The Study Of TCF8 RNA Interference On EMT Induced By High Concentration Of Glucose And Angiotensin Ⅱ In Podocyte

In the kidneys,podocytes are attached to the outside of the glomerular basement membrane.Podocytes have long processes that interdigitate and that are separated by very narrow spaces that are bridged by a membrane called the slit diaphragm.This diaphragm constitute the filtration barrier of the kidney.Therefore,podocytes play an important role in protecting the integrity of the glomerular filtration barrier.Ultrastructural changes in podocytes and reduced expression of associated molecules is closely related to renal impairment and can lead to glomerulosclerosis and renal interstitial fibrosis.The epithelial-mesenchymal transition(EMT)is a process by which polarized epithelial cells undergo numerous biochemical changes to obtain a mesenchymal cell phenotype.Podocytes are epithelial cells.so when they are affected by different conditions(such as TGF-β,a high concentration of glucose,doxorubicin,or some other stimuli),EMT may occur.When EMT occurs,epithelial phenotypic markers such as E-cadherin,P-cadherin,nephrin,podocalyxin.and synaptopodin are downregulated,and mesenchymal phenotypic markers such as desmin.FSP-1,MMP-9,ILK,fibronectin,vimentin,a-SMA.collagen I.and snail are upregulated.EMT is considered to be the key factor for an impaired kidney in renal fibrosis,which may lead to renal interstitial fibrosis.Podocytes,which are also known as glomerular visceral epithelial cells,are cells affected by different types of injury.EMT in podocytes will result in damage to the glomerular filtration barrier and proteinuria;in severe cases,it may lead to podocyte detachment from the glomerular basement membrane and apoptosis,causing a decrease in the number of podocytes.thereby aggravating proteinuria and glomerular sclerosis.EMT in podocytes is a key starting point for studying the mechanisms by which proteinuria and renal fibrosis develop.AIM:to examine EMT in podocytes in the presence of a high concentration of glucose and angiotensin II and to examined the effects of TCF8 on EMT in podocytes.METHODS:Differential podocytes were incubited with Ang II or glucose for 24 h,protein expression was detected by Western blotting method.The migration of podocyte was measured with a transwell assay and the invasion by podocytes was measured with a Matrigel assay.TCF8 mRNA expression was measured using qRT-PCR.RESULTS:1.The effects on protein expression of E-cadherin,α-catenin,N-cadherin and vimentin stimulated by Ang Ⅱ and high concentration glucose in podocyteThe expression of E-cadherin and a-catenin were decreased and the expression of N-cadherin and Vimentin were increased.2.The effects on protein expression of TCF8 stimulated by Ang Ⅱ and high concentration glucose in podocyteThe expression of TCF8 was increased in concentration dependent manner.3.The effects on podocyte migration and invasion induced with a high concentration of glucose or angiotensin ⅡThe podocyte migration and invasion were inhenced in the presence of Ang II or glucose.4.Sreening of siRNA for silencing TCF8The result showed that the expression of TCF8 was decreased significantly,especially the#1 siRNA.5.The effects on protein expression of E-cadherin,a-catenin,N-cadherin and vimentin stimulated by Ang II and high concentration glucose in podocyte with silenced TCF8The expression of E-cadherin and a-catenin were decreased and the expression of N-cadherin and Vimentin were increased in podocytes with silenced TCF8 induced with glucose and angiotensin Ⅱ.The results showed that the EMT of podocytes could be inhibited after the silencing of TCF8.TCF8 could promote EMT in podocytes.6.The effects on cell migration and invasion induced with a high concentration of glucose or angiotensin II in podocyte with silenced TCF8The cell migration and invasion were decreased in the presence of Ang Ⅱ or glucose in podocyte with silenced TCF8.CONCLUSION:The results suggest that high concentration glucose and angiotensinⅡ may promote EMT in podocytes via TCF8.