Role Of Connective Tissue Growth Factor Antisense Oligodeoxynucleotides In Tubular Epithelial-myofibroblast Transdifferentiation

OBJECTIVE: Tubular epithelial-myofibroblast transdifferentiation(TEMT) plays an important role in tubulointerstitial fibrosis, which is a common pathway of progressive renal disease. Transforming growth factor-β (TGF- β )is one of the important cytokines that induce TEMT. Connective tissue growth factor(CTGF) is a TGF- β downstream mediator. Multiple lines of evidence have shown a close relationship between the expression of CTGF and TEMT. Antisense oligodeoxynucleotides (AS-ODNS) play an inhibiting role in the target gene expression. Whether CTGF AS-ODNS can inhibit CTGF-induced TEMT remains unknow. In this study, we investigated the effect of blockade of endogenous CTGF on TGF- β₁ induced TEMT and the secretion of type â…¢ collagen by CTGF AS-ODNS .METHODS: Cultured normal rat kidney tubular epithelial(NRK-52E)cells were divided into control group, TGF- β₁—stimulated group, cationic lipofection group, control reverse ODNS group and AS-ODNS group. After CTGF AS-ODNS and control reverse ODNS were transfected into cells by cationic lipofection, cells were stimulated with TGF- β₁ for 48h. InhibitedCTGF gene expression was assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The morphology of transdifferentiate tubular cells were observed under scanning electron microscopy and phase-contrast microscopy. The expression of a -smooth muscle actin( a -SMA) was detected by RT-PCR> immunohistochemistry and flowcytometry. Fibrogenic factor type III collagen was measured by ienzyme-linked immunosorbent assay (ELISA).RESULTS: (1) Cultured NRK52E cells in the presence of TGF- 0 ,lOng/ml showed a clear fibroblast-like morphology. The levels of CTGF mRNA and a -SMA mRNA were elevated significantly 48 hours after treatment with TGF-P i(P<0.05). The percentage of a —SMA+ cells and mean channel fluorescence(MCF) levels in the TGF-13 ]—stimulated group were much higer than those of normal control group(P<0.05). The level of type III collagen in the culture supernatant was also elevated(P<0.05). (2) There was no differences in the measured indexes among TGF- 0 , — stimulated group > cationic lipofection group and control reverse ODNS group(P>0.05). (3) CTGF AS-ODNS effectively inhibited TGF- P ,-induced CTGF mRNA expression(P<0.05). In this condition , the TGF- P ,-induced NRK52E cells morphology transformation and a -SMA expression was significantly inhibited by CTGF antisense treatment. TGF- {3 i—induced type III collagen expression was also significantly attenuated with AS-ODNS transfection compared with control reverse ODNS(P<0.05). (4) CTGF mRNA and a -SMA mRNA(r=0.970 P<0.01) as well as CTGFmRNA and type III collagen(r=0.815 PO.01) were positively correlated.CONCLUSIONS: (l)It was certificated by AS-ODNS that CTGF couldinduce TEMT and the secretion of type III collagen in cultured NRK-52E cells, and this role belonged to one of downstream effects of TGF- P . (2) In cultured NRK-52E cells , CTGF AS-ODNS transfection effectively inhibited TGF- f3 i-induced CTGF mRNA expression and further attenuated TEMT .It suggested that CTGF blockade by AS-ODNS could be a possible therapeutic strategy against tubulointerstitial fibrosis.