Preventing Renal IschemiaReperfusion Injury Using Small Interfering RNA by Targeting Complement 3 Gene

Abstract

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-a . 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.

Gene Silencing of Complement C5a Receptor Using siRNA for Preventing Ischemia/Reperfusion Injury

“…Kidneys can express massive amounts of complement, as in the case of rejecting allografts. 22 On the other hand, using a mouse I/R injury model, we have demonstrated that the renal expression or deposition of C9 occurs early after reperfusion and is localized to cellular debris and injured tubular epithelial cells, which is consistent with our previous study 7,9 and is supported by the view that the complement pathway is crucially involved in renal I/R injury. 16,[23][24][25] In our present study, we extend the link between renal I/R injury and complement by demonstrating that specific blockade of C5aR expression using siRNA can prevent renal I/R injury.…”

“…3,15,16 We previously showed that inhibiting C3 with siRNA protected renal from I/R injury. 7,9,10 In this study, we have demonstrated that silencing the C5aR gene using siRNA can inhibit the renal expression of C5aR and that successful delivery of C5aR-siRNA to the kidneys results in the prevention of renal I/R injury.…”

“…4 expression vector that encodes a green fluorescent protein reporter gene as described previously. 7 To validate gene silencing efficiency, we co-transfected the C5aR siRNA with C5aR cDNA into L929 cells that do not express C5aR gene. The silencing efficiency was assessed at the level of mRNA using real-time PCR.…”

“…In our previous study, we found that complement activation affects TNF-␣ production 7 and treatment with C3 siRNA prevented the expression of TNF-␣ induced by I/R. To determine whether C5aR siRNA affected renal tubular cell expression of TNF-␣, we measured the expression of TNF-␣ in kidneys using real-time PCR.…”

Aloperine Protects Mice against Ischemia-Reperfusion (IR)-Induced Renal Injury by Regulating PI3K/AKT/mTOR Signaling and AP-1 Activity

“…Morphological changes in the cor tex and medulla were scored in a blinded fashion by two pathologists according to the severity of tubular necrosis, integrity of brush border and basement membrane, and cast formation. The scoring included 4 scales: 0) <10%; 1) 10% to 25%; 2) 25% to 50%; 3) 50% to 75%; and 4) >75% (36). Each section was assessed with randomly selected 10 highpower fields (magnifica tion, × 400), and three mice were included in each study group.…”