Modulation by cyclic AMP and phorbol myristate acetate of cephaloridine-induced injury in rat renal cortical slices

Intracellular signaling pathways of cAMP and protein kinase C (PKC) have been suggested to modulate the generation of free radicals. We investigated the effects of cAMP and phorbol myristate acetate (PMA), a PKC activator, on cephaloridine (CER)-induced renal cell injury, which has been reported to be due to the generation of free radicals. Incubation of rat renal cortical slices with CER resulted in increases in lipid peroxidation and lactate dehydrogenase (LDH) release and in decreases in gluconeogenesis and p-aminohippurate (PAH) accumulation in rat renal cortical slices, suggesting free radical-induced injury in slices exposed to CER. A derivative of cAMP ameliorated not only the increase in lipid peroxidation but also the renal cell damage induced by CER. This amelioration by a cAMP derivative of lipid peroxidation and renal cell damage caused by CER was blocked by KT 5720, a protein kinase A (PKA) inhibitor. Lipid peroxidation and the indices of cell injury were increased by PMA. PMA also enhanced CER-induced lipid peroxidation and cell damage in the slices. This enhancement by PMA of CER-induced injury was blocked by H-7, a PKC inhibitor. These results indicated that intracellular signaling pathways of cAMP and PKC modulate free radical-mediated nephrotoxicity induced by CER.     

Role of oxidative stress in nickel chloride-induced cell injury in rat renal cortical slices.

Nickel chloride (NiCl2) induced lactate dehydrogenase (LDH) release and lipid peroxidation (LPO) in rat renal cortical slices in vitro in a concentration- (0-2 mM) and time- (0-4 hr) dependent manner, with initial significant LDH release occurring as early as 1 hr, whereas significant increase in LPO started 3 hr after exposure, suggesting that LPO results from renal cell injury. Both NiCl2-induced LDH release and LPO were prevented significantly by glutathione and dithiothreitol, suggesting that NiCl2-induced renal cell injury is dependent on thiols. However, such injury is not dependent solely on thiols, because (a) these thiols failed to inhibit completely the uptake of Ni2+ by the renal cortex, and (b) diethylmaleate pretreatment failed to increase NiCl2-induced cell injury further. Superoxide dismutase partially reduced the NiCl2-induced LDH release without affecting LPO and glutathione, whereas catalase did not affect such LDH release and LPO. Dimethylthiourea and DMSO completely prevented NiCl2-induced LPO, but only partially reduced LDH release. Deferoxamine prevented NiCl2-induced renal cell injury without affecting LPO and without significantly reducing Ni2+ uptake by the renal cortex, suggesting that nickel chelation is not important in such prevention of injury. NiCl2-induced inhibition of para-aminohippurate uptake was prevented significantly by thiols, deferoxamine, and dimethylthiourea. NiCl2-induced loss of cellular glutathione content was prevented significantly by thiols and deferoxamine, but not by superoxide dismutase and dimethylthiourea. These results suggest that LPO was not related to NiCl2-induced lethal renal cell injury, whereas such injury may be caused by the induction of the Fenton reaction, generating hydroxyl radicals.     

Targeting the Raf/MEK/ERK pathway with small-molecule inhibitors

Mutations occur in some cancer cells and result in elevated expression or constitutive activation of various growth factor receptors. The Raf/MEK/ERK pathway is often activated by mutations in these growth factor receptors. This pathway is regulated by upstream Ras, which is mutated in 20 to 30% of human cancers. B-Raf is also activated by mutation, especially in melanoma and thyroid cancers. Many of the events elicited by the Raf/MEK/ERK pathway have direct effects on survival and proliferative pathways. Aberrant regulation of the Raf/MEK/ERK pathway can contribute to uncontrolled cell growth and lead to malignant transformation. The effective targeting of this pathway may result in the suppression of cell growth, and death of malignant cells. This review focuses on targeting the Raf/MEK/ERK pathway with small-molecule inhibitors for the treatment of cancer.     

Transport of 2,4-dichlorophenoxyacetate by rat renal cortical slices

The herbicide, 2,4-dichlorophenoxyacetate (2,4-D), has been shown previously to be accumulated by renal cortical slices in a manner similar to p-aminohippurate (PAH). To characterize further the mechanism of 2,4-D transport, the efflux of 2,4-D from preloaded renal slices was studied. The efflux curve for 2,4-D has a fast (k = 0.254 min^?1) and a slow (k = 0.016 min^?1) component. Both rate constants are increased by certain competitive inhibitors and metabolic inhibitors (probenecid, 2,4-dinitrophenol, and iodoacetamide). Unlike PAH efflux, no biphasic response was noted with increasing concentrations of probenecid. SITS, another competitive inhibitor, increased only the slow efflux component. The efflux of 2,4-D into a Ca²⁺-free medium was unaltered; however, when a K⁺-free medium was used, the slow component was increased. As with PAH efflux, the slow component of 2,4-D efflux was decreased in the presence of lactate, but this effect was not noted for the fast phase which was increased. The slow component was unaltered when the temperature was decreased from 25 to 15°C, although the rate was increased when the temperature was changed from 25 to 35°C. The rate of the fast component was moderately greater at 15 and 35°C than that at 25°C. The rate of initial influx of 2,4-D when compared to 2,4-D efflux was rapid enough to account for the fact that this compound, at low media concentrations, continues to be concentrated within the tissue and does not exhibit a steady state level of transport. Moreover, this herbicide is an extremely potent inhibitor of the organic anion system since a concentration of approximately 20 μm 2,4-D produced a 50% inhibition of PAH transport. These results give a further indication that the renal transport of 2,4-D has characteristics different from PAH. Such differences appear to be consistent with the in vivo excretion pattern of this herbicide.     

Analysis of gentamicin uptake by rat renal cortical slices.

Slices from rat renal cortex accumulated gentamicin (GM) against a concentration gradient. At least part of this accumulation appeared to be due to active transport as evidenced by dependence on oxygen. Classical inhibitors (both competitive and noncompetitive) of organic acid and base transport in the kidney did not reduce in vitro GM tissue to medium equilibrium ratios suggesting that GM is not transported by classical renal organic ion transport mechanisms. High concentrations of GM in vitro inhibited renal transport of the organic anion p-aminohippurate and the organic cation N¹-methylnicotinamide as well as the rate of tissue respiration.     

Neuroprotection afforded by diazepam against oxygen/glucose deprivation-induced injury in rat cortical brain slices

The aim of the present investigation was to assess neuroprotection exerted by diazepam (0.1-25 microM) in rat cortical brain slices subjected to oxygen-glucose deprivation and reoxygenation. Neuronal injury and neuroprotection were assessed by measuring the release of glutamate and lactate dehydrogenase and tissue water content. Results demonstrate that diazepam exerted neuroprotective effects according to a "U-shaped", hormetic-like, concentration-response curve, with an efficacy window of 0.5-5 microM concentration. Flumazenil (20 microM) fully antagonised neuroprotection afforded by 5 microM diazepam. In conclusion, the hormetic response of diazepam should be taken into consideration when designing experiments aimed at assessing diazepam neuroprotection against ischemia/reoxygenation injury.     

Characterization of prostaglandin E1 transport by rat renal cortical slices

The purpose of this study was to examine prostaglandin E1 (PGE1) transport in rat kidney. [3H]PGE1 administered intravenously was accumulated most abundantly in the renal cortex. Infusion of probenecid and bromcresol green (BCG) decreased [3H]PGE1 accumulation in the renal cortex after injection of [3H]PGE1. To further investigate PGE1 transport in the kidney, [3H]PGE1 uptake by renal cortical slices was examined. Probenecid and BCG inhibited [3H]PGE1 uptake by the slices. Unlabeled PGE1 decreased [3H]PGE1 uptake by renal cortical slices in a concentration-dependent manner. The inhibitory effect of various dicarboxylates with different carbon atoms on [3H]PGE1 uptake was maximal at 6 carbon atoms. Preloading cortical slices with glutarate significantly increased [3H]PGE1 uptake. [3H]PGE1 uptake was inhibited by various eicosanoids and compounds with other structures (p-aminohippurate, benzylpenicillin, estrone-3-sulfate, etc.). These findings suggest that PGE1 uptake by renal cortical slices may be mediated by the members of the organic anion transporter family.     

Involvement of peroxynitrite and hydroxyradical generated from nitric oxide in hypoxia/reoxygenation injury in rat cerebrocortical slices

The changes in nitric oxide (NO) formation during hypoxia and reoxygenation were measured in slices of rat cerebral cortex, and the possible involvement of NO and its decomposition products, including peroxynitrite and hydroxyradical, in the hypoxia/reoxygenation injury was subsequently investigated. NO formation estimated from cGMP accumulation in the extracellular fluids was enhanced during hypoxia and to a lesser extent in the reoxygenation period. The mRNA for inducible NO synthase (NOS) was detected 3-5 h after reoxygenation, although neuronal NOS mRNA decreased after reoxygenation. Several NOS inhibitors such as N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine blocked not only the NO formation but also the hypoxia/reoxygenation injury as determined by lactate dehydrogenase (LDH) leakage. The hypoxia/reoxygenation injury was prevented by peroxynitrite scavengers including deferoxamine and uric acid, or several hydroxyradical scavengers such as dimethylthiourea, 2-mercaptopropionylglycine and D(-) mannitol. In addition, the hypoxia/reoxygenation injury was attenuated by poly(ADP-ribose)synthetase inhibitors such as banzamide, 3-aminobenzamide and 1,5-isoquinolinediol. On the other hand, both N-morpholinosidnonimine, a peroxynitrite generator, and hydroxyradical-liberating solution containing FeCl(3)-ADP and dihydroxyfumarate caused a marked LDH leakage in normoxic slices. These findings suggest that the enhanced formation of NO causes hypoxia/reoxygenation injury after degradation to peroxynitrite and hydroxyradical and the resultant activation of poly(ADP-ribose)synthetase.     

异槲皮苷对HepG2细胞中Raf/MEK/ERK信号通路的干预作用

目的研究异槲皮苷对Hep G2细胞中Raf/MEK/ERK信号通路的干预作用。方法采用异槲皮苷(0、40、80、160、320μmol·L~(-1))作用于Hep G2细胞,MTT法检测异槲皮苷对Hep G2细胞增殖的影响;倒置显微镜下观察24、48 h后,细胞形态及生长情况;流式细胞术检测异槲皮苷(0、40、80、160μmol·L~(-1))作用Hep G2细胞48 h后细胞周期情况;荧光定量PCR及Western blot检测异槲皮苷作用Hep G2细胞后Ras、Raf、MEK、ERK mRNA及相关蛋白的表达。结果MTT检测发现异槲皮苷对Hep G2细胞生长有明显抑制作用,且与异槲皮苷的浓度及作用时间呈正相关;不同浓度的异槲皮苷作用Hep G2细胞24、48 h后,倒置显微镜下观察发现随着浓度的增高及作用时间的延长,细胞生存数量逐渐减少,且细胞形态发生明显变化;流式细胞术检测发现随着异槲皮苷浓度的增高,细胞被阻滞在G1期的数量逐渐增加;荧光定量PCR及Western blot结果均表明,与空白组相比,异槲皮苷(80μmol·L~(-1))作用Hep G2细胞后Ras、Raf、MEK、ERK mRNA及其相关蛋白的表达均明显降低(P<0.05),差异有统计学意义。结论异槲皮苷有诱导Hep G2细胞凋亡的作用,其作用机制可能与对Raf/MEK/ERK信号通路中相关因子的干预有关。     

Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery

Since the discovery that activating mutations of the Ras GTPase were associated with 30% or more of human cancers, the RAF/MEK/ERK pathway has been the focus of intense drug discovery effort. Within the new class of molecularly targeted anti-cancer agents progressing through the late stages of clinical development, BAY 43-9006 and PD0325901 have shown considerable promise.     

胞外信号调节激酶1/2通路在阿霉素引起的心肌细胞损伤中的作用

目的探讨胞外信号调节激酶1/2(ERK 1/2)通路在阿霉素(doxorubicin,DOX)引起的心肌细胞损伤中的作用。方法应用DOX处理H9c2心肌细胞建立细胞损伤的体外模型,在DOX处理前应用ERK 1/2抑制剂PD98059抑制ERK 1/2的活化;检测细胞存活率、ERK 1/2的活化、胞内活性氧(ROS)水平、线粒体膜电位(MMP)以及胱硫醚γ-裂解酶(CSE,为内源性硫化氢的合成酶)的表达。结果 5μmol·L-1DOX处理H9c2心肌细胞1～6 h,呈时间依赖性地促进ERK1/2的活化;5μmol·L-1DOX对心肌细胞具有明显的损伤作用,表现为细胞存活率下降、ROS水平升高、MMP丢失及CSE表达降低;在DOX处理H9c2心肌细胞前30min,应用ERK1/2抑制剂10μmol·L-1PD-98059预处理能明显拮抗DOX对心肌细胞的损伤作用,使ROS水平降低,细胞存活率MMP和CSE表达水平均升高。结论 ERK1/2通路参与DOX对H9c2心肌细胞的损伤作用。     

ERK1/2通路参与ET_A和ET_B受体介导的离体肠系膜上动脉收缩研究

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号转导通路在内皮素1(ET-1)的两个G蛋白偶联受体ET_A和ET_B介导的收缩机制。方法用大鼠肠系膜上动脉器官培养模型,以敏感的离体药理学实验方法记录培养前后血管平滑肌张力,实时定量的PCR测定培养前后受体mRNA表达水平的变化。结果S6c不引起新鲜的肠系膜上动脉收缩,培养后ETB受体mRNA表达水平上调,介导的收缩明显增强(P<005);而ETA受体介导的收缩功能和mRNA均变化不大。低浓度的SB386023(10-5mol·L-1)降低S6c引起的最大收缩(Emax从239%±26%降至89%±13%,P<001),而对ET1引起的最大收缩并无影响(Emax271%±19%vs251%±16%,P>005);高浓度的SB386023(10-4mol·L-1)明显抑制ETA受体介导的收缩。结论ET-1通过ET_A受体介导新鲜动脉的收缩;动脉培养后表达ET_B受体;ERK1/2信号转导通路对ET_B受体的作用强于ET_A受体。     

MUC15 promotes growth and invasion of glioma cells by activating Raf/MEK/ERK pathway

MUC15 is a novel mucin associated with the cell membrane that is overexpressed in human gliomas. Its function in glioma is unclear. In this study, high MUC15 levels were detected in glioma tissues and cells. We found that transfection with MUC15 siRNA in U251 and T98G cells reduced MUC15 expression and decreased cell proliferation, invasion, and migration (P < .05). After transfecting U251 and T98G cells with pcDNA3.1-myc-His-MUC15 plasmid to overexpress MUC15, MUC15 expression was significantly upregulated and cell proliferation, invasion, and migration were increased (P < .05). MUC15 activated the Raf/MEK/ERK signalling pathway and the ERK inhibitor PD98059 partly reversed MUC15-enhanced proliferation, invasion, and migration of glioma cells (P < .05). The results indicate that MUC15 plays a part in glioma tumorigenesis, and the Raf/MEK/ERK signalling is involved in the regulation of MUC15 on glioma cell activity.     

Use of precision-cut renal cortical slices in nephrotoxicity studies

1.?Unlike cell lines and primary cells in culture, precision-cut tissue slices remain metabolically differentiated for at least 24–48?h and allow to study the effect of xenobiotics during short-term and long-term incubations.

2.?In this article, we illustrate the use of such an experimental model to study the nephrotoxic effects of (i) chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, (ii) of cobalt chloride, a potential leakage product of the cobalt-containing nanoparticles, and (iii) of valproate, a widely used antiepileptic drug.

3.?Since all the latter test compounds, like many toxic compounds, negatively interact with cellular metabolic pathways, we also illustrate our biochemical toxicology approach in which we used not only enzymatic but also carbon 13 NMR measurements and mathematical modelling of metabolic pathways.

4.?This original approach, which can be applied to any tissue, allows to predict the nephrotoxic effects of milligram amounts of test compounds very early during the research and development processes of drugs and chemicals. This approach, combined with the use of cells that retain their in vivo metabolic properties and, therefore, are predictive, reduces the risk, the time and cost of such processes.     

Evaluation of organic nephrotoxins in rabbit renal cortical slices

An in vitro cortical-slice system was used to assess the toxicity of several organic nephrotoxins that require transport and/or bioactivation in order to induce toxicity. The toxins cephaloridine, hexachlorobutadiene,

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and gentamicin all produce site-specific proximal tubular injury when administered in vivo. Damage was assessed in vitro by observing alterations in intracellular potassium, intracellular lactate dehydrogenase, and organic anion and cation accumulation. Histopathology was studied to assess the localization of injury. All four compounds produced dose- and time-dependent decreases in the biochemical parameters as well as site-specific lesions in the S₃ region. The results illustrate the usefulness of renal cortical slices in acute studies of organic nephrotoxins.     

Toxicity of a sevoflurane degradation product incubated with rat liver and renal cortical slices

Compound A (2-fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene) is a degradation product of the anesthetic sevoflurane which is created in closed-circuit anesthetic machines. Past in vivo and in vitro studies have implied that Compound A is nephrotoxic via bioactivation through the cysteine conjugate beta-lyase pathway. Although glutathione (GSH) conjugates of Compound A have been reported, it is not clear if they are formed enzymatically or via direct reaction with GSH. To determine if these metabolites are produced and toxic, a tissue slice system that first exposes male Fischer 344 rat liver slices to volatilized Compound A followed by exposure of rat kidney slices to the liver incubate was employed. Liver slices exposed to volatilized Compound A (6-12 microM medium conc.; approximately 23 ppm) exhibited a loss of K+ by 6 h, which was not seen in kidney slices exposed to Compound A. Aminobenzotriazole, a cytochrome P 450 suicide inhibitor, initially inhibits the cytotoxicity of Compound A to liver slices (at these times and concentrations). The sequential liver/kidney slice experiments using Compound A have not demonstrated nephrotoxic results. GSH conjugates were synthesized and was found to be nephrotoxic at concentrations above 91 microM (18 h), with higher concentrations showing toxicity at earlier times. Additionally, non-enzymatic reactions of Compound A with GSH or sulfhydryl-containing medium produces nephrotoxic products. These studies show that Compound A is directly toxic to the liver, possibly via P 450 activation, and Compound A can react with sulfhydryls directly to produce a nephrotoxic.     

Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.