细胞外信号调节激酶在血管瘤组织中的表达及活性

目的:探讨细胞外信号调节激酶1/2(ERK1/2)及其活性形式磷酸化ERK1/2(p-ERK1/2)在血管瘤组织中的蛋白表达及其意义。方法:应用免疫组织化学SP法,检测23例增生期血管瘤组织及19例退化期血管瘤组织ERK1/2和p-ERK1/2的蛋白表达。结果:增生期血管瘤组织与退化期血管瘤组织ERK1/2的蛋白表达无统计学差异(Z=-0.305,P>0.05);增生期血管瘤组织p-ERK1/2的蛋白表达显著高于退化期血管瘤组织的表达(Z=-4.271,P<0.01)结论:增生期血管瘤组织p-ERK1/2呈高表达,血管瘤的增生与ERK通路的激活有关。     

Anosmin-1蛋白通过成纤维生长因子1型受体激活ERK1/2信号通路增强嗅鞘细胞的迁移

目的探究Anosmin-1蛋白对嗅鞘细胞迁移的影响及对成纤维生长因子(FGF)1型受体和ERK1/2信号通路的影响。方法选择SPF级成年雌性SD大鼠40只,7～10周龄,体重250～300 g。提取大鼠嗅球的嗅鞘细胞并纯化,纯化后细胞培养7 d后,采用随机数字表法将所有细胞平均分为四组:对照组(C组),FGF 1型受体激动剂(FGF2)组(F组),Anosmin-1蛋白组(A组)和Anosmin-1蛋白+FGF 1型受体抑制剂(Su5402)组(S组)。C组为空白对照组,培养液中不加任何药物处理;F组为阳性对照组,于培养液中加入FGF2 25 ng/ml; A组于培养液中加入Anosmin-1蛋白2 nmol/L;S组于培养液中加入Anosmin-1蛋白2 nmol/L和Su5402 30μmol/L。四组细胞处理24 h后,采用Transwell法检测嗅鞘细胞迁移能力,Western blot法检测嗅鞘细胞磷酸化ERK1/2(p-ERK1/2)蛋白、磷酸化AKT(p-AKT)蛋白和神经型钙粘连蛋白(N-cadherin)含量,免疫荧光染色法检测N-cadherin荧光强度。结果纯化后的嗅鞘细胞占总细胞的85.6%。与C组比较,F组和A组迁移至下室的嗅鞘细胞明显增多(P0.05),p-ERK1/2蛋白、N-cadherin含量明显升高(P0.05)。与A组比较,S组迁移至下室的嗅鞘细胞明显减少(P0.05),p-ERK1/2蛋白、N-cadherin含量明显降低(P0.05)。S组和C组间、F组和A组间迁移至下室的嗅鞘细胞数量、p-ERK1/2蛋白、N-cadherin含量差异无统计学意义。四组p-AKT蛋白含量差异无统计学意义。结论 Anosmin-1蛋白通过FGF 1型受体激活ERK1/2信号通路,从而上调N-cadherin,增强嗅鞘细胞的迁移能力。     

ERK1/2激活参与乳化异氟醚后处理的脑保护作用

目的观察细胞外信号调节激酶1/2(ERK1/2)的激活在8%乳化异氟醚后处理对大鼠局灶性脑缺血-再灌注损伤中的作用。方法健康成年雄性SD大鼠48只,随机均分为六组:假手术组(S组)、缺血-再灌注组(IR组)、乳化异氟醚后处理组(EI组)、ERK抑制剂PD98059组(PD组)、乳化异氟醚后处理+PD98059组(EP组)、溶媒DMSO组(D组)。除S组外,均采用大脑中动脉阻闭2h,再灌注24h建立局灶性脑缺血-再灌注模型。缺血2h恢复再灌注即刻,EI组、EP组腹腔注射8%乳化异氟醚10.5ml/kg,其余各组注射生理盐水10.5ml/kg。PD组、EP组和D组于再灌注前30min侧脑室分别注入PD98059和DMSO。再灌注24h时进行神经功能缺陷评分(NDS评分),并观察组织形态学变化及细胞凋亡、p-ERK1/2阳性表达。结果与IR组相比,EI组NDS评分降低,凋亡细胞减少,磷酸化ERK1/2(p-ERK1/2)表达上调(P<0.05);与EI组相比,EP组NDS评分增高,凋亡细胞显著增加,p-ERK1/2表达下调(P<0.05)。结论 8%乳化异氟醚后处理通过激活ERK1/2信号通路对抗局灶性脑缺血-再灌注损伤。     

ERK1/2对高糖环境下足细胞凋亡的作用及来氟米特对其的影响

目的：观察ERK1/2对高糖环境下足细胞凋亡的作用及来氟米特对其的影响。方法：培养的人足细胞分为正常糖组、高渗对照组、高糖组,高糖组按不同刺激时间又分为0.5h、1h、6h、12h、24h5组,应用Western印迹分析法检测各组足细胞ERK1/2信号途径中ERK1/2蛋白活化的变化。足细胞凋亡检测：将足细胞分为高糖对照组、抑制剂组、抑制剂＋来氟米特（A771726）组、A771726组,将以上各组足细胞培养24h后,分别进行ERK1/2信号途径的检测同前及流式细胞仪检测足细胞的凋亡率。结果：高糖组30min可以活化ERK1/2蛋白,6h开始达高峰,持续活化到24h开始降至基础水平。正常糖及高渗对照组未能活化ERK1/2。与高糖组相比,ERK1/2蛋白抑制剂（PD98059）抑制了ERK1/2的磷酸化,使磷酸化的ERK1/2（P-ERK1/2）蛋白表达明显减少（P〈0.01）,从而影响了下游多种核转录因子的活化,影响目的基因的表达而增加了足细胞的凋亡（P〈0.01）。A771726可以增加P-ERK1/2的表达量（P〈0.01）,从而减少足细胞的凋亡（P〈0.05）。而PD98059可以影响A771726的作用,使其对足细胞的保护作用减弱,其凋亡率比高糖组明显增加（P〈0.01）。结论：ERK1/2信号途径参与了来氟米特对足细胞的保护作用。     

丙泊酚对神经元缺血再灌注损伤的保护与磷酸化蛋白激酶信号通路的关系

目的:探讨丙泊酚在脑缺血/再灌注损伤(CI/RI)中的作用及可能机制。方法:采用氧糖剥夺再灌注(OGD/RP)法体外构建缺血/再灌注细胞模型,将细胞分为对照组、OGD/RP组和丙泊酚+OGD/RP联合组。采用MTT法检测皮质神经元存活率,Annexin V-PI检测细胞凋亡情况,RT-PCR方法检测碱性成纤维细胞生长因子(b FGF)的m RNA表达,Western blot检测丙泊酚对皮质神经元内b FGF、磷酸化蛋白激酶B(p PKB)和磷酸化细胞外信号调节激酶1/2(p ERK1/2)表达。采用小干扰RNA构建b FGF沉默的细胞。结果:丙泊酚能够显著促进CI/RI后神经元的存活,抑制其凋亡,OGD/RP处理组神经元凋亡率为43.2%,以10 mg/L的丙泊酚预处理后细胞的凋亡率即降为19.5%(P<0.05)。与对照组(1.02±0.03)相比较,OGD处理后细胞中b FGF的含量(0.78±0.06)显著下调(P<0.05),丙泊酚处理的皮质神经元中b FGF含量(1.43±0.04)显著高于OGD处理组(P<0.05)。沉默的b FGF或者施加蛋白激酶B(PI3K-p PKB)以及p ERK1/2信号通路抑制剂都会导致细胞存活率显著下降(P<0.05),抑制PI3K-p PKB以及ERK1/2的激活。结论:丙泊酚可以通过上调b FGF的表达,激活PI3K-p PKB和ERK1/2的信号通路,减轻体外培养神经元凋亡/再灌注损伤,从而增加皮质神经元的存活。     

电针对大鼠吗啡耐受时脊髓背角细胞外信号调节激酶1/2活性的影响

目的 探讨电针对大鼠吗啡耐受时脊髓背角细胞外信号调节激酶1/2(ERK1/2)活性的影响.方法 取鞘内置管成功的雄性SD大鼠25只,采用随机数字表法,将其分为5组(n=5):生理盐水组(NS组)鞘内注射生理盐水10μl;吗啡组(M组)鞘内注射吗啡10 μg;错义寡核苷酸组(MO组)鞘内注射吗啡10μg+ ERK1/2错义寡核苷酸10μg;反义寡核苷酸组(AO组)鞘内注射吗啡10 μg+ERK1/2反义寡核苷酸10 μg;电针组(EA组)鞘内注射吗啡10 μg,同时每日首次给药后电针大鼠阳陵泉和足三里(频率2 Hz,波宽1 ms,电流强度3 mA,刺激时间30 min).各组注射药物容量均为10μl,2次/d,连续7d.于鞘内给药前、鞘内给药2、4、6d和鞘内给药结束后1 d(T0-4)测定机械痛阈.于T4时机械痛阈测定结束后,取脊髓背角组织,采用Western blot法测定大鼠ERK1/2和磷酸化ERK1/2(p-ERK1/2)的表达.结果 M组、MO组和AO组和EA组发生了吗啡耐受,EA组吗啡耐受程度最轻.与NS组比较,M组和MO组p-ERK1/2表达上调,AO组总ERK1/2表达下调(P＜0.05),EA组上述指标差异无统计学意义(P＞0.05);与M组和MO组比较,AO组p-ERK1/2和总ERK1/2表达下调,EA组p-ERK1/2表达下调(P＜0.05);与AO组比较,EA组p-ERK1/2和总ERK1/2表达上调(P＜0.05).结论 电针可抑制慢性吗啡给药所导致的脊髓背角ERK1/2活性升高,从而缓解吗啡耐受的形成.     

MicroRNA-155对甲状腺乳头状癌细胞生物学行为的作用及机制研究

目的探讨miR-155在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)细胞生长、侵袭与转移过程中的作用及可能的作用机制。方法构建人的miR-155类似物并体外转染PTC BCPAP细胞,通过CCK8及transwell试验观察细胞增殖及侵袭能力的变化。miR-155类似物体外转染BCPAP细胞并用Western blot方法检测MAPK通路相关蛋白本底及磷酸化表达。给予ERK通路抑制剂U0126观察能否逆转miR-155过表达造成的甲状腺癌细胞异常增殖及侵袭能力增强。结果过表达miR-15548 h后通过CCK8试验检测发现BCPAP细胞明显增殖,过表达miR-15524h、48 h后通过transwell试验发现甲状腺癌细胞侵袭能力明显增强(P<0.05);利用Western blot检测MAPK信号通路相关蛋白JNK、ERK、P38的表达均明显上调(P<0.05)。同时检测细胞内p-ERK蛋白表达升高(P<0.05),利用ERK通路抑制剂U0126与miR-155共同处理细胞发现p-ERK表达较miR-155组明显降低(P<0.05)。同时,我们检测各组细胞的增殖及侵袭情况,发现U0126能逆转miR-155造成的促增殖及促侵袭作用。结论miR-155能通过激活MAPK通路的ERK通路,进而促进PTC BCPAP细胞的增殖以侵袭能力,为治疗甲状腺癌提供了潜在的靶点。     

骨化三醇对高糖诱导的肾小管上皮细胞肾素高表达的影响

目的探讨骨化三醇对高糖状态下肾小管上皮细胞肾素表达的影响及其机制。方法按随机数字表法将24只6~8周龄Wistar大鼠随机分为对照组、糖尿病组、骨化三醇组(0.03μg·kg~(-1)·d~(-1)),各组8只。16周后处死各组大鼠,检测尿蛋白、尿肌酐、内生肌酐清除率(endogenous creatinine clearance rate,CCr),并应用实时荧光定量法(real time-polymerase chain reaction,RT-PCR)和蛋白印迹法(Western blot)检测肾脏组织肾素及磷酸化细胞外信号调节激酶(phosphorylated extracellularsignal-regulated kinase,p-ERK)/细胞外信号调节激酶(extracellularsignal-regulated kinase,ERK)表达情况。NRK-52E细胞分组:①正常对照组;②高糖组(糖浓度为25 mmol/L,不同时间点);③高糖+骨化三醇(10~8 mol/L、10~9 mol/L、10~10 mol/L)组;④高糖+ERK抑制剂(FR18020410~5 mol/L)组。RT-PCR法检测肾素mRNA表达,Western印记法检测肾素蛋白、p-ERK/ERK蛋白表达。结果与对照组比较,糖尿病组大鼠尿β2微球蛋白(β2-microglobulin,β2-MG)、尿白蛋白/尿肌酐升高(P0.05),CCr降低(P0.05);与糖尿病组比较,骨化三醇组大鼠尿β2-MG、尿白蛋白/肌酐降低(P0.05),CCr升高(P0.05)。与对照组比较,糖尿病组大鼠肾组织肾素、p-ERK/总ERK表达均明显升高,骨化三醇能显著减轻糖尿病大鼠上述蛋白的高表达(P0.05)。高糖可诱导NRK-52E细胞ERK蛋白磷酸化(P0.05),诱导肾素mRNA及蛋白表达上调,并呈时间依赖性(P0.05)。骨化三醇可浓度依赖性地降低高糖诱导的NRK-52E细胞ERK蛋白磷酸化及肾素mRNA、蛋白的高表达(P0.05)。ERK抑制剂能部分抑制高糖诱导的肾素蛋白高表达(P0.05)。结论骨化三醇降低糖尿病大鼠蛋白尿,具有肾脏保护作用,其机制可能与抑制ERK通路降低高糖诱导的肾素高表达有关。     

急性内脏痛大鼠脊髓背角神经元ERK1/ERK2磷酸化水平的变化

目的 探讨脊髓背角神经元细胞外信号调节激酶(ERK)是否参与急性内脏痛的形成.方法 第一部分成年雌性SD大鼠30只,随机分为5组(n=6),假手术组(S组)不行宫颈扩张,UCD25组、UCD50组、UCD75组和UCD100组分别采用25、50、75、100 g的强度进行宫颈扩张10 s,10 min后处死大鼠,采用免疫组化方法测定颈段(C5～8)、胸段(T5～8)、胸腰段(T12～L2)及腰骶段(L6～S1)脊髓背角神经元磷酸化ERK1/ERK2(p-ERK1/ERK2)表达水平.第二部分成年雌性SD大鼠20只,宫颈扩张(强度为75 g)10 s,于宫颈扩张后10、30、60及120 min时分别处死5只大鼠,测定胸腰段(T12～L2)p-ERK1/ERK2表达水平.结果 与S组比较,其它各组胸腰段p-ERK1/ERK2表达上调,其中UCD75组和UCD100组最明显(P<0.05),其他脊髓段p-ERK1/ERK2表达差异无统计学意义(P>0.05).宫颈扩张后60 min时胸腰段p-ERK1/ERK2表达达高峰(P<0.05).结论 胸腰段脊髓背角神经元ERK参与了宫颈扩张诱发大鼠急性内脏痛的形成.     

MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用

目的 探讨MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖过程中的作用. 方法 体外培养雪旺细胞,S-100免疫荧光鉴定;Western blot检测MEK下游因子ERK1/2磷酸化激活形式(p-ERK1/2)的表达;MEK抑制剂(PD98059)阻断该通路后检测p-ERK1/2的表达;MTT法检测经PD98059阻断MEK通路后雪旺细胞的增殖情况. 结果 吡咯喹啉醌可激活雪旺细胞内MEK/ERK信号通路,在加入吡咯喹啉醌1 h后p-ERK1/2表达最高;吡咯喹啉醌在1～500 nmol/L范围内可使p-ERK1/2表达增加,1 000 nmol/L时与对照组比较差异无统计学意义,10 000 nmol/L时则表现为抑制作用(P＜0.05);经PD98059阻断MEK通路后p-ERK1/2的上调效应消失(P＜0.05).而且加入PD98059阻断MEK通路后吡咯喹啉醌对雪旺细胞的促增殖效果减弱. 结论 吡咯喹啉醌可激活雪旺细胞MEK/ERK信号通路,且该通路在吡咯喹啉醌促雪旺细胞增殖过程中发挥作用.     

Glial cell line-derived neurotrophic factor and its receptor ret is a novel ligand-receptor complex critical for survival response during podocyte injury

Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair.     

氟伐他汀联合氯沙坦对血管紧张素Ⅱ诱导的人足细胞血管内皮生长因子表达的影响及机制研究

目的 探讨氟伐他汀联合氯沙坦对血管紧张素Ⅱ( AngⅡ)诱导的人足细胞血管内皮生长因子(VEGF)表达的影响及机制.方法 体外培养人足细胞株,采用不同浓度的AngⅡ(10-9～10-5mol/L)刺激人足细胞,另予氟伐他汀(10-7、10-6、10-5 mol/L)和(或)氯沙坦(10-7、10-6、10-5 mol/L)、ERK特异性阻断剂PD98059( 5× 10-5 mol/L)干预.Western印迹法检测VEGF、磷酸化(p)ERK1/2蛋白的表达.RT-PCR法检测VEGF mRNA的表达.结果 AngⅡ刺激后,人足细胞VEGF mRNA和蛋白的表达显著增加(P＜0.05),p-ERK1/2增加(P＜0.05).氟伐他汀、氯沙坦及PD98059均可下调AngⅡ诱导的VEGF mRNA和蛋白表达及ERK1/2磷酸化(P＜0.05),而氟伐他汀和氯沙坦联合干预较单独作用抑制效果更为显著(P＜0.01).结论 氟伐他汀和氯沙坦均可抑制AngⅡ诱导足细胞VEGF的过表达,且两者联合具有协同作用.抑制ERK信号通路可能是实现其联合作用的机制之一.     

Role of IQGAP1 in mediating angiotensinⅡ-induced apoptosis of podocytes and its underlying mechanism

Objective To investigate the role of IQ domain GTPase-activating protein 1 (IQGAP1) in angiotensinⅡ(AngⅡ) -induced podocyte apoptosis and the underlying mechanism. Methods Differentiated mouse podocytes were exposed to AngⅡ at different concentrations for 6 h or at 10-8 mol/L for variable incubation time. Podocyte apoptosis was assessed by flow cytometry. Expression of IQGAP1 was analyzed by immunofluorescence and Western blotting. IQGAP1 siRNA and MAPK pathway inhibitors(10 μmol/L SB202190, 25 μmol/L SP600125, 10 μmol/L U0126) were further introduced to investigate the role of IQGAP1 and MAPK signalings in the process. And co-immunoprecipitation was used to evaluate the interaction between ERK1/2 and IQGAP1. Results (1) AngⅡ promoted podocyte apoptosis in a dose- and time-dependent manner. (2) IQGAP1 was located in celluar membrane and cytoplasm of cultured podocytes. Exposure to AngⅡ stimulated IQGAP1 expression in a dose- and time-dependent manner, and elevated phosphorylation of p38, JNK, and ERK1/2 simultaneously. (3) Pretreatment with SB202190, SP600125, or U0126 dramatically prevented AngⅡ-promoted podocyte apoptosis respectively (P＜0.05). However, the protein level of IQGAP1 was not altered. (4) Knockdown of IQGAP1 with siRNA obviously prevented AngⅡ-induced apoptosis of podocytes(P＜0.05) and reduced AngⅡ-induced phosphorylation of ERK1/2(P＜0.05), but not that of p38, JNK. This was accompanied by a reduced interaction between ERK1/2 and IQGAP1(P＜0.05). Conclusion IQGAP1 contributes to AngⅡ-induced podocyte apoptosis by interacting with the ERK1/2 signaling protein.     

普伐他汀抑制羧甲赖氨酸修饰白蛋白诱导足细胞表达单核细胞趋化蛋白1

目的研究普伐他汀对羧甲基赖氨酸修饰白蛋白（CML-BSA）诱导的小鼠足细胞单核细胞趋化蛋白1（MCP-1）表达的干预作用。方法使用RT-PCR和ELISA方法检测MCP-1的表达水平及细胞上清液MCP-1含量。共聚焦显微镜测量对二氯荧光黄敏感的细胞内活性氧（ROS）的产量。Western印迹法和免疫组化技术检测活化的细胞外信号调节蛋白激酶（ERK）、核因子κB（NF-κB）和转录因子Sp1的表达。结果CML-BSA呈时间和浓度依赖的方式诱导MCP-1的表达。0.1或1.0 mmol/L普伐他汀可抑制CML-BSA诱导的MCP-1 mRNA和蛋白的表达。CML-BSA可迅速诱导足细胞内ROS的生成。普伐他汀不影响细胞ROS生成。CML-BSA诱导足细胞磷酸化ERK（p-ERK）表达升高,而普伐他汀可以浓度依赖方式对此加以阻断。Western印迹法和免疫组化实验结果均提示普伐他汀预处理足细胞可以阻断CML-BSA诱导的NF-κB和Sp1的易位。结论普伐他汀能够通过调节足细胞内ERK、NF-κB和Sp1信号途径,阻断CML-BSA诱导MCP-1的表达。     

Role of the secretory PLA2-IB and PLA2R in human podocyte injury and its possible mechanism

Objective To investigate the role of the group IB secretory phospholipase A2 (sPLA2-IB) and M-type phospholipase A2 receptor (PLA2R) in human podocyte injury and its possible signal transduction pathway. Methods Differentiated human podocytes were exposed to PBS or different sPLA2-IB concentration conditions (10-9, 10-7, 10-5 mol/L) for 2 hours. The wound healing assay was used to measure cell migration rate; Apoptosis in cultured human podocytes was assessed by Hoechst 33342 staining and flow cytometry; Western blot was used to analyze the protein expression of p-cPLA2α, p-p38, and p53. Then control siRNA or PLA2R siRNA were transfected to podocytes. Podocytes were divided into normal control group, negative control siRNA group and PLA2R siRNA group. Twenty four hous later, the cells were stimulated by 10-5 mol/L sPLA2-IB for 2 hours. The protein expression of p-cPLA2α, p-p38, and p53 were detected by Western blot. Results Compared to PBS control group, the migration ability of podocytes decreased when stimulated with sPLA2-IB (10-7 mol/L-10-5 mol/L), and the apoptosis of podocytes increased in a concentration-dependent manner, the protein level of p-cPLA2α, p-p38 and p53 protein increased too. After the knockdown of PLA2R by PLA2R siRNA transfection, stimulated the podocytes with the same dosage of sPLA2-IB, the protein expression of p-cPLA2α, p-p38 and p53 all decreased. Conclusion sPLA2-IB stimulation can increase human podocyte apoptosis and decrease its migration ability. The possible mechanism might be through p38-cPLA2α-p53 pathway.     

The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis

BACKGROUND: Reduced podocyte number is a critical determinant in the development of glomerulosclerosis. Transforming growth factor-beta1 (TGF-beta1) induces podocyte apoptosis, but the cell cycle events are not known. The cyclin-dependent kinase (CDK) inhibitor p21 increases in podocytes in diseases where TGF-beta increases. Accordingly, we studied the role of p21 in podocyte apoptosis. METHODS: Immortalized and primary p21+/+ and p21-/- mouse podocytes were used. Apoptosis was measured by Hoechst 33342 staining and caspase-3 activity following the exposure to TGF-beta1 or puromycin aminonucleoside. p21 and specific Bcl-2-related family proteins levels were measured by Western blot analysis. To prove a role for p21, we reconstituted p21 expression in p21-/- podocytes utilizing an adenovirus vector. RESULTS: TGF-beta1 increased the protein levels of p21 in p21+/+ podocytes, and this coincided with apoptosis. In contrast, TGF-beta1 did not induce apoptosis in p21-/- podocytes. Restoring p21 expression increased apoptosis in p21-/- podocytes following exposure to TGF-beta1. TGF-beta1 increased the protein levels of an anti-apoptotic Bcl-2 in p21-/- podocytes, but not in p21+/+ podocytes. Moreover, TGF-beta1 did not increase Bcl-2 expression in p21-/- podocytes in which p21 expression was restored. Finally, puromycin aminonucleoside also induced apoptosis in p21+/+ podocytes, but not in p21-/- podocytes. CONCLUSION: Podocyte apoptosis induced by TGF-beta1 and puromycin aminonucleoside requires p21, and Bcl-2 plays a crucial role downstream of p21 in mediating this effect. These results suggest that p21 may play a critical role in the decrease in podocyte number in disease status accompanied by increased TGF-beta1.     

Influence of dexamethasone on the expression and distribution of transient receptor potential cation channel 6 in glomerular podocytes

Objective To observe the changes of foot processes, expression and distribution of transient receptor potential cation channel 6 (TRPC6) in podocytes by puromycin aminonucleoside (PAN) and dexamethasone (DEX) intervention, then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases. Methods Podocytes cultured in vitro were divided into three group: control group, PAN stimulation group and DEX intervention group. Mouse podocyte cell line (MPC5) were cultured in 0.02% dimethyl sulfoxide (DMSO) in control group, subjected to PAN (50 μg/ml) treatment alone or with DEX (1 μmol/L) in other two groups for 8 h, 24 h, 48 h. The podocyte morphology was observed and took pictures by phase - contrast microscope, then the differences of morphology and areas were analyzed. The distribution, mRNA expression and protein expression of TRPC6 were detected by indirect immunocyto-fluorescence, real-time quantitative PCR and Western blotting, respectively. Results The well - developed podocyte arborization and interconnection was formed in control group, but PAN led to significant shrinkage of podocytes (P＜0.05), together with podocyte foot process retraction, effacement and loss of cell contact. DEX significantly prevented the shrinkage and apoptosis of podocytes. The apoptosis rate was significantly increased after PAN stimulated 48 h (P＜0.05). Real-time quantitative PCR and Western blotting found TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group (P＜0.05). The distribution of TRPC6 becamed abnormal in PAN group. DEX decreased TRPC6 mRNA and protein expression at 48 h compared with PAN group (P＜0.05). The abnormal distribution of TRPC6 was also alleviated by the protection of DEX. Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm against podocyte injury, and alleviates proteinuria via stabilizing mRNA, protein expression and distribution of TRPC6.     

Protective effect ofACTH4-10 on adriamycin-induced podocyte injury

Objective To observe the influence of adrenocorticotropic hormone (ACTH4-10) in the changes of podocyte proliferation, apoptosis and expression of nephrin and podocin on adriamycin(ADR) -induced podocyte injury and investigate the protective effect ofACTH4-10. Methods All podocytes were randomly divided into following groups: normal control, ADR-induced group andACTH4-10 intervention group (low, middle and high concentration). Normal control group was not treated, ADR-induced group was induced to set the model of podocyte injury by ADR (1 μmol/L) for 24 hours andACTH4-10 intervention groups were intervened by 1 μg/L, 10 μg/L and 100 μg/LACTH4-10 for 1 hours respectively, prior to setting the model of podocyte injury. Cell counting kit (CCK-8) was used to detect the multiplication of podocytes and TUNEL apoptosis detection kit was used to detect podocyte apoptosis. Real-time PCR and Western blotting were used to examine the expression of nephrin and podocin. Results Compared with control group, podocyte proliferation and expression of nephrin and podocin was decreased significantly in ADR-induced group (P＜0.05), meanwhile podocyte apoptosis was increased obviously (38.14% vs 5.12%). Compared with ADR-induced group, podocyte proliferation and expression of nephrin and podocin was increased generally with concentration ofACTH4-10. Although podocyte apoptosis rates (20.45%, 17.39%, 11.02%) were increased inACTH4-10 intervention group (low, middle and high concentration) while comparing with normal control group, podocyte apoptosis decreased obviously while comparing with ADR-induced group. ConclusionsACTH4-10 can stabilize the expression of nephrin and podocin on slid diaphragm, and has the protective effect on podocyte injury induced by ADR, while the effect depends on the concentration ofACTH4-10.