Rat kidneys were dissected and fixed in 4% paraformaldehyde and embedded in paraffin, and 4 μm serial sections were then prepared for histological analysis under a light microscope or fluorescence microscope. TdT-mediated dUTP nick-end labeling (TUNEL) was done with an apoptosis detection kit (Servicebio) following the manufacturer’s instructions. Hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS)[27 (link)] staining were carried out using standard protocols. A random sample of three glomeruli from each rat was analyzed using image analysis software (Image-Pro plus 6.0) to determine the percentage of PAS-positive areas, expressed as a mesangial index. Immunohistochemical (IHC) analyses were carried out using the rabbit anti-AGEs (1:300; bs-1158R; Bioss, Woburn, MA, United States) primary antibodies. HRP-conjugated goat anti-rabbit IgG (1:500; GB23303; Servicebio) was used to detect primary antibodies. The nuclei were subsequently stained with 3,3’-diaminobenzidine, and three fields of view from each rat were digitized. The integrated optical density from all fields was calculated using Image-Pro Plus 6.0.