Aldosterone i125 radioimmunoassay

Plasma was collected in a stress-free environment by rapid non-terminal retro-orbital blood draw using thin hematocrit capillaries (Fisher Scientific). Aldosterone levels were determined on heparinized plasma (pre-diluted 2.5 times in PBS + 0.1% BSA) using an aldosterone I125 radioimmunoassay according to the manufacturer’s instructions (Tecan). Plasma renin activity was determined on EDTA-collected plasma using a competitive radioimmunoassay for Angiotensin I following the manufacturer’s instructions (Tecan). To rule out the impact of limiting amounts of angiotensinogen substrate in the plasma, an excess (about 180 ng) of angiotensinogen 1–14 renin substrate porcine (Sigma) was added to every sample, calibrator and control before quantification.

Methods for urine and plasma analysis were conducted as previously described^{30 (link)}. Urinary aldosterone was measured using an aldosterone I¹²⁵ radioimmunoassay (Tecan, Morrisville, NC), and standardized to urinary creatinine (Jaffe’ colorimetric assay, Cayman Chemicals, Ann Arbor, MI) in order to account for differences in GFR and urine volume. Plasma renin concentration was determined by RIA (Diasorin, Stillwater, MN). Blood chemistries were determined from retro-orbital sinus sampling using heparinized capillary tubes for analysis with an iStat hand held analyzer (EC8+ cartridge, Heska, Fort Collins, CO). Urinary Na⁺ and K⁺ were measured by flame photometry (IL943 Automatic Flame Photometer, Instrumentation Laboratory, Inc.).