Fluorometric activity assay

We used a fluorometric activity assay from Sigma (St. Louis, MO) to measure the activity of soluble ACE2 enzyme in plasma following the manufacturer’s instruction: Plasma samples were diluted 1:5 in ACE2 Lysis Buffer. 5μl were incubated with 45 μl of assay buffer for 15 min. 50 μl of ACE2 substrate mix containing a peptide-MCA (4-methylcoumarin-7-acetate) conjugate was added and the change in fluorescence recorded (Ex 320 nm/Em 420 nm) over 30 min on a SpectramaxM5 (Molecular Devices: San Jose, CA). Lysis Buffer was used as background control, recombinant ACE2 used as positive control. To distinguish ACE2 activity from other proteolytically active enzymes, activity of samples was also measured in the presence of a specific ACE2 inhibitor. For the negative control ACE2 inhibitor was added to positive controls. ACE2 activity of the samples was calculated based on a standard curve created by recording fluorescence of serial dilutions of MCA at Ex320nm/Em420nm.

Paraffin-embedded livers were sectioned into 3 μm sections and mounted onto positively charged slides (VWR, Radnor, PA, USA). Slides were deparaffinized and stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA, USA) for 1 min followed by staining for 1 min with eosin Y (Amresco, Solon, OH, USA) and rinsed in 95% ethanol. The slides were then dipped into 100% ethanol and subsequently through two xylene washes. Coverslips were mounted onto the slides using Vectamount mounting media (Vector Laboratories). The slides were viewed and imaged using an Olympus BX40 microscope with an Olympus DP25 imaging system (Olympus, Center Valley, PA, USA).
Serum alanine aminotransferase (ALT) and bilirubin were assessed using commercially available kits. ALT measurement was performed using a fluorometric activity assay (Sigma-Aldrich). Total bilirubin was assayed using a total bilirubin ELISA (CusaBio, Wuha, China). All assays and subsequent analyses were performed according to manufacturers’ instructions.