Protection of renal ischemia injury using combination gene silencing of complement 3 and caspase 3 genes.
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.
Zheng
Xiufen
X
Department of Surgery, University of Western Ontario, London, Ontario, Canada.
Zhang
Xusheng
X
Sun
Hongtao
H
Feng
Biao
B
Li
Mu
M
Chen
Gang
G
Vladau
Costin
C
Chen
Dong
D
Suzuki
Motohiko
M
Min
Lisa
L
Liu
Weihua
W
Zhong
Robert
R
Garcia
Bertha
B
Jevnikar
Anthony
A
Min
Wei-Ping
WP
D000818:Animals; D053148:Caspase 3 / Q000235:genetics; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D003176:Complement C3 / Q000037:antagonists & inhibitors* / Q000235:genetics; D015316:Genetic Therapy; D007668:Kidney / Q000098:blood supply* / Q000473:pathology; D016030:Kidney Transplantation; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000627:therapeutic use; D015427:Reperfusion Injury / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*
D061945:Caspase Inhibitors; D003176:Complement C3; D034741:RNA, Small Interfering; D053148:Caspase 3