*2.5. Parameters of Oxidative Stress*

Oxidative stress was evaluated according to the activities of antioxidant enzymes, concentrations of reduced and oxidized glutathione, and levels of lipoperoxidation products TBARS and conjugated dienes [20]. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) were analyzed using Cayman Chemicals assay kits (Ann Arbor, MI, USA). Catalase (CAT) activity was measured spectrophotometrically. Lipoperoxidation products were determined by assaying the reaction with thiobarbituric acid. The levels of conjugated dienes were analyzed by extraction in the media (heptane:isopropanol = 2:1) and measured spectrophotometrically in heptane's layer. Concentrations of reduced (GSH) and oxidized (GSSG) forms of glutathione were determined using an HPLC diagnostic kit with fluorescence detection (ChromSystems, Gräfelfing, Germany).

#### *2.6. Urine Collection and Microalbuminuria*

Rats were placed in metabolic cages for 16 h to obtain urine samples for the analysis of urinary excretion of albumin, creatinine, sodium, and glucose. The level of albumin in urine was analyzed by HPLC method with UV-VIS detection and adjusted for creatinine concentration. Urinary glucose levels were analyzed by glucose oxidase assay (Erba-Lachema, Brno, Czech Republic). Urinary protein was measured using the Folin method with bovine serum albumin as a standard [21].
