Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy

Background: Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.<br/><br/>Methods: Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.<br/><br/>Results: A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P&lt; 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.<br/><br/>Conclusions: These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.© 2013 Kavanagh et al.; licensee BioMed Central Ltd.

Cardiomyopathy and Diastolic Dysfunction in the Embryo and Neonate of a Type 1 Diabetic Mouse Model

<p style="margin: 5px 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-style: inherit; font-size: inherit; line-height: 1.5; text-align: inherit; vertical-align: baseline;">Objective: The purpose of this study was to examine the effect of maternal type 1 diabetes on the structure and function of the embryonic and neonatal mouse heart.</p><p style="margin: 5px 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-style: inherit; font-size: inherit; line-height: 1.5; text-align: inherit; vertical-align: baseline;">Methods: Type 1 diabetes was induced in female C57BL6/J mice using streptozotocin. Embryonic (n = 105) and neonatal hearts (n = 46) were examined using high-frequency ultrasound (US) and a cohort of E18.5 (n = 34) and 1-day-old pup hearts (n = 27) underwent histological examination.</p><p style="margin: 5px 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-size: inherit; line-height: 1.5; text-align: inherit; vertical-align: baseline;">Results: Global cardiac hypertrophy in late gestation (E18.5) was evident on US in the diabetic group compared to controls with increased interventricular septal (IVS) thickness (0.44 ± 0.08 mm vs 0.36 ± 0.08 mm, P &lt; .05) and increased left ventricular wall thickness (0.38 ± 0.04 mm vs 0.29 mm ± 0.05, P &lt; .01). Isovolumetric relaxation time was initially prolonged in the diabetic group but resolved by E18.5 to control values. Histological examination at E18.5 demonstrated increased transverse measurements (2.42 ± 0.72 mm/g vs 1.86 ± 0.55 mm/g, P &lt; .05) and increased IVS thickness (0.64 ± 0.20 mm/g vs 0.43 ± 0.15 mm/g, P &lt; .05) in diabetic embryos compared to control embryos.</p><p style="margin: 5px 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-style: inherit; font-size: inherit; line-height: 1.5; text-align: inherit; vertical-align: baseline;">Conclusion: Maternal hyperglycemia has severe effects on offspring with evidence of cardiac impairment and cardiac hypertrophy in the embryo. These effects persisted in the 1-day old but attenuated in the 1-week old suggesting cardiac remodeling after the hyperglycemic milieu of pregnancy is removed</p>

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Maternal Vitamin D Status in Type 1 Diabetic Pregnancy: Impact on Neonatal Vitamin D Status and Association with Maternal Glycaemic Control

<p>Objective: The first aim of this study was to assess 25-hydroxy vitamin D (25OHD) concentrations in women with type 1 diabetes (T1DM) during pregnancy, post-delivery and also foetal (cord blood) 25OHD concentrations and to examine relationships between these. The second aim of the study was to investigate potential interactions between maternal body mass index (BMI) and foetal vitamin D status. A further study aim was to examine potential relationships between maternal 25OHD and glycosylated haemoglobin (HbA_1c) throughout pregnancy.</p><p>Research Design and Methods: This was an observational study of 52 pregnant controls without diabetes and 65 pregnant women with T1DM in a university teaching hospital. Maternal serum 25OHD was measured serially throughout the pregnancy and post-delivery. Cord blood 25OHD was measured at delivery. 25OHD was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS).</p><p>Results: Vitamin D deficiency (25OHD &lt;25 nmol/L) was apparent in both the T1DM subjects and controls at all 3 pregnancy trimesters. Vitamin D levels in all cord blood were &lt;50 nmol/L. Maternal 25OHD correlated positively with cord 25OHD at all 3 trimesters in the T1DM group (p= 0.02; p&lt;0.001; p&lt;0.001). 25OHD levels within cord blood were significantly lower for women with diabetes classified as obese vs. normal weight at booking [normal weight BMI &lt;25 kg/m² vs. obese BMI &gt;30 kg/m^2Â(nmol/L±SD); 19.93±11.15 vs. 13.73±4.74, p= 0.026]. In the T1DM group, HbA_1c at booking was significantly negatively correlated with maternal 25OHD at all 3 trimesters (p= 0.004; p = 0.001; p= 0.05).</p><p>Conclusion: In T1DM pregnancy, low vitamin D levels persist throughout gestation and post-delivery. Cord blood vitamin D levels correlate with those of the mother, and are significantly lower in obese women than in their normal weight counterparts. Maternal vitamin D levels exhibit a significant negative relationship with HbA_1c levels, supporting a potential role for this vitamin in maintaining glycaemic control. </p>

Lipoproteins in the DCCT/EDIC cohort:associations with diabetic nephropathy

Lipoproteins may contribute to diabetic nephropathy. Nuclear magnetic resonance (NMR) can quantify subclasses and mean particle size of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), and LDL particle concentration. The relationship between detailed lipoprotein analyses and diabetic nephropathy is of interest.

Lipoprotein subclass profiles of hyperlipidemic diabetic mice measured by nuclear magnetic resonance spectroscopy

Dyslipidemia accelerates vascular complications of diabetes. Nuclear magnetic resonance (NMR) analysis of lipoprotein subclasses is used to evaluate a mouse model of human familial hypercholesterolemia +/- streptozotocin (STZ)-induced diabetes. A double knockout (DKO) mouse (low-density lipoprotein receptor [LDLr] -/-; apolipoprotein B [apoB] mRNA editing catalytic polypeptide-1 [Apobec1] -/-) was studied. Wild-type (WT) and DKO mice received sham or STZ injections at age 7 weeks, yielding control (WT-C, DKO-C) and diabetic (WT-D, DKO-D) groups. Fasting serum was collected when the mice were killed (age 40 weeks) for Cholestech analysis (Cholestech Corp, Hayward, CA) and NMR lipoprotein subclass profile. By Cholestech, fasting triglyceride and total cholesterol increased in DKO-C versus WT-C. Diabetes further increased total cholesterol in DKO. High-density lipoprotein cholesterol (HDL-C) was similar among all groups. NMR revealed that LDL in all groups was present in a subclass the size of large human LDL and was increased 48-fold in DKO-C versus WT-C animals, but was unaffected by diabetes. HDL was found in a subclass equivalent to large human HDL, and was similar among groups. In conclusion, NMR analysis reveals lipoprotein subclass distributions and the effects of genetic modification and diabetes in mice, but lack of particles the size of human small LDL and small HDL may limit the relevance of the present animal model to human disease.

Metabolism of very low- and low-density lipoproteins isolated from normolipidaemic type 2 (non-insulin-dependent) diabetic patients by human monocyte-derived macrophages

The very low- and low-density lipoprotein fractions were isolated from 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients in good to fair glycaemic control and from corresponding age-, sex-, and race-matched, non-diabetic control subjects. Rates of cholesteryl ester synthesis averaged 268 +/- 31 vs 289 +/- 40 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for very low- and 506 +/- 34 vs 556 +/- 51 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for low-density lipoproteins isolated from the Type 2 diabetic patients and control subjects, respectively, when they were incubated with human macrophages. A group of approximately one-third of the patients was selected for separate analyses because very low-density lipoproteins isolated from these patients did stimulate more cholesteryl ester synthesis when incubated with macrophages. There were no significant differences in the lipid composition of the lipoproteins isolated from the three groups of subjects. The relative proportion of apoprotein C to apoprotein E was significantly decreased (p less than 0.002) in the very low-density lipoproteins from diabetic patients and was further decreased in samples from these selected diabetic patients. The apoprotein C-I content of very low-density lipoproteins isolated from diabetic patients was increased compared to control subjects and was further increased in samples from the selected diabetic patients (p less than 0.02). There were no significant differences in the proportions of apoproteins C-III-0, C-III-1, or C-III-2 among the three groups. These studies suggest that in normolipidaemic Type 2 diabetic patients, the apoprotein composition of VLDL is abnormal and this may alter VLDL macrophage interactions and thus contribute to the increased prevalence of atherosclerosis in diabetic patients.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density less than 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 +/- 1.01 v 1.99 +/- 0.39 nmol 14C-cholesteryl oleate synthesized/mg cell protein/20 h; mean +/- SEM, P less than .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P less than .05). The increase in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 +/- 0.23 v 2.12 +/- 0.20 micrograms degraded/mg cell protein/20 h) and accumulation (283 +/- 35 v 242 +/- 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycated Hemoglobin and Risk of Death in Diabetic Patients Treated With Hemodialysis: A Meta-analysis

Background: Studies investigating the association between glycated hemoglobin (HbA) level and mortality risk in diabetic patients receiving hemodialysis have shown conflicting results. <br/>Study Design: We conducted a systematic review and meta-analysis using MEDLINE, EMBASE, Web of Science, and the Cochrane Library. <br/>Setting &amp; Population: Diabetic patients on maintenance hemodialysis therapy. <br/>Selection Criteria for Studies: Observational studies or randomized controlled trials investigating the association between HbA values and mortality risk. Study authors were asked to provide anonymized individual patient data or reanalyze results according to a standard template. <br/>Predictor: Single measurement or mean HbA values. Mean HbA values were calculated using all individual-patient HbA values during the follow-up period of contributing studies. <br/>Outcome: HR for mortality risk. <br/>Results: 10 studies (83,684 participants) were included: 9 observational studies and one secondary analysis of a randomized trial. After adjustment for confounders, patients with baseline HbA levels =8.5% (=69 mmol/mol) had increased mortality (7 studies; HR, 1.14; 95% CI, 1.09-1.19) compared with patients with HbA levels of 6.5%-7.4% (48-57 mmol/mol). Likewise, patients with a mean HbA value =8.5% also had a higher adjusted risk of mortality (6 studies; HR,1.29; 95% CI, 1.23-1.35). There was a small but nonsignificant increase in mortality associated with mean HbA levels =5.4% (=36 mmol/mol; 6 studies; HR, 1.09; 95% CI, 0.89-1.34). Sensitivity analyses in incident (=90 days of hemodialysis) and prevalent patients (&gt;90 days of hemodialysis) showed a similar pattern. In incident patients, mean HbA levels =5.4% also were associated with increased mortality risk (4 studies; HR, 1.29; 95% CI, 1.23-1.35). <br/>Limitations: Observational study data and inability to adjust for diabetes type in all studies. <br/>Conclusions: Despite concerns about the utility of HbA measurement in hemodialysis patients, high levels (=8.5%) are associated with increased mortality risk. Very low HbA levels (=5.4%) also may be associated with increased mortality risk. 

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA. © 2013 Macmillan Publishers Limited All rights reserved.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams &amp; Wilkins.