Necroptosis contributes to the cyclosporin A-induced cytotoxicity in NRK-52E cells

Cyclosporin A (CsA) induces renal tubular epithelial cells apoptosis and necrosis following in vitro exposure. The mechanisms of CsA-induced apoptosis have been studied intensively, whereas the mechanisms of necrosis remain to be elucidated. Necroptosis has been described as programmed necrosis. This study investigated the ability of CsA to induce necroptosis in the rat tubular cell line NRK-52E. The NRK-52E cells were incubated with CsA for 24 hours with or without necrostatin-1 (Nec-1). The majority of the NRK-52E cells died of necrosis as indicated by LDH leakage, Hoechst 33342/PI staining, and flow cytometry analysis. Cell death was significantly reduced by Nec-1 pretreated before CsA exposure. CsA-induced apoptosis and necrosis were also compared in NRK-52E cells with or without knockdown of receptor interaction protein 3 (RIP3) expression using small interfering RNA. Moreover, the role of reactive oxygen species (ROS) in CsA-induced cell death was also attempted. The result suggests that necroptosis contributes to the CsA-induced cytotoxicity in NRK-52E cells. Meanwhile, RIP3 and ROS are involved in CsA-induced necroptosis. To our knowledge, this is the first report on necroptosis in CsA-induced renal tubular cell death pathways, which might offer a novel protective target for CsA nephrotoxicity.     

Oxidative stress and cyclooxygenase-2 induction mediate cyanide-induced apoptosis of cortical cells

Cyanide (KCN)-induced generation of reactive oxygen species (ROS) involves cyclooxygenase-2 (COX-2)-mediated reactions in some neurons. The present study examines the extent to which COX isoforms are involved in KCN-induced apoptotic cell death processes of cultured cortical cells. After treatment with KCN (10-300 microM), COX-2 was expressed in a time- and concentration-dependent manner increasing markedly over a 4-h period. However, no significant changes were observed in COX-1 levels at any cyanide concentration. Correlated with COX-2 up-regulation, KCN induced a time-dependent apoptotic death. TUNEL staining showed that the COX-2 inhibitor NS-398 (30 microM) blocked KCN-induced apoptosis, whereas the selective COX-1 inhibitor valeryl salicylate did not affect the level of apoptotic cell death. Exposure of cells to KCN (300 microM) for 24 h resulted in DNA fragmentation, which was also reduced by NS-398. Prostaglandin E(2) (PGE(2)) accumulation in cell culture supernatants was increased by KCN and NS-398 blocked PGE(2) generation. PCR studies further confirmed that COX-2 expression was increased by KCN. Antioxidants phenyl-N-test-butylnitrone, superoxide dismutase, and catalase significantly inhibited KCN-induced COX-2 up-regulation and subsequent apoptosis. N(G)-nitro-L-arginine methylester an inhibitor of nitric oxide synthase, blocked KCN-induced PGE(2) production and apoptosis, but not COX-2 expression. Increased nitric oxide levels caused by cyanide may directly activate the COX-2 enzyme. These data show that cyanide treatment of cortical cells involves increased COX-2 expression, PGE(2) accumulation, and ROS generation, resulting in apoptotic cell death.     

Cadmium toxicity is caused by accumulation of p53 through the down-regulation of Ube2d family genes in vitro and in vivo

Cadmium (Cd) causes renal dysfunction with damage to kidney proximal tubule cells; however, the precise mechanisms of the toxicity remain unclear. Previously, we found that the expression of Ube2d4 gene, which is a member of the ubiquitin-conjugating enzyme Ube2d family, is suppressed by Cd in NRK-52E rat renal tubular epithelial cells. To investigate the mechanisms of Cd-induced renal toxicity, we examined the effects of Cd on the ubiquitin-proteasome system, particularly the expression and function of Ube2d family members in the NRK-52E cells and mice. Cd markedly decreased the expression of Ube2d1, Ube2d2, Ube2d3 and Ube2d4 prior to the appearance of cytotoxicity in the NRK-52E cells. Cd also dramatically increased p53 protein levels in the cells, without stimulation of p53 gene expression or inhibition of proteasome activity. In addition, Cd induced phosphorylation of p53 and caused apoptosis in the NRK-52E cells. In vivo, we examined the effect of orally administrated Cd for 12 months on the expression of Ube2d genes and accumulation of p53 in the mouse kidney. Chronic Cd exposure also caused suppression of Ube2d genes expression and accumulation of p53. Cd did not induce severe kidney injury, but caused apoptosis in the renal tubules. These results suggest that the Cd-induced accumulation of p53 may be due to inhibition of p53 degradation through the down-regulation of Ube2d family genes, and that Cd induces p53-dependent apoptosis in renal tubular cells. Moreover, Ube2d family members may be one of the critical targets of renal toxicity caused by Cd.     

Accelerative effect of a selective cyclooxygenase-2 inhibitor on Fas-mediated apoptosis in human neutrophils

Inflammatory stimuli, such as cytokines, can induce cyclooxygenase-2 (COX-2) expression in neutrophils. Selective, anti-inflammatory COX-2 inhibitors have been developed for patients with acute inflammatory diseases. Recent work has shown that selective COX-2 inhibitors interfere with tumor cell growth. The purpose of this study was to examine the capability of selective COX-2 inhibitors on Fas-mediated apoptosis in cytokine-stimulated neutrophils. Tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced prostaglandin E2 (PGE2) release through the induction of COX-2 in neutrophils. This effect was not seen with either interleukin (IL)-1beta or IL-8. TNF-alpha-and GM-CSF-induced PGE2 release was blocked by the addition of the selective COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398; 1 microM). GM-CSF, IL-1beta and IL-8 suppressed Fas-mediated apoptosis in neutrophils; however, this effect was not seen with TNF-alpha. The anti-apoptotic effect of cytokines on Fas-mediated neutrophil apoptosis was attenuated by the addition of NS-398 (100 microM). These results suggest that NS-398 operates via two distinct mechanisms for regulating apoptosis and COX-2 activation in neutrophils. This distinction is indicated by the difference in concentration of NS-398 required for acceleration of Fas-mediated neutrophil apoptosis, and the inhibition of PGE2 synthesis. Moreover, NS-398 suppressed the anti-apoptotic activity of IL-8 and IL-1beta, but did not induce COX-2; therefore, the pro-apoptotic mechanism of the selective COX-2 inhibitor may be unrelated to COX-2 activity. Thus, a selective COX-2 inhibitor may contribute to the reduction of acute inflammation through the enhancement of neutrophil apoptosis.     

Effect of Sedum sarmentosum Bunge Extract on Aristolochic Acid– Induced Renal Tubular Epithelial Cell Injury

Aristolochic acid (AA) is known as a potent mutagen that induces significant cytotoxic and mutagenic effects on renal tubular epithelial cells. Clinically, the persistent injury of AA results in the infiltration of inflammatory cells, epithelial-to-mesenchymal transition (EMT), and renal tubulointerstitial fibrosis. There are no truly effective pharmaceuticals. In this study, we investigated the potential role of the extract of Sedum sarmentosum Bunge (SSB), a traditional Chinese herbal medicine, on rat tubuloepithelial (NRK-52E) cells after AA injury in vitro. Evidence revealed that AA induced mitochondrial-pathway–mediated cellular apoptosis, accompanied by cell proliferation in a feedback mechanism. Treatment with SSB also induced cells to enter early apoptosis, but inhibited cell proliferation. In cultured NRK-52E cells, AA induced the imbalance of MMP-2/TIMP-2 and promoted EMT and ECM accumulation. SSB treatment significantly alleviated AA-induced NRK-52E cells fibrosis-like appearance, inhibited the induction of EMT, and deposition of ECM. SSB also decreased the activity of the NF-κB signaling pathway, resulting in down-regulated expression of NF-κB–controlled chemokines and pro-inflammatory cytokines, including MCP-1, MIF, and M-CSF, which may regulate the macrophage-mediated inflammatory reaction during renal fibrosis in vivo. Therefore, these findings suggest that SSB exerts protective effects against AA-induced tubular epithelial cells injury through suppressing the synthesis of inflammatory factors, EMT, and ECM production.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

The protective effect of prostacyclin on adriamycin-induced apoptosis in rat renal tubular cells

Adriamycin-induced nephrosis in rats is a commonly used experimental model for pharmacological studies of human chronic renal diseases. Adriamycin-induced apoptosis of renal tubular cells has been reported in adriamycin-treated rats. In addition, prostacyclin (PGI(2)) is known to have various protective effects on many kinds of cells. To investigate the protective effect of PGI(2) on cells undergoing adriamycin-induced apoptosis, this study selectively augmented PGI(2) production via adenovirus-mediated transfer of genes for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) (two key enzymes of PGI(2) synthesis) to renal tubular cells. This PGI(2) overexpression protected rat renal tubular cells from adriamycin-induced apoptosis. Ad-COX-1/PGIS transfection was found to reduce the adriamycin-stimulated activities of caspase-3 and caspase-9, inhibit adriamycin-induced release of cytochrome c, elevate the expression of Bcl-x(L), and suppress the activation and translocation of nuclear factor-kappaB (NF-kappaB) in adriamycin-treated renal tubular cells. Our results reveal that selective augmentation of PGI(2) production can protect rat renal tubular cells from adriamycin-induced apoptosis via the NF-kappaB signaling pathway. This implies the therapeutic potential of combined COX-1 and PGIS gene transfer in gene therapy for chronic renal diseases.     

牛磺酸对肾NRK-52E细胞缺氧/复氧损伤的保护及初步机制

目的观察牛磺酸(Tau)对NRK-52E细胞缺氧/复氧(H/R)损伤的保护作用及初步机制。方法用NRK-52E细胞缺氧8 h复氧12 h培养建立了H/R模型,流式细胞仪检测凋亡率和胞内游离钙离子浓度,RT-PCR和Western blot检测GRP78、Caspase-12、Caspase-3 mRNA和蛋白表达。结果与对照组比较,H/R组细胞凋亡率上升,GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均明显增高(P<0.01),胞质游离钙离子浓度也升高(P<0.01);与H/R组比较,各浓度牛磺酸处理组的细胞Caspase-3活性和凋亡率明显下降,胞质游离钙和GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均有明显降低(P<0.01)。结论牛磺酸对NRK-52E细胞H/R损伤有较好的抑制作用,且呈一定的剂量依赖性,其机制可能是调节胞内钙稳态、抑制GRP78、Caspase-12及Caspase-3表达,减少了细胞凋亡。     

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Interleukin-1beta (IL-1beta) has been implicated in the pathogenesis of inflammatory diseases of the airway. In this study, we investigated the regulation of MUC2 and MUC5AC expression and of their regulatory mechanisms through cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Cells activated by IL-1beta showed increased COX-2, MUC2, and MUC5AC expressions at both the mRNA and protein levels. Mucin production was blocked by the selective COX-2 inhibitor NS398, and PGE(2) directly induced MUC2 and MUC5AC expression at both the mRNA and protein levels in a dose-dependent manner. These results suggest a role for PGE(2) in IL-1beta-induced mucin synthesis in NCI-H292 cells. To investigate the roles of molecules upstream of COX-2 in mucin regulation, we examined the role of mitogen-activated protein kinases (MAPKs). Cells activated by IL-1beta showed increased extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, and IL-1beta-induced MUC2 and MUC5AC production was blocked by the ERK pathway inhibitor PD98059 or the p38 inhibitor SB203580. The inhibition of both MAPKs reduced IL-1beta-induced COX-2 expression and PGE(2) synthesis. Furthermore, the addition of PGE(2) to cells overcame the inhibitory effects of both MAPK inhibitors in IL-1beta-induced mucin production. These results indicate that in human pulmonary epithelial cells, IL-1beta activates ERK or p38 to induce COX-2 production, which in turn induces MUC2 and MUC5AC production.     

Rofecoxib对肾间质纤维化大鼠TGF-及肾小管上皮细胞凋亡的影响

目的 探讨Rofecoxib对肾间质纤维化大鼠的肾脏保护机制。方法以UUO建立肾间质纤维化大鼠模型,随机分为假手术组、UUO组及Rofecoxib干预组,检测不同时间点肾脏COX-2、TGF-1及肾小管上皮细胞凋亡的表达。结果与假手术组相比,UUO组随着模型时间延长,TGF-β、COX-2蛋白表达明显增加,可检测到较多的凋亡细胞（P〈0．01）,用药组与UUO组平行相比TGF-β,、COX-2蛋白表达明显减少（P〈0．01）,凋亡细胞明显减少（P〈0．01）。结论特异性COX-2抑制剂Rofecoxib可能通过阻断UUO大鼠肾组织COX-2活性,下调TGF-的蛋白合成,减少肾小管细胞的过度凋亡,起到肾脏保护的作用。     

激活素A及卵泡抑素对肾小管上皮细胞转分化的作用

目的探讨激活素A(ACTA)对大鼠肾小管上皮细胞(NRK-52E)转分化的影响及卵泡抑素(FS)的干预作用。方法 NRK-52E细胞生长于达氏修正伊氏培养基(DMEM)/F12培养基。实验A:将培养的NRK-52E细胞按rh-ACTA浓度不同分为4组:ACTA浓度分别为0,1,10,100ng/ml。实验B:分为4组:对照组;ACTA组(A组),rh-ACTA浓度10ng/ml;rh-FS组(F组),rh-FS浓度为100ng/ml;ACTA+FS组(A+F组):rh-ACTA浓度10ng/ml,rh-FS浓度为100ng/ml。24h后收集各组细胞及细胞上清液。蛋白印迹法检测各组细胞α-平滑肌肌动蛋白(α-SMA)和纤维连接蛋白(FN)的表达;酶联免疫吸附法(ELISA)检测各组细胞上清液转化生长因子-β(TGF-β)的含量。结果 ACTA能剂量依赖性刺激肾小管上皮细胞α-SMA及FN的表达(P<0.05),而对TGF-β的表达没有影响。FS能抑制ACTA的上述效应,而单纯FS对肾小管上皮细胞α-SMA及FN的表达无影响(P>0.05)。结论 ACTA能刺激NRK-52E细胞转分化及FN的合成;FS可通过阻断ACTA的上述效应产生有效的肾脏保护作用。     

Berberine protects renal tubular cells against hypoxia/reoxygenation injury via the Sirt1/p53 pathway

Berberine (BBR) has been demonstrated to protect against renal ischemia/reperfusion injury; however, the underlying molecular mechanism is largely unknown. In the present study, we examined the role of silent information regulator 1 (Sirt1)/p53 in the protective effect of BBR on hypoxia/reoxygenation (H/R)-mediated mitochondrial dysfunction in rat renal tubular epithelial cells (NRK-52E cells). NRK-52E cells were preconditioned with small interfering RNA targeting Sirt1 (Sirt1-siRNA) and BBR before subjected to H/R. Cell damage was assessed by CCK8 assay and detection of oxidative parameters. The apoptotic rate was determined by flow cytometry and Hoechst 33258 staining. The expression of apoptotic markers, Sirt1, p53 and the translocation of p53 were examined by Western blotting assay. Nuclear p53 deacetylation by Sirt1 was detected using immunoprecipitation. Compared with the H/R group, BBR pretreatment increased cell viability and inhibited mitochondrial oxidative stress and apoptosis. Protein expression of Sirt1 was also enhanced along with a reduction of p53. Furthermore, both nuclear translocation of p53 and its acetylation were inhibited in NRK-52E cells pretreated with BBR. However, the knockdown of Sirt1 counteracted the renoprotection of BBR. BBR preconditioning protects rat renal tubular epithelial cells against H/R-induced mitochondrial dysfunction via regulating the Sirt1/p53 pathway.     

选择性COX-2抑制剂NS-398对人胃腺癌细胞增殖和凋亡的影响

目的 研究环氧化酶2(COX-2)抑制剂NS-398对胃癌培养细胞系BGC-823增殖及凋亡的影响.方法 应用MTT法检测NS-398对胃癌细胞BGC-823细胞增殖的影响;应用流式细胞术、逆转录聚合酶链反应(RT-PCR)和Western blot法检测NS-398对胃癌细胞BGC-823细胞凋亡的影响.结果 NS-398随剂量的增加及作用时间延长对胃癌细胞呈抑制作用;体外NS-398能减少BGC-823细胞株COX-2的表达,对BGC-823有细胞毒作用,可增加细胞凋亡率.结论 NS-398可抑制胃癌BGC-823细胞的增殖,促进胃癌BGC-823细胞的凋亡.其可能机制是与抑制COX-2表达有关.     

Renal epithelial gene expression profile and bismuth-induced resistance against cisplatin nephrotoxicity

Nephrotoxicity is the most important dose-limiting factor in cisplatin based anti-neoplastic treatment. Pretreatment with bismuth salts, used as pharmaceuticals to treat gastric disorders, has been demonstrated to reduce cisplatin-induced renal cell death in clinical settings and during in vivo and in vitro animal experiments. To investigate the genomic basis of this renoprotective effect, we exposed NRK-52E cells, a cell line of rat proximal tubular epithelial origin, to 33 microM Bi3+ for 12 hours, which made them resistant to cisplatin-induced apoptosis. Differentially expressed genes in treated and untreated NRK-52E cells were detected by subtraction PCR and microarray techniques. Genes found to be down regulated (0.17-0.31-times) were cytochrome c oxidase subunit I, BAR (an apoptosis regulator), heat-shock protein 70-like protein, and three proteins belonging to the translation machinery (ribosomal proteins S7 and L17, and S1, a member of the elongation factor 1-alpha family). The only up-regulated gene was glutathione S-transferase subunit 3A (1.89-times). Guided by the expression levels of these genes, it may be possible to improve renoprotective treatments during anti-neoplastic therapies.