Renal Histology Evaluation in Mice

For renal histology, mouse kidneys were fixed in 10% formalin and embedded in paraffin for Periodic Acid-Schiff (PAS), H&E staining and Masson-Trichrome (MT) staining. Tissue blocks were sliced into 4-μm thickness. Glomerular injury score, inflammatory cell infiltration, and tubulointerstitial fibrosis score were assessed as described previously [12 (link)]. Briefly, for assessment of glomerular injury, renal sections were stained with PAS. To evaluate the glomerular injury score, more than 50 PAS-stained random glomeruli per mouse (n = 4 mice) were examined, and scored from 0 to 4 (0, no lesion; 1, expansion of mesangial area; 2, expansion of Bowman’s epithelial cells and adhesion of glomeruli and Bowman’s capsule; 3, sclerotic area in 50% - 75% of glomerulus; 4, sclerotic area in 75% - 100% of glomerulus). Double blind scoring was performed and values were computed and presented in a graph as percentage. For tubulointerstitial fibrosis score, MT-stained kidneys were evaluated using Bio-Revo imaging and analysis software (Keyence, Japan). MT-stained area vs unstained area was calculated and presented as % fibrotic region.