Genomic damage and circulating AGE levels in patients undergoing daily versus standard haemodialysis

Evangelia Fragedaki1, Michael Nebel2, Nicole Schupp1, Katarina Sebekova3, Wolfgang Völkel1, André Klassen4, Monika Pischetsrieder5, Matthias Frischmann5, Toshimitsu Niwa6, Jörg Vienken7, August Heidland4 and Helga Stopper1

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Patients with end-stage renal failure, whether on conservative or haemodialysis therapy, have a high incidence of DNA damage. It is not known if improved control of the uraemic state by daily haemodialysis (DHD) reduces DNA lesions.

Methods. DNA damage in peripheral blood lymphocytes (PBLs) was evaluated in a cross-sectional study of 13 patients on DHD (2–3 h, 6 times/week), 12 patients on standard haemodialysis (SHD) therapy (4–5 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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
End stage renal failure is characterized by a high incidence of cardiovascular disease and cancer [1,2]. Among the mechanisms underlying this phenomenon, uraemia, microinflammation, and oxidative and carbonyl stress are of fundamental importance [3,4]. Augmented levels of reactive oxygen species and carbonyls lead to their enhanced reaction with various cellular constituents, e.g. proteins, membrane lipids and DNA [5]. Growing interest has focused on DNA damage, because of its genetic consequences. DNA lesions have been linked to premature aging, neurodegenerative diseases, diabetes mellitus, atherosclerosis and, recently, to atherosclerotic plaques [6]. Mutations of critical genes may also represent one of the mechanisms of carcinogenesis [7]. In various malignant diseases, more DNA damage was found in cancerous tissues than in surrounding, histologically normal tissues [7]. Assays of DNA damage in peripheral blood lymphocytes (PBLs) are used widely in occupational and environmental settings as biomarkers of early effects of genotoxic carcinogens. Long-term studies showed the high predictivity of chromosomal aberrations in lymphocytes for an increased overall cancer risk [8].

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 [13–15]. 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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We performed a cross-sectional study in patients on DHD (for 2–3 h, 6–7 times/week) and on SHD therapy. As a biomarker of DNA damage, MN frequency was used. Moreover, selected plasma parameters of microinflammation and oxidative stress were assessed: C-reactive protein (CRP), interleukin-6 (IL-6), neopterin and advanced oxidation protein products (AOPPs), as well as homocysteine. The following plasma AGEs were analysed: methylglyoxal-derived imidazolinone (MGI), carboxymethyllysine (CML), imidazolone A (3-deoxyglucosone-derived imidazolinone) and AGE-associated fluorescence.

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 4–4.5 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 2–2.5 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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Table 1. Characteristics of the patients on two different schedules of dialysis

 
Twelve healthy subjects (seven male, five female) with a mean age of 52.9±10.7 years served as control. They had normal routine blood chemistries (data not shown).

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 cycle—after 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 40–44 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 3–8 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 50–230 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 ({lambda}ex 350 nm/{lambda}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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Fig. 1. MN frequencies in peripheral lymphocytes of patients on DHD or SHD. Shown are the individual values with standard deviation (A) based on four independent sampling times (controls: one sampling) and mean values with standard deviations (B). **P<0.01.

 

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Table 3. Plasma parameters of the indicators of inflammation and oxidative stress (mean±SD)

 

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Table 4. Levels of advanced AGEs in the plasma of HD patients and controls (mean±SD)

 


   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The blood chemistry data of the two dialysis groups are compared in Table 2. In the DHD patients, serum urea was significantly lower than in the SHD patients, while creatinine concentrations were comparable. The Kt/V levels per week were identical in both dialysis groups. Total protein was significantly higher in the DHD group, while albumin showed no difference. Serum transferrin was slightly but not significantly higher in the DHD than in the SHD patients. Nearly identical concentrations of haemoglobin, haematocrit and serum iron were found in both dialysis groups. The percentage of transferrin saturation averaged 27.8% in the DHD patients and 30.1% in the SHD patients. Serum ferritin was insignificantly elevated in the DHD patients, compared with the SHD group. Plasma concentrations of calcium and inorganic phosphate were not different between the two groups; intact parathyroid hormone levels were insignificantly higher in the DHD group. Systolic and diastolic blood pressures were identical in the DHD and SHD groups; however, the average number of antihypertensive medications per patient was lower in the DHD group than in the SHD group (1.15±1.75 vs 2.0±2.48). The doses of erythropoetin were identical in both dialysis groups.


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Table 2. Blood chemistry parameters of the patients on daily and standard haemodialysis (mean±SD)

 
Individual MN values are shown in Figure 1A and the mean±standard deviation in Figure 1B. As can be seen, all individual values of the DHD group are consistently lower when compared with those of the SHD patients. The mean number of MN/1000 BN cells in the DHD group was 14.8±4.0, 29.1±5.9 (P<0.01) in the SHD group. Healthy subjects had an average MN frequency/1000 BN cells of 13.2±3.04, not significantly different from the patients on DHD.

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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Fig. 2. Correlation of the MN frequency and the plasma concentration of imidazolone A in the lymphocytes of either patient group (DHD, squares, n = 13, and SHD, triangles, n = 11). The average value for the control group is also shown (circle).

 


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Fig. 3. Correlation of the micronuclei frequency and the plasma CML concentration in lymphocytes of either patient group (DHD, squares, n = 13, and SHD, triangles, n = 11). The average value for the control group is also shown (circle).

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This is the first study to measure genomic damage in PBLs of patients on DHD. The number of patients investigated is low since DHD is not established as a routine form of renal replacement therapy. To date, DHD is available only to a limited number of patients. Nevertheless, according to our cross-sectional study in a sample of these patients, DNA damage—as assessed by MN frequency—was significantly lower in the patients on DHD than in those on SHD. In the latter, the higher degree of DNA damage corresponded to our earlier findings [9,10]. Although the average age of the SHD group was higher than that of the DHD group (57.5±12.9 years vs 47.9±11.5 years), it seems unlikely that age caused the genomic differences between the groups. If one would exclude the two oldest patients from the SHD group and the two youngest from the DHD group, this would give in the SHD group a mean age of 54.4±11.7 years with an MN frequency of 31.2±5.9 versus 50.3±10.9 years and 15.6±3.4, respectively, in the DHD group. So, the genomic damage would still be highly significantly different between those two groups adjusted for age. The DHD patients were on HD 4 years longer than the SHD patients, so the lesser DNA damage in the DHD group was unrelated to time on dialysis. It was also unrelated to the underlying diseases, BMI, type of dialyzer, blood pressure or concomitant drug therapy. Similar to the findings of other investigators [13], the number of antihypertensive medications, including ACE inhibitors and angiotensin receptor blockers (ARBs), was lower in our cohort. Therefore the beneficial effect that renin-angiotensin system blockers potentially could have cannot be the sole cause of the lower DNA damage in the DHD group.

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 AGEs—such as deletions, insertions and transposon activation—have 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.



   Acknowledgments
 
We thank M. Kessler and M. Scheurich for their expert technical assistance.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Herzog CA, Ma JZ, Collins AJ. Poor long-term survival after acute myocardial infarction among patients on long-term dialysis. N Engl J Med 1998; 339: 799–805[Abstract/Free Full Text]
  2. Maisonneuve P, Agodoa L, Gellert R et al. Cancer in patients on dialysis for end-stage renal disease: an international collaborative study. Lancet 1999; 354: 93–99[CrossRef][ISI][Medline]
  3. Tepel M, Echelmeyer M, Orie NN, Zidek W. Increased intracellular reactive oxygen species in patients with end stage renal failure: Effect of hemodialysis. Kidney Int 2000; 58: 867–872[CrossRef][ISI][Medline]
  4. Miyata T, Kurokawa K, Van Ypersele De Strihou C. Advanced glycation and lipoxidation end products: role of reactive carbonyl compounds generated during carbohydrate and lipid metabolism. J Am Soc Nephrol 2000; 11: 1744–1752[Free Full Text]
  5. Imlay JA, Linn S. DNA damage and oxygen radical toxicity. Science 1988; 240: 1302–1309[ISI][Medline]
  6. Martinet W, Knaapen MW, De Meyer GR, Herman AG, Kockx MM. Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques. Circulation 2002; 106: 927–932[Abstract/Free Full Text]
  7. Loft S, Poulsen HE. Cancer risk and oxidative DNA damage in man. J Mol Med 1996; 74: 297–312[CrossRef][ISI][Medline]
  8. Hagmar L, Strömberg U, Bonassi S et al. Impact of types of lymphocyte chromosomal aberrations on human cancer risk: results from Nordic and Italian cohorts. Cancer Res 2004; 64: 2258–2263[Abstract/Free Full Text]
  9. Stopper H, Meysen T, Böckenförde A, Bahner U, Heidland A, Vamvakas S. Increased genomic damage in lymphocytes of patients before and after long term maintenance hemodialysis therapy. Am J Kidney Dis 1999; 34: 433–437[ISI][Medline]
  10. Stopper H, Boullay F, Heidland A, Vienken J, Bahner U. Comet assay analysis identifies genomic damage in lymphocytes of uremic patients. Am J Kidney Dis 2001; 38: 296–301[ISI][Medline]
  11. Tarng DC, Huang TP, Wei YH et al. 8-Hydroxy-2'-deoxyguanosine of leukocyte DNA as a marker of oxidative stress in chronic hemodialysis patients. Am J Kidney Dis 2000; 36: 934–944[ISI][Medline]
  12. Vamvakas S, Bahner U, Becker P, Steinle A, Gotz R, Heidland A. Impairment of DNA repair in the course of long-term hemodialysis and under cyclosporine immunosuppression after renal transplantation. Transplant Proc 1996; 28: 3468–3473[ISI][Medline]
  13. Ting GO, Kjellstrand C, Freitas T, Carrie BJ, Zarghamee S. Long-term study of high-comorbidity ESRD patients converted from conventional to short daily hemodialysis. Am J Kidney Dis 2003; 42: 1020–1035[ISI][Medline]
  14. Williams AW, Chebrolu SB, Ing TS et al. Early clinical, quality-of-life, and biochemical changes of ‘daily hemodialysis’ (6 dialyses per week). Am J Kidney Dis 2004; 43: 90–102[CrossRef][ISI][Medline]
  15. Floridi A, Antolini F, Galli F, Fagugli RM, Floridi E, Buochristiani U. Daily haemodiaylsis improves indices of protein glycation. Nephrol Dial Trasnplant 2002; 17: 841–847
  16. Stopper H, Müller SO. Micronuclei. Biological end point for genotoxicity. Vitro 1997; 11: 661–667[CrossRef]
  17. Fenech M. The cytokinesis–block micronucleus technique: A detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 1993; 285: 35–44[CrossRef][ISI][Medline]
  18. Witko-Sarsat V, Friedlander M, Capeillere-Blandin C et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 1996; 49: 1304–1313[ISI][Medline]
  19. Thornalley PJ, Battah S, Ahmed N et al. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem J 2003; 375: 581–592[CrossRef][ISI][Medline]
  20. Niwa T, Katsuzaki T, Miyazaki S et al. Immunohistochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of diabetic patients. J Clin Invest 1997; 99: 1272–1280[Abstract/Free Full Text]
  21. Münch G, Keis R, Wessels A et al. Determination of advanced glycation end products in serum by fluorescence spectroscopy and competitive ELISA. Eur J Clin Chem Clin Biochem 1997; 35: 669–677[ISI][Medline]
  22. Pischetsrieder M, Seidel W, Münch G, Schinzel R. N2(1-Carboxylethyl)deoxy-guanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies. Biochem Biophys Res Com 1999; 264: 544–549[CrossRef][ISI][Medline]
  23. Stopper H, Schinzel R, Sebekova K, Heidland A. Genotoxicity of advanced glycation end products in mammalian cells. Cancer Lett 2003; 190: 151–156[CrossRef][ISI][Medline]
  24. Perna AF, Ingrosso D, De Santo NG, Galletti P, Zappia V. Mechanism of erythrocyte accumulation of methylation inhibitor S-adenosylhomocysteine in uremia. Kidney Int 1995; 47: 247–253[ISI][Medline]
  25. D’Intini V, Bordoni V, Bolgan I et al. Monocyte apoptosis in uremia is normalized with continuous blood purification modalities. Blood Purif 2004; 22: 9–12[CrossRef][ISI][Medline]
Received for publication: 19. 5.04
Accepted in revised form: 8. 3.05





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Articles by Fragedaki, E.
Articles by Stopper, H.