Role Of Egr-1 In Hypoxia-induced Renal Emt And The Underlying Mechanisms

【Background】Renal fibrosis is a common pathological feature for kinds of chronic kidney disease (CKD). Its progression will result in renal dysfunction and end- stage renal disease (ESRD) . Therefore , it become the prominent problems for academia to understand the mechanism of renal fibrosis and look for clinical therapeutic targets to prevent or even stop its progression early. In recent years, studies have confirmed that chronic hypoxia -induced renal EMT play an important role in renal fibrosis, and Twist, BVR, URG11, CTGF, HIF-1αas well as other molecules are involved in this pathological process. However , treatments targeting these molecules can not effectively inhibit the tubulointerstitial injury and fibrosis in progress. This suggests us that the underlying molecular mechanisms are not yet fully understood. Therefore, its necessary for us to explore new molecules inducing EMT, and clarify its potential mechanisms involved in the pathological process.【Aims】To explore the role of early growth response factor1 in hypoxia-induced renal EMT and underlying molecular mechanisms, and providing new theoretical basis for clinical treatment of renal fibrosis.【Methods】1.The 5/6 subtotal nephrectomy was performed in rats to construct the model of renal fibrosis. Collectthe clinical sample for CKD. Immunohistochemistry was used to detect the expression of Egr-1, Snail, E-cadherin and Fsp-1 in rats and human kidney tissue. 2. HK-2 cells were cultured in hypoxia condition for different times. Then, Immunocytochemical analysis, Quantitative reverse transcription PCR and Western blot assay were employed to detect the expression of Egr-1 under hypoxia. 3. Plasmids for pcDNA3.1-Egr-1 and pcDAN3.1 were transfected into HK-2 cells with Lipofectamine2000. After transfection , observing the morphologic change in transfected cells, detecting the expression of Egr-1, E-cadherin and Fsp-1with Western Blot analysis. Transfecting siEgr-1,psilencer3.1 into HK-2 cells, using Western Blot analysis to detect the expression of Egr-1,E-cadherin and Fsp-1. 4. PCK or ERK inhibitor, calphostinC,PD98059,were used to block the PKC/ERK pathway. Western blot assay were employed to detect the expression of PKC,pERK,ERK,Egr-1 after hypoxia for 1h. 5. HK-2 cells were cultured in hypoxia condition for different times. Quantitative reverse transcription PCR and Western blot assay were employed to detect the expression of Egr-1,Snail,E-cadheirn,Fsp-1 under hypoxia. Transfecting siEgr-1,psilencer3.1 into HK-2 cells, using Western Blot analysis and Quantitative reverse transcription PCR to detect the mRNA and protein level of Egr-1,Snail, E-cadherin and Fsp-1 .【Results】1. Immunohistochemistry staining showed that Egr-1 was highly expressed in the cytoplasm of renal tubules cells and partly in the nucles in 5/6 - nephrectomized rats, and overexpression of Snail was also detected in the cytoplasm of HK-2 cells, whereas there was almost no staining in sham - operated kidneys. Meanwhile ,in renal tubular epithelial cells, the staining of E-cadherin was found decreased , Fsp-1 increased after subtotal nephrectomy. 2.Paraffin-embedded renal tissues from patients with IgAN and normal kidney tissue were examined for Egr-1,E-cadherin,Fsp-1 and Snail. We observed that compared with controls, the expression of Egr-1 increased and mainly located in cytoplasm and nucles of renal tubules cells, the expression of E-cadherin decreased, Fsp-1 increased, and we also found high expression of Snail in the cytoplasm, and no staining was found in normal kidneys. 3. Hypoxia can induce the expression of Egr-1 in HK-2 cells both in mRNA and protein levels. 4. After transfection ,as the overexpression of Egr-1,the expression of E-cadherin decreased, Fsp-1 increased, compared with control. Whereas, when knockout the Egr-1 with siRNA, those EMT phenotype were efficiently reversed. Both parental and empty vector-transfected cells displayed a typical orbicular -ovate epithelial shape, whereas Egr-1-transfected cells were elongated in shape and larger than control cells, just like the morphology of myofibroblasts. These results confirmed that Egr-1 involved in the hypoxia-induced renal EMT. 5. We treated HK-2 cells with PD98059, a specific inhibitor to MAPK/ERK, or calphostin C, a specific PKC inhibitor. After hypoxia for 1hour, the levels of PKC , phosphorylated-ERK and Egr-1 increased. Whereas, when treated with PD98059 or calphostin C followed by 1 hour hypoxic condition, HK-2 cells decreased the expression of Egr-1. This date suggested that in renal epithelial cells, hypoxia induced Egr-1 expression by the same PKC/ERK pathway. 6. When HK-2 cells were cultured in hypoxia condition for different times, as the high expression of Egr-1,the Snail mRNA and protein level were gradually upregulated. We also detected the decreased expression of E-cadherin and the increased expression of Fsp-1. Whereas, when downregulation of Egr-1 with siRNA, levels of Snail were downregulated, and the expression of E-cadheirn increased, Fsp-1 decreased.【Conclusions】Egr-1 is induced under hypoxia through PKC/ERK1/2 pathway, and further promotes tubular EMT via activation of Snail.