Background. In our previous study, naked caspase-3 siRNA infused into the renal artery during cold preservation was effective, but did not protect the auto-transplant kidneys in a porcine model. The systemic complementary effects were noticed in the siRNA preserved auto-transplant kidneys with increased caspase-3, inflammation and apoptosis. Therefore, there is a warranty to further elucidate whether the siRNA activates innate immune responses and which signaling pathways are involved.

Materials and Methods. The left kidney was retrieved from mini pigs and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA into the renal artery with the renal artery and vein clamped for 24-h cold storage. After right nephrectomy, the left kidney was auto-transplanted into the right for 48-h without the systemic treatment of siRNA. The protein expression was detected by western blotting, while the mRNA expression was detection by qPCR.

Results. The protein level of toll like receptor (TLR)-3 and TLR-7, as well as their main adapters, TRIF and MyD88, was up-regulated by the siRNA in the auto-transplant kidneys. Furthermore, in the siRNA preserved auto-transplant kidneys, the mRNA level of inflammatory transcription factors involved in the TLR signaling pathway, such as NF-ΚB and c-Jun, were also increased, which resulted in the enhanced mRNA of pro-inflammatory cytokines, including IL-1Β and IL-6, TNF-Α and interferon (IFN)-Α, Β and Γ. The protein of RIG-1, an non-TLR RNA sensor, was not affected by the siRNA.

Conclusion. Naked caspase-3 siRNA administered into the kidney activated TLR mediated innate immune responses, and then amplified inflammation. The systemic complementary effects after applied naked siRNA locally via the TLR signaling pathway provided valuable evidence to guide future pre-clinic studies.

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