Wnt-7a蛋白通过wnt/β-catenin通路抑制单侧输尿管梗阻小鼠肾脏上皮细胞-间充质细胞转分化

目的:观察Wnt-7a蛋白对单侧输尿管梗阻模型小鼠肾脏纤维化的拮抗作用及其可能机制。方法:雄性C57BL/6小鼠随机分为假手术组、UUO模型组和Wnt-7a蛋白治疗组,术后7 d行Masson染色观察肾间质纤维化程度,免疫组化染色检测肾组织E-钙黏素、α-平滑肌肌动蛋白、波形蛋白的表达,免疫印迹法测定肾皮质中E-钙黏素、α-平滑肌肌动蛋白及β-连环蛋白(β-catenin)的蛋白质表达。结果:与模型组相比,治疗组肾组织间质纤维化相对面积显著减少,α-平滑肌肌动蛋白及波形蛋白的表达显著减少,E-钙黏素表达显著增多(P0.05)。同时,治疗组肾组织中β-catenin表达较模型组显著减少(P0.05)。结论:Wnt-7a蛋白可以抑制肾脏上皮细胞-间充质细胞转分化的发生而减轻肾脏纤维化,其可能是通过抑制经典的wnt/β-catenin通路。     

槲皮素对单侧输尿管梗阻大鼠肾脏α-平滑肌肌动蛋白表达的影响

目的:观察槲皮素(Que)对单侧输尿管梗阻大鼠肾脏病理和α-平滑肌肌动蛋白(α-SMA)表达的影响,探讨槲皮素对肾间质纤维化的保护作用及作用机制.方法:采用单侧输尿管结扎(UUO)致肾间质纤维化大鼠模型.将36只大鼠随机分为3组(n=12):假手术组(Sham组)、模型组(UUO组)、槲皮素(剂量100 mg·kg-1·d-1)治疗组(Que组).术后10 d以化学比色法测定梗阻侧肾组织匀浆中羟脯氨酸(Hyp)的含量;HE和Masson染色观察大鼠肾脏病理变化;采用免疫组织化学法、Western blot法检测各组大鼠梗阻侧肾脏α-SMA的表达部位及蛋白表达水平.结果:HE和Masson染色联合图像分析显示大鼠UUO术后10 d梗阻侧肾脏呈现肾间质纤维化,经 Que治疗梗阻侧肾脏病理改变减轻(P＜0.05).免疫组织化学和免疫蛋白印记分析显示经Que治疗梗阻侧肾脏α-SMA染色阳性表达范围和相对表达丰度明显减少(P＜0.01).同时经Que治疗后肾组织匀浆中的Hyp含量降低(P＜0.05).结论:槲皮素可以减轻UUO大鼠肾间质纤维化,其作用机制与抑制肾小管上皮细胞转化成肌成纤维细胞有关.     

糖肾方改善单侧输尿管梗阻小鼠肾间质纤维化的机制研究

目的:观察糖肾方对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的作用,并初步探讨其机制。方法:45只雄性C57BL/6小鼠随机分为假手术组(Sham)、UUO模型组(UUO)、糖肾方组(TSF);预先给药7 d,除Sham组外其余各组行UUO手术,术后7 d取肾组织进行HE染色及Masson染色观察肾组织病理改变,免疫组化染色检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(Collagen typeⅠ,ColⅠ)和Ⅲ型胶原(Collagen typeⅢ,ColⅢ)表达,Western Blot法检测转化生长因子-β_1(transforming growth factor-β_1,TGF-β_1)、E-钙连素(E-cadherin)和波形蛋白(vimentin)表达。结果:与Sham组比,UUO组小鼠肾小管间质损伤评分及纤维化面积百分比均显著升高;与UUO组比,TSF组肾小管间质损伤评分及纤维化面积百分比均显著降低。免疫组化与Western blot结果显示,与Sham组比,UUO组小鼠肾组织TGF-β_1及ColⅠ、ColⅢ表达水平显著升高;肾小管上皮细胞间质转分化(epithelial-mesenchymal transition,EMT)标志性分子α-SMA和vimentin蛋白表达水平明显升高,E-cadherin蛋白表达水平明显降低,TSF可显著逆转UUO小鼠肾组织上述蛋白表达。结论:糖肾方可改善UUO小鼠肾间质纤维化,其机制与其抑制TGF-β_1,减少EMT发生有关。     

依贝沙坦对单侧输尿管梗阻小鼠肾脏整合素连接激酶表达的影响及意义

目的研究依贝沙坦（Irb）对单侧输尿管梗阻（UUO）小鼠肾脏整合素连接激酶（ILK）表达的影响,并探讨其与肾小管上皮间充质转化的关系。方法将雄性CD-1小鼠随机分为假手术对照组（C,n=20）、UUO组（UUO,n=40）和Irb治疗组（UUO＋Irb,n=40）,分别于术后1、3、7和14d处死小鼠。Masson染色观察肾间质纤维化。免疫组化检测小鼠肾组织ILK、上皮细胞钙黏蛋白（E-cadherin）和α平滑肌肌动蛋白（α-SMA）表达。Western印迹观察小鼠肾组织ILK蛋白表达。荧光实时定量PCR（real-time PCR）检测ILK、E-cadherin、α-SMA和纤连蛋白（FN）mRNA表达。结果与C组相比,UUO组术后1d ILK mRNA及蛋白表达均增高;术后3d FN mRNA表达上调,E-cadherin mRNA及蛋白表达明显减少,α-SMA mRNA及蛋白表达显著增加。与UUO组相同时间点比较,Irb治疗组ILK、FN及α-SMA表达均被显著抑制（P均＜0．05）;E-cadherin表达则增高（P＜0．01）。ILK蛋白表达与α-SMA蛋白表达呈正相关（r =0．707,P＜0．01）,与E-cadherin蛋白表达呈负相关（r=-0．919,P＜0．01）。结论Irb能减轻肾脏纤维化,抑制肾小管上皮细胞转分化,这可能与其抑制肾小管细胞ILK表达有关。     

输尿管逆向注射重组腺病毒介导的BMP-7对大鼠肾间质纤维化的作用

目的：构建出具有转染后能表达活性的骨形成蛋白-7（BMP-7）的重组腺病毒,将其直接导入肾纤维化大鼠肾脏,观察其抗肾纤维化的作用。方法：无菌级雄性SD大鼠48只随机分为假手术组、单侧输尿管梗阻（UUO组）组、BMP-7治疗组,以免疫组织化学方法检测肾组织中α-SMA蛋白的表达水平,以逆转录-多聚酶链反应技术检测TGF-β1的表达水平。结果：免疫组化结果示：BMP-7组肾小管间质α-SMA表达显著减少（P〈0.05）。RT-PCR结果显示：TGF-β1随梗阻时间延长而逐渐增多,第28天,有所下降,但无统计学意义。在治疗组,BMP-7组比同一识相点UUO组TGF-β1表达减少（P〈0.05）。结论：BMP-7能够明显减轻肾间质纤维化,下调TGF-β1的表达,从而发挥其在单侧输尿管结扎模型中对肾脏的保护作用,延缓和抑制了肾间质纤维化的发展。     

骨形态发生蛋白-7在幼年大鼠肾小管间质损伤中的表达及意义

目的：探讨单侧输尿管梗阻（UUO）幼年大鼠肾间质纤维化形成过程中骨形态发生蛋白-7（BMP-7）的表达趋势及其与转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）的相关关系;观察血管紧张素转换酶抑制剂（ACEI）和血管紧张素受体拮抗剂（ARB）的干预作用。方法：采用单侧输尿管结扎制备UUO模型。3～4周龄幼年Wistar雄性大鼠随机分为对照组、模型组和干预组。于实验第3、7、14、28天取大鼠8只处死。HE及Masson染色观察肾组织的病理改变;免疫组化半定量检测各组大鼠肾组织BMP-7、TGF-β1及α-SMA蛋白表达。了解BMP-7与TGF-β1、α-SMA、肾间质纤维化程度的关系。结果：随梗阻时间延长,模型组BMP-7表达逐渐下降,TGF-β1、α-SMA表达进行性增高,干预组BMP-7表达较模型组显著增加（P〈0.05）;TGF-β1、α-SMA表达较模型组明显减少（P〈0.05）。模型组肾小管间质TGF-β1、α-SMA表达明显增高（P〈0.05）,BMP-7表达显著减少（P〈0.05）。与模型组相比,干预组肾小管间质TGF-β1、α-SMA表达显著减少（P〈0.05）,而BMP-7表达显著增多（P〈0.05）。BMP-7与TGF-β1、α-SMA、肾间质纤维化程度成负相关（r分别为-0.844、-0.787、-0.952,P均〈0.01）。结论：BMP-7表达减少伴随着肾小管上皮细胞转分化出现,提示BMP-7可能具有维持小管上皮细胞表型作用。苯那普利联合氯沙坦可能通过下调TGF-β1、α-SMA蛋白的异常高表达,上调BMP-7蛋白的异常低表达,直接或间接负性调控肾小管上皮细胞转分化,阻止肾间质纤维化进展。     

辛伐他汀减轻UUO大鼠肾纤维化及可能机制的研究

目的本研究旨在探讨辛伐他汀改善输尿管梗阻(UUO)大鼠肾组织纤维化以及可能的机制。方法 30只SD大鼠,随机分为假手术对照组(Sham组)、模型组(UUO组)和辛伐他汀治疗组(Sim组),治疗组术前3 d予20 mg·kg~(-1)·d~(-1)辛伐他汀生理盐水混悬液3 mL灌胃治疗,对照组及模型组予等体积生理盐水灌胃。成模后7 d处死各组大鼠。行HE、Masson、PAS染色方法光镜下观察大鼠肾脏病理改变;免疫组化观察Wnt4和β-catenin蛋白在肾组织的表达; Western blotting检测各组大鼠肾皮质β-catenin、Wnt4、GSK-3β、p-GSK-3β、CollagenⅠ、α-SMA和E-cadherin蛋白在各组大鼠肾组织的相对表达量。结果与Sham组相比,UUO组大鼠肾组织纤维化病变明显,肾组织β-catenin、Wnt4、α-SMA、p-GSK-3β、CollagenⅠ蛋白的表达明显增多(P 0.05),E-cadherin蛋白表达减少(P 0.01),与UUO组相比,Sim组肾组织α-SMA、CollagenⅠ、Wnt4、β-catenin和p-GSK-3β蛋白表达减少(P 0.05),E-cadherin蛋白表达多(P 0.05)。结论辛伐他汀能改善肾脏纤维化病变,可能与其抑制Wnt4/β-catenin信号通路的激活和传导有关。     

缬沙坦对单侧输尿管梗阻大鼠肾脏氧化应激的影响

目的：探讨血管紧张素Ⅱ-1型受体拮抗剂（AT1Ra）缬沙坦对单侧输尿管梗阻（UUO）大鼠肾脏氧化应激的影响。方法：Wistar大鼠行左侧输尿管结扎术,分为UUO模型组（n=11）,缬沙坦治疗组（n=11）,同时设假手术对照组（n=7）。术后第14天处死各组大鼠,进行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织丙二醛（MDA）和超氧化物歧化酶（SOD）含量;免疫组织化学方法测定α-平滑肌肌动蛋白（α-SMA）和转化生长因子-β1（TGF-β1）的表达。结果：UUO组与假手术组大鼠比较,肾脏病理改变加重,肾组织MDA含量明显升高（P〈0.01）,SOD含量下降（P〈0.05）,肾组织α-SMA和TGF-β1的表达显著增加（P〈0.01）。缬沙坦治疗组与UUO组大鼠比较,肾间质纤维化减轻（P〈0.05）,肾组织MDA含量降低（P〈0.05）,SOD含量升高（P〈0.05）;同时肾组织α-SMA和TGF-β1的表达降低（P〈0.05）。结论：缬沙坦可通过减少UUO组肾组织脂质过氧化物的产生,增加抗氧化酶的含量,下调TGF-β1的表达,从而显著改善UUO所致的肾间质纤维化。     

维甲酸抑制大鼠单侧输尿管梗阻模型肾间质纤维化

目的探讨维甲酸对大鼠单侧输尿管梗阻(UUO)模型肾间质纤维化的影响。方法建立大鼠UUO模型前2d治疗组和对照组分别每天给予10mg/kg全反式维甲酸或溶媒皮下注射。观察模型第3、7和12天肾小管损害百分比、肾间质纤维化程度、肾间质巨噬细胞数、肾间质α-平滑肌肌动蛋白(α-SMA)、胶原Ⅲ和单核细胞趋化蛋白-1(MCP-1)mRNA的表达。结果维甲酸显著减轻肾小管损害和肾间质纤维化(P<0.01)。治疗组肾间质巨噬细胞数和肾间质α-SMA表达显著低于对照组(P<0.01)。维甲酸显著抑制胶原Ⅲ和MCP-1mRNA表达(P<0.01)。结论维甲酸减少大鼠UUO模型肾间质巨噬细胞浸润、降低胶原Ⅲ和MCP-1mRNA表达、抑制α-SMA蛋白的表达,从而减轻肾间质纤维化。     

冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化的抑制作用及机制

目的 探讨冬虫夏草菌粉对5/6肾大部切除术大鼠肾脏纤维化的抑制作用及其可能机制.方法 30只雄性SD大鼠随机分为3组:假手术组(Sham组,n=10)、5/6肾大部切除模型组(SNx组,n=10)以及5/6肾大部切除+冬虫夏草菌粉干预组(CS组,n=10).术前及术后4、8、12周分别检测大鼠体质量、尿蛋白量变化,并于术后第12周末处死大鼠,检测血尿素氮、肌酐变化,取肾组织切片行HE、Masson染色观察肾脏病理变化,免疫组化观察转化生长因子β1(TGF-β1)及其Ⅰ型受体(TβR Ⅰ)、Ⅱ型受体(TβR Ⅱ)的表达,免疫荧光观察E-c adherin、α-SMA的表达,Western印迹法检测肾脏组织TGF-β1、TβR Ⅰ、TβR Ⅱ、磷酸化(p)Smad2/3、Smad7、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)的表达.结果 术后CS组大鼠的体质量高于SNx组,尿蛋白量及血尿素氮、血肌酐低于SNx组.肾脏组织病理分析显示,CS组肾小球硬化、肾小管间质损伤程度均显著低于SNx组(均P＜0.01).CS组TGF-β1、TβR Ⅰ、TβR Ⅱ、p-Smad2/3蛋白表达量均显著低于SNx组(均P＜0.05),E-cadherin蛋白表达量显著高于SNx组(P＜0.05),α-SMA蛋白表达量显著低于SNx组(P＜0.05),Smad7蛋白表达量显著高于SNx组(P＜0.05).结论 冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化具有明显的抑制作用,其机制可能是与其抑制TGF-β1及其下游信号通路以及抑制EMT的发生有关.     

A porcine model of relief of 3 days unilateral ureteral obstruction: study on the damage and self-repairing capability of the kidney

Objective To explore the reversibility of early stage tubular interstitial injury as well as the timing of reparation through the pig relief of unilateral ureteral obstruction (R-UUO) model. Methods Eight three-month-old female Guangxi BA-MA mini pigs were selected for the construction of R-UUO models. Five time points were set which were UUO 0 day, UUO 3 days, R-UUO 7 days, R-UUO 14 days, and R-UUO 21 days. Renal function, histological structure, and protein expressions of α-smooth muscle actin (α-SMA), vimentin and E-cadherin were evaluated at different time points. Results After 3 days of UUO, compared with UUO 0 day, serum creatinine levels were increased obviously and the kidney tissues presented varying degrees of damage. The expressions of α-SMA and vimentin were increased and E-cadherin expression was decreased (P＜0.05). Following R-UUO after 3 days of UUO, compared to UUO, serum creatinine levels were significantly decreased. Renal pathological tissue damage was repaired. The expressions of α-SMA and vimentin were decreased and E-cadherin expression was increased (P＜0.05). Conclusions The pig R-UUO animal model may provide a good platform to study the kidney injury and repair. The tubular injury may be fully reversed and repaired when removing the pathogenic factors if the renal tubular injury was at an earlier stage.     

Protective effect of emodin on renal interstitial fibrosis in mice of unilateral ureteral obstruction via PI3K/Akt/mTOR signaling pathway

Objective To investigate the effect and mechanism of emodin (EM) in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), UUO operation group (n=8), UUO operation+losartan (LST) group (n=8) and UUO operation+EM group (n=8). The mice in each group were ingested the suspensions by gavage for 14 days after surgery. Mice in UUO+LST and UUO+EM groups were given 10 mg?kg-1?d-1 LST and 20 mg?kg-1?d-1 EM, respectively. LST and EM were mixed with 0.5% sodium carboxymethyl cellulose. Mice in sham group and UUO group were given 0.5% sodium carboxymethyl cellulose. The mice were sacrificed at the 14th day. Interstitial fibrosis was observed by HE, Masson and PAS stain. Real-time PCR was used to detect LC3, Beclin-1 and mTOR mRNA. Protein expressions of TGF-β1, α-SMA, E-cadherin, LC3, Beclin-1, PI3K, p-Akt and mTOR were detected by Western blotting. The autophagy was observed with transmission electron microscopy in the renal tissue. Results Compared with sham mice, UUO mice at the 14th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of TGF-β1 and α-SMA (all P＜0.01), and decreased expressions of E-cadherin (P＜0.01). Their renal expressions of PI3K, p-Akt and mTOR were also raised (all P＜0.01). Compared with those in UUO group, in UUO+LST group and UUO+EM group, expressions of autophagy protein LC3 and Beclin-1 were increased (all P＜0.01), and the number of autophagic was increased. Additionally, expressions of TGF-β1 and α-SMA were reduced in UUO+LST group and UUO+EM group (all P＜0.01), while the expression of E-cadherin was increased by emodin treatment (P＜0.05). And expressions of PI3K, p-Akt and mTOR were decreased in UUO+LST group and UUO+EM group (all P＜0.05), meanwhile renal tissue fibrosis significantly reduced. Conclusions Emodin can promote autophagy, ameliorate renal interstitial fibrosis and protect renal function through PI3K/Akt/mTOR signaling pathway.     

大黄蒽醌提取物缓解小鼠肾组织纤维化作用的实验研究

目的 通过观察大黄蒽醌提取物抑制小鼠病变肾组织纤维化的作用，探讨大黄治疗肾脏病的作用机制。方法 采用左侧输尿管结扎的方法建立单侧输尿管梗阻(UUO)雄性CD-1小鼠动物模型。用形态学半定量方法评价组织学病变；酸水解-比色法测定肾组织胶原的含量；蛋白印迹技术检测胶原α表达的水平。以α-平滑肌肌动蛋白(α-SMA)作为上皮细胞转分化的观察指标。结果 大黄提取物(50 mg/kg体重)能够显著地减少肾间质的病变，降低肾组织中胶原的聚积，与对照组相比两者差异均有统计学意义。大黄提取物(25、50 mg/kg体重)能够降低病变肾组织中胶原α的表达水平及减少梗阻肾组织中α-SMA的表达，抑制上皮细胞转分化。结论 大黄蒽醌化合物能够改善肾脏的纤维化，其作用可能与降低肾组织内胶原的沉积以及抑制上皮细胞转分化有关。     

Inhibition of Src kinase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of Src kinase in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), sham operation+PP2 group (n=8), UUO operation group (n=8) and UUO operation+PP2 group (n=8). The mice were injected 2 mg/kg PP2 by intraperitoneal everyday after surgery in sham+PP2 group and UUO+PP2 group. PP2 dissolved in 1% DMSO (formulated with normal saline). Sham and UUO group were given equal 1% DMSO. The mice were sacrificed at 7th day. Renal collagen was observed with Sirius red stain. The activities of Src, protein kinase B (PKB, AKT), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and the protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting. The expression of collagen I (COLⅠ) was detected by immunohistochemistry and the expressions of matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6) were measured by ELISA. Results Compared with sham mice, UUO mice on 7th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of COLⅠ and FN, and activities of AKT, ERK and p38 MAPK (all P＜0.05). Their renal expressions of α-SMA, TGF-β1, MMP-9, TIMP-1, MCP-1 and IL-6 were also raised (all P＜0.05). Compared with those in UUO group, in UUO+PP2 group the activities of Src, AKT, p38 MAPK and ERK, and expressions of TGF-β1, MCP-1 and IL-6 decreased (all P＜0.05). Additionally, expressions of COLⅠ, FN and α-SMA, collagen deposition and renal fibrosis receded in UUO+PP2 group (all P＜0.05). However, the expressions of MMP-9 and TIMP-1 were not influenced by PP2 treatment. Conclusions Src kinase promotes myofibroblasts accumulation and inflammatory reaction through activating its downstream signaling pathway in the progressing of renal interstitial fibrosis.     

肝细胞生长因子负性调控细胞转分化在肾间质纤维化中的作用

目的 研究肝细胞生长因子(HGF)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的保护作用及其可能机制&#65377;方法 大鼠随机分为UUO组&#65380;HGF治疗组和假手术组&#65377;用实时荧光定量RT-PCR&#65380;Western杂交和免疫组化检测术后大鼠肾组织结缔组织生长因子(CTGF)和骨形成蛋白7(BMP7)表达量&#65377;免疫组化检测大鼠肾组织TGF-β1&#65380;FN及α-SMA表达&#65377;结果 与假手术组相比,UUO组及HGF治疗组CTGF mRNA&#65380;TGF-β1&#65380;α-SMA&#65380;FN&#65380;CTGF蛋白表达均增高,且UUO组明显高于治疗组;UUO组及HGF治疗组BMP7 mRNA和蛋白表达均减少,且UUO组显著低于治疗组&#65377;结论 HGF能减轻肾间质纤维化,负性调控肾小管上皮细胞-肌成纤维细胞转分化,调节CTGF及BMP7表达可能是其作用途径&#65377;     

参附注射液对单侧输尿管梗阻大鼠肾脏肝细胞生长因子表达的影响

目的：探讨单侧输尿管梗阻后大鼠肾间质纤维化发生过程中肝细胞生长因子（HGF）的表达及中药参附注射液对其的影响。方法：采用单侧输尿管结扎（UUO）制造梗阻性肾病模型，将56只大鼠随机分为对照组（假手术组）、手术组（UUO组）和治疗组（UUO＋参附），术后7d、14d观察肾组织病理改变，应用免疫组织化学方法检测肾组织中HGF的表达。结果：与对照组相比，手术组肾间质出现了明显的纤维化，HGF的表达在术后第7天明显增加，第14天较第7天减弱，与手术组相比，治疗组肾间质纤维化明显减轻，而且HGF的表达在术后第7天明显上调，第14天较第7天上调更明显，有统计学差异（P〈0．05）。结论：参附注射液可以上调肾组织HGF的表达，减轻肾小管一间质纤维化，发挥肾保护作用。     

血管内皮生长因子C参与肾间质纤维化的进程

目的探讨血管内皮生长因子C（VEGF-C）在大鼠。肾小管上皮细胞向间充质细胞转化（EMT）过程中的变化及其作用通路,并利用动物模型研究血管紧张素Ⅱ受体拮抗剂替米沙坦对VEGF-C的影响,从而探讨VEGF-C在肾间质纤维化中的作用。方法体外培养大鼠肾小管上皮细胞（NRK52E）,用转化生长因子B1（TGF-β1）孵育不同时间,观察其对VEGF-C、上皮型钙黏蛋白（E-cadherin）、波形蛋白（vimentin）、磷酸化AKT（P-AKT）等表达的影响,并在TGF-131作用同时加入PBK抑制剂Wortmannin,观察上述指标的变化;用单侧输尿管结扎术（UUO）制作SD大鼠肾间质纤维化模型,将21只大鼠随机分为假手术组、模型组和替米沙坦治疗组,每组7只。2周后,用免疫组织化学法检测α平滑肌肌动蛋白（mSMA）及VEGF-C在肾组织的分布,用RT-PCR和Westernblot—ring法检测其mRNA和蛋白表达。结果TGF-β1促进EMT的同时促进VEGF-C的表达增加。加入Wortmannin后EMT被抑制,同时VEGF-C的表达减低。动物模型组α-SMA和VEGF-C表达较假手术组高,替米沙坦治疗组α-SMA和VEGF-C表达较模型组低。结论TGF-β1可通过P13K—AKT通路促进VEGF-C的表达,VEGF-C与α-SMA的变化有同步性,提示VEGF-C可能参与肾间质纤维化的进程。     

Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis

Unilateral ureteral obstruction (UUO) is a well-characterized hydronephrosis model exhibiting interstitial inflammatory-cell infiltration and tubular dilatation followed by tubulointerstitial fibrosis of the obstructed kidney. Our recent report indicates that rapamycin is effective for 50% of transplant recipients with chronic allograft nephropathy. In this study, we investigate the effect of rapamycin on UUO-induced renal fibrosis. UUO or sham-operated rats were randomly assigned to rapamycin or vehicle and were killed on days 7 and 14 after UUO or sham operation. Rapamycin decreased cross-sectional and gross-morphology changes in the obstructed kidney significantly. Rapamycin markedly blunted the increase in weight of the obstructed kidney, obstructed kidney length, and the obstructed/non-obstructed kidney weight ratio (by 74.6, 42.8, and 61.6% on day 14, respectively, all P<0.01). The scores for tubular dilatation, interstitial volume, interstitial collagen deposition, and alpha-smooth muscle actin (alpha-SMA) after UUO were significantly reduced by rapamycin. Rapamycin also decreased the number of infiltrative anti-ED1-positive cells and the gene expression of transforming growth factor (TGF)-beta1 (84.8 and 80.2% on day 7) after UUO (both P<0.01). By double immunostaining and Western analysis, rapamycin blocked the TGF-beta1-induced loss of E-cadherin expression and de novo increase of the expression of alpha-SMA in a dose-dependent manner. In conclusion, rapamycin significantly attenuated tubulointerstitial damage in a UUO-induced rat model of renal fibrosis, suggesting that rapamycin may have the potential to delay the progression of tubulointerstitial renal fibrosis.     

STAT3抑制剂S3I－201对实验性肾小管间质纤维化的保护作用

目的探讨STAT3抑制剂S3I－201对小鼠实验性肾小管间质纤维化的保护作用。 方法采用单侧输尿管梗阻手术的方法建立肾小管间质纤维化模型。将实验小鼠随机分为药物假手术组(Sham＋S3I－201),安慰剂假手术组(Sham＋Vehicle),药物造模组(UUO＋S3I－201),安慰剂造模组(UUO＋Vehicle)4组,通过腹腔注射S3I－201溶液(药物)或0.05%DMSO PBS(安慰剂)给药,每天给药一次。造模第7天时留取肾脏标本,用Masson染色和颜色面积测算法评估胶原蛋白沉积的情况。用qRT－PCR法检测肾组织内趋化因子配体16(CXCL16),白介素－1β(IL－1β),细胞间黏附分子1(ICAM－1),转化生长因子－β(TGF－β),肿瘤坏死因子(TNF－α)的mRNA表达,用免疫组化法染色和免疫印迹法检测PDGFRβ蛋白在梗阻肾脏内的表达。 结果UUO＋Vehicle小鼠的肾间质胶原蛋白沉积显著高于Sham＋Vehicle组(P<0.05)。UUO＋Vehicle小鼠肾组织CXCL16,IL－1β,ICAM－1,TGF－β,TNF－α的mRNA表达显著高于Sham＋Vehicle组(P<0.05),UUO＋Vehicle小鼠肾组织血小板来源生长因子受体β(PDGFRβ)蛋白表达显著高于Sham＋Vehicle组(P<0.05)。经过S3I－201治疗7 d后,UUO＋S3I－201小鼠的上述各项指标均显著低于UUO＋Vehicle(P<0.05)。 结论S3I－201通过抑制多种细胞因子的mRNA表达,以及降低PDGFRβ蛋白的表达,减轻实验性肾小管间质纤维化小鼠的肾间质炎症反应,从而发挥肾脏保护作用。     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

目的 探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。 方法 健康雄性Wistar大鼠25只,按随机数字表法分为UUO模型组（n=18）和假手术组（n=7）,UUO模型组行左侧输尿管结扎术,假手术组仅分离输尿管不结扎,分别于术后3 d、7 d、14 d处死各组大鼠,行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织羟脯氨酸（HYP）含量;免疫组化法检测α平滑肌肌动蛋白（α-SMA）;原位末端标记法（TUNEL）与DNA电泳观察肾小管间质细胞凋亡情况;RT-PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化;Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase-3）和GRP78的蛋白表达变化。 结果 与假手术组比较,UUO模型组肾脏病理改变加重,肾间质纤维化程度随梗阻时间延长逐渐加重,肾组织HYP含量显著升高（P < 0.05）,肾组织α-SMA也在肾小管间质细胞广泛表达,TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义（均P < 0.01）;在此后观察期间内GRP78 mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3 d即有显著上调（P < 0.05）,且随着梗阻时间延长进行性升高,于术后7 d、14 d增多更为显著,与假手术组差异均有统计学意义（P < 0.05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r = 0.657,P < 0.01;r = 0.714,P < 0.01）。 结论 UUO早期即可诱导ERS标志蛋白表达变化,触发ERS。长期ERS可诱导肾小管间质细胞凋亡;caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。