Adrenomedullin ameliorates podocyte injury induced by puromycin aminonucleoside in vitro and in vivo through modulation of Rho GTPases

International Urology and Nephrology - Podocyte injury is a key event in proteinuric kidney disease and eventually glomerular scarring. While adrenomedullin (AM), a potent vasodilatory peptide, has...     

Melatonin decreases apoptosis and expression of apoptosis-associated proteins in acute puromycin aminonucleoside nephrosis.

BACKGROUND: The anti-apoptotic properties of melatonin have been demonstrated previously in several in vivo and in vitro studies. Previous reports have shown increased apoptosis during puromycin aminonucleoside nephrosis (PAN). The aim of this study was to determine if melatonin (MEL) can prevent apoptosis and modify oxidative stress, an apoptosis inducer, in this experimental model. METHODS: Rats were injected intraperitoneally with puromycin aminonucleoside. In addition, by the intragastric route they received 1 mg/kg/day of MEL or vehicle 3 days before puromycin injection and throughout the experiment. Animals were sacrificed at weeks 1 and 2 of nephrosis and frozen renal sections were studied for apoptosis by TUNEL, for apoptosis-associated proteins by monoclonal and polyclonal antibodies, and for superoxide anion (O(2)(-)) by a histochemical method. Nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH), and the activities of superoxide dismutase (SOD) and catalase were measured in homogenized kidney tissue by appropriate biochemical and enzymatic methods. RESULTS: Increases in apoptosis, p53, Fas and Fas-ligand were observed in nephrotic animals. MEL treatment decreased apoptosis at weeks 1 and 2 in the glomerular, interstitial and tubular compartments. This was accompanied by decreased expression of p53 (glomerulus, week 1; tubules, weeks 1 and 2), Fas (glomerulus and interstitium, week 2; tubules, weeks 1 and 2) and Fas-ligand (interstitum and tubules, week 2). Increased expression of Bcl-2-positive cells was observed at week 2 in all renal compartments in MEL-treated animals. High levels of O(2)(-) and NO generation and lipid peroxidation (MDA) were found in nephrotic animals. SOD and GSH remained unchanged, and only decreased catalase activity (week 1) was observed in PAN animals. Tendencies toward decreased values of O(2)(-) and MDA content along with recovery of catalase activity (week 1) were observed in MEL-treated nephrotic animals, but were insignificant in magnitude. MEL, however, did significantly downregulate pro-apoptotic genes and upregulated anti-apoptotic genes. CONCLUSIONS: The data demonstrate that, in PAN, melatonin has anti-apoptotic effects, which might in part be independent of the modulation of the oxidative status.     

Salvia przewalskii extract of total phenolic acids inhibit TLR4 signaling activation in podocyte injury induced by puromycin aminonucleoside in vitro

Background: TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling.

Methods: The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB?+?RA or tacrolimus for 30?min, followed by PAN (100?μg/mL) for 24?h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway.

Results: (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24?h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components.

Conclusions: SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes.     

Bcl-2基因家族在睾丸生殖细胞凋亡中的作用

睾丸生殖细胞的凋亡是一种重要的生理机制,通过凋亡来调节生精小管中生殖细胞的数目。正常生精过程中, 存在着生殖细胞的主动退化现象,有很多因素参与调节这一过程,其中Bcl-2基因家族在生殖细胞的凋亡过程中起着重要的调节作用。     

大鼠骨骼肌缺血再灌注损伤的细胞凋亡及相关基因表达

目的研究缺血性损伤和缺血再灌注损伤的发生情况和病理改变，探讨细胞凋亡及相关基因p53、p21、Bax、Bcl-2的表达规律。方法建立大鼠后肢缺血和缺血再灌注实验模型，光镜下观察缺血和缺血再灌注早期的骨骼肌组织病理学变化，检测缺血和缺血再灌注过程中细胞凋亡现象的发生及相关基因p53、p21、Bax、Bcl-2的表达。结果缺血5h的大鼠骨骼肌全部存活，而缺血9h者未获存活。缺血和缺血再灌注损伤引起骨骼肌细胞水肿、坏死和细胞凋亡，并于再灌注过程观察到微循环障碍和中性粒细胞趋化浸润现象。缺血7h细胞凋亡率最高，相关基因p53、p21、Bax表达与缺血时间成正比，而Bcl-2表达与缺血时间成反比。结论细胞凋亡是缺血和缺血再灌注损伤的重要病理学改变。微循环障碍和中性粒细胞趋化浸润是缺血再灌注损伤的重要原因之一。相关基因表达与凋亡的发生关系密切。     

Prognostic factors in resectable pancreatic cancer: p53 and Bcl-2

The p53 tumor suppressor gene and the Bcl-2 proto-oncogene regulate cell cycle progression and apoptosis. We evaluated the expression of these molecular markers with standard pathologic prognostic variables in patients who received multimodality therapy for resectable adenocarcinoma of the pancreas to study the effect of p53 and Bcl-2 on survival duration. Immunohistochemical staining of archival material was performed to determine levels of expression of p53 and Bcl-2 proteins in 70 patients with adenocarcinoma of pancreatic origin. All patients underwent a potentially curative pancreaticoduodenectomy and standardized pathologic analysis of resected specimens. Potential pathologic and molecular prognostic variables were assessed for their effect on survival duration. Nuclear staining for p53 was observed in 33 (47%) of 70 specimens. Immunostaining for Bcl-2 was observed in 23 specimens (33%). A trend toward improved survival duration was seen in patients whose tumors stained positive for either p53 or Bcl-2. Negative staining for both markers predicted short survival (P = 0.01). By univariate and multivariate analyses, no single pathologic factor was associated with survival duration. Immunohistochemical staging using both p53 and Bcl-2 significantly predicted survival duration by univariate and multivariate analysis; patients whose tumors stained positively for p53 and/or overexpressed Bcl-2 had a significantly longer survival than those whose tumors stained negative for both proteins. Supported by grants from the National Institutes of Health (RO1 CA75517-01) and the Various Donors Fund for Pancreatic Cancer Research at M.D. Anderson Cancer Center. Presented to the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La., May 17–20, 1998.     

Role of CD2-associated protein in podocyte apoptosis and proteinuria induced by angiotensin II

Podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Proteinuria is the most common clinical manifestation of glomerular diseases. Losartan is the traditional renin–angiotensin system (RAS). However; the precise mechanisms underlying the beneficial effects of Losartan on podocytes are still unknown. This study tested the hypothesis that podocytes were exposed to Angiotensin II (Ang II) to induce apoptosis and Proteinuria. Losartan directly reduces the rate of apoptotic podocytes induced by Ang II. Our results showed that apoptotic podocytes may be related to the decrease of CD2AP mRNA and protein expressions, Losartan reduced the apoptosis and Proteinuria by stabilizing the CD2AP mRNA and protein expressions. In this study, we explored the role of CD2-associated protein in podocyte apoptosis and Proteinuria induced by Ang II. Our findings provide some possible clues for further exploring the pharmacological targets to the proteinuria.     

表浅性膀胱移行细胞癌中p53和Bcl-2同时表达的临床意义

目的 :探讨表浅性膀胱移行细胞癌中Bcl 2和 p5 3的同时表达与膀胱癌预后之间的关系。 方法 :应用免疫组化技术 (SABC法 )检测 4 2例表浅性膀胱癌蜡块标本中Bcl 2和p5 3的表达。患者术后平均随访 36个月 ,14例复发 ,其中复发 2次以上 9例 ,有转移者 4例。结果 :4 2例中 ,31例 (73.8% ) p5 3阳性表达 ,与G1(6 2 .5 % )和G2 (72 .2 % )相比较 ,G3 (81.2 % )更多见 ;pT1期 (78.6 % )p5 3阳性率较 pTa期 (6 4 .3% )高。 12例(2 8.6 % )发现有Bcl 2表达 ,Bcl 2表达阳性率G3 (37.5 % )明显高于G2 (2 2 .2 % )和G1(2 5 .0 % ) ,与分期无关 (P>0 .0 5 )。膀胱癌复发率p5 3基因表达阳性者高于阴性者 (P <0 .0 5 ) ,而Bcl 2基因表达阳性者与阴性者的差别无统计学意义 (P >0 .0 5 ) ,但 p5 3和Bcl 2同时呈阳性表达者 (8例 )明显高于其他患者 (P <0 .0 1)。Bcl 2和p5 3的阳性表达与肿瘤侵袭性和淋巴结及远处转移有关。结论 :p5 3和Bcl 2同时阳性表达的肿瘤患者预后较差 ,两者同时检测对判断膀胱癌复发及预后更具有指导意义。     

化疗诱导人肝癌细胞凋亡中p53和bcl-2基因的调控

目的 建立化疗药物丝裂霉素诱导肝癌细胞凋亡的模型,初步探讨在该凋亡过程 中重要凋亡相关基因ｐ５３和ｂｃｌ－２的调控作用。方法 用丝裂霉素诱导人肝癌细胞系ＨｅｐＧ２凋亡,我显微镜和透射电镜观察肝癌细胞的形态学改变,琼脂糖凝胶电泳检测凋亡细胞ＤＮＡ片段的梯状条带,用流式细胞仪免疫荧光法检测凋亡不同时期Ｐ５３和Ｂｃｌ－２蛋白的表达量。结果 ８ｍｇ／ｍｌ丝裂霉素作用１２,２４,４８ｈ后,Ｐ５３蛋白表达量明显增高,尤以１２ｈ明显,而后逐渐下降。Ｂｃｌ－２蛋白在整个凋亡过程中无明显变化。结论 在化疗药物丝裂霉素诱导肝癌细胞凋亡过程中,ｐ５３基因参与了调控,并为凋亡信号传导途径中的早期事件。丝裂霉素诱导的肝癌细胞凋亡途径为ｐ５３依赖方式,而在该凋亡过程中,ｂｃｌ－２基因的调控并不重要。     

Livin mRNA及Bcl-2，p53蛋白在胰腺癌中的表达

目的:探讨凋亡抑制蛋白Livin在胰腺癌组织中的表达及其与Bcl-2和p53蛋白表达的关系。 方法:采用逆转录-多聚酶链反应（RT-PCR）法检测52例胰腺癌和12例正常胰腺组织中Livin mRNA的表达；用免疫组织化学法检测胰腺癌组织中Bcl-2和p53蛋白的表达。 结果:Livin mRNA在胰腺癌中的表达率为71.2%，明显高于正常胰腺组织（P＜0.01）。Livin基因表达与胰腺癌的分化程度和淋巴结转移有关（P＜0.05）。Livin基因表达与Bcl-2蛋白表达有关，而与p53蛋白无关。 结论:Livin基因在胰腺癌的发生发展中起重要作用；Livin与Bcl-2的异常表达可能在胰腺癌的发生中起协同作用；Livin基因过表达与抑癌基因p53的失活无关。     

Resveratrol induced serine phosphorylation of p53 causes apoptosis in a mutant p53 prostate cancer cell line

PURPOSE: Resveratrol (Calbiochem, La Jolla, California) is a naturally occurring stilbene reported to cause apoptosis in various cultured cancer cells. In the current study the effect of resveratrol was determined in the androgen insensitive DU 145 prostate cancer cell line. Induction of apoptosis and activation of apoptosis related signal transduction pathways were measured. MATERIALS AND METHODS: DU 145 cells were treated with resveratrol and apoptosis was measured by determining nucleosome content. Activation of mitogen activated protein kinase (MAPK) (extracellular signal-regulated kinase 1/2), p53 content and serine-15 phosphorylation of p53 were measured by immunoblot. Electrophoretic mobility shift assay of p53 binding to DNA, and measurement of p21 and glyceraldehyde-3-phosphate dehydrogenase messenger RNA were also done. RESULTS: Resveratrol induced apoptosis in DU 145 cells. The stilbene activated MAPK and caused increased abundance of p53 and serine-15 phosphorylated p53. Resveratrol induced serine-15 phosphorylation of p53 was blocked by PD 98059 (Calbiochem), a MAPK kinase inhibitor, implicating MAPK activation in the phosphorylation of p53. PD 98059 also inhibited resveratrol induced apoptosis. These results suggest that apoptosis induction by resveratrol in DU 145 cells requires serine-15 phosphorylation of p53 by MAPK. Inhibition of MAPK dependent serine-15 phosphorylation resulted in reduced p53 binding to a p53 specific oligonucleotide on electrophoretic mobility shift assay. Pifithrin-alpha (Calbiochem), a p53 inhibitor, blocked resveratrol induced serine-15 phosphorylation of p53 and p53 binding to DNA. Resveratrol caused a p53 stimulated increase in p21 messenger RNA. Transfection of additional wild-type p53 into DU 145 cells induced apoptosis, which was further enhanced by resveratrol treatment. CONCLUSIONS: Resveratrol causes apoptosis in DU 145 prostate cancer cells. This action depends on the activation of MAPK, increase in cellular p53 content, serine-15 phosphorylation of p53 and increased p53 binding to DNA.     

Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease

BACKGROUND: Apoptosis is a characteristic feature of human autosomal-dominant polycystic kidney disease (ADPKD). The Han:Sprague-Dawley (SPRD) rat model closely resembles human ADPKD and presents an opportunity to investigate the apoptotic pathway in the pathogenesis of this disease. METHODS: Han:SPRD rats were studied during the early stages of ADPKD (newborn, 2 and 6 weeks old). Apoptotic cells were detected by the TUNEL (Tdt-mediated dUTP nick end-labeling) assay. Caspase-3 activity was measured using the fluorescent substrate DEVD-AMC and cleavage of poly (ADP-ribose) polymerase [PARP]. Expression of pro- and anti-apoptotic B-cell lymphoma (Bcl-2) proteins was detected on Western blot analysis. RESULTS: TUNEL (+) cells, caspase-3 activity and caspase-mediated PARP breakdown were significantly increased in 2-week-old heterozygous (Cy/+) and homozygous (Cy/Cy) rat kidneys compared to normal littermate controls. In Cy/+ rat kidneys, decreased expression of anti-apoptotic Bcl-XL coincided with increased caspase-3 activity at 2 weeks of age while expression of Bcl-2, another anti-apoptotic protein, increased at 6 weeks of age. In Cy/Cy rat kidneys, decreased expression of Bcl-XL and increased expression of Bcl-2 was present at 2 weeks of age. Pro-apoptotic Bax and Bad expression was unchanged at 2 weeks of age in both Cy/+ and Cy/Cy rat kidneys. CONCLUSIONS: Activation of caspase-3 and dysregulation of the balance between pro- and anti-apoptotic Bcl-2 family members, specifically a down-regulation of anti-apoptotic Bcl-XL, correlates with increased apoptosis in polycystic Han:SPRD rat kidneys.     

化疗药物诱导胰腺癌细胞凋亡过程中p53和bcl—2基因表达量？…

目的 本文通过观察化疗药物话导胰腺癌细胞凋亡过程中,Ｐ５３和ＢＣＬ－２基因表达量的变化,探讨化疗药物诱导胰腺癌细胞发生凋亡过程与相关基因表达的关系。方法 采用斑点杂交及激光密度扫描技术对化疗药物配伍;５－氟尿嘧喧（５－ＦＵ）、丝裂霉素（ＭＭＣ）和表柔比星（Ｅ－ＡＤＭ）诱导胰腺癌细胞株细胞发生凋亡过程中Ｐ５３和ＢＣＬ－２基因表达量的变化进行了检测分析。结果 化疗药物诱导胰腺癌细胞株细胞发生凋亡过程中     

Bcl-2 protein expression associated with resistance to apoptosis in clear cell adenocarcinomas of the vagina and cervix expressing wild-type p53

Background: Clear cell adenocarcinomas (CCAs) of the vagina and cervix are rare tumors that often overexpress wild-type p53. In vitro, expression of protooncogene bcl-2 can block p53-mediated apoptosis. The objective of this study was to determine if bcl-2 is expressed in CCAs and whether this expression is associated with inhibition of apoptosis. Methods: Twenty-one paraffin-embedded clear cell adenocarcinomas were immunohistochemically stained for bcl-2 (antibody M 887, Dako, Carpinteria, CA) and DNA fragmentation (Apop-Tag, Oncor, Gaithersburg, MD), a marker for apoptosis. Fifteen tumors were associated with in utero exposure to diethylstilbestrol (DES). Prior p53 gene analysis had indicated the presence of wild-type p53 in each tumor. Human lymphoid tissue containing bcl-2-expressing lymphocytes and DNase I-exposed CCA tissue sections were used as positive controls for the bcl-2 and apoptosis assays, respectively. Expression of bcl-2 and DNA fragmentation was classified (0 to 3+) according to percentage of positive cells and intensity of staining. Results: Expression of bcl-2 was identified in each CCA examined, and was strongly positive (2+ to 3+) in 18 of 21 samples. Despite the presence of wild-type p53, only 4 of 21 tumors showed evidence of apoptosis as assessed through DNA fragmentation. Conclusions: DNA damage leads to increased intracellular p53 levels. Overexpression of p53 induces apoptosis as a means of protecting organisms from the development of malignancy. CCAs of the vagina and cervix, which contain wild-type p53 genes and often overexpress p53 protein, presumably have evolved mechanisms to avoid p53-induced apoptosis. Our observations are consistent with the hypothesis that overexpression of bcl-2 can inhibit p53-mediated apoptosis and suggest a mechanism by which these rare tumors can arise without mutation of the p53 gene. Supported by American Cancer Society Career Development Award 93–95 and NIH U01-CA-62912.     

环王巴明通过调节bcl-2的表达影响Du145细胞的凋亡

目的 研究环王巴明诱导Du145细胞凋亡作用,并探其机制.方法 不同浓度环王巴明处理细胞,透射电镜下观察细胞超微结构变化.同时行DAPI染色后荧光显微镜下观察细胞核形态变化;应用western blot 方法 检测Bc1-2蛋白水平变化.结果 10μmol/L环王巴明作用72h后,DAPI染色后荧光显微镜下见Du145细胞核内有致密的颗粒荧光,固缩或片段化的细胞核.透射电镜下可见细胞核固缩,染色质边集,细胞膜微绒毛减少等典型的凋亡形态学改变.而对照组的细胞形态正常,细胞膜表面可见许多微绒毛.10 μmol/L组Du145细胞Bcl-2蛋白表达显著降低,与空白对照组及DMSO组相比差异有统计学意义(P<0.05).结论 环王巴明通过调节bcl-2蛋白的表达,从而影响Du145细胞的凋亡.     

MicroRNA-503 regulates high-glucose induced apoptosis of renal tubular epithelial cells by targeting Bcl-2

Objective To investigate the expression vibration of microRNA-503(miR-503) and its effect on target gene Bcl-2, caspase enzyme activity and apoptosis of human renal tubular epithelial cells (HK-2) induced by high glucose, and to clarify the pathogenesis of renal tubular injury induced by high glucose. Methods HK-2 cells were cultured in normal glucose group (NG), mannitol hypertonic control group (MA), and high glucose group (HG). The morphology of apoptotic cells was observed using inverted microscope. The expression of miR-503 was determined using real-time quantitative PCR. The apoptosis rate of HK-2 cells was detected by Annexin Ⅴ-FITC double dye using flow cytometry instrument. The expression of Bcl-2 and cleaved caspase-9 were detected by Western blotting. Results In the high glucose and mannitol groups HK-2 cell, an obviously increased apoptotic rate was observed under inverted microscope compared with normal glucose group (P＜0.05). MA and HG up-regulated miR-503 expression (P＜0.01), down-regulated anti-apoptotic protein Bcl-2 expression (P＜0.05) and up-regulated cleaved caspase-9 (P＜0.05). Conclusions The expression of miR-503 increases in HK-2 cells cultured by high glucose and mannitol. MiR-503 promotes apoptosis of HK-2 cells via activating mitochondrial apoptotic pathways and enhancing cleaved caspase-9 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. The miR-503 may be involved in diabetic tubular injury and may be a new therapeutic target of DN.     

甲状腺肿瘤中Bcl-2及p53基因产物的表达及意义

Ｂｃｌ－２及ｐ５３基因可以调控细胞凋亡。我们应用Ｂｃｌ－２及ｐ５３基因产物单克隆抗体．标记５３例甲状腺腺癌及４１例甲状腺癌。结果发现，甲状腺癌和部分甲状腺腺瘤有Ｂｃｌ－２及ｐ５３基因产物表达。阳性产物分别定位于肿瘤细胞浆和细胞核。在甲状腺癌，二种基因产物阳性表达率在恶性度较高的未分化癌和滤泡状癌或临床ＴＮＭ分期Ⅲ、Ⅳ期的病例增高显著。提示Ｂｃｌ－２及突变型ｐ５３基因产物与甲状腺肿瘤的发生、发展及预后有关。