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Tumor necrosis factor-alpha (TNF-α) has been found to be centrally involved in the development of ischemia-reperfusion injury (IRI)-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interfering RNA (siRNA) may protect renal IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. TNF-α siRNA was administered intravenously to silence the expression of TNF-α. The therapeutic effects of siRNA were evaluated in terms of renal function, histological examination, and overall survival following lethal IRI. A single systemic injection of TNF-α siRNA resulted in significant knockdown of TNF-α expression in ischemia-reperfusion injured kidney. In comparison with control mice, levels of BUN and serum creatinine were significantly reduced in mice treated with siRNA. Pathological examination demonstrated that tissue damage caused by IRI was markedly reduced as a result of TNF-α siRNA treatment. Furthermore, survival experiments showed that nearly 90% of control mice died from lethal IRI, whereas more than 50% of siRNApretreated mice survived until the end of the eight-day observation period. We have demonstrated for the first time that silencing TNF-α by specific siRNA can significantly reduce renal IRI and protect mice against lethal kidney ischemia, highlighting the potential for siRNA-based clinical therapy.

Background: Ischemia-reperfusion (I/R) injury is the major cause of delayed renal graft function in kidney transplantation. To date, there are no effective therapeutic approaches for preventing I/R injury. We previously reported that treatment of animals with small interference RNA (siRNA) would prevent warm I/R injury in nontransplant models and cold I/R injury in heart transplantation. In the present study, we further explore the feasibility of protecting grafts from extended cold I/R injury as applied to kidney transplantation by downregulating I/R-associated genes using siRNA., Methods: Donor kidneys were intra-arterially perfused with siRNA containing solution during donor excision and preserved in siRNA containing solution. The siRNA-treated donor organs were then implanted into syngeneic recipient mice, and the 2 original kidneys were removed from the recipient. The effect of siRNA solution on extended cold I/R injury was determined by assessing renal function, histopathological change, cell apoptosis, and inflammation., Results: The perfused siRNA solution knocked down the expression of complement 3, RelB, and Fas in the kidney at the mRNA and protein levels. Administration of siRNA solution reduced the levels of blood urea nitrogen and serum creatinine as compared with control groups. The siRNA cocktail decreased cell apoptosis and histopathological changes in the kidney and prolonged graft survival. The siRNA cocktail also reduced the expression of proinflammatory cytokines, IL-6, and TNFα., Conclusions: In conclusion, this is the first demonstration that perfusing donor organs with an siRNA cocktail solution can induce gene silencing in the kidney and prevent kidneys from extended cold I/R injury in kidney transplantation, highlighting the promise of the clinical application of siRNA-based therapies in the preservation of donor organs.

Background: Tumor necrosis factor-alpha (TNF-α) is a central mediator in the hepatic response to ischemia/reperfusion. Short hairpin RNA (shRNA) has been proven to be an effective means of harnessing the RNA interference pathway in mammalian cells. In the current study, we investigated whether silencing TNF-α gene with shRNA can prevent liver ischemic reperfusion injury (IRI)., Methods: Male BalB/c mice were randomized to TNF-α shRNA, scramble shRNA, or sham operation groups. TNF-α shRNA and scramble shRNA groups were injected 48 h before inducing IRI. IRI was induced via microaneurysm clamps applied to the left hepatic artery and portal vein. Six hours after reperfusion, IRI injury was examined by serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology, MPO, and MDA level, as well as by relative quantities of TNF-α mRNA., Results: TNF-α expression induced by ischemia reperfusion in the liver was significantly suppressed after treatment with TNF-α shRNA compared with the group treated with scramble shRNA (P < 0.001). Mice treated with TNF-α shRNA showed lower peak values of AST and ALT than scramble shRNA treated mice (P < 0.001). On histopathologic slides, mice treated with TNF-α shRNA had significantly less ischemia/reperfusion injury based on Suzuki score than the scramble shRNA group, 3.57 ± 2.30 and 8.83 ± 0.98 respectively (P < 0.001), while the sham group was not significantly different from the TNF-alpha shRNA group, 0 ± 0 and 3.57 ± 2.30, respectively (P = 0.075). Liver tissue MDA levels were significantly lower in mice treated with TNF-α shRNA as compared with the group treated with scramble shRNA (P < 0.01). Immunohistochemical staining for MPO was significantly lower in mice treated with TNF-α shRNA compared with the group treated with shRNA (compared with treated with scramble shRNA group.), Conclusions: Liver IRI can be minimized through gene silencing of TNF-α. This may represent a novel therapy in the setting of transplantation and in other conditions associated with IRI of the liver., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) is an endogenous mechanism of cellular RNA control through degradation of specific messenger RNA sequences. This process of gene silencing may be exploited by the use of small interfering RNA (siRNA) to mediate precise control of targeted cellular functions. The nature of transplantation leads invariably to tissue injury, as organs are damaged by the loss of blood supply and resultant ischemia associated with the procurement procedure. Upon reperfusion, an inflammatory program is activated, and subsequent injury results in delayed graft function and, potentially, organ failure. Many of the molecular components in ischemia-reperfusion injury (IRI) have been identified, but effective therapeutics are not currently available. Accumulating evidence supports a role for siRNA in controlling IRI, as siRNA is specific, relatively low in toxicity, and limited in duration of effect. The capacity of siRNA to control IRI-related transcription factors, cell death and apoptosis, complement factors, and oxidative stress molecules supports the concept that RNAi-based therapeutics represent a novel and promising strategy for the control of IRI. However, there are issues of RNAi strategies, including siRNA design, "off-target" effects, and delivery that merit consideration in approaching IRI with gene silencing. This review will provide an overview of current concepts in RNAi and the potential application to IRI in solid organ transplantation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of endogenous cellular pathways of RNA degradation, resulting in selective and potent silencing of genes that have homology to the double strand. Much of the excitement surrounding small interfering RNA (siRNA)-mediated therapeutics arises from the fact that this approach overcomes many of the shortcomings previously experienced with alternative approaches to selective blocking that use antibodies, antisense oligonucleotides or pharmacological inhibitors. Induction of RNAi through administration of siRNA has been successfully applied to the treatment of hepatitis, viral infections, and cancer. Increased success in addressing issues of siRNA delivery and efficiency will permit this approach to evolve as a new paradigm in clinical therapeutics. In this chapter, we present applications of RNAi in tissue injury, and the possibilities of using this highly promising approach in the context of transplantation.

Background: Nuclear factor kappaB (NF-kappaB) has been found to be critical to the pathogenesis of renal ischemia-reperfusion injury (IRI). Using small interfering RNA (siRNA) to silence the expression of RelB, a component of the transcription factors Rel/nuclear factor kappaB, may protect renal IRI. Here, we report an siRNA-based treatment of preventing IRI., Methods: Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. The therapeutic effects of siRNA were evaluated in renal function, histologic examination, and overall survival after lethal IRI., Results: A single injection of RelB siRNA resulted in knockdown of renal RelB expression. In comparison with control mice, levels of blood urea nitrogen and serum creatinine were significantly decreased in mice treated with siRNA. Pathologic examination demonstrated that tissue injury caused by IRI was markedly reduced as a result of RelB siRNA treatment. Additionally, with RelB siRNA treatment, immunohistochemistry showed a significant attenuation of tumor necrosis factor-alpha expression. Furthermore, survival experiments revealed that more than 90% of control mice died from lethal IRI, whereas 80% of siRNA-pretreated mice survived until the end of the 8-day observation period., Conclusion: Silencing RelB, using siRNA, can significantly attenuate IRI-induced renal dysfunction and protect mice against lethal kidney ischemia, highlighting the potential for siRNA-based clinical therapy.

Renal ischemia-reperfusion injury (IRI) can result in acute renal failure with mortality rates of 50% in severe cases. NK cells are important participants in early-stage innate immune responses. However, their role in renal tubular epithelial cell (TEC) injury in IRI is currently unknown. Our data indicate that NK cells can kill syngeneic TEC in vitro. Apoptotic death of TEC in vitro is associated with TEC expression of the NK cell ligand Rae-1, as well as NKG2D on NK cells. In vivo following IRI, there was increased expression of Rae-1 on TEC. FACS analyses of kidney cell preparations indicated a quantitative increase in NKG2D-bearing NK cells within the kidney following IRI. NK cell depletion in wild-type C57BL/6 mice was protective, while adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2(-/-)gamma(c)(-/-) mice with IRI. NK cell-mediated kidney injury was perforin (PFN)-dependent as PFN(-/-) NK cells had minimal capacity to kill TEC in vitro compared with NK cells from wild-type, FasL-deficient (gld), or IFN-gamma(-/-) mice. Taken together, these results demonstrate for the first time that NK cells can directly kill TEC and that NK cells contribute substantially to kidney IRI. NK cell killing may represent an important underrecognized mechanism of kidney injury in diverse forms of inflammation, including transplantation.

Ischemia/reperfusion (I/R) injury in organ transplantation significantly contributes to graft failure and is untreatable using current approaches. I/R injury is associated with activation of the complement system, leading to the release of anaphylatoxins, such as C5a, and the formation of the membrane attack complex. Here, we report a novel therapy for kidney I/R injury through silencing of the C5a receptor (C5aR) gene using siRNA. Mice were injected with 50 microg of C5aR siRNA 2 days before induction of ischemia. Renal ischemia was then induced through clamping of the renal vein and artery of the left kidney for 25 minutes. The therapeutic effects of siRNA on I/R were evaluated by assessment of renal function, histopathology, and inflammatory cytokines. siRNA targeting C5aR efficiently inhibited C5aR gene expression both in vitro and in vivo. Administering C5aR siRNA to mice preserved renal function from I/R injury, as evidenced by reduced levels of serum creatinine and blood urea nitrogen in the treated groups. Inhibition of C5aR also diminished in vivo production of the pro-inflammatory cytokine tumor necrosis factor-alpha and chemokines MIP-2 and KC, resulting in the reduction of neutrophils influx and cell necrosis in renal tissues. This study demonstrates that siRNA administration represents a novel approach to preventing renal I/R injury and may be used in a variety of clinical settings, including transplantation and acute tubular necrosis.

Background: Ischemia/reperfusion (I/R) injury occurs in clinical kidney transplantation, which results in graft dysfunction and rejection. It has been documented that I/R injury is associated with complement activation and renal cell apoptosis. The purpose of this study was to develop a strategy to prevent I/R injury using small interfering RNA (siRNA) that target complement 3 (C3) and caspase 3 genes., Methods: siRNA-expression vectors were constructed to target C3 and caspase 3 genes. Gene silencing efficacy was assessed using real-time polymerase chain reaction. In vivo gene silencing was performed by hydrodynamic injection with C3 and caspase 3 siRNA. Renal I/R injury was induced through clamping the renal vein and artery for 25 min. I/R injury was evaluated using kidney histopathology, blood urea nitrogen (BUN), serum levels of creatinine, and survival., Results: Effective gene silencing was first confirmed in vitro. Notably upregulated expression of C3 and caspase 3 genes was observed from 2 to 48 hr after I/R injury, which were effectively and specifically inhibited by C3 and caspase 3 siRNA. In comparison with control mice, serum levels of creatinine and BUN were also significantly decreased in C3 and caspase 3 siRNA-treated mice. Furthermore, the therapeutic effect of siRNA was assessed in a severe, lethal I/R injury experiment, in which siRNA treatment significantly reduced mortality. Tissue histopathology showed an overall reduction in injury area in siRNA-treated mice., Conclusions: This is the first demonstration that renal I/R injury can be prevented through silencing the complement gene and apoptosis gene, highlighting the potential for siRNA-based clinical therapy.

Background: Apoptotic pathways mediated by caspases play a critical role in renal ischemia-reperfusion injury (IRI). Downregulation of the caspase cascade, using small interfering RNA (siRNA) to silence the expression of caspase 3 and caspase 8, may have substantial therapeutic potential for limiting renal injury., Methods: IRI was induced in mice by clamping of the renal vein and artery for 25 or 35 min at 37 degrees C. Caspase 3 and caspase 8 (caspase 3/8) siRNA was administrated by hydrodynamic injection. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to analyze the gene silencing efficacy, and the therapeutic effects of siRNA were evaluated by renal function analysis, histological examination, and overall survival of mice suffering from IRI., Results: In this study, we have shown, using quantitative PCR, that IRI is associated with increased levels of renal caspase 3/8 mRNA. Mice treated with caspase 3/8 siRNA showed a significant down-regulation in kidney expression of caspase 3/8 at both, transcriptional and protein levels. Kidney function in IRI was protected by siRNA therapy, as levels of blood urea nitrogen and creatinine were significantly reduced in mice treated with siRNA. Histological examination demonstrated that tissue injury caused by IRI was significantly reduced as a result of caspase 3/8 siRNA treatment. Furthermore, survival data showed that more than 70% of mice in siRNA-treated groups survived until the end of the eight-day observation period., Conclusion: Herein, we have demonstrated the therapeutic potential of using siRNA to knock down the expression of caspases and prevent acute renal injury.

The complement system is one of the important mediators of renal ischemia-reperfusion injury (IRI). We hypothesized that efficient silencing of C3, which is the central component on which all complement activation pathways converge, could be achieved using small interfering RNA (siRNA), and that this would result in overall inhibition of complement activation, thereby preventing IRI in kidneys. A series of experiments was conducted, using a mouse model of IRI and vector-delivered C3-specific siRNA. We demonstrated the following: (1) renal expression of C3 increases as a result of IRI; (2) by incorporation into a pRNAT U6.1 vector, siRNA can be delivered to renal cells in vivo; (3) systemically delivered siRNA is effective in reducing the expression of C3 in an experimentally induced mouse kidney model of IRI; (4) similarly, siRNA reduces complement-mediated IRI-related effects, both in terms of renal injury (as evidenced by renal function and histopathology examination) and mouse mortality and (5) silencing the production of C3 diminishes in vivo production of TNF-alpha. This study implies that siRNA represents a novel approach to preventing IRI in kidneys and might be used in a variety of clinical settings, including transplantation and acute tubular necrosis.