Background and Objectives: obesity and blood pressure disorders are one of the main risk factors for antenatal, intra, postpartum, and neonatal complications. In preeclampsia (PE), the placental hypoxia leads to vascular endothelium dysfunction, cell necrosis, and apoptosis. This condition is associated with the release of free fetal DNA (cffDNA) circulating in plasma. The disturbance of the efficiency of vasodilatation and blood pressure regulation in PE can be confirmed by analyzing the apelin, salusin, and prosalusin. This study aimed to assess the influence of obesity on cffDNA, and the effectiveness of maintaining normal blood pressure in patients with preeclampsia and gestational hypertension. Material and Methods: the research material was blood serum and oral mucosa swabs, obtained from 168 patients. Pregnant women were divided into the following: a control group (C)—67 women; a gestational hypertension group (GH)—35 patients; a preeclampsia with obesity group (PE + O) (pre-gravid BMI > 30)—23 patients. The rest were lean preeclamptic women (PE)—66 patients—(pre-gravid BMI < 25 in 43 women). Results: the cffDNA was observed in 1.50% of women in the C group, in 2.45% in the GH group, but in 18.18% of lean patients with preeclampsia. The cffDNA was detected in 58% of obese pregnant women with PE. The greater the placental hypoxia was in preeclampsia, the less efficient the hypotensive mechanisms, according to an analysis of the studied adipokines. The prosalusin concentration was significantly lower in the PE group with cffDNA than in the PE group without it (p = 0.008). Apelin was higher in the PE group with cffDNA (p = 0.006) compared to other groups. The same results were also observed in the subgroup with obesity. Conclusion: in preeclamptic women, obesity seems to act as an additive factor of placental damage by means of the dysregulation of hypotensive mechanisms. Obesity in pregnant women is associated with a significant risk of gestational diabetes, blood pressure disturbance, fetal growth restriction or over nutrition, birth complications and an increased number of caesarean sections. Therefore, in recent decades, it has been a particular concern in global perinatal health care . A high The influence of obesity on the course of pregnancy is mediated by the action of adipocytes. The cells secrete hormones, which regulate metabolism, and may increase the production of pro-inflammatory agents, regulate reproductive functions, vasoconstriction, and dilatation. Thus, fat tissue has a major role in processes involved in the pathogenesis of preeclampsia.Hypertensive disorders of pregnancy are classified into one of several categories: chronic hypertension present before 20 weeks gestation, preeclampsia–eclampsia, chronic hypertension with superimposed preeclampsia and gestational hypertension. The hypothesis of inadequate trophoblast invasion leading to incomplete remodeling of the uterine spiral arteries has been considered to be a primary cause of the placental ischemia/hypoxia. However, at present, other interesting cardiovascular effector systems are being researched, to see whether they are responsible for smooth muscle relaxation in the systemic vessels. The efficiency of the hypotensive mechanisms, such as vasodilatation and blood pressure regulation, in the pregnant body can be confirmed by adipokines such as apelin and salusin. Apelin, identified in 1998, is a peptide encoded by the APLL gene and an endogenous ligand for angiotensin receptor-like 1 (APJ receptor) . This prIn many scientific reports, the usefulness of assessing the occurrence of cffDNA in the mother’s circulation when screening for fetal genetic defects has been confirmed . CurrentMaternal obesity is a major health concern. According to the WHO in Europe, 54.3% of women over the age of 18 are overweight, and 24.5% of them are obese . MultiplThis study aimed to determine if obesity could influence the percentage of cffDNA in preeclamptic women, and if this effect could be mediated by the disturbance of hypotensive mechanisms.The pregnant women hospitalized between 2008 and 2015 in the Department of Obstetrics and Pathology of Pregnancy in Lublin, Poland were divided into groups: a control group (C) with 67 pregnant women, a gestational hypertension group (GH) with 35, and group with preeclampsia (PE) with 66 patients. The samples were taken for investigation between the 25th and 41st weeks’ gestation.The women with preeclampsia were further divided into two subgroups: obese (O) (pre-gravid BMI > 30) and lean (pre-gravid BMI < 25). The first subgroup contained 23 pregnant women and the second included 43. All pregnant women with diagnosed hypertension received methyldopa at therapeutic doses to control their blood pressure.Normal blood pressure before pregnancy.No general diseases .A physiological single pregnancy .No fetal structural or chromosomal abnormalities detected in prenatal tests and after pregnancy.The occurrence of hypertension ≥ 140/90 mmHg after the 20th week of pregnancy.A proteinuria of 1+ or higher in a single urine sample, or ≥300 mg in a 24-h urine collection.No history of hypertensive disorders during previous pregnancies.2.Body mass index calculation was defined as the body mass divided by the square of the body height, in units of kg/mThe general criteria for selecting the patients undergoing the examination were as follows:Hypertension was first diagnosed after the 20th week of pregnancy.Proteinuria was found.The presence of other systemic or fetal abnormalities was excluded.Pre-gravid BMI < 25.The criteria for including patients in the study PE group were determined as follows:The criteria for preeclampsia with the obesity group included all of the above as well as a pre-gravid body mass index over 30.Hypertension was first diagnosed after the 20th week of pregnancy.The presence of other systemic or fetal abnormalities was excluded.Absence of significant proteinuria.The criteria for including lean patients (BMI < 25) in the group with gestational hypertension were as follows:Normal blood pressure before pregnancy.9.No systemic diseases.10.The current correct course of a single pregnancy.11.No fetal structural or chromosomal abnormalities detected in prenatal tests and after pregnancy.12.No history of hypertensive disorders during previous pregnancies.13.Pre-gravid BMI < 25.The criteria for the control group:In 33 patients, the mild form of preeclampsia was present. The same number of pregnant women developed a severe form of this disease. Division into two groups was carried out based on the occurrence of one or more of the following symptoms :In seven patients, the first symptoms of hypertension and proteinuria were observed before 34 weeks of gestation. The rest of the pregnant women had late preeclampsia (59 patients).The research material was the blood serum and oral mucosa swabs obtained from 168 Caucasian patients hospitalized in the Department of Obstetrics and Pregnancy Pathology at the Medical University of Lublin. All the patients were analyzed for apelin, salusin-alpha, prosalusin and cell-free fetal DNA.g. In order to extract the microparticles, the centrifuged samples were centrifuged again for 90 min at 16,000× g. The microparticles were centrifuged in the lower fraction of the sample after centrifugation.The research material was pregnant plasma collected from the patients who qualified for the study. In order to verify the genetic material, a swab was taken from the mucosa of the inner side of the cheek in the mother and from the newborn after delivery. The test material was plasma, obtained from the blood of the patients and collected on citrate anticoagulant (0.109 M). In order to separate the plasma from the morphotic elements, the whole blood was centrifuged twice using a rotor, for 15 min at the angular velocity of 2500× The determination of the human prosalusin level was performed using the ELISA Human ELISA Kit  from Wuhan EIAab Science Co., LTD . The measuring scale ranged from 0.31 to 20.0 ng/mL. The concentration of Apelin-36 and Salusin-α in the blood serum was determined using test kits from Phoenix Europe GmbH , namely the Apelin-36 (Human) EIA Kit, catalog number: EK-057-15. The analytical sensitivity of the test was 0.1 ng/mL, and the measurement scale ranged from 0 to 100 ng/mL. Salusin-α (Human) EIA Kit, Catalog No.: EK-010-67 The analytical sensitivity of the test was 0.25 ng/mL and the measurement scale was within the range of from 0 to 100 ng/mL.The tests were carried out according to the procedure attached by the manufacturer of each set. The results were read in a Microplate Reader model 680 plate reader . Samples were stored at –80 °C and frozen in liquid nitrogen for isolation. Total cellular DNA was isolated and purified from about 200 mL of blood using commercially available DNA isolation kits DNeasy/RNeasy Mini Kitnr  according to the manufacturer’s instructions. The DNA quality was estimated by agarose gel electrophoresis.Based on the polymorphism analysis of the autosomal short tandem repeat (STR), microsatellite loci and genes were located in the Y-chromosome. Such repetitions occur in a population of a variable number (from 5 to 100). The AmpFLISTER NGM PCR Amplification Kit  was used for amplification of the DNA, allowing for analysis of the following microsatellite repeats: D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11 (located on different chromosomes in the human genome) and, in the FGA gene (fibrinogen), vWA (von Willbrand factor) and TH01 (tyrosine heptaxylase). Genotyping was performed using an ABI 310 analyzer .In order to determine the STR genotype for the 15 microsatellite loci and amelogenin of the sex marker when establishing the biological material of the offspring of the mothers, the AmpFlSTR NGM PCR Kit (Thermo Fisher Scientific) was used. The PCR reaction was carried out in a final volume of 5 μL containing 1× AmpFlSTR NGM Master Mix, 1× AmpFlSTR NGM DNA primers and, quantitatively, ranging from 0.5 to 2 ng. In the case of a low concentration of the entry DNA matrix, the volume of the PCR reaction was increased to 10 μL to ensure an adequate amount of template in the reaction mixture. The conditions of the thermal cycles were in accordance with the manufacturer’s recommendations. An analysis of the size of the PCR products was carried out in an ABI 3130xl automatic sequencing apparatus; . The size of the PCR products was determined in relation to the internal standard size of GS600LIZ .Turk. J. Med. Sci. 2000, 30, 501–503). The products of amplifications were separated in 1.55 agarose gel and by sequencing.Identification of the coding sequence of the SRY9MBI Fermentas0 gene: the SRY gene is located on the Y-chromosome and encodes a transcription factor that initiates the activation of genes responsible for male gender differentiation. The DNA, isolated from the peripheral blood, was a matrix to identify the coding sequence of a gene in a PCR reaction. SRY F: 5′GAATATTCCCCGCTCTCCGGAG-3′ and SRY R: 5′-ACCTGTTGTCCAGTTGCACT-3′ were used to amplify a 418 bp fragment. The PCR mixture contained 0.2 ug of genomic DNA, 2.5 U of Taq polymerase, 0.5 umol/L of each primer, 100 mm/L dNTPs, 3.0 Mm MgCl2 and 1× PCR buffer in a final volume of 50 µL. The PCR condition was 2 min at 94 °C for pre-heating, 35 cycles of 94 °C for 30 s, 570 °C for 30 s and 72 °C for 1 min and 72 °C for 10 min, as described  Each patient received an informed consent form and a standardized questionnaire.t-student test. For the variables tested, which did not show normal distribution, non-parametric tests were used for further analyses. The concentrations of apelin, salusin-alpha, prosalusin were compared using the U-Mann–Whitney test and the Pearson Chi2 test. The relationship between individual substances and clinical data was found using the Spearman correlation test. Statistical significance was set at p ≤ 0.05. Results which were statistically insignificant were defined by the abbreviation “ns”. Statistical calculations were based on Statistica 10 .A statistical analysis of clinical data was carried out using the arithmetic mean standard deviation (SD) medians (ME) using the Shapiro test and the Kruskal–Wallis H test. A comparison of the differences between the control group and the study group, and between the subgroups of patients, was carried out with the Control group—67 patients (39.88%);Patients with PE—66 patients (39.29%): including obese patients (PE + O) (pre-gravid BMI > 30)—23 patients (34.85%)—and lean with PE—43 patients (65.15%);Patients with GH—35 patients (20.83%).The study involved 168 pregnant women, who were divided into three groups:A general statistical analysis was performed of baseline characteristics . These cp = 0.33); vaginal labor was significantly more frequent in the control group, while there were more caesarean sections in the PE, obesity, and GH group ; the gestity in the individual groups of patients did not differ significantly ; the parity in the individual groups of patients did not differ significantly ; term at delivery was significantly different between groups. ; the newborn weight was statistically significantly different between groups . Statistically significant differences occurred between the birth weight of the child of the control patient and the PE and obesity groups.Maternal age did not differ between groups .p = 0.18).There were no statistically significant correlations between obesity and early, late, or severe preeclampsia and the occurrence of cffDNA  between the control group and obesity with preeclampsia and between the control and the group with gestational hypertension ((1). The values of prosalusin were statistically significantly different  between patients with obesity and preeclampsia and those with gestational hypertension ((2). Salusin-alpha values were statistically significantly different .(3). Apelin’s values differed statistically significantly  . TherefoThis study has shown correlations between obesity and the mechanisms responsible for blood pressure control. The studied proteins were chosen for analysis because of their properties of vasodilation and blood pressure regulation. The salusins are bioactive peptides, with hemodynamic effects. Their role in hypertension, atherogenesis and cardiovascular diseases is related to the regulation of blood pressure. Apelin causes systemic vasodilatation and increased cardiac contractility in humans. In our study, it was confirmed that an increased BMI in PE increases the concentration of apelin.Studies have confirmed the involvement of apelin in the process of regulating blood pressure in animals as well as in humans . Apelin Our study found higher concentrations of apelin in patients with preeclampsia. Similar results to ours were observed by other researchers ,25,26. ISalusins and their precursor, prosalusin, are multifunctional bioactive peptides, synthesized in many human tissues, lowering hypertension without affecting the synthesis of nitric oxide. The evaluation of the concentration of the above substances in our study aimed to determine their impact on the course of preeclampsia, which is characterized by an increased contraction of blood vessels, hypoperfusion and tissue necrosis. In several publications on the occurrence of salusin, slightly higher values were found in pregnant women with preeclampsia than in physiological pregnancies . In our The precursor to salusin in the human body is prosalusin, and our study is the first one to determine the concentration of prosalusin in pregnancies with abnormal blood pressure during pregnancy. It was observed that its values were, statistically, the lowest in the group with preeclampsia as compared to the control group and that with gestational hypertension. In addition, there was a correlation between the occurrence of fetal DNA and the low concentration of prosalusin in preeclamptic pregnancies. This is confirmed by the failure of mechanisms that lower blood pressure with a co-existing loss of protein. However, these results are the opposite of those found for salusin-alpha. Further studies should be carried out to determine how preeclampsia affects the biosynthesis of this protein.Cell-free fetal DNA may provide information about the placenta, and potentially be used to predict clinical problems. Studies on the first trimester of pregnancy and cffDNA predictive properties provide conflicting results. According to some researchers, the assessment of fetal DNA between 11–13 and 20–24 weeks of pregnancy is not a good predictor of preeclampsia ,34,35. HIn a study established on a mouse model of maternal-diet-induced obesity, the placentas from obese dams released significantly less cfDNA compared to placentas from lean dams at time 0 in culture . AccordiIn the latest study, the association between cell-free DNA and gestational diabetes (GDM) in a cohort of women presenting for screening for fetal aneuploidy was determined. Although fetal fraction was lower among women diagnosed with GDM, this relationship was not statistically significant in obese patients. The researchers associate obesity with a lower cell-free fetal fraction, as a result of the dilution effect on the placental cfDNA due to an increased amount of maternal cfDNA. Maternal cfDNA levels might be increased in obese women as a result of greater necrosis and apoptosis of adipose tissue and an increased maternal plasma volume. Obesity is a known risk factor for GDM; therefore, for the authors, there was no association between fetal fraction of cfDNA and the development of GDM .Our research is the first to estimate the increase in purely fetal cffDNA due to obesity with preeclampsia patients.The limitations of many research tests are the possibility of determining fetal DNA only in pregnancies with a male fetus. In our study, the determination of amelogenin allowed us to avoid omitting female fetuses. It has been suggested that the test determining the sex with the amelogenin gene is usually sufficient, while other markers of the Y-chromosome  can be used to identify gender in more ambiguous cases . The misper se. Obesity is a multifactorial disease, associated with complex metabolic and physiological changes. Adipose tissue is not only fat storage but rather a hormonally active tissue producing cytokines, e.g., adipokines. Our study did not explore which substance or mechanism is responsible for increasing the risk of preeclampsia, placental damage nor blood pressure regulation. The BMI index itself is also is an imperfect indicator for adiposity. The measurement of body composition, including body fat percentage, might identify the obese woman at risk of preeclampsia more accurately.Another limitation of our research is the performance of analyzes in obese patients In sum, since there is no effective treatment for preeclampsia other than delivery to improve the prenatal care standard, low-dose aspirin prophylaxis is the most useful preventive pharmacologic intervention, especially in pregnant women with obesity.Obesity in preeclampsia is an independent risk factor for placental damage and disturbances in maintaining normal blood pressure. This study found the presence of cell-free fetal DNA in the serum of pregnant women with obesity and preeclampsia to be significantly more common and associated with inefficient hypotensive mechanisms. Understanding the pathomechanism of hypertension in pregnancy is crucial in the development of new therapeutic strategies against preeclampsia.


It is debated whether patients with IgAN with heavy proteinuria and decreased eGFR benefit from aggressive treatment consisting of corticosteroids alone or combined with immunosuppressive agents.2. These patients were assigned to receive supportive care alone or supportive care plus immunosuppressive therapy. The primary outcome was defined as the first occurrence of a 50% decrease in eGFR or the development of ESKD.A retrospective study was performed between January 2008 and December 2016 on patients with IgAN who had urinary protein excretion > 1.0 g/d and an eGFR between 15 and 59 mL/min/1.73 mp < .001). The median annual rate of eGFR decline in the immunosuppression group was −2.0 (−7.3 to 4.2), compared with −8.4 (–18.9 to −4.1) mL/min/1.73 m2 in the supportive care group (p < .001). In multivariate Cox regression analyses, immunosuppressive therapy was associated with a lower risk of progression to ESKD, independent of age, sex, eGFR, proteinuria, MAP, kidney histologic findings and the use of RASi agents . Among the adverse events, infection requiring hospitalization occurred at similar rates in both groups (p = .471).All 208 included patients were followed for a median of 43 months, and 92 (44%) patients experienced the primary outcome. Cumulative kidney survival was better in the immunosuppression group than in the supportive care group (Immunosuppressive therapy attenuated the rate of eGFR decline and was associated with a favorable kidney outcome in IgAN patients with heavy proteinuria and decreased eGFR, and the side effects were tolerable. IgAN is recognized as the world’s most common primary glomerular disease with variable clinical presentation and progression rates that are dependent on the clinicopathological phenotype and duration of follow-up. Overall 4–40% of patients progress to end-stage kidney disease (ESKD) by 10 years ,3, a num2 at the time of biopsy and/or proteinuria >3.5 g/d [IgAN has characteristics of immune complex-mediated glomerulonephritis, and the dominant deposition of IgA in the mesangial area has been suggested as a critical factor in the pathogenesis of IgAN . Cortico>3.5 g/d . On accoThis retrospective study was performed to evaluate the effectiveness and safety of supportive care and immunosuppressive combination strategies in IgAN patients with proteinuria >1.0 g and stage 3 or 4 chronic kidney disease. We estimated the risk of progression based on the 50% decline in eGFR and the development of ESKD.2), and a follow-up time of 6 months or more. The exclusion criteria were as follows: systemic disease known to be associated with secondary IgAN , crescent IgAN (defined as >50% crescentic glomeruli on kidney biopsy), minimal change disease with IgA mesangial area deposits, acute interstitial nephritis, acute kidney injury, or any prior treatment with immunosuppressants. The study protocol was approved by the Ethics Committee of our hospital .This study was a retrospective cohort study that was carried out at Xijing Hospital. We collected all kidney biopsy-proven IgAN patients between January 2008 and December 2016 in the Department of Nephrology. The indications for kidney biopsy were proteinuria >0.5 g, decreased eGFR, newly emerging hypertension, and/or hematuria, and the clinical experience of nephrologists. The inclusion criteria were as follows: IgAN proven by kidney biopsy, age between 16 and 80 years old, urinary protein excretion >1.0 g/d, impaired kidney function (defined as an eGFR between 15 and 59 mL/min/1.73 mn = 167) had immunosuppression therapy. Immunosuppressive regimens included corticosteroids , cyclophosphamide , mycophenolate mofetil (17%), and calcineurin inhibitor (3%). The corticosteroid monotherapy group consisted of patients who received oral prednisone at a dose of 0.6–1.0 mg/kg/day for 2 months with subsequent gradual reduction . The low-dose CS + immunosuppressant therapy group consisted of patients who received oral prednisone at a dose of 0.4–0.6 mg/kg/day for 2–3 months with subsequent gradual reduction . Oral CTX was prescribed at 50 mg/day for 5 months, and pulse CTX was prescribed at 0.8 g per month for 6 months. The starting dose of MMF was 1–1.5 g/d for 6 months and then dosed by half for 6 months. Immunosuppressants other than CS were used in combination with CS as a first intention in 87% of patients. Otherwise, they were added after an initial course of CS.Originally, 1680 biopsy-confirmed IgAN patients were screened in our study. Finally, 208 IgAN patients who met the inclusion criteria were included . Among aBaseline demographic and clinical data were collected from electronic medical records at the time of the biopsy or the time closest to the patient's biopsy. Age, sex, and systolic and diastolic blood pressure were recorded. All follow-up data were updated to January 2019, and the results of laboratory tests of serum creatinine and 24 h urinary protein levels were also recorded. Urine protein excretion was quantified every 3 months until month 12 after the kidney biopsy, and every 6–12 months until the last follow-up. The eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation . Patholo2, or initiation of chronic sustained dialysis for three months), or death due to kidney disease. The secondary outcomes included proteinuria changes evaluated by time-averaged proteinuria , the rate of kidney function decline (slope of eGFR), or death due to any cause across the study visits.The primary outcome was kidney survival rate, defined as the first occurrence of a 50% decrease in eGFR, the development of ESKD .Data are presented using descriptive statistics such as the mean ± standard deviation, medians and interquartile ranges, and numbers with proportions. Intergroup differences were compared using an independent t test, nonparametric tests as appropriate, and Pearson’s chi-squared test for categorical variables. Kaplan–Meier curves were used to estimate cumulative kidney survival, and the log-rank test was used to compare between groups. Univariate and multivariable-adjusted Cox proportional hazards analyses were carried out to estimate which variables were associated with kidney survival. Variables were brought into a multivariable-adjusted Cox proportional hazards model using an enter method, and the hazard ratio (HR) resulted from adjusted models: model 1 was adjusted for demographic and clinical data; model 2 was adjusted for the variables in model 1 plus RASi; model 3 was adjusted for the variables in model 1 plus histological data; and model 4 was adjusted for the variables in model 3 plus RASi. The results are presented as hazard ratios (HRs) and 95% confidence intervals (CIs). Statistical significance was defined as a two-sided p = .004), but age, systolic, diastolic, and mean arterial pressure (MAP) did not differ between groups at the time of kidney biopsy (p > .05). The mean eGFR was 39.4 ± 12.0 mL/min/1.73 m2 in the immunosuppression group and 39.4 ± 14.4 mL/min/1.73 m2 in the supportive care group (p = .188). Likewise, the mean urinary protein excretion did not differ between the two groups, as it was 2.8 ± 2.4 g in the immunosuppressive therapy group and 2.5 ± 1.8 g in the control group (p = .992).The baseline clinical and pathological characteristics of the included patients are shown in p = .005, The pathological data are also listed in p = .001). The cumulative kidney survival rate from baseline was better in the immunosuppression therapy group than in the control group  mL/min/1.73 m2 in the supportive care group  progressed to the primary outcome. As shown in p < .001); multivariate analysis adjusted for the demographic and clinical factors of sex, age, mean arterial pressure, proteinuria, and eGFR showed that immunosuppressive therapy was significantly associated with a decreased risk of kidney outcome  (p < .001)  , Model 1 < .001) , Model 42) and the use of RASi agents (2) or the nonuse of RASi agents , age (<60 y), sex , kidney function . In the immunosuppression group, 12 patients were hospitalized with infections . One patient who received mycophenolate mofetil plus glucocorticoid therapy in the immunosuppression group died of sepsis. Three patients in the immunosuppression group underwent hemodialysis for acute-on-chronic kidney disease, while one patient was dependent on dialysis. Other patients did not receive kidney replacement therapy. We did not observe more hepatotoxic events, serious femoral head necrosis, new-onset diabetes mellitus, or leukopenia in the immunosuppression group than in the supportive care group.Over the mean period of 43 months of follow-up, 12 patients (29%) with at least one first AE were observed in the supportive care group, and 59 patients (35%) were observed in the immunosuppression group , and there was no control arm of patients not receiving immunosuppressive therapy in either study. A recently published retrospective analysis of a large European cohort VALIGA study [2 as well as those with proteinuria above 3 g/d. In contrary, the STOP-IgAN [2) and/or a rapidly progressive clinical course. Finally, the immunosuppressive regimen was strongly associated with the kidney survival rate in patients with a significant reduction in eGFR. Nevertheless, the STOP-IgAN study [2 per year before treatment in the intervention group. These results demonstrated that the superior benefits of immunosuppressive therapy on kidney survival might not be more remarkable than those of supportive care management in non-progressive IgAN. The rate of eGFR decrease among patients in the supportive care group of our study was higher than that in the STOP-IgAN study (−8.4 vs −1.6 mL/min/1.73 m2 per year). Possible reasons for the difference include the fact that participants in this trial were at higher risk progressively because of higher mean baseline proteinuria, and also because individuals of Eastern Asian  origin may have more rapid rates of eGFR decline and renal failure [A number of clinical studies have demonstrated a positive effect of immunosuppressive combination therapy in IgAN ,10,11. FGA study  suggesteTOP-IgAN  trial suTOP-IgAN  includedAN study  excluded failure . The ann failure  and a re failure , both fr failure  study. I failure , the ove2, immunosuppressive therapy had protective effects on the composite renal failure outcome in the eGFR ≥ 30 mL/min/1.73 m2 subgroup, which may partly be due to poor prognosis of decreased eGFR. Likewise, subgroup analysis found that in patients who did not use RASi agents, immunosuppressants did not have more kidney protection advantages than the control group. These results might be explained by the worse kidney function in the group that did not use RASi agents , which was consistent with previous studies [Moreover, subgroup analysis showed that compared with patients who had eGFR < 30 mL/min/1.73 m studies ,23. HencThough an effective treatment regimen for patients with IgAN, immunosuppressants are associated with many adverse events, including inducing or aggravating infection, hepatotoxic events, metabolism disorders, and leukopenia . Both imp = .037). Third, due to the limitation of sample size, we did not further analyze the response of patients with eGFR < 30 mL/min/1.73 m2 to immunotherapy. Finally, we could not show the results of appropriate comparison between immunosuppression and supportive care because of lower number in the supportive care group and the strong bias resulting from the selection of therapeutic protocols at our institutions. Although ethnic differences in response to treatment and the longer follow-up, the rapid decline eGFR of supportive care group may still have a bias for the study’s results. Therefore, a large prospective cohort study should be performed to verify our findings.Although our results are promising, several limitations should be acknowledged. First, this was a single-center retrospective observational study, and missing data were inevitable. There were some differences in histological backgrounds between the cohorts, although they were not significant after matching. Second, nothing is known about compliance with treatment, the occurrence of adverse events with either therapy, and the doses of corticosteroids and RASi agents; only 57% of patients received RASi agents in all patients. The use of RASi agents was not distributed evenly between the supportive care group and the immunosuppression group in the original cohort (42% verse 60%, We showed that immunosuppressive therapy slowed the rate of kidney function decline and improved long-term kidney outcomes in IgAN patients with heavy proteinuria and decreased eGFR. Although the adverse events directly related to immunosuppressive therapy are tolerable, side effects still need to be closely monitored during medication. These findings have practical clinical implications in the treatment of IgAN patients with chronic kidney disease stage 3 or 4. Further multicenter clinical trials should be performed to verify our findings.


The objective was to analyze the association of changes in pre-dialysis systolic and diastolic blood pressure with air temperature in a municipality in the Brazilian Pantanal, a tropical climate area. Longitudinal panel study, with analysis of mixed effects models of 133 hemodialysis patients in the city of Cáceres-Mato Grosso in 2014. Air temperature showed an inverse association with pre-dialysis systolic and diastolic blood pressure. With each increase of 1 °C in the mean air temperature, the pre-dialysis systolic blood pressure decreases -0.730mmHg (p ≤ 0.000) and the pre-dialysis diastolic blood pressure decreases -0.280mmHg (p ≤ 0.000). The estimated effect was greater for systolic blood pressure, but both pre-dialysis blood pressure measures are reduced with an increase in lag (up to two days), even when adjusted for relative air humidity. Air temperature is determinant for changes in pre-dialysis systolic and diastolic blood pressure in hemodialysis patients. The temperature effect was greater for systolic blood pressure than for diastolic blood pressure. Renal dialysis, Biometeorology, Bioclimatology, Temperature, Environmental health. Hypertension can be the cause or the consequence of renal dysfunction, and the control of hypertension is essential for the prevention of the disease . Air temWith the initiation of hemodialysis treatment, some patients reverse SAH by removing excess fluids. However, some patients continue to present with hypertension , so muchThere are no limits on ideal and safe blood pressure values for patients on HD. On the other hand, the blood pressure values that are indicated for the general population, when applied to patients undergoing HD treatment, result in an increase in mortality in this group , 12.Most studies show an inverse association between air temperature and blood pressure in hemodialysis patients , 23, 24.The Intergovernmental Panel on Climate Change-IPCC  motivateKnowing that patients undergoing hemodialysis have uncontrolled blood pressure, it is relevant to understand the effects of variations in air temperature on the blood pressure of patients on hemodialysis residing in a tropical climate area such as the Brazilian Pantanal region. Quantifying the effect of air temperature on blood pressure changes will guide the adoption of practices to minimize blood pressure changes and their consequences for patients undergoing hemodialysis. The aim of this study was to analyze the association of changes in pre-dialysis systolic and diastolic blood pressure with air temperature in a municipality in the Pantanal of Mato Grosso.2parda" brown color [This research was designed as a longitudinal retrospective epidemiological study, with repeated measures, defined as a panel study. This analysis was carried out with chronic renal patients undergoing HD in 2014 in the city of Cáceres - Mato Grosso, Brazilian Pantanal. In terms of ethnicity, the population of the state of Mato Grosso is of mixed race, like most Brazilians. During the last census, 90% of the population self-classification black or “wn color , 30.For the study, data were collected from the medical records of 133 patients who signed the Free and Informed Consent Form. All patients were of legal age (over 18 years) and had been undergoing HD for more than three months. Data were collected in January 2014 at the only hemodialysis center in the region, located in the city of Cáceres. These studies are non-existent in Brazil due to the difficulty in obtaining data on patients undergoing treatment, so our study was limited to this group of patients from southwestern Mato Grosso.The patients lived in 14 municipalities in the southwestern region of Mato Grosso, with a total population of 206,356 inhabitants . Among tAll patients had a prescription for hemodialysis three times a week. Eventually, there could be absences for some patients or extra sessions when requested by the medical team. From these patients, data were obtained on variables related to individual characteristics that do not change over time, including date of birth, sex, color/race, underlying disease, initial diagnosis, use of antihypertensive drugs. Information about the initial diagnosis is related to the diagnosis that led the individual to seek the nephrology service. This fact is not necessarily involved in starting hemodialysis, and in some cases, it may be the secondary diagnosis, as there is no such field in the medical record.Monthly variations (hemoglobin measurement (g/dL)), free calcium (mg/dL), phosphorus measurement (mg/dL), glutamic-pyruvic transaminase - ALT (u/L), anti-HCV (reagent and not-reagent), or daily, such as: day of the hemodialysis session, blood pressure (mmHg) recorded  and pre-dialysis weight (kg). The patients were weighed at the entry and exit of the HD sessions using a digital scale, with weight checks in kg. In patients using a wheelchair and bedridden, no weighing was performed due to the lack of an appropriate weight scale (6.3% of the set of measures).The dependent variables were pre-dialysis Systolic Blood Pressure (pre-SBP) and pre-dialysis Diastolic Blood Pressure (pre-DBP), whose measurements were taken before the hemodialysis procedure. Blood pressure measurements were performed by an indirect method, using the auscultatory technique with the use of an aneroid sphygmomanometer. This procedure was done in the armchair that patients underwent hemodialysis.Adjustment variables include the use of antihypertensive drugs, diagnosis of disease , sex, age, and weight of entry, hemoglobin, calcium, TGP and anti-HCV.For meteorological data, daily air temperature  and relative humidity  from January to December 2014 were considered. This information was obtained through the automatic meteorological station of the National Meteorological Institute (INMET)  installeThe association between air temperature and pre-SBP and preDBP was estimated using mixed effects models. These models allow both fixed and random effects; thus, they are appropriate for data analysis required in this study which in considering the independence between patients and the dependence in the time of the repeated measurements of each patient.(I)Adjustment of the time trend;(II)Inclusion of variables related to individual characteristics fixed over time;(III)Inclusion of variables that change over time (month and day);(IV)Adjustment of the variance function of the random error;(V)Adjustment of residual autocorrelation;(VI)Evaluation of the effect of temperature and/or humidity.Adjusting the mixed effects model follows these steps:In adjusting the trend, initially the variable that represents the observation day was centered on the midpoint of the study period. The trend was adjusted by a cubic spline, with a random effect on the parameters. The variance function of the random error was adjusted by the variables sex and age (categorized) and the residual autocorrelation by a common autocorrelation structure (AR(1)). Compound Symmetry structure was also evaluated, but AR(1) presented lower AIC.nlme library [The Likelihood-Ratio Test and the Akaike Information Criterion (AIC) were used to assess the entry of explanatory variables in the model and the quality of the adjustment. At the 5% significance level, the significant variables for adjusting the model were: use of antihypertensive drugs; disease diagnosis , sex, age, weight of entry (with random effect in the parameter), hemoglobin, calcium, ALT, anti-HCV and relative humidity to assess the effect of air temperature. To adjust the meteorological variables, lag0 and lag1 measurements were used. For the main exposure variables, lag0 to lag3 and Arithmetic Moving Averages (MMA) were considered for the calculations. In order to estimate the cumulative effect of exposure to temperature, the Polynomial Distributed Lag Model (PDLM) technique was also considered. In this study, quadratic polynomials of the effects lagged up to 3 days were calculated. The data were analyzed in the R program (version 3.3.5)  through  library .The study was approved by the Research Ethics Committee of the State University of Mato Grosso, and Certificate of Presentation for Ethical Appreciation (CAAE): 49487815.0.0000.5166, in November 2015.3In 2014, the average air temperature in Cáceres was 25.9 °C. The average maximum temperature was 31.7 °C, and the average minimum temperature was 21.7 °C. The records of minimum temperatures were concentrated between June and August, which registered 12.2 °C as the lowest temperature in this period. Regarding the maximum temperature, it increases from August, with a higher record of 38.7 °C in October. The average relative air humidity in the period was 76.2%, reaching a maximum of 92.8% in May and a minimum of 22% in September. The period of lowest record of relative humidity was from August to October .Figure 1An inverse relationship is observed between air temperature, and pre-SBP, and pre-DBP. The months of May, June, and July were the months with the lowest temperature averages in the year and also the months whose mean systolic and diastolic blood pressure was highest .Figure 2Most patients were men (61%), with a mean age of 55 (SD14.9) years. The mean pre-SBP was 133 mmHg (60–240 mmHg), the mean pre- DBP was 76.2 mmHg (30–150 mmHg) and the mean pre-dialysis weight was 67 kg (36–110 kg). For monthly laboratory tests, hemoglobin averaged 10.27g/dl (SD 2.05), hematocrit was 30.7% (SD 5.91), calcium dosage of 4.78mg/dl (SD 0.35), the ALT of 23.53U/l (SD 21.19) and 5 (3.4%) patients had the reagent Anti-HCV .Table 1DThe models of basic mixed effects for systolic and diastolic blood pressure, without the inclusion of meteorological variables were analyzed. The use of antihypertensive drugs was associated with an increase in the mean pre- SBP of 8.45 mmHg (p = 0.017). The diagnosis of end-stage renal disease was associated with an increase of 41.13 mmHg (p = 0.013) in pre-dialysis SBP, as well as the diagnosis of unspecified chronic renal failure with a positive association of 39.26 mmHg (p = 0.018). Pre-dialysis weight showed an association of 0.53mmHg  in the increase in pre- SBP; in which, for a gain of 1kg there is an increase in mean SBP of 0.53mmHg. With regard to laboratory tests, it appears that with each increase of a unit in hemoglobin (HB) there is an increase of 0.48mmHg (p = 0.002) in the mean of the SBP; and the increase of one unit in the ALT is associated with an increase of 0.02mmHg (p = 0.021) in the mean of the SBP .Table 2BAs for clinical variables, it is observed that the diagnosis of unspecified acute nephrotic syndrome, chronic nephrotic syndrome, unspecified nephrotic syndrome, congenital kidney malformation, sex, age and calcium concentration did not show statistically significant associations with pre- SBP, but were kept in the model due to the quality of the adjustments.For pre-DBP, it is observed that the diagnoses are positively associated, with the unspecified acute nephrotic syndrome associated with an increase of 27.58mmHg (p < 0.005) of the pressure; the diagnosis of chronic nephrotic syndrome at an increase of 36.73mmHg  in pressure; the diagnosis of end-stage renal disease with an increase of 33.65mmHg  in pressure. And the diagnosis of other congenital malformations specific to the kidney with an increase of 20.02mmHg (p = 0.048) in pressure.The age of patients had an inverse association of -0.36mmHg (p ≤ 0.000) with pressure. Thus, with each increase of one year of age, the mean pre-DBP decreased by -0.36mmHg. Pre-dialysis weight showed an association of 0.14 mmHg  in the increase in pre- DBP. Thus, for every 1 kg gain by the patient, there was an increase in the pre- DBP. The results of laboratory tests of hematocrit and anti-HCV reagent also showed a positive association with pre-SBP. With each increase of one unit in hematocrit (HT) there was an increase of 0.16mmHg  in the mean of the DBP. And anti-HCV reactive patients are associated with an increase of 10.85mmHg (p = 0.007) in the mean DBP. Thus, the patient having hepatitis C increases the pre- DBP.The clinical variables gender, age and ALT did not show statistically significant associations with pre- DBP, but were maintained in the model for better adjustment .After adjusting the basic model, the variables temperature and relative humidity were included for the analysis. To assess the effect of temperature, the variable average relative humidity on the current day (lag 0) or lagged one day (lag 1) was included in the model as a covariate. The final model included the basic model, relative humidity on the current day and temperature  . For SBPFor DBP, when adjusted for relative air humidity at lag 0, with a 1 °C increase in the average temperature, a reduction in pre-dialysis DBP was observed on the day of the hemodialysis session (lag 0) ; the day before the hemodialysis session (lag 1) ; in the two days preceding the hemodialysis session (lag 2) ; in the three days preceding the hemodialysis session (lag 3)  and in the two-day moving average  .By the approach of the distributed lag polynomial models, the global effect of exposure to temperature in lags 1, 2, 3 and current day was significant for both systolic and diastolic blood pressure, with a reduction in pre-SBP of 0.956mmHg  and reduction in the pre-DBP of 0,385mmHg  for each 1 °C increase in the average temperature . The fin4This is the first study to quantify the changes in SBP and DBP of patients undergoing hemodialysis about temperature variations in a tropical climate area. The analysis shows that the greater the daily variations in temperature, the greater the changes in systolic and diastolic blood pressure during the pre-dialysis period. The analysis shows that the greater the daily variations in temperature, the greater the changes in pre-dialysis systolic and diastolic blood pressure.Although the scientific literature has demonstrated the inverse relationship between air temperature and blood pressure  for patiFor the general population, a study conducted with data from 16 countries (Europe and North America), it was found that the increase of 1 °C in the average external temperature decreased -0.19 mmHg  of the SBP . In a meIn the present study, patients undergoing hemodialysis, the relationship found was even more evident, implying a reduction of -0.730mmHg  in SBP and -0.280mmHg  in pre-dialysis DBP. Although the methods used are different, this difference can indicate the biological vulnerability to temperature changes in patients on hemodialysis when compared to the general population and also to patients with other chronic diseases.Variations in SBP and DBP influenced by air temperature can further impair the evolution of patients undergoing hemodialysis whose risk of cardiovascular mortality in these people was associated with either a reduction or an increase in blood pressure , 44. ThuAlthough the link between air temperature and blood pressure has been established, the mechanisms underlying this relationship have not been fully understood to date, and there are likely to be multiple , 45, 46.When the body is exposed to low temperatures, the first physiological response will be peripheral vasoconstriction., which will reduce thermal conduction through the skin and trigger tremors and reduce diuresis. This response mediated by sympathetic activation increases heart rate, renin-angiotensin activity, and aldosterone levels, which raise blood pressure , 48, 49.Other physiological responses involved in the increase in blood pressure due to the decrease in temperature are platelet activation , erythroThe response to heat (temperature increased) is more systemic when compared to cold (temperature decrease), whose first reaction of the organism is peripheral vasodilation and sweating to lower the core temperature . With thPre-SBP is generally used for the management of hypertension in patients undergoing hemodialysis . The assElucidating the temperature response on the pre-SBP and DBP, a moment when the influence of the external air temperature is higher because the patient has recently been in the environment of the session, which is air-conditioned.In the present study, pre- SBP and DBP were associated with clinical and laboratory variables. Both pressures were positively associated with chronic kidney disease diagnoses. The influence of diagnoses on blood pressure can be explained by the pathophysiological mechanism of the disease .Pre-dialysis weight was positively associated with pre SBP and DBP, and the increase in blood pressure is related to fluid overload, which is a consequence of CKD , 46. It The relationship between increased ALT concentration and SBP has not yet been described in the literature, and therefore requires further clarification. There were also no studies that related the association of increased DBP in patients with hepatitis C in hemodialysis. ALT is used as a marker of liver function, and elevation is indicative for screening for infection by hepatitis B and C viruses , 60. ThePre-SBP was positively associated with hypertensive patients. The relationship between hypertension and chronic kidney disease can be related to the cause or consequence of the disease . In BrazThe inverse association between age and DBP was contrary to the findings of other studies, whose association found was positive for patients undergoing hemodialysis , 62.Knowing the association between air temperature and blood pressure, it is evident that we must analyze weather conditions, focusing on local temperature and the possible impacts of climate change for this group of patients, who are vulnerable to changes in blood pressure as the rise in air temperature.The projections for the Pantanal region in the scenario projected by the IPCC  will be The increase in temperature will lower blood pressure, but the increase in heat associated with a decrease in the relative humidity of the air increases the feeling of thirst, which can increase fluid intake  and blooThe missing data represented 12.7% of the records, a situation for which we judged the data to be robust. As a limitation of the study, the meteorological data and the blood pressure measurements were collected in the municipality of treatment. Despite this limitation, it is noteworthy that approximately 52% of patients live in the municipality of treatment and 48% in neighboring municipalities. This region, as described in the materials and methods section, presents the same climatic characteristics, according to the Köppen classification.However, this is the municipality that concentrates half of the study population. Although the adjustments of the models may have been influenced, the observations in lag 0 reinforce our findings. It was impossible to perform stratified analyzes by gender and age group due to the number of participants. The temperature data series refers to meteorological data, as it covers only one year. However, the results are extremely relevant to public health due to the clinical implications of this group's extreme biological vulnerability.Our findings show the relevance of the change in air temperature in the diagnosis and treatment of these patients. Health professionals must be prepared for the adjustments of medications, liquid control conducts, and temperature changes , 24, 65.Due to the difficulties in reaching goals and controlling blood pressure in patients undergoing hemodialysis , 67, theLongitudinal epidemiological studies of the effects of air temperature and other meteorological factors on health - in addition to the direct impacts of meteorological variables - will provide a more accurate measure of health impacts and will help in planning and allocating resources to priorities.5It is concluded that air temperature is inversely associated with pre-dialysis systolic and diastolic blood pressure in patients undergoing hemodialysis treatment. The influence of temperature variation on systolic blood pressure is greater than diastolic and the acute effect is greater than the cumulative effect.Shaiana Vilella Hartwig and Eliane Ignotti: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Sandra de Souza Hacon and Raniere Flávio Viana Sousa: Contributed reagents, materials, analysis tools or data; Wrote the paper.Beatriz Fátima Alves de Oliveira and Ludmilla da Silva Viana Jacobson: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.10.13039/501100002322Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and the 10.13039/501100005286Research Support Foundation of the State of Mato Grosso - Brasil (FAPEMAT), by providing the research grant. And it had a partnership with Rede Clima - Brasil, nº 550022/2014-7 and FINEP/ Climate Network (01.13.0353.00).This work was supported by the The authors are unable or have chosen not to specify which data has been used.The authors declare no conflict of interest.No additional information is available for this paper.


High-throughput techniques allow us to measure a wide-range of phospholipids which can provide insight into the mechanisms of hypertension. We aimed to conduct an in-depth multi-omics study of various phospholipids with systolic blood pressure (SBP) and diastolic blood pressure (DBP). The associations of blood pressure and 151 plasma phospholipids measured by electrospray ionization tandem mass spectrometry were performed by linear regression in five European cohorts (n = 2786 in discovery and n = 1185 in replication). We further explored the blood pressure-related phospholipids in Erasmus Rucphen Family (ERF) study by associating them with multiple cardiometabolic traits (linear regression) and predicting incident hypertension (Cox regression). Mendelian Randomization (MR) and phenome-wide association study (Phewas) were also explored to further investigate these association results. We identified six phosphatidylethanolamines  and two phosphatidylcholines (PC 32:1 and PC 40:5) which together predicted incident hypertension with an area under the ROC curve (AUC) of 0.61. The identified eight phospholipids are strongly associated with triglycerides, obesity related traits , diabetes related traits  and prevalent type 2 diabetes. The genetic determinants of these phospholipids also associated with many lipoproteins, heart rate, pulse rate and blood cell counts. No significant association was identified by bi-directional MR approach. We identified eight blood pressure-related circulating phospholipids that have a predictive value for incident hypertension. Our cross-omics analyses show that phospholipid metabolites in the circulation may yield insight into blood pressure regulation and raise a number of testable hypothesis for future research. Pervious study showed that the patients with essential hypertension have abnormal sodium-lithium counter transport across the red cell membrane, and that the level of transport is heritable2. Phosphatidylcholines (PC), phosphatidylethanolamines (PE), lysophosphatidylcholines (LPC), PE-based plasmalogens (PLPE), ceramides (CERs) and sphingomyelin (SPM) are groups of phospholipids that have a key function in the bilayer of (blood) cell membranes3. Although changes of membrane phospholipids in essential hypertension have been recognized and studied for a long time4, these previous studies either focused on animal models or overall phospholipid groups with limited resolution in the measurement. More detailed characterization of phospholipids in relation to hypertension at the population level is lacking.Long-term high blood pressure, of which 90–95% essential hypertension, is a major risk factor for cardiovascular diseases, e.g. coronary artery disease, stroke, heart failure, atrial fibrillation, etc11. Despite these developments, the number of studies on the role of phospholipid profiles in hypertension and blood pressure is still limited. Very few studies of blood pressure and hypertension have investigated phospholipid profile in high resolution15. The study by Kulkarni et al. examined 319 (phospho)lipids in 1192 Mexican-Americans12 and found that diacylglycerols (DG) in general and DG 16:0/22:5 and DG 16:0/22:6 in particular are significantly associated with systolic (SBP), diastolic (DBP) and mean arterial pressures as well as the risk of incident hypertension12. Stefan et al. studied 135 cases and 981 non-cases of incident hypertension in a European study and identified four phospholipids and two amino acids which could improve the predictive performance of hypertension in addition to the known risk markers15. To our knowledge, no large-scale epidemiological study of blood pressure and/or hypertension with high-throughput measured phospholipids has been performed with replication in an independent study or has studied in detail the mechanism of the associations.In recent decades, high-throughput mass spectrometry (MS) has offered the opportunity to determine phospholipids on the chemical molecular level with high resolution at a low price. Thus, phospholipid panels with detailed characterisation are increasingly adopted in large epidemiological studiesThe aim of this study was to conduct an in-depth multi-omics study of the associations and causality of the associations of phospholipids with SBP and DBP, which are the diagnostic variables of hypertension, through metabolomics, genomics and phenomics. To this end, we investigated the association of blood pressure and 151 quantified phospholipids including 24 SPMs, 9 CERs, 57 PCs, 15 LPCs, 27 PEs, and 19 PLPEs, in 3971 individuals from five European populations. Using Mendelian Randomization (MR), we further investigated the causality in these relationships. The potential genetic pleiotropy between phospholipids and blood pressure was also explored.16, (2) the Erasmus Rucphen Family (ERF) study, conducted in the Netherlands (n = 717)17, (3) the Northern Swedish Population Health Survey (NSPHS) in Norrbotten, Sweden (n = 678)18, (4) the Orkney Complex Disease Study (ORCADES) in Scotland (n = 681)19, and finally (5) the MICROS study from the South Tyrol region in Italy (n = 1185)20 which was included for replication. Fasting blood samples were collected for the biochemical measurements. All studies were approved by the local ethics committees and all participants gave their informed consent in writing.This study was conducted using five populations throughout Europe. The individuals with both blood pressure and phospholipid measure available were included: (1) the CROATIA-Vis study conducted on the island of Vis, Croatia (n = 710)The association tests of phospholipids and blood pressure were performed on the same baseline data, for each of the five studies. The predictive analysis was performed in ERF study in which we collected follow-up data from March 2015 to May 2016 (9–14 years after baseline visit). During the follow-up, a total of 572 participants’ records from the 717 individuals included in the baseline analysis were scanned for common diseases in general practitioner’s databases.22 and a brief description can be found in the As part of the European Special Populations Research Network (EUROSPAN) project, the absolute concentrations (µM) of 151 lipid traits in plasma were centrally measured by electrospray ionization tandem mass spectrometry (ESIMS/MS), including 24 SPMs, 9 CERs, 57 PCs, 15 LPCs, 27 PEs and 19 PLPEs. The methods used have been validated and described previously23.Supplementary Table 28. As all of the five cohorts included closely related individuals, family relationship based on the genotype was adjusted for in the analysis by extracting polygenic residuals for the phenotypic traits, by using the polygenic option in GenABEL package in R29.Lipids were natural log-transformed and standardized (mean-centered and divided by their standard deviation). We corrected blood pressure levels for antihypertensive medication use by adding 15 mmHg to the SBP and 10 mmHg to the DBP of users of antihypertensive medication30. To correct for multiple testing, we used Bonferroni correction using the number of 70 independent components extracted from the 151 directly measured phospholipids . Matrix Spectral Decomposition was separately used to calculate the number of independent equivalents31 in each of the four discovery studies. Bonferroni correction was done for 70 tests which was the highest number obtained in CROATIA-Vis. We did not correct for the number of blood pressure variables as SBP and DBP are highly correlated .In each study, we used linear regression to examine the association between each of the phospholipids and blood pressure individually. Blood pressure variables were used as dependent variables and phospholipids were used as independent variables. We performed a discovery analysis in CROATIA-Vis, ERF, NSPHS, and ORCADES, adjusting for age and sex (model 1). Results from the four discovery populations were meta-analyzed with inverse-variance weighted fixed-effects model using the METAL softwareWe replicated our findings in MICROS (n = 1185) using the same statistical framework as in the discovery analysis and using a Bonferroni correction for the independent number of tested associations, i.e., equivalents of the significant phospholipids.In a combination of all five cohorts, we examined a further model (model 2) to assess the impact of potential confounders and mediators by additionally adjusting for BMI, HDL-C, TC, lipid-lowering medication and type 2 diabetes (T2D) status. We checked the pairwise Pearson's correlation matrices of the blood pressure related phospholipids in ERF adjusting for age, sex and family relationships.32. Integrated Discrimination Improvement test (IDI) and continuous Net Reclassification Improvement test (NRI) were performed to compare different joint models.The phospholipids significantly associated with SBP or DBP were tested for the association with the occurrence of hypertension during the follow-up in ERF. The incident cases were defined as the participants free of hypertension at baseline who were diagnosed with hypertension at follow-up by general practitioners. Time-to-event was defined as the time from the enrollment date at baseline to either the onset date of disease, date of death, date of censoring (moving away) or date of follow-up collection. Cox proportional regression analysis was used to evaluate the individual effect of phospholipids considering of the follow-up time (time-to-event). To determine the joint effect of the phospholipids on the discrimination of future hypertension patients, we calculated the area under the receiver operator characteristics (ROC) curve (AUC). We further determined whether the addition of the listed phospholipids increase the AUC value of the factors in the Framingham risk score for hypertension which includes age, sex, SBP, DBP, BMI, cigarette smoking and parental hypertension (Framingham model)38. The distributions of adiponectin, insulin, leptin, triglycerides, C-reactive protein, HOMA-IR and resistin are skewed and therefore were log-transformed before performing the analysis. We used the standardized residuals of natural-log-transformed phospholipid levels as the dependent variable, adjusted for age, sex and family relationship. A hierarchical clustering approach was used to cluster the cardiometabolic traits39. We estimated the false discovery rate less than 0.05 by Benjamini & Hochberg method considering of the gathering of categorical and continuous variables.In ERF, we further examined the association of these identified phospholipids with known cardiometabolic traits, including adiponectin, albumin, alcohol consumption, anti-diabetic and anti-hypertensive medications, BMI, creatinine, C-reactive protein, glucose, HDL-C, insulin, intima-media thickness, low-density-lipoprotein cholesterol (LDL-C), heart rate, homeostatic model assessment-insulin resistance (HOMA-IR), leptin, lipid-lowering medication, metabolic syndrome, plaque score, pulse wave velocity, resistin, smoking status, TC, total fat percentage, triglycerides, T2D, waist circumference and waist-to-hip ratio. The description and measurement methods of the above mentioned cardiometabolic traits can be found in our previous reports40. We performed a two-sample bi-directional MR of the 11 significant associations of phospholipids and SBP or DBP. We used summary statistics level data of blood pressure and phospholipids28 utilizing the pipeline in the R-package TwoSampleMR41. In brief, the genetic instrument was based on the top genetic determinant SNPs with linkage disequilibrium R2 < 0.05 within 500kbps clumping distance. The proportion of variance in the exposure explained by the genetic variance (R2) and F statistics were calculated to estimate the statistic power of MR. As the sample size in the phospholipid GWAS in the ESIMS/MS platform is small (n = 4034), to increase the explained variance of the instrumental variable, P-value < 1.0 × 10−7 was used to define the genetic determinants of phospholipids. The GWAS summary statistics of the same phospholipids available in Biocrates metabolomics platform were also used additionally to increase the statistical power (n = 7478)42. For genetic determinants of blood pressure, the genome-wide significance level  was used. Inverse-variance weighted MR was used with weighted median, sample mode and weighted mode methods as sensitivity to investigate pleiotropy. MR-Egger regression was used to control the directional horizontal pleiotropy, and the Egger estimates on the intercept was used for the heterogeneity tests44. The Bonferroni corrected P-value with independent equivalents of phospholipids was used as the significance level.MR is a statistical method which uses the effect of genetic variants determining an exposure and test its association with the outcome under study, based on the assumption that the genetic variant is inherited independent of the confounding variables45. For the top SNPs of either phospholipids used in MR, we looked up their pheno-wide associations in GWAS ATLAS45. We estimated the false discovery rate less than 0.05 by Benjamini & Hochberg method.We further studied the pleiotropic effect of the genetic determinants of the identified phospholipids using phenome-wide association study (Phewas) by data-mining from previous publicationsCROATIA-VIS: All subjects were asked to provide written consent, after being informed on the study goals and main approaches, in accordance with the Declaration of Helsinki. The study was approved by the ethics committees of the University of Zagreb (No. 018057) and the University of Split School of Medicine (No. 2181-198-A3-04110-11-0008), Croatia and the Multi-Centre Research Ethics Committee for Scotland (No. 01/0/71). ERF: The study protocol was approved by the medical ethics board of the Erasmus Medical Center Rotterdam, the Netherlands. All participants gave their informed consent in writing. MICROS: A detailed information sheet and a form for the written informed consent were provided to each prospective participant to approve. The study was approved by the Ethics Committee of the Autonomous Province of Bolzano. NSPHS: The NSPHS study was approved by the local ethics committee at the University of Uppsala  in compliance with the Declaration of Helsinki. All participants gave their written informed consent to the study. For participants of under legal age, a legal guardian also signed. The procedure used to obtain informed consent and the respective informed consent form has been recently discussed according to current ethical guidelines. ORCADES: ORCADES received ethical approval from the appropriate research ethics committees in 2004. Data collection was carried out in Orkney between 2005 and 2007. Informed consent and blood samples were provided by 1019 Orcadian volunteers who had at least one grandpa rent from the North Isles of Orkney.Figure Baseline characteristics of the five participating cohorts are shown in Table P-value thresholds: 0.017 for SBP and 0.013 for DBP  years old in NSPHS to 56.6 (with standard deviation 15.6) years old in CROATIA-Vis. and the proportion of females ranged from 53.2% in NSPHS to 57.0% in ORCADES. The means and standard deviations of the concentration of the 151 phospholipids across the five cohorts are shown in Supplementary Table The significant associations were generally attenuated upon adjustment for BMI, HDL-C, TC, lipid-lowering medication and T2D status in model 2, with the proportion of the effect estimate decreased ranged from 4.5% for the association between PC 32:1 and SBP to 46.2% for the association between PE 40:6 and DBP, but all of the associations remained significant  were individually significantly associated with incident hypertension in our study , 471 SNPs for SBP (R2 = 4.0%) and 506 SNPs for DBP (4.3%). The F-statistics ranged from 55.1 for the MR in PE 40:4 to DBP to 105.7 in PE 40:5 to SBP and DBP. The two PCs, i.e. PC 32:1 and PC 40:5 were also performed using the summary statistics of Biocrates platform. However, no significant results were found in either MR test , T2D and related traits . Meanwhile, the genetic determinants of these phospholipids also highly and genome-widely associated with lipoproteins, blood cell counts, heart rate, pulse rate, glucose and many potential pleiotropic traits, e.g. male pattern baldness, height, etc. No significant association was identified between the genetic susceptibility of blood pressure and phospholipids by MR approach, in either direction.12; among the 11 replicated associations in the current study, ten associations were replicated in the Mexican–American population using the current Bonferroni P-value adjustment  are replicated but also validated by a previous study12. This provides evidence that the associations of the specific phospholipids and blood pressure are genetically driven. FADS1, FADS2 and TMEM258 are all located on chromosome 11 and band q12.2 (11q12.2) in linkage disequilibrium. Our findings that they are also genome-widely significantly associated with heart and pulse rate raise the chance that phospholipids metabolism may be implicated in the relationship with blood pressure through the pleiotropic effect of genes located in 11q12.2. An in-depth study in the (pleiotropic) role of gene FADS/TMEM258 on the association of phospholipids, blood pressure and these traits is highly suggested.All the blood pressure-related phospholipids identified in the current study have side chains that include poly-unsaturated fatty acids. For example, phospholipid identified with PE 40:5 as for the number of carbon: double bond includes a fraction of 18:0 (sn-1)/22:5(sn-2) with docosapentaenoic acid (DPA) in the sn-2 position. The same PE 40:5 measurement with our MS method also includes a fraction of 18:0/22:4, but also 18:1/20:4 which is commonly found as arachidonic acid (omega-6) and associated with unfavorable cardiovascular outcomes52. In the current study, we found a significant association of anti-hypertensive drug intake and the blood pressure related phospholipids. Following the route of the previous large GWAS study of blood pressure which adjusted for anti-hypertensive drugs intake and using MR to overcome confounders in the association of blood pressure and phospholipids, we still cannot fully exclude the effect of anti-hypertensive drugs on phospholipids. Indeed, one of the genes we identified, ALDH1A2 has been implicated in coronary artery calcification53 and is known to interact with atenolol, a beta blocker that is prescribed to treat high blood pressure and irregular heartbeats (arrhythmia)54.This asks for more careful exploration of the difference between the effect of hypertension and the effect of anti-hypertensives.The strengths of this study include the use of detailed characterized phospholipid data in a large sample size, as well as the use of replication panels. A multi-omics approach and the integration of genomic, metabolomic and epidemiologic data were performed to maximize the in-depth research of the mechanism. One of the limitations is the small number of incident hypertension cases in the current study. However, the integration of genetic data has raised an interesting hypothesis to be tested in future pathophysiological studies, in human beings and animals. To our knowledge, this is the first study performing MR on phospholipids and blood pressure. Though no significant findings were identified in the current study, the development of high-throughput technology on lipidomics will facilitate the discovery of more genetic determinants for the phospholipids and improve the strength of the instrumental variables. Previous studies reported that some anti-hypertensive drugs may have an effect on metabolism as wellIn conclusion, we show eight phospholipids in the circulation that significantly associate with blood pressure and show strong clustering with components of cardiometabolic disease. These phospholipids collectively associate with incident hypertension and improve the discrimination effect of previous prediction model. Our cross-omics analyses show that phospholipid metabolites in circulation may yield insight into blood pressure regulation and raise a number of testable hypothesis for future research.All methods were carried out in accordance with relevant guidelines and regulations in the method section.Supplementary Information.Supplementary Figures.Supplementary Tables.


To evaluate a possible influence of anti‐cyclic citrullinated peptide autoantibodies (ACPA) ‐ positive rheumatoid arthritis (RA) on visual field (VF) testing in patients with arterial hypertension (aHT).®) and flicker perimetry (Pulsar®). Results were analysed for a possible correlation with blood pressure or RA‐activity.We conducted an observational cross‐sectional study comparing patients with ACPA‐positive RA and aHT, patients with aHT and healthy subjects. Further inclusion criteria were visual acuity (VA) of 0.8 or better and age between 40 and 60 years. VF testing was performed with standard automated achromatic perimetry (SAP), short wavelength automated perimetry (SWAP) (Octopus 300p = 0.001) and with hypertensive patients . In SAP we observed a significant difference between patients with RA and healthy subjects regarding loss variance (LV) . Flicker perimetry did not demonstrate significant differences between groups. A correlation of VF changes with blood pressure level or RA‐activity was not observed.Twenty subjects with RA and aHT, 26 patients with aHT and 22 healthy participants were examined. Significant differences were found for mean sensitivity (MS) in SWAP comparing RA‐patients with healthy participants (ΔMS −3.06, Patients with ACPA‐positive RA and aHT showed significant impairment of VF performance in SWAP compared to patients with aHT alone and healthy subjects. SAP also revealed a significant difference of LV between RA‐patients and healthy subjects. aHT does not seem to induce functional changes in VF testing alone. Systemic comorbidities are very common in elderly ophthalmology patients, although they might not be the actual reason for a consultation. Arterial hypertension (aHT) has become a worldwide public health challenge, particularly in patients over 50 years of age. According to recent epidemiological data, 55% of the German population is affected by this condition.Rheumatoid arthritis is primarily characterised by inflammation and swelling of multiple synovial joints. However, extra‐articular organ manifestations such as heart, lung, kidney, skin and eye involvement can occur either at the beginning or during the course of the disease. Ophthalmic involvement is very common in chronic inflammatory rheumatic diseases and can, in principle, affect every part of the eye.Interestingly, we still know very little about how these two common comorbidities affect visual physiology. Indeed, morphological changes of the retina in association with aHT have been identified clinically in funduscopy and described in the classification of hypertensive retinopathy.et al. did not show changes in white‐on‐white perimetry function in patients with aHT alone.Endothelial dysfunction caused by chronic hypertensive conditions resulting in atherosclerotic vascular changes could impede microhemodynamics in retinal vessels.Therefore, the aim of this study was to investigate whether early retinal perfusion related damage to the VF can be detected through flicker perimetry or short wavelength automated perimetry, and if RA as an additional vascular risk factor enhances these VF defects in a linear or nonlinear manner. A secondary aim is to explore a possible correlation between perimetric examinations, RA disease activity and blood pressure. We assume that morphological retinal changes occur very late in the disease process, and so functional visual examinations can be used for the early diagnosis of vascular comorbidities.The study protocol was approved by the Clinical Ethics Committee of the medical faculty, Charite University, Berlin and adhered to the tenets of the Declaration of Helsinki. The recruitment of all subjects was conducted at the Hospital Schlosspark Klinik, Berlin in the Department of Internal Medicine and Rheumatology and via a bulletin to recruit the control group. A patient information sheet and a consent form were signed by all participants.Table A total of 68 patients who fulfilled inclusion and exclusion criteria were included in this cross‐sectional observational study. Each participant was allocated to one of three groups: 20 patients with RA and aHT, 26 patients with aHT solely and 22 control subjects. The distribution of female and male subjects in our groups was unequal in favour of female subjects, particularly in the rheumatoid arthritis cohort. Testing was performed on one eye per patient, which was either chosen at random if visual acuity (VA) was equal in each eye, or the eye with better VA was selected. Demographics are displayed in All subjects were between 40 and 60 years of age, in order to achieve a homogeneous patient collective. With increasing age, loss of accommodation as well as cloudiness of the optical media such as age‐related cataract leading to reduced VA and contrast are anticipated; these confounding variables were excluded by the age restriction and ocular examination. The fundamental precondition for the aHT group was aHT requiring pharmaceutical treatment. Inclusion in the RA group required a confirmed diagnosis based on the classification criteria of the American College of Rheumatology (ACR), the presence of anti‐cyclic citrullinated peptide autoantibodies (ACPAs) and coexistent aHT with medication‐based treatment.In order to avoid bias effects, exclusion criteria were other comorbidities and risk factors leading to vascular changes such as peripheral arterial occlusive disease, current smokers or cessation of smoking within one year, hyperlipidemia, coronary heart disease, stroke history, diabetes, chronic obstructive pulmonary disease and substance abuse . We used the Cut Down‐Annoyed‐Guilty‐Eye Opener (CAGE) questionnaire adapted to include drug use (CAGE‐AID) as an anamnestic screening tool for detecting problematic consumption at a cut‐off grade of 2 or higher.Distance VA was measured through the manifest refractive correction determined in a standardised ophthalmological examination to ensure subjects met the exclusion criteria. This examination included measurement of intraocular pressure (Goldmann applanation tonometry) and a clinical slit lamp examination with funduscopy to check for the presence of any abnormalities specified as exclusion criteria.Rheumatologic examination and disease activity evaluation were conducted on patients in the RA‐group. As recommended by the ACR for clinical practice, rheumatic disease activity was calculated using the following score systems: DAS‐28 (Disease Activity Score 28) and CDAI . In addition, blood samples were taken to measure ACPAs and other inflammatory parameters and blood pressure was measured using the Riva‐Rocci method).The aim of this study was to compare and correlate VF indices such as mean sensitivity (MS) and loss variance (LV) with three perimetric techniques in the three cohorts namely aHT, rheumatoid arthritis in combination with aHT and a control group.®  was used as the randomisation method to allocate each patient to one of six groups, which included each possible sequence for the three measurements. The tests were performed by the same trained personnel. To mitigate against any bias rising from a learning effect, all participants performed three other visual field tests before the actual experimental testing. Sufficient time was allowed between the tests to reduce the effects of fatigue and stress. Tests were termed reliable with <20% fixation losses, false positives and false negatives.Patients underwent three different visual field techniques within the central 30 degrees. Testing was performed in random order to prevent a sequence effect. The ‘randbetween’ function in Excel 2007www.haag‐streit.com). Using the Octopus 300 dG1 program, the threshold for the local differential luminance sensitivity (DLS) was assessed, which took 4 to 6 min to complete. Mean sensitivity (MS) was described as the average of the threshold limit values in the VF. Values were recorded in decibels and indicated diffuse distributed retinal deficiencies in the VF. Loss variance (LV) was described as the variance of deviations from the age‐related standard and was a measure for visual defect irregularity. Individual defects that represented strong outliers would lead to pathological LV values.Standard automated achromatic perimetry was performed using the SAP, Octopus 300 . Sixty six moving flicker stimuli having different frequencies were presented to measure the temporal visual function, spatial resolution and contrast sensitivity. Flicker perimetry was performed with a background luminance of about 31.8 cd m−2 and a 500 ms stimulus duration. The amplitude of flicker decreased towards the periphery. Again, outcome values were MS and LV.Flicker perimetry was performed using the Pulsar . Using the Octopus 300 cG1 programme, blue stimuli on a yellow background selectively stimulated the blue cones. SWAP testing was conducted with a background luminance of 100 cd/m2 and a 200 ms stimulus duration. The setup and procedure for this perimetric technique was similar to the SAP measurement. Again, outcome parameters were MS and LV. LV values were obtained by squaring the machine‐generated square root of loss variance (sLV), which was defined as the standard deviation of the measured deviations from the age‐related standard for all three perimetric examinations.Short wavelength automated perimetry (SWAP) or blue‐on‐yellow perimetry was carried out using the Octopus 300 , R®  and SPSS . p‐values less than 0.05 were considered as statistically significant.Statistical analysis was carried out using Excel 2007anova and post hoc t‐test. When comparing the three groups, normally distributed variables with equal variances were compared using one‐way anova and post hoc unpaired t‐test. Variables with unequal variances were compared using the non‐parametric Kruskal‐Wallis test. We applied the Mann‐Whitney‐U test for independent samples and the Wilcoxon test for dependent samples.The Shapiro‐Wilk test was used to determine whether a data set was normally distributed. Descriptive statistics were presented as mean and standard deviation (S.D.) for normally distributed variables, and median and mean absolute deviation about the median (MAD) for non‐normally distributed variables. Given a normal distribution with equal variances, perimetric variables were compared using one‐way 2 test. Pearson correlation for normally distributed samples and Spearman correlation for unequally distributed samples were used to assess the relationship between disease activity or blood pressure and perimetric parameters. To counteract the family‐wise error‐rate of multiple comparisons, the Bonferroni‐Holm correction method was applied.Nominal variables were compared using the χTable Investigation outcomes were assessed and analysed for a total of 68 patients fulfilling the inclusion and exclusion criteria, divided into three observational groups as summarised in Seven out of 26 aHT patients and five out of 20 RA patients, demonstrated degree I hypertensive fundus changes based on the classification of Keith‐Wagener.Table Table The mean (S.D.) duration of aHT was comparable in the AhT [7.6(7.6) years] and RA [9.8(8.1) years] cohorts. Therapeutic control of arterial hypertension was also similar as shown in Mean (S.D.) relative elevation level of ACPA in the RA cohort was 31.58 (42.62). Sixty percent of patients were sero‐positive for rheumatoid factor, 20% were sero‐negative and 20% had unknown status. With regard to disease activity in the RA patients, the commonly used activity scales DAS‐28 and CDAI revealed that the majority of the patient collective had a moderate to high disease activity .Table In all three perimetric strategies, no significant differences between groups were observed for the secondary indices . p = 0.004) , but not with systolic  or diastolic blood pressure .Comparison of the non‐normally distributed MS and LV values of SAP across groups with the Kruskal‐Wallis test revealed no significant difference in MS. However, a significant increase in LV was observed with in the RA group compared with the controls  and aHT groups . The Kruskal‐Wallis test was used for non‐normally distributed SWAP LV values which showed a significant effect. Hence, the Wilcoxon test was performed, which revealed a significant difference in the RA group compared to the controls  . Therefore, this cohort showed more localised deep defects in SAP than the control and aHT groups. In order to interpret this observation, it is necessary to remember that SAP measures a broad cell community and is functionally dependent on the parvocellular and magnocellular ganglion cell systems. This determines the relatively low sensitivity of this method, identifying defects only after a loss of 25‐50% of the ganglion cells.No correlation was seen between the higher LV with SAP in RA patients with blood pressure levels. One explanation might be due to the efficient blood flow autoregulation in the retinal and chorioidal vesselsWhile SAP was only capable of revealing slight differences in perimetric indices, SWAP was able to demonstrate significant differences in the main MS and LV indices in RA patients. This was not surprising because this technique assessed the functionality of blue cones and the downstream localised koniocellular ganglion cells. These are a small neuronal cell population of the human retina  and a highly specialised cell system that selectively perceives blue‐yellow stimuli.Finally, the results from flicker perimetry did not demonstrate significant changes in perimetric indices between the groups. Flicker perimetry also belongs to the group of new, non‐conventional perimetric tests providing earlier detection of VF damage by selectively stimulating certain visual pathways to overcome potential redundancy.Side effects of the numerous combination therapies in our aHT and RA patients on VF procedures were difficult to evaluate. We assume that conventional as well as biological disease‐modifying anti‐rheumatic drugs (DMARDs) and antihypertensive treatments exert a positive regulatory influence on microvessels. In contrast, non‐steroidal anti‐inflammatory drugs and glucocorticoids could worsen systemic blood pressure regulation and contribute to VF defects. It was not possible to eliminate the possible impact of medication on our results.Currently, it is not possible to differentiate between VF disturbances caused by a primary ophthalmological disease and the overlapping effects of coexisting systemic diseases. Our findings demonstrated that ACPA‐positive RA patients with aHT showed VF anomalies with highly sensitive perimetric procedures such as SWAP. However, aHT alone did not affect SAP, SWAP or flicker perimetry. Further, no clinically relevant correlation between functional VF impairment and blood pressure level or RA disease activity was detected. Nevertheless, the extent to which functional impairment in sensitive perimetric procedures such as SWAP may be a diagnostic means of monitoring interdisciplinary disease remains to be elucidated. Furthermore, it remains evident that eye care practitioners must consider systemic disorders when interpreting perimetric results, although aHT does not appear to be a confounder in the perimetric methods tested here.The authors report no conflicts of interest and have no proprietary interest in any of the materials mentioned in this article.Jascha Wendelstein: Writing‐original draft ; Writing‐review & editing . Barbara Fuchs: Writing‐original draft ; Writing‐review & editing . Sarah Schlittgen: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Software ; Visualization . Robert Zielke: Data curation ; Investigation ; Resources . Jeanette Brünner: Investigation ; Resources ; Supervision ; Validation ; Writing‐original draft . Matthias Bolz: Supervision . Rieke Alten: Data curation ; Investigation ; Methodology ; Resources . Carl Erb: Conceptualization ; Project administration ; Resources ; Supervision ; Validation .