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

Wysocki, Jan; Schulze, Arndt; Batlle, Daniel

To test the systemic effect of the short ACE2 variants 1-605 and 1-619 in vivo, we used an established protocol for acute Ang II-induced hypertension [6, 7] . Using this protocol previously, we have demonstrated that lowering plasma Ang II using native rACE2 effectively mitigated an increase in blood pressure caused by Ang II administration [7] . Similar to native ACE2 [6, 7] , both small ACE2 variants blunted the peak increase in blood pressure after Ang II infusion which normalized faster within five minutes or less. This shows that both small ACE2 variants efficiently degrade the excess of systemic circulating Ang II, thereby enhancing blood pressure recovery. Having demonstrated the systemic effect on blood pressure, we further studied whether both small ACE2 variants have any added effect to their systemic action at the local urinary and kidney level. ACE2 is normally present in the urine, and to better assess whether there is any increase in urinary ACE2 activity after administration of the short ACE2 variants, ACE2-deficient mice were used. In contrast to native rACE2 injection, both small recombinant variants resulted in a gain in urinary ACE2 activity. L-lysine, a tubular reabsorption blocker [28, 29] , further increased urinary ACE2 activity, suggesting that the two short ACE2 variants are both filtered and reabsorbed by the tubules. That the short ACE2 protein variant that is taken up by the kidney is indeed enzymatically active was shown by the presence of ACE2 activity in kidneys from ACE2 deficient mice that had been injected with ACE2 1-619. In addition, kidney cortex lysates from ACE2 1-619-injected mice were able to form significantly more Ang (1-7) from Ang II than kidney lysates from PBS-or native rACE2-injected mice. In the aggregate, these data show that mouse short ACE2 variants are active, and sufficiently small to be filtered by the kidney and, moreover, capable of increasing kidney ACE2 activity to the extent that Ang 1-7 formation from Ang II is increased ( Figure 10 ). As a limitation of this study, we acknowledge that our studies were not performed in a blinded fashion and that a potential bias in the measurements of activity of ACE2 in the urine and at the kidney level were not prevented by stratified randomization of the treated animals. In this regard, the ACE2 knockout allowed us to demonstrate urine and kidney activity after injection of our short ACE2 variants relatively easily and show that this was not the case after the infusion of native ACE2. The use of native rACE2 as a control enhances the results obtained with the novel short ACE2 variants and is a strength of our design. Even though the pharmacokinetic data show the clear superiority of short rACE2 variants as compared to the native rACE2, we caution to emphasize that this effect may be dependent on the artificial substrate used to measure ACE2 activity.