Quantifying Tubular Injury and Inflammation

The kidneys were fixed and embedded in paraffin. The blocks were sectioned and stained with periodic acid-Schiff (PAS). Tubular injury was assessed in 5 random fields (400×) per sample. The percentage of damaged area was evaluated and scored by using a semiquantitative scale: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [25 (link)]. The sections were also immunostained with antibodies against neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and 4-hydroxynonenal (4-HNE; Abcam, Cambridge, MA, USA). The percentage of area stained with NGAL or 4-HNE was analyzed by examining five random fields (400×) per sample using the i-Solution DT software version 11.0 (IMT i-Solution, Coquitlam, BC, Canada). The number of F4/80-stained cells was counted in 10 random fields (400×) per sample.