Increased global placental DNA methylation levels are associated with gestational diabetes.

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. METHODS: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. RESULTS: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1-4 (2.9-5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. CONCLUSIONS: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.

Dual endothelin-converting enzyme/neutral endopeptidase blockade in rats with D-galactosamine-induced liver failure.

Secondary activation of the endothelin system is thought to be involved in toxic liver injury. This study tested the hypothesis that dual endothelin-converting enzyme / neutral endopeptidase blockade might be able to attenuate acute toxic liver injury. - Male Sprague-Dawley rats were implanted with subcutaneous minipumps to deliver the novel compound SLV338 (10 mg/kg*d) or vehicle. Four days later they received two intraperitoneal injections of D-galactosamine (1.3 g/kg each) or vehicle at an interval of 12 hours. The animals were sacrificed 48 hours after the first injection. - Injection of D-galactosamine resulted in very severe liver injury, reflected by strongly elevated plasma liver enzymes, hepatic necrosis and inflammation, and a mortality rate of 42.9 %. SLV338 treatment did not show any significant effect on the extent of acute liver injury as judged from plasma parameters, hepatic histology and mortality. Plasma measurements of SLV338 confirmed adequate drug delivery. Plasma concentrations of big endothelin-1 and endothelin-1 were significantly elevated in animals with liver injury (5-fold and 62-fold, respectively). Plasma endothelin-1 was significantly correlated with several markers of liver injury. SLV338 completely prevented the rise of plasma big endothelin-1 (p<0.05) and markedly attenuated the rise of endothelin-1 (p = 0.055). - In conclusion, dual endothelin-converting enzyme / neutral endopeptidase blockade by SLV338 did not significantly attenuate D-galactosamine-induced acute liver injury, although it largely prevented the activation of the endothelin system. An evaluation of SLV338 in a less severe model of liver injury would be of interest, since very severe intoxication might not be relevantly amenable to pharmacological interventions.

Pathophysiology of the endothelin system - lessons from genetically manipulated animal models.

Shortly after discovery of ET-1 in 1988, the entire endothelin system was characterized. The endothelin system consists of the three peptides ET-1, ET-2 and ET-3, their G-protein-coupled receptors endothelin receptor A and B (ETRA and ETRB) and the two endothelin-converting enzymes (ECE-1 and ECE-2). Genetically modified animal models are an important tool in biomedical research. Here we describe the key findings obtained from genetically modified animal models either over-expressing compounds of the ET system or lacking these compounds (knockout mice). Results from the different transgenic and knockout models disclose that the ET system plays a major role in embryonic development. Two ET system-dependent neural crest-driven developmental pathways become obvious: one of them being an ET-1/ETAR axis, responsible for cardio-renal function and development as well as cranial development; the other seems to be an ET-3/ETBR mediated signalling pathway. Mutations within this axis are associated with disruptions in epidermal melanocytes and enteric neurons. These findings led to the discovery of similar findings in humans with Hirschsprung disease. In adult life the ET system is most important in the cardiovascular system and plays a role in fibrotic remodelling of the heart, lung and kidney as well as in the regulation of water and salt excretion.

Renal phenotype of ET-1 transgenic mice is modulated by androgens.

INTRODUCTION: Activation of the endothelin (ET) system promotes inflammation and fibrosis in various tissues including the kidney. Male ET-1 transgenic mice are characterized by chronic kidney inflammation and renal scarring. We hypothesized that this renal phenotype might be modulated by androgens. Thus the aim of our study was to elucidate the impact of gonadectomy in ET-1 transgenic mice on kidney function and morphology. - METHODS: Male ET-1 transgenic mice at the age of 10 weeks were randomly allocated to the following groups: normal ET transgenic mice (ET; n = 17) and ET transgenic mice that underwent castration (ET + cas; n = 12). Study duration was 9 months. Creatinine clearance and protein excretion was monitored. At study end animals were sacrificed and kidneys were harvested for histology/immunhistochemistry. RESULTS: Castration significantly ameliorated glomerulosclerosis in ET-1 transgenic mice (ET glomerulosclerosis-score: 3.0 +/- 0.17 vs ET+cas: 2.4 +/- 0.17; p < 0.05) as well as renal perivascular fibrosis (ET fibrosis-score: 3.0 +/- 0.14 vs ET + cas: 2.2 +/- 0.14; p < 0.05). However, interstitial fibrosis and media/lumen-ratio of renal arteries remained unaffected by castration. Regarding inflammation, castration significantly reduced the number of CD4-positive cells in renal tissue of ET-1 transgenic mice (ET CD4-positive cells/10000 cells: 355 +/- 72 vs ET + cas: 147 +/- 28; p < 0.05). Renal tissue contents of CD8 positive cells as well as of macrophages were not affected by castration. Regarding kidney function castration significantly reduced proteinuria in ET-1 transgenic mice whereas creatinine clearance did not differ between study groups. CONCLUSION: Our study demonstrates that the renal histopathological phenotype in male ET-1 transgenic mice with regard to glomerulosclerosis, proteinuria, perivascular fibrosis and immune cell immigration is ameliorated by castration. We thus conclude that the effects of ET-1 overexpression on renal tissue injury are modulated by androgens.

Tissue specific activation of the endothelin system in severe acute liver failure.

The endothelin system has been implicated in the pathogenesis of acute liver failure. However, it has not yet been assessed in a tissue specific manner. - Acute liver failure was induced in rats by two intraperitoneal injections of galactosamine (1.3 g/kg, interval of 12 hours, n = 20). The animals were sacrificed after 48 hours. - Plasma measurements demonstrated that animals receiving galactosamine had a laboratory constellation of severe liver injury and they histologically presented with hepatic necrosis and inflammation. Plasma concentrations of endothelin-1 were elevated 60-fold in the animals receiving galactosamine (p = 0.005). In contrast endothelin-1 tissue contents were decreased in the kidneys and unchanged in the liver. Western blot analysis showed that animals receiving galactosamine had a significantly lower endothelin B receptor concentration in liver and kidney tissue, whereas no differences were detected for endothelin A receptors. - This study demonstrates that the local endothelin system of liver and kidneys is not responsible for the increase of plasma endothelin-1 concentrations in acute liver failure. Since it is well established that the endothelin B receptor acts as a clearance receptor, its decreased density might contribute to the strongly elevated plasma endothelin-1 concentrations seen in this model of acute liver injury.

Impact of maternal endothelial nitric oxide synthase gene polymorphisms on blood pressure, protein excretion and fetal outcome in pregnancy.

A genetic association study was conducted to assess whether genetically determined alterations of the nitric oxide system are associated with clinical markers of pre-eclampsia. A large number of Caucasian women were consecutively included after delivery and genotyped for the endothelial nitric oxide synthase gene (NOS3) polymorphisms G894T, T789C (n=1502) and intron 4a/b (n=2186). There are no significant differences in mean blood pressure (BP), protein excretion or new-onset peripheral oedema between any of the genotypes over the course of pregnancy. Neither particular haplotypes nor the combined presence of any two alleles is associated with those markers of pre-eclampsia. The maternal polymorphisms do not seem to influence fetal growth, birth weight or the incidence of congenital malformations. We demonstrate in a large Caucasian population that maternal polymorphisms of the NOS3 gene are not related to clinical markers of pre-eclampsia. The functional relevance of the NOS3 variants alone does not seem to be strong enough to affect BP regulation during pregnancy.

High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.

Maternal low-protein diet during pregnancy is a risk factor for cardiovascular disease of the offspring in later life. The impact of high-protein diet during pregnancy on the cardiovascular phenotype of the offspring, however, is still unknown. We examined the influence of a high-protein diet during pregnancy and lactation on the renal, hemodynamic, and metabolic phenotype of the F1 generation. Female Wistar rats were either fed a normal protein diet (20% protein: NP) or an isocaloric high-protein diet (40% protein: HP) throughout pregnancy and lactation. At weaning, the offspring were fed with standard diet, and they were allocated according to sex and maternal diet to four groups: normal-protein male (NPm, n = 25), normal-protein female (NPf, n = 19), high-protein male (HPm, n = 24), high-protein female (HPf, n = 29). During the experiment (22 wk), the animals were characterized by repeated measurement of body weight, food intake, blood pressure, glucose tolerance, energy expenditure, and kidney function. At the end of the study period histomorphological analyses of the kidneys and weight measurement of reproductive fat pads were conducted. There were no differences in birth weight between the study groups. No influence of maternal diet on energy expenditure, glucose tolerance, and plasma lipid levels was detected. Blood pressure and glomerulosclerosis were elevated in male offspring only, whereas female offspring were characterized by an increased food efficiency, higher body weight, and increased fat pads. Our study demonstrates that a high-protein diet during pregnancy and lactation in rats programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.

Rapid immunochromatographic strip test for the detection of albuminuria and brief literature review on albuminuria screening.

Albuminuria is a powerful predictor of cardiovascular events in diabetic and hypertensive patients as well as in the general population. In the present study we evaluated the diagnostic performance of the new PreventID Albumin test, a semi-quantitative immunochromatographic dipstick device for the rapid detection of low concentrations of urinary albumin (microalbuminuria). 88 randomly selected fresh urine samples from the central clinical laboratory of the Charité, Berlin/Germany were analysed. The diagnostic accuracy of the PreventID Albumin test for the detection of urinary albumin excretion >18 mg/l was assessed by comparing the results with urinary albumin excretion determined by immunoturbidimetry as golden standard. In comparison with immunoturbidimetry the PreventID Albumin test had a sensitivity of 96.2% and specificity of 97.1%. False negative results were found in 2.3% and false positive results were obtained in 1.1% of specimens. These findings suggest that the PreventID Albumin test may be a useful and valid method for the screening of albuminuria. However, it should not be regarded as a diagnostic test. Positive results should be followed by quantification of urinary albumin or albumin/creatinine ratio by a laboratory based method.