Proteosilver plus kit

Urine samples containing 5 μg of creatinine were solubilized in SDS buffer (2% SDS, 10% glycerol, 0.1% bromophenol blue, 50 mM Tris, pH 6.8), and the proteins were separated by SDS-PAGE using 12% polyacrylamide gels. Following electrophoresis, gels containing urine samples were silver stained using the ProteoSilver Plus kit (Sigma-Aldrich Chemical, St. Louis, Missouri, USA) to detect the urinary proteins.

Serum creatinine concentration was measured using the Jaffé method. The animals were classified as AKI according to the AKIN (Mehta et al. 2007 (link)), in which the diagnosis of AKI is defined by an increase in serum creatinine level above 150–200% from baseline.
Serum urea was measured by UV UREASI/GLDH kinetic. Urinary and serum sodium, potassium, chloride, urinary pH, and partial pressure of carbon dioxide (pCO₂) in urine were measured on a Radiometer ABL800Flex (Radiometer Medical, Bronshoj, Demmark). Urinary bicarbonate concentration was calculated using the Handerson–Hasselbach equation. All these data were used to calculate the fractional excretion of ions. Urine protein excretion was quantified using a Sensiprot kit (Labtest, Minas Gerais, Brazil) and qualified by 10% SDS‐PAGE containing 10 μg of creatinine and 2 μg of bovine serum albumin (BSA) as a positive control, silver stained using a Proteosilver Plus kit (Sigma). Urinary glucose and creatinine were measured before ischemia (0), during ischemia (60 min), and post reperfusion (135, 360, 780, and 1080 min).