As previously described [15 (link)], immunohistochemistry was performed on formalin-fixed 3-μm-thick paraffin-embedded sections. Briefly, sections were deparaffinized, rehydrated using an ethanol series, blocked with normal horse serum, treated with primary antibodies at 4°C overnight, and incubated for 30 minutes with a secondary antibody (ImmPRESS HRP reagent kit, Vector Laboratories, Burlingame, CA, USA). The slides were developed using 3,3′-diaminobenzidine (DAB; Vector Laboratories) and counterstained with Harris hematoxylin. The primary antibodies used were anti-phosphorylated Akt1 (p-Akt1) (ab5954, 1:200, Abcam, Cambridge, UK), anti-E-cadherin (#3195, 1:400, Cell Signaling Technology, Danvers, MA, USA), anti-transforming growth factor-β1 (TGF-β1) (ab190503, 1:500, Abcam), anti-phosphorylated glycogen synthase kinase-3β (p-GSK-3β) (ab131097, 1:100 dilution, Abcam), anti-β-catenin (#9562, 1:400, Cell Signaling Technology), and anti-Snail (sc-271977, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Staining intensity was semi-quantitatively graded in a blinded manner using the following scoring scale: 0 = absence of specific staining; 1, < 25% area with specific staining; 2, 25% to 50% staining; 3, 50% to 75% staining; 4, > 75% staining. At least 20 fields of renal cortex were investigated [16 (link)].