Hematoxylin and eosin (H&E) staining

H&E staining was performed to observe the kidneys in the control and DN groups. After 72 h of injection, the rats were sacrificed and kidney tissue samples were collected at the end of week 1, 2 and 3. Kidney tissues from rats were immediately fixed in formalin for 24 h at room temperature. Next, tissues were dehydrated with alcohol, embedded in paraffin, cut into 7-µm uniform sections and placed on glass slides coated with 3-aminopropyltriethoxysilane. The sections were then deparaffinized using xylene, hydrated and stained with H&E reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For staining, sections were incubated with hematoxylin at room temperature for 5 min, washed and then incubated with eosin at room temperature for 1-3 min. Subsequent to washing with gradient ethanol, neutral gel was used for sealing. The mean glomerular volume was calculated by measuring the maximum diameter of the glomerulus in 10 random fields.