Myeloperoxidase promoter polymorphism -463G is associated with more severe clinical expression of cystic fibrosis pulmonary disease.

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.

Changes in glycation and oxidation markers in patients starting peritoneal dialysis: a pilot study.

BACKGROUND: The high incidence of cardiovascular disease in uremic patients makes it a major cause of morbidity and mortality in those patients. Uremia is associated with carbonyl and oxidative stress, which result in the enhanced formation of glycation and oxidation products respectively. In the present study, the blood levels of advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs) were investigated in uremic patients prior to and after initiation of peritoneal dialysis (PD). METHODS: 22 patients [11 nondiabetic (G1) and 11 diabetic (G2) subjects] were enrolled in a single-center prospective study. Prior to starting PD (TO) and 6 and 12 months later, changes in AGE and AOPP levels were analyzed in the total study population and in each group (Friedman test, intragroup). At each time point, a comparison was made between the levels of the above-mentioned products in G1 and G2 (Mann-Whitney test, intergroup). Correlations between AGE or AOPP levels and residual renal function, peritoneal creatinine clearance, glucose peritoneal equilibration test, or daily dextrose exposure were analyzed using the Pearson test. RESULTS: At TO, no significant difference was found between the two groups for AGE or AOPP levels. Initiation of PD was followed by an increase in AGE levels in all patients (p < 0.01 at 6 and 12 months). AGE Levels were higher in G2 than in G1 at 12 months after the start of PD (p < 0.05). In contrast to G2 results, initiation of PD in G1 led to reduced AOPP Levels (at 6 and 12 months, p = 0.01 and p < 0.05 respectively). However, no correlation between AGE or AOPP levels and residual renal function, peritoneal creatinine clearance, glucose peritoneal equilibration test, or daily dextrose exposure could be established. CONCLUSION: This study demonstrates that PD is associated with an increase in levels of blood glycation end products, particularly in diabetic patients, but also with a decrease in oxidative products such as AOPPs, especially in nondiabetic subjects.

Respective role of uraemic toxins and myeloperoxidase in the uraemic state.

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 (H(2)O(2)). 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 H(2)O(2) 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.

The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.

BACKGROUND: Cardiovascular disease is the most frequent cause of mortality in chronic renal failure (CRF). Therefore, it is important to identify appropriate treatment measures. The antioxidant N-acetylcysteine (NAC) has been shown to reduce cardiovascular events in hemodialysis patients. Here we examine a possible direct effect of NAC supplementation on uremia-enhanced atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS: Uremia was induced surgically in 8-week-old female apoE(-/-) mice. Two weeks after creation of CRF mice were randomized to receive either NAC (daily oral gavage with 200 mg/kg for 8 weeks) or placebo. They were compared to a control group of sham-operated apoE(-/-) mice receiving placebo. After 8 weeks of treatment, the mice were sacrificed, and the cross-section surface area of atherosclerotic plaques was measured in aortic root and descending aorta. RESULTS: At 10 weeks following surgery, atherosclerotic lesions were significantly larger in uremic apoE(-/-) mice than in nonuremic controls. This accelerated atherosclerosis was associated with an increase in aortic nitrotyrosine expression and collagen plaque content. NAC treatment inhibited the progression of atherosclerotic lesions and plaque collagen content compared with placebo treatment. In addition, plaques from NAC-treated uremic animals showed a significant decrease in nitrotyrosine expression whereas the degree of macrophage infiltration was comparable in both uremic groups. There was no difference in mean arterial blood pressure between the three groups. CONCLUSION: We show for the first time that the antioxidant NAC is capable of reducing atheroma progression, in an animal model of uremia-enhanced atherosclerosis, probably via a decrease in oxidative stress.

Advanced oxidation protein products as risk factors for atherosclerotic cardiovascular events in nondiabetic predialysis patients.

BACKGROUND: Inflammation and oxidative stress are established risk factors for atherosclerosis, but whether they contribute to the accelerated atherogenesis associated with chronic kidney disease (CKD) remains to be assessed at the predialysis stage. METHODS: We prospectively examined the relationship between plasma levels of C-reactive protein (CRP), fibrinogen, and advanced oxidation protein products (AOPPs), as selected markers of inflammation and oxidative stress, and incident first occlusive atherosclerotic cardiovascular (CV) events (ASCVEs) in a single-center cohort of 80 uremic predialysis patients without diabetes with a creatinine clearance ranging from 20 to 40 mL/min/1.73 m2 . RESULTS: During follow-up (median, 7 years), 21 patients developed coronary, cerebral, or peripheral artery occlusive accidents, an incidence of 44/1,000 patient-years. Except for older age, their conventional risk factors did not differ compared with the 59 patients who remained free of such accidents. Conversely, plasma levels of CRP (4.3 +/- 2.7 versus 2.3 +/- 2 mg/L; P = 0.005), fibrinogen (5.6 +/- 1.4 versus 4.4 +/- 1.2 mg/L; P = 0.0009), and AOPPs (58 +/- 20 versus 42 +/- 14 micromol/L; P = 0.0002) were significantly greater at baseline, although serum creatinine levels did not differ between the 2 groups. By multivariate Cox regression analysis, age and CRP, fibrinogen, and AOPP levels were significant independent predictors of ASCVEs. Risk factor-adjusted hazard ratios were as follows: age, 1.13 (95% confidence interval, 1.04 to 1.22; P = 0.002); CRP level, 1.37 (95% confidence interval, 1.05 to 1.79; P = 0.02); fibrinogen level, 2.23 (95% confidence interval, 1.20 to 4.13; P = 0.011); and AOPP level, 1.68 (95% confidence interval, 1.12 to 2.51; P = 0.011). CONCLUSION: CRP, fibrinogen, and AOPP levels independently predict ASCVEs in patients with CKD in the predialysis phase and might directly contribute to the uremia-associated accelerated atherogenesis.

Early prediction of IgA nephropathy progression: proteinuria and AOPP are strong prognostic markers.

BACKGROUND: Inflammation and oxidative stress have been incriminated in the pathogenesis of IgA nephropathy (IgAN). The aim of the present study was to assess whether markers reflecting these pathophysiologic processes, namely C-reactive protein (CRP) and advanced oxidation protein products (AOPP), would allow-in conjunction with clinical and histopathologic parameters-to predict disease progression. METHODS: Between 1994 and 1997, 120 adult patients with biopsy-proven IgAN were included in a prospective cohort study, and followed until the end of 2002 or start of dialysis. In every patient, we determined plasma levels of CRP and AOPP. These parameters were included, together with clinical data, in a multivariate Cox proportional hazard regression analysis, with halving of baseline creatinine clearance as the primary renal end point. RESULTS: A total of 51 patients reached the renal end point, including 30 who had to start dialysis. With multivariate analysis, the most potent independent risk factors of poor renal outcome were proteinuria > or =1 g/day [proportional hazard risk (HR) = 23.7, P= 0.0001], hypertension (HR = 8.13, P= 0.008), and AOPP plasma level (HR = 1.09 per 10 micromol/L, P= 0.042), whereas angiotensin II inhibitors were protective (HR = 0.19, P= 0.001). CONCLUSION: Our data support the role of oxidative stress in the pathogenesis of IgAN and suggest that patients with proteinuria > or =1 g/day should be eligible for early implemented antioxidant and/or anti-inflammatory therapeutic strategies, with AOPP plasma level as a surrogate marker to evaluate their effects.

Biochemical and spectrophotometric significance of advanced oxidized protein products.

We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The present study was carried out to further investigate how myeloperoxidase (MPO)-catalyzed reactions could contribute to AOPP generation in the plasma. First, patterns of plasma protein oxidation obtained after in vitro incubation of control plasma with hypochlorous acid (HOCl) were compared to those from HD patients and control plasma. The use of various analytical techniques enabled localising and identifying the main oxidized proteins with albumin (HSA) after protein separation by size-exclusion chromatography and SDS-PAGE electrophoresis. The characterization of the oxidation level of the individual plasma proteins in terms of carbonyl groups and 3-nitrotyrosine formations was performed by immunoblotting. Secondly, to highlight the significance of AOPP index monitored by spectrophotometry, spectra were established for plasma fractions from HD patients and compared to data for control plasma and HOCl-treated plasma. The corresponding absorbance difference spectra were matched with external standards such as dityrosine, nitrotyrosine and pentosidine and elaborated chromophoric probe models. Indeed, HSA was chlorinated by HOCl reagent or HOCl generated via the MPO/H(2)O(2)/Cl(-) system and was nitrated by tetranitromethane. Increased absorbances at the range of 340 nm were observed both with chlorinated and nitrated HSA. Finally, our results indicate that HOCl, and not NO(2)(*), generated via MPO activity, could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes.

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: a potential target for N-acetylcysteine treatment in dialysis patients.

UNLABELLED: AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target forN-acetylcysteine treatment in dialysis patients. BACKGROUND: Oxidative stress largely contributes to hemodialysis-associated lethal complications, thus explaining the urgent need of antioxidant-based therapeutic strategies in hemodialysis patients. We previously identified advanced oxidation protein products (AOPP) in the uremic plasma as exquisite markers of oxidative stress and potent mediators of monocyte activation. The present study was aimed at searching whether (1) AOPP can also trigger activation of polymorphonuclear neutrophils (PMN), and (2) whether AOPP-induced activation could be inhibited by N-acetylcysteine (NAC), a widely used compound which has been shown to prevent oxidative injury to kidney. METHODS: Both human serum albumin (HAS) AOPP (i.e., HOCl-modified HSA in vitro preparations and AOPP extracted from plasma of hemodialysis patients) were tested for their capacity to trigger phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent activities as measured by lucigenin- and luminol-amplified chemiluminescence (CL), respectively, as compared to receptor-dependent [opsonized zymosan or receptor-independent phorbol myristate acetate (PMA)]. The effect of PMN priming by platelet-activating factor (PAF), and the effect of NAC on normal monocyte and on normal or hemodialysis patient's (N = 16) PMN oxidative responses were compared. RESULTS: HSA-AOPP triggered in a HOCl dose-dependent manner both NADPH-oxidase- and MPO-dependent CL of PMN. This latter was further enhanced by PAF priming. Plasma-derived AOPP obtained from hemodialysis patients also triggered PMN respiratory burst. NAC significantly reduced HSA-AOPP-mediated responses of normal monocyte and of normal and uremic PMN but had no significant effect on opsonized zymosan- or PMA-induced CL responses. CONCLUSION: This dual potential of NAC to inhibit phagocyte oxidative responses induced by HSA-AOPP without affecting those mediated by compounds mimicking pathogens supports the proposal of a therapeutic trial with NAC aimed at reducing oxidative stress-related inflammation in hemodialysis patients.

Review on uremic toxins: classification, concentration, and interindividual variability.

BACKGROUND: The choice of the correct concentration of potential uremic toxins for in vitro, ex vivo, and in vivo experiments remains a major area of concern; errors at this level might result in incorrect decisions regarding therpeutic correction of uremia and related clinical complications. METHODS: An encyclopedic list of uremic retention solutes was composed, containing their mean normal concentration (CN), their highest mean/median uremic concentration (CU), their highest concentration ever reported in uremia (CMAX), and their molecular weight. A literature search of 857 publications on uremic toxicity resulted in the selection of data reported in 55 publications on 90 compounds, published between 1968 and 2002. RESULTS: For all compounds, CU and/or CMAX exceeded CN. Molecular weight was lower than 500 D for 68 compounds; of the remaining 22 middle molecules, 12 exceeded 12,000 D. CU ranged from 32.0 ng/L (methionine-enkephalin) up to 2.3 g/L (urea). CU in the ng/L range was found especially for the middle molecules (10/22; 45.5%), compared with 2/68 (2.9%) for a molecular weight <500 D (P < 0.002). Twenty-five solutes (27.8%) were protein bound. Most of them had a molecular weight <500 D except for leptin and retinol-binding protein. The ratio CU/CN, an index of the concentration range over which toxicity is exerted, exceeded 15 in the case of 20 compounds. The highest values were registered for several guanidines, protein-bound compounds, and middle molecules, to a large extent compounds with known toxicity. A ratio of CMAX/CU <4, pointing to a Gaussian distribution, was found for the majority of the compounds (74/90; 82%). For some compounds, however, this ratio largely exceeded 4 [e.g., for leptin (6.81) or indole-3-acetic acid (10.37)], pointing to other influencing factors than renal function, such as gender, genetic predisposition, proteolytic breakdown, posttranslation modification, general condition, or nutritional status. CONCLUSION: Concentrations of retention solutes in uremia vary over a broad range, from nanograms per liter to grams per liter. Low concentrations are found especially for the middle molecules. A substantial number of molecules are protein bound and/or middle molecules, and many of these exert toxicity and are characterized by a high range of toxic over normal concentration (CU/CN ratio). Hence, uremic retention is a complex problem that concerns many more solutes than the current markers of urea and creatinine alone. This list provides a basis for systematic analytic approaches to map the relative importance of the enlisted families of toxins.

Are advanced oxidation protein products potential uremic toxins?

Oxidative stress, defined as a disruption of the equilibrium between the generation of oxidants and the activity of anti-oxidant systems, plays a significant role in the development of the inflammatory syndrome associated with chronic renal failure and hemodialysis. In our recent work, the aim of which was to better characterize oxidative stress in dialysis patients, we described the presence of oxidized protein products, which we have termed advanced oxidation protein products (AOPP), in the plasma of dialysis patients and we proposed AOPP as new markers of oxidative stress and potential inflammatory mediators. AOPP represent an exquisite marker of phagocyte-derived oxidative stress, and their role in the pathophysiology of chronic renal failure and dialysis-related complications might be of great importance. Regarding the mechanisms of generation of AOPP, we pointed out the importance of myeloperoxidase and the subsequent generation of chlorinated oxidants, previously considered solely as microbicidal agents, in the formation of AOPP. Indeed, AOPP appear to act as true inflammatory mediators since they are able to trigger the oxidative burst and the synthesis of inflammatory cytokines in neutrophils, as well as in monocytes. Thus, it could be hypothesized that the AOPP, which arise from the reaction between chlorinated oxidants and plasma proteins, constitute new uremic toxins with pro-inflammatory effects.

Iron therapy, advanced oxidation protein products, and carotid artery intima-media thickness in end-stage renal disease.

BACKGROUND: Increased common carotid artery intima-media thickness (CCA-IMT) is a marker of early atherosclerosis. Low-grade inflammation is associated with the pathogenesis of atherosclerosis. Low-grade inflammation and increased CCA-IMT are observed in end-stage renal disease (ESRD). Oxidative stress is involved in uremia-related inflammation. Advanced oxidation protein products (AOPP) are markers of oxidant-mediated protein damage in ESRD. Intravenous iron given to patients on hemodialysis (HD) might induce oxidative stress. We investigated the relationships between AOPP, iron therapy, and CCA-IMT in stable HD patients. METHODS AND RESULTS: Plasma AOPP and blood chemistry, including iron status, were analyzed in a cohort of 79 ESRD patients on HD. Measurements of CCA-IMT and CCA diameter, as assessed by B-mode ultrasonography, were obtained in 60 patients. AOPP levels were elevated in ESRD patients, and in univariate (r=0.42, P<0.0001) and multivariate analyses (r=0.38, P<0.001), they correlated with serum ferritin and with the intravenous iron dose received during the 12 months preceding the study (ferritin, P<0001; AOPP, P<0.01). Univariate and multivariate analyses identified the AOPP concentration as being significantly associated with CCA-IMT (P=0.0197) and CCA wall-to-lumen ratio (r=0.560, P<0.0001). Independently of AOPP concentration, cumulative iron dose was positively related to CCA-IMT (P=0.015) in patients <60 years. CONCLUSION: In ESRD patients, CCA-IMT and CCA wall-to-lumen ratio were associated with plasma AOPP, serum ferritin, and the annual intravenous iron dose administered. These findings support the concept of a role of oxidative stress in the early atherosclerosis of ESRD patients, which may be increased by the usually recommended doses of intravenous iron.

Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease, potentiates cell proliferation.

In this study, we present evidence for the critical role of proteinase-3 (PR3) in the proliferation of myeloid cells via the proteolytic regulation of the cyclin-dependent kinase inhibitor p21(waf1). Expression of recombinant PR3 in rat (RBL) or human (HMC1) mast cell lines increased bromodeoxyuridine incorporation and CDK2 activity compared with RBL and HMC1 cells transfected with an enzymatically inactive PR3 mutant (PR3(S203A)) or with human neutrophil elastase. Western blot analysis of p21(waf1) showed an absence of detectable protein, despite normal levels of p21 mRNA. Ectopic overexpression of p21 restored normal levels of p21 in the RBL/PR3/p21 double transfectants and reverted the proliferative effect of PR3. Inhibition of the 26 S proteasome by lactacystin or of caspases by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not inhibit p21 proteolysis. p21 cleavage correlated with PR3 expression in HMC1 cells infected with recombinant adenoviral vector Ad/PR3. During in vitro studies, purified p21 was cleaved by PR3, resulting in a 10-kDa p21 fragment. Employing double immunofluorescence confocal microscopy, subcellular fractionation, and co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-alpha, which induces PR3 re-expression, we observed that p21 disappeared and was reversed by Pefabloc, a serine proteinase inhibitor. The physiopathological implications of the cleavage of p21 by PR3 have to be determined.

Immune system dysregulation in uremia: role of oxidative stress.

Immune system dysregulation in end-stage renal disease patients is a multifactorial process combining profound immunodeficiency with a state of cellular activation. While at the origin of the deficiency uremic toxins are thought to play a prominent role, the dialysis procedure is the main factor for the genesis of a recurrent cellular activation process leading to a chronic inflammation state dominated by oxidative stress and its related severe complications, e.g. beta(2)-microglobulin amyloid arthropathy and accelerated atherosclerosis. The recent identification of advanced oxidation protein products (AOPPs) in the plasma of uremic patients and the following demonstration that AOPPs act as both potential uremic toxins and proinflammatory mediators, have opened novel areas of research on these novel molecular bases of oxidative stress and on therapeutic strategies aimed at reducing its most deleterious effects in hemodialysis patients.