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.     

Histone methyltransferase MLL1 accelerates the development of renal fibrosis via promoting the epithelial-mesenchymal transition of HK-2 cells

Objective To investigate the possible effects of histone methyltransferase MLL1 on renal interstitial fibrosis and epithelial-mesenchymal transition (EMT). Methods Forty-two male SD rats were randomly divided into normal group, sham operation group and unilateral ureteral occlusion (UUO) group, and then UUO group was further divided into group 1 d, 1 week, 2 week, 3 week and 4 week after operation. The expression of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 in UUO rat kidney tissue as well as TGF-β1 stimulated HK-2 cells were detected by real-time PCR and Western blotting. siRNA-MLL1 plasmids was used to inhibit the expression of MLL1 and the protein levels of MLL1, α-SMA, Vimentin, E-cadherin, Col3α1 and H3K4me3 induced by TGF-β1 stimulation were detected by Western blotting. The level of H3K4me3 in promoter region of EMT related genes was observed by chromatin immunoprecipitation (CHIP). Results Compared with normal and sham operated groups, the loss of renal function in UUO group was more obvious with the obstruction time (P＜0.05). The renal fibrosis was most obvious 1 week and 2 weeks after the rats underwent the UUO operation (all P＜0.05), with the highest protein expressions of MLL1, E-cadherin, α-SMA, Vimentin and Col3α1 (all P＜0.05). Compared with the control group, 3 ng/ml TGF-β1 induced the highest expression of MLL1 and the most obvious EMT in HK-2 cells (all P＜0.05). Moreover, the EMT and the high level of H3K4me3 in HK-2 triggered by TGF-β1 were all inhibited by siRNA-MLL1 plasmids transfection (all P＜0.05). Conclusions MLL1 can enhance the occurrence of EMT induced by TGF-β1 in HK-2 cells by increasing the level of H3K4me3 in the promoter region of α-SMA and Vimentin.     

肾间质纤维化中DJ-1抑制抗纤维化因子PTEN的表达

目的 观察肾小管上皮细胞转分化过程中PTEN的表达和分布,并研究上调DJ-1 对 PTEN 表达和分布及 PI3K-Akt 通路活化的影响。 方法 以人肾小管上皮细胞为研究对象,10 μg/L TGF-β1 刺激72 h 诱导人肾小管上皮细胞转分化;Western印迹法检测正常组和 TGF-β1 干预组细胞内 PTEN、E-cadherin 和α-SMA 蛋白表达;RT-PCR 法检测两组细胞内 PTEN mRNA 表达水平。脂质体法介导 pEGFP-N1-DJ-1 或空载体转染人肾小管上皮细胞,倒置荧光显微镜和Western印迹鉴定转染效率后,Western印迹法检测正常组、pEGFP-N1-DJ-1 转染组和空载体转染组细胞内 PTEN 蛋白表达;RT-PCR 法检测各组细胞内 PTEN mRNA 表达。pEGFP-N1-DJ-1 转染前 1 h 用 PI3K 抑制剂 LY294002 预处理,Western印迹检测正常组、pEGFP-N1-DJ-1 转染组和空载体转染组及 LY294002 预处理1 h 后 pEGFP-N1-DJ-1 转染组的 p-Akt 和Akt蛋白的表达。激光共聚焦显微镜下观察正常组、TGF-β1 干预组和 pEGFP-N1-DJ-1 转染组细胞内 PTEN 蛋白的分布。 结果 正常组细胞表达 E-cadherin 和 PTEN,几乎不表达α-SMA;TGF-β1干预组α-SMA 表达显著高于正常组（P < 0.05）,而 E-cadherin表达显著低于正常组（P < 0.05）,PTEN mRNA 和蛋白表达均显著低于正常组（P < 0.05）。pEGFP-N1-DJ-1和空载体转染后,细胞绿色荧光表达均在 80% 以上;pEGFP-N1-DJ-1 转染组细胞内 DJ-1 表达显著高于正常组（P < 0.05）,而 PTEN mRNA 和蛋白表达均显著低于正常组（P < 0.05）;pEGFP-N1-DJ-1 转染组p-Akt 表达显著高于正常组（P < 0.05）,但经 LY294002 干预后与正常组表达基本一致。正常组细胞内PTEN 分布于细胞质和细胞核;TGF-β1 干预组细胞质内 PTEN 几乎完全消失,而细胞核PTEN 略有增加;pEGFP-N1-DJ-1 转染组细胞核表达PTEN,但细胞质几乎无 PTEN 表达,这与 TGF-β1干预组相似。 结论 肾间质纤维化中高表达 DJ-1 可抑制 PTEN 表达,并促进 PI3K-Akt 通路活化。     

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。     

单侧输尿管梗阻大鼠肾脏转化生长因子β受体亚型表达的变化

目的 探讨转化生长因子β（TGF-β）Ⅰ型受体（RⅠ）、Ⅱ型（RⅡ）受体以及下游Smad蛋白在单侧输尿管梗阻（UUO）大鼠模型肾脏中表达及意义。 方法 90只雌性Wistar大鼠随机分为正常对照组（CON组）、假手术组（SOR组）和单侧输尿管梗阻组（UUO组）,分别于术后1、3、7、14、21 d处死,检测各组大鼠肾功能;PAS与Masson染色观察大鼠肾间质病理形态改变;实时定量PCR基因芯片分析正常大鼠和肾间质纤维化大鼠肾组织TGF-βⅠ、Ⅱ、Ⅲ型受体及Smad蛋白家族表达。筛选出差异表达的受体亚型,进一步应用实时荧光定量PCR、蛋白免疫印迹法、免疫荧光法检测和验证筛选出的差异受体亚型在不同分期肾间质纤维化大鼠肾组织的分布和表达。 结果 与CON组相比,UUO组大鼠的Scr及BUN于术后3 d开始升高（P < 0.05）,第21天达峰值（P < 0.01）;UUO组术后3 d肾间质可见明显炎性细胞浸润;14 d后出现明显肾小管萎缩;21 d可见明显肾间质纤维化。UUO组肾组织TGF-βⅠ型受体ALK-5、ALK-7和TGF-βRⅡ的mRNA表达于术后3 d上升并随梗阻时间延长逐渐增加（P < 0.05）,于14 d达到峰值（均P < 0.01）;ALK-6的mRNA表达于术后3 d下降（P < 0.05）并随梗阻时间延长逐渐减少,于14 d达谷值（P < 0.01）。ALK-5、ALK-6、ALK-7和TGF-βRⅡ蛋白表达与基因表达一致。Smad2/3及磷酸化（p）-Smad2/3的蛋白表达于术后3 d上升（均P < 0.05）并随梗阻时间延长逐渐增加,于14 d达到峰值（均P < 0.01）。 结论 在肾间质纤维化进展中不同TGF-β受体亚型存在不同的变化规律并与肾间质纤维化进展密切关联。     

High uric acid induces phenotypic transition of renal tubular cells via PI3K/Akt signaling pathway

Objective To investigate the effect and the mechanism of epithelial-mesenchymal transition (EMT) in renal tubular cells induced by uric acid. Methods Normal rat kidney tubular cell line (NRK-52E) were exposed to different concentrations of uric acid (100, 200, 400, 600, 800 μmol/L UA) for 48 hours to induce EMT. Morphological changes of the NRK-52E cells were examined under an inverted phase contrast microscope. The protein expression of E-cadherin, α-SMA, p-Akt and Akt were detected by Western blotting. The distribution of E-cadherin and α-SMA were detected by immunofluorescence. NRK-52E cells were pretreated by different concentrations of LY294002(0, 2.5, 5, 10, 15 μmol/L), the inhibitor of PI3K/p-Akt signaling pathway, and then processed by uric acid (400 μmol/L) for 48 hours. Western blotting was used to detect the protein expression of p-Akt and Akt. NRK-52E cells were then divided into four groups: normal group (N), uric acid group (UA), LY294002 group (LY), uric acid with LY294002 group (UA+LY). The protein expression of E-cadherin and α-SMA were detected by Western blotting, the distribution of E-cadherin, α-SMA and p-Akt were detected by immunofluorescence. Results There was abundant cellular expression of E-cadherin in unstimulated renal tubular cells whereas its expression was significantly decreased in uric acid-stimulated cells (P＜0.05). In addition, uric acid induced de novo expression of α-SMA in contrast to almost negative staining in untreated cells (P＜0.05). p-Akt were obviously increased in high uric acid group (P＜0.05) and Akt changed not significantly (P＞0.05). NRK-52E cells transformed into elongated fibroblast-like cells from cuboidal clustered epithelial cells. These indicated that uric acid has induced EMT and activated PI3K/p-Akt signaling pathway in NRK-52E cells. However, the above effects of uric acid were abolished when p-Akt was blocked by the PI3K inhibitor (10, 15 μmol/L LY294002), indicated that LY294002 has reversed the trend of EMT. Conclusions High uric acid induces phenotypic transition of renal tubular cells probably via activating PI3K/Akt signaling pathway.     

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.     

CIP4对转化生长因子β1诱导的人肾小管上皮细胞-间充质转分化的影响

目的 观察骨架调节蛋白CIP4（Cdc42 interacting protein 4）对转化生长因子β1（TGF-β1）诱导的人肾小管上皮细胞-间充质转分化（EMT）的影响,并探讨其产生的机制。 方法 10 ?滋g/L TGF-β1刺激72 h诱导人近端肾小管上皮细胞（HK-2细胞）向间充质转分化。Western印迹法检测各组细胞内E-cadherin和α-SMA蛋白的表达。倒置显微镜观察细胞形态的变化。根据GenBank人CIP4的完全cDNA序列,设计1条特异性干扰CIP4表达的RNA片段（CIP4-siRNA）和含野生型CIP4的重组真核表达质粒（pcDNA3.1-hCIP4）,利用lipofactamine 2000将其转染HK-2细胞。Western 印迹法检测对照组、TGF-β1刺激组、CIP4-siRNA转染组、pcDNA3.1-CIP4转染组细胞内CIP4、E-cadherin和α-SMA蛋白的表达,共聚焦显微镜观察 E-cadherin和α-SMA蛋白的分布改变;用PI3K-Akt特异性抑制剂渥曼青霉素（wortmannin） 1 μmol/L干预TGF-β1刺激的HK-2细胞48 h,Western 印迹法检测对照组和干预组CIP4表达的变化。 结果 TGF-β1干预后HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,细胞形态由典型的上皮细胞向肌成纤维细胞转变,表明TGF-β1诱导肾小管上皮细胞EMT模型成功。CIP4-siRNA抑制TGF-β1诱导的HK-2细胞表达CIP4后,E-cadherin蛋白表达显著增多（P ＜ 0.05）,α-SMA蛋白表达显著减少（P ＜ 0.05）,部分逆转了上述TGF-β1诱导的肾小管上皮细胞EMT。pcDNA3.1-hCIP4转染使CIP4高表达后,HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,诱导了肾小管上皮细胞EMT。用渥曼青霉素干预TGF-β1刺激的HK-2细胞48 h,CIP4可能蛋白表达显著减少（P ＜ 0.05）。 结论 TGF-β1通过PI3K-Akt途径上调CIP4表达,CIP4可能进一步参与TGF-β1诱导的肾小管上皮细胞EMT过程。     

Effects of renal sympathetic denervation on renal interstitial fibrosis in rats with unilateral ureteral obstruction

Objective To observe the influence of renal sympathetic denervation (RSD) on renal interstitial fibrosis and transforming growth factor beta 1(TGF-β1) and microRNA-21 (miR-21) in rats with unilateral ureteral obstruction(UUO). Methods 40 male Wistar rats were randomly divided into UUO group (A group, n=10), sham UUO group (B group, n=10), RSD+UUO group (C group, n=10) and RSD+sham UUO group (D group, n=10). Rats in A group and C group underwent unilateral ureteral ligation, while those in B group and D group underwent sham operation. Rats in C group and D group were followed by RSD. Rats were sacrificed at 21 days after the operation to evaluate the fibrosis by Masson staining. Immunohistochemical staining and Western blotting were used to detect the expressions of collagen I (COL-I), collagen Ⅲ(COL-Ⅲ) and TGF-β1 in four groups. The expression of miR-21 was detected by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (RT-qPCR). Results A large amount of collagen deposition was observed in the renal interstitial area in A and C group compared to either B or D group (P＜0.05), but the change in C group was decreased significantly than that in A group (P＜0.05). Similarly, the expressions of COL-I, COL-Ⅲ, TGF-β1 and miR-21 were obviously higher in A and C group compared to either B or D group (P＜0.05), but those change in C group were decreased significantly than those in A group (P＜0.05). The above indexes were not significantly different between B group and D group (P＞0.05). Conclusion RSD may relieve the renal interstitial fibrosis in UUO rats, and down-regulate the expression of TGF-β1 and miR-21.     

泽泻对单侧输尿管梗阻大鼠肾组织补体C_3及肾纤维化的影响

目的：探讨泽泻对单侧输尿管梗阻（unilateral uretreal obstructire,UUO）大鼠肾小管上皮细胞间充质转分化（epithelial-to-mesenchymal transition,EMT）的作用及其机制。方法：采用单侧输尿管梗阻方法制作大鼠肾间质纤维化模型。30只雄性SD大鼠随机分为假手术组、UUO组和泽泻治疗组。术前3d开始,泽泻治疗组给予中药泽泻9g·kg-1·d-1饮片溶于适量生理盐水中灌胃用。术后14d处死大鼠,观察梗阻侧肾组织病理损害和间质纤维化,免疫组化检测α-SMA、E-cadherin、补体成分C3在肾组织中的表达。结果：UUO组大鼠肾间质纤维化明显,泽泻干预后肾间质纤维化程度减轻。免疫组化结果表明UUO组大鼠C3和α-SMA表达明显增加,E-cadherin表达明显减少;泽泻干预后,C3和α-SMA的表达明显低于UUO组（P〈0.05）,E-cadherin的表达高于UUO组（P〈0.05）。结论：UUO大鼠肾小管上皮细胞的C3表达增加,中药泽泻能减轻UUO大鼠C3的表达,抑制肾小管上皮细胞EMT。     

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.     

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。     

Effect of NADPH oxidases inhibition on the development of interstitial fibrosis in unilateral ureter obstruction rats

ObjectiveTo clarify whether the NADPH oxidases (NOXs) family contributed to the reactive oxygen species (ROS) production and subsequent interstitial fibrosis in unilateral ureter obstruction (UUO) rats. MethodsMale Wistar rats were randomly divided into sham operation group (n＝8), sham operation + apocynin treatment group (n＝8), UUO operation group (n＝8) and UUO operation+apocynin treatment group (n＝8). Either vehicle or apocynin (100 mg/kg per day) were given by gavage for 7 days after surgery. Rats were sacrificed at 7th day. ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT), and the level of 8-iso-prostaglandin F2alpha (8-iso- PGF2α) in renal tissue. Western blotting was used to detect the protein expressions of NADPH oxidase subunit NOX2 and NOX4, α- smooth muscle actin(α-SMA), collagen I (COL-I) and the level of ERK1/ 2 phosphorylation (p-ERK1/2). ResultsUUO rats with vehicle displayed increased oxidative stress, as measured by renal tissue 8-iso-PGF2α, accompanied with increased renal expression of NADPH oxidases (NOX2, 1.5-fold and NOX4, 1.7-fold, respectively), compared with sham-operated rats (P＜ 0.05). Furthermore, vehicle treated UUO rats showed increased renal COL - I and α - SMA levels, compared with sham-operated rats (P＜0.05). ERK1/2 was also activated as detected by p-ERK1/2 expression in UUO rats with vehicle (P＜0.05). Apocynin treatment significantly decreased renal tissue 8-iso-PGF2α level and expressions of NOX2 (-28.7%) and NOX4 (-31.0%) in UUO rats, respectively, compared with vehicle treated rats (P＜0.05). And significant decrease of COL-I (-26.4%) and α-SMA expression (-80.0%) were also observed (P＜0.05). The activation of ERK1/2 in UUO rats was greatly inhibited by apocynin treatment (P＜0.01). Despite the pronounced dysregulation of pro – oxidative NOXs family, no compensatory increase of antioxidative enzyme activities occurred. ConclusionThe NOXs family contributes largely to the production of ROS and subsequent interstitial fibrosis after ureter ligation, and inhibition of the NOXs family may be a choice for preventing interstitial fibrosis.     

Role of JLP on the epithelial to mesenchymal transition in renal tubular epithelial cells

Objective To observe the effect of JLP on transdifferentiation of human renal proximal tubular epithelial cells (HK-2), and to investigate the role of p38 MAPK signaling pathway in this process. Methods The knock-down plasmids of JLP were constructed. HK-2 cells were randomly divided into four groups: negative control cells (Ctrl-shRNA group), knock-down jlp cells (jlp-shRNA group), negative control cells with FGF-2 treatment (FGF-2 group) and knock-down jlp cells with FGF-2 treatment(jlp-shRNA+FGF-2 group). The expressions of JLP, E-cadherin, TGF-β1, α-SMA, p-p38 MAPK protein were detected by Western blotting．After the induction of FGF-2 for 24 hours, the expressions of α-SMA, COL-I, FN were detected by immunocytochemistry. Results Compared with Ctrl-shRNA group, the expression of JLP protein was significantly down-regulated in FGF-2 group. Compared with FGF-2 group, the expressions of TGF-β1, α-SMA, p-p38 MAPK protein were significantly up-regulated, while E-cadherin protein was significantly down-regulated (P＜0.05). Compared with FGF-2 group, the expressions of α-SMA, COL-I, FN immunostaining increased markedly in jlp-shRNA+FGF-2 group. Conclusion Scaffolding protein JLP is critical in preventing EMT in the course of fibrosis through the inhibition of p-p38 activation in HK-2 cells.     

JNK-c-Jun信号通路下调连接蛋白43的表达引起肾小管上皮细胞转分化

目的 观察c-Jun氨基末端激酶（JNK）-c-Jun通路对连接蛋白43（Cx43）表达的影响及在转化生长因子（TGF）β1诱导的肾小管上皮细胞-肌成纤维细胞转分化（TEMT）中的作用。 方法　大鼠肾小管上皮细胞（NRK-52E）随机分成3组：对照组、TGF-β1（10 μg/L）组和TGF-β1（10 μg/L）+JNK选择性抑制剂SP600125（50 μmol/L）组。用免疫细胞化学、Western印迹检测JNK、c-Jun、连接蛋白43（Cx43）、上皮细胞标志物E-钙黏蛋白（E-cadherin）和肌成纤维细胞标志物α-SMA的表达。用RT-PCR检测Cx43的mRNA水平。用激光共聚焦显微镜荧光漂白恢复（FRAP）技术检测NRK-52E细胞间通讯功能。 结果 TGF-β1引起肾小管上皮细胞α-SMA、JNK、c-Jun表达上调（均P < 0.05）,Cx43、E-cadherin表达下调（均P < 0.05）,Cx43的mRNA水平下降（P < 0.05）,细胞间通迅功能下降 （P < 0.05）。JNK抑制剂处理后,上述改变明显减轻。 结论 TGF-β1引起肾小管上皮细胞内JNK表达上调,增加c-Jun活性,从而抑制Cx43的表达和降低细胞间通迅功能,导致TEMT。     

mTOR通路调控肾间质成纤维细胞活化的机制

目的 探讨mTOR信号通路在肾间质成纤维细胞增生活化过程中的调控作用,并研究其抑制剂在抗肾纤维化治疗中的可行性.方法 用8周龄雌性C57BL/6小鼠构建单侧输尿管结扎(UUO)肾间质纤维化动物模型(n=30),按数字随机法分为雷帕霉素组(n=15)及UUO组(n=15).雷帕霉素组术前1d开始腹腔注射雷帕霉素(2 mg·kg-1·d-1)至实验结束;UUO组注射生理盐水.分别于术后1、3、7、14 d处死小鼠(n=3),留肾组织进行相关检测.同时,体外实验评估雷帕霉素对TGF-β诱导鼠成纤维细胞株(NIH3T3细胞)活化的干预作用.结果 UUO小鼠肾组织中活化的肌成纤维细胞[α肌动蛋白(α-SMA)阳性]高表达mTOR通路下游效应因子pS6K.雷帕霉素显著抑制pS6K表达及肾间质中肌成纤维细胞的活化,改善肾小管间质损伤及纤维化程度.实时荧光定量PCR结果提示雷帕霉素组小鼠肾皮质组织中成纤维细胞特异蛋白1 (FSP1)、转化生长因子β(TGF-β)、结缔组织因子(CTGF)及Ⅳ型胶原蛋白基因α1 (Col 4A1)的mRNA水平显著下降.体外实验结果示TGF-β诱导小鼠成纤维细胞株( NIH3T3)的mTOR通路显著活化,并大量合成α-SMA.雷帕霉素能够明显抑制mTOR通路活性,降低细胞的纤维化活性.结论 肾间质纤维化过程中成纤维细胞内的mTOR信号通路高度活化.抑制mTOR通路能够显著降低成纤维细胞的活性,改善肾间质纤维化程度.     

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.     

Effect and mechanism of soluble epoxide hydrolase inhibitor in renal fibrosis mice model

Objective To investigate the effect and mechanism of soluble epoxide hydrolase inhibitor (sEHI) for NF-κB pathway and cell circle arrest of tubular epithelial cell in unilateral ureteral obstruction (UUO) mice model. Methods Thirty-two healthy C57BL/6 male mice performed UUO surgery to induce renal interstitial fibrosis. Animals were randomly divided into 4 groups: sham group (n=8), sEHI (1 mg?kg-1?d-1) group (n=8), UUO group (n=8) and UUO+sEHI (1 mg?kg-1?d-1) group (n=8). Daily sEHI [1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea, TUPS] or 2% DMSO was applied to mice by oral gavage from day 1 to day 14 after surgery. All mice were sacrificed at day 14 and kidneys were harvested for further analysis. The changes of renal tissue morphology and pathology were observed by Hematoxylin and eosin (HE) and sirius red staining. The expressions of sEH, nuclear factor κB p65 (NF-κB p65) and IκB were measured by Western blotting. The expressions of TNF-α, IL-1β, MCP-1, IL-6, TGF-β, CTGF, collagen-IV and α-SMA were analyzed by real-time PCR. Immunofluorescence staining of phospho-histone H3 (p-HH3) and Ki67 was performed to determine the stage of cell cycle G2/M arrest. Results The expression and activity of sEH increased in UUO group (P﹤0.05). Administration of sEHI inhibited activity of sEH and infiltration of inflammatory cell in tubular interstitial, as well as attenuated tubular damage and tubular interstitial fibrosis. Western blotting analysis revealed administration of sEHI inhibited up-regulated NF-κB p65 and down-regulated IκB in UUO group (P﹤0.05). Real-time PCR demonstrated that administration of sEHI obviously decreased the mRNA expression of cytokines and fibrosis markers, including of TNF-α, IL-1β, MCP-1, IL-6, TGF-β, CTGF, Collagen-IV, α-SMA (P﹤0.05). Immunofluorescence staining showed that there were much more p-HH3 and Ki67 double positive nuclear tubular epithelial cells and interstitial cells in UUO group, compared with Sham group (P﹤0.05). Administration of sEHI reduced the number of double positive nuclear cell only in tubular epithelial cells (P﹤0.05), but not in interstitial cells. Conclusions In UUO tubular interstitial fibrosis model, sEHI inhibits the activation of NF-κB pathway by down-regulating p65 and up-regulating IκB and ameliorates the infiltration of inflammatory cells. In addition, sEHI plays anti-fibrosis effect by moderating cell cycle G2/M arrest and reducing the excrete of pro-fibrosis factors of tubular epithelial cells.     

JAK-STAT通路在小鼠单侧输尿管梗阻模型肾间质纤维化中的作用

目的 探讨Janus蛋白酪氨酸激酶-信号转导子和转录激活子(JAK-STAT)通路在小鼠单侧输尿管梗阻(UUO)模型.肾间质纤维化过程中的作用.方法 选用30只雄性Balb/c小鼠建立小鼠UUO模型(n=24)和假手术小鼠(n=6),术后第1、4、7和14天检测JAK-STAT磷酸化情况.另把18只雄性Balb/c小鼠随机分为假手术组、UUO模型组和治疗组,每组各6只.治疗组在建模前2 h开始给予选择性JAK2抑制剂AG490治疗,每天1次;模型组仅注射溶媒.术后第14天处死动物.组织学评估肾小管损伤和.肾间质纤维化程度;免疫组化检测肾脏巨噬细胞浸润和α-SMA表达;RT-PCR检测Ⅲ型胶原和单核细胞趋化蛋白(MCP)1 mRNA表达;Western印迹检测JAK2和STATl磷酸化.结果 JAK2-STAT1在UUO模型中被激活,其磷酸化水平与病情、肾小管组织学损害以及.肾间质纤维化相一致.AG490能显著抑制JAK2和STAT1的磷酸化(P＜0.01).AG490治疗显著减轻肾小管损害[(21.7±1.7)%比(49.4±1.0)%]和肾间质纤维化(1.0±0.1比2.3±0.2)、α-SMA表达(0.9±0.1比2.1±0.2)和巨噬细胞积聚[(13.3±1.6)细胞/HPF比(34.4±1.0)细胞/HPF](均P＜0.01).AG490治疗显著抑制Ⅲ型胶原和MCP-1 mRNA表达.结论 JAK-STAT信号通路在肾小管间质炎性反应和纤维化中发挥重要作用.     

银杏叶提取物对单侧输尿管梗阻大鼠肾脏的TGF-β_1表达影响

目的：观察银杏叶提取物（EGB）对单侧输尿管梗阻（UUO）大鼠肾组织转化生长因子-β1（TGF-β1）参与的肾间质纤维化的影响。方法：建立UUO大鼠模型,分组：UUO组、治疗组（UUO＋EGB）、对照组（假手术组）。治疗组给予EGB200mg·kg^-1.d^-1灌胃,用免疫组化方法检测术后7d、10d、14d的肾组织TGF-β1表达量并观察肾脏病理改变及测定肾功能、血TGF-β1等指标。结果：治疗组与UUO组相比,治疗组的肾组织TGF-β1表达及血TGF-β1水平均明显降低（P〈0.01）,肾间质炎性细胞浸润、肾小管扩张、萎缩及肾间质纤维化均较UUO组减轻,并且血TGF-β变化与病理轻重相平行。结论：银杏叶提取物可通过下调肾组织TGF-β1减轻UUO术后肾组织间质纤维化。