Clinical Dialysis

Profound Mishandling of Protein Glycation Degradation Products in Uremia and Dialysis

Stamatina Agalou, Naila Ahmed, Roya Babaei-Jadidi, Anne Dawnay and Paul J. Thornalley
JASN May 2005, 16 (5) 1471-1485; DOI: https://doi.org/10.1681/ASN.2004080635

Abstract

The aim of this study was to define the severe deficits of protein glycation adduct clearance in chronic renal failure and elimination in peritoneal dialysis (PD) and hemodialysis (HD) therapy using a liquid chromatography-triple quadrupole mass spectrometric detection method. Physiologic proteolysis of proteins damaged by glycation, oxidation, and nitration forms protein glycation, oxidation, and nitration free adducts that are released into plasma for urinary excretion. Inefficient elimination of these free adducts in uremia may lead to their accumulation. Patients with mild uremic chronic renal failure had plasma glycation free adduct concentrations increased up to five-fold associated with a decline in renal clearance. In patients with ESRD, plasma glycation free adducts were increased up to 18-fold on PD and up to 40-fold on HD. Glycation free adduct concentrations in peritoneal dialysate increased over 2- to 12-h dwell time, exceeding the plasma levels markedly. Plasma glycation free adducts equilibrated rapidly with dialysate of HD patients, with both plasma and dialysate concentrations decreasing during a 4-h dialysis session. It is concluded that there are severe deficits of protein glycation free adduct clearance in chronic renal failure and in ESRD on PD and HD therapy.

Both peritoneal dialysis (PD) and hemodialysis (HD) therapy as practiced currently have high mortality and morbidity—the median survival time from commencement of dialysis therapy is 5 to 8 yr, depending on other exacerbating factors (old age and diabetes). Mortality and morbidity are associated with an increased risk for cardiovascular disease. Inadequate removal of uremic toxins is a primary cause of uremia associated vascular disease (1). Protein glycation adducts are a class of uremic toxin (2), but it is currently unclear how effective conventional HD and PD therapies are in removing glycation adducts.

Glycation of proteins is a complex series of parallel and sequential reactions collectively called the Maillard reaction. It occurs in all tissues and body fluids. Early stage reactions with glucose lead to the formation of the early glycation adduct fructosyl-lysine (FL), and later stage reactions form advanced glycation end products (AGE) (3). FL degrades slowly to form AGE. Glyoxal (G), methylglyoxal (MG), and 3-deoxyglucosone (3-DG) are also potent glycating agents that are formed by the degradation of glycated proteins, glycolytic intermediates, and lipid peroxidation. They react with proteins to form AGE directly (Figure 1, a and b). Important AGE quantitatively are hydroimidazolones derived from arginine residues modified by glyoxal, MG and 3-DG, G-H1, MG-H1, and 3DG-H, respectively (Figure 1c). Other important and widely studied AGE are Nε-carboxymethyl-lysine (CML), Nε-carboxyethyl-lysine (CEL), and pentosidine (4) (Figure 1, d and e). Proteins also suffer oxidative and nitrosative damage forming methionine sulfoxide (MetSO) (5) and 3-nitrotyrosine (3-NT) (6) (Figure 1f). Glycation adduct residues are formed by the physiologic glycation of endogenous cellular and extracellular proteins and are also present in ingested food (7). Glycation free adducts are found in plasma, urine, and other physiologic fluids. They originate from the turnover of endogenous glycated proteins by cellular proteolysis and from food (4,8). Glycation reactions are increased in uremia, where there are increased concentrations of many α-oxoaldehydes, particularly glyoxal, MG, and 3-DG (9,10). α-Oxoaldehydes are also present in PD fluids, formed by thermal sterilization (11), and introduced into the peritoneal cavity, they add to the increased glycation potential in ESRD.

Figure 1.
Figure 1.

Protein glycation in physiologic systems. (a) Pathways for the formation of advanced glycation endproducts (AGE). (b) α-Oxoaldehyde glycating agents. (c) Hydroimidazolone AGE: glyoxal-H1 (G-H1; 214 Da), methylglyoxal-H1 (MG-H1; 228 Da), and 3-deoxyglucosone-H (3DG-H; 318 Da). (d) Monolysyl glycation adducts: fructosyl-lysine (FL; 308 Da), Nε-carboxymethyl-lysine (CML; 204 Da), and Nε-carboxyethyl-lysine (CEL; 218 Da). (e) Cross-links and fluorophores: pentosidine (379 Da), (methylglyoxal-derived lysine dimer (MOLD; 341 Da), and argpyrimidine (254 Da). (f) Oxidation and nitration markers: methionine sulfoxide (MetSO; 165 Da) and 3-nitrotyrosine (3-NT; 226 Da). (c through f) Protein glycation, oxidation, and nitration residues are shown. For the corresponding free adducts at physiologic pH, the N-terminal amino group is protonated −NH3+ and the C-terminal carbonyl is a carboxylate −CO2− moiety.

The normal high renal clearance of AGE free adducts (4) is impaired markedly in ESRD; hence, the concentrations of free AGE in plasma are expected to increase. In this study, we evaluated the characteristics of glycation free adduct excretion of patients who had chronic renal failure (CRF) with mild uremia and patients who had ESRD with severe uremia and were on HD and PD therapies.

Materials and Methods

Clinical Sampling: Patient Characteristics and Blood and Urine Sample Collection

Venous blood samples were drawn from normal healthy subjects, untreated CRF patients, and ESRD patients into tubes with heparin anticoagulant. Subject characteristics are given in Table 1. Renal creatinine clearance for all dialysis patients was <10 ml/min. Blood samples and aliquots of dialysate from HD patients (three with diabetes) were collected at the start and at the end of a 4-h dialysis session using a polysulfone membrane. Blood samples from PD patients (two with diabetes) were collected 2 h after introduction of PD fluid into the peritoneal cavity during a peritoneal equilibration test (PET) with peritoneal dialysate collected after 0, 2, and 4 h of dwell time. Peritoneal dialysate was also collected for approximately 12 h during the night preceding the test and for the 24 h before this. Peritoneal dialysate from four exchanges was pooled to estimate the 24-h excretion flux. The PD fluids used by patients in this study were single-compartment fluids (Dianeal; Baxter Healthcare Corporation, Deerfield, IL) that contained glucose osmolyte. Analyte estimates for PD and HD patients with diabetes were not outliers from PD and HD patients without diabetes, respectively. Blood cells were sedimented by centrifugation (2000 × g, 10 min), and the plasma was removed and immediately frozen at −80°C. Urine samples were collected at ambient temperature over 24 h from normal healthy control subjects, CRF patients and PD patients with residual diuresis (<10% of analyte amounts were lost during this period). Plasma and urine samples were stored at −80°C before analysis. The study was approved by East London and The City Health Authority Research Ethics Committee (London, UK), and written informed consent was given by all participants and conformed to the Declaration of Helsinki.

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Table 1

Clinical data for normal healthy control subjects and patients with CRF and ESRD on PD or HD therapya

Other Clinical Markers

Protein glycation adducts were assayed by liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS) (4). The interbatch coefficients of variation were ≤10%, and analyte estimates corroborated with independent measurements when these were available (6,12). Free glycation adducts were determined by assay of analytes in ultrafiltrate (12 kD filter cutoff, 50-μl aliquot) of plasma, urine, and dialysate. Glycation adduct residues of plasma protein were determined in exhaustive enzymatic digests (50-μg protein equivalent) (4,13). Routine clinical service methods were used for urine and plasma creatinine (Jaffe rate method) and serum albumin (BCG colorimetric assay) on an Olympus analyzer. Plasma monocyte chemotactic protein-1 (MCP-1) and TGF-β were assayed in platelet free plasma by double-antibody ELISA (R & D Systems, Abingdon, UK).

Statistical Analyses

Renal clearance of analytes (ml/min) for control subjects and CRF patients was determined as [Analyte]urine × urine volume/([Analyte]plasma× urine collection time). Clearance of analytes in PD patients was calculated similarly using both urine and peritoneal dialysate outputs. Significance of difference between mean and median AGE concentrations was determined using t test and the Mann Whitney U test, respectively. Correlation analysis was performed by calculating Spearman ρ statistic. Mass transfer area coefficients (MTAC) were deduced by the simplified Garred equation (14).

Results

Detection of Glycation, Oxidation, and Nitration Free Adducts by LC-MS/MS in Plasma and Urine of Control Subjects and CRF Patients and Plasma, Urine, and Dialysate of ESRD Patients on HD or PD Therapy

Protein glycation, oxidation, and nitration free adducts were detected by LC-MS/MS in plasma and urine of normal healthy control subjects, patients with CRF, and ESRD patients on PD and HD therapy. They were also detected in peritoneal dialysate and hemodialysate of ESRD patients (Figure 2, a through h). Analytical chromatograms of the hydroimidazolone MG-H1 showed the expected partially resolved pair of epimers (Figure 2, c and d), and the hydroimidazolone 3DG-H showed the expected three resolved structural isomers (4) (Figure 2, e and f). The concentrations of 3-NT were similar to those reported by others using mass spectrometric techniques considered to be artifact-free (6).

Figure 2.
Figure 2.

Specimen analytical chromatograms in the determination of protein glycation adducts by liquid chromatography with triple quadrupole mass spectrometric detection in ESRD patients. CML (a) and [13C6]-CML (10 pmol; b) in plasma protein hydrolysate of a hemodialysis (HD) patient, Rt 7.9 min. MG-H1 (c) and [15N2]-MG-H1 (50 pmol; d) in peritoneal dialysis (PD) dialysate at 2 h of dwell time, Rt 24.0 (MG-HA) and 24.2 (MG-HB) min. 3DG-H (e) and [15N2]-3DG-H (50 pmol; f), isomers 1, 2, and 3 (as indicated) in hemodialysate, Rts 24.6, 25.1, and 23.8 min, respectively. Argpyrimidine (g) and [15N2]argpyrimidine (50 pmol; h) in HD patient plasma filtrate after a dialysis session, Rt 10.1 min. Chromatographic conditions were described in the Materials and Methods section.

Glycation, Oxidation, and Nitration Free Adducts in Plasma and Urine of Control Subjects and CRF Patients

The AGE free adducts MG-H1 and 3DG-H were found in the highest concentration in blood plasma of control subjects, 122 nM, which was 133- and 46-fold higher than the concentrations of pentosidine and 3-NT free adduct (Table 2). Most glycation free adducts were increased in CRF patients, with respect to normal control subjects. The increases were two-fold for FL, four-fold for CML, four-fold for CEL, four-fold for G-H1, five-fold for MG-H1, two-fold for 3DG-H, and two-fold for argpyrimidine. This was associated with a marked decline in renal clearance of glycation adducts but similar 24-h excretion rate, with respect to control subjects (Table 3). The plasma concentration of glycation and oxidation free adducts correlated negatively with creatinine clearance for CML (r = −0.94, P < 0.01), MG-H1 (r = −0.79, P < 0.05), and 3DG-H (r = −0.75, P = 0.05), consistent with a decrease in GFR leading to the accumulation of glycation free adducts in plasma. Glycation free adducts are the major form of glycation adduct excretion in the urine of healthy subjects (4). The major glycation free adducts quantitatively excreted in the urine of control subjects and CRF patients were FL, hydroimidazolones, CML, and CEL.

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Table 2

Protein glycation, oxidation, and nitration free adduct concentrations in plasma of normal, healthy control subjects and patients with CRF and ESRD on PD or HD therapya

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Table 3

Renal clearance of free glycation, oxidation, and nitration adducts of normal healthy control subjects and CRF patients, and urinary excretion rates of normal healthy control subjects, CRF patients, and ESRD patients on PD therapya

Glycation, Oxidation, and Nitration Free Adducts in Plasma, Urine, and Dialysate of PD Patients

In PD patients, the concentrations of most protein glycation free adducts in blood plasma were increased, with respect to control subjects. The increases were six-fold for CML, 10-fold for CEL, four-fold for G-H1, 18-fold for MG-H1, eight-fold for 3DG-H, five-fold for pentosidine, and 51% for 3-NT (Table 2). Protein glycation, oxidation, and nitration free adducts were excreted by transfer into the peritoneal cavity and elimination in the peritoneal dialysate and by residual diuresis. Although the clearance of most protein glycation, oxidation, and nitration free adducts in PD patients was decreased with respect to control subjects, the 24-h excretion rates were increased: G-H1 35%; FL, CML, CEL, argpyrimidine, and methylglyoxal-derived lysine dimer (MOLD) approximately two-fold; 3DG-H and pentosidine three-fold; 3-NT four-fold; MG-H1 nine-fold; and MetSO 16-fold (Table 3). The proportion of protein glycation, oxidation, and nitration free adduct excreted in the peritoneal dialysate was 65% for FL, 76% for CML, 71% for CEL, 78% for G-H1, 78% for MG-H1, 75% for 3DG-H, 90% for argpyrimidine, 74% for MOLD, 46% for pentosidine, 96% for MetSO, and 65% for 3-NT (two anuric patients were excluded from this calculation).

The concentration of most protein glycation, oxidation, and nitration free adducts in peritoneal dialysate increased with increasing dwell time, except for MOLD and 3-NT, which did not increase significantly (Figure 3 and Figure 4). From the 4-h PET, MTAC were deduced for 3DG-H and pentosidine free adducts where peritoneal dialysate concentrations were consistent with equilibration with the plasma concentration. For these and other analytes, dialysate/plasma concentration (D/P) ratios were deduced (Table 4). At the 4- and 12-h dwell times, the concentrations of glycation free adducts FL, CML, CEL, G-H1, MG-H1, and MetSO in the peritoneal dialysate exceeded greatly the concentrations of the corresponding free adduct in blood plasma determined at the 2-h dwell time point; hence, the D/P ratios were >1, and there were high peritoneal clearances (Table 4). The concentration of 3DG-H free adduct in peritoneal dialysate at 12 h of dwell time exceeded the concentration of 3DG-H free adduct in blood plasma. These data suggest intraperitoneal formation of glycation and oxidation free adducts within the PD fluid dwell time.

Figure 3.
Figure 3.

Concentrations of protein glycation free adducts in peritoneal dialysate of PD patients showing the effect of PD fluid dwell time. (a) FL. (b) CML. (c) CEL. (d) G-H1. (e) MG-H1. (f) 3DG-H. (g) pentosidine. (h) MOLD. Data are mean ± SEM (n = 8). The dotted line is the corresponding free adduct concentration in plasma at the 2-h dwell time in the peritoneal equilibration test (PET).

Figure 4.
Figure 4.

Protein oxidative and nitrosative markers in plasma of normal healthy control subjects and chronic renal failure (CRF) patients and plasma and dialysate of PD patients. (A and B) Concentration of MetSO and 3-NT free adducts in peritoneal dialysate with dependence on PD fluid dwell time. The dotted line is the corresponding free adduct concentration in plasma at the 2-h dwell time in the PET. (C and D) Concentration of MetSO and 3-NT residues of plasma protein. CON, controls; PD (2), PD patients at 2 h in the PET; HD (0) and HD (4), HD patients before and after a 4-h dialysis session, respectively. Data are mean ± SEM (n = 7 to 8). *P < 0.05, **P < 0.01, and ***P < 0.001, with respect to control subjects.

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Table 4

Dialysate/plasma concentration ratios and mass transfer area coefficients for the PETa

Glycation, Oxidation, and Nitration Free Adducts in Plasma and Dialysate of HD Patients

In HD patients, the plasma concentrations of glycation free adducts before a dialysis session were increased markedly, with respect to control subjects. The increases were three-fold for FL, 12-fold for CML, 21-fold for CEL, seven-fold for G-H1, 40-fold for MG-H1, 10-fold for 3DG-H, four-fold for argpyrimidine, three-fold for MOLD, and five-fold for pentosidine (Table 2). Similar high concentrations of glycation free adducts were found in the HD dialysate at the start of the dialysis session, except for MG-H1 and MOLD, reflecting rapid equilibration of glycation free adducts across the dialysis membrane (Figure 5). Dialysis for 4 h normalized the plasma concentrations of pentosidine and MOLD free adducts and reversed the increases of other glycation free adducts in blood plasma by 54 to 93% but failed to normalize them (Table 2, Figure 5). The plasma concentrations of MetSO and 3-NT free adducts in HD patients were increased but not significantly (Table 2).

Figure 5.
Figure 5.

Concentrations of protein glycation free adducts in plasma and hemodialysate of HD patients. (a) FL. (b) CML. (c) CEL. (d) G-H1. (e) MG-H1. (f) 3DG-H. (g) Pentosidine. (h) MOLD. Key: PCON, control plasma ultrafiltrate; PHD (0), plasma ultrafiltrate of HD patients at the start of dialysis; PHD (4), plasma ultrafiltrate of HD patients after 4 h of dialysis; DL (0), dialysate at the start of dialysis; DL (4), dialysate after a 4-h dialysis session. Data are mean ± SEM (n = 8). **P < 0.01 and ***P < 0.001, plasma concentrations with respect to control subjects. oP < 0.05, ooP < 0.01, and oooP < 0.001 for plasma and dialysate at the end of a dialysis session with respect to the corresponding concentration at the start of the dialysis session; +P < 0.05 and +++P < 0.001 for dialysate at the start of a dialysis session with respect to plasma at the start of dialysis.

The glycation free adduct reduction ratio (RR) was calculated as the fall in concentration between the start and the end of dialysis, expressed as a percentage of the concentration at the start. Comparison was made with the RR for urea, which is an established measure of dialysis adequacy in a single session with a minimum target of 65%. The urea RR was 69 ± 8%. Free adduct RR values were 84 ± 5% for CML and 77 ± 9% for MOLD (greater than urea, P < 0.001 and P < 0.05); 74 ± 8% for MG-H1, 74 ± 10% for 3DG-H, and 72 ± 9% for pentosidine (all not significant); and 39 ± 19% for FL, 54 ± 17% for CEL, 52 ± 9% for G-H1 (lower than urea, P < 0.01, P < 0.05, and P < 0.001).

Glycation, Oxidation, and Nitration Adduct Residues in Plasma Proteins of Normal Control Subjects, CRF Patients, and ESRD Patients on HD or PD Therapy

The plasma protein content of FL residues in control subjects was 0.83 mmol/mol lys. This was decreased to 0.34 mmol/mol lys (−59%) in CRF patients and to 0.39 mmol/mol lys (−53%) in HD patients but was not decreased significantly in PD patients (Figure 6a). FL residues degrade to form CML residues (15). The plasma protein content of CML residues was 0.039 mmol/mol lys in control subjects and increased two-fold in CRF and PD patients and three-fold in HD patients. The plasma protein CML residue content of HD patients did not change during a dialysis session, but it was significantly higher than in CRF and PD patients (Figure 6b). There was a lower plasma protein content of CEL residues than CML residues in control subjects (0.021 mmol/mol lys), and this was increased two-fold in all uremic patients (Figure 6c). The plasma protein content of G-H1 residues was 0.049 mmol/mol arg in control subjects and was increased significantly in HD patients only after a dialysis session (51%; Figure 6d). There was a high plasma protein content of MG-H1 residues in control subjects: 0.67 ± 0.13 mmol/mol arg. This was not increased in CRF patients but was increased approximately two-fold in PD and HD patients. The plasma protein content of MG-H1 residues was also decreased to control levels during a dialysis session in HD patients (Figure 6e). The plasma protein content of 3DG-H residues in control subjects was lower than MG-H1 residues—0.37 ± 0.04 mmol/mol arg—and was increased two-fold in CRF and PD patients and three-fold in HD patients before dialysis (Figure 6f). The protein cross-links, pentosidine, and MOLD were minor glycation adduct residues quantitatively in plasma protein of control subjects (0.0104 ± 0.0016 mmol/mol lys and 0.0087 ± 0.0024 mmol/mol lys, respectively). The plasma protein content of pentosidine residues was increased three-fold in CRF patients, two-fold in PD patients, and four-fold in HD patients before dialysis (Figure 6g). In contrast, there was no significant difference in the plasma protein content of MOLD residues in uremic patients (Figure 6h). The plasma protein content of MetSO residues of control subjects was 0.78 ± 0.05 mmol/mol met and was increased approximately two-fold in CRF patients, PD patients, and HD patients before HD. The plasma protein content of MetSO residues was not increased significantly after an HD session (Figure 4c). The plasma protein content of 3-NT residues of control subjects was 0.0031 ± 0.0009 mmol/mol tyr and was increased three-fold in PD patients and HD patients before HD. The plasma protein content of 3-NT residues was decreased during HD (Figure 4d).

Figure 6.
Figure 6.

Concentrations of glycation adduct residues in plasma protein of normal control subjects, CRF patients, and ESRD patients with PD and HD therapy. (a) FL. (b) CML. (c) CEL. (d) G-H1. (e) MG-H1. (f) 3DG-H. (g) Pentosidine. (h) MOLD. CON, controls; PD (2), PD patients 2 h after change of PD fluid; HD (0) and HD (4), HD patients before and after a 4-h dialysis session, respectively. Data are mean ± SEM (n = 7 to 8). *P < 0.05, **P < 0.01, and ***P < 0.001, with respect to control subjects; oP < 0.05 with respect to CRF and PD patients; +P < 0.05 with respect to HD (0).

Plasma Concentrations of MCP-1 and TGF-β

The plasma concentration of the chemokine MCP-1 was increased significantly in PD and HD patients, with respect to control subjects (Figure 7A); estimates in HD patients after dialysis were corrected for loss of plasma water by normalizing to albumin concentration predialysis. The plasma concentration of soluble TGF-β was increased in HD patients only, before and after a dialysis session, with respect to control subjects and CRF and PD patients (Figure 7B).

Figure 7.
Figure 7.

Plasma concentrations of monocyte chemotactic protein-1 (MCP-1) and TGF-β of normal control subjects, CRF patients, and ESRD patients with PD and HD therapy. (a) MCP-1. (b) TGF-β. CON, controls; PD (2), PD patients 2 h after change of PD fluid; HD (0) and HD (4), HD patients before and after a 4-h dialysis session, respectively. Data are mean ± SEM (n = 7 to 8). *P < 0.05 and **P < 0.01 with respect to control subjects. A correction factor using the individual changes in serum albumin concentrations before and after dialysis was applied to post-HD (4) data to allow for plasma water loss during HD (mean correction factor = 0.88).

Correlation Analysis of Protein Glycation, Oxidation, and Nitration Adducts

The relationships between glycation, oxidation, and nitration adduct residues and free adducts in normal control subjects and CRF, PD, and HD patients were examined. For HD patients, analyte values were predialysis estimates. Homotypic correlations are correlations of glycation, oxidation, and nitration adducts of the same type in different forms or locations, namely plasma protein residues and free adducts in plasma and urine. There were no homotypic correlations in control subjects and CRF patients. In HD and PD patients, the plasma concentration of CML free adduct correlated positively with the concentration of CML residues (r = 0.51, P < 0.05), and the plasma concentration of 3DG-H free adduct correlated positively with the concentration of 3DG-H residues (r = 0.53, P < 0.05). Other correlation analyses of protein glycation, oxidation, and nitration adduct analytes and related variables are given for control subjects (Tables 5 through 7), CRF patients (Tables 8 through 10), and PD and HD patients (Tables 11 and 12). These are provided, without detailed explanation, for substantiation and interpretation in future studies.

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Table 5

Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in normal, healthy control subjects

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Table 6

Correlation of protein glycation, oxidation, and nitration free adducts in plasma in normal, healthy control subjects

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Table 7

Correlation of protein glycation, oxidation, and nitration free adduct urinary excretion rates in plasma in normal, healthy control subjects

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Table 8

Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in CRF patients

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Table 9

Correlation of protein glycation, oxidation, and nitration free adducts in plasma in CRF patients

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Table 10

Correlation of protein glycation, oxidation, and nitration free adduct urinary excretion rates in CRF patients

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Table 11

Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in HD (predialysis) and PD patients

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Table 12

Correlation of protein glycation, oxidation, and nitration free adducts in plasma protein in HD (predialysis) and PD patients

Discussion

In this study, we report the marked accumulation of protein glycation free adducts in CRF and ESRD. Glycation free adducts normally represent <5% of the total glycation adducts in plasma, but this increases substantially for some glycation adducts in uremia—particularly MG-H1. Overall, the total concentration of glycation free adducts in blood plasma increased in the order control<CRF<PD<HD (predialysis) subjects. This pattern of high plasma concentrations of glycation free adducts in uremic patients reflects marked decreased clearance of glycation free adducts in CRF patients. Marked decreased clearance was also generally found in PD patients, in whom the effect was exacerbated by increased glycation free adduct formation—as indicated by increased glycation free adduct excretion. The increased plasma concentration of glycation free adducts in HD may also be due to increased formation and decreased clearance of glycation free adducts.

In CRF patients, compared with creatinine, we found a disproportionate increase in plasma concentration of glycation free adducts and precipitous decline in their clearance while excretion rates were normal—except for FL and pentosidine. This may indicate that there is active tubular secretion of free adducts and this is impaired in CRF. Some other uremic toxins were actively secreted in renal tubules by organic anion transporters (16). The plasma concentration of glycation free adducts correlated negatively with creatinine clearance, but there was no correlation of glycation free adduct clearances with molecular mass (data not shown). This is probably because glycation free adducts all have low molecular mass (<400 Da) and hence are freely filtered by the glomerulus.

In PD therapy, excretion of protein glycation, oxidation, and nitration free adducts occurs mainly by elimination in the peritoneal dialysate—except for pentosidine. D/P ratios were >1 for most analytes (FL, CML, CEL, G-H1, MG-H1, argpyrimidine, and MetSO) at the end of the 4-h PET. Glycation and oxidation free adducts are proposed to pass through the hypothesized small 5-nm pores of micromolecular transport (17), as indeed do amino acids (18), although additional active transport may enhance solute entry into the peritoneal cavity; cf the role of aquaporin-1 in the transport of water (19). The probable explanations for glycation and oxidation free adduct concentrations in the peritoneal dialysate being higher than in plasma are (1) formation of glycation and oxidation free adducts in the peritoneal cavity by glycation and oxidation of amino acids and matrix proteins (with subsequent proteolysis) by glucose degradation products in the PD fluid and (2) active transport of glycation and oxidation free adducts across the endothelium of peritoneal capillaries and vessels. Increased FL (as indicated by the assay of furosine), 3DG-H, other AGE, and oxidative markers in protein of peritoneal dialysate, vascular wall, and mesothelium have been reported (20–24) and were decreased by use of low glucose degradation products that contained PD fluid (24). Glycation and oxidation free adducts probably utilize amino acid transporters to cross the capillary endothelium of the peritoneum (25). This transport may contribute to amino acid loss into the peritoneal dialysate in PD therapy (18). The MTAC value for creatinine was similar but pentosidine was higher than reported values (20); the MTAC for 3DG-H was lower than that of creatinine.

The excretion of most protein glycation, oxidation, and nitration free adducts was increased in PD patients, with excretions of MetSO and MG-H1 exceptionally high. This may reflect increased protein modification in carbonyl, oxidative, and nitrosative stress of uremia. With reduced functional kidney mass and elimination via the peritoneal cavity, MetSO escapes metabolism by renal MetSO reductase (26); hence, the excretion rate and clearance of MetSO were increased markedly (16-fold). The nine-fold increase in excretion of MG-H1 in PD patients was the most marked increase found for the excretion of a glycation free adduct. Increased protein glycation by MG in ESRD is expected because of the high concentrations of MG found in the plasma of ESRD patients and also in PD fluids (9–11,27).

HD therapy of ESRD patients removed glycation free adducts from the circulation by passage through the dialysis membrane. Protein glycation and oxidation free adducts have molecular masses <400 Da and therefore are small molecular mass uremic toxins (2). At the end of the dialysis session, protein glycation free adducts were decreased equally in both plasma and hemodialysate but not normalized to control levels—except for pentosidine and MOLD. RR values of glycation free adducts ranged from 39 to 84%, demonstrating heterogeneity of clearance, and were lower than urea for only FL, CEL, and G-H1. Increased frequency of HD with higher flux may improve the elimination of glycation free adducts.

Most protein glycation, oxidation, and nitration adduct moieties in plasma are modified amino acid residues of plasma protein and are not eliminated efficiently by PD and HD therapy. The glycation adduct residue content of plasma protein may be affected by hypoalbuminemia in renal failure and related changes in albumin turnover (Table 1). Hypoalbuminemia in PD patients is caused by loss of albumin in urine and peritoneal dialysate and is countered by increased albumin synthesis—inflammation and poor nutrition limit this response (28). This leads to a decreased half-life of albumin. Glycation of proteins in CRF and ESRD is expected to increase because of increased concentrations of α-oxoaldehyde glycating agents (9,10,27). The concentration of FL residues may be decreased in CRF and HD patients by increased oxidative stress with conversion of FL to CML residues (15). High levels of glycoxidation adducts (pentosidine and CML) in HD may reflect increased oxidative stress, relative to PD (29). The concentrations of MG-H1, MetSO, and 3-NT residues were normalized in HD patients after dialysis. 3-NT–modified albumin undergoes preferential endothelial transcytosis (30), which may be increased in the inflammatory response to dialysis. MetSO residues may be reduced to methionine residues by MetSO reductase during endothelial transcytosis. MG-H1–modified proteins may be bound by activated monocytes in the dialysis session and degraded (31). The concentration of MG-H1 residues in plasma protein of PD and HD patients correlated negatively with the plasma concentration of albumin (Table 11). Decreased albumin concentration is associated with poor survival of ESRD patients (23).

The few homotypic correlations of glycation adduct protein residues and free adducts reflect important nonvascular sources of glycation free adduct formation: Degradation of glycated tissue proteins (4), adsorption from digested food protein (8), and glycation of amino acids (13). Major sources of glycation and oxidation free adducts are likely to be cellular proteolysis and digested food (4,32).

The plasma concentration of MCP-1 was increased significantly in PD and HD patients, and the plasma concentration of soluble TGF-β was increased in HD patients only, confirming previous reports (33–36). MCP-1 is associated with macrophage infiltration into the peritoneal cavity of PD patients and correlates with soluble adhesion molecule expression in HD (33,37). TGF-β is a key mediator of renal fibrosis in progressive renal failure and ESRD. The concentration of MG-H1 and 3DG-H residues in plasma protein correlated positively with TGF-β in CRF patients. Increased expression of TGF-β in CRF is associated with a progressive decline of residual renal function and increased fibrosis leading to ESRD. Although protein glycation, oxidation, and nitration free adducts are eliminated by dialysis, plasma MCP-1 and TGF-β concentrations were maintained by dialysis therapy. The inflammation of dialysis and the increase AGE may be linked, but there is no direct relationship with the clearance of AGE free adducts.

Acknowledgments

We thank Baxter Healthcare Inc (Deerfield, IL) for support for a PhD studentship for S.A. and the Wellcome Trust for support for our protein biomarker research. We thank the staff and patients of the Renal Unit, St. Bartholomew's Hospital, for their participation in this study.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Profound Mishandling of Protein Glycation Degradation Products in Uremia and Dialysis
Stamatina Agalou, Naila Ahmed, Roya Babaei-Jadidi, Anne Dawnay, Paul J. Thornalley
JASN May 2005, 16 (5) 1471-1485; DOI: 10.1681/ASN.2004080635
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