腹膜透析患者肾素血管紧张素Ⅱ水平变化及其在腹膜纤维化中的意义

目的 探讨持续性非卧床腹膜透析（CAPD）患者血浆及腹腔局部肾素血管紧张素系统（RAS）的变化及其在腹膜纤维化中的意义。 方法 选取腹透感染组和非感染组CAPD患者25例，非感染患者根据透析龄分为≥6月组和＜6月组。应用放射免疫法检测CAPD患者腹透透出液和血浆中肾素及血管紧张素Ⅱ（AngⅡ）的浓度。以不同浓度D-葡萄糖（0.3%、1.5%、2.5%）、甘露醇（1.5%、2.5%）及AngⅡ（0、1、10、100、1000 nmol/L）刺激大鼠腹膜间皮细胞（RPMC），放射免疫法检测细胞上清液AngⅡ浓度变化；实时定量PCR方法观察AngⅡ 1型受体（AT1）、血管紧张素转化酶（ACE）、转化生长因子β1（TGF-β1）、结缔组织生长因子（CTGF）mRNA的表达； Western印迹法检测TGF-β1及CTGF蛋白的表达；ELISA法检测上清液中TGF-β1的变化。 结果 CAPD患者腹透透出液中未检测到肾素。感染及≥6月组的CAPD患者透出液中AngⅡ的浓度分别为（151.99±114） pmol/L和（14.17±41.5） pmol/L，分别为非感染[（7.98±12.69） pmol/L]及＜6月组[（2.18±5.62） pmol/L]的19倍及6.5倍（P < 0.01或P < 0.05）。高糖腹透液（2.5％）注入4 h后，CAPD患者循环中肾素及AngⅡ浓度分别为[（4.30±8.48）和（54.19±34.43） pmol/L]，是基础水平的4.06倍和1.21倍（P < 0.01）。高糖组（2.5％）RPMC AT1、ACE mRNA和上清液AngⅡ浓度分别为低糖组（0.3％）的2.61、2.62和2.01倍（P <0.05）。AngⅡ可在mRNA和蛋白水平上调RPMC表达TGF-β1与CTGF，呈剂量依赖性，100 nmol/L组TGF-β1 mRNA及蛋白水平分别为对照组的2.8和2.19倍（P < 0.05）；1000 nmol/L组CTGF mRNA及蛋白水平分别为对照组的4.48倍和2.82倍（P < 0.05）。AngⅡ促进RPMC分泌TGF-β1，呈时间依赖性，刺激24 h组TGF-β1的表达量为基础值的3.65倍（P < 0.05）。 结论 CAPD尤其是腹透感染或长期腹透患者存在RAS活化状态。RAS的主要效应因子AngⅡ可通过上调间皮细胞表达和分泌致纤维化细胞因子参与腹膜纤维化。阻断RAS可能成为预防和治疗腹膜纤维化的新靶点。     

高糖对大鼠肾小球内皮细胞肾素-血管紧张素系统的影响及机制

目的 探讨高糖对大鼠肾小球内皮细胞肾素-血管紧张素系统（RAS）的影响及机制。 方法 5 mmol/L、30 mmol/L葡萄糖（加或不加卡托普利、糜蛋白酶抑制剂）分别作用于细胞12、24、48、72 h后,用ELISA法检测上清与细胞内的血管紧张素Ⅱ（AngⅡ）水平;实时荧光定量PCR法检测血管紧张素原、肾素mRNA的表达;Western印迹法检测细胞AngⅡ1型受体（AT1R）、AngⅡ2 型受体（AT2R）、血管紧张素原及肾素的表达;激光共聚焦间接免疫荧光法检测AT1R、AT2R、肾素在细胞内的分布及改变。 结果 与对照组相比,高糖刺激细胞12、72 h时,上清AngⅡ水平均升高（P < 0.05）;而在细胞内AngⅡ水平只在72 h时有升高 （P < 0.05）;在高糖刺激24、48 h时,细胞内外AngⅡ水平均未发生明显变化。加入卡托普利、糜蛋白酶抑制剂预处理,然后高糖刺激细胞72 h,上清和胞内AngⅡ水平较不加抑制剂时显著下降（P < 0.05）。高糖可使血管紧张素原mRNA表达上调、肾素 mRNA表达下调（均P < 0.05）。同时,高糖可使血管紧张素原蛋白表达上调、AT1R蛋白表达下调,而AT2R、肾素蛋白表达量无明显变化,但激光共聚焦间接免疫荧光观察到AT2R发生由细胞核到细胞质的转移。 结论 高糖可激活大鼠肾小球内皮细胞内的RAS,这种作用至少与血管紧张素转换酶（ACE）和非ACE途径有关。高糖可通过增加底物水平引起肾小球内皮细胞AngⅡ的改变,同时AngⅡ受体在高糖刺激下也发生相应的改变。     

原发性IgA肾病患者肾脏局部血管紧张素Ⅱ表达与肾脏纤维化相关

目的 研究原发性IgA肾病患者肾脏局部肾素-血管紧张素系统（RAS）组分的表达及其相互调节,探讨肾内血管紧张素Ⅱ（AngⅡ）表达与临床病理损伤指标间的关系。 方法 采用免疫组织化学方法评价肾脏局部RAS组分的表达。分析36例原发性IgA肾病患者肾脏局部RAS组分表达之间的相关性以及肾内AngⅡ表达与血压、估算肾小球滤过率（eGFR）、24 h尿蛋白量和Katafuchi肾脏病理评分之间的相关性。 结果 肾内肾素、血管紧张素原与AngⅡ表达呈正相关（r ＝ 0.43,P < 0.01;r ＝ 0.34,P < 0.05）。肾内AngⅡ表达与eGFR呈负相关（r ＝ -0.61,P < 0.01）,并与病理慢性积分及间质炎性细胞浸润积分呈正相关（ρ ＝ 0.39,P < 0.05;ρ ＝ 0.52,P < 0.05）。 结论 IgA肾病患者肾内AngⅡ表达与肾内肾素、血管紧张素原表达相关,并且肾内AngⅡ表达与肾脏纤维化程度相关。     

连续性血液透析滤过对多器官功能障碍犬器官功能和细胞因子的影响

观察连续性血液透析滤过（CVVHDF）对多器官功能障碍综合征(MODS)犬器官功能和细胞因子的影响，并探讨CVVHDF治疗MODS的机制。 方法 15只Beagle犬采用失血性休克+复苏灌注+内毒素血症复制MODS模型，随机分为CVVHDF组(n=8)和MODS组(n=7)。内毒素注射完毕后CVVHDF组接受CVVHDF治疗12 h；MODS组未接受CVVHDF治疗。检测各时间点动脉血氧分压(PaO2)、动脉血二氧化碳分压(PaCO2)、谷丙转氨酶（ALT）、谷草转氨酶（AST）、Scr、BUN的水平。ELISA检测血浆TNF-α、IL-6、IL-10浓度。 结果 CVVHDF治疗后血浆IL-6、IL-10显著下降, 在3、6、9及12 h时间点显著低于MODS组，差异均有统计学意义（P < 0.01）；血浆TNF-α浓度无明显变化，两组间各时间点差异无统计学意义。超滤液中检出IL-6、TNF-α，筛选系数分别为0.27±0.13、0.1±0.1，但未能检出IL-10。CVVHDF组各主要器官功能均明显改善, 平均动脉压基本保持在正常水平，尤其在6、9及12 h时间点显著高于MODS组，差异均有统计学意义（P < 0.01）。PaO2逐渐升高，与MODS组在3、6、9及12 h时间点比较，差异均有统计学意义（P < 0.01）。 结论 连续性血液透析滤过能有效降低血浆IL-6、IL-10水平,使抗炎反应和促炎反应两方面趋于动态平衡；能明显改善内毒素诱导的低血压，提高动脉血氧分压。     

血管紧张素Ⅱ受体拮抗影响肝纤维化的实验研究

目的 观察血管紧张素Ⅱ及其受体拮抗剂对体外培养的肝星状细胞合成转化生长因子-β1(TGF-β1),以及金属蛋白酶组织抑制因子-1(TIMP-1)mRNA表达的影响. 方法 采用HSC-T6肝星状细胞系作为活化的肝星状细胞的研究模型.将培养的肝星状细胞随机分为对照组、血管紧张素Ⅱ(AngⅡ)组、受体拮抗剂(AT1RA)组和血管紧张素Ⅱ+受体拮抗剂(AngⅡ+AT1RA)组.采用ELISA法检测细胞培养上清液中TGF-β1蛋白的含量.RT-PCR法检测肝星状细胞中TIMP-1 mRNA的表达. 结果 细胞培养上清液中TGF-β1蛋白的含量,对照组、AngⅡ组和AngⅡ+AT1RA组分别为(7.531±0.654)pg/mL、(9.855±1.485)pg/mL和(7.719±0.329)pg/mL,AngⅡ组高于对照组(P＜0.05),AngⅡ+AT1RA组显著低于AngⅡ组(P＜0.05).肝星状细胞TIMP-1mRNA的表达水平,对照组、AngⅡ组和AngⅡ+AT1RA组分别为3.387±0.042、4.870±0.061和3.837±0.042,AngⅡ组高于对照组(P＜0.05),AngⅡAT1RA组显著低于AngⅡ组(P＜0.05). 结论 血管紧张素Ⅱ能够促进肝星状细胞TGF-β1蛋白的合成以及TIMP-1 mRNA的表达,而血管紧张素Ⅱ1型受体拮抗剂能够明显抑制这一作用.     

血管紧张素Ⅱ调节肾脏球旁器颗粒细胞合成肾素的机制研究

目的 观察血管紧张素Ⅱ（AngⅡ）对肾脏球旁器颗粒细胞肾素分泌的调节作用。 方法 密度梯度离心原代分离小鼠肾小球球旁器颗粒细胞（JGC）。实时定量PCR法检测JGC内血管紧张素转换酶（ACE）、AngⅡ受体（AT）1型和2型（AT1和AT2） mRNA表达。不同浓度的AngⅡ与球旁器细胞共孵育后,放射免疫法测定上清中肾素活性;实时定量PCR法测定细胞内肾素mRNA表达;化学发光法测定细胞内、外环磷酸腺苷（cAMP)水平的剂量和时间效应。改变细胞内钙离子浓度后,观察JGC内和上清中cAMP浓度改变。实时定量PCR法检测AngⅡ对JGC内腺苷酸环化酶（AC）5和AC6 mRNA表达的影响。 结果 成功分离的JGC存在ACE、AT基因表达。AngⅡ可抑制JGC肾素分泌[（370.6±36.9）比（299.6±25.7） ng AngI&#8226;ml-1&#8226;h-1,P = 0.014],并能抑制基础状态和前列腺素E2、去甲肾上腺素诱导的肾素mRNA表达。AngⅡ剂量依赖地降低JGC内和上清中cAMP水平。内质网上的Ca-ATP泵抑制剂毒胡萝卜内酯和环盐酸吗甲吡嗪酸均能剂量依赖地降低cAMP水平。细胞内钙离子螯合剂BAPTA-AM可降低细胞内钙离子浓度,进而使细胞内cAMP水平显著升高[（11.09±0.48）比（3.55±0.47） nmol/L,P < 0.01]。AngⅡ能通过减少42.12%的AC5 mRNA 表达,进而抑制肾素分泌。 结论 AngⅡ直接抑制肾素分泌可能是通过影响AC5,进而抑制cAMP表达来实现的。JGC内调节肾素分泌过程中,钙离子途径和GSα-cAMP两大信号传导途径中存在交联对话。     

连续性血液透析滤过对MODS犬肝肾IL-6和IL-10 mRNA表达的影响

目的：探讨连续性血液透析滤过（CVVHDF）后多器官功能障碍综合征（MODS）犬肝、肾组织白细胞介素-6（IL-6）和白细胞介素-10（IL-10）mRNA表达水平的变化及意义。方法：15只雄性Beagle犬,采用失血性休克＋复苏灌注＋内毒素血症复制MODS模型,随机分为CVVHDF组（n=8）和MODS组（n=7）,CVVHDF组在内毒素注射完毕后给予CVVHDF治疗12h,MODS组不给CVVHDF治疗。测定各器官功能相关指标,同时应用半定量逆转录-聚合酶链反应（RT-PCR）测定两组肝、肾组织中IL-6、IL-10mRNA表达水平。结果：CVVHDF组治疗后肝、肾功能有关指标水平均有不同程度的改善;与MODS组相比,在内毒素注射后3h及以后各时间点,血清肌酐（Scr）、尿素氮（BUN）水平显著降低（P〈0.05）;器官衰竭发生率较MODS组明显降低（37.5%vs85.7%,P〈0.05）;MODS组肝、肾组织IL-6mRNA表达水平显著高于正常对照组和CVVHDF组（P〈0.01）,而CVVHDF组肝、肾组织IL-10 mRNA表达水平显著高于正常对照组和MODS组（P〈0.01）。结论：连续性血液透析滤过能明显改善肾功能,CVVHDF早期应用可以降低MODS肝、肾组织IL-6/IL-10mRNA比值,有助于重建机体免疫系统内稳状态。     

肾素-血管紧张素系统在慢性环孢素肾病中的作用

目的测定肾素、血管紧张素Ⅱ(AngⅡ)在慢性环孢素(CsA)肾病肾移植患者肾组织、血浆中的水平,探讨肾素-血管紧张素系统(RAS)在慢性CsA肾病中的作用.方法(1)采用免疫组化、放射免疫和荧光偏振法,测定13例慢性CsA肾病患者肾组织中肾素表达及血浆AngⅡ、CsA早晨服药后2 h血浓度(C2)水平,并结合临床表现进行分析.(2)体外培养人脐静脉内皮细胞株(HUVEC)、大鼠系膜细胞株(MC),经不同浓度CsA(0、250、500、1000 μg/L)孵育细胞后,用免疫组化和放免方法测定肾素的表达及AngⅡ的含量.结果慢性CsA肾病组肾素指数与对照组比较显著增高(P＜0.01);血浆AngⅡ、C2水平高于对照组,但无显著性差异[(122.69±26.73)pg/ml比(121.88±36.35)pg/ml,P=0.977,(719.04±55.89)pg/ml比(658.80±90.78)pg/ml,P=0.196].CsA在一定浓度范围内呈剂量依赖刺激HUVEC、MC分泌AngⅡ及上调肾素的表达.结论移植肾活检可避免慢性CsA肾病漏诊或误诊.慢性CsA肾病肾移植患者肾组织局部RAS活化,导致血浆AngⅡ水平与肾组织局部表达不一致.CsA刺激血管内皮细胞、肾小球系膜细胞分泌AngⅡ,上调肾素的表达.阻断RAS可能有益于延缓慢性CsA肾病的进程.     

白蛋白对肾小管上皮细胞血管紧张素转换酶表达的影响

目的 观察白蛋白对肾小管上皮细胞血管紧张素转换酶（ACE） mRNA和蛋白水平表达的影响，并探讨尿蛋白激活肾脏局部肾素-血管紧张素系统（RAS）的机制。 方法 分别采用2.5、5、10 g/L的牛血清白蛋白（BSA）刺激人近端肾小管上皮细胞株（HK-2） 6 h和12 h。并分别采用实时定量PCR和Western印迹检测ACE mRNA和蛋白水平的表达。 结果 与对照组相比，随着BSA刺激浓度的增加，HK-2细胞ACE mRNA表达均显著增加（均P <0.05)。同时，Western印迹显示ACE蛋白表达也均显著增加(均P < 0.05)。另外，BSA 10 g/L作用于HK-2细胞6 h和12 h后，ACE mRNA表达显著增加(均P < 0.05)；Western印迹显示ACE蛋白也显著增加（均P < 0.05）。 结论 BSA可显著增加HK-2细胞ACE表达，此作用可能是导致肾间质局部AngⅡ蓄积从而启动肾小管间质纤维化的重要机制之一。     

Angiotensin II up-regulates the release of inflammation factors via Toll-like receptor 4 signal in rat mesangial cells under high glucose

Objective To observe the regulation of Toll-like receptor 4 (TLR4) signal and the release of inflammation factors after angiotensin II (AngⅡ) stimulation in rat mesangial cells under high glucose condition, revealing the innate immune-related mechanism of injury by AngⅡ on mesangial cells under high glucose. Methods After synchronization, cells incubated with AngⅡ(10-7 mmo/L) and/or high glucose (25 mmol/L) were used as the stimulation group, cells without stimulation were as normal control (5.6 mmol/L glucose). To determine the role of TLR4 and the adaptor myeloid differentiation factor 88 (MyD88), equal number of HBZY-1 cells were added with 10-5 mmol/L irbesartan and/or TLR4 blocker (10 mg/L) for 1 h and then incubated with AngⅡ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively. Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h. Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4, MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1, IL-6 in cell supernatant respectively. Results Compared with normal control group, TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or AngⅡ-induced HBZY-1 cells (P＜0.01), TLR4, MyD88 and NF-κB protein as well as MCP-1, IL-6 were also up-regulated significantly (P＜0.01). Compared with high glucose or AngⅡ group, MyD88 and NF-κB protein as well as MCP-1, IL-6 were further up-regulated markedly in AngⅡ and high glucose costimulated group (P＜0.01)．In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker, TLR4 and MyD88 protein expression were obviously inhibited, IL-6 and MCP-1 production were also decreased remarkably compared with high glucose and/or AngⅡ group (P＜0.01). Conclusions High glucose and AngⅡ stimulate the release of proinflammatory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway. This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal. AngⅡ has the positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.     

Effects of B7-1 on the cytoskeleton rearrangement in podocytes induced by angiotensin Ⅱ

Objective To observe the effect of costimulatory molecule B7-1 on cytoskeleton rearrangement in mouse podocytes induced by angiotensinⅡ(AngⅡ), and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes. Methods All cultivation of conditionally immortalized mouse podocytes (MPC) in vitro were divided into the following groups: normal control group, CTLA-4 group, AngⅡgroup (10-6 mmol/L 12 h, 24 h; 10-8 mmol/L 12 h, 24 h) and CTLA-4 with AngⅡgroup. Transfect B7-1 RNA interference fragment (siRNA) to the mature podocytes, and then restimulated by AngⅡ(10-6 mmol/L 12 h), the change of podocyte cytoskeleton after AngⅡ stimulation were observed. The expression of B7-1 in each group was assayed by flow cytometry and Western blotting. The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting. At the same time, the podocyte cytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy. Results Flow cytometry and Western blotting showed that B7-1 was not expressed in the normal control group. AngⅡshowed a concentration and time dependent induction of B7-1 expression in mouse podocytes (P＜0.05). Western blotting indicated that AngⅡinduced B7-1 protein expression (P＜0.05). Expression of nephrin and p-nephrin was significantly down-regulated by AngⅡ(P＜0.05). Compared with the normal control group, the expression of podocyte protein nephrin and p-nephrin in Ang II stimulation group was significantly reduced (P＜0.05). Using FITC phalloidin fluorescence staining showed that CTLA-4+AngⅡ stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score (mCFS) decreased compared with AngⅡ group (P＜0.05), suggesting that AngⅡ led to the disorder of the podocytes cytoskeleton and the destruction of the cytoskeleton of podocytes by AngⅡ could be improved after B7-1 blocking. Compared with the AngⅡ stimulation, transfection of B7-1 siRNA+AngⅡ stimulation group improved F-actin cytoskeletal rearrangement, and mCFS also decreased significantly (P＜0.05), suggesting that transfection of B7-1 siRNA might improve the damage of AngⅡ on podocytes cytoskeleton. Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.     

血管紧张素Ⅱ灌注诱导大鼠肾脏血管紧张素转换酶2表达改变

目的 研究血管紧张素Ⅱ(AngⅡ)灌注后大鼠肾脏血管紧张素转换酶2(ACE2)表达的改变及其意义。 方法 雄性SD大鼠随机分为AngⅡ灌注组(400 ng&#8226;kg-1&#8226;min-1)、生理盐水灌注组和健康对照组，每组6只。测定28 天内大鼠血压及尿蛋白量变化。于第28天处死动物取肾，观察组织学改变，并用免疫组化、RT-PCR及Western印迹法检测ACE2表达及分布变化；凝胶电泳迁移率分析法(EMSA)检测核因子(NF)-κB的DNA结合活性变化。 结果 (1) AngⅡ灌注组大鼠血压升高，并出现明显蛋白尿。AngⅡ灌注后第28天，部分肾小球出现轻中度系膜增生，少数有节段性硬化；部分肾小管上皮细胞变性、坏死或萎缩，间质灶性炎性细胞浸润。(2) 健康大鼠肾脏ACE2主要分布于肾小管，以近端肾小管刷状缘分布最多。AngⅡ灌注后第28天，肾皮质ACE2 mRNA及蛋白表达均明显下降(P均< 0.05)，NF-κB结合活性显著增加(P < 0.05)。相关分析表明，ACE2表达与NF-κB结合活性之间呈负相关(r = -0.64，P < 0.01)。 结论 AngⅡ灌注可致大鼠肾脏ACE2表达明显下降，后者与肾损害的程度密切相关。肾脏ACE2表达下降可能是AngⅡ引起肾损害的重要机制     

先兆子痫大鼠循环、胎盘及肾脏局部血管紧张素Ⅱ及其1型受体的表达

目的 研究先兆子痫大鼠模型循环系统、胎盘及肾脏局部血管紧张素Ⅱ（AngⅡ）及其1型受体（AT1）的表达。 方法 采用一氧化氮合酶抑制剂亚硝基左旋精氨酸甲酯（L-NAME）制备大鼠先兆子痫模型，分别比较先兆子痫大鼠、正常妊娠大鼠、未孕对照组大鼠动脉收缩压（SBP）、尿蛋白量(24 h)、肝肾功能，并对各组大鼠肾组织进行光镜检查。ELISA法和放射性免疫法分别测定各组大鼠血浆及肾脏局部匀浆液AngⅡ水平。Western印迹法检测大鼠胎盘局部AT1表达。免疫组化法及Western印迹法测定大鼠肾脏局部AT1的表达。 结果 在先兆子痫大鼠中，SBP及尿蛋白量(24 h)均较未孕对照组显著升高（P < 0.05）。先兆子痫大鼠血浆AngⅡ显著高于正常妊娠组[（0.706±0.086） ng/L比(0.540±0.085) ng/L，P < 0.05]；胎盘局部AT1表达比正常妊娠组升高46％（P < 0.05）；肾脏局部AngⅡ明显低于正常妊娠组[(65.543±40.634) ng/g比(165.543±33.078) ng/g，P < 0.05]；肾脏AT1表达减少，仅为正常妊娠组及未孕对照组的33％及59％（P < 0.05）。 结论 先兆子痫中，胎盘局部RAS表达增强，循环AngⅡ表达增高，肾脏局部RAS表达下调。     

血管紧张素Ⅱ通过TLR4-MyD88途径诱导大鼠肾小管上皮细胞炎性因子释放

目的 观察血管紧张素Ⅱ（AngⅡ）刺激肾小管上皮细胞（NRK-52E）后肿瘤坏死因子α（TNF-α）和热休克蛋白47（HSP47）的表达,分析Toll样受体4（TLR4）信号通路的变化与上述因子的关联,探讨AngⅡ促进NRK-52E细胞炎性反应、纤维化的天然免疫机制。 方法 细胞同步化后,将其分为4组：对照组、AngⅡ（10-7 mmol/L）组、坎地沙坦（10-5 mmol/L）+AngⅡ组、TLR4阻断剂（20 mg/L）+AngⅡ组,培养6 h后RT-PCR法检测TLR4及转接信号髓分化因子88（MyD88）mRNA表达水平;12 h后免疫荧光法检测细胞表面TLR4蛋白表达;24 h后以ELISA法检测细胞上清液TNF-α及HSP47的浓度。 结果 与对照组相比,AngⅡ显著上调NRK-52E细胞 TLR4、MyD88 mRNA和TLR4蛋白表达（均P < 0.01）,并诱导细胞TNF-α和HSP47的释放（均P < 0.01）。与AngⅡ组相比,TLR4阻断剂和坎地沙坦干预均显著抑制 AngⅡ对细胞TLR4、MyD88的刺激效应（均P < 0.01）;坎地沙坦抑制AngⅡ诱导的细胞 TNF-α、HSP47的释放（均P < 0.01）,TLR4阻断剂对细胞 TNF-α、HSP47的下调呈剂量依赖性。 结论 AngⅡ对NRK-52E细胞天然免疫信号TLR4、MyD88具有激活效应,该信号激活可能是AngⅡ促进肾小管细胞炎性相关因子释放的重要机制之一。坎地沙坦抑制肾小管细胞炎性因子的体外效应也与其调节该信号通路有关。     

Escherichia coli lipopolysaccharide inhibits renin activity in human mesangial cells

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.     

全反式维甲酸对5/6肾大部切除大鼠肾素-血管紧张素系统的影响

目的 观察全反式维甲酸（atRA）对残余肾肾素-血管紧张素系统（RAS）的影响。 方法 采用5/6肾大部切除大鼠模型，分别给予5 mg·kg-1·d-1（n＝8）、10 mg·kg-1·d-1（n＝8）、20 mg·kg-1·d-1（n＝8）的atRA灌胃。单纯肾大部切除非干预组（NX组，n＝8）和假手术组（sham组，n＝8）为对照。放射免疫法检测大鼠肾皮质肾素、血管紧张素水平。Western印迹检测肾组织血管紧张素1型受体（AT1R）水平。实时定量PCR方法检测激活蛋白1（AP-1）亚单位c-jun和c-fos mRNA的表达水平。 结果 atRA 灌胃10周时， 3种剂量atRA组收缩压明显低于NX组（P < 0.05），但不同剂量atRA组间差异无统计学意义。NX组大鼠肾皮质肾素和血管紧张素Ⅱ水平均显著高于sham组（P < 0.05）。atRA治疗组肾皮质肾素和血管紧张素Ⅱ水平均显著低于NX组，其中20 mg·kg-1·d-1组二者水平显著低于其他两个剂量组。atRA抑制残肾皮质AT1R表达约30％，而20 mg·kg-1·d-1剂量组AT1R表达水平最低。atRA组AP-1亚基c-jun和c-fos mRNA表达显著低于NX组（P < 0.05）。 结论 atRA能明显抑制5/6肾大部切除大鼠肾皮质RAS主要成分的高表达。     

血管紧张素Ⅱ对大鼠肺微血管内皮细胞APJ mRNA和Apelin mRNA表达的影响

目的 探讨血管紧张素Ⅱ(AngⅡ)对肺微血管内皮细胞APJ mRNA和Apelin mRNA表达的影响.方法 培养并鉴定24 h新生SD大鼠肺微血管内皮细胞,取2～4代培养细胞,细胞密度5×105个/ml,培养至80%融合后,进行药物干预.第一部分实验分为5组(n=4):加入AngⅡ至终浓度分别为10-9 mol/L(Ⅱ组)、10-1 mol/L(Ⅲ组)、10-7mol/L(Ⅳ组)、10-6 mol/L(Ⅴ组),Ⅰ组不加入AngⅡ,24 h后采用RT-PCR技术测定Apelin mRNA和APJ mRNA的表达水平.第二部分实验分为6组(n=4):加入AngⅡ至终浓度为10-7mol/L,分别在孵育即刻(Ⅰ组)、1 h(Ⅱ组)、6 h(Ⅲ组)、12 h(Ⅳ组)、24 h(Ⅴ组)、48 h(Ⅵ组)时,采用RT-PCR技术测定Apelin mRNA和APJ mRNA的表达水平.结果 第一部分实验结果:与Ⅰ组比较,Ⅱ组Apelin mRNA表达上调,APJ mRNA表达下调,Ⅲ组～V组Apelin mRNA和APJ mRNA表达下调(P<0.05或0.01),与Ⅱ组比较,Ⅲ组～Ⅴ组Apelin mRNA和APJ mRNA表达下调,呈浓度依赖性(P<0.01).第二部分实验结果:Apelin mRNA在10-7mol/L AngⅡ孵育6 h内表达上调,孵育1 h时达高峰(P<0.05或0.01),孵育6 h后Apelin mRNA表达下调,且呈时间依赖性(P<0.01);而APJ mRNA在孵育12 h后呈时间依赖性持续下调(P<0.01).结论 AngⅡ呈浓度和时间依赖性地下调Apelin mRNA和APJ mRNA的表达,可能是其参与肺微血管内皮细胞损伤的发生机制.     

Effect of angiotensinⅡ type 1 receptor blocker on the 12-lipoxygenase activity and P-cadherin expression in type 2 diabetic rat glomeruli

Objective To investigate the effect of angiotensinⅡ(AngⅡ) type 1 receptor blocker (ARB) on 12-lipoxygenase (12-LO) activity and P-cadherin expression in type 2 diabetic rat glomeruli. Methods Podocytes were stimulated by 10-7 mol/L AngⅡ for 24 hours. 12(S)-HETE (1 mg•kg-1•d-1) and AngⅡ (400 ng•kg-1•min-1) were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively. Rats fed with high fat diet received low dose streptozotocin (STZ) to make type 2 diabetes and divided into 2 groups: low dose STZ (DN group), low dose STZ＋ARB treatment (Losartan group). Rats fed with regular chow were used as control group. All the rats were sacrificed after 6 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. ELISA, RT-PCR and Western blotting for related target were performed respectively. Results AngⅡincreased 12(S)-HETE levels in podocytes and glomeruli (all P＜0.01). AngⅡ levels in the glomeruli were significantly increased by 12(S)-HETE stimulation (P＜0.01). Blood glucose, kidney/body weight and 24 hour urinary protein were increased in DN group compared with that in control group (all P＜0.01). However, urine protein, Kidney/body weight were decreased in Losartan group compared with DN group (all P＜0.05). Increment of 12(S)-HETE content and decrement of P-cadherin expression were observed in DN glomeruli compared with that in control group(all P＜0.01). These abnormalities were prevented by administration of the losartan (all P＜0.05). Conclusions AngⅡ can down-regulate glomerular P-cadherin expression via activation of 12-LO.ARB can ameliorate the progression of DN via up-regulation of glomerular P-cadherin through inhibition of 12-LO activation in type 2 DN rats.     

Effects of continuous venovenous hemodiafiltration on plasma LPS,IL -1β, IL -4, HMGB -1 in multiple organ dysfunction syndrome dogs

Objective To investigate the effects of continuous venovenous hemodiafiltration (CVVHDF) on plasma lipopolysaccharide (LPS), interleukin(IL) 1β, IL-4, high mobility group box 1 (HMGB-1) in multiple organ dysfunction syndrome (MODS ) dogs, to explore the therapeutic mechanism of CVVHDF. Methods Dogs were subjected to hemorrhagic shock plus resuscitation and endotoxemia to establish MODS model, then they were randomly divided into 2 groups: CVVHDF group (n=6) and MODS group (n=6). After endotoxin injection completed, the CVVHDF group received CVVHDF treatment for 24 h; MODS group did not receive CVVHDF treatment. The LPS, IL-1β, IL-4, HMGB-1 levels in plasma and ultrafiltrate were measured at different time points(before operation, before intravenous infusion of endotoxin, 0 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h after intravenous infusion completion of endotoxin), then the sieving coefficient (SC) was calculated. Results Compared with MODS group, the LPS, IL-1β, IL-4, HMGB-1 levels in plasma were significantly decreased in the CVVHDF group at 9 h, 12 h, 18h,24h(P ＜0.05).SomeconcentrationofIL-1β,IL-4andHMGB-1wasdetectedintheultrafiltrate,the SC for IL-1β, IL-4 and HMGB-1 was 0.60±0.12, 0.83±0.11, 0.70±0.09 respectively, and LPS could not be detected in the ultrafiltrate, its SC was 0. After treatment, the damage level of lung and renal had been significantly reduced in CVVHDF group. Conclusion CVVHDF can effectively reduce the levels of pro-inflammatory mediator and anti-inflammatory cytokine related to MODS, cut off the vicious circle of cytokine network and reduce the damage level of organism in the treatment of MODS.