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2008). The rate of substrate cleavage was determined by comparison to a standard curve of the free fluorophore, 4-amino-methoxycoumarin (Sigma, St Louis, MO, USA) and expressed as nanomoles of substrate cleaved per milligram of protein per hour. For plasma ACE2 activity, blood collected into heparinized tubes was centrifuged at 4��C and plasma assayed. Results are expressed as nanomoles of substrate per millilitre of plasma per hour. Immunohistochemical staining for cardiac ACE2 (polyclonal antibody, T17, from Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1:100) was performed as described previously (Burrell et al. 2005). Sections (4 ��m thick) were rehydrated and blocked with 3% (v/v) H2O2 and 10% (v/v) Entinostat in vivo normal goat serum before incubation with primary antibody at 4��C overnight. The secondary antibody was goat anti-rabbit at a dilution of 1:400. Antibody labelling was visualized using an avidin�Cbiotin complex (ABC kit; Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine Reelin before counterstaining with Haematoxylin. Negative control sections were incubated in the absence of primary antibody. Images were acquired on an Olympus BX50 microscope (objective lens, ��20/0.50), using a Leica DFC480 camera and Leica IM50Image Manager (Leica, North Ryde, NSW, Australia). Data are shown as means �� SEM. Data were analysed using Student's unpaired t test (SPSS Statistics version 17; SPSS Inc., Chicago, IL, USA). Plasma ACE and Ang-(1�C7) were not normally distributed and were analysed using the Mann�CWhitney U test. Data were compared between control sham-operated rats and STNx rats using Student's unpaired t test to confirm that STNx led to kidney failure. Vehicle-treated STNx rats were then compared with STNx rats treated with ramipril to determine the effect of ACE inhibition in rats with CKD. Two-tailed P value check details the effect of STNx on body weight, fluid balance and renal function compared with control rats. Rats with STNx had compensatory hypertrophy of the left kidney (P