Synthetic small interfering RNA down‐regulates caspase‐3 and affects apoptosis, IL‐1β, and viability of porcine proximal tubular cells

Yang, Bin; Elias, Joshua E.; Bloxham, Maureen; Nicholson, Michael L.

doi:10.1002/jcb.23050

Cited by 20 publications

(16 citation statements)

References 37 publications

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“…As shown here (Fig. 4A), also others have shown that pro-caspase 3 levels increase following inflammatory processes such as renal injury [34] and decrease after caspase-3 siRNA addition within the injured kidney [35]. We have observed similar differential effects in Hsp70's interaction with elements of the NF-κB signaling pathway [21].…”

Section: Discussionsupporting

confidence: 86%

Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

Aschkenasy¹,

Bromberg²,

Raj

et al. 2011

PLoS ONE

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The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury.Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFα. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co- immunoprecipitation and a caspase-3 activity assay.TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes.We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery.

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Section: Discussionsupporting

confidence: 86%

Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

Aschkenasy¹,

Bromberg²,

Raj

et al. 2011

PLoS ONE

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“…Significant evidence indicates that caspase-3 is either partially or totally responsible for the proteolytic cleavage of many key proteins, facilitates cellular disassembly, and serves as a marker of cells undergoing apoptosis. 34,35 In this study, caspase-3 activity increased significantly after renal I/R, reached a peak at 24 h, and increased slightly at 72 h. The elevated caspase-3 activity was significantly reduced by rhALR administration. The variation trend of caspase-3 activity was the same as that of apoptosis rate detected by TUNEL.…”

Section: Discussionmentioning

confidence: 46%

Augmenter of Liver Regeneration Attenuates Tubular Cell Apoptosis in Acute Kidney Injury in Rats: The Possible Mechanisms

Liao

Chen

et al. 2012

Renal Failure

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Augmenter of liver regeneration (ALR), the expression of which increased in rat kidneys after renal ischemia/reperfusion (I/R) injury, enhances renal tubular cell regeneration in vivo and in vitro. We aimed to investigate the effects of ALR on apoptosis of renal tubular cells after renal I/R injury in vivo and consider the possible mechanisms. Rats that were subjected to bilateral renal ischemia for 60 min followed by reperfusion were administered with either vehicle or recombinant human ALR (rhALR). Renal dysfunction and histologic injury were assessed by the measurement of serum biochemical markers and histological grading. Apoptosis was assessed by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick-end labeling (TUNEL). Caspase-3 activity was measured using a colorimetric protease assay. Expression of Bcl-2, Bax Fas, phosphorylated-Akt (p-Akt), and phosphorylated-p53 (p-p53) was determined by western blotting. Compared with vehicle-treated rats, renal dysfunction and histologic injury were significantly attenuated by administration of rhALR. The number of TUNEL-positive tubular cells and caspase-3 activity were decreased, Bcl-2 and p-Akt expression was up-regulated, and Bax and p-p53 expression was down-regulated by administration of rhALR. However, administration of rhALR had no effect on Fas protein expression. These results indicate that the protective effect of rhALR on renal I/R injury is associated with its anti-apoptotic action in renal tubular cells. RhALR inhibits apoptosis by increasing the ratio of Bcl-2 to Bax and by decreasing the activity of caspase-3. The activation of Akt and inactivation of p53 are involved in the rhALR anti-apoptosis process.

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“…The most effective sequences: 5′-GGGAGACCUUCACAAACUUtt-3′ and 5′-AAGUUUGUGAAGGUCUCCCtg-3′, were selected in LLC-PK1 cells [9]. The in vivo ready custom caspase-3 siRNA (Silencer®) with selected sequences and optimized dosage were then verified in our ex vivo porcine kidney preservation study [1].…”

Section: Methodsmentioning

confidence: 99%

Innate immunity activation involved in unprotected porcine auto-transplant kidneys preserved by naked caspase-3 siRNA

Yang

Xue

et al. 2013

J Transl Med

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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.

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Synthetic small interfering RNA down‐regulates caspase‐3 and affects apoptosis, IL‐1β, and viability of porcine proximal tubular cells

Cited by 20 publications

References 37 publications

Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

Augmenter of Liver Regeneration Attenuates Tubular Cell Apoptosis in Acute Kidney Injury in Rats: The Possible Mechanisms

Innate immunity activation involved in unprotected porcine auto-transplant kidneys preserved by naked caspase-3 siRNA

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