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Originally published In Press as doi:10.1074/jbc.M202634200 on March 23, 2002

J. Biol. Chem., Vol. 277, Issue 23, 20309-20315, June 7, 2002
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Advanced Glycation End Product-induced Apoptosis and Overexpression of Vascular Endothelial Growth Factor and Monocyte Chemoattractant Protein-1 in Human-cultured Mesangial Cells*

Sho-ichi YamagishiDagger §, Yosuke InagakiDagger , Tamami OkamotoDagger , Shinjiro AmanoDagger , Kohachiro KogaDagger , Masayoshi Takeuchi, and Zenji MakitaDagger

From the Dagger  Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, Kurume 830-0011, Japan and the  Department of Biochemistry, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa 920-1181, Japan

Received for publication, March 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Diabetic nephropathy is a leading cause of end-stage renal disease and accounts for disabilities and high mortality rates in patients with diabetes (1, 2). The development of diabetic nephropathy is characterized by glomerular hyperfiltration and thickening of glomerular basement membranes followed by an expansion of extracellular matrix in mesangial areas and increased albumin excretion rate. Diabetic nephropathy ultimately progresses to glomerular sclerosis associated with renal dysfunction (3).

Reducing sugars including glucose, fructose, and trioses can react non-enzymatically with the amino groups of proteins to form reversible Schiff bases and then Amadori products. These early glycation products undergo further complex reactions such as rearrangement, dehydration, and condensation to become irreversibly cross-linked heterogeneous fluorescent derivatives termed advanced glycation end products (AGE)1 (4, 5). The formation and accumulation of AGE in various tissues are known to progress during normal aging and at an extremely accelerated rate in diabetes mellitus. Recent understandings of this process have confirmed that AGE are implicated in the pathogenesis of diabetic microvascular and macrovascular complications (6-14). In fact, there is compelling evidence that the formation and accumulation of AGE mediates the progressive alteration in renal architecture and loss of renal function (15-17). However, whether AGE are involved in the pathogenesis of the early phase of diabetic nephropathy remains to be elucidated.

We have recently found that AGE can arise not only from glucose but also from short chain-reducing sugars and dicarbonyl compounds in serum of diabetic patients (18). Therefore, we prepared three immunochemically distinct AGE in vitro by incubating bovine serum albumin (BSA) with glucose (glu)-AGE-BSA, glyceraldehyde (glycer)-AGE-BSA, or glycolaldehyde (glycol)-AGE-BSA. We then investigated the effects of AGE-BSA on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We demonstrated that all of the different types of AGE not only caused apoptotic cell death in mesangial cells but also elicited hyperfiltration and microalbuminuria by stimulating secretion of VEGF and MCP-1 proteins and thus were involved in the pathogenesis of the early phase of diabetic nephropathy.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- BSA (fraction V), 2,4,6-trinitrobenzenesulfonic acid, N-acetylcysteine (NAC), Tris-HCl, EGTA, and EDTA were purchased from Sigma. D-Glyceraldehyde, glycolaldehyde, Triton X-100, sucrose, NaF, and protease inhibitor cocktails were from Nakalai Tesque (Kyoto, Japan). D-Glucose was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Pyrraline, pentosidine, and argpyrimidine were kindly provided by Mitsubishi Kagaku Bio-clinical Laboratories, Inc. (Tokyo, Japan). 3-Deoxyglucosone hydroimidazolones, glyoxal-lysine dimer, and methylglyoxal-lysine dimer were generously provided by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). [3H]Thymidine, [gamma -32P]ATP, [alpha -32P]UTP, Hybond-N+ nylon membrane, CDP-Star chemiluminescence system, enhanced chemiluminescence system, and prostacyclin (PGI2) enzyme-linked immunosorbent assay (ELISA) system were from Amersham Biosciences (Buckinghamshire, United Kingdom). Reverse transcriptase and T4 polynucleotide kinase were from Takara (Kyoto, Japan). VEGF and MCP-1 ELISA systems were from R&D systems (Minneapolis, MN).

Preparation of AGE Proteins-- AGE-BSA was prepared as described previously (18). BSA was incubated under sterile conditions with D-glucose for 8 weeks or with D-glyceraldehyde or glycolaldehyde for 7 days. Unincorporated sugars were then removed by dialysis against phosphate-buffered saline. Control non-glycated BSA was incubated in the same conditions with the exception of the absence of reducing sugars. Preparations were tested for endotoxin using Endospecy ES-20S system (Seikagaku Co., Tokyo, Japan) where no endotoxin was detectable. Carboxylmethyllysine (CML) and Carboxylethyllysine were prepared as described previously (18). The extent of chemical modification was determined as described with 2,4,6-trinitrobenzenesulfonic acid as a difference in lysine residues of modified and unmodified protein preparations (19). The extent of lysine modification (%) of modified BSA preparations was 42% for glu-AGE-BSA, 65% for glycer-AGE-BSA, and 90% for glycol-AGE-BSA.

Cells-- Mesangial cells from human kidney were maintained in MsGM medium supplemented with 5% fetal bovine serum and GA-1000 according to the manufacturer's instructions (Clonetics Corp., San Diego, CA). AGE treatments were carried out in a medium containing 0.5% fetal bovine serum and GA-1000. Medium was changed every 2 days. Human glomerular endothelial cells were purchased from Cell Systems Corporation (Kirkland, WA) and maintained in CS-C medium supplemented with 10% fetal bovine serum according to the manufacturer's instructions. Cells at 2-4 passages were used for the experiments.

Measurements of Growth and [3H]Thymidine Incorporation in Mesangial Cells-- Human mesangial cells cultured for the indicated time periods in the presence or absence of various types of AGE-BSA were dislodged with trypsin, and the number of viable cells was counted by the dye exclusion method (20). [3H]Thymidine incorporation was determined as described previously (21).

Measurement of Apoptotic Cell Death in Mesangial Cells-- Human mesangial cells were incubated with various types of AGE-BSA for 4 or 6 days in the presence or absence of 1 mM NAC. Cells were then lysed, and the supernatant was analyzed in an ELISA for DNA fragments (Cell Death Detection ELISA, Roche Molecular Biochemicals).

Preparation of Antibodies against glu-AGE-BSA-- Antibodies against glu-AGE-BSA was prepared as described previously (18).

Characterization of Antibodies against glu-AGE-BSA-- Reactivity of antibodies to several AGE-modified BSA or non-glycated BSA was examined by competitive ELISA systems as described previously (22).

Western Blot Analysis for p53 in Mesangial Cells-- Human mesangial cells treated with or without various types of AGE-BSA for 4 days were lysed in 30 mM Tris-HCl (pH 7.4) containing 10 mM EGTA, 5 mM EDTA, 1% Triton X-100, 250 mM sucrose, 1 mM NaF, and protease inhibitor cocktails. 15 µg of proteins were loaded per lane and separated by SDS-PAGE (15%) and transferred onto a nitrocellulose membrane. The filter was then treated with mouse monoclonal antibody against human p53 (Santa Cruz Biotechnology, Santa Cruz, CA), and the resultant immunocomplexes were visualized with CDP-Star chemiluminescence system according to the manufacturer's instructions.

RNase Protection Assay for Bax, Bcl2, and L32-- RNase protection assay was performed according to the manufacturer's instructions (RiboQuant multi-probe RNase protection assay system, BD PharMingen). Single-stranded antisense 32P-labeled probes were prepared from bax, bcl-2, and L32 templates, and then they were hybridized in excess to target RNA in solution, after which free probes and other single-stranded RNA were digested with RNase. The remaining RNase-protected probes are purified, resolved on denaturing polyacrylamide gels, and quantified by autoradiography.

Western Blot Analysis for Bax in Mesangial Cells-- Human mesangial cells treated with or without various types of AGE-BSA for 4 days were lysed in 30 mM Tris-HCl (pH 7.4) containing 10 mM EGTA, 5 mM EDTA, 1% Triton X-100, 250 mM sucrose, 1 mM NaF, and protease inhibitor cocktails. 15 µg of proteins were loaded per lane and separated by SDS-PAGE (15%) and transferred onto a nitrocellulose membrane. The filter was then treated with a polyclonal antibody against human Bax (Daco Corporation, Carpinteria, CA), and the resultant immunocomplexes were visualized with an enhanced chemiluminescence system as described previously (23).

Co-culture Systems-- Human glomerular endothelial cells were plated in a 0.4-µm pore-sized 24-mm diameter well (Corning Coster Corporation, Cambridge, MA). The next day, the medium was changed to MsGM medium (Clonetics Corp., San Diego, CA) supplemented with 0.5% fetal bovine serum and GA-1000, and then the Transwell was inserted into a 6-well dish in which mesangial cells had been grown. After 24 h, the conditioned medium in the Transwell was corrected and analyzed in an ELISA for 6-keto-PGF1alpha .

Measurement of 6-keto-PGF1alpha -- 6-keto-PGF1alpha , a stable metabolite of PGI2, released into media was measured with an ELISA system according to the manufacturer's instructions.

Primers and Probes-- Sequences of the upstream and downstream primers and the internal probe used in quantitative reverse transcription (RT)-PCR for detecting human VEGF and beta -actin mRNAs were the same as described previously (24).

Quantitative RT-PCR-- Poly(A)+ RNAs were isolated (24) from cells treated with or without AGE-BSA for the various time periods, and they were analyzed by RT-PCR as described previously (24). 10-µl aliquots of each RT-PCR reaction mixture were electrophoresed on a 1.2% agarose gel, transferred to a Hybond-N+ nylon membrane, and the membrane was hybridized with the respective 32P end-labeled probes (25). The amounts of poly(A)+ RNA templates (30 ng) and cycle numbers (35 cycles) for amplification were chosen in quantitative ranges where reactions proceeded linearly, which had been determined by plotting signal intensities as the functions of the template amounts and cycle numbers (24). Signal intensities of hybridized bands were measured by microcomputer-assisted NIH Image (Version 1.56).

Measurement of VEGF and MCP-1-- Human mesangial cells were incubated with various types of AGE-BSA for 4 days, and then VEGF and MCP-1 proteins released into media were measured with ELISA systems according to the manufacturer's instructions (26).

Statistical Analysis-- All values were presented as the means ± S.E. Statistical significance was evaluated using the Student's t test for paired comparison. p < 0.05 was considered significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of AGE-BSA on Growth of Mesangial Cells-- Mesangial cells were cultured with various types of AGE-BSA or non-glycated BSA, and viable cell numbers were determined at days 2 and 4 after AGE addition. As shown in Fig. 1A, the growth curve was found to be shifted downwards by the treatments of AGE. Viable cell numbers of mesangial cells decreased to 40% of those of control cells when exposed to glycer-AGE for 4 days. All of the AGE-BSA also significantly inhibited DNA synthesis in mesangial cells (Fig. 1B). Although CML was present in trace amounts in these AGE-BSA preparations, carboxylmethylated BSA added to the culture medium failed to inhibit DNA synthesis in human mesangial cells (data not shown).


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  		Fig. 1.   Effects of AGE-BSA on growth of human mesangial cells.  A, mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml of non-glycated BSA. The culture period after the addition of AGE-BSA is indicated on the abscissa, and the viable cell number is on the ordinate. B, mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml of non-glycated BSA. After incubation for 4 days, [3H]thymidine was added, and then its incorporation into the cells was assayed. The percentage of [3H]thymidine incorporation is indicated on the ordinate and related to the value of the control with non-glycated BSA. *, p < 0.01 compared with the value of the control with non-glycated BSA (Student's t test).

Effects of AGE-BSA on Apoptotic Cell Death in Mesangial Cells-- We next investigated whether AGE-BSA could induce apoptotic cell death in cultured mesangial cells. Apoptosis is characterized by DNA fragmentations because of endogenous endonuclease activation (27). Therefore, we quantitatively measured DNA fragments in the cytoplasm of mesangial cells exposed to AGE-BSA or non-glycated BSA. As shown in Fig. 2, various types of AGE-BSA slightly but significantly induced apoptotic cell death in mesangial cells.


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  		Fig. 2.   Effects of AGE-BSA on apoptotic cell death in mesangial cells. Mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml non-glycated BSA. Cells then were lysed, and the supernatant was analyzed in an ELISA for DNA fragments. The percentage of absorbance at 405 nm is indicated on the ordinate and related to the value of the control with non-glycated BSA. *, p < 0.01 compared with the value of the control with non-glycated BSA (Student's t test).

Characterization of Antibodies against glu-AGE-BSA-- We prepared polyclonal antibodies against glu-AGE-BSA and then examined the reactivity of antibodies to several structurally identified AGE-modified BSA including pyrraline-BSA, pentosidine-BSA, argpyrimidine-BSA, 3-deoxyglucosone imidazolone-BSA, CML-BSA, carboxylethyllysine-BSA, glyoxal-lysine dimer, or methylglyoxal-lysine dimer. As shown in Fig. 3, A and B, the antibodies were found not to cross-react with these structurally well defined AGE epitopes.


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  		Fig. 3.   Immunoreactivity of AGE antibodies with various AGE preparations. 50 µl of various AGE preparations were added to each glu-AGE-BSA-coated well as a competitor for 50 µl of AGE antibodies followed by incubation for 2 h at room temperature. The immunoreactivity of antibodies with various AGE preparations then was determined by competitive ELISA. Results were expressed as B/B0, calculated as (experimental optical density - background optical density)/(total optical density - background optical density).

Effects of Antibodies against glu-AGE-BSA on glu-AGE-induced Apoptotic Cell Death in Mesangial Cells-- We next investigated whether the antibodies could neutralize the glu-AGE-induced apoptotic cell death in mesangial cells. As shown in Fig. 4A, the antibodies were found to prevent the glu-AGE-induced mesangial cell apoptosis in a dose-dependent manner. The results demonstrated that the structurally well defined AGE epitopes could not be involved in the AGE-induced apoptotic cell death in mesangial cells.


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  		Fig. 4.   Effects of AGE antibodies (A) and NAC (B) on glu-AGE-induced apoptotic cell death in mesangial cells. A, mesangial cells were treated with 100 µg/ml glu-AGE-BSA or 100 µg/ml non-glycated BSA in the presence or absence of AGE antibodies for 6 days. B, mesangial cells were treated with 100 µg/ml glu-AGE-BSA or 100 µg/ml non-glycated BSA in the presence or absence of 1 mM NAC for 4 days. Cells then were lysed, and the supernatant was analyzed in an ELISA for DNA fragments. The percentage of absorbance at 405 nm is indicated on the ordinate and related to the value of the control with non-glycated BSA. *, p < 0.01 (Student's t test).

Effects of NAC on glu-AGE-induced Apoptotic Cell Death in Mesangial Cells-- We next investigated whether reactive oxygen species were involved in apoptotic cell death in mesangial cells. As shown in Fig. 4B, an antioxidant, NAC was found to completely inhibit the glu-AGE-induced apoptotic cell death in mesangial cells.

Effects of AGE-BSA on p53 Accumulation in Mesangial Cells-- The tumor suppressor gene p53 not only controls the transition of cells from the G1 to S phase, but it is also capable of inducing programmed cell death (28). Therefore, we investigated the effects of AGE-BSA on p53 protein accumulation in mesangial cells. As shown in Fig. 5A, various types of AGE-BSA significantly increased p53 accumulation in mesangial cells.


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  		Fig. 5.   Effects of AGE-BSA on p53 accumulation in mesangial cells. A, mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml non-glycated BSA for 4 days. Proteins then were extracted and analyzed by Western blotting as described under "Experimental Procedures." Arrow indicates the position of p53 protein. Similar results were obtained in two independent experiments. B, quantitative representation of p53 protein induction.

Effects of AGE-BSA on Bax Gene and mRNA Expression in Mesangial Cells-- Proteins of Bcl-2 family are well known inducers and integrators of survival and death signals (29, 30), and pro-apoptotic molecule Bax has also been shown to be a p53 target (31). Therefore, we next studied whether AGE-BSA could affect the expression of Bax in mesangial cells. As shown in Fig. 6A, the level of bax mRNA was increased by the treatments of various types of AGE-BSA. When standardized to L32 mRNA-derived signals, the bax mRNA level in mesangial cells exposed to glycol-AGE for 4 days was ~2-fold higher than that in controls. However, pro-survival molecule bcl-2 gene was found not to be expressed in cultured mesangial cells (data not shown). Western blot analysis also showed that various types of AGE actually increased the expression of the Bax protein in mesangial cells (Fig. 6C).


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  		Fig. 6.   Effects of AGE-BSA on expressions of bax gene and mRNA in mesangial cells. A, RNase protection assay for bax gene in mesangial cells. Poly(A)+ RNAs were prepared from cells that were treated with 100 µg/ml of different types of AGE-BSA or 100 µg/ml of non-glycated BSA for 4 days. mRNAs were hybridized with bax and L32 riboprobes as described under "Experimental Procedures." Similar results were obtained in two independent experiments. B, quantitative representation of bax gene induction. Data were normalized by the intensity of L32 mRNA-derived signals and related to the value with non-glycated BSA. C, mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml of non-glycated BSA for 4 days. Proteins then were extracted and analyzed by Western blotting as described under "Experimental Procedures." Arrow indicates the position of Bax protein. Similar results were obtained in two independent experiments. D, quantitative representation of Bax protein induction.

Effects of Mesangial Cells on PGI2 Production by Co-cultured Glomerular Endothelial Cells-- We next investigated whether human mesangial cells could affect the ability of glomerular endothelial cells to produce PGI2, the key substance endowing the endothelium with antithrombogenic activities (32). Human glomerular endothelial cells were co-cultured with mesangial cells for 1 day, and the culture medium was assayed for 6-keto-PGF1alpha , a stable metabolite of PGI2. As shown in Fig. 7, the amount of 6-keto-PGF1alpha released from glomerular endothelial cells was significantly elevated when they were co-cultured on the mesangial feeder layer (mesangial cells caused a 3-fold increase in PGI2 production by co-cultured glomerular endothelial cells).


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  		Fig. 7.   Effects of human mesangial cells on PGI2 production by co-cultured glomerular endothelial cells. Human glomerular endothelial cells were cultured with or without mesangial cells for 1 day, and the culture medium was assayed for 6-keto-PGF1alpha . *, p < 0.01 compared with the value without mesangial cells (Student's t test).

Mesangial Cells Express mRNA for Secretory Forms of VEGF in Response to AGE-BSA-- Poly(A)+ RNAs were isolated from mesangial cells treated with various types of AGE-BSA for various time periods and analyzed by a quantitative RT-PCR technique to determine the effects of AGE on the expression of VEGF genes. It has been reported that there are five alternatively spliced products from the single VEGF gene, VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 (33). Because Northern blot analysis can not clearly discriminate the five mRNA products, we employed a more sensitive RT-PCR technique as described previously (24). In these experiments, 486- and 618-base pair-long cDNA products would be amplified from mRNAs for VEGF121 and VEGF165, respectively (24). As shown in Fig. 8, A and B, human mesangial cells expressed mRNAs for VEGF121 and VEGF165, the secretory forms of VEGF. The VEGF mRNA levels began to increase at 2 h and reached a maximum at 4 h in the presence of various types of AGE-BSA. the peak value was 3-fold higher than the basal level when exposed to glycer-AGE-BSA. To confirm whether AGE actually increased the secretion of VEGF proteins, we further performed an ELISA for VEGF proteins in a medium of cells that had been treated with or without 100 µg/ml of various types of AGE-BSA for 4 days. As shown in Fig. 8C, AGE were found to stimulate the secretions of VEGF proteins by mesangial cells.


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  		Fig. 8.   Effects of AGE-BSA on expressions of VEGF gene and mRNA in mesangial cells. A, time course of VEGF mRNA induction by various types of AGE-BSA. Cells were treated with or without 100 µg/ml of different types of AGE-BSA, and then 30 ng of poly(A)+ RNAs were transcribed and amplified by PCR at the indicated times. Each of the lower panels shows the expression of beta -actin genes. PCR amplification for beta -actin mRNA was performed for 20 cycles. Similar results were obtained in two independent experiments. B, quantitative representation of VEGF gene induction. Data were normalized by the intensity of beta -actin mRNA-derived signals and related to the value with non-glycated BSA. C, mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml of non-glycated BSA for 4 days. Medium was collected, and VEGF content in the medium was analyzed in an ELISA kit. *, p < 0.01 compared with the value of the control with non-glycated BSA (Student's t test).

Effects of AGE-BSA on MCP-1 Productions by Mesangial Cells-- MCP-1 is a C-C chemokine for monocytes that can mediate monocyte infiltration in mesangial areas (34). We investigated the effects of AGE on MCP-1 production by mesangial cells. As shown in Fig. 9, various types of AGE-BSA significantly stimulated the secretion of MCP-1 proteins by mesangial cells.


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  		Fig. 9.   Effects of AGE-BSA on MCP-1 expression in mesangial cells. Mesangial cells were treated with 100 µg/ml of various types of AGE-BSA or 100 µg/ml of non-glycated BSA for 4 days. Medium was collected, and MCP-1 content in the medium was analyzed in an ELISA kit. *, p < 0.01 compared with the value of the control with non-glycated BSA (Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have demonstrated for the first time that various types of AGE, non-enzymatically glycated protein derivatives formed at an accelerated rate in diabetes (18), not only inhibit DNA synthesis but also induce apoptotic cell death in human mesangial cells. Mesangial cells occupy a central anatomical position in the glomerulus, playing crucial roles in maintaining structure and function of glomerular capillary tufts (35). In fact, they actually provide structural support for capillary loops and modulate glomerular filtration by its smooth muscle activity (35-37). Therefore, AGE-induced mesangial cell loss and the resultant mesangial cell-altered contractility may contribute in part to glomerular hyperfiltration, an early renal dysfunction in diabetes (3).

In this study, AGE antibodies that did not cross-react with well defined AGE epitopes completely neutralized the cytopathic effects of glu-AGE-BSA on mesangial cells. Therefore, it is unlikely that such structurally identified AGE proteins are relevant epitopes in the preparations of AGE-BSA. Furthermore, we have recently found that structural epitopes of in vitro prepared AGE actually existed in the serum of diabetic patients, and glu-AGE acted on mesangial cells at the physiological concentration of diabetic patients (18, 22). The results suggest that structurally unidentified AGE epitope in the preparations of AGE-BSA might be involved in the development of diabetic nephropathy. In this study, CML added to the culture medium failed to inhibit DNA synthesis in mesangial cells (data not shown). Because CML adducts of proteins have been reported to be ligands for receptor for AGE termed RAGE (38), it is probable that AGE receptors other than RAGE might mediate the AGE effects.

We have found in this study that various types of AGE increased p53 accumulation in mesangial cells. Furthermore, pro-apoptotic molecule bax mRNA and protein were induced by treatments with AGE. Because p53 has been reported to mediate programmed cell death through Bax protein expression in response to a number of stress signals (31, 39), AGE might induce apoptotic cell death in mesangial cells through p53-dependent Bax expression. In this study, NAC completely prevented the glu-AGE-induced mesangial cell apoptosis. Therefore, reactive oxygen species generation induced by AGE might also be involved in mesangial cell apoptosis. Recently, Sandau et al. (40) reported that superoxide induced p53 and Bax accumulation during mesangial cell apoptosis, supporting our observation.

We have previously shown that AGE exerted a growth inhibitory effect and a cell type-specific immediate toxicity on retinal pericytes, the microvascular counterpart of mesangial cells, and have proposed a novel mechanism for pericyte loss, the earliest histopathological hallmark in diabetic retinopathy (8). The results suggest that AGE-induced loss of vascular wall cells in kidney and retina may be an initial event that leads to the progression of diabetic microangiopathies. Recently, Bax is reported to be increased in the retina of diabetic subjects and associated with apoptosis of pericytes (30). Therefore, it is probable that AGE may be mainly involved in apoptotic cell death of pericytes through the induction of Bax in diabetic retinopathy.

There is a growing body of evidence that platelet activation may participate in the pathogenesis of diabetic vascular complications by promoting microthrombus formation (41-43). In addition, antiplatelet agents such as thromboxane synthetase inhibitor and dipyridamole have been reported to decrease microalbuminuria and retard the progression of diabetic nephropathy (44, 45). In this study, we found for the first time that mesangial cells can stimulate the production of PGI2, an antithrombogenic prostanoid, by co-cultured glomerular endothelial cells. Pericytes also possess the ability to stimulate PGI2 production by endothelial cells (21). Therefore, AGE-induced vascular wall cell loss could predispose the neighboring endothelial cells to thrombogenesis by impairing PGI2 production and thus be implicated in the development and progression of diabetic microangiopathies. Recently, a novel bioactive peptide PGI2-stimulating factor was cloned, and its mRNA expression found to be suppressed in the kidney of streptozotocin-induced diabetic rats (46). Mesangial cells could contribute to the maintenance of glomerular hemostasis by producing PGI2-stimulating factor.

VEGF is a specific mitogen to endothelial cells, also known as vascular permeability factor, and is generally thought to be involved in the pathogenesis of diabetic retinopathy (47). Recently, antibodies against VEGF have been found to improve hyperfiltration and albuminuria in experimental diabetes (48). Therefore, AGE-induced VEGF overproduction by mesangial cells might play a central role in the early phase of diabetic nephropathy (49). In support of our observations, pronounced immunostaining of mesangial VEGF was observed in diabetic nephropathy, and significant correlation was found between VEGF excretion and the level of microalbuminuria in patients with diabetes (50). Furthermore, we have recently found that all of the AGE preparations used in these experiments increased levels of VEGF mRNAs in bovine retinal pericytes (51). These observations suggest that AGE could induce similar metabolic and functional abnormalities in vascular wall cells such as mesangial cells and pericytes and thus participate in the development of diabetic microangiopathies.

MCP-1 is a specific chemokine that recruits and activates monocytes from circulation to inflammatory sites (34). Increased MCP-1 expression associated with monocyte infiltration in mesangium has been observed in the early phase of diabetic nephropathy (52). AGE accumulation in glomerulus could be implicated in the initiation of diabetic nephropathy by promoting the secretion of MCP-1 in mesangial cells.

In conclusion, AGE could not only disturb glomerular homeostasis by inducing apoptotic cell death in mesangial cells but also elicit hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins and thereby could be involved in the pathogenesis of the early phase of diabetic nephropathy.

    FOOTNOTES

* This work was supported in part by grants from Venture Research and Development Centers of the Ministry of Education, Culture, Sports, Science and Technology, Japan; the Suzuken Memorial Foundation, Japan; the Hokuriku University Foundation, Japan; and the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-942-31-7563; Fax: 81-942-35-8943; E-mail: shoichi@med.kurume-u.ac.jp.

Published, JBC Papers in Press, March 23, 2002, DOI 10.1074/jbc.M202634200

    ABBREVIATIONS

The abbreviations used are: AGE, advanced glycation end products; BSA, bovine serum albumin; glu, glucose; glycer, glyceraldehyde; glycol, glycolaldehyde; VEGF, vascular endothelial growth factor; MCP-1, monocyte chemoattractant protein-1; NAC, N-acetylcysteine; PGI2, prostacyclin; ELISA, enzyme-linked immunosorbent assay; CML, carboxylmethyllysine; 6-keto-PGF1alpha , 6-keto-prostaglandin F1alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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