Dulbecco’s modified Eagle’s medium, MCDB131, trypsin and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Vascular endothelial growth factor was purchased from BD Biosciences (San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-phalloidin was obtained from Molecular Probes Life Technologies (Grand Island, NY, USA). FITC-labeled goat anti-rabbit antibody, human serum albumin (HSA) and FITC-bovine serum albumin were from Sigma-Aldrich (St. Louis, MO, USA). DAPI was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and 2′-O-methyladenosine-3′,5′-cyclic monophosphate (O-Me-cAMP) was purchased from Aladdin Reagents Co., Ltd (Shanghai, China). Transwell was purchased from Corning Life Sciences (Tewksbury, MA, USA) and a Rac1 Activity Detection kit was purchased from EMD Millipore (Billerica, MA, USA). A Bradford kit was purchased from Shenneng Bocai Biotechnology Co., Ltd. (Shanghai, China).

The isolation and culture of primary GECs was conducted as previously described (17–19). All studies were approved by the Ethics Committee of Anhui Provincial Hospital (Hefei, China) for Animal Experiments and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Primary GECs were isolated from the kidneys of two male Wistar rats (body weight, 80–120 g) (Animal Department of Anhui Medical University, China; certificate no. 003). Primary GECs were cultured in MCDB131 medium supplemented with 10% FBS in a humidified atmosphere with 5% CO₂. All operations were performed under 10% chloral hydrate (Hechang Chemical Company, Wuhan, China) and all efforts were made to minimize suffering.

AGE-HSA was prepared by incubating HSA with glucose, as previously described (20–23). The reaction system contained 1.5 g HSA and 3.0 g D-glucose (Meilun Bio, Dalian, China), which were dissolved in 10 ml phosphate-buffered saline (PBS; 0.2 mol; pH 7.4; Gibco Life Technologies) and filtered with 0.22 μm microporous membranes (EMD Millipore). The solution was then maintained in a container filled with nitrogen, which was sealed, protected from light and incubated at 37°C for three months. The unbound materials were removed using a dialysis bag (molecular weight, 10,000; Corning, Inc., Corning, NY, USA). The same procedure was completed without the addition of D-glucose for the control. The AGE value of the samples (1 mg/ml) was detected by fluorescence scanning (BX43; Olympus Corp., Tokyo, Japan) and samples were stored at −20°C.

Staining was conducted as previously described (24–27). The slides were washed in PBS twice and fixed in 4% paraformaldehyde at room temperature for 30 min. Subsequently, the slides were treated with 0.1% Triton X-100 (Sangon Biotech Co., Ltd, Shanghai, China) for 15 min (F-actin staining). Subsequent to blocking with 1% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h at room temperature, the samples were incubated with FITC-phalloidin for 1 h at room temperature in the dark (F-actin staining), or with rabbit anti-mouse monoclonal cortactin antibody (SAB1305513; 1:100) overnight at 4°C. For cortactin staining, samples were then incubated with DyLight 605-labeled goat anti-rabbit antibody (SAB4600398; 1:500) for 1 h at room temperature in the dark following washing three times with PBS for 5 min. Antibodies were obtained from Santa Cruz Biotechnology, Inc. (Beijing, China) and samples were incubated with antibodies for 2 h at room temperature. Glycerol (50%; Xilong Chemical Company, Guangzhou, China) was used to mount the glass slides with cells, and the images were captured using a confocal microscope.

Racl activity was analyzed using a pull-down assay, as previously described (9,28). The protein was extracted using a chemical cleavage method, and the protein concentration was detected using the Bradford assay. Samples (50 μg) underwent SDS-PAGE (10%) and were subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were incubated with RAC1-GTP antibody and were then incubated with horseradish peroxidase-conjugated secondary antibodies (Zhongshan Jinqiao Company, Beijing, China). The chemiluminescent images were obtained using a Kodak Image Station 2000R system (Kodak, Rochester, NY, USA) and the results were analyzed using ImageJ software version 1.44e (National Institutes of Health, Bethesda, MD, USA).

Using a previously reported method (29), GECs were seeded into the top compartment of a Transwell chamber with FITC-albumin (100 μl; 1 mg/ml; Sigma-Aldrich). Subsequent to incubation, the fluorescence intensity of samples was analyzed using a HTS-7000 Bio Assay Reader (BioAssay Systems, Hayward, CA, USA) with 495 nm excitation and 520 nm emission filters. The apparent permeability coefficient [(P_a) = (F/t)(1/A)(v/L)] was used, where F indicates the fluorescence intensity in the bottom chamber, t indicates time (sec), A is the membrane area (cm²), v is the solution volume in the bottom chamber and L indicates the fluorescence intensity in the top chamber. The results are expressed as a percentage [P_a% = (experimental P_a value/control P_a value) × 100%]. The experiments were repeated a minimum of five times.

Statistical analysis was performed using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard error and were analyzed by a one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Under normal conditions, endothelial cells appear smooth and intact (30). F-actin is filamentous among the long axis and near cell junctions and is present in reticular, intact and continuous lines, predominantly in the edges of cells and the inner membranes. A small amount of reticular nuclear matrix is also present around the nucleus. Cortactin is predominantly distributed in the cytoplasm and is occasionally also present in the cell membrane. With an increase in AGE-HSA concentration or treatment time, the edge of the F-actin peripheral dense band was observed to become rough and irregular, with a jagged appearance. F-actin was observed to be diffusely distributed in cells and the number of stress fibers, composed of a single row of non-polar actin filaments, increased. The distribution of cytoplasmic cortactin became disorganized and it was unclear in the cortex and membrane. Cells became round and retracted when treated with AGE-HSA for 8 h at a concentration of 100 μg/ml; however, HSA alone did not produce these effects (Figs. 1 and 2).

The reconfiguration of the structure of F-actin, formation of central stress fibers and retraction in GECs induced by AGE-HSA was markedly inhibited by pre-treatment with O-Me-cAMP. In addition, similar inhibitory effects were observed in AGE-HSA-induced cortactin disorganization and cell retraction with O-Me-cAMP-pre-treatment. However, HSA alone did not produce this inhibitory effect (Fig. 3). These observations further suggested an involvement of the Rac1 signaling pathway in the development of AGE-induced morphological and structural alterations in GECs.

The levels of Rac1 activity (but not total Rac1 levels) in the AGE-HSA (100 μg/ml) treatment group were significantly reduced compared with those in the control group (P=0.002), whereas HSA alone did not produce this effect (Fig. 4). These results suggested that the Rac1 signaling pathway is important in the mediation of AGE-induced functional alterations in endothelial cells.

The permeability of GECs to FITC-BSA was significantly increased with AGE-HSA-treatment (P<0.05), but not with HSA alone (P>0.05). This effect was inhibited by O-Me-cAMP, as demonstrated by reduction in P_a from 189.32±6.16 to 128.52±3.53% subsequent to treatment with O-Me-cAMP (Fig. 5).