Histopathological Analysis of Rat Kidneys

Both kidneys were removed carefully after sacrificing rats without any damage and fixed with 10% neutral buffered formalin. Paraffin-embedded blocks were then made and sectioned (5 µm in thickness) for H&E, PAS, and Masson's trichrome staining to examine histopathological changes, glomerular basement membrane, and interstitial fibrosis, respectively (21 (link)). Images of stained kidney sections were taken using a Leica DM 2500 microscope (Leica Microsystems, Germany). For H&E-stained sections, ×400 magnification was used. For PAS- and Masson's trichrome-stained sections, ×1,000 magnifications were used. For each rat, separate slides were prepared. Ten different fields of the same slide were then examined. Two experienced renal pathologists assessed histopathological changes via quantitative tubulointerstitial injury measurement by counting numbers of apoptotic and necrotic cells, determining loss of tubular brush border, tubular dilatation, cast formation, and neutrophil infiltration, and examining glomeruli basement membrane thickness in a double-blinded fashion. The scoring was done based on level of damage: 0=none; 1=0-10%; 2=11-25%; 3=26-45%; 4=46-75%; and 5=76-100% (22 (link)).