Plasma Concentrations of Trimethylamine-N-Oxide, Choline, and Betaine in Patients With Moderate to Advanced Chronic Kidney Disease and Their Relation to Cardiovascular and Renal Outcomes.

OBJECTIVES: Trimethylamine N-oxide (TMAO) is a gut bacteria-mediated liver metabolite of dietary betaine, choline, and carnitine, which is excreted by glomerular filtration. We studied whether TMAO is excreted by cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). METHODS: Among 478 patients with CKD stage G2 (n = 104), G3a (n = 163), G3b (n = 123), and G4 (n = 88), we studied the association between fasting plasma concentrations of TMAO, choline, or betaine at baseline and kidney function, prevalent CVD, and future renal outcomes during a mean follow-up of 5.1 years. RESULTS: Decreased glomerular filtration rate was associated with higher plasma concentrations of TMAO, choline, and betaine. Baseline concentrations of TMAO were higher in participants with preexisting CVD compared to those without CVD (8.4 [10.1] vs. 7.8 [8.0] µmol/L; P = .047), but the difference was not significant after adjusting for confounders. During the follow-up, 147 participants experienced CVD or died, and 144 reached the predefined renal endpoint. In the adjusted regression analyses, TMAO or choline concentrations in the upper three quartiles (vs. the lowest quartile) were not associated with any of the study's clinical endpoints. In contrast, the adjusted hazard ratio of plasma betaine in the highest quartile versus the lowest quartile was 2.14 (1.32, 3.47) for the CVD endpoint and 1.64 (1.00, 2.67) for the renal endpoint. CONCLUSIONS: Elevated plasma TMAO concentrations were explained by impaired kidney function. Elevated plasma concentrations of betaine, but not those of TMAO or choline, constituted a risk factor for adverse outcomes. TMAO might not be an appropriate target to reduce CVD or renal outcomes in patients with preexisting CKD.

ELOVL2-methylation and renal and cardiovascular event in patients with chronic kidney disease.

BACKGROUND: Methylation of the Elongation Of Very Long Chain Fatty Acids-Like 2 (ELOVL2) gene promoter may predict premature ageing and cardiovascular risk. METHODS: We studied the cross-sectional associations between blood ELOVL2-methylation and cardiovascular risk factors in 350 patients with chronic kidney disease (CKD) stage G2-G4 aged between 22 and 90 years. In a follow-up study for a mean of 3.9 years, we investigated the association between baseline ELOVL2-methylation and renal or cardiovascular events including death. RESULTS: ELOVL2-methylation at seven CpG cites increased with age (the correlation coefficients between 0.67 and 0.87, p < 0.001). The ELOVL2-CpGs methylation was lower in patients with CKD stage G2 versus those in stage G3a, G3b and G4, but the differences were explained by age. ELOVL2-CpGs methylation showed no correlations with cardiovascular risk factors after adjusting for age. During the follow-up, 64 patients showed deterioration in renal function or died and 77 showed cardiovascular events or died. The hazard ratio and 95% confidence intervals for renal or cardiovascular events according to baseline ELOVL2-CpGs methylation were not significant after adjustment for covariates. CONCLUSIONS: ELOVL2-hypermethylation showed a strong association with age, but was not independently associated with cardiovascular risk factors or with future renal or cardiovascular events in patients with CKD. ELOVL2 gene methylation is not likely to be itself a cause for ageing or illnesses, but it could be rather influenced by other upstream processes that deserve investigation.

Assessment of a serum calcification propensity test for the prediction of all-cause mortality among hemodialysis patients.

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.

Time-resolving characterization of molecular weight retention changes among three synthetic high-flux dialyzers.

BACKGROUND: 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. METHODS: In plasma recirculation experiments over 240 min, sieving coefficients (SC) for ß2-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. RESULTS: SC decreased over time for all dialyzers (mean relative decrease across all dialyzers: ß2-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). CONCLUSIONS: 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.

FGFR4 and Klotho Polymorphisms Are Not Associated with Cardiovascular Outcomes in Chronic Kidney Disease.

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.

Evolution of body composition and wasting indicators by time of day of haemodialysis.

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.

Complement activation by dialysis membranes and its association with secondary membrane formation and surface charge.

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.

HDL functionality and cardiovascular outcome among nondialysis chronic kidney disease patients.

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.

L-Homoarginine and its AGXT2-metabolite GOCA in chronic kidney disease as markers for clinical status and prognosis.

Plasma concentrations of L-homoarginine (hArg) are an emerging marker for clinical status and prognosis in renal and cardiovascular disease. Lowered hArg concentrations are associated with higher risk for these conditions, although a clear pathophysiological explanation for this association has not been established. Baseline plasma samples of patients with different stages of chronic kidney disease (CKD) (n = 527) were obtained from the CARE FOR HOMe study and were analyzed for hArg and, for the first time, its metabolite 6-guanidino-2-oxocaproic acid (GOCA) by isotope dilution LC-MS/MS methods. GOCA is converted from hArg by the enzyme alanine:glyoxylate aminotransferase 2 (AGXT2), which is also in the focus of current cardiovascular research. hArg levels ranged from 0.20-4.01 µmol/L with a median of 1.42 µmol/L, whereas GOCA levels were 0.08-25.82 nmol/L with a median of 1.45 nmol/L. hArg levels in the highest tertile (≥ 1.71 µmol/L) were associated with significantly lower risk for reaching the renal (hazard ratio 0.369, 95% confidence interval 0.028-0.655) or cardiovascular (HR 0.458, CI 0.295-0.712) endpoints in univariate Cox regression analysis. Inversely, GOCA levels in the highest tertile (≥ 2.13 nmol/L) were associated with increased renal (HR 3.807, CI 1.963-7.381) and cardiovascular (HR 1.611, CI 1.041-2.495) risk. A decreased ratio between hArg and GOCA predicted even more pronounced the risks for renal (HR 0.178, CI 0.087-0.363) and cardiovascular (HR 0.447, CI 0.281-0.709) events. However, adjustment for the confounders eGFR and albuminuria attenuated these findings. A pathophysiological role of an increased activity of AGXT2 in CKD should be evaluated in future clinical studies.

slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation.

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.

Impact of individual intravenous iron preparations on the differentiation of monocytes towards macrophages and dendritic cells.

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.

Comparison of two different strategies for human monocyte subsets gating within the large-scale prospective CARE FOR HOMe Study.

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.

Immunosuppression and monocyte subsets.

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 (ß = 0.331; P < 0.001), CD14(++)CD16(-) monocytes (ß = 0.374; P < 0.001), CD14(++)CD16(+) monocytes (ß = 0.221; P = 0.010) and CD14(+)CD16(++) monocytes (ß = -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.

Lower Apo A-I and lower HDL-C levels are associated with higher intermediate CD14++CD16+ monocyte counts that predict cardiovascular events in chronic kidney disease.

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: ß=-0.171; P<0.001; high-density lipoprotein cholesterol: ß=-0.138; 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-1ß, 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.

Distinct immunologic effects of different intravenous iron preparations on monocytes.

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-1ß) 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.

The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation incorporating both cystatin C and creatinine best predicts individual risk: a cohort study in 444 patients with chronic kidney disease.

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 m2); 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) of -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 CKD-EPIcreat. Addition of albuminuria marginally improved risk classification for those who had the event.

Hydrophilic Modification of Dialysis Membranes Sustains Middle Molecule Removal and Filtration Characteristics.

While efficient removal of uremic toxins and accumulated water is pivotal for the well-being of dialysis patients, protein adsorption to the dialyzer membrane reduces the performance of a dialyzer. Hydrophilic membrane modification with polyvinylpyrrolidone (PVP) has been shown to reduce protein adsorption and to stabilize membrane permeability. In this study we compared middle molecule clearance and filtration performance of nine polysulfone-, polyethersulfone-, and cellulose-based dialyzers over time. Protein adsorption was simulated in recirculation experiments, while ß2-microglobulin clearance as well as transmembrane pressure (TMP) and filtrate flow were determined over time. The results of this study showed that ß2-microglobulin clearance (-7.2 mL/min/m2) and filtrate flow (-54.4 mL/min) decreased strongly during the first 30 min and slowly afterwards (-0.7 mL/min/m2 and -6.8 mL/min, respectively, for the next 30 min); the TMP increase (+37.2 mmHg and +8.6 mmHg, respectively) showed comparable kinetics. Across all tested dialyzers, the dialyzer with a hydrophilic modified membrane (FX CorAL) had the highest ß2-microglobulin clearance after protein fouling and the most stable filtration characteristics. In conclusion, hydrophilic membrane modification with PVP stabilizes the removal capacity of middle molecules and filtration performance over time. Such dialyzers may have benefits during hemodiafiltration treatments which aim to achieve high exchange volumes.

SuperSAGE evidence for CD14++CD16+ monocytes as a third monocyte subset.

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.

Clinical relevance of epigenetic dysregulation in chronic kidney disease-associated cardiovascular disease.

Across the spectrum of clinical medicine, the field of epigenetics has gained substantial scientific interest in recent years. Epigenetics refers to modifications in gene expression which are not explained by changes in DNA sequence. Classical components of epigenetic regulation comprise DNA methylation, histone modifications and RNA interference. In chronic kidney disease (CKD), several features of uraemia, such as hyperhomocysteinemia and inflammation, may contribute to changes in epigenetic gene regulation. It has been suggested that these changes may affect genes related to cardiovascular disease. Thereby, a uraemia-associated disturbance in epigenetic regulation may contribute to the substantial increase in cardiovascular morbidity in CKD patients. The present review aims to summarize current knowledge of epigenetic dysregulation in cardiovascular disease from a nephrological perspective, with a special focus on DNA methylation. We first describe the impact of altered epigenetic regulation in non-CKD-associated arteriosclerosis, and next characterize uraemic features which may affect epigenetic gene regulation in the context of cardiovascular disease. Finally, we conclude that substantial additional work is needed before epigenetic regulatory mechanisms may become therapeutic targets in CKD-associated cardiovascular disease.

Hemodiafiltration: Technical and Medical Insights.

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.