Mmp12

Western blotting was performed essentially as previously described (17 (link)). The expression of CD137 (1:500, rat, Abcam, Cambridge, UK), ASK1 (1:2,000, rabbit, Abcam), phospho-ASK1 [1:500, rabbit, Cell Signaling Technology (CST), Danvers, MA, USA], p38 (1:1,000, rabbit, CST), phospho-p38 (1:1,000, rabbit, CST), JNK (1:1,000, rabbit, CST), phospho-JNK (1:1,000, rabbit, CST), IκBα (1:1,000, rabbit, CST), phospho-IκBα (1:1,000, rabbit, CST), MMP9 (1:1,000, rabbit, Abcam), MMP12 (1:2,000, rabbit, Abcam), and β-actin (1:1,000, rabbit, CST) in MH-S cells or lung tissues was measured by western blot. Protein expression was normalized to β-actin.

After deparaffinization in xylene and subsequent rehydration in graded alcohol series, tissue sections were blocked by exposure to 3% H₂O₂ and boiled in a citrate buffer (pH 5.9–6.2) at 95°C for 20 min. After blocking in 5% BSA for 1 h at room temperature, tissue sections were incubated at 4°C overnight with antibodies to CD68 (1:200, rat, Abcam), MMP9 (1:200, rabbit, Servicebio, Wuhan, China), MMP12 (1:50, rabbit, Abcam), or collagen I (1:200, rabbit, Abcam). The sections were washed with phosphate-buffered saline, and then incubated with horseradish peroxidase secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 h. A DAB kit (Santa Cruz) was used to perform the positive staining. Nuclei were stained by hematoxylin.

Frozen kidney sections (5-μm thick) that were collected on slides were fixed and processed by hematoxylin and eosin and periodic acid schiff (PAS) staining (Sigma-Aldrich, St. Louis, MO, USA). The mesangial area was analyzed to determine the percentage of glomerular area (10 glomeruli per kidney per animal) using Image J software (National Institutes of Health, Bethesda, MD, USA). For immunohistochemistry, sections were fixed in pre-cooled acetone (−20 °C) for 5 min; after three washes in phosphate-buffered saline and a 10-min treatment with 3% H₂O₂, sections were serum-blocked and incubated with antibodies against MMP-12, CD68 (both from Abcam, Cambridge, MA, USA), FN, collagen IV (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), or nitrotyrosine (EMD Millipore, Billerica, MA, USA), followed by incubation with biotinylated anti-rabbit, anti-rat, or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA), a streptavidin-horseradish peroxidase conjugate, and finally diaminobenzidine substrate for visualization (Vector Laboratories). For glomerular assessment, the percentage of mesangial area stained as glomeruli was quantitated using 10 glomeruli per kidney per animal, using Image J software.