Xia Zhiyin, Abe Katsushige, Furusu Akira, Miyazaki Masanobu, Obata Yoko, Tabata Yasuhiko, Koji Takehiko, Kohno Shigeru
Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Am J Nephrol. 2008;28(1):34-46. doi: 10.1159/000108759. Epub 2007 Sep 21.
BACKGROUND/AIM: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model.
The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting.
Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA.
Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.
背景/目的：单侧输尿管梗阻（UUO）是一种公认的肾小管间质纤维化模型。在肾小管间质纤维化进展过程中，观察到肾小管间质区域胶原蛋白合成上调以及随后胶原蛋白的积累。热休克蛋白47（HSP47）是一种胶原蛋白特异性分子伴侣，在调节胶原蛋白合成中起重要作用。我们设计了针对HSP47 mRNA的小干扰RNA（siRNA）序列，以研究HSP47是否参与小鼠UUO模型中肾小管间质纤维化的进展。
在制备UUO时，通过输尿管一次性注射HSP47 siRNA。我们还应用了一种使用阳离子化明胶微球的siRNA新基因递送系统。在UUO后7天和14天收获肾脏。通过免疫组织化学和蛋白质印迹分析HSP47以及I、III和IV型胶原蛋白的表达水平。
UUO后7天，梗阻肾脏或绿色荧光蛋白siRNA处理的梗阻肾脏中HSP47以及I、III和IV型胶原蛋白的表达水平显著上调。注射HSP47 siRNA显著降低了蛋白表达水平，并显著减轻了伴随的间质纤维化。此外，作为递送系统的阳离子化明胶微球增强并延长了HSP47 siRNA的抗纤维化作用。
我们的结果表明，HSP47是预防肾小管间质纤维化的候选靶点，并且使用siRNA选择性阻断HSP47表达可能是肾病患者一种潜在有用的治疗方法。
