Animals and adriamycin-induced nephropathy model
Mouse adriamycin nephropathy (AN) was induced by a single injection of adriamycin (10.2 mg/kg, with physiological saline for controls) through tail vein in MMP-9 knockout (MMP-9-/- mice crossed into a BALB/c background, obtained from Prof Zena Werb of University of California San Francisco) and wild-type control (BALB/c mice crossed with MMP-9-/- mice of BALB/c background, the MMP-9+/+ phenotype was chosen as control mice. Both MMP-9-/- and control mice, male healthy with body weight between 20 to 25 gram, are randomly selected control (saline) and adriamycin injection group, 5 mice each group, total 20 miced, standard 4 mice each standard filtered PVC cage, with standard light/dark cycle, temperature control, free access to water and standard normal food. Injection was performed in specific pathogen free PC2 operation room of animal housing facility. All animals were euthanased by CO2 at 4 weeks after injection, to collect tissue samples. Experiments were carried out in accordance with the protocols approved by the Animal Ethics Committee of Western Sydney Local Health District.
Urinary proteinuria concentration and kidney function
Urinary albumin to creatinine ratio was used to evaluate proteinuria. Urinary albumin concentration was determined by nephelometric method as reported [12]. Urinary creatinine concentration was determined by enzymatic method. Blood samples were taken from mice before sacrifice.
For calculation of GFR, mice were acclimatised to the metabolism cages for 48 h prior to 24 h urine collection. Urine samples were collected in metabolism cages 24 h before sacrifice. Serum creatinine levels were determined using a creatinine assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Calculated GFR was evaluated by creatinine clearance using the standard formula.
Histological analysis
Four weeks after adriamycin treatment, paraffin-embedded kidney sections (4 μm) were deparaffinised with xylene and rehydrated through a descending ethanol gradient. Histological sections were examined following Sirius red (SR) or Gomori trichrome (GT) staining. Quantification of pulmonary and kidney fibrosis was performed as we described previously [13]. All scoring was performed in a blinded manner.
Immunofluorescence analysis
Frozen kidney blocks were cut into 7 µm sections and fixed with ice-cold acetone for 10 min at -20°C and blocked with 2% BSA for 1 h. Double immunofluorescence staining was performed using combinations of antibodies against P-cadherin and desmin, VE-cadherin and α-SMA, respectively. Tissue sections were then incubated with its corresponding fluorescence-conjugated secondary antibody. After washing with PBS, sections were counterstained with DAPI for 5 min before mounting with fluorescence mounting medium. Images were obtained using a confocal microscope (Olympus FV1000) at ×40 magnification. For quantitative analysis, the percentage of the area stained positive for P-cadherin and desmin, or VE-cadherin and α-SMA, were counted on high power fields (HPFs) in a blinded manner.
Statistical analysis
Results were expressed as means ± SEM. Statistical significance was evaluated using unpaired two-tailed t-test for comparison between two groups. The level of significance was set at p<0.05.