【作者】 赵龙；

【导师】 关广聚； 杨天新；

【作者基本信息】 山东大学 ， 内科学（肾病）， 2017， 博士

【摘要】 研究背景与研究目的糖尿病肾病(diabetic nephropathy,DN)在糖尿病患者中的患病率大约为20%～40%,是糖尿病的重要并发症和主要死亡原因。近年来随着糖尿病患者人数的快速增长,DN的发病率逐年上升,在一些国家或地区已经成为终末期肾病(ESRD)的首位原因。DN的发病机制极为复杂,其确切机制尚未明确,多种因素通过各自作用以及相互交叉作用共同导致DN的发生和发展。对DN发病机制更深一步的研究,将对发掘DN有效的预防和治疗方法产生深远而重要的意义。足细胞是肾小球滤过膜极其重要的组成部分,其电荷屏障作用是阻止蛋白质漏出的关键。DN时足细胞发生EMT,足突融合、消失,随后发生阴离子电荷减少、微绒毛形成、体积缩小,足细胞最终从肾小球基底膜(GBM)上分离剥脱至肾小囊中,发生足细胞分离。通常认为足细胞EMT是足细胞损伤的起始环节,可以导致足细胞运动性增强、蛋白尿和肾小球纤维化。在EMT的过程中,足细胞标志物synaptopodin、紧密连接蛋白-1(zonula occludens,ZO-1)、P-cadherin 和nephrin降低,间质细胞标志物胶原蛋白-1、纤连蛋白(fibronectin,FN)、成纤维细胞特异性蛋白-1(Fibroblast specific protein-1,FSP-1)以及 α-平滑肌激动蛋白(α smooth muscle actin,α-SMA)升高。尽管传统观点认为DN是继发性肾小球疾病,现在越来越多的证据表明肾功能损伤的程度与肾小管间质纤维化的程度更加相关,肾小管损伤在DN进展中作用不容忽视。在肾小管上皮细胞(Renal tubular epithelial cells,TECs)EMT的过程中,上皮细胞标志物E-cadherin和ZO-1降低,间质细胞标志物FN、collagen-1、α-SMA、FSP-1 升高。Snail、Zeb 和 helix loop helix(HLH)家族都是引发EMT的重要转录因子,它们能够抑制E-cadherin的表达并促进间质细胞标志物的表达。血清反应因子(serum response factor,SRF)是高度保守的顺式作用因子,属于MADS-box转录因子家族。与SRF结合的DNA位点为血清反应元件(Serum response element,SRE),存在于几乎所有物种的基因组中,控制着细胞骨架和收缩蛋白的表达。SRF促进中胚层细胞迁移,在胚胎体轴延长中发挥重要作用,表明SRF对胚胎发育至关重要。根据目的基因的不同,SRF可以通过两条途径被激活(见图1):Ras-细胞外信号调节激酶(extracellular regulated kinase,ERK)-三重复合物因子(ternary complex factors,TCFs);Rho-肌动蛋白-心肌蛋白相关转录因子(myocardin related transcription factors,MRTFs/MAL)。大量研究表明SRF在上皮肿瘤(如肝细胞癌、胃癌、前列腺癌、胰腺癌、乳腺癌等)的EMT和转移过程中发挥重要作用。在转基因小鼠,过表达的SRF可以导致肌细胞肥大、间质纤维化和肌纤维损伤。相反的,SRF冠状动脉平滑肌细胞敲除可以降低血管平滑肌细胞的迁移和增殖能力。此外,SRF促进转化生长因子-β(transforming growth factor β,TGF-β)介导的肺肌成纤维细胞分化,应用蛋白激酶A降低SRF的表达和活性可以抑制这一作用。近期王汉民等研究表明,SRF在大鼠单侧输尿管结扎模型中可导致肾小管上皮细胞EMT;SRF通过snail信号转导通路加重高糖透析液引起的腹膜间皮细胞EMT。根据以上背景,我们提出如下假说:在高血糖情况下,大鼠肾脏中SRF和磷酸化SRF(pSRF)表达升高,并且从细胞质向细胞核转位增加,通过Snail信号转导通路导致足细胞和肾小管上皮细胞EMT,加重肾脏纤维化和蛋白尿;小分子抑制剂CCG-1423可以直接抑制SRF和pSRF的表达,减轻足细胞和肾小管上皮细胞EMT,改善糖尿病大鼠的肾脏纤维化和蛋白尿。为了验证此假说,我们通过1型糖尿病大鼠模型和体外培养永生化足细胞、近端小管上皮细胞,分别探讨SRF在DN肾小球纤维化和肾小管间质纤维化中的作用及相关机制,从而发掘DN新的治疗靶点。研究方法为了明确SRF在DN肾小球纤维化中的作用及相关机制,体外实验采用小鼠永生化足细胞,CCG-1423(1 uM或2μM)预处理1h后,进行高糖刺激72h;为了探索SRF是否促进足细胞EMT,我们采用SRF上调质粒转染足细胞,并使用Transwell小室迁移分析评估足细胞的迁移能力。体内实验采用一次性腹腔注射STZ(60 mg/kg BW)构建糖尿病大鼠模型,STZ注射后每日腹腔注射CCG-1423(0.01或0.02 mg/kg BW),持续8周。实验结束前对大鼠称重并放入代谢笼收取24h尿,然后抽血、灌流、取肾,随后检测和计算各项指标。采用western blot、实时荧光定量PCR(QPCR)和半定量PCR方法检测小鼠足细胞和大鼠肾皮质SRF、pSRF、synaptopodin、ZO-1、collagen-1、α-SMA 和 FSP-1 等的表达;免疫荧光染色检测足细胞中SRF的表达量和定位;实验室方法检测大鼠血糖、血肌酐、血尿素氮和24h尿蛋白定量;免疫组织化学染色(免疫组化)方法检测大鼠肾小球中SRF、P-cadherin、FN等的表达;PAS染色和masson染色评估大鼠肾小球纤维化的程度。为了明确SRF在DN肾小管间质纤维化中的作用及相关机制,体外实验采用永生化人近端小管上皮细胞(HK-2细胞),CCG-1423(1μM or 2μM)预处理1h后,进行高糖刺激72h;为了探索SRF是否促进HK-2细胞EMT,我们采用SRF上调质粒转染HK-2细胞,并使用Transwell小室迁移分析评估HK-2细胞的迁移能力。体内实验采用一次性腹腔注射STZ(60 mg/kg BW)构建糖尿病大鼠模型,STZ注射后每日腹腔注射CCG-1423(0.01或0.02 mg/kg BW),持续8周。实验结束前对大鼠称重并放入代谢笼收取24h尿,然后抽血、灌流、取肾,随后检测和计算各项指标。采用western blot和QPCR方法检测大鼠肾髓质和HK-2细胞SRF、pSRF、E-cadherin、ZO-1、collagen-1、α-SMA 和 FSP-1 等的表达;免疫荧光染色检测HK-2细胞中SRF的表达量和定位;实验室方法检测大鼠血糖、血肌酐、血尿素氮和24h尿蛋白定量;免疫组化方法检测大鼠肾髓质中SRF、E-cadherin、FN等的表达;PAS染色评估大鼠肾小管间质纤维化的程度。研究结果1.SRF在DN足细胞EMT中的作用及相关机制1.1高糖刺激导致足细胞EMT和SRF表达升高与正常组和甘露醇对照组相比,高糖刺激72h后,足细胞中SRF和pSRF的蛋白表达和基因表达均明显升高,足细胞标志物(synaptopodin、ZO-1)表达明显下降,间质细胞标志物(collagen-1,FN,FSP-1和α-SMA)表达明显升高。免疫荧光染色显示,高糖不仅导致SRF表达升高,而且诱导SRF向足细胞核内转移。1.2在足细胞中过表达SRF,导致EMT和肾小球滤过膜损伤与转染空白质粒(pcDNA3.1)的足细胞相比,转染SRF上调质粒(pcDNA-SRF)的足细胞中 synaptopodin 表达下降,collagen-1、FN、FSP-1、α-SMA 和 Snail表达升高。在Transwell小室迁移分析中,pcDNA-SRF组迁移数量较pcDNA 3.1组明显增加。在白蛋白滤过实验中,pcDNA-SRF组白蛋白滤过量较pcDNA 3.1组明显升高。1.3 CCG-1423对高糖诱导的足细胞EMT的作用与高糖组相比,经CCG-1423预处理的足细胞SRF、pSRF和Snail的蛋白表达和基因表达均降低,synaptopodin表达升高,collagen-1、FN、FSP-1和α-SMA表达降低。免疫荧光染色显示,CCG-1423不仅可以降低SRF的表达,而且还能抑制SRF向足细胞核内转移,从而进一步抑制SRF的作用。1.4糖尿病大鼠肾皮质中SRF的变化随着糖尿病时间的延长,大鼠肾皮质中SRF的蛋白表达和基因表达均逐渐升高。免疫组化表明,随着糖尿病病程的延长,肾小球中SRF的表达较正常组逐渐升高。1.5 CCG-1423对糖尿病大鼠生化指标的影响与正常组相比,DM组血糖、血肌酐、血尿素氮、24h尿蛋白定量和肾重体重比升高,血清自蛋白降低,所有糖尿病大鼠造模成功。与DM组相比,CCG-1423治疗后的糖尿病大鼠血清白蛋白明显升高,但是血糖、血肌酐、血尿素氮和肾重体重比没有变化。1.6 CCG-1423对糖尿病大鼠肾小球纤维化和蛋白尿的影响与DM组相比,CCG-1423治疗后糖尿病大鼠24h尿蛋白定量降低约500%,肾皮质SRF、pSRF、collagen-1、FSP-1表达量明显降低,synaptopodin表达量明显升高。免疫组化显示,与DM组相比,DM+CCG-1423组肾小球α-SMA和FN的表达降低,P-cadherin表达升高。Masson染色和PAS染色均表明,CCG-1423可以使糖尿病大鼠的肾小球纤维化程度明显减轻,肾小球硬化指数降低约500%。2.SRF在DN肾小管上皮细胞EMT中的作用及相关机制2.1高糖刺激导致HK-2细胞EMT和SRF表达升高与正常组和甘露醇对照组相比,高糖刺激72h后,HK-2细胞中SRF和pSRF的蛋白表达和基因表达均明显升高,上皮细胞标志物(E-cadherin和ZO-1)表达明显下降,间质细胞标志物(collagen-1,FN,α-SMA和FSP-1)表达明显升高。免疫荧光染色显示,高糖不仅导致SRF表达升高,而且诱导SRF向HK-2细胞核内转移,从而发挥调节转录的作用。2.2在HK-2细胞中过表达SRF导致EMT与正常组和pcDNA 3.1组相比,转染SRF上调质粒(pcDNA-SRF)的HK-2细胞中 E-cadherin 表达下降,collagen-1、FN、α-SMA、FSP-1 和 Snail 表达升高。在Transwell小室迁移分析中,pcDNA-SRF组迁移细胞数量较正常组和pcDNA 3.1组明显增加。2.3 CCG-1423对高糖诱导的HK-2细胞EMT的作用与高糖组相比,经CCG-1423预处理的HK-2细胞SRF、pSRF和Snail的蛋白表达和基因表达均降低,E-cadherin表达升高,collagen-1、FN、α-SMA和FSP-1表达降低。免疫荧光染色显示,CCG-1423不仅可以降低SRF的表达,而且还能抑制SRF向HK-2细胞核内转移,从而进一步抑制SRF的作用。2.4糖尿病大鼠生化指标的变化与正常组相比,随着糖尿病时间的延长,大鼠血糖、血肌酐、血尿素氮、24h尿蛋白定量和肾重体重比均逐渐升高,血清白蛋白逐渐降低,实验组所有糖尿病大鼠造模成功。2.5糖尿病大鼠肾髓质中SRF的变化随着糖尿病时间的延长,大鼠肾髓质中SRF的蛋白表达和基因表达均逐渐升高。免疫组化表明,随着糖尿病病程的延长,肾小管中SRF的表达较正常组逐渐升高。2.6 CCG-1423对糖尿病大鼠肾小管间质纤维化和白蛋白尿的影响与DM组相比,CCG-1423治疗后糖尿病大鼠肾髓质SRF、pSRF、collagen-1、α-SMA、FSP-1和Snail表达量明显降低,E-cadherin表达量明显升高,24h尿白蛋白定量降低约36.4%。免疫组化显示,与DM组相比,DM+CCG-1423组肾小管FN、FSP-1和Snail表达降低,E-cadherin表达升高。PAS染色表明,CCG-1423可以使糖尿病大鼠的肾小管间质纤维化程度明显减轻。结论1.高血糖激活大鼠肾脏中SRF,使SRF和pSRF表达升高,并且从细胞质向细胞核转移增加,通过Snail信号转导通路导致足细胞和肾小管上皮细胞EMT,导致蛋白尿、肾小球纤维化和肾小管间质纤维化。2.使用CCG-1423抑制SRF,可以使足细胞和肾小管上皮细胞EMT减轻,丢失减少,保护肾小球和肾小管的功能。3.使用CCG-1423抑制SRF,可以减轻DN的肾小球纤维化和肾小管间质纤维化,降低蛋白尿,升高血清白蛋白,延缓DN的进展,有望成为DN的有效治疗方法。更多还原

【Abstract】 Background and ObjectiveThe incidence of diabetic nephropathy(DN)in diabetes mellitus(DM)patients is approximately 20%～40%,and DN is the most severe complication and chief cause of death in DM.With the rapid increase in the number of DM patients,the incidence of DN rises year by year.It has become the first reason leading to end-stage renal disease(ESRD)in some countries.The pathogenesis of DN is so complicated that the exact mechanism is not known yet.Many factors act not only alone but also together to cause and to aggravate DN.Further study of the pathogenesis of DN is needed to seek an efficient treatment of DN.Podocytes play a fundamental role in glomerular filtration and its charge barrier can effectively prevent the leakage of protein.In DN,podocyte EMT leads to the fusion and effacement of foot process.Eventually,podocyte separated from the glomerular basement membrane(GBM)to the renal capsule.Podocyte EMT is considered as the beginning process of podocyte injury,which can enhance the motility of podocytes and lead to proteinuria and glomerular fibrosis.In the process of EMT,epithelial markers(synaptopodin,ZO-1,P-cadherin)are downregulated along with upregulation of mesenchymal markers(collagen-1,fibronectin,FSP-1 and a-SMA).Although DN was traditionally considered as a primarily glomerular disease,it is now widely accepted that the rate of deterioration of renal function correlates best with the degree of renal tubulointerstitial fibrosis.Renal tubular epithelial cells(TECs)epithelial-mesenchymal transition(EMT)is considered as the beginning process of tubulointerstitial fibrosis.In the process of EMT,epithelial markers(E-cadherin,ZO-1)are downregulated along with upregulation of mesenchymal markers(fibronectin,collagen-1,a-SMA,FSP-1).Snail,Zeb and HLH family are important transcriptional repressors of E-cadherin and other about EMT.SRF is a highly conserved DNA binding protein of MADS-box transcription factor family and a main switch for the transcription of cytoskeletal and contractile genes in almost all cells across various species.SRF induces mesoderm cells migration and plays an important role in the extension of embryonic body axis,which suggests that SRF is critical for embryonic development.SRF can be activated through two different ways according to the sensitivity of target genes to the Ras-extracellular regulated kinase(ERK)-ternary complex factors(TCFs)or Rho-actin-myocardin related transcription factors(MRTFs).SRF has been shown to have an essential role in tumor progression,especially in the EMT and metastasis of epithelial tumor cells,such as hepatocellular carcinoma,gastric cancer and prostate cancer.In transgenic mice,high SRF expression can cause myocyte hypertrophy,interstitial fibrosis and muscle fiber damage.And SRF knockout of coronary arterial smooth muscle cells(SMCs)could reduce the migration and proliferation ability of SMCs.In addition,SRF plays a key role in TGF-β induced lung myofibroblast differentiation,and using PKA to reduce SRF expression and activity can inhibit this process.It is reported by Wang HM that SRF provokes EMT of TECs in unilateral uretereal obstruction(UUO)model and accelerates the high glucose-induced EMT in human peritoneal mesothelial cells,suggesting a close connection between upregulation of SRF and DN.However,how SRF regulates EMT of DN remains largely unknown.According to the information above,our hypotheses are:SRF is upregulated and translocates from cytoplasm to nuclei in the kidney of DN rats,which results in EMT of podocytes and TECs,renal fibrosis and proteinuria through Snail signaling pathway;CCG-1423(a small molecule inhibitor of SRF)can relieve EMT of podocytes and TECs and improve renal fibrosis and proteinuria in DN rats.To verify our hypotheses,we used Type 1 diabetic rats model,mouse conditionally immortalized podocyte cell line(mpc5)and human proximal renal tubular epithelial cells(HK-2 cells)to explore the role and mechanism of SRF in glomerular fibrosis and renal tubulointerstitial fibrosis.Furthermore,the therapeutic potential of this signaling was also investigated.MethodsTo evaluate the role and mechanism of SRF in glomerular fibrosis,mpc5 was pretreated with CCG-1423(1μM or 2μM)for 1h followed by high glucose treatment for 72h;To illustrate whether SRF provokes podocyte EMT,we used plasmids containing full-length complementary DNA(cDNA)sequence of mice SRF and transwell chamber migration assay to determine the migration and motility of podocytes.In vivo,rats obtained a single intraperitoneal injection of 60 mg/kg STZ.CCG-1423 was administered by daily intraperitoneal injection at doses of 0.01 and 0.02 mg/kg body weight for 8 weeks since the day after STZ injection.At the end of the study,rats were weighed and housed in metabolic cages to collect 24-h urine.Blood samples of rats were collected followed by systemic perfusion.Kidneys were quickly removed,decapsulated and weighed.Western blot and Quantitative reverse transcription polymerase chain reaction(QPCR)were used to measure the expression of SRF,pSRF,ZO-1,collagen-1,a-SMA and FSP-1 in mpc5 and renal cortex of rats.Immunofluorescence staining was used to determine the expression and localization of SRF in podocytes.Serum glucose,serum creatinine(Scr),blood urea nitrogen(BUN)and 24-h urinary protein(24-h UP)were measured by the Department of Pathology;The expression of SRF,cadherin,fibronectin in glomeruli of rats was detected through immunohistochemical staining.Periodic acid-Schiff(PAS)staining and Masson staining were used to demonstrate glomerular fibrosis.To evaluate the role and mechanism of SRF in renal tubulointerstitial fibrosis,HK-2 cells were pretreated with CCG-1423(1μM or 2μM)for 1h followed by high glucose treatment for 72h;To illustrate whether SRF provokes EMT of HK-2 cells,we used plasmids containing full-length complementary DNA(cDNA)sequence of human SRF and transwell chamber migration assay to determine the migration and motility of HK-2 cells.In vivo,rats obtained a single intraperitoneal injection of 60 mg/kg STZ.CCG-1423 was administered by daily intraperitoneal injection at doses of 0.01 and 0.02 mg/kg body weight for 8 weeks since the day after STZ injection.At the end of the study,rats were weighed and housed in metabolic cages to collect 24-h urine.Blood samples of rats were collected followed by systemic perfusion.Kidneys were quickly removed,decapsulated and weighed.Western blot and QPCR were used to measure the expression of SRF,pSRF,ZO-1,collagen-1 a-SMA and FSP-1 in HK-2 cells and renal medulla of rats.Immunofluorescence staining was used to determine the expression and localization of SRF in HK-2 cells.Serum glucose,serum creatinine(Scr),blood urea nitrogen(BUN)and 24-h urinary protein(24-h UP)were measured by the Department of Pathology;The expression of SRF,cadherin,fibronectin in renal medulla of rats was detected through immunohistochemical staining.PAS staining and Masson staining were used to demonstrate renal tubulointerstitial fibrosis.Result1.The role of SRF in podocyte EMT of DN1.1 High glucose mediated podocyte EMT and SRF upregulation Substantial increase in protein and mRNA level of SRF and pSRF was observed at 72 h after high glucose treatment.High glucose suppressed epithelial synaptopodin and ZO-1 expression,and induced collagen-1,FN,FSP-1 and a-SMA expression as shown by western blot or QPCR analysis.SRF transferred from podocyte cytoplasm to nuclei obviously and the quantity of SRF was also increased after high glucose treatment,according to immunofluorescence staining.1.2 SRF overexpression mediated podocyte EMT and barrier dysfunctionOverexpression of exogenous SRF reduced epithelial synaptopodin expression,and increased collagen-1,FN,FSP-1,α-SMA and Snail expression in podocytes,as shown by western blot and QPCR.Transwell chamber migration assay showed that overexpression of SRF significantly upregulated the migration of podocytes across the pores of transwell filters.Albumin filtration assay showed that albumin easily diffused across the monolayer of podocytes transfected with SRF cDNA,in contrast to that transfected with empty pcDNA 3.1 vectors.1.3 Inhibition of SRF preserved podocyte phenotypes in vitroCCG-1423 selectively suppressed the expression of pSRF and SRF in podocytes after high glucose stimulation for 72 h.Besides,simultaneous treatment of podocytes with CCG-1423 also significantly abolished the reduction of synaptopodin expression and the induction of collagen-1,FN,FSP-1,a-SMA and Snail expression.Immunofluorescence staining suggested that CCG-1423 not only inhibited SRF expression,but also reduced the transfer of SRF from cytoplasm to nucleus in podocytes treated with high glucose.1.4 SRF was upregulated and activated in renal cortex of diabetic ratsWestern blot analysis,quantitative RT-PCR and immunohistochemical staining were carried out using renal cortex tissues from control group or DM group.SRF was significantly upregulated in DM group compared to control group in a time-dependent manner in vivo.1.5 Biochemical analysis of ratsThe DN model was proved by biochemical test results,which showed that serum glucose,scr,BUN,24-h UP and KW/BW were increased in the DM group,while serum albumin was decreased.However,compared to DM group,only serum albumin was improved after CCG-1423 treatment.1.6 Inhibition of SRF ameliorated glomerular fibrosis and proteinuria in vivoCCG-1423 significantly reduced 24-h UP by about 50%,compared with the vehicle control.Besides,inhibition of SRF with CCG-1423 also significantly abrogated the reduction of synaptopodin expression and the induction of SRF,collagen-1.α-SMA,FSP-1 expression in renal cortex tissues,according to western blot analysis.Masson and PAS staining demonstrated that renal fibrosis was present in DM group,and after 8 weeks of treatment with CCG-1423,renal fibrosis and glomerulosclerosis index were dramatically ameliorated compared with vehicle controls.In addition,CCG-1423 significantly preserved glomerular P-cadherin expression and suppressed a-SMA and FN expression in DN rats according to immunohistochemical staining.2.The role of SRF in EMT of TECs in DN2.1 High glucose induced EMT and SRF upregulation in HK-2 cellsSubstantial increase in protein and mRNA level of SRF and pSRF was observed at 72 h after high glucose treatment.High glucose suppressed epithelial E-cadherin and ZO-1 expression,and induced collagen-1,FN,FSP-1 and a-SMA expression as shown by western blot or QPCR analysis.SRF transferred from cytoplasm to nuclei obviously and the quantity of SRF was also increased in HK-2 cells after high glucose treatment,according to immunofluorescence staining.2.2 SRF overexpression mediated EMT and migration of HK-2 cellsOverexpression of exogenous SRF reduced epithelial E-cadherin expression,and increased collagen-1,FN,FSP-1,a-SMA and Snail expression in HK-2 cells,as shown by western blot and QPCR.Transwell chamber migration assay showed that overexpression of SRF significantly upregulated the migration of HK-2 cells across the pores of transwell filters.2.3 Inhibition of SRF preserved phenotypes of HK-2 cells in vitroCCG-1423 selectively suppressed the expression of pSRF and SRF in HK-2 after high glucose stimulation for 72 h.Besides,simultaneous treatment of HK-2 cells with CCG-1423 also significantly abolished the reduction of E-cadherin expression and the induction of collagen-1,FN,FSP-1,a-SMA and Snail expression.Immunofluorescence staining suggested that CCG-1423 not only inhibited SRF expression,but also reduced the transfer of SRF from cytoplasm to nucleus in HK-2 cells treated with high glucose.2.4 Biochemical analysis of ratsThe DN model was proved by biochemical test,which showed that serum glucose,Scr,BUN,24-h UP and KW/BW were increased in the DM group,while serum albumin was decreased.2.5 SRF was upregulated and activated in renal medulla of diabetic ratsWestern blot analysis,quantitative RT-PCR and immunohistochemical staining were carried out using renal medulla tissues from control group or DM group.SRF was significantly upregulated in DM group compared to control group in a time-dependent manner in vivo.2.6 Inhibition of SRF improved renal tubulointerstitial fibrosis and albuminuria in vivoInhibition of SRF with CCG-1423 significantly abrogated the reduction of E-cadherin expression and the induction of SRF,snail,collagen-1,a-SMA and FSP-1 expression in renal medulla tissues,according to western blot analysis.PAS staining demonstrated that renal tubulointerstitial fibrosis was present in DM group,and after 8 weeks treatment with CCG-1423,renal tubulointerstitial fibrosis was dramatically ameliorated compared with vehicle controls.In addition,CCG-1423 significantly preserved renal tubular E-cadherin expression and suppressed Snail,FN and FSP-1 expression in DN rats according to immunohistochemical staining.More importantly,CCG-1423 at 0.02 mg/kg BW dramatically decreased 24-h urinary albumin excretion(UAE)by about 36.4%,compared with the vehicle control.Conclusion1.SRF is activated and upregulated and translocates from cytoplasm to nuclei in the kidney of DN rats,which results in EMT of podocytes and TECs,renal fibrosis and proteinuria through Snail signaling pathway.2.Inhibition of SRF with CCG-1423 can ameliorate EMT and the loss of podocytes and TECs,resulting in improved renal glomerular and tubular function of DN.3.Inhibition of SRF by CCG-1423 can attenuate renal glomerular and tubulointerstitial fibrosis with improved proteinuria and serum albumin,which can delay the progress of DN.That may be an attractive therapeutic strategy for DN.更多还原