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.