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The Ang II (Angiotensin II)-Angiotensin-(1-7) axis of the Renin Angiotensin System encompasses 3 enzymes that form Angiotensin-(1-7) [Ang-(1-7)] directly from Ang II: ACE2 (angiotensin-converting enzyme 2), PRCP (prolylcarboxypeptidase), and POP (prolyloligopeptidase). We investigated their relative contribution to Ang-(1-7) formation in vivo and also ex vivo in serum, lungs, and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In wild-type (WT) mice, infusion of Ang II resulted in a rapid increase of plasma Ang-(1-7). In ACE2 −/− / PRCP −/− mice, Ang II infusion resulted in a similar increase in Ang-(1-7) as in WT (563±48 versus 537±70 fmol/mL, respectively), showing that the bulk of Ang-(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a POP inhibitor, Z-Pro-Prolinal reduced the rise in plasma Ang-(1-7) after infusing Ang II to control WT mice. In POP −/− mice, the increase in Ang-(1-7) was also blunted as compared with WT mice (309±46 and 472±28 fmol/mL, respectively P =0.01), and moreover, the rate of recovery from acute Ang II-induced hypertension was delayed ( P =0.016). In ex vivo studies, POP inhibition with ZZP reduced Ang-(1-7) formation from Ang II markedly in serum and in lung lysates. By contrast, in kidney lysates, the absence of ACE2, but not POP, obliterated Ang-(1-7) formation from added Ang II. We conclude that POP is the main enzyme responsible for Ang II conversion to Ang-(1-7) in the circulation and in the lungs, whereas Ang-(1-7) formation in the kidney is mainly ACE2-dependent.

ACE2 is an enzyme with a molecular size of more than 100 kDa which produces Angiotensin (1-7) from Angiotensin II (1-8). Attempts to target kidney RAS using ACE2 to treat kidney disease are hampered by its large molecular size which precludes its glomerular filtration and subsequent tubular uptake. We tested two ACE2 truncates (1-605 and 1-619AA) that we generated through truncation from the C terminus. ACE2 enzyme activity measured using Mca-APK-Dnp substrate was as high for each of them as native rACE2 (1-740 AA). The two truncates had an apparent MW of ~70 kDa, as expected from the amino acid sequence, as shown by western blot. For radioimaging each purified truncate was labeled with 99m Tc to study kidney uptake. After i.v. injection of Tc99m -labeled rACE2, there was kidney cortex retention for Tc99m 1-605 [8.6±0.7% of whole body (WB) radioactivity] and for Tc99m 1-619 (4.2±2.1% WB) as compared to only trace retention after injection of native Tc99m -rACE2 1-740 (1.2±0.2% WB)(figure). When infused to mice with genetic ACE2 deficiency, a single i.v. injection of 1-619 resulted in detectable ACE2 activity in urine and kidneys (23.1±4.3 and 1.96±0.73 RFU/ug prot/hr, respectively). In addition, the kidneys of ACE2-nul mice infused with 1-619 studied ex vivo formed more Ang (1-7) from exogenous Ang II than those of uninfused animals (p We conclude that our novel ACE2 truncates undergo glomerular filtration which is associated with kidney uptake of enzymatically active protein that can enhance formation of Angiotensin 1-7 from Angiotensin II (1-8). These truncates may offer a potentially useful approach to target kidney RAS overactivity to combat kidney disease.