*2.5. Histological Analysis*

Kidney was isolated, fixed in 10% formaldehyde solution, dehydrated, and then embedded in para ffin. Sections of renal tissues were cut into 5 μm thickness and stained with hematoxylin and eosin (H&E) through removal of para ffin in xylene and rehydration in alcohol, as per concentration of the series. The stained tissues on slide glass were mounted with histological mounting medium (Histomount, Atlanta, GA, USA) after drying. All images were taken using an optical microscope (Nikon ECLIPSE Ci, Nikon Instrument, Tokyo, Japan).

#### *2.6. Protein Extraction and Western Blot Analysis*

The kidneys were homogenized in the hypotonic lysis bu ffer (1.5 mM MgCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM KCl, 0.05% nonidet P-40 (NP40), 0.5 mM dithiothreitol (DTT), and distilled water) with protease and phosphotease inhibitor (Thermo Fisher, Waltham, MA, USA), shacked on ice for 1 h, and centrifuged at 1945× *g* at 4 ◦C for 10 min. The supernatants were centrifuged again at 9078× *g* at 4 ◦C for 30 min and final supernatants were used as a cytosol extract for Western blot analysis. The remaining pellets were re-homogenized in hypertonic lysis bu ffer (1.5 mM MgCl2, 5 mM HEPES, 0.5 mM DTT, 0.2 mM EDTA, 26% glycerol, and distilled water) with 4.6 M NaCl on ice. After shaking on ice for 1 h, the homogenates were centrifuged at 9078× *g* at 4 ◦C for 20 min. Then, supernatants were used as nuclear extract for Western blot analysis. Total protein amount of the extract was quantified by bovine serum albumin (BCA) protein assay (ThermoFisher Scientific, Grand Island, NY, USA).

Protein samples were separated with SDS-PAGE and transferred onto poly-vinylidine fluoride (PVDF) membranes (Millipore, Marlborough, MA, USA). After blocking in 3% bovine serum albumin (BSA) in phosphate-bu ffed saline–0.1% Tween 20 (PBS-T), the membranes were incubated at 4 ◦C with primary antibodies. Then, the membranes were washed with PBS-T and incubated with respective horseradish peroxide (HRP)-conjugated secondary antibodies for 1 h, and then washed with PBS-T again. The chemiluminescent signals were developed using ECL solution (Bio-rad, Hercules, CA, USA). Images of the developed bands were recorded and quantified with the Syngene G box (Syngene, Cambridge, UK).
