Loss of receptor interacting protein kinases 3 and caspase-8 augments intrinsic apoptosis in tubular epithelial cell and promote kidney ischaemia-reperfusion injury.

Background: Ischaemia-reperfusion injury (IRI) is associated with programmed cell death that promotes inflammation and organ dysfunction. Necroptosis is mediated by members of receptor interacting protein kinases (RIPK1/3). Inhibition of RIPK1/3 provides a pro-survival benefit in kidney IRI. Caspase-8 initiates apoptosis and contributes to IRI. We studied whether inhibiting both RIPK3 and caspase-8 would provide an additional benefit in kidney IRI.

Methods: A clamp was applied to the left kidney pedicle for 45 min followed by right kidney nephrectomy. Kidney and serum from wild type, RIPK3 , and RIPK3 caspase-8 double knockout (DKO) mice were collected post-IRI for assessment of injury. Tubular epithelial cells (TEC) isolated from wild type, RIPK3 , and DKO mice were treated with interferons-γ and interleukin-1β to induce apoptotic death.

Results: Kidney IRI of DKO mice did not show improvement over RIPK3 mice. We have found that DKO triggered 'intrinsic' apoptosis in TEC in response to interleukin-1β and interferons-γ. Up-regulation of the B-cell lymphoma 2 (Bcl-2)-associated death promoter, the Bcl-2-homologous antagonist killer and Bcl-2-associated X protein and enhanced activation of caspase-3 and 9 were found in DKO TEC. TEC infected with Murine cytomegalovirus that encodes multiple cell death inhibitors resist to death.

Conclusion: We show that the deletion of both RIPK3 and caspase-8 does not provide additive benefit in IRI or TEC death and may enhance injury by up-regulation of intrinsic apoptosis. This suggests blocking multiple death pathways may be required for the prevention of kidney IRI clinically.