2.5 Histopathological analysis
Kidney and liver tissues were formalin fixed and paraffin embedded. The paraffin embedded tissues were cut into 4μm sections for histological analysis. Tissue sections were deparaffinized, re-hydrated, and kidney tissue slices were stained with Periodic Acid-Schiff (PAS) and Picrosirius Red (PSR). Glomerulosclerosis and mesangial matrix expansion were scored from kidney sections stained with PAS staining using methods described earlier (Hye Khan et al., 2018,2019). Histological analysis was done at a magnification of 400X to assess glomerular injury, and renal tubular proteinaceous cast was assessed at a magnification of 200X using Nikon NIS Elements Software (Nikon Instruments Inc., Melville, NY, USA). The percentage area positive for proteinaceous cast was calculated from the mean of eight cortical and five medullary fields for each animal. Fibrosis in the kidney and liver were determined from kidney and liver sections stained with PSR and examined at a magnification of 200X. In the kidney, the percentage area positive for collagen was calculated as the fibrotic area from the mean of eight cortical and five medullary fields for each kidney sample. The mean percentage of collagen positive liver fibrotic areas were calculated from 20 fields in each liver sample. In assessing liver steatosis, frozen liver sections (10 μm) were stained with Oil Red O dye according to manufacturer’s protocol (Abcam, Cambridge, MA, USA). The percent of liver tissue area with lipid accumulation was calculated as described earlier (Hye Khan et al., 2018). The percentage area positive for lipid accumulation was calculated from the mean of 20 fields (at 200X magnification) for each animal using Nikon NIS Elements Software. Histological scoring was carried out by two observers who were blinded to the identity of the experimental groups.