| OBJECTIVE: Renal interstitial fibrosis which has been believed to be the common final pathway leading to end-stage renal failure is a progressive course independent on the reason. Renal tubular epithelial cell is directly responsible for the collection of extracellular matrix through epithelial-myofibroblast transdifferentiation ( EMT ) , which has been identified as an important mechanism promoting renal interstitial fibrosis. TGF- β is one of the key fibrogenic cytokines and can induce renal interstitial fibrosis by regulating EMT. Recently, Pioglitazone has been believed to negatively regulate the fibrosis of many organs. The objective of our experiment is to investigate whether pioglitazone can inhibit TGF- β -induced phenotypic remodeling of renal tubular epithelial cell and effect the expression of CTGF , which is a down-stream cytokine of TGF- β , to illuminate the anti-fibrotic mechanism of pioglitazone, and to provide a new method and academic foundation for blocking the renal intestitial fibrosis.METHODS: Cultured NRK52E cells were divided into controlled group, TGF- β -treated group, TGF- β and DMSO-treated group, TGF- β andpioglitazone-treated groups with different doses. The morphological change of cultured tubular cells was observed under scanning electron microscopy and phase-contrast microscopy. Immunohistochemistry and flowcytometry were used to assess the a—SMA+ cells, the expression of a—SMAmRNA was measured by RT-PCR. The expression of CTGFmRNA and protein were evaluated by immunohistochemistry and RT-PCR. The level of collagen type III in the culture supernatant was measured by ELISA.RESULTS: ?Confluent NRK52E cells cultured in medium with 5% FCS showed a normal epithelial morphological state and cobblestone pattern. All cells almost had no expression of a-SMA and CTGF. Conversly, the addition of 3ng/ml of TGF- P to the NRK52E cells resulted in the presence of strong cytoplasmic staining for a-SMA, and significant increase of the level of a-SMAmRNA expression and MCF (PO.05). Many a-SMA+ cells showed marked hypertrophy, elongated shape, which indicated transdifferentiating into myofibroblasts. the level of CTGFmRNA expression in TGF- P -treated group cells was higher than that of the normal control group(P<0.05). Compared with controlled group, there was a significant increase of content of collagen type III in supernatant (PO.05). ?There were no differences in the measured indexes between the TGF- P -treated group and the TGF- P +DMSO treated group. (3)The phenotypic changes induced by TGF- P were prevented by incubation with pioglitazone. Pioglitazone inhibited in a dose-dependent manner a-SMAmRNA and CTGFmRNA expression of NRK52E cells(p<0.05). Content of collagen type III in supernatant was significantly reduced by pioglitazone (PO.05). ?CTGFmRNA expression of NRK52E cells was significantly associated with a-SMAmRNAexpression(r=0.975, PO.01), MCF of a-SMA(r=0.943 P<0.01) and content of collagen type III(r=0.988, P<0.01). Expression of a-SMAmRNA was correlated with content of collagen type III(r=0.952, P<0.01).CONCLUSION: CD TGF- 0 can promote excretion of collagen type III by inducing tubular epithelial-myofibroblast transdifferentiation and expression of CTGF, which may be the one of fibrogenic mechanisms of TGF- 3 ; (2) Pioglitazone blocked the EMT and excretion of collagen type III; (3) Pioglitazone may negatively regulate EMT through inhibiting the expression of CTGF.
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