糖尿病大鼠肾脏组织中磷酸化p38丝裂原活化蛋白激酶的表达特征及意义

目的:探讨糖尿病(DM)大鼠肾组织p38丝裂原活化蛋白激酶(MAPK)及其下游转录因子cAMP反应元件结合蛋白(CREB)的表达特征及与肾小球肥大、细胞外基质积聚的关系.方法:70只雄性Wistar2周后,随机分为两组:右肾切除对照组(CN)和DM组.DM组经腹腔注射链脲佐菌素(STZ,65 mg/kgDM.CN组注射等量枸橼酸缓冲液.两组分别于注射后第1、2、4、8和12周各取6只大鼠,收集24 h尿液,测定尿蛋白(Upro)、肌酐(UCr);股动脉放血分离血清,测血糖(Glu)、血肌酐(SCr)、尿素氮(BUN),并计算肌酐清除率(CCr);免疫组化法检测肾皮质磷酸化p38 MAPK (P-p38 MAPK)及?CREB(P-CREB)的表达特征,并检测转化生长因子-β1(TGF-β1)、纤维粘连蛋白(FN)及层粘连蛋白(LN)的表达,应用图像分析系统进行定量分析.流式细胞术检测P-p38 MAPK和P?CREB蛋白的表达.结果:与CN组比较,DM组P-p38 MAPK在1、2、4和8周升高(P均<0.01),第12周降至正常.DM组P-CREB在第2、4和8周增高(P均<0.01),在12周时降至正常.TGF-β1、FN、LN分别于第2、4周开始升高.结论:肾小球p38 MAPKCREB活性在早期糖尿病肾病大鼠肾组织中升高,p38 MAPK途径的激活有可能在糖尿病肾病;的早期肾小球肥大和细胞外基质积聚中发挥一定作用.     

p38MAPK和JNK在大鼠肾缺血-再灌注损伤模型中的表达及意义

目的 观察p38MAPK、JNK在肾缺血-再灌注损伤大鼠肾组织中的表达和活化,从而探讨p38MAPK、JNK信号转导通路与肾缺血-再灌注损伤的关系.方法 用缺血1 h再灌注1 h 制备缺血-再灌注模型,20只健康雄性SD大鼠[体质量(200±20)g]随机分为假手术组(n=10)、缺血-再灌注损伤组(n=10).检测肾组织丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性及血尿素氮(BUN)、肌酐(Cr)浓度.观察肾组织光镜、电镜下的形态学变化,用RT-PCR技术检测两组肾组织p38MAPK、JNK mRNA的表达情况,Western印迹法观察p38MAPK、JNK的活化情况.结果 缺血-再灌注损伤后,大鼠肾功能和肾小管上皮细胞明显受损,BUN、Cr、MDA浓度均高于假手术组(P<0.01),SOD活性低于假手术组(P<0.01),p38MAPK、JNK磷酸化蛋白在假手术组呈少量散在表达或不表达,缺血-再灌注损伤后磷酸化p38MAPK、JNK呈阳性表达(P<0.01).p38MAPK、JNK mRNA在缺血-再灌注损伤组的表达较假手术组显著增高(P<0.01).结论 肾缺血-再灌注可增加p38MAPK、JNK的磷酸化水平及p38MAPK、JNK mRNA的转录水平,p38MAPK、JNK可能在肾缺血-再灌注损伤中起到至关重要的作用.     

吡格列酮对高糖培养大鼠肾小球系膜细胞p38丝裂原活化蛋白激酶和转化生长因子β-1的影响

目的观察吡格列酮(PIO)对高糖(HG)培养的肾小球系膜细胞(MCs)p38丝裂原活化蛋白激酶(p38MAPK)和转化生长因子β-1(TGF-β1)表达的影响,探讨PIO肾保护作用及机制。方法体外培养MCs,取对数生长期的细胞以2×105/孔的密度接种于6孔细胞培养板,同步化后随机分为正常对照(NG)组、HG组、p38MAPK抑制剂(S)组及PIO(P)组。Western blot测定磷酸化p38MAPK(p-p38)和总p38MAPK(t-p38)蛋白含量,半定量RT-PCR测定细胞TGF-β1 mRNA表达情况。结果与NG组比较,HG组细胞内p-p38MAPK含量和TGF-β1mRNA表达增强(P0.01);与HG组比较,p38MAPK抑制剂显著抑制HG刺激的细胞内p38MAPK活性和TGF-β1表达(P0.05);与HG组比较,P组细胞内上述变化亦明显降低(P0.05),与S组相似;p38MAPK活性和TGF-β1表达呈正相关(r=0.587,P0.01)。结论 PIO可抑制肾小球系膜p38MAPK通路,降低TGF-β1表达,该作用可能与其肾脏保护部分有关。     

参芪地黄汤对系膜增生性肾小球肾炎大鼠的作用及对p38MAPK/NF-κB信号通路的影响

目的]探讨参芪地黄汤对系膜增生性肾小球肾炎(MsPGN)大鼠的保护作用及对38丝裂原激活蛋白激酶/核转录固子Kappa B(p38MAPK/NF-xB)通路的影响。【方法】将40只大鼠随机分为假手术组、模型组及参芪地黄汤低剂量组和高剂量组,每组10只,采用胃肠道综合免疫法建立MsPGN大鼠模型(假手术组大鼠麻醉后仅暴露左肾,其余操作同模型组),其中参芪地黄汤低剂量组、高剂量组大鼠于模型建立成功后分别给予参芪地黄汤生药量(1.4 mg/mL、2.8 mg/mL溶液)10 mL/(kg·d)灌胃给药(连续4周),假手术组、模型组大鼠同时问灌胃等量生理盐水,检测各组尿液中的24 h尿蛋白及血尿素氮、血肌酐含量。ELISA法检测各组大鼠肾脏组织中白介素-8(IL-8)、肿癌环死因子α(TNF-α)水平;免疫印法检测各组大鼠肾脏组织中p38MAPK/NF-xB通路蛋白(p38MAPK、P-p38MAPK、NF-xB p65)水平,【结果】与假手术组比较,模型组及低剂量组、高剂量组大鼠24 h尿蛋白、尿素氮、血肌酐水平及肾组织中IL-8、TNF-α水平、P-p38MAPK/p38MAPK、NF-kB p65蛋白水平均升高(P<0.05);与模型组比较,低剂量组、高剂量组大鼠24 h尿蛋白、尿素氮、血肌酐水平及肾组织中IL-8、TNF-α水平、P-p38MAPK/p38MAPK、NF-xB p65蛋白水平均降低(P<0.05);与低剂量组比较,高剂量组大鼠各指标水平均降低(P<0.05)。【结论】参芪地黄汤对MsPGN大鼠有保护作用,可能是通过抑制p38MAPK/NF-kB通路活化,进而抑制肾脏组织炎症反应实现。     

来氟米特对糖尿病大鼠肾组织单核趋化蛋白1、转化生长因子β1表达的影响

目的探讨来氟米特对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠肾组织单核趋化蛋白1(MCP-1)、转化生长因子β1(TGF-β1)表达的影响,进一步探索来氟米特对糖尿病大鼠肾脏的保护作用。方法健康雄性SD大鼠54只随机分为3组:正常对照组(NC组)、糖尿病组(DM组)、来氟米特干预组(DM+LEF组),每组18只。应用STZ诱导糖尿病模型,成模后,DM+LEF组给予来氟米特5mg.kg-1.d-1溶液灌胃,于实验第4、6、8周末,各组分别取6只大鼠留取24小时尿量测24小时尿蛋白定量,内眦静脉采血测血肌酐、尿素氮,光镜观察肾组织病理变化,采用链霉菌抗生物素蛋白-过氧化物酶连结法(streptavidin-perosidase,SP)观察肾组织中MCP-1、TGF-β1表达,采用反转录聚合酶链反应(RT-PCR)检测肾组织MCP-1、TGF-β1mRNA的表达。结果与DM组比较,DM+LEF组大鼠24小时尿蛋白定量明显减少(P<0.01),DM+LEF组比同期DM组病理改变明显减轻,经来氟米特干预后,DM+LEF组大鼠肾组织MCP-1、TGF-β1mRNA表达明显减少。结论来氟米特可能通过减少肾组织MCP-1、TGF-β1表达,减轻肾损害,起到保护糖尿病肾脏的作用。     

有氧运动对衰老大鼠肾脏纤维化的影响

目的 观察规律有氧运动对衰老大鼠肾脏纤维化的影响并探讨其作用机制。 方法 采用随机数字表法将30只雄性SD大鼠分为青年对照组（YC组）、老年对照组（OC组）及老年运动组（OE组）,每组10只大鼠。YC组及OC组大鼠均置于鼠笼内安静饲养,OE组大鼠则给予8周中等强度跑台有氧运动。于实验结束后检测各组大鼠24 h尿蛋白、血尿素氮（BUN）及血清肌酐（SCr）含量,采用Masson染色检测肾脏纤维化程度,采用Western Blot技术检测转化生长因子β1（TGF-β1）、p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化p38MAPK（p-p38MAPK）、E-粘钙素及α-平滑肌肌动蛋白（α-SMA）表达水平。 结果 与YC组比较,OC组大鼠24 h尿蛋白、BUN及SCr明显升高（P<0.05）,肾脏纤维化程度增加（P<0.05）,TGF-β1、p-p38MAPK和α-SMA蛋白表达上调（P<0.05）,E-粘钙素表达下调（P<0.05）;与OC组比较,OE组大鼠24 h尿蛋白、BUN及SCr明显降低（P<0.05）,肾脏纤维化程度减轻（P<0.05）,TGF-β1、p-p38MAPK和α-SMA蛋白表达下调（P<0.05）,E-粘钙素表达增加（P<0.05）。 结论 有氧运动能缓解衰老大鼠肾脏纤维化,其作用机制可能与抑制TGF-β1/p38MAPK信号通路及上皮-间质转变有关。     

乌司他丁经p38MAPK通路对脓毒症大鼠急性肾损伤影响的研究

目的研究乌司他丁（UTI）经p38MAPK通路对脓毒症大鼠急性肾损伤（AKI）的影响。 方法采用脂多糖（LPS）诱导脓毒症AKI大鼠模型,将32只SD大鼠随机分为4组,空白对照组、脂多糖组（LPS组）、乌司他丁处理组（LPS+UTI组）、p38MAPK阻断剂干预组（LPS+SB组）,采用酶联免疫吸附试验（ELISA）法检测肾组织中肿瘤坏死因子α（TNF-α）和转化生长因子（TGF-β1）的表达,采用蛋白质免疫印迹试验（Western Blotting）检测肾组织中磷酸化p38MAPK蛋白的表达,并在光镜和电镜下观察肾小球及肾小管的变化。 结果与空白对照组相比,LPS组光镜下肾小球充血肿胀,肾球囊扩张,近曲肾小管上皮细胞肿胀明显,细胞核淡染,部分出现核浓缩、破碎甚至溶解现象,肾间质充血、水肿、大量炎性细胞浸润;电镜下肾小球毛细血管内皮细胞窗孔消失或闭塞,基底膜部分断裂消失,足细胞足突广泛融合消失,近曲肾小管上皮细胞质内线粒体排列紊乱,结构模糊,高度肿胀,有空泡样现象;均提示脓毒症肾损伤严重,且肾组织TNF-α、TGF-β1和p38MAPK蛋白的表达明显升高。与LPS组相比,LPS+UTI组肾组织TNF-α、TGF-β1和p38MAPK蛋白的表达均较LPS组下降［TNF-α（pg/ml）：4915.00±267.06 vs 8836.00±739.51;TGF-β1（pg/ml）：257.71±23.88 vs 354.39±29.44;p-p38MAPK/β-actin：0.158±0.022 vs 0.300±0.044,均P＜0.05］,LPS+SB组肾组织中TNF-α、TGF-β1和p38MAPK蛋白的表达较LPS组均下降［TNF-α（pg/ml）：4856.75±167.23 vs 8836.00±739.51;TGF-β1（pg/ml）：249.56±23.42 vs 354.39±29.44;p-p38MAPK/β-actin：0.136±0.017 vs 0.300±0.044,均P＜0.05］,但LPS+UTI组与LPS+SB组差异无统计学意义（P＞0.05）。 结论乌司他丁对脓毒症急性肾损伤有保护作用,且可能是通过抑制TGF-β1/p38MAPK信号转导通路来实现的。     

p38信号通路对单侧输尿管结扎大鼠RANTES的影响

目的探讨p38有丝分裂素激活蛋白激酶(MAPK)在单侧输尿管结扎(UUO)大鼠肾组织的表达及活化状态。方法采用免疫组织化学法和Western印迹分析法检测UUO大鼠肾组织p38MAPK的磷酸化水平和RANTES蛋白水平,逆转录-聚合酶链反应(RT-PCR)方法检测RANTES mRNA的表达。结果RANTES蛋白和RANTES mRNA随着梗阻时间的延长,表达明显上调,同时p38MAPK蛋白活性增高,两者呈显著正相关(P＜0．05)。结论UUO大鼠肾组织中p38MAPK的活化可上调RANTES的表达。     

氯沙坦对糖尿病大鼠肾小管间质P38MAPK表达的影响

【目的】探讨P38丝裂原活化的蛋白激酶（P38MAPK）与糖尿病肾病（DN）肾小管间质病变的关系,及氯沙坦对其的影响。【方法】雄性Wistar大鼠,随机分为正常对照组和模型组,后者经STZ诱导糖尿病模型成功后再随机分为糖尿病模型组和氯沙坦干预组。8周后检测各组大鼠24 h尿蛋白、血肌酐;留取肾组织行HE和MASSON染色,观察肾小管间质损伤指数、肾间质胶原面积;免疫组化检测肾小管间质磷酸化P38MAPK以及TGFβ1表达,并做半定量分析。【结果】①与对照组相比,糖尿病模型组大鼠尿蛋白排泄量、肾小管间质损伤指数和肾间质胶原面积明显增加（P〈0.01）;②糖尿病组大鼠肾小管间质磷酸化P38MAPK、TGFβ1表达均显著高于对照组（P〈0.01）;③氯沙坦组尿蛋白排泄量、肾小管间质损伤程度较糖尿病组减轻,肾小管间质磷酸化P38MAPK、TGFβ1表达较糖尿病组显著下调（P〈0.01）。【结论】糖尿病肾病小管间质病变的病理过程中存在P38MAPK的活化;氯沙坦可阻抑糖尿病大鼠肾小管间质P38MAPK活化,下调TGFβ1表达而发挥肾脏保护作用。     

miR-188-5p通过靶向调控丝裂原活化蛋白激酶信号通路对大鼠移植肾慢性排斥反应的作用

目的探讨miR-188-5p通过靶向调控丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)信号通路对大鼠移植肾慢性排斥反应的作用。方法 SPF级F344大鼠20只和Lewis大鼠40只,其中以Lewis大鼠供体10只、受体10只进行肾移植为对照组;以F344大鼠20只为供体,Lewis大鼠20只为受体行肾移植建立大鼠慢性排斥反应模型,并分为模型组和过表达组。3组均于术后连续注射环孢素A 1.5 mg/kg 10 d。过表达组术后当天同时注射miR-188-5p高表达慢病毒颗粒0.1 mL,对照组与模型组分别注射等量生理盐水。术后12周观察3组大鼠存活情况,比较3组大鼠肾功能指标;观察3组大鼠移植肾组织病理变化情况,采用慢性移植肾损伤指数评分系统评价移植肾损伤情况;采用反转录PCR法检测移植肾组织miR-188-5p、p38MAPK、细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2, ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)mRNA相对表达量,采用Western blot法检测p38MAPK、ERK1/2、JNK蛋白及其磷酸化蛋白相对表达量,并比较p-p38MAPK/p38MAPK、(p-ERK1/2)/(ERK1/2)、p-JNK/JNK。结果术后12周3组大鼠均存活。模型组和过表达组血清肌酐水平、24 h尿蛋白定量、慢性移植肾损伤指数评分高于对照组(P0.05),模型组高于过表达组(P0.05)。移植肾组织病理学观察,对照组存在交界性改变,仅少量炎症细胞浸润;模型组慢性排斥反应病理改变明显,过表达组较轻。过表达组移植肾组织miR-188-5p相对表达量(1.90±0.22)高于模型组(0.62±0.18)和对照组(1.02±0.25)(P0.05),对照组高于模型组(P0.05);模型组p38MAPK、ERK1/2、JNK mRNA相对表达量,p-p38MAPK、p38MAPK、p-ERK1/2、ERK1/2、p-JNK蛋白相对表达量及p-p38MAPK/p38MAPK、(p-ERK1/2)/(ERK1/2)、p-JNK/JNK高于过表达组和对照组(P0.05),过表达组高于对照组(P0.05)。结论 miR-188-5p可有效抑制大鼠移植肾慢性排斥反应,其机制可能与靶向调控MAPK信号通路中相关基因的表达及蛋白磷酸化过程有关。     

二十味沉香丸调控糖尿病肾病大鼠肠道菌群益生菌构成的机制研究

目的分析二十味沉香丸调控糖尿病肾病大鼠肠道菌群益生菌构成的机制。 方法选取50只SPF级8~10周龄雄性SD大鼠,10只作为对照组,其余40只建立糖尿病肾病模型,之后将40只大鼠随机分为模型组、二十味沉香丸低剂量组、二十味沉香丸中剂量组、二十味沉香丸高剂量组各10只。建模成功后开始灌胃,对照组、模型组给于3 ml生理盐水,二十味沉香丸低剂量组给予生理盐水+0.5 g/kg二十味沉香丸,二十味沉香丸中剂量组给予生理盐水+1 g/kg二十味沉香丸,二十味沉香丸高剂量组给予生理盐水+2 g/kg二十味沉香丸。观察每组肝功能指标、炎症因子表达水平、肠道菌群多样性与丰富度、p38丝裂素活化蛋白激酶（p38MAPK）通路蛋白表达量。 结果与对照组相比,模型组、二十味沉香丸低剂量组、二十味沉香丸中剂量组、二十味沉香丸高剂量组天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、超敏C-反应蛋白（hs-CRP）、肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）、白介素-1β（IL-1β）、p38MAPK、磷酸化cAMP反应元件结合蛋白（P-CREB）、纤维连接蛋白（FN）通路蛋白升高,AST/ALT水平、OTU数量、chaol指数、shannon指数、simpson指数降低（P＜0.05）;与模型组相比,二十味沉香丸低剂量组、二十味沉香丸中剂量组、二十味沉香丸高剂量组AST、ALT、hs-CRP、TNF-α、IL-6、IL-1β水平、p38MAPK、P-CREB、FN通路蛋白降低,AST/ALT水平、OTU数量、chaol指数、shannon指数、simpson指数升高（P＜0.05）;与二十味沉香丸低剂量组相比,二十味沉香丸中剂量组、二十味沉香丸高剂量组AST、ALT、hs-CRP、TNF-α、IL-6、IL-1β水平、p38MAPK、P-CREB、FN通路蛋白水平降低,AST/ALT水平、OTU数量、chaol指数、shannon指数、simpson指数升高（P＜0.05）;与二十味沉香丸中剂量组相比,二十味沉香丸高剂量组AST、ALT、hs-CRP、TNF-α、IL-6、IL-1β水平、p38MAPK、P-CREB、FN通路蛋白水平降低,AST/ALT水平、OTU数量、chaol指数、shannon指数、simpson指数升高（P＜0.05）。 结论高剂量二十味沉香丸可改善大鼠肝功能,缓解机体炎症反应,维持肠道菌群益生菌稳定。     

p38丝裂原活化蛋白激酶对大鼠肾脏分泌单核细胞趋化因子-1的影响

目的 动态观察单核细胞趋化因子-1(MCP-1)在单侧输尿管结扎(UUO)大鼠肾小管的表达,探讨它与p38丝裂原活化蛋白激酶(p38MAPK)、核转录因子-кB(NF-кB)及肾损害之间的关系. 方法 雌性SD大鼠共36只,体质量150～170 g.随机分为2组:假手术组作为对照组;手术组根据梗阻时间分3组,每组6只.皮下注射水合氯醛(4 mg/kg)麻醉,开腹后结扎右侧输尿管,缝合腹腔,即制备UUO模型.假手术组开腹后不结扎输尿管,缝合腹腔.采用免疫组织化学检测NF-кB、MCP-1,Western印迹分析法检测p38MAPK的磷酸化水平和lVICP-1蛋白水平,逆转录-聚合酶链反应(RT-PCR)方法检测MCP-1 mRNA的表达.光镜检查肾组织的形态改变.生化方法测定血尿素氮、血肌酐. 结果 与对照组比,随着梗阻时间的延长,MCP-1蛋白表达明显上调,[对照组:0.401±0.039,UUO组8h:0.894±0.137;24 h:1.416±0.135;72 h:1.894±0.143,与对照组相比,P<0.05],MCP-1 mRNA表达明显上调,[对照组:50.08±3.210,UUO组8 h:108.25±4.325;24 h:179.34±3.237;72 h:230.12±3.026,与对照组相比,P<0.05],同时NF-кB、p38MAPK蛋白活性增高,[p38MAPK蛋白对照组:110.65±9.734,UUO组8 h:200.15±8.326;24 h:272.74±7.244;72 h:549.11±9.544,与对照组相比,P<0.05],肾小管MCP-1的表达与p38MAPK、NF-кB呈显著正相关(r=0.74、r=0.81,P<0.01). 结论 UUO大鼠.肾小管MCP-1表达增加参与了UUO大鼠.肾小管间质损害的发病机制;p38MAPK可能介导了NF-кB的表达,进而调节MCP-1的表达增多.     

基质金属蛋白酶在大鼠肾小球硬化中的表达

目的检测基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）在大鼠局灶节段肾小球硬化中的表达,探讨其与细胞外基质沉积的关系。方法12只雄性Wistar大鼠,实验组7只,一次性静脉注射氨基核苷嘌呤霉素9mg/100g体重,对照组5只,静脉注射生理盐水一次,动态观察尿蛋白定量变化。第20周末宰杀,腹主动脉取血,检测血浆白蛋白、血脂、肾功能。留取肾脏,制备病理标本。应用免疫组织化学法观察两组大鼠肾组织中MMP-2、MMP-9和FN的表达,分析其在肾组织中的相对含量,及与蛋白尿程度的相关性。结果FSGS模型组大鼠用药后尿蛋白定量显著增高,血总胆固醇显著增高。肾脏病理变化示部分肾小球局灶节段硬化或全球性硬化,细胞外基质节段性增多。MMP-9在正常大鼠肾小球上皮细胞和内皮细胞有少量表达,在模型组大鼠肾组织中的表达显著减少（P〈0.05）;MMP-9的表达与尿蛋白定量之间无显著相关性。MMP-2在正常大鼠的肾小球动脉和肾小管间质有少量表达,在模型组大鼠肾组织中,MMP-2的表达略有减少（P〉0.05）;MMP-2的表达与尿蛋白的定量之间无显著相关性。在正常大鼠肾组织中FN表达于肾小球系膜区,基底膜及肾间质处,在模型组大鼠肾组织中FN的表达明显增多（P〈0.05）。FN的表达与尿蛋白定量、MMP-2的表达无显著相关性,与MMP-9的表达呈直线负相关。结论局灶节段肾小球硬化时,MMP-9的表达下调,可以引起FN降解减少,细胞外基质沉积,促进肾小球硬化的发生。     

促红细胞生成素对大鼠肾脏缺血再灌注损伤的抗炎保护作用及其机制

目的探讨促红细胞生成素（EPO）对大鼠肾脏缺血再灌注损伤（瓜I）的抗炎保护作用及其机制。方法36只雄性SD大鼠随机分为假手术（SOR）组、IRI组和EPO组,每组12只。于再灌注2h、6h、12h、24h各时相点观察肾脏病理改变,检测血尿素氮（BUN）和肌酐（Scr）水平,ELISA法检测肾组织匀浆肿瘤坏死因子-a（TNF-a）含量,Western印迹法检测磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）蛋白表达。结果IRI组血BUN、Scr水平和肾组织匀浆TNF=Ⅱ含量显著升高,并出现明显的肾脏病理改变;肾组织p-p38MAPK蛋白表达明显增强,于再灌注12h达到峰值,24h表达减弱。而在EPO组,BUN、Scr和TNF=a水平显著低于IRI组（P〈0．05）,在各时相点的肾脏病理改变与同期IRI组比较明显减轻,p-p38MAPK表达在各时相点较IRI组明显减弱。结论EPO可显著改善IRI造成的肾脏病理和肾功能异常,其肾脏保护作用可能与抑制p38MAPK活化、降低TNF．Ct水平、减轻炎性损伤有关。     

骨桥蛋白在糖尿病大鼠肾组织中的表达及意义

目的 观察糖尿病大鼠肾组织中骨桥蛋白(OPN)的表达及其与巨噬细胞(CD68)、细胞增殖核抗原(PCNA)之间的关系,初步探讨其在糖尿病肾损害中的意义.方法 雄性Wistar大鼠被随机均分为对照组和糖尿病组,每组24只.腹腔单次注射链脲佐菌素(STZ)诱发糖尿病大鼠模型,采用免疫组化和蛋白质免疫印迹法(Western blotting)分别检测OPN、CD68及PCNA在肾组织的表达,原位杂交检测肾组织中OPN mRNA的表达.结果 与对照组比较,糖尿病组大鼠在注射STZ后1、2、4和8周,血糖、尿素氮(BUN)和24 h尿蛋白定量显著增高,肌酐清除率(CCr)显著降低;OPN表达呈进行性增加并明显高于对照组(P均＜0.05);1周和2周时PCNA表达高于对照组,4周和8周时CD68表达较对照组略有增高.结论 OPN在糖尿病肾损害过程的早期可能与肾小管上皮细胞增殖有关,后期可能趋化巨噬细胞浸润.     

Urinary excretion and renal production of prostaglandins E2, F2 alpha, and thromboxane B2 in experimental diabetes mellitus

To delineate the urinary excretion of prostaglandins E2 (PGE), F2 alpha (PGF), and thromboxane B2 (TxB) in diabetic rats, we treated male Sprague-Dawley rats with streptozocin 60 mg/kg or with vehicle. Plasma glucose and creatinine concentration and 24-hour urine collections for determination of TxB, PGE, PGF, creatinine, and protein excretion were measured at 2, 8, and 14 weeks after streptozocin. Creatinine clearance was significantly decreased in diabetic rats at 2 and 8 weeks, whereas the urinary protein excretion was three to five times that of control animals at all times. The 24-hour excretion of TxB was elevated twofold to threefold in diabetic rats, whereas PGE and PGF excretion were both significantly decreased. This alteration in excretion was not caused by increased urine flow rate, inasmuch as mannitol-induced osmotic diuresis caused an increase in PGE and PGF excretion. Eight weeks after streptozocin, a group of diabetic and control rats were sacrificed, and the production of PGE, PGF, and TxB by the renal papillae and glomeruli determined. An additional five diabetic rats were treated with protamine zinc insulin for 1 week before sacrifice to determine the effect of insulin treatment on the production of PGE, PGF, and TxB by the glomeruli and renal papillae. Papillary production of PGE, PGF, and TxB was decreased in diabetic rats, as was glomerular production of PGE and PGF. Insulin treatment increased PGF production by the renal papillae and increased PGE, PGF, and TxB production by glomeruli. These results indicate that changes in prostaglandin excretion accompany the development of marked proteinuria and a decrease in creatinine clearance.(ABSTRACT TRUNCATED AT 250 WORDS)     

p38 MAPK inhibitors ameliorate target organ damage in hypertension: Part 1. p38 MAPK-dependent endothelial dysfunction and hypertension

Numerous mediators, believed to play a role in endothelial dysfunction (e.g., neurohormones, cytokines, hypoxia, and stretch), have been shown to activate p38 mitogen-activated protein kinase (MAPK) in a variety of cell types. The purpose of the present study was to examine the regulation of p38 MAPK in endothelium and its role in endothelial dysfunction and salt sensitivity. In cultured human umbilical vein endothelial cells (HUVECs), tumor necrosis factor-alpha and lipopolysaccharide increased phosphorylation of p38 MAPK (P-p38 MAPK) and increased ICAM-1 expression. Preincubation with highly selective p38 MAPK inhibitors, 1-(1,3-dihydroxyprop-2-yl)-4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl] imidazole (SB-239063AN) or SB-239063, dose dependently reduced intercellular adhesion molecule-1 expression in HUVECs. In spontaneously hypertensive-stroke prone rats (SHR-SP), P-p38 MAPK was localized by immunohistochemistry to the aortic endothelium and adventitia but was undetectable in aortae from normotensive rats. Introduction of a salt/fat diet (SFD) to the SHR-SP strain induced endothelial dysfunction (ex vivo vascular reactivity analysis), albuminuria, and an increase in blood pressure within 4 weeks. Chronic dietary dosing (approx. 100 mg/kg/day) with SB-239063AN inhibited the SFD diet-induced hypertension. In addition, delayed treatment also significantly improved survival and restored nitric oxide-mediated endothelium-dependent relaxation in SFD-SHR-SPs with established endothelial dysfunction. These results suggest an important role for p38 MAPK in endothelial inflammation and dysfunction as well as providing the first evidence for p38 MAPK-dependent hypertension.     

Exogenous activated protein C inhibits the progression of diabetic nephropathy

Summary. Background: Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown. Objectives: We investigated the therapeutic potential of APC in mice with streptozotocin‐induced diabetic nephropathy. Methods: Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month. Results: APC‐treated mice had a significantly improved blood nitrogen urea‐to‐creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein‐1 (MCP‐1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet‐derived growth factor (PDGF), transforming growth factor‐β1 and connective tissue growth factor (CTGF) were significantly lower in APC‐treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT‐1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment. Conclusion: Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease.     

Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats.

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.