Role Of Connective Tissue Growth Factor Involved In Epithelial-mesenchymal Transition Of Human Proximal Tubular Cells

AIM: Tubulointersitial fibrosis is the final common way to end-stage renal disease despite of various causes. Recent studies have demonstrated that tubular epithelial-mesenchymal transition is the key event of tubulointerstitial fibrosis. CTGF(connective tissue growth factor) comprises four individual modules and presents as isoforms in different length. It has been discovered that CTGF is involved in a wide variety of biological activities, although many of them contradict with others. As considering the relationship of structure to biological activities, some pioneers have investigated the structure-function relationship of CTGF. Some data have shown that module 4 may be the most hopeful candidate of major function of CTGF. CTGF is implicated in many chronic kidney diseases. Furthermore, it has been demonstrated that CTGF is involved in many key events of tubulointerstitial fibrosis such as proliferation, EMT, and over-deposition of ECM etc. Nonetheless, the basis for the divergence of these biological responses to CTGF remains unclear. The present work uses different fragments of rhCTGF to directly study the role and mechanism of CTGF involved in EMT and subsequent TIF, at the same time identify the structure basis of these functions.METHODS: The human proximal tubular cell line (HK-2) was grown in DMEM containing 10% heat inactivated FCS. After rested in serum-free medium for 24 hours, different dose of rhCTGF(C) (consisted of module 4) was added into the medium, and then the proliferation index was monitored by MTT assay. And the most appropriate dose-time dot had been selected by this mean. Fluorescence-activated cell sorter(FACS) was applied to investigate the influence of anti-CTGF antibody on rhCTGF(C) induced cell cycle changes. The response of CK(cytokeratin)&VIM(vimentin) mRNA and protein expression to the stimulation of rhCTGF were observed by RQ-PCR and immunochemistry . At the same time, the morphogenic changes were observed by microscope, and FN expression was detected by laser confocal microscope. In sequence , the dose & time response of ILK mRNA and protein expression to the stimulation of rhCTGF(C) were also obsevered by RQ-PCR & immunochemistry. Totally, these effects were compared with rhCTGF(N) (consisted of module1,2,3) and observed in a condition with addition of anti-CTGF antibody.Results: The result showed rhCTGF(C) could dose-dependently induced HK-2 proliferation under the concentration of 50ng/ml, while it could significantly inhibit HK-2 growth in a dose-dependent manner from 100 to 1000 ng/ml. Furthermore, Flow cytometer study showed that rhCTGF promoted the cells to pass G1-S checkpoint, which was significantly reversed by anti-CTGF antibody (P<0.01) . By stimulated HK-2 using 50 ng/ml rhCTGF(C) for different time , the HK-2 cells in a time-dependent manner decreased CK expression and increased VIM expression both in mRNA and protein. HK-2 cells lost their cobbule-stone morphology and apical-basal polarity, gained an elongated shape. Confocal microscope study showed that rhCTGF(C) couldmarkedly increase FN expression in a time-dependent manner. The ILK mRNA and protein expression was responsed to rhCTGF(C) stimulation in a dose and time dependent manner. Totally, cocultured with anti-CTGF could abrogate most of the rhCTGF(C) effects, while rhCTGF(C) showed no significant variability compared with the control group.Conclusions: 1. rhCTGF(C) time and dose dependently induce the proliferation of HK-2 cells in a specific range of concentration, and facilitate HK-2 cell to transfer from G1 phase to S phase. 2. rhCTGF(C) could induce HK-2 EMT, while rhCTGF(N) could not induce HK-2 EMT . 3. rhCTGF(C) could promote HK-2 to synthesis of ECM, while rhCTGF(N) had not the same effect. 4. rhCTGF(C) could directly induce the expression of ILK mRNA and protein in a dose and time dependent manner, while rhCTGF(N) could not increase the expression of ILK.