[Role of connective tissue growth factor in human renal tubular epithelial cell transdifferentiation in vitro]

To observe the effect of connective tissue growth factor (CTGF) on the transdifferentiation of human renal tubular epithelial cells and to explore the influence of CTGF antisense oligodeoxynucleotide (ASODN) transfection on the transdifferentiation process induced by transforming growth factor-beta1 (TGF-beta1).

Human renal tubular epithelial cells of the strain HKC were cultured and divided into 3 groups: (1) negative control group, (2) low dose CTGF group, treated with recombinant human CTGF (rhCTGF) with the terminal concentration of 2.5 microg/L, and (3) high dose CTGF group, treated with rhCTGF with the terminal concentration of 5.0 microg/L). To evaluate the contribution of CTGF to the transdifferentiation induced by TGF-beta1, Another HKC cells were divided into 4 groups: (1) untreated control group (Group C), (2) Group T, stimulated by TGF-beta1 (10.0 microg/L), (3) Group S, stimulated by sense ODN transfection + TGF-beta1 (10.0 microg/L), and (4) Group A, stimulated by antisense ODN transfection + TGF-beta1 (10.0 microg/L). RT-PCR was used to detect the mRNA expression of alpha-smooth muscle actin (alpha-SMA) and collagen type IV (col IV) mRNA. Indirect immunofluorescence assay and flow cytometry were used to assess the level of intracellular alpha-SMA protein. ELISA was used to determine the concentration of col IV in the media.

The normal HKC cells were round and the HKC cells stimulated with rhCTGF became elongated. Upon the stimulation of different concentrations of rhCTGF, the expression of alpha-SMA mRNA increased markedly (both P < 0.01), while the mRNA expression of collagen type IV gene was down-regulated significantly (both P < 0.01). The percentage of alpha-SMA positive cells was significantly higher in the stimulated groups than that in negative control with significant difference among any 2 groups (38.9%, 65.5% vs. 2.4% respectively, all P < 0.01). Under this condition, collagen type IV secreted into the culture medium was lowered markedly upon the induction of CTGF (P < 0.01). RT-PCR analysis showed that the CTGF gene expression was upregulated by TGF-beta1 stimulation and peaked in 3 hours, and the alpha-SMA expression was upregulated by TGF-beta1 stimulation, however, peaked in 6 hours. The CTGF mRNA expression of the HKC cells transfected with CTGF ASODN that was stimulated by TGF-beta1 10 microg/L was significantly suppressed (P < 0.01) and the alpha-SMA mRNA expression induced by TGF-beta1 10 microg/L was significantly inhibited by CTGF ASODN transfection (P < 0.01). Indirect immunofluorescence assay showed that normal HKC cells did not express alpha-SMA, 48 hours after stimulation of TGF-beta1 10 microg/L the HKC cells showed expression of alpha-SMA in the cytoplasm, and the intracytoplasmic alpha-SMA expression was significantly down-regulated by the transfection of CTGF ASODN (P < 0.01).

CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (MyoF) in vitro, and CTGF blockade results in a dramatic inhibition of TGF-beta-induced transdifferentiation of renal tubular cells. So CTGF may be a crucial factor in promoting tubular-epithelial myofibroblast transdifferentiation.