Chronic Kidney Disease

Enhanced Expression of Receptor for Advanced Glycation End Products in Chronic Kidney Disease

Fan Fan Hou, Hao Ren, William F. Owen, Zhi Jian Guo, Ping Yan Chen, Ann Marie Schmidt, Toshio Miyata and Xun Zhang
JASN July 2004, 15 (7) 1889-1896; DOI: https://doi.org/10.1097/01.ASN.0000131526.99506.F7

Abstract

ABSTRACT. Inappropriate chronic inflammation associated with progressive, chronic kidney disease (CKD) reflects sustained activation of immunocompetent cells, like monocytes/macrophages. Advanced glycation end products (AGE) accumulate in CKD, but it is unclear if they stimulate monocytes by binding with the receptor for AGE (RAGE). Posited was the notion that RAGE plays a contributory role to monocyte-mediated systemic inflammation of progressive CKD. Peripheral blood monocytes were isolated from 102 patients without diabetes with varying severity of CKD. RAGE expression on peripheral blood monocytes increased with worsening CKD (r2 = 0.73) and was strongly correlated with plasma levels of pentosidine, a marker for AGE (r = 0.71). Strongly positive statistical correlations were observed in patients with CKD between monocyte RAGE and plasma levels of tumor necrosis factor alpha (TNF-α) (r = 0.61), the monocyte activation marker, neopterin (r = 0.65), and the systemic acute phase reactant, C-reactive protein (r = 0.44). Monocytes obtained from patients with CKD showed a monotonic increase in the number and affinity of specific AGE binding sites and increased production of TNF-α under stimulation of AGE. All these upregulatory responses in uremic monocytes could be largely blocked by an anti-RAGE antibody. It was concluded that RAGE expression was upregulated on monocytes from patients with CKD. Enhanced RAGE may amplify AGE-induced monocytes perturbation and contribute to monocyte-mediated systemic inflammation in progressive CKD.

Progressive, chronic kidney disease (CKD) and ESRD are associated with chronic, clinical, and serologic signs of systemic inflammation that appear independent of the etiology of the kidney disease (1,2⇓). Patients with noninflammatory as well as inflammatory causes of CKD, and patients with ESRD without clinically identifiable reasons for systemic inflammation, both demonstrate serologic, pathologic, and clinical signs of a systemic inflammatory state. Common examples of these abnormalities associated with CKD and ESRD include progressively declining nutritional status, accelerated atherosclerosis, increased severity and frequency of cardiovascular events in the absence of conventional atherosclerotic vascular disease risk factors, elevated levels of acute phase reactants like C-reactive protein (CRP), and reduced blood levels of reverse phase reactants like albumin and transferrin that are disproportionately low for the protein-caloric intake (1–6⇓⇓⇓⇓⇓). Like for the general population, these abnormalities are independently associated with an increased cardiovascular and/or all-cause risks of death (4,6–8⇓⇓⇓).

A unifying hypothesis for these observations in CKD and patients with ESRD is that there is abnormal, persistent, and deleterious activation of the effector limb of adaptive immunity (1,2⇓). Supporting this premise is the typical finding of substantial elevation of blood levels of proinflammatory cytokines like TNF-α and IL-1β in CKD and ESRD, which are disproportionately raised for the degree of renal insufficiency. Moreover, peripheral blood monocytes from such patients have increased production of these cytokines in comparison to healthy individuals. Taken together, inappropriate activation of the monocyte/macrophage system and/or inadequate clearance of their products have been posited to be causal (9–11⇓⇓).

The source of immunological agonists for peripheral blood monocytes has not been fully elucidated. In hemodialysis (HD) patients, the dialysis procedure itself has been posited to be causal. Chronic maintenance HD is associated with repeated exposure to several immunological provocateurs that stimulate monocytes/macrophages (12,13⇓). For example, direct contact of circulating mononuclear cells with selected dialyzer membrane materials (like cuprophane), or the back-flux of LPS or LPS fragments from nonsterile dialysate into the blood, may provoke cytokine synthesis.

Elevated levels of selected inflammatory cytokines, like patients with ESRD, are also found in predialysis patients with CKD. But in progressive CKD before dialysis begins, putative immunological provocateurs are less evident. Therefore, it is posited that pathobiological changes associated with CKD per se can contribute to the proinflammatory state (9,11⇓)—i.e., immunological provocateurs accumulate as a consequence of decreased excretory function and/or the milieu of CKD is associated with their aberrant formation.

Nonenzymatic glycation of proteins, leading to the formation of advanced glycation end products (AGE), have been increasingly scrutinized as immunological provocateurs in conditions like CKD and ESRD. In vitro studies demonstrate that AGE initiate a range of monocytes/macrophages-mediated inflammatory responses, including chemotaxis (14), synthesis of proinflammatory cytokines (14–17⇓⇓⇓), and delayed apoptosis (17). In vivo, the relevance of AGE in monocyte-mediated inflammatory syndrome associated with CKD is supported by the observations that AGE formation occurs at an accelerated rate in CKD (18), and AGE correlate with neopterin, and TNF-α levels in patients with CKD (19). Moreover, the accumulation of AGE has been correlated with the development of ESRD complications, such as dialysis-related amyloidosis and accelerated atherosclerosis (14–16⇓⇓).

Many, but not all, of the effects of AGE are mediated by their engagement with the receptor for AGE (RAGE), a member of the immunoglobulin superfamily that is a signal transduction receptor (20). RAGE is present at low levels on a range of cell types, including endothelium, smooth muscle cells, fibroblasts, and monocyte/macrophages (16,17,21–23⇓⇓⇓⇓). However, at sites of accumulated RAGE ligands, there is a marked increase in RAGE-expressing cells with functional receptor upregulation, suggesting receptor induction by AGE (24).

Although CKD is associated with increased levels of TNF-α, monocyte activation markers, and AGE, there has been little characterization of RAGE expression on peripheral blood monocytes from patients with CKD. Nor has there been a substantial cross-sectional profiling of this panel of immunologic changes in patients with varying severity of kidney failure, which could offer statistical support for the assignment of causality. The study presented here examined the expression of RAGE on circulating monocytes from healthy patients, patients with CKD, and patients with ESRD to test the association between RAGE expression and chronic monocyte-mediated systemic inflammation of CKD.

Materials and Methods

Study Subjects

The study subjects included patients without diabetes with varying degrees of CKD. As standard practice, because of the absence of community-based subspecialty care for CKD, all patients who received a presumed diagnosis of CKD were hospitalized for evaluation of reversible causes of kidney disease and a treatment plan (if possible). All final subjects had CKD and no confounding conditions. To confirm the relative stability of their CKD and absence of comorbid conditions, all blood samples obtained for this study were drawn after 4 wk or more of hospitalization for CKD workup. Study subjects were recruited for study from June to December 2001. Patients on HD were accrued from the same period.

Subjects diagnosed with diabetic nephropathy or diabetics with other forms of kidney diseases were excluded by their clinical history and laboratory assessment (fasting blood glucose ≥7.0 mmol/L or 2-h postprandial blood glucose ≥11.1 mmol/L during an oral glucose tolerance test). Patients with acute or chronic infections, active liver diseases, autoimmune diseases, nephrotic-range proteinuria, or immunosuppressant therapy were also excluded. A total of 102 patients were enrolled after giving informed consent. Sixty patients with CKD not receiving dialysis were categorized as having mildly to moderately decreased GFR (CKD stage 2 to 3, GFR 30 to 89 ml/min/1.73 m2, n = 19), severely decreased GFR (CKD stage 4, GFR 15 to 29 ml/min/1.73 m2, n = 22), and kidney failure (CKD stage 5, GFR <15 ml/min/1.73 m2, n = 19), according to the disease stratification issued by the National Kidney Foundation (NKF) in 2002 (25,26⇓). Among the 60 patients, 48 had never received medical treatment, and 12 had been treated with antihypertensive drugs (β-receptor blockers and/or calcium channel blockers). The other 42 patients were on maintenance HD for >90 d (dialysis vintage 32.7 ± 18.5 mo) and received 4 to 5 h of HD three times a week (single pool Kt/V ≥ 1.2). They were dialyzed with polysulfone dialyzers (Fresenius Medical Care) with a bicarbonate dialysate. Patients’ characteristics are listed in Table 1. HD patients were receiving recombinant erythropoietin, but none of them had been treated with intravenous iron dose (intravenous iron dose was not available in China before 2003). Thirty randomly selected healthy adults served as control subjects.

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Table 1. Patient characteristicsa

Monocyte Isolation

Whole blood was collected from fasting participants in sterile polystyrene tubes containing 5 mM EDTA. In HD patients, blood sampling was conducted before dialysis. After low-speed centrifugation of blood, plasma was aspirated and stored at −80°C. Plasma assays were conducted on duplicate samples thawed once. Mononuclear cells were separated from the cell pellet by Ficoll-Paque sedimentation, and monocytes were purified by CD14-positive immunomagnetic selection (18).

Quantitation of RAGE Expression

Freshly isolated monocytes were washed, resuspended in PBS with 0.5% human serum albumin (HSA), and incubated for 45 min in ice with 1:25 diluted rabbit anti-RAGE IgG or nonimmune rabbit IgG (Sigma, St. Louis, MO) (27). Primary antibodies were detected with 1:50 diluted FITC-conjugated goat anti-rabbit IgG (PharMingen, San Diego, CA). Mean fluorescence intensity (MFI) were analyzed with FAScan, and the results were presented as the magnitude increase of MFI (MFI units of tested antibody staining/MFI units of control antibody staining) (28).

Ligand Binding Assays

AGE-modified HSA (AGE-HSA) was prepared in vitro by incubating HSA (Sigma) with 200 mM glucose in PBS for 8 wk at 37°C. The amount of AGE formed was assessed by an ELISA and fluorescence spectra. Endotoxin was eliminated as described previously (15). Endotoxin levels of all materials used was <0.5U/ml as measured by the E-Toxat kit (Sigma).

For binding studies of AGE-HSA on fresh isolated monocytes, AGE-HSA was radioiodinated with carrier-free 125I (Institute of Atomic Energy of China, Peking, China) by the Iodogen method to a specific activity of approximately 800 cpm/ng protein (29). The amount of radiolabeled material specifically bound was measured and analyzed by Scatchard analysis (30). Nonspecific binding was always ≤25% of the total binding. Where indicated, monocytes were preincubated with 50 μg/ml of rabbit anti-RAGE IgG or nonimmune rabbit IgG for 2 h at 4°C before addition of radiolabeled AGE-HSA. The concentration of anti-RAGE was based on previous studies demonstrating a plateau of inhibition at 50 μg/ml (23,27,31⇓⇓). The uptake and intracellular degradation of AGE-HSA was measured as described previously (29).

Quantitation of TNF-α Production

Monocytes (2.5 × 105 cells/well) were incubated in RPMI 1640 with 10% AB human serum containing selected concentrations of AGE-HSA at 37°C for 24 h. The supernatants were harvested and centrifuged at 400 × g for 5 min. TNF-α was quantitated by ELISA (BioSource International, Camarillo, CA,) with a detection limit of <0.09 pg/ml. Where indicated, monocytes were preincubated with 50 μg/ml of rabbit anti-RAGE IgG or nonimmune rabbit IgG for 2 h. For these experiments, the monolayers were then washed three times and incubated with 50 μg/ml of AGE-HSA for 24 h.

Quantitation of Plasma Pentosidine

The plasma concentrations of pentosidine were determined by competitive ELISA (32). BSA modified with synthetic pentosidine (BSA-pentosidine containing 75 pmol pentosidine per microgram albumin by HPLC) and anti–pentosidine-KLH rabbit IgG were prepared as described previously. Multiple-well plates were coated overnight at 4°C with BSA-pentosidine and blocked with 4% skim milk at 37°C for 1 h. A limiting dilution of 1:200 anti-pentosidine rabbit IgG was preincubated with an equal volume of plasma hydrolysate or standard BSA-pentosidine for 1 h. The mixture was added to the wells and incubated overnight at 4°C. After washing, the wells were reacted with peroxidase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL), developed in citrate buffer containing 0.04% O-phenylene-diamine dihydrochloride and 0.09% hydrogen peroxide, and the absorbance measured at 492 nm.

Quantitation of Plasma Inflammatory Markers

Plasma neopterin levels were measured by RIA (Behring Diagnostic, Rueil Malmaison, France), TNF-α by ELISA (BioSource), and CRP levels by ELISA (BioCheck, Burlingame, CA). The minimum detectable concentration of CRP was 0.1 mg/L. Other hematologic values were determined with standard laboratory equipment (Olympus AU800, Japan), methods, and reagents.

Statistical Analyses

We used SPSS software, version 10.0, to evaluate differences between means by Student’s paired or two-sample t test, or ANOVA for more than two groups. Multiplicative interaction terms were included to evaluate for interaction among explanatory variables. Relationships between variables were tested by simple (Pearson’s correlation coefficient) or curve (exponential correlation coefficient) regression analysis. Two-tailed P values <0.05 were considered significant. Values are reported as mean ± SEM.

Results

RAGE Expression on Monocytes from Patients with CKD

Pentosidine, a marker of the advanced glycation process, is a readily measurable surrogate for the heterogeneous AGE that bind to RAGE (33). The plasma levels of pentosidine were significantly higher in patients with CKD than in healthy subjects and increased progressively with worsening CKD (Table 2). Because chronic AGE exposure has been reported to induce RAGE (24), we examined RAGE expression on freshly isolated peripheral blood monocytes from individuals with varying degrees of renal insufficiency. Freshly isolated monocytes from healthy subjects constitutively expressed RAGE (Table 2). RAGE expression increased monotonically at stage 4 with worsening CKD (Table 2) and inversely correlated with GFR (R2 = 0.725, P < 0.001, Figure 1A). A strong statistical relationship was observed between RAGE expression and pentosidine levels in CKD (n = 60, r = 0.710, P < 0.001, Figure 1B). This correlation remained significant by multiple regression analysis after adjustment of RAGE and pentosidine values for GFR (r = 0.711, P < 0.001 and r = 0.644, P < 0.001, respectively); these findings suggest an independent association between RAGE and pentosidine. The same directional association was observed in HD patients (n = 42, r = 0.389, P = 0.011, Figure 1C), but was less prominent.

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Table 2. RAGE expression and plasma levels of RAGE, pentosidine, CRP, and monocyte activation markers in patients with CKD

Figure1

Figure 1. Influence of renal function (A) and plasma levels of pentosidine on monocyte receptor for advanced glycation end products (RAGE) expression in 60 nondialyzed patients with chronic kidney disease (CKD) (B) and 42 hemodialysis (HD) patients (C). Exponential curve was used to fit the relationship between RAGE expression and GFR. The correlation coefficients (r) by linear regression analysis between RAGE expression and pentosidine levels were statistically significant at the indicated P values.

Functionality of RAGE on Monocytes from Patients with CKD

Binding Properties and Degradation of AGE.

To begin to characterize the specificity and functionality of monocyte RAGE, 125I-AGE-HSA binding studies were performed on monocytes isolated from 5 healthy subjects, 5 HD patients, and 15 patients with CKD (5 each with CKD stage 2, stage 3, and stage 4). All patients were randomly chosen. Monocyte RAGE demonstrated saturable binding at 4°C. Twice the amount of 125I-AGE-HSA per microgram protein was needed to saturate monocyte RAGE in progressing from healthy to ESRD (Figure 2A). There was a parallel increase in the amount of intracellular degraded 125I-AGE-HSA, measured as TCA soluble radioactivity (Figure 2B). No significant change was observed in the binding and degradation of 125I-normal-HSA (data not shown). The maximal specific binding of AGE-HSA was inhibited by approximately 80% when monocytes were preincubated with a blocking, anti-RAGE antibody. The intracellular degradation of 125I-AGE-HSA was diminished to a lesser extent by the anti-RAGE treatment. Treatment of monocytes with nonimmune IgG did not affect the binding or degradation of AGE-HSA (data not shown).

Figure2

Figure 2. Maximal specific binding (A) and intracellular degradation (B) of 125I-radiolabeled advanced glycation end product–modified human serum albumin (125I-AGE-HSA) (25 μg/ml) by monocytes. Peripheral blood monocytes were obtained from random patients with varying severity of chronic kidney disease (CKD) (n = 15), patients maintained on hemodialysis (HD) (n = 5), and healthy subjects (n = 5). 125I-AGE-HSA binding and degradation by freshly isolated monocytes (▪) or by cells preincubated with 50 μg/ml of anti–receptor for advanced glycation end products (RAGE) for 2 h (□) were analyzed as described in the text. Data are expressed as the mean ± SEM. ANOVA, P < 0.001 (A), P < 0.001 (B); the anti-RAGE–treated groups are significantly different from the untreated groups (paired sample t test, P < 0.001).

By Scatchard analysis, the number of specific binding sites on monocytes from healthy subjects was 0.37 ± 0.02 × 105 per cell with an apparent binding affinity (Ka) of 1.57 ± 0.03 × 106 M−1 (Table 3). A monotonic increase in the binding sites and Ka were observed on freshly isolated monocytes from patients with worsening CKD.

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Table 3. Number of AGE binding sites (R) and binding affinity (Ka) for 125I-AGE-HSA on peripheral blood monocytes from patients with CKD

RAGE-Stimulated TNF-α Release.

Because RAGE-stimulation of monocytes induces TNF-α (27), and we demonstrated that monocytes from patients with CKD have greater RAGE, we posited that monocytes from patients with CKD also produce greater amounts of TNF-α when AGE stimulated. Monocytes from five random patients with GFR <15 ml/min, but not yet on dialysis, were incubated with incremental concentrations of AGE-HSA (0 to 100 μg/ml). The constitutive production of TNF-α by monocytes from patients with CKD versus healthy subjects was 9.98 ± 0.41 versus 4.84 ± 0.22 pg/105 cells, respectively. In a dose-dependent manner with an apparent plateau of 50 to 100 μg/ml AGE-HSA, monocytes from CKD versus healthy subjects stimulated with 100 μg/ml AGE-HSA produced 90.52 ± 2.82 and 26.38 ± 1.54 pg/105 cells, respectively (ANOVA, P < 0.001; subjects, P < 0.001; AGE-HSA dose, P < 0.001; subjects × dose interaction, P < 0.001).

To further confirm the role of RAGE, rather than another binding domain for AGE, monocytes were preincubated with rabbit anti-RAGE or nonimmune rabbit IgG before interaction with AGE-HSA. Preincubation with anti-RAGE significantly diminished stimulated TNF-α production by monocytes from patients with CKD from 90.52 ± 2.82 to 17.86 ± 1.05 pg/105 cells and from healthy subjects from 26.38 ± 1.54 to 6.76 ± 0.20 pg/105 cells. HSA did not increase TNF-α production above constitutive levels; nonimmune IgG did not reduce stimulated TNF-α production (ANOVA, P < 0.001; SNK: α-RAGE+AGE-HSA is significantly different from AGE-HSA alone and HSA P < 0.05).

Relationship between RAGE Expression and Systemic Measures of Inflammation

To survey the relationship between declining renal function and laboratory measures of inflammation, plasma was analyzed from cohorts of patients with worsening degrees of renal insufficiency. Plasma levels of neopterin, TNF-α, and CRP increased in parallel with worsening renal function (Table 2). Toward relating these inflammatory abnormalities to the monocyte cellular perturbations described earlier, correlations were tested between monocyte RAGE or plasma pentosidine levels. Highly significant positive correlations between RAGE expression or pentosidine levels and plasma concentrations of neopterin, TNF-α, and CRP were observed in patients with CKD (Figure 3). Toward imputing causality, these associations were analyzed after adjustment for GFR; the correlations still remained highly significant (RAGE: r = 0.580, P < 0.001; r = 0.538, P < 0.001; r = 0.403, P = 0.001; pentosidine: r = 0.622, P < 0.001; r = 0.615, P < 0.001; and r = 0.459, P < 0.001, respectively). In HD patients, a significant positive correlation was found between RAGE expression and plasma TNF-α levels (r = 0.337, P = 0.029). However, in HD patients, no significant relationship could be found between RAGE expression or pentosidine and neopterin or CRP concentrations (data nor shown).

Figure3

Figure 3. Relationships between receptor for advanced glycation end products (RAGE) expression or pentosidine levels and C-reactive protein (CRP) or monocyte activation markers in 60 nondialyzed patients with chronic kidney disease (CKD). Pearson’s correlations coefficients between RAGE expression (left) or pentosidine (right) and TNF-α, neopterin, or CRP were significant at the indicated P values.

The bivariate associations between selected variables were examined to further analyze other relationships. In patients with CKD, CRP was inversely associated with serum albumin (r = −0.381, P = 0.003) and hemoglobin concentration (r = −0.434, P = 0.001), and was positively correlated with white blood cell count (r = 0.372, P = 0.003). Serum albumin and hemoglobin concentrations correlated inversely with RAGE expression (r = −0.260, P = 0.045 and r = −0.431, P = 0.001). Both correlations remained significant by multiple regression analysis after adjustment of serum albumin and hemoglobin levels for CRP (r = −0.391, P = 0.002 and r = −0.432, P = 0.001). Significant inverse correlations were also observed between hemoglobin and pentosidine (r = −0.388, P = 0.002), even after adjustment of hemoglobin for CRP (r = −0.275, P = 0.03). No relationship was found between RAGE expression or pentosidine levels and white blood cell count or transferrin saturation. In HD patients, CRP correlated inversely with serum albumin (r = −0.332, P = 0.03) and hemoglobin (r = −0.394, P = 0.01), but correlated directly with white blood cell count (r = 0.354, P = 0.02). However, none of these values correlated with RAGE expression or pentosidine concentration in HD patients.

Discussion

CKD is complicated by accumulation of AGE-modified proteins in plasma and tissues (14,19,22⇓⇓). The cellular effects of AGE are largely mediated by their interaction with specific cell surface receptors, like RAGE (24). Developmentally, high levels of RAGE are present in the brain, where it may serve as a receptor for amphoterin (21,34⇓). With maturity, RAGE expression decreases to low levels in a range of cell types, including monocytes/macrophages. However, in certain pathologic processes associated with ligand-rich microenvironments, RAGE expression increases stably. For example, in diabetic vasculature, cells expressing high levels of RAGE are often juxtaposed to areas in which AGE are abundant (20). A similar relationship has been shown in synovium from patients with dialysis-related amyloidosis, in which RAGE mediates cellular perturbations by AGE-β2 microglobulin (22,35⇓).

The study presented here demonstrates for the first time that expression of RAGE on peripheral blood monocytes is upregulated in patients with nondiabetic CKD. The increased expression of RAGE was evident at stage 4 of CKD and became more prominent as renal function deteriorated. RAGE expression was prominent in HD patients, despite HD being performed with biocompatible dialyzer membrane materials and bicarbonate buffered dialysate. As described by others, we observed that plasma pentosidine, a well known and characterized AGE structure, increased markedly with progressive CKD. In patients with CKD, monocyte RAGE expression was strongly correlated with pentosidine levels. This correlation remained significant when the values were adjusted for GFR, suggesting that the increment in RAGE was not simply a consequence of impaired urinary excretion of AGE. These in vivo observations suggest that increased AGE during progressive CKD might upregulate RAGE expression on monocytes. Supporting this premise, in vitro studies have demonstrated that engagement of RAGE by AGE results in activation of nuclear factor-kappa B (NF-κB), and NF-κB sites in the RAGE promoter affect ligand-associated upregulation of RAGE (36,37⇓).

AGE-modified proteins can be internalized and degraded by monocytes/macrophages through interaction with several cell surface-binding sites such as p60/p90 (38) and type A macrophage scavenger receptor (39). Monocytes obtained from patients with CKD exhibited significant increases in the binding and degradation of 125I-AGE-HSA, compared with healthy subjects. Anti-RAGE antibody diminished the enhanced binding of AGE by monocytes from patients with CKD, suggesting that these increased functional binding sites are mainly RAGE. Anti-RAGE was less effective in reducing intracellular degradation of AGE, consistent with the observation that RAGE is less involved in mediating endocytosis and degradation of bound ligands (37) because non-RAGE binding sites are also involved.

In view of the observation that AGE-RAGE interaction mediates monocyte chemotaxis and TNF-α production (15), and the increased RAGE on monocytes from patients with CKD exhibited specificity and functionality, we examined AGE-induced TNF-α release in patients with varying severity of CKD. Constitutive and AGE-induced production of TNF-α by monocytes from patients with CKD was substantially greater than the amounts from healthy subjects. The increase in TNF-α production was greatly attenuated by blocking of RAGE, suggesting that this cellular perturbation is mainly mediated by RAGE. It is interested to note that interaction of monocytes with TNF-α enhances expression of AGE receptors via an autocrine manner (40). Moreover, both AGE and TNF-α diminish monocyte apoptosis, and so offer an additional mechanism for disordered regulation of monocytes-mediated inflammatory responses (17). Therefore, we propose that AGE accumulated in progressive CKD stimulate peripheral monocytes to release TNF-α through interaction with RAGE. Engagement of RAGE by AGE, and/or interaction of monocytes with TNF-α, enhance RAGE expression and may extend survival of a pro-inflammatory monocyte phenotype. A resulting positive feedback loop is triggered in which increased RAGE enhances the capacity of the monocytes for subsequent binding of AGE and TNF-α production. These events stand out in sharp contrast to the acute and self-limited host-responses to terminate cellular activation.

To further determine the role of AGE-RAGE in inflammation associated with CKD, monocyte activation markers were evaluated. In patients with CKD not undergoing HD, RAGE expression and plasma levels of pentosidine, were closely associated with neopterin and TNF-α concentrations. A close correlation was also observed between RAGE or AGE and CRP, even when the values were corrected for GFR. Furthermore, an inverse correlation was found between RAGE expression and serum albumin concentration, which decreases in inflammatory states as a reverse acute phase reactants (1). The relationships between RAGE expression and neopterin or CRP were NS in HD patients. The absence of a strong association is not counterintuitive because the monocyte agonists are likely different and expanded in HD, e.g. monocyte stimulation can be provoked by interaction with bioincompatible dialyzer membranes (13) or exposure of blood to dialysate LPS (12). Neopterin levels in HD patients were more than fourfold higher than that measured in Stage 5 CKD patient. Moreover, a close correlation between RAGE expression and neopterin was observed even after adjustment of neopterin levels for GFR. Therefore, the increase in neopterin could not be ascribed only to a deterioration of renal function.

In conclusion, we used a substantial cohort of patients with varying degrees of renal insufficiency to demonstrate that RAGE-dependent binding and TNF-α production by circulating monocytes is upregulated in CKD. The upregulation of these functional monocyte receptors was tightly associated with circulating monocyte activation markers, systemic cytokine levels, and nonspecific measures of systemic inflammation, and matched the severity of the kidney disease. These data suggest that enhanced RAGE in CKD may amplify AGE-induced monocyte perturbation, and so contribute to monocyte-mediated systemic inflammation associated with renal failure. These findings suggest a novel therapeutic target for disordered systemic immunity in CKD and ESRD. [Printer: Reference (26) is cited here for parsing. Please delete this bracketed information.]

Acknowledgments

This work was supported by a National Nature and Science Grant of China (No. 30330300; 30270622) to Dr. Hou; the Team Collaboration Project of Guangdong (No. 10717); and Guangdong Nature and Science Grant (No. 013076) to Dr. Hou.

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Enhanced Expression of Receptor for Advanced Glycation End Products in Chronic Kidney Disease
Fan Fan Hou, Hao Ren, William F. Owen, Zhi Jian Guo, Ping Yan Chen, Ann Marie Schmidt, Toshio Miyata, Xun Zhang
JASN Jul 2004, 15 (7) 1889-1896; DOI: 10.1097/01.ASN.0000131526.99506.F7
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