The Effect And Mechanism Of Caspase-3siRNA On Donor Kidney In Porcine Auto-transplantation Model

Background:Caspase-3plays key roles in ischemia reperfusion injury. The efficacy of naked caspase3small interfering RNA (siRNA) has been proved in an isolated porcine kidney perfusion model but not in auto-transplantation.Methods:(1) The left kidney was retrieved from mini pigs and infused with the University of Wisconsin (UW) solution with/without0.3mg of caspase3siRNA (n=5) into the renal artery with the renal artery and vein clamped for24-h cold storage (CS). After right nephrectomy, the left kidney was auto-transplanted into the right for48h without systemic treatment of siRNA.(2) The left kidney was retrieved and infused by UW solution with/without0.3mg caspase-3or negative siRNA (n=5) into the renal artery for24-h cold storage (CS). After intravenously injected0.9mg siRNA and the right nephrectomy, the left kidney was auto-transplanted for2weeks.Results:(1) Fluorescent dye labeled caspase3siRNA was visualized in the post-CS kidneys but was weakened after transplantation. The expression of caspase3messenger RNA and precursor was down-regulated by siRNA in the post-CS kidneys. In the siRNA-preserved posttransplant kidneys, however, the caspase3mRNA and active subunit were up-regulated with further decreased precursor but increased active caspase-3+cells, apoptotic cells, and myeloperoxidase+cells. Moreover, the renal tissue damage was aggravated by siRNA, whereas the renal function was not significantly changed.(2) The effectiveness of caspase-3siRNA was confirmed by down-regulated caspase-3mRNA, precursor and active subunit in the post-CS and/or post-transplant kidneys. The down-stream events were also affected with reduced apoptosis and inflammation represented by less myeloperoxidase+cells, decreased IL-1β, IL-6, NF-κB, IFN-α, IFN-β and IFN-y mRNA, and lowered HMGB1protein. The minimal off-target effect of caspase-3siRNA was demonstrated by unchanged TLR3and TLR7. Most importantly, renal function and tissue damage were improved by caspase-3siRNA.Conclusion:(1) The short-acting naked caspase3siRNA administered into the kidney was effective in cold preservation but not enough to protect posttransplant kidneys, which might be because of systemic complementary responses overcoming local effects.(2) the2-week auto-transplant kidneys were protected by the novel long-acting serum stabilized naked caspase-3siRNA administrated locally and systemically, which provides invaluable data for future clinical trials.(3) the novel long-acting serum stabilized naked caspase-3siRNA was safe without the activation of TLR-dependent innate immune response.