Adult (9 to 11 weeks old) male Lewis rats were purchased from Harlan Teklad (Madison, WI, USA). Animals were housed in the animal care facility at the William Middleton Veterans Affairs Hospital (VAH) in Madison, WI, USA, and the procedures were performed in accordance with the animal care policies at the VAH and the University of Wisconsin. The UUO procedure was performed under general anesthesia with isoflurane as described previously . Briefly, the left ureter was ligated with 6-0 silk at two points and then severed between the ligatures to prevent retrograde urinary tract infection. Control animals underwent surgery and received vehicle (10% ethanol) (n = 5). Juglone was administered intraperitoneally for 14 days at 0.25 (n = 5) and 1 mg/kg/24 h diluted in 1 ml of vehicle (n = 5). Animals were killed after 2 weeks by exsanguination through cardiac puncture under general anesthesia. Both kidneys were harvested and sectioned longitudinally in half. Half was snap frozen immediately and used for immunoblot analysis and the other half was formalin fixed and paraffin embedded for immunohistochemical analyses. The right kidney served as the control to the left obstructed kidney.
Western blotting was performed on protein lysates obtained from whole kidney tissue or cell lysates as described previously . Briefly, after separation by SDS-PAGE (10% to 20% gradient PAGE, Bio-Rad, Hercules, CA, USA) proteins were transferred electrophoretically (100 V, 30 min) to nitrocellulose membranes (Bio-Rad) that were then blocked with a solution containing 5% non-fat milk, 50 mM Tris, HCl, pH 7.4, NaCl 150 mM, Tween 20 0.05% (TBS-Tween) overnight at 4°C. Membranes were incubated the next day with antibodies against α-SMA (2,000-1), Vimentin (100-1), collagen type III (100-1), phospho-HSP27 (0.25-1), phospho-smad2 (2,500-1), Pin 1 (200-1), and GAPDH (1:5,000). Binding of primary antibodies was followed by incubation for 1 h at room temperature with a secondary horseradish peroxidase (HRP)-conjugated IgG in 1% non-fat milk. Signals were visualized by enhanced chemiluminescence signals captured on x-ray films. Data was normalized to GAPDH. Densitometry was performed using the NIH Image J software http://rsbweb.nih.gov/ij/.
A portion of the kidney tissue was excised promptly after the animals were killed. It was immediately placed in 10% neutral-buffered formalin. Tissue was fixed overnight in formalin and processed for paraffin embedding following standard protocols and then sectioned for antibody staining. Double staining of E-cadherin/α-SMA and Nox-2/MnSOD was performed after sections were deparaffinized and hydrated. Heat-induced antigen retrieval was performed using a 5 mM ethylenediaminetetra-acetic acid (EDTA) solution (pH = 8.0) and a 10 mM citrate solution (pH = 6.0), respectively, at 25 psi for 2 min in a decloaking chamber. Non-specific staining was blocked using Sniper (Biocare Medical, Concord, CA, USA) for 9 min. Slides were incubated overnight with p-smad2 (500-1), E-cadherin (50-1) or Nox-2 (50-1) then washed and incubated with 3% hydrogen peroxide for 30 min. MACH 2 HRP polymer detection system (Biocare Medical, Concord, CA, USA) and 3,3'-diaminobenzidine (DAB) substrate were used to tag and stain the first primary antibody brown. Slides were then incubated with the second primary antibody α-SMA (50,000-1) or MnSOD (5,000-1) at room temp for 1 h. MACH 2 HRP polymer detection system and VIP substrate (Vector Laboratories, Burlingame, CA, USA) were used to tag and stain the second primary antibody purple. Tissue sections were washed in distilled water, counterstained with hematoxylin, dehydrated through an ethanol series and mounted with cover slips. Harris hematoxylin and 1% alcohol eosin were used to assess overall kidney injury and morphology. A total of 25 μM dihydroethidine dye (Molecular Probes, Carlsbad, CA, USA) was used for superoxide anion staining according to the manufacturer's recommendations.
Cortical staining intensity was scored on a scale of 0 to 3 (0 no staining, 1 mild, 2 moderate, 3 intense) for E-cadherin (distal tubules), α-SMA (interstitium), MnSOD (tubules), Nox-2 (interstitium), p-smad2 and superoxide (tubulointerstitium). Five high magnification fields were evaluated per kidney and results were expressed as mean and standard error bars.
Pin 1 activity assay
Pin 1 activity was measured in whole kidney protein lysates as described previously . Briefly, tissue lysates were prepared by five freeze-thaw cycles in a buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 100 mM NaCl (pH 7.0). Total protein (10 μg) in 10 μl was mixed with 70 μl of the HEPES/NaCl buffer supplemented with 2 mM dithiothreitol (DTT) and 0.04 mg/ml bovine serum albumin (BSA). Then, 5 μl of chymotrypsin (60 mg/ml in 0.001 N HCl) was added and thoroughly mixed. Finally, 5 μl of the substrate Suc-AEPF-pNa (provided by Peptides International, Louisville, KY, USA) dissolved in dimethyl sulfoxide (DMSO) and prepared at 100 μg/ml in 480 mM LiCl/trifluoroethanol was added. The absorption at 390 nM, which detects the formation of free p-nitroanilide (pNA), was monitored using a Beckman Coulter
DU 800 spectrophotometer (Brea, CA, USA). All of the reagents and materials were kept at 4°C during the procedure. Mean values and standard error bars from UUO and control kidneys are represented for each time point.
Juglone in vitro experiments
Normal rat kidney proximal epithelial cells (NRK52E) were obtained from the American Type Culture Collection (ATCC, Rockwell, MD, USA) and maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were seeded at 2.5 × 105 cells per well into six-well culture plates in Dulbecco modified Eagle medium (DMEM; high glucose) containing 5% heat inactivated fetal bovine serum (FBS), 44 mM NaHCO3, 5,000 IU penicillin and 5,000 μg/ml streptomycin (Cellgro, VA, USA). At 80% confluency, media was changed to serum free DMEM supplemented with 0.1% BSA for 12 h to arrest growth and synchronize cell activity. Cells were treated with juglone (1 μM) or vehicle (0.01% ethanol) for 48 h. Studies were performed in triplicates. Western blots for α-SMA, phospho-smad2 and β-actin were performed as described above.
The Student t test and the non-parametric Mann-Whitney rank sum test (Sigma Stat Software, Jandel Scientific, Chicago, IL, USA) were utilized when appropriate to compare differences in Pin 1 activity and gene and protein expression between groups. P values ≤ 0.05 were considered significant.