Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE) (original) (raw)

Cell culture. The well-characterized normal rat kidney epithelial cell line (NRK-52E) was obtained from the American Tissue Culture Collection (Rockville, Maryland, USA). NRK-52E cells are believed to be of a proximal tubular origin on the basis of patterns of collagen secretion, C-type natriuretic peptide secretion, and the presence of EGF receptors (20). Cells were maintained in DMEM containing 4.5 g/l glucose (Sigma Chemical Co., St. Louis, Missouri, USA) with 10% FCS at 37°C in a 5% CO2 atmosphere and passaged twice a week.

In vitro preparation of ligands. AGE-BSA and AGE-RNase were prepared by incubating BSA (10 mg/ml) or RNase (10 mg/ml) at 37°C for 6 weeks with D-glucose (90 g/l) (Sigma Chemical Co.) in a 0.4-M phosphate buffer containing azide (21). Control preparations were treated identically except that glucose was omitted. Finally, preparations were extensively dialyzed against phosphate buffer to remove free glucose. The extent of advanced glycation was assessed by characteristic fluorescence (excitation 370, emission 440 nm) (3). Advanced glycation was associated with an approximately tenfold increase in fluorescence compared with controls.

Carboxymethyl lysine–modified (CML-modified) BSA was prepared as described previously (22). Briefly, 50 mg/ml aliquots of BSA were incubated with increasing concentrations of glyoxylic acid (5–90 mM) in the presence of approximately fivefold molar excess of sodium cyanoborohydride. Control proteins were prepared under the same conditions, except that glyoxylic acid was omitted. The extent of chemical modification of lysine residues was determined as described previously using 2,4,6-trinitrobenzenesulfonic acid (23). The extent of lysine modification was up to 34% for CML-BSA preparations and 77% for AGE-modified proteins.

Iodination of AGE-BSA. AGE-BSA was iodinated by incubating AGE-BSA with chloramine-T (24). Bound 125I was separated from free 125I using a Biogel (Bio-Rad Laboratories Inc., Hercules, California, USA) P6DG desalting gel. Specific activity of the tracer was 400 Ci/mM.

Membrane preparation. Membranes were prepared based on the method of Skolnik (25). NRK-52E cells were grown to confluence in 150-cm2 tissue-culture flasks. Cells were washed twice with PBS, then detached from plates using a HEPES (100 mM) solution containing BSA (0.1%) and Triton X-100 (0.1%) with EDTA (5 mM), leupeptin (1 μM), and PMSF (2 mM). Cells were centrifuged for 5 minutes at 2,000 g and resuspended in the above buffer before disruption by ultrasound. Cell debris was removed by further centrifugation (10 m at 2,000 g) and the supernatant ultracentrifuged at 100,000 g (Beckman Instruments, Oakleigh, Australia) for 1 hour at 4°C. The supernatant was discarded, and the cell membranes in the precipitate were resolubilized in the above buffer. Protein concentrations were determined by the method of Bradford (26). The membrane preparation was used in binding studies and for ligand and Western blot analysis.

Binding studies. Cell membrane extracts were incubated with 125I-AGE-BSA (0.5 nM) and increasing concentrations of unlabeled AGE-BSA (0.015–7.46 μmol) for 3 hours at 4°C in HEPES (100 mM) binding buffer with BSA (0.1%), Triton X-100 (0.1%), leupeptin (1 μM), and PMSF (2 mM). A Brandel cell filter (Biomedical Research and Development Laboratories, Gaithersburg, Maryland, USA) was used to separate bound and free radioligand. A Tris-HCl (10 mM) polyethylene glycol (6.6%) buffer was used to wash components through the apparatus onto glass filter papers, which were counted in a γ-counter (Wallac, Turku, Finland) for 1 minute. Binding experiments were performed in duplicate in five separate experiments, and the specificity of binding was assessed in further experiments using unlabeled, unmodified BSA (1 μmol) as the competitor. Binding data were analyzed using a specific binding program (LIGAND; ref. 27).

Ligand and Western blot analysis. Cell membrane extracts (20 μg of membrane protein per lane) were subjected to nonreducing SDS (12–15%) PAGE and electroblotted onto nitrocellulose membranes (Hybond; Amersham Pharmacia Biotech, Castle Hill, Australia). For ligand blotting the membranes were blocked overnight at 4°C in a Tris (10 mM, pH 7.4), NaCl (150 mM), Tween (0.1%) buffer containing BSA (2.5%), before incubation with 125I-AGE-BSA (110 ng/ml) for 2 hours at room temperature. After washing with Tris (10 mM, pH 7.4), NaCl (150 mM), and Tween (0.1%), the membrane was exposed to Kodak Biomax MS film (Eastman Kodak Co., Rochester, New York, USA) for 1–4 hours. Receptor-ligand binding specificity was studied in competitive experiments where unlabeled AGE-BSA, AGE-RNase, BSA, and RNase (all 100 μg/ml) were added as competing ligands.

In Western blotting experiments, the nitrocellulose membrane was incubated at room temperature for 1 hour with a polyclonal goat Ab against human RAGE 1:2,000 (gift of M. Neeper, Merck, West Point, Pennsylvania, USA) (28, 29). Membranes were washed before a 15-minute incubation with a biotinylated secondary Ab (DAKO Corp., Carpinteria, California, USA) and a streptavidin-horseradish peroxidase conjugate (Vector Laboratories, Burlingame, California, USA). Immunoreactivity was detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) and exposure to Kodak Biomax MR film. Recombinant RAGE (gift of K. Jansen, Merck, West Point, Pennsylvania, USA), 900 ng per lane, and bovine lung extract, 30 μg, were loaded onto gels as positive controls. Primary Ab’s were omitted in experiments as negative controls and RAGE Ab specificity was confirmed by preincubation of the membrane with recombinant RAGE before the addition of RAGE Ab.

To further explore the lower-molecular-weight band seen on ligand blotting, Western blotting experiments were performed with a primary Ab to lysozyme, 1:1,000 (RB-372-A1; Lab Vision NeoMarkers, Fremont, California, USA). Lysozyme (10 μg) was added to the gel as a positive control.

Transdifferentiation. The cells were grown to confluence on eight-well glass slides (Nalge Nunc International, Naperville, Illinois, USA). The media was changed to DMEM supplemented with 1% FCS. Cells were then cultured for 2, 4, and 6 days in the presence of AGE-modified BSA (AGE-BSA, 5–40 μM), AGE-ribonuclease (AGE-RNase, 100 μM), or with the same nonglycated proteins as controls. In further experiments, cells were incubated for 6 days with variously CML-modified BSA (200 μM) or control. In separate experiments, anti-human RAGE Ab (1:200) (28), or neutralizing Ab to TGF-β (MAB 240, 10–20 μg ml; R&D Systems Inc., Minneapolis, Minnesota, USA) were added to evaluate the role of RAGE and TGF-β in transdifferentiation. Azide-free control mouse monoclonal or rabbit polyclonal Ab’s (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) were added at identical Ab concentrations. Fresh media containing AGE-proteins and Ab’s were replaced at 3 days.

Light microscopy and immunocytochemistry. At the end of the incubations described above, cells were examined for changes in cell morphology by light microscopy (×200–400) and antigen expression by immunohistochemistry. Cells were stained for the myofibroblast marker alpha-smooth muscle actin (α-SMA) (30, 31). As a further marker of transdifferentiation, cells were examined for the presence of the epithelial junctional protein E-cadherin in parallel experiments, as described previously (20).

Immunohistochemistry. Ab’s used were to α-SMA (1A4; DAKO Corp.), a well-characterized Ab recognizing AGE (28), and RAGE (gift of M. Neeper) (28, 29). Ab’s to TGF-β (MAB 1835; R&D Systems Inc.) and E-cadherin (C37020; Transduction Laboratories, San Diego, California, USA) were used at 5 μg/ml.

Cells were fixed in 4% paraformaldehyde by the acid-shift method (32). To aid E-cadherin antigen retrieval fixed cells were treated with 10 minutes of microwave oven heating in citrate buffer (10 mM, pH 6.0) at 2,450 MHz and at 800 W of power.

Kidney tissues examined for α-SMA, AGE, and RAGE were formalin fixed, and tissues used for TGF-β staining were fixed in mercuric chloride.

In general, staining followed standard procedures using an avidin-biotin–based Ab system. Nonspecific binding was prevented by incubation with 10% normal goat serum, and cells and tissues were incubated sequentially with the primary Ab, 0.3% hydrogen peroxide in PBS, and a biotinylated secondary Ab (DAKO Corp.). Finally, streptavidin-conjugated horseradish peroxidase (Vector Laboratories Inc.) was added, followed by signal development using diaminobenzidine (DAB; DAKO Corp.) for AGE, RAGE, SMA, and TGF-β or novo red (E-cadherin) as substrate.

In vivo transdifferentiation. Two normotensive rat models were studied, Wistar Kyoto (WKY) and Sprague Dawley (SD) rats, to allow comparison of transdifferentiation at various time points. Both strains were chosen because our group has previously characterized the evolution of diabetic nephropathy in detail in these animals (33, 34). At age 8 weeks, rats were randomized into two groups, control and diabetic. Diabetes was induced by intravenous injection of streptozotocin (STZ) 50 mg/kg, after an overnight fast. Only rats with plasma glucose of more than 15 mmol/l were considered diabetic. All diabetic animals received 2 U insulin/zinc suspension (Ultratard HM; Novo Nordisk, Bagsvaerd, Denmark) injected subcutaneously three times a week to maintain body weight and improve survival. The SD rats were further randomized to receive the cross-link breaker, ALT-711, at 10 mg/kg/day or nothing by gavage (14). These thiazolium compounds have been shown previously to specifically cleave preformed AGEs (11) and are associated with reduced AGE accumulation (12).

WKY rats were sacrificed after 16 and 24 weeks and the SD rats at 32 weeks. Kidneys were formalin fixed and paraffin embedded. Sections from diabetic and control animals (n ≥ 5) at each time point were stained for α-SMA as per cell culture experiments and counterstained with periodic acid-Schiff reagent (PAS) to aid the identification of basement membranes and brush borders.

One animal that failed to become diabetic after STZ administration was used as a control for nonspecific effects of STZ.

In addition, a number of human biopsy samples from type 1 diabetic subjects with nephropathy were stained and examined for tubular α-SMA immunostaining. A number of postmortem samples from patients with diabetic nephropathy were also obtained and examined for the presence of AGE and RAGE.

Quantitation of immunocytochemical staining. Slides were blinded for identity and more than ten high-power fields (×400, approximately 300 cells per field) were counted with the aid of a graticule to identify cells expressing α-SMA or E-cadherin.

In animal experiments tubules containing α-SMA positive (+ve) cells were counted with more than 40 high-power fields per section and expressed as a proportion of total tubule number. In the ALT 711 study the proportion of α-SMA +ve cells in each affected tubule was also quantitated. The observer was blinded as to the status of each section. Results are shown as the percentage of α-SMA +ve tubules of total tubules counted and percentage of α-SMA +ve cells per affected tubule.

Tubular TGF-β immunostaining performed in the SD rat group was quantitated using a computerized imaging system as described previously (35). Areas including tubules only were selected, and proportional immunostaining was assessed in more than 40 high-power fields, with observers masked as to slide identity. Results are expressed as the proportional stained area.

TGF-β analysis. NRK 52E cells were passaged into six-well plates and treated as above with AGEs or unmodified proteins in the presence or absence of Ab’s. After 3 days, the media was removed and analyzed for TGF-β using a sandwich ELISA kit (Promega Corp., Annandale, Australia) per the manufacturer’s instructions. Total TGF-β was measured in duplicate samples from three separate experiments and final TGF-β concentrations adjusted for cell number.

Statistical analysis. Values are means plus or minus SEM. Data were analyzed by ANOVA and compared using Tukey post hoc test. A two-tailed unpaired t test was used to compare control and diabetic animals for α-SMA staining. P values less than 0.05 were considered significant.