Lumbricus terrestris erythrocruorin (LtEc) as a red blood cell substitute in normothermic ex vivo kidney perfusion - PROJECT SUMMARY The ability to maintain the viability and function of organs outside the body (ex vivo) at body temperature (normothermia) is an emerging field of innovation. Normothermic ex vivo perfusion systems have been developed to maintain hearts, lungs, and livers in this fashion for up to several days and have been a valuable tool in transplantation medicine to extend the longevity of donor organs. The potential for these technologies to be utilized in other ways is now also being recognized. Normothermic ex vivo kidney perfusion (NEVKP), in comparison, has been more challenging to develop. The kidney is highly susceptible to damage from the products of red blood cell (RBC) degradation that are generated by machine perfusion. Because of this, existing NEVKP systems cannot reliably sustain the viability of kidneys beyond 24 hours. The goal of this project is to investigate whether this challenge can be overcome using a promising RBC alternative: the naturally acellular hemoglobin (erythrocruorin) of the earthworm Lumbricus terrestris. We aim to use this novel oxygen carrier to extend the duration of NEVKP, making it a more viable tool for kidney disease research. L. terrestris erythrocruorin (LtEc) has many properties suggesting it would be an ideal oxygen carrier for NEVKP. Having evolved to transport oxygen in an extracellular environment, LtEc is molecularly stable in a wide range of temperatures, has a low oxidation rate, and does not scavenge nitrous oxide (NO), an important vasodilator. Its size (30 nm) is favorable for perfusing the kidney as it would likely not get lost to glomerular filtration (6-8 nm cutoff) but remain able to perfuse vasoconstricted capillaries that are inaccessible to RBCs (8 µm). Together with preliminary results from our lab demonstrating that LtEc can sustain respiration in NEVKP, these properties strongly justify investigating the utility of LtEc in NEVKP. In this proposal, we will first evaluate LtEc by testing different LtEc concentrations in an NEVKP system against autologous porcine RBCs as a control. We will investigate the ability of LtEc to support kidney function and viability while monitoring adverse effects including oxidative stress, inflammation, and kidney injury. These experiments will help determine the concentration that best supports respiration and minimizes toxicity during NEVKP. LtEc will also be evaluated against other HBOCs to determine whether the advantageous molecular properties of LtEc confer an empirical benefit in NEVKP. We will then attempt to maintain NEVKP for up to 7 days using an LtEc-based perfusate. Optimizing the use of LtEc for NEVKP of porcine kidneys could result in two major breakthroughs: 1) the development of long-term NEVKP, a tool that would enable many avenues for novel discovery in kidney disease; and 2) the validation of LtEc as an effective and sustainable oxygen carrier in a mammalian organ, the impact of which could be relevant to numerous fields across medicine.
