Neprilysin (NEP) Activity

Renal and urinary NEP activity were measured using an indirect fluorogenic assay as described before (Alawi et al., 2020) with some modifications. The cleavage of the fluorogenic substrate, Succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin (Suc-Ala-Ala-Phe-AMC) (Sigma-Aldrich, St. Louis, MO, United States) by NEP generates hydrophobic residue (phenyalanine-methylcouramin). For each reaction, kidney lysates (0.75 μg protein) or urine samples equivalent (1 µg of creatinine) were incubated with assay buffer (50 mM Tris, 5 mM ZnCl₂, 150 mM NaCl2 and 10 µM lisinopril) in the presence and absence of the NEP inhibitor, thiophen (10 μM), in black-colored 96-well plate (Corning Inc.) for 15 min. Then 100 μL of the reaction substrate (4 mM, Suc-Ala-Ala-Phe-AMC) was added to each well and the plate was incubated for 1 h at 37°C. After cleavage of the NEP substrate, 5 µl of 4 mM leucine aminopeptidase was added to each well followed by 2 µl of 1 mM phosphoramidon which liberates the fluorescent molecule AMC. The fluorescence was measured at excitation (λ ex): 390 nm and emission (λ _em): 460 nm using Biotek Synergy H1 plate reader (Winooski, VT, United States).