Histological analyses

Sections (thickness 4 μm) were cut from 10% formalin‐fixed, paraffin‐embedded kidney samples taken at 20 weeks of age and subjected to periodic acid–Schiff (PAS) staining to evaluate expansion of the mesangial matrix. PAS‐positive area was calculated as described previously (Terami et al. 2014). Kidney sections were also stained with Picrosirius Red F3BA Liquid (Polysciences, Warrington, PA) for 60 min to evaluate interstitial fibrosis. To quantify percentage renal fibrosis, 10 views of the renal cortex were selected randomly in each slide. Renal expression of type IV collagen, macrophages, and NADPH oxidase 4 (Nox4) was also analyzed, as described previously (Terami et al. 2014).