[Show abstract][Hide abstract] ABSTRACT: Reduction of inflammation profile in human THP-1 monocytes and Peripheral Blood Mononuclear Cells - secondary effects of serum and dialysates obtained from chronic hemodialysis patients maintained on high-cutoff membranes
1Bogusz Trojanowicz, 1Christof Ulrich, 1Roman Fiedler, 1Eric Seibert, 2Marcus Storr, 3Daniel Zickler, 3Ralf Schindler, 1Matthias Girndt
1Department of Internal Medicine II, Martin-Luther-University Halle-Wittenberg, Germany
2Department of Research and Development, Gambro Dialysatoren GmbH, Hechingen, Germany
3Department of Nephrology, Charité Virchow Clinic, Berlin, Germany
AIMS: Patients with chronic kidney disease maintained on intermittent hemodialysis suffer from systemic chronic inflammation which is causally associated with high mortality. Inflammation mediators of 15-45 kDa range cannot be effectively removed by conventional dialysis-membranes. METHODS: In this study we tested the influence of serum and dialysates obtained from 19 patients maintained on High-Cutoff (HCO) and Polyflux (PF) membranes on inflammation profile of human THP-1 monocytes and Peripheral Blood Mononuclear Cells (PBMC). RESULTS: Treatment of freshly isolated PBMC or THP-1 monocytes with HCO serum led to significant reduction of TNFa and IL-6 expression as well as inflammation-related osteopontin and osteocalcin as compared to PF membrane treatment. Furthermore, treatment with HCO dialysates revealed development of pro-apoptotic phenotype of the cells which demonstrated significantly increased percentage of 7-AAD (7-aminoactinomycin D) and Annexin V positivity. Transcriptional screening of serum-treated cells conducted with RT-Profiler assay (84 inflammation-related targets), revealed noticeably decreased inflammation profile under HCO serum as compared to PF treatment. CONCLUSIONS: Taken together, these data demonstrate that HCO membranes eliminate a wide spectrum of mediators possessing high inflammation and cytotoxic properties
6. Jahrestagung der Deutschen Gesellschaft für Nephrologie; 09/2014
[Show abstract][Hide abstract] ABSTRACT: Maillard α-dicarbonyl compounds are known as central intermediates in advanced glycation endproduct (AGE) formation. Glucose is the primary source of energy for the human body whereas L-threo-ascorbic acid (vitamin C) is an essential nutrient, involved in a variety of enzymatic reactions. Thus, the Maillard degradation of glucose and ascorbic acid is of major importance in vivo. To understand the complex mechanistic pathways of AGE formation, it is crucial to extend the knowledge on plasma concentrations of reactive key α-dicarbonyl compounds, e.g. 1-deoxyglucosone. With the present work we introduce a highly sensitive LC-MS/MS multi-method for human blood plasma based on derivatization with o-phenylenediamine under acidic conditions. The impact of workup and reaction conditions, particularly of pH, was thoroughly evaluated. A comprehensive validation provided the limit of detection, limit of quantitation, coefficients of variation and recovery rates. The method includes the α-dicarbonyls 1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederer's glucosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypentosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-deoxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal, the α-keto-carboxylic acids pyruvic acid and glyoxylic acid and the dicarboxylic acid oxalic acid. The method was then applied to the analyses of 15 healthy subjects and 24 uremic patients undergoing hemodialysis. The comparison of the results revealed a clear shift in the product spectrum. In most cases, the plasma levels of target analytes were significantly higher. Thus, this is the first time that a complete spectrum of α-dicarbonyl compounds relevant in vivo was established. The results provide further insights into the chemistry of AGE formation and will be helpful to find specific markers to differentiate between the various precursors of glycation.
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
Current hemodialysis membranes lack clearance of molecules within the 15-45 kD molecular weight segment. This leads to accumulation of several substances that enhance chronic inflammation in dialysis patients. The PERCI trial uses a high cut-off dialyser membrane for treatment of chronic end stage renal disease (ESRD) patients to overcome this limitation and to reduce chronic inflammation.
Although the primary end-point of reduced monocyte CD162 expression was missed in the prospective cross-over treatment trial, secondary end-points such as leukocyte cytokine transcription rates indicate that anti-inflammatory effects of high cut-off dialysis are achievable. Albumin loss was too high for long term treatment, which justifies the ongoing development of highly selective membranes.
51st Congress of the European Renal Association and European Dialysis and Transplant Association 2014; 08/2014
[Show abstract][Hide abstract] ABSTRACT: Aims
Elevated expression levels of monocytic-ACE have been found in haemodialysis patients. They are not only epidemiologically linked with increased mortality and cardiovascular disease, but may also directly participate in the initial steps of atherosclerosis. To further address this question we tested the role of monocytic-ACE in promotion of atherosclerotic events in vitro under conditions mimicking those of chronic renal failure.
Methods and Results
Treatment of human primary monocytes or THP-1 cells with uremic serum as well as PMA-induced differentiation led to significantly up-regulated expression of ACE, further increased by additional treatment with LPS. Functionally, these monocytes revealed significantly increased adhesion and transmigration through endothelial monolayers. Overexpression of ACE in transfected monocytes or THP-1 cells led to development of more differentiated, macrophage-like phenotype with up-regulated expression of Arg1, MCSF, MCP-1 and CCR2. Expression of pro-inflammatory cytokines TNFa and IL-6 were also noticeably up-regulated. ACE overexpression resulted in significantly increased adhesion and transmigration properties. Transcriptional screening of ACE-overexpressing monocytes revealed noticeably increased expression of Angiotensin II receptors and adhesion- as well as atherosclerosis-related ICAM-1 and VCAM1. Inhibition of monocyte ACE or AngII-receptor signalling led to decreased adhesion potential of ACE-overexpressing cells.
Taken together, these data demonstrate that uremia induced expression of monocytic-ACE mediates the development of highly pro-atherogenic cells via an AngII-dependent mechanism.
PLoS ONE 07/2014; 9(7):e102137. DOI:10.1371/journal.pone.0102137 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background/Aims: Although most hemodialysis patients share a significant 25-hydroxyvitamin D [25(OH)D] deficiency, supplementation is controversially discussed. A potential influence on monocyte and T lymphocyte dysfunction advocates blood level-adapted supplementation as recommended by K/DOQI guidelines. This was a prospective double-blind randomized placebo controlled trial examining immune effects of 12 weeks of cholecalciferol supplementation. Methods: We initiated serum level-adapted de novo cholecalciferol supplementation in 38 hemodialysis patients. Outcome measures were: monocyte subset cell counts (CD14+CD16++ vs. CD14++CD16+ vs. CD14++CD16-), lymphocyte Th1/Th2 differentiation frequencies, serum inflammatory proteins CRP and TNFα, parathyroid hormone (PTH), FGF-23, and α-Klotho. Results: At baseline, the mean 25(OH)D serum level in the study population was 31.7 ± 14.3 nmol/l, and only 3% of patients had levels within the normal range. At 12 weeks, 25(OH)D levels were normalized in the verum group (87.8 ± 22.3 vs. placebo 24.6 ± 8.0 nmol/l, p < 0.0001). In parallel, 1,25(OH)2D levels increased in the verum group. Monocyte subset cell counts as well as Th1 and Th2 lymphocyte frequencies did not change significantly after 12 weeks of cholecalciferol supplementation. There was also no significant difference in PTH, alkaline phosphatase, calcium, phosphate, TNFα, FGF-23, α-Klotho and CRP levels. Conclusions: Oral cholecalciferol supplementation according to the K/DOQI recommendations normalizes 25(OH)D levels without relevant side effects such as hyperphosphatemia or hypercalcemia. However, beneficial pleiotropic effects on monocyte subset cell counts, T cell differentiation, or cytokine production could not be confirmed at least at the used dosage. PTH and FGF23 levels were not affected during cholecalciferol administration.
Nephron Clinical Practice 08/2013; 123(3-4):209-219. DOI:10.1159/000354717 · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 68 Hämodialysepatienten (HD) und 42 gesunden altersgematchten Kontrollprobanden (KO) wurden Blutproben mit dem PAX-Blutabnahme-Set (Becton-Dickinson) entnommen und RNA isoliert. Mittels Real Time PCR wurde die Expression der Zielgene in Relation zu einem Housekeeping-Gen quantifiziert. Der dargestellte RQ-Wert entspricht der x-fachen Expression im Vergleich zu einem internen Kalibrator. Zur Evaluierung einer klinisch manifesten Atherosklerose wurde die Intima Media Dicke (IMT) der Arteriae carotides communes bds. mittels B-Mode-Ultraschall gemessen und der Atherosklerose-Schweregrad mit Hilfe eines Plaque-Score-Systems (CJASN 2011; 6:505-511) definiert.
119. Kongress der Deutschen Gesellschaft für Innere Medizin e.V.;6.-9. April 2013; 04/2013
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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 < 0.001) monocytes predicted the primary endpoint, whereas nonclassical monocytes did not (p = 0.158). After full adjustment for confounders, CD14++CD16+ monocytes remained the only monocyte subset independently related to cardiovascular events (fourth vs. first quartile: hazard ratio: 3.019; 95% confidence interval: 1.315 to 6.928; p = 0.009).
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.
Journal of the American College of Cardiology 10/2012; 60(16):1512–1520. DOI:10.1016/j.jacc.2012.07.019 · 15.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: Atherosclerotic plaques progress in a highly individual manner. Plaque eccentricity has been associated with a rupture-prone phenotype and adverse coronary events in humans. Endothelial shear stress (ESS) critically determines plaque growth and low ESS leads to high-risk lesions. However, the factors responsible for rapid disease progression with increasing plaque eccentricity have not been studied. We investigated in vivo the effect of local hemodynamic and plaque characteristics on progressive luminal narrowing with increasing plaque eccentricity in humans.
Methods: Three-dimensional coronary artery reconstruction using angiographic and intravascular ultrasound data was performed in 374 patients at baseline (BL) and 6-10 months later (FU) to assess plaque natural history as part of the PREDICTION Trial. A total of 874 coronary arteries were divided into consecutive 3-mm segments. We identified 408 BL discrete luminal narrowings with a throat in the middle surrounded by gradual narrowing proximal and distal to the throat. Local BL ESS was assessed by computational fluid dynamics. The eccentricity index (EI) at BL and FU was computed as the ratio of max to min plaque thickness at the throat. Mixed-effects logistic regression was used to investigate the effect of BL variables on the combined endpoint of substantial worsening of luminal narrowing (decrease in lumen area >1.8 mm2 or >20%) with an increase in plaque EI.
Results: Lumen worsening with an increase in plaque EI was evident in 73 luminal narrowings (18%). Independent predictors of worsening lumen narrowing with plaque EI increase were low BL ESS (<1 Pa) distal to the throat (odds ratio [OR] =2.2 [95% CI: 1.3-3.7]; p=0.003) and large BL plaque burden (>51%) at the throat (OR=1.7 [95% CI: 1.0-2.8]; p=0.051). The incidence of worsening lumen narrowing with increasing plaque eccentricity was 30% in the presence of both predictors versus 15% in luminal narrowings without this combination of characteristics (OR=2.4 [95% CI: 1.4-4.3]; p=0.002).
Conclusions: Low local ESS independently predicts areas with rapidly progressive luminal narrowing and increasing plaque eccentricity. Coronary regions manifesting an abrupt anatomic change, i.e., at highest risk to cause an adverse event, can be identified early by assessment of ESS and plaque burden.
[Show abstract][Hide abstract] ABSTRACT: Chronic inflammation in hemodialysis (HD) patients is associated with cardiovascular complications and mortality. Circulating immune active proteins in the molecular range 15-45 kD that cannot be efficiently cleared by high-flux (HF) dialysis may be causally involved. We intended to test the feasibility of using a high cutoff (HCO) dialyzer in chronic HD patients and its influence on inflammation and monocyte activation. The Gambro HCO1100 dialyzer was compared to a conventional HF membrane in a randomized double-blind crossover trial in 19 chronic HD patients selected for the presence of elevated serum C-reactive protein levels. Patients were treated for six consecutive dialysis sessions (2 weeks) with each membrane. Safety analysis recorded adverse events and albumin losses through the protein-leaking membranes. Efficacy analysis observed reductions in the number of proinflammatory (CD14+CD16+) monocyte subpopulations in circulating blood. Treatment with the HCO membrane was well tolerated, although the number of adverse events was slightly higher. Despite significant serum albumin loss (from 34.1 ± 2.7 to 29.6 ± 3.0 g/L; P < 0.01), there was no need to supplement albumin, and rising activity of cholinesterase during HCO treatment indicated compensation by enhanced hepatic synthesis. The HCO membrane cleared high amounts of proinflammatory cytokines, but did not reduce predialysis inflammatory monocytes and markers. Although the time of HD session was extended, the study was hampered by a lower Kt/V in the HCO compared to the HF period. Treatment of chronic HD patients with this HCO dialyzer for 2 weeks is tolerable in terms of albumin loss and able to clear proinflammatory cytokines; however, this was not sufficient to decrease monocyte activation. Therefore, a more selective, less albumin-leaking membrane is desirable to allow prolonged high-efficient dialysis with more effective cytokine clearance.
[Show abstract][Hide abstract] ABSTRACT: The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N(6)-acetyl lysine, N(6)-formyl lysine, N(6)-lactoyl lysine, and N(6)-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N(1)-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N(6)-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N(6)-acetyl lysine, parallels enzymatic processes.
[Show abstract][Hide abstract] ABSTRACT: Vitamin D deficiency and protein-energy wasting (PEW) are highly prevalent in hemodialysis (HD) patients. The goal of our study was to investigate if a lack of vitamin D influences mortality and hospitalization of HD patients with or without PEW.
In 81 chronic HD patients with different nutritional status assessed by the Malnutrition Inflammation Score (MIS), vitamin D deficiency (25-OH-vitamin D(3) levels ≤30 nmol/l or ≤12 ng/ml) was prospectively investigated for its prognostic impact on mortality and hospitalization. Over a 3-year follow-up, all-cause mortality and hospitalization were determined. The predictive value of low vitamin D levels and PEW as well as their combined effect were evaluated using a multivariate Cox regression model.
Vitamin D deficiency was frequent in HD patients with and without PEW. It significantly increased mortality rate in HD patients (HR 2.76 (1.33-5.73), p < 0.01), which was aggravated by concomitant PEW (HR 5.88 (2.29-15.09), p < 0.001). The hospitalization rate, however, was not influenced independently by nutritional status.
Low 25-OH-vitamin D(3) concentration is an independent predictor for survival, but not for hospitalization of HD patients. It is not merely a malnutrition-associated finding, although a MIS ≥8 further impaired survival prognosis.
Nephron Clinical Practice 08/2011; 119(3):c220-6. DOI:10.1159/000328927 · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 'Functional foods' supplemented with plant sterol esters (PSE) and plant stanol esters (PSA) are therapeutic options for the management of hypercholesterolaemia. However, their effects on blood monocytes, endothelial function, atherogenesis, and sterol tissue concentrations are poorly understood.
Male apoE-/- mice (n= 30) were randomized to three different diets for 6 weeks (n= 10 per group): high-cholesterol (1.25%) western-type diet (WTD), WTD + 2% PSE, and WTD + 2% PSA. Both supplements reduced serum cholesterol. WTD + PSE resulted in increased plant sterol serum concentrations and increased inflammatory Ly-6C(high) monocyte numbers. WTD + PSA increased plant stanol serum concentrations and Ly-6C-monocyte numbers, but decreased vascular superoxide release, lipid hydroperoxides, and inflammatory cytokines in aortic tissue, in plasma, and in circulating monocytes. Despite reduced serum cholesterol concentrations, both supplements impaired endothelial vasodilation compared with WTD. WTD + PSA reduced the development of atherosclerotic lesions compared with WTD alone (12.7 ± 3.7 vs. 28.3 ± 3.5%), and WTD + PSE was less effective (17.5 ± 3.7%). WTD + PSE and WTD + PSA reduced the cholesterol content in the liver, but not in the brain. However, WTD + PSE and WTD + PSA increased plant sterol and plant stanol concentrations in the liver as well as in the brain.
PSE and PSA supplementation reduced serum cholesterol, but increased plant sterol and plant stanol concentrations. Elevated levels of PSE and PSA were associated with endothelial dysfunction and increased central nervous system depositions. Atherosclerotic lesion retardation was more pronounced in WTD + PSA, coinciding with higher regenerative monocyte numbers, decreased oxidative stress, and decreased inflammatory cytokines compared with WTD + PSE.
Cardiovascular Research 02/2011; 90(3):484-92. DOI:10.1093/cvr/cvr020 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating monocytes can be divided into functionally distinct subpopulations according to their surface expression of CD14 and CD16. Monocytes with high-level expression of both antigens (CD14(++)CD16(+), Mo2 cells) are associated with cardiovascular morbidity and mortality in hemodialysis patients. These cells express angiotensin converting enzyme (ACE) on their surface. They are involved in the association of chronic inflammation and cardiovascular disease in kidney patients. Cardiovascular morbidity results from atherosclerosis (plaque-forming, vessel occluding disease) and arteriosclerosis (loss of arterial dampening function). It is unknown whether ACE-expressing proinflammatory monocytes are related to atherosclerosis, arteriosclerosis, or both.
During baseline examination for a prospective study on monocyte ACE expression and mortality, 60 chronic hemodialysis patients of an academic outpatient center were screened for atherosclerosis by carotid artery ultrasound, for arteriosclerosis by pulse pressure measurement, and for ACE expression on Mo2 cells by flow cytometry.
ACE expression on Mo2 monocytes was significantly higher in patients with severe compared with those with little or no carotid atherosclerosis. Mo2 ACE correlated with a score to semiquantify atherosclerosis and remained a significant predictor of carotid plaques in multivariate analysis including the other univariately associated variables of age, hemoglobin A1c, and albumin. Mo2 ACE was not related to pulse pressure.
ACE expression on Mo2, although being a known predictor of mortality and cardiovascular disease in end-stage renal disease patients, may act via enhancement of atherosclerosis rather than arteriosclerosis.
Clinical Journal of the American Society of Nephrology 12/2010; 6(3):505-11. DOI:10.2215/CJN.06870810 · 5.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The relationship of cholesterol homeostasis and carotid intima-media thickness (cIMT) is unknown. To address this, we assessed markers of cholesterol homeostasis (serum plant sterols and cholesterol precursor concentrations as surrogate measures of cholesterol absorption and synthesis, respectively) and cIMT in a middle-aged, statin-naive population.
In this prospective study of primary prevention cIMT was measured by ultrasound in 583 hospital employees aged 25-60 years without prevalent cardiovascular disease or lipid-modifying medication. The serum concentrations of plant sterols (as markers of cholesterol absorption) were measured by gas-liquid chromatography. Lathosterol serum concentrations were quantitated to assess hepatic cholesterol synthesis.
cIMT correlated positively with serum cholesterol (r = 0.22, P<0.0005) and lathosterol-to-cholesterol (r = 0.18, P<0.001). In contrast, plant sterols, as markers of cholesterol absorption, showed a weak negative correlation to cIMT measurements (r = -0.18; P<0.001 for campesterol-to-cholesterol). Stratifying subjects by serum sterol levels, we found that cIMT increased continuously over quintiles of serum cholesterol (P<0.0005) and was positively associated to serum lathosterol-to-cholesterol levels (P = 0.007), on the other hand, plant sterol levels showed a weak negative association to cIMT (P<0.001 for campesterol-to-cholesterol).
In this population without prevalent cardiovascular diseases or lipid-modifying medication, markers of increased endogenous cholesterol synthesis correlated positively with cIMT, while markers of cholesterol absorption showed a weakly negative correlation. These data suggest that not only total serum cholesterol levels but also differences in cholesterol homeostasis are associated with cIMT.
PLoS ONE 10/2010; 5(10):e13467. DOI:10.1371/journal.pone.0013467 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with chronic kidney disease (CKD) pose a worldwide growing burden to health care systems due to accelerated atherosclerosis and subsequent high cardiovascular (CV) morbidity. Atherogenesis is prominently driven by monocytes and monocyte-derived macrophages. The expression of CD14 and CD16 characterizes three monocyte subsets: CD14(++)CD16(-), CD14(++)CD16(+), and CD14((+))CD16(+) cells; the latter two are often denoted as 'proinflammatory' CD16(+) monocytes. Despite an association between CD16(+) monocyte counts and higher CV risk in cross-sectional cohorts, the prognostic impact of elevated CD16(+) monocyte counts is poorly understood.
We assessed monocyte heterogeneity using flow cytometry in 119 patients with non-dialysis CKD, who were prospectively followed for a median of 4.9 (inter-quartile range 4.8-5.0) years for the occurrence of CV events. In addition, we assessed expression of chemokine receptors on monocyte subsets. CD14(++)CD16(+) monocyte were independently associated with CV events [hazard ratio (for an increase of 10 cells/μL) 1.26 (confidence interval: 1.04-1.52; P = 0.018)] after adjustment for variables that significantly affected CD14(++)CD16(+) cell counts at baseline. Across the spectrum of CKD, CD14(++)CD16(+) monocytes selectively expressed CCR5.
We found that CD14(++)CD16(+) monocytes were independently associated with CV events in non-dialysis CKD patients. Our results support the notion that CD16(+) monocytes rather than CD16(-) monocytes are involved in human atherosclerosis.
European Heart Journal 10/2010; 32(1):84-92. DOI:10.1093/eurheartj/ehq371 · 14.72 Impact Factor