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The stained sections were viewed with an Olympus BX40 microscope (Olympus America, Melville, NY, USA) on bright-field setting fitted with a digital camera (Olympus DP70; Olympus America). For quantification of CD3 and ED-1 staining, positive cells were counted in 20 randomly selected 200?��?200?��m fields of kidney cortex per rat by an individual blinded to the treatment group. Positive cells in the outer and inner medulla were similarly counted in 20 and 10 randomly selected 200?��?200?��m fields for each region, respectively. Results are expressed as the number of positive cells per mm2. A semiquantitative analysis of Gomori��s Trichrome-stained slides was conducted in a blinded fashion from four animals per group to assess the morphology of the glomeruli and the presence of proteinaceous tubular casts. Glomeruli were given a score of 0 (>?90% normal), 1 (70% normal), 2 (30% normal) or 3 (selleck inhibitor and the presence of tubular casts throughout the kidney rated at low, moderate or high. Urinary concentrations of sodium (ion-sensitive electrode, EasyLyte Na/K/CL/Li Analyser; Medica, Bedford, MA, USA), protein (Bradford assay; Bio-Rad, Hercules, CA, USA) and albumin (enzyme immunoassay (EIA); Cayman SERCA Chemical, Ann Arbor, MI, USA) were determined at baseline and weekly during the 21?day experimental period and 24?h excretion rates calculated. Urinary and plasma creatinine concentrations were determined from samples collected on Day 21 (picric acid method adapted for microtitre plates23) and creatinine clearance was calculated according to the standard formula. Data are presented as the mean?��?SEM. P?��?0.05 was considered significant. Statistical analyses were performed using Graphpad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data collected at multiple time points were analysed by analysis of variance with repeated measures, testing for main effects of time and treatment group, with the Bonferroni post hoc test used to examine differences between pairs of groups at individual time points. Data collected at one time point were analysed by one-factor analysis of variance and the Newman�CKeuls�� multiple comparison test. Data for 24?h average MAP at the mid-point of each week are presented in Fig.?1. As expected, DOCA-salt treatment induced robust hypertension, with the average MAP on Day 18 of treatment reaching PLK inhibitor 180?��?3?mmHg (Fig.?1). Treatment of DOCA-salt rats with MMF significantly attenuated the development of hypertension, with this difference being significant on Days 11 and 18 and MAP reaching 146?��?7?mmHg on Day 18 (P?
