Studies Of The Role Of HDAC1in The Developing In AAN

Aristolochic acid nephropathy (AAN) is a renal tubulointerstitial disorder caused by long-term or overdose intake of traditional Chinese herbal medicines containing aristolochic acid (AA).Aristolochic acid I (AAI) is the main and most toxic component of AA; it can induce the transdifferentiation of epithelial cells into myofibroblasts. AAI also causes necrosis/apoptosis of renal tubular epithelial cells.thereby leading to renal interstitial fibrosis.During fibrosis, epithelial cells transdifferentiate into myofibroblasts via epithelial-endothelial mesenchymal transition (EMT/EnMT),a process now believed to be a key factor in the development of renal interstitial fibrosis.The signals for EMT are regulated by various growth factors and cytokines.Among them, transforming growth factor-β (TGF-β) is one of the most potent stimulants of myofibroblast formation by EMT.It is generally believed that TGF-β receptors,Smad proteins (Smads),cofactors,and their target genes form a TGF-β signaling pathway.Smads are divided into three subtypes: receptor-regulated Smads (R-Smads1,2,3,5,8),common mediator Smads (Co-Smad4), and antagonistic Smads (I-Smads6,7).I-Smads are chiefly characterized by their roles in protein degradation and regulation of TGF-β signaling.Acetylated Smad7can become deacetylated by histone deacetylases (HDACs). After deacetylation, Smad7undergoes further ubiquitination and then participates in the degradation of proteins by proteinases. When HDACs are overexpressed and recruited by transcriptional factors, aberrant inhibition of target genes and consequent development of diseases occurs.Therefore,it is important to clarify the actual location of the formation of protein-Smad7complexes in cells,and the locations of Smad7deacetylation/acetylation, final degradation,and trafficking under the assistance of receptors.This previous study successfully developed a mouse AAN model,and investigated the effects of AAI dose on renal interstitial fibrosis in mice.In the present study,we adopted the mouse AAN model and the optimum doses determined in that study to further analyze the effects of duration of treatment on AAN development in mice. We also investigated the expression of factors involved in AAI-induced renal interstitial fibrosis in these mice,and the expression of genes and proteins associated with TGF-β1/Smads signaling pathway.Our objectives were to gain a better understanding of the pathogenic mechanisms of AAI-induced renal interstitial fibrosis in mice and the influence of the TGF-β1/Smads signaling pathway in this process, in an attempt to identify target genes.The role of AA I in AAN and the TGF-β1/Smads signaling pathwayThe aim of this study was to study the changes in the expression of transforming growth factor β1(TGF-β1), Smad7and HDAC1in mice with aristolochic acid nephropathy(AAN),and analyze HADC1and Smad7expression in renal fibrosis occurrence and development of the functions and the relationship between them. CK-18, α-SMA,HDAC1and Smad7protein were localized and detected by immunohistochemistry method.RT-PCR was applied to detect the mRNA levels of TGF-betal, Smad7and HDAC1in kidneys.The TGF-betal concentrations were determined by ELISA.The tissue section technique was used to observe the histopathologic changes, such as kidney damage and renal tubules interstitial fibrosis degree.The interaction between HDAC1and Smad7was studied by coimmunoprecipitation and western blotting.In mice with induced AAN,the mRNA levels for TGF-β1and HDAC1were increased with treatment time.The mRNA levels of TGF-β1were higher than that of their control (normal mice without treatment)(P<0.05).Starting from28day, mRNA of HDAC1became-significantly higher than the level of the control (P<0.01).The levels of Smad7protein and its mRNA were decreased with time,and lower than that of control. HDAC1protein expression was increased with time post treatment.Compared with the control group, HDAC1positive stains were mainly distributed in the epithelial and stromal cell nucleus.Histopathologic changes of kidney showed acute tubular interstitial damage,such as turbidity,swollen,degeneration and fall off of renal tubular. With extension of treatment time,renal tubular damage was aggravated progressively. Immunoprecipitation using Smad7as the bait protein identified HDAC1, and using HDAC1as the bait protein identified Smad7.HDAC1participates in the TGF-β/Smad7signaling pathway by forming a complex with Smad7.During the development of AAN in mice,AAI can cause renal tubular damage,impare renal tubular renewable repair ability,restrain Smad7mRNA expression. The weak expression of Smad7can promote TGF-beta1signal transduction, help the fibre the formation and maintain the proliferation condition.One of the mechanism happened is about HDAC1,which mRNA and protein high expression plays an important role in the development aristolochinc acid nephropathy,may be a new potential target.Determination of related factor change in the TGF-β1/Smads signaling pathway with kidney fibrosisTo investigate the role of AA I on mouse renal tubular epithelial cells (RTECs) in vitro,and whether TGF-β1/Smads signal transduction is involved in AA I induced RTECs-myofibroblast transdifferentiation,and to find out the target gene of AA I.It would provide help to reveal the mechanism of renal interstitial fibrosis induced by AA I.This study aimed to determine changes in the expressions of transforming growth factor β1(TGF-β1),the TGF-β1receptor (T βR-I),Smad7,and histone deacetylase1(HDAC1) on mouse RTECs with aristolochic acid nephropathy (AAN),and analyze the relationship between HDAC1and Smad7. Mouse primary RTECs were cultured and identificated by morphology observation. After RTECs treated with AA I,immunofluorescence staining used to analyze CK-18and α-SMA distribution.Real-time PCR was used to analyze TGF-β1/Smads signal moleculars expression in AAN mice RTECs.The TGF-β1concentrations were determined by ELISA.AA I in culture medium was added to RTECs simultaneously, immunofluorescence staining was used to observe changes of cells morphology. Mouse RTECs could be successfully cultured by mechanical separation and collageanse I digestion.High doses of AA I caused death of RTECs;as time increased,1μg/mL of AA I stimulated RTECs to transdifferentiate into myofibroblast As showed by immunofluorescence staining,CK18exprssion was down-regulated, α-SMA expression was up-regulated in RTECs treated with AA I.AA I stimulated TGF-β1mRNA expression and protein secretion;AA I induced T βR-I and HDAC1mRNA expression,while significantly inhibited Smad7expression in dose-dependence manner. Mouse primary culture is successfully cultured in this study.RTECs are confirmed to be target cells of AA I.TGF-β1/Smads signal transduction is involved in EMT induced by AA I.Smad7is one of the target genes in RTECs of AA IInterference effects of siRNA to HDAC1on the AAN of RTECsTo transfect histone deacetylases1(HDAC1)-small interferencing RNA (siRNA) into mouse renal tubular epithelial cells (RTECs) by liposome transfection and to investigate its inhibitory effect.Analyze the TGF-β1/Smads signal moleculars expressionlt would provide help to reveal the mechanism of renal interstitial fibrosis induced by AA I.It was transient into RTECs by liposome transfection.Fluorescently labeled siRNA (FAM-siRNA)screening transfected proportion,real-time fluorescence quantitative polymerase chain reaction (RealTime-PCR) to determine the optimal conditions and testing the various sites (374,525and822) HDAC1-siRNA inhibition effect,MTT method to detect the influence of the HDAC1-siRNA on suppression RTECs proliferation in different time points,and compare with the effect of trichostatin A(TSA) on RTECs.The protein expression of HDAC1and Smad7was studied by western blotting. Expression of fluorescence and Real-Time PCR results show that30pmol siRNA:1.5mL LipofectamineTM2000for optimal transfection conditions.Real-Time PCR results show that HDAC1-374of the most obvious interference effect.The MTT testing results of HDAC1-374and TSA are consistent:HDAC1-siRNA can effectively restrain RTECs proliferation. The test using liposomes transiently transfected siRNA method, successful inhibition the expression of HDAC1in RTECs.HDACl-siRNA could inhibition RTECs proliferation and induced apoptosis,as the role of TSA. HDAC1-siRNA restrain HDAC1mRNA and protein expression,lifted the AAN Smad7over-deacetylation, and increased Smad7protein expression to antifibrotic significance.HDAC1can be used as a new target of the AAN gene therapy,and HDACi can be used to control the occurrence of the AAN.