1 Department of Internal Medicine IV, Friedrich Schiller University of Jena, Germany, 2 Institute of Cell and Protein Engineering, Medical Park Hannover, Germany, 3 Kuratorium für Dialyse und Nierentransplantation, Jena, Germany and 4 Fresenius Medical Care, Bad Homburg, Germany
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Abstract |
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Methods. In our study, 10 non-diabetic and 10 diabetic ESRD patients were on haemodialysis with low-flux membranes (LF) followed by a cross-over haemodialysis with high-flux or super-flux polysulfone membranes (HF, SF) for 6 months each. We measured the protein-bound pentosidine and free pentosidine serum levels by high-performance liquid chromatography (HPLC) as well as the serum AGE peptide, AGE-ß2-microglobulin and ß2-microglobulin concentrations, using ELISA assays.
Results. All parameters investigated were significantly higher in dialysis patients than in healthy subjects. The reduction rates during a single dialysis session were found to be higher using the SF than those obtained with the HF (free pentosidine 82.4±7.3 vs 76.6± 8.7%; AGE peptides 79.7±7.7 vs 62.3±14.7%; AGE-ß2-microglobulin 64.0±16.5 vs 45.4±17.7%; ß2-microglobulin 70.5±5.6 vs 58.2±6.0%). The protein-bound pentosidine levels remained constant over the respective dialysis sessions. In the 6-month treatment period with the SF, decreased pre-dialysis serum levels of protein-bound pentosidine, free pentosidine and AGE peptides were observed in non-diabetics and diabetics as compared with values obtained with the LF. The respective pre-dialysis AGE-ß2-microglobulin concentrations decreased insignificantly, whereas those of ß2-microglobulin were significantly lower. Using the HF dialyser, only moderate changes of the parameters measured were noted.
Conclusion. Treatment with the biocompatible polysulfone SF dialyser seems to be better suited to lower serum AGE levels and to eliminate their precursors.
Keywords: advanced glycation end-products; haemodialysis; high-flux polysulfone
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Introduction |
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In renal failure, the increased oxidative stress enhances the formation of AGE precursors along with an impaired clearance and may cause their accumulation. These processes may further result in the generation and accumulation of AGEs.
Serum AGEs circulate in the form of modified proteins or peptides and, as in the case of pentosidine, also in its free form. Using immunological determination or fluorescence detection methods, serum AGE levels have been reported to be increased in diabetic as well as in non-diabetic patients with end-stage renal failure (ESRD) [811].
We investigated the influence of maintenance haemodialysis treatment on a subset of serum AGE levels (AGE peptides <30 kDa, AGE-modified ß2-microglobulin, pentosidine) using high-flux or super-flux biocompatible polysulfone membranes in a cross over study comprising 20 ESRD patients. This study aimed to find out whether AGE-modified proteins or peptides could be effectively removed through internal filtration combined with increased convectional forces and whether highly permeable membranes in haemodialysis or haemodiafiltration could be a new therapeutic approach in treating ESRD.
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Subjects and methods |
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Pre- and post-dialysis serum samples were obtained at the start and the end of dialysis sessions.
As controls, serum samples of age-matched healthy subjects were used.
Serum protein concentration was determined according to the Biuret method and serum albumin by colorimetric end-point measurement using routine autoanalyser methods.
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Pentosidine measurement by HPLC |
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Protein-bound pentosidine (expressed in pmol per mg protein) was calculated as follows:
[Total serum pentosidine (pmol/ml)-Free serum pentosidine (pmol/ml)] / Serum protein concentration (mg/ml)
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AGE peptide and AGE-ß2-microglobulin measurement by competitive ELISA |
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AGE-KLH: Keyhole limpet haemocyanin (KLH, 2 mg/ml, Sigma, Germany) was incubated with PBS (pH 7.4) containing 1 M glucose (Sigma, Germany) at 37°C in the dark under aerobic and aseptic conditions for 6 weeks. After incubation, unbound glucose was removed by gel filtration using PBS as an elution solvent. The extent of lysine-modified residues of KLH reacting with glucose determined according to the method described by Habeeb [15] was found to be 83%.
Immunological techniques
Polyclonal anti-AGE antibody: Polyclonal AGE antibodies were obtained by immunizing New Zealand rabbits receiving a primary immunization (500 µg of AGE-KLH at two sites of the back) with Freund's complete adjuvant. Booster injections with Freund's incomplete adjuvant (250 µg) were given each month over a 3-month period. After immunization controlled by Ouchterlony double diffusion, both antisera yielded a titre of 1:2500 dilution. The polyclonal antibodies were purified by affinity chromatography, using protein A (Affi-Gel, Pierce, USA) in the first and AGE-collagen (AminoLink Plus Immobilization Kit, Pierce, USA) in the second step.
The major immunological epitope of the antibody has been found to be a CML-protein adduct (70%), following the method described by Ikeda [14]. Thirty per cent of the antigens recognized by the antibody were identified as non-CML adducts. The anti-AGE antibody did not react with early Amadori products, pentosidine or caproyl pyrraline as well as with glycated proteins incubated under antioxidative conditions.
Monoclonal anti-AGE antibody: The production of the monoclonal anti-AGE antibody has been performed as described by Niwa et al. [16]. Six-week-old female BALB/c mice were given intraperitoneal injections of AGE-KLH (0.1 mg, four times at 2-week intervals) prepared as described with Freund's adjuvant. The culture of the antibody-secreting fused cells has been performed according to Köhler [17]. Thus, the antibody-secreting cells were injected into 8-week-old female BALB/c mice. The ascitic fluid was collected after 2 weeks, and the IgG fraction was purified using DEAE-cellulose column chromatography (Amersham Pharmacia Biotech, Germany).
The subclass of the antibody was determined as IgG2a and tested for its identity with the monoclonal AGE-antibody produced by T. Niwa (kindly provided by Dr T. Niwa, Nagoya University Daiko Medical Center, Japan). Using a competitive assay, it could be shown that the antibody exclusively recognized the proteins glycated under aerobic conditions. The antibody did not react with glycated proteins incubated under antioxidative condition. Furthermore, the anti-AGE antibody was unreactive to early Amadori products, pentosidine and caproyl pyrraline. CML-BSA significantly inhibited the recognition of AGE-BSA by the antibody, suggesting that CML was the major existing antigenic determinant.
AGE peptides
Sample preparation: pre- and post-dialysis serum samples were diluted 10-fold with PBS solution and filtered through 30-kDa-membrane filter units (Millipore, USA).
The AGE peptide levels in the filtrates were measured using the polyclonal rabbit anti-AGE antibody. The wells (96-well MaxiSorp ELISA plate, Nunc, Denmark) were coated at 4°C with AGE-BSA (1 µg AGE-BSA in 100 µl of 50 mM carbonate buffer per well, pH 9.6) overnight, washed with phosphate-buffered saline containing 0.5% Tween-20 (pH 7.4) and blocked at room temperature with 200 µl of 0.5% gelatine in 50 mM carbonate buffer for 1 h. After washing, 50 µl-aliquots of samples or standards and 50 µl-aliquots of the anti-AGE antibody (500 ng/ml) were added to each well and incubated at room temperature for 1 h, then washed and subsequently incubated with 100 µl of alkaline phosphatase-conjugated mouse anti-rabbit IgG (1 h, room temperature, Biorad, USA). The plates were washed again and developed with 100 µl of alkaline phosphatase substrate (Biorad, USA). The absorbance at 405 nm was measured by a microplate reader after a 30-min reaction time. The effect of polyclonal anti-AGE antibody on AGE-BSA was used as a standard. Results were expressed in UI per ml (1 UI/ml represents the amount of AGE contained in the samples, which is equivalent to the inhibition of the AGE antibody binding to the antigen).
AGE-ß2-microglobulin
Sample preparation: pre- and post-dialysis serum samples were diluted 10-fold with PBS solution and filtered through 30-kDa-membrane filter units (Millipore, USA). The ß2-microglobulin fractions of the filtrates were isolated by affinity chromatography using a polyclonal anti-ß2-microglobulin antibody (DAKO, Denmark). The AGE-ß2-microglobulin levels were measured directly in the ß2-microglobulin fractions.
After activating the carboxy-terminal portion of AGE-ß2-microglobulin (10 µg/ml) with 0.1 M EDAC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccininmide; Sigma, Germany) at a ratio of 1:1 at room temperature for 30 min, the activated protein was diluted with 1 M carbonate buffer (pH 8.6), and 100 µl per well were used to coat the plate (Covalink-NH ELISA plates, Nunc, Denmark) at 4°C for 30 min. The plates were washed four times with distilled water followed once with a PBS-rinse containing 0.5% Tween-20. The reaction was blocked at room temperature with 200 µl of 0.5% gelatine in PBS-Tween (pH 7.4) for 1 h. After washing the plates three times with PBS-Tween, 50 µl of test samples and 50 µl of monoclonal mouse anti-AGE antibody (100 ng/ml) were added to each well and incubated at room temperature for 1 h, washed, followed by incubation with 100 µl of alkaline phosphatase-conjugated anti-mouse IgG (1 h at room temperature; Biorad, USA). The plates were washed and developed with 100 µl of alkaline phosphatase substrate. The absorbance at 405 nm was measured by a microplate reader after a 30-min reaction time. The results were expressed in UI per ml. An average of 0.5 µg/ml AGE-ß2-microglobulin inhibited about 50% of the antibody binding to the antigen.
ß2-Microglobulin
ß2-Microglobulin levels in pre- and post-dialysis serum samples were measured nephelometrically using a latex-enhanced ß2-microglobulin kit (BNII Kit, The Binding Site, UK).
Calculations and statistics
Data are expressed as means±SD. To exclude the influence of haemoconcentration, all post-dialysis serum concentrations measured were corrected according to the method described by Bergstrom and Wehle [18]. Statistics were performed using the Wilcoxon test for comparison of paired samples, the MannWhitney U test for comparison of unpaired samples and the Pearson correlation test for estimating relationships between variables. Statistical significance was achieved when the P-value was <0.05.
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Results |
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As shown in Figure 2, a moderate decrease in protein-bound serum pentosidine levels was observed, being less than 15% in non-diabetic patients using the HF dialyser (months 16). In contrast, in the following period of dialysis with the SF (months 712) these levels decreased significantly in months 8, 9 and 10 as compared with values obtained with the LF membranes. Likewise, in the diabetic patient group, treatment with the SF (months 16) was associated with significant decreases in months 1 and 2, followed by persistently lower values in the HF-phase afterwards. In both groups, SF dialysis caused a reduction of protein-bound pentosidine levels by nearly 30%.
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AGE peptides
All AGE peptide levels measured in this study were significantly higher than those in healthy subjects (1.1±0.3 UI/ml, n=20).
Contrary to pentosidine, the initial pre-dialysis AGE-peptide values were significantly lower in the non-diabetic than in the diabetic group (41.1±18.9 vs 61.5±19.0 UI/ml; P=0.01). AGE peptides were cleared by all membranes used in the study (e.g. pre- and post-dialysis SF values of the diabetic patient group in month 6: 41.5±9.0 and 8.9±4.2 UI/ml, respectively); the SF was characterized by a significantly higher reduction rate than the HF (79.7±7.7 vs 62.3±14.7%).
During dialysis with the SF membrane, the pre-dialysis AGE peptide serum levels decreased significantly in the non-diabetic patient group (months 912) as compared with the month-7-value as well as in the diabetic patient group (months 26) as compared with the LF-value (Figure 4). No significant changes could be observed during the HF dialyser period of application in both patient groups.
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AGE-ß2-microglobulin
AGE-ß2-microglobulin was not detectable in healthy subjects. In dialysis patients, the initial pre-dialysis levels were insignificantly lower in non-diabetics than in diabetics (1.72±1.08 vs 2.93±1.9 UI/ml, n.s.). In both patient groups, a 6-month treatment with the SF dialyser was associated with decreases in the pre- and post-dialysis concentrations (Figure 5). The AGE-ß2-microglobulin reduction rate induced by the SF was found to be significantly higher than that of the HF (64.0±16.5 vs 45.4±17.7%).
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ß2-Microglobulin
All ß2-microglobulin levels determined in dialysis patients were significantly elevated as compared with healthy subjects (1.17±0.4 µg/ml, n=26).
The initial pre-dialysis ß2-microglobulin levels in the non-diabetic and the diabetic patient group were nearly identical (55.0±7.9 vs 48.0±19.0 µg/ml). A significant reduction could be obtained at the end of dialysis session when the HF or SF membranes were used (e.g. pre- and post-dialysis SF values of the diabetic patient group in month 1: 31.0±8.1 and 9.7±4.2 µg/ml, respectively). The SF was characterized by a significantly higher reduction rate for ß2-microglobulin than the HF dialyser (70.5±5.6 vs 58.2±6.0%). During the application period of both, the HF and SF dialyser, the ß2-microglobulin pre-dialysis levels decreased significantly in the non-diabetic as well as in the diabetic patient group as compared with the LF-values (Figure 6).
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Albumin
The serum albumin concentrations did not change significantly during dialysis using all three membranes. In the 12-month study period, the pre-dialysis mean values ranged from 43.1 to 40.6 g/l in the non-diabetic and from 43.6 to 38.0 g/l in the diabetic patient group, respectively.
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Discussion |
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Nonetheless, as in the case of dialysis-related amyloidosis, several groups are looking for strategies to decrease the amount of accumulated ß2-microglobulin as well as AGE products and to lower the risk of AGE generation per se [26]. As reported recently, circulating pentosidine and carboxymethyllysine levels are influenced by membrane characteristics [27,28]. The free form of pentosidine (molecular weight 379 Da) is totally excreted via the glomerulus in patients with normal renal function. Its accumulation is the result of a decreased glomerular filtration rate. More than 95% of the circulating pentosidine is linked to serum proteins, i.e. almost exclusively to albumin (>95%) and cannot be eliminated by standard haemodialysis [12]. The pre-dialysis total pentosidine levels were found to be lower in HD patients dialysed with high-flux polysulfone membranes than those treated with classical cellulose membranes [29].
In our study, we have investigated whether a new biocompatible polysulfone membrane, the so-called Superflux F800S (SF) (Fresenius Medical Care, Germany) enabling increased convectional forces and internal filtration through a modified geometry of the fibre and increased permeability, is able to remove significant amounts of AGEs in comparison with another high-flux membrane (HF) (F60S, Fresenius Medical Care, Germany) and with non-biocompatible, low-flux membranes consisting of polycarbonate or cellulose, respectively.
Our results demonstrate that in dialysis patients serum protein-bound pentosidine levels, representing their albumin-linked form, can be lowered by means of polysulfone HF membranes, with the highest reduction obtained using the SF. The similarity of the pre- and post-dialysis levels suggests that pentosidine was not cleared by all the membranes used in this study. As cited elsewhere, it may be possible that the formation of pentosidine-modified proteins is lower when using biocompatible polysulfone membranes, especially the SF dialyser [28]. On the other hand, the SF is characterized by the highest reduction rate of free pentosidine, of AGE peptides with a molecular weight <30 kDa and of AGE-ß2-microglobulin as compared with the HF and LF dialysers. Moreover, free pentosidine levels correlate closely with those of protein-bound pentosidine. These observations may indicate possible links between the removal of low-molecular-weight AGEs during dialysis and the decreased protein-bound pentosidine levels resulting from treatment with biocompatible membranes. This is supported by our finding that the pre-dialysis levels of free pentosidine and AGE peptides decreased significantly in the period of dialysis with the SF dialyser, suggesting a higher removal rather than the formation of new AGE products. Furthermore, the high reduction rate of free pentosidine and AGE peptides may reflect that not only these AGE structures but also other low-molecular-weight AGEs as well as reactive carbonyl compounds, as a source of protein modification, are more efficiently eliminated by the SF than by the other membranes.
Interestingly, contrary to the pre-dialysis protein-bound pentosidine and free pentosidine levels, AGE peptide concentrations were significantly higher in the diabetic group. This difference may indicate a possible influence of hyperglycaemia on the generation of CML-modified peptides. This is supported by the findings of Henle, who found the pre-dialysis plasma concentrations of the Amadori product fructoselysine, a major precursor of CML, significantly higher in diabetic than in non-diabetic patients [30].
In agreement with recent data, the AGE-ß2-microglobulin and ß2-microglobulin have been effectively removed by the HF membranes in each dialysis session for both patient groups studied, with an advantage of the SF [31].
To the best of our knowledge, no data on serum AGE-ß2-microglobulin and serum ß2-microglobulin levels in haemodialysis patients measured simultaneously have been reported yet. Treatment with both polysulfone HF membranes was associated with lower pre- and post-dialysis ß2-microglobulin levels. For AGE-ß2-microglobulin, such changes could only be observed in the application period of the SF dialyser.
In conclusion, treatment with the SF dialyser seems to be advantageous for the removal of free pentosidine, AGE-modified low-molecular-weight proteins and peptides. This observation indicates that this biocompatible polysulfone membrane may be superior to LF membranes.
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Acknowledgments |
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Notes |
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References |
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