1 Institute of Pharmacology and Toxicology, University of Würzburg, Germany, 2 Kuratorium for Dialysis and Kidney Transplantation, Cologne, Germany, 3 Slovak Medical University, Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 4 Department of Internal Medicine, University of Würzburg, Germany, 5 Institute of Pharmacy and Food Chemistry, University of Erlangen-Nürnberg, Germany, 6 Daiko Medical Center, Nagoya University, Nagoya, Japan and 7 Fresenius Medical Care, Bad Homburg, Germany
Correspondence and offprint request to: Prof. Dr Helga Stopper, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacherstr. 9, D-97080 Würzburg, Germany. Email: stopper{at}toxi.uni-wuerzburg.de
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Abstract |
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Methods. DNA damage in peripheral blood lymphocytes (PBLs) was evaluated in a cross-sectional study of 13 patients on DHD (23 h, 6 times/week), 12 patients on standard haemodialysis (SHD) therapy (45 h, 3 times/week) and 12 healthy age-matched volunteer controls. The biomarker of DNA damage used was micronucleus frequency. The assessed plasma parameters of microinflammation and oxidative stress were C-reactive protein (CRP), interleukin-6 (IL-6), neopterin, advanced oxidation protein products (AOPP), and homocysteine. We also measured plasma concentrations of the circulating advanced glycation end products (AGEs) MGI (methylglyoxal-derived imidazolinone), CML (carboxymethyllysine), imidazolone A (3-deoxyglucosone-derived imidazolinone) and AGE-associated fluorescence.
Results. Compared to SHD, DHD was associated with significantly lower DNA damage, approaching the normal range. Micronuclei (MN) frequency averaged 29.1 MN±5.9/1000 binucleated (BN) cells in the SHD group, which is significantly elevated (P<0.01), 14.8 MN±4.0/1000 BN cells in the DHD group, and 13.2 MN±3.04/1000 BN cells in the controls. CRP and AOPP were in the normal range (and similar between the dialysis groups). In contrast, IL-6 and neopterin were significantly elevated, with lower values associated with DHD as compared with SHD. The increased levels of AGEs tended to be lower in the DHD group, reaching significance for CML and imidazolone A.
Conclusions. Overall, it was found that genomic damage in PBLs is lower in patients on DHD than in those on SHD. Lower plasma concentrations of uraemic toxins, including circulating AGEs, may account for the differences. To confirm these data, prospective clinical trials need to be performed.
Keywords: advanced glycation endproducts; daily haemodialysis; micronuclei frequency; oxidative stress
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Introduction |
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In patients with chronic renal failure, whether pre-dialysis or in dialysis, genomic damage has been demonstrated by various methods, such as identifying micronucleus (MN) frequency [9], single-cell gel electrophoresis [10] in PBLs, and measuring 8-hydroxy 2'-deoxyguanosine (8-OH-dG) content in leukocyte DNA [11]. Studies of DNA repair, after exposure to UV light, in PBLs from patients on maintenance haemodialysis (MHD) showed a decreased level of repair activity after more than 10 years treatment duration [12].
Since a clear relationship between the severity of renal impairment and the degree of genomic damage has been demonstrated in pre-dialysis patients [10], we hypothesized that the improved uraemic state, induced in MHD patients by daily haemodialysis (DHD), should result in fewer DNA lesions as compared with standard haemodialysis (SHD). With DHD, the plasma concentrations of various toxins are lower, due to shorter interdialytic intervals as well as increased removal of small solutes [13,14]. At the same time, lower plasma urea levels along with a better control of circulating advanced glycation end products (AGEs) are observed [15]; and nutritional state, fluid volume, hypertension, renal anaemia and quality of life are beneficially influenced [1315]. To ascertain the effect of dialysis frequency we measured DNA damage and markers of microinflammation and oxidative stress in a cross-sectional study in patients on SHD and DHD.
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Subjects and methods |
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Subjects
Two groups of patients with end stage renal failure treated with either SHD or DHD were assessed. Their demographic data are given in Table 1. Each of the following criteria was a basis for exclusion: diabetes, bacterial or viral infections (HCV-, HBV-, HIV-), malignancies, hepatic insufficiency, and treatment with anti-inflammatory, cytostatic or immunosuppressive drugs. The SHD group consisted of 12 patients (five males and seven females; mean age of 57.5±12.8 years) and dialyzed for 44.5 h, 3 times/week for an average of 3.6±1.82 years. The DHD group consisted of 13 patients (10 males, three females; mean age of 48±11.5 years) dialyzed at home for 22.5 h, 6 times/week. Their average time on DHD was 3.3±1.37 years, with an overall MHD time of 7.4±8.34 years. All patients were treated with synthetic, biocompatible, non-complement-activating polyethersulfone membranes. Of the 13 patients on DHD, seven were treated with a high-flux dialyzer (FX 60), and six with a low-flux dialyzer (Polyflux). Four SHD patients were dialyzed with an FX 60, the other eight with a Polyflux dialyzer. The Kt/V value (double pool model) averaged 4.50±0.74 per week in the DHD group and 4.51±0.63 per week in the SHD group. Ultrapure dialysate was used in all patients. Blood samples were always taken before the dialysis session. The pre-dialysis blood pressure in both dialysis groups was in the normal range; seven of the 13 patients in the DHD group and three of the 12 in the SHD group did not need antihypertensive drugs. Erythropoetin dosage was identical in both groups. DHD and SHD were performed at the same dialysis centre.
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The study was carried out in accordance with the Declaration of Helsinki. All participants gave their written informed consent.
Blood collection and lymphocyte isolation
PBLs were separated from 10 ml samples of heparinized blood on Histopaque 1077 gradients, washed twice in RPMI 1640 medium (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) supplemented with 15% heat-inactivated foetal calf serum (Biochrom KG Berlin), and then re-suspended in RPMI 1640 medium.
Routine chemistry tests were performed on the day of blood collection; for the other analyses, aliquots of plasma were stored at 20°C.
Micronucleus frequency assay
MN are expressed in dividing cells that either contain chromosome breaks (resulting from unrepaired double-strand breaks) or by whole chromosomes that are unable to travel to the spindle poles during mitosis [16]. They are observed in cells with completed nuclear division and are counted in the binucleated stage of the cell cycleafter using the cytokinesis inhibitor, cytochalasin B. Our MN frequency assay was performed according to Fenech [17], with slight modifications. Briefly, lymphocytes were cultured at 37°C in a humidified atmosphere containing 5% CO2. Phytohemagglutinin (Sigma-Aldrich Chemie GmbH) was added in a concentration of 5 µg/ml, to stimulate the lymphocytes into traversing through the cell cycle. The addition of cytochalasin B (Sigma-Aldrich Chemie GmbH) in a concentration of 4.5 µg/ml 4044 h after stimulation, prevented cytokinesis, and resulted in binucleated (BN) cells. Cells were cultured for an additional 24 h and then transferred onto glass slides by cytospin centrifugation. The slides were fixed in a methanol solution (20°C) for 1 h.
For staining, the slides were incubated for 5 min in 62.5 µg/ml acridine orange (Sigma-Aldrich Chemie GmbH), diluted in Sörensen buffer (67 mmol/l of Na2HPO4/KH2PO4; pH 6.8). Afterwards, the slides were washed twice for 5 min in Sörensen buffer. MN were counted in 1000 BN cells, three slides per patient, using a 400-fold magnification. Objects were classified as MN if they appeared separated from the nucleus, were round or oval, showed staining characteristics similar to those of the nuclei, and had an area less than one-quarter of the area of the average normal nucleus.
MN frequencies were determined for each of the four samples (control group: one sampling) taken at 38 week intervals, and are given as a mean±standard deviation (SD) for each determination. Group averages were calculated from these individual averages and are given as mean±standard of all patients in a group.
Blood chemistry parameters
Duplicate determinations of AOPP were made spectrophotometrically, on a microplate reader (MRX; Dynatech, USA) and according to the method of Witko-Sarsat et al. [18], of plasma IL-6 and neopterin by ELISA (Immunotech, Marseille, France) and of homocysteine concentrations by HPLC. CRP was assessed using the fixed-point immunorate method (Vitros 250 analyser; J&J, Rochester, USA). Iron, ferritin, transferrin and the transferrin saturation ratio were determined according to standard procedures.
Plasma AGE levels
MGI levels were analysed by liquid chromatography/mass spectrometry. To identify free MGI, 0.3 ml of plasma samples were filtered with a 10.000 Da cut-off filter to remove plasma proteins. Of each eluate, 5 µl was injected onto an X-Terra RP18 HPLC column (2.1 mm x 50 mm; 2.5 µm, 100 A; Waters, Eschborn, Germany), using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump. Separation was performed by an isocratic elution with water (Roth, Germany) within 7 min at a flow rate of 250 µl. An enhanced product ion scan (EPI) was performed on a linear ion-trap mass spectrometer (QTrapTM Applied Biosystems, Darmstadt, Germany) equipped with a TurboIonSprayTM interface connected to the HPLC-system. To record spectral data, a vaporizer temperature of 400°C and a TurboIonSpray voltage of 5.5 kV in the positive ionisation mode was applied. Declustering potential was set at 30 V, collision energy was 40 V, N2 was used as collision gas with the CAD set at high. The precursor ion was 229.2 amu (M+H)+; and product ions in the range of 50230 amu were monitored using a Q3 entry barrier of 8 V, a fill time of 200 ms, and a scan rate of 4000 amu/s. An EPI spectrum of a plasma sample demonstrated identical fragment ions, as previously described by Thornalley et al. [19]. For the relative quantification of MGI in plasma samples, the same procedure was performed as described above. Analytes were detected in the positive ion mode at a vaporizer temperature of 400°C and a TurboIonSpray voltage of 5.5 kV. Spectral data were recorded, with N2 as the collision gas (CAD = 4), in the multiple reaction monitoring mode with a dwell time of 1000 ms and a declustering potential of 30 V; and a collision energy of 40 V was used for all transitions: 229.2 142.0 as quantifier, 229.2
114.0 and 229.2
96.0 as qualifiers.
CML and imidazolone A
CML was measured by ELISA using the non-commercial CML-specific monoclonal antibody 4G9 (provided by Roche Diagnostics GmbH, Penzberg, Germany). A 6-(N-carboxymethylamino) caproate standard was used for quantification. In contrast to the protocol of Roche, protein digestion with proteinase K was not performed, resulting in lower absolute values that, however, strongly correlate with data obtained after protein digestion. CML levels could not be determined in the controls due to technical problems.
Imidazolone A was measured by ELISA using a non-commercial monoclonal antibody that is specific for imidazolone A [20], with imidazolone A-HSA (which was prepared by the reaction of HSA with 3-deoxyglucosone) used as standard, and the analyses were performed in triplicate.
AGE-associated fluorescence
AGE-associated fluorescence was measured by fluorescence spectroscopy (ex 350 nm/
em 450 nm) of 50-fold diluted plasma samples in triplicate (Fluorite 1000; Dynatech, USA), as described previously [21].
Statistics
Data are presented as means ± SD. The means were compared for significance between dialysis groups as well as healthy controls using the two-sided Student's t-test. Analysis of variance (ANOVA) was used for comparison of the SHD, DHD and control groups (Figure 1, Tables 3 and 4). P-values <0.05 were required to accept results as significant.
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Results |
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The parameters of inflammation and oxidative stress are presented in Table 3. The plasma concentrations of CRP and AOPP were in the normal range in all groups, while IL-6 and neopterin were significantly enhanced in the dialysis patients compared with the control group. In the DHD group, IL-6 tended to be higher than in the SHD patients, without reaching statistical significance, while neopterin was significantly lower in the DHD group than in the SHD group. Also, plasma homocysteine was significantly higher in the SHD group compared with the control. In the DHD group, homocystein, although significantly higher than in the control group, showed a trend to lower levels than in the SHD group.
Data on different AGE measurements are shown in Table 4. AGE-associated fluorescence, CML and imidazolone A were significantly elevated in the SHD group (data of 11 of the 12 patients) compared with the controls. Significantly reduced levels of AGE-associated fluorescence, CML and imidazolone A were observed in the DHD group compared with the SHD group. Free MGI was not different between the two dialysis groups.
The DNA damage in PBLs showed no relationship with blood urea concentration. There was no dependence on BMI or the type of haemodialyzer (high- vs low-flux). However, an association could be found between MN frequency and various circulating AGE levels. Thus there was a direct correlation between MN frequency and imidazolone A (Figure 2) or CML concentrations (Figure 3). The correlation between fluorescent AGEs and MN frequency was r = 0.42, p<0.05. No relationship could be found in healthy subjects, between MN frequency and the narrow range of normal plasma imidazolone A or fluorescent AGE levels (data not shown); however, the average values of the control group were located at the lower end of the theoretical extension of the linear regression of the patient samples (Figures 2 and 3).
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Discussion |
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We assume that the lesser DNA damage in the DHD group is a consequence of the improved uraemic state. The peak of the uraemic toxin levels in DHD is lower than in SHD, because the interdialytic intervals are shorter. Moreover, DHD may be more efficient than SHD, as a consequence of better small solute clearance [14]. It provides a greater dialysis dose due to more rapid urea decrease, resulting in lower average urea concentrations over time [13,14]. In our study plasma urea concentration was significantly lower in the DHD group than in the SHD group. We also observed significant declines in the levels of the circulating protein-bound AGEs CML, imidazolone and AGE-associated fluorescence. These data are in line with the results of the prospective study of Floridi et al. [15], who showed that various AGEs were significantly decreased after long-term DHD. They also observed a reduction of free pentosidine, while the amount of the free AGE measured in this study, MGI, was the same in DHD and SHD.
Among the compounds potentially contributing to DNA damage in uraemia, numerous ones have to be discussed, including circulating AGEs and carbonyl compounds. These substances can react with DNA in a similar way as with proteins, resulting in the formation of DNA-bonded AGEs. Mutagenic effects of DNA-bonded AGEssuch as deletions, insertions and transposon activationhave been demonstrated in bacterial models [22]. Our group recently investigated the genotoxicity of unspecific AGE-BSA and the BSA-bound AGEs, MGI and CML, in cultured kidney cells. In this study, dose-dependent DNA damage was observed, as evaluated in the comet assay [23]. If our recent in vitro findings are representative for dialysis patients, the higher genomic damage in the SHD group could be a consequence of the marked accumulation of reactive AGEs and carbonyl compounds, while the reduction in genomic damage in the DHD group might be, at least in part, due to a lowering in these toxic substances during long-term therapy. The association we found between the levels of circulating AGEs (CML and imidazolone) and the DNA damage in lymphocytes (MN frequency) could be a hint in favour of a causal link between AGEs and genomic damage.
Another possible cause of the lessened genomic damage in the DHD group could be enhanced DNA repair, something that needs to be analysed in further studies.
An important factor in the induction of genomic damage is oxidative stress [5], a frequent complication in end stage renal failure [3,4]. A relationship between DNA damage (as evaluated by 8-OH-dG) and increased oxidative stress in peripheral leukocytes of HD patients has been demonstrated [11]. Surprisingly, in our DHD and SHD patients the plasma indicators of oxidative stress and microinflammation, such as CRP and AOPPs, were in the normal range. Other markers of inflammation such as IL-6 and neopterin were elevated significantly in the dialysis patients. With regard to neopterin (with its low molecular weight of around 250 Da), its reduction in the DHD group, compared with the SHD group, might be explained by an enhanced removal in DHD.
Moreover, since genomic damage due to hypomethylation has been described repeatedly in the past, the toxic effects of hypomethylation in uraemia have to be taken into consideration. In end stage renal failure, the intracellular accumulation of the natural methyltransferase inhibitor S-adenosylhomocysteine (S-Ado-Hcy), which impairs methylation-dependent repair processes, has been shown [24]Ado-Hcy was directly related to the blood concentration of homocysteine in end stage renal disease. In our cohort of dialysis patients, the markedly elevated homocysteine levels were slightly (19%) but not significantly lower (in the DHD group), and could therefore make only a minor contribution to explaining the decreased DNA damage.
We did not address specifically the potential role of apoptosis. It has been shown that uraemia directly causes apoptosis of mononuclear cells. Apoptosis may be further enhanced by the exposure of blood to dialysis membranes, in particular to those with low biocompatibility. It is conceivable that, because of more continuous blood purification, the degree of apoptosis is less in patients on DHD than in those on SHD. Such a relationship was suggested recently by the results of in vitro investigations that showed a nearly normal apoptosis rate in patients on continuous ambulatory peritoneal dialysis (CAPD) [25]. It is a task for future investigations to evaluate the role of apoptosis.
In conclusion, our study suggests that, in patients with end-stage renal failure, DHD is associated with lower genomic damage than is SHD. The improvement may be the result of an enhanced removal of toxic substances, including AGEs. If this lower genomic damage in PBLs corresponds also to lower DNA damage in other organs, such as the kidney and liver, the incidence of cancer should be less among patients on long-term DHD. The data generated in the present cross-sectional study should be confirmed in prospective clinical investigations.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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