Department of Pediatric Nephrology, Shengjing Hospital, China Medical University, Shenyang, China.
Ren Fail. 2012;34(10):1288-96. doi: 10.3109/0886022X.2012.723662.
It was found that PAX2 (Paired Box 2) re-expression in renal tubular epithelial cells correlated with renal interstitial fibrosis of rats with obstructive nephropathy. The purpose of the present study was to identify whether RNA interference (RNAi) induced by polyethylenimine (jetPEI) could inhibit PAX2 gene re-expression and impact renal interstitial fibrosis of rats with obstructive nephropathy.
Four pairs of small interfering RNA (siRNA) interference sequences and a negative control were designed and synthesized according to the whole PAX2 gene mRNA sequence. siRNA was then transfected into renal capsule in a rodent model of unilateral ureteral obstruction (UUO), using in vivo jetPEI as delivery vector. The expression of PAX2 mRNA and protein in renal tissue was determined by real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. A carboxyfluorescein (FAM)-labeled negative control was also synthesized at the same time to identify transfection in vivo. Renal interstitial fibrosis was observed under light microscope after PAX2 gene silenced.
Green fluorescence was observed around renal tubule epithelial cells by fluorescence microscopy 3 days after the procedure validating the success of transfection. While the PAX2 gene expression was inhibited by all siRNA interference sequences, the strongest inhibition was observed with siRNA3. The PAX2 mRNA and protein were inhibited by 55% and 81%, respectively. Renal tubular damage and renal interstitial fibrosis were remitted obviously after PAX2 gene silenced.
siRNA was able to inhibit the expression of PAX2 in tubular epithelial cells in the UUO model. The siRNA may thus provide therapeutic potential for retarding the pathological progression of UUO.
研究发现配对盒基因 2（PAX2）在肾小管上皮细胞中的重新表达与梗阻性肾病大鼠肾间质纤维化有关。本研究旨在确定聚乙烯亚胺（jetPEI）诱导的 RNA 干扰（RNAi）是否可以抑制 PAX2 基因的重新表达，并影响梗阻性肾病大鼠的肾间质纤维化。
根据 PAX2 全长 mRNA 序列设计并合成了 4 对小干扰 RNA（siRNA）干扰序列和阴性对照。然后，将 siRNA 转染到单侧输尿管梗阻（UUO）动物模型的肾被囊中，使用体内 jetPEI 作为递送载体。通过实时定量聚合酶链反应（PCR）和 Western blot 分别检测肾组织中 PAX2mRNA 和蛋白的表达。同时还合成了羧基荧光素（FAM）标记的阴性对照以鉴定体内转染。沉默 PAX2 基因后，在光镜下观察肾间质纤维化。
荧光显微镜观察到肾小管上皮细胞周围 3 天有绿色荧光，证实转染成功。虽然所有 siRNA 干扰序列均抑制 PAX2 基因的表达，但 siRNA3 的抑制作用最强。PAX2mRNA 和蛋白的抑制率分别为 55%和 81%。沉默 PAX2 基因后，肾小管损伤和肾间质纤维化明显减轻。
siRNA 能够抑制 UUO 模型中肾小管上皮细胞中 PAX2 的表达。因此，siRNA 可能为延缓 UUO 的病理进展提供治疗潜力。
