糖基化终产物对人肾小球系膜细胞氧化应激及MCP-1表达的影响及机制

目的：探讨体外培养条件下糖基化终产物（AGEs）对人肾小球系膜细胞（HRMCs）中糖基化终产物受体（RAGE）、氧化应激及单核细胞趋化因子-1（MCP-1）表达的影响。方法：将HRMCs与不同浓度的糖化牛血清白蛋白（AGE-BSA）和牛血清白蛋白（BSA）共同培养,或与同一质量浓度的AGE-BSA和BSA共同培养不同时间,以中和抗RAGE抗体封闭细胞膜上RAGE;采用细胞免疫化学法检测AGEs对HRMCs中RAGE表达的影响,流式细胞术检测细胞内活性氧（ROS）,半定量逆转录-聚合酶链反应（RT-PCR）法检测MCP-1 mRNA的表达。结果：在HRMCs中AGE-BSA能够促进RAGE的表达,并以时间和剂量依赖方式促进HRMCs中ROS及MCP-1的表达;ROS及MCP-1的表达水平在加入不同浓度（50、100、200、400 mg/L）的AGE-BSA作用48 h后以及加入质量浓度为200 mg/L的AGE-BSA作用不同时间（12、24、48、72 h）后,较相应质量浓度或时间的BSA组和对照组均明显升高（P〈0.05）;抗RAGE抗体干预后能够部分抑制AGE-BSA诱导ROS及MCP-1的表达,而人IgG没有这种作用。结论：AGEs通过RAGE激活氧化应激效应诱导MCP-1的表达上调,是糖尿病肾病发生发展的可能机制。     

Advanced glycation end products inhibit adhesion ability of differentiated podocytes in a neuropilin-1-dependent manner

Podocyte injury can occur by a number of stimuli. Maintaining of an intact podocyte structure is essential for glomerular filtration; therefore, podocyte damage severely impairs renal function. Recently, we have reported that addition of glycated BSA [advanced glycation end products (AGE)-BSA] to differentiated murine podocytes inhibited neuropilin-1 (NRP1) expression and dramatically influenced podocyte migration ability (Bondeva T, Ruster C, Franke S, Hammerschmid E, Klagsbrun M, Cohen CD, Wolf G. Kidney Int 75: 605-616, 2009; Bondeva T, Wolf G. Am J Nephrol 30: 336-345, 2009). The present study analyzes the influence of AGEs and NRP1 on podocyte adhesion and cytoskeleton reorganization. We show that treatment with AGE-BSA significantly reduced podocyte adhesion to collagen IV, laminin, and fibronectin compared with Co-BSA (nonglycated BSA)-incubated cells, which was further augmented by transient inhibition of NRP1 expression using NRP1 short interference (si) RNA. On the other hand, forced overexpression of NRP1 markedly increased the adhesion ability of podocytes to the ECMs despite the AGE-BSA treatment. No changes were observed when podocyte adhesion to collagen I was assayed. These findings were also manifested with disorganization of podocyte actin stress fibers and decreased lamellipodia formation processes due to AGE-BSA treatment or NRP1 suppression. In addition, AGE-BSA or suppression of NRP1 both reduced the phosphorylation of focal adhesion kinase (FAK) and Erk1/2 in PMA-stimulated differentiated podocytes. Analysis of RhoA family GTPase activity demonstrated that treatment with AGE-BSA or NRP1 depletion inhibited as well the activation of the Rac-1 and Cdc42 but did not affect RhoA activity. All these effects were reversed by forced overexpression of full-length NRP1 cloned into the pcDNA3 vector in differentiated podocytes. Our study demonstrates that AGEs, in part via suppression of NRP1 expression, decreased podocyte adhesion and contribute to reduction of Rac-1 and Cdc42 GTPase activity. These effects may be further responsible for the podocytes damage and loss in diabetic nephropathy. Our findings suggest a role for NRP1 in regulating the podocyte actin cytoskeleton, and therefore reduction of NRP1 expression could be critical for podocyte function.     

The amino-terminal domains of the ezrin,radixin, and moesin (ERM) proteins bind advanced glycation end products,an interaction that may play a role in the development of diabetic complications

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography. NH2-terminal and internal sequencing identified the proteins as the NH2-terminal domains of ezrin, radixin, and moesin (ERM proteins). Using BIAcore biosensor analysis, human N-ezrin-(1-324) bound to immobilized AGE-BSA with a KD of 5.3 +/- 2.1 x 10 -7 m, whereas full-length ezrin-(1-586) and C-ezrin-(323-586) did not bind. Other glycated proteins such as AGE-RNase, N in -carboxymethyllysine (CML)-BSA, and glycated human serum albumin isolated from hyperglycemic diabetic sera competed with the immobilized AGE-BSA for binding to N-ezrin, but non-glycated BSA and RNase did not. Thus N-ezrin binds to AGEs in a glycation- and concentration-dependent manner. Phosphorylated ezrin plays a crucial role in cell shape changes, cell attachment, and cell adhesion. The effect of AGE-BSA on ezrin function was studied in a tubulogenesis model in which LLC-PK1 cell tubule formation is dependent on phosphorylated ezrin. Addition of AGE-BSA completely inhibited the ability of the cells to produce tubules. Furthermore, in vitro tyrosine phosphorylation of N-ezrin and ezrin was also inhibited by AGE-BSA. These proteins represent a novel family of intracellular binding molecules for glycated proteins and provide a potential new target for therapeutic intervention in the prevention or treatment of diabetic complications.     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA.     

Lack of recognition of Nepsilon-(carboxymethyl)lysine by the mouse liver reticulo-endothelial system: implications for pathophysiology

Advanced glycation end products (AGEs) are known to be associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer's disease, uremia, as well as with normal aging. This study was undertaken to investigate whether Nepsilon-(carboxymethyl)lysine (CML), a major structure among numerous AGEs, engenders hepatic AGE clearance. For this purpose uptake of BSA substituted with heterogeneous AGEs or with CML only was monitored in vivo and in cultured hepatic scavenger cells. Here, we show that following intravenous administration of 125I-AGE-BSA and 125I-CML-BSA, blood radioactivity was reduced by 50% after 50s and >100 min, respectively. Recoveries from the circulation at 6 min after injection were: 5% for AGE-BSA, 95% for CML-BSA. More than 80% of the injected AGE-BSA was recovered from the liver. AGE-BSA, but not CML-BSA, was avidly endocytosed by cultured liver scavenger cells. Our results suggest that CML does not engender AGE-BSA clearance. Macromolecules substituted with CML only may escape elimination and cause pathological effects.     

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.     

Oxidative stress-inducing carbonyl compounds from common foods: novel mediators of cellular dysfunction

BACKGROUND: The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE. MATERIALS AND METHODS: Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNFalpha), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250 degrees C, for 10 min, along with synthetic AGEs. RESULTS: Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA), AGE-BSA, and AGE-low density lipoprotein (AGE-LDL), induced a dose- and time-dependent depletion of GSH (()60-75%, p, 0.01) and an increase in GPx activity (()500-600%, p < 0.01), consistent with marked TNFalpha and cross-link formation (p < 0.05); this contrasted with the low bioreactivity of starch/vegetable AGE-extracts, which was similar to that of control BSA and CML- BSA and BSA (p:NS). Anti-AGE-R1,2,3 and -RAGE IgG each inhibited cell-associated (125) I-dAGE by approximately 30-55%; GSH/GPx were effectively blocked by N-acetyl-cysteine (NAC, 800 uM, p < 0.01) and aminoguanidine-HCl (AG, 100 uM, p < 0.01). CONCLUSION: Thus, food-derived AGE, prior to absorption, contain potent carbonyl species, that can induce oxidative stress and promote inflammatory signals.     

Identification of AGE-precursors and AGE formation in glycation-induced BSA peptides

The glycation of BSA leads to protein/peptide modifications that result in the formation of AGEs. AGEs react with the amino groups of N-terminal amino acid residues, particularly arginine and lysine residues. Enhanced AGE formation exists in the blood and tissues of diabetics, as well as in aging and other disorders. The Identification of AGEs is of great importance. Mass spectrometry has been applied to identify and structurally elucidate AGEs. Here, we report on the identification of AGE- peptides and AGE-precursors based on relative mass changes as a result of specific AGE formation. HPLC-ESIMS, ESI-MS/MS, and the Mascot database were used. The relative mass changes due to the specific type of AGE formation were added to the identified peptides followed by a manual search of the glycated samples, which resulted in the identification of seven peptides for the formation of five AGEs, namely CML, pyrraline, imidazolone A, imidazolone B, and AFGP. Four glycated peptides (FPK, ECCDKPLLEK, IETMR, and HLVDEPQNLIK) were identified in the formation of AGE-precursors.     

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.     

Advanced glycation end-product receptor interactions on microvascular cells occur within caveolin-rich membrane domains.

Advanced glycation end products (AGEs) have an important role in diabetic complications, with many responses mediated through AGE-receptors. The current study has investigated the binding and uptake of AGEs by retinal microvascular endothelium in an attempt to understand the nature of AGE-interaction with receptors on the cell surface. There has been special emphasis placed on the R1, R2, and R3 components of AGE-receptor complex (AGE-RC) and their localization to caveolin-rich membrane domains. Retinal microvascular endothelial cells (RMECs) were exposed to either AGE-modified BSA (AGE-BSA) or native BSA conjugated to colloidal gold (gAGE, gBSA) for various time periods, fixed, and processed for transmission electron microscopy (TEM). Localization of AGE-RC components in caveolae was investigated using confocal microscopy and ultrastructural immunogold labeling. Caveolae were extracted from RMECs using differential Triton X-100 solubility, and Western analysis was conducted to test for caveolae enrichment and the presence of AGE-RC complex components. Ligand blots determined 125I-AGE-BSA binding to caveolae-enriched extracts. Colloidal gold conjugates of AGE-BSA bound to caveolae and were internalized to be trafficked to lysosomal-like compartments. AGE-receptor complex components were significantly enriched within caveolae. The data suggest that AGEs interact with their receptors within caveolae. It is significant that the AGE-R complex localizes to these organelles, because this may have implications for AGE binding, internalization, signal transduction, and the modulation of AGE-receptor-mediated vascular cell dysfunction.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched.Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner.Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.     

Eriodictyol and naringenin inhibit the formation of AGEs: An in vitro and molecular interaction study

Maillard reaction occurs between the carbonyl group of reducing sugars and the free amino groups of protein, which eventually results in the formation and accumulation of advanced glycation end products (AGEs) irreversibly. Excessive production of AGEs is associated with many diseases, such as Alzheimer disease, neuropathy, retinopathy, and nephropathy. In this study, the effects of eriodictyol and naringenin on the inhibition of AGEs were studied with bovine serum albumin (BSA)–methylglyoxal (MGO) model by spectroscopic techniques and molecular docking methods. The fluorescence spectroscopy results suggested that eriodictyol and naringenin could inhibit the formation of AGEs. Circular dichroism (CD) studies indicated that eriodictyol and naringenin could stabilize the structure of BSA and inhibit the formation of AGEs. The molecular docking results demonstrated that eriodictyol formed two hydrogen bonds with Lys 350 and Leu 480 and the main forces were hydrogen bonding and hydrophobic interactions. However, naringenin interacted with Arg 484 of BSA, and the main force was hydrophobic interaction. It can be concluded that eriodictyol and naringenin can inhibit the formation of AGEs and eriodictyol has stronger inhibitory activity of AGEs than that of naringenin, which is probably due to the additional hydroxyl group in the position C‐3′ of B ring of eriodictyol.     

Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.     

Albumin extravasation rates in tissues of anesthetized and unanesthetized rats

Bovine serum albumin (BSA) labeled with 131I was injected intravenously in chronically prepared, unanesthetized rats and into pentobarbital-anesthetized rats that had received 2 ml 5% BSA to help sustain plasma volume. Initial uptake rates (clearances) in skin, skeletal muscles, diaphragm, and heart (left ventricle) were measured over 1 h. BSA labeled with 125I was injected terminally to correct for intravascular 131I-BSA. Observed clearances were in the following order in both groups of animals: heart much greater than diaphragm approximately equal to skin greater than resting skeletal muscles. Differences between unanesthetized and anesthetized animals were small and inconsistently directed. Our results suggest that the lower albumin clearances reported in the literature for anesthetized rats are not the result of their immobility or any direct effect of anesthesia on albumin transport in these tissues. The lower transport rates appear to result indirectly from changes produced by anesthesia and/or surgery in controllable parameters such as plasma volume and intravascular protein mass.