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1.
Objective To observe the effects of 7,8-dihydroxyflavone (7,8-DHF) on hypoxia induced endoplasmic reticulum stress (ERS) in human proximal tubular epithelial cells (HK-2). Methods The mRNA level of ERS associated biomarkers was evaluated by RT-PCR assay in cell hypoxia damaged model. And HK-2 cells were pretreated with different concentrations of 7,8-DHF through CCK-8 assay; meanwhile CCAAT/enhancer-binding protein homologous protein (CHOP), Cyr61, Akt and p-Akt were determined by western blotting assay. Moreover, HK-2 cells were pretreated by LY294002, a kind of PI3K/Akt inhibitor, to inhibit the PI3K/Akt signaling, and its effects on protein level induced by 7,8-DHF was detected. HK-2 cells was then over-expressed Cyr61 and exposed to hypoxia Apoptosis rate and CHOP expression were determined. Results Compared to hypoxia group (P<0.01), Hypoxia for 12h was effective in inducing ERS (P<0.01), while pretreatment with 7,8-DHF (100 μmol/L) increased cell proliferation significantly . The protein expressions of Cyr61 and p-Akt in H+7,8-DHF group were higher, but the level of CHOP was decreased (P<0.05). With LY294002 pretreated, the expression of Cyr61, p-Akt was down-regulated (all P<0.05) while the expression of CHOP was up-regulated (P<0.05). In comparison to empty plasmid group, when cells were transfected with over-expression of Cyr61 plasmid and exposed to hypoxia, the number of apoptotic tubular cells was decreased (P<0.01). And over-expression of Cyr61 significantly reduced CHOP expression compared with the empty plasmid group (P<0.01). Conclusion Pretreatment of 7,8-DHF could protect cells from hypoxia injury and inhibit ERS, which may involve the Akt-Cyr61 signaling pathway.  相似文献   

2.
Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2). Methods Cyr61 cDNA was cloned into pEGFP-N2, then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine. The cell proliferation was measured by MTT. The expression level of Cyr61, p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting. The cell cycle and cell apoptosis were analyzed by flow cytometry. Results The recombinant plasmid pEGFP-N2-Cyr61 could be transfected into HK-2 efficiently. After transfection, the proliferative activity was significantly increased, the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly, the level of cell apoptosis decreased markedly (all P<0.01). The expressions of Cyr61, p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P<0.01). Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway, resulting in the promotion of HK-2 cells entering into S phase, cell proliferation and the reduction of cell apoptosis.  相似文献   

3.
目的 探讨丝裂原激活蛋白激酶类(MAPKs)对缺氧条件下人近端肾小管上皮细胞(HKC)中富含半胱氨酸蛋白61(Cyr61)基因转录活性的调控机制。方法 缺氧培养HKC,Northern印迹检测Cyr61mRNA表达;Western印迹检测Cyr61、p38、细胞外信号调节激酶(ERK1/2)、c—Jun—N末端蛋白激酶(JNK)以及缺氧诱导因子1c(HIF-1α)的表达。构建含有人Cyr61基因启动子的报告基因Cyr61-luc质粒,将其单独或者分别与表达活性MAPKs的质粒Ca—MEK1和Ca—MKK6共同瞬时转染HKC。通过荧光素酶活性检测观察缺氧、MAPKs抑制剂和MAPKs活性酶对Cyr61基因转录活性的调控。结果 缺氧时HKC表达cyr61、HIF-1α增高,ERK1/2、JNK、p38总量不变,而其各自的磷酸化形式均明显增加。HKC转染Cyr—luc后,p38通路抑制剂SB203580和ERK通路抑制剂PD98059显著抑制缺氧时Cyr61的转录活性,两者协同作用时抑制作用显著增强。Ca—MEK1与Cyr—luc共转染HKC后,Cyr61转录活性无改变;而Ca—MKK6与Cyr—luc共转染后,Cyr61转录活性显著增高。对缺氧培养的HKC,PD98059处理使HIF-1α和Cyr61蛋白表达显著降低;SB203580处理可显著降低Cyr61蛋白表达,但对HIF-1α无影响。结论 在HKC中,缺氧可通过p38通路直接上调Cyr61基因启动子活性,也可通过ERK1/2途径促进HIF-1α表达,间接调节Cyr61基因启动子活性。  相似文献   

4.
Objective To investigate the effect and mechanism of cysteine-rich protein 61(Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia. Methods Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups:control group, Cyr61 group, MAPK inhibitor group (Cyr61+PD98059), p38 inhibitor group (Cyr61+SB203580) and PI3K inhibitor group (Cyr61+Wortmannin). Each group was pretreated for 12 h and then injured by anoxia.The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry. The cellular ROS level was measured by spectro-fluorometry. The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test. The expression of Nrf2 in HK-2 cells was detected by Western blotting. Results Anoxia enhanced the expression of ROS and Nrf2, decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P<0.05). Cyr61 increased the expression of p-Akt, Nrf2, SOD and CAT in HK-2, and decreased the expression of ROS, at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P<0.05). Wortmannin inhibited the expression of p-Akt,Nrf2, SOD and CAT, meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P<0.05). PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P>0.05). Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2, which enhances the expression of SOD and CAT, and decreases the expression of ROS. Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.  相似文献   

5.
目的探讨重组大鼠肝再生增强因子(rrALR)对体外培养的肾小管上皮细胞增殖及凋亡的影响。方法将体外培养的人肾小管上皮细胞株(HK2)分组,分别加入不同浓度的庆大霉素(GM)或(和)m~LR,^3H-胸腺嘧啶核苷(^3H-TdR)掺入法检测HK2细胞的增殖:吖啶橙/溴乙啶(AO/EB)染色和钙磷脂结合蛋白/碘化丙啶(annexin V/PI)双标记流式细胞术检测上述各组HK2细胞的凋亡。结果(1)rrALR对常规培养条件下的HK2细胞有直接的促增殖作用。并具有量效关系(在25ng/ml~50μg/ml的浓度范围内逐渐增强,P〈0.01)和时效关系(培养12h即有该作用,48h达到高峰,以后逐渐下降,P〈0.05);(2)rrALR能够促进GM损伤后的HK2细胞增殖,有明显剂量依赖性(P〈0.05);(3)rrALR呈剂量依赖性抑制GM诱导的HK2细胞凋亡(P〈0.01)。结论rrALR能够促进体外常规培养和GM损伤后的肾小管上皮细胞增殖.抑制GM诱导的小管上皮细胞凋亡,提示ALR可能对小管上皮细胞的中毒性损伤有改善作用。  相似文献   

6.

OBJECTIVE

To examine whether hypoxia (one of the many components of ischaemic preconditioning) can induce a protective response in culture renal tubular cells, and thus determine if non‐lethal periods of hypoxia could confer protection against apoptotic injury to human proximal tubular cells during cold storage and subsequent cytotoxic insult, and establish the cellular mechanisms by which this protection is induced.

MATERIALS AND METHODS

Human proximal tubular cells (HK‐2) were pre‐incubated for 24 h in normoxic or hypoxic conditions and then incubated at 4 °C for 6 h to mimic cold storage, before being returned to normal conditions and exposed to varying concentrations of cyclosporine A (CSA). Cell viability and apoptosis were measured using propidium iodide staining and flow cytometry. The expression of heat‐shock protein (HSP)‐70 was determined by Western blotting.

RESULTS

Hypoxia had no effect on cell viability or apoptosis. Pre‐exposure of cells to hypoxia significantly protected against CSA‐induced damage even after a period of cold storage. Western blotting analysis showed that hypoxia up‐regulated the anti‐apoptotic protein HSP‐70. HK‐2 cells over‐expressing HSP‐70 mimicked hypoxia preconditioning, in that they were protected during cold storage and CSA‐induced apoptosis.

CONCLUSION

Exposure of renal tubular cells to a sequential model of cold storage, reperfusion and incubation with CSA resulted in apoptotic cell death. Preconditioning these cells with hypoxia induced a protective response and up‐regulation of the anti‐apoptotic protein HSP‐70. There was a similar response in non‐preconditioned cells over‐expressing HSP‐70. Further understanding of the cellular changes occurring during this period of preconditioning will allow the development of more targeted, clinically relevant methods of preconditioning in renal transplantation.  相似文献   

7.
RNA干扰抑制Cyr61表达对人脑胶质瘤细胞生物学行为的影响   总被引:1,自引:1,他引:0  
目的 观察RNA干扰技术沉默Cyr61基因表达对人脑胶质瘤U251细胞生物学行为的影响.方法 针对Cyr61 mRNA的序列设计合成小干扰RNA(siRNA)的DNA模板,构建pRNAT-Cyr61重组质粒,转染人脑胶质瘤U251细胞;RT-PCR和Western blot检测其对U251细胞内源性Cyr61表达的影响;用噻唑蓝(MTT)比色法观察U251细胞体外增殖活性的变化;用Transwell 小室法检测U251细胞体外侵袭能力的改变;流式细胞仪检测U251细胞凋亡并用透射电镜观察凋亡后的细胞形态学变化.结果 pRNAT-Cyr61重组质粒在mRNA及蛋白水平分别显著抑制Cyi61基因表达,抑制率分别最高达到74.87%和78.23%;U251细胞的体外生长抑制率最高达68.15%,侵袭细胞数下降至(46.00±2.82)个;U251细胞凋亡率最高达53.16%.结论 pRNAT-Cyt61可抑制Cyr61在人脑胶质瘤U251细胞中的表达,并抑制细胞的增殖活性和侵袭能力,促进细胞凋亡.  相似文献   

8.
【摘要】 目的 探讨脂多糖(LPS)对大鼠NRK-52E肾小管上皮细胞增殖的影响及MEK/ERK1/2信号途径的调节作用。方法 不同剂量LPS按不同时间刺激NRK-52E细胞,并以MEK特异性抑制剂U0126干预。使用cell counting kit-8(cck-8)试剂检测细胞增殖,Western blot检测NRK-52E细胞ERK1/2、Akt蛋白磷酸化水平。结果 LPS在0.001、0.01 mg·L-1作用72 h对NRK-52E细胞增殖无明显影响,在0.1、1.0 mg·L-1抑制NRK-52E细胞增殖;MEK特异性抑制剂U0126显著抑制NRK-52E细胞增殖,其作用在2.5、5、10、20 ?滋M浓度之间无显著差异。U0126预处理加强LPS对NRK-52E细胞增殖的抑制作用。LPS诱导NRK-52E细胞ERK1/2和Akt磷酸化;U0126单独或联合LPS处理NRK-52E细胞72 h阻断ERK1/2蛋白磷酸化,伴有pAkt蛋白水平上调。结论 MEK/ERK1/2可能在LPS抑制NRK-52E细胞增殖的过程中起保护作用。  相似文献   

9.
目的:研究缺氧复氧后肾小管上皮细胞凋亡的表达及N-乙酰半胱胺酸(NAC)对其的影响。方法:选用人肾小管上皮细胞(HK2)建立缺氧复氧损伤模型,设正常对照组、单纯缺氧复氧组、不同浓度(1mmol/L、5mmol/L、10mmol/L)NAC干预组。流式细胞术检测细胞内氧化应激水平与细胞凋亡表达。结果:缺氧复氧后HK2细胞内氧化应激水平提高,HK2细胞凋亡细胞和死亡细胞数量增多,且随缺氧时间的延长,细胞内氧化应激水平逐渐升高,凋亡细胞和死亡细胞数量逐渐增多;NAC呈剂量关系抑制细胞内氧化应激水平,同时呈剂量关系减少凋亡细胞和坏死细胞的数量。结论:缺氧复氧诱导细胞内氧化应激的产生,诱导HK2细胞凋亡和死亡;NAC可以通过抑制细胞内氧化应激减少细胞和坏死细胞的数量。  相似文献   

10.
目的  探讨环状RNA SNRK(circSNRK)在缺血-再灌注损伤(IRI)中的作用及机制。方法  构建缺氧-复氧(IRI)细胞模型,检测IRI处理后circSNRK的表达情况及过表达circSNRK对细胞增殖和细胞凋亡的影响。分析circSNRK的作用靶点。将HK2细胞分为空白组(Mock组)、IRI组、对照质粒+IRI组(IRI+NC组)、过表达人circSNRK+IRI组(IRI+circSNRK组)、过表达人circSNRK+IRI+蛋白激酶B(Akt)抑制剂组(IRI+circSNRK+MK2206组)、对照质粒组(NC组)。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞增殖及凋亡情况。进行circSNRK作用靶点的基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)聚类分析。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞CDKN1A、Akt、B细胞淋巴瘤(Bcl)-2、半胱氨酸天冬氨酸蛋白酶(Caspase)-9信使RNA(mRNA)表达水平,p21、Bcl-2、Caspase-9、Akt、p-Akt蛋白表达水平。检测NC组、IRI+NC组、IRI+circSNRK组、IRI+circSNRK+MK2206组细胞增殖及凋亡情况。结果  与Mock组比较,IRI组circSNRK表达水平较低,HK2细胞增殖能力下降,细胞凋亡增多。IRI+circSNRK组细胞增殖能力较IRI+NC组升高,细胞凋亡较IRI+NC组减少。circSNRK可通过51个微小RNA(miRNA)作用于648个靶点。GO富集分析显示,circSNRK作用靶点主要富集于细胞过程和生物调节等生物学过程,细胞部分、细胞和细胞外部分等细胞成分,以及结合、结合蛋白和酶等分子功能。KEGG聚类分析显示,circSNRK作用靶点主要富集在癌症信号通路、磷脂酰肌醇-3-激酶(PI3K)-Akt信号通路和癌症miRNA等相关通路。与Mock组比较,IRI组CDKN1A和Caspase-9 mRNA相对表达量较高,miR-99a-5p RNA表达水平较高,Akt和Bcl-2 mRNA相对表达量较低;与IRI+NC组比较,IRI+circSNRK组CDKN1A和Caspase-9 mRNA相对表达量较低,Akt和Bcl-2 mRNA相对表达量较高,miR-99a-5p RNA表达水平较低,差异均有统计学意义(均为P < 0.05)。IRI组p21和Caspase-9蛋白表达较Mock组增多,p-Akt、Akt和Bcl-2蛋白表达较Mock组减少;IRI+circSNRK组p21和Caspase-9蛋白表达较IRI+NC组减少,p-Akt、Akt和Bcl-2蛋白表达较IRI+NC组增多。circSNRK和Akt上存在miR-99a-5p结合位点。与NC组比较,IRI+NC组细胞增殖能力下降;与IRI+NC组比较,IRI+circSNRK组细胞增殖能力升高;与IRI+circSNRK组比较,IRI+circSNRK+MK2206组细胞增殖能力下降(均为P < 0.05)。IRI+NC组细胞凋亡水平较NC组高;IRI+circSNRK组细胞凋亡水平较IRI+NC组低;IRI+circSNRK+MK2206组细胞凋亡水平较IRI+circSNRK组高。结论  在IRI条件下,circSNRK可影响HK2细胞增殖和凋亡,可能是通过Akt通路发挥作用。  相似文献   

11.
目的:观察不同病理类型肾病综合征(NS)患者尿蛋白对肾小管上皮细胞(RTECs)增殖和凋亡的影响,以进一步明确尿蛋白所致肾小管-间质损害的机制.方法:(1)从局灶-节段性肾小球硬化症(FSGS)、膜性肾病(MN)、微小病变肾病(MCN)三种不同病理类型的NS患者尿液中提取尿蛋白,经成份分析、灭菌等处理后以0.5 mg/ml、1.0 mg/ml、2 mg/ml、4 mg/ml、8 mg/ml浓度分别刺激体外培养的HK-2细胞,另设空白对照组.(2)MTT法检测不同病理类型NS患者尿蛋白刺激后细胞的增殖情况.(3)乳酸脱氢酶(LDH)释放实验检测不同病理类型NS患者尿蛋白的细胞毒作用.(4)Western Blotting法检测Fas蛋白表达.结果:各病理类型所提取的尿蛋白成分相同,主要为白蛋白、转铁蛋白、IgG等,但各病理类型组成比例不同;肾小管上皮细胞MTT值低浓度有明显增殖作用,高浓度时细胞过度增殖则导致凋亡;肾小管上皮细胞LDH释放率和Fas蛋白的表达水平随尿蛋白浓度的升高而升高;以上各项检测指标中FSGS患者尿蛋白对HK-2细胞的作用最强,MN次之,MCD最弱.结论:在体外条件下,尿蛋白对RTECs呈剂量依赖性的细胞毒作用,低剂量尿蛋白诱导RTECs异常增殖,较高剂量尿蛋白可诱导RTECs凋亡;除尿蛋白的量决定了损伤严重程度外,尿蛋白的性质也决定了损伤的严重程度.  相似文献   

12.
Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor -β1 (TGF-β1)-activated renal fibroblasts (NRK-49F), and to explore its effect and mechanism. Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0, 0.5, 1.0, 2.0, 5.0 μg/L). Western blotting was used to detect the expression of Cyr61 protein, and CCK-8 assay was used to test the proliferative activity of NRK-49F cells. (2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection. The cells were divided into control group (null vector transfection), over-expression group and low-expression group. The proliferation was discovered by CCK-8 assay after 24, 48 and 72 h. Further, 5.0 μg/L TGF-β1 activated these three groups. The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry. The mRNA expressions of fibrosis markers (Col1α1, Col3α1, MMP9, MMP13) and factors of cell senescence signal pathway (p53, p21, Rb, p16) were ascertained by real time PCR, and the protein expressions of Col3 and MMP9 were detected by Western blotting. Results (1) Compared with 0.0 μg/L TGF-β1 group, the proliferation of NRK-49F cells was enhanced in 0.5, 1.0, 2.0 and 5.0 μg/L TGF-β1 groups (all P<0.05), while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P<0.05). (2) The proliferation of over-expression group was lower than that of control group after 24, 48 and 72 h (all P<0.05), which was in a time-dependent manner. (3) Compared with control group activated by TGF-β1, the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1) and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P<0.05). Simultaneously, the proportion of cells bogged down in G1 phases, as well as the expressions of p53, p21 and Rb mRNA increased (all P<0.05). The above effects of low-expression group were just opposite to over-expression group. Moreover, there was no significant difference in the expression of p16 gene among the three groups (P>0.05). Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts, thereafter slowing down the process of renal fibrosis. The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.  相似文献   

13.
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&#65377; 方法 将培养的大鼠肾小管细胞NRK-52E分别与不同浓度(10&#65380; 20&#65380; 30 mg/ml)的去脂无内毒素牛血清白蛋白(BSA)共同孵育6&#65380; 12&#65380; 18和24 h&#65377;透射电镜&#65380;共聚焦激光显微镜和流式细胞仪检测细胞凋亡&#65377;BSA 20 mg/ml刺激NRK-52E细胞15&#65380; 30&#65380; 60和120 min后, Westen印迹测定p38&#65380;氨基末端激酶(JNK)和细胞外信号调节激酶(ERK)活性&#65377;将SB202190(20 μmol/L, p38抑制剂)&#65380;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&#65377;结果 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡&#65377;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&#65377;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&#65377;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&#65377;  相似文献   

14.
15.
BACKGROUND: Hyperplasia is attributed to enhanced tubular cell proliferation with unbalanced cell death. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors induce apoptosis in a variety of cell lines, including proximal tubular cells. However, the mechanisms by which statins induce apoptosis in tubular cells have not been fully addressed. METHODS: Apoptosis induced by simvastatin was measured in murine tubular cells with and without overexpressing Bcl-xL. Expression of genes implicated in cell death was studied by Northern and Western blot. RESULTS: The treatment of proliferating murine tubular cells (MCT) with simvastatin induced apoptosis in a time- and dose-dependent manner (0.1 to 1 micromol/L). Apoptosis was correlated with Bcl-xL mRNA and protein down-regulation. By contrast, the treatment with simvastatin did not modify the expression of the proapoptotic protein Bax. Simvastatin treatment was associated with cytochrome C release from the mitochondria to the cytosol. We also observed the presence of active caspase 9 and 3 during apoptosis induced by simvastatin. These effects were reversed by mevalonate, farnesylpyrophosphate (FPP), and geranylgeranylpyrophosphate (GGPP), suggesting the involvement of protein prenylation. Simvastatin appears to alter the balance between cell-life and death-promoting genes, as reflected by the decreased Bcl-xL/Bax ratio. Supporting this hypothesis, overexpression of Bcl-xL reduced the amount of apoptosis induced by simvastatin by 80% when compared with control vector-expressing cells. The overexpression of Bcl-xL also prevented the activation of caspase 9 and 3. CONCLUSION: Our results indicate that down-regulation of Bcl-xL expression mediates apoptosis induced by statins in tubular cells. These results may be relevant to the treatment of disorders characterized by altered tubular proliferation.  相似文献   

16.
目的:探讨瑞芬太尼对缺氧/复氧诱导的PC12细胞增殖、凋亡和氧化应激的影响及分子机制。方法:将PC12细胞按照随机数字表法分为缺氧/复氧组(缺氧15 h后复氧5 h),空白组(正常培养的细胞),瑞芬太尼低、中、高浓度组(2、10、50 mg/L瑞芬太尼处理缺氧/复氧诱导的PC12细胞),瑞芬太尼+LY294002组(1...  相似文献   

17.
BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton. The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known. METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells. RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis. A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis. A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells. Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis. The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor. CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis. Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.  相似文献   

18.
Hypoxia that is caused by vascular defects or disruption is commonly associated with renal diseases. During cisplatin nephrotoxicity, hypoxic regions are identified in the outer medulla and the renal cortex. However, the regulation of cisplatin injury by hypoxia is unclear. Previous work has demonstrated the cytoprotective effects of hypoxia against apoptotic injury. This study further examines the cytoprotective mechanisms in models of cisplatin-induced tubular cell apoptosis. In cultured renal tubular cells, 20 microM cisplatin induced approximately 60% apoptosis within 16 h. The rate of apoptosis was suppressed to < 20%, when the incubation was conducted under hypoxia (2% O2). Mitochondrial events of apoptosis, namely Bax accumulation and cytochrome c release, also were ameliorated. During cisplatin treatment, cell ATP was maintained in both normoxic and hypoxic cells. Hypoxic incubation lowered extracellular pH, but prevention of the pH decrease did not restore cisplatin-induced apoptosis. The cytoprotective effects of hypoxia also were independent of hypoxia-inducible factor 1 (HIF-1). Cobalt, as hypoxia, activated HIF-1 yet did not suppress cisplatin-induced apoptosis. Moreover, hypoxia suppressed cisplatin-induced apoptosis in HIF-1-deficient mouse embryonic stem cells and renal proximal tubular cells. Conversely, mitochondrial inhibitors, particularly inhibitors of respiration complex III (antimycin A and myxothiazol), mimicked hypoxia in apoptosis suppression. The effects of hypoxia and mitochondrial inhibitors were not additive. It is interesting that both hypoxia and complex III inhibitors ameliorated cisplatin-induced p53 activation. Therefore, the cytoprotective effects of hypoxia are independent of changes in cell ATP, pH, or HIF but may involve mitochondrial inhibition and the suppression of p53.  相似文献   

19.
目的 探讨脯胺酰羟化酶抑制剂3,4-二羟基苯甲酸乙酯(EDHB)对白蛋白诱导的低氧条件下培养的肾小管上皮细胞(NRK-52E)凋亡的影响。 方法 NRK-52E细胞在以下各组孵育24 h:常氧(5%CO2+空气)组,低氧(1%O2+5%CO2+94%N2)组,常氧+白蛋白(30 g/L)组及低氧+白蛋白(30 g/L)组,观察各组NRK-52E细胞的凋亡情况。然后NRK-52E细胞在以下各组孵育24 h:常氧组、低氧组,低氧+白蛋白(30 g/L)组,低氧+EDHB(500 μmol/L)组,EDHB预处理组(培养细胞中先加入EDHB 500 μmol/L,0.5 h后再加入BSA 30 g/L,低氧培养),观察EDHB对白蛋白诱导低氧培养NRK-52E凋亡的影响。流式细胞技术检测细胞凋亡;RT-PCR检测凋亡相关蛋白bcl-2、bax 及血管内皮生长因子(VEGF)mRNA表达;Western印迹检测VEGF蛋白表达。 结果 NRK-52E细胞凋亡率在常氧组与低氧组差异无统计学意义 (P > 0.05),但低氧+白蛋白组细胞凋亡率显著高于常氧+白蛋白组(37.36%±4.95%比25.59%±3.32%,P < 0.05)。低氧+白蛋白组bax mRNA表达显著高于常氧+白蛋白组(P < 0.05),而bcl-2 mRNA的表达则显著低于常氧+白蛋白组(P < 0.05)。EDHB预处理可显著抑制低氧+白蛋白组细胞凋亡率的增高(P < 0.05)、bax mRNA表达的增高(P < 0.05)以及bcl-2 mRNA表达的降低(P < 0.05)。低氧组NRK-52E细胞VEGF mRNA和蛋白表达显著高于常氧组(P < 0.05),而低氧+白蛋白组则显著低于低氧组(P < 0.05)。EDHB预处理可显著抑制低氧+白蛋白组细胞VEGF mRNA和蛋白表达的降低(P < 0.05)。 结论 白蛋白和低氧联合刺激可显著增加NRK-52E细胞凋亡,EDHB预处理可改善该病变,可能与其提高VEGF的表达有关。  相似文献   

20.
Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.  相似文献   

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