Urea ce kit

Analysis of renal function was performed according to previously published studies (Silva-Aguiar et al., 2018 (link); Teixeira et al., 2020 (link); Peruchetti et al., 2021 (link); Farias et al., 2023 (link); Peres et al., 2023 (link)). Briefly, 24-h urine samples were quantified to determine urinary volume (mL) and urinary flow (mL/min). Urine samples were then centrifuged 5 times (10,000 × g for 10 min) to remove urine sediments. Plasma was obtained by centrifuging whole blood (2,500 × g for 5 min). All parameters analyzed in urine and plasma were measured using commercial kits following the manufacturers’ instructions. The levels of creatinine, blood urea nitrogen (BUN), and urinary proteinuria in both urine and plasma were measured, respectively, using the creatinine kit (ref. 35-100), urea CE kit (ref. 27-500), and sensiprot kit (ref. 36) purchased from Labtest (Lagoa Santa, MG, Brazil). The levels of urinary and plasma sodium were measured using the enzymatic sodium kit (ref. BT1201100) purchased from BioTecnica (Varginha, MG, Brazil). These results were then utilized to calculate parameters such as creatinine clearance (CCr), sodium clearance, renal fractional excretion of sodium (FE_Na⁺), and urinary protein to creatinine ratio (UPCr).

Blood samples were collected by retroocular access in anesthetized animals. SUN was determined using an enzymatic-colorimetric method (Urea CE kit, Labtest, MG, Brazil).

For the biochemical characterization and inhibition experiments, 0.2 μg/mL ARGLi was incubated with 50 mM L-arginine in 50 mM CHES buffer (pH 9.5) at 37 °C for 5 min. The amount of urea produced was determined by the UREA CE kit (Labtest), according to analytical procedures described by the manufacturer. Briefly, 10 μL of reaction mixture was incubated with 500 μL of urease solution (13 KU/L) diluted in buffer [20 mM sodium phosphate (pH 6.9), 62.4 mM sodium salicylate, 3 mM sodium nitroprusside] at 37 °C for 5 min. Subsequently, 500 μL of oxidising solution [140 mM sodium hydroxide, 6 mM sodium hypochlorite] was added and the indophenol blue formed was measured at 600 nm. The concentration of product was calculated using a standard curve of urea. Enzymatic assays were performed in triplicate.