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*
Institut National de la Santé et de la Recherche Médicale, Unit 90 and Department of Nephrology, Necker Hospital, Paris, France;
University Hospitals, Cleveland, OH 44106;
Centre National de la Recherche Scientifique, Unité de Recherche Associée 400, Paris, France; and
§
Division of Immunology and Allergy, University Hospital, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF soluble receptors (TNF-sR55 and
TNF-sR75). To determine the mechanisms by which AOPP and AGE could be
directly involved in monocyte activation, AOPP-human serum albumin
(HSA) and AGE-HSA were produced in vitro by treating HSA with oxidants
or glucose, respectively. Spectroscopic analysis confirmed that
AOPP-HSA contains carbonyls and dityrosine. Both AOPP-HSA and AGE-HSA,
but not purified dityrosine, were capable of triggering the oxidative
burst of human monocytes in cultures. The AOPP-HSA-induced respiratory
burst was dependent on the chlorinated nature of the oxidant and on the
molar ratio HSA/HOCl. Collectively, these data first demonstrate that
AOPP act as a mediator of oxidative stress and monocyte respiratory
burst, which points to monocytes as both target and actor in the immune
dysregulation associated with chronic uremia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, by activated
monocytes. Recent studies suggest that both the oxidative stress
induced by ROS and the proinflammatory effects of these cytokines are
reinforced by profound defects in antioxidant (6, 7) and anticytokine
systems (3, 4) and largely contribute to ß2m amyloid
arthropathy (8, 9) and accelerated atherosclerosis (10, 11), which
remain leading causes of morbidity and mortality in dialysis patients
(12). The exquisite vulnerability of proteins to ROS is now well documented (13, 14, 15, 16). Oxidation of amino acid residues such as tyrosine, leading to the formation of dityrosine, protein aggregation, cross-linking, and fragmentation, is an example of ROS-mediated protein damage in vitro. In contrast, evidence for the presence of such oxidatively damaged proteins in vivo and their possible clinical significance was still lacking until recently (15, 17). Indeed, in the search for whether such protein oxidative damage could reflect the dialysis-associated oxidative stress, we were able to isolate and characterize dityrosine-containing protein cross-linking products in the plasma of dialysis patients, which we designated advanced oxidation protein products (AOPP) (17).
The contribution of uremia per se to the chronic inflammatory state has been suggested, and consistent evidence has been afforded that both monocyte activation and a defect in antioxidant systems occur early in the course of chronic renal failure and gradually increase with its progression to end-stage renal disease (4, 7). Interest has focused on the role of "uremic toxins" generated during the course of chronic renal failure, some of which having known effects on neutrophil and monocyte functions (18, 19). Among these, growing efforts are being devoted to the potential toxicity of advanced glycation end products (AGE) (19, 20, 21, 22, 23, 24).
The formation of AGEs has been widely documented during diabetes and aging and has been held to be responsible for tissue degradation. The presence of increased plasma levels of AGEs has been observed in dialysis patients, independently of diabetes, and it is likely that, like ROS, AGEs contribute to ß2m deposits (21, 22). Interestingly, the hypothesis that oxidative stress is implicated in AGE formation might also be relevant to the uremic toxicity syndrome (24, 25). More recently, we reported that the well-characterized AGE-pentosidine accumulates with progression of chronic renal failure in close relationship with neopterin, a well-characterized monocyte activation marker (26).
In the search for whether AOPP could act as mediators in the monocyte-mediated inflammatory process associated with chronic renal failure and participate in the monocyte activation state, we determined 1) in vivo, AOPP levels in the plasma of uremic patients with varying degrees of renal failure in relationship with immunologic markers; and 2) in vitro, the potency of AOPP to induce the monocyte respiratory burst.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One hundred and sixty-two nondialyzed chronic renal failure
patients at various stages of renal failure were enrolled in the study
after giving informed consent. They included mild renal failure defined
by creatinine clearance (Ccr) of 41 to 80 ml/min (n =
73), moderate renal failure with Ccr of 20 to 40 ml/min
(n = 53), and advanced renal failure with Ccr <20
ml/min (n = 36). Primary renal diseases are indicated
in Table I
. Patients suffering from
diabetes mellitus, systemic lupus erythematosus, malignant tumors, or
acute infection or receiving immunosuppressive therapy at the time of
blood sampling were excluded from the study. Controls consisted of 31
healthy adults recruited among blood donors from our blood transfusion
center.
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Venous blood (5–10 ml) was collected in standard sterile polystyrene vacuum tubes, with 5 mM EDTA. After centrifugation (600 x g for 10 min), the plasma was stored in 500-µl aliquots at -70°C until use. Assays were conducted on duplicate samples thawed once.
To investigate the effect of AOPP on monocyte respiratory burst, monocytes were isolated from blood (20 ml) of normal volunteers by a two-step procedure involving erythrocyte sedimentation on dextran followed by leukocyte-rich plasma layer sedimentation on Nycoprep (1.068 solution; Nycomed Pharma, Oslo, Norway) according to the supplier’s instructions.
Determination of AOPP
AOPP were determined in the plasma using the semiautomated method previously devised in our laboratory (17). Briefly, AOPP were measured by spectrophotometry on a microplate reader (model MR 5000, Dynatech, Paris, France) and were calibrated with chloramine-T (Sigma, St. Louis, MO) solutions that in the presence of potassium iodide absorb at 340 nm (27). In test wells, 200 µl of plasma diluted 1/5 in PBS was placed on a 96-well microtiter plate (Becton Dickinson Labware, Lincoln Park, NJ), and 20 µl of acetic acid was added. In standard wells, 10 µl of 1.16 M potassium iodide (Sigma) was added to 200 µl of chloramine-T solution (0–100 µmol/liter) followed by 20 µl of acetic acid. The absorbance of the reaction mixture is immediately read at 340 nm on the microplate reader against a blank containing 200 µl of PBS, 10 µl of potassium iodide, and 20 µl of acetic acid. The chloramine-T absorbance at 340 nm being linear within the range of 0 to 100 µmol/liter, AOPP concentrations were expressed as micromoles per liter of chloramine-T equivalents.
Spectroscopic analysis of AOPP-HSA and of dityrosine prepared in vitro
Human serum albumin (HSA; type V; Sigma) was exposed to HOCl (Fluka, Buchs, Switzerland) as described previously (17). Briefly, HOCl stock solution (100 mM) was freshly prepared in PBS, and the concentration was measured by spectrophotometry using a molar extinction coefficient of 350 M-1 cm-1 at 290 nm at pH 12. Various concentrations of oxidants of HOCl were added to HSA at the indicated HSA/HOCl molar ratio. The AOPP-HSA preparation was incubated for 30 min at room temperature and then dialyzed overnight against PBS and tested for AOPP content. AOPP-HSA were also prepared by exposing purified HSA (100 mg/ml) to chloramine-T or hydrogen peroxide (H2O2) at the indicated concentrations and was dialyzed overnight against PBS. Dityrosine synthesis was adapted from the Anderson method (28). Briefly, 270 mg of tyrosine was dissolved in 250 ml of 0.2 M borate buffer, pH 9.5, in the presence of 6 mM H2O2. The reaction was started by the addition of 6.3 mg of horseradish peroxidase and performed for 18 h at 37°C. Then the mixture was concentrated almost to dryness in a rotatory evaporator under vacuum at 35°C. The brown powder was suspended in water acidified by concentrated HCl. A precipitated was removed by filtration through a glass filter funnel (G4; Millipore, Bedford, MA). Further separation from unreacted L-tyrosine and impurities was performed by chromatography on a cation exchanger. The brown solution was then applied to a fibrous cellulose phosphate (Sigma; 50–100 µm) column (1.5, 25 cm) equilibrated with 0.2 N acetic acid. After washing, the elution was performed with 0.5 M NaCl in 0.2 N acetic acid. Fractions of 3 ml were collected and evaluated after dilution in 0.1 N NaOH spectrophotometrically at 280 and 315 nm. Fluorescence detection (lex = 320 nm, lem = 410 nm) was also monitored. Fractions exhibiting dityrosine fluorescence were pooled and concentrated by lyophilization. The concentrate was solubilized in distilled water, filtrated, then loaded on a Dowex 50W-X8 (Bio-Rad) column previously soaked in 1 M HCl. The column was extensively washed with water to remove all NaCl and acetic acid. The dityrosine was subsequently eluted with 2 M ammonium hydroxide. Fluorescent fractions were concentrated in a rotatory evaporator under vacuum. The final fraction was reprecipitated several times in methanol-ether and stored under ether. Finally, a slightly yellow dityrosine was obtained (yield, 12%) and characterized by UV spectra, fluorescence emission, and magnetic resonance spectroscopy (29, 30).
Spectroscopic analyses on HSA, AOPP-HSA, and purified dityrosine were performed on a Kontron SF 25 spectrophotometer; UV and visible spectra were recorded in PBS at pH 7.5 at room temperature. Absorption and emission spectra were recorded in denaturing 6 M urea buffer at pH 7.5. Protein-bound dityrosine production was assayed in plasma or HSA samples by fluorescence measurements after dilution of the sample in 20 mM phosphate buffer, pH 7.5, in the presence of 7 M urea (10). After a 30-min incubation, the fluorescence emission spectra of dityrosine was recorded from 550 to 350 nm following excitation at 320 nm using a Kontron SF 25 spectrophotometer (Kontron, Zurich, Switzerland) and was measured at its maximum at 410 nm. The assay was calibrated by means of external standardization using a calibration curve generated in the same urea medium with authentic dityrosine. Its concentration was monitored spectrophotometrically at 315 nm, E = 5 mM-1 cm-1 at pH 7.5 (29, 30).
Determination of carbonyl residues
Carbonyl residues were determined as previously described (31)
using dinitrophenylhydrazine. Briefly, samples were submitted to 10 mM
dinitrophenylhydrazine in 2.5 M HCl for 1 h, followed by
deproteinization with 20% TCA. The pellet was washed three times in
ethanol/ethyl acetate and solubilized in guanidine 6 M. The carbonyl
concentration was measured by spectrophotometry at an OD of 370 nm with
370 = 22 mM-1 cm-1. The
protein concentration was determined in parallel using OD at 280 nm in
reference to BSA.
AGE preparation and determination
The AGE-HSA used in vitro was prepared by incubating HSA (type V; Sigma; 50 mg/ml) with 500 mM glucose in PBS for 65 days at 37°C under sterile conditions. The AGE-pentosidine, as a marker of nonenzymatic glycation of proteins, was measured using a modification of the method described by Odetti et al. (32). Briefly, plasma proteins or AGE-HSA were precipitated on TCA. The pellets were hydrolyzed in 2 ml of 6 N HCl and dissolved in 250 µl 0.01 M heptafluorobutyric acid (Sigma). The equivalent of 4 mg of plasma protein was injected into an HPLC system (Waters Division of Millipore, Marlborough, MA). A 25- x 0.46-cm C18 Vydac type 218TP (10 µm) column was used (Separations Group, Hesperia, CA). HPLC was programmed with a linear gradient of 10 to 17% acetonitrile from 0 to 35 min. Pentosidine was eluted at approximately 30 min as monitored by fluorescence excitation at 335 nm and emission at 385 nm. Pentosidine prepared according to the method of Sell and Monnier (33) was used as standard, and results were given in picomoles per milligram of protein.
Measurement of thiobarbituric acid-reacting substances
Thiobarbituric-reacting substances, including malondialdehyde (MDA), were determined using a commercially available kit according to the supplier’s instructions (Sobioda, Grenoble, France). Briefly, tetraethoxypropane was used as standard; each molecule of tetraethoxypropane hydrolyzes to yield one molecule of MDA under assay conditions. One hundred microliters of plasma was mixed with a mixture of thiobarbituric and perchloric acid and boiled for 1 h, then butanol (Carlo Erba, Milan, Italy) was added. Tubes were vortexed and centrifuged to extract MDA. Fluorometric measurements (excitation at 532 nm and emission at 553 nm) were performed on supernatants, using a Kontron SFM 25.
Determination of glutathione peroxidase (GSH-Px) activity
The level of selenium-dependent plasma GSH-Px was determined according to the method of Anderson (34) and expressed as micromoles of NADPH oxidized per milliliter.
Measurement of cell activation markers, cytokines, and cytokine inhibitors
Commercially available kits were used for measuring plasma
levels of neopterin (monocyte activation marker; RIA, Behring
Diagnostic, Rueil-Malmaison, France), IL-1R antagonist (IL-1Ra; ELISA
Quantikine, R&D Systems, Minneapolis, MN), soluble CD23 (B cell
activation marker; ELISA, BioSource Europe, Fleurus Belgium), and
TNF- (ELISA, BioSource). Plasma levels of TNF soluble receptors,
TNF-sR55 and TNF-sR75, were determined as described previously (35)
with specific mAbs provided by Dr. H Gallati (F. Hoffmann-La Roche,
Basel, Switzerland). The plasma level of soluble CD25 (T cell
activation marker) was determined with a kit (EIA, Cobas Core, sIL-2R,
Hoffmann-La Roche) provided by M. E. Nobile (Roche, Neuilly,
France).
Measurement of monocyte respiratory burst
The capacity of AOPP-HSA or AGE-HSA to activate the monocyte respiratory burst was measured by chemiluminescence, using dimethylbiacridinium (lucigenin) as the chemoluminigenic substrate. In this system the reductive dioxygenation of lucigenin that yields luminescence is strictly NADPH-oxidase dependent (36). One hundred microliters of monocyte suspension (2 x 105/ml) was automatically injected into tubes containing 100 µl of HBSS (basal activity), the tested preparations at the indicated concentrations (native HSA, AOPP-HSA or AGE-HSA, or human AB serum-opsonized zymosan (2 x 109 yeast cell wall units/ml), or PMA (Sigma). Chemiluminescence production was measured in duplicate in a luminometer (LB953 Berthold, Wildbad, Germany), and luminescence intensity was expressed in counts per minute.
Statistical analysis
The data were analyzed using standard statistical methods (Statistica Software, Tulsa, OK). Differences between means were evaluated using Student’s paired or unpaired t test where appropriate or ANOVA for comparing more than two groups. Relationships between variables were tested using simple (Pearson’s r correlation coefficient) or multiple linear regression analysis as indicated. All values are reported as the mean ± SEM. Statistical significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mean plasma level of AOPP was significantly higher in chronic
renal failure patients than in healthy control subjects (52 ± 2
vs 29 ± 5 µmol/l; p < 0.001). AOPP
concentrations increased over a nearly threefold range from the
incipient to the advanced stage of chronic renal failure
(p < 0.001; Table II). An inverse relationship between AOPP
levels and creatinine clearance was seen (r = -0.46,
p < 0.001; Fig. 1).
However, no significant effect of the underlying nephropathy on
circulating levels of AOPP was identified (Table III).
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The mean plasma level of MDA, the lipid peroxidation product, was
significantly higher at the incipient stage of chronic uremia than in
controls, but remained stable during the progression of renal failure
(Table II
). Thus, no relationship was found between AOPP and MDA levels
(r = 0.16, p = NS). In contrast, the
plasma level of GSH-Px, a major antioxidant enzymatic system, decreased
significantly with the progression of renal failure, reaching half its
initial level at an advanced stage of chronic renal failure (Table II;
p < 0.001). An inverse relationship was found between
AOPP and GSH-Px levels (r = -0.34, p
< 0.001). However, this correlation was not significant after these
two parameters were adjusted for creatinine clearance
(r = 0.16, p = NS).
Both AOPP and AGE-pentosidine are closely related to the monocyte activation state in chronic renal failure patients
A study of the relationships among AOPP, AGEs, and cell activation
markers was performed in a representative group of 56 patients, equally
distributed across the range of chronic renal failure (Table IV). Plasma AGE-pentosidine levels
gradually increased over a fourfold range with progression from early
to advanced chronic renal failure (p < 0.001),
and a close relationship was observed between AOPP and AGE-pentosidine
levels (r = 0.52, p < 0.001; Fig. 2). This correlation remained significant
after these two parameters were adjusted for creatinine clearance
(r = 0.42, p < 0.01).
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). While no relationship was found between
plasma AOPP and soluble CD25 or soluble CD23 levels, a highly
significant correlation was observed between neopterin and AOPP
(r = 0.55, p < 0.0001) or
AGE-pentosidine levels (r = 0.84, p <
0.0001; Fig. 3). Both correlations
remained significant by multiple regression analysis after adjustment
of AOPP and AGE-pentosidine values for creatinine clearance
(r = 0.32, p = 0.007 and
r = 0.67, p < 0.0001,
respectively).
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IL-1ß was only rarely present in chronic renal failure patients,
mostly in those with advanced disease (data not shown). In contrast,
IL-1Ra plasma levels were higher in chronic renal failure patients than
in controls (p < 0.01; Table IV), and weak
correlations were observed between IL-1Ra and AOPP levels
(r = 0.30, p < 0.01) or
AGE-pentosidine (r = 0.26, p = 0.05);
these correlations were not significant by multiple regression analysis
when IL-1Ra and AOPP values were adjusted for creatinine clearance.
In all chronic renal failure patients TNF- was detected and
increased with deterioration of renal function (Table IV). Significant
correlations were observed between TNF- and AOPP (r
= 0.36, p = 0.004) or AGE-pentosidine levels
(r = 0.50, p = 0.0001; Fig. 3).
Likewise, both TNF-sR55 and TNFsR75 levels increased with the
progression of chronic renal failure (Table IV), and significant
correlations were observed with the levels of AOPP (r =
0.41, p = 0.002 and r = 0.55,
p = 0.0001, respectively) or AGE-pentosidine
(r = 0.46, p = 0.0004 and
r = 0.60, p = 0.0001, respectively;
Fig. 4). However, when all values were
adjusted for creatinine clearance, r correlation
coefficients remained significant only with TNF-sR75. As previous
studies, including ours (3, 4), have shown that the final determination
of a cytokine’s biologic activity is best reflected by the ratio of
the cytokine to its inhibitor, we determined the molar ratios
TNF-sR55/TNF- and TNF-sR75/TNF- and performed regression analysis
between these ratios and AOPP or pentosidine values. For AOPP, the
r correlation coefficients were 0.15
(p = 0.07) and 0.07 (p
= NS) with TNF-sR55/TNF- and TNF-sR75/TNF-, respectively, and for
pentosidine the r values were 0.06
(p = NS) and 0.18 (p =
NS), respectively.
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We first determined the optimal conditions for HSA oxidation as
assessed by AOPP, dityrosine, and carbonyl concentrations and with
respect to the nature and concentration of the oxidant. As shown in
Figure 5, treatment of HSA with HOCl or
chloramines induced a dose-dependent increase in AOPP, dityrosine, and
protein carbonyl concentrations, whereas such an increase was not
obtained with H2O2. The ineffectiveness of
H2O2 remained consistent at 1- or 10-M
concentrations (data not shown).
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. They illustrate
the relative absorbance contribution of native HSA and AOPP-HSA in the
200 to 450 nm range. Below 250 nm, the spectra showed typical
absorbance of the peptide bonds in both native and AOPP-HSA.
Significant differences occurred in the 250 to 300 nm region typical of
aromatic residues. In native HSA, this peak at 280 nm was sharp and
well individualized, whereas in AOPP-HSA, it was considerably altered,
demonstrating a modification in the content or characteristics of
tyrosine and/or tryptophan residues induced by chlorinated oxidants. In
addition, above 300 nm, the presence of an absorbance shoulder at 315
nm appeared to be a feature of HSA treated with chlorinated oxidants,
as shown in AOPP-HSA. Interestingly, in the same pH conditions,
purified dityrosine showed a peak at the same wavelength, at 315 nm. Of
note, no band was detectable in native HSA.
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B). Both purified dityrosine and AOPP-HSA presented the
same emission fluorescence with a maximum at 410 nm. Flat spectra were
observed for both HSA and control urea buffer. Excitation spectra
recorded at a fixed wavelength of 410 nm (Fig. 2B),
indicated an overlapping between dityrosine and AOPP-HSA within the 320
to 350 nm, whereas the contribution of native HSA was close to that of
the control urea buffer. One interesting feature in native HSA is the
presence of the peak typical of aromatic residues around 280 nm (as
previously mentioned for absorbance spectra), which is not visible in
either AOPP-HSA or dityrosine. Taken together, these spectral
characteristics clearly demonstrate the presence of dityrosine in
AOPP-HSA. AOPP-HSA trigger the monocyte respiratory burst
As shown in Figure 7, HOCl-induced
AOPP-HSA triggered a respiratory burst in isolated human monocytes, as
measured by lucigenin-amplified chemiluminescence. The
chemiluminescence production increased with the HSA/HOCl molar ratio,
reaching a maximum at 1:60. AOPP-HSA at a HSA/HOCl molar ratio of 1:120
induced a lower chemiluminescence production, and this was observed as
a significant increase in both AOPP and dityrosine and a moderate
increase in carbonyl concentrations compared with AOPP-HSA at a
HSA/HOCl molar ratio of 1:60. In contrast, no oxidative burst
was triggered by native HSA or by purified dityrosine at the
concentration range of 1 to 100 µM, which is the same as that found
in the optimal concentration of AOPP for inducing respiratory burst.
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).
Interestingly, this AGE-HSA preparation, adjusted to a 20 mg/ml protein
concentration, contained AOPP (55 µmol/l); this concentration was
similar to that found in AOPP-HSA obtained after HSA treatment with
HOCl using a 1:60 molar ratio. In contrast, the latter AOPP-HSA, which
induced the maximal chemiluminescence production, contained the same
amount of AGE-pentosidine as native HSA, thus ruling out the
contribution of AGEs in AOPP-induced monocyte respiratory burst.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, plasma levels of AOPP are significantly elevated in uremic patients compared with those in healthy individuals. The increase starts at an early stage of chronic renal failure and gradually rises with the progression of renal failure, as emphasized by the highly significant inverse relationship between plasma concentrations of AOPP and the glomerular filtration rate. Second, and more importantly, the accumulation of AOPP during the course of chronic renal failure is not only related to the decrease in kidney excretory function, but is also closely associated with several immunoinflammatory markers. However, whether the level of AOPP is related to the rate of progression of renal failure needs further investigation by a sequential follow-up of AOPP levels in patients with the same type of nephropathy but with distinct rates of decline in glomerular filtration.
The present finding that AOPP accumulation coexists with decreased GSH-Px level, while the plasma concentration of malondialdehyde remains stable, supports the contention that AOPP are more accurate markers of oxidative stress than lipid peroxidation products (17). The occurrence of oxidative stress in chronic renal failure patients whether on dialysis or not has been suggested in studies showing an imbalance between oxidant and antioxidant systems and their cofactors (6, 7). Recent reports have stressed the role of such an oxidative stress in the accelerated atherosclerosis process associated with end-stage renal disease (12).
To determine the role of AOPP in uremia-associated immune
dysregulation, we analyzed their relationships with cell activation
markers, with emphasis on monocytes as a potential source of oxidants
and proinflammatory cytokines. While no relationship was found between
AOPP and T or B cell activation markers, a close correlation was
observed between AOPP and neopterin, the monocyte activation marker.
This selective relationship between AOPP and monocyte activation was
further established with positive correlations between AOPP and TNF-
and its soluble receptors, and, to a lesser degree, with IL-1Ra,
although these correlations tended to be of only borderline
significance when values were corrected for creatinine clearance. These
latter findings suggested that the relationship was not related to
cytokine or cytokine inhibitor biologic activities, and this was
further evidenced by the absence of correlation between AOPP and the
TNF-sR55/TNF- or TNF75/TNF- molar ratios, which may better
reflect the biologic activity of TNF- (3, 4).
Of note, in another state of profound immune dysregulation, in HIV patients, we observed very high plasma levels of AOPP. In this clinical condition, characterized by a pronounced oxidative stress in the absence of renal failure, AOPP was an exquisite marker of oxidative stress correlating tightly with the degree of monocyte activation (37).
Another important aspect of the present study was to further investigate the relationship between AOPP and advanced glycosylation proteins as assessed by AGE-pentosidine. We and other investigators have previously documented that chronic renal failure is associated with increased AGE formation independent of diabetes or aging (24, 25, 26). In the present study, the close correlation observed between plasma AGE-pentosidine and AOPP demonstrates that this relationship already exists in uremic patients not yet undergoing dialysis and further suggests that AGE and AOPP may share common mechanisms of formation and/or common biologic activities in vivo. Moreover, several studies pointed to the involvement of oxidative pathways in the formation of AGE, notably in patients with end-stage renal failure (24, 38).
Our previous observations demonstrated that, in vivo, AOPP result from oxidant-induced protein cross-linking and correlate with dityrosine levels. To determine the mechanisms by which AOPP could be involved in monocyte activation, the potential proinflammatory effects of AOPP-HSA produced in vitro by exposing HSA to chlorinated oxidants were studied. As evidenced in the case of carbonyl and dityrosine levels in oxidant-treated HSA, chlorinated oxidants appear very efficient in inducing AOPP. Spectroscopy studies reveal that AOPP-HSA could be identified as follows: in UV-visible spectra, by perturbation of the aromatic residue band associated with a large band at 320 nm, the latter property being used for routine quantification of AOPP; in fluorescence spectra by the loss of tryptophan and tyrosine emission and the appearance of the dityrosine contribution clearly visualized in the excitation spectrum. Therefore, the spectral measurement of AOPP, which is maximum at 320 nm, is the result of an overlapping between chromophores, among which dityrosine is identified as a predominant component.
We then demonstrated that AOPP-HSA, but not purified dityrosine, possesses the ability to activate isolated monocytes in vitro, emphasizing three remarkable features of the induced respiratory burst: 1) its induction is dependent on the chlorinated nature of the oxidant, i.e., HOCl and chloramine, but not H2O2; 2) its intensity is directly related to the level of protein oxidation as defined by the molar ratio of protein to oxidant and ascertained by dityrosine and carbonyl measurements; and 3) it is related to the protein cross-linking structure, since purified dityrosine alone has no effect on monocytes.
In the past, studies on the interactions between proteins and oxidants have focused on the structural changes induced by oxidants generated by water pulse radiolysis, including superoxide and hydroxyl radicals (13, 14). Such studies have demonstrated that structural modifications of proteins (selective loss of an amino acid, fragmentation, or aggregation) were highly dependent on the nature of the oxidant. Since AOPP formation is optimal with chlorinated oxidants, it is interesting to speculate that its formation in vivo might result from enzymatic activity of phagocyte-derived MPO (39). Interestingly, although the best-characterized product of MPO is HOCl, MPO-derived enzymatic activity can also generate dityrosine (16), a compound that correlates with AOPP levels, and aldhehyde compounds, which may exert a potent biologic effect at the site of inflammation (40). MPO can also convert L-serine to glycolaldehyde, which mediates the formation of carboxymethylysine, described as a byproduct of protein glycation (41). The pathophysiologic relevance of MPO-derived chlorinated oxidant is also illustrated by its role in the formation of oxidatively modified low density lipoproteins (42).
Another important aspect of the present study was to further
investigate the relationship between AOPP and AGEs. Both AOPP-HSA and
AGE-HSA triggered monocyte NADPH oxidase activation, leading to
superoxide anion production. Of note, while AOPP-HSA did not contain
AGE-pentosidine, significant concentrations of AOPP were found in
AGE-HSA preparations. Taken together, these data suggest that
AGE-proteins contain some structural motif that results from an
oxidative process and support the hypothesis that AGE-mediated biologic
activities might depend on their level of oxidation (43). The
interaction between AGEs and macrophages is now well established.
Macrophages may internalize AGE-modified proteins via a specific
receptor, or RAGE (21, 44), which is also expressed by endothelial
cells (45). Interestingly, the macrophage RAGE can be up-regulated by
TNF- (21, 46). How macrophages process AOPP-HSA remains elusive, but
taking into account a possible structural resemblance between AGE and
AOPP, the involvement of a similar receptor-mediated process is
plausible.
In conclusion, AOPP may represent a novel class of proinflammatory mediators acting as a mediator of oxidative stress and monocyte respiratory burst. The monocyte is thus, at the same time, the elective cellular target of AOPP and a potential source of oxidants inducing AOPP.
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Acknowledgments |
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Footnotes |
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2 A portion of this work was presented at the 29th Annual Meeting of the American Society of Nephrology, New Orleans, LA.
3 Address correspondence and reprint requests to Dr. Béatrice Descamps-Latscha, Institut National de la Santé et de la Recherche Médicale, Unit 90, Hôpital Necker, 161 rue de Sèvres, 75015 Paris, France. E-mail address:
4 Abbreviations used in this paper: ROS, reactive oxygen species; AOPP, advanced oxidation protein products; AGE, advanced glycation end product; HSA, human serum albumin; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; IL-1Ra, interleukin-1 receptor antagonist.
Received for publication February 12, 1998. Accepted for publication April 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and their specific inhibitors in chronic renal failure and maintenance dialysis: relationships with activation markers of T cells, B cells, and monocytes. J. Immunol. 154:882.[Abstract]
and interleukin-1. J. Clin. Invest. 93:521.
-hydroxy and , ß-unsaturated aldehydes by phagocytes at sites of inflammation. J. Clin. Invest. 99:424.[Medline]
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 M. G.H. Betjes, W. Weimar, and N. H.R. Litjens CMV Seropositivity Determines Epoetin Dose and Hemoglobin Levels in Patients with CKD J. Am. Soc. Nephrol., December 1, 2009; 20(12): 2661 - 2666. [Abstract] [Full Text] [PDF]
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 Y.-F. Lin, V.-C. Wu, W.-J. Ko, Y.-S. Chen, Y.-M. Chen, W.-Y. Li, N.-K. Chou, A. Chao, T.-M. Huang, F.-C. Chang, et al. Residual Urine Output and Postoperative Mortality in Maintenance Hemodialysis Patients Am. J. Crit. Care., September 1, 2009; 18(5): 446 - 455. [Abstract] [Full Text] [PDF]
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 A. Servettaz, C. Goulvestre, N. Kavian, C. Nicco, P. Guilpain, C. Chereau, V. Vuiblet, L. Guillevin, L. Mouthon, B. Weill, et al. Selective Oxidation of DNA Topoisomerase 1 Induces Systemic Sclerosis in the Mouse J. Immunol., May 1, 2009; 182(9): 5855 - 5864. [Abstract] [Full Text] [PDF]
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 X. F. Wei, Q. G. Zhou, F. F. Hou, B. Y. Liu, and M. Liang Advanced oxidation protein products induce mesangial cell perturbation through PKC-dependent activation of NADPH oxidase Am J Physiol Renal Physiol, February 1, 2009; 296(2): F427 - F437. [Abstract] [Full Text] [PDF]
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 N. Rabbani and P. J. Thornalley QUANTITATION OF MARKERS OF PROTEIN DAMAGE BY GLYCATION, OXIDATION, AND NITRATION IN PERITONEAL DIALYSIS Perit. Dial. Int., February 1, 2009; 29(Supplement_2): S51 - S56. [Abstract] [Full Text] [PDF]
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Y. Iwao, K. Nakajou, R. Nagai, K. Kitamura, M. Anraku, T. Maruyama, and M. Otagiri CD36 is one of important receptors promoting renal tubular injury by advanced oxidation protein products Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1871 - F1880. [Abstract] [Full Text] [PDF]
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 K. Amann Media Calcification and Intima Calcification Are Distinct Entities in Chronic Kidney Disease Clin. J. Am. Soc. Nephrol., November 1, 2008; 3(6): 1599 - 1605. [Abstract] [Full Text] [PDF]
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R. Vanholder, U. Baurmeister, P. Brunet, G. Cohen, G. Glorieux, J. Jankowski, and for the European Uremic Toxin Work Group A Bench to Bedside View of Uremic Toxins J. Am. Soc. Nephrol., May 1, 2008; 19(5): 863 - 870. [Abstract] [Full Text] [PDF]
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 X. Y. Shi, F. F. Hou, H. X. Niu, G. B. Wang, D. Xie, Z. J. Guo, Z. M. Zhou, F. Yang, J. W. Tian, and X. Zhang Advanced Oxidation Protein Products Promote Inflammation in Diabetic Kidney through Activation of Renal Nicotinamide Adenine Dinucleotide Phosphate Oxidase Endocrinology, April 1, 2008; 149(4): 1829 - 1839. [Abstract] [Full Text] [PDF]
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 H.-Z. Pan, H. Zhang, D. Chang, H. Li, and H. Sui The change of oxidative stress products in diabetes mellitus and diabetic retinopathy Br J Ophthalmol, April 1, 2008; 92(4): 548 - 551. [Abstract] [Full Text] [PDF]
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 R. Vanholder, S. V. Laecke, F. Verbeke, G. Glorieux, and W. V. Biesen Uraemic toxins and cardiovascular disease: in vitro research versus clinical outcome studies NDT Plus, February 1, 2008; 1(1): 2 - 10. [Full Text] [PDF]
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 D. Simar, D. Malatesta, S. Badiou, A. M. Dupuy, and C. Caillaud Physical Activity Modulates Heat Shock Protein-72 Expression and Limits Oxidative Damage Accumulation in a Healthy Elderly Population Aged 60 90 Years J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2007; 62(12): 1413 - 1419. [Abstract] [Full Text] [PDF]
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 R. Barazzoni, A. Bernardi, F. Biasia, A. Semolic, A. Bosutti, M. Mucci, F. Dore, M. Zanetti, and G. Guarnieri Low fat adiponectin expression is associated with oxidative stress in nondiabetic humans with chronic kidney disease--impact on plasma adiponectin concentration Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R47 - R54. [Abstract] [Full Text] [PDF]
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A. Y.-M. Wang THE "HEART" OF PERITONEAL DIALYSIS Perit. Dial. Int., June 1, 2007; 27(Supplement_2): S228 - S232. [Abstract] [Full Text] [PDF]
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 G. Marsche, M. Semlitsch, A. Hammer, S. Frank, B. Weigle, N. Demling, K. Schmidt, W. Windischhofer, G. Waeg, W. Sattler, et al. Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway FASEB J, April 1, 2007; 21(4): 1145 - 1152. [Abstract] [Full Text] [PDF]
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 K. Sebekova, T. Eifert, A. Klassen, A. Heidland, and K. Amann Renal Effects of S18886 (Terutroban), a TP Receptor Antagonist, in an Experimental Model of Type 2 Diabetes Diabetes, April 1, 2007; 56(4): 968 - 974. [Abstract] [Full Text] [PDF]
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H. Y. Li, F. F. Hou, X. Zhang, P. Y. Chen, S. X. Liu, J. X. Feng, Z. Q. Liu, Y. X. Shan, G. B. Wang, Z. M. Zhou, et al. Advanced Oxidation Protein Products Accelerate Renal Fibrosis in a Remnant Kidney Model J. Am. Soc. Nephrol., February 1, 2007; 18(2): 528 - 538. [Abstract] [Full Text] [PDF]
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M. A. Verkade, C. J. van Druningen, L. M. B. Vaessen, D. A. Hesselink, W. Weimar, and M. G. H. Betjes Functional impairment of monocyte-derived dendritic cells in patients with severe chronic kidney disease Nephrol. Dial. Transplant., January 1, 2007; 22(1): 128 - 138. [Abstract] [Full Text] [PDF]
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 N.C. Edwards, R.P. Steeds, C.J. Ferro, and J.N. Townend The treatment of coronary artery disease in patients with chronic kidney disease QJM, November 1, 2006; 99(11): 723 - 736. [Abstract] [Full Text] [PDF]
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K. E. Jie, M. C. Verhaar, M.-J. M. Cramer, K. van der Putten, C. A. J. M. Gaillard, P. A. Doevendans, H. A. Koomans, J. A. Joles, and B. Braam Erythropoietin and the cardiorenal syndrome: cellular mechanisms on the cardiorenal connectors Am J Physiol Renal Physiol, November 1, 2006; 291(5): F932 - F944. [Abstract] [Full Text] [PDF]
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G. Zalba, A. Fortuno, and J. Diez Oxidative stress and atherosclerosis in early chronic kidney disease Nephrol. Dial. Transplant., October 1, 2006; 21(10): 2686 - 2690. [Full Text] [PDF]
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K. Amann, C. Wanner, and E. Ritz Cross-Talk between the Kidney and the Cardiovascular System J. Am. Soc. Nephrol., August 1, 2006; 17(8): 2112 - 2119. [Abstract] [Full Text] [PDF]
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C. Capeillere-Blandin, V. Gausson, A. T. Nguyen, B. Descamps-Latscha, T. Drueke, and V. Witko-Sarsat Respective role of uraemic toxins and myeloperoxidase in the uraemic state Nephrol. Dial. Transplant., June 1, 2006; 21(6): 1555 - 1563. [Abstract] [Full Text] [PDF]
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 S. X. Liu, F. F. Hou, Z. J. Guo, R. Nagai, W. R. Zhang, Z. Q. Liu, Z. M. Zhou, M. Zhou, D. Xie, G. B. Wang, et al. Advanced Oxidation Protein Products Accelerate Atherosclerosis Through Promoting Oxidative Stress and Inflammation Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 1156 - 1162. [Abstract] [Full Text] [PDF]
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R. A. Ward Protein-Leaking Membranes for Hemodialysis: A New Class of Membranes in Search of an Application? J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2421 - 2430. [Abstract] [Full Text] [PDF]
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 J. D Kopple The phenomenon of altered risk factor patterns or reverse epidemiology in persons with advanced chronic kidney failure Am. J. Clinical Nutrition, June 1, 2005; 81(6): 1257 - 1266. [Abstract] [Full Text] [PDF]
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M. Saemann, T Weichhart, M Zeyda, G Staffler, M Schunn, K. Stuhlmeier, Y Sobanov, T. Stulnig, S Akira, A von Gabain, et al. Immunoregulation in Urinary Tract Inflammation--A Role of Tamm-Horsfall Glycoprotein: Tamm-Horsfall Glycoprotein Links Innate Immune Cell Activation with Adaptive Immunity via a Toll-Like Receptor-4-Dependent Mechanism. J Clin Invest 115: 468-475, 2005 J. Am. Soc. Nephrol., April 1, 2005; 16(4): 829 - 836. [Full Text] [PDF]
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A. Y.-M. Wang, J. Woo, M. Wang, M. M.-M. Sea, J. E. Sanderson, S.-F. Lui, and P. K.-T. Li Important differentiation of factors that predict outcome in peritoneal dialysis patients with different degrees of residual renal function Nephrol. Dial. Transplant., February 1, 2005; 20(2): 396 - 403. [Abstract] [Full Text] [PDF]
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 L. G. Bongartz, M. J. Cramer, P. A. Doevendans, J. A. Joles, and B. Braam The severe cardiorenal syndrome: 'Guyton revisited' Eur. Heart J., January 1, 2005; 26(1): 11 - 17. [Abstract] [Full Text] [PDF]
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 A Heidland, K Sebekova, A Frangiosa, L S De Santo, M Cirillo, F Rossi, M Cotrufo, A Perna, A Klassen, R Schinzel, et al. Paradox of circulating advanced glycation end product concentrations in patients with congestive heart failure and after heart transplantation Heart, November 1, 2004; 90(11): 1269 - 1274. [Abstract] [Full Text] [PDF]
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 C. Yazici, K. Kose, M. Calis, S. Kuzuguden, and M. Kirnap Protein oxidation status in patients with ankylosing spondylitis Rheumatology, October 1, 2004; 43(10): 1235 - 1239. [Abstract] [Full Text] [PDF]
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J. Himmelfarb, E. McMonagle, S. Freedman, J. Klenzak, E. McMenamin, P. Le, L. B. Pupim, T. A. Ikizler, and The PICARD Group Oxidative Stress Is Increased in Critically Ill Patients with Acute Renal Failure J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2449 - 2456. [Abstract] [Full Text] [PDF]
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F. Santangelo, V. Witko-Sarsat, T. Drueke, and B. Descamps-Latscha Restoring glutathione as a therapeutic strategy in chronic kidney disease Nephrol. Dial. Transplant., August 1, 2004; 19(8): 1951 - 1955. [Full Text] [PDF]
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A. Y.-M. Wang, M. Wang, J. Woo, C. W.-K. Lam, S.-F. Lui, P. K.-T. Li, and J. E. Sanderson Inflammation, Residual Kidney Function, and Cardiac Hypertrophy Are Interrelated and Combine Adversely to Enhance Mortality and Cardiovascular Death Risk of Peritoneal Dialysis Patients J. Am. Soc. Nephrol., August 1, 2004; 15(8): 2186 - 2194. [Abstract] [Full Text] [PDF]
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F. F. Hou, H. Ren, W. F. Owen Jr, Z. J. Guo, P. Y. Chen, A. M. Schmidt, T. Miyata, and X. Zhang Enhanced Expression of Receptor for Advanced Glycation End Products in Chronic Kidney Disease J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1889 - 1896. [Abstract] [Full Text] [PDF]
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K. Amann, M.-L. Gross, and E. Ritz Pathophysiology Underlying Accelerated Atherogenesis in Renal Disease: Closing in on the Target J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1664 - 1666. [Full Text] [PDF]
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 C. Koechlin, A. Couillard, D. Simar, J. P. Cristol, H. Bellet, M. Hayot, and C. Prefaut Does Oxidative Stress Alter Quadriceps Endurance in Chronic Obstructive Pulmonary Disease? Am. J. Respir. Crit. Care Med., May 1, 2004; 169(9): 1022 - 1027. [Abstract] [Full Text] [PDF]
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 C. Koechlin, A. Couillard, J.P. Cristol, P. Chanez, M. Hayot, D. Le Gallais, and C. Prefaut Does systemic inflammation trigger local exercise-induced oxidative stress in COPD? Eur. Respir. J., April 1, 2004; 23(4): 538 - 544. [Abstract] [Full Text] [PDF]
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J. van de Kerkhof, C. G. Schalkwijk, C. J. Konings, E. C. Cheriex, F. M. van der Sande, P. G. Scheffer, P. M. ter Wee, K. M. Leunissen, and J. P. Kooman N {epsilon}-(carboxymethyl)lysine, N {epsilon}-(carboxyethyl)lysine and vascular cell adhesion molecule-1 (VCAM-1) in relation to peritoneal glucose prescription and residual renal function; a study in peritoneal dialysis patients Nephrol. Dial. Transplant., April 1, 2004; 19(4): 910 - 916. [Abstract] [Full Text] [PDF]
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T. Boure and R. Vanholder Which dialyser membrane to choose? Nephrol. Dial. Transplant., February 1, 2004; 19(2): 293 - 296. [Full Text] [PDF]
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M. Kalousova, S. Sulkova, L. Fialova, J. Soukupova, I. M. Malbohan, P. Spacek, M. Braun, L. Mikulikova, M. Fortova, M. Horejsi, et al. Glycoxidation and inflammation in chronic haemodialysis patients Nephrol. Dial. Transplant., December 1, 2003; 18(12): 2577 - 2581. [Abstract] [Full Text] [PDF]
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F. Locatelli, B. Canaud, K.-U. Eckardt, P. Stenvinkel, C. Wanner, and C. Zoccali Oxidative stress in end-stage renal disease: an emerging threat to patient outcome Nephrol. Dial. Transplant., July 1, 2003; 18(7): 1272 - 1280. [Abstract] [Full Text] [PDF]
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M. E. Suliman, O. Heimburger, P. Barany, B. Anderstam, R. Pecoits-Filho, E. Rodriguez Ayala, A. R. Qureshi, I. Fehrman-Ekholm, B. Lindholm, and P. Stenvinkel Plasma Pentosidine Is Associated with Inflammation and Malnutrition in End-Stage Renal Disease Patients Starting on Dialysis Therapy J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1614 - 1622. [Abstract] [Full Text] [PDF]
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K. Amann, C. Ritz, M. Adamczak, and E. Ritz Why is coronary heart disease of uraemic patients so frequent and so devastating? Nephrol. Dial. Transplant., April 1, 2003; 18(4): 631 - 640. [Abstract] [Full Text] [PDF]
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R. Vanholder, G. Glorieux, and N. Lameire Uraemic toxins and cardiovascular disease Nephrol. Dial. Transplant., March 1, 2003; 18(3): 463 - 466. [Full Text] [PDF]
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 R. De Smet, J. Van Kaer, B. Van Vlem, A. De Cubber, P. Brunet, N. Lameire, and R. Vanholder Toxicity of Free p-Cresol: A Prospective and Cross-Sectional Analysis Clin. Chem., March 1, 2003; 49(3): 470 - 478. [Abstract] [Full Text] [PDF]
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 M. G. Shlipak, L. F. Fried, C. Crump, A. J. Bleyer, T. A. Manolio, R. P. Tracy, C. D. Furberg, and B. M. Psaty Elevations of Inflammatory and Procoagulant Biomarkers in Elderly Persons With Renal Insufficiency Circulation, January 7, 2003; 107(1): 87 - 92. [Abstract] [Full Text] [PDF]
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T. Drueke, V. Witko-Sarsat, Z. Massy, B. Descamps-Latscha, A. P. Guerin, S. J. Marchais, V. Gausson, and G. M. London Iron Therapy, Advanced Oxidation Protein Products, and Carotid Artery Intima-Media Thickness in End-Stage Renal Disease Circulation, October 22, 2002; 106(17): 2212 - 2217. [Abstract] [Full Text] [PDF]
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 S. Delbosc, J.-P. Cristol, B. Descomps, A. Mimran, and B. Jover Simvastatin Prevents Angiotensin II-Induced Cardiac Alteration and Oxidative Stress Hypertension, August 1, 2002; 40(2): 142 - 147. [Abstract] [Full Text] [PDF]
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W. H. Horl Hemodialysis Membranes: Interleukins, Biocompatibility, and Middle Molecules J. Am. Soc. Nephrol., January 1, 2002; 13(90001): S62 - 71. [Abstract] [Full Text] [PDF]
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 L. M. Ruilope, D. J. van Veldhuisen, E. Ritz, and T. F. Luscher Renal function: the Cinderella of cardiovascular risk profile J. Am. Coll. Cardiol., December 1, 2001; 38(7): 1782 - 1787. [Abstract] [Full Text] [PDF]
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G. F. Kormoczi, U. M. Wolfel, A. R. Rosenkranz, W. H. Horl, R. Oberbauer, and G. J. Zlabinger Serum Proteins Modified by Neutrophil-Derived Oxidants as Mediators of Neutrophil Stimulation J. Immunol., July 1, 2001; 167(1): 451 - 460. [Abstract] [Full Text] [PDF]
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G. A. KAYSEN The Microinflammatory State in Uremia: Causes and Potential Consequences J. Am. Soc. Nephrol., July 1, 2001; 12(7): 1549 - 1557. [Abstract] [Full Text] [PDF]
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T. Nguyen-Khoa, Z. A. Massy, J. P. De Bandt, M. Kebede, L. Salama, G. Lambrey, V. Witko-Sarsat, T. B. Drueke, B. Lacour, and M. Thevenin Oxidative stress and haemodialysis: role of inflammation and duration of dialysis treatment Nephrol. Dial. Transplant., February 1, 2001; 16(2): 335 - 340. [Abstract] [Full Text] [PDF]
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A. Wynckel, C. Randoux, H. Millart, C. Desroches, P. Gillery, E. Canivet, and J. Chanard Kinetics of carbamylated haemoglobin in acute renal failure Nephrol. Dial. Transplant., August 1, 2000; 15(8): 1183 - 1188. [Abstract] [Full Text] [PDF]
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P. Stenvinkel, O. Heimburger, B. Lindholm, G. A. Kaysen, and J. Bergstrom Are there two types of malnutrition in chronic renal failure? Evidence for relationships between malnutrition, inflammation and atherosclerosis (MIA syndrome) Nephrol. Dial. Transplant., July 1, 2000; 15(7): 953 - 960. [Full Text] [PDF]
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M. Morena, J.-P. Cristol, T. Dantoine, M.-A. Carbonneau, B. Descomps, and B. Canaud Protective effects of high-density lipoprotein against oxidative stress are impaired in haemodialysis patients Nephrol. Dial. Transplant., March 1, 2000; 15(3): 389 - 395. [Abstract] [Full Text] [PDF]
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