Death of kidney parenchymal cells during transplantation occurs through counterbalanced mechanisms of cellular death, apoptosis and necrosis. Members of the receptor interacting protein family (RIP1, RIP3) are regulators of newly described pro-inflammatory programmed necrosis, necroptosis, which can be upregulated by blocking caspase-8 mediated apoptosis. We tested the effect of caspase-8 inhibition and RIP3 in kidney allograft injury. Previously we have shown caspase-8 shRNA silencing reduced kidney ischemia-reperfusion injury at 48h. To test caspase-8 shRNA silencing on alloimmunity, in the present study we transplanted C57BL/6 (H-2b) kidneys into fully nephrectomized Balb/c (H-2d) recipients without immunosuppression. Surprisingly, donor kidney inhibition of apoptosis using caspase-8 shRNA reduced allograft survival compared to controls (33.3±8.7 vs 68.3±10.9 days, p=0.0003, n=7-9/group). Quantification of necrosis showed increased necrotic death in caspase-8 shRNA kidneys at day 4 post-transplant. To test whether enhanced necrosis was linked to caspase-8 inhibition, we exposed renal tubular epithelial cells (TEC) to pro-inflammatory cytokines and assessed cell death by Annexin-V/PI co-labeling. Indeed, Annexin-V/PI co-positivity increased in TNF-Α/IFN-Γ treated and caspase-8 inhibited (zVAD) TEC at 48h (from 9% to 17%) which was blocked by RIP3 inhibition using Nec-1. Similarly, TNF-Α/IFN-Γ treated RIP3-/- TEC had decreased Annexin-V/PI co-positivity compared to wild type controls (22% vs. 51%). We then directly tested the role of RIP3 in allograft survival using donor kidneys from RIP3-/- mice. Absence of donor RIP3 prolonged allograft survival compared to wild type control recipients (94.6±2.0 vs 43.0±10.4 days, p=0.01, n=8-11/group). As well, RIP3-/- transplants had lower serum creatinine levels compared to controls (31±6.6 vs 86.2±24.1 Μmol/l, p=0.03, n=7-9/group) at study end (day 100 or earlier termination). Interestingly, while a trend of reduced tubular injury was noted in RIP3-/- allografts, reduced vasculopathy was the most prominent difference in histological injury (0.5±0.4 vs. 1.6±0.2, p=0.02,n=4-5/group). These data demonstrate for the first time that loss of RIP3 mediated necroptosis improves kidney allograft survival in non-immunosuppressed recipients.
To cite this abstract in AMA style:Lau A, Wang S, Haig A, Yin Z, Zhang Z, Jevnikar A. Inhibition of Receptor Interacting Protein 3 (RIP3) Promotes Long Term Renal Allograft Survival [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/inhibition-of-receptor-interacting-protein-3-rip3-promotes-long-term-renal-allograft-survival/. Accessed November 21, 2019.
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