Inhibition ofPAX2Gene Expression by siRNA (Polyethylenimine) in Experimental Model of Obstructive Nephropathy

Abstract

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

Nano-sized carriers in gene therapy for renal fibrosisin vivo

“…Polyethylenimine (PEI) nanoparticles, liposome nanoparticles, and cationic gelatin nanoparticles have been demonstrated to show effective delivery of genes to the kidney and have promising treatment effects in animal models of renal fibrosis [49–54]. …”

“…Small interfering RNA (siRNA) targeted paired box2 (PAX2) was reported to be delivered to the kidney with PEI nanoparticles via an intrarenal capsule injection in a mouse model of renal fibrosis induced by UUO [49]. PAX2–siRNA–PEI nanoparticles inhibited PAX2 mRNA and PAX2 protein in the kidney, and ameliorated renal fibrosis [49].…”

“…Small interfering RNA (siRNA) targeted paired box2 (PAX2) was reported to be delivered to the kidney with PEI nanoparticles via an intrarenal capsule injection in a mouse model of renal fibrosis induced by UUO [49]. PAX2–siRNA–PEI nanoparticles inhibited PAX2 mRNA and PAX2 protein in the kidney, and ameliorated renal fibrosis [49]. Delivery of microRNA-146a mimic with PEI nanoparticles was reported to over-express microRNA-146a in the fibrotic kidney induced by UUO, and inhibited renal fibrosis by inhibiting TGF-β 1 and NF-κB signaling pathways in vivo [49–54].…”

“…PAX2–siRNA–PEI nanoparticles inhibited PAX2 mRNA and PAX2 protein in the kidney, and ameliorated renal fibrosis [49]. Delivery of microRNA-146a mimic with PEI nanoparticles was reported to over-express microRNA-146a in the fibrotic kidney induced by UUO, and inhibited renal fibrosis by inhibiting TGF-β 1 and NF-κB signaling pathways in vivo [49–54]. …”

PAX2 regulates NRP-1 expression in renal tubular epithelial cellsto promote epithelial-to-mesenchymal transition and renal fibrosis

“…PAX2 is not expressed in mature kidneys, but it is expressed following renal injury [5] . Our preliminary study indicated that PAX2 can causes RIF via EMT [6,7] , and we subsequently found that PAX2 could induce EMT in normal renal tubular epithelial cells in vitro [8] , which indicates that PAX2 is associated with the development of RIF. However, the mechanisms involved in how PAX2 induces EMT are not well understood.…”