Vascular Endothelial Growth Factor (vegf) In Renal Tubular Epithelial - Mesenchymal Transformation Of The Role And Mechanism Of The Interstitial Cells Of Discussion

Part 1.Effects of vascular endothelial growth factor on TGF-β1 induced epithelial-myofibroblast transiation(EMT) of HK-2 cells and the expression of BMP-7 and IdBackground Epithelial-myofibroblast transiation(EMT) is a major pathway leading to generation of the renal interstitial fibrosis.EMT is regulated by numerous growth factors.Overexpression of TGF-β1 plays an important role in the initiation and progression of EMT,but bone morphogenetic protein-7(BMP-7) may block EMT. Inhibitors of differentiation,Id2 and Id3,expressions of epithelial cells showed long-term repression by TGF-βand sustained induction by BMP-7.Vascular endothelial growth factor(VEGF) is a potential endothelial,angiogenic factor and enhancer of vascular permeability.It plays an important role in the development and maintenance of the early vasculature of the kidney.In this study,we examined the effects of VEGF on TGF-β1 induced EMT and expressions of BMP-7,Id2 and Id3 of HK-2 cells.Methods This study includes three experiments.1.To examine the effects of different concentrations VEGF on TGF-β1 induced EMT of HK-2 cells and expressions of BMP-7 and Id,the cultured HK-2 cells were divided into three groups:a,negative control,b,treated with TGF-β1(5ng/ml) as positive control,c,co-treated with TGF-β1 (5ng/ml) and VEGF₁₆₅(0.1,1,10,100ng/ml) for 48h.α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK-2 cells were assessed with double-stain immunocytochemistry method,α-SMA,BMP-7,Id2,Id3 protein expressions were assessed with Western Blot.2.To examine the effects of VEGF and VEGFR1 antibody on TGF-β1 induced EMT of HK-2 cells and expressions of BMP-7 and Id,the cultured HK-2 cells were divided into six groups:a,negative control,b,treated with TGF-β1(5ng/ml) as positive control,c,treated with VEGF₁₆₅(100ng/ml),d,co-treated with TGF-β1(5ng/ml) and VEGF₁₆₅(100ng/ml),e,treated with VEGFR1 antibody(10ug/ml),f,co-treated with TGF-β1(5ng/ml) and VEGFR1 neutralized antibody(10ug/ml) for 48h.α-SMA and E-cadherin expressions of HK-2 cells were assessed with double-stain immunocytochemistry method,α-SMA,BMP-7,Id2,Id3 mRNA and protein expressions were assessed with Real-time PCR and Western Blot respectively.3.To examine the effect of VEGF on TGF-β1 induced EMT of HK-2 cells and expression of Id2 after neutralizing endogenous BMP-7,The cultured HK-2 cells were divided into eight groups:a,negative control,b,treated with TGF-β1(5ng/ml) as positive control,c,treated with VEGF165(100ng/ml),d,treated with Alk-6/Fc Chimera(2μg/ml) to neutralize endogenous BMP-7,e,co-treated with TGF-β1(5 ng/ml) and VEGF₁₆₅(100 ng/ml),f,co-treated with TGF-β1(5ng/ml) and Alk-6/Fc Chimera(2μg/ml),g,co-treated with VEGF₁₆₅(100ng/ml) and Alk-6/Fc Chimera (2μg/ml),h,co-treated with TGF-β1(5ng/ml),VEGF₁₆₅(100 ng/ml) and Alk-6/Fc Chimera(2μg/ml) for 48h,α-SMA and Id2 protein expressions were assessed with Western blot.Results By double-stain immunocytochemistry method,Real-time PCR and Western Blot analysis,α-SMA expression significantly increased and E-cadherin expression significantly decreased in HK-2 cells treated with TGF-β1(5ng/ml) compared with negative controls(P＜0.05),BMP-7,Id2,Id3 mRNA and protein expressions significantly decreased simultaneouly(P＜0.05).VEGF₁₆₅ dramatically abrogated TGF-β1 inducedα-SMA expression and restored the E-cadherin expression in HK-2 cells in a dose-dependent manner.At the concentrations of 10,100ng/ml,VEGF₁₆₅ almost completely blockedα-SMA mRNA and protein expression induced by TGF-β1(5ng/ml)(P＜0.05),and BMP-7,Id2 mRNA and protein expressions of HK-2 cells were upregulated concomitantly(P＜0.05),but Id3 expression was not interruptted,α-SMA expression increased and E-cadherin expression decreased further in HK-2 cells co-treated with TGF-β1(5ng/ml) and VEGFR1 antibody (10μg/ml) compared with positive controls(P＜0.05),and BMP-7,Id2 mRNA and protein expressions also decreased further(P＜0.05).However,Id3 expression did not change significantly.At last,neutralization of endogenous BMP-7 in HK-2 cells co-treated with TGF-β1(5ng/ml) and VEGF₁₆₅(100ng/ml) raised the expression ofα-SMA,but did not interrupt the expression of Id2.Conclusions The results documented that VEGF₁₆₅ may partially inhibit TGF-β1-induced EMT in HK-2 cells in vitro,and this effect is related to upregulated expressions of BMP-7 and Id2,but not related to Id3.Id2 may be upregulated directly by VEGF₁₆₅,but not related to upregulation of BMP-7. Part 2.Downregulation of Smad2/3 in supression of TGF-β1 induced epithelial-myofibroblast transiation(EMT) of HK-2 cells by vascular endothelial growth factorBackground Vascular endothelial growth factor(VEGF) is an endothelial-specific growth factor that promotes endothelial cell proliferation,differentiation and survival. Smad2 and Smad3 play critical roles in the process of epithelial-myofibroblast transiation(EMT) that TGF-β1 induced.VEGF attenuates TGF-βaction in HUVECs, specifically at the level of Smad2/3 phosphorylation.In this study,we examined the effects of VEGF on TGF-β1 induced EMT and Smad2/3 transduction pathway of HK-2 cells.Methods This study includes two experiments.1.To examine the effects of different concentrations VEGF on TGF-β1 induced EMT of HK-2 cells and the levels of phosphorylation of Smad2/3,the cultured HK-2 cells were divided into three groups: a,negative control,b,treated with TGF-β1(5ng/ml) as positive control,c,co-treated with TGF-β1(5ng/ml) and VEGF₁₆₅(0.1,1,10,100ng/ml) for 48h.α-SMA, p-Smad2/3 and Smad2/3 protein expressions were assessed with Western blot.2.To examine the effects of VEGF and VEGFR1 antibody on TGF-β1 induced EMT of HK-2 cells and levels of phosphorylation of Smad2/3,the cultured HK-2 cells were divided into six groups:a,negative control,b,treated with TGF-β1(5ng/ml) as positive control,c,treated with VEGF₁₆₅(100ng/ml),d,co-treated with TGF-β1 (5ng/ml) and VEGF₁₆₅(100ng/ml),e,treated with VEGFR1 antibody(10ug/ml),f, co-treated with TGF-β1(5ng/ml) and VEGFR1 neutralized antibody(10ug/ml) for 48h.α-SMA mRNA,p-Smad2/3 and Smad2/3 protein expressions were assessed with Real-time PCR and Western Blot respectively.Results By Western Blot analysis,α-SMA expression significantly increased in HK-2 cells treated with TGF-β1(5ng/ml) compared with negative controls(P＜0.05),the ratio of p-Smad2/3 and Smad2/3 significantly increased simultaneouly(P＜0.05). VEGF₁₆₅ dramatically abrogated TGF-β1 inducedα-SMA expression and phosphorylation of Smad2/3 in HK-2 cells in a dose-dependent manner.At the concentrations of 10,100ng/ml,VEGF₁₆₅ almost completely blockedα-SMA protein expression and phosphorylation of Smad2/3 induced by TGF-β1(5ng/ml)(P＜0.05).α-SMA expression and the ratio of p-Smad2/3 and Smad2/3 increased further in HK-2 cells co-treated with TGF-β1(5ng/ml) and VEGFR1 antibody(10μg/ml) compared with positive controls(P＜0.05).Conclusions The results documented that VEGF₁₆₅ may partially inhibit TGF-β1-induced EMT in HK-2 cells in vitro,and this effect is related to suppression of phosphorylation of Smad2/3. Part 3.Effects of Mycophenolate Mofetil(MMF) on Renal Interstitial Fibrosis and Epithelial-Myofibroblast Transiation(EMT) in Adenine-induced CRF RatsObjective Mycophenolate Mofetil(MMF),the 2-4-morpholino ethyl ester of mycophenolic acid(MPA),the biologically active component,was introduced as a novel immunosuppressive agent.MPA is a highly selective,non-competitive,and reversible inhibitor of the enzyme inosine monophosphate dehydrogenase(IMPDH). There is a growing body of evidence pointing to therapeutic applications of MMF other than immunosuppression,in particular the prevention of fibrosis.The aim of this study is to examine the effect of MMF on Epithelial-Myofibroblast Transiation (EMT) in adenine-induced chronic renal failure(CRF) rat model and the role of vascular endothelial growth factor(VEGF) and inhibitor of differentiation(Id2 and Id3) in EMT in the rat kidney.Methods Sixty-four male Wistar rats were randomly assigned to the following groups: normal control(n=16),CRF(n=24) and CRF+MMF(n=24).CRF was induced by gastric gavage of adenine(125mg·kg^-1·d^-1) to rats for eight weeks.CRF rats were treated with MMF(15mg·kg^-1·d^-1) as "CRF+MMF" group.The rats were sacrificed at week 2,4,6 and 8,respectively.Urinary protein and serum creatinine levels were measured,and the histopathologic degrees of interstitial fibrosis were evaluated in Masson-stained sections.Expressions ofα-smooth muscle actin (α-SMA),transforming growth factor-β1(TGF-β1),VEGF and Id(Id2 and Id3) in the kidney tissue were assessed by Immunohistochemistry,RT-PCR and/or Western blot.Results The urinary protein level in CRF+MMF group was evidently lower than that in CRF group(P＜0.01),whereas no statistically significant difference was observed in serum creatinine level between the two groups.Renal interstitial fibrosis was ameliated significantly with MMF treatment(P＜0.01).Expression ofα-SMA in CRF+MMF group was lower than that in CRF rats at week 6,8(P＜0.01),while expression of TGF-β1 was decreased markedly at week 2,4,6(P＜0.01).The expressions of VEGF in CRF+MMF rats were increased significantly at week 6,8 (P＜0.01),and Id2,Id3 in CRF+MMF rats were increased significantly at week 4,6 (P＜0.05).Conclusions MMF may ameliorate chronic renal fibrosis and EMT in adenine induced CRF rats.This effect of MMF on EMT is probably related to upregulation of VEGF,Id2 and Id3 expressions and suppressing overexpression of TGF-β1 in renal tissue.The exact mechanism needs to be studied further.