Nephrat kit

Urinary albumin was measured using a commercial Nephrat kit (Exocell, Philadelphia, PA, USA), and total protein was determined via the Bradford method (Bio-Rad Protein Assay kit, Bio-Rad Laboratories, Hercules, CA, USA). A modified kinetic Jaffe reaction was performed to determine the urine creatinine level. The ratio of urine protein to creatinine was calculated by dividing the urine protein concentration by the creatinine concentration, both expressed in mg/dL.

In the last week of treatment protocol, the rats were placed in individual metabolic cages (Tecniplast 304) for 48 hours for the analysis of metabolic parameters. The first 24 hours were used for adaptation, and the following 24 hours were used to record food and water intake and to collect all the urine produced. NS, LS or HS chows were given ad libitum to the respective experimental groups. Urine produced in 24 h was measured by gravimetry and samples were immediately frozen at -20°C for posterior analysis. Creatinine concentration in blood and urine was determined by a kinetic method (Labtest, Minas Gerais—Brazil) based on the Jaffé reaction. The glomerular filtration rate (GFR) was estimated by the creatinine clearance. Sodium and potassium excretion was measured on a radiometer ABL800 Flex121 blood gas analyzer (Radiometer Medical, Brønshøj, Denmark). Protein excretion was determined using a spectrophotometric assay with a Sensiprot kit (Labtest, Minas Gerais—Brazil). The concentration of albumin in the urine samples was determined by using an ELISA kit (Nephrat kit Exocell, Philadelphia, PA). All kits were used following the manufacturer’s instructions.

Fasting glucose levels were measured using the ACCU-CHECK^® Performa meter (Roche Diagnostics GmbH, Mannheim, Germany). The serum urea was measured by colorimetric enzymatic hydrolysis by the urease system using a Labtest kit (Labtest, Minas Gerais, Brazil). Serum and urinary creatinine concentrations were measured using a kinetic method (Labtest) and a ThermoPlate Analyzer Plus (ThermoPlate, São Paulo, Brazil). The urinary protein excretion was determined using a Sensiprot kit (Labtest). The urinary albumin concentration was determined using an ELISA kit specific for rat urine albumin (Nephrat kit; Exocell, Philadelphia, PA, USA). The experiments were carried out in accordance with the manufacturer’s instructions.