Histological Analysis of Kidney Injury

After harvesting, kidneys were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned and stained with hematoxylin & eosin (H&E) stain, periodic acid Schiff (PAS) stain, and Masson’s trichrome stain. Images were captured using a confocal microscope (Nikon, Tokyo, Japan). Tubular injury in PAS-stained sections was assessed and scored at a ×200 magnification using 10 randomly selected fields for each kidney as follows: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [18 (link)]. For IHC staining, the sections were incubated with primary antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxynonenal (4-HNE; Abcam), galectin-3 (Abcam), α-smooth muscle actin (α-SMA; Sigma-Aldrich), or collagen I (Abcam) overnight at 4 °C and then probed with a secondary antibody for 30 min at room temperature. All the sections were counterstained with hematoxylin. The percentage of positive staining was assessed in 5 randomly selected fields (×400 magnification) per each kidney using i-Solution Lite V.9.1 image analysis software (IMTechnology, Vancouver, BC, Canada). Galectin 3 positive cells or CD4 positive cells were counted in 5 randomly selected fields (×400 magnification) per each kidney.