The anthropometric parameters, systolic blood pressure (SBP) and diastolic blood pressure (DBP), serum malondialdehyde (MDA), nitric oxide (NO) and tumor necrosis factor α (TNFα) were assessed at baseline and after 8 weeks of intervention. After 8 weeks of intervention, combined symbiotic and alpha-tocopherol, symbiotic and alpha-tocopherol alone administration, compared with the placebo, resulted in significant decreases in SBP , serum MDA , serum TNFα  concentrations. A significant decrease in serum AST , ALT  and ALP  was seen. Variations in DBP and serum NO concentration were not significant. Alpha-tocopherol and symbiotic supplementation among patients with NAFLD resulted in decreased SBP, serum MDA, TNFα levels and enzymes liver; however, they did not affect DBP and serum NO concentration.Non-alcoholic fatty liver disease (NAFLD) has been suggested to be well correlated with altered blood pressure. This study was conducted to determine the effects of symbiotic and vitamin E supplementation on blood pressure and inflammatory indices of patients with NAFLD. This randomized, double-blind, placebo-controlled trial was performed among 60 NAFLD patients aged 25 to 64 years old. Participants were randomly divided into four groups to receive a 400 IU alpha-tocopherol and 2 × 10 NAFLD is one of the most common chronic liver diseases in the world today, occurring when fat is deposited (steatosis) in the liver . The prNot only insulin resistance and oxidative stress status are considered as roots of non-alcoholic fatty liver disease, but also intestinal flora is responsible for NAFLD. Small intestinal bacterial overgrowth (SIBO) is proposed to be responsible for the inflammation occurred through the production of cytokine, yet the exact mechanism is still unknown . Some pRecent literature supports the role of a gut-liver interaction in the pathogenesis of portal hypertension as a novel therapeutic focus; the prevailing hypothesis is that bacterial translocation stimulates the release of pro-inflammatory cytokines and the activation of the vasodilator nitric oxide results in a more pronounced deterioration of the baseline hyperdynamic circulatory state .Subjects with NAFLD and elevated liver enzyme levels are at an increased risk of developing blood pressure and metabolic syndrome due to the oxidative stress status; in fact, decreased level of antioxidants in patients with NAFLD has effects on the measures of blood pressure. TNFα is also responsible for the pathogenesis of NAFLD via effecting on mitochondrial radical formation which might finally lead to cell death . Some iFurther, antioxidant agents have been proposed as a potentially effective treatment; vitamin E is a potent antioxidant compound that has been tested in pediatric NAFLD due to the absence of side effects. Conflicting results have been reported in clinical trials, among both children and adults  aged 25 to 64 years old, who were recruited from Hazrat-e-Rasoul Medical Complex  in Tehran, Iran. This study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from all participants. The research was approved by the ethics committee of IUMS and was recorded in the Iranian website for registration of clinical trials (www.irct.ir: IRCT201111082709N22).2.All eligible patients with NAFLD were recruited Figure 1. The diaExclusion criteria comprising any pathologic conditions affecting liver as viral hepatitis, alcohol consumption, hypothyroidism, Wilson disease, acute systemic disease, cystic fibrosis, coeliac disease and alpha-1-antitrypsin deficiency as well as a history of cancer, metabolic disorders, cardiovascular disease, autoimmune diseases and drug or alcohol abuse. Diabetes mellitus, pregnancy, lactation, menstruation at the time of blood sampling, infectious diseases during the study, using non-steroidal anti-inflammatory drugs, antibiotics, and probiotics and food supplements prior to the enrolment of the study were also considered as exclusion criteria.Physical examinations, medical history, diet and physical activity level of each patient were assessed at the beginning of the study. Subjects were asked not to consume any probiotic containing food, yogurt, or its products during an initial 2 week run-in period before the study. They were also asked not to consume other probiotic products during the intervention. At the end of the run-in period, eligible patients were randomly assigned to one of the named groups. Symbiotic Group (n=15) who consumed symbiotic and alpha-tocopherol-like placebo capsule, alpha-tocopherol group (n=15) who received alpha-tocopherol and symbiotic-like placebo, symbiotic + alpha-tocopherol group (n=15) who consumed symbiotic and alpha-tocopherol supplementation and the final 15 patients were given symbiotic-like placebo and an alpha-tocopherol-like placebo supplementation as control group.Lactobacillus casei, Lactobacillus rhamnosus, Stretococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus bulgaricus and prebiotic (fructooligosaccharide). The concentration of each probiotic strain was 2 × 108 CFU/g per capsule. Patients were asked to consume 2 symbiotic capsules per day (each capsule contained 1 g) orally after the main meal. Two identical-appearing placebo capsules  were taken daily by participants assigned to either the placebo or the alpha-tocopherol group. Justification for choosing this dosage was based on the earlier study . Alpha-Randomization was carried out according to Balanced Block Randomization procedure while participants, nutrition specialists, and outcome assessors were all blinded to the interventions into which the individuals were allocated. This study was granted by Iran National Science Foundation.Compliance was monitored by phone calls weekly and verified using capsule counts (number of capsules left in the capsule bottle at the end of the study).2).Weight  was measured at study baseline and after 8 weeks of intervention. Height was measured using a non-stretched tape measure . BMI was calculated using the height and weight measurements . Plasma MDA levels were determined by thiobarbituric acid reactive substance spectrophotometric test . PlasmaRandomization assignment was performed using computer-generated random numbers. Randomization and allocation were blinded to the researchers and participants until the final analyses were completed. The randomized allocation sequence as well as enrolling participants and allocating them to interventions were conducted by a trained staff at the department of nutrition.To examine the normal distribution of variables, we used Kolmogorov-Smirnov test. The analyses were conducted based on intention-to-treat (ITT) approach. One-way analysis of variance (ANOVA) was used to detect differences in general characteristics, dietary intakes, chemical biomarkers and blood pressure at the study initiation between the four groups. A Pearson χ2 test was used for comparison of categorical variables. Repeated measure ANOVA was used to determine the effects of symbiotic plus alpha-tocopherol administration on blood pressure and the used biomarkers, we adjusted the analysis for biochemical parameters, age and BMI, to assess their confounding effects. P values less than 0.05 were considered as statistically significant. All statistical analyses were done using the Statistical Package for Social Science version 17 .The raw data are provided as an attachment . 65 NAFLD patients were recruited in the study at baseline; however, 2 subjects were excluded as one of them was not a local resident (n = 1) and the other had a sudden heart attack during the study (n = 1); furthermore, in the symbiotic group, 2 subjects [death (1 = 3)], s (n = 1)] were excluded from the alpha-tocopherol group, due to personal reason and death. Moreover, 2 persons [death (n = 1) and withdrawal due to personal reasons (n = 1)] were excluded from the placebo group. Finally, 60 participants  completed the trial. However, as the analysis was performed based on intention-to-treat principle, all 60 patients (15 in each group) were included in the final analysis. No side effects were recorded following the administration of symbiotic, alpha-tocopherol, and joint symbiotic and alpha-tocopherol supplements in patients with NAFLD throughout the study.There were no initial differences in the mean measures of age , height, body weight, SBP, DBP and BMI between all the groups. Table 1In addition, mean values of height, weight and BMI were not statistically different between the four groups after the treatment period.According to the 3-day dietary records that were obtained throughout the intervention, no statistically significant differences were seen between the four groups in terms of macro- and micro-nutrient intakes Table 2.Joint supplementation with symbiotic and alpha-tocopherol had no significant effects on DBP and serum NO levels. After 8 weeks of intervention, all three groups of alpha-tocopherol only, individual symbiotic, and combined symbiotic and alpha-tocopherol experienced a significant improvements in SBP compared with the placebo group (P=0.01); however, improvements were much greater in the individual symbiotic (-16.07±3.56 mmHg) and combined symbiotic and alpha-tocopherol (-17.07±2.1 mmHg) groups compared with alpha-tocopherol group alone (-1.73±2.25 mmHg) Table 1.A significant decrease in serum MDA was shown in all intervention groups . Reduction in the levels of TNFα among individuals taking co-supplements of symbiotic and alpha-tocopherol, was greater in comparison to the other groups .Furthermore, a significant decrease in serum AST level was seen among both individual and combined intervention with symbiotic and alpha-tocopherol groups , ALT  and ALP  Table 3.When the analyses were adjusted for baseline values including biochemical parameters, age and BMI, significant changes in our findings were observed except for NO (P = 0.82) and DBP (P = 0.82) levels Table 4.This study demonstrated that administration of symbiotic and alpha-tocopherol supplements for 8 weeks among patients with NAFLD had beneficial effects on SBP, liver enzymes, serum TNFα and MDA levels compared with the control group, while they did not affect DBP and serum NO levels. To the best of our knowledge, this is the first study evaluating the favorable synergistic effects of symbiotic and alpha-tocopherol supplementation on blood pressure and biomarkers of inflammation and oxidative stress among patients with NAFLD. 8 CFU/g symbiotic and 400 IU of alpha-tocopherol for 8 weeks among patients with NAFLD compared with the placebo group led to significant reductions in SBP.Our study revealed that consumption of 2 × 10Lactobacillus acidophilus yoghurt in spontaneously hypertensive rats (SHR) has shown that milk fermented with L. heveticus and casein hydrolysates, which were produced by this organisms, have anti-hypertensive activity. Cell wall polysaccharide-glycoprotein complexes of L. casei have also been reported to have antihypertensive activity in SHR and hypertensive rats  oral adal., 1990; Furushial., 1990), it hasal., 1990), there Data on diastolic blood pressure needs to be interpreted with caution, and future studies with larger sample size would hopefully provide valuable information concerning this topic.Findings from this study have shown that consumption of the symbiotic and alpha-tocopherol for 8 weeks among NAFLD patients significantly reduced serum TNFα compared with the placebo.Bifidobacterium longum and fructo-oligosaccharides among patients with non-alcoholic steatohepatitis. This protective effect is as a result of reduced hepatic exposure to intestinal products, such as lipopolysaccharides (LPS), which induced the release of TNFα from hepatic macrophage. TNFα is a cytokine that promotes cell death in the liver and increases inflammation and thus blood pressure .It is logical to assume that similar effects of intestinal flora and NAFLD also exist, since TNFα is the key mediator of inflammation and so hypertension in this disease. Currently, there are two mechanisms in which intestinal flora may increase the hepatic oxidative stress and thus the blood pressure; the first is the increased endogenous ethanol production, and the second is the direct activation of inflammatory cytokines in luminal epithelial cells, non-parenchymal liver cells (macrophages) or both of them via releasing LPS, which may activate the production of TNFα in Kupffer cells and thus induce hepatic inflammation, oxidative stress and high blood pressure . High amounts of bacterial DNA and their derivatives induce the production of TNFα, IL-2, IL-6, IL-12 , the cual., 2010).Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 could significantly decrease serum MDA concentration in type 2 diabetic patients; moreover, Daga et al.  found aOxidative stress is now believed to be an important factor in the development of hypertension in non-alcoholic fatty liver disease; increased blood pressure is found to be associated with NAFLD in patients with increased alanine transaminase concentrations . AlthouThis study assessed the supplementation of both symbiotic and alpha-tocopherol on serum NO concentration in people with NAFLD; although the plasma NO levels were increased in all intervention groups, the changes were not significant. The beneficial effects of vitamin E supplementation on serum NO and so blood pressure among patients with metabolic syndrome have been previously evaluated . AntioxNO plays an important role in regulating systemic vascular resistance, arterial relaxation, and dispensability; collectively, these actions reduce cardiac hemodynamic load, which thereby reduce myocardial hypertrophy and left ventricular dysfunction and finally protect target organs  and IL-6 as well as the biomarkers of oxidative stress such as catalase and superoxide dismutase in NAFLD patients. Future studies with crossover design, longer duration of intervention and larger sample size are needed to confirm the validity of our findings.Lactobacillus casei, Lactobacillus rhamnosus, Stretococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium longum, and Lactobacillus bulgaricus for 8 weeks among patients with NAFLD had beneficial effects on systolic SBP, MDA and TNFα but did not affect DBP and serum NO levels compared with the control group. Findings of the current study suggest that simultaneous supplementation of vitamin E and symbiotic may confer advantageous therapeutic outcomes for patients with NAFLD.Overall, administration of 400 IU alpha-tocopherol and symbiotic supplements containing Funding: This study was funded by Iran national science foundation (grant number: 90005246).Conflict of interest: The authors declare that they have no conflict of interest.Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent: Informed consent was obtained from all individual participants included in the study.The authors thank the participants in the study for their important contributions.


Nonalcoholic steatohepatitis (NASH) is an active form of nonalcoholic fatty liver disease. Risk factors for NASH include type 2 diabetes mellitus (T2DM) and obesity. Sodium–glucose cotransporter 2 (SGLT2) inhibitors used to treat T2DM prevent glucose reabsorption in the kidney and increase glucose urinary excretion. Dapagliflozin is a potent, selective SGLT2 inhibitor that reduces hyperglycemia in patients with T2DM and has been demonstrated to reduce some complications associated with NASH in rodent models.To assess the efficacy and safety profile of dapagliflozin for the treatment of NASH-associated with T2DM.In this single-arm, nonrandomized, open-label study, 16 patients with percutaneous liver biopsy-confirmed NASH and T2DM were enrolled to be prescribed dapagliflozin 5 mg/d for 24 weeks. Of these, 11 patients were evaluable. Patients with chronic liver disease other than NASH were excluded. Body composition, laboratory variables related to liver tests and metabolism, and glucose homeostasis were assessed at baseline and periodically during the study. Changes from baseline were evaluated with the Wilcoxon signed-rank test.P < 0.01), waist circumference (P < 0.01), and waist-to-hip ratio (P < 0.01). Changes in body composition were driven by reductions in body fat mass (P < 0.01) and percent body fat (P < 0.01), without changes in lean mass or total body water. Liver tests  also significantly improved during the study. Insulin concentrations decreased (P < 0.01 by Week 24) in combination with significant reductions in fasting plasma glucose (P < 0.01) and glycated hemoglobin (P < 0.01) levels and increases in adiponectin (P < 0.01) levels from Week 4 onward.Administration of dapagliflozin for 24 weeks was associated with significant decreases in body mass index (Dapagliflozin was associated with improvements in body composition, most likely a reduction in visceral fat, which occurred together with improvements in liver tests and metabolic variables in patients with NASH-associated with T2DM.UMIN Clinical Trial Registry identifier: UMIN000023574. Nonalcoholic steatohepatitis (NASH) is an active form of non-alcoholic fatty liver disease (NAFLD). NASH is characterized by steatosis, liver fibrosis, ballooned hepatocytes, and lobular inflammation. In some patients, NASH may progress to cirrhosis of the liver and hepatocellular carcinoma.4The preferred therapeutic approach is to encourage lifestyle changes to reduce a patient’s weight by ≥7% through changes in diet and lifestyle habits, including regular exercise.Sodium–glucose co-transporter 2 (SGLT2) inhibitors prevent the reabsorption of glucose in the kidney and increase urinary excretion of glucose,Dapagliflozin is a potent and selective SGLT2 inhibitor that has been shown to reduce hyperglycemia in patients with T2DM.The study was a prospective, open-label, uncontrolled pilot study. No formal sample size calculation was undertaken; however, the number of patients was estimated to be 20.This clinical study was performed after obtaining approval from the Ethics Committee of Shimane University School of Medicine, as well as written informed consent from all participating patients. This study complied with the Declaration of Helsinki and applicable laws and requirements. The trial was registered at the University Hospital Medical Information Network under the registration number UMIN000023574. All costs were covered by patients because the study was carried out as part of standard care in daily clinical practice under Japanese health insurance. This was an investigator-initiated study; the sponsor only provided funding for writing support.Patients were screened for metabolic syndrome, T2DM, NASH, dyslipidemia, and hypertension, with all current medications recorded. Metabolic syndrome was defined as described previously, with minor modifications.Body composition was measured at baseline and at Weeks 2, 4, 8, 12, 16, 20, and 24 by segmental multifrequency bioimpedance analysis with an InBody720 .9/L] × ALT [U/L]).We analyzed the following serum liver tests and metabolic variables: aspartate transaminase (AST), alanine transaminase (ALT), FPG, insulin, HbA1c, HDL-C, LDL-C, and triglycerides. In addition, the following laboratory tests were performed: type IV collagen 7S (T4C7S), ferritin, and adiponectin, as well as the NAFIC score and Fibrosis-4 index, γ-glutamyl transpeptidase, and high sensitivity C-reactive protein. The NAFIC score is useful for predicting steatohepatitis in nonalcoholic fatty liver disease and is calculated from the levels of ferritin, fasting insulin, and T4C7S.P < 0.05 was considered to indicate statistical significance of changes versus baseline.Data are expressed as the median (interquartile range). The Wilcoxon signed-rank test was used to compare the values obtained at baseline with those obtained at Week 24 in patients who completed the study. Sixteen patients (9 men and 7 women) with a median age of 58 years were enrolled during the study period. Five patients discontinued or were excluded from the final analyses for the following reasons: 1 patient reported severe hunger and another patient reported epigastric discomfort, both after 4 weeks of treatment with dapagliflozin; 1 patient was excluded after Week 12 owing to poor compliance (it was determined during the consultation that the drug was being taken approximately once every 3 days); 1 patient received glimepiride before starting this study and continued administration during the study ; and 1 patient was confirmed to have colon cancer after Week 20. Therefore, 11 patients (6 men and 5 women) aged 46 to 78 years  completed the study .Figure 1The characteristics of these 11 patients are summarized in Metabolic syndrome was diagnosed in 8 patients and hypertension was diagnosed in 7 patients . DyslipiOf 7 patients diagnosed with hypertension, 4 had been prescribed antihypertension drugs . Of 7 paP values < 0.01). The waist-to-hip ratio significantly decreased from 1.02  at baseline to 1.00  at Week 24 (P < 0.01). Waist circumference decreased significantly from 101.4 cm  at baseline to 94.2 cm  at Week 24 (P < 0.01) (P < 0.01) (P < 0.01). The percentage of skeletal muscle mass significantly increased from 30.9%  at baseline to 34.1%  at Week 24 (P < 0.01) (As indicated in  < 0.01) . Body fa < 0.01)  as did t < 0.01) . There wP < 0.05). There was no change in the skeletal muscle mass index.Appendicular skeletal muscle (ASM) mass relative to body weight increased significantly from 23.3%  at baseline to 25.5%  at Week 24  (P < 0.05). Serum adiponectin concentrations increased significantly from 5.40 µg/mL  at baseline to 7.0 µg/mL  at Week 24 (P < 0.01) (aseline) . These caseline) . The NAF < 0.01) . High seIn terms of the evaluation of hepatic steatosis using abdominal ultrasound, distinct improvement was found in hepatorenal contrast, deep attenuation, or unclear vessels in 8 out of 11 patients, although no clear change was observed in 3 out of 11 patients.P < 0.01). Likewise, HbA1c decreased significantly from 7.4%  to 6.7%  (P < 0.01). Serum glucagon concentrations did not change significantly from baseline to Week 24, although serum HDL-C concentrations significantly increased from 52 mg/dL  to 55 mg/dL  (P = 0.04), there were no notable changes in LDL-C or triglyceride concentrations.As indicated in Systolic blood pressure decreased from 126 mm Hg  at baseline to 120 mm Hg  at Week 24. Diastolic blood pressure decreased from 84 mm Hg  at baseline to 70 mm Hg  at Week 24.In this study, we showed that the administration of dapagliflozin, a potent and selective SGLT2 inhibitor, was associated with improvements in liver tests and metabolic laboratory variables in patients with NASH and T2DM over the course of 24 weeks. These patients experienced significant reductions in body weight and BMI, which were driven by marked reductions in body fat . Although the percent skeletal muscle mass increased, the actual skeletal muscle mass remained unchanged. Moreover, there were no marked changes in total body water, protein, or soft lean mass during the treatment period.There were significant decreases in median values of waist circumference and waist-to-hip ratio, which are also suggestive of reduced visceral fat mass. This is important because higher waist circumferences and waist-to-hip ratios caused by visceral fat mass have been strongly associated with an increased risk factor for metabolic and cardiovascular disease in adults.The hepatic effects of dapagliflozin were assessed in terms of the changes in liver enzymes, serum ferritin, insulin, and T4C7S. All of these variables showed significant improvements during the 24-week study. We also calculated the NAFIC score and Fibrosis-4 index.Serum ferritin concentration is an independent predictor of hepatic iron overload, which is associated with NASH and advanced hepatic fibrosis.The serum insulin concentrations were significantly lower at Weeks 8, 16, and 24 than at baseline. Insulin resistance is a common factor in NASH and T2DM, and the decrease in insulin secretion coupled with improvements in glycemic control  likely suggests that there was an improvement in insulin resistance, which might have been partly mediated by the reduction in visceral fat mass and increased adiponectin.It is also intriguing to note that there were reductions in systolic blood pressure and diastolic blood pressure during the study. The underlying mechanism is unclear; however, it might involve changes in body composition or metabolic variables. It is notable that the changes in blood pressure were not accompanied by significant changes in total body water. The improvement in blood pressure may be particularly relevant to patients with hypertension.The percentage of skeletal muscle mass increased in this study. A recent Asian study reported that sarcopenia is an independent risk factor for NASH and significant fibrosis.A recent retrospective study compared the efficacies of administration of an SGLT2 inhibitor or a DPP4 inhibitor for 24 weeks in Japanese patients with T2DM and biopsy-confirmed NAFLD.The safety profile of dapagliflozin was evaluated in terms of general laboratory variables, but there were no clinically significant changes in any of these variables in our cohort during the study. We think that the slight increase in hematocrit was not a result of dehydration, but rather reflective of an increase in red blood cells, because no changes in blood urea nitrogen, creatinine, or estimated glomerular filtration rate were observed. Furthermore, there were no changes in total body water during the study. Thus, dehydration was likely not a complication in the present study. These findings suggest that dapagliflozin did not have any untoward effects on clinically relevant laboratory variables in this small group of selected patients.The limitations of this study include the open-label design, short treatment period, and the small number of patients. Furthermore, liver biopsies were not performed. Further large-scale, randomized, controlled studies are needed to validate the efficacy of dapagliflozin in the treatment of NASH and T2DM.In this small, open-label, uncontrolled study, dapagliflozin improved the body composition of patients with NASH associated with T2DM by reducing body fat, most likely visceral fat mass, and was associated with improvements in liver tests and metabolism. Longer-term, larger studies are needed to verify these results and enable the use of dapagliflozin to treat NASH associated with T2DM.Dapagliflozin was developed by AstraZeneca, who provided funding for publication fees and development of the manuscript, but did not play any other role in the study design; collection, analysis, and interpretation of data; writing of the manuscript; or the decision to submit the manuscript for publication. HT and YK have received honoraria from AstraZeneca K.K. Funding to pay for editorial support and the Open Access publication charges for this article were provided by AstraZeneca K.K. and Ono Pharmaceutical Co, Ltd. The authors have indicated that they have no other conflicts of interest regarding the content of this article.


SCNN1A, SCNN1B, and SCNN1G) as additive components by using Plink. Twenty-four-hour sodium excretions were estimated from spot urine samples using the Tanaka formula. The general linear model (GLM) was applied to assess the association between SNPs and urinary sodium excretion or blood pressure. In the SCNN1G gene, six SNPs  were significantly different in 24-h urinary sodium excretion according to gene variants. However, no difference was found in blood pressure among participants with gene variants of ENaC. Our finding indicated that 24-h urinary sodium excretions were different according to variants of the SCNN1G gene in large samples. Further studies to replicate these findings are warranted.This study was performed to investigate whether genetic variation in the epithelial sodium channel (ENaC) is associated with 24-h urinary sodium excretion and blood pressure. A total of 3345 participants of the KoGES_Ansan and Ansung study were eligible for this study. Genomic DNA samples were isolated from peripheral blood and genotyped on the Affymetrix Genome-Wide Human SNP Array 5.0. Thirty-four single nucleotide polymorphisms (SNPs) were extracted for gene regions ( SCNN1A, SCNN1B, SCNN1G, and SCNN1D, respectively).The epithelial sodium channel (ENaC) is expressed in several excretory organs such as the salivary glands, kidney, and lung ,3,4,5,6.ENaC is located in the distal nephron and plays a crucial role in controlling sodium balance. ENaC is mostly expressed in the luminal membrane of connecting tubule cells and plays an important role in renal sodium reabsorption and excretion ,4. ExcesENaC is located within taste cell membranes and is the primary mediator of salt taste. ENaC seems to be responsible for the appetitive behavioral responses caused by salt taste ,2. TasteSCNN1A appears to be essential in fluid transport in the lung. Genetic variants of SCNN1A were associated with the risk of neonatal respiratory distress syndrome [ENaC has been reported to play an important role in lung fluid clearance . SCNN1A syndrome .SCNN1A, SCNN1B, and SCNN1G) is associated with 24-h urinary sodium excretion and blood pressure in a Korean adult population.Previous studies have suggested that genetic variants of ENaC subunits may influence blood pressure or hypertension . The resn = 5020) and Ansung (n = 5018). Ansan is an urban area located in the southwest of Seoul , and Ansung is a rural area located south of Seoul. The baseline examination was conducted between 2001 and 2002, and the participants were followed up biennially. The details of the study design and procedures are described in a previous report [The KoGES_Ansan and Ansung study, an ongoing community-based cohort, was initiated to investigate the trends in diabetes and the related risk factors as part of the Korean Genome Epidemiology Study (KoGES) in 2001. At baseline, 10,038 participants aged 40–69 years were recruited from two communities, Ansan (s report .n = 4425). In addition, participants who were diagnosed by a doctor as having hypertension, diabetes, myocardial infarction, heart failure, coronary artery disease, stroke, renal disease, or cancer were excluded. Finally, a total of 3345 participants were included in the final data analysis. This study was approved by the electronic institutional review board (e-IRB) of the Korea National Institute for Bioethics Policy (KoNIBP).DNA samples obtained from 10,004 participants were genotyped. After performing the sample and SNP quality control, 8840 participants were included in the data analysis. The detailed process has been described elsewhere . Among 82). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured at least twice in a supine position, and then an average value was calculated.Interview-based questionnaires were performed by interviewers trained with a standardized manual to obtain demographic information, medical history, family history, and lifestyles. Weight was measured in kilograms to the nearest 0.1 kg, and height was measured within 0.1 cm without shoes. Body mass index (BMI) was defined as the body weight in kilograms divided by the square of the height in meters  for measuring the quantities of sodium. Estimation of 24-h sodium excretion was performed using the Tanaka formula .p value < 10−6 were discarded. More details on the genotype calling, quality-control, and imputation processes are described in a previous study [Genomic DNA samples were isolated from peripheral blood and genotyped on the Affymetrix Genome-Wide Human SNP Array 5.0 . Genotype calling was conducted with the Bayesian Robust Linear Modeling using the Mahalanobis Distance (BRLMM) Genotyping Algorithm  for 500,568 SNPs, and 352,228 SNPs remained after quality control. SNP imputation was conducted using the IMPUTE program ,16. Baseus study .http://pngu.mgh.harvard.edu/~purcell/plink2/), we extracted SNP genotype data for gene regions  as additive  components. A total of 34 SNPs were identified. Association analyses between SNPs and urinary sodium excretion or blood pressure were conducted using SAS software .By using Plink  was used to assess the association between SNPs and urinary sodium excretion or blood pressure according to the number of minor alleles after being adjusted for age, sex, BMI, and smoking status (non-smoker and current smoker). 2 and 24.5 kg/m2. The proportions of the subjects with a family history of hypertension were 14.3% for male subjects and 15.6% for female subjects. SCNN1G gene, individuals homozygous for the C allele of rs4073291 (A>C) showed lower 24-h urinary sodium excretion than those with either the AA or AC genotype. Individuals with homozygous for the C allele of rs12934362 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. Individuals homozygous for the T allele of rs7404408 (C>T) showed lower 24-h urinary sodium excretion than those with either the CC or CT genotype. Individuals homozygous for the G allele of rs4494543 (A>G) showed lower 24-h urinary sodium excretion than those with either the AA or AG genotype. Individuals homozygous for the C allele of rs5735 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. Individuals homozygous for the C allele of rs6497657 (T>C) showed lower 24-h urinary sodium excretion than those with either the TT or TC genotype. In addition, as using the general linear model with an additive model after adjusting for age, sex, BMI, and smoking status, 24-h urinary sodium excretion showed significant decreasing trends in the SCNN1G gene .The associations of genetic variation in the ENaC gene with estimated 24-h urinary sodium excretion obtained with the Tanaka formula are presented in The associations of genetic variation in the ENaC gene with systolic blood pressure and diastolic blood pressure are shown in SCNN1A, SCNN1B, and SCNN1G) with urinary sodium excretion and blood pressure in Korean adults. Polymorphisms of six SNPs in the genes that code for the ENaC γ subunit (SCNN1G) may modify urinary sodium excretion. However, no difference was found in blood pressure among the gene variants of ENaC.The present study was performed to determine the association of genetic variation in ENaC  were significantly different in 24-h urinary sodium excretion and 11 SNPs  showed significant decreasing trends in an additive model. However, there was no difference in the SCNN1A and SCNN1B genes.Homozygotes for the minor alleles of all SNPs in the ENaC is located in the distal nephron. Aldosterone and vasopressin regulate ENaC activity. A low-sodium diet stimulates the renin-angiotensin-aldosterone system (RAAS), and aldosterone increases sodium reabsorption by activating ENaC in the distal nephron. Vasopressin is secreted in response to increases in plasma osmolality, which means a water deficit. Vasopressin stimulates ENaC activity and increases water conservation by increasing sodium reabsorption . In addiSCNN1G gene may reabsorb more sodium in the kidney or consume less sodium than the persons having carriers of major alleles. Excessive sodium reabsorption by the kidney has been known to increase the risk of hypertension. In a South African study, there were no differences in urinary sodium excretion between the wild type and carriers of the R563Q variant of the ENaC [SCNN1B showed greater Na retention in normotensive young people (both black and white) [SCNN1G tended to excrete less sodium in healthy white adults [SCNN1B and SCNN1G, may be associated with reabsorption of more sodium, but there may be racial differences.Low 24-h urinary sodium excretion can be associated with high sodium reabsorption in the kidney or low sodium intake. Thus, the persons who are homozygous for the minor alleles of six SNPs  of the the ENaC . Howeverd white) . In addie adults . TherefoSCNN1G may consume less sodium than those who are carriers of major alleles. These differences seem to be related to taste perception. Salt taste perception has been the focus of studies on salt taste receptors such as the ENaC and transient receptor potential cation channel subfamily V member 1 (TRPV1) [SCNN1B modified suprathreshold taste sensitivity. In the TRPV1 gene, one SNP (rs8065080 (C>T)) polymorphism modified salt taste perception. Carriers of the T allele perceived salt solutions stronger than those homozygous for the C allele. Even though the types of taste receptors were not the same as in the present study, genetic variation in salt taste receptors seems to modify taste perception, which results in the differences in salt intake. However, according to a twin study, salt taste perception appears to be determined more by environmental influences [Twenty-four-hour urinary sodium excretion can reflect dietary sodium intake; thus, the people who are homozygous for the minor alleles of SNPs in the  (TRPV1) . ENaC is (TRPV1) , two SNPfluences . FurtherSCNN1G gene was associated with 24-h urinary sodium excretion but was not related to blood pressure. The SCNN1A and SCNN1B genes were not associated with blood pressure either. The present study was performed to focus on urinary sodium excretion reflecting salt intake. Hypertensive patients were excluded from the study since they tried to modify their sodium intake. Previous studies reported conflicting results about the associations of ENaC with blood pressure [SCNN1A gene was associated with diastolic blood pressure (DBP) and that rs12447134 in the SCNN1B gene was associated with systolic blood pressure (SBP) under a codominant model. In addition, 5 SNPs in SCNN1G and 4 SNPs in SCNN1B were associated with SBP, DBP, or mean arterial pressure (MAP) under the additive model. In addition, in the other GenSalt study, SCNN1A SNP rs11064153 and SCNN1G SNP rs4401050 were related to longitudinal changes in SBP [In the present study, the pressure ,27,28,29pressure  conducteThere are some limitations to consider when interpreting the results of this study. First, 24-h urinary sodium excretion was estimated using the Tanaka formula with spot urine samples. The 24-h urine collection is hardly realistic in large-sample epidemiologic studies. Second, sodium reabsorption in the kidney is affected by hormones, such as aldosterone and vasopressin, but they were not measured in the present study. Third, sodium intake and salt perception were not measured; thus, it is not known whether the difference in urinary sodium excretion according to ENaC gene variants is due to sodium intake  or sodium reabsorption in the kidney. Fourth, persons with hypertension were excluded in the present study because hypertension patients tended to try a low sodium diet and we were more interested in sodium intake according to gene variants.SCNN1G gene in large samples, and this difference did not appear for blood pressure. Further studies are needed to determine whether the difference in 24-h urinary sodium excretion according to the ENaC gene variant is due to salt taste perception differences or sodium reabsorption differences in the kidney. In addition, since ENaC activity is also related to urinary potassium excretion, urinary Na/K ratio should be compared by genetic variation in the ENaC gene.Despite these limitations, this study confirmed that there was a difference in 24-urinary sodium excretion according to variants of the


To assess the value of serum homocysteine (Hcy) in differential diagnosis of white coat hypertension (WCH).P < 0.001) compared to WCH patients. Serum Hcy levels were positively correlated with 24-h mean systolic blood pressure, r = 0.1378, P < 0.001. The results of the receiving operating characteristic (ROC) curve showed that the AUC value of Hcy was 0.80 , the cut-off value was 13.8 μmol/L, the sensitivity was 68.58% and the specificity 87.21%. In the prospective study, the AUC value of Hcy was 0.73 (95% CI: 0.67–0.78), higher than N - terminal pro - brain natriuretic peptide(NT-pro-BNP)  and cystatin C (Cys-C) . Hcy, NT-proBNP and Cys-C combined, provided a better indication of a differential diagnosis of WCH, than Hcy alone.In this retrospective study, serum Hcy levels were elevated in hypertensive patients  with elevated clinical blood pressure (BP) were recorded. Pearson correlation analysis was used to test the correlation between Hcy and BP. The ROC curve was used to analyze the value of measuring Hcy levels in differential diagnosis of WCH.Serum Hcy was decreased in WCH patients and therefore could be a biomarker for differential diagnosis of WCH. Hypertension is a modifiable risk factor for cardiovascular events, and anti-hypertensive treatment is the cornerstone for the management . HoweverWhite coat hypertension (WCH) also referred to as isolated office or isolated clinic hypertension, is used to define patients with elevated clinic BP at repeated visits, but who show normal BP outside the doctor's office. This difference is detected either through ambulatory BP monitoring (ABPM) or home BP monitoring . WCH occPrevious studies found that WCH patients were at a substantially reduced risk of morbidity compared with sustained mild hypertension patients , 7. The 24-h-ABPM monitors the patient at multiple times a day. It can provide comprehensive data not only about the mean BP, but also its variability, thus enabling the identification of the white coat effect. ABPM also has a stronger prognostic value in relation to cardiovascular outcomes and provides more accurate BP measurements . So, ABPRecently, Courand et al. demonstrated the diagnostic value of N - terminal pro - brain natriuretic peptide (NT-proBNP) to rule out WCH in a prospective study that included 26 WCH patients and 1133 sustained hypertension patients . These sN = 681) according to the 2013 ESH/ESC guidelines for the management of arterial hypertension [Data from 1920 patients who underwent 24-h ABPM were collected. Out of 1920 patients, 767 patients met the inclusion criteria and divided into the WCH group (N=86) and the hypertensive group .As shown in Table Correlation analyses between levels of serum biochemical factors and BP were performed. The serum Hcy level was positively correlated with the 24-h average BP(ABP)  of 0.80 (95% CI: 0.77–0.83), serum Hcy seems to be a good biomarker for differentially diagnosing WCH from sustained hypertension. When using a best cut-off value of 13.8 μmol/L, its sensitivity was 68.58%, and the specificity was 87.21%  and WCH group (N = 46).From January 1, 2015 to March 31, 2017, data from 347 patients with clinical BP, SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg were collected. As shown in Figure The proportion of male patients in the hypertensive group (51.8%) was much higher than that in the WCH group. This may be due to women being more prone to nervousness, and their BP therefore being more affected by entering the clinic. This result was consistent with other research , 19. In P < 0.001. Pure diastolic WCH appears to be exceedingly rare. This result indicated that most WCH presented with high SBP and normal DBP. This could imply that WCH is caused by sympathetic nerves, since the sympathetic nervous system mainly raises SBP [P < 0.05).As shown in Table ises SBP , 23. Wher = 0.1928, P = 0.0029). This may be because the sudden increase in SBP caused by the white coat effect, affected the heart systolic and diastolic functions, inducing NT-proBNP secretion. Hence, the higher the SBP, the more NT-proBNP was present in the blood. Serum Hcy level was positively correlated with a 24-h daytime SBP  and 24-h average SBP . In addition, we found that Cys-C was positively correlated with a 24-h average DBP  individually. When we had a cut-off value of 1.82 for this score, the sensitivity was 61.5% and the specificity was 91.3%. Hcy, NT-proBNP and Cys-C combined gave a better performance than Hcy alone  and the WCH group .The retrospective study sample population was collected from July 1, 2010 to March 31, 2014 according to strict inclusion and exclusion criteria. The inclusion criteria were as follows: first, patients must undergo 24-h ABPM at the department of dynamic blood pressure monitoring. Second, the clinical BP of the patients must be SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg. Third, none of the patients could have received anti-hypertension therapy. Patients more than 70 years old or with a history of coronary heart disease, heart failure, renal insufficiency or diabetes mellitus or those who had not been given the serum Hcy level test were excluded. After these criteria were applied, 767 patients were included. According to 2013 ESH/ESC Guidelines for the management of arterial hypertension , these 7N = 191) and the WCH group (N = 46).The prospective study started January 1, 2015 and ran until March 31, 2017. The inclusion and exclusion criteria were the same as the retrospective study. 237 patients were included and then divided into the hypertensive group , traditional cardiovascular risk factors ) and existence or history of any clinical disease .ABPM was performed using a non-invasive automated device with the cuff fitted on the non-dominant arm, as described in previous studies . The ABPBlood samples were obtained from each subject and then used for biochemical analysis. Centralized analysis of NT-proBNP and creatinine was performed using blood samples obtained at admission. Plasma concentration of NT-proBNP was measured by microparticle enzyme immunoassay. Serum creatinine, Cys C and Hcy was analyzed using commercially available kits from R&D Systems company.2-test for categorical variables. Associations between serum biochemical factors and BP levels were tested using the Pearson correlation. To assess the diagnostic efficiency of the serum biochemical factors, the area under the receiver operating characteristic curve (auROC) was calculated, since this is a measure of discrimination. Furthermore, the standard indexes of validity, such as the Youden index, sensitivity and specificity were calculated according to ROC results.Continuous variables were summarized as mean ± standard deviation (SD), and categorical variables were displayed as counts or percentages. Student-t test was used for continuous variables and χ


Iloprost, a stable prostacyclin analog, is used as a rescue therapy for severe peripheral arterial disease (PAD). It has systemic vasodilatory and anti-aggregant effects, with severe vasodilatation potentially causing organ ischaemia when severe atherosclerosis is the underlying cause. In this study, we retrospectively analysed renal outcomes after iloprost infusion therapy in 86 patients.Eighty-six patients with PAD who received iloprost infusion therapy were retrospectively analysed. Clinical and biochemical parameters were recorded before , during , and after  treatment. Acute kidney injury (AKI) was defined according to KDIGO guidelines as a ≥ 0.3 mg/dl (26.52 μmol/l) increase in creatinine levels from baseline within 48 hours.p = 0.02 and p = 0.008, respectively) of AKI during iloprost treatment. On the third infusion day, patients’ urinary output significantly increased (1813.30 ± 1123.46 vs 1545.17 ± 873.00 cm3) and diastolic blood pressure significantly decreased (70.07 ± 15.50 vs 74.14 ± 9.42 mmHg) from their initial values.Cr2 (1.46 ± 0.1 mg/dl) (129.06 ± 8.84 μmol/l) and Cr3 (1.53 ± 0.12 mg/dl) (135.25 ± 10.61 μmol/l) creatinine levels were significantly higher compared to the initial value (1.15 ± 0.6 mg/dl) (101.66 ± 53.04 μmol/l). AKI was observed in 36 patients (41.86%) on the third day of iloprost infusion. Logistic regression analysis revealed smoking and not using acetylsalicylic acid as primary predictors (While iloprost treatment is effective in patients with PAD who are not suitable for surgery, severe systemic vasodilatation can cause renal ischaemia, resulting in nonoliguric AKI. Smoking, no acetylsalicylic acid use, and lower diastolic blood pressure are the clinical risk factors for AKI during iloprost treatment. Iloprost is a stable epoprostenol  analog that mimics the effects of prostacyclin in the microvascular blood flow, namely, inhibition of platelet aggregation, leukocyte–vessel interaction and vasodilatation.Renoprotective effects of iloprost have been reported in contrast-induced nephropathy and I/R injury.10Because of iloprost’s hypotension-inducing potent systemic vasodilatory effect, several potential safety issues should be considered in these patients.We retrospectively analysed patients with severe PAD who received iloprost infusion therapy at a dose of 1 ng/kg/min between January 2011 and January 2012 at Baskent University Hospital, Ankara, Turkey. Severe PAD was detected with non-invasive tests, including ankle–brachial index < 0.40 and absent blood flow on duplex ultasonography. Among these, 86 patients were selected according to the following exclusion criteria: (1) malignant disease, (2) rheumatological or chronic inflammatory disease of unknown origin, (3) history of systemic vasculitis, (4) unstable heart failure (ejection fraction < 50%) during infusion therapy, (5) chronic liver failure, (6) systemic infective or non-infective inflammatory diseases, (7) evidence of end-stage renal disease (ESRD), (8) younger than 18 years of age. An informed consent was obtained from all subjects of the study.According to the treatment protocol, patients received iloprost infusion at a dose of 1 ng/kg/min for 10–14 days. Iloprost in a 100-ml isotonic solution was infused during a six-hour period via the intravenous route. Nausea, flushing, headache and thrombophlebitis were recorded as drug-related side effects. In accordance with the Kidney Disease Improving Global Outcomes (KDIGO) guidelines, acute kidney injury (AKI) was defined as ≥ 0.3 mg/dl (26.52 μmol/l) increase in creatinine levels from baseline within 48 hours.12The following parameters were collected retrospectively from clinical charts: (1) age; (2) gender; (3) smoking status; (4) presence of diabetes, hypertension, dyslipidaemia [serum highdensity lipoprotein cholesterol (HDL-C) < 40 mg/dl (1.04 mmol/l) and/or low-density lipoprotein cholesterol (LDL-C) > 130 mg/dl (3.37 mmol/l) and/or triglycerides > 300 mg/dl (3.39 mmol/l)], or ischaemic heart disease; (4) urinary output; (5) use of statins, acetylsalicylic acid (ASA), clopidogrel, low-molecularweight heparin (LMWH), angiotensin converting enzyme inhibitors (ACEI), or angiotensin receptor blockers (ARB); (6) daily systolic, diastolic and mean arterial (2 × diastolic pressure + systolic pressure)/3) blood pressure measurement data; (7) haemoglobin, sodium, potassium, calcium, phosphorus, albumin, blood urea nitrogen (BUN), creatinine, and estimated glomerular filtration rate (eGFR) (MDRD equation) values.Clinical and biochemical parameters were collected before (baseline), during (third day of infusion therapy) and after  iloprost treatment, and mean values were determined as arithmetic means. Baseline values were defined as those measured at admission to the in-patient clinic. Office blood pressure levels were recorded.n = 50), and presence of AKI, as the AKI group (n = 36).Statistical analyses were performed using SPSS software . Subjects were grouped according to the absence of AKI, as the normal renal function group , or paired samples t-test. Numerical variables with a skewed distribution were compared using the Mann–Whitney U- and Kruskal–Wallis tests. Spearman and Pearson correlation tests were used for correlation analyses. A binary logistic regression analysis was performed to assess the major determinant of AKI between correlated variables. A Kaplan–Meier survival analysis was used to compare 30-day patient survival between the two groups. A p-value < 0.05 was considered statistically significant.Normality of data was analysed using the Kolmogorov–Smirnov test. All numerical variables with normal distributions were expressed as means ± standard deviations (SD), while variables with skewed distributions were expressed as medians and interquartile ranges (IR). Categorical variables were expressed as percentages and compared using the chi-squared test. Normally distributed numerical variables were analysed by the independent samples The clinical features of the patients are summarised in p = 0.001 for both]. The BUN level recorded on the third day (30.0 ± 20.7 mg/dl) was significantly higher than the baseline level , as was the serum C-reactive protein (CRP) level . On the third day of the infusion, urinary output was significantly increased from the baseline value  while eGFR values were significantly lower compared to baseline levels  (p = 0.011). Patients’ mean arterial pressures were significantly decreased on the third day of therapy .When the entire study group was analysed, serum creatinine levels recorded on the third and 28th day of treatment  (135.25 ±10.61 and 129.06 ± 8.84 μmol/l) were significantly higher than the baseline level [1.15 ± 0.6 mg/dl (101.66 ± 53.04 μmol/l),  = 0.01) . All pat2, p = 0.01) (p = 0.025). A non-significant trend towards a lower blood pressure on the third and 28th days was observed (p > 0.05)  . All pat > 0.05) . Accordi > 0.05) .p = 0.032) and CRP (p = 0.012) levels and significantly lower eGFR values (p = 0.05) at baseline compared to patients without AKI (p = 0.001) and creatinine (p = 0.001) levels and lower eGFR (p = 0.001) and systolic (p = 0.015), diastolic (p = 0.014) and mean arterial (p = 0.039) blood pressure values on the third day of infusion compared to patients without AKI (Patients who developed AKI had significantly higher serum creatinine (hout AKI . Those whout AKI .p = 0.042) and to the value of the third day of patients without AKI (p = 0.037) (p = 0.001), BUN (p = 0.012), CRP (p = 0.001) and urinary output (p = 0.005) levels on the third day of infusion compared to baseline values. Among patients who developed AKI, systolic (p = 0.002), diastolic (p = 0.023) and mean arterial pressures (p = 0.003) as well as eGFR (p = 0.0001) values were significantly lower on the third day of infusion compared to the baseline value  . Patientne value .p = 0.02, p = 0.003 and p = 0.008, respectively) for the development of AKI during iloprost treatment.Drug-related side effects were similar in both patient groups . A binarp = 0.005) and AKI (p = 0.012) are significant determinants of mortality in patients undergoing iloprost infusion therapy. The Kaplan– Meier analysis revealed a significant difference in survival between patients with AKI and those without AKI  We also evaluated factors associated with 30-day mortality and compared survival ratios between the patient groups. The Cox regression analysis revealed that diabetes mellitus  and a significant decline in eGFR. Patients who developed AKI were more likely to have worse renal function at the initiation of therapy than other patients. In the multivariate analysis, diastolic hypotension, smoking and lack of ASA treatment were independently associated with an increased risk of developing AKI. In addition, AKI was associated with a higher mortality rate at the 30-day follow up.In the kidney, prostaglandins uphold the balance between vasodilation and vasoconstriction to maintain homeostasis and physiological kidney function.15et al., iloprost was successfully used to prevent contrast-mediated nephropathy.19In experimental animal studies, iloprost preserved kidney function against anoxia in rabbits,Hypotension is the principal, dose-dependent side effect of iloprost. There is evidence that such hypotension is a risk factor for the development of AKI and it is a commonly encountered problem in elderly patients with AKI,26et al. showed an independent association between the relative decrease in systolic blood pressure and the development of AKI.et al.We observed that patients who received iloprost had a significant decrease in systolic, diastolic and mean arterial pressure compared to baseline, and that relative diastolic hypotension was a significant risk factor for the development of AKI. In their study, Liu In patients with chronic kidney disease (CKD), the risk of developing AKI is significantly increased.Consistent with these findings, smoking and the lack of ASA use were significant independent predictors for the development of AKI in our patients. Smoking is a major preventable risk factor for atherosclerosis. Exposure to cigarette smoke activates a number of mechanisms predisposing to atherosclerosis, including thrombosis, vascular inflammation, abnormal vascular growth and angiogenesis.33ASA, the fundamental therapy given for PAD, reduces the risk of cardiovascular events and arterial occlusion. The use of ASA for primary and secondary prevention of cardiovascular events in most patients with PAD is supported by excellent clinical evidence.The mortality rate in AKI patients with CKD was 3.3 times higher than that of patients without CKD.This study has several limitations, many of which are inherent in its retrospective design, including the possibility of missing risk factors that could contribute to a confounding bias. In addition, renal imaging studies were unavailable for all patients. However, the association between iloprost-induced hypotension and AKI was independent and clear. Our results clearly illustrate that relative hypotension may play a key role in the development of AKI during iloprost infusion therapy in patients with altered renal function.We conducted a retrospective study to evaluate the risk factors for AKI development and mortality in patients with severe PAD treated with iloprost. We found that patients who developed AKI were more likely to have relative decreases in systolic, diastolic and mean arterial pressures and worse baseline renal function than unaffected patients. We suggest that patients with a smoking habit and those not using ASA are at an increased risk for AKI. In this group of patients we advise iloprost dose reduction and close follow up for evidence of AKI, and discontinuation of iloprost in patients with severe hypotension. As patients with AKI have a higher mortality risk, we suggest that iloprost treatment should be given to selected patients. From our findings, we advise that iloprost should be avoided as it is very likely to cause AKI in patients with CKD or low blood pressure. In addition, we recommend iloprost dose reduction or possible discontinuation for patients receiving iloprost who show evidence of AKI or hypotension. Ultimately, prospective, randomised studies will be needed to address the effects of iloprost infusion therapy on renal outcomes in patients with severe PAD.


Young patients with diabetes present with reduced compensatory responses to hypovolemic stress. Less compliant veins could be a contributing factor, since roughly two-thirds of the blood volume resides in the venous system as a blood reservoir, adjusting proper venous inflow to the heart. The aim of this study was to measure venous compliance and lower limb blood pooling during hypovolemic stress, and to correlate them to indices of diabetes severity and glucose control.Fifteen young women with type 1 diabetes (DW) and 18 healthy age-matched women (C) were subjected to lower body negative pressure (LBNP) (11–44 mmHg), creating hypovolemic stress. Lower limb blood pooling was measured with strain gage technique and venous compliance calculated as the relationship between ∆V/∆P.P = 0.003). Calculated venous compliance was also reduced in DW , and P = 0.002). A progressive reduction in both venous compliance (P < 0.007) and blood pooling (P < 0.005) was seen with increasing level of HbA1c, and furthermore, less strongly associated with presence of microvascular disease (signs of retinopathy).DW presented with reduced blood pooling , and Women with type 1 diabetes present with both reduced venous compliance and blood pooling. The reductions were particularly present in patients with long-standing poor glycemic control. Orthostatic hypotension is more common in people with type 1 diabetes . DiabeteA1c (Lower body negative pressure (LBNP) is a well-proven technique to mimic orthostatic and central hypovolemic stress by pooling blood in the lower part of the body . ReducedA1c . Female A1c . FurtherA1c . ReducedA1c . CollectThe aim of this present study was to assess venous compliance and lower limb blood pooling in healthy women and in young women with type 1 diabetes and, furthermore, to correlate venous compliance with known risk factors associated with type 1 diabetes . We hypothesized that venous compliance and blood pooling would be reduced in women with type 1 diabetes, and that the reductions would be associated with markers of diabetes severity.S-technique and then calculated to IFCC values using the formula: HbA1c IFCC (mmol/mol) = 10.45 ×  – 10.62. HbA1c in DW were 66 ± 2 mmol/mol (range 47–82 mmol/mol), reference value <45 mmol/mol, with background data in DW presented in Table 1c from 5 years preceding the study was calculated ; moderate (n = 5) [HbA1c5 range 61–70 mmol/mol (mean 67 ± 2 mmol/mol)]; poor (n = 5) [HbA1c5 range > 70 mmol/mol (mean 77 ± 2 mmol/mol)] . Microvascular disease was present in seven DW , comprising the subgroup of RET+, while the remaining eight DW without signs of retinopathy formed RET−. All women were scheduled in the middle part of the menstrual cycle, with 9 C and 6 DW on oral contraceptives. No impact of menstrual cycle or oral contraceptives have been seen on venous compliance  were included in the study. They were all on a multiple-dose insulin regime or treated with insulin pump without any other chronic cardiovascular medication. HbA1c was analyzed with Mono ogy with , 18] beimpliance , 19. EacThe experiments started 1 h after a light meal randomly in the morning or afternoon. No circadian variations have been seen in previous, similar experiments in our lab . Room teTo measure the amount of blood pooling evoked by LBNP, calf volume changes (ml/100 ml) were measured with mercury-in-silicone strain gage plethysmography applied at the maximal circumference of the right calf. To avoid any confounding external tissue pressure, the lowest part of the calf was 2 cm above the floor of the LBNP chamber. The subjects rested in the supine position for at least 30 min to ensure stable calf volume and arterial inflow prior to the start of experiments. Interindividual supine resting venous pressure has been shown to be fairly constant .Momentarily after LBNP initiation, a rapid increase in calf volume is seen (blood pooling) followed by a slower, but continuous rise caused by net capillary fluid filtration from blood to surrounding tissue. At cessation, there is a rapid decrease in calf volume corresponding with the increase at onset of LBNP . Fully dAfter at least 30 min of complete rest in the supine position, LBNP of 11 mmHg was applied for 4–6 min followed by complete rest for 5–10 min. LBNP of 22 and 44 mmHg was then applied in similar fashion with rest in between. Continuous recordings of calf volume ensured that basal calf volume was restored and stable before each LBNP session. Continuous, online recordings confirmed a clear and stable filtration slope for at least 2 min used to separate blood pooling from net fluid filtration. The above protocol was in the majority of cases repeated, and the mean blood pooling was calculated. The lower LBNP pressure (11 mmHg) was chosen since change in calf volume below LBNP pressures of 10 mmHg may be dependent on other parameters than venous filling, not reflecting venous wall properties . The upp0 is the y-intercept, and β1 and β2 are characteristics of the slope of the volume–pressure curve. This equation showed an excellent mathematical fit to the measured data points ] was then calculated, creating a linear compliance–pressure curve  was measured by a modified version of the technique developed by Olsen and Lanne  and preve Figure .In conjugation with the experiment described above, cardiovascular parameters were monitored, e.g., brachial blood pressure  and forearm blood flow (FBF) [standard venous occlusion strain gage plethysmography ] .t-test. Bonferroni corrections were applied in subgroup analyses. One-way ANOVAs were applied to assess differences in continuous parameters  when studying subgroups as nominals . Two-way repeated measures ANOVAs were applied to assess group differences in blood pooling over transmural pressure of 9–36 mmHg and rate of blood pooling during the first 60 s of LBNP. Regression analyses were applied to assess correlations between continuous parameters of diabetes . No differences were seen in body height  or body weight , with body mass index (BMI) slightly higher in DW, but well within normal range . Please see Table P < 0.0001) and increased pulse pressure (P < 0.001).DW were slightly older than C . Figure P < 0.05) and LBNP of 44 mmHg  as well as overall reduced during the whole pressure range  and a more flat slope with increased transmural pressure in DW (P < 0.0001). The rate of calf volume increase (reflecting rate of blood pooling) during the first minute of LBNP 22 mmHg was similar in DW and C (P = 0.70). Figure P = 0.0007) as well as during calculated compliance between transmural pressure of 15–35 mmHg . The two separate components for venous compliance calculation were: β1, 0.081 ± 0.009 in DW and 0.092 ± 0.007 in C; β2, −0.00088 ± 0.00015 in DW and −0.00084 ± 0.00015 in C.Figure 1c the last 5 years (HbA1c5). Blood pooling was progressively reduced in groups with increasing HbA1c5 (P = 0.005). Women with normal HbA1c5 had significantly greater blood pooling during LBNP of 44 mmHg than women with moderate and severe HbA1c5 , but no statistical differences were seen between the other three groups. Figure 1c5 (P < 0.0001). A calculated venous compliance during transmural pressure of 20 mmHg was likewise progressively reduced in a similar fashion with increasing HbA1c5 (P = 0.007).Figure 1c5 and the rate of calf volume increase (ml/100 ml/sec) 30–60 s after LBNP initiation in DW, with a negative correlation seen , i.e., the higher the HbA1c5 value, the slower the blood pooling rate.Figure 1c5 tended to be increased in RET+ compared to RET− (P = 0.06). Figure P = 0.003), a pattern also recurring at LBNP-induced blood pooling of 44 mmHg (P = 0.008). Figure P < 0.0001). A calculated venous compliance during transmural pressure of 20 mmHg was also correspondingly reduced (P = 0.003). No significantly detectable differences were seen between RET+ and RET− in blood pooling (P = 0.12) or venous compliance (P = 0.16).HbAR = −0.37, P = 0.03) and reduced venous compliance in all women studied (P = 0.04). Although some significant correlations fell out randomly between the parameters and blood pooling/venous compliance, no consistent patterns or correlations were seen.Blood pooling and venous compliance were also compared with parameters stated in Table The main findings in the present study were: blood pooling and venous compliance in the lower limb were reduced in women with type 1 diabetes. Worse glycemic control, but also the presence of microvascular disease and signs of autonomic dysfunction, were associated with further reduction in blood pooling and venous compliance.Patients with diabetes are prone to hemodynamic instability and present with reduced tolerance to hypovolemia, most prominent in the short term during change from supine to erect body position and during anesthesia –4. The pVenous compliance was reduced in DW Figure B, in anaMen with type 1 diabetes present with reduced blood pooling only when microvascular disease was present , 28. Venn = 5), and as such, the results must be interpreted with caution. Nevertheless, significant findings in line with the hypothesis of the study and the pathophysiology of diabetes were found.The reduced venous compliance could in part explain the seen reduction in hypovolemic stress-induced compensatory mobilization of capacitance blood in diabetes , 14. Bot1c5 . However, no statistically significant difference was seen between RET− and RET+ when comparing venous compliance (P = 0.16) and blood pooling (P = 0.12). This is in contrast to a previous study in men with diabetes, presenting reduced blood pooling with presence of microvascular disease (Clinical examination revealed evidence of background retinopathy in seven DW, with four of these categorized as slight or minimal and none with proliferative retinopathy. As such, none of the DW had more than mild microvascular engagement, and no one presented with clinical signs of neuropathy or nephropathy Table . Both re disease . However disease .DW presented with increased heart rate at rest Table , a sign Cardiovascular autonomic neuropathy is common in diabetes patients and increases with duration of disease, although difficult to diagnose in its early stages , 32. No The diabetes care in Linköping is of high international standard, and the participating young women with type 1 diabetes presented with overall good glycemic control and were free of aggravated microvascular disease (Table Calf venous compliance was measured in accordance with previously published work from our laboratory , byVenous compliance was reduced in women with type 1 diabetes. Blood pooling at transmural pressures relevant to upright posture was also reduced in women with type 1 diabetes. Worse glycemic control over time was correlated with further aggravated reduction in venous compliance. These data suggest pathophysiological incorporation of advanced glycemic end products in the vessel wall contributing to the reduction in venous compliance. A less compliant venous system could contribute to the hemodynamic instability associated with diabetes.ML was responsible for the study design, performed the experiments, compiled and interpreted the data, and drafted the manuscript.The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest. No other conflict of interest exist.


The main focus of the Genetic Analysis Workshop 19 (GAW19) is identification of genes related to the occurrence of hypertension in the cohort of patients with type 2 diabetes mellitus (T2DM). The aim of our study was to predict dynamics of the future hypertension incidence, based on gene expression profiles, systolic and diastolic blood pressure changes in time, sex, baseline age, and cigarette smoking status.We analyzed data made available to GAW19 participants, which included gene expression profiles of peripheral blood mononuclear cells (PBMCs) from the diabetic members of 20 Mexican American families.On the basis of mid blood pressure measurements at several time points, the coefficient of regression (slope) was calculated for each individual. We corrected the slope value in patients treated with antihypertensive medications. Feature preprocessing methods were used to remove highly correlated probes and linear dependencies between them. Subsequently, multiple linear regression model was used to associate gene expression with the regression coefficient calculated for each T2DM patient. Tenfold cross-validation was used to validate the model. We used linear mixed effects model and kinship coefficients to account for the family structure. All calculations were performed in R.RTP4, FXYD6, GDF11, IFNAR1, NOX3, and HLA-DQ2, associated with dynamics of future hypertension incidence. Two of them, IFNAR1 and NOX3 were previously implicated in pathogenesis of hypertension.This analysis allowed us to identify 6 well-annotated genes: There is no obvious mechanism that links all detected genes with dynamics of hypertension incidence. Identification of possible connection with hypertension needs further investigation. As a result of civilization factors, the incidence of hypertension is increased . HyperteThe San Antonio Family Heart Study collected phenotype data for 1371 patients and expression data were made available to Genetic Analysis Workshop 19 (GAW19) participants for 647 patients with T2DM /2) as a dependent variable and time of subsequent measurements as an independent variable.The group was divided as described above and we determined the median MBP value for each group.We adjusted slope of every patient from group 3  or using available genotype data (King software) and used kinship matrix in a linear mixed effects model.From the initial study group we selected 340 patients without hypertension phenotype. Median values of the MBP slopes were 0.41; 1.77; 0.65 for first, second, and third group, respectively. Size of the consecutive groups was 220, 38, and 82 cases for the first, second, and third group, respectively.The first step was to calculate regression coefficient (slope), which represents dynamics of progression to hypertension. We used a linear regression model with mid baseline blood pressure data (MBP defined as [SBP + DBP]/2) as a dependent variable and measurement time data as an independent variable. Then we divided study group into 3 different phenotypes. First group consisted of patients who did not develop hypertension (NoHTN) and who do not take any blood pressure medications (NoBPM); the second group consisted of patients who developed hypertension (HTN) but are not treated for HTN (NoBPM); and the third group consisted of patients with developed HTN who take blood pressure medication (BPM). We determined median for each group . This means that T2DM patients who later developed HTN, already had a tendency to have higher blood pressure at baseline, although still within the normal limits. However, based on these differences we could not provide a diagnostic test that supports clinical decision making. We can see the trend, but to create a clinically useful predictor we need a stronger discriminator, such as a set of genes.We assume that patients from a third group would have a higher blood pressure at later measurements and, in consequence, a higher regression coefficient (slope), provided they do not take any medication. So, we adjusted slope of every patient from group 3 by adding the difference between median of the second and third groups’ slopes.Use of more than 20,000 gene expression probes for modeling the response in 340 subjects in the form of a full model or univariate screen of probes does not provide a stable model. Therefore, we used feature preprocessing capabilities implemented in caret package of R to decrease the dimensionality of the data. First, we removed probes with highly correlated expressions, using as the cutoff point for probe removal correlations above 0.75. Subsequently, we removed linear dependencies between the probes. This resulted in reduction of the number of the probes used for further modeling from 20,634 – 340.p value of association of less than 0.1. We also forced into the model environmental covariates: age at the time of first examination, sex, and smoking status. The first predictor variable is age at the time of the first blood pressure measurements, which is identical to the time of blood drawing for gene expression analysis. As expected, the age was substantially higher in the second and third groups, in agreement with the tendency of pressure increase with age . The next predictors are cigarette smoking and sex, included in the model as recorded during the first visit.To construct the final model of dynamic progression to HTN, starting with preprocessed 340 probes, we performed a univariate screen of all probes with a linear regression model (lm function of R) against the slope. We included in the final model all probes with a p value of less than 0.1. Together with 3 environmental variables, the final model includes 23 variables. The multiple R2 for this model is 0.2106, adjusted R2 is 0.1532 with a p value of 9.11 × 10−8. Seven probes were significantly associated with the slope, these include probes for the following genes: RTP4, FXYD6, GDF11, IFNAR1, NOX3, and HLA-DQA2, as well as 1 probe with lack of annotation  or using genotype data (KING: kinship based inference for GWAS [genome-wide association studies]). Genotype data were available for 200 individuals from our data set. Kinship coefficients calculated with these 2 approaches were nearly identical. No misparenting events, sample mixup, or hidden family structure were detected (data not shown). To avoid decrease in our sample size, we used kinship coefficients calculated from pedigree structure in all further analyses.coxme package of R with a function lmekin. No random effects of family structure were associated with the slope in this model  and 1 probe that does not correspond to any gene, which fulfill that criterion and remain significantly associated with our outcome after cross-validation.We performed our analysis so as to identify genes whose expression can predict the dynamics of future HTN incidence in T2DM patients. We identified 6 well-annotated genes  are an adequate source of information in this context. Maybe a liver, adipose tissue, muscle or pancreatic biopsy, being closer to the pathogenesis of diabetes, could be a better source of information than PBMCs? Possibly a renal tissue would be more informative for HTN? Unfortunately, these tissue fragments are very hard to obtain. On the other hand, mononuclear cells are involved in the development of inflammation, which is one of the causal pathways of HTN. Nevertheless, this data set provides an interesting opportunity to check the modeling strategy in prediction of the events distant in time.We decided to construct the outcome variable that captured scattered-in-time information about blood pressure measurements. The slope of blood pressure changes allows us to deal efficiently with the situation in which time of consecutive patient visits and blood pressure changes was quite irregular. Our concern was that the slope of blood pressure changes was rather flat among patients identified as hypertensive based on antihypertensive treatment they used - quite efficiently for the majority of them, as the data show. We decided that the best available correction would be to adjust their slope by a factor seen in patients identified as hypertensive based on their blood pressure.Another difficulty we encountered was a large number of features  to be analyzed with a rather modest number of subjects (only 340). To avoid instability of the model created in such a situation, we implemented feature reduction techniques, which reduced the number of probes to several hundred. We removed highly correlated probes. The univariate screen provided us with a list of 20 probes, 6 of which were significantly associated with the slope and remained significant after 10-fold cross-validation. Linear mixed model, with family structure as a random effect, showed that no family membership is a significant factor for this dynamics.NOX3 is one of the isoforms of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzyme-producing reactive oxygen species associated with renal function and a risk of diabetic nephropathy and HTN in Africans [(IFNAR1) was implicated in pulmonary HTN [We could not explain the potential association of all found genes with dynamics of HTN incidence. Interestingly, however, 2 of these genes were previously associated with HTN in the available literature. Africans . Recentlnary HTN . It is nThese results encourage us to claim that gene expression data generated from PBMCs may be with careful modeling strategy associated with events distant in time.We think that our model is useful for analyzing combined transcriptomic and genomic data in complex diseases such as asthma, allergies, cancer, and obesity. Our strategy demonstrates using a modest-size phenotypic dataset to find associations with gene expression. We used a slope (regression coefficient) as an unbiased proxy for the blood pressure changes during development of the complex trait like hypertension. Apart from environmental factors, which are still the biggest causative agent of hypertension, we found genes that might be useful in predicting the progression of this disease.


Pre-eclampsia (PE) is related to an impaired endothelial function. Endothelial dysfunction accounts for altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity. Impaired endothelial function originates from production of inflammatory and cytotoxic factors by the ischemic placenta and results in systemic oxidative stress (OS) and an altered bioavailability of nitric oxide (•NO). The free radical •NO, is an endogenous endothelium-derived relaxing factor influencing endothelial function. In placental circulation, endothelial release of •NO dilates the fetal placental vascular bed, ensuring feto-maternal exchange. The Endopreg study was designed to evaluate in vivo endothelial function and to quantify in vitro OS in normal and pre-eclamptic pregnancies.2•) and placental concentration of •NO will be determined using EPR (electron paramagnetic resonance). Endothelial function and arterial stiffness will be evaluated using respectively Peripheral Arterial Tonometry (PAT), Flow-Mediated Dilatation (FMD) and applanation tonometry. Placental expression of eNOS  will be determined using immune-histochemical staining. Target recruitment will be 110 patients for the longitudinal study and 90 patients in the case-control study.The study is divided into two arms, a prospective longitudinal study and a matched case control study. In the longitudinal study, pregnant patients ≥18 years old with a singleton pregnancy will be followed throughout pregnancy and until 6 months post-partum. In the case control study, cases with PE will be compared to matched normotensive pregnant women. Maternal blood concentration of superoxide (O2•− and eNOS measurements provide further inside in the pathophysiology of PE.The results of Endopreg will provide longitudinal information on in vivo endothelial function and in vitro OS during normal pregnancy and PE. Adoption of these vascular tests in clinical practice potentially predicts patients at risk to develop cardiovascular events later in life after PE pregnancies. •NO, ONCT02603913. Registered October 2015.This trial has been registered on clinicaltrials.gov. ClinicalTrials.gov Identifier:  Pre-eclampsia (PE) is a potentially life-threatening pregnancy related vasculopathy characterized by hypertension and proteinuria. PE results in high morbi-mortality for both mother and her unborn child. Between five and 10 % of pregnancies are complicated by hypertensive disorders and worldwide the incidence of PE has increased by 25% in the past two decades  or > 300 mg/day or at least 1 g/L [‘2 +’] on dipstick testing)renal insufficiency (creatinine > 90 μmol/L)liver involvement neurological complications hematological complications Other maternal organ dysfunctionUteroplacental dysfunction: fetal growth restriction◦ Hypertension greater than or equal to 140 mmHg systolic or 90 mmHg diastolic, must be confirmed at least 4 h apart. Hypertension greater than or equal to 160 mmHg systolic or 110 mmHg diastolic can be confirmed after a short interval (minutes) to facilitate antihypertensive treatment .Exclusion criteria are  diabetes, multiple pregnancies, fetal abnormalities, hypercholesterolemia, renal disease, auto-immune disorders and connective tissue disease. Intake of low-dose aspirin or vitamin C supplements (> 500 mg/day) is an exclusion criterion. Since in Belgium folic acid is an advised pre- and periconceptional therapy, this will not act as an exclusion criterion.The use of other medication/supplements will be listed in the patients record to find eventual confounders afterwards.The gold standard for non-invasive assessment of endothelial function is FMD, measuring NO-dependent vasodilation of the brachial artery in response to reactive hyperaemia . An ultrFMD and PAT measurements will be performed simultaneously and patients will be asked 24 h prior to examination not to eat high-fat substances nor drink caffeine or alcohol and to refrain from smoking at least 6 h prior to examination. Fingernails had to be short and no nail polish applied. Patients were studied in a quiet, temperature-controlled room (21-24 °C) and stressful situations for the patient were avoided  Patients will not by measured in active labour.PAT is recorded using the Endo-PAT2000®  and the disposable fingertip probes  in accordance with the manufacturer’s recommendations. PAT is a less operator-dependent and more reproducible technique. The system uses pneumatic finger probes which assess digital volume changes accompanying pulse waves. Reactive hyperaemia is induced as described for FMD and measurements are performed simultaneously to FMD. The ratio of the average amplitude of the PAT signal over a 1 min period starting 1 min after cuff deflation (maximum pulse amplitude) divided by the average amplitude of the PAT signal over a 3.5 min period before cuff inflation (baseline pulse amplitude) is calculated. The control arm is used to correct for confounding factors . The result is expressed as the reactive hyperaemia index (RHI) . To calculate PWV, two pressure waveforms must be measured at a known distance apart and the distance between measurement sites is divided by the propagation time. Aortic PWV is measured by carotid-femoral PWV (cfPWV) as it is the ‘gold standard’ measurement of the stiffness of the aorta. Measurements of cfPWV will be performed using a pressure tonometer to transcutaneously record the pressure pulse waveform in the underlying artery. The tonometer contains a micromanometer that provides a very accurate recording of the pressure within the artery. The carotid and femoral PWV will be assessed by gently compressing respectively the carotid artery and the femoral artery with the tip of the tonometer at the site of maximal pulsation. The Sphygmocor device will automatically calculate the cfPWV.Pulse wave analysis will calculate AIx by placing the tonometer at the radial artery (site of maximum pulsation). A generalized transfer function will derive the aortic pressure waveform from the radial artery waveform. From the aortic pressure waveform, the augmentation pressure (AP) and augmentation index (AIx) can be calculated. The AP (ΔP) is defined as the height of the late systolic peak above the inflection point on the waveform. The AIx is defined as AP expressed as a percentage of the aortic PP. As AIx is affected by heartrate, it will be standardized to a heart rate of 75 bmp (AIx-75).Uterine artery Doppler examinations will be performed using trans-abdominal colour directed pulsed wave Doppler . Pulsatility index of both uterine arteries will be obtained on either side of the cervix before 14 weeks’ gestation and at the apparent crossover with the external iliac arteries after 14 weeks , 29.Three similar consecutive waveforms must be obtained before calculating the PI. The mean PI of the two vessels was calculated. Observation on the presence or absence of a bilateral early protodiastolic notch will be performed. A notch is defined as a persistent decrease in blood flow velocity below the diastolic peak velocity, in early diastole.At the same moment basic fetal biometry parameters will be measured: bi-parietal diameter, head circumference, abdominal circumference, femur length and expected fetal weight using Hadlocks formula .Performing a complete blood count (CBC), the neutrophil/lymphocyte ratio (NLR), mean platelet volume (MPV) and platelet count will be obtained using a ADVIA 120 Hematology System  –33.SBP, DBP and MAP after 10 min rest in a sitting position, will be measured using a Mindray VS 900 monitor .EPR (electron paramagnetic resonance) is derived from magnetic resonance spectroscopy and uses microwave radiation to detect molecules with an unpaired electron number, like radicals. When an magnetic field is created by the EPR spectrometer, all radicals will align. The EPR spectrometer sends out a radio frequent microwave, causing the electrons to jump from a low to a high energy state. This energy absorption can be measured and is directly correlated to the amount of free radicals in the sample. A ‘spin trap’ will be added to scavenge the very reactive radicals and to prolong their half live. EPR spectra will be obtained using the Bruker EMX 1273 spectrometer equipped with an ER 4119HS high-sensitivity resonator (=cavity) and 12-kW power supply operating at X band frequencies. The Bruker WINEPR -post processing system software  will be used to analyse the spectra .3 placental tissue will be taken avoiding placental infarcts. The sample will be rinsed with saline (NaCl) and immediately added to the spin trap 750 μl FeSO4 + 750 μl DETC (iron (II) diethyldithiocarbamate solution). After 1 h of incubation at 37 °C, the sample will be snap frozen and stored in − 80 °C until analysis with EPR [Placental tissue will be obtained within 2 min after  delivery. At a standardized central location, a viable sample of 1cmwith EPR .Since labour causes ischemia-reperfusion injury at the site of the placenta, which will influence placental NO concentrations, samples will be subdivided in labour  versus no labour (elective caesarean section) .Maternal blood will be obtained at 12 weeks and 24-28 weeks in a heparin tube (BD vacutainer) and transported on ice. After 15 min, 30 μl of spin trap CMH  will be added to 30 μl blood. The whole will be incubated on ice and transferred into a capillary after 5 min. The sample will then be snap frozen and stored in − 80 °C until analysis .Placental tissue will be obtained shortly after delivery and stained for eNOS and iNOS as previously described by Du et al. As sample size determining factor, we took the RHI since this is an important variable in our study, there is normal distribution and the population standard deviation is known.For the physiologic study of the RHI in pregnancy we calculate that for a 95% confidence interval, a population standard deviation of 0.5 (as described in other populations for RHI) and a tolerable standard error of the mean (SEM) value of 0.1, 97 women have to be followed . Taking (http://www.stat.ubc.ca/~rollin/stats/ssize/n2.html); which we consider the sample size for our cross-sectional study comparing pre-eclamptic patients with normotensive controls. The sample size of this study will be 90 patients, 45 in each group.In a pilot study by Yinon  the RHI 2•− will be analysed.For the physiologic study of the RHI in pregnancy we will calculate the reference values and 95% confidence interval. Longitudinal data will be plotted and a linear mixed-effects model with random intercept will be fitted. Percentiles for RHI, FMD and PWV/PWA will be calculated based on this model. Correlation coefficients between baseline RHI, FMD PWV/PWA, UA Doppler PI, fetal biometry, NLR, MPV, MAP, birth-weight percentile, eNOS, •NO and O2•− concentration and other continuous variables in PE versus healthy pregnancies will be tested for normality using the Shapiro Wilk Test. If there is normality, they will be expressed as mean, standard deviations and 95% confidence intervals and compared using two sided T test. If not, they will be expressed as median and interquartile ranges and compared using Mann Whitney U Test.RHI, FMD, PWV/PWA, UA Doppler PI, fetal biometry, NLR, MPV, MAP, birth-weight, eNOS, •NO and OPE is responsible for 11.5% of maternal deaths in Flanders. Ten percent of early neonatal deaths are caused by maternal hypertensive disorders . This prAs stated before, the gold standard for non-invasive assessment of endothelial function is FMD . PreviouBoth in normal pregnancy and PE are inflammatory effects present, which can be objectified by higher neutrophil to lymphocyte ratio (NLR) and higher mean platelet volume (MPV) . Increas2•−, hydrogen peroxide (H2O2), hydroxyl radical (•OH), nitric oxide (•NO), and peroxynitrite (ONOO−) are involved in the pathophysiology of placental pregnancy disorders. OS at the site of the placenta causes placental damage and this ischemic placenta releases cytotoxic, anti-angiogenic and inflammatory markers in the circulation resulting in systemic endothelial dysfunction and peripheral organ damage [2•−, the most abundant free radical, encloses an important role in the beneficial effects of OS. Studies measuring O2•− concentration longitudinally in normal pregnancy and during PE are lacking, creating a gap in the knowledge of OS in pregnancy and PE. Evidence for OS at the placenta and in the maternal circulation in PE has led to the suggestion that anti-oxidant therapy can improve or even prevent PE. Vitamins E and C, L-arginine (precursor of •NO) and •NO donors have been proposed to limit both endothelial injury and for the prevention of PE with conflicting results [Previous research has demonstrated that markers of OS, like On damage . O2•−, t results –55. SubsENDOPREG is the first clinical study comparing in vivo measurements of endothelial function with in vitro markers of endothelial dysfunction in a longitudinal and case-control setting. To our knowledge, serial changes in maternal endothelial function have not been evaluated previously in pregnancy using two different methods, i.e. PAT/FMD and EPR, simultaneously. The main strength of our study is the longitudinal design. Little studies determined maternal •NO or reactive oxygen species concentration at the moment of diagnosis  and only