Detailed Kidney Histopathology Assessment

Kidney specimens were processed by light and immunofluorescence microscopic examination. For the light microscopy, the right kidneys from each animal were fixed in 10% phosphate-buffered formalin solution and embedded in paraffin. Sections of 2 μm thickness were cut and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). To evaluate the glomerular hypercellularity, at least 10 glomeruli were examined for each animal, the number of cells in each glomeruli (including endothelial cells, mesangial cells, and podocytes) was counted and average number was calculated; meanwhile, the incidence of glomerular basement membrane thickening or mesangial proliferation among 100 glomeruli was calculated too.
To assess the tubulointerstitial damage, a semiquantitative method of renal histology using a grading scale of 0–4 was applied: 0: normal; 1: lesions in <25% of the area; 2: lesions in 25% to 50% of the area; 3: lesions in >50% of the area; and 4: lesions involving the entire area [9 (link), 10 (link)]. Tubular atrophy, dilation, casts, interstitial inflammation, and fibrosis were assessed in 10 kidney fields at a magnification of ×100.
For immunofluorescence microscopy, tissue blocks from the left kidney were instantaneously frozen in n-hexane precooled to −70°C, and 4 μm cryostat sections were stained with fluorescein isothiocyanate- (FITC-) conjugated anti-rat IgG. The degree of deposition of immune complex was calculated quantitatively on the basis of the staining intensity and distribution.