Histological analysis and assessment of glomerular injury.

Periodic acid-Schiff (PAS) staining was performed to evaluate glomerular histology and glomerulosclerosis, which was indicated by the glomerular score. Paraffin-embedded kidney sections (5 μm) were stained with PAS (Sigma-Aldrich, St. Louis, MO). Images were captured using an Olympus DP70 digital camera with DP manager software (version 2.2.1). The glomerular scores in PAS-stained sections were graded by a blind observer using a scale of 0–4: 0 was assigned to normal glomeruli, 1 was assigned to glomeruli with mesangial expansion, 2 was assigned to glomeruli in which sclerosis encompassed <50% of the glomerulus, 3 was assigned to glomeruli with lesions encompassing 50–75% of the glomerulus, and 4 was assigned to glomeruli with lesions encompassing >75% of the glomerulus or fully collapsed glomeruli. For each mouse, the glomerular score was evaluated in >50 glomeruli from 5 sections (>10 glomeruli/section), and those individual scores were averaged to obtain a mean value for that mouse.