Immunohistochemical detection of an AGE,a ligand for macrophage receptor,in peritoneum of CAPD patients

BACKGROUND: In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), ultrafiltration (UF) capacity of peritoneal membrane may be impaired due to accumulation of advanced glycation end products (AGEs). This study aimed to elucidate the characteristics of a novel anti-AGE antibody, ODI-GLC19, and to demonstrate AGE accumulation in the peritoneum of CAPD patients using the antibody. METHODS: A monoclonal anti-AGE antibody (ODI-GLC19) was prepared by immunizing female balb/c mice using D-glucose-modified keyhole limpet hemocyanin. The characteristics of ODI-GLC19 were determined by enzyme-linked immunosorbent assay and receptor binding inhibition assay. Immunohistochemistry using ODI-GLC19 was performed to detect AGE in peritoneal tissues obtained from patients with nonrenal disease, and CAPD patients with normal and low UF. RESULTS: ODI-GLC19 reacted with glycolaldehyde-modified BSA (GA-BSA) and glucose-modified BSA (GLC-BSA), but not with imidazolone or N epsilon-(carboxymethyl)lysine. GA-BSA and GLC-BSA strongly bound to cultured macrophages. Time-dependent recognition of newly formed GA-BSA by ODI-GLC19 was similar to that by macrophages. The binding of GA-BSA to macrophages was inhibited by ODI-GLC19 in a dose-dependent manner. Immunohistochemical studies revealed that ODI-GLC19-positive AGE was exclusively detected in peritoneal cells including macrophages, and its staining intensity was more prominent in the peritoneum of CAPD patients, especially with low UF, than in patients with nonrenal disease. CONCLUSIONS: A novel monoclonal anti-AGE antibody, ODI-GLC19, recognizes a ligand for an AGE receptor on macrophages. Incorporation of AGE into peritoneal cells including macrophages may be involved in progressive peritoneal dysfunction in CAPD patients.     

Differential effects of advanced glycation end-products on renal tubular cell inflammation

Aim: The authors recently showed that advanced glycation end‐products (AGE) in the form of glycated albumin (GA) upregulated renal tubular expression of interleukin (IL)‐8 and soluble intercellular adhesion molecule‐1 (sICAM‐1), but not other important cytokines known to mediate diabetic nephropathy. This implies that other molecules such as the carbonyl intermediates of AGE or other modified protein lysine‐albumin may participate in diabetic tubular injury. Methods: Human proximal tubular epithelial cells (PTEC) were growth‐arrested and exposed to methylglyoxal (MG), MG‐bovine serum albumin (BSA)‐AGE, carboxymethyllysine (CML)‐BSA, AGE‐BSA or BSA with or without prior addition of rosiglitazone that was previously shown to attenuate the pro‐inflammatory effect of GA alone. Results: MG‐BSA‐AGE and AGE‐BSA upregulated tubular expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)‐β, and vascular endothelial growth factor (VEGF), whereas CML‐BSA stimulated expression of IL‐6, CCL‐2, CTGF, TGF‐β and VEGF. These AGE compounds also activated nuclear factor (NF)‐κB and their effects were attenuated by pre‐incubation with anti‐RAGE antibody. MG and BSA did not affect the expression of any of these molecules. Rosiglitazone did not affect the in vitro biological effects of MG, MG‐BSA‐AGE, AGE‐BSA or CML‐BSA on PTEC. Conclusion: AGE exhibit differential inflammatory and fibrotic effects on PTEC via RAGE activation and NF‐κB signal transduction. Rosiglitazone had no effect on these responses. Further investigations on compounds that nullify the downstream effects of these AGE are warranted.     

Autoantibody against N(epsilon)-(carboxymethyl)lysine: an advanced glycation end product of the Maillard reaction.

Prolonged incubation of proteins with reducing sugar produces advanced glycation end products (AGEs), which are implicated as factors for aging and diabetic complications. We previously demonstrated the presence of N(epsilon)-(carboxymethyl)lysine (CML), one of the main AGE structures, in human and animal tissues using a monoclonal anti-CML antibody (6D12). These findings suggest that CML structures present in vivo could serve as immunogens to generate autoantibodies. This suggestion was tested in the present study. First, plasma samples from diabetic rats reacted positively with AGE bovine serum albumin (BSA). These reactivities increased with the duration of diabetic states and were inhibited specifically by CML-BSA. Second, a fraction purified from plasma of diabetic patients, which bound to AGE-BSA, showed a positive reaction to CML-BSA and furthermore also to human lens proteins, which are known to undergo CML modification in vivo. Finally, patients with renal failure caused by diabetes or nondiabetic pathologies had a higher autoantibody activity against CML structure than that in normal subjects or diabetic patients without renal failure. These results indicate that CML accumulated in vivo serves as an immunological epitope to generate an autoantibody specific for CML that might be used as a potential marker for diabetic nephropathy or chronic renal failure.     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

Production of N(epsilon)-(carboxymethyl)lysine is impaired in mice deficient in NADPH oxidase: a role for phagocyte-derived oxidants in the formation of advanced glycation end products during inflammation

Advanced glycation end products (AGEs) derived from glucose are implicated in the pathogenesis of diabetic vascular disease. However, many lines of evidence suggest that other pathways also promote AGE formation. One potential mechanism involves oxidants produced by the NADPH oxidase of neutrophils, monocytes, and macrophages. In vitro studies have demonstrated that glycolaldehyde, a product of serine oxidation, reacts with proteins to form N(epsilon)-(carboxymethyl)lysine (CML), a chemically well-characterized AGE. We used mice deficient in phagocyte NADPH oxidase (gp91-phox(-/-)) to explore the role of oxidants in AGE production in isolated neutrophils and intact animals. Activated neutrophils harvested from wild-type mice generated CML on ribonuclease A (RNase A), a model protein, by a pathway that required L-serine. CML formation by gp91-phox(-/-) neutrophils was impaired, suggesting that oxidants produced by phagocyte NADPH oxidase contribute to the cellular formation of AGEs. To determine whether these observations are physiologically relevant, we used isotope-dilution gas chromatography/mass spectrometry to quantify levels of protein-bound CML in mice suffering from acute peritoneal inflammation. Phagocytes from the gp91-phox(-/-) mice contained much lower levels of CML than those from the wild-type mice. Therefore, oxidants generated by phagocyte NADPH oxidase may play a role in AGE formation in vivo by a glucose-independent pathway.     

晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响

目的：探讨晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响,以期揭示晚期糖化终末产物在骨质疏松发病中的作用。方法：用体外制备的AGE-牛血清白蛋白（AGE-BSA）加入人成骨细胞培养体系,观察不同浓度AGE和不同作用时间,对人成骨细胞增殖及分泌白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的影响。结果：加入AGE-BSA24h后,低浓度抑制成骨细胞增殖;培养48、72h后,浓度依赖性的促增殖作用增加。加入AGE-BSA48h,100μg/ml AGE-BSA促进成骨细胞分泌IL-6。200μg/ml-100μg/ml对IL-6及TNF-α的分泌均无显影响。结论：晚期糖化终末产物对人成骨细胞表现出在较高浓度时,仍具有很强的促增殖活性,提示人对高浓度AGE的耐受性增强。AGE在较高浓度对人成骨细胞分泌IL-6和TNF-α无显影响。提示由随龄增加的AGE在老年人引起的成骨细胞介导的骨吸收没有明显增加,与老年性骨质疏松为低转换型结果相一致。     

Radioreceptor assay for advanced glycosylation end products.

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1 mu CI/ml [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 micrograms/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid-BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of the inhibitory effect of OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-yla cetanilide] on advanced glycation end product and advanced lipoxidation end product formation

The accumulation in uremic plasma of reactive carbonyl compounds (RCO) derived from both carbohydrates and lipids ("carbonyl stress") contributes to uremic toxicity by accelerating the advanced glycation and lipoxidation of proteins. It was previously demonstrated that OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo- thiazolidin-5-ylacetanilide] inhibited the in vitro formation of advanced glycation end products (AGE) in uremic plasma. This study was designed to elucidate the mechanism of action of OPB-9195 by further delineating the AGE and advanced lipoxidation end product (ALE) precursors targeted by this drug. The inhibitory effects of OPB-9195 on the formation of two AGE (N:epsilon-carboxymethyllysine and pentosidine) on bovine serum albumin incubated with various AGE precursors were examined. Inhibition of N:epsilon-carboxymethyllysine and pentosidine formation with OPB-9195 was more efficient than with aminoguanidine. OPB-9195 also proved effective in blocking the carbonyl amine chemical processes involved in the formation of two ALE (malondialdehyde-lysine and 4-hydroxynonenal-protein adduct). The efficiency of OPB-9195 was similar to that of aminoguanidine. When glucose-based peritoneal dialysis fluid was incubated in the presence of OPB-9195, a similar inhibition of AGE formation was observed. The direct effect of OPB-9195 on major glucose-derived RCO in peritoneal dialysis fluids was then evaluated. The effects of OPB-9195 could be accounted for by its ability to trap RCO. The concentrations of three major glucose-derived RCO (glyoxal, methylglyoxal, and 3-deoxy-glucosone) were significantly lower in the presence of OPB-9195 than in its absence. Aminoguanidine had a similar effect. In conclusion, OPB-9195 inhibits both AGE and ALE formation, probably through its ability to trap RCO. OPB-9195 might prove to be a useful tool to inhibit some of the effects of RCO-related uremic toxicity.     

Advanced glycation end-products and peritoneal sclerosis

Long-term continuous ambulatory peritoneal dialysis (CAPD) often causes peritoneal fibrosis and sclerosis with a loss of function, and some CAPD patients develop sclerosing encapsulating peritonitis. Glucose-based peritoneal dialysis fluids readily produce glucose degradation products by heat sterilization, and glucose degradation products accelerate the formation of advanced glycation end-products (AGE) in the peritoneal cavity. The accumulation of AGE is observed in peritoneal mesothelial and submesothelial layers in CAPD patients, accompanied by enhanced expression of various growth factors and peritoneal thickening. The expression of transforming growth factor-beta1 (TGF-beta1), macrophage-colony stimulating factor, and vascular endothelial growth factor (VEGF) is distributed in the peritoneum similarly to that of AGE. In CAPD patients with low ultrafiltration (UF) capacity, peritoneal membrane is thickened owing to an increase in the number of cells such as fibroblasts and macrophages and collagen in the submesothelial layer. AGE is detected in the fibroblasts and macrophages as well as degenerated collagen. These cells in the submucosal layer are almost positive for the receptor for AGE (RAGE) and uptake AGE. The intensity of AGE accumulation and the expression of growth factors are associated with the severity of UF impairment. In fact, the accumulation of AGE and the expression of growth factors are recognized most markedly in the peritoneum of CAPD patients with low UF and sclerosing encapsulating peritonitis. In conclusion, long-time CAPD with heat-sterilized peritoneal dialysis fluid promotes AGE accumulation in the peritoneal membrane and alteration in peritoneal cell function and dialysis quality, followed by peritoneal sclerosis, and, finally, sclerosing encapsulating peritonitis.     

Glycation free adduct accumulation in renal disease: the new AGE

Glycation adducts formed in the later stages of protein glycation reactions, advanced glycation endproducts (AGEs), are a class of uraemic toxin. Protein glycation was viewed originally as a post-translational modification that accumulated mostly on extracellular proteins. We now know that AGE residues are also formed on short-lived cellular and extracellular proteins. Cellular proteolysis forms AGE free adducts from these proteins, which are released into plasma for urinary excretion. AGE free adducts are also absorbed from food. AGE free adducts are the major molecular form by which AGEs are excreted in urine. They normally have high renal clearance, but this declines markedly in chronic renal failure patients, leading to profound increases in plasma AGE free adducts. Accumulation of plasma AGE free adducts is increased further in end stage renal disease patients on peritoneal dialysis and haemodialysis by increased AGE formation. The impact of AGEs absorbed from food is probably most marked for undialysed patients with mild uraemia. The toxicity of AGEs has been associated with resistance of the extracellular matrix to proteolysis and AGE receptor-mediated responses. AGE free adducts may also contribute to vascular disease in uraemia. They represent an important new age for glycation research in nephrology.     

Restriction of dietary glycotoxins reduces excessive advanced glycation end products in renal failure patients

Advanced glycation endproduct (AGE) levels are elevated in renal failure patients and may contribute to the excessive cardiovascular disease in this population. Diet-derived AGE are major contributors to the total body AGE pool. It was postulated that a reduction in dietary AGE intake might impact on the high circulating AGE levels in renal failure patients. Twenty-six nondiabetic renal failure patients on maintenance peritoneal dialysis were randomized to either a high or a low AGE diet for 4 wk. Three-day dietary records, fasting blood, 24-h urine, and dialysis fluid collections were obtained at baseline and end of study. AGE levels were determined by ELISA for N(epsilon)-carboxymethyl-lysine (CML) and methylglyoxal-derivatives (MG). Eighteen patients completed the study. Low dietary AGE intake decreased serum CML (34%; P < 0.002), serum MG (35%; P < 0.008), CML-LDL (28%; P < 0.011), CML-apoB (25%; P < 0.028), dialysate CML (39%; P < 0.03), and dialysate MG output (40%; P < 0.04). High dietary AGE intake increased serum CML (29%; P < 0.028), serum MG (26%; P < 0.09), CML-LDL (50%; P < 0.011), CML-apoB (67%; P < 0.028), and dialysate CML output (27%; P < 0.01). Serum AGE correlated with BUN (r = 0.6, P < 0.002 for CML; r = 0.4, P < 0.05 for MG), serum creatinine (r = 0.76, P < 0.05 for CML; r = 0.55, P < 0.004 for MG), total protein (r = 0.4, P < 0.05 for CML; r = 0.4, P < 0.05 for MG), albumin (r = 0.4, P < 0.02 for CML; r = 0.4, P < 0.05 for MG), and phosphorus (r = 0.5, P < 0.006 for CML; r = 0.5, P < 0.01 for MG). It is concluded that dietary glycotoxins contribute significantly to the elevated AGE levels in renal failure patients. Moreover, dietary restriction of AGE is an effective and feasible method to reduce excess toxic AGE and possibly cardiovascular associated mortality.     

Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells.

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.     

Effect of dwell time on carbonyl stress using icodextrin and amino acid peritoneal dialysis fluids

BACKGROUND: Deterioration of the peritoneal membrane limits the technical survival of peritoneal dialysis (PD). Advanced glycation of the membrane has been incriminated in this evolution. Advanced glycation end products (AGEs) develop under the influence of glucose and of its degradation products, mainly reactive carbonyl compounds (RCOs) such as glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG). The present study was undertaken to evaluate the impact of recently developed glucose-free PD fluids on AGE generation. METHODS: Recently developed glucose-free PD fluids containing either icodextrin or amino acids were investigated. GO, MGO, and 3-DG [high-performance liquid chromatography (HPLC)] and total RCOs (spectrophotometry) were measured in fresh solutions and in effluents after various dwell duration. The AGE formation potential of PD fluids and effluents was assessed by incubation at 37 degrees C, for one week, with bovine serum albumin and by the eventual measurement of pentosidine (HPLC) and Nepsilon-carboxymethyllysine (CML; gas chromatography/mass spectrometry). RESULTS: GO, MGO, and 3-DG (P < 0. 001) as well as total RCOs levels (P < 0.01) were significantly lower in icodextrin and amino acid PD fluid than in commercial, heat-sterilized, 1.36% glucose PD fluid. Pentosidine and CML generation were also significantly lower (P < 0.001) in icodextrin and amino acid PD fluid than in conventional 1.36% glucose PD fluid. The levels of total RCOs, however, increased in icodextrin and amino acid PD fluid effluents with dwell time. AGE formation potential rose accordingly, as demonstrated by a parallel increase in the generation of pentosidine and CML during incubation of PD effluents. CONCLUSION: The present data demonstrate lower RCO contents and AGE formation potential in fresh icodextrin and amino acid PD fluids than in fresh heat-sterilized glucose PD fluids. However, this difference decreases progressively during dwell time, mainly as a result of the influx of total RCOs.     

Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions

Advanced glycation end products (AGE) include a variety of protein adducts whose accumulation has been implicated in tissue damage associated with diabetic nephropathy (DN). It was recently demonstrated that among AGE, glycoxidation products, whose formation is closely linked to oxidation, such as carboxymethyllysine (CML) and pentosidine, accumulate in expanded mesangial matrix and nodular lesions in DN, in colocalization with malondialdehyde-lysine (MDA-lysine), a lipoxidation product, whereas pyrraline, another AGE structure whose deposition is rather independent from oxidative stress, was not found within diabetic glomeruli. Because CML, pentosidine, and MDA-lysine are all formed under oxidative stress by carbonyl amine chemistry between protein amino group and carbonyl compounds, their colocalization suggests a local oxidative stress and increased protein carbonyl modification in diabetic glomerular lesions. To address this hypothesis, human renal tissues from patients with DN or IgA nephropathy were examined with specific antibodies to characterize most, if not all, carbonyl modifications of proteins by autoxidation products of carbohydrates, lipids, and amino acids: CML (derived from carbohydrates, lipids, and amino acid), pentosidine (derived from carbohydrates), MDA-lysine (derived from lipids), 4-hydroxynonenal-protein adduct (derived from lipids), and acrolein-protein adduct (derived from lipids and amino acid). All of the protein adducts were identified in expanded mesangial matrix and nodular lesions in DN. In IgA nephropathy, another primary glomerular disease leading to end-stage renal failure, despite positive staining for MDA-lysine and 4-hydroxynonenal-protein adduct in the expanded mesangial area, CML, pentosidine, and acrolein-protein adduct immunoreactivities were only faint in glomeruli. These data suggest a broad derangement in nonenzymatic biochemistry in diabetic glomerular lesions, and implicate an increased local oxidative stress and carbonyl modification of proteins in diabetic glomerular tissue damage ("carbonyl stress").     

糖基化终产物对肾间质成纤维细胞DNA损伤的影响

目的 探讨糖基化终产物(AGE)对肾间质成纤维细胞DNA损伤的影响及抗氧化剂的干预作用&#65377; 方法 不同浓度AGE修饰的牛血清白蛋白(AGE-BSA)作用于NRK-49F细胞24 h,普通培养基和牛血清白蛋白 (BSA)作为对照&#65377;N-乙酰半胱氨酸(NAC)预处理细胞以观察抗氧化剂的干预作用&#65377;AlamarBlue还原法测定细胞增殖活力,单细胞凝胶电泳(彗星实验)测定细胞DNA损伤&#65377; 结果 与对照组比较,AGE作用后的细胞增殖活力下降;DNA迁移距离(彗星尾长)增加,差异有统计学意义,且2者呈剂量依赖关系&#65377;各浓度BSA作用后对细胞增殖活力无明显影响;彗星尾长无明显变化&#65377;与相同浓度BSA相比, 400&#65380;800 mg/L AGE分别使AlamarBlue还原率下降14%和15%(P < 0.05);200&#65380;400&#65380;800 mg/L AGE组彗星尾长分别为BSA组的1.45&#65380;2.12&#65380;2.71倍(P < 0.05)&#65377;与未用NAC预处理组相比,NAC预处理可使AlamarBlue还原率上升[(45.15±0.93)% 比(38.40±0.81)%,P < 0.05];彗星尾长变短[(10.02±4.54) μm比(13.48±5.32) μm,P < 0.05]&#65377; 结论 AGE可导致肾间质成纤维细胞DNA损伤,使用抗氧化剂可有效减轻AGE导致的DNA损伤&#65377;细胞内氧化应激增强可能是AGE引起肾间质成纤维细胞DNA损伤的作用机制之一。     

Reduced systemic advanced glycation end products in children receiving peritoneal dialysis with low glucose degradation product content.

BACKGROUND: Glucose degradation products (GDP) in peritoneal dialysis (PD) solutions are toxic to the peritoneal membrane and promote the formation of advanced glycation end products (AGE), which contribute to accelerated atherosclerosis and amyloidosis. Double chamber PD solutions have a markedly reduced GDP content. METHODS: We analysed GDP and AGE kinetics in 21 children (7 months to 18 years) on automated PD in a prospective multicentre trial with randomized administration of single chamber, high-GDP and double-chamber, low-GDP dialysis solution for 12 weeks each. Total AGE fluorescence, carboxymethyllysine (CML, ELISA) and 3-deoxyglucosone (3-DG, HPLC) were measured in plasma and PD effluent during a 4 h peritoneal equilibration test. Plasma AGE profiles were assessed by size selective gel permeation chromatography and compared with 23 healthy controls. RESULTS: Initial effluent 3-DG concentrations were 140+/-55 and 25+/-4 micromol/l with high- and low-GDP PD fluid, respectively and declined to 53+/-32 and 7+/-2 micromol/l within 4 h dwell time (P<0.001). The ex vivo AGE generating capacity was three times higher with the high-GDP solution and decreased significantly with dwell time. Plasma AGE levels were 1.8-7.4-fold above those of healthy controls; the elevation was most marked for the small molecular fraction (<2 kDa). Plasma AGE and CML levels were significantly higher after 12 weeks exposure to high-GDP solution (20991+/-4145 AU and 1505+/-617 ng/ml) than following treatment with low-GDP fluid (17518+/-4676 AU and 1151+/-438 ng/ml; both P<0.05). Four hour AGE clearance was higher with low-GDP solution (0.74+/-0.3 vs 0.44+/-0.15 ml/min*1.73 m2, P<0.01). CONCLUSION: GDP are rapidly absorbed from the peritoneal cavity. Administration of PD solutions with low-GDP content reduces plasma AGE levels and may thus improve the cardiovascular risk profile of dialysed children.