Effects Of Static Subnormothermic Preservation Following Warm Ischemia on Ischemia-Reperfusion Injury and Long-Term Outcome of the Renal Allograft in CLAWN Miniature Swine

Abstract

Objective: Normothermic organ machine perfusion has been suggested in place of hypothermic preservation for kidneys from donors after cardiac death and other marginal donors. Here we attempted to evaluate the efficacy of static subnormothermic preservation on renal grafts exposed to periods of warm ischemia time (WIT) in a preclinical large animal model using MHC inbred miniature swine. Methods: Nine MHC-inbred CLAWN miniature swine received MHC-matched renal allografts with 12 days of FK506 (days 0-11). In Group 1, two animals received kidneys without WIT. In Groups 2 and 3, the grafts were subjected to 60-min of WIT, and then preserved at 4°C (Group 2; n=3) or 22°C (Group 3; n=4) for 60-min in ET-Kyoto solution. In 2 additional groups (4 and 5), WIT was extended to 2 hours. Renal function was monitored by serum creatinine (sCr) and biopsies. Serum levels of TNF-α and IL-6 were measured to characterize inflammatory responses. Results: In Group 1, both animals survived over 60 days with stable renal function and minimal rise in sCr (2.4 ± 0.8mg/dl around day 2). In Group 2, all animals showed moderate elevations of sCr (4.1 ± 0.8mg/dl), peaking at a median of 2 days; renal function recovered by day 7 in all three animals and remained stable for over 60 days in 2 animals. sCr in the remaining animal gradually increased after 30 days and the animal was sacrificed at day 60 (sCr=20.5 mg/dl). In Group 3, sCr increased to 3.6±1.0mg/dl, peaking at day 2 and recovered by day 6. One animal died from infection at day 24, but the other 3 recipients survived 60 days. Systemic early pro-inflammatory cytokine levels (TNF-α, IL-6) increased in both Group 2 and Group 3, peaking 6 hours after reperfusion (Group 2 vs. 3; TNF-α: 152 ± 9 vs. 122 ± 6 pg/ml; IL-6: 2357 ± 367 vs. 1823 ± 487 pg/ml). Although there was no statistical difference in peak levels of sCr, renal function as well as the peak cytokine concentration for animals receiving 22°C (vs 4°C) preservation was better across all three parameters. One animal underwent 2-hr of WIT plus 1-hr 4°C preservation (Group 4) and died of irreversible graft non-function on day 5. In contrast, 2 animals, underwent 2-hr of WIT plus 1-hr of 22°C preservation (Group 5) and maintained graft function >45 days (ongoing) although both had transient rise in sCr around day 3. Conclusion: We demonstrated that subnormothermic (22°C) organ preservation of kidney grafts exposed to 60-min of WIT was as effective as hypothermic (4°C) preservation, in miniature swine. These data also suggest a possible benefit of subnormothermic preservation for organs which experience of up to 2-hr of WIT. To our knowledge, this is the first demonstration of the applicability of static subnormothermic storage in preclinical large animals. Although further optimization is required, this preservation method may contribute to an expansion of the donor pool through improvement of extended criteria organs.