Animal models
Male Wistar rats (8 to 10 weeks of age, 200–250 g) were obtained from the Animal Center of National Cheng Kung University Medical College. To induce diabetes rats were given a single intravenous injection of 60 mg/kg streptozotocin (STZ; Sigma-Aldrich, Inc., St. Louis, Mo., USA). Animals were considered to be diabetic if they had plasma glucose concentrations of 350 mg/dl or greater, in addition to polyuria and other diabetic features. All studies were carried out two weeks after the injection of STZ. All animal procedures were performed according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (United States), as well as the guidelines of the Animal Welfare Act. The study was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at Tajen University (approval number: IACUC 99–24; approval date: December 23, 2011).
Treatment protocols
STZ-diabetic rats in the treatment group were dosed with 0.3, 1.0 or 3.0 mg/kg ruscogenin (≥ 98%; Chengdu Biopurify Phytochemicals Ltd., Chengdu, Sichuan, China; Cat. No. 472-11-7) in distilled water (1.5 ml/kg) by oral gavage once daily for eight weeks. The dosage regimen was selected based on a previous report demonstrating that ruscogenin at the indicated dosage regimen was potentially effective in inhibiting lipopolysaccharide-induced inflammation in mice [15]. Another group of STZ-diabetic rats was treated orally for eight weeks with 5 mg/kg/day rosiglitazone (purity ≥ 99.0%, Sigma-Aldrich, Inc.). The dose of rosiglitazone was based on studies with long-term treatment in STZ-diabetic rats [19]. A vehicle-treated groups of STZ-diabetic rats and normal rats were give 1.5 ml/kg distilled water by oral gavage over the same period. Animals had free access to standard rat diet (Harlan Teklad, Madison, WI, USA; Cat. No. 2018) and water throughout the entire treatment period. Treatment was continued even though the plasma glucose of STZ-diabetic rats was lower than 350 mg/dl during the eight-week treatment period. At the end of the eight-week treatment, the rats were weighed, and blood samples were collected from a tail vein. The evening prior to blood sample collection, animals were restricted to 3 g of chow (given at 18:00 h), which was consumed immediately, and thereafter had access to only water. The animals were transferred to metabolic cages (Shineteh Instruments Co., Ltd, Taipei, Taiwan), and urine was collected for 24 hours under a layer of toluene (to inhibit bacteria growth) and stored at 4°C for later analysis. Toluene had no detectable effect on the estimation of albumin and creatinine in the urine samples. Following urine collection, rats were sacrificed using an intraperitoneal injection of sodium pentobarbital (50 mg/kg).
The kidneys were dissected and rinsed with cold isotonic saline and weighed. The right kidney was stored immediately at −80°C in liquid nitrogen for biochemical determinations and Western blot analyses. Other kidney tissues were fixed in 10% neutralized formalin for histology.
Blood sampling and analysis
Blood samples were centrifuged at 2 000 × g for 10 minutes at 4°C, and plasma was divided into aliquots for subsequent analyses. Plasma glucose concentration was determined using a diagnostic kit from BioSystem (Barcelona, Spain; Cat. No. COD12503). Serum creatinine (Scr) concentration was determined using a commercial assay kit purchased from Diagnostic Chemicals Limited (Connecticut, USA; Cat. No. 221–30). Blood urea nitrogen (BUN) was determined by kinetic reagent (Diagnostic Chemicals Limited, Cat. No. 283–30). Commercial enzyme-linked immunosorbent assay kits were used to quantify glycosylated hemoglobin (HbA1c) levels (Integrated Bio Ltd., Taipei, Taiwan; Cat. No. CSB-E08140r). All analyses were performed in accordance with the instructions provided by the manufacturers.
Analysis of urine parameters
The 24-hour urine samples collected from each diabetic rat and age-matched control was centrifuged at 2 000 × g for 10 minutes. Urinary albumin concentrations were measured with the Nephrat II ELISA kit (Exocell, PA, USA; Cat. No. NR002). The concentration of creatinine in pooled urine samples was determined using a commercial assay kit (Diagnostic Chemicals Limited; Cat. No. 221–30). All analyses were performed in accordance with the manufacturer’s instructions. Creatinine clearance (Ccr) was calculated in individual rats using the relationship: Ccr = urine creatinine × (urine volume/plasma creatinine) × time [20].
NF-κB activity measurement
Nuclear extracts of kidney from the above-mentioned groups were prepared using the nuclear extract kit (Active Motif, CA, USA; Cat. No. 40010). Twenty micrograms of nuclear extract were used for the determination of NF-κB activity with the TransAM® NF-κB p65 transcription factor assay kit (Active Motif; Cat. No. 40096) according to the manufacturer’s instruction.
Renal cytokines determination
Renal tissue was homogenized in 10 mmol/L Tris–HCl buffered solution (pH 7.4) containing 2 mol/L NaCl, 1 mmol/L EDTA, 0.01% Tween 80, 1 mmol/L PMSF, and centrifuged at 9 000 × g for 30 min at 4°C [21]. The resultant supernatant was used for cytokine determination. ELISA kits for the determination of tumor necrosis factor-α (TNF-α) (Cat. No. ab46070), interleukin (IL)-6 (Cat. No. ab100772), and IL-1β (Cat. No. ab100768) were obtained from Abcam Inc. (Cambridge, MA, USA). Samples were assayed in duplicates according to manufacturer's instructions. The protein concentrations of kidney filtrate were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Japan) and bovine serum albumin (BSA) as a standard.
Renal histological analysis
For histological analysis, the kidney was removed and embedded in paraffin to prepare 4-μm tissue slices. The tissue slices were stained with hematoxylin and eosin (H&E). The mesangial expansion index was scored in four levels from 0 to 3, with the index scores defined as follows [22]: 0, normal glomeruli; 1, matrix expansion occurred in up to 50% of a glomerulus; 2, matrix expansion occurred in 50 to 75% of a glomerulus; 3, matrix expansion occurred in 75 to 100% of a glomerulus. Scores were assigned for at least 30 glomeruli from kidney slices from each animal, and the means were calculated. Each slide was scored by a pathologist who was unaware of the experimental details.
Immunohistochemistry
Formalin-fixed, paraffin-embedded kidney tissue sections were used for immunohistochemical staining. After deparaffinization and hydration, the slides were washed in Tris-buffered saline (TBS; 10 mmol/l Tris HCl, 0.85% NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating the slides in methanol and 0.3% H2O2 in methanol. After overnight incubation with mouse monoclonal anti-monocyte/macrophage antibody (anti-ED-1) (Santa Cruz Biotechnology Inc. CA, USA; Cat. No. sc-59103), goat polyclonal anti-ICAM-1 antibody (Santa Cruz Biotechnology, Inc.; Cat. No. sc-1511), rabbit polyclonal nti-MCP-1 antibody (Abcam plc, Cambridge, UK; Cat. No. ab7202), mouse monoclonal anti-TGF-β1 antibody (Santa Cruz Biotechnology, Inc.; Cat. No. sc-52893), or rabbit polyclonal anti-fibronectin antibody (Abcam plc, Cat. No. ab2413) at 4°C, the slides were washed in TBS. Horseradish peroxidase-conjugated secondary antibody was then added, and the slides were incubated at room temperature for an additional 1 hour. The slides were washed in TBS, incubated with diaminobenzidine tetrahydrochloride as the substrate, and counterstained with hematoxylin. A negative control without primary antibody was included in the experiment to verify antibody specificity.
Sections were counterstained with haematoxylin for 15 seconds. Brownish yellow granular or linear deposits were interpreted as positive areas. Intraglomerular ED1-positive cells were counted in 30 glomeruli per animal at 400 × magnification by two independent observers with no prior knowledge of the experimental design [23]. The average number per glomerulus was used. Semi-quantitative assessments of the immunostaining of ICAM-1, MCP-1, TGF-β1, and fibronectin expression were scored using 4 levels, and an average value was obtained from analyses of more than 30 glomeruli per rat. The degree of ICAM-1, MCP-1, TGF-β1 and fibronectin expression in five rats from each group was graded as follows: 0, absent or < 25% staining; 1, 25% to 50% positive staining; 2, 50% to 75% positive staining; and 3, > 75% positive staining [22].
Western blotting
Protein extraction of isolated kidney was performed as follows [24]. The sample was homogenized in ice-cold in 1 ml of hypotonic buffer A [10 mmol/l HEPES (pH 7.8), 10 mmol/l KCl, 2 mmol/l MgCl2, 1 mmol/l DTT, 0.1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonylfluoride]. The cells were then lysed with 12.5 μl 10% Nonidet P-40. The homogenate was centrifuged, and supernatant containing the cytoplasmic extracts was stored frozen at −80°C. The nuclear pellet was resuspendedin 25 μl ice-cold nuclear extraction buffer. After 30 minutes of intermittent mixing, the extract was centrifuged, and supernatants containing nuclear extracts were secured.
Before immunoblotting, and the protein concentration of each tissue was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Japan) and BSA as a standard, to ensure equal loading among lanes. Nnuclear extracts (50 μg total protein) were separated on a 7.5-15% polyacrilamide gel and electophoretically transferred to nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline Tween (20 mmol/l Tris, pH 7.6, 137 mmol/l NaCl, and 0.1% Tween 20) for 3 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: p-NF-κB p65 (Ser276) (Santa Cruz Biotechnology Inc.; Cat. No. sc-101749), NF-κB p65 (Cell Signaling Technology, USA; Cat. No. 4764,). The level of lamin A (Santa Cruz Biotechnology Inc.; Cat. No. sc-20680) was estimated for equal loading of nuclear sample. Three times after washing with Tris-buffered saline Tween 20 (TBST), incubation with appropriate horseradish peroxidase-conjugated secondary antibodies were performed for 1 h at room temperature. After three additional TBST washes, the immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. Band densities were determined using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan). All experimental sample values were then expressed relative to this adjusted mean value. Tissue sections were sampled from 4 independent experiments.
Statistical analysis
The results are presented as the mean ± standard deviation (SD) for each group of animals at the number (n) indicated. Statistical analysis was performed with one-way analysis of variance (ANOVA). The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. The renal morphohistology and the morphologic analysis for PAS staining were analyzed statistically using the Kruskal-Wallis Test and Dunn’s Multiple Comparisons Test. Values of P < 0.05 were considered statistically significant.