Search

Background Vascular calcification is a major contributor to the high cardiac burden among hemodialysis patients. A novel in vitro T50-test, which determines calcification propensity of human serum, may identify patients at high risk for cardiovascular (CV) disease and mortality. We evaluated whether T50 predicts mortality and hospitalizations among an unselected cohort of hemodialysis patients. Methods This prospective clinical study included 776 incident and prevalent hemodialysis patients from 8 dialysis centers in Spain. T50 and fetuin-A were determined at Calciscon AG, all other clinical data were retrieved from the European Clinical Database. After their baseline T50 measurement, patients were followed for two years for the occurrence of all-cause mortality, CV-related mortality, all-cause and CV-related hospitalizations. Outcome assessment was performed with proportional subdistribution hazards regression modelling. Results Patients who died during follow-up had a significantly lower T50 at baseline as compared to those who survived (269.6 vs. 287.7 min, p = 0.001). A cross-validated model (mean c statistic: 0.5767) identified T50 as a linear predictor of all-cause-mortality (subdistribution hazard ratio (per min): 0.9957, 95% CI [0.9933;0.9981]). T50 remained significant after inclusion of known predictors. There was no evidence for prediction of CV-related outcomes, but for all-cause hospitalizations (mean c statistic: 0.5284). Conclusion T50 was identified as an independent predictor of all-cause mortality among an unselected cohort of hemodialysis patients. However, the additional predictive value of T50 added to known mortality predictors was limited. Future studies are needed to assess the predictive value of T50 for CV-related events in unselected hemodialysis patients.

The dialyzer is the core element in the hemodialysis treatment of patients with end-stage kidney disease (ESKD). During hemodialysis treatment, the dialyzer replaces the function of the kidney by removing small and middle-molecular weight uremic toxins, while retaining essential proteins. Meanwhile, a dialyzer should have the best possible hemocompatibility profile as the perpetuated contact of blood with artificial surfaces triggers complement activation, coagulation and immune cell activation, and even low-level activation repeated chronically over years may lead to undesired effects. During hemodialysis, the adsorption of plasma proteins to the dialyzer membrane leads to a formation of a secondary membrane, which can compromise both the uremic toxin removal and hemocompatibility of the dialyzer. Hydrophilic modifications of novel dialysis membranes have been shown to reduce protein adsorption, leading to better hemocompatibility profile and performance stability during dialysis treatments. This review article focuses on the importance of performance and hemocompatibility of dialysis membranes for the treatment of dialysis patients and summarizes recent studies on the impact of protein adsorption and hydrophilic modifications of membranes on these two core elements of a dialyzer.

Introduction: High plasma fibroblast growth factor 23 (FGF-23) predicts cardiovascular events in chronic kidney disease (CKD) patients. Experimental evidence suggests FGF receptor 4 (FGFR4) activation by FGF-23, and deficiency of the soluble form of its co-receptor Klotho promotes left-ventricular hypertrophy (LVH). To evaluate the clinical relevance of these findings, a Mendelian randomization study analyzed the association of genetic variants of FGFR4 and Klotho with echocardiographic parameters and cardiac events in CKD patients. Methods: The prospective Cardiovascular and Renal Outcome in CKD 2–4 Patients–The Fourth Homburg Evaluation study recruited CKD G2–G4 patients, of whom 519 consented to SNP genotyping (FGFR4: rs351855; Klotho: rs9536314). Echocardiographic examinations at baseline and 5 years later assessed prevalence of LVH by measurement of left-ventricular mass index (LVMI). Patients were followed for 5.1 ± 2.1 years for the primary endpoints of cardiac decompensation and atherosclerotic cardiovascular disease (ASCVD). Results: Carriers of the different alleles did neither differ in baseline LVMI (rs351855: p = 0.861; rs9536314: p = 0.379) nor in LVMI changes between baseline and follow-up (rs351855: p = 0.181; rs9536314: p = 0.995). Hundred and four patients suffered cardiac decompensation, and 144 patients had ASCVD. Time to cardiac decompensation (rs351855: p = 0.316; rs9536314: p = 0.765) and ASCVD (p = 0.508 and p = 0.800, respectively) did not differ between carriers of different alleles. Discussion/Conclusion: rs351855 and rs9536314 were not associated with LVMI or cardiac events. These findings do not provide evidence for a relevant clinical role of either FGFR4 stimulation or soluble form of Klotho deficiency in LVH development.

Background It has been a long-standing clinical concern that haemodialysis (HD) patients on afternoon shifts (ASs) are more prone to protein-energy wasting (PEW) than those on morning shifts (MSs), as their dialysis scheme and post-dialysis symptoms may interfere with meal intake. We evaluated the effect of time of day of HD on the evolution of body composition changes and PEW surrogates. Methods We conducted a retrospective study among 9.963 incident HD patients treated in NephroCare centres (2011–16); data were routinely collected in the European Clinical Database. The course of multi-frequency bioimpedance determined lean and fat tissue indices (LTI and FTI) between patients in MSs/ASs over 2 years were compared with linear mixed models. Secondary PEW indicators were body mass index, albumin, creatinine index and normalized protein catabolic rate. Models included fixed (age, sex, vascular access and diabetes mellitus) and random effects (country and patient). Results Mean baseline LTI and FTI were comparable between MSs (LTI: 12.5 ± 2.9 kg/m2 and FTI: 13.7 ± 6.0 kg/m2) and ASs (LTI: 12.4 ± 2.9 kg/m2 and FTI: 13.2 ± 6.1 kg/m2). During follow-up, LTI decreased and FTI increased similarly, with a mean absolute change (baseline to 24 months) of −0.3 kg/m2 for LTI and +1.0 kg/m2 for FTI. The course of these malnutrition indicators did not differ between dialysis shifts (P for interaction ≥0.10). We also did not observe differences between groups for secondary PEW indicators. Conclusions This study suggests that a dialysis shift in the morning or in the afternoon does not impact the long-term nutritional status of HD patients. Regardless of time of day of HD, patients progressively lose muscle mass and increase body fat.

Toxin removal capacity (i.e., performance) of a dialyzer is not constant but diminishes during treatment, as the adsorption of proteins to the membrane provides an additional barrier to uremic solutes. We investigated time-resolving molecular weight retention changes among synthetic high-flux dialyzers and compared the results with recent data from a randomized controlled trial.In plasma recirculation experiments over 240 min, sieving coefficients (SC) for β2-microglobulin, myoglobin, and albumin were determined for the FX CorAL (Fresenius Medical Care), ELISIO (Nipro), and xevonta (B. Braun). Molecular weight retention (MWR) curves were generated and the shifts over 120 min were characterized. Effective pore radius was determined, and the predicted albumin loss was compared with clinical data.SC decreased over time for all dialyzers (mean relative decrease across all dialyzers: β2-microglobulin: 8.0% (120 min); myoglobin: 56.6% (240 min); albumin: 94.1% (240 min)). FX CorAL (7.3%, 52.6% and 91.1%) and ELISIO (7.7%, 51.0%, and 93.8%) showed a lower decrease than xevonta (9.0%, 66.2%, and 97.4%). For all dialyzers, MWR curves shifted toward lower molecular weight, with the lowest shift for FX CorAL (by 0.23 nm at SC50%, 120 min) and highest for xevonta (0.50 nm). FX CorAL had the highest slope over time and the smallest decrease in the effective pore radius (2 min: 2.31 nm, 120 min: 2.08 nm). Predicted albumin loss over 4 h was highest for xevonta (609.3 mg) and comparable between ELISIO (283.6 mg) and FX CorAL (313.3 mg).Substantial differences in the temporal performance profile of dialyzers exist. The present approach allows the characterization of dialyzer permeability changes over time using standard, clinically relevant protein markers.

Despite the significant medical and technical improvements in the field of dialytic renal replacement modalities, morbidity and mortality are excessively high among patients with end-stage kidney disease, and most interventional studies yielded disappointing results. Hemodiafiltration, a dialysis method that was implemented in clinics many years ago and that combines the two main principles of hemodialysis and hemofiltration—diffusion and convection—has had a positive impact on mortality rates, especially when delivered in a high-volume mode as a surrogate for a high convective dose. The achievement of high substitution volumes during dialysis treatments does not only depend on patient characteristics but also on the dialyzer (membrane) and the adequately equipped hemodiafiltration machine. The present review article summarizes the technical aspects of online hemodiafiltration and discusses present and ongoing clinical studies with regards to hard clinical and patient-reported outcomes.

Background Dialyzers should be designed to efficiently eliminate uraemic toxins during dialysis treatment, given that the accumulation of small and middle molecular weight uraemic solutes is associated with increased mortality risk of patients with end-stage renal disease. In the present study we investigated the novel FX CorAL dialyzer with a modified membrane surface for performance during online hemodiafiltration (HDF) in a clinical setting. Methods comPERFORM was a prospective, open, controlled, multicentric, interventional, crossover study with randomized treatment sequences. It randomized stable patients receiving regular post-dilution online HDF to FX CorAL 600 (Fresenius Medical Care Deutschland), xevonta Hi 15 (B. Braun) and ELISIO 150H (Nipro) each for 1 week. The primary outcome was β2-m removal rate (β2-m RR) during online HDF. Secondary endpoints were RR and/or clearance of β2-m and other molecules. Albumin removal over time was an exploratory endpoint. Non-inferiority and superiority of FX CorAL 600 versus comparators were tested. Results Fifty-two patients were included and analysed. FX CorAL 600 showed the highest β2-m RR (75.47%), followed by xevonta Hi 15 (74.01%) and ELISIO 150H (72.70%). Superiority to its comparators was statistically significant (P = 0.0216 and P Conclusions FX CorAL 600 efficiently removed middle and small molecules and was superior to the two comparators in β2-m RR. Albumin sieving kinetics point to reduced formation of a secondary membrane.

Background and Aims Activation of the complement system may occur during blood-membrane interactions in hemodialysis and contribute to chronic inflammation of patients with end-stage renal disease (ESRD). Hydrophilic modification with polyvinylpyrrolidone (PVP) has been suggested to increase the biocompatibility profile of dialysis membranes. In the present study we compared complement activation of synthetic and cellulose-based membranes, including the polysulfone membrane with α-tocopherol-stabilized, PVP-enriched inner surface of the novel FX CorAL dialyzer, and linked the results to their physical characteristics. Method Eight synthetic and cellulose-based dialyzers (FX CorAL, FX CorDiax [Fresenius Medical Care]; Polyflux, THERANOVA [Baxter]; ELISIO, SUREFLUX [Nipro]; xevonta [B. Braun]; FDX [Nikkisio Medical]) were investigated in the present study. Complement activation (C3a, C5a, sC5b-9) was evaluated in a 3h ex vivo recirculation model with human blood. Albumin sieving coefficients were determined over a 4h ex vivo recirculation model with human plasma as a surrogate of secondary membrane formation. Zeta potential was measured as an indicator for the surface charge of the membranes. Results The FX CorAL dialyzer induced the lowest activation of the three complement factors (C3a: -39.4%; C5a: -57.5%; sC5b-9: -58.9% compared to the reference). Highest complement activation was found for the cellulose-based SUREFLUX (C3a: +154.0%) and the FDX (C5a: +335.0%; sC5b-9: +287.9%) dialyzers. Moreover, the FX CorAL dialyzer had the nearest-to-neutral zeta potential (-2.38 mV) and the lowest albumin sieving coefficient decrease over time. Albumin sieving coefficient decrease was associated with complement activation by the investigated dialyzers. Conclusion Our present results indicate that the surface modification implemented in the FX CorAL dialyzer reduces secondary membrane formation and improves the biocompatibility profile. Further clinical studies are needed to investigate whether these observations will result in a lower inflammatory burden of hemodialysis patients.

Background and Aims Hydrophilic modification with polyvinylpyrrolidone (PVP) increases the biocompatibility profile of synthetic dialysis membranes. However, PVP may be eluted into patient’s blood, which has been discussed as a possible cause for adverse reactions rarely occurring with synthetic membranes. We now investigated the content of PVP and its elution from the blood-side surface from commercially available dialyzers, including the novel FX CorAL with α-tocopherol-stabilized, PVP-enriched membrane, and link the results to the level of platelet loss during dialysis as a maker of biocompatibility. Method Six synthetic, PVP containing, dialyzers (FX CorAL, FX CorDiax [Fresenius Medical Care]; Polyflux, THERANOVA [Baxter]; ELISIO [Nipro]; xevonta [B. Braun]) were investigated in the present study. The content of PVP on blood-side surface was determined with X-ray photoelectron spectroscopy (XPS). The amount of elutable PVP was measured photometrically after 5h recirculation. The level of platelet loss was evaluated in an ex vivo recirculation model with human blood. Results Highest PVP content on the blood-side surface was found for the polysulfone-based FX CorAL (26.3%), while the polyethersulfone-based THERANOVA (15.6%) had the lowest PVP content. Elution of PVP was highest for the autoclave steam sterilized THERANOVA (9.1 mg/1.6m² dialyzer) and Polyflux (9.0 mg/1.6m² dialyzer), while the lowest PVP elution was found for the INLINE steam sterilized FX CorAL and FX CorDiax (< 0.5 mg/1.6m² dialyzer, for both). Highest platelet loss was found for xevonta (+164.4% compared to the reference) and the lowest for the FX CorAL (-225.2%) among the polysulfone-based dialyzers; among the polyethersulfone-based dialyzers, THERANOVA (+95.5%) had the highest and ELISIO (-52.1%) the lowest platelet loss. Conclusion PVP content and elution differs between commercially available dialyzers and was found to be linked to the membrane material and sterilization method. The amount of non-eluted PVP on the blood-side surface may be an important determinant for the hemocompatibility of dialyzers.

Background and aims There are safety issues regarding plant sterol ester-enriched functional food. Oxidized plant sterols, also called oxyphytosterols, are supposed to contribute to plant sterol atherogenicity. This study aimed to analyze associations of plasma oxyphytosterol levels with cardiovascular events. Methods Plasma cholesterol was measured by gas chromatography-flame ionization detection. Plasma campesterol and sitosterol and their 7-oxygenated metabolites were analyzed by gas chromatography-mass selective detection. Results In 376 patients admitted for elective coronary angiography, who were not on lipid-lowering drugs, 82 cardiovascular events occurred during a follow-up period of 4.2 ± 1.8 years. Patients with cardiovascular events had significantly higher 7α-hydroxycampesterol plasma levels (median, 0.46; [interquartile range (IQR) 0.22–0.81] nmol/L vs. median, 0.25 [IQR, 0.17–0.61] nmol/L; p = 0.003) and 7α-hydroxycampesterol-to-cholesterol ratios (median 0.08 [IQR, 0.04–0.14] nmol/mmol vs. median, 0.05 [IQR 0.03–0.11] nmol/mmol; p = 0.005) than controls without such events. Patients above the median were characterized by higher cumulative event rates in Kaplan-Meier-analysis (Logrank-test p = 0.084 and p = 0.025) for absolute and cholesterol corrected 7α-hydroxycampesterol, respectively. After adjustment for influencing factors and related lipids, the hazard ratios per one standard deviation of the log-transformed variables (HR) were 1.19 [95% confidence interval (CI), 0.95–1.48], p = 0.132 for 7α-hydroxycampesterol and HR, 1.18 [95% CI, 0.94–1.48], p = 0.154 for 7α-hydroxycampesterol-to-cholesterol ratio. None of the other investigated oxyphytosterols showed an association with cardiovascular events. Conclusions In patients not on lipid-lowering drugs, absolute plasma levels of 7α-hydroxycampesterol and their ratios to cholesterol are associated with cardiovascular events. Further research is required to elucidate the role of OPS in cardiovascular diseases.

INTRODUCTION Hydrophilic modification with polyvinylpyrrolidone (PVP) increases the biocompatibility profile of synthetic dialysis membranes. However, PVP may be eluted into the patient's blood, which has been discussed as a possible cause for adverse reactions rarely occurring with synthetic membranes. We investigated the content of PVP and its elution from the blood-side surface from commercially available dialyzers, including the novel FX CorAL, with PVP-enriched and α-tocopherol-stabilized membrane, and link the results to the level of platelet loss during dialysis as a maker of biocompatibility. METHODS Six synthetic, PVP containing, dialyzers (FX CorAL, FX CorDiax [Fresenius Medical Care]; Polyflux, THERANOVA [Baxter]; ELISIO [Nipro]; xevonta [B. Braun]) were investigated in the present study. The content of PVP on blood-side surface was determined with X-ray photoelectron spectroscopy (XPS). The amount of elutable PVP was measured photometrically after 5 h recirculation. The level of platelet loss was evaluated in an ex vivo recirculation model with human blood. FINDINGS Highest PVP content on the blood-side surface was found for the polysulfone-based FX CorAL (26.3%), while the polyethersulfone-based THERANOVA (15.6%) had the lowest PVP content. Elution of PVP was highest for the autoclave steam-sterilized THERANOVA (9.1 mg/1.6 m2 dialyzer) and Polyflux (9.0 mg/1.6 m2 dialyzer), while the lowest PVP elution was found for the INLINE steam sterilized FX CorAL and FX CorDiax (

Activation of the complement system may occur during blood-membrane interactions in hemodialysis and contribute to chronic inflammation of patients with end-stage renal disease. Hydrophilic modification with polyvinylpyrrolidone (PVP) has been suggested to increase the biocompatibility profile of dialysis membranes. In the present study we compared the complement activation of synthetic and cellulose-based membranes, including the polysulfone membrane with α-tocopherol-stabilized PVP-enriched inner surface of the novel FX CorAL dialyzer, and linked the results to their physical characteristics. Eight synthetic and cellulose-based dialyzers (FX CorAL, FX CorDiax [Fresenius Medical Care]; Polyflux, THERANOVA [Baxter]; ELISIO, SUREFLUX [Nipro]; xevonta [B. Braun]; FDX [Nikkisio Medical]) were investigated in the present study. Complement activation (C3a, C5a, and sC5b-9) was evaluated in a 3 hours ex vivo recirculation model with human blood. Albumin sieving coefficients were determined over a 4 hours ex vivo recirculation model with human plasma as a surrogate of secondary membrane formation. Zeta potential was measured as an indicator for the surface charge of the membranes. The FX CorAL dialyzer induced the lowest activation of the three complement factors (C3a: -39.4%; C5a: -57.5%; and sC5b-9: -58.9% compared to the reference). Highest complement activation was found for the cellulose-based SUREFLUX (C3a: +154.0%) and the FDX (C5a: +335.0% and sC5b-9: +287.9%) dialyzers. Moreover, the FX CorAL dialyzer had the nearest-to-neutral zeta potential (-2.38 mV) and the lowest albumin sieving coefficient decrease over time. Albumin sieving coefficient decrease was associated with complement activation by the investigated dialyzers. Our present results indicate that the surface modification implemented in the FX CorAL dialyzer reduces the secondary membrane formation and improves the biocompatibility profile. Further clinical studies are needed to investigate whether these observations will result in a lower inflammatory burden of hemodialysis patients.

Background and Aims Vascular calcification as cause of vascular stiffness is a major contributor to the high cardiac burden among stage 5 chronic kidney disease dialysis (CKD5HD) patients. Early identification of patients with high calcification propensity is crucial for proper risk stratification and management of these patients. Recently, a novel in vitro test (T50-test) was developed which determines calcification propensity of human serum and predicts mortality among ND-CKD (non-dialysis chronic kidney disease) patients, kidney transplant recipients and CKD5HD patients suffering from secondary hyperparathyroidism. We now evaluated whether these results can be confirmed in an unselected cohort of CKD5HD patients and can be used in the future for improving care of these patients. Method This prospective clinical study included 776 incident and prevalent hemodialysis patients from 8 Fresenius Medical Care NephroCare centers in Cataluña (Spain). T50 was determined at Calciscon AG, all other clinical data were retrieved from the European Clinical Database (EuCliD®). After their baseline T50 measurement, patients were followed for two years for the occurrence of the primary endpoint all-cause mortality. The association between T50 and all-cause mortality was examined by using proportional subdistribution hazards regression modelling accounting for kidney transplantation as competing event. In sensitivity analyses we adjusted for age, sex, vascular access (fistula/graft, catheter), treatment modality (HD, HDF), Charlson comorbidity index and dialysis vintage. Results Mean age of the study population was 72.2 years and 63.8% were male. 42.5% had diabetes and 55.0% past history of cardiovascular disease. Mean T50 was 283.4 min among the total cohort. During follow-up, 185 (23.8%) patients died. Patients who reached the endpoint had a significantly lower T50 at baseline as compared to those who survived during follow-up (269.6 vs 287.7 min, p = 0.001). A cross-validated model (mean c statistic: 0.5767) identified T50 as a linear predictor of all-cause-mortality (subdistribution hazard ratio (per min): 0.9957, 95% CI [0.9933;0.9981]). T50 remained a significant predictor of all-cause mortality after adjusting for relevant confounding in sensitivity analyses. Conclusion In this prospective study, we confirmed that T50 is an independent predictor of all-cause mortality among a large unselected cohort of hemodialysis patients. T50 may be used to identify patients with high vascular calcification propensity and mortality risks and may help physicians to implement a personalized and more precise renal replacement therapy option.

BACKGROUND: High-flux dialyzers effectively remove uremic toxins, are hemocompatible to minimize intradialytic humoral and cellular stimulation, and have long-term effects on patient outcomes. A new dialyzer with a modified membrane surface has been tested for performance and hemocompatibility. METHODS: This multicenter, prospective, randomized, crossover study involved the application of the new polysulfone-based FX CorAL 600 (Fresenius Medical Care, Bad Homburg, Germany), the polyarylethersulfone-based Polyflux 170H (Baxter Healthcare Corporation, Deerfield, IL), and the cellulose triacetate–based SureFlux 17UX (Nipro Medical Europe, Mechelen, Belgium), for 1 week each, to assess the noninferiority of the FX CorAL 600’s removal rate of β2-microglobulin. Performance was assessed by removal rate and clearance of small- and medium-sized molecules. Hemocompatibility was assessed through markers of complement, cell activation, contact activation, and coagulation. RESULTS: Of 70 patients, 58 composed the intention-to-treat population. The FX CorAL 600’s removal rate of β2-microglobulin was noninferior to both comparators (P

Introduction Although high serum uric acid (SUA) has been consistently associated with an increased risk of death in the general population and in persons with nondialysis chronic kidney disease (CKD), studies in patients undergoing dialysis are conflicting. It has been postulated that low SUA simply reflects poor nutritional status in dialysis patients. We here characterize the association between SUA and the risk of death in a large dialysis cohort and explore effect modification by underlying nutritional status as reflected by body composition. Methods In this retrospective cohort study, we included 16,057 hemodialysis (HD) patients treated during 2007 to 2016 in NephroCare centers as recorded in the European Clinical Database (EuCliD). The association between SUA, all-cause, and cardiovascular (CV)−related mortality was evaluated with competing risk models and characterized with splines. Effect modification was explored by lean tissue index (LTI) and fat tissue index (FTI). Results During a mean of 1.8 years of follow-up, 2791 patients (17.4%) died. We found a multivariable-adjusted U-shaped pattern between SUA and all-cause mortality. Patients with SUA levels of 6.5 mg/dl (387 μmol/l) were at the lowest risk of death (subdistribution hazard ratio = 0.94 [confidence interval {CI} 0.91; 0.96]). The form of association was not meaningfully affected by underlying LTI and FTI. Conclusion We found a U-shaped pattern between SUA levels and all-cause mortality among HD patients, which was independent of the patients’ body composition., Graphical abstract

Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes. By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of

A more precise identification of patients at “high cardiovascular risk” is preeminent in cardiovascular risk stratification. To investigate the relationships between markers of cholesterol homeostasis, cardiovascular events and all-cause mortality. We quantified markers of cholesterol homeostasis by gas chromatography–mass spectrometry in 377 subjects with suspected coronary artery disease, who were not on lipid-lowering drugs at baseline. All patients were followed for occurrence of cardiovascular events and mortality over a period of 4.9 +/− 1.7 years. The standardized mortality ratio (SMR) was calculated as the ratio of the observed and the expected deaths based on the death rates of the Regional Databases Germany, and Poisson regression (rate ratio, RR) was used to compare subgroups. The SMR and RR were standardized for sex, age category and calendar period. In addition, Cox regression (Hazard ratio, HR) was used to determine the effect of co-variables on (cardiovascular) mortality within the cohort. Cardiovascular events, cardiovascular mortality and all-cause mortality. A total of 42 deaths were observed in 1818 person-years corresponding with an SMR of 0.99 (95% CI 0.71–1.33; p = 0.556). A fatal cardiovascular event occurred in 26 patients. Lower levels of lathosterol were associated with increased cardiovascular mortality (HR 1.59; 95% CI: 1.16–2.17; p = 0.004) and excess all-cause mortality (HR 1.41; 95% CI: 1.09–1.85; p = 0.011). Lower lathosterol tertile compared to the adjacent higher tertile was associated with 1.6 times higher all-cause mortality risk (RR 1.60; 95% CI 1.07–2.40; p for trend = 0.022). This corresponded with a 2.3 times higher mortality risk of a lathosterol–LDL ratio equal to or below the median (RR 2.29; 95% CI 1.19–4.43; p = 0.013). None of the other cholesterol homeostasis markers were associated with cardiovascular and all-cause mortality. In patients not on lipid-lowering agents, low serum lathosterol correlated with increased risk of cardiovascular events and excess all-cause mortality.

Background Treatment of iron deficiency with intravenous (i.v.) iron is a first-line strategy to improve anaemia of chronic kidney disease. Previous in vitro experiments demonstrated that different i.v. iron preparations inhibit differentiation of haematopoietic stem cells to monocytes, but their effect on monocyte differentiation to macrophages and mature dendritic cells (mDCs) has not been assessed. We investigated substance-specific effects of iron sucrose (IS), sodium ferric gluconate (SFG), ferric carboxymaltose (FCM) and iron isomaltoside 1000 (IIM) on monocytic differentiation to M1/M2 macrophages and mDCs. Methods Via flow cytometry and microRNA (miRNA) expression analysis, we morphologically and functionally characterized monocyte differentiation to M1/M2 macrophages and mDCs after monocyte stimulation with IS, SFG, FCM and IIM (0.133, 0.266 and 0.533 mg/mL, respectively). To assess potential clinical implications, we compared monocytic phagocytosis capacity in dialysis patients who received either 500 mg IS or IIM. Results Phenotypically, IS and SFG dysregulated the expression of macrophage (e.g. CD40, CD163) and mDC (e.g. CD1c, CD141) surface markers. Functionally, IS and SFG impaired macrophage phagocytosis capacity. Phenotypic and functional alterations were less pronounced with FCM, and virtually absent with IIM. In miRNA expression analysis of mDCs, IS dysregulated miRNAs such as miR-146b-5p and miR-155-5p, which are linked to Toll-like receptor and mitogen-activated protein kinase signalling pathways. In vivo, IS reduced monocytic phagocytosis capacity within 1 h after infusion, while IIM did not. Conclusions This study demonstrates that less stable i.v. iron preparations specifically affect monocyte differentiation towards macrophages and mDCs.

CVD remains the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD profoundly affects HDL composition and functionality, but whether abnormal HDL independently contributes to cardiovascular events in CKD patients remains elusive. In the present study, we assessed whether compositional and functional properties of HDL predict cardiovascular outcome among 526 nondialysis CKD patients who participate in the CARE FOR HOMe study. We measured HDL cholesterol, the content of HDL-associated proinflammatory serum amyloid A (SAA), and activities of the HDL enzymes paraoxonase and lipoprotein-associated phospholipase A2 (Lp-PLA2). In addition, we assessed the antioxidative activity of apoB-depleted serum. During a mean follow-up of 5.1 ± 2.1 years, 153 patients reached the predefined primary endpoint, a composite of atherosclerotic cardiovascular events including cardiovascular mortality and death of any cause. In univariate Cox regression analyses, lower HDL-cholesterol levels, higher HDL-associated SAA content, and lower paraoxonase activity predicted cardiovascular outcome, while Lp-PLA2 activity and antioxidative capacity did not. HDL-cholesterol and HDL-paraoxonase activity lost their association with cardiovascular outcome after adjustment for traditional cardiovascular and renal risk factors, while SAA lost its association after further adjustment for C-reactive protein. In conclusion, our data suggest that neither HDL quantity nor HDL composition or function independently predict cardiovascular outcome among nondialysis CKD patients.

Monocytes are heterogeneous cells consisting of (at least) three subsets: classical, intermediate, and nonclassical monocytes. Correct enumeration of cell counts necessitates well-defined gating strategies, which are essentially based upon CD14 and CD16 expression. For the delineation of intermediate from nonclassical monocytes, a “rectangular gating (RG) strategy” and a “trapezoid gating (TG) strategy” have been proposed. We compared the two gating strategies in a well-defined clinical cohort of patients with chronic kidney disease (CKD). Within the ongoing CARE FOR HOMe study, monocyte subsets were reanalyzed in 416 CKD patients, who were followed 3.6 ± 1.6 years for the occurrence of a cardiovascular event. Gating was performed by either RG or TG. We analyzed the expression of surface markers, and compared the predictive role of cell counts of monocyte subsets, as defined by RG and TG, respectively. With both gating strategies, higher intermediate monocyte counts predicted the cardiovascular endpoint in Kaplan-Meier analyses (P

Chronic kidney disease (CKD) is associated with increased risk of renal and cardiovascular events. It has been claimed that endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are contributing factors. However, earlier studies were partly contradictory and mainly focused on prevalent dialysis patients. Moreover, the potential contribution of degradation products, such as acetylated ADMA and SDMA (AcADMA and AcSDMA) and other methylarginines including L-NG-monomethylarginine (LNMMA) remains unknown. To better understand their potential pathophysiological contribution to renal and cardiovascular events, we aimed to provide a comprehensive analysis of methylarginines in a cohort of patients with non-dialysis CKD. Blood samples of 528 patients with CKD KDIGO G2 to G4 were obtained from the CARE FOR HOMe study. Baseline plasma levels of ADMA, SDMA, AcADMA, AcSDMA, and LNMMA were measured by liquid chromatography—tandem mass spectrometry. All patients were followed annually for CKD progression and for incident atherosclerotic cardiovascular events. During 5.1 ± 2.1 years follow-up, 80 patients displayed CKD progression and 145 patients developed incident atherosclerotic cardiovascular events. In univariate Cox regression analyses, elevated plasma levels of all five metabolites were associated with both CKD progression and atherosclerotic cardiovascular disease. However, adjustment for confounders attenuated the prognostic implications of ADMA, LNMMA, AcADMA and AcSDMA. In contrast, patients in the highest tertile of plasma SDMA remained at highest risk for CKD progression and incident atherosclerotic cardiovascular events in fully adjusted Cox regression analyses. Our results underline a potential pathophysiological role of SDMA in CKD progression and atherosclerotic cardiovascular disease among non-dialysis CKD patients. SDMA predicts CKD progression and future atherosclerotic cardiovascular events more consistently than other methylarginines. Future experimental and clinical studies should therefore focus upon SDMA rather than upon ADMA.

Introduction and Aims: Serum p-cresyl sulfate associates with cardiovascular disease in patients at different stages of chronic kidney disease. p-Cresyl sulfate concentrations are determined by intestinal uptake of p-cresol, human metabolism to p-cresyl sulfate and renal clearance. Whether intestinal uptake of p-cresol itself is associated with cardiovascular disease in patients with renal disease has not been studied to date. Methods: We performed a prospective study in patients with chronic kidney disease stage 1-5 (clinicaltrials.gov NCT00441623). Intestinal uptake of p-cresol, under steady state conditions, was estimated from 24h urinary excretion of p-cresyl sulfate. Primary endpoint was time to first cardiovascular event, i.e. cardiac death, myocardial infarction/ischemia, ventricular arythmia, cardiovascular surgery, cerebrovascular accident or symptomatic peripheral arterial disease. Statistical analysis was done using Kaplan Meier estimates and Cox proportional hazard analyses. Results: In a cohort of 200 patients, median 24h urinary excretion of p-cresyl sulfate was 457.47 µmol (IQR 252.68-697.17). After a median follow-up of 52 months, 25 patients reached the primary endpoint (tertile 1/2/3: 5/6/14 events, log rank P 0.0368, see figure). Higher urinary excretion of p-cresyl sulfate was related with cardiovascular events (univariate hazard ratio per 100 µmol increase: 1.112, P 0.0015). In multivariate analysis, urinary excretion of p-cresyl sulfate remained a predictor of cardiovascular events, independent of markers of renal function (Hazard ratio 1.120, P 0.0022) and in different models with other cardiovascular risk factors (Framingham risk factors, cardiovascular history, diabetes mellitus and biochemical parameters). The independent association between urinary excretion of p-cresyl sulfate and outcome persisted after correction for serum p-cresyl sulfate. Conclusions: Intestinal uptake of p-cresol associates with cardiovascular disease independent of renal function. Insights into mechanisms governing intestinal generation and absorption of p-cresol may lead to identification of novel therapeutic targets to reduce cardiovascular disease risk in patients with chronic kidney disease.

Background Iron deficiency contributes to anaemia in patients with chronic kidney disease. I.v. iron is therefore widely used for anaemia treatment, although it may induce oxidative stress and activate monocytes. Different i.v. iron preparations are available, but interestingly their substance-specific immunologic effects are poorly studied. Methods We analysed the effect of iron sucrose, ferric carboxymaltose, iron isomaltoside 1000, low-molecular-weight iron dextran and ferumoxytol on classical, intermediate and nonclassical monocyte biology. We therefore stimulated in vitro mature monocytes and haematopoietic CD34+ stem cells during their differentiation into monocytes with different concentrations (0.133, 0.266, 0.533 mg/mL) of i.v. iron preparations. Alterations of monocyte subset distribution, expression of surface markers (CD86, CCR5, CX3CR1), as well as production of pro-inflammatory cytokines (TNF-α, IL-1β) and reactive oxygen species were measured using flow cytometry. Additionally, we analysed phagocytosis and antigen presentation capacity. Results We found specific immunologic effects after stimulation with iron sucrose which were not induced by the other iron preparations. Iron sucrose activated monocyte subsets leading to significantly increased CD86 expression. Simultaneously CD16 and CX3CR1 expression and monocytic phagocytosis capacity were decreased. Additionally, differentiation of monocytes from haematopoietic CD34+ stem cells was almost completely abolished after stimulation with iron sucrose. Conclusions Our findings demonstrate that specific immunologic effects of distinct i.v. iron preparations exist. The clinical relevance of these findings requires further investigation.

Background— Accelerated atherosclerosis is a hallmark of chronic kidney disease (CKD). Although the role of epigenetic dysregulation in atherosclerosis is increasingly appreciated, only a few studies focused on epigenetics in CKD-associated cardiovascular disease, virtually all of which assessed epigenetic dysregulation globally. We hypothesized that gene-specific epigenetic dysregulation in CKD exists, affecting genes pertinent to inflammation and atherosclerosis. Methods and Results— Ten clinically stable patients undergoing hemodialysis therapy and 10 healthy age- and sex-matched controls were recruited. Genome-wide analysis of DNA methylation was performed by SuperTAG methylation-specific digital karyotyping, in order to identify genes differentially methylated in CKD. Analysis of 27 043 436 tags revealed 4288 genomic loci with differential DNA methylation ( P –10 ) between hemodialysis patients and control subjects. Annotation of UniTags to promoter databases allowed us to identify 52 candidate genes associated with cardiovascular disease and 97 candidate genes associated with immune/infection diseases. These candidate genes could be classified to distinct proatherogenic processes, including lipid metabolism and transport (eg, HMGCR , SREBF1 , LRP5 , EPHX2 , and FDPS ), cell proliferation and cell-cycle regulation (eg, MIK67 , TP53 , and ALOX12 ), angiogenesis (eg, ANGPT2 , ADAMTS10 , and FLT4 ), and inflammation (eg, TNFSF10 , LY96 , IFNGR1 , HSPA1A , and IL12RB1 ). Conclusions— We provide a comprehensive analysis of genome-wide epigenetic alterations in CKD, identifying candidate genes associated with proatherogenic and inflammatory processes. These results may spur further research in the field of epigenetics in kidney disease and point to new therapeutic strategies in CKD-associated atherosclerotic disease.

Atherosclerosis has been characterized as an inflammatory process, in which monocytes and monocyte-derived macrophages are of paramount importance. Contrasting with their established role in atherosclerosis, monocytes have not unanimously been found to predict cardiovascular events in large epidemiological studies. However, in these studies human monocyte heterogeneity has been largely overlooked so far. Three human monocyte subsets can be distinguished: classical CD14(++)CD16(-), intermediate CD14(++)CD16(+) and nonclassical CD14(+)CD16(++) monocytes. Of note, correct enumeration of subset counts requires appropriate staining and gating strategies that encompass a pan-monocytic marker (e.g. HLA-DR or CD86). In experimental studies on murine atherogenesis a monocyte subset-specific contribution to atherosclerosis has been established. However, major interspecies differences in atherogenesis itself, as well as in the immune system (including monocyte subset phenotype and distribution) preclude a direct extrapolation to human pathology. Experimental and pilot clinical studies point to a prominent involvement of intermediate CD14(++)CD16(+) monocytes in human atherosclerosis. Future clinical studies should analyze monocyte heterogeneity in cardiovascular disease. If a specific contribution of intermediate monocytes should be confirmed, immunomodulation of this monocyte subset could represent a future therapeutic target in atherosclerosis.

Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes. By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of

Objectives The aim of this study was to analyze the yet ill-defined relationship of distinct human monocyte subsets with cardiovascular outcomes in a broad patient population at cardiovascular risk. Background Monocytes, the most abundant immune cell type found in atherosclerotic plaques, are crucial promoters of atherogenesis. Three distinct human monocyte subsets exist: classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Immunomodulation of distinct monocyte subsets has recently been discussed as a new therapeutic avenue in atherosclerosis. Methods Cardiovascular events in 951 subjects referred for elective coronary angiography were prospectively analyzed. Monocyte subset analysis was performed using flow cytometry, blinded to patients’ clinical characteristics, and patients were categorized according to quartiles of total monocyte and monocyte subset counts. The primary endpoint was defined a priori as the first occurrence of cardiovascular death, acute myocardial infarction, or nonhemorrhagic stroke. Endpoint adjudication was done blinded to monocyte subset distribution. Results During a mean follow-up period of 2.6 ± 1.0 years, 93 patients experienced the primary endpoint. In univariate Kaplan-Meier analysis, counts of total (p = 0.010), classical CD14++CD16− (p = 0.024), and intermediate CD14++CD16+ (p Conclusions CD14++CD16+ monocytes independently predicted cardiovascular events in subjects referred for elective coronary angiography. Future studies will be needed to elucidate whether CD14++CD16+ monocytes may become a target cell population for new therapeutic strategies in atherosclerosis.

Recent clinical studies have shown a strong correlation between in vitro cholesterol efflux capacity (CEC) and cardiovascular disease prevalence [(1)][1] and incidence [(2,3)][2] that seems to be independent of high-density lipoprotein cholesterol (HDL-C) levels. We tested the hypothesis that CEC

Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14++CD16+ monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14++CD16+ monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14++CD16+ monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. After CD14++CD16+ monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.

Progression of CKD toward ESRD is heterogeneous. The Kidney Failure Risk Equation (KFRE) was developed to identify CKD patients at high risk of ESRD. We aimed to externally validate KFRE and to test whether the addition of predefined Duplex ultrasound markers - renal resistive index (RRI) or difference of resistive indices in spleen and kidney (DI-RISK) - improved ESRD prediction.The prospective Cardiovascular and Renal Outcome in CKD 2-4 Patients-The Fourth Homburg evaluation (CARE FOR HOMe) study recruits CKD stage G2-G4 patients referred to a tertiary referral center for nephrologic care. Four hundred three CARE FOR HOMe participants enrolled between 2008 and 2012 had available RRI measurements at study inclusion; they were subsequently followed for a mean of 4.4±1.6 years. This subcohort was used to validate KFRE and to assess the added value of the ultrasound markers (new models KFRE+RRI and KFRE+DI-RISK). Model performance was assessed by log-likelihood ratio test, c-statistic, integrated discrimination improvement metrics (for study participants without subsequent ESRD [IDI No ESRD] and for patients with ESRD [IDI ESRD]), and calibration plots. If either new model improved on KFRE, we determined to validate it in an independent cohort of 162 CKD patients.KFRE predicted ESRD in CARE FOR HOMe participants with a c-statistic of 0.91 (95% confidence interval, 0.83 to 0.99). Adding RRI improved the KFRE model (P0.001), and the KFRE+RRI model was well calibrated; however, the c-statistic (0.91 [0.83-1.00]) was similar, and overall sensitivity (IDI No ESRD=0.05 [0.00-0.10]) or overall specificity (IDI ESRD=0.00 [0.00-0.01]) did not improve. Adding DI-RISK did not improve the KRFE model. In the external validation cohort, we confirmed that the KFRE+RRI model did not outperform KFRE.Routine Duplex examinations among CKD patients did not improve risk prediction for progression to ESRD beyond a validated equation.

Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.

Monocytes are heterogeneous cells consisting of (at least) three subsets: classical, intermediate, and nonclassical monocytes. Correct enumeration of cell counts necessitates well-defined gating strategies, which are essentially based upon CD14 and CD16 expression. For the delineation of intermediate from nonclassical monocytes, a "rectangular gating (RG) strategy" and a "trapezoid gating (TG) strategy" have been proposed. We compared the two gating strategies in a well-defined clinical cohort of patients with chronic kidney disease (CKD). Within the ongoing CARE FOR HOMe study, monocyte subsets were reanalyzed in 416 CKD patients, who were followed 3.6 ± 1.6 years for the occurrence of a cardiovascular event. Gating was performed by either RG or TG. We analyzed the expression of surface markers, and compared the predictive role of cell counts of monocyte subsets, as defined by RG and TG, respectively. With both gating strategies, higher intermediate monocyte counts predicted the cardiovascular endpoint in Kaplan-Meier analyses (P 0.001 with RG; P 0.001 with TG). After correction for confounders, intermediate monocyte counts remained independent predictors in Cox-Regression analyses (HR = 1.013 [95% CI: 1.006-1.020; P 0.001] with RG; HR = 1.015 [95% CI: 1.006-1.024; P = 0.001] with TG). NRI was 3.9% when reclassifying patients from quartiles of intermediate monocyte counts with RG strategy toward quartiles of intermediate monocytes counts with TG strategy. In expression analysis, those monocytes which are defined as intermediate monocytes by the RG strategy and as nonclassical monocytes by the TG strategy share characteristics of both subsets. In conclusion, intermediate monocytes were independent predictors of cardiovascular outcome irrespective of the applied gating strategy. Future studies should aim to identify markers that allow for an unequivocal definition of intermediate monocytes, which may further improve their power to predict cardiovascular events.

Despite significant improvements in our health care systems, cardiovascular disease (CVD) remains a major cause of death across the world. All features of epigenetic regulation, i.e., DNA methylation, histone modifications, and miRNAs, have now been characterized in the context of CVD. In aggregate, there is substantial evidence that epigenetic mechanisms contribute centrally to the development and progression of CVD.

BACKGROUND Monocytes are critical in innate immunity and transplantation. Three monocyte subsets exist, CD14(++)CD16(-), CD14(++)CD16(+) and CD14(+)CD16(++) monocytes; cell counts of CD14(++)CD16(+) and CD14(+)CD16(++) monocytes are increased in pre-transplant chronic kidney disease. Interestingly, the effect of immunosuppressants on monocyte heterogeneity has not been well studied. METHODS The impact of immunosuppressants on monocyte subsets was studied: (i) in 152 kidney transplant (KTx) recipients to characterize subset distribution in the steady state, (ii) in patients after autologous (n = 10) versus allogenic (n = 9) haematopoietic stem cell transplantation (HSCT) to analyse monocyte subset development and (iii) in an in vitro model to compare the effect of immunosuppressants on monocyte subset biology. RESULTS In KTx, steroid intake was associated with higher total, CD14(++)CD16(-) and CD14(++)CD16(+) monocyte counts, but fewer CD14(+)CD16(++) monocytes, whereas intake of mycophenolate, calcineurin inhibitors (CNI) and mammalian target of rapamycin inhibitors (mTORI) did not affect monocyte (subset) counts. In linear regression analysis, only steroid intake was a significant determinant of monocyte (subset) counts: total monocytes (β = 0.331; P < 0.001), CD14(++)CD16(-) monocytes (β = 0.374; P < 0.001), CD14(++)CD16(+) monocytes (β = 0.221; P = 0.010) and CD14(+)CD16(++) monocytes (β = -0.169; P = 0.049). After HSCT, CD14(++)CD16(-) monocytes were the first to arise, followed by CD14(++)CD16(+) and later by CD14(+)CD16(++) monocytes. Monocyte subset distribution did not differ significantly in patients after allogenic compared with autologous transplantation. CNI, mycophenolate and methotrexate did not influence monocyte subset development, but modified surface receptor expression (CCR2, HLA-DR, ENG, TEK and TLR4) in allogenic HSCT. CONCLUSION Chronic low-dose steroids are associated with monocytosis and higher counts of CD14(++)CD16(-) and of proinflammatory CD14(++)CD16(+) monocytes.

Objective— Patients with chronic kidney disease (CKD) display impaired cholesterol efflux capacity and elevated CD14 ++ CD16 + monocyte counts. In mice, dysfunctional cholesterol efflux causes monocytosis. It is unknown whether cholesterol efflux capacity and monocyte subsets are associated in CKD. Approach and Results— In 438 patients with CKD, mediators of cholesterol efflux capacity (high-density lipoprotein cholesterol/apolipoprotein A-I) and monocyte subsets were analyzed as predictors of cardiovascular events. Monocyte subset-specific intracellular lipid content, CD36, CD68, and ABCA1 were measured in a subgroup. Experimentally, we analyzed subset-specific cholesterol efflux capacity and response to oxidized low-density lipoprotein cholesterol stimulation in CKD. Epidemiologically, both low Apo-I and low high-density lipoprotein cholesterol were associated with high CD14 ++ CD16 + monocyte counts in linear regression analyses (apolipoprotein A-I: β=−0.171; P P =0.005), but not with counts of other monocyte subsets. In contrast to apolipoprotein A-I or high-density lipoprotein cholesterol, higher CD14 ++ CD16 + monocyte counts independently predicted cardiovascular events (hazard ratio per increase of 1 cell/μL: 1.011 [1.003–1.020]; P =0.007). Experimentally, CD14 ++ CD16 + monocytes demonstrated preferential lipid accumulation, high CD36, CD68, and low ABCA1 expression and, consequently, displayed low cholesterol efflux capacity, avid oxidized low-density lipoprotein cholesterol uptake, and potent intracellular interleukin-6, interleukin-1β, and tumor necrosis factor-α production. Conclusions— Taken together, mediators of cholesterol efflux are associated with CD14 ++ CD16 + monocyte counts, which independently predict adverse outcome in CKD.

Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/.

Although homocysteine has been proposed as a cardiovascular risk factor, interventional trials lowering homocysteine have not consistently demonstrated clinical benefit. Recent evidence proposed the homocysteine metabolite S-adenosylhomocysteine (SAH) rather than homocysteine itself as the real culprit in cardiovascular disease. Of note, SAH is predominantly excreted by the kidneys, and cannot be lowered by vitamin supplementation. Due to its cumbersome measurement, data from large studies on the association between SAH, kidney function and cardiovascular disease are not available.We recruited 420 apparently healthy subjects into our I Like HOMe FU study. Among all study participants, we assessed parameters of C1 metabolism (homocysteine, SAH and S-adenosylmethionine), renal function (estimated glomerular filtration rate [eGFR]) and subclinical atherosclerosis (common carotid intima-media-thickness [IMT]). eGFR was estimated by the CKD-EPIcreat-cys equation.Traditional cardiovascular risk factors and subclinical atherosclerosis were associated with SAH, but not with homocysteine (IMT vs SAH: r = 0.129; p = 0.010; IMT vs homocysteine: r = 0.009; p = 0.853). Moreover, renal function was more closely correlated with SAH than with homocysteine (eGFR vs SAH: r = -0.335; p0.001; eGFR vs homocysteine: r = -0.250; p0.001). The association between eGFR and SAH remained significant after adjustment for traditional cardiovascular risk factors.In summary, cardiovascular risk factors, subclinical atherosclerosis and eGFR are more strongly associated with SAH than with homocysteine in apparently healthy subjects. Thus, SAH might represent a more promising target to prevent cardiovascular disease than homocysteine.

Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD.

Background. The recently introduced CKD-EPIcreat-cys equation surpassed creatinine-based equations for GFR estimation in a large cross-sectional analysis. However, its performance to predict individual risk of CKD progression and death in patients with various underlying CKD etiologies is unknown. Methods. We recruited 444 patients with CKD GFR categories 2–4 (eGFR 15–89 mL/min/1.73 m 2 ); baseline eGFR was estimated by the established MDRD and CKD-EPIcreat equations and by the novel CKD-EPIcreat-cys equation. Results. Patients were followed for 2.7 ± 1.2 years for the occurrence of the combined predefined endpoint event: death, need for renal replacement therapy or halving of eGFR. The endpoint occurred in 62 patients. Reclassification from MDRD determined categories to CKD-EPIcreat-cys categories yielded net reclassification improvements for those with the endpoint event (NRIevents) of 27.4% (95% CI: 16.7–40.0%) and for those without the event (NRInon-events )o f−3.1% (−8.2 to 1.6%). Similarly, reclassification from CKD-EPIcreat categories to CKD-EPIcreat-cys categories yielded an NRIevents of 22.6% (10.2–34.3%) and NRInon-events of −11.3% (−15.9 to −6.5%). Addition of albuminuria to each eGFR equation increased the calculated risk of the outcome for a net 26–32% of those who subsequently reached the endpoint, and reduced the calculated risk in a net 21–23% in non-event patients, but only minimally. Conclusions. The CKD-EPIcreat-cys equation assigned patients who went on to have the event to more appropriate CKD risk categories than MDRD and CKD-EPIcreat, but patients without the event to less appropriate categories than CKDEPIcreat. Addition of albuminuria marginally improved risk classification for those who had the event.