Nephrat 2

The rats were individually placed in metabolic cages, and urine samples were collected for 24 h on the last week of the study to quantify UAE levels. Albumin concentrations were measured using an ELISA kit (Nephrat II; Exocell, Philadelphia, PA, USA). Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Kanagawa, Japan). Urinary and plasma creatinine concentrations were measured with an enzymatic Creatinine Plus version 2 reagent kit, using an automatic analyzer (COBAS INTEGRA 400 plus; Roche, Basel, Switzerland). Urinary corticosterone and aldosterone levels were measured using an enzyme immunoassay system (Cayman Chemical, Ann Arbor, MI, USA). UAE, UPE, urinary corticosterone and aldosterone levels were normalized to the urinary creatinine concentration.

After 24 weeks, the rats underwent the analysis of renal function. The serum fasting plasma glucose (FPG) level, serum creatinine and urinary creatinine were detected by an automatic biochemical analysis instrument (DVI 1650; Bayer, Leverkusen, Germany). The level of glycosylated hemoglobin A_1c (HbA_1c) was measured using the high performance liquid chromatography method (HA-8160 HbA_1c Analyzer; Menarini Diagnostics, Florence, Italy). To measure advanced glycation end products (AGEs) in serum, a fluorescence spectrophotometer (Hitachi F-2500; Hitachi, Tokyo, Japan) was used to determine specific fluorescence by measuring 440 nm emissions after excitation at 370 nm [10 (link)]. To collect urine samples, the rats had free access to tap water but were deprived of food for 24 h in individual cages before being killed. The urine samples were centrifuged (1500 × g, 15 min) and supernatants were stored in poly-ethylene containers at –20°C. Urinary albumin concentrations in fresh urine samples were determined by enzyme-linked immunosorbent assay (ELISA, Nephrat II; Exocell, Philadelphia, PA), and this value was multiplied by urine volume to get the total 24-h urine albumin excretion (mg).