Inflammation and Oxidative Stress in Patients on Hemodiafiltration

Abstract

Cardiovascular disease remains the most common cause for the excess of morbidity and mortality in end-stage renal disease (ESRD) patients [1]. The annual mortality rate of ESRD patients managed with three times weekly hemodialysis (HD) is 20% with over 50% of deaths due to cardiovascular complications [2]. Although traditional cardiovascular risk factors (such as old age, hypertension, diabetes, smoking, dyslipidemia, left ventricular hypertrophy, heart failure and physical inactivity) are overrepresented in this population, they cannot fully explain the accelerated cardiovascular risk [3]. Chronic inflammation and increased oxidative stress, as non-traditional cardiovascular risk factors, are common features in HD patients and contribute substantially to endothelial dysfunction and accelerated atherosclerosis [4].The causes of inflammation in dialysis patients include both factors arising from dialysis itself (efficiency and biocompatibility issues) as well as other non-dialysis-related causes [5]. ESRD patients have increased levels of inflammation-related proteins, such as interleukin-6 (IL-6) and C-reactive protein (CRP), and are subjected to enhanced oxidative stress, as a result of both insufficient antioxidant defense mechanisms and excessive generation of oxidant compounds [3, 6]. Despite rapid improvements in dialysis technology during the last 20 years, the mortality rate in ESRD patients treated with dialysis has remained unacceptably high; therefore studies on the effects of various anti-inflammatory treatment strategies are warranted to improve outcomes in this patient population [3].Hemodiafiltration (HDF) offers a highly effective dialysis modality expanding the spectrum of removed uremic toxins from small to middle-sized molecular solutes by combining ultrafiltration (convective clearance for removing larger solutes) with diffusion (for removal of small solutes) [7]. Observational results from the Dialysis Outcomes and Practice Patterns Study (DOPPS) in five European countries, which followed 2,165 patients from 1998 to 2001 and compared characteristics and outcomes for patients receiving HDF versus HD, suggest that HDF, in particular when large infusion volumes are used, may improve patient survival [8]. However, solid evidence on the influence of HDF on oxidative stress and inflammation is still lacking and there are only scanty data suggesting that convective dialysis modalities ameliorate the chronic inflammatory status observed in ESRD patients [9]. The aim of the present study was to examine, prospectively, the effect of 9-month post-dilution HDF on oxidative stress and inflammatory biomarkers in stable HD patients.After a 6-month run-in period of standard bicarbonate HD with a synthetic membrane, 9 clinically stable patients were treated for 9 months with post-dilution HDF. The main characteristics of the patients studied are shown in table 1.Diabetics and patients on ACE inhibitors, statins and corticosteroids were excluded. None of the patients presented with evidence of infectious disease during at least 1 month prior to blood sampling for the biochemical determinations. All patients received low-molecular-weight heparin, erythropoietin, iron and carnitine IV, folic acid and sevelamer hydrochloride, as phosphate binder, per os. The formulation used for IV iron infusion was iron sucrose (Venofer).High-flux polyamide dialyzers (Polyflux H, Gambro, Hechingen, Germany) with 1.7–2.1 m2 membrane surface area were used and 10 liters of replacement fluid were infused in the post-dilution mode. The dialyzers were not reused. The blood flow rate was 350 ml/min with a dialysate flow rate of 500 ml/min. The colony count of microorganisms in the water used to prepare the dialysis fluid was <200 colonies/ml.Predialysis blood samples were drawn at the beginning, 3 months later and at the end of the study. Hemoglobin (Hb), white cell and platelet counts, total protein, albumin, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, ferritin and uric acid were measured monthly with standard methods. Urea kinetics were performed monthly at a midweek session according to the Daugirdas formula with post-dialysis urea samples obtained immediately after the end of the dialysis session by using a slow-flow method [10].The study was performed in accordance with the Declaration of Helsinki and with the approval of the local ethics committee. An informed written consent was obtained from all patients before they entered the study.Total antioxidant capacity (TAC) was measured spectrophotometrically (Hitachi U-1800 UV-Vis spectrophotometer). Briefly, TAC was determined using the manual method of Benzie and Strain [11], which measures the ferric to ferrous iron reduction in the presence of antioxidants (ferric reducing ability of plasma [FRAP] assay). The final result was expressed as mmol/l. The FRAP assay was performed in triplicate on each sample, and the mean of the three values was used in the analysis. The intra- and interassay coefficients of variation were 3.5 and 10.2% respectively.Reactive oxygen metabolites and derivatives (d-ROMs) were evaluated spectrophotometrically by the d-ROMs Test™ FRAS 4 (Diacron International, Grosseto, Italy). This test is based on the ability of transition metals to catalyze, in the presence of peroxides, the formation of free radicals which are then trapped by an alchylamine. The alchylamine reacts forming a colored radical detectable at 505 nm through a kinetic reaction which is linear up to 500 U CARR (Carratelli units). The determination of free radicals can be made with a normal spectrophotometer. The normal range has been determined as 250–300 U CARR [12].Superoxide dismutase (SOD) activity was determined using a commercially available determination kit (Fluka Chemie GmbH, Buchs, Switzerland). The kit allows SOD assaying based on the capacity of SOD or SOD-like materials to inhibit the reduction of water-soluble tetrazolium salt to a water-soluble formazan dye by the xanthine/xanthine oxidase system. The development of the reaction was monitored spectrophotometrically at 450 nm using a microplate reader (Expert 96, ASYS Hitech, Eugendorf, Austria). One SOD unit was defined as the amount of enzyme that inhibited the reduction of cytochrome c by 50% in a coupled system with xanthine oxidase at pH 7.8 at 25°C in a 3.0-ml reaction volume. The final result was expressed as SOD units per milliliter. The SOD assay was performed in duplicate on each sample, and the mean of the two values was used in the analysis. The intra- and interassay coefficients of variation were 4.7 and 7.9% respectively.Serum high-sensitivity CRP (hs-CRP) and IL-6 levels were measured using commercially available enzyme-linked immunosorbent assays (ELISA). The hs-CRP assay uses a kit specific for human CRP (Bender MedSystems, Vienna, Austria) according to the manufacturer’s instructions. The CRP assay was performed in duplicate on each sample, and the mean of the two values was used in the statistical analysis. The results are expressed in milligrams per liter of sample. The sensitivity of the technique allows the detection of levels as low as 3.0 pg/ml. The intra- and interassay coefficients of variation were 3.8 and 4.7% respectively.IL-6 levels in the samples were measured using sandwich ELISA kits specific for human IL-6 (BioMedix IVD, Athens, Greece), according to the manufacturer’s instructions. The assay was performed in duplicate on each sample, and the mean of the two values was used in the statistical analysis. The results are expressed in picograms per milliliter of sample. The sensitivity of the technique allows the detection of levels as low as 1.6 pg/ml. The intra- and interassay coefficients of variation were 5.5 and 7.6% respectively.After overnight fasting, blood samples were drawn from arteriovenous fistulas prior to the second dialysis session of the week. Blood samples were collected at baseline, 3 and 9 months. Samples were immediately centrifuged and the plasma was stored at –80°C until analysis. Iron supplementation was at least 2 weeks away from blood sampling for determination of inflammatory and oxidative stress biomarkers.Data are presented as mean ± SD. Non-parametric tests (Wilcoxon-matched pairs test and Mann-Whitney U test) were applied for statistical analyses. Statistical significance was set at p < 0.05.Kt/V urea estimated by the Daugirdas formula was not significantly different at baseline (1.4 ± 0.05), 3 months later (1.6 ± 0.29) and at the end of the study (1.2 ± 0.05), as shown in table 2. Kt/V urea was thus maintained well within the NKF-KDOQI 2006 target levels [13], namely Kt/V urea ≥1.2, throughout the study. No significant variations in Hb, white cell and platelet counts, total protein, albumin, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, ferritin and uric acid were observed throughout the study (table 2). Hb was also kept within the target range valid at the time when our study was performed, namely 11–13 g/dl. The erythropoietin requirements (IU/kg/week) remained unchanged during the study (table 2). Finally, it is of interest to note that serum albumin showed a non-significant rise, while serum LDL cholesterol values showed a non-significant trend to decrease throughout the study (table 2).d-ROM levels showed a significant reduction at 3 months of HDF, compared to baseline values (323.0 ± 81.9 vs. 275.0 ± 62.0 U, p < 0.008) and remained low (267.9 ± 45.8 U) to the end of the study (fig. 1). Nine months of HDF increased serum TAC levels significantly in comparison to baseline values (1.3 ± 0.1 vs. 1.4 ± 0.1 mmol/l, p < 0.03), as shown in figure 2. Concerning SOD activity (fig. 3), an increase was observed in SOD plasma levels during the study, but it did not reach statistical significance (13.1 ± 9.8 vs. 18.7 ± 9.4 U/ml, p = 0.06).Inflammatory status as evidenced by plasma CRP levels determined using highly sensitive CRP assay showed a significant decrease at the end of the study compared to CRP baseline values (16.3 ± 11.4 vs. 6.0 ± 5.1 µg/ml, p < 0.02), as depicted in figure 4. Of note, the difference in CRP values was non-significant after 3 months of HDF treatment. On the contrary, IL-6 levels showed no significant differences (16.9 ± 9.3 vs. 12.0 ± 7.3 pg/ml, p = 0.2) but there was a trend towards a substantial decrease in IL-6 values during the study (fig. 5).A chronic inflammatory state and an increase in oxidative stress have been widely documented in patients with chronic kidney disease (CKD) and are more pronounced in those on HD [3]. Components of the inflammatory response associated with the HD method include neutrophil activation, cytokine release (IL-1β, IL-6, TNF-α), oxidative stress with free radical production, lipid and protein oxidation [3]. There is a strong relationship between inflammation and oxidative stress in this patient population, as both are related to endothelial dysfunction, and reactive oxygen species (ROS) are generated in response to inflammatory stimuli [14]. Data increasingly suggest that these non-traditional cardiovascular risk factors are a potentially important source of patient morbidity and mortality, although this knowledge is not yet applicable in daily clinical practice [15].In CKD and ESRD, the balance between pro- and antioxidant factors is shifted towards an increased oxidative stress [16]. Several deficiencies in different components of the antioxidant defense mechanisms have been demonstrated, including reduced levels of vitamins C and E (primarily due to dietary restrictions and losses during dialysis), reduced SOD activity in erythrocytes and deficiency in the glutathione-scavenging system [3]. At the same time, pro-oxidant activity is increased. Factors contributing to this include characteristics of this patient population, such as advanced age and diabetes, renal disease and uremia per se, chronic inflammatory state and factors associated with dialysis process, such as bioincompatibility of dialysis membranes and solutions, poor quality of dialysis water, intravenous iron therapy and presence of foreign bodies (access graft, intravenous HD catheter, peritoneal dialysis catheter). While alterations in pro- and antioxidant capacity start during the early stages of CKD, they are highly prevalent in patients on dialysis [17].During the last years, the quality and technology of HD have undoubtedly improved in relation to biocompatibility of the materials used and the ability to remove larger size uremic toxin molecules [7]. However, the role of convective-based dialysis modalities such as HDF in amelioration of inflammatory and oxidative stress status has not been yet fully clarified. To the best of our knowledge, research or clinical studies on this issue are limited [9].The results of our study demonstrate that post-dilution HDF treatment suppresses oxidative stress by rapid lowering of d-ROMs, improves antioxidant capacity by increasing TAC (significantly) and SOD (non-significantly) and reduces inflammation by decreasing hs-CRP (significantly) and IL-6 (non-significantly). The most plausible explanation may come from the reduction of pro-inflammatory cytokines and related ROS due to improved biocompatibility and better clearance of middle- and large-molecular-weight uremic toxins, a fact that has been suggested in previous studies [18]. In this context, HDF, by combining convection and diffusion and by using high-flux synthetic membrane with low bioreactive profile, results in more sufficient removal of larger size uremic toxins and in optimized biocompatibility of the extracorporeal circuit.Biocompatibility of the dialysis membrane influences the inflammatory and oxidative response to dialysis. Switching from a cellulose acetate to a low-flux polysulfone dialysis membrane for a year or more may decrease the level of protein oxidation suggesting a decrease in oxidative stress and greater biocompatibility of the polysulfone membrane [19]. Flux properties of dialysis membranes may also affect levels of oxidized molecules. Short-term dialysis with a high-flux membrane decreases oxidized low-density lipoprotein concentration, an indicator of oxidative stress, independent of membrane material and has, at the same time, a potent positive effect on uremic dyslipidemia [20]. However, results from studies on the long-term impact of high-flux dialysis on inflammation and oxidative stress are conflicting. Thus, Pupim et al. [21] examined changes in inflammation and oxidative stress in a prospective cohort study of 50 patients starting HD with high-flux polysulfone dialysis membranes and demonstrated that a 12-month period of HD had no significant influence on plasma concentrations of CRP and IL-6 as well as plasma protein carbonyl content as a marker of oxidative stress. On the contrary, another well-designed study from the USA documented normalization of neutrophil oxygen radical production during high-flux HD which is associated with improvement in some markers of oxidative stress [22].Additionally, the recently completed HD study was a clinical that the role of high-flux membranes and dialysis on the morbidity and mortality of HD patients treated three times per week. The results showed no effects of an increase in dialysis the standard and no improvement in mortality with of high-flux membranes in prevalent HD patients followed up to However, convective-based were not the role of large removal and effect on outcomes is no to the inflammatory state in with kidney as a positive protein, and albumin, as a protein, are used as markers of inflammatory state in CKD and ESRD patients as well as in normal At the same time, albumin is the plasma protein in patients on HD and oxidation of albumin decrease plasma antioxidant However, factors other the of inflammation also affect serum albumin, in particular status In it is well documented in studies that serum ferritin levels are increased in HD patients not only due to inflammation but also of or increased iron Thus, it is that markers albumin or ferritin are not of inflammatory a fact that may explain the non-significant variation of these observed in our studies demonstrated increased levels of CRP and pro-inflammatory cytokines as well as markers of oxidative stress in HD CRP is to in HD patients as compared to and a study showed an increase in CRP levels in of the patients during a dialysis session In the as HD to be associated with increased CRP levels Of note, more dialysis such as daily HD and daily HD, to have effects on plasma CRP levels during the 12-month period data on the long-term effect of HDF on CRP levels are However, a study evaluated CRP levels in long-term HD patients on different dialysis modalities including HDF, at baseline and after months of treatment. The results of this study demonstrated that CRP values did not significantly both in patients treated with HD as well as in those on HDF during a period of months in this study the mean CRP values were significantly in HDF with low infusion volumes liters per in standard HD and HDF with infusion volumes liters per session On the contrary, the results of our study a long-term effect of HDF on inflammation by significant decrease in CRP an inflammatory CRP is the best of it is measured with a high-sensitivity assay as in our study, a marker of and cardiovascular mortality in the population as well as in HD patients is well documented that HD patients pro-inflammatory such as IL-6 and a fact that is when membranes are used The HD treatment itself as a pro-inflammatory contributing to a increase in cytokine at the end of each session In our study, there was a in IL-6 levels during 9-month HDF, but the difference did not reach statistical This result can be to the fact that detection of cytokine changes in HD patients is by the and the low plasma concentrations of most cytokines some cytokine only be in which are with strong and in determination of cytokine plasma is by is of interest to note the of markers of inflammatory such as white cell albumin and observed in our patients through the present study. However, it be that a non-significant in albumin values was observed during our study. On the contrary, CRP levels showed a significant decrease after 9 months of HDF treatment. there for this Data are scanty but it is that and is a more sensitive and marker of inflammatory status the factors, which can be by other the inflammatory itself In studies in patients with advanced kidney disease have suggested after for is not a risk for cardiovascular disease clinical of oxidative stress is based on the of different stable oxidized such as lipid and advanced oxidation protein advanced and acid oxidation derivatives or oxidized such as low-density lipoprotein [3]. At the same time, both antioxidants and antioxidants vitamins C and uric and inflammatory proteins, such as and albumin can be evaluated [16]. However, there is no on an marker of oxidative stress. a of as it is the in our study, may be better of oxidative alterations in plasma of patients long-term HD treatment is by the results of studies d-ROMs, which are in HD patients and a increase the dialysis are the oxidative stress biomarkers more the oxidative alterations in HD while determination of plasma such as which was used in well-designed to oxidation during the HD The determination of TAC in HD patients is by the in plasma levels and therefore However, in our study no significant was observed in plasma The FRAP method of TAC which is used in our study, the most sensitive of plasma antioxidant capacity in HD according to a study the biomarkers of oxidative stress in this population results from the suggest that HDF, in particular when large infusion volumes are used, may improve patient survival [8]. However, the mechanisms in this result are associated with the of HDF as compared to HD and a effect of HDF on inflammation and oxidative stress status has not been study from in patients on HD demonstrated results with a effect of HDF on markers of oxidative stress. and which were used as oxidative stress biomarkers in this study, increased in standard HD, they showed a significant decrease in HDF. stress effects are to be by effective removal of radical protein, protein, and protein in HDF and by of radicals by of replacement study from that convective dialysis modalities in oxidative stress by but this effect was to dialysis fluid used in the study patients In a of data on the influence of standard HDF on inflammatory and oxidative stress biomarkers was documented from a of the available studies on the impact of HDF on inflammatory and oxidative stress biomarkers are the results of studies recently have suggested the of HDF to HD by removal of and other middle-sized as well as by significant reduction of pro-inflammatory The enhanced convective clearance by HDF is the for removing larger uremic such as inflammatory biomarkers has also been shown that HDF reduces the levels of that are as markers of oxidative stress and are in the complications of long-term dialysis patients and variation of HDF, HDF with of which the of convection and in well-designed studies to levels of cytokines such as IL-6 and CRP and to have a positive impact on oxidative stress as compared with standard dialysis In both were shown to be compared to standard bicarbonate HD in reducing the inflammatory state of dialysis patients study is only in with these the inflammatory biomarkers only hs-CRP values significantly at the end of the study, while IL-6 showed no significant our results suggest that post-dilution HDF for a period of 9 months suppresses oxidative stress by rapid lowering of d-ROMs and improves antioxidant capacity by increasing TAC (significantly) and SOD (non-significantly) while it reduces inflammation by decreasing (significantly) hs-CRP and IL-6 (non-significantly). of our study is the small of the in a population a of inflammation from as well as the fact that this is not a However, this to our knowledge, a study on the long-term impact of HDF on inflammation and oxidative stress. On this data from research and clinical are scanty in the of and markers of oxidative stress in our study is of results as a for studies with a larger of patients as well as for study to demonstrate the of HDF on oxidative morbidity and mortality and the results of the present was presented in at the of the European Dialysis and