failure model by direct kidney injection of doxorubicin in rats.

Several experimental chronic renal failure (CRF) models are available for testing new drugs. A CRF model induced by the intravenous injection of 2 mg/kg of doxorubicin (DXR) twice during a 20-day interval reportedly results in pathological characteristics similar to glomerular sclerosis seen clinically. However, it normally takes more than 16 weeks to create this CRF model. We used three methods of direct drug injection into the kidney of rats to determine the method that would induce CRF within 4 weeks; Method A: DXR was injected directly into both kidneys; Method B: DXR was injected directly into the left kidney immediately after right nephrectomy; Method C: DXR was injected directly into the left kidney 1 week before right nephrectomy, and DXR was injected again directly into the left kidney. As a result, urinary protein, blood urea nitrogen (BUN), creatinine and creatinine clearance were significantly changed >1 week after the injection of DXR by Method C. Quantification of tissue transforming growth factor-beta1 (TGF-beta1), which is a prime fibrogenic cytokine in renal fibrosis, significantly increased in the kidney. A light microscopic image showed glomerular decrement, tubular dilation and atrophy and vacuolation of parenchyma. In conclusion, the results of this study demonstrate that the DXR model using Method C develops CRF within 4 weeks.

A genetic variation of the transcription factor 7-like 2 gene is associated with risk of type 2 diabetes in the Japanese population.

AIMS/HYPOTHESIS: It has been suggested that transcription factor 7-like 2 protein (TCF7L2) plays an important role in glucose metabolism by regulating the production level of glucagon-like peptide-1, a hormone which modifies glucose-dependent insulin secretion. Recently, variants of TCF7L2 gene were reported to confer an increased risk of type 2 diabetes in three different samples from European and European-origin populations. We studied whether the single nucleotide polymorphisms (SNPs) in TCF7L2 were associated with type 2 diabetes in samples from a Japanese population. METHODS: Five SNPs were genotyped in three different sample sets. Association with type 2 diabetes was investigated in each, as well as in combined sample sets. RESULTS: The SNP rs7903146 was nominally associated with type 2 diabetes in the initial (p = 0.08) and two replication sample sets (p = 0.05 and 0.06). For the combined sample set, in which we successfully genotyped 1,174 type 2 diabetes patients and 823 control subjects, rs7903146 showed a significant association with type 2 diabetes (odds ratio = 1.69 [95% CI 1.21-2.36], p = 0.002) with the same direction as the previous reports in samples from European and European-origin populations. SNPs rs7903146 and rs7901695 were in complete linkage disequilibrium. The rest of the five SNPs (rs7895340, rs11196205 and rs12255372) did not show any significant associations with type 2 diabetes. CONCLUSIONS/INTERPRETATION: The consistent association between rs7903146 in TCF7L2 and type 2 diabetes in different ethnic groups, including the Japanese population, suggests that TCF7L2 is a common susceptibility gene for type 2 diabetes.

Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [(3)H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-beta receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 microM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 microM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 microM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 microM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-beta receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.