Date: Monday, May 4, 2015
Session Time: 2:15pm-3:45pm
Presentation Time: 3:03pm-3:15pm
Location: Room 119-A
[Background] Ischemia-reperfusion injury (IRI), a complex limiting factor in organ transplant survival, is related to multiple death pathways. Eliminating the key necroptotic molecule (RIPK3) improves survival in IRI and kidney transplantation, while results of caspase-8 inhibition vary. We aimed to test the potential combined benefit of both RIPK3/caspase-8 deletion (DKO) in a mouse renal IRI model. [Results] 48-hour post renal clamping, DKO mice did not gain additional benefit over RIPK3-/- alone, as their serum creatinine levels were not different (74±33 vs. 52±20, umol/L, n=7, p=0.91) but improved over wildtype. Alternate forms of cell death can be enhanced with perturbations of death pathways. The activation of caspase-9 by IL-1β+IFNγ was observed in wildtype, RIPK3-/- and DKO TECs, but enhanced only in DKO (DKO vs. RIPK3-/-, 19,367±2397 vs 10,670±1881, by luciferase assay, n=3, p=0.046). BAX inhibitor peptide (BIP) attenuated this enhancement (11,070±1342 vs 10,670±1881, n=3, p=0.87), though not to basal levels (<6261±467), indicating augmented intrinsic apoptosis in DKO. Consistently, DKO TEC survival by MTT was reduced compared to RIPK3-/- (18±3 vs 36±2%, n=3, p=0.0032) and was restored with BIP (62±5 vs 46±6 %, n=3, p=0.122) at 24 hr. Basal mRNA levels of mitochondrial apoptotic promoters were increased in DKO than RIPK3-/- TEC (BAX 2.12-fold, n=3, p=0.0059; BAK 3.84-fold, n=3, p<0.0001) by RT-PCR. Murine CMV (MCMV) encodes inhibitors for both BAX and BAK and in preliminary experiments, was able to completely block IL-1β+IFNγ induced intrinsic apoptosis in wildtype, RIPK3-/- and DKO TEC in vitro. [Conclusion] For the first time, we demonstrate elimination of both caspase-8 and RIPK3 can augment TEC death by unanticipated up-regulation of caspase-9 mediated apoptosis, which attenuates the pro-survival benefit of RIPK3-loss. While inhibition of caspase-8 apoptosis and regulated necrosis may be useful to protect from organ injury, these results highlight the complex biology resulting from perturbations of death pathways. Inhibition of multiple forms of cell death will likely be required to maximize clinical benefit.
To cite this abstract in AMA style:Sung B, Su Y, Lau A, Jiang J, Haig A, Green D, Zhang Z, Jevnikar A. Loss of RIPK3 and Caspase-8 Enhance Intrinsic Apoptosis in Tubular Epithelial Cell (TEC) Death and Contributes to Kidney Ischemia Reperfusion Injury (IRI) [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/loss-of-ripk3-and-caspase-8-enhance-intrinsic-apoptosis-in-tubular-epithelial-cell-tec-death-and-contributes-to-kidney-ischemia-reperfusion-injury-iri/. Accessed January 15, 2021.
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