Short Hairpin RNA Knockdown of Connective Tissue Growth Factor by Ultrasound-Targeted Microbubble Destruction Improves Renal Fibrosis.
The purpose of this study was to evaluate whether ultrasound-targeted microbubble destruction transfer of interfering RNA against connective tissue growth factor (CTGF) in the kidney would ameliorate renal fibrosis in vivo. A short hairpin RNA (shRNA) targeting CTGF was cloned into a tool plasmid and loaded onto the surface of a cationic microbubble product. A unilateral ureteral obstruction (UUO) model in mice was used to evaluate the effect of CTGF knockdown. Mice were administered the plasmid-carrying microbubble intravenously, and ultrasound was applied locally to the obstructed kidney. Mice undergoing a sham UUO surgery and untreated UUO mice were used as disease controls, and mice administered plasmid alone, plasmid with ultrasound treatment and microbubbles and plasmid without ultrasound were used as treatment controls. Mice were treated once and then evaluated at day 14. CTGF in the kidney was measured by quantitative reverse transcription polymerase chain reaction and Western blot. Expression of CTGF, transforming growth factor β1, α smooth muscle actin and type I collagen in the obstructed kidney was evaluated by immunohistochemistry. The cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA and protein expression of CTGF (p < 0.01). Furthermore, CTGF gene silencing decreased the interstitial deposition of transforming growth factor β1, α smooth muscle actin and type I collagen as assessed in immunohistochemistry, as well as reduced renal fibrosis in pathologic alterations (p < 0.01). No significant changes in target mRNA, protein expression or disease pathology were observed in the control cohorts. A single treatment of ultrasound-targeted microbubble destruction is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in disease severity. This technique may be a potential therapy for treatment of renal fibrosis.
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China.
Shuping
S
Wei
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China.
Chaoli
C
Xu
Targeson, Inc., San Diego, California, USA.
Joshua J
JJ
Rychak
Targeson, Inc., San Diego, California, USA.
Alice
A
Luong
Department of Pharmacological Study, Origin Biosciences, Inc., Nanjing, Jiangsu Province, China.
Yu
Y
Sun
Department of Pharmacological Study, Origin Biosciences, Inc., Nanjing, Jiangsu Province, China.
Zhijian
Z
Yang
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China.
Mingxia
M
Li
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China.
Chunrui
C
Liu
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China.
Ninghua
N
Fu
Department of Ultrasound, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, China. Electronic address: yb12yx@hotmail.com.
Bin
B
Yang
D000818:Animals; D015153:Blotting, Western; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D005355:Fibrosis / Q000628:therapy; D007674:Kidney Diseases / Q000235:genetics / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014464:Ultrasonic Therapy / Q000379:methods*
D034741:RNA, Small Interfering; D055513:Connective Tissue Growth Factor
Cationic microbubbles; Connective tissue growth factor; Gene delivery; RNA interfering; Renal fibrosis; Short hairpin RNA; Sonoporation; Transforming growth factor-β; Ultrasound-targeted microbubble destruction; Unilateral ureteral obstruction