Gaucher disease (GD) is a lysosomal storage disorder that responds well to enzyme replacement therapy (ERT). Certain laboratory parameters, including blood concentration of glucosylsphingosine (Lyso-Gb1), the lyso-derivate of the common glycolipid glucocerebroside, correlate with clinical improvement and are therefore considered candidate-monitoring biomarkers. Whether they can indicate a reduction or loss of treatment efficiency, however, has not been systematically addressed for obvious reasons. We established and validated measurement of Lyso-Gb1 from dried blood spots (DBSs) by mass spectrometry. We then characterized the assay’s longitudinal performance in 19 stably ERT-treated GD patients by dense monitoring over a 3-year period. The observed level of fluctuation was accounted for in the subsequent development of a unifying data normalization concept. The resulting approach was eventually applied to data from Lyso-Gb1 measurements after an involuntary treatment break for all 19 patients. It enabled separation of the “under treatment” versus “not under treatment” conditions with high sensitivity and specificity. We conclude that Lyso-Gb1 determination from DBSs indicates treatment issues already at an early stage before clinical consequences arise. In addition to its previously shown diagnostic utility, Lyso-Gb1 thereby qualifies as a monitoring biomarker in GD patients. GBA gene )The second part of our study builds on an unforeseen discontinuation of treatment. Such “treatment holidays” have been known from case reports e.g., ,25,26) a,26 a25,2The present study was based on a cohort of 19 mainly unrelated Gaucher patients for whom Lyso-Gb1 had regularly been monitored over up to 40 months. It was approved by the Albanian Ethic Committee ). As monitoring of the first patients started in November 2016, we defined November 1 of this year as day 1 of the study in all analyses. While monitoring of all patients is ongoing, the end of the forced treatment break (see below for details) was arbitrarily defined as the endpoint for the data reported in the present publication.GBA gene. With 13 occurrences among the 19 patients (68%), the predominant genotype was compound heterozygosity for c.1226A > G (p.(N409S), i.e., one of the most frequent Gaucher-associated GBA variants [All patients were followed at a single center in Albania by the same team of expert physicians. Twelve of the patients were male, and seven were female. Their ages at the start of the study ranged from 6 to 78 years (median 22 years). They all manifested with typical symptoms of the disease and had received a definite genetic diagnosis based on identification of biallelic pathogenic or likely pathogenic variants in the variants ) and c.[variants ). Detail2O, 1:1) and 100 μL internal standard solution. Samples were then mixed using a multitube vortex device at 2500 rpm for 30 s, kept at 37 °C for 30 min under agitation at 700 rpm, and sonicated for 1 min at maximum power. Two hundred microliters of the sonicated sample were transferred to an AcroPrep filter plate with polytetrafluoroethylene (PTFE) membrane  placed on a 96-well, V-shaped-bottom plate . Any solid particles were removed by filtration ).DBS samples (CentoCard) were prepared from ethylendiamine tetraacetic acid (EDTA) blood drawn just before administration of the enzyme. From one DBS, three punches of diameter 3.2 mm were obtained using a DBS puncher , plated in 2.2 mL round-bottom tubes , and layered with 50 μL extraction solution  coupled with an ABSciex 5500 Triple Quad mass spectrometer . The following transitions were monitored in the mass spectrometric analysis: 624.4→282.2 for the internal standard and 464.4→282.2 for Lyso-Gb1. A water-based calibration curve was added to each batch for all analyses performed for this study, with concentration ranging from 0 to 1000 ng/mL. For each batch, a quality check utilized samples with known Lyso-Gb1 concentrations ; the analyses were considered valid only when all these controls were in the predetermined range.Validation of Lyso-Gb1 quantification from DBSs followed International Organization for Standardization (ISO) 15189 .The present study validates Lyso-Gb1 quantification from DBSs. It also reveals the extent of variation of this biomarker over comparatively short time intervals and introduces concepts for how to address the resulting challenges. It eventually reveals that monitoring of Lyso-Gb1 enables flagging of absent/insufficient treatment before clinical consequences arise. With DBSs facilitating self-sampling  and with


Our results demonstrate that, in people with CF, longer fT > MIC can be achieved with a simple modification of meropenem dosing. Further studies are needed to clarify if this may translate into improved microbiological and clinical outcomes, in particular in adults with difficult-to-treat chronic infection by carbapenem-resistant Pseudomonas aeruginosa.Anti-infective treatment of pulmonary exacerbations is a major issue in people with cystic fibrosis (CF). Individualized dosing strategies and adaptation of infusion times are important concepts to optimize anti-infective therapy. In this prospective non-randomized controlled open-label trial, we compared pharmacokinetics of meropenem in 12 people with CF experiencing a pulmonary exacerbation, of whom six received parenteral meropenem 2 g tid as short infusion over 30 min and six extended infusion over 120 min. We measured blood concentrations of meropenem at five predetermined time points over 240 min and calculated differences in the percentages of the time above the minimal inhibitory concentration (fT > MIC) for meropenem concentrations >16 and >32 mg/L, respectively. Mean percentages of fT > 16 and fT > 32 mg/L were higher in the extended compared to the short infusion group (83 and 56% vs. 59% and 34%), with a statistically significant prolongation of the fT > 32 mg/L (mean 134 vs. 82 min;  Pseudomonas aeruginosa (PA) [Pulmonary exacerbations frequently occur in people with cystic fibrosis (CF). The most important pathogen chronically infecting the airways of people with CF is osa (PA) , which mosa (PA) ,3,4. Theosa (PA) . In peoposa (PA)  due to aosa (PA) ,8. Antibosa (PA) , and oftosa (PA) . Althougosa (PA) . Applyinosa (PA) . In patiIn this investigator-initiated prospective non-randomized controlled open-label trial, we enrolled 12 adults with CF and normal renal function (glomerular filtration rate ≥60 mL/min), who were admitted to our hospital due to a pulmonary exacerbation. The first 6 patients received meropenem in a dose of 2 g over 30 min tid, while the second 6 patients received the same dose regimen over 120 min. Patients were not stratified for clinical characteristics or laboratory findings. On the second day of antibiotic treatment, we measured blood concentrations of meropenem at predetermined time points . Measurements were conducted via a certified high performance liquid chromatography (HPLC) method  in both short and extended infusion regimens. The fT > MIC has repeatedly been shown to be the pharmacodynamic variable most closely linked to bactericidal activity . BecauseAll patients were consecutively recruited between October 2019 and September 2020. Institutional Review Board approval (No. 8702_BO_K_2019) was given to our study according to German regulations and all patients provided written and informed consent. All patients fulfilled the following inclusion criteria: (i) a confirmed diagnosis of CF according to European standards of care ; (ii) agp-values are two sided. p-values < 0.05 were considered statistically significant.We used SPSS Statistics 27.0  and Stata 13.0  statistical software to analyze the data. Continuous variables are shown as median and interquartile range or mean and standard deviation, as appropriate. Categorical variables are shown as numbers and percentage (%). For group comparisons, Fisher’s exact test, Chi-squared test, or Mann–Whitney U-test were used, as appropriate. Normal distribution of continuous variables was checked by Kolmogorov–Smirnov test. Linear equations were used to calculate the assumed time point of reaching therapeutic plasma levels. All reported A total of 15 consecutive adults with CF experiencing a pulmonary exacerbation were screened for eligibility of whom 12 were included in this analysis and three were excluded because of impaired renal function. A total of 12 consecutive adults were included in this analysis, of whom six received meropenem over 30 and 120 min, each. Baseline characteristics were obtained prior to meropenem treatment initiation and were comparable between groups .p = 0.037; The mean percentages of fT > 16 and fT > 32 were higher among patients who received meropenem over 120 min (extended infusion group) compared to 30 min . However, a statistically significant prolongation could only be shown for the time above 32 mg/L , whereas peak meropenem concentrations were significantly higher in the short infusion group. We preferred an infusion time of two hours rather than a 3-h or 4-h infusion as a comparator as this is more feasible to implement in a HIVAT schedule using an elastomeric infusion pump system  for self-application, according to our local standard.In this study, we analyzed the pharmacokinetics of meropenem, administered via two different infusion regimens in adults with CF experiencing a pulmonary exacerbation. The percentage of fT > 16 and fT > 32 mg/L was higher in the extended infusion group, with statistically significant prolongation of the fT > 32 mg/L (Current international guidelines for Antimicrobial Stewardship recommend individualized dosing strategies and adaptation of doses according to measured serum levels as concepts to optimize anti-infective therapy in the critically ill ,16. LikeThe use of meropenem for treatment of pulmonary exacerbation has significantly contributed to improving the quality of life and life expectancy of people with CF . It has Our study has several limitations. First, we measured the plasmatic drug concentrations, which might be different from concentrations in the lungs as the site of infection. Second, our trial had a small sample size and a single-center design.Pseudomonas aeruginosa, and to prove its feasibility in HIVAT, applying commonly used elastomer pumps for self-application.Our results demonstrate that in people with CF, longer fT > MIC can be achieved with a simple modification of meropenem dosing. Further randomized long-term studies are needed to clarify if this may translate into improved microbiological and clinical outcomes, in particular in adults with difficult-to-treat chronic infection by carbapenem-resistant


In otolaryngologic surgery, ankle is frequently used for monitoring anesthesia in place of brachial when the patient doesn´t need invasive arterial cannulation. If there is a clinically useful and Predictable link between the two readings in hemodynamic normal patient, this difference during otolaryngologic surgery, was not evaluated. We aimed to investigate the reliability and the acceptability of non invasive blood pressure measurements at the ankle compared to those obtained concurrently at the arm during otolaryngologic surgery.Eighty ASA grade I and II patients who had to be operated under general anesthesia were taken as subjects for our study. Blood pressures were measured simultaneously in the 2 limbs before induction and then every 10 minutes until the end of the surgical procedure. Readings were initiated concurrently. Statistical analysis was performed with PASW Statistics 13.There were 41 males (51.2 %) and 39 females (48.8 %). Bland-Altman analysis of mean difference between the ankle and arm (95 % limits of agreement) was -11.47 (- 23.77 to 0.82) mmHg for systolic blood pressure (SBP), -7.89 (-19.16 to 3.36) mmHg for diastolic blood pressure (DBP) and - 9.09 (18.19 to 0.00) mmHg for mean arterial pressure (MAP). Non-parametric analysis showed that 67.5 % of SBP, 46.2 % of DBP and 56.2 % of MAP measurements differed by > 10mmHg.Ankle BP cannot be used routinely in otolaryngological surgery. Although, the ankle can be used as an alternative where the arm cannot be used taking into account a difference. Good surgical field visibility is one of the basic prerequisites for a precise and safe otolaryngologic surgery and the main obstacle to good visibility is excessive perioperative bleeding. Studies have shown a variety of methods reducing bleeding in the surgical field through: lowering the mean arterial pressure (MAP), lowering heart rate (HR), local anemization with adrenaline, preoperative use of steroids and reversing the trendelenburg position, which reduces blood supply in the surgical field . Controlet al. have shown in their study that inadequate BP monitoring is associated with inadequate intraoperative blood transfusions, vasopressor infusions and antihypertensive medication administration [Given the particularity of cephalic surgery and the difficult access to the head and upper limb, ankle BP allows a better maneuverability for the anesthetists. Lack of ankle-arm BP differences, can lead to error in the anesthetic management. Wax stration . Due to Our study was approved by our Institutional Ethics Committee over a period of two months (April and May 2019). After obtaining written informed consent from all participants, eighty ASA grade I and II patients who had to be operated under general anesthesia were taken as subjects for our study. Patients with significant cardiovascular pathology, hypertension, with BP above 160/110 mmHg, musculoskeletal abnormalities, pregnant women, child under 14 and patients known to have peripheral arterial disease were excluded. After the patient was shifted to the operating theater, he was asked to relax for five minutes. The circumferences of the left arm immediately proximal to the antecubital fossa and the left ankle immediately above the malleolus were used to select appropriately sized NIBP cuffs. The cuff bladder was sited anteriorly on the arm and posteriorly on the ankle. The standard position was made with the arm resting on the operation theatre table, at the level of the heart. Blood pressures were measured simultaneously in the two limbs with a GE healthcare carescape V100 vital signs monitor before induction and then every 10 minutes until the end of the surgical procedure.Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were recorded by a nurse single researcher, who did not have clinical responsibility for the case. Readings were initiated concurrently. Any readings that took a prolonged period to measure were noted. Drugs used for induction of anesthesia were the same in whole patients . The attending anesthetist was blinded to the ankle blood pressure readings. In addition to the registration of the BP values, the following data were collected: demographic characteristics, medical background, ASA physical status, type of otolaryngologic operation, the need for deepening anesthesia, the wake-up delay and the existence of a wake-up incident. The paired t-test was used for comparisons of paired parametric data and the McNemar test for paired categorical data. The t-test was used for unpaired parametric data and Fisher´s exact test for unpaired categorical data. Modified Bland-Altman analysis partitioning between - and within - patient agreement was used to compare arm and ankle blood pressure measurements [Eighty patient candidates for otolaryngologic elective surgery were enrolled into the study. The data on 80 patients were included for analysis. There were 41 males (51.2 %) and 39 females (48.8 %). The basic demographic data are summarized in As early as 1925, it was found that the systolic BP was 20 - 40 mmHg higher in the leg than in the arm in normal subjects at rest . If therThis shows that this difference is statistically and clinically significant and translates to a non-detection of significant variation of BP. Particularly when comparing SAP between the two sites of measurements, the degree of discrepancy in unacceptable for ankle NIBP to be used routinely. DBP fared best, with the mean difference between the arm and ankle being - 7.89mmHg. Although it seems very small, reading at opposite extremes of the 95% limits of agreement of arm and ankle could differ by as much as 22.52mmHg, with the actual CI range being -19.16; 3.36mmHg and only 53% of patients had a mean difference lower than 10mmHg. This shows that this difference is statistically significant and clinically not significant, within the clinically acceptable error range of 10mmHg. The MAP was also statistically significantly different between the arm and the ankle, with higher MAPs noted in the ankle. The MAP actual mean difference between the arm and the ankle fell within the clinically acceptable error range of 10mmHg but was 9mmHg higher than the mean arm MAP. Extreme of the 95% limits of agreement of arm and ankle could differ by as much as 18.19 for MAP.et al. [Knowing that the MAP is the perfusion pressure of the organs, this indicates that in our study, the value of this pressure found on the ankle is always higher than that found at the arm. The basis of NIBP measurement by oscillometry is the determination of MAP from the maximum pulse oscillation during cuff deflation . Systoliet al.  in a stuet al., [et al. compared arm and ankle blood pressure under spinal anesthesia for cesarean section [The agreement was closer for DBP and MBP measurements. The difference in BP between the upper and lower limbs in pregnant women has been subject of several studies. Zahn et al.,  found th section  and foun section  concludeIn children 8 years and younger, BPs were found to be lower in the leg than in the arm , whereaset al. concerning the impact of controlled induced hypotension on cognitive functions of patients undergoing functional endoscopic sinus surgery (FESS), they found that the degree of controlled intraoperative hypotension during FESS did not influence the results of psychometric tests as well as no complications in the functions of kidneys, lungs, nervous system and cardiovascular system were observed [Despite that, no neurological disturbances were found in any of the patients in the postoperative period. Also, in the study conducted by Stanistaw observed . It seemobserved . In our observed .A comparison of NIBP in the arm and ankle in admitted patients to otolaryngologic surgery has shown that NIBP readings taken from the ankle tend to be higher when compared with those taken from the arm. The least significant difference found was the DBP, which unfortunately does not have many clinical applications as a target in the resuscitation setting. Given the great lack of precision and the wide limits of agreement, ankle BP cannot be used routinely especially in otolaryngologic surgery, whose hypotension controlled with the maintenance of good hemodynamic stability, is the basis of good anesthetic management. Although, the ankle can be used as an alternative site for measurement of BP where the arm cannot be used, taking into account a difference.There is a difference in arterial pressure between the arm and ankle;Controlled hypotension is a common procedure during anesthesia applied to patients undergoing otolaryngologic surgery.In otolaryngologic surgery ankle NIBP tend to be higher when compared with those taken from the arm;Ankle BP cannot be used routinely especially in otolaryngologic surgery.The authors declare no competing interests.


Patients with severe refractory asthma (SRA), even when using high doses of multiple controller medications in a regular and appropriate way, can have persistent complaints of exercise limitation.2) < 80% of predicted at peak exercise, without cardiac impairment or ventilatory limitation. Exercise-induced bronchoconstriction (EIB) was defined as a forced expiratory volume in one second ≥ 10% lower than the baseline value at 5, 10, 20, and 30 minutes after CPET.This was a cross-sectional study involving patients with SRA , who presented no signs of a lack of asthma control other than exercise limitation. We also evaluated healthy controls, matched to the patients for sex, age, and body mass index. All participants underwent cardiopulmonary exercise testing (CPET) on a cycle ergometer, maximum exertion being defined as ≥ 85% of the predicted heart rate, with a respiratory exchange ratio ≥ 1.0 for children and ≥ 1.1 for adolescents. Physical deconditioning was defined as oxygen uptake (VO2 (VO2peak), there was no significant difference between the patients and controls, (P = 0.10). Among the patients, we observed isolated EIB in 30%, isolated physical deconditioning in 25%, physical deconditioning accompanied by EIB in 25%, and exercise-induced symptoms not supported by the CPET data in 15%.We evaluated 20 patients with SRA and 19 controls. In the sample as a whole, the mean age was 12.9 ± 0.4 years. The CPET was considered maximal in all participants. In terms of the peak VOPhysical deconditioning, alone or accompanied by EIB, was the determining factor in reducing exercise tolerance in patients with SRA and was not therefore found to be associated with a lack of asthma control. Children and adolescents with asthma should be encouraged to engage in regular physical activity; one of the objectives of adjusting the treatment regimen is to allow the patients to engage in such activities without any limitations . Before 2)  is the gold standard for the investigation of exercise limitation, which, in individuals with asthma, can be attributed to airway obstruction, ventilatory limitation, an increased level of perception of dyspnea, or exercise-induced bronchoconstriction (EIB) . For the2) . A reducitioning .Studies attempting to determine whether children and adolescents with asthma are less active than are their healthy peers have produced conflicting results. In some studies, asthma was not associated with physical inactivity , whereasAlthough some studies have evaluated the level of physical activity in patients with asthma using objective measures  few haveThe present study aims to determine the causes of exercise limitation in children and adolescents with severe asthma that was refractory to regular, appropriate pharmacological treatment. The specific focus was on exercise limitation as the only manifestation of a lack of asthma control.Hospital das Clínicas and the Laboratory for Evaluation and Research in Cardiorespiratory Performance, both of the Federal University of Minas Gerais, in the city of Belo Horizonte, Brazil. All medications necessary for the treatment of the children and adolescents with asthma, as well as the spacers for the inhalers, are made available free of charge via the “Wheezy Child” Program of the city of Belo Horizonte and via the Minas Gerais State Public Health Care System , exhibiting appropriate inhaler techniques, having no unaddressed comorbidities, having a confirmed rate of treatment adherence ≥ 80% in the last 4 months, and having had no other complaints indicative of a lack of asthma control. We determined the rate of adherence to the use of the inhaled corticosteroid, in accordance with the protocol of the facility , by calcWe also evaluated a group of 21 healthy controls, recruited from public and private schools in the city of Belo Horizonte, who were matched to the patients for sex, age, and body mass index. Asthma and allergic rhinitis were excluded by using the International Study of Asthma and Allergies in Childhood questionnaire , on whicThe study was approved by the Research Ethics Committee of the Federal University of Minas Gerais . All patients and their parents or legal guardians gave written informed consent.In the patients, the level of asthma control was assessed by applying the GINA criteria, which involve questioning subjects about daytime and nighttime symptoms, use of short-acting bronchodilators and any activity limitation due to asthma . The resDermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Alternaria alternata, Aspergillus fumigatus, cat epithelium, dog epithelium, and cockroach allergens (from Periplaneta americana and Blattella germanica).For the diagnosis of allergic rhinitis, we applied the criteria established in the Allergic Rhinitis and its Impact on Asthma guidelines . Skin pr1) of 12% or 200 mL after administration of a bronchodilator  in a previous spirometry examination was considered indicative (1 in one or more measurements during the 30-minute post-CPET period was considered indicative of EIB (1 (from the pre-exercise level) is > 10% but < 25%, > 25% but < 50%, or > 50%, respectively  . The IPAvis-à-vis the responses on the IPAQ-SF, the participants were classified as active or inactive.Current guidelines for physical activity in childhood and adolescence recommend that young people engage in ≥ 60 minutes/day of moderate exercise on five or more days a week, for a weekly total of at least 300 minutes of moderate exercise or 150 minutes of vigorous exercise . On the The CPET was performed on a cycle ergometer coupled to a metabolic cart . For the first 2 minutes, the participant was allowed to cycle with no resistance (at 0 w). Subsequently, the workload was increased once every minute and the participant was instructed to maintain a constant cycling speed of 50–70 rpm . The pro2), respiratory exchange ratio, carbon dioxide output (VCO2), the ventilatory equivalents (VE/VO2 and VE/VCO2) and the equivalent of the peak VO2 over the workload in watts (VO2peak/w), the ratio between VO2peak and heart rate (VO2peak/HR), and the ratio between VE and maximal voluntary ventilation  reached at peak exercise minute ventilation (VE), oxygen uptake (VOtilation . Those volerance . The pre2peakVO (mL/min) = 52.8 × body weight − 303.4for boys—2peakVO (mL/min) = 28.5 × body weight + 288.2for girls—Blood pressure, HR, and peripheral oxygen saturation were measured continuously, being recorded before the test, every 2 minutes during the test, immediately after the test, and 2 minutes after the test. Before and after the test, dyspnea and lower-limb fatigue were assessed with the modified Borg scale .Participants were instructed to eat lightly on the day of the test, as well as not to drink coffee, tea, of caffeinated soda in the last 2 hours prior to the test. The asthma patients were instructed to suspend their use of long-acting bronchodilators for 12 hours prior to the test, of anticholinergics for 8 hours prior, of short-acting antihistamines for 24 hours prior, and of long-acting antihistamines for 72 hours prior .2peak was <80% of the predicted value, the patient was categorized as having ventilatory limitation.The CPET was considered maximal when the HR was ≥85% of the predicted value and the respiratory exchange ratio was appropriate (≥1.0 for children and ≥1.1 for adolescents), assuming that the patient had achieved the maximum effort (as evidenced by sweating and fatigue) during the test . When th2peak/HR ratio, not achieving 80% of the maximum predicted HR, not showing a ≥ 10 mmHg increase in systolic blood pressure at peak exercise, and showing a reduction in VO2peak to below 80% of the predicted value were categorized as having cardiac impairment. Hyperventilation was defined as a VO2peak ≥ 80% of the predicted value, an increased VE/VCO2 at the anaerobic threshold (>34), an elevated respiratory rate (>55 breaths/min), and normal peripheral oxygen saturation (>95%). Physical deconditioning was defined as a reduction in VO2 to below 80% of the predicted value, without evidence of cardiac impairment or ventilatory limitation . The distribution of the variables was evaluated with the Shapiro–Wilk test. Continuous variables are expressed as mean and standard deviation or as median and interquartile range, whereas categorical variables are expressed as absolute and relative frequencies. For comparisons between groups (patients vs. controls), the unpaired Student’s Of the 60 patients in the cohort, 40 were excluded: 36 because they exhibited signs of a lack of asthma control other than exercise limitation (nighttime or daytime symptoms) or had used a short-acting bronchodilator in the last 4 weeks and 4 because they did not complete the protocol . Of the 21 controls recruited, two were excluded: one because of an abnormal electrocardiogram finding and one for not having met the criteria for a maximal CPET. Therefore, the final sample comprised 20 patients and 19 controls.The clinical and functional characteristics of the study sample are shown in P = 0.04). The VO2peak/workload ratio was indicative of less ventilatory efficiency in the patients than in the controls (P = 0.01). 2peak/workload ratio at peak exercise. The results of the CPET at anaerobic threshold (AT) are in a The results of the CPET are shown in 1 was observed, the mean reduction was 15.4 ± 6.9% in the asthma group and 15.2 ± 1.9% in the control group (Among the participants in which a > 10% post-CPET reduction in FEVol group .P = 0.082). The CPET identified the cause of the exercise limitation in 85% of the patients. Vocal cord dysfunction was not identified in any of the participants.The most important finding of the present study was that the presence of physical deconditioning, with or without EIB, was the cause of the exercise limitation in 55% of the asthma patients in our sample. When we analyzed EIB in isolation, we found a prevalence of only 30%, lower than that reported in the literature, in which overall prevalence of EIB has been found to be 81% among individuals with severe asthma  and appr2, to be 31 and 55%, respectively, among individuals with severe asthma, higher than the 25% observed in the present study for physical deconditioning in isolation. However, those authors did not evaluate EIB. As mechanisms for limiting exercise tolerance, physical deconditioning and EIB can act in isolation or in concert. In our study, we found that the frequency of EIB accompanied by physical deconditioning, as assessed through CPET, was twice as high in the patients as in the controls. In addition, of the 60% of our patients that presented EIB, half were physically deconditioned. As previously mentioned, none of the patients in our sample were obese.In a population-based study conducted in Greece , free ruThe World Health Organization (2011) recommends that children engage in moderate to vigorous physical activity for a minimum of 60 minutes per day, although it does not provide any guidance regarding the exact proportion of vigorous activity , which hChildren tend to alternate between short periods of vigorous physical activity and longer periods of light to moderate physical activity. Physiological indicators of physical conditioning are important biomarkers for monitoring overall cardiorespiratory, muscle, and bone health . HoweverIn the present study, the patients with asthma consumed the same amount of oxygen but completed the test with a 25% lower workload in comparison with the controls, suggesting that individuals with asthma have less aerobic capacity. In addition, the patients reported less dyspnea, according to the Borg scale, than did the controls, possibly due to the lower workload they endured or the poor perception of symptoms that some asthma patients have .1, before or after bronchodilator use, does not correlate with VO2peak, the anaerobic threshold, or peripheral oxygen saturation . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.LL, EM, RF, and VG conceptualized the study. EM, LL, RF, and VG contributed to the study methodology. EM, LL, RF, VG, FL, MQ, and LO contributed to the formal analysis. LL, EM, and FL contributed to the software. All authors contributed to the data curation, validation, investigation, and writing . All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Patients with diabetes mellitus are at a high risk of developing postoperative acute kidney injury. For patients receiving laparoscopic surgery, standard-pressure pneumoperitoneum (SPP) currently applied in clinical practice also undermines renal perfusion. Several studies have shown that low-pressure pneumoperitoneum (LPP) might reduce pressure-related ischemic renal injury. However, LPP may compromise the view of the surgical field. Previous studies have indicated that deep neuromuscular blockade (NMB) can ameliorate this issue. However, the conclusion is still uncertain. The hypothesis of this study is that the joint use of LPP and deep NMB can reduce perioperative renal injury in diabetic patients undergoing laparoscopic pelvic surgery without impeding the view of the surgical field.This is a double-blinded, randomized controlled trial using a 2 × 2 factorial trial design. A total of 648 diabetes patients scheduled for major laparoscopic pelvic surgeries at Peking Union Medical College Hospital will be randomized into the following four groups: SPP (12–15 mmHg) + deep-NMB (post-tetanic count of 1–2) group, LPP (7–10 mmHg) + deep-NMB group, SPP + moderate-NMB (train-of-four of 1–2) group, and LPP + moderate-NMB group. The primary outcome is serum cystatin C level measured before insufflation, after deflation, 24 h postoperatively, and 72 h postoperatively. The secondary outcomes are serum creatinine level, intraoperative urine output, erythrocytes in urinary sediment, renal tissue oxygen saturation, Leiden’s surgical condition rating scale, surgery duration, and occurrence of bucking or body movement.This study will provide evidence for the effect of LPP on renal function protection in patients with diabetes undergoing laparoscopic pelvic surgery. The trial can also help us to understand whether deep NMB can improve surgical conditions.ClinicalTrials.gov: NCT04259112. Prospectively registered on 5 February 2020. Globally, the number of diabetes mellitus patients has quadrupled in the last 30 years, and China has the fastest growing epidemic of type 2 diabetes . More thPostoperative acute kidney injury (AKI) affects 13–20% of patients undergoing major surgery , 7 and iPatients undergoing abdominal surgery are more likely to develop AKI than those undergoing other noncardiac, nonvascular surgeries , 18. PneLow-pressure pneumoperitoneum  is recommended to minimize the ischemic organ injury caused by standard-pressure pneumoperitoneum  . LPP hasversus SPP on renal function protection in patients with diabetes undergoing laparoscopic pelvic surgery and to evaluate whether deep NMB can improve surgical conditions compared with moderate NMB. We hypothesize that the use of LPP, facilitated by deep NMB, can improve postoperative renal function after laparoscopic pelvic surgeries.Therefore, the objective of this study is to assess the effect of LPP This is a double-blinded, randomized controlled trial using a 2 × 2 factorial trial design, aiming to investigate the effect of pneumoperitoneum pressure and the depth of NMB on renal function in patients with diabetes undergoing laparoscopic pelvic surgery. Participants will be divided into the following four groups: 1) standard PP + deep NMB (S + D), (2) low PP + deep NMB (L + D), (3) standard PP + moderate NMB (S + M), and (4) low PP + moderate NMB (L + M) , which is a tertiary general teaching hospital with 2200 beds in Beijing, China. An average of 20 elective pelvic surgeries are performed by five surgeons on each working day. The Ethics Committees of this hospital (reference number: ZS-1654) approved the trial.All patients scheduled for elective laparoscopic pelvic surgeries under general anesthesia will be screened for eligibility by an investigator who will not be involved in the process of randomization, intervention, data collection, or outcome assessment. Patients will be considered eligible if they meet the inclusion criteria and do not meet the exclusion criteria. Written informed consent will be obtained after evaluation.Age 18–70 yearsAmerican Society of Anesthesiologist (ASA) Classification II-IIIDiagnosis of diabetes mellitusEstimated surgery duration > 3 h. The estimated surgery duration will be determined by the average duration of the same type of surgery by the same surgeon in the last year.The inclusion criteria are as follows:Not willing to participate in the study or unable to sign informed consentDiagnosis of any kidney diseases other than diabetic kidney diseasesSevere renal insufficiency defined as serum Cr > 2 times the upper limit of normal, urine output < 0.5 ml/kg/h, or estimated glomerular filtration rate (eGFR) < 60 ml/hSevere liver, lung or heart dysfunctionKnown or suspected neuromuscular diseaseUse of drugs that may affect neuromuscular block monitoringSevere diabetic neuropathy or any other peripheral neuropathyKnown or suspected allergy to general anestheticsFamily history of malignant hyperthermiaPrevious history of pelvic surgeryThe exclusion criteria are as follows:After enrollment, just prior to the surgery, the patients will be randomized by a statistician without the knowledge of random allocation in a balanced manner to one of the four groups. The allocation sequence will be computer-generated using random block sizes and will be kept in an opaque sealed envelope. These assignment envelopes will only be opened by a nurse, who will set the pneumoperitoneum pressure, prepare the muscle relaxant, and monitor the depth of NMB. Therefore, participants, anesthesiologists, surgeons, and outcome assessors will all be blinded to the allocation throughout the trial.2), invasive arterial blood pressure (ABP), stroke volume (SV) and stroke volume variation (SVV) measured by LiDCO-rapid™, inspiratory and expiratory gas analysis, pressure of end tidal carbon dioxide (PetCO2), tidal volume, airway pressure, bispectral index (BIS), cerebral oxygen saturation, renal tissue oxygen saturation, nasopharyngeal temperature, and peripheral skin temperature. Anesthesia will be induced via the target-controlled infusion (TCI) of propofol (at an effect-site concentration of 6 μg/ml) and a bolus injection of 2–3 μg/kg fentanyl or 0.2–0.3 μg/kg sufentanil. Endotracheal intubation will be performed after the administration of a predefined dose of rocuronium based on the grouping. Anesthesia will be maintained with a TCI of propofol and continuous infusion of remifentanil. Mean blood pressure and heart rate fluctuations will be maintained within 20% of baseline, and the targeted BIS value will be 50–60. Fluid infusion will be adjusted to maintain SV < 10% and SVV < 13. Patients will be mechanically ventilated using a volume-controlled pattern with a tidal volume of 7–9 ml/kg and a frequency of 10–13/min. PetCO2 will be maintained between 35 and 45 mmHg. Forced-air warming will be used to maintain central and peripheral skin temperatures ≥ 36 °C and 32 °C, respectively.An intravenous (IV) cannula will be inserted for the administration of anesthetic drugs. All enrolled patients will receive total intravenous anesthesia (TIVA). Intraoperative monitoring will include electrocardiography (ECG), pulse oximetry  . The armAfter successful calibration, 0.6 mg/kg rocuronium will be administered in the two moderate-NMB groups (S + M group and L + M group) and 1 mg/kg in the two deep-NMB groups (S + D group and L + D group). The TOF ratio will be measured every 1 min during anesthesia. When there is no response to TOF, the PTC pattern will be applied every 5 min, which is a single-twitch stimulation (2 Hz for 0.2 ms) starting 3 s after a tetanic stimulus (50 Hz for 5 s). In the two moderate-NMB groups, rocuronium will be continuously infused at an initial rate of 0.3 mg/kg/h, and the rate will be adjusted to maintain a TOF of 1–2. In the two deep-NMB groups, the infusion rate will be 0.6 mg/kg/h at first and then titrated for a targeted PTC of 1–2. Rocuronium infusion will be discontinued when pneumoperitoneum deflates.Rocuronium (10 mg/ml) will be diluted by the nurse aware of the allocation. The concentration used at induction will be 0.1 × body weight (Wt) mg/ml in the deep-NMB groups and 0.06 × Wt mg/ml in the moderate-NMB groups; thus, 10 ml will be given in all four groups. The concentrations used for continuous infusion in the deep-NMB and moderate-NMB groups will be 0.06 × Wt mg/ml and 0.03 × Wt mg/ml, respectively; thus, the initial rate should be 10 ml/h. Therefore, one cannot tell the grouping from the injection dose or pump rate. An opaque curtain will be used to cover the NMB monitor. Only the nurse will be allowed to access the monitor and see the screen. If the NMB depth deviates from the target, the nurse will ask the anesthesiologist to adjust the pump rate.The nurse aware of the allocation will be the first to enter the operating room. She will initially set the pneumoperitoneum pressure at 7 mmHg in the LPP group and 12 mmHg in the SPP group. The pneumoperitoneum pressure can be increased to a maximum of 10 mmHg in the LPP group and 15 mmHg in the SPP group, if the surgery cannot proceed due to poor surgical sight. All the screens displaying intra-abdominal pressure will be covered with opaque tape; thus, the patients, surgeons, and data collectors will be blinded. The pneumoperitoneum pressure will remain unchanged during surgery.A well-validated, 5-point scale, Leiden’s surgical condition rating scale Table  33], wi, wi33], During the surgery, the surgeon will evaluate with the scale every 15 min starting from the introduction of the first trocar. At the same time, a 1-min video will be recorded by a camera connected to the endoscopic probe. The other four surgeons who will not perform this surgery will evaluate by watching videos independently. If the surgical condition suddenly worsens, additional assessments will be performed.Patients will stay in the postanesthesia care unit for 15–20 min and then return to the surgical ward, where they will spend at least 3 days before discharge. Nephrotoxic medications will be avoided in the postoperative period.The primary outcome is serum CysC level, which is a sensitive biomarker of renal injury, and this will be measured 30 min before pneumoperitoneum insufflation (T1), 30 min after pneumoperitoneum deflation (T2), 24 h postoperatively (T3), and 72 h postoperatively (T4). Among them, T1 will be the baseline. The mean difference of the serum CysC between groups will be used as the effect measure. We will compare the serum CysC between groups throughout T2, T3, and T4, using mixed-effect model which can provide an estimate for the overall effect in the whole timeframe.The secondary outcomes include four indicators of renal function: (1) serum Cr level measured at T1, T2, T3, and T4; (2) intraoperative urine output; (3) the presence of erythrocytes in urinary sediment on postoperative days 1 and 3; and (4) renal tissue oxygen saturation measured every 15 min following pneumoperitoneum insufflation, and cerebral oxygen saturation will be used as an internal reference to adjust individualized differences. The other three secondary outcomes selected to evaluate surgical conditions are (1) surgical condition rating scale measured every 15 min starting from the introduction of the first trocar by five surgeons independently, (2) duration of surgery, and (3) the occurrence of bucking or body movement during the surgery. The details of the secondary outcomes are demonstrated in Table The following demographic data and previous medical history will be collected by a data collector preoperatively from the medical records: sex, age, height, weight, duration of diabetes, and the presence of any complications of diabetes.Peripheral venous blood samples will be extracted at T1, T2, T3, and T4, and urine samples will be obtained on the morning of postoperative days 1 and 3. The samples will be sent immediately to the clinical laboratory in PUMCH and will be analyzed by the staff there. Blood samples will be centrifuged at room temperature to obtain serum. Serum CysC concentration will be determined by sandwich enzyme-linked immunosorbent assay (ELISA), and Cr will be measured using an enzymatic method. Urine samples will be analyzed by automated urine microscopy analysis. All the samples will be handled within one hour since their arrival at the laboratory. We do not plan to store the samples after the study.During the surgery, in addition to the secondary outcomes, two data collectors will record the heart rate, ABP, SV, and SVV every 15 min. They will also record the amount of crystalloid and colloid infused, the amount of transfusions, and the estimated blood loss.There is an anesthesiologist and a surgeon in charge of monitoring the adherence to the protocol. Any suspected nonadherence during participant recruitment, randomization, intervention, data collection, or outcome assessment will be recorded. Based on the intention-to-treat (ITT) principle, all the randomized participants will be analyzed, despite any nonadherence to the protocol.Data will be collected in the paper-based case report form (CRF) and then entered into a digital database by two independent investigators. Each patient will be distinguished by a unique serial number, and his or her name will be concealed. The database will be secured by a password, which will be restricted to the Data Safety Monitoring Committee (DMC), which is composed of two anesthesiologists with more than 10 years of experience. They will check the completeness and accuracy of the data, and the auditing will be independent from that of the other investigators. The information recorded in medical charts and saved in monitors will be traced to correct and supplement the data. The database will be locked up after the entry of the last case. The allocation will not be unblinded until all data analysis is completed; thus, the data analyst will also be blinded.A previous study  reportedWe will first compare the two SPP groups with the two LPP groups and then compare the two deep-NMB groups with the two moderate-NMB groups. Finally, all four groups will be compared.The baseline characteristics and intraoperative factors of the enrolled patients will be evaluated using descriptive analysis. Continuous variables will be summarized as the mean  ±  standard deviation (SD) or median  as appropriate, whereas categorial variables will be summarized as the number (percentage). The baseline characteristics will be compared between groups using the standardized difference, and a standardized difference < 0.2 will be considered an acceptable balance between groups. Unbalanced variables at baseline will be included in the ANOVA or mixed-effects model to adjust for the potential confounding effect.Considering the multiple measures of CysC, Cr, surgical condition rating scale, and renal tissue oxygen saturation at different time points, the effect of intervention in the whole timeframe will be analyzed by a mixed-effects model. Other continuous variables will be compared by two-sided independent Student’s t tests or the Mann–Whitney U test, as appropriate. Categorical variables will be analyzed by a Chi-square test or Fisher’s exact test, as appropriate. The Bonferroni method will be used to correct the potential increase in the probability of a type I error when analyzing the differences between the four groups.We will also investigate the interaction between pneumoperitoneum pressure and NMB on postoperative renal function. The difference in serum Cysc levels at T2 between the S + D and L + D groups will be compared with that between the S + M and L + M groups. If there is no significant difference, we will assume there is no interaction.P values < 0.05 will be considered significant.We will perform statistical analysis using R (version 3.4.4). Two-sided Based on ITT principle, we will analyze all the post-randomization cases, including the ones with missing data. The missing data of the repeatedly measured outcomes will be imputed using the “last observation carried forward” method, while the missing data of other outcomes will be imputed by baseline data. Sensitivity analysis will be performed to assess the bias that may be introduced due to nonadherence to protocol or missing data.We anticipate that the potential risk of this study is minimal. First, to minimize the risks of failure in performing surgeries under LPP, we will exclude patients with a history of pelvic procedures, which may lead to adhesions and may increase the manipulation difficulty. Furthermore, all five surgeons involved have at least 10 years of experience in performing laparoscopic surgeries. Second, SPP has been safely used in PUMCH for more than 20 years. Patients with severe liver, heart, lung and kidney dysfunction will be excluded; thus, the potential risk of irreversible ischemic organ injury caused by SPP should be extremely low. Third, rocuronium is mainly metabolized in the liver, and no evidence exists to show that the dosage used in deep NMB has renal toxicity in patients without ESRD. Finally, to avoid residual paralysis, 6 mg neostigmine will be given at the end of surgery to antagonize the residual muscle relaxant, and patients will not be extubated until the TOF ratio is > 90%.Adverse events information will be collected spontaneously from the participants during the follow-up and recorded in CRFs with details . Serious adverse events will be reported to the medical ethics committee.To the best of our knowledge, this is the first trial to date assessing the effects of pneumoperitoneum pressure and NMB on postoperative renal function in diabetes patients. Accumulating evidence shows the benefits of combined LPP and deep NMB in reducing postoperative pain and enhancing recovery , 34. HowOur study is limited by the following aspects. First, we will exclude patients with ASA IV or with severe organ dysfunction; thus, the conclusion of this study cannot be generalized to critical cases. Second, since the surgeons involved in this study are experienced in laparoscopic procedures, they may have lower requirements for pneumoperitoneum pressure. Third, for the concern of workload, the diagnosis of diabetes will be based on the patients’ medical records rather than the “gold criteria,” the oral glucose tolerance test (OGTT). Therefore, some patients with undiagnosed early diabetes may be inadvertently excluded. Finally, unexpected events may occur, such as an actual surgery duration > 3 h or conversion to open surgery. Based on the ITT principle, the data of these cases should also be collected and analyzed. Considering the equal likelihood of the occurrence of non-adherence in each group, the effect on the results should be low.In conclusion, our study will investigate the effects of LPP and deep NMB on postoperative renal function and surgical conditions. We hope this study can shed light on intraoperative renal protection in diabetes patients receiving laparoscopic surgeries.ClinicalTrials.gov  on February 5, 2020. The recruitment will start on August 1, 2020, and will be completed on December 31, 2021. Data assessment should be completed in April 2022.The study protocol corresponds to the first version of the protocol, as submitted to Additional file 1. Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist.


Data on clinical characteristics of nonalcoholic fatty liver disease (NAFLD) in patients with chronic kidney disease (CKD) are scarce. We investigated the clinical features and risk factors of NAFLD using noninvasive serum markers in CKD patients and attempted the temporal validation of a predictive model for CKD based on NAFLD.n = 567) and validation  groups.This retrospective cross-sectional study was conducted in a single tertiary center. We enrolled 819 CKD patients and evaluated the predictive performance of relevant clinical and laboratory markers for the presence of NAFLD in both derivation , 28.0 ml/min). BMI, hemoglobin, serum alanine aminotransferase, eGFR, and triglyceride-glucose index were used to derive a prediction model for the presence of NAFLD. Using the cutoff value of 0.146, the area under the receiver operating characteristic curve (AUROC) for the prediction of NAFLD was 0.850. In the validation group, NAFLD was observed in 51 patients . Using the same prediction model and cutoff value, the AUROC was 0.842. NAFLD prevalence in CKD patients was comparable to that in the general population, increasing over time.In the derivation group, NAFLD was observed in 89 patients , 24.6 kg/mOur model using BMI, renal function, triglyceride-glucose index, serum alanine aminotransferase, and hemoglobin accurately predicted the presence of NAFLD in CKD patients. Given that its global prevalence is more than 10% of the adult population,chronic kidney disease (CKD) is a global health problem. CKD is a more serious problem in developed countries such as the United States, where the prevalence exceeds 25% of people above 65 years , 4. MoreNAFLD is an independent risk factor for CKD and is also the most rapidly increasing indication for simultaneous liver-kidney transplantation . Additio2 according to the Kidney Disease Improving Global Outcomes (KDIGO) guidelines   × (√ALT [IU/L])  , 18. Theg/dL)/2] . The diag/dL)/2] . For eacg/dL)/2] . CKD was/1.73 m2 . Among sn (g/dL) . The BARth NAFLD . FIB-4 s [IU/L]) , 26.P < 0.05 were subsequently included in the multivariate analysis. The selected variables for the logistic regression analysis included clinical characteristics, laboratory parameters, and noninvasive fibrosis markers. Forward and backward stepwise selection procedures were sequentially used to select the best-fitted model. For the selected model, the predictive performance was confirmed based on the AUROCs. We examined goodness of fit by comparing the observed response with the expected response, estimated from the risk score with a Hosmer-Lemeshow test. An optimal cutoff value was determined based on Youden’s index to maximize both sensitivity and specificity for risk stratification. Based on the cutoff value, we calculated sensitivity, specificity, positive predictive value, and negative predictive value. The model was internally validated and optimized with 1000 times bootstrapping. Finally, using the data collected between 2015 and 2016, a temporal validation of the performance of the prediction model was performed as described. Subgroup analysis for patients with NAFLD was conducted to evaluate the severity of liver fibrosis. A two-sided P-value < 0.05 was considered statistically significant. All statistical analyses were performed using SAS software ver. 9.4 .Baseline characteristics are presented as mean ± standard deviation for normally distributed continuous variables, and as median with interquartile ranges for continuous variables with a skewed distribution. Discrete variables are summarized as numbers with percentages. To compare baseline characteristics by the presence of NAFLD, we used the Student t-test or Mann-Whitney U-test, as appropriate. Skewed distribution variables were transformed into natural logarithms and used for further analysis. Categorical variables were compared using the chi-square and Fisher’s exact tests. The entire cohort was divided into two parts to derive and validate a risk prediction model. In the derivationgroup (data collected between 2011 and 2014), simple logistic regression analysis was used to identify relevant features associated with the presence of NAFLD, and variables with n = 567) and validation (n = 252) groups. In the derivation group (data collected between 2011 and 2014), the mean age was 64.7 ± 15.3 years, and the prevalence of NAFLD was 15.7%. Patients in the NAFLD group had a significantly higher body mass index , triglyceride (192 vs. 119 mg/dL), and total cholesterol (178.7 ± 42.4 vs. 163.2 ± 44.9 mg/dL) than those in the non-NAFLD group . Although the prevalence of hypertension and diabetes was similar between the groups, dyslipidemia was significantly higher in the NAFLD group (P < 0.001). For biochemical markers of kidney function, patients in the NAFLD group had a lower serum creatinine level (1.7 vs. 2.4 mg/dL) and a higher median estimated glomerular filtration rate . Stages of CKD were categorized according to eGFR as follows: G3a (45–59 ml/min), n = 102 (18.0%); G3b (30–44 ml/min), n = 165 (29.1%), G4 (15–29 ml/min), n = 139 (24.5%); and G5 (< 15 ml/min), n = 161 (28.4%). Patients in the NAFLD group were more likely to have higher plasma fasting glucose levels, HbA1c, and TyG index compared to those in the non-NAFLD group.This study enrolled 819 patients with CKD. Table P < 0.001), hemoglobin , ALT , eGFR , and TyG index . A prediction model for the presence of NAFLD was derived based on these variables as follows: − 21.0935 + 0.2204 × hemoglobin + 1.590 × TyG + 0.4974 × log (ALT) + 1.263 × eGFR category  + 1.1779 × BMI category . Using 0.146 as the cutoff value, the AUROC for the prediction of NAFLD in the derivation group was 0.850  . Additionally, the sensitivity, specificity, accuracy, positive- and negative likelihood ratios were 0.805, 0.772, 0.393, and 0.956, respectively.Multivariate logistic regression analyses were performed in the derivation group to identify relevant clinical and laboratory factors associated with the presence of NAFLD Table . Predict2, and the median eGFR was 36.0 ml/min. Patients were categorized according to their eGFR as follows: G3a, 70 (27.8%); G3b, 89 (35.3%), G4, 62 (24.6%); and G5, 31 (12.3%). Unlike the derivation group, there were no significant differences in baseline characteristics between the control and NAFLD patients, except for age. The model remained well-calibrated in the validation group (Hosmer-Lemeshow test: P = 0.324). The predictive performance of the model in the validation set improved using the same cutoff value  , we checked the potential interaction between erythropoietin use and hemoglobin as a risk factor for NAFLD. No significant interaction was revealed. Several previous studies have suggested the putative role of hemoglobin in the development of NAFLD in the non-CKD population. A Chinese study reported that a higher hemoglobin level was an independent predictor of NAFLD in non-obese patients , 54. AnoSerum ALT levels were significantly higher in NAFLD patients with CKD. The mechanism of NAFLD development is explained by insulin resistance, which activates lipolysis resulting in the accumulation of non-esterified fatty acids . This enIn our CKD population, NAFLD was more frequently observed in patients with relatively preserved renal function. This was an unexpected finding because the severity of hepatic steatosis in NAFLD was associated with increased CKD risk and severity in a previous study . Given tCaution is needed to interpret the results of the present study due to the following limitations. First, although we found relevant risk factors for the presence of NAFLD, which are biologically plausible, the retrospective nature of the study could have affected the results, especially concerning a potential selection bias. However, we attempted a temporal validation, and the performance of our prediction model was maintained in the validation. Second, the lack of histological or radiological data such as ultrasound-based or magnetic resonance-based elastography prevented the refined categorization of NAFLD severity. Instead, we employed widely used and validated serum marker-based indices such asNAFLD fibrosis score and FIB-4 . TherefoIn conclusion, this study identified relevant predictors of the presence of NAFLD in CKD patients. The predictors of the presence of NAFLD showed an accurate predictive performance, which was confirmed in a temporal validation. Given the absence of specific noninvasive predictive tools for the presence of NAFLD as a comorbidity of CKD patients to date, screening for NAFLD based on these risk factors seems reasonable in CKD patients. This is because CKD patients are prone to liver-related morbidity or mortality, and all-cause or cardiovascular mortality on top of underlying CKD and other related outcomes. These results need further validation, including assessment for liver disease severity and liver- and kidney-related outcomes in a prospective setting.


Chronic kidney disease (CKD) involves many factors that can cause frailty and oral hypofunction. We aimed to investigate the prevalence of frailty and oral hypofunction and to examine the associations among kidney function, frailty, and oral function in adults with CKD in Japan.This cross-sectional study was conducted at two institutions. The participants included 109 patients with CKD stages 3–5 who visited outpatient clinics or were admitted for inpatient treatment. Frailty was evaluated using the Japanese version of the Cardiovascular Health Study frailty criteria. Oral function was evaluated by assessing oral motor skills [oral diadochokinesis (ODK) rate], masticatory ability, and the repetitive saliva swallowing test. The estimated glomerular filtration rate (eGFR) was used to indicate kidney function. We examined the associations among kidney function, frailty, and oral function using binomial logistic regression analysis.p = 0.048] and ODK rate . However, no significant association was found between CKD severity and masticatory or swallowing function.In total, 31 participants (28.4%) were classified as being frail. Univariate analysis showed that age, body mass index, eGFR, and haemoglobin level were significantly associated with frailty. ODK and swallowing function were significantly associated with frailty. Multivariate analysis revealed that frailty was significantly associated with eGFR [odds ratio (OR) 0.96, 95% confidence interval (CI) 0.92–1.00, We found a high prevalence of frailty in patients with CKD and a significant association between frailty and oral motor skills, affecting the swallowing function of patients with nondialysis CKD. The high prevalence of frailty among patients with CKD suggests that routine assessment of frailty is necessary to prevent the development of severe complications. In addition, oral and kidney function should be carefully evaluated, and oral health education and interventions should be performed for patients with CKD. Chronic kidney disease (CKD) is a common disease worldwide  and is aFrailty is a common geriatric syndrome that has been gaining increased attention. In 2001, Fried et al.  reportedThe prevalence of frailty among patients with nondialysis CKD ranged from 7.9% , whereasRecent studies have indicated that oral function is significantly associated with physical frailty, sarcopenia, disabilities, and mortality in the future , 14. In This cross-sectional study was conducted at two institutions . Data were collected from patients with CKD who visited outpatient clinics or who were admitted for inpatient treatment between July 2018 and May 2019. This study was approved by the ethics committees of both institutions and was conducted in accordance with the ethical principles of the Declaration of Helsinki. All of the participants provided written informed consent .Patients with CKD who met the inclusion criteria were consecutively enrolled in the survey. The inclusion criteria included the following: age ≥ 20 years old, CKD stages 3–5 , currentThe frailty phenotype, oral function, and number of teeth were evaluated, and medical records were reviewed. Frailty evaluations and oral function tests were performed by a nurse or dental hygienist who received training on the study protocol and function tests prior to performing the surveys.To evaluate frailty, we used the Japanese version of the Cardiovascular Health Study frailty criteria (J-CHS) . The J-CWeight loss: “Yes” in response to the question “Have you lost 2 kg or more in the past 6 months?”Muscle weakness: Grip strength of < 26 kg in men or < 18 kg in women as measured using N-FORCE HG200 .Endurance: “Yes” in response to the question “In the past 2 weeks, have you felt tired without a reason?”Gait speed: The participants walked a total of 6 m at a usual pace, and the time necessary to traverse 6 m was measured. A walking speed of < 1.0 m/s was defined as low-level mobility.Physical activity: “No” in response to either of the following questions: “Do you engage in moderate-level physical exercise or sports aimed at health? Do you engage in low-level physical exercise aimed at health?”Frailty was defined as having 3–5 of the aforementioned domains, as pre-frailty by having 1 or 2, and as robust by having none.Oral motor skillOral function was evaluated according to the following three aspects as recommended by the Japanese Society of Gerodontology : oral mo2)Masticatory ability testOral motor skills were assessed using the oral diadochokinesis (ODK) test. In the ODK test, a participant pronounces sounds as quickly as possible to allow for the evaluation of the lip (/Pa/), tongue tip (/Ta/), and tongue back (/Ka/) movements, collectively referred to as slickness. We measured the ODK rate according to a previously described method . The par3)Repetitive saliva swallowingThe masticatory ability test involves the use of a gummy candy containing glucose . Each paSwallowing function was assessed using the repetitive saliva swallowing test (RSST) , 24. TheDental status was evaluated as a factor related to oral function. The numbers of present teeth and functional teeth were counted. We defined present teeth as naturally grown teeth and functional teeth as both present and missing teeth replaced by prostheses, such as dentures and dental implants. Teeth with severe decay and those with stumps were excluded from the category of functional teeth because they were not used for mastication.2) = 194 × (serum creatinine)− 1.094 × (age)− 0.287 (× 0.739 for women) , laboratory test results , comorbidities , CKD stage, body height, body weight, body mass index (BMI), and primary kidney disease. eGFR was calculated using the equation from the new Japanese coefficient-modified Modification of Diet in Renal Disease study: eGFR (mL/min/1.73 mr women) .t-test for parametric variables, the Mann-Whitney U-test for non-parametric variables, and the chi-square test for categorical variables. The Jonckheere-Terpstra test was performed to compare CKD stage and oral function. In multivariate analysis, to determine the associations among kidney function, frailty, and oral function, we performed binomial logistic regression analysis. Due to the small number of robust patients and the multiple independent variables used in the study model, we made the dependent variable, i.e., frailty, a binary value (robust and pre-frail vs. frail), and the independent variables used were kidney and oral functions. Variables that could be considered confounding factors based on previous studies [p-value of < 0.05 was considered statistically significant.Demographic data were verified for normal distributions. Descriptive characteristics are presented as the mean and standard deviation (SD) or the median and interquartile range (IQR) for continuous data and as frequencies and percentages for categorical data. In bivariate analysis, the participants were divided into robust and pre-frail groups (non-frail groups) and compared with the frail group; differences between the two groups were evaluated using the unpaired Student’s  studies –28 were Among the 119 patients who met the inclusion criteria, 109 (91.6%) consented to participate in this study. Table p < 0.05, all). Significant differences were also observed in the ODK rate and RSST findings, with declining values in the frail group. The number of functional teeth differed significantly according to the frailty phenotype (p < 0.05). However, no significant differences were observed in sex, albumin level, triglyceride level, blood glucose level, presence of primary kidney disease, or complications between the two groups.The variables that differed significantly between the frail and non-frail groups were age, BMI, eGFR, and haemoglobin level . However, no significant association was found between frailty and either masticatory or swallowing function.Table To our knowledge, this study was the first to examine the associations among kidney function, frailty, and oral function, with a specific focus on the functions necessary for swallowing, mastication, and oral motor skills among patients with nondialysis CKD in Japan. The prevalence of frailty in our participants was 28.4%, and kidney function significantly decreased in the frail group compared with the non-frail group. Frailty was significantly associated with decreased oral motor skills and swallowing function according to univariate analysis. However, the significant decline in oral function was not associated with increased severity of CKD. Multivariate analysis showed that decreased kidney function and oral motor skills were independent factors associated with frailty, even after controlling for the effects of age, sex, and BMI.The prevalence of frailty was much higher in our study than that reported in a previous national survey   and was Similar to the findings of previous studies , 14, fraHowever, when oral function was examined according to CKD stage, no significant differences were found among different CKD stages. Previous studies have primarily focused on periodontal disease and oral dryness in patients with CKD. Decreased saliva volume has been associated with a decline in swallowing function, and periodontal disease can lead to weakened masticatory ability –40. ManyMultivariate analysis showed a significant association among frailty, kidney function, and tongue motor skills. The ODK /ta/ test reflects the movement of the tongue, which carries food to the back of the mouth and is associated with swallowing , 49. In The results of this study suggest that oral function is associated with frailty in patients with CKD and must be carefully monitored. Although patients with CKD have been reported to have lower rates of dental visits than the general population , regularThe present study has several limitations. First, the study design was cross-sectional; therefore, the evidence for causality was limited. Second, although the participants were recruited from two institutions, a small number of participants were enrolled, and they were not necessarily representative of all patients with this disease; therefore, the results might not be generalizable to other populations. In addition, potential factors related to frailty, kidney function, and oral function might not have all been examined and therefore should be further studied with an increased sample size and assessment variables in the future. Third, to understand the nutritional status of patients with CKD, both serum levels and comprehensive nutritional indicators must be evaluated, such as the Subjective Global Assessment score , 54 and Despite these limitations, this study was the first to show the actual state of oral function in patients with CKD, and we believe that long-term studies will clarify the associations between CKD and not only frailty but also mortality and other outcomes. In the future, the comparison between patients with CKD and those without CKD will be necessary to determine precautions specific to CKD and to provide appropriate interventions.The findings of this study indicate a high prevalence of frailty among patients with CKD and a significant association between frailty and the oral motor skills that affect swallowing in patients with nondialysis CKD. Recognizing the high prevalence of frailty in patients with CKD and performing routine assessments of frailty will be necessary to prevent the development of severe complications. Moreover, oral function and kidney function should be carefully examined, and early education and interventions regarding oral health should be performed for patients with CKD.


Fetal exposure to phthalates and bisphenols may lead to vascular developmental adaptations, which program later cardiovascular disease. We examined the associations of fetal exposure to phthalates and bisphenols with childhood blood pressure.In a population-based, prospective cohort study among 1,064 mother-child pairs, we measured maternal urine phthalate and bisphenol concentrations in first, second and third trimester of pregnancy. We measured childhood blood pressure at the mean age of 9.7 years (standard deviation 0.2 years) old. Analyses were performed for the total group, and for boys and girls separately.Maternal urine phthalate concentrations were not associated with childhood blood pressure among boys. Higher third trimester maternal urine concentrations of high molecular weight phthalates (HMWP), di-2-ehtylhexylphthalate (DEHP) and di-n-octylphthalate (DNOP) were associated with lower systolic and diastolic blood pressure among girls . Also, higher second trimester maternal urine total bisphenol and bisphenol A concentrations were associated with higher systolic blood pressure among boys , but tended to be associated with a lower diastolic blood pressure among girls.Our results suggest sex-dependent associations of maternal urine phthalate and bisphenol concentrations during pregnancy with childhood blood pressure. Further studies are needed to explore the underlying mechanisms and long term consequences. PhthalatThe associations of phthalates and bisphenol A (BPA) with cardiovascular risk factors, such as obesity, hypertension and diabetes, have mainly been studied in cross-sectional designs among adult populations and showed inconsistent results.  PreviousWe hypothesized that fetal exposure to higher levels of maternal phthalate and bisphenol concentrations lead to developmental vascular adaptations and subsequent higher blood pressure during childhood. We assessed, in a population-based cohort study from fetal life onwards among 1,064 mother–child pairs, the sex-specific associations of maternal phthalate and bisphenol urine concentrations in first, second and third trimester of pregnancy with childhood blood pressure.2.2.1.This study was embedded in the Generation R Study, a population-based prospective cohort study from early fetal life onwards in Rotterdam, the Netherlands.  Phthalat2.2.Phthalate and bisphenol concentrations were measured in spot urine samples obtained from each woman at three time points during pregnancy (median 12.9 weeks of gestation (25th-75th percentiles 12.1 – 14.5); median 20.4 weeks of gestation (25th-75th percentiles 19.9 – 20.9); median 30.2 weeks of gestation (25th-75th percentiles 29.9 – 30.8)). These periods were considered as first, second and third trimester. Urine samples were collected between February 2004 and July 2005. The analyses of phthalate, bisphenol and creatinine concentrations were performed at the Wadsworth Center, New York State Department of Health, Albany, New York, USA, using previously described methods.  Urine bi2.3.As described previously, children were invited to visit our research center around the age of 10 years.  We measu2.4.Information on maternal characteristics, including age, ethnicity, pre-pregnancy body mass index, use of folic acid supplementation, educational level, parity, and maternal smoking habits and alcohol consumption  were obtained from questionnaires during pregnancy. Child’s sex was obtained from midwife and hospital records at birth. A Directed Acyclic Graph (DAG) showing the hypothesized relationships between fetal exposure to phthalates and bisphenols, childhood blood pressure and the covariates is presented in 2.5.t-test or Mann-Whitney U test, when applicable. We used linear regression models to assess the associations of maternal urine phthalate and bisphenol concentrations  with childhood blood pressure. Non-linearity of the relationship was visually assessed and ruled out. First, models were adjusted for child’s sex- and age-adjusted height SDS, age and sex only (basic model). Subsequently, potential confounders were identified based on the graphical criteria for confounding by visualizing a DAG. Of these potential confounders, we included those in the models that changed the effect estimates > 10% for at least one of the outcomes.  to ease the interpretation of effect sizes. We constructed standard deviation scores (SDS) [/standard deviation (SD)] of the blood pressure based on the sample distribution to enable comparisons of effect sizes. To assess possible bias due to loss-to-follow-up, participants were compared to non-participants using a Chi-squared test, student’s utcomes.  To examiutcomes. . Previouutcomes.  Because utcomes.  Since weutcomes.  To correutcomes.  To maint3.3.1.3.2.3.3.4.4.1.We observed in a population-based prospective cohort study that the associations of exposure to phthalates and bisphenols during pregnancy with blood pressure at 10 years old differ between boys and girls. Higher fetal exposure to phthalates is associated with lower blood pressure among girls, while higher fetal exposure to bisphenols is associated with higher blood pressure among boys at age 10 years.4.2.Because of the wide-spread use of phthalates and bisphenols and their ability to cross the placenta, exposure to these potential harmful metabolites probably starts before birth. Exposure to phthalates and bisphenols during fetal life may lead to vascular developmental adaptations, with subsequently an increase in the risk of hypertension and cardiovascular disease in later life.Few studies assessed the associations of fetal exposure to phthalates with childhood blood pressure.  In a stuPrevious studies examining the associations of maternal bisphenol concentrations with childhood blood pressure have only focused on BPA. No association between maternal urine BPA concentrations and childhood blood pressure was found in 235 Greek mothers and their 4-year-old children.  In anothOur results suggest that exposure to endocrine disruptors during fetal life may have long-lasting effects on blood pressure during childhood. Due to the observational design of this study, we cannot draw conclusions about causality. The observed effect estimates were small but are of interest from a developmental perspective since blood pressure tends to track into adulthood and even subclinical differences during childhood are related to the development of cardiovascular disease in later life. 4.3.Numerous underlying mechanisms have been proposed for the hypothesized adverse effects of exposure to phthalates and bisphenols on blood pressure. It has been suggested that exposure to phthalates causes an adverse inflammatory environment through oxidative stress.  This hyp4.4.An important strength of this study is the population-based cohort design from fetal life onwards, with repeated measurements of multiple maternal phthalate and bisphenol concentrations and measures of childhood blood pressure in a large number of mother–child pairs. Selection bias due to selective loss to follow-up would be of concern in any study if the associations of prenatal phthalate and bisphenol concentrations with childhood blood pressure are different between participants and non-participants. This seems unlikely, but cannot be excluded with certainty. Furthermore, we measured maternal urine phthalate and bisphenol concentrations at one moment per trimester. It is possible that one urine sample per trimester does not accurately represent the concentration during the whole trimester because of the reported short biological half-lives of phthalates and bisphenols.  However,5.In this population-based prospective cohort study, we found sex-specific differences in the association of maternal urine phthalate and bisphenol concentrations during pregnancy with childhood blood pressure at 10 years old. Higher fetal exposure to (high molecular weight) phthalates is associated with lower blood pressure among girls and higher fetal exposure to bisphenols is associated with higher blood pressure among boys. These results should be considered as hypothesis generating and further studies are needed for replication and identification of underlying mechanisms.SUpplementary material


Hypertension is a major modifiable risk factor for arteriosclerosis that can lead to target organ damage (TOD) of heart, kidneys, and peripheral arteries. A recent epigenome-wide association study for blood pressure (BP) identified 13 CpG sites, but it is not known whether DNA methylation at these sites is also associated with TOD.In 1218 African Americans from the Genetic Epidemiology Network of Arteriopathy (GENOA) study, a cohort of hypertensive sibships, we evaluated the associations between methylation at these 13 CpG sites measured in peripheral blood leukocytes and five TOD traits assessed approximately 5 years later.P < 0.05). Mendelian randomization analysis suggests that methylation at two sites (cg16246545 and cg14476101) in PHGDH may causally influence UACR.Ten significant associations were found after adjustment for age, sex, blood cell counts, time difference between CpG and TOD measurement, and 10 genetic principal components (FDR q < 0.1): two with estimated glomerular filtration rate , six with urinary albumin-to-creatinine ratio , and two with left ventricular mass indexed to height . All associations with eGFR and four associations with UACR remained significant after further adjustment for body mass index (BMI), smoking status, and diabetes. We also found significant interactions between cg06690548 and BMI on UACR, and between 3 CpG sites  and diabetes on UACR (FDR q < 0.1). Mediation analysis showed that 4.7% to 38.1% of the relationship between two CpG sites (cg19693031 and cg00574958) and two TOD measures (UACR and LVMI) was mediated by blood pressure (Bonferroni-corrected In conclusion, we found compelling evidence for associations between arteriosclerotic traits of kidney and heart and previously identified blood pressure-associated DNA methylation sites. This study may lend insight into the role of DNA methylation in pathological mechanisms underlying target organ damage from hypertension. More than 100 million Americans have high blood pressure, which costs more than 50 billion dollars a year , 3. TOD N = 17,010) identified 13 significant CpG sites [Genome-wide association studies have identified genetic variation that influences TOD traits –13. EpigpG sites . In a crpG sites . HoweverHere, we investigate the association between the 13 DNA methylation sites associated with blood pressure and TOD of the heart, kidneys, and peripheral arteries in a cohort of largely hypertensive African Americans, and investigate whether these effects are modified by other risk factors for arteriosclerosis including body mass index (BMI), smoking status, and diabetes or are mediated by blood pressure.The Genetic Epidemiology Network of Arteriopathy (GENOA) is a community-based study designed to identify genes influencing blood pressure . In the N = 316 from 450 K and N = 1100 from EPIC). The Minfi R package [2 ratio of the intensity of the methylated probe versus the unmethylated probe [ComBat in R [DNA methylation was assessed, and bisulfite converted from genomic DNA extracted from stored peripheral blood leukocytes collected at GENOA Phase 1 using Illumina Infinium HumanMethylation BeadChips  less than 0.01. Imputation was performed separately by chip (Affymetrix or Illumina) using the 1000G Phase3 v5 reference panel, and post-imputation comparison between the two groups revealed no substantial differences in genotype and allele frequencies. SNPs with imputation quality less than 0.8 were removed before MR analysis.2.7, LVMI), and a measure of the peripheral arteries . TOD measurement methods are described in detail elsewhere [Measures of systolic blood pressure (SBP) and diastolic blood pressure (DBP) were the average of the last two of three sequential readings taken in a sitting position for at least 5 min at Phase 1 . SBP andlsewhere . BMI wasOutliers greater than four standard deviations from the mean of each TOD outcome and covariate were removed prior to analysis, and UACR was natural log transformed as ln (UACR+ 1). Ten genotype principal components (PCs) were included in all models to adjust for population stratification. Analyses were conducted in R (Version 3.4.1)  and SAS N = 422) were included. To examine whether the blood pressure-associated CpG sites from Richard et al. (2017) were also associated with SBP and DBP in the full GENOA sample with methylation data and TOD traits (N = 1212), we used a linear mixed effects model while accounting for family structure. Covariates included age at Phase 1, sex, BMI, smoking status, and 10 PCs.In the Richard et al. (2017) EWAS, a subset of GENOA African Americans with 450 K methylation data , we tested for interaction between the CpG site and each of the three risk factors  on the TOD measure. For each significant interaction, we plotted an interaction using a scatter plot by risk factor status (e.g. diabetes VS non-diabetes). The y-axis of each plot was a predicted value of TOD measure estimated from each linear regression model.mediation R package to examine whether the effects of the CpG sites on TOD measures are mediated through blood pressure [For significant associations between CpG sites and TOD measures prior to adding arteriosclerosis risk factors (FDR q < 0.1), formal mediation analyses were conducted using the pressure . We usedP < 0.1) both with a CpG site and a TOD measure, we performed a formal mediation analysis. We used a linear mixed effects model adjusted for family structure with covariates including sex, age when gene expression was measured, and 10 PCs. When DNA methylation M-value or gene expression level was the independent variable, it was pre-adjusted for sex and age before conducting the association analysis. In formal mediation analysis, we tested the indirect association between a CpG site and a TOD measure through changes in proximal gene expression levels using the bootstrapping method in the mediation R package [To examine whether the associations between CpG sites and TOD measures prior to adding arteriosclerosis risk factors (FDR q < 0.1) are mediated by gene expression levels, we evaluated the associations between 1) CpG sites and gene expression levels from genes that were located within ±1 Mb of the corresponding CpG sites, and 2) between the gene expression levels and TOD traits, separately. For gene expression levels that had at least a marginal association ( package .N = 1151 and FDR q < 0.05) [2 = 0.1 and physical distance threshold = 100 kb). The associations between IVs with CpGs and TOD measures were examined to obtain the beta and standard error estimates in GENOA African Americans. Inverse-variance weighting (IVW) was used to evaluate causation for each CpG on TOD measures. If the IV is comprised of only one variant, the Wald-ratio MR method was used to examine the causal effect [TwoSampleMR R package [P-value to be significent (P < 0.005). For each significant MR result, we created a scatter plot to depict the relationship of the SNP effects on the exposure against the SNP effects on the outcome using the same package. A line was drawn in each plot and the slope corresponds to the estimated causal effect from the IVW method. We used the MR Egger test to examine pleiotropy because IVW MR is invalid in the presence of horizontal pleiotropic effects of IVs [To investigate whether the previously identified CpG sites (FDR q < 0.1 prior to adding arteriosclerosis risk factors) might contribute causally to TOD measures, we performed Mendelian randomization (MR) analysis using 1000G imputed SNPs in GENOA African Americans. Since public mQTL databases and TOD GWAS data are limited in African Americans, we performed one-sample MR analysis using data from GENOA African Americans. Instrumental variables (IVs) for DNA methylation were drawn from cis-mQTL analysis in GENOA using SNPs within ±1 Mb of each corresponding CpG site using RVTEST ( < 0.05)  and cluml effect . Causal  package . We conss of IVs .2 (SD: 6.7), 12% of participants were current smokers, and 31% had diabetes.Descriptive statistics at Phase 1 (SBP and DBP) and Phase 2 (other variables) are shown in Table P < 0.05) with SBP and DBP, respectively, after adjustment for age, sex, BMI, and smoking status: cg19693031 and cg00574958 with both SBP and DBP; cg18120259 with SBP only (Table P < 0.0038) . Effect directions for nine CpG sites for SBP and 12 CpG sites for DBP were negative in our analysis, while the effect directions for all of the 13 CpG sites were negative in Richard et al. (2017).Three and two out of the 13 evaluated CpG sites had at a least marginal association  on TOD measures, we added interaction terms in the baseline association models. We found four significant interactions on UACR: cg06690548-BMI, cg14476101-diabetes, cg19693031-diabetes, and cg06690548-diabetes  through changes in blood pressure. Mediation analysis showed that seven associations between CpG sites and two TOD measures (UACR and LVMI) were significantly mediated by blood pressure  with both the corresponding CpG site and the TOD trait . We then performed a formal mediation analysis for these five associations but found no significant (P < 0.05) mediating effect of gene expression on the associations.Using the 10 significant CpG site-TOD measure associations prior to adding arteriosclerosis risk factors (FDR q < 0.1), we conducted a 2-step association analysis to identify potential mediating effects of gene expression level: an association between each CpG site and expression of proximal genes, and an association between gene expression and the TOD measure. A total of five genes with expression data that were located within 1 Mb of a corresponding CpG site were at least marginally associated  and cg14476101  tests to examine forward causality of DNA methylation on TOD. The results of MR analysis of CpG-TOD association are shown in the Table Scis gene expression mediating the associations between CpG sites and TOD measures. MR analysis provided supporting evidence of the forward direction of association for 2 CpG sites located near PHGDH with UACR.We identified ten significant associations between previously identified CpG sites associated with blood pressure and TOD measures including eGFR, UACR, and/or LVMI in a sample of 1218 African Americans. Half of these associations remained significant even after further adjustment for other TOD risk factors including BMI, smoking status, and diabetes. Among the CpG sites from the ten significant associations, one significant interaction with BMI and three significant interactions with diabetes on UACR were identified in an interaction analysis. Seven associations between CpG sites and two TOD measures were partially mediated by blood pressure (4.7 to 38.1%), but there was no significant Only a small portion of previously identified CpG sites from Richard et al. (2017) were also significantly associated with blood pressure in GENOA after multiple testing correction (three out of 13 CpG sites). Syme et al. (2019) found three out of the 13 CpG sites had significant associations with blood pressure in 135 adolescents and 110 middle-aged adults . Most ofResults from the mediation analysis suggests the significant associations between the CpG sites and TOD are not always mediated through blood pressure. Since the GENOA participants were selected based on their own or their sibling’s hypertension status, the average blood pressure in GENOA was elevated compared to the general population. Some of these CpG sites from Richard et al. (2017) may operate at a lower blood pressure level, but may have weaker effects at higher blood pressures, so associations may not be as pronounced in GENOA. Despite the lower rate of replication with blood pressure, we found 10 significant associations with TOD measures in the kidney and heart .SLC7A11), cg19693031 (TXNIP), and cg00574958 (CPT1A) and TOD in the kidney even further after adjustment for BMI, smoking status, and diabetes. cg06690548 (SLC7A11), cg19693031 (TXNIP) and cg00574958 (CPT1A) have previously been associated with triglyceride levels, in addition to blood pressure, in EWAS studies [PHGDH that may causally influence affect UACR. PHGDH encodes an enzyme which facilitates amino acid L-serine biosynthesis. Disturbances in serine metabolism are associated with endothelial dysfunction, inflammation, and atherosclerosis in the kidney, and there is evidence that the mechanism of these effects may be through the 1-carbon (DNA methylation) metabolism pathway [We identified significant associations between cg10601624, cg06690548  or those who were obese (cg06690548 in SLC7A11). PHGDH is previously reported to be involved in the progression of glucose intolerance through serine biosynthesis in mice adipose tissues [TXNIP is increased in the presence of high glucose, which promotes oxidative stress and β-cell apoptosis [The inverse association between DNA methylation and UACR was stronger in those with diabetes , we ascertained the relationships between TOD measures and previously identified DNA methylation levels associated with blood pressure using state-of-the-art analytic methods in a population with a high burden of hypertension. In GENOA, TOD traits were measured approximately 5 years later than methylation, thus helping to establish the temporal relationship between changes in methylation and TOD. We were also able to characterize the direction of association using MR techniques. However, for two CpG sites (cg06690548 and cg10601624), we were not able to examine the causal direction of the association because of the lack of appropriate instruments. Also, despite of the five-year lag between measurements, the level of TOD trait could have been changed before methylation level changed. In addition, we lacked the ability to account for the duration of hypertension, which preceded DNA methylation for over two thirds of the study population. When we adjusted for hypertension in the baseline association models, the significance remained for eGFR and UACR, but attenuated in LVMI. Studies with longitudinal measures of DNA methylation at multiple timepoints may help better elucidate these temporal relationships.PHGDH in the kidney, potentially through endothelial dysfunction, inflammation, and atherosclerosis. Future research should further confirm these initial findings in populations of other ancestries. This study may lend insight into the role of DNA methylation in complex pathological mechanisms underlying target organ damage from hypertension and provide potential clinical targets for treatment and prevention.In conclusion, we found compelling evidence for associations between arteriosclerotic traits of kidney and heart and previously identified blood pressure-associated DNA methylation sites in a cohort of older African Americans. For the identified associations, we additionally examined the interactions between CpGs and other risk factors, potential mediation by blood pressure, and the causal relationship connecting CpG sites and TOD measures. We found evidence supporting the causal relationship of epigenetic regulation of Additional file 1: Table S1. Interactions between CpG sites and target organ damage risk factors including BMI, current smoking, and diabetes on target organ damage phenotypes in GENOA African ancestry population. Table S2. Results of mediation analysis of CpG sites-systolic blood pressure-target organ damage phenotypes in GENOA African ancestry population. Table S3. Results of mediation analysis of CpG sites-systolic blood pressure-target organ damage phenotypes adjusted for BMI, current smoking, and diabetes in GENOA African ancestry population. Table S4. Mendelian randomization results showing the inverse-variance weighted effects of multiple cis-SNP used as instrumental variables in the association of DNA methylation and target organ damage measures in GENOA African ancestry population. Figure S1. Interaction plots between a CpG site and the traditional risk factors for arteriosclerosis (BMI and diabetes) on urine albumin-to-creatinine ratio (UACR). Figure S2. Mendelian randomization (MR) scatterplot of urine albumin-to-creatinine ratio vs. CpG with estimates from inverse-variance weighted (IVW) MR using cis-SNPs within ±1 Mb of corresponding CpG site in GENOA African Americans.