血管紧张素1-7对糖尿病大鼠肾小管间质纤维化的影响

目的 研究血管紧张素1-7(Ang1-7)对糖尿病大鼠肾小管间质纤维化的影响及其可能机制.方法 32只雄性Wistar大鼠被随机分为4组:健康对照组(NC组)、模型组(DM组)、替米沙坦组(TM组)、治疗组(T组).建模成功后第9周末检测各组大鼠24 h尿蛋白量、尿NAG/Cr、血糖、血胰岛素、三酰甘油(TG)、总胆固醇(TC)、BUN、Scr、血K+及血Na+ ;PAS染色观察肾脏病理改变 ;实时定量PCR法检测各组大鼠肾脏组织中转化生长因子β1(TGF-β1)、过氧化物酶体增殖物激活受体(PPAR)γ、α平滑肌肌动蛋白(α-SMA)mRNA水平 ;Western印迹法检测PPARγ、α-SMA、TGF-β1蛋白表达.结果 (1)第9周末,DM组大鼠血压、尿蛋白量、肾质量/体质量较NC组显著升高(P＜0.05),TM组及T组较DM组显著降低(P＜0.05),且T组变化更明显.(2)DM组第9周末肾间质损伤指数显著高于NC组(P＜0.05),TM组及T组则低于DM组(P＜0.05).(3)实时定量PCR结果显示,DM组TGF-β1、α-SMAmRNA水平显著升高(P＜0.05),PPARγ mRNA水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA mRNA水平均显著下降(P＜0.05),PPARγ mRNA水平显著上升(P＜0.05),且T组变化更明显.(4)Western印迹结果显示,DM组TGF-β1、α-SMA蛋白水平显著升高(P＜0.05),PPARγ蛋白水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA蛋白水平均显著下降(P＜0.05),PPARγ蛋白水平显著上升(P＜0.05),且T组变化更明显.结论 Ang1-7在体内可通过上调PPARγ表达,抑制α-SMA表达,对糖尿病大鼠肾小管间质纤维化可能具有抑制作用.     

肾衰饮对肾纤维化大鼠TGF-β_1/Smad信号通路的作用研究

目的:探讨肾衰饮抗肾纤维化分子生物学机制。方法:采用腺嘌呤肾病模型,随机将60只SD大鼠分为空白组、模型组、肾衰饮组与尿毒清组,采用RT-PCR观察肾衰饮对CRF大鼠TGF-β1mRNA表达的影响;ELISA法检测Smad2、Smad3、Smad7蛋白表达;免疫组化法检测α-SMA蛋白表达。结果:模型组与正常组比较TGF-β1mRNA表达增强,Smad2、Smad3水平升高,Smad7下降,治疗后肾衰饮组TGF-β1mRNA表达明显减少,Smad2、Smad3水平下降,Smad7升高,同时肾组织α-SMA表达下降,明显优于尿毒清(P0.05)。结论:肾衰饮抑制TGF-β1mRNA表达,提高Smad7水平,通过调节TGF-β1/Smad信号通路,以抑制肾纤维化的发生、发展。     

地黄提取物对Smads信号通路影响的实验研究

目的：探讨中药地黄对大鼠肾纤维化的作用及可能的机制。方法：将实验动物分成正常对照组、模型组、地黄组,采用一次性尾静脉注射阿霉素复制大鼠肾病模型。治疗8周后观察尿蛋白、尿素氮、血肌酐及肾脏病理改变;同时观测肾皮质中转化生长因子β受体Ⅰ（TβRⅠ）、转化生长因子β受体Ⅱ（TβRⅡ）和Smad2、7的表达。结果：地黄组各项指标与模型组相比,均有统计学差异,形态学观察也显示其组损害轻于模型组。且模型组肾组织TβRⅠ、Smad2 mRNA显著上调,地黄组肾组织TβRⅠ、Smad2 mRNA显著下调,而模型组肾组织Smad7蛋白表达显著下调。同时TβRⅡ mRNA的表达无明显变化。结论：在阿霉素肾病大鼠模型中,地黄提取物对大鼠肾脏有一定的保护作用,其信号转导机制至少部分与它能下调TβRⅠ、Smad2及上调Smad7的表达有关。     

依那普利对单侧尿路梗阻大鼠肾组织Smad 2/3活性的影响

目的:研究血管紧张素转换酶抑制剂(ACEI)依那普利对单侧尿路梗阻大鼠肾组织Smad2/3信号蛋白活性的影响.方法:采用单侧输尿管结扎(UUO)模型,治疗组从造模前24 h至造模后28 d以依那普利10 mg·kg-1·d-1灌胃,另设假手术组作正常对照.分别于造模后1,3,7,14,21和28 d取肾组织,应用免疫组化法检测肾组织TGF-β1、磷酸化Smad2/3和α-SMA表达.结果:正常大鼠肾组织具有基础的TGF-β1(4.32±1.72)%和磷酸化Smad 2/3(19.31±5.37)个/mm2的表达,α-SMA只表达于血管平滑肌,肾小管-间质无表达.UUO术后1 d,TGF-β1的表达无明显增加(5.15±2.08)%,第3 d明显增加(13.55±6.33)%,第7 d达高峰(26.78±8.77)%,此后表达减少;UUO术后3 d,磷酸化Smad2/3的表达明显增加(67.95±13.87)个/mm2,并持续增加到第7 d(150.61±27.34)个/mm2,此后表达减少;而UUO术后3 d,肾组织α-SMA表达亦明显升高(5.58±1.23)%,第7 d达到高峰(13.43±3.32)%,14 d表达开始下调.依那普利治疗可以明显抑制肾组织TGF-β1信号蛋白Smad2/3活性(降低39%～55%,P＜0.01),显著下调肾组织α-SMA的表达(降低44%～58%,P＜0.01),减轻肾间质纤维化.结论:依那普利可显著抑制梗阻肾肾组织α-SMA的表达,减轻梗阻肾间质纤维化,这一作用可能与其抑制肾组织TGF-β1信号蛋白Smad2/3的活性有关.     

高糖通过转化生长因子β信号诱导肾小球内皮细胞向肌成纤维细胞转分化

目的 探讨高糖(HG)能否通过转化生长因子β(TGF-β)途径诱导大鼠肾小球内皮细胞向肌成纤维细胞转分化(EndMT).方法 体外培养大鼠肾小球内皮细胞(GEnC),分为正常对照组(NG,5.5 mmol/L)、高糖组(HG,15、30 mmol/L)、TGF-β抑制剂组(HG+LY36,30 mmol/L葡萄糖+10 μmol/L LY364947)以及高渗对照组(M,5.5 mmol/L葡萄糖+25.5 mmol/L甘露醇)和溶剂对照组(D,5.5 mmol/L葡萄糖+1 ml/LDMSO).采用Western印迹法检测各组细胞内皮细胞标志物claudin5和肌成纤维细胞标志物α-SMA表达变化;实时定量PCR法检测细胞TGF-β1和TGF-β2 mRNA表达改变;免疫荧光法观察细胞形态学变化以及血管内皮细胞标志物VE-cadherin和肌成纤维细胞标志物α-SMA的表达.结果 与NG组比较,HG组claudin5蛋白的表达量随葡萄糖浓度增加而降低(P＜0.05),α-SMA蛋白表达量随葡萄糖浓度增加而升高(P＜0.05),TGF-β1和TGF-β2 mRNA表达均升高(P＜0.05).与HG组比较,TGF-β抑制剂组claudin5蛋白表达量升高(P＜0.05),α-SMA蛋白表达降低(P＜0.05).高渗对照组和溶剂对照组改变差异无统计学意义.激光共聚焦免疫荧光结果显示,高糖处理可引起细胞形态由卵圆形向梭形改变,VE-cadherin表达减少,α-SMA表达增加;TGF-β抑制剂组细胞形态无明显改变.与HG组比较,TGF-β抑制剂组VE-cadherin表达增加,α-SMA表达降低(P＜0.05).结论 高糖诱导大鼠肾小球内皮细胞TGF-β表达增加及内皮细胞-肌成纤维细胞转分化.抑制TGF-β可抑制高糖引起的转分化,提示TGF-β参与了高糖引起的肾小球内皮细胞转分化过程.     

他克莫司和环孢素A对大鼠肾脏转化生长因子及其受体表达影响的比较

目的比较他克莫司（FK506）和环孢素A（CsA）所致慢性肾毒性大鼠模型肾组织转化生长因子目（TGF-1β）及其受体（TβRⅡ）的表达。方法分别以FK506和CsA灌胃复制大鼠FK506和CsA慢性肾毒性模型，观察大鼠的一般情况，计算肌酐清除率，观察大鼠肾组织病理变化，以免疫组织化学法检测肾组织中TGF-1β、TβR Ⅰ、TBRⅡ蛋白表达的变化，原位杂交法检测肾组织中TβR Ⅰ mRNA及TβR Ⅱ mRNA表达的变化。结果CsA组和FK506组的肾小管、小管间质和人球小动脉均有损伤，但CsA组的损伤明显较FK506组重（P〈0．05）。正常对照组大鼠肾组织中仅见少量TGF-[3，、TβR I和T13RⅡ表达，CsA组和FK506组的TGF-β，、TβR I和TβR Ⅱ表达均明显增加，但FK506组稍轻；正常对照组大鼠肾组织中仅见少量TβR I mRNA、TβR Ⅱ mRNA表达，CsA组和FK506组TβR I mRNA、TβR Ⅱ mRNA明显表达，但FK506组较CsA组为轻。结论FK506的慢性肾毒性弱于CsA，它所诱导的大鼠肾组织中TGF-β，及其受体TβR I和TβR Ⅱ的表达均低于CsA。     

己酮可可碱对肾小管上皮细胞转分化抑制作用的实验研究

目的:探讨己酮可可碱(PTX)对单侧输尿管梗阻(UUO)模型大鼠肾间质平滑肌肌动蛋白(α-SMA)表达的影响,并在体外研究其对肾小管上皮细胞转分化及其对Smad 7表达的作用.方法:将实验大鼠分为假手术组、UUO对照组和UUO PTX治疗组.用免疫组化法测定各组动物肾组织α-SMA表达.体外培养的人肾小管上皮细胞(HK-2),经TGF-β1及PTX干预后,用蛋白印迹检测α-SMA和Smad 7表达,用ELISA检测培养上清中Ⅰ型胶原含量.结果:假手术组大鼠肾组织仅血管壁α-SMA免疫染色阳性.UUO对照组第3 d、第7 d、第14 d,α-SMA表达程度分别为(2.90±0.71)%、(8.50±0.97)%、(17.9±2.70)%,PTX治疗组则分别为(2.70±0.62)%、(6.90±0.83)%、(13.8±1.9)%,术后第7 d(P<0.05)、第14 d(P<0.01)两组有统计学差异.TGF-β1(8 ng/ml)刺激HK-2细胞72 h后,α-SMA表达强度为(87.7±7.0)%,培养上清中Ⅰ型胶原浓度为(2 560.49±95.03) ng/ml;而加入PTX 10、50、100、300、500 μg/ml干预72 h后,α-SMA表达强度(F＝174.998,P<0.001)和培养上清中的Ⅰ型胶原(F＝460.883,P<0.001)呈浓度依赖性减少.在TGF-β1刺激前24 h、TGF-β1刺激后0 h、12 h、24 h、36 h或48 h后分别加入PTX(500 μg/ml),α-SMA表达强度(F＝144.131,P<0.001)和培养上清中的Ⅰ型胶原(F＝444.557,P<0.001)则呈时间依赖性减少.TGF-β1刺激的同时,分别加入PTX 10、50、100、300、500 μg/ml干预72 h后,Smad 7表达强度分别为(50.6±3.1)%、(49.4±2.9)%、(48.9±2.5)%、(51.4±1.8)%、(49.5±3.0)%,与阳性对照组(46.9±3.5)%比较无统计学差异(P＞0.05).结论:(1)PTX可减少UUO大鼠肾间质α-SMA表达;(2)在体外,PTX可抑制TGF-β1诱导的肾小管上皮细胞转分化,该作用与Smad 7表达无关,其机制有待于进一步研究.     

γ-干扰素对梗阻性积水肾间质纤维化的治疗作用

目的 探讨γ干扰素(IFN-γ)对梗阻性肾积水肾间质纤维化的抑制作用及其可能的机制.方法 将65只雄性SD大鼠随机分成4组:治疗组(20只)、模型组(20只)、药物对照组(20只)、假手术组(5只).在建模和给药后的第3、7、14、21、28天,每组各随机处死动物4只(假手术组1只).采用苏木素-伊红(HE)和Masson染色观察病变肾脏间质纤维化的情况;采用聚合酶链反应(RT-PCR)技术检测肾组织中转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(Col-Ⅰ)的mRNA表达情况;免疫组织化学法观察以上三种蛋白的变化.结果 模型组大鼠术后肾间质逐渐出现纤维化改变,并随梗阻时间的延长逐渐加重,TGF-β1、α-SMA和Col-Ⅰ的mRNA和蛋白表达亦逐渐升高.第14天,模型组TGF-β1和α-SMA的表达量达到高峰,28 d时各指标的表达均达到最高,TGF-β1、α-SMA和Col-Ⅰ分别为51.84%、72.59%和68.73%.治疗组各指标在不同时间点均低于模型组,第28天时,三项指标依次为33.84%、32.59%和48.73%,与模型组比较P＜0.05.Banff评分显示治疗组间质纤维化程度明显减轻(P＜0.01).其余两组未见肾间质纤维化改变.结论 IFN-γ具有减轻积水后肾间质纤维化程度的作用,该作用与其下调TGF-β1的表达、抑制肌成纤维细胞(MyoF)激活和减少Col-Ⅰ生成有关.     

转化生长因子-β转导通路在良性胆管狭窄形成中的作用

目的 观察转化生长因子-β1(TGF-β1)/Smad/结缔组织生长因子(CTGF)通路中各因子在良性狭窄胆管组织中的表达,探讨其信号转导通路在良性胆管狭窄形成机制中的作用.方法 采用免疫组织化学SABC法和原位杂交法检测TGF-β1,TGF-β受体I(TβR Ⅰ),TGF-β受体Ⅱ(TβRⅡ),Smad4,Smad7,CTGF蛋白以及CTGF mRNA在23例良性胆管狭窄组织及6例正常胆管组织中的表达,分别计算阳性表达率,并分析TGF-β1,Smad4及CTGF之间的相关性.结果 TGF-β1、TβR Ⅰ、TβRⅡ、Smad4、CTGF蛋白和CTGFmRNA在23例胆管良性狭窄患者中的阳性表达率分别为91.3%、82.6%、87.O%、78.3%、82.6%、65.2%,高于其在6例正常胆管中的表达且差异有统计学意义(P＜0.05),Smad7在胆管良性狭窄患者中的阳性表达为70.0%,与正常胆管比较升高但差异无统计学意义(P＞0.05).TGF-β1、Smad4及CTGF在良性狭窄胆管的阳性表达呈正相关.结论 TGF-β1/Smad/CTGF信号转导通路高表达是导致良性胆管狭窄形成的重要因素.     

缺血后处理减轻肾缺血再灌注损伤后肾小管间质的纤维化

目的 观察缺血后处理可以减轻大鼠肾缺血再灌注损伤(IPI)后肾小管间质纤维化的作用及其机制.方法 建立原位大鼠单侧肾缺血再灌注动物模型,摘除右肾后对左肾行缺血后处理,即10s再灌注,10 s缺血,6次循环后再灌注12周.实验动物分为缺血再灌注损伤组(IRI),缺血后处理组(IPO)和假手术组(Sham),全自动生化分析仪检测血尿素氮(BUN)和肌酐(Cr),Masson特殊染色法观察小管间质纤维化程度;Western blotin法测定α平滑肌肌动蛋白(α-SMA)、转化生长因子(TGF-β)和磷酸化Smad2蛋白表达(p-Smad2);免疫组织化学法观察肾脏α-SMA和TGF-β1的分布.结果 IRI组和IPO组较Sham组出现了明显肾间质纤维化,但是IPO组肾间质纤维化与IRI组比较有所减轻;α-SMA、TGF-β1和p-Smad2的水平明显减低,各组间差异有统计学意义(P＜0.05).结论 肾脏缺血再灌注损伤可以引起小管间质纤维化.缺血后处理可以影响此病理改变,其保护机制可能为缺血后处理抑制TGF-β1和p-Smad2信号通路激活.     

Protective effect of resveratrol on 5/6 nephrectomized rats and its mechanism

Objective To investigate the protective effect of resveratrol (RSV) on 5/6 nephrectomized rats and its mechanism. Methods Fifty male SD rats were randomly divided into three groups: sham operated (Sham, n＝10）, 5/6 nephrectomy (Nx, n＝20), and 5/6 nephrectomy＋RSV 20 mg/kg (Nx＋RSV, n＝20). RSV or normal saline was administered one week after 5/6 nephrectomy. Proteinuria was detected every 4 weeks. Serum creatinine and the renal pathological changes were measured after 12 weeks. Immunohistochemisty staining of fibronectin (FN), collagenⅠ, transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) were used to analyze the changes of renal fibrosis. Western blotting was used to measure the expression of Smad3, phospho-Smad3, and acety-Smad3. Immunoprecipitation was used to detect the interaction between Sirt1 and Smad3. Results Compared with the sham operated rats, subtotal nephrectomy significantly increased proteinuria [(152.14±30.49) mg/24 h vs (25.34±7.54) mg/24 h], serum creatinine[(111.60±21.50) μmol/L vs (53.90±11.59) μmol/L], glomerular sclerosis index (1.56±0.34 vs 0.35±0.08) and the expressions of fibronectin, collagenⅠ, TGF-β and CTGF in renal tissue at 12 weeks after operation (all P＜0.01), and RSV treatment significantly inhibited the above up-regulations (all P＜0.01). Compared with the sham operated rats, subtotal nephrectomy increased the expression of phospholylation and acetylation of Smad3. RSV treatment significantly reduced the expression of acety-Smad3, but had no effect on the phospho-Smad3. Immunoprecipitation revealed a binding effect of Smad3 with Sirt1. Conclusions RSV treatment can attenuate proteinuria, protect renal function and inhibit renal fibrosis in 5/6 nephrectomized rats. This renal protective effect is associated with reduced Smad3 acetylation and activation of Sirt1, which suggesting that Sirt1 may be a potential therapeutic target of CKD.     

支架蛋白JLP在单侧输尿管梗阻小鼠肾间质纤维化中的作用及其机制探讨

Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO), and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy. Methods jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups: jlp+/+- and jlp-/--sham-operated groups(jlp-/--sham group and jlp+/+-sham group), jlp+/+- and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp-/--UUO group and jlp+/+-UUO group). Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining.The expression of JLP in jlp+/+renal tissue was assayed by immunohistochemistry staining, immunofluorescence and Western blotting. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), collagen Ⅰ(COL-Ⅰ), collagen Ⅲ(COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups. Besides, the α-SMA, COL-Ⅰ, COL-Ⅲ, TGF-β1, p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups. Results The expression of JLP was mainly demonstrated in the renal tubules of mice. A large amount of collagen deposition was observed in the renal interstitial area in jlp-/--UUO group compared to jlp+/+-UUO group. Similarly, the expression of α-SMA, COL-Ⅰ, COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp-/-- UUO-operated groups. Meanwhile, Western blotting showed that the expression of α-SMA, COL-Ⅰ, COL-Ⅲ, and TGF-β1 protein was obviously higher in jlp-/--UUO group. Moreover, the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/--UUO group. Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.     

Circulating miRNA-130b-3p promotes epithelial-to-mesenchymal transition in renal tubular epithelial cells by inhibiting expression of ERBB2IP and PPARγ

Objective To investigate a possible molecular mechanism of MiRNA-130b-3p involved in renal damage. Methods Human renal tubular epithelial cells (HK-2) were transfected with MiR-130b-3p mimics or normal control mimics. Then HK-2 cells were stimulated with 10 μg/L recombinant TGF-β1 for 72 h. After 72 h, the mRNA and protein expression of Collegen Ⅳ, α-smooth muscle actin (α-SMA), Collegen Ⅰand E-cadherin were quantified by real-time PCR and Western blotting. The mRNA and protein expression of ERBB2IP and PPARγ were also detected. The reporter plasmids containing ERBB2IP 3'-UTR and PPARγ 3'-UTR were constructed. The activity of ERBB2IP and PPARγ were detected by dual luciferase report system. Results Compared to NC mimic group,transfection of HK-2 cells with MiR-130b-3p mimics resulted in significantly increased expression of mRNA and protein of Collegen Ⅳ, α-SMA, Collegen Ⅰ, and decreased expression of E-cadherin after stimulating by TGF-β1 (all P＜0.05). And MiR-130b-3p mimic could significantly down-regulate the mRNA and protein expression of ERBB2IP and PPARγ in HK-2 cells (all P＜0.05) whether in the presence of TGF-β1 or not. The dual luciferase reporter assay showed that MiR-130b-3p induced decreased ERBB2IP 3'-UTR luciferase activity compared to NC mimic group, but there was no significant difference between NC mimic group and mut-MiR-130b-3p mimic group. MiR-130b-3p mimic+mut-PPARγ-3'UTR cotransfection group had lower PPARγ luciferase activity than NC mimic + mut-PPARγ-3'UTR group , and MiR-130b-3p+PPARγ-3'UTR group got lower further (all P＜0.01). Conclusions MiR-130b-3p promotes epithelial-to-mesenchymal transition (EMT) in renal tubular epithelial cells by directly targeting at the 3'-UTR of ERBB2IP and PPARγ, which may play an important role in renal damage of early stage lupus nephritis.     

Triptolide inhibits the tubulointerstitial fibrosis in rats of unilateral ureteral obstruction by up-regulating Ski expression

Objective To investigate the effects of triptolide on tubulointerstitial fibrosis in rats kidneys with the unilateral ureteral obstruction (UUO) by examining the expression of collagen type Ι (Col-Ι), Ski, Smad3, TGF-β1. Methods Sixty male SD rats were divided into three groups: Sham operation group (Sham group), UUO group and triptolide (0.2 mg•kg-1•d-1) treatment group. The levels of blood urea nitrogen (BUN), serum creatinine (Scr), pathological changes were measured. Col-Ι, Ski and Smad3 expressions were assessed by immunohistochemistry. Protein and mRNA expressions of Ski, Smad3, TGF-β1 were assessed by Western blotting and real-time PCR. Results Compared with Sham group, Scr and BUN increased significantly in UUO group (P＜0.05). Interstitial fibrosis was prominent and renal interstitial injury score increased significantly in UUO group (P＜0.05). The expressions of Col-Ι and Smad3 were increased in UUO group (P＜0.05). Compared with Sham group, the protein expressions of TGF-β1 and Smad3 were increased, the Ski protein was decreased in UUO group (P＜0.05). In triptolide group, the morphological changes were notably reduced (P＜0.05). Comparison with UUO group, triptolide could increase the protein and mRNA expressions of Ski significantly, and decreased the protein and mRNA expressions of Smad3 and TGF-β1 (P＜0.05). Conclusion Triptolide can reduce the tubulointerstitial fibrosis by up-regulating Ski, and down-regulating TGF-β1 and Smad3.     

Effect of suramin on the epithelial-mesenchymal transition in peritoneal mesothelial cells induced by high concentration glucose

Objective To explore the effect of suramin on the epithelial-mesenchymal transition (EMT) and the excretion of transforming growth factor-β1 (TGF-β1) in peritoneal mesothelial cells (PMCs) induced by high concentrations of glucose solution (GS). Methods Cultured PMCs were divided into three groups: (1) normal control group; (2) GS-treated group: cells were treated with 1.5%, 2.5%, 4.25% GS for 12 h, 24 h, 48 h, respectively; (3) Suramin-treated group: PMCs cultured with 4.25% GS were exposed to different doses of suramin (25, 50, 100 μmol/L) for 48 h. Expression levels of α-smooth muscle actin (α-SMA) and E-cadherin were detected by Western blotting and the concentration of TGF-β1 in the culture supernatant was determined by ELISA. Results Compared with normal control group, GS-treated PMCs exhibited a time-dependent increase in the expression of α-SMA, and decrease in the expression of E-cadherin. GS also stimulated PMCs to secrete TGF-β1. In the presence of suramin, GS-induced α-SMA expression and TGF-β1 production were reduced, E-cadherin expression was increased. Conclusions Suramin can inhibit high glucose-induced EMT of PMCs by down-regulating the expression of TGF-β1. Suramin may be a novel therapeutic agent for the treatment of peritoneal fibrosis.     

Effect of intermedin on microvascular injury of unilateral ureteral obstruction rats

Objective To observe the effect of intermedin(IMD) on microvascular injury of renal fibrosis in unilateral ureteral obstruction (UUO) rat model. Methods Seventy-two male Wistar rats were randomly divided into two groups: the sham - operation group (n=24) underwent the left ureteral dissection, the other 48 rats were made as unilateral ureteral obstruction models and sub - divided into model group(UUO, n=24) and IMD group (n=24). At the 7, 14, 21, 28 day after the operation, 6 randomly - selected rats from each of the three groups respectively were blooded by abdominal arotic and their obstructive kidneys were taken out. The renal histopathological changes were observed through HE and Masson staining, the contents of BUN, Scr and cystatin C (CysC) of the obstructive kidneys were determined, the expressions of transforming growth factor - β1 (TGF - β1), α-SMA, bone morphogenetic protein-7 (BMP-7), E-cadherin, thrombospondin 1 (TSP-1) and vascular endothelial growth factor (VEGF) were detected by RT - PCR and immunohistochemistry. Results Compared with the sham-operated group, the pathological changes of kidney in the model group showed that the degree of fibrosis was obvious, tubular interstitial damage aggravated, the levels of BUN, Scr, CysC in the model group increased (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA, TSP-1 increased (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF decreased (P＜0.05). Compared with the UUO group, renal tubular damage, interstitial fibrosis in the IMD group were lighter, the levels of BUN, Scr, CysC in the IMD group were lower (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA,TSP-1 were down-regulated (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF were up-regulated (P＜0.05). Conclusion IMD can ameliorate the renal interstitial fibrosis, and the mechanism may be related to the fact that VEGF mediated by IMD can reduce vascular injury.     

Norcantharidin inhibits renal interstitial fibrosis through down-regulating protein phosphatase-2Ac

Objective To observe the relationship between protein phosphatase-2Ac (PP2Ac) and renal interstitial fibrosis, and to investigate the effects and the underlyingmechanism of norcantharidin (NCTD) on renal fibrosis in unilateral ureteral obstruction (UUO) rats. Methods Sixteen male Sprague-Dawley rats were randomly divided into four groups: Sham operation group, UUO day 3 group, UUO day 7 group and UUO day 14 group. The rats were sacrificed at day 1, 3, 7, 14 respectively and kidneys were harvested. Immunohistochemical staining were used to detect the expression of fibronectin (FN), type I collagen (Col-I) and PP2Ac. Another twenty male Sprague-Dawley rats were also randomly divided into four groups: Sham operation group, UUO group, low dose NCTD treatment group (0.05mg·kg-1·d-1) and high dose NCTD treatment group (0.1 mg·kg-1·d-1). The rats in NCTD treatment groups were injected with norcantharidin into abdominal cavity 1 day before operation, while the Sham and UUO group were injected with equal normal saline. Rats were sacrificed at day 14 after surgery and the kidneys were harvested. Immunohistochemical staining, Western blotting and real-time PCR were used to detect the expression of FN, Col-I, α-SMA, E-cadherin and PP2Ac. Results (1)Compared with sham group, the expression of PP2Ac, FN and Col-I increased with the development of ureteral obstruction (all P＜0.05). The expression of PP2Ac was positively correlated with the expression of FN and Col-I (r＝0.894 and 0.887, all P＜0.05). (2)Compared with sham group, the expression of FN, Col-I, α-SMA increased and the E-cadherin decreased in UUO group, the expression of PP2Ac also increased (all P＜0.05). After the treatment of NCTD, the above changes were all alleviated in a dose dependent manner, and the expression of PP2Ac was down-regulated (all P＜0.05). Conclusion NCTD can ameliorate tubulointerstitial fibrosis and its anti-fibrosis effect may be related to its inhibition to PP2Ac.     

Sonic hedgehog signaling is involved in aristolochic acid-induced renal tubular epithelial cells injury in vitro

Objective To investigate the molecular mechanism of aristolochic acid (AA)-induced renal tubular epithelial cells (NRK-52E) injury, and to elucidate possible role of sonic hedgehog (Shh) signaling in this process. Methods The proliferation inhibition rate was measured by WST-1 assay in vitro after NRK-52E cells were treated with AA (1, 10, 100 mg/L) for 12, 24, and 48 h, respectively. Cell apoptosis was determined by Hoechst 33258 staining. mRNA expressions of Smo, Ptch1, α-SMA, E-cadherin, TGF-β1, and type III collagen were detected by real-time PCR. The levels of Shh and TGF-β1 were detected by ELISA assay. The expressions of bcl-2, bax, α-SMA, and E-cadherin were examined by Western blotting. Results WST-1 assay showed that AA significantly inhibited the proliferation of NRK-52E cells after 24 h, which was in a dose-dependent pattern (r＝0.817, P＝0.047). Evidence from Hoechst 33258 staining revealed that lower concentration (1, 10 mg/L) AA induced cell apoptosis, but higher concentration (100 mg/L) AA induced cell necrosis. In AA-treated cells (10 mg/L), apoptosis was induced, which was partly mediated by the mitochondrial pathway with decreased bcl-2 and enhanced bax expression. The over-expression of Shh protein and Smo mRNA, and down-regulation of Ptch1 mRNA expression were found, indicating that Shh signaling was activated. In addition, the expressions of α-SMA, TGF-β1, and type Ⅲ collagen were significantly increased, but E-cadherin expression was decreased, suggesting that epithelial-to-mesenchymal transition and fibrosis occurred. Conclusions AA can significantly inhibit proliferation, and induce apoptosis and necrosis in NRK-52E cells. In this process, Shh signaling is activated, which promotes epithelial-to-mesenchymal transition and fibrosis.     

Effects of silibinin on expression of integrin linked kinase,transforming growth factor β1 and α-smooth muscle actin in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of silibinin on the expression of integrin linked kinase (ILK), transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose(1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 12, 24, 48, 72 hours, high glucose (2.5%) for 24 hours after silibinin (5, 10, 20 mg/L) preincubate for 2 hours. ILK and α-SMA mRNA were detected by real-time PCR. ILK protein was detected by Western blotting. TGF-β1 protein in supernatants was detected by ELISA. Results Compared with the control group, the expresssion of ILK, TGF-β1 and α-SAM was significantly increased in groups stimulated by high glucose (all P＜0.05). Silibinin could significantly decrease the expression of ILK, TGF-β1 and α-SMA induced by high glucose (all P＜0.05). Conclusions High glucose can up-regulate the expression of ILK, TGF-β1 and α-SMA. Silibinin can reverse these changes.