Quantifying Mouse Plasma Renin Activity

Renin enzymatic activity from EDTA-aprotinin supplemented mouse plasma samples (described in the Section 4.2) were measured in a 96-well microplate (Synergy HT reader and Gen5 v1.09 software, BioTek Instruments, Inc., Winooski, VT, USA) and quantified using exogenous fluorescence resonance transfer (FRET) peptide substrates of renin FRET-QXL™520/5-FAM, optimized for mouse renin (SensoLyte 520 mouse renin assay kit, AnaSpec, Fremont, CA, USA) as previously reported [25 (link),27 (link),32 (link),78 (link),79 (link),80 (link),81 (link)]. Cleavage of the FRET substrate by mouse renin results in the recovery of quenched fluorescence of 5-FAM, which was detected at excitation/emission = 490/520 nm with minimum autofluorescence of plasma samples. The 5-FAM fluorescent reference standard curve was used for results quantification. It is important to note that the plasma renin activity concentration assay differs from plasma renin activity (PRA) and active renin concentration (ARC)/ active plasma renin concentration (APRC) which have been historically used to report active renin in clinical trials [32 (link)].