Igf-i And Ctgf In Vitro Studies Of Human Renal Tubular Epithelial Cell Differentiation And Extracellular Matrix Synthesis Effects

Various kinds of renal diseases progressing to the end stage of renalfailure strongly result from the renal tubulointerstitial fibrosis(RIF). Expertsalso pay attention to the epithelial-myofibroblast transdifferentiation(EMT)as an important pathogenesis of RIF. The growth factors regulate the cells'proliferation, differention and function intensively as the endogenous signalprotein.When they bind with the membrane receptors, many signaltransduction process can be started. Then they regulate the gene expressionand the protein synthesis. Recent years, people have thought highly of theeffects of the growth factor in the RIF.Growth factors include transforminggrowth factor-β(TGF-β), insulin-like growth factor(IGF), connective tissuegrowth factor(CTGF), epidermal growth factor(EGF), vascular endothelialgrowth factor(VEGF), fibroblast growth factor(FGF), platelet-derivedgrowth factor(PDGF) and so on. Our past studies have indicated that CTGFmay play a important role in the EMT and extracellular matrix(ECM)synthesis.The part will be continued in the following.The present studieshave confirmed the positive interaction between CTGF and IGF-â… in theRIF.But we know a little about the mechanism. Maybe they have thepositive interaction in the EMT.To address the possibility, we examine theinterrelation ship of IGF-â… and EMT as a foundation research for the studyof the interaction between CTGF and IGF-â… in the EMT.The present studyconsists of two parts.Partâ… : Effects of exogenous growth factorâ… on thetransdifferentiation and extracellular matrix synthesis of the human renal tubular epithelial cells.Objective To investigate the role of insulin:like growth factorâ… (IGF-â… )in transdifferentiation and extracellular matrix (ECM) synthesis of humanrenal tubular epithelial cell line(HK₂) in vitro.Methods Cultured HK₂ were divided into two groups: (1) control cells;(2) treated with IGF-â… (25,50,100,200 ng/ml,respectively).The cellmorphological changes were traced with inverted microscope, and theexpression of E-cadherin,Keratin,α-smooth muscle actin (α-SMA),Vimentin, fibronectin (FN), collagenâ… and collagenâ…¢mRNAs weredetected by RT-PCR. Concentration of collagenâ… secreted into the culturesupernatant was determined by ELISA.Results: When IGF-â… stimulated HK2 cells, the oval-to-fusiformtransdifferentiation in morphology was found.At the same time,itdownregulated E-cadherin and Keratin mRNA expression (P＜0.05).Vimentin,α-SMA and FN mRNAs expression were upregulated (P＜0.01).Different concentrations of IGF-â… upregulated collagenâ… and collagenâ…¢mRNA expression (P＜0.05). Secreted collagenâ… level was also up-regulateby IGF-â… in the concentration of 100 and 200 ng/mL recpectively.Conclusions(1) IGF-â… can promote the transdifferentiation of human renal tubularepithelial cells. (2) IGF-â… can stimulate the synthesis of extracellular matrix.Partâ…¡: Effects of exogenous connective tissue growth factor on thetransdifferentiation and extracellular matrix synthesis of the humanrenal tubular epithelial cells.Objective To investigate the role of exogenous connective tissue growthfactor(CTGF) in transdifferentiation and extracellular matrix (ECM)synthesis of human renal tubular epithelial cell line HK₂ in vitro.Methods Cultured HK₂ cells were divided into three groups: (1) controlgroup;(2)low dose CTGF-treated group (2.5ng/ml);(3)high doseCTGF-treated group(20ng/ml).The cell morphological changes were tracedwith inverted microscope, and MTT assay was used to detect the CTGFeffects on cells proliferation. The expression of E-cadherin,α-smooth muscleactin (α-SMA),Fibronectin(FN) and collagenâ… amRNA was detected byRT-PCR. Immunohistochemistry stain was used to assess the levels ofintracellular FN and collagenâ… protein.Results In HK₂ cells, CTGF stimulated the morphlolgical oval-to-fusiformtransdifferentiation of the cells, increased the cell proliferativeactivity. Different concentrations of CTGF downregulated E-cadherin mRNAexpression (P＜0.05), upregulatedα-SMA and FN mRNAs expression (P＜O.05). CollagenⅠαmRNA expression was upregulated by highconcentration of CTGF (P＜0.05).The protein expression of FN wasincreased with the low concentration of CTGF (P＜0.05). High concentrationof CTGF increased the protein expression of FN and collagenâ… (P＜0.05).Conclusions(1) CTGF can promote the transdifferentiation of human renal tubularepithelial cells.(2) CTGF can stimulate the synthesis of FN in vitro. High concentration ofCTGF increases the expression of collagenâ… protein.