Nestin, A Intermediate Filament Protein, Is Expressed In The Course Of Epithelial-myofibroblast Transition

PartⅠNestin,a intermediate filament protein,is expressed in the course of epithelial-myofibroblast transition of unilateral ureteral obstruction rats.BackgroundTubulointerstitial fibrosis is a common final pathway leading to end-stage renal disease,irrespective of the nature of the initial renal injury.Tubular epithelial-myofibroblast transition(EMT) is an important event during progression of renal tubulointerstitial fibrosis.However,the mechanism of EMT is not very clear. Nestin is a classⅥintermediate filament and may play a role in connecting the three components of the cytoskeleton and coordinate changes in cell dynamics.We have reported that Nestin was transiently expressed in immature proximal tubular epithelial cells of newborn kidney and disappeared after birth.Dedifferentiation of the tubular epithelial cell and enhanced cell migration and invasion are key events of EMT.So, we hypothesis that Nestin will be re-expressed in the course of EMT in unilateral ureteral obstruction rats.MethodsUnilateral ureteral obstruction(UUO) was performed in male Sprague-Dawley rats to establish a classical tubulointerstitial animal model.Sham-operated rats had their ureters exposed,manipulated but not ligated.Rats were sacrificed at different time points after surgey and the kidneys were removed.Morphological analyses were performed with Masson staining.The expression of TGFβ1,epithelial marker E-cadherin,myofibroblastic markerα-smooth muscle actin(αSMA) and Nestin were demonstrated by immunostaining and Western blot analyses.Messenger RNA expressions of Nestin was detected by real-time PCR.ResultsAfter UUO,the extent of interstitial fibrosis increased with time course. Immunohistochemical localization studies revealed that the expression of TGFβ1 was specifically induced in renal tubular epithelia and interstitial areas of obstructed kidneys.Western blot demonstrated a time-dependent alteration in the expression of E-cadherin andαSMA with complete loss of E-cadherin expression 14 days after UUO and de novo expression ofαSMA 3 days after UUO.Immunohistochemical localization studies also revealed that the expression of Nestin was induced in interstitial areas and tubular epithelia.Co-labeling showed Nestin was expressed in some E-cadherin negative epithelial cells and was co-localized withαSMA.The messenger RNA and protein levels of Nestin were dramatically increased in a time-dependent manner(P＜0.05).The expression of Nestin were positively correlated to the extent of interstitial fibrosis(r=0.816,P＜0.05) and the expression ofαSMA (r=0.97,P＜0.05) and negatively correlated to the expression of E-cadherin(r=-0.816, P＜0.05 ).Conclusion1.Tubular epithelial-myofibroblast transition(EMT) presented in the diseased kidneys in an animal model of renal interstitial fibrosis.2.Nestin,a marker of immature proximal tubular epithelial cell was re-expressed in the course of EMT in unilateral ureteral obstruction rats.3.The expression of Nestin significantly correlated to the extent of interstitial fibrosis and the change of cell phenotype.. PartⅡNestin is re-expressed in the course of epithelial-myofibroblast transition of cultured human proximal tubular epithelial cells.BackgroundAccording to their higher metabolic demands,proximal tubular cells(PTCs) are more susceptible to various injuries such as hypoxic stimuli,proteinuria, immunoinflammations,and so on.Unusual responses of PTCs may change their phenotypes by a progression of epithelial-myofibroblast transition(EMT).As a result, the transformed cells acquire the capacity of migration and invation.As a special class of intermediate filament,Nestin has been shown to regulate the organization of the three kinds of cytoskeletons.We have reported that Nestin was transiently expressed in immature proximal tubular epithelial cells of newborn kidney and had some effects on tubular epitheli's differentiation.In the first part,we also found that Nestin was re-expressed in the course of EMT in UUO tubulointerstitial fibrosis rats.This study, therefore,is conducted to further confirm the Nestin expression in the course of EMT in vitro with human proximal epithelial cells(HKC).We will focus on the relationship between the Nestin expression and the change of cell phenotype.MethodsHKC were cultured in DMEM/F12 medium supplemented with 10%fetal bovine serum.The recombinant TGFβ1 was added at a final concentration of 5 ng/ml.The cells were collected at different time points for further characterization.The expression of E-cadherin,αSMA and Nestin were demonstrated by immunostaining and Western blot analyses.Messenger RNA expressions of Nestin was detected by real-time PCR.Cell migration was evaluated using Boyden chamber motogenicity assay with tissue culture-treated transwell filters.ResultsTGFβ1-treated HKC lost the typical cobblestone pattern of an epithelial monolayer and displayed a spindle-shape,fibroblast-like morphology.In addition,we investigated the time course of loss of E-cadherin expression,which was completely disappeared after 48 hours of TGFβ1 incubation.After 36 hours of incubation,the de novo expression ofαSMA happened.Incubation of HKC with TGFβ1 for 72 hours began to induce cell migration across the pores of the transwell filters.The migrated cells was 15.37 folds of control HKC cells(P＜0.05).At the same time,the expression of Nestin took place about 24 hours after TGFβ1 treatment and undergone with time course(P＜0.05).The expression of Nestin was earlier than the disappearance of E-cadherin and the expression ofαSMA.Conclusion1.HKC incubated with TGFβ1 undergone epithelial-myofibroblast transition with changed cell phenotype and enhanced cell migration.2.Nestin was re-expressed in the course of HKC EMT.The expression of Nestin was earlier than the disappear of E-cadherin and the expression ofαSMA. PartⅢNestin enhances the migration capacity of transformed human proximal tubular epithelial cells.BackgroundBecause tubular epithelial cells and myofibroblasts are separated by TBM in vivo,it is essential for the transformed cells to acquire the motile and invasive capacity to eventually migrate into interstitium.The reorganization of cytoskeleton may provide a structure foundation not only for defining the morphology of the transformed cells, and even acquire the above capacity.Nestin plays a role in connecting the three components of the cytoskeleton and coordinatse changes in cell dynamics.This study, therefore,is conduced to investigate whether Nestin modulate the migration capacity of transformed HKC cells use the technique of RNA interference.MethodsWe constructed a plasmid expressing Nestin short hairpin RNA mediated by pRNAT-U6.1 plasmid vector and transferred it into HKC cells with Lipofectin.And the incubated HKC cells with TGFβ1.The efficiency of transfection was determined by Nestin western blot.The expression of cell phenotype and the capacity of cell migration were ievaluated by western blot and Boyden chamber motogenicity assay with tissue culture-treated transwell filters,respectively.ResultsAmong the 3 shRNA-Nestin plasmid,the third one named 14-32 can inhibit the Nestin protein expression by 35%(P＜0.05 vs control plasmid).Neither E-cadherin norαSMA was affected by the downregulation of Nestin expression.While,Nestin RNAi could significantly reduced the number of migrated cells compared with non-transfected cells(128.40±12.4 vs 193.73±13.51 cells/field,P＜0.05).ConclusionNestin enhances the migration capacity of transformed human proximal tubular epithelial cells in the course of epithelial-myofibroblast transition.