Juglone reduced fibrogenesis after UUO

Male Lewis rats (3 months old) underwent UUO of the left kidney for 2 weeks. There were three groups receiving vehicle, juglone 0.25 mg/kg/day or juglone 1 mg/kg/day for 2 weeks starting the day of surgery. There was no animal death associated with treatment. Treated animals had a 10% weight loss in the first week after surgery, which resolved by the end of week 2. Immunoblot analyses for Pin 1, biomarkers of matrix remodeling (α-smooth muscle actin (SMA), collagen type III and vimentin) and signaling pathways involved in fibrogenesis (phospho-smad2) and stress response (phospho-heat shock protein (HSP)27) demonstrated that juglone therapy decreased α-SMA, collagen type III, vimentin, p-smad2 and p-HSP27 levels (Figure 1). There was no difference in Pin 1 levels between treatment and control groups suggesting that juglone inhibits fibrogenesis independently of Pin 1 levels in the UUO model.

Juglone and UUO had similar inhibitory effects on Pin 1 activity

We next examined Pin 1 activity in unobstructed and obstructed kidneys in control or juglone-treated rats. These analyses would help us determine whether the antifibrotic properties of juglone resulted from Pin 1 blockade. The studies demonstrated that juglone effectively inhibited Pin 1 activity in unobstructed right kidneys (Figure 2). However, Pin 1 activity was significantly decreased in left obstructed kidneys regardless of treatment status (Figure 2). Pin 1 activity in obstructed kidneys was reduced to the same level as in unobstructed kidneys treated with juglone. In aggregate, these studies suggest that the antifibrotic effects of juglone are independent from Pin 1 blockade during UUO.

Juglone reduced EMT and oxidative stress after UUO

To further define the effects of juglone on fibrogenesis, we assessed EMT using double-staining immunohistochemical analyses for E-cadherin (pink, epithelial marker) and α-SMA (brown, mesenchymal marker) in normal right and UUO left kidneys in control or juglone-treated rats (Figure 3a-c). These studies showed pink basolateral E-cadherin staining in distal tubules of control kidneys (Figure 3a). UUO resulted in downregulation of E-cadherin (no basolateral staining) and greater brown interstitial staining for α-SMA (Figure 3b). Treatment with juglone preserved distal E-cadherin expression and decreased interstitial α-SMA levels (Figure 3c), suggesting that juglone may attenuate EMT during UUO. Aggregate semiquantitative scoring is presented as a bar graph in the right panel (Figure 3).

We evaluated oxidative stress using dual-staining analyses for antioxidant (Mn superoxide dismutase (SOD)) and pro-oxidant (NAPDH oxidase 2 (Nox-2)) enzymes (Figure 3d-f). MnSOD is a superoxide scavenger while Nox-2 is one of the key generators of superoxide in the kidney [7]. Normal kidneys showed strong tubular MnSOD staining and rare patchy areas of monocytic infiltration positive for Nox-2 (Figure 3d). UUO resulted in reduced tubular MnSOD and increased tubulointerstitial Nox-2 (Figure 3e) consistent with significant monocyte/macrophage infiltration and a pro-oxidant milieu. Juglone attenuated these changes suggesting that treatment decreases oxidative stress (Figure 3f). Consistent with these findings, dihydroethidine staining for superoxide anion was significantly decreased with juglone compared to vehicle (Figure 3g, h). Aggregate semiquantitative scoring is presented as a bar graph in the right panel (Figure 3).

The effects of juglone on fibrogenesis may be mediated by smad2

To determine whether juglone inhibited TGFβ 1 activity we evaluated phospho-smad2 activity in normal kidneys compared to obstructed kidneys treated or not with juglone (Figure 4). These studies demonstrated that nuclear p-smad2 was significantly increased after UUO and that juglone prevented nuclear p-smad2 activity. Lastly, we evaluated the effects of juglone on α-SMA and activated smad2 (p-smad2) levels in proximal tubular epithelial cells. Juglone (1 μM) significantly reduced α-SMA and p-smad2 levels, consistent with our in vivo studies and suggesting that juglone may inhibit smad2 phosphorylation and activation in tubular epithelial cells (Figure 5).

Animals

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 [14]. 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.

Immunoblotting

Western blotting was performed on protein lysates obtained from whole kidney tissue or cell lysates as described previously [14]. 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/.

Immunohistochemical analyses

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 [18]. 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.

Statistical analysis

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.