白蛋白诱导的肾小管细胞凋亡与丝裂素激活蛋白激酶信号通路

目的 探讨白蛋白诱导肾小管上皮细胞凋亡以及诱导凋亡的信号传导机制｡ 方法 将培养的大鼠肾小管细胞NRK-52E分别与不同浓度(10､ 20､ 30 mg/ml)的去脂无内毒素牛血清白蛋白(BSA)共同孵育6､ 12､ 18和24 h｡透射电镜､共聚焦激光显微镜和流式细胞仪检测细胞凋亡｡BSA 20 mg/ml刺激NRK-52E细胞15､ 30､ 60和120 min后, Westen印迹测定p38､氨基末端激酶(JNK)和细胞外信号调节激酶(ERK)活性｡将SB202190(20 μmol/L, p38抑制剂)､SP600125(10 μmol/L, JNK抑制剂)和PD98059(20 μmol/L, ERK抑制剂)分别与白蛋白和NRK-52E细胞共同孵育24 h后检测细胞凋亡｡结果 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡｡白蛋白与NRK-52E细胞共孵育后,p38和JNK活性明显升高,ERK活性显著降低｡SB202190和SP600125可分别抑制白蛋白诱导NRK-52E细胞凋亡,而PD98059促进白蛋白诱导的NRK-52E细胞凋亡｡结论 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡,而p38和JNK激活与ERK抑制介导了白蛋白诱导的肾小管细胞凋亡｡     

代谢综合征及其代谢因子与慢性肾损害相关性的临床研究

目的 探讨代谢综合征及其各组成因子与包括轻度肾损害在内的慢性肾损害的相关性｡ 方法 收集我院2003年1月至2003年12月心内科､ 肾内科和内分泌科符合入选标准的住院患者966例进行回顾性分析｡按有､ 无慢性肾脏病(CKD)或轻度肾损害分组, 比较代谢综合征各因素与慢性肾脏损害的关系｡ 统计学处理包括单变量t检验､卡方检验和Logistic多因素回归分析｡结果 (1)CKD组的年龄､ 身体质量指数(BMI)､ 总胆固醇(TC)､ 甘油三酯(TG)､ 高血糖､ 高血压和尿酸水平, 冠心病､ 脑卒中的患病率均高于无CKD组, 而高密度脂蛋白(HDL)水平明显低于无CKD组患者; (2)随着代谢综合征因子数量的增多, CKD发病率上升; (3)代谢综合征中各因子并存较各因子单独存在的CKD的危险性增加, 与高血糖并存的频率最高; (4)BMI增加也是CKD的重要危险因素; (5)高血糖患者发生轻度肾损害的风险最大(优势比OR=7.698)｡结论 代谢综合征及其各组成因子是包括轻度肾损害在内的CKD的重要危险因素｡随着代谢综合征因子的增多, CKD的危险也随之增加｡ 除了高血糖和高血压, BMI增加也是其中重要的影响因子｡     

通过基因敲低探讨多个足细胞分子作用及分子间反应

目的 研究足细胞裂孔隔膜(SD)复合体分子nephrin､podocin和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应｡方法 针对nephrin､ podocin､CD2AP和α-actinin-4 mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以 “敲低”其表达｡免疫荧光染色观察其分布方式｡半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达｡ 结果 (1) podocin敲低组(siPod966和siPod54):未检测到podocin及nephrin mRNA,其蛋白分别下降了92%､79%及82%､67%｡而CD2AP mRNA和蛋白分别增加了62%､42%及71%､46%｡α-actinin-4无变化｡(2)nephrin敲低组(siNep492):未检测到nephrin mRNA和蛋白｡而CD2AP mRNA和蛋白分别增加了35%､48%｡Podocin和α-actinin-4无变化｡(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2AP mRNA,其蛋白分别下降了92%和83%｡Nephrin mRNA和蛋白分别下降了60%､48%及76%､72%;而podocin mRNA和蛋白分别增加了38%､22%及56%､44%｡Α-actinin-4无变化｡(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin 的mRNA分别下降了69%､58%及64%､49%;蛋白分别下降了81%和55%以及71%､64%｡而podocin以及CD2AP mRNA分别增加了50%､34%及45%､28%;蛋白分别增加了64%､46%及65%､42%｡(5)敲低nephrin､podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主｡α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中｡结论 (1)在SD复合体分子中,nephrin可能具有相对独立的作用｡(2) α-actinin-4对nephrin､podocin和CD2AP有直接或间接的作用｡(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”､也可能是“双向的”｡(4)nephrin､podocin､CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应｡     

两种病理类型肾病患者尿IgG对人肾小管上皮细胞表达巨噬细胞移动抑制因子的影响

目的 分离纯化表现为肾病综合征(NS)的微小病变型(MCD)及膜性肾病(MN)患者尿IgG，比较它们对人近端小管上皮细胞(HK-2)表达巨噬细胞移动抑制因子(MIF)的影响。方法 采用硫酸铵沉淀&#65380;蛋白G亲和层析纯化尿中IgG，并经SDS-PAGE、Western印迹分析鉴定&#65377;用不同浓度(0&#65380;0.5&#65380;1.0&#65380;2.5&#65380;5.0&#65380;10.0 mg/ml)的上述两种患者的尿IgG分别刺激HK-2细胞6 h，应用RT-PCR检测细胞表达MIF mRNA的变化;应用Western印迹检测细胞中MIF的蛋白水平&#65377; 结果 纯化的尿IgG经SDS-PAGE分析显示其分解为4个片段,以兔抗人IgG抗体进行免疫印迹鉴定，证实这些蛋白条带均为IgG成分&#65377; 两种不同病理类型NS患者的尿IgG均可上调HK-2细胞MIF 的基因及蛋白表达，并呈剂量依赖性&#65377;MN患者的尿IgG 0.1 mg/ml即可明显上调HK-2细胞MIF mRNA和蛋白表达(P < 0.01);而MCD患者的尿IgG需达到2.5 mg/ml才具有显著上调效应&#65377; 结论 呈NS的MCD和MN患者尿IgG可上调HK-2细胞表达MIF&#65377;MN患者尿IgG的作用强于MCD患者,提示这两种不同病理类型患者尿IgG可能存在结构或功能上的差异&#65377;     

肾病综合征患者肾组织podocin的表达

目的 观察不同病理类型&#65380; 不同激素反应性肾病综合征(NS)患者肾小球足细胞中podocin的表达和分布特征&#65377;方法 肾组织作冰冻切片免疫荧光双染色, 以激光共聚焦显微镜采集图像&#65377;与定位标志Ⅳ型胶原α3链比较&#65377; 受检NS患者21例, 其中局灶节段性肾小球硬化(FSGS)12例(难治性7例, 非难治性5例)&#65380; 微小病变(MCD)5例&#65380; 膜性肾病(MN)4例;正常肾组织对照3例&#65377;采用LSM 510图像处理系统进行处理&#65377;荧光强度以吸光度表示&#65377; 结果 (1)podocin在正常肾组织沿肾小球基底膜呈连续&#65380; 线形分布&#65377;部分FSGS患者podocin的分布呈点状&#65380; 短线条状, 少数患者未见podocin沉积&#65377;(2)FSGS患者肾小球中podocin表达量(80.5±33.5)与对照正常肾组织(138.4±38.1)比较,显著减少(P=0.0211), 其中难治性FSGS(67.2±30.5)与正常肾组织的差异有统计学意义(P=0.0131); 非难治性FSGS患者(99.0±31.0)与正常肾组织的差异无统计学意义(P=0.1585)&#65377;(3)MCD(112.1±47.6)&#65380; MN(92.5±34.8)患者podocin的表达量亦降低,与正常肾组织比较,无统计学意义(P=0.4497, P=0.1570)&#65377;(4)不同病理类型各组NS患者肾小球中podocin表达量的差异均无统计学意义(P > 0.05)&#65377;结论 FSGS的NS患者肾小球中podocin的表达减少, 难治性FSGS患者尤为明显, 部分FSGS患者podocin分布发生改变&#65377;podocin的检测可能在FSGS激素疗效评价方面有一定价值&#65377;     

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;     

血液透析&#65380;腹膜透析和肾移植的成本-效果分析

目的 探讨我院行血液透析(HD)&#65380;腹膜透析(CAPD)和肾移植(KT)3种终末期肾脏病(ESRD)替代疗法第1&#65380;第2年的成本-效果比&#65377;方法 回顾性研究上述3种疗法患者开始治疗两年内的成本&#65380;对工作的影响&#65380;以及现阶段的生活质量(用SF-36量表),并进行有关分析&#65377;结果 KT组第1年的费用高于另外两组(P < 0.001);在第2年则明显低于另外两组(P=0.005),后两组间差异无统计学意义&#65377;KT组的睡眠质量&#65380;回返工作的比例均优于另外两组&#65377;在精神健康&#65380;生理职能和精力方面,KT组与CAPD组均优于HD组&#65377;在生理机能&#65380;一般健康状况&#65380;社会功能&#65380;情感职能上,KT组优于CAPD与HD组,后两组差异无统计学意义&#65377;结论 KT组从第2年开始体现其费用上的优势,而CAPD与HD两组之间在医疗成本上无显著差异&#65377;肾移植的治疗效果在整体上优于CAPD和HD,CAPD的治疗效果略优于HD&#65377;随着KT近期和远期存活率的提高,KT应是成本-效果比最好的ESRD替代治疗方法&#65377;     

替米沙坦对糖尿病大鼠肾小球表达整合素α3β1的影响

目的 观察整合素α3β1在糖尿病大鼠肾小球的表达以及替米沙坦对其影响&#65377;方法 制备糖尿病大鼠模型,随机将动物分为糖尿病组&#65380;治疗组, 另设对照组&#65377;治疗组给予替米沙坦3 mg·kg^-1·d^-1&#65377;6周后,检测各组24 h尿白蛋白定量&#65380;肌酐清除率&#65380;血糖&#65380;血胰岛素&#65380;血压&#65380;肾重/体重;免疫组化法检测肾小球整合素α3β1表达部位及表达水平&#65377;分离肾小球,Western印迹法检测肾小球整合素α3β1蛋白表达水平&#65377; RT-PCR 检测肾小球TGF-β1mRNA 的表达&#65377;光镜下观察肾组织病理改变;电镜下观察肾组织超微结构变化&#65377;结果 免疫组化结果显示,正常大鼠整合素α3β1主要沿肾小球血管袢呈线性表达,系膜区有少量表达&#65377;糖尿病组肾小球整合素α3β1表达比正常对照组明显减弱;替米沙坦治疗组整合素α3β1表达较糖尿病组明显增加,24 h尿白蛋白定量及其它肾功能指标以及病理改变明显改善,血压无明显变化, 肾小球TGF-β1mRNA表达比糖尿病组显著下降&#65377;结论 替米沙坦可以减少糖尿病肾病大鼠早期的尿蛋白,改善病理变化,保护肾功能,其作用机制可能部分通过减少TGF-β1表达,上调整合素α3β1表达而实现&#65377;     

Wilms瘤1基因在肾小管上皮细胞转分化中的表达及作用

目的 探讨Wilms 肿瘤1基因(WT1)在肾小管上皮细胞(TEC)向肌成纤维细胞转分化中的可能作用&#65377;方法 将体外原代培养TEC分别置含IL-1α(10 ng/ml)&#65380; IL-1α+抗WT1中和抗体(10 μg/ml)的DMEM/F12培养基中培养, 观察TEC的形态变化及免疫学特征&#65377;采用RT-PCR检测各组TEC中WT1及α-SMA的表达&#65377;结果 经IL-1α掺入培养5 d后,体外培养的TEC形态特征趋于成纤维细胞化,细胞拉长&#65380;梭形变,失去原有的呈铺路石样的生长方式&#65377;电镜下细胞极性丧失,表面微绒毛消失&#65377;正常情况下,成年TEC不表达WT1&#65377;经IL-1α掺入培养1 d后,TEC重新表达WT1,同时伴有α-SMA的表达;3 d后WT1表达消失,呈一过性特征,而α-SMA的表达则随时间的延长而逐渐增强&#65377;在培养中掺入抗WT1抗体以中和WTl基因产物后,尽管仍给予IL-1α刺激,TEC大都保持原有特征不变,α-SMA及WT1 mRNA仅呈微弱表达&#65377;结论 高浓度IL-1α可导致TEC向肌成纤维细胞的转分化&#65377;在TEC的转分化过程中,WT1基因的重新&#65380;一过性表达可能起着重要作用&#65377;成年TEC重新获得WT1表达,可能是其发生转分化的内在启动机制&#65377;体外中和WT1的基因产物可明显抑制TEC的转分化进程,并可能籍此影响肾脏的纤维化发生&#65377;     

糖基化终产物对肾间质成纤维细胞DNA损伤的影响

目的 探讨糖基化终产物(AGE)对肾间质成纤维细胞DNA损伤的影响及抗氧化剂的干预作用&#65377; 方法 不同浓度AGE修饰的牛血清白蛋白(AGE-BSA)作用于NRK-49F细胞24 h,普通培养基和牛血清白蛋白 (BSA)作为对照&#65377;N-乙酰半胱氨酸(NAC)预处理细胞以观察抗氧化剂的干预作用&#65377;AlamarBlue还原法测定细胞增殖活力,单细胞凝胶电泳(彗星实验)测定细胞DNA损伤&#65377; 结果 与对照组比较,AGE作用后的细胞增殖活力下降;DNA迁移距离(彗星尾长)增加,差异有统计学意义,且2者呈剂量依赖关系&#65377;各浓度BSA作用后对细胞增殖活力无明显影响;彗星尾长无明显变化&#65377;与相同浓度BSA相比, 400&#65380;800 mg/L AGE分别使AlamarBlue还原率下降14%和15%(P < 0.05);200&#65380;400&#65380;800 mg/L AGE组彗星尾长分别为BSA组的1.45&#65380;2.12&#65380;2.71倍(P < 0.05)&#65377;与未用NAC预处理组相比,NAC预处理可使AlamarBlue还原率上升[(45.15±0.93)% 比(38.40±0.81)%,P < 0.05];彗星尾长变短[(10.02±4.54) μm比(13.48±5.32) μm,P < 0.05]&#65377; 结论 AGE可导致肾间质成纤维细胞DNA损伤,使用抗氧化剂可有效减轻AGE导致的DNA损伤&#65377;细胞内氧化应激增强可能是AGE引起肾间质成纤维细胞DNA损伤的作用机制之一。     

饰胶蛋白聚糖与转化生长因子β1对大鼠肾系膜细胞生长及相关信号途径的影响

目的探讨饰胶蛋白聚糖（DCN）与转化生长因子β1（TGF-β1）对大鼠肾系膜细胞（MsC）生长及相关信号途径的影响。方法经脂质体介导将DCN基因转染体外培养的大鼠肾MsC,经筛选和鉴定后,收集阳性细胞克隆的培养上清（DCN上清）。采用流式细胞仪检测经TGF-β1（2μg/L）和（或）DCN上清作用后MsC细胞周期的变化。采用Western印迹法分别检测两者单独或联合作用后,MsC磷酸化丝裂原活化蛋白激酶（p-MAPK）、磷酸化Smad2 （p-Smad2）和p21蛋白表达情况。采用免疫共沉淀方法检测阳性细胞克隆上清中DCN与TGF-β1的结合情况。结果TGF-β1或DCN上清单独作用时均可抑制MsC的增殖,G_2-M期细胞数分别为对照组的81%及86.5%,而两者共同作用时G_2-M期细胞数与对照组差异无统计学意义。经TGF-β1和DCN上清单独作用12 h后,p-ERK1/2表达为对照组的4.4、3.7倍和3.2、3.7倍;p-SAPK/JNK表达为对照组的2.0、1.5倍和1.9、1.5倍;而两者共同作用组p-ERK1/2和p-SAPK/JNK表达与对照组差异无统计学意义。TGF-β1单独作用12 h后,MsC p-Smad2的表达为对照组的2.6倍,而DCN单独作用组及2者联合作用组其表达与对照组差异无统计学意义。DCN单独作用12 h后p21蛋白的表达为对照组的2.6倍,而TGF-β1单独作用及2者联合作用组其表达与对照组差异无统计学意义。阳性细胞克隆上清中DCN可以直接与TGF-β1结合。结论TGF-β1与DCN单独作用均可抑制MsC增殖及激活MAPK信号途径,但2者共同作用时其作用相互抵消。TGF-β1激活Smad信号途径作用可被DCN阻断;DCN上调p21蛋白作用可被TGF-β1阻断。2者通过不同信号分子抑制细胞生长,其作用可能与两者相互结合有关。     

Overexpression of decorin induces apoptosis and cell growth arrest in cultured rat mesangial cells in vitro

Background: Decorin (DCN) is a small leucine-rich proteoglycan that plays an important role in the regulation of intercellular contact, cell migration and proliferation. DCN suppresses cell growth and induces apoptosis in various tumour cells. The aim of this study was to investigate whether overexpression of DCN could induce apoptosis and cell growth arrest in mesangial cells (MsCs) in vitro. Methods: PcDNA3.1A-DCN plasmid was transfected into cultured rat MsCs, and positive clones stably expressing DCN (MsC/DCN) were selected. SiRNA was used for blocking DCN expression in MsC/DCN. Apoptosis and cell growth of MsCs were assayed by flow cytometry. Hoechst staining was used for observing apoptotic cells. Expressions of active Caspase-3, epidermal growth factor receptor (EGFR), P21 and transforming growth factor-β (TGF-β1) were analyzed using Western blot. Results: Overexpression of DCN in MsCs induced apoptosis and arrested cells in the G₀/G₁ phase. The protein level of active Caspase-3 was significantly elevated in MsC/DCN (P < 0.01). DCN transfection induced downregulation of EGFR and up-expression of P21. In addition, the expression of TGF-β1 was significantly inhibited. DCN-siRNA transfection remarkably blocked the expression of DCN and reversed the downregulatory effects of DCN on MsC's proliferation. Conclusion: Overexpression of DCN could inhibit MsCs proliferation by inducing apoptosis and cell growth arrest in vitro and it also downregulates expression of TGF-β1. These results suggest novel strategies for regulating the proliferation of MsC in glomerular diseases.     

Aldose reductase regulates TGF-beta1-induced production of fibronectin and type IV collagen in cultured rat mesangial cells

AIM: To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC). METHODS: The vector, pcDNA3-AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC. CONCLUSION: AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1.     

肾病综合征氧化低密度脂蛋白脂质肾损伤的实验研究

目的 通过观察氧化低密度脂蛋白（oxidized low-density lipoprotein,ox-LDL）对系膜细胞（Mesangial Cells,MCs）分泌炎症介质功能的影响及MAPK信号通路和核因子-κB（nuclear factor kappa B,NF-κB）活性的改变,进一步阐明脂质在肾损伤中的作用机制.方法 利用ox-LDL诱导大鼠系膜细胞增殖,分别采用ELISA、real-time PCR、western blot技术检测MCs炎症介质、MAPK通路相关蛋白（p38、JNK、ERK）及NF-κB的表达水平.结果 利用ox-LDL诱导大鼠系膜细胞增殖并加入CXCR6受体后,其表面炎症因子[CXCL16、CD36、ADAM10、ADAM17、干扰素（IFN）、白细胞介素（IL6）、肿瘤坏死因子（TNF-α）]的表达水平以及MAPK信号通路（p38、JNK、ERK）、NF-κB的磷酸化水平显著升高（P〈0.01）.结论 ox-LDL可促使系膜细胞释放CXCL16、CD36、ADAM10、ADAM17、IFN、IL6、TNF-α等炎症介质,CXCR6可介导这一途径.ox-LDL激活MAPK信号转导通路,使p38、ERK1/2、SAPK/JNK的磷酸化水平升高,激活了NF-κB p65的活性,CXCR6-CXCL16介导MAPK信号途径.     

Prostaglandin E2 receptor subtype 3-siRNA reduces the mesangial cell damage induced by TGF-β1through inhibiting MAPK pathway in mice

Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 (EP3) on transforming growth factor β1 (TGF-β1)-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into: (1)control group; (2)TGF-β1 (10 μg/L) group; (3)NC-siRNA plus TGF-β1 (10 μg/L) group; (4) EP3-siRNA group; (5)EP3-siRNA plus TGF-β1 (10 μg/L). Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP in cell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin (FN), connective tissue growth factor (CTGF), cyclooxygenase-2 (COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) were detected by real-time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was decected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased (P＜0.05), the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated (all P＜0.05). Compared with TGF-β1 group, the cell proliferation in EP3-siRNA plus TGF-β1 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated (all P＜0.05), the phosphorylation of ERK1/2, p38 MAPK were also declined (P＜0.05). Conclusion EP3-siRNA may reduce TGF-β1-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.     

Promotion of survival and prevention of apoptosis in rat mesangial cells by a membrane-anchored form of heparin-binding EGF-like growth factor

Background. We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) was expressed in rat mesangial cells and was involved in the progression of of glomerulonephritis in human and animal models. Soluble HB-EGF promotes cultured rat mesangial cell proliferation and the synthesis of type I and type III collagen mRNA. However, the precise role of proHB-EGF in mesangial cell function has not yet been clarified. In the present study, we address this question. Methods. ProHB-EGF-transfected rat mesangial cells (MsC; MsC^proHB-EGF) and empty vector-transfected rat MsC (MsC^vector) were constructed. Cells were cultured in fetal calf serum (FCS)-containing (10%) or FCS-deficient medium, and the growth rates and numbers of surviving cells were determined. To elucidate the anti-apoptotic effect of proHB-EGF, cells were exposed to hydrogen peroxide (H₂O₂) or dexamethasone (DEX). Apoptosis was identified by qualitative and quantitative analysis. Expression of Bcl-2 protein in MsC^proHB-EGF after exposure to apoptosis inducers was also evaluated. Results. When cells were plated in a medium containing 10% FCS, the growth rate of MsC^proHB-EGF was not different from that of MsC transfected with a plasmid alone (MsC^vector). When cultured in the absence of FCS, MsC^vector showed decreased cell numbers, indicating apoptotic cell death. In contrast, MsC^proHB-EGF formed small colonies, although they did not show increased cell numbers, while cell viability was 90%–100% of the initial cell number after subculture. When quiescent MsC were exposed to DEX or H₂O₂, MsC^vector exhibited significant DNA laddering, whereas MsC^proHB-EGF showed resistance to these stimuli. The release of caspase-3 from MsC^proHB-EGF after exposure to DEX or H₂O₂ was significantly lower than that from MsC^vector. Bcl-2 protein expression was weak in MsC^proHB-EGF at the baseline, but this expression was significantly upregulated after exposure to these stimuli. Confluent MsC^proHB-EGF spontaneously expressed high level of p21 protein. Northern blot analysis revealed that MsC^proHB-EGF expressed increased levels of type I and type III collagen mRNA and proteins compared with those of MsC^vector. Conclusions. These results indicate that proHB-EGF contributes to mesangial cell survival by promoting cell viability and by inhibiting apoptosis. The anti-apoptotic effect of proHB-EGF could be closely related to the upregulation of Bcl-2 and p21 expression. Received: November 28, 2000 / Accepted: March 31, 2001     

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils.

BACKGROUND: Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS: PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS: LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS: These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.     

Expression of mitogen-activated protein kinase family in rat renal development

BACKGROUND: Among mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) promotes proliferation or differentiation, whereas c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) are thought to inhibit cell growth and induce apoptosis. MAPK phosphatase-1 (MKP-1) inactivates and modulates MAPKs. During renal development, large scale proliferation and apoptosis occur. We investigated the temporal and spatial expression patterns of MAPKs and MKP-1 in rat kidney during development. METHODS: Western blot analysis and immunohistochemistry were performed in the developing and mature kidney of the rat. RESULTS: The expression of ERK, p38, and MKP-1 were high in developing kidney. On the other hand, JNK was abundantly expressed in adult kidney. Active forms of ERK, p38, and JNK correlated with the protein expression levels. Immunohistochemical studies revealed that ERK was strongly expressed by blastema cells, mesenchymal cells, and ureteric bud tips in nephrogenic zone of embryonic kidney. In neonatal kidney, ERK was more abundant in the deep cortex and the medulla corresponding to tubule maturation. p38 and MKP-1 were detected uniformly in mesenchymal cells, mesangial cells, and ureteric bud epithelia of fetal kidney without an obvious correlation with the occurrence of apoptosis. JNK was expressed by tubular cells and podocytes of adult kidney. CONCLUSIONS: ERK, p38, and MKP-1 are strongly expressed in developing kidney, and JNK is detected predominantly in adult kidney. Both the temporal and spatial expression of ERK coincides with the maturation of the kidney.