Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetic patients. To prevent the development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE) have been reported to be effective in treating DN, while little is known about the functional protein changes. In this study, we used streptozotocin (STZ) to induce diabetic rats. GSPE (250 mg/kg body weight/day) were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin, and advanced glycation end products were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate kidney protein profiles among the control, untreated and GSPE treated diabetic rats. Twenty-five proteins were found either up-regulated or down-regulated in the kidneys of untreated diabetic rats. Only nine proteins in the kidneys of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in oxidative stress, glycosylation damage, and amino acids metabolism. Our findings might help to better understanding of the mechanism of DN, and provide novel targets for estimating the effects of GSPE therapy.     

Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract.

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants such as hydrogen peroxide mostly contributed by neutrophils and macrophages. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. This study provides first evidence showing that natural extracts such as grape seed proanthocyanidin extract containing 5000 ppm resveratrol (GSPE) facilitates oxidant-induced VEGF expression in keratinocytes. Using a ribonuclease protection assay (RPA), the ability of GSPE to regulate oxidant-induced changes in several angiogenesis-related genes were studied. While mRNA responses were studied using RPA, VEGF protein release from cells to the culture medium was studied using ELISA. Pretreatment of HaCaT keratinocytes with GSPE upregulated both hydrogen peroxide as well as TNF-alpha-induced VEGF expression and release. The current results suggest that GSPE may have beneficial therapeutic effects in promoting dermal wound healing and other related skin disorders.     

Protective effect of grape seed polyphenols against high glucose-induced oxidative stress

We aimed to clarify whether grape seed polyphenols (GSPs) are candidates therapeutic agents against diabetes mellitus, and to determine what degree of GSP oligomerization has the most potent efficacy. We studied the protective effects of various molecular weight GSPs (monomer, oligomer, polymer, and oligonol) on high glucose-induced cytotoxicity. In the present study, a high concentration of glucose (30 mM) induced cytotoxicity and oxidative stress (reactive oxygen species and nitric oxide) in cultured LLC-PK1 cells, but treatment with GSPs, especially oligomer GSPs, had potent protective effects against high glucose-induced oxidative stress. In addition, high glucose induced nuclear translocation of nuclear factor-kappa B, and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and bax, but GSP treatment inhibited them. These results indicate that GSPs have protective effects against high glucose-induced cytotoxicity, and among them, oligomer GSPs have more potent effects than other GSPs (monomer, polymer, and oligonol) on high glucose-induced renal cell damage.     

Mechanistic pathways of antioxidant cytoprotection by a novel IH636 grape seed proanthocyanidin extract

To understand the bioavailability and mechanistic pathways of cytoprotection by IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) a series of in vitro and in vivo studies were conducted. Comparative protective abilities of GSPE, and vitamins C and E, singly and in combination, were assessed against smokeless tobacco extract (STE)-induced oxidative stress, DNA fragmentation and apoptotic cell death in a primary culture of normal human oral keratinocytes. GSPE protected against STE-induced oxidative stress, DNA damage and apoptotic cell death, and provided better protection as compared to vitamins C and E, singly and in combination. The bioavailability and protective ability of GSPE were examined against acetaminophen (AP)-induced hepato- and nephrotoxicity, amiodarone (AM)-induced lung toxicity, doxorubicin (DX)-induced cardiotoxicity and dimethylnitrosamine (DM)-induced spleenotoxicity in mice. GSPE-fed animals were compared with GSPE-untreated mice to evaluate the protective ability of GSPE against these structurally diverse drugs/chemicals. Serum chemistry changes, histopathology and DNA damage were evaluated. Results indicate that GSPE preexposure prior to the drugs/chemicals such as AP, AM, DX or DM treatment, provided near complete protection in terms of serum chemistry changes and inhibition of both forms of cell death, e.g., apoptosis and necrosis. DNA damage in various tissues triggered by these agents was significantly reduced in GSPE-fed animals. Histopathological examination of multiple target organs provided similar data. The results suggest that GSPE exposure is bioavailable and provides significant multiorgan protection against structurally diverse drug- and chemical-induced toxic assaults. Further, these studies exhibited a series of mechanistic information including free radical scavenging ability, anti-endonucleolytic activity, cytochrome P450 2E1 inhibitory activity, anti-necrotic, anti-apoptotic and anti-carcinogenic activities, modulatory effects on antioxidative and apoptotic regulatory genes such as Bcl2, c-myc and p53, which may be responsible for the novel chemoprotective properties exhibited by GSPE.     

Downregulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 by grape seed proanthocyanidin extract

Urokinase plasminogen activator (uPA) system, comprising of uPA, its receptor uPAR and inhibitor, type 1 plasminogen activator inhibitor (PAI-1), plays a vital role in various biological processes involving extracellular proteolysis, fibrinolysis, cell migration and proliferation. The timely occurence of these processes are essential for normal wound healing. This study examines the regulation of uPA and PAI-1 by a natural polyphenol-rich compound, grape seed extract (GSE). GSE is reported to have beneficial effects in promoting wound healing. Fibroblast cells exposed to different doses of GSE for 18 hours were processed for further studies such as ELISA, RT-PCR, western blotting, fibrinolytic assay, cell surface plasmin activity assay and in vitro wound healing assay. GSE treatment caused a significant downregulation of uPA and PAI-1 expression, both at the RNA and protein levels. ELISA also revealed a dose-dependent decrease in uPA and PAI-1 activities. Functional significance of the downregulation was evident in decreased fibrinolytic activity, concomittant with decreased cell-surface plasmin activity. In vitro wound healing studies showed that GSE also retarded the migration of cells towards the wounded region.     

Protective effects of grape seed extract against oxidative and nitrative damage of plasma proteins

Oxidative stress, vascular inflammation, endothelial dysfunction plays a crucial role in the pathogenesis of cardiovascular diseases. The aim of our in vitro study was to examine the antioxidative properties of grape seed extract, and its potential protective effect on the haemostatic function of human fibrinogen under oxidative stress conditions, induced by peroxynitrite (100 μM). The preincubation of plasma with the tested extract (0.5-50 μg/ml or 0.5-300 μg/ml) reduced the formation of 3-nitrotyrosine and diminished oxidation of thiol groups in plasma proteins. The low concentrations (0.5-50 μg/ml) of grape seed extract also decreased the level of carbonyl groups, however at higher concentrations (100-300 μg/ml) this effect was not observed. Furthermore, grape seed extract counteracted the inhibitory effect of peroxynitrite on human plasma clotting. The results obtained in this study indicate that components of the grape seed extract posses antioxidative properties and may be promising substances for the creation of new dietary supplements.     

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells

This study was conducted to investigate the effects of aging on collagen and collagenase expression by human dermal fibroblasts. To evaluate this effect, the expression of these ECM was determined and compared between either fetal and adult fibroblasts or dermal fibroblasts at various passages. A total of 13 cell strains, 8 fetal foreskin and 5 adult dermal fibroblasts, were grown to 80-90% confluency and their rates of cell proliferation and expression of mRNA for collagenase (MMP-1) and pro 1(I) chain of type I collagen was determined and compared. Fetal cells had a significantly higher rate of proliferation relative to adult fibroblasts evaluated within 10 days of culture. Northern analysis was used to evaluate the steady state levels of mRNA in these cells. The result of these experiments revealed a significantly greater expression of mRNA for collagenase (58.6 ± 7.7 vs. 9.9 ± 1.5, p < 0.05) in strains of adult fibroblasts. This was consistent with collagenase activity of conditioned medium derived from adult cells relative to fetal fibroblasts. However the expression of pro 1(I) chain of type I collagen mRNA was not significantly (56.2 ± 5.2 vs. 58.5 ± 3.5) different between adult and fetal fibroblasts. This finding was confirmed by measuring total collagen production present in conditioned medium of these cells using hydroxyproline as an index for collagen production. The cellular response to IGF-1 and IFN-2b as representatives of fibrogenic and anti-fibrogenic factors were also evaluated. When expression of collagenase was used as an indication for cellular response, the degree of this response to IGF-1 but not IFN-2b was significantly greater in fetal relative to adult cells. Serial passage was also used as an in vitro model for aging fibroblasts and found a gradual reduction in pro 1(I) chain of type I collagen mRNA and hydroxyproline formation due to passaging. In conclusion, a slower rate of proliferation, a greater collagenase activity and expression of collagenase mRNA by aging fibroblasts could be some of the main reasons for attenuation of wound healing in elderly patients.     

Protection against oxidative stress by vitamin D in cone cells

Photoreceptor degeneration (PD) refers to a group of heterogeneous outer retinal dystrophies characterized by the death of photoreceptors. Both oxidative stress and inflammation are involved in the pathogenesis of PD. We investigate whether vitamin D has a potential for the treatment of PD by evaluating the anti‐oxidative stress and anti‐inflammatory properties of the active form of vitamin D₃, 1,α, 25‐dihydroxyvitamin D₃, in a mouse cone cell line, 661W. Mouse cone cells were treated with H₂O₂ or a mixture of H₂O₂ and vitamin D; cell viability was determined. The production of reactive oxygen species (ROS) in treated and untreated cells was measured. The expression of key anti‐oxidative stress and inflammatory genes in treated and untreated cells was determined. Treatment with vitamin D significantly increased cell viability and decreased ROS production in 661W cells under oxidative stress induced by H₂O₂. H₂O₂ treatment in 661W cells can significantly down‐regulate the expression of antioxidant genes and up‐regulate the expression of neurotoxic cytokines. Vitamin D treatment significantly reversed these effects and restored the expression of antioxidant genes. Vitamin D treatment also can block H₂O₂ induced oxidative damages. The data suggested that vitamin D may offer a therapeutic potential for patients with PD.     

Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress.

Glutathione (GSH), the major cellular protectant against reactive oxygen and nitrogen species, is compartmentalized in a cytosolic (c) and a mitochondrial (mt) pool. We investigated how c-GSH and mt-GSH are differentially affected by endogenously produced nitric oxide (NO). Microglial cell line (N9) cultures were immunostimulated with lipopolysaccharide/interferon-gamma to elicit the inducible isoform of NO synthase (iNOS). Despite a significant reduction in total GSH, the mt-GSH remained nearly unaffected by iNOS-mediated NO production. To investigate possible consequences of GSH depletion on the mitochondrial membrane potential, we used buthionine sulfoximine (BSO) to reduce separately the c-GSH, whereas ethacrynic acid (EA) was applied to deplete both mt-GSH and c-GSH. The mitochondrial membrane potential was more vulnerable to NO exposure in EA-pretreated cultures than in BSO-pretreated cultures, indicated by a potentiated release of tetramethylrhodamine from mitochondria into the cytosol. To relate the EA-mediated decrease in mitochondrial membrane potential to the oxidant buildup after GSH depletion, we loaded the cells with the oxidant-sensitive fluorochrome 2',7'-dihydrodichlorofluorescein (DCF) diacetate. EA treatment caused an increase in DCF fluorescence over time that was potentiated when the iNOS expression was stimulated. Inhibition of NO production abolished this effect. We conclude that endogenous NO production in microglial cells does not compromise the mt-GSH pool which, in turn, might explain the ability of these cells to combat high-output NO production.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

An aqueous extract of Zingiber officinale Roscoe protects mouse primary hepatic cells against hydrogen peroxide-induced oxidative stress

Hepatocytes exposed to an oxidative stressor such as hydrogen peroxide (H₂O₂) are potentially sensitized to cell death; thus, reactive oxygen species (ROS) are considered to be critical mediators of liver damage. Zingiber officinale Roscoe (ZO), also known as ginger, is cultivated commercially in China, India, Korea, and other parts of the world. In addition, it is used as a spice and flavoring agent and is also purported to possess a number of medicinal properties. In the present study, we examined the protective effect of ZO against cell damage caused by H₂O₂-induced oxidative stress. ZO reduced H₂O₂-induced apoptotic signals and the levels of intracellular ROS. ZO pretreatment also increased the phosphorylation of c-Jun, and JNK kinase. The expression of heme oxygenase-1 (HO-1) and heat shock protein 72 (HSP72) were increased by ZO pretreatment more than H₂O₂ treatment. In addition, siRNA-mediated knockdown of HO-1 and HSP72 decreased protective effect of ZO pretreatment. Our data suggest that ZO decreases ROS levels and the expressions of HO-1 and HSP72 are involved in the hepatocyte protective function of ZO against H₂O₂.     

Amelioration of cisplatin-induced nephrotoxicity by grape seed extract and fish oil is mediated by lowering oxidative stress and DNA damage

Cisplatin (CP) is a chemotherapeutic drug used in treatment of malignancies. However, its clinical utility is limited by nephrotoxicity. The purpose of the present study was to investigate the protective role of grape seed proanthocyanidin extract (GSPE) (100 mg/kg/day) or fish oil (FO) (5 ml/kg/day) against cisplatin induced nephrotoxicity in terms of biochemical parameters, oxidative stress and DNA damage. CP nephrotoxiciy is manifested by increased levels of serum creatinine, urea and uric acid, accompanied by their decrease in urine. Na, K and Ca levels were altered in both serum and urine. In addition, cisplatin caused a decrease in renal GSH, SH-group, SOD, GST, and Na–K–ATPase levels. However the levels of MDA, H₂O₂ and NO were increased. Also, we assessed the renal genotoxic potential of cisplatin as manifested by an increase in the tail length of DNA, tail intensity (DNA %) and tail moment. On the other hand, administration of GSPE or FO pre-cisplatin treatment ameliorated the current changes in most of the above tested parameters, particularly oxidative stress, endogenous antioxidant defense system and DNA damage indicating their curative effect. Thus, it can be concluded that the consumption of GSPE or FO might be useful for preventing nephrotoxicity caused by cisplatin treatment.     

Grape seeds proanthocyanidin extract as a hepatic-reno-protective agent against gibberellic acid induced oxidative stress and cellular alterations

The present study aims to investigate the heptonephro-protective effect of grape seeds proanthocyanidin extract (GSPE) against the risks induced by gibberellic acid (GA3) in male rats. The results recorded that GA3 caused a significant increase in total lipids, total cholesterol, triglycerides and LDL-C levels in serum, concomitant with a significant decrease in serum HDL-C. A significant increase in serum AST, ALT, urea and creatinine, while, a significant decrease in total protein content in serum was observed in rats given GA3. Hepatic and renal lipid peroxidation product (MDA) was significantly increased, meanwhile, total antioxidant capacity (TAC), glutathione, and catalase levels were significantly decreased. In addition, there was a negative change in liver structure including dilatation in the central veins with degeneration of endothelium cells and cellular injury around the veins as well as in the kidney structure such as lesion in both glomeruli and tubules, detachment of the Malpighian corpuscles from the Bowman’s capsule’s epithelium, shrinkage in the glomerular capillary network. However, almost all of these adverse effects seemed to be ameliorated by oral administration of GSPE with GA3 to rats for 2 month indicating the protective effect of grape seeds GSPE on GA3 induced oxidative stress in rats.     

Protection against ischemia-reperfusion induced oxidative stress by vitamin E treatment.

The rat heart protection offered by vitamin E against oxidative stress after ischaemia-reperfusion was studied by using a new methodological approach. Functional recovery of hearts from ischaemia-reperfusion was correlated with a traditional index of oxidative stress such as lipid peroxidation and with antioxidant capacity and susceptibility to oxidants of the tissue evaluated by enhanced chemiluminescence techniques. Rats were treated with ten daily i.m. injections of 100 mg/kg body weight of vitamin E. The functional recovery during reperfusion (20 min, following 45 min ischaemia) of Langendorff preparations from control (vehicle-injected) and vitamin E treated rats was evaluated in terms of heart rate, left ventricular developed pressure (LVDP), double product (= heart rate. LVDP) and coronary flow recovery. Vitamin E treatment significantly improved functional recovery of heart rate, LVDP, double product and coronary flow. It also increased the level of vitamin E and reduced the levels of both malondialdehyde and hydroperoxides in the heart tissue at the end of the ischaemia-reperfusion protocol. In contrast, it did not affect the antioxidant capacity and the response of heart homogenates to in vitro oxidative stress measured after ischaemia-reperfusion. These results show a protective action of vitamin E treatment against lipid peroxidation and cardiac dysfunction associated with ischaemia-reperfusion. Although the precise mechanism of this protection is not evident, our model in part suggests a role of vitamin E other than as a free radical scavenger.     

Reducing oxidative/nitrosative stress: a newly-discovered genre for melatonin

The discovery of melatonin and its derivatives as antioxidants has stimulated a very large number of studies which have, virtually uniformly, documented the ability of these molecules to detoxify harmful reactants and reduce molecular damage. These observations have clear clinical implications given that numerous age-related diseases in humans have an important free radical component. Moreover, a major theory to explain the processes of aging invokes radicals and their derivatives as causative agents. These conditions, coupled with the loss of melatonin as organisms age, suggest that some diseases and some aspects of aging may be aggravated by the diminished melatonin levels in advanced age. Another corollary of this is that the administration of melatonin, which has an uncommonly low toxicity profile, could theoretically defer the progression of some diseases and possibly forestall signs of aging. Certainly, research in the next decade will help to define the role of melatonin in age-related diseases and in determining successful aging. While increasing life span will not necessarily be a goal of these investigative efforts, improving health and the quality of life in the aged should be an aim of this research.     

Renal and cardiac oxidative/nitrosative stress in salt-loaded pregnant rat

Sodium supplementation given for 1 wk to nonpregnant rats induces changes that are adequate to maintain renal and circulatory homeostasis as well as arterial blood pressure. However, in pregnant rats, proteinuria, fetal growth restriction, and placental oxidative stress are observed. Moreover, the decrease in blood pressure and expansion of circulatory volume, normally associated with pregnancy, are prevented by high-sodium intake. We hypothesized that, in these pregnant rats, a loss of the balance between prooxidation and antioxidation, particularly in kidneys and heart, disturbs the normal course of pregnancy and leads to manifestations such as gestational hypertension. We thus investigated the presence of oxidative/nitrosative stress in heart and kidneys following high-sodium intake in pregnant rats. Markers of this stress [8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)) and nitrotyrosine], producer of nitric oxide [nitric oxide synthases (NOSs)], and antioxidants [superoxide dismutase (SOD) and catalase] were measured. Then, molecules (Na(+)-K(+)-ATPase and aconitase) or process [apoptosis (Bax and Bcl-2), inflammation (monocyte chemoattractant protein-1, connective tissue growth factor, and TNF-alpha)] susceptible to free radicals was determined. In kidneys from pregnant rats on 1.8% NaCl-water, NOSs, apoptotic index, and nitrotyrosine expression were increased, whereas Na(+)-K(+)-ATPase mRNA and activity were decreased. In the left cardiac ventricle of these rats, heightened nitrotyrosine, 8-iso-PGF(2alpha), and catalase activity together with reduced endothelial NOS protein expression and SOD and aconitase activities were observed. These findings suggest that oxidative/nitrosative stress in kidney and left cardiac ventricle destabilizes the normal course of pregnancy and could lead to gestational hypertension.     

Protection against oxidative stress in liver of four different vertebrates.

The possible relation between respiratory capacity and antioxidant capacity and susceptibility to oxidative stress of the liver has been investigated in Rattus norvegicus, Gallus gallus domesticus, Lacerta s. sicula, and Rana esculenta. Accordingly, we measured oxygen consumption and cytochrome oxidase activity, glutathione peroxidase and glutathione reductase activity and overall antioxidant capacity, and lipid peroxidation and response to oxidative stress in vitro in liver. The order of liver oxygen consumption and cytochrome oxidase activity among the different species was rat > chick > lizard > frog. The antioxidant defenses supplied by the combined action of glutathione peroxidase and glutathione reductase were not adapted to the respiratory capacities. In particular, there was no correlation either between the activities of two enzymes or between their activities and oxygen consumption. In contrast, the overall antioxidant capacity of the liver appeared to be related to its oxidative capacity, and the malondialdehyde formation, an indirect measure of lipid peroxidation, was inversely related to antioxidant capacity. The response to oxidative stress in vitro indicated that the liver susceptibility to oxidative challenge is higher in ectothermic than in endothermic species. Such higher susceptibility appeared to depend on both lower antioxidant capacity and higher levels of free radical producing species. This finding is apparently in contrast with a higher content of cytochromes in endotherms, which are able to determine both respiratory characteristics and sensitivity to pro-oxidants. However, it could indicate the existence of species-related differences in the tissue content of either preventive antioxidants or hemoproteins able to trap the radicals produced at their active center. J. Exp. Zool. 284:610-616, 1999.     

Quercetin protects C6 glial cells from oxidative stress induced by tertiary-butylhydroperoxide

The anti-oxidant and cyto-protective activity of quercetin against tertiary-butylhydroperoxide (t-BOOH) induced oxidative stress on C6 glial cells is reported. Exposure of the cells to t-BOOH resulted in a significant increase in cytotoxicity, reactive oxygen species (ROS) generation and lipid peroxidation. There was a significant increase in DNA strand breaks and fall in reduced GSH levels in cells exposed to t-BOOH. A significant increase in calcium ion influx was noticed in cells exposed to t-BOOH. Pre-treatment of cells with quercetin, vitamin C (vit C), Trolox, and deferoxamine (DFO) significantly inhibited t-BOOH induced cytotoxicity and ROS generation. Pretreatment of cells with quercetin, Trolox and DFO inhibited the DNA damage, maintained higher GSH levels and prevented calcium influx significantly. Although vit C protected the cells from cytotoxicity induced by t-BOOH, the intracellular Ca²⁺ levels were significantly higher than the control cells. However, anti-oxidants like butylated hydroxy toluene (BHT), vitamin E (vit E), N-acetyl cysteine (NAC) did not have significant cytoprotection against t-BOOH induced oxidative injury in C6 glial cells.     

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.     

Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation

We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca2+ regulation, MAPK activation, and cytoprotection in LLC-PK1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H2O2-induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca2+ concentration that normally follows H2O2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H2O2-induced injury, and prevented the development of tolerance to H2O2 that normally occurs with preconditioning. ERK and JNK were transiently (30-60 min) phosphorylated in response to H2O2. ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H2O2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H2O2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H2O2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkAS-grp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H2O2-induced cell injury in LLC-PK1 cells by preventing an increase in intracellular Ca2+ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.