Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the Kidney Renin Angiotensin System

Wysocki, Jan; Schulze, Arndt; Batlle, Daniel

Kidney cortex total protein fractions were extracted as previously described [23, 24] . Urines (500 µL; each containing 1-5 mg total protein) and kidney cortex lysates (each containing 1-2.5 mg total protein) from ACE2-deficient mice (C57BL/6 genetic background) were mixed with 50 mM HEPES, pH 7.4, NaCl (150 mM), Triton X-100 (0.5%), ZnCl 2 (12.5uM end-concentration) and incubated with 1 µg of native mouse mrACE2 (10 nM) at 37 • C constantly shaking at 800 rpm for up to 48 h. At specified time points of incubation, a 50 µL aliquot was taken and mixed with an equal volume of PBS buffer containing EDTA-free inhibitor cocktail (Roche, Basel, Switzerland) and immediately frozen. Each of the aliquots was then used for measuring ACE2 activity using Mca-APK-Dnp substrate and to detect ACE2 protein on Western blot. For Western blot, proteins were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked in non-fat dry milk (5-7% wt/vol) solubilized in Tris-buffered saline solution containing 0.1% Tween 20. The nitrocellulose membranes were incubated with primary anti-ACE2 antibody [AF3437 (R&D Systems, Minneapolis, MN, USA) or Ab38888 (Abcam, Cambridge, MA, USA)] and horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The specificity of the ACE2 antibodies was confirmed by the absence of any bands in Western blot using ACE2KO samples, as previously reported [12] . Bands were visualized using chemiluminescence system (Super Signal Pico, Pierce, Rockford, IL, USA).