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NDT Advance Access originally published online on February 13, 2006
Nephrology Dialysis Transplantation 2006 21(6):1555-1563; doi:10.1093/ndt/gfl007
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Clinical Nephrology

Respective role of uraemic toxins and myeloperoxidase in the uraemic state

Chantal Capeillère-Blandin1, Valérie Gausson, Anh Thu Nguyen, Béatrice Descamps-Latscha, Tilman Drüeke and Véronique Witko-Sarsat

INSERM U507, Université Paris 5, Necker Hospital, Paris 75015 and 1 Laboratoire de Chimie et Biochimie pharmacologiques et toxicologiques, CNRS UMR 8601, Université Paris 5, 45 rue des Saints Pères, 75270 Paris Cedex 06, France

Correspondence and offprint requests to: Dr Chantal Capeillère-Blandin, Université Paris 5, CNRS UMR 8601, 45 rue des Saints Pères, 75270 Paris Cedex 06, France. Email: Chantal.Capeillere-Blandin{at}univ-paris5.fr



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. In haemodialysis (HD) patients, advanced oxidation protein products (AOPP) were previously ascribed to oxidized plasma proteins, resulting mainly from increased myeloperoxidase (MPO) activity. The aim of the present study was to assess the mechanisms leading to the generation of AOPP during the course of chronic kidney disease including end-stage renal disease, with particular focus on AOPP and MPO characterization in the plasma at decreasing levels of kidney function.

Methods. Phagocyte activation was evaluated by whole blood NADPH oxidase and MPO activities. In plasma, MPO protein concentration was quantified by ELISA and catalytic activity assayed by the spectrophotometric detection of phenol and 4-aminoantipyrine (AAP) co-oxidation in the presence of hydrogen peroxide (H2O2).

Results. In HD patients, plasma AOPP concentration was linked to neutrophil oxidative activity. Such an association was not found in control subjects or predialysis patients, suggesting that in the latter, AOPP generation did not mainly result from MPO released by activated neutrophils. Similarly, plasma AOPP correlated with plasma MPO protein concentration in HD patients, but not in control subjects or predialysis patients, suggesting that in the latter AOPP did not predominantly result from MPO activity. This interpretation was supported by the observation of a greater degree of co-oxidation of phenol and AAP in the absence of H2O2 in predialysis patients than in HD patients or control subjects. The contribution of MPO dramatically differed between predialysis and HD patients (2±5 vs 46±6%; P<0.001).

Conclusion. Our observations suggest that AOPP generation in predialysis patients mainly results from MPO-independent oxidation mechanisms.

Keywords: AOPP; chronic kidney disease; haemodialysis; myeloperoxidase; oxidative stress; plasma proteins



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
In the course of chronic kidney disease (CKD), a chronic inflammatory state develops which is characterized by oxidative stress and sustained monocyte activation, as part of a complex picture of immune dysregulation [1–4]. Although a myriad of compounds which accumulate in CKD may act as potential ‘uraemic toxins’, none of these has been shown to be specifically at the origin of the observed immune disturbances and oxidative stress associated with the uraemic state [5,6].

Uraemia represents a unique clinical condition in which immune dysregulation appears to be closely associated with phagocyte-derived oxidative stress. The latter is dramatically exacerbated by haemodialysis (HD) treatment [4,7]. This aggravation is commonly attributed to the recurrent activation of neutrophils during blood passage through the dialysis circuits and subsequent generation of activated complement components, following the contact with bioincompatible membranes and/or the retrodiffusion of endotoxins from the dialysate [8,9]. Activated neutrophils generate a cascade of highly reactive oxygen species resulting from NADPH oxidase complex activity [10]. In addition, phagocytic cells contain the haem enzyme myeloperoxidase (MPO) which catalyses the reaction of chloride ion with hydrogen peroxide (H2O2) to generate large amounts of hypochlorous acid (HOCl), a powerful oxidizing and microbicidal agent. The deleterious effect of HOCl is directed not only at microorganisms [11] but also at bystander host cells during inflammation [12–14]. Activated monocytes are mainly responsible for the production of potent proinflammatory cytokines, including IL-1ß, TNF-{alpha} and IL-6 [3,15,16], which, together with oxidants and proteases, largely contribute to the excess of morbidity (ß2-microglobulin amyloidosis [17,18], accelerated atherosclerosis [19,20]) and mortality observed in dialysis patients.

In previous studies, we identified in the plasma of chronic HD patients the presence of oxidized proteins, designated as advanced oxidation protein products (AOPP) by analogy with the well-characterized advanced glycation end products (AGE) to which AOPP appeared to be closely related [21]. A new chromogen was identified previously, which caused increased absorbance at 340 nm, and its spectrophotometric determination was proposed as a novel index of oxidative stress measuring the level of AOPP [21]. In contrast to control plasma, direct spectrophotometric recordings of the absorbance of plasma from HD patients showed a significant absorbance band in the range 380–300 nm, with its intensity being correlated with the AOPP index [22]. It was verified that this absorbance band was not modified by pH variations in the range from 1.9 to 9.7.

AOPP is not only a marker of CKD progression but also a mediator of phagocyte activation. In HD patients, plasma AOPP levels were correlated with markers of monocyte activation such as neopterin, TNF-{alpha} and its soluble receptors [3]. In vitro, human serum albumin (HSA) treated with HOCl was capable of activating monocytes and neutrophils and triggering respiratory burst [3]. Moreover, biochemical analyses showed that plasma AOPP were cross-linked oxidized proteins, mostly albumin, whose concentration was related to plasma carbonyl groups and dityrosine, but not nitrotyrosine. Finally, plasma AOPP are associated with oxidized HSA and mainly dependent on MPO chlorination activity [22].

In fact, plasma AOPP proved to be a reliable and relevant marker for the monitoring of various pathological conditions involving oxidative stress. In addition to uraemia, plasma levels of AOPP were elevated in patients with coronary artery disease [23], in pre-term neonates [24,25], in patients with diabetes [26,27] and in patients with systemic sclerosis [28]. A significant positive association was even observed between plasma levels of AOPP and thiobarbituric acid reactive substances (TBARS) under conditions of oxidative stress [29].

The aim of the present study was to determine: (i) to what extent MPO contributed to the generation of AOPP in predialysis patients and (ii) whether the underlying mechanisms resulting in AOPP generation in vivo in these patients were the same as those observed in HD patients.



   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
In this study, 184 non-diabetic CKD patients were enrolled after giving informed consent. Clinical characteristics of predialysis patients (n = 115) were stratified based on the level of kidney failure according to CKD stages: (CKD stage 2: Ccr ≥60 ml/min; CKD stage 3: 30<Ccr<59 ml/min; CKD stage 4: Ccr<30 ml/min) (Table 1). Among HD patients, 53 were dialysed with modified cellulose membranes (Baxter Laboratories, Chicago IL, USA) (n = 53) and 16 with polyacrylonitrile AN69 (Hospal, Lyon, France) membranes. The dialysate was of standard composition and the bicarbonate buffer was used in all patients. Patients suffering from diabetes mellitus, systemic lupus erythematosus, malignant tumours, acute infection or receiving immunosuppressive therapy at the time of blood sampling were excluded from the study. Control subjects consisted of 53 healthy volunteers recruited among blood donors of the local blood transfusion centre.


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  		Table 1. Age, sex, creatinine clearance, plasma AOPP levels and primary kidney disease in predialysis patients at various CKD stages and HD patients

 
Blood (5–10 ml in the presence of 5 mM EDTA) was taken from predialysis patients at the time of routine laboratory investigations. Creatinine clearance (Ccr) was estimated using the Cockroft–Gault formula. In HD patients, blood was drawn just before the start of a dialysis session. Following centrifugation (600 g x 10 min) the plasma was stored in 500 µl aliquots at –70°C until use. Assays were carried out on duplicate samples thawed once.

Determination of AOPP
AOPP were determined in the plasma using the semi-automated method previously devised in our laboratory [3] which was recently shown to be as reliable as the method based on a conventional spectrophotometer with a 1 cm light-path cuvette [22]. Briefly, AOPP are measured by spectrophotometry at 340 nm on a microplate reader (Model MR 5000, Dynatech, Paris, France) under acidic conditions. The absorbance measurement has to be performed just after the addition of acetic acid (<5 min). The AOPP formation is calibrated by the formation of tri-iodide ion upon oxidation of potassium iodide with chloramine-T (Sigma Chemical Co., St Louis, MO) [30]. The tri-iodide ion absorbance at 340 nm gives a linear calibration curve within the range of 0–100 µmol/l of chloramine-T. AOPP concentrations are expressed in micromoles per litre of chloramine-T equivalents.

Measurement of neutrophil NADPH oxidase and MPO activities in whole blood
Basal phagocyte NADPH oxidase activity was measured by the luminescence of the product of reaction of lucigenin with Formula and basal phagocyte MPO activity was measured by the luminescence of the product of reaction of luminol with HOCl as previously described [31]. Briefly, EDTA-anticoagulated whole blood was tested within 1 h of collection. Basal NADPH oxidase and MPO peroxidase activities were measured through the luminometer injector (Autolumat LB953, EG&G Berthold, Wildbad, Germany) following addition of 0.1 ml of diluted blood (1 µl whole blood equivalent) into tubes containing 0.5 ml of 0.2 mM lucigenin or 0.15 mM luminol, respectively. Luminescence activities were measured at 37°C in duplicate over a 20 min interval normalized with respect to neutrophil count per microlitre of blood.

Determination of plasma MPO by ELISA
Measurement of immunoreactive MPO was performed as previously described [31]. MPO used as standard was purified (purity index RZ ≥ 0.7) according to a previously reported method [32]. Briefly, 96-well plates were coated with the anti-MPO polyclonal antibody (Calbiochem, La Jolla, CA) diluted 1:500 in carbonate buffer, pH 9.6. After saturation with 1% borine serum albumin in a phosphate buffer saline, various dilutions of plasma samples (1:100–1:600) or MPO standard solutions ranging from 0 to 100 ng/ml were added and incubated for 1 h at 37°C. The second antibody was the biotinylated anti-MPO polyclonal antibody diluted 1:1000. The final detection system was an alkaline phosphatase–streptavidin complex (Amersham, Buckinghamshire, UK) using p-nitrophenyl phosphate (Sigma) as substrate and making measurements at OD 405 nm.

Determination of plasma MPO activity by formation of quinoneimine
MPO activity was measured spectrophotometrically in 96-well microtitre plates, by a peroxidase-coupled assay system involving phenol, 4-aminoantipyrine (AAP) and H2O2 as recommended in [33]. Briefly, 130 µl of 2.5 mM AAP (Sigma) and 20 mM phenol were placed in each well, followed by 150 µl of 1.7 mM H2O2. Ten microlitres of plasma or MPO standard solution ranging from 0 to 3 µg/ml were then added. In the presence of H2O2 as oxidizing agent, MPO catalysed the oxidative coupling of phenol and AAP yielding a coloured product, quinoneimine, with a maximum absorbance at 500 nm, according to the following overall reaction:

Formula

The formation of quinoneimine was read 30 min later at 492 nm in a microplate reader. The absorbance was stable between 15 and 60 min. The comparison of these absorbance readings with data obtained from assays performed in spectrometer cuvettes of 1 cm optical path led to the determination of a microtitre plate pathlength of 0.85 cm. The millimolar absorbance coefficient for the quinoneimine was determined to be {Sigma} = 14±0.1/mM–1 cm–1 (n = 2), close to the previously reported values [34]. The results were expressed in micromolar of quinoneimine produced at 30 min.

Statistical analysis
Statistical analysis was performed using Statistica® software package (Statsoft Tulsa, OK, USA). Data are expressed as mean±standard error of the mean (SEM). Comparisons were made by analysis of variance (ANOVA) or Student's t-test, paired or unpaired, and the chi-square or Fisher's exact test and, in the case of multiple comparisons, P-values obtained were multiplied by the number of comparisons made. Simple regression analysis and Pearson's r correlation coefficient were used to determine the relationships between variables.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Circulating AOPP levels and blood neutrophil oxidative activities
Plasma AOPP were significantly higher in predialysis CKD patients (n = 115, 50.3±2.3 µM) than in control subjects (n = 28, 23.5±2.4 µM; P<0.0001) and culminated in HD patients (n = 69, 97.2±4.5 µM; P<0.0001). Among predialysis patients, it was significantly lower in those with CKD stage 2 (n = 18, 38±5 µM) than in those with CKD stage 4 (n = 36, 65±4 µM, P<0.0001) (Table 1). An inverse relationship was found by regression analysis between plasma AOPP (n = 115) and Ccr (r = 0.39, P<0.0001) (data not shown), thus confirming that plasma AOPP is a reliable marker of kidney disease, as previously shown in an independent study [3].

Neutrophil basal NADPH oxidase activity (as measured by lucigenin-amplified chemiluminescence, CL) was found within the range of controls, regardless of the stage of CKD in predialysis patients, but was significantly increased in HD patients (P<0.001, Figure 1A). Likewise, neutrophil MPO activity (as measured by luminol-amplified CL) remained within the normal range in predialysis patients, whatever the degree of CKD, but was markedly higher in HD patients (P<0.001) (Figure 1B). These results indicated that, in contrast to what was observed in HD patients, activated neutrophils were not activated in predialysis patients. The low basal activities that were observed in controls or in CKD may arise from neutrophil adherence to polystyrene tubes during the isolation process, thus triggering a minimal NADPH assembly and MPO mobilization. Besides, in HD patients basal chemiluminescence activities, either lucigenin or luminol, may result from in vivo activation.


Figure 1
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  		Fig. 1. Relationships between neutrophil oxidant activities and plasma AOPP concentrations. Neutrophil oxidant activities were measured in diluted blood using either lucigenin (panels on the left) or luminol (panels on the right) as a chemiluminigenic probe to assess NADPH oxidase or MPO activities, respectively. These measurements were performed in plasma from either controls or predialysis patients (stratified with the level of their kidney failure in CKD stage 2: Ccr≥60 ml/min; CKD stage 3: 30<Ccr<59 ml/min; CKD stage 4: Ccr<30 ml/min) or HD patients. Regression analyses were used as a best fit model to evaluate the relationships between AOPP levels and neutrophil oxidant activities, either NADPH oxidase (panels C and E) or MPO activities (panels D and F), respectively. The correlation coefficients (r) were significant at the indicated p value for HD patients (n = 40) in panels C and D and not significant for predialysis patients (n = 56) in panels E and F.

 
Plasma AOPP and neutrophil oxidative activity in HD patients (n = 40) were found to be directly related, as shown by a significant correlation between AOPP and NADPH oxidase (r = 0.39, P = 0.01) (Figure 1C), slightly higher than that between AOPP and MPO activity (r = 0.35, P = 0.03) (Figure 1D). In predialysis patients (n = 56), no significant correlation was found between AOPP and neutrophil oxidative activities (Figure 1E and F), suggesting that AOPP generation does not result from MPO released by activated neutrophils in these patients.

Circulating MPO protein levels
Plasma MPO protein concentration, measured by ELISA, did not significantly differ between predialysis and control subjects (15.3±1.1 ng/ml, n = 48 vs 13.3±0.5 ng/ml, n = 15, NS) nor among predialysis patients, between those with CKD stage 2 or CKD stage 4 (16.3±1.7 ng/ml, n = 7 vs 13.6±1.8 ng/ml, n = 17, NS). The latter is close to the previously reported serum MPO level of 0.2 nM (15 ng/ml) [12]. In contrast, MPO protein concentration was markedly increased in HD patients (39±4 ng/ml, n = 14, P<0.001), as compared to control subjects or predialysis patients (Figure 2A). Plasma MPO protein was correlated with plasma AOPP in HD patients (r = 0.57, n = 14, P = 0.03) (Figure 2B). No such correlation was found in predialysis patients (r = 0.10, n = 48, P = 0.47) or control subjects (r = 0.12, n = 15, P = 0.66), as illustrated in Figure 2C and D, respectively.


Figure 2
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  		Fig. 2. Influence of the progression of CKD and HD on MPO protein measured by ELISA. (A) Histograms showing the plasma MPO concentration (in ng/ml) measured by ELISA using a polyclonal MPO antibody as described in ‘Materials and Methods’ in uraemic patients stratified with the level of their kidney failure in CKD stage 2, CKD stage 3 and CKD stage 4 and in HD patients. (B–D) Regression analyses between AOPP concentrations and MPO protein values in HD (n = 14), predialysis patients (n = 48) and control subjects (n = 15), respectively. The correlation coefficients (r) were significant at the indicated P-value.

 
MPO activity measured by quinoneimine formation
As measured by quinoneimine concentrations after 30 min, plasma MPO catalytic activity was significantly high in predialysis patients (17.0±0.4 µM, n = 115). However, the highest activity was found in HD patients (22.0±0.9 µM, n = 69, P<0.001) (Figure 3). Among predialysis patients, it was lower in CKD stage 2 (16.0±0.4 µM, n = 65) than in those with more advanced stages of CKD (CKD stage 3, 19.0±0.8 µM, n = 30, P<0.001).


Figure 3
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  		Fig. 3. Influence of the progression of CKD and HD on MPO activity measured by co-oxidation of phenol–AAP. (A) Histograms showing the plasma MPO activity measured by the concentration of quinoneimine (in µM) produced by the coupling oxidation of phenol and AAP catalysed by MPO in the presence of H2O2 in uraemic patients stratified with the level of their kidney failure in CKD stage 2, CKD stage 3 and CKD stage 4 and in HD patients. Regression analyses were used as a best fit model to evaluate the relationships between AOPP levels and MPO activity values in HD patients (n = 69) (panel B) and in predialysis patients (n = 115) (panel C) the correlation coefficient (r) was statistically significant at the indicated P-value.

 
A positive correlation was found between plasma AOPP and quinoneimine formation in HD patients (r = 0.24, n = 69, P = 0.078) (Figure 3B). Surprisingly, the strongest correlation was found in predialysis patients (r = 0.45, n = 115, P<0.0001) (Figure 3C).

MPO-independent quinoneimine formation
To understand the discrepancy in CKD plasma between low immunoreactive MPO levels detected by ELISA and high MPO enzymatic activity levels measured by quinoneimine formation, we investigated whether the oxidative coupling of phenol and AAP resulted mainly from MPO activity in the presence of H2O2 or whether oxidative plasma components were directly involved in this oxidation. In order to differentiate between MPO-catalysed oxidation and chemical coupling oxidation, comparative assays were performed in the presence or absence of H2O2, respectively (Table 2). Lower levels of oxidation were found in control subjects and in HD patients in the absence of added H2O2 than in its presence. The oxidative coupling was performed by MPO present in the plasma of HD patients, and the remaining H2O2 present at a lower level than the one obtained by adding H2O2. In contrast, similar quinoneimine levels were found in predialysis patients, corroborating the hypothesis that in CKD patients, AOPP did not result mainly from MPO activity released by neutrophil activation.


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  		Table 2. Plasma redox activity determined by formation of quinoneimine

 


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
The major finding of the present study is the lack of association of plasma AOPP concentration with circulating MPO activity and circulating MPO protein concentration in predialysis patients, in contrast to the strong association found in HD patients. These observations suggest that in CKD patients, the generation of AOPP occurs mainly via an MPO-independent pathway involving other agents, such as uraemic toxins or active redox compounds.

MPO-dependent generation of AOPP in HD patients
Using a combined spectrophotometric and biochemical approach, we recently provided evidence that both in vitro generated AOPP and in vivo generated plasma AOPP result mainly from enhanced MPO activity [22]. The present study confirms this finding in that in HD patients, plasma AOPP concentration correlated with the activity of neutrophil MPO. It is important to stress that the increase in basal neutrophil MPO activity is not related to soluble plasma MPO but is due to intracellular MPO activity. Likewise, we have previously reported an increase in basal oxidase and MPO activities in isolated neutrophils from HD patients, in the absence of plasma components [15]. Moreover, plasma AOPP concentrations were related to both MPO protein concentration and MPO activity measured by the oxidative coupling of phenol and AAP in the presence of H2O2. These findings suggest that during the HD session, activated blood neutrophils release active MPO which oxidizes plasma proteins resulting in enhanced AOPP generation. Increased MPO activity would thus be part of the inflammatory reactions associated with the HD procedure as suggested previously [35].

Recent studies point to the importance of MPO in kidney disease [36,37]. MPO and HOCl-modified proteins were detected in the glomerular basement membrane of patients with membranous glomerulonephritis, suggesting a pathogenic role for MPO in situ, in glomerular changes [38]. The description of a polymorphism in the promoter of MPO at position –463 (G–A), linked with differences in MPO expression [39,40], was followed by numerous reports which implicated MPO in a variety of biological events unrelated to host defense. In particular, MPO polymorphism was shown to influence the course of MPO-ANCA vasculitis in women [41] and the G/G genotype was shown to be associated with the risk of cardiovascular disease in uraemic patients [42], confirming previous studies which showed that high plasma levels of AOPP were associated with coronary and carotid artery disease in patients with or without CKD [20,23,43].

In fact, plasma AOPP proved to be a relevant marker for the monitoring of various pathological conditions involving oxidative stress, including diabetes [27], preterm neonates [25] and dendritic cell injury [44]. This strongly supports the view that AOPP is a clinically relevant parameter. However, before considering as reliable for all clinical disease states plasma AOPP determination, it remains to be checked whether marked dyslipidaemia or haemolysis interfere with its measurement.

MPO-independent generation of AOPP in CKD
In the predialysis phase of CKD, the main mechanism of AOPP generation clearly appeared to differ from the one acting in HD patients. The activity of neutrophil MPO was not increased as compared with control. In addition, there was no significant correlation between plasma AOPP levels and either the enzymatic activity of neutrophil MPO or the amount of MPO protein. These findings are consistent with the interpretation that AOPP does not directly result from MPO activity associated with neutrophil activation. However, the close correlation between AOPP and quinoneimine levels suggested that, in CKD patients, plasma proteins could be directly oxidized by an MPO-independent mechanism. These findings suggest for the first time that uraemia-related factors per se are able to induce or potentiate the oxidation of plasma proteins.

The use of the oxidative coupling of phenol–AAP allows one to differentiate enzymatic oxidation from chemical oxidation. Peroxidases catalyse one-electron oxidation of organic substances to free radicals which are known to participate in a variety of non-enzymatic reactions acting as diffusible oxidants to oxidize secondary molecules [45,46]. The phenoxyl radical P-O• produced mediates the oxidation of AAP to the radical cation AAP+• and their oxidative coupling leads to the chromophoric compound quinoneimine.

In the plasma of predialysis patients which is devoid of peroxidase, the oxidation level measured by quinoneimine formation remained constant irrespective of both H2O2 and MPO presence, suggesting mediation by other oxidative plasma components. Indeed, the formation of a coloured product by co-oxidation of phenol and AAP in the presence of a chemical oxidizing reagent is well documented [47,48]. Exogenous compounds, such as uraemic toxins or active redox compounds, could mediate the oxidative coupling of phenol and AAP, leading to quinoneimine formation, independently of MPO enzymatic activity.

Interestingly, plasma aminothiols including homocysteine, cysteine and glutathione, which have been found to be excessively oxidized in uraemia [49] could constitute good candidates to perform redox mediation of aminopyrine and phenol, and thus stimulate the spectrophotometric detection of quinoneimine. Consistent with this hypothesis, the HD procedure was shown to restore the redox status of the aminothiols [50] and, in the present study, to decrease the apparent MPO activity measured in the absence of added H2O2, which was lower in HD than in predialysis patients. One possible explanation is the elimination during dialysis of chemical reagents having the ability to co-oxidize phenol and AAP. Another possibility implies an in vivo generation of H2O2 followed by MPO catalysis, since the enzyme is still present in HD plasma. Thus, the present assay based on the oxidative coupling of phenol–AAP is an objective measurement of the plasma redox status of predialysis patients, independently of MPO catalysis.

Our study points to the potential importance of uraemic toxins in the generation of AOPP and in the activation of monocytes in CKD patients. It may be hypothesized that uraemic toxins are the initial stimulus for the generation of AOPP, which promote the monocyte-driven inflammatory process, as evidenced by the close correlation between AOPP and neopterin, a marker of monocyte activation [3]. Notably, we recently reported that high AOPP levels were predictive of progression in IgA nephropathy [51]. The close correlation between AGE and AOPP in predialysis patients strongly suggests that uraemic toxins potentiate both AOPP and AGE formation in CKD.

In conclusion, the recent data show that during the course of CKD, the oxidative coupling of phenol and AAP probably results from exogenous compounds associated with uraemic toxicity. They further demonstrate that the oxidant burden gradually increases with kidney failure, in the absence of MPO released from activated neutrophils. In contrast, in HD patients, both MPO activity and concentration are increased as compared with control subjects, almost certainly because of a massive liberation of MPO from activated neutrophils triggered by the HD procedure. Interestingly, the exogenous oxidants responsible for MPO-independent phenol–AAP co-oxidation appear to be removed by the dialysis procedure.



   Acknowledgments
 
This work was supported by the Baxter Extramural Grant Program (V.W.-S.) and a grant from AMGEN. The authors are indebted to Dr Malik Touam and Prof. Paul Jungers for recruitment of patients and follow-up and thank Mrs Nadya Mothu for valuable laboratory assistance.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 20. 9.05
Accepted in revised form: 10. 1.06


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