Abstract
BackgroundThe naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. The mechanisms involved, in particular, whether siRNA or complementary systemic feedback eliciting innate immune responses are worthy to be further investigated.MethodsThe protein and mRNA expression of innate immunity-related molecules were detected by western blotting and quantitative PCR in the tissues previously collected from 48 h auto-transplant kidneys. The donor kidneys were retrieved from mini pigs and cold preserved by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA for 24 h.ResultsThe protein level of Toll like receptor (TLR) 3, TLR7, and their main adapters, TRIF and MyD88, was up-regulated in the siRNA preserved auto-transplant kidneys. The mRNA level of NF-κB and c-Jun was increased, as well as pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and interferon (IFN)-α, β and γ. In addition, the non-TLR RNA sensor PKR protein, but not RIG1, was also increased in the siRNA preserved auto-transplant kidneys.ConclusionsThe activation of innate immunity with amplified inflammatory responses in the caspase-3 siRNA preserved auto-transplant kidneys are associated with increased TLR3, TLR7 and PKR, which might be due to complementary systemic feedback, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely ruled out. These results provided valuable evidence to guide future siRNA design and pre-clinic studies.
Highlights
The naked caspase-3 small interfering RNA infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study
In order to show a whole profile of this study, the results from our previous experiments using the same model were summarized as the following: the expression of caspase-3 mRNA precursor and active subunit was decreased in the post-Cold storage (CS) kidneys preserved by caspase-3 small interfering RNA (siRNA), but the auto-transplanted kidneys were not protected, in which increased caspase-3 mRNA and its active subunit, inflammation [myeloperoxidase (MPO)+ cell staining], apoptosis [in situ end labeling (ISEL) fragmented DNAs] and tubulointerstitial damage (TID, assessed by hematoxylin and eosin staining) were demonstrated, even though the renal function was not significantly changed (Table 2) [4]
TLR3 and TLR7 protein expression To determine whether the Toll like receptors (TLR)-dependent innate immunity was activated, the expression of TLR3, TLR7, MyD88 and TRIF protein in the post-transplant kidneys was detected by western blotting (Figure 1A-D)
Summary
The naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. In our previous study, naked synthetic caspase-3 siRNA delivered into the isolated porcine kidney and hemoperfusate during cold preservation improved ischemia reperfusion injury (IRI) in post-reperfused kidneys [1]. The RNA-sensing pattern recognition receptors (PRRs) are the most important components in the innate immunity. The response of PRRs to siRNA is mediated by either Toll like receptors (TLR) or non-TLR [5]. Non-TLR-mediated responses are triggered by siRNA binding to proteins such as dsRNA-binding protein kinase (PKR) associated with caspase-3, retinoic acid inducible gene 1 (RIG1) and NF-κB within the cytoplasm. TLR or non-TLR PRRs activation induces excessive cytokine release and inflammation [6]. High-mobility group box 1 protein (HMGB1) regulates gene expression acting as a nuclear transcription factor after cytoplasmic-nuclear translocation [7], which linked to caspase-3 activation, apoptosis and inflammation [8]
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