Machine Perfusion of Mice Kidneys to Deliver a Protective Bioengineered Soluble ACE2 Protein

Background: Machine perfusion of donor kidneys has emerged as a promising approach to improve transplant outcomes by decreasing storage-related injury and reducing the occurrence of delayed graft function (DGF). Activation of the renin-angiotensin-system (RAS) has been shown to play a role in the pathophysiology of DGF and countering this overactivity may improve transplant outcomes. Here we investigated the delivery of a short soluble ACE2 protein, bioengineered in our lab and termed ACE2-618-ABD, to mice kidneys using hypothermic machine perfusion as a strategy to reduce RAS activity. Methods: Kidneys from c57bl6 ACE2 Knock-Out (ACE2-KO) and c57bl6 wildtype (WT) mice were harvested and the renal artery cannulated. We adapted a perfusion system previously used for rats to be used with mice kidneys. The mice kidneys were perfused with phosphate buffered saline (PBS) either alone or with added ACE2 618-ABD for four hours at four degrees celsius through the cannulated renal artery in a closed-circuit perfusion system using a peristaltic pump. ACE2 enzymatic activity was then measured using a fluorogenic assay and ACE2 protein levels determined by Western Blot. Results: While ACE2 enzymatic activity and protein were undetectable in non-perfused ACE2-KO kidneys, enzymatic activity and protein by Western Blot were found in ACE2-618-ABD perfused ACE2-KO kidneys. In WT kidneys perfused with ACE2-618-ABD, Western Blot showed two distinct bands for ACE2 protein at ~100kD and ~85kD. The ~100kD band reflects the native ACE2, the ~85kD represents the perfused short soluble ACE2-618-ABD which was not found in the PBS- and non-perfused WT kidneys. In kidney lysates, the cytosolic compartment of ACE2-618-ABD perfused kidneys had increased ACE2 enzymatic activity and protein demonstrating intracellular uptake. Conclusion: Delivery of a bioengineered, short soluble ACE2 protein to mice kidneys can be accomplished via machine perfusion of the renal artery which results in passage of the glomerular filtration barrier and tubular uptake. Ex-vivo perfusion of kidneys with ACE2-618-ABD results in cytosolic uptake as demonstrated by increased protein and enzymatic activity. This approach may result in improved quality of kidneys prior to transplantation. Funding: Other NIH Support - NIAID Grant to Daniel Batlle, Private Foundation Support