Angiotensinogen

Rat renin was kindly provided by Dr. Jan Danser, Erasmus University, the Netherlands. Angiotensinogen was from Sigma Chemical Co., Saint Louis, MI, USA, and Aliskiren was kindly provided by Novartis Pharmaceuticals. Enalaprilat was from Merck.

Blood was collected via cardiac puncture with EDTA and centrifuged to isolate plasma (n=6–7 per group). For the renin activity assay, 25 μl of plasma was added to 95 μl of buffer (20 mM Tris/Cl, 30 mM EDTA, 0.2% BSA, pH 7.4 at 37°C). Synthetic renin substrate (angiotensinogen, Sigma–Aldrich, Oakville, ON) was added to a final concentration of 1 μM and PMSF protease inhibitor was added to a final concentration of 1 mM. Tubes were incubated on ice to assess native renin via endogenous angiotensin I levels or incubated for 1 h at 37°C to assess active renin levels. Angiotensin I was measured using ELISA as per manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY).