Experimental animal models
Experimental diabetes was induced in 6-week-old male Sprague–Dawley (SD) rats (200–250 g) by intravenous injection of STZ (50 mg/kg body weight) in sodium citrate buffer pH 4.5, following an overnight fast . Rats with plasma glucose concentrations in excess of 15 mmol/L were included in this study. Vehicle-injected control (Ctrl) animals (n = 15) were followed concurrently. Diabetic rats were randomized into two groups and followed for 32 weeks; one group (n = 15) received a vehicle, and the other an ACE inhibitor, ramipril (1 mg/kg body weight/day in drinking water; generously provided by Sanofi, Bridgewater, NJ) for 32 weeks (n = 16) . Two units of ultralente insulin (Ultratard HM, Novo Industries, Bagsvaerd, Denmark) were administrated daily to diabetic animals to prevent ketoacidosis and avoid death. In addition, non-diabetic normal male SD rats (6-week-old) were infused intraperitoneally with AGE-modified rat serum albumin (AGE-RSA) (n = 10) or RSA (n = 9) at a dose of 20 mg/kg body weight/day for 16 weeks by an osmotic pump (Alzet osmotic pumps, model 1004, Cupertino, CA, USA).
Systolic blood pressure (SBP) was measured by tail-cuff plethysmography as described previously . Glomerular filtration rate (GFR) was evaluated by 99Tc-DTPA, and urinary albumin excretion (UAE) levels by an enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Montgomery, TX, USA). Other clinical valuables were measured as described previously . All animal procedures were in accordance with guidelines set by the Baker IDI Heart and Diabetes Institute Ethics Committee and the National Health and Medical Research Council of Australia.
Preparation of AGE-RSA and AGE-modified bovine serum albumin (AGE-BSA)
AGE-RSA and AGE-BSA were prepared by incubating RSA or BSA (Fraction V, Sigma Chemical Co, St. Louis, MO, USA) with 0.5 M D-glucose in PBS at 37°C for 3 months as previously described . After sterilization using 0.2 μm micropore filters, unincorporated glucose was removed by dialysis against phosphate buffer saline (PBS) at 4°C for 48 hr. Samples were passed through Detoxigel column (Pierce Biotechnology Inc., Rockford, IL, USA) in order to remove endotoxin. Preparations were tested for endotoxin using Limulus Amebocyte Lysate validity testing (AMS Laboratories, Sydney, Australia); no endotoxin was detected. Finally, the solution was filtered through 0.2 μm micropore filter in sterile conditions, and percentage of lysine modifications and carboxymethyllysine (CML) moieties were determined by Selective Ion Monitoring Gas chromatography–mass spectrometry as previously described . Control non-glycated RSA or BSA was incubated in the same conditions except for the absence of glucose.
Isolation of renal tubules from kidneys
The cortical tissue was minced and gently pushed through a 250 μm steinless steel mesh with 0.9% sodium chloride solution, and sieved stepwise through 125 μm and 75 μm meshes. The tubules were collected on the 125 μm mesh as described previously . Tubular structures were confirmed by light microscopy. After centrifugation, the tubules were re-suspended in the lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate) containing a complete protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) without ethylenediaminetetraacetic acid (EDTA). Then the samples were sonicated and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology Inc.).
Twenty μg protein isolated from renal tubules or cell culture media were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg/ml gelatin (Labchem, Auburn, NSW, Australia), and active MMP-2 levels were evaluated by zymography as previously described .
Measurement of AGE levels in tubules
AGE levels in the isolated tubules were measured with CML ELISA system as previously described . In brief, 1 μg of samples diluted in 50 mM carbonate buffer (pH 9.6) (1:800) or standards were added to a microtitre plate (Nunc-Immuno MaxiSorp, Nunc, Kamstrup, Roskilde, Denmark) and incubated overnight at 4°C. After washing three times with PBS (pH 7.4) containing 0.1% Tween-20, wells were blocked for 1 hr with PBS containing 1% BSA, and then 100 μl of a rabbit polyclonal anti-CML antibody (5 μg/ml) was added . After 1 hr incubation and washing, 100 μl of 0.2 μg/ml goat-anti-rabbit IgG biotinylated antibody (Dako Corporation, Carpinteria, CA, USA) was added to each well. After 1 hr of shaking, wells were washed and 100 μl of streptavidin horseradish peroxidase (Dako Corporation) was added for 30 min. The wells were washed and 100 μl of Tetramethylbenzidine (Sigma Chemical Co.) was added, the reaction being terminated after 15 min using 100 μl 1.8 M H2SO4. The absorbance was quantitated using a microtitre plate reader at 450 nm (EMax, Molecular Devices Corporation, Sunnyvale, CA, USA).
Western blot analysis
Ten μg protein isolated from renal tubules was separated by 10% SDS-PAGE electrophoresis, and then transferred to a polyvinylidene fluoride membrane (Hybond P; Amersham, Buckinghamshire, UK). Membranes were incubated with mouse anti-RAGE (1:1000) or rabbit anti-α-tubulin (1:1000) antibodies overnight, and then the secondary antibody horseradish peroxidase-conjugated mouse or rabbit IgG for 1 hr. Bound antibodies were detected by reaction with an enhanced chemiluminescence kit (Pierce Biotechnology Inc.). All antibodies were obtained from Chemicon (Santa Cruz, SA, USA).
Quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)
About 4 μg of total RNA extracted from each kidney cortex or RPTCs were used to synthesize cDNA with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsband, CA, USA) as previously described . Quantitative real-time RT-PCR was performed using Assay-on-Demand and TaqMan 5 fluorogenic nuclease chemistry (Applied Biosystems, Foster City, CA, USA) according to the supplier’s recommendation. The forward, reverse primers and specific probes for rat RAGE and MMP-2 genes were 5′-TCCTGGTGGGACCGTGAC-3′, 5′-GGGTGTGCCATCTTTTATCCA-3′, and FAM5′-TGTGCCATCTCTGC-3′-MGB, and 5′-GCCCCTATCTACACCTACACCAA-3′, 5′-TGGATCCCCTTGATGTCATCA-3′, and FAM-5′-AACTTCCGATTATCCC-3′-MGB, respectively. TaqMan Ribosomal RNA Control Reagents (18S) was used as an endogenous control (Applied Biosystems).
Isolation and characterization of RPTCs
RPTCs were isolated from rat kidney as previously described . In brief, kidneys were removed from male SD rats (200–250 g), and renal cortex was minced and centrifuged at 250 g, 4°C for 5 min. Final pellets were digested in Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (1:1) containing 1 mg/mL collagenase (Type-2, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 30 min at 37°C with constant agitation. This suspension was filtered through a 75 μm pore size metal sieve to remove glomeruli, and re-suspended in a 50% Percoll solution (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) followed by centrifugation at 26,500 g at 4°C for 30 min. The lowest band was retrieved as it is enriched for proximal tubule fragments at a purity of greater than 98% as described previously . RPTCs were characterized as cobble stone-like appearance and immunocytochemical characteristics with positive staining for cytokeratin and P. vulgaris lectin in the absence of CD90.1 (thy1.1) using confocal microscopy. Treatments with AGE-BSA, Ang II (Sigma Chemical Co.), an inhibitor of ACE, ramiprilat, or an inhibitor of IκB-α phosphorylation, BAY11-7082 ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile) (Biomol international, Lp, USA)  were carried out in Minimum Essential Medium containing D-valine instead of L-valine without fetal calf serum in humidified 5% CO2/95% air atmosphere at 37°C.
Intracellular ROS generation
RPTCs were treated with 100 μg/ml AGE-BSA or BSA in the presence or absence of 10-7 M ramiprilat for 24 hr, and then intracellular ROS generation was measured using the fluorescent probe 5,6-chloromethyl- 2′,7′-dichlorohydrofluorescein diacetate (Molecular Probes, Eugene, OR, USA) as described previously .
Results are expressed as mean ± standard error. Data for albuminuria were not normally distributed, therefore analyzed as logarithmic transformation. One-way ANOVA followed by the Tukey-test or unpaired t-test was performed for statistical comparisons; p < 0.05 was considered significant. All statistical analyses were performed with SPSS system (Ver. 20, SPSS, Chicago, IL, USA).