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Sodium concentrations in serum and urine samples were measured using a Sherwood-420 flame photometer (Sherwood Scientific, Cambridge, UK) and the creatinine concentration was determined by Jaffe's kinetic method without deproteinization in an EOS Bravo W automated analyser (Hospitex Diagnostics, Firenze, Italy). Urinary concentrations of PGE2, cAMP and cGMP were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Measurements of optical density (OD) and calculations of PGE2, cAMP and cGMP concentrations were performed on an ELx800 absorbance microplate reader (BIO-TEK Instruments, Winooski, VT, USA). Diuresis (V; mL/min per kg), creatinine clearance (CCr; mL/min per kg) and urinary sodium excretion (UNaV; ��mol/min per [http://www.selleckchem.com/products/mi-773-sar405838.html SAR405838] kg) were computed according to standard formulas and normalized for bodyweight.[19] Formulas for the calculation of solute-free water clearance and the excretion rates of PGE2 and second messengers are listed below. Solute-free water clearance: where V is diuresis, UOsm is urine osmolality, POsm is serum osmolality (287?��?2 and 275?��?2?mOsmol/kg H2O in control and [http://en.wikipedia.org/wiki/Resiquimod Resiquimod] water-loaded rats, respectively) and UPGE2, UcAMP and UcGMP are urinary concentrations of PGE2, cAMP and cGMP, respectively. The total amount excreted over the first 30?min of the experiment was computed according to the following formulas:30?min urine volume: Urine samples obtained during the 0�C60?min interval were taken into account. Weighted mean values of renal functional parameters during 15 or 30?min periods (e.g. 0�C15, 16�C30 or 0�C30?min) were calculated for each animal and then averaged within the experimental group. Data were analysed using STATISTICA 6.0 software (StatSoft, Tulsa, OK, USA). All values are presented as the mean?��?SEM. All values for a specific experimental group within each protocol were compared by one-way repeated-measures analysis of variance (anova) followed by Holm�CSidak post hoc analysis for specific mean comparisons. All [http://www.selleckchem.com/products/lee011.html LEE011 research buy] measurements between groups were compared by one- or two-way anova followed by the t-test with Bonferroni correction for multiple comparisons with the control group or by Holm�CSidak post hoc analysis for specific mean comparisons, respectively. Two-tailed P?