Serum Of Hemodialysis Patients Research Articles

In-situ synchrotron imaging, experimental, and computational investigations on the efficiency of Trametes versicolor laccase on detoxification of P-Cresyl Sulfate (PCS) Protein Bound Uremic Toxin (PBUT).

Protein bound uremic toxins (PBUTs) are small substances binding to larger proteins, mostly human serum albumin (HSA), and are challenging to remove by hemodialysis (HD). Among different classes of PBUTs, p-cresyl sulfate (PCS) is the most widely used marker molecule and major toxin, as 95 % is bound to HSA. PCS has a pro-inflammatory effect and increases both the uremia symptom score and multiple pathophysiological activities. High-flux HD to clear PCS leads to serious loss of HSA, which results in a high mortality rate. The goal of the present study is to investigate the efficacy of PCS detoxification in serum of HD patients using a biocompatible laccase enzyme from Trametes versicolor. Molecular docking was used to gain an in-depth understanding of the interactions between PCS and the laccase to identify the functional group(s) responsible for ligand-protein receptor interactions. UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to assess the detoxification of PCS. GC-MS was used to identify the detoxification byproducts and their toxicity was assessed using docking commutations. In situ synchrotron radiation micro-computed tomography (SR-µCT) imaging available at the Canadian Light Source (CLS) was conducted to assess HSA binding with PCS before and after detoxification with laccase and undertake the corresponding quantitative analysis. GC-MS analyses confirmed the detoxification of PCS with laccase at a concentration of 500mg/L. The potential pathway of PCS detoxification in the presence of the laccase was identified. Increasing laccase concentration led to the formation of m-cresol, as indicated by the corresponding absorption in the UV-Vis spectra and a sharp peak on the GC-MS spectra. Our analysis provides insight into the general features of PCS binding on Sudlow site II, as well as insights into PCS detoxification product interactions. The average affinity energy for detoxification products was lower than that of PCS. Even though some byproducts showed potential toxicity, the level was lower than for PCS based on toxicity indexes (e.g., LD50/LC50, carcinogenicity, neurotoxicity, mutagenicity). In addition, these small compounds can also be more easily removed by HD compared to PCS. SR-µCT quantitative analysis showed adhesion of the HSA to a significant reduced extent in the presence of the laccase enzyme in bottom sections of the polyarylethersulfone (PAES) clinical HD membrane tested. Overall, this study opens new frontiers for PCS detoxification.

MO556: Markers of Oxidative Stress and Coronary Calcifications in Chronic Hemodialysis Patients

Abstract BACKGROUND AND AIMS Cardiovascular morbidity and mortality are increased in hemodialysis patients. Oxidative stress is considered to be an important factor in these cardiovascular complications in these patients. The aim of this research is to establish the link between the levels of advanced oxidation protein products (AOPP) in the serum of hemodialysis patients and coronary calcifications. METHOD This is a longitudinal study involving 67 hemodialysis patients, all patients underwent a Coro-CT scan to assess coronary calcifications according to the Agatston coronary calcium score (SCCA). We divided the patients into three groups according to their low (0–100), medium (101–400) and severe calcification score > 400, we measured the levels of AOPP in the three groups and we studied the correlation between the rate of AOPP at the level of the different groups. RESULTS The mean age of the patients is 55 ± 12.5 years, the sex ratio is 1.3, the mean us-CRP level is 12.5 mg/L, the mean NO, AOPP, MDA level, MPO and LDLox are respectively (62.188 ± 5.598, 70.82 ± 10, 6.97 ± 0.488 µmol/L, 61.466 ± 8.82 IU/mL and 31.755 ± 3.9 µmol/l), we divided the patients into three groups according to the agtston coronary calcium score (SCCA), we found 27% of the patients had a low score (0–100), 48% had a moderate sore (101–400) and 25% had a severe score >400. We found that the rate of oxidative stress markers worsened with increasing calcification score, patients with a low score had levels of NO, AOPP, MDA, MPO and LDLox are respectively (67.2 ± 6.5, 64.9 ± 7, 3.24 ± 0.32, 57.8 ± 6.9 IU/mL and 10.17 ± 1.9 µmol/l), the mean score had respective levels of NO, AOPP, MDA, MPO and LDLox are respectively (62.5 ± 3.2, 72.7 ± 12, 7.2 ± 0.24 µmol/L, 60.34 ± 5.7 IU/mL and 27.6 ± 2.1 µmol/L) and patients with a severe calcification score had NO, AOPP, MDA, MPO and LDLox levels respectively (59.3 ± 2.4, 82.82 ± 12, 9.45 ± 0.34 µmol/L, 74.23 ± 9.7 IU/mL and 42.89 ± 5.19 µmol/L), there are positive correlations between the rate of coronary calcifications and the levels of markers oxidative stress NO, AOPP, MDA, MPO and LDLox) which are respectively (r = 0744, P = 0.0001; r = 0.777, P = 0.000; r = 0.318, P = 0.000; r = 0.620, P = 0.000; r = 0.526, P = 0.0001). CONCLUSION Markers of oxidative stress are higher in hemodialysis patients with vascular calcifications, they can be predictors of cardiovascular complications in hemodialysis patients.

Detection of Cytomegalovirus Antibodies in the Serum of Hemodialysis Patients.

The infection with Cytomegalovirus (CMV) is one of the main causes of morbidity and mortality in hemodialysis patients. This study aims to investigate CMV serostatus in renal failure patients undergoing hemodialysis. The study included a total of 134 cases (55 females and 79 males) with an age range of 35 - 68 years (mean age 37.43 ± 13.42 years). HCMV-IgG and IgM were determined in subjects' sera. Eighty-seven out of 134 (65%) were undergoing hemodialysis, while 47 (35%) had normal kidney functions without HCV infection. Sixty-eight (78.2%) and 31 (35.6%) cases out 87 hemodialysis patients were positive for CMV-IgG and IgM antibodies, respectively. No significant differences were noted between females and males in terms of seroprevalence rates. High rate of positive CMV-IgG was observed among hemodialysis patients who were 48 - 64 years old. Our data suggest that seroprevalence of CMV antibodies among hemodialysis cases is high and causes complications for these patients.

Pro-calcifying analysis of uraemic serum from patients treated with medium cut-off membrane in a prospective, cross-over study.

BackgroundThe retention of a large number of solutes that are normally excreted or metabolized by the kidney is responsible for the symptoms typical in uraemic patients. These molecules are defined as uraemic toxins and can be classified into three groups: small water-soluble molecules, middle molecules and protein-bound toxins. Recently, efforts were put towards developing dialysis membranes that allow the removal of large middle molecules without clinically relevant albumin loss. These membranes are the medium cut-off (MCO) membranes that allow the removal of middle molecules up to ∼50 000 Da.MethodsWe performed a prospective, open-label, controlled, cross-over pilot study comparing expanded haemodialysis (HDx) (novel MCO membrane Theranova 400) and conventional haemodialysis (HD) in 20 prevalent HD patients. Ten patients used conventional HD high-flux dialyser and 10 patients used HDx for 3 months; later the patients switched and received the other treatment for a further 3 months. We then analysed the pro-calcifying effect of uraemic serum in a model of high phosphate(Pi)–induced calcification in vascular smooth muscle cells (VSMCs).ResultsIn this study, every patient was the control of himself and, interestingly, we found a tendency of less pro-calcifying potential from HDx-treated patients’ serum compared with HD. Studying pathogenetic processes involved in high Pi–induced calcium deposition, we found that uraemic serum of HDx-treated patients induced less VSMC necrosis compared with uraemic serum of HD patients. Nevertheless, no differences were found between the different dialytic treatments in the serum potential to induce apoptosis and to modulate the expression of a panel of genes involved in VSMC simil-osteoblastic differentiation such as bone morphogenetic protein 2, runt-related transcription factor 2, osteocalcin, matrix Gla protein, osteopontin, elastin and collagen I α1. In an effort to characterize the difference in uraemic toxin profile during the two different dialytic treatments, we measured a panel of 10 uraemic toxins and 3 precursors, finding a significant increased removal during HDx of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, tryptophane and some of its metabolites, such as 3-indoxyl sulphate, indole 3-acetic acid and kynurenine.ConclusionsThese preliminary data are promising, although larger patients’ groups are needed to better understand the effects of HDx on vascular calcification.

A simple liquid chromatography/differential ion mobility spectrometry tandem mass spectrometry method for the determination of trimethylamine-N-oxide in human serum: An application in dialysis patients.

Trimethylamine-N-oxide (TMAO) is a potential indicator of cardiovascular disease and chronic kidney disorders. It is important to monitor the TMAO level in plasma or serum of hemodialysis patients. A simple liquid chromatography/differential ion mobility spectrometry tandem mass spectrometry (HPLC/DMS-MS/MS) method was established and validated for the determination of TMAO in the serum of hemodialysis patients. Chromatographic separation was performed on a Waters Atlantis HILIC silica column (2.1 × 50 mm, 3 μm). The gradient mobile phase consisted of 10 mM ammonium formate buffer and acetonitrile with 0.1% formic acid in both solvents. The serum sample was precipitated with acidic acetonitrile prior to HPLC/DMS-MS/MS analysis and TMAO-d9 was used as the internal standard. Data acquisition was performed in positive ion mode with a DMS system before the electrospray ionization source. The selected reaction monitoring transitions were m/z 76.0 → 58.0 and m/z 85.2 → 66.1 for TMAO and the internal standard, respectively. Excellent linearity was observed over the calibration range 0.05-20 μg/mL (r2 > 0.995). The method was validated for good specificity and sensitivity. The inter-run and intra-run precision and accuracy were less than 13.6% and 10.7%, respectively. We established a novel and robust HPLC/DMS-MS/MS method for the quantification of TMAO in human serum samples. The validated assay was simple, rapid, sensitive and reliable. The developed method could be applied to the assay of serum samples from patients with kidney disease who are undergoing hemodialysis.

Excessive ADAM17 activation occurs in uremic patients and may contribute to their immunocompromised status.

IntroductionWe previously reported that fibroblast growth factor 23 (FGF23)‐klotho signaling plays a role in B cell immunity. Despite high serum levels of FGF23, a decline in immunity is frequently observed in patients on hemodialysis (HD); thus, abnormalities in the FGF23‐klotho signaling pathway in immune cells may occur in these patients.MethodsWe analyzed the number of klotho‐positive cells in peripheral blood mononuclear cells from 10 male and 6 female patients on HD and 5 healthy male subjects using flow cytometry. We analyzed the abundance of cleaved klotho protein in the murine B cell line, A20, and in the serum of HD patients and healthy subjects (HS) using flow cytometry and Western blotting. The serum level of A disintegrin and metalloprotease 17 (ADAM17) was measured in HD patients and HS using enzyme‐linked immunosorbent assay.ResultsThe number of klotho‐positive B cells was reduced in HD patients. Serum ADAM17 was responsible for the reduction in klotho, as a specific ADAM17 inhibitor reversed this change. The total serum levels of ADAM17 were similar in HD patients and HS; however, activated ADAM17 was increased in the serum of HD patients.ConclusionsWe concluded that abnormal ADAM17 activation could contribute to the immunocompromised status in patients on HD, in line with the reported role of ADAM17 as an anti‐inflammatory and immunosuppressive factor.

Elevated serum soluble interleukin-2 receptor levels increase malignancy-related risk in patients on chronic hemodialysis.

Patients on chronic hemodialysis (HD) have an increased incidence of malignancy due to decreased immunity. Soluble interleukin-2 receptor (sIL-2R), as an immunomodulator, seemed to have an effect in the process of malignancy. In this study, we aimed to evaluate the clinical significance of increased sIL-2R in the course of malignancy among HD patients. Patients who undergoing chronic hemodialysis were followed for 24months. Risk factors for malignancy events and malignancy-related mortality during the 2-year follow-up period were investigated among various clinicopathological variables. Of the 363 patients included in this research, 47 patients (12.95%) had a prior history of treated malignancy. During the 2-year follow-up period, malignancy events were detected in 15 (4.12%) patients. Sixty-seven patients died during the study period, of which nine patients (13.43%) were died of malignancy. Malignancy events reduced 2-year mortality significantly (log-rank = 23.02, P < 0.0001). Both high sIL-2R levels ( ≥ 2-fold upper limit of the normal value) (OR 6.6, P = 0.006) and a prior history of treated malignancy (OR 4.12, P = 0.018)were identified by multivariate logistic analysis as independent determinants for malignancy events. However, only the levels of sIL-2R (used as a continuous variable) had the significantly predictive effect on malignancy events and malignancy-related mortality in the following 2years. Elevated sIL-2R levels was commonly seen in serum of HD patients. And this elevated level increased the risk of malignancy. Aside from its role as a biomarker, sIL-2R may also exert biological effects in the course of malignancy.

Significance of Cu/Zn-Superoxide Dismutase Levels in Hemodialysis Patients: A Mini Review

Cu/Zn-superoxide dismutase (Cu/Zn-SOD) is an enzyme that is ubiquitously present in the cytoplasm and causes dismutation of superoxide radicals, therefore Cu/Zn-SOD is primarily used as an antioxidant marker. Levels of Cu/Zn-SOD are higher in the serum of hemodialysis patients than in serum of healthy volunteers. The increase of serum Cu/Zn-SOD levels is related to the decrease of kidney function with aging and arteriosclerosis in hemodialysis patients. Moreover, infection, vascular puncture, and hemostasis may be related to the increase in serum Cu/Zn-SOD levels. As it is associated with numerous factors in hemodialysis patients, Cu/Zn-SOD may serve as a complex marker for arteriosclerosis, vascular, and inflammatory conditions. It is important to investigate various agents that decrease serum Cu/Zn-SOD levels to improve the life-span of hemodialysis patients.

Syndecan-4 Is an Independent Predictor of All-Cause as Well as Cardiovascular Mortality in Hemodialysis Patients

BackgroundLeft ventricular hypertrophy is associated withincreased mortality in hemodialysis (HD) patients.Syndecan-4 plays a role in many processes that are involved in the heart fibrosis and hypertrophy.We designed this study to prospectively determine whether syndecan-4 was predictive of mortality in a group of HD patients.MethodsIn total, 191 HD patients were included. Clinical, biochemical and echocardiographic parameters were recorded. HD patients were followed-up for 23.18 ± 4.02 months.ResultsSyndecan-4 levels correlated strongly with geometrical echocardiographic parameters and ejection fraction. Relations with pressure-related parameters were weak and only marginally significant. Using the receiver operating characteristics the optimal cut-off points in predicting all-cause as well as cardiovascular (CV) mortality were evaluated and patients were divided into low and high syndecan-4 groups. A Kaplan–Meier analysis showed that the cumulative incidences of all-cause as well as CV mortality were higher in high serum syndecan-4 group compared with those with low serum syndecan-4 (p<0.001 in both cases).A multivariate Cox proportional hazards regression analysis revealed syndecan-4 concentration to be an independent and significant predictor of all-cause (hazard ratio, 2.99; confidence interval, 2.34 to 3.113; p<0.001)as well as CV mortality (hazard ratio, 2.81;confidence interval, 2.28to3.02; p<0.001).ConclusionsSerum syndecan-4 concentration reflects predominantly geometrical echocardiographic parameters. In HD patients serum syndecan-4 concentration is independently associated with all-cause as well as CV mortality.

Abstract 11103: Increase of Oxidative Stress Precipitated by Selenium Deficiency Plays a Role in Cardiac Hypofunction in Uremic Cardiomyopathy

Introduction: Various trace elements are deficient in hemodialysis patients, but the relationship of such deficiencies with cardiac hypofunction is unclear. We examined the deficiencies of trace elements in the blood and hair of hemodialysis patients diagnosed with uremic cardiomyopathy and investigated cardiac hypofunction mechanism from myocardial tissue. Methods: Using blood samples, we measured 25 elements including Se, Mn, Cu, and Zn by high-resolution inductively coupled plasma (ICP) mass spectrometry and ICP emission spectral analysis. We also compared glutathione peroxidase (GSHPx) activation and radical formation potential between the uremic cardiomyopathy patient group (n = 22) and a control group with normal cardiac function (n = 30). Using myocardial tissue, we assessed the exacerbation of oxidative stress by 8-hydroxy-2-deoxyguanosine, 4-hydroxy-2-nonenal, and GSHPx staining and microcirculatory disorder by the perivascular fibrosis area ratio (PVFR). We also measured concentrations of elements in hair at the Super Photon ring-8 GeV (SPring-8) facility. Results: Compared with the control group patients, the uremic cardiomyopathy patients had lower selenium (99.7 ± 21.4 vs. 146 ± 23.3 ng/ml, p &lt; 0.00001) and manganese concentrations (0.39 ± 0.15 vs. 0.60 ± 0.12 ng/ml, p &lt; 0.001); they also showed decreased activation of GSHPx (121 ± 33 vs. 328 ± 44 μmol/min/L, p &lt; 0.00001), an antioxidant enzyme involving selenium, with only selenium concentration correlating to left ventricular ejection fraction (p = 0.0009). Oxidative stress marker staining, radical formation potential in serum, and PVFR were all augmented in the uremic cardiomyopathic patients (p &lt; 0.001). Conclusions: Selenium concentration decrease in the serum of hemodialysis patients may reflect a decrease in GSHPx activation, increasing oxidative stress, and precipitating a coronary microcirculatory disorder involved in the cardiac hypofunction seen in uremic cardiomyopathy.

Effects of Serum of Hemodialysis Patients on TF and PAI-1 Secretion by Human Vascular Endothelial Cells During Treatment with Atorvastatin and Anticoagulant

Effects of Serum of Hemodialysis Patients on TF and PAI-1 Secretion by Human Vascular Endothelial Cells During Treatment with Atorvastatin and Anticoagulant

Uremic pruritus

Uremic pruritus

Determination of Aluminium by Electrothermal Atomization Atomic Absorption Spectrometry in Serum to Characterize Hemodialysis Toxicity

A simple and cost-effective method is described for the determination of aluminum by electrothermal atomic absorption spectrometry (ET-AAS) in serum of hemodialysis patients and healthy subjects. The only preparative step required is the dissolution of the serum sample in 0.2% magnesium nitrate matrix modifier and separate diluents 0.01 M EDTA and 0.1% Triton X-100. The calibration curve was linear from 20 to 100 µg/L with correlation coefficients of 0.9993 and 0.9998 for EDTA and Triton X-100, respectively. The sensitivity of the method for aluminum at the 309.3 nm line was 74 pg. The instrumental and method limits of detection were 2.2 µg/L and 4.4 µg/L, respectively. The aluminum concentrations of forty serum samples from hemodialysis patients and healthy subjects were determined and the mean values were 170.9 ± 6.8 µg/L and 47.3 ± 9.3 µg/L, respectively, whereas the permissible limit for aluminum in blood serum is 10 µg/L. The high level of Al in serum was related to oral phosphate binding agents and dialysis treatment.

Development and validation of a high performance liquid chromatography assay for the determination of temocillin in serum of haemodialysis patients

Therapeutic drug monitoring of β-lactams can be useful for the optimization of therapy, especially when little reference data exist on actual pharmacokinetic profiles such as in patients undergoing haemodialysis. There is no reported validated method for temocillin assay in serum samples, and preliminary studies evidenced potential for interferences by acidic metabolites and co-administered drugs in patients with advanced kidney failure. This paper describes a fully validated method for the determination of temocillin in human serum, and its applicability in haemodialysis patients. Temocillin was extracted from human serum by a solid phase extraction methodology, and then assayed by reversed-phase HPLC with UV-detection. The method was validated according to the accuracy profile methodology, using total error to verify the trueness, precision and overall accuracy. It showed high specificity and precision and was accurate in the concentration range of 5–400mg/L. LOD and LOQ were 1.2 and 5mg/L, respectively. No interference with 30 co-administered drugs was evidenced. The method was successfully applied to clinical samples from haemodialysis patients, showing a high degree of dialysability of temocillin.

Damage-associated molecular patterns derived from mitochondria may contribute to the hemodialysis-associated inflammation

Inflammation is common in hemodialysis (HD) patients. Mitochondrial damage-associated molecular patterns (DAMPs) are released during cell necrosis or apoptosis and induce inflammation. Cell apoptosis is increased in HD patients. The mitochondrial protein cytochrome c, as a marker of released mitochondrial DAMPs, and interleukin-6 (IL-6), as a marker of inflammation, were evaluated in HD patients. Thirty-four HD patients and 20 controls were enrolled in the study. Serum cytochrome c and IL-6 were measured by means of enzyme-linked immunosorbent assay. Compared to controls, cytochrome c was markedly increased in HD patients (1392.88 ± 905.24 pg/mL vs. 212.95 ± 91.71 pg/mL). IL-6 was also significantly increased in HD patients (50.32 ± 35.89 pg/mL vs. 14.27 ± 6.83 pg/mL). In HD patients serum IL-6 was positively related to serum cytochrome c (r = 0.458). Both circulating cytochrome c and IL-6 are markedly increased in HD patients. Cytochrome c is positively related to IL-6.

Perilipin‐1 in Hemodialyzed Patients: Association With History of Coronary Heart Disease and Lipid Profile

Perilipin-1 surrounds lipid droplets in both adipocytes and in atheroma plaque foam cells and controls access of lipases to the lipid core. In hemodialysis (HD) patients, dyslipidemia, malnutrition, inflammation and atherosclerosis are common. Thirty-six HD patients and 28 healthy volunteers were enrolled into the study. Ten HD patients suffered from coronary heart disease (CHD). Perilipin-1, triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), body mass index, albumin, geriatric nutritional risk index, normalized protein catabolic rate, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured. Perilipin-1 did not differ between HD patients and healthy volunteers. IL-6 and TNF-α were higher in HD patients. The evaluated nutritional markers and the markers of inflammation did not differ between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels. Regarding the lipid profile, only HDL-C differed between HD patients with high perilipin-1 levels and HD patients with low perilipin-1 levels, and it was higher in the first subgroup. Perilipin-1 was significantly higher in HD patients without CHD. Perilipin-1 is detectable in the serum of HD patients and it is associated with increased HDL-C and decreased incidence of CHD.

Effect of IFN-γ and IL-4 levels on the expression of Fas and Bcl-2 in peripheral blood lymphocytes in hemodialysis patients

To study the correlation between the levels of IFN-γ and IL-4 and the expression of Fas and Bcl-2 in peripheral blood lymphocytes (PBL) in hemodialysis patients, the indirect immune fluorescein labeling method of flow cytometry and solid sandwich enzyme-linked immunosorbent assay were performed for detecting the expression of Fas and Bcl-2 in PBL and the levels of IFN-γ and IL-4 in the serum of 30 hemodialysis patients, respectively. It was found that the expression of Fas in PBL and the level of IL-4 in the serum of hemodialysis patients were significantly higher (P<0.01), whereas Bcl-2 in PBL and IFN-γ in the serum were significantly lower (P<0.01) than those of the normal controls. According to statistical analysis, the expression of Fas in PBL had a negative correlation with the level of IFN-γ, but a positive correlation with IL-4 in the serum of hemodialysis patients. Contrarily, the expression of Bcl-2 had a positive correlation with IFN-γ, but a negative correlation with IL-4 in the serum of hemodialysis patients. These results suggest that hemodialysis patients have a suppressed secretion of Th1-associated cytokine IFN-γ, but an increased secretion of Th2-associated cytokines IL-4, and these two aspects may play an important role in the abnormal apoptosis of PBL and its accompanying immune deficiency.

Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand and Vascular Calcification

Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non-uremic and uremic wild type and apolipoprotein knockout (Apo E(-/-) ) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non-uremic ApoE(-/-) mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate-induced mineralization of hVSMCs in a dose-dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.

Hemopexin Is Protective Against Intravascular Hemolysis–Induced Vascular Damage

AbstractAbstract 3207 Background:Intravascular hemolysis renders hemoglobin (Hb), usually protected by the red blood cell environment, susceptible to oxidation to its ferric (metHb) form. Haptoglobin (Hp) provides first aid to protect the vasculature from acellular metHb havoc. However, once Hp is consumed, ferrous Hb readily undergoes oxidation to metHb, in which the globin to heme affinity is reduced. MetHb releases the loosely bound heme allowing it to execute its oxidative activity. While nucleated cells, like endothelial cells, are equipped with the heme oxygenase-1 defence system, the nucleus lacking red blood cells (RBC) and low density lipoproteins (LDL) particles have no defence from heme-induced oxidation. Therefore the sole defence from heme oxidative damage remains the plasma protein hemopexin (Hx), which binds hemin with high affinity comparable to that of globin. Following hemin binding, Hx undergoes conformational changes rendering the heme inaccessible thereby inactive. Aim:The current study is focused on the ability of plasma Hx to defend the vasculature from acellular Hb induced damage. Results:Hx defence from metHb damage was studied in the following projects: a) preventing in vitro LDL oxidation by metHb, b) decreasing in vitro lysis of red cells created by hypotonic and mechanical stress. (c) Exploring consumption of Hx in hemodialysis (HD) treated patients by measuring their Hx levels. (a) Methemoglobin iron is a central cause of LDL oxidation. This process initiates formation of blood vessel plaques and atherosclerosis development. This part of the study examined the effect of apoHx (lacking heme) on LDL oxidation by metHb. Incubation of isolated native LDL (nLDL) or vitamin E depleted (dLDL) with low concentration of metHb range (1-10μM) resulted in covalent apoB inter-particle crosslinking in both LDL types, dLDL being more sensitive than nLDL to the oxidative crosslinking. Addition of apoHx abolished metHb-induced oxidation even in the dLDL. This Hx activity (comparable to that of haptoglobin) was confirmed by both SDS-PAGE and fluorescent assay of bityrosines formation. The fact that Hx protects LDL, especially vitamin E depleted particles, shed light on the controversial antioxidant vitamin E properties by indicating its efficacy in defence against acellular Hb induced LDL oxidation. (b) Like LDL the red cell membrane is exposed to metHb-induced oxidative damage. Acellular Hb was formed by hypotonic shock or mechanical shear stress to fresh whole blood of healthy donors. The effect of Hx on acellular Hb levels was measured spectrophotometrically. Reduction of osmotic pressure in fresh blood to 40% of its normal value resulted in formation of ∼300 μM acellular Hb. Supplementation of exogenous apoHx (20μM) to blood before exposure to hypotonicity reduced acellular Hb formation by half. The specificity of Hx was proved by addition of up to 80μM albumin, which had no effect on acellular Hb concentration. Acellular Hb was formed also by applying shear stress on circulating fresh blood in narrow channels using a peristaltic pump. Within 4 hours acellular Hb reached ∼500μM. However, in presence of 20μM apoHx hemolysis was significantly reduced (up to 50%). This reduction may be attributed to Hx's ability to block the hemolysis amplifying effect of metHb on the RBC membrane. (c) In HD treated patients RBCs are exposed to the effects of recurrent mechanical shear stress. The last part of the research was dedicated to quantisation of apoHx levels (by titration with hemin) in sera of HD patients (n=60) prior to and immediately after a single dialysis procedure versus normal controls (n=20). Sera apoHx levels in HD patients prior to a dialysis session were lower compared to healthy controls (10.91±3.19 μM vs. 14.95±2.63 μM, p<0.05) and were even lower (7.49±3.8 μM) immediately after the treatment. These findings indicate that in the plasma of HD treated patients Hx consumption rate exceeds synthesis rate leading to a chronically low level of Hx. Moreover, reduction of Hx was even more prominent in patients on long term HD treatment of above five years. Conclusion:Hx is specifically effective in attenuation of acellular Hb damage to the vasculature. These results point to the option that treating HD patients with exogenous Hx may slow down the rate of atherosclerotic cardiovascular disease development, the major cause of morbidity and mortality in these patients. Disclosures:No relevant conflicts of interest to declare.

Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD

Fetuin-A is an important inhibitor of extraosseous calcification, but some of the studies that used ELISAs did not identify a significant relationship between serum fetuin-A levels and vascular calcification in patients with chronic kidney disease (CKD). Here, we used centrifugation to separate a fetuin-mineral complex (FMC) composed of fetuin-A, fibrinogen, fibronectin-1, and calcium from the serum of hemodialysis patients. In addition, we analyzed serum fetuin-A levels of 73 patients with diabetes and CKD (predialysis) after centrifugation. Fetuin-A concentrations were significantly lower in supernatants than in serum from patients at any stage of CKD, indicating systemic circulation of FMC in these patients. With greater severity of CKD, the contribution of FMC to total fetuin-A increased. Despite the absence of a correlation between serum fetuin-A and coronary artery calcification scores (CACS), supernatant fetuin-A negatively correlated with CACS and the extent to which centrifugation reduced fetuin-A (reduction ratio [RR]) positively correlated with CACS. In a longitudinal study of 12 hemodialysis patients with secondary hyperparathyroidism, parathyroidectomy and cinacalcet therapy each significantly reduced the RR without changing supernatant fetuin-A levels after 1 month, suggesting a reduction in FMC. Moreover, the magnitude of cinacalcet-induced reduction in parathyroid hormone correlated with the decrease in RR but not with changes in serum or supernatant fetuin-A. These data suggest that a quantitative measure of FMC, not supernatant or serum fetuin-A as measured in previous studies, reflects extraosseous calcification stress.