[Show abstract][Hide abstract] ABSTRACT: Purpose:
The Nutrition Societies in Germany, Austria, and Switzerland recommend a daily intake of 20 µg vitamin D3 for adults when endogenous synthesis is absent. The current study aimed to elucidate whether this vitamin D3 dose impacts cardiovascular risk markers of adults during the winter months.
The study was conducted in Halle (Saale), Germany (51(o) northern latitude) as a placebo-controlled, double-blinded, randomised trial (from January to April). A total of 105 apparently healthy subjects (male and female, 20-71 years old) were included. Subjects were randomly allocated to two groups. One group received a daily 20-µg vitamin D3 dose (n = 54), and the other group received a placebo (n = 51) for 12 weeks. Outcome measures included blood pressure, heart rate, concentrations of renin, aldosterone, serum lipids and vascular calcification markers, and haematologic variables such as pro-inflammatory monocytes.
Blood pressure and systemic cardiovascular risk markers remained unchanged by vitamin D3 supplementation, although serum 25-hydroxyvitamin D3 increased from 38 ± 14 to 73 ± 16 nmol/L at week 12. The placebo and vitamin D groups did not differ in their final cardiovascular risk profile.
Daily supplementation of 20 µg vitamin D3 during winter is unlikely to change cardiovascular risk profile.
Full-text · Article · Nov 2015 · European Journal of Nutrition
[Show abstract][Hide abstract] ABSTRACT: Background:
Hemodialysis patients suffer from chronic systemic inflammation and high incidence of cardiovascular disease. One cause for this may be the failure of diseased kidneys to eliminate immune mediators. Current hemodialysis treatment achieves insufficient elimination of proteins in the molecular weight range 15-45kD. Thus high cut-off dialysis might improve the inflammatory state.
In this randomized cross-over trial 43 hemodialysis patients were treated for three weeks with high cut-off or high-flux dialysis. Inflammatory plasma mediators, monocyte subpopulation distribution, and leucocyte gene expression were quantified.
High cut-off dialysis supplemented by a low-flux filter did not influence the primary end-point, expression density of CD162 on monocytes. Nevertheless, treatment reduced multiple immune mediators in plasma. Such reduction proved - at least for some markers - to be a sustained effect over the interdialytic interval. Thus, for example soluble TNF-Receptor1 concentration predialysis was reduced from median 13.3 (IQR 8.9-17.2) to 9.7 (IQR 7.5-13.2) ng/mL with high cut-off while remaining constant with high-flux treatment. The expression profile of multiple proinflammatory genes in leucocytes was significantly dampened. Treatment was well tolerated although albumin losses in high cut-off dialysis would be prohibitive against long-term use.
The study shows for the first time that a dampening effect of high cut-off dialysis on systemic inflammation is achievable. Earlier studies had failed due to short study duration or insufficient dialysis efficacy. Removal of soluble mediators from the circulation influences cellular activation levels in leucocytes. Continued development of less albumin leaky membranes with similar cytokine elimination is justified. This article is protected by copyright. All rights reserved.
No preview · Article · Oct 2015 · European Journal of Clinical Investigation
[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
[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.
Preview · Article · Aug 2014 · Journal of Biological Chemistry
[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.
[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.
[Show abstract][Hide abstract] ABSTRACT: Introduction and Aims: Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been suggested to be involved in the mechanism of renal fibrosis.
Previously, we have shown the direct effects of S1P on the fibrotic process in the unilateral ureteral obstruction (UUO) model
using nude mice which were characterized by deficit of immune response. To get more insight into roles for S1P and receptor
subtype effects in vitro, we performed using antagoists and siRNAs knockdown of receptor subtypes.
Methods: Normal rat kidney interstitial fibroblast (NRK-49F) cells were stimulated with exogenous S1P and the expressions (mRNA/Western
blotting) of a-SMA, E-cadherin, collagen type 1 (COL1), collagen type 4 (COL4), tissue inhibitor of matrix metalloproteinase-1
(TIMP1) and plasminogen activator inhibitor-1 (PAI1) were examined. To specify the kidney specific signal pathway, antagonists
and siRNAs targeted to S1P receptor subtypes were generated. Then, the fibrotic changes of the NRK-49F cells after stimulation
by S1P were examined (3 days, 7 days). The growth and migration of cultured cells were quantified by using CL-Quant software
to analyze time-lapse images in a Nikon BioStation CT. The real-time images of cell migration were monitored for 2 days. And
also NRK-49F cells were stimulated with S1P after the addition of FTY720 (S1P 1, 3, 4, 5 agonist), or DMS (sphingosine kinase
inhibitor) were evaluated.
Results: S1P stimulated fibrosis of NRK-49F cells in a dose- and time-dependent manner as previously observed, and induced morphological
changes (elongation of the cell shape with spindle-like extension, increased migration) of the NRK-49F cells. Migration of
NRK49F cells was accelerated and increase in a-SMA, COL1, COL4, TIMP1 and PAI1 expressions and decrease in E-cadherin expression
were observed by addition of S1P. Antagonist and siRNA transfection to NRK-49F cells of Sphingosine 1-phosphate receptor-3
(S1PR-3) attenuated cell growth and migration, in addition, the expression of fibrotic markers was also diminished by antagonist
and siRNA transfection to NRK-49F cells of S1PR-3. And also in the presence of FTY720 and DMS, fibrosis and migration induced
by S1P were suppressed.
Conclusions: These results suggest that activation of S1P signaling mediated by S1PR-3 results in chronic pathological fibrosis, such
as in chronic kidney disease (CKD).
Full-text · Article · May 2014 · Nephrology Dialysis Transplantation
[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.
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.
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.
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.
No preview · Article · Aug 2013 · Nephron Clinical Practice
[Show abstract][Hide abstract] ABSTRACT: Introduction: Microalbuminuria is a risk factor for cardiovascular events in patients with chronical kidney disease. Currently, the evidence for the importance of microalbuminuria in predicting the progression of coronary heart disease (CHD) is sparse. There has been discordance in defining a proper cut-off value to distinguish between normal and elevated cardiovascular risk. The objective of this study is to evaluate the significance of microalbuminuria as a marker of cardiovascular risk in patients with pre-existing heart disease and to evaluate a suitable reference value of albumin excretion.
Methods: Data were taken from the prospective PHAMOS-Study (ClinicalTrials.gov Identifier: NCT01442948). We investigated albumin-creatinine-ratio (ACR) in 673 patients with angiographically documented CHD, submitted to the Department of Medicine III and the Department of Cardiothoracic Surgery of the University Clinic Halle. Patients were followed up for 52 weeks.
Results: In 59 cases the predefined combined primary endpoint consisting of acute myocardial infarction or death due to cardiovascular disease occurred during follow-up. ROC-analysis showed that ACR had a significant predictive value in assessing one-year event rate (AUC = 0.738, p < 0.001). The optimal discriminator for distinction between normal and elevated one-year event risk was found at an ACR of 10 mg/g. Kaplan-Meier analysis revealed a significantly higher event risk when microalbuminuria (30-299 mg/g) or low-level microalbuminuria (> 10 mg/g) were present. Multivariate analysis revealed an independent association between an elevated one-year event rate and microalbuminuria (HR 2.39 [CI 1.00-5.70]; p = 0.049) as well as low-level microalbuminuria (HR 2.59 [CI 1.01-6.66]; p = 0.048), respectively.
Conclusion: ACR measurement is considered to be of significant value in evaluating disease progression in patients with CHD. Substantial risk elevation in this specific risk population is present already at much lower values of ACR than the commonly applied conventional cut-off levels in our analyses.
Full-text · Article · Aug 2013 · European Heart Journal
[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.
[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.
Full-text · Article · Oct 2012 · Journal of the American College of Cardiology
[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 N6-acetyl lysine, N6-formyl lysine, N6-lactoyl lysine, and N6-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 N1-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N6-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 N6-acetyl lysine, parallels enzymatic processes.
Preview · Article · Nov 2011 · Journal of Biological Chemistry
[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.
No preview · Article · Aug 2011 · Nephron Clinical Practice