Abstract 480: ACE2 Independent Pathways of Angiotensin 1-7 Formation in the Murine Kidney

Abstract

Fluid balance, salt retention and tissue remodeling is hormonally regulated by the Renin-Angiotensin-System. The sophisticated enzyme network serially cleaves peptide hormones generating various effector hormones including the vasoconstrictor Angiotensin 1-8 and its counterbalancing downstream metabolite Angiotensin 1-7. LC-MS/MS based analysis of kidney and plasma of ACE2 knockout mice revealed unchanged Angiotensin 1-7 concentrations compared to wild type animals pointing to alternative pathways of Angiotensin 1-7 formation and suggesting a minor role for ACE2 in the formation of Angiotensin 1-7 in vivo. Moreover, we observed a decrease in the Angiotensin 1-8/Angiotensin 1-10 ratio in ACE2KO-mice indicating a changed conversion rate from Angiotensin 1-10 to Angiotensin 1-8. We analyzed Angiotensin 1-7 metabolism in kidney homogenates by spiking natural substrates in the absence and presence of specific enzyme inhibitors. The metabolism of ex vivo spiked Angiotensins in kidney homogenates in presence and absence of the Prolyl-Carboxypepdidase (PCP) and Prolyl-Endopeptidase (PEP) Inhibitor ZPP and the ACE2-Inhibitor MLN-4760 revealed stronger contribution of PCP and PEP to Angiotensin 1-7 formation than ACE2. Analyzing the formation of Angiotensin 1-8 from Angiotensin 1-10 in renal cortex and medulla homogenates of WT and ACE2KO mice in the absence and presence of an ACE-Inhibitor confirmed our observations on endogenous peptide levels and the ratio between Angiotensin 1-8 and Angiotensin 1-10 in plasma, revealing a reduced ACE activity in the renal cortex of ACE2KO. Our study raises questions concerning the relevance of renal ACE2 for the local formation of Angiotensin 1-7. Moreover, the reduced ACE expression and activity in renal cortex might be a physiologically and therefore phenotype relevant consequence of the knockdown of ACE2 in these mice. The possible crosstalk between ACE and ACE2 expression and activity might give rise to further studies addressing the underlying mechanisms and might be important for our understanding of RAS dynamics in regard to therapeutic interventions.