The Role Of Focal Adhesion Kinase On Renal Interstitial Fibrosis

Objective To investigate the expression of Focal adhesion kinase (FAK) in epithelial-mesenchymal transition (EMT) of TGF-β1-stimulated HK-2 cells and the effect of FAK knockdown by small interfering on EMT.Methods HK-2 cells were grown in DMEM/F12 medium supplemented by 10% fetal bovine serum (FBS). HK-2 cells were cultrued in free serum medium for 24h, then were stimulated by TGF-β1 (10μg/L). The expression of E-cadherin,α-SMA, FAK mRNA and protein were detected by RT-PCR, western blot and immunofluorescence, respectively. The expression level of phosphorylated FAK-Tyr397 was detected by western blot. HK-2 cells were transfected with 200nM of FAK-siRNA or negative control-siRNA using Lipofectamine 2000. Then the expression of E-cadherin,α-SMA, FAK protein were detected by western blot.Results The expression of E-cadherin mRNA and protein were markedly decreased in HK-2 cells induced by TGF-β1, and The expression ofα-SMA mRNA and protein were dramatically increased. western blot analysis demonstrated that then protein levels of FAK and p-FAK (Tyr397) were progressively increased in a time-dependent manner in response to TGF-β1 treatment in HK-2 cells. After siRNA transfection, FAK mRNA expression was inhibited by 61%. Fifty-two percent of reduction in FAK protein expression was shown after 48h stimulation. Knockdowned the expression of FAK led to a severe blockage of TGF-β1 induced E-cadherin suppression andα-SMA induction.Conclusions The expression of FAK were up-regulated in HK-2 cells stimulated by TGF-β1. But the EMT induced by TGF-β1 in HK-2 cells was inhibited by FAK knockdown, which suggested that FAK play an important role in TGF-β1-induced tubular EMT and renal fibrosis. Objective Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which plays a key role in many cellular processes such as cell adhesion, migration, proliferation, and cell survival. However, the role of FAK in renal tubular EMT is still unknown. In the present study, we investigated the potential role of FAK in TGF-β1-induced renal tubular EMT.Methods FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-β1). The expression of FAK, E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV(COL-IV) were detected by western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay.Results The expression of FAK was up-regulated in HK-2 cells when incubated with TGF-β1(10μg/L), which was accompanied by reduced expression of E-cadherin. All these changes were restored by knocking-down of FAK with small interfering RNA (siRNA). Akt phosphorylation was induced by the treatment with TGF-β1, which was also attenuated by knockdown of FAK using siRNA. TGF-β1-induced decline of E-cadherin was also recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-β1 induced an increase in MMP-9 expression, and a decreased expression of TIMP-1 and collagen IV were all restored by the FAK siRNA transfection. Furthermore, FAK-siRNA significantly reduced TGF-β1-induced cells migration as demonstrated by using a transwell assay system.Conclusion FAK may play a crucial role in mediating TGF-β1-induced EMT through the activation of Akt pathway. Objective To explore the effects of Focal Adhesion Kinase (FAK) on renal tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in rats.Methods 16 Sprague-Dawley (SD) rats were randomly divided into sham-operated group and UUO group and established of the animal model. Serum potassium, sodium and creatinine were evaluated on the 14th day after UUO. Masson Trichrome staining, quantitative measured of tubulointerstitial fibrosis degree, immunohistochemical studies and western blot detected the expression of FAK,α-SMA and E-cadherin respectively.Results Compared to sham group, serum potassium and sodium were analogous (P>0.05), but the creatinine in UUO group was significantly higher (P<0.05). The degree of renal tubulointerstitial fibrosis, interstitial expression of FAK andα-SMA increased significantly in UUO group (P<0.05), and the expression of E-cadherin decreased (P< 0.05).Conclusion FAK participated in the development of renal tubulointerstitial fibrosis.