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.     

纤维蛋白肽Bβ15～42对大鼠缺血再灌注损伤肾脏局部炎性反应的影响

目的 观察纤维蛋白肽Bβ15～42(the fibrin-derived peptide Bβ15-42,FgBβ15～42肽)对大鼠肾脏缺血再灌注损伤(IRI)后肾脏局部炎性反应的影响并探讨其机制.方法 将SD大鼠随机分成假手术组(Sham组)、IRI组、阴性治疗组和FgBβ15 ～ 42肽治疗组.Sham组:分离肾动脉后关闭腹腔;IRI组:采用双侧肾动脉夹闭的方法制作肾脏IRI模型;阴性治疗组:于肾脏再灌注后立即尾静脉注射随机肽段3.6 mg/kg; FgBβ15～42肽治疗组:于肾脏再灌注后立即尾静脉注射FgBβ15～ 42肽3.6 mg/kg.后3组按照再灌注24h、48 h分为两个亚组,Sham组与各亚组均为8只大鼠.常规生化法检测肾功能;HE、PAS染色观察肾脏组织学改变;免疫组化、实时荧光定量PCR法及Western印迹检测肾组织白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)的mRNA及蛋白表达.结果 与Sham组相比,IRI组的Scr和BUN水平均显著增加(均P ＜0.05),肾小管及间质病理损伤显著,以再灌注48 h更为明显;与IRI组相比,FgBβ15～ 42肽治疗组Scr和BUN显著下降(均P＜0.05),小管间质损伤程度明显减轻(P＜0.05).与Sham组相比,IRI组IL-1β和ICA M-1的mRNA和蛋白水平于再灌注24h显著上升,48 h稍微下降,但仍维持在较高水平;FgBβ15～ 42肽治疗组大鼠肾组织IL-1β和ICAM-1的表达于再灌注24h、48 h显著低于同时间点的IRI组(均P＜0.05),但仍明显高于Sham组.上述各指标在阴性治疗组和IRI组之间的表达差异无统计学意义.结论 FgBβ15～42肽对肾脏IRI具有保护作用,其作用机制可能与其减少炎性因子IL-1β、黏附分子ICAM-1的表达有关.     

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.     

Expression and function of Erbin protein in renal ischemia-reperfusion injury

Objective To investigate the expression of ErbB2 interacting protein (Erbin) in renal ischemia-reperfusion injury (IRI) in vivo and in vivo, and the effect of Erbin over-expression on IRI. Methods (1) In vivo, the model of renal IRI was constructed in mice, and set up sham group and reperfusion 3, 6, 12, 24 and 48 h IRI group. BUN and Scr were detected and PAS staining was used to observe the pathology change of renal tissues. Cell apoptosis was detected by TUNEL staining. Erbin and NF-κB expression in renal tissue was detected by Western blotting, and immunohistochemistry was used to detect the distribution of Erbin. (2) In vivo, IRI model in HK2 cells was constructed and cells were harvested after culturing in normal medium for 3, 6, 12 and 24 h. Erbin expression was detected by Western blotting. Flow cytometry and ELISA were used to evaluate the level of cell apoptosis and inflammatory cytokine secretion respectively. HK2 cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, and were divided into control group, IRI group, Erbin group and Erbin+IRI group. The protein expression of Erbin and NF-κB, cell apoptosis and inflammatory cytokine secretion was detected. Results (1) Compared with sham group, serum BUN and Scr were dramatically increased in IRI model, especially in 24 h after reperfusion (P＜0.05). Moreover, PAS staining showed that a lot of renal tubular epithelial cells were necrosis and fell off, and many protein cast were formed, renal injury score and apoptotic index were higher in 6 h, 12 h, 24 h, 48 h IRI model than those in sham group (all P＜0.05). The expression of Erbin, which was expressed in renal tubules, and nuclear NF-κB in 24 h IRI model were significantly increased, as compared with sham group (all P＜0.05). (2) Compared to those in control group, nuclear NF-κB expression, apoptosis and inflammatory cytokine secretion were significantly increased in IRI group. Meanwhile, Erbin expression was also induced and peaked at 24 h (P＜0.05). Compared to those in IRI group, cell apoptosis, the expression of nuclear NF-κB, inflammatory cytokine IL-6 and TNF-α were decreased in Erbin+IRI group (all P＜0.05). Conclusions Erbin expression is up-regulated in renal IRI, and over-expression of Erbin can partly inhibit NF-κB activation, cell apoptosis and inflammatory cytokine secretion in IRI group, which indicates Erbin may playing a protective role in renal IRI.     

外源性硫化氢在大鼠肾脏缺血再灌注损伤中的保护作用

目的 观察不同剂量外源性硫化氢(H2S)供体硫氢化钠对大鼠肾脏缺血再灌注损伤( IRI)的保护作用.方法 健康雄性Wistar大鼠28只随机分为4组,即假手术组( Sham)、肾缺血再灌注(IR)组、硫氢化钠(NaHS)高剂量组、硫氢化钠低剂量组.大鼠右肾切除后,以NaHS作为硫化氢的供体,NaHS高、低剂量组分别经左肾动脉插管,按照1.5 μmol/min、300 nmol/min的剂量连续15 min给药,假手术组及IR组给予同体积生理盐水.停药5 min 后,NaHS组和IR组用无损伤微动脉夹夹闭左侧肾蒂45 min后解除阻断,建立大鼠急性IRI模型,假手术组不夹闭左肾动脉,其他操作同模型组.于肾脏恢复血流24h时留取血和肾组织标本,检测血清尿素氮(BUN)、血肌酐(Scr);半定量分析肾脏病理损伤;检测肾组织H2S生成率;采用实时定量PCR法检测胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE )mRNA表达.结果 与假手术组相比,IR组H2S生成率显著降低(P＜0.01);CBS、CSE mRNA表达显著下降(P＜0.01 );Scr、BUN显著升高(P＜0.01);肾脏病理表现为急性肾小管坏死,且最严重.与IR组相比,NaHS预处理组H2S生成率升高(P＜0.05);CBS、CSE mRNA表达升高(P＜0.01 );Scr、BUN降低(P＜0.01);病理损伤明显减轻.NaHS两个剂量组之间差异无统计学意义.结论 外源性H2S对大鼠IRI具有保护作用.     

TLR4／NF—κB信号通路与肾脏缺血后处理的肾脏保护作用的关系

目的探讨小鼠。肾脏缺血后处理（ischemic postconditioning,IPC）对缺血再灌注损伤（ischemia-reperfusion injury,IRI）的保护作用及与Toll样受体（toll—like receptor,TLR）4／核因子（nuclear factor,NF）-κB信号通路的关系。方法BALB／c小鼠42只随机分组：假手术组6只、IRI组18只与IPC组18只,IRI组与IPC组均设术后1d、3d、5d亚组,每个亚组各6只小鼠。建立肾脏IRI及IPC模型;于术后1d、3d、5d取血检测血清肌酐（Scr）、血尿素氮（BUN）,取肾组织行病理学检查,计算术后1d急性肾小管损伤的Jablonski评分,采用蛋白质印迹（Western blot）法检测术后1d、3d、5d的TLR4、NF—κB表达水平。结果术后各时点,IRI组的Scr、BUN水平均明显高于假手术组（均为P〈0．05）;术后1d,IPC组的Scr、BUN水平均明显低于IRI组（均为P〈0．05）,且于术后5d两指标明显下降并恢复至正常范围。与假手术组比较,IRI组和IPC组肾小管损伤程度较为严重（均为P〈0．05）;与IRI组比较,IPC组的肾小管损伤程度显著减轻（P〈0．05）。术后1d、3d,IPC组的TLR4表达水平较IRI组明显降低（均为P〈0．05）。术后1d、3d、5d,IPC组的NF—κB表达水平较IRI组明显降低（均为P〈0．05）。结论IPC可减轻缺血再灌注对肾脏的损伤,其保护作用可能通过抑制TLR4／NF—κB信号通路实现。     

外源性硫化氢抑制TLR2和TLR4表达并减轻大鼠肾脏缺血再灌注损伤

目的观察外源性硫化氢(H₂S)供体硫氢化钠(NaHS)能否抑制Toll样受体2(TLR2)和Toll样受体4(TLR4)表达、减轻大鼠肾脏缺血再灌注损伤(IRI)。 方法24只6~8周龄雄性SD大鼠随机分为3组：假手术(Sham)组、肾脏缺血再灌注(I/R)组、NaHS+I/R组。采用右肾切除联合左肾动脉夹闭45 min后再灌注24 h的方法诱导肾IRI。夹闭左肾动脉前，NaHS+I/R组给予NaHS(300 nmol/min)连续输注10 min，Sham组和I/R组则给予等体积生理盐水。分别留取各组腹主动脉血及肾组织标本。Western印迹法检测肾组织TLR2、TLR4蛋白的表达；免疫组织化学法检测肾组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的表达；比色法检测血尿素氮(BUN)、血肌酐(Scr)。HE染色观察肾脏组织学改变；TUNEL法检测肾组织细胞凋亡。 结果与Sham组比较，I/R组的TLR2、TLR4、IL-6、TNF-α表达均增加(P<0.05)，BUN、Scr亦明显升高(P<0.05)，肾小管上皮损伤评分较高(P<0.05)，肾组织凋亡细胞增加(P<0.05)。与I/R组比较，NaHS+I/R组的TLR2、TLR4、IL-6、TNF-α表达均减少(P<0.05)，BUN、Scr亦明显下降(P<0.05)，肾小管上皮损伤评分较低(P<0.05)，肾组织凋亡细胞减少(P<0.05)。 结论外源性H₂S可以抑制TLR2、TLR4途径，减少炎症因子释放及细胞凋亡，减轻大鼠肾脏IRI。     

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.     

Erbin在肾间质纤维化中的表达和作用

目的 探讨Erbin在肾脏间质纤维化中表达量的变化及上调Erbin对转化生长因子β1（TGF-β1）诱导大鼠近端肾小管上皮细胞（NRK52E）转分化的影响。 方法 体内实验采用SD大鼠5/6肾切除法建立肾纤维化动物模型,收集并检测各组血清中Scr、BUN水平;Masson染色观察肾间质纤维化程度;免疫组化及Western印迹检测Erbin的分布与表达。 体外实验采用TGF-β1（10 μg/L）刺激NRK52E细胞72 h建立上皮细胞-间充质转分化（EMT）细胞模型;免疫荧光及Western印迹法检测E钙黏蛋白（E-cadherin）和α平滑肌肌动蛋白（α-SMA）的表达变化;RT-PCR及Western 印迹法检测Erbin的表达变化。用脂质体2000将质粒 Prk5-myc-Erbin瞬时转染至NRK52E细胞,Western印迹法观察上调Erbin表达后对上述各种指标的影响。 结果 （1）假手术组大鼠肾功能正常[Scr（33.96±7.28） μmol/L、BUN（8.11±2.55） mmol/L],Masson染色未见肾间质纤维化,Erbin在肾小管表达较少;模型组大鼠Scr [（140.52±61.11） μmol/L]、BUN[（34.23±7.66） mmol/L] 均显著高于假手术组（均P < 0.05）,肾间质可见明显纤维化,Erbin 在肾小管表达也明显增加,是假手术组的2.9倍（P < 0.01）。（2）正常NRK52E 细胞表达E-cadherin,少量表达Erbin和α-SMA。TGF-β1刺激后,NRK52E细胞E-cadherin表达显著减少,Erbin和α-SMA则表达增加（均P < 0.05）;而转染质粒Prk5-myc-Erbin可逆转TGF-β1诱导的NRK52E细胞E-cadherin表达下调,并可抑制α-SMA表达上调（均P < 0.05）。 结论 Erbin在肾间质纤维化中表达增加,上调Erbin表达可抑制TGF-β1诱导NRK52E发生EMT, 提示Erbin在肾脏纤维化中可发挥保护作用。     

Erythropoietin mobilizes renal progenitor cells to reduce ischemic reperfusion injury in rats with nephron-sparing surgery

Objective To investigate the effects of the erythropoietin (EPO) on ischemia reperfusion injury (IRI) in rats with nephron-sparing surgery (NSS). Methods Fifty-four Sprague Dawley rats were divided into 3 groups randomly after right kidney nephrectomy: Sham group, NSS group (PBS+NSS) and EPO group (EPO+NSS). During NSS, renal artery was clamped for 40 min to induce IRI. Sham group just adopted exposure renal artery without vascular clamped. Rats in NSS group were injected intraperitoneally with PBS for 3 days before NSS. Rats in EPO group were injected intraperitoneally with EPO for 3 days before NSS. After 12 h, 24 h, 72 h, blood sample and renal tissues were collected. The serum creatinine (Scr) and urea nitrogen (BUN) were evaluated. The pathology injury was evaluated by HE staining. The CD24/CD133 double-positived renal progenitor cells (RPCs) were tested by flow cytometry. The CD133 and PCNA protein were quantified by immunohistochemical staining. The expressions of Wnt7b and β-catenin protein were detected by Western blotting. Results Rats in NSS group had more elevated Scr, BUN and pathology injury scores 12 h, 24 h and 72 h after operation than those in Sham group (all P＜0.05). Compared with those in the NSS group, the Scr and BUN in the EPO group were significantly lower 24 h after the surgery (all P＜0.05), and the pathology injury score also decreased (P＜0.05). The proportion of RPCs, expressions of CD133 and PCNA, and expressions of Wnt7b and β-catenin protein were significantly higher after 24 h of the surgery in NSS group than those in the Sham group (all P＜0.05). While compared with those in the NSS group, the proportion of RPCs and expressions of CD133, PCNA, Wnt7b and β-catenin increased at the EPO group (all P＜0.05). Conclusions EPO can reduce the IRI after NSS, and its mechanism may be related to the mobilization of the RPCs by the Wnt7b/β-catenin signal pathway.     

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.     

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.     

Astragaloside IV attenuates renal tubulointerstitial fibrosis by inhibiting p38 MAPK signaling

Objective To investigate the effect of astragaloside IV (AS-IV) on renal tubulointerstitial fibrosis and its regulation on p38 MAPK signaling. Methods In vivo, UUO model with renal tubulointerstitial injury was constructed. Mice in AS-IV group were orally administrated AS-IV 20 mg•kg-1•d-1 for 7 days after operation, and mice in other groups were administrated the equal volume vehicle. Bilateral kidneys were collected in 7 and 14 days after operation. Transverse kidney slices were stained with Masson trichrome to evaluate the severity of renal tubule injury. In vitro, normal human renal tubular epithelial cells (HK-2) were stimulated with recombinant TGF-β1 (10 ng/ml) and simultaneously treated with different concentrations of AS-IV (0, 50, 100, 200 μg/ml) for 24 h. SB203580 (10 μmol/L) was also ultilized to pre-treat HK-2 cells for 1 h to inhibit phosphorylation of p38 MAPK signaling. The expression of FN, Col IV, and α-SMA were investigated by western blotting and real-time PCR. The expression of p-p38 MAPKs were also observed by Western Blotting. Results Astragaloside IV morphologically ameliorated renal tubulointerstitial fibrosis. The proteins and mRNA expression of FN, Col IV, α-SMA, and TGF-β1 were also increased significantly in UUO kidney tissues (all P＜0.05), which could be reversed by AS-IV administration (all P＜0.05). In vitro, the expression of FN, Col IV, and α-SMA were up-regulated by TGF-β1 after stimulating for 24 h (all P＜0.05), which were decreased by AS-IV. The inhibition effect on FN and α-SMA were similar between AS-IV and MAPK inhibitor SB203580. AS-IV inhibited p-p38 MAPK signals both in vivo and in vitro. Conclusions AS-IV could attenuate renal tubulointerstitial fibrosis induced by UUO and TGF-β1 through reducing FN、Col IV、α-SMA expression in renal tubular cells. The mechanism of AS-IV protective effect might be associated with inhibition of p38 MAPK phosphorylation.     

益肾胶囊对糖尿病肾病大鼠肾组织JAK/STAT信号通路及α-SMA、FN的影响

目的:探讨益肾胶囊对糖尿病肾病(DN)大鼠24h尿蛋白定量、尿素氮(BUN)、血肌酐(Scr)、JAK/STAT及α-平滑肌激动蛋白(α-SMA)、纤维连接蛋白(FN)的影响及其机制。方法:SD大鼠55只,随机留取10只为正常对照组(N)。利用一次性腹腔注射链脲佐菌素的方法建立DN大鼠模型。造模成功大鼠按随机数字表法分为糖尿病肾病模型组(DN)、益肾胶囊治疗组(DN+Y)、氯沙坦钾治疗组(DN+L)、氯沙坦钾+益肾胶囊治疗组(DN+L+Y),每组10只,连续灌胃12周。于实验的12周检测24h尿蛋白定量;12周末处死大鼠,检测血糖、BUN、Scr;取大鼠肾组织做组织病理学观察;免疫组化法检测肾组织磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)、α-SMA、FN的表达。结果:实验12周末,与DN组比较,益肾胶囊治疗组24h尿蛋白定量、BUN、Scr明显降低(P<0.05),肾组织形态学改变较轻,p-JAK2、p-STAT3、α-SMA、FN的表达显著降低(P<0.05)。氯沙坦钾联合益肾胶囊治疗组与氯沙坦钾治疗组比较,差异有统计学意义(P<0.05)。结论:益肾胶囊可能通过抑制糖尿病肾病大鼠肾组织JAK/STAT信号通路的活化,下调α-SMA、FN的表达,延缓了糖尿病肾病的进展;益肾胶囊联合氯沙坦钾可更好地保护肾脏功能,提示二者联合使用有协同作用。     

Metformin alleviated EMT and fibrosis after renal ischemia–reperfusion injury in rats

Purpose The purpose of this study is to assess the potential effects of metformin on the development of EMT and tubulointerstitial fibrosis 12 weeks after acute renal ischemia–reperfusion. Methods Male Sprague–Dawley rats were randomly assigned to four groups: Sham, IRI, transient administration of metformin (TAM), and continuous administration of metformin (CAM). Metformin was administered i.p. at a dose of 125?μg kg?^??1 d^???1 3 d prior to suffering from IRI (TAM), or from 3 d before suffering from IRI to 12 weeks after reperfusion (CAM). Renal function, histology, and expressions of IL-6, TNF-α, α-SMA, TGF-β1, Vimentin, and E-cadherin were analyzed. Results Tubulointerstitial fibrosis worsened further in IRI, accompanied by the increased expressions of interleukin-6, TNF-α, α-SMA, TGF-β1, Vimentin, and loss of E-cadherin. Although there were no significant differences between IRI and TAM (p?>?0.05). Compared with the IRI, expressions of IL-6, TNF-α, α-SMA, TGF-β1, and Vimentin were reduced and the expression of E-cadherin was restored in CAM (p?0.05). CAM also significantly promoted activation of AMPK (p?0.05), which showed no difference among Sham, IRI, and TAM (p?>?0.05). Conclusions CAM significantly attenuated tubulointerstitial fibrosis and EMT in rats, potentially via activation of AMPK and down-regulation of TGF-β1.     

STF-083010对肾脏缺血-再灌注损伤的保护作用

目的 观察STF083010对急性肾缺血-再灌注损伤的作用,探讨其损伤保护作用的机制.方法 选择健康雄性SD大鼠30只,随机分为假手术组(打开腹腔)、I-R组(建立大鼠肾I-R损伤模型)与STF083010组.分别在缺血-再灌注24h后处死大鼠,取血液和肾组织.全自动生化仪检测各组血清尿素氮(BUN)及肌酐(Scr)水平.PAS染色观察大鼠肾组织病理变化,免疫组化检测肾脏组织中XBP1、GRP78蛋白的表达.Quantitative real-time PCR(QPCR)测定大鼠肾组织标本中XBP1、GRP78 mRNA水平.结果 I-R组肌酐、尿素氮水平与假手术组相比差异均有统计学意义(P＜0.05).STF-083010组与I-R组相比,差异亦有统计学意义(P＜0.05).PAS病理图片可见STF-083010组肾小管损伤较I-R组明显减轻(P＜0.05);免疫组化检测显示STF-083010组XBP1的表达较I-R组明显降低(P＜0.05),STF-083010组GRP78蛋白的表达较I/R组明显升高;QPCR结果显示STF-083010组XBP1 mRNA水平较I-R组明显降低(P＜0.05),STF-083010组GRP78 mRNA较I-R组明显升高(P＜0.05).结论 STF-083010可以对大鼠肾脏缺血-再灌注损伤性保护作用.     

洋川芎内酯A 对单侧输尿管梗阻大鼠肾间质纤维化的作用及机制研究

目的:探讨洋川芎内酯A(SenA)对单侧输尿管梗阻(UUO)大鼠肾脏病变的保护作用。方法:50只雄性SD大鼠随机分为假手术组、模型组、SenA低剂量(20 mg/kg)组、SenA高剂量(40 mg/kg)组及阳性对照(厄贝沙坦,20 mg/kg)组,行UUO术制备单侧输尿管模型,并于术后21天处死大鼠。根据试剂盒说明书检测血清尿素氮(BUN)、肌酐(Scr)的含量,HE染色法观察肾脏病理改变,免疫组化法检测肾组织α-SMA及CollagenⅠ蛋白的表达,Western blotting检测各组大鼠肾皮质Wnt4、β-catenin、p-GSK-3β、E-cadherin的表达水平。结果:与模型组比较,SenA低、高剂量组及阳性对照组大鼠血清中Scr、BUN含量明显降低(P<0.05),肾脏病变情况得到不同程度的改善。同时,SenA可明显减少大鼠肾组织中α-SMA和CollagenⅠ蛋白的分泌(P<0.05),下调Wnt4、β-catenin及p-GSK-3β的表达水平(P<0.05),并伴有E-cadherin蛋白表达显著上调(P<0.01)。结论:SenA可通过下调Wnt4/β-catenin信号通路,抑制肾组织细胞外基质沉积并改善肾功能,最终改善UUO模型大鼠肾间质纤维化的病变进程。     

intermedin预处理对大鼠肾脏缺血再灌注损伤修复和再生过程的作用

目的 探讨intermedin （IMD）预处理对大鼠肾脏缺血再灌注（IR）损伤修复和再生过程的作用。 方法 将Wistar大鼠按随机数字表法分为4组：假手术组（sham）、IR组、转空质粒组和转IMD组。在切除右肾后,转IMD组用超声微泡造影剂介导的基因转染方法将IMD真核质粒转染到大鼠肾组织,用RT-PCR和Western印迹法检测转染效率。转染成功后,制作肾脏IR损伤模型,分别于再灌注后1 d、2 d、3 d、4 d、7 d和14 d 6个时间点各取6只大鼠,留取血清及肾组织标本,常规检测血清BUN和Scr;HE和PAS染色观察肾组织的病理变化;免疫组化法观察肾小管上皮细胞的增殖程度。 结果 （1）转IMD组比转空质粒组的IMD蛋白和mRNA表达均增多（均P < 0.05）,且转IMD组7 d时表达最多,与转IMD组4 d时差异无统计学意义;（2）与sham组相比,IR组1 d和2 d时Scr和BUN均显著增高（P < 0.05）;与IR组相比,转IMD组显著下降（P < 0.05）;转空质粒组与IR组相比差异无统计学意义（P > 0.05）。（3）IR组、转空质粒组和转IMD组大鼠的肾小管均受损,但转IMD组的损伤较轻,均以2 d时病理损伤最重。（4）sham组肾小管和肾小球内几乎没有增殖细胞核抗原（PCNA）阳性细胞的表达;IR组和转空质粒组的PCNA阳性数在IR损伤1 d时开始增加,7 d时最多;转IMD组的PCNA阳性细胞数在IR损伤1 d时开始增加,3 d时最多。与IR组1~4 d相比,转IMD组的PCNA阳性细胞数显著增加（P < 0.05）;与IR组7 d相比,转IMD组7 d的PCNA阳性细胞数显著减少（P < 0.05）。 结论 IMD预处理可以促进肾小管上皮细胞增殖,加速肾脏IR损伤修复和再生。     

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.     

钙三醇抑制肾间质成纤维细胞的活化

目的 通过观察钙三醇对肾间质成纤维细胞增殖和凋亡的影响，及其对转化生长因子（TGF）β1诱导的成纤维细胞转分化和细胞外基质(ECM)合成的干预作用，旨在探讨钙三醇抗肾间质纤维化的潜在效应及相关机制。 方法 体外培养大鼠肾间质成纤维细胞NRK-49F，分别以不同浓度TGF-β1（1、2、5和10 μg/L）和（或）不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）进行干扰。MTT法观察细胞增殖情况；流式细胞术分析细胞周期和细胞凋亡改变；实时定量PCR和Western印迹法分别检测细胞内α平滑肌肌动蛋白（α-SMA）、结缔组织生成因子（CTGF）及纤连蛋白（FN）的mRNA和蛋白表达。 结果 钙三醇能明显抑制正常和TGF-β1（5 μg/L）诱导的NRK-49F细胞增殖（P < 0.01）。经不同浓度钙三醇（10－4、10－5、10－6、10－7、和10－8 mmol/L）作用48 h后，NRK-49F细胞G2/S期细胞比例较TGF-β1刺激组均明显减少（分别为25.88%、21.81%、21.73%、23.28%、23.61%比27.42%，均P < 0.05），但对细胞凋亡无明显影响。NRK-49F细胞经TGF-β1刺激后，其α-SMA、CTGF、FN mRNA表达量显著上升，并在TGF-β1 1~5 μg/L的范围内呈剂量依赖效应。钙三醇能明显抑制TGF-β1诱导的α-SMA、CTGF、FN mRNA表达上调（均P < 0.05），而不同钙三醇浓度干预组之间差异无统计学意义。钙三醇能显著降低TGF-β1诱导的α-SMA和FN蛋白表达（P < 0.05）。 结论 钙三醇可通过G1期停滞抑制大鼠肾间质成纤维细胞增殖，但不影响凋亡。钙三醇具有拮抗 TGF-β1致肾间质纤维化的潜在作用。