Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of arachidonic acid metabolic inhibitors on hypoxia/reoxygenation-induced renal cell injury

The present study was undertaken to examine the role of arachidonic acid (AA) metabolites in hypoxia/reoxygenation (H/R)-induced renal cell injury in rabbit renal cortical slices using AA metabolic inhibitors. Inhibitors of cyclooxygenase (indomethacin and diclofenac sodium) and lipoxygenase pathways (nordihydroguaiaretic acid, caffeic acid, and eicosapentaenoic acid) reduced H/R-induced LDH release in a dose-dependent manner, whereas an inhibitor of cytochrome P-450 monooxygenase pathway ethoxyresorufin was not effective. AA increased LDH release in control slices, and the effect was not altered by indomethacin and nordihydroguaiaretic acid. The protective effect of indomethacin was not affected by addition of PGE2, a main product of cyclooxygenase pathway in the kidney. H2O2-induced LDH release was prevented by inhibitors of lipoxygenase but not by inhibitors of cyclooxygenase and cytochrome P-450 monooxygenase H/R-induced LDH release was not altered by iron chelators, phenanthroline and deferoxamine, and a potent antioxidant, N,N'-diphenyl-p-phenylenediamine, suggesting that the H/R-induced cell injury is not attributed to a generation of reactive oxygen species. Morphological studies showed that H/R-induced structural changes including cell necrosis were significantly prevented by indomethacin. These results suggest that inhibitors of cyclooxygenase and lipoxygenase pathways exert a direct protective effect against the H/R-induced cell injury in renal tubules. Whether these effects are mediated by alterations of AA metabolic pathways is not certain.     

Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney tubular cells during cold storage

BACKGROUND: Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation. METHODS: Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4 degrees C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe. RESULTS: Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG. CONCLUSION: The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.     

Salviae radix extract prevents cisplatin-induced acute renal failure in rabbits.

The present study was carried out to determine if salviae radix extract (SRE) exerts a beneficial effect against cisplatin-induced renal failure in rabbits. Rabbits were pretreated with SRE orally for 7 days, followed by cisplatin injection (5 mg/kg i.p.). Cisplatin injection caused a reduction in GFR, which was accompanied by an increase in serum creatinine levels. The fractional Na+ excretion was increased by cisplatin injection. PAH uptake by renal cortical slices was inhibited by the administration of cisplatin. Such changes were prevented by SRE pretreatment. Cisplatin injection increased lipid peroxidation, which was prevented by SRE pretreatment. The protective effect of SRE was supported by morphological studies. Cisplatin injection reduced renal blood flow that was not affected by SRE pretreatment. Cisplatin treatment in vitro in renal cortical slices increased LDH release and lipid peroxidation, which were prevented by 0.05% SRE. These results indicate that lipid peroxidation plays a critical role in cisplatin-induced acute renal failure. SRE exerts a protective effect against renal cell injury induced by cisplatin, and its effect may be attributed to its antioxidant action. However, the underlying mechanism by which SRE has antioxidant action remains to be defined.     

Effects of radical scavengers and antioxidant on ischemic acute renal failure in rabbits

This study was undertaken to determine whether reactive oxygen species (ROS) are involved in the pathogenesis of ischemic acute renal failure (IARF) in rabbits. Renal ischemia was induced by clamping bilateral renal arteries for 60 min. Animals were pretreated with combination of xanthine oxidase inhibitor (allopurinol), hydrogen peroxide scavenger (catalase), and hydroxyl radical scavenger (sodium benzoate). Serum creatinine level significantly increased 24 h after ischemia and remained higher to 72 h. Ischemia caused a reduction of GFR and an increase of FENa. Such changes were significantly attenuated by scavenger pretreatment. The uptake of p-aminohippurate in cortical slices and microsomal Na(+)-K(+)-ATPase activity were depressed in kidneys subjected to 72 h of reflow following ischemia, indicating impairment of tubular transport function, which were significantly attenuated by scavenger treatment. Renal blood flow 72 h after reflow was markedly reduced and it was restored by scavenger pretreatment. When animals were pretreated with a potent antioxidant DPPD, lipid peroxidation in cortex and medulla was significantly inhibited. However, ischemia-induced impairment of renal function was not attenuated by pretreatment of the antioxidant. These results suggest that radical scavengers may exert a protective effect against ischemia acute renal failure by other actions rather than ROS scavenging. Thus, the data do not support involvement of ROS in IARF in rabbits.     

Diverse effects of natural antioxidants on cyclosporin cytotoxicity in rat renal tubular cells.

BACKGROUND: As is well known, the use of the immunosuppressive drug cyclosporin A (CsA) is partially restricted by its nephrotoxic effects, which include early changes in haemodynamics followed by irreversible injuries to the renal tubules. Although the mechanisms responsible for these side effects are poorly understood, an involvement of reactive oxygen species (ROS) has been suggested. In this study, we selected three natural antioxidants, resveratrol, hydroxytyrosol and vitamin E, on the basis of their scavenging capabilities, and tested their protective effects against CsA toxicity. METHODS: Immortalized rat tubular cells (RPTc) were used as the model system. Cell viability was checked with trypan blue assay, and free radical formation was measured using the fluorescent probe 2,7-dichlorofluorescein (DCF). We evaluated several oxidative stress parameters, including phospholipid peroxidation products, glutathione levels and oxygenase expression. RESULTS: Incubation of RPTc with 25 muM CsA induced a significant decrease in cell viability paralleled by intracellular ROS formation and alterations in lipid peroxidation. There was also an imbalance of glutathione redox state as well as upregulation of heme oxygenase-1 (HO-1). The three antioxidants, at micromolar concentration, quantitatively prevented the ROS-activated DCF fluorescent signal and membrane lipid peroxidation. Both hydroxytyrosol and resveratrol strengthened the CsA induction of HO-1 expression. Moreover, vitamin E and resveratrol counteracted CsA-induced changes in the glutathione redox state via different mechanisms, whereas hydroxytyrosol was completely ineffective. Similarly, CsA-dependent nephrotoxicity was prevented by vitamin E, while resveratrol only exerted partial protection, and hydroxytyrosol showed no protective effects. CONCLUSION: Our results indicate that the diverse cytoprotective effects of the antioxidants tested in these studies were not directly related to their scavenging capabilities. These findings confirm a key role for glutathione in protecting cells from CsA-induced adverse effects and do not support a direct link between CsA-mediated ROS generation and adverse renal effects.     

Effect of cyclosporine A on accumulation of tetraethylammonium and p-aminohippurate, and on lipid peroxidation in rat renal microsomes and cortical slices

The effect of cyclosporine A (CsA) on lipid peroxidation (LPO) was assessed in renal cortical slices and renal microsomes. Cortical slices were incubated with 1500 micrograms/ml CsA and microsomes with 0.5-20 micrograms/ml under identical conditions (pH 7.4, 37 degrees C) for 3 hours, and LPO monitored by the formation of malondialdehyde (MDA). CsA at concentrations of 3 micrograms/ml and higher caused a significant increase MDA in microsomes and renal cortical slices showed a time dependent release of MDA into the incubation medium. The influence of CsA on tetraethammonium (TEA) and p-aminohippurate (PAH) accumulation in renal cortical slices was investigated for up to 3 hours with concentration of CsA from 10 to 1000 micrograms/ml. CsA caused a time- and concentration-dependent decrease of TEA accumulation and higher concentrations of CsA decreased PAH accumulation in renal cortical slices. The results add further evidence to the suggestion that lipid peroxidation participate in CsA-induced impairment of kidney function.     

Effect of Cyclosporine a on Accumulation of Tetraethylammonium and p-Aminohippurate,and on Lipid Peroxidation in Rat Renal Microsomes and Cortical Slices

The effect of cyclosporine A (CsA) on lipid peroxidation (LPO) was assessed in renal cortical slices and renal microsomes. Cortical slices were incubated with 1500 μg/ml CsA and microsomes with 0.5–20 μg/ml under identical conditions (pH 7.4,37d`C) for 3 hours, and LPO monitored by the formation of malondialdehyde (MDA). CsA at concentrations of 3 μg/ml and higher caused a significant increase MDA in microsomes and renal cortical slices showed a time dependent release of MDA into the incubation medium. The influence of CsA on tetraethammonium (TEA) and p-ami-nohippurate (PAH) accumulation in renal cortical slices was investigated for up to 3 hours with concentration of CsA from 10 to 1000 μg/ml. CsA caused a time-and concentration-dependent decrease of TEA accumulation and higher concentrations of CsA decreased PAH accumulation in renal cortical slices. The results add further evidence to the suggestion that lipid peroxidation participate in CsA-induced impairment of kidney function.     

Amphotericin B and sodium deoxycholate induced impairment of renal p-aminohippurate accumulation (PAH) and effect on lipid peroxidation in the rat kidney.

The influence of amphotericin B on PAH transport as well as on lipid peroxidation in rat renal cortical slices was studied in vitro and ex vivo. In vitro, renal cortical slices were incubated with different amphotericin B (AmB) concentrations (2-60 micrograms/mL) or with the corresponding vehicle concentrations of sodium deoxycholate (NaDo) (1.64-49.2 micrograms/mL) and time dependently (15-30-60 min) with 30 micrograms/mL AmB or 24.6 micrograms/mL NaDo. Ex vivo, PAH transport of renal cortical slices was investigated following a 3-day intravenous AmB administration with 3 mg/kg per day or 2.46 mg/kg/day NaDo, respectively. In vitro AmB as well as NaDo decreased PAH transport dose and time dependently. At the highest AmB concentration of 60 micrograms/mL, PAH uptake decreased to 17.6%. The corresponding NaDo concentration (49.2 micrograms/mL) decreased PAH uptake to 33.3%. Time dependently AmB decreased PAH uptake to 25% after 60 min. NaDo caused a decrease to 69%. Administration of AmB for 3 days resulted in a PAH decline to 81%; NaDO decreased PAH uptake to 77%. In vitro as well as in vivo, AmB or its vehicle did not induce lipid peroxidation in renal cortical tissue. In summary, the results show that AmB and its vehicle, NaDo, decrease PAH uptake by renal cortical cells, reflecting a direct effect of AmB on tubular function. The impairment of the PAH transport is not due to enhanced lipid peroxidation.     

Reactive oxygen species and rat renal epithelial cells during hypoxia and reoxygenation.

To study the importance of oxygen free radical production by and injury to proximal tubule epithelial cells, an in vitro model was established. Rat renal proximal tubule epithelial cells in primary culture were subjected to normoxic conditions or 60 minutes of hypoxia and 30 minutes of reoxygenation. Under normoxic conditions, these cells produced superoxide radical, hydrogen peroxide, and hydroxyl radical. During hypoxia and reoxygenation, there was an increase in the production of these reactive oxygen species, detected in the extracellular medium, of 252, 226, and 45 percent, respectively. The production rate of superoxide radical was most markedly increased in the first five minutes of reoxygenation. Studies employing 2,7-dichlorofluorescein which fluoresces when oxidized by peroxides revealed a seven-fold increase in cellular fluorescence in cells studied after hypoxia and reoxygenation compared with control cells. That increased production of reactive oxygen species played a role in cellular injury was demonstrated by an increase in lipid peroxidation during hypoxia and reoxygenation, as well as substantial injury during hypoxia and reoxygenation which could be largely prevented by the addition of superoxide dismutase, catalase, dimethylthiourea, or deferoxamine to the cells. These studies demonstrate that proximal tubule epithelial cells produce reactive oxygen species in increased amounts during hypoxia and reoxygenation, and that these reactive oxygen species are injurious to the cells under these conditions.     

Role of iron in postischemic renal injury in the rat

To determine whether iron participates in free radical-mediated postischemic renal injury and lipid peroxidation, we examined the effects of removal of endogenous iron or provision of exogenous iron following renal ischemia, as well as the effects of renal ischemia and reperfusion on renal venous and urinary "free" iron. Rats underwent 60 minutes of renal ischemia and were studied after either 24 hours (inulin clearance) or 15 minutes (renal malondialdehyde content) of reperfusion. Infusion of the iron chelator deferoxamine (200 mg/kg/hr) during the first 60 minutes of reperfusion resulted in a marked improvement in renal function (inulin clearance: 879 +/- 154 vs. 314 +/- 74 microliter/min; P less than 0.025) and a reduction in lipid peroxidation (renal malondialdehyde: 0.449 +/- 0.06 vs. 0.698 +/- 0.08 mmol/mg prot; P less than 0.05) compared to control animals. Infusion of 50 mg/kg/hr deferoxamine also protected renal function after ischemia (inulin clearance: 624 +/- 116 vs. 285 +/- 90 microliter/min; P less than 0.05) and resulted in less histologic injury. Iron-saturated deferoxamine had no protective effect. Conversely, infusion of the iron complex EDTA-FeCl3 during reperfusion exacerbated postischemic renal dysfunction and lipid peroxidation. Following renal ischemia there was no detectable increase in "free" iron in arterial or renal venous plasma. However, urinary "free" iron increased 10- to 20-fold following reperfusion. Iron chelators which underwent filtration and gained access to this free iron in the urine (free deferoxamine or inulin-conjugated deferoxamine) provided protection, whereas a chelator confined to the vascular space (dextran-conjugated deferoxamine) did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactive oxygen metabolites in endotoxin-induced acute renal failure in rats

Based on recent reports that reactive oxygen metabolites may play a role in endotoxin-induced injury in other tissues, we postulated that reactive oxygen metabolites may be important mediators of endotoxin-induced acute renal failure. Superoxide dismutase, a scavenger of superoxide, or catalase, which destroys hydrogen peroxide, did not protect against endotoxin-induced renal failure. Similarly, neither the hydroxyl radical scavenger dimethylthiourea nor the iron chelator deferoxamine (which presumably would act by preventing the generation of hydroxyl radical via the iron-catalyzed Haber-Weiss reaction) prevented the endotoxin-induced fall in renal function. In separate experiments, we found no increase in renal cortical lipid peroxidation (a marker of reactive oxygen metabolite-mediated tissue injury) in endotoxin-treated rats, providing further evidence against a role for reactive oxygen metabolites in endotoxin-induced renal injury. Finally, using the aminotriazole-induced inhibition of catalase (a measure of in vivo changes in the hydrogen peroxide generation) we found no evidence of enhanced hydrogen peroxide generation in the renal cortex in endotoxin-treated rats. Taken together, the data from these three separate experimental approaches suggest that reactive oxygen metabolites are not important mediators of endotoxin-induced acute renal failure.     

Visualizing tubular lipid peroxidation in intact renal tissue in hypertensive rats

An imbalance between production of reactive oxygen species (ROS) and antioxidant defense is involved in the pathogenesis of diverse chronic parenchymatous diseases. To identify the primary site of such increased oxidative stress, a lipophilic ROS-sensitive probe (C11-Bodipy 581/591) is introduced, which allows the visualization and quantification of oxidative injury using confocal fluorescence microscopy in living cells. The properties of this probe are such that its emission wavelength irreversibly shifts from red to green upon oxidation. This probe was used to identify the spatiotemporal distribution of lipid peroxidation in the rat kidney during chronic NOS inhibition, a model associated with hypertension and proteinuria. Chronic NOS inhibition resulted in increased lipid peroxidation in renal tubules but hardly any in glomeruli or blood vessels. This peroxidation preceded the loss of renal function characteristic of the model and was accompanied by parallel changes in thiobarbituric acid reactive substances in the renal cortex. Furthermore, the increase in oxidation was dependent on angiotensin II and NADPH oxidase and prevented by vitamin E. Induction of cytoprotective heat-shock protein 70 preceded lipid peroxidation, rise in BP, or proteinuria. These findings challenge the paradigm that the vascular wall is the source and target of oxidative stress in chronic parenchymatous renal disease associated with hypertension.     

Production of reactive oxygen species by tubular epithelial cells in culture

Reactive oxygen species (ROS) have been implicated in the pathogenesis of toxic, ischemic and immunologically-mediated renal injury. Although substantial evidence exists for the production of ROS by glomerular cells, little is known about production of these reactive oxygen metabolites by renal tubular cells. We examined the ability of cultured cells from different segments of the rabbit nephron to elaborate ROS. Under basal conditions, cells of the proximal tubule, cortical collecting duct, and papillary collecting duct produced superoxide anion and hydrogen peroxide. Exposure to opsonized zymosan or heat-aggregated gamma globulin significantly increased ROS production by all three tubular cell types. The production of superoxide anion and hydrogen peroxide was time dependent and increased with increasing concentrations of the stimulating factors. These experiments indicate that renal tubular cells have the potential to participate in renal injury via elaboration of highly-reactive oxygen metabolites.     

氧自由基在异氟醚诱导小鼠前脑细胞凋亡中的作用

目的 观察小鼠暴露于异氟醚后前脑Caspase-3蛋白和氧自由基的变化,以了解氧自由基(reactive oxygen spceies,ROS)在异氟醚神经损伤中的作用.方法 56只雄性C57BL/6J小鼠按随机数字表法分为异氟醚组(Iso组,n=20)、二甲基硫脲±异氟醚组(DMTU±Iso组,n=8)、二甲基硫脲组...     

Delayed treatment of hemoglobin neurotoxicity

Hemoglobin is an oxidative neurotoxin that may contribute to cell injury after CNS trauma and hemorrhagic stroke. Prior studies have demonstrated that concomitant treatment with iron-chelating antioxidants prevents its neurotoxicity. However, the efficacy of these agents when applied hours after hemoglobin has not been determined, and is the subject of the present investigation. Consistent with prior observations, an increase in reactive oxygen species generation, detected by 2',7'-dichlorofluorescin oxidation, was observed when mixed neuronal/astrocyte cultures prepared from mouse cortex were exposed to hemoglobin alone. However, this oxidative stress developed slowly. A significant increase in the dichlorofluorescein signal compared with control, untreated cultures was not observed until four hours after addition of hemoglobin, and was followed by loss of membrane integrity and propidium iodide staining. Treating cultures with the 21-aminosteroid U74500A or the ferric iron chelator deferoxamine four hours after initiating hemoglobin treatment markedly attenuated reactive oxygen species production within 2 h. Continuous exposure to 5 micro M hemoglobin for 24 h resulted in death of about three-quarters of neurons, without injuring astrocytes. Most neuronal loss was prevented by concomitant treatment with U74500A; its effect was not significantly attenuated if treatment was delayed for 2-4 h, and it still prevented over half of neuronal death if treatment was delayed for 8 h. Similar neuroprotection was produced by delayed treatment with deferoxamine or the lipid-soluble iron chelator phenanthroline. None of these agents had any effect on neuronal death when added to cultures 12 h after hemoglobin. These results suggest that hemoglobin is a potent but slowly-acting neurotoxin. The delayed onset of hemoglobin neurotoxicity may make it an attractive target for therapeutic intervention.     

Use of allopurinol and deferoxamine in cellular protection during ischemia.

During cellular ischemia and death, many changes occur in the cell. These include the build-up of purines and lipid peroxidation. In this study, we evaluated the effectiveness of allopurinol, which blocks purine breakdown, and deferoxamine, which inhibits lipid peroxidation, as cytoprotective agents. Rats were subjected to middle cerebral artery occlusion and were treated with high or low doses of allopurinol or high or low doses of deferoxamine, with normal saline used as a control. Treatments were given 1 hour before, 1 hour after, or 5 hours after occlusion. The outcome was based on neurological status and infarct size. Both infarct size and neurological status were found to be improved in all treatment groups when compared to controls. This study suggests that the use of these agents prevents cellular damage during ischemia.     

Aminoglycosides induce acute cell signaling and chronic cell death in renal cells that express the calcium-sensing receptor

The aminoglycoside antibiotics (AGAs) are calcium-sensing receptor (CaR) agonists that are toxic to the renal proximal tubule. Proximal tubule-derived opossum kidney (OK) cells express CaR-like proteins and respond to AGAs with intracellular Ca2+ mobilization and extracellular regulated protein kinase (ERK) phosphorylation. To examine the possible cellular basis of AGA toxicity, acute and chronic responses to AGA treatment in OK cells and in CaR stably transfected HEK-293 cells (CaR-HEK) were studied. Changes in cell-fate signaling, proliferation, and cell death were detected by semiquantitative Western blotting, Hoechst staining, cell counting, and FACS analysis. Confocal microscopy was used to study the relative internalization of fluorophore-labeled gentamicin in CaR-transfected and -nontransfected cells. Here it is reported that the AGA neomycin and gentamicin elicit acute, phosphatidylinositol-3 kinase-dependent phosphorylation of Akt, glycogen synthase kinase 3beta, and p38 mitogen-activated protein kinase. After 24 h of gentamicin treatment, OK cell proliferation was observed, whereas after 4 d, the OK cells underwent cell death, an effect that was mimicked by the CaR agonists spermine and polyarginine. Furthermore, gentamicin elicited substantially more cell death in CaR-HEK cells than in nontransfected HEK-293 cells. The pan-specific caspase inhibitor Z-VAD significantly inhibited cell death in both OK and CaR-HEK cells. Finally, the intracellular uptake of Texas Red-labeled gentamicin was equivalent in both CaR-transfected and vector-transfected HEK-293 cells, suggesting that the CaR does not enhance drug uptake. Together, these observations demonstrate that the AGAs induce both acute and chronic cell fate changes in OK cells and CaR-HEK cells and that the proximal tubular CaR is likely to contribute to signaling underlying the renal toxicity of the AGAs.     

铁死亡在器官缺血再灌注损伤中的作用机制研究进展

缺血再灌注损伤（IRI）是导致器官功能障碍和移植失败的重要因素之一,其机制复杂,迄今未完全阐明,且缺乏有效治疗手段。IRI过程中,多种细胞死亡方式被激活,如凋亡、焦亡、自噬、程序性坏死等。铁死亡作为一种新型程序性细胞死亡方式,以铁依赖性的活性氧（ROS）自由基和脂质过氧化物堆积为主要特征,已被证实在IRI中发挥重要作用,通过调节铁、糖、氨基酸和脂质代谢及信号通路,加剧器官IRI。阻止铁死亡过程在多个器官中被证实能有效降低IRI的破坏,但与其他程序性细胞死亡方式相比,铁死亡在IRI中的作用机制研究仍较少。IRI与ROS产生密切相关,ROS通过诱发脂质过氧化反应,损害细胞膜结构,与铁死亡存在紧密联系。本文结合细胞铁死亡过程中的铁代谢、脂质过氧化、抗氧化系统及其他多种调节因子的调控途径,探讨铁死亡在器官IRI中扮演的多重角色,以期为IRI相关实验及临床治疗提供参考。     

Iron and Oxidative Stress in Cold-Initiated Necrotic Death of Rat Hepatocyte

Iron chelators and antioxidants have been shown to prevent hypothermia-induced apoptosis in hepatocytes. This study examined whether iron chelation and antioxidants could also prevent hypothermia-induced necrosis. Isolated rat hepatocytes were incubated at 4°C for 6 hours and then rewarmed at 37°C for 18 hours with or without the iron chelator deferoxamine and a selection of antioxidants. There was no evidence of increased cell death or adenosine triphosphate depletion during hypothermic incubation. After hypothermia and rewarming, the majority of rat hepatocytes died of necrosis as indicated by the absence of DNA fragmentation, caspase 3 activity, and apoptotic bodies. Cell death was significantly reduced if deferoxamine or a selection of antioxidants were present during hypothermia and rewarming. Deferoxamine was more effective in preventing cell death when added prior to hypothermia, indicating cell death processes were likely initiated during hypothermia.