Fecal ACE and ACE2 activities reflect intestinal shedding and microbiota modulation of the renin–angiotensin system

Angiotensin-converting enzymes (ACE and ACE2) are key components of the renin–angiotensin–aldosterone system (RAAS) and are present in the gastrointestinal tract and intestinal content, preserving their catalytic activity, and may interact with the gut microbiota. The present study aimed to determine the origin of fecal ACE and ACE2 activity. Fecal pellets from germ-free, ACE and ACE2 knockout (KO) mice, and from the corresponding controls were analyzed using fluorimetric enzyme activity assays. ACE activity was assessed using Hippuryl-His-Leu and Z-Phe-His-Leu as substrates; ACE2 activity was assessed using Mca-APK (Dnp), with and without the ACE2 inhibitor MLN-4760. Germ-free mice showed increased fecal ACE and ACE2 activity compared to controls. ACE2-KO mice lacked fecal ACE2 activity, whereas ACE activity was unaffected. In ACE-KO mice, fecal ACE activity was reduced, but not abolished, while ACE2 activity remained similar to controls. In ACE C- and N-domain KO mice, ACE activity was similar to controls, and inhibition with captopril completely abolished fecal ACE activity using Hippuryl-His-Leu, but not Z-Phe-His-Leu, in those animals. These findings indicate that fecal ACE and ACE2 activity results from combined intestinal shedding and microbiota-related mechanisms, supporting a modulatory role of the gut environment on luminal RAAS activity.