Influence of connective tissue growth factor antisense oligonucleotide on angiotensin II-induced epithelial mesenchymal transition in HK2 cells1

Abstract

The present study was designed to further investigate the effect of connective tissue growth factor antisense oligonucleotide (CTGF-AS) on angiotensin II (Ang II)-induced tubular cell epithelial mesenchymal transition (EMT) in vitro. The human proximal tubular cell line (HK2) was grown in Dulbecco's modified Eagle's medium containing 10% heat inactivated fetal calf serum. After being rested in serum-free medium for 24 h, the influence of CTGF-AS (20 mug/mL) on Ang II-induced (0.1 micromol/L) CTGF mRNA and the protein expression were examined by using reverse transcription-polymerase chain reaction and indirect-immunofluorescence. The effect of CTGF-AS on Ang II-induced cellular ultrastructure was observed using a transmissive electronic microscope. The expression of alpha-smooth action (alpha-SMA) was assayed by immunocytochemistry. In all experiments, the control group was treated with scrambled oligonucleotide. It was shown that Ang II significantly induced the increasing expression of CTGF mRNA and protein (P<0.01, respectively), which were significantly abolished by treatment with CTGF-AS. After stimulating cells with Ang II, the cellular ultrastructure showed mesenchymal features. These effects were partially inhibited by CTGF-AS. Ang II significantly resulted in the expression of alpha-SMA in time dependent manner, which was markedly attenuated by the treatment with CTGF-AS (P<0.01, respectively). In contrast, no similar effects were observed in the control group treated with scrambled oligonucleotide. Ang II-induced EMT in human proximal tubular epithelial cells (PTC) can be attenuated by treatment with CTGF-AS. Our data provides further evidence that CTGF might be involved in Ang II-induced EMT in PTC.