Modulating the Endoplasmic Reticulum Stress Response with trans-4,5-Di
http://www.100md.com
《毒物学科学杂志》
Department of Chemistry, Clarkson University, Potsdam, New York
Toxicology and Drug Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, Indiana 46146
Renal Division, Brigham and Women's Hospital, Boston, Massachusetts
Department of Medicine, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Agents that disrupt functions of the endoplasmic reticulum (ER) induce expression of ER stress-response genes including ER chaperones. Increased expression of the major ER chaperone, Grp78, protects cells, including renal epithelial cells, from chemically induced injury and death in vitro. In this study, we determined if pharmacological manipulation of the ER stress-response gene is an effective strategy to protect the kidney from chemical stress in vivo. Treatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), a novel inducer of ER stress proteins, stimulated a time-and dose-dependent increase in Grp78 expression in the kidney, but it did not cause detectable injury. Furthermore, prior treatment with DTTox protected the proximal tubular epithelium against a subsequent challenge with the nephrotoxicant S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC). In contrast, activating a heat shock response did not have a protective effect. Prior treatment with DTTox did not reduce covalent binding of radiolabeled reactive metabolites of 35S-TFEC to renal proteins, indicating that protection was not due to an effect on the metabolic activation of TFEC to the reactive metabolite(s) responsible for renal injury. Antisense grp78 expression in the renal epithelial cell line LLC-PK1 blocked the DTTox-induced Grp78 increase and ablated the protective effect against TFEC damage, indicating that the induction of grp78 expression and the ER stress response were critical for the protective effect of DTTox. These findings suggest that increased expression of Grp78 plays a major role in the protection of renal epithelial cells from reactive intermediate–induced chemical injury in vivo and that pharmacological manipulation is an effective strategy to prevent damage by some classes of nephrotoxicants.
Key Words: ER stress; Grp78; renal injury; nephrotoxicant; S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC); trans-4,5-dihydroxy-1,2-dithiane (DTTox).
INTRODUCTION
Physical and chemical stresses activate stress response genes in a wide range of cell types and tissues. This molecular stress response is important physiologically because it protects cells against injury and provides tolerance to subsequent toxic insults (Kaufman, 2002; Lee, 2001; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004). Among the many stress-inducible proteins are members of the heat shock proteins (Hsp) and glucose-regulated proteins (Grp), families defined by their induction by heat shock and glucose deprivation, respectively (Lee, 2001; Takayama et al., 2003). The exact mechanism by which these stress proteins confer protection is not entirely clear, but their ability to confer tolerance to injury is well documented (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). Genes, such as hsp70, hsp90, grp78, and grp94, are an important class of stress-inducible genes as they encode chaperone proteins that monitor and repair damage to the proteome by directing the refolding and/or degradation of denatured and damaged proteins (Lee, 2001; Takayama et al., 2003). Heat shock proteins mediate cytosolic protein folding, whereas the Grps are responsible for folding and assembly of proteins that traverse the endoplasmic reticulum and Golgi apparatus (Lee, 2001; Takayama et al., 2003). It is generally accepted that chaperones mediate cellular tolerance by preventing the accumulation of cytotoxic aggregates of damaged proteins, i.e., proteotoxic stress (Hightower and Ryan, 1997; Rutkowski and Kaufman, 2004; Takayama et al., 2003).
The transcriptional control of stress-gene expression is sometimes regulated by a direct feedback loop that balances the amount of stress protein available against the need for increased expression in response to a new insult. For example, heat shock factor (HSF) regulates hsp70 transcription through a feedback loop in which the demand for chaperones to fold nascent and damaged proteins is linked to HSF activation through negative regulation by free Hsps (Morimoto, 2002). This feedback regulation is a common theme in the response to proteotoxic stress.
Regulation of the ER stress response, also called the "unfolded protein response" (UPR) is more complex, but it also involves feedback regulation by chaperones; recent reviews summarize three sensing mechanisms that converge on a consensus CCAAT(N9)CCACG endoplasmic reticulum stress-response element (ERSE) in genes such as xbp-1, chop/gadd153, grp78, grp94, and calreticulin (Kaufman, 2002; Lee, 2001; Oyadomari et al., 2002; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004). The first sensing mechanism involves IRE1, a transmembrane protein with a serine-threonine kinase domain in the ER lumen and a cytoplasmic tail with site-specific endonuclease activity (Nikawa and Yamashita, 1992; Yoshida et al., 2001). Grp78 interacts with the IRE1 kinase domain, but increasing demand to fold client ER proteins releases Grp78 activating the IRE1 kinase and RNase activities (Wang et al., 1998, 2000). Unspliced X-box DNA binding protein-1 (XBP1) message is a target for active IRE1 RNase activity; cleavage results in a translation frame-shift producing a protein that binds to the opposite strand complement of the CCACG sequence in the ERSE. The second sensing mechanism requires ATF6, a 90 kDa transmembrane ER protein that is cleaved to release an active p50 DNA-binding protein that binds the CCACG in ERSE when the CCAAT motif is occupied by NF-Y/CBF (Yoshida et al., 2000). Glucose-regulated protein 78 binds p90 ATF6, preventing trafficking to the Golgi, where proteolytic processing to active ATF6 p50 occurs (Shen et al., 2002). Unfolded proteins in the ER bind Grp78, releasing ATF6 for proteolytic processing to active p50. The third mechanism involves a third ER transmembrane protein, the PKR-like kinase (PERK) (Harding et al., 2000; Ma et al., 2002). The PERK is also held in check by Grp78; upon release, the cytoplasmic serine-threonine kinase phosphorylates the translation initiation factor eIF2, blocking translation of most mRNAs. Thus, all three mechanisms may be controlled by a feedback loop that depends on the interaction of Grp78, and possibly other chaperones, with three proximal sensors, IRE1, ATF6, and PERK. Only the IRE1 pathway is replicated in yeast. The presence of multiple sensors in mammalian cells provides increased levels of complexity and flexibility in the mammalian UPR (Halleck et al., 1997; Hung et al., 2003; Kaufman, 2002; Lee, 2001; Liu et al., 1997,1998; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004; Takayama et al., 2003; van de Water et al., 1999).
Nephrotoxic cysteine conjugates (NCC) generate reactive acylating species in the proximal tubule epithelium and are a well-characterized model of chemically induced renal injury in vitro and in vivo. Prior treatment of LLC-PK1 cells with DTTox, a novel and nontoxic inducer of ER stress (Halleck et al., 1997) prevents toxicity caused by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), an NCC, and the penultimate toxic metabolites derived from glutathione conjugation of the nephrotoxic gas tetrafluoroethylene (Hayden and Stevens, 1990). Therefore we used TFEC to study the role of ER stress in protection against chemical damage to the kidney in vivo, and determined if DTTox was suitable as a pharmacological modulator of ER stress in the kidney. Amphetamine, which activates a heat shock response in vivo by increasing core temperature, was used to investigate the effect of the heat shock response on TFEC-induced renal injury. The results suggest that the ER stress response, but not the heat shock stress response, prevents NCC-induced renal injury in vivo.
MATERIALS AND METHODS
Animals and reagents.
All chemicals, unless otherwise stated, were purchased from Sigma Chemical Company (St. Louis, MO). Unlabeled and 35S-radiolabeled TFEC were synthesized and characterized as previously described (Hayden and Stevens, 1990). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were obtained from GIBCO (Grand Island, NY).
Male Fischer 344 rats were purchased from Taconic Farms (Germantown, NY). The animals were housed in temperature- and humidity-controlled animal quarters with a 12-h light/dark cycle with free access to food and water at all times. The rats were allowed to acclimatize for 2 weeks before any experimental procedures. All animal experiments were conducted according to approved Institutional Animal Care and Use Committee protocols.
Antibodies.
The following antibodies were purchased from the companies indicated: monoclonal antibodies against Grp78 (SPA-827, anti-KDEL; from StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) and Hsp70 (Hsp72/73, Ab-1; from Oncogene Sciences, Cambridge, MA); polyclonal antibody against Grp78 (PA1-014; from Affinity Bioreagents Inc., Golden, CO). Polyclonal antisera against the porcine Grp78 were produced as described below. Secondary goat anti-rabbit and anti-mouse antibodies were purchased from Amersham Biosciences (Piscataway, NJ).
Antibodies recognizing porcine Grp78 were raised using two peptides designated C13L and C18L. The peptides consist of the C-terminal 13 and 18 amino acids of pig Grp78 sequence (GenBank accession no. X92446) with an additional cysteine at the amino terminal. Peptides were synthesized by the solid phase method using an automated synthesizer (model 431A, Applied Biosystems, Foster City, CA). Synthesis was performed on the 0.1 mmole level using the Fmoc (flouren-9-methoxycarbonyl) protocol. The quality of the crude peptide preparations (data not shown) was evaluated by reverse phase high performance liquid chromatography using a C-18 column eluted with a mobile phase consisting of a gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid and electrospray spectrometry on a PerkinElmer Sciex AP1300 triple quadrupole mass spectrometer (Concord, Thornbill, Canada). The crude synthetic preparations contained the desired peptide as a major component and were used to generate specific anti-peptide polyclonal antisera. The KLH-peptide immunogens were prepared according to the standard protocol supplied by Pierce (Rockford, IL). Briefly, 2 mg of each peptide was mixed with 2 mg of the maleimide activated KLH in 0.5 ml of PBS, pH 7.2 and allowed to react at room temperature for 2 h. The KLH-peptide conjugates were purified by dialysis and gel filtration on a column provided in a SulfoLink kit. The KLH-peptide conjugates were emulsified in Freund's complete medium (0.2 mg/injection) and injected intradermally into several sites in New Zealand white rabbits. Initial booster injections in incomplete Freund's adjuvant were administered sc after 1 and 2 weeks and im after 4 weeks. The first bleed was collected 6 weeks after the first injection. Further booster injections were administered every 4 weeks.
Chemical treatments and stress protein expression.
To assess the effect of various treatments on stress protein level in rat kidney, animals weighing 200–250 g were injected ip with vehicle or various chemicals as described below. At the appropriate times, blood and kidney samples were collected from the animals under anesthesia (pentobarbital, ip; 60–75 mg/kg). After removal of the kidney, a 2–4-mm-thick piece of kidney tissue was excised by lateral slicing. The kidney slice was further dissected to obtain the "cortex," which included the outer cortex and the outer stripe of the medulla (OSOM), the medulla (section between papilla and OSOM), and the papilla as previously described (Ichimura et al., 1995). Remaining kidney samples were snap frozen in liquid nitrogen and stored at –70°C for later use.
TFEC, DTTox, and amphetamine were administered as follows: Rats were injected ip with vehicle or 20 mg/kg of TFEC in phosphate buffered saline (PBS). At 6, 12, 24, and 48 h blood and kidneys were collected. To determine the concentration-dependent effect of DTTox on Grp78 expression, rats were treated ip with 0, 100, 200, and 400 mg/kg DTTox, and kidneys were collected after 24 h for Western analysis. The time-dependent effect on Grp78 expression was also determined using cortex sections from rats treated with a single 200 mg/kg dose of DTTox. Amphetamine was used to induce a heat shock response in vivo. Rats were treated ip with 7.5 mg/kg of amphetamine. Kidneys were collected at 0, 6, and 24 h after treatment, and Hsp70 levels were determined by Western blot analysis.
To determine the effect of prior DTTox and amphetamine exposure on TFEC toxicity, rats were pretreated with a single ip injection of 200 mg/kg of DTTox or 7.5 mg/kg of amphetamine, followed by 20 mg/kg TFEC 24 h later. Forty-eight hours after TFEC injection, blood and kidney samples were collected. Kidney samples were prepared for Western analysis as described above, and a section of kidney tissue was collected from the contralateral kidney of each animal for histological evaluation.
Renal injury.
The extent of protection afforded by stress-response–inducing agents against TFEC-induced renal damage was determined both by histological examination and by measurement of blood urea nitrogen (BUN) content. For histopathology, a transverse section of the kidney was fixed in Carnoy's solution and processed for paraffin embedding. Sections measuring 5-μm in thickness were mounted and stained with hematoxylin and eosin for histopathological examination by light microscopy. The extent of TFEC-induced renal damage was graded according to the following criteria: score 1, no exfoliated cells or casts; score 2, few sloughed cells and no casts; score 3, sloughed cells mostly in cortical S3 segment (S3C), less damage (exfoliated cells) in medullary S3 segment (S3M) and minor cast involvement; score 4, damage to S3C and S3M, casts; score 5, severe damage in both S3C and S3M, casts in both the medullary and cortical regions, dilated collecting ducts. Blood collected from the vena cava was allowed to coagulate at room temperature. The serum fraction was separated and the BUN concentrations were measured with the diacetyl monoxime assay according to the procedure provided with the kit (535A; Sigma).
Covalent binding of TFEC to renal protein.
35S-labeled TFEC was used to measure the effect of DTTox pretreatment on the covalent binding of TFEC to renal proteins. Rats were injected (ip) with saline or 200 mg/kg of DTTox in saline followed by 20 μCi of 35S-TFEC in a dose of 20 mg/kg. At 6, 12, and 24 h after 35S-TFEC treatment, animals were anesthetized with 60 mg/kg of pentobarbital, and the kidneys were perfused with PBS. The kidneys were removed and homogenized in 5 ml of water. Protein was precipitated by addition of cold 20% trichloroacetic acid. After 15 min on ice, the protein pellet was collected by centrifugation and washed three times each with 5% trichloroacetic acid, methanol, and hexane. The final pellet was dried in a dessicator (overnight) and dissolved in 1 ml of 1 N potassium hydroxide at 80°C for 2 h. Ecoscint (10 ml) was added to 100 μ1 of the protein solution, neutralized with phosphoric acid, and left at room temperature overnight. The radioactivity in each sample was determined by liquid scintillation spectrometry (LKB Wallac, 1211 Rackbeta, Finland). The amount of 35S-TFEC bound is expressed as nanomoles 35S-label bound to protein per kidney.
Cell culture.
LLC-PK1 cells, an epithelial cell line derived from porcine kidney (Hull et al., 1976; Stevens et al., 1986), were purchased from American Type Culture Collection (Manassas, VA). LLC-PK1 cells were cultured as previously described (Liu et al., 1996) and maintained in DMEM supplemented with 10% fetal bovine serum (complete medium). pkASgrp78, and pkNEO clones generated from LLC-PK1 cells transfected with antisense grp78 mRNA or empty vector, respectively (Liu et al., 1997), were cultured in complete medium during the experimental procedures. The pkASgrp78, and pkNEO clones were seeded and grown to confluence in complete medium supplemented with 400 μg/ml of G418 for routine maintenance prior to the tolerance experiments.
To produce ER stress preconditioning, confluent cells were treated with DTTox (10 mM in EBSS, for 3 h) and thapsigargin (300 nM in complete medium, for 12 h) as described elsewhere (Halleck et al., 1997; Liu et al., 1997, 1998). Cells were then rinsed and processed for Western analysis as described below. Heat shock proteins were induced in confluent pkASgrp78 (clones 5, 8, and 10), and pkNEO (clones 2, 9, and 10) cells were grown in 6-well dishes as previously described (Liu et al., 1997). To determine if prior ER or heat shock protein induction would confer protection, chemically pretreated or heat shocked cells were subsequently exposed to 0.5 mM of TFEC (in EBSS) for 4 h, followed by 2 additional h in medium without TFEC (6 h total). DTTox-pretreated cells were allowed to recover in complete medium for 12 h before TFEC treatment, whereas thapsigargin-treated cells were dosed immediately after the EBSS wash step. Media aliquots were removed at 4 h, before the cells were washed free of TFEC, and again after the cells were returned to complete medium for 2 h. The samples were then combined, and cytotoxicity was assessed by measuring the amount of lactate dehydrogenase (LDH) released into the pooled medium according to the protocol described in Liu et al. (1996).
Immunoblot analysis.
Regions of the kidney prepared by microdissection were directly homogenized in sodium dodecyl sulfate (SDS) sample preparation buffer (Laemmli) to which a mixture of protease inhibitors containing 5 μg/ml each of leupeptin, aprotinin, and antipain and 1 mM phenylmethylsulfonyl fluoride (PMSF) was added. Protein concentrations were determined using the BioRad (BioRad Laboratories, Richmond, CA) protein assay kit with bovine immunoglobulin G (IgG) as the standard. The sample concentrations were kept low in the assay buffer to avoid interference from the SDS in the homogenization buffer. Equal amounts (10–25 μg) of each sample were resolved on 7.5% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose and immunostained with the appropriate antibodies. Bands were visualized by enhanced chemiluminescence (Amersham Biosciences) and quantitated by scanning densitometry with a Bio Image Densitometer (Bio Image Laboratories).
For Western analysis of cell culture samples, cells were washed with cold PBS followed by cold Tris/sucrose buffer (10 mM Tris/HCl, 250 mM sucrose, 1 mM EGTA; pH 7.4). Rinsed cells were scraped into Tris/sucrose buffer containing leupeptin (10 μg/ml), aprotinin (10 μg/ml) and dithiothreitol (1 mM) and PMSF (1 mM) and sonicated to break up the cells. Cell lysates were solubilized in Laemmli sample preparation buffer and Western blots were performed as described for the in vivo studies above.
Statistical analysis.
Data are presented as means ± standard deviation and were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keul's test when multiple means were compared with the exception of the data for TFEC-induced stress protein expression, which were compared by least significant differences using Student's t-test. A p value under 0.05 was considered significant. Letter designations are used to indicate significant difference. Means with the same letter designation are not different, whereas those with a different letter designation are significantly different from all other means with different letter designations. Means with two-letter designations are not different from groups with either letter designation.
RESULTS
TFEC-Induced Expression of Stress Proteins in Rat Kidney
Exposure to chemical stress can activate stress response genes in renal epithelial cell lines (Liu et al., 1996). TFEC is a well-characterized nephrotoxicant that damages proximal tubule epithelial cells in rat kidney (Hayden et al., 1991). To determine if TFEC treatment alone activated either the heat shock or ER stress responses in vivo, we measured Hsp70 and Grp78 protein expression in protein extracts from the outer medulla region of the kidney at various times after treating rats with a single 20 mg/kg dose of TFEC (Fig. 1). Hsp70 was present in low levels in kidneys from control animals (data not shown; see Fig. 3 below), but increase markedly by 12 h post-TFEC. Although results with the two different antibodies cannot be compared directly, Grp78 expression appeared to be more abundant than Hsp70 in control rat kidneys but also increased after TFEC treatment. Relative to Hsp70, there was a later increase in Grp78 (2–3-fold). These results showed that both cytosolic and ER stress response proteins levels increase after TFEC treatment.
Time Course and Concentration Dependence of DTTox Induction of Grp78
Activation of either the heat shock or ER stress response can protect cells from injury (Rutkowski and Kaufman, 2004; Takayama et al., 2003). Since DTTox can induce an ER stress response and prevent cell injury and has been reported to be well tolerated in mice, we determined if DTTox is a useful tool to activate an ER stress response and Grp78 expression in rat kidneys in vivo (Halleck et al., 1997). Rats were treated with a single dose of 200 mg/kg DTTox and kidney extract prepared 0, 6, 12, 24, 48, and 72 h later (Fig. 2A). There was a time-dependent increase in Grp78 expression with a maximum induction of about twofold, similar to the magnitude seen with TFEC (Fig. 1) 12 h to 24 h post-treatment. Grp78 levels returned to control levels by 48 h. The effect was dose dependent at 24 h over a range of 0–400 mg/kg DTTox (Fig. 2B). DTTox did not induce Hsp70 (Fig. 3). Thus, DTTox induces a reversible increase in Grp78, but not Hsp70, in rat kidney in vivo.
Induction of Hsp70 Expression by Amphetamine
Amphetamine treatment raises core temperature and is a noninvasive way to activate Hsp70 expression in rat liver (Cairo et al., 1985). Amphetamine pretreatment also protects against hepatotoxicants (Salminen et al., 1997). To determine if amphetamine increases Hsp70 expression in the kidney, we treated rats with amphetamine (7.5 mg/kg) and then measured Hsp70 expression in kidney extracts 6 and 24 h later (Fig. 3). There was a marked induction of Hsp70, comparable to that seen with TFEC treatment by 6 h (data not shown); the response was maintained through 24 h. Amphetamine did not induce Grp78 (Fig. 3). Thus, amphetamine treatment is an effective inducer of heat shock stress response but not an ER stress response in rat kidney.
DTTox, but Not Amphetamine Pretreatment Causes Tolerance to TFEC in Rat Kidney
To determine if inducing either an ER or a heat shock stress response produces tolerance to TFEC treatment, rats were pretreated with 200 mg/kg DTTox or 7.5 mg/kg amphetamine and then challenged with 20 mg/kg of TFEC 24 h later. The extent of damage was evident histologically as a typical coagulative necrosis as noted previously (data not shown; Hayden et al., 1991). Examination of kidney sections showed that DTTox pretreatment resulted in a substantial protection against TFEC-induced kidney damage (Table 1). DTTox pretreatment significantly reduced the TFEC-induced increase in BUN (Fig. 4). DTTox alone had no observable effect on histopathology or BUN. In contrast, amphetamine pretreatment did not confer proteBUN measurements.
Effect of DTTox on TFEC Binding
Although we suspected that DTTox treatment prevented renal injury by uncoupling injury from cell death, we could not exclude the possibility that DTTox pretreatment interfered with TFEC absorption, distribution, or metabolism. To exclude this possibility, rats were pretreated with DTTox or vehicle and then challenged with radiolabeled 35S-TFEC. The amount of radiolabel covalently bound to kidney macromolecules, a measure of the amount of reactive metabolite produced, was determined 6, 12, and 24 h later. DTTox pretreatment had no effect on covalent binding of 35S-radiolabeled TFEC at any time point (Table 2). Because inhibition of absorption, distribution, or metabolism would lead to a reduction in binding, we concluded that DTTox was not preventing TFEC activation but was, in fact, uncoupling damage caused by the reactive metabolite from cell death.
Blocking GRP78 Expression Prevents DTTox-Induced Protection
Although the data suggested a cause-and-effect relationship between Grp78 induction and protection in vivo, the association was only correlative. Previously, we have shown that stable expression of a 0.5-kb antisense grp78 fragment in LLC-PK1 cells—so called pkASgrp78 cells—disrupts the induction of Grp78 and the ability of ER stress to protect against the toxicity of iodoacetamide, t-butylhydroperoxide, and hydrogen peroxide (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). To determine the effect of the antisense grp78 on basal and inducible Grp78, we measured Grp78 content in pkASgrp78 and pkNEO cell lysates (pkNEO cells contain only the empty vector with the selectable marker). As shown in Figure 5, pkASgrp78 cells have normal, constitutive levels of Grp78 but are unable to increase expression after treatment with DTTox or thapsigargin, another classic Grp78 activator (Liu et al., 1997). However, induction of Hsp70 expression by heat shock remained intact (data not shown). Because pkASgrp78 cells were unable to increase Grp78 expression after DTTox treatment, we determined if they developed tolerance to TFEC. Three clones of the pkASgrp78 and the pkNeo lines were treated with DTTox and thapsigargin before a challenge with TFEC (Fig. 6). The pkASgrp78 cells were more sensitive to TFEC relative to pkNEO or nontransfected LLC-PK1 cells, as noted with other toxicants (Liu et al., 1997, 1998), suggesting that the inability to increase grp78 expression upon toxicant exposure sensitizes cells to injury. In addition to the enhanced sensitivity of pkASgrp78 cells to TFEC, DTTox and thapsigargin treatment prevented cell death only in pkNEO or LLC-PK1 but not in pkASgrp78 cells. The data suggest that the ability of DTTox to prevent TFEC toxicity and the cellular response to TFEC treatment depend on an increase in the expression of ER stress proteins. In contrast, heat shock conferred no protection against TFEC toxicity in pkASgrp78, pkNEO cells, or LLC-PK1 cells (data not shown), corroborating the results in vivo. These findings suggest that the induction of Grp78 after ER stress plays a major role in protecting proximal tubules from chemically induced cell damage in vitro and in vivo.
DISCUSSION
Heat shock protects cells against a wide variety of insults (Takayama et al., 2003), but less is known about the role of the ER stress response in cytoprotection and tolerance. Nonetheless, evidence is accumulating that the ER stress response is important in the cellular response to heavy metals, oxidative stress, ischemia, toxic prostaglandins, reactive chemical species, and excitotoxicity (Lee, 2001; Ron, 2002; Rutkowski and Kaufman, 2004). In addition, the ER stress response plays an important role in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, which are mediated by mutations in vital cellular proteins resulting in processing and/or functional defects (Kaufman, 2002). The kidney, and specifically the proximal tubular epithelium, is a major target for chemical injury; previous data suggest that the ER stress response prevents injury to renal epithelial cells in culture (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). In the LLC-PK1 renal epithelial cell line, treatment with the alkylating agent iodoacetamide increased expression of hsp70 and grp78 (Liu et al., 1996). Prior ER stress, but not heat shock, prevents cell death from a subsequent challenge with iodoacetamide, hydrogen peroxide or t-butylhydroperoxide and 11-deoxy-16,16-dimethyl prostaglandin E2 (DDM-PGE2) (Halleck et al., 1997; Hung et al., 2003; Jia et al., 2004; Liu et al., 1997, 1998; van de Water et al., 1999). Although ER stress can prevent injury to renal epithelial cells in culture, the importance of the ER stress pathway in vivo is less clear.
Previously, we had shown that ER stress prevents injury to renal epithelial cells from damage by chemical toxicants in vitro (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). We now show that DTTox is an effective inducer of an ER stress response in rat kidney in vivo. Prior exposure to DTTox prevented chemical injury in a manner consistent with a mechanism involving increased expression of grp78 and, perhaps, other ER stress proteins. It is interesting that this is not the first time that DTTox has been shown to protect against injury in vivo. Falconi et al. (1970) showed that DTTox protects mice against ultraviolet (UV) irradiation. Although it is possible that DTTox may prevent UV damage directly, or after reduction to DTT (Falconi et al., 1970; Halleck et al., 1997), our studies suggest that protection could also be due to induction of an ER stress response. Regardless, the fact that modulating the ER stress response provides protection from injury in vivo suggests that the ER stress response pathway should be considered a likely candidate as a general mechanism of cellular resistance to stress. A number of key points are worth additional consideration.
The mechanism by which ER stress protects against chemical damage in vivo is not entirely clear, but the accumulated data support a role for intracellular calcium. Studies using LLC-PK1 cells show that an increase in cellular calcium is an important contributor to cell death (Chen et al., 1994; Hung et al., 2003; Liu et al., 1997, 1998). Both alkylating agents and oxidants can disrupt the ability of cells to regulate intracellular calcium levels in renal epithelial cells. Moreover, prior ER stress prevents the increase in cellular calcium and oxidative stress (Hung et al., 2003; Liu et al., 1997, 1998). We have proposed that the ER with increased stress proteins may be better able to buffer intracellular calcium level during stress and thereby prevent calcium-mediated adverse consequences such as activation of proteases, caspases, and phosphorylation of JNKs (Hung et al., 2003). Better regulation of intracellular calcium protects mitochondrial function by preventing the accumulation of calcium in the mitochondria, which otherwise would disrupt mitochondrial function (Orrenius et al., 2003). Thus, it is plausible to propose that a similar mechanism of protection is active in protecting the TFEC treated-kidneys in vivo. Thus, it seems reasonable to propose, based on our own data (Hung et al., 2003; Liu et al., 1997, 1998), that an effect on intracellular calcium may underpin the protection noted in vivo.
The heat shock response has received considerable attention as a protective response (Goldberg, 2003; Takayama et al., 2003). Several studies have shown that amphetamine not only increases core temperature and induces expression of heat shock proteins but also protects against toxicant injury and cardiac ischemia–reperfusion injury (Maulik et al., 1995; Salminen et al., 1997). Although amphetamine treatment produced a heat shock response in rat kidney, it provided only modest protection as reflected in the small, but statistically significant, reduction in BUN levels in the amphetamine-pretreated TFEC group relative to the saline-pretreated TFEC-treated group. This is consistent with studies in LLC-PK1 cells showing that heat shock does not protect against iodoacetamide-induced (Liu et al., 1997) and t-butyl-hydroperoxide-induced (Liu et al., 1998) cell death. Nonetheless, Hsp70 was induced in rat kidney after TFEC treatment, suggesting that the heat shock response was induced. Although Hsp70 induction did not correlate with protection against chemically induced damage in vitro or in vivo, the results do not necessarily exclude a role for Hsp70. Indeed, unpublished work (B. van de Water and J. L. Stevens) indicates that when cells are protected by prior ER stress, one consequence is a more robust induction of heat shock proteins in response to an insult that would be lethal to nave cells. Thus, it is likely that these two stress responses play complementary but distinct roles in the cellular response to stress.
Thus, the induction of Grp78 by DTTox protects against chemically induced renal damage both in vitro and in vivo. Endoplasmic reticulum stress response as a cellular defense mechanism may serve as an important adaptive response accounting for the preconditioning response our lab (Park et al., 2001, 2002, 2003) and others have previously reported in the kidney. This relationship between an upregulated ER stress response and protection against nephrotoxin raises possibilities for therapeutic interventions directed toward these responses in the prophylaxis against nephrotoxic injury.
NOTES
2 Present Address: Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294.
ACKNOWLEDGMENTS
We acknowledge Dr. Joe Bonventre for critical reading of the manuscript and constructive comments. This work was supported in part by National Institutes of Health grant DK46267.
REFERENCES
Cairo, G., Bardella, L., Schiaffonati, L., and Bernelli-Zazzera, A. (1985). Synthesis of heat shock proteins in rat liver after ischemia and hyperthermia. Hepatology 5, 357–361.
Chen, Q., Jones, T. W., and Stevens, J. L. (1994). Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates. J. Cell. Physiol. 161, 293–302.
Falconi, C., Scotto, P., and De Franciscis, P. (1970). Radioprotection and recovery by dithiothreitol. Experientia 26, 172–3.
Goldberg, A. L. (2003). Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–9.
Halleck, M. M., Liu, H., North, J., and Stevens, J. L. (1997). Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells. J. Biol. Chem. 272, 21760–21766.
Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H., and Ron, D. (2000). Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904.
Hayden, P. J., Ichimura, T., McCann, D. J., Pohl, L. R., and Stevens, J. L. (1991). Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts. J. Biol. Chem. 266, 18415–18418.
Hayden, P. J., and Stevens, J. L. (1990). Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria. Mol. Pharmacol. 37, 468–476.
Hightower, L. E., and Ryan, J. A. (1997). Are stress proteins part of a cell's solution to toxicity or are they part of the problem Hepatology 25, 1279–1281.
Hull, R. N., Cherry, W. R., and Weaver, G. W. (1976). The origin and characteristics of a pig kidney cell strain, LLC-PK. In Vitro 12, 670–677.
Hung, C. C., Ichimura, T., Stevens, J. L., and Bonventre, J. V. (2003). Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J. Biol. Chem. 278, 29317–29326.
Ichimura, T., Maier, J. A.-M., Maciag, T., Zhang, G., and Stevens, J. L. (1995). Fibroblast growth factor-1 (acidic FGF) in normal and regenerating kidney: Expression in mononuclear, interstitial and regenerating epithelial cells. Am. J. Physiol. 269, F653–F662.
Jia, Z., Person, M. D., Dong, J., Shen, J., Hensley, S. C., Stevens, J. L., Monks, T. J., and Lau, S. S. (2004). Grp78 is essential for 11-deoxy-16,16-dimethyl PGE2-mediated cytoprotection in renal epithelial cells. Am. J. Physiol. Renal Physiol. 287, F1113–F1122.
Kaufman, R. J. (2002). Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 110, 1389–1398.
Lee, A. S. (2001). The glucose-regulated proteins: Stress induction and clinical applications. Trends Biochem. Sci. 26, 504–510.
Liu, H., Bowes, R. C., III, van de Water, B., Sillence, C., Nagelkerke, J. F., and Stevens, J. L. (1997). Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J. Biol. Chem. 272, 21751–21759.
Liu, H., Lightfoot, R., and Stevens, J. L. (1996). Activation of heat shock factor by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. J. Biol. Chem. 271, 4805–4812.
Liu, H., Miller, E., van de Water, B., and Stevens, J. L. (1998). Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death. J. Biol. Chem. 273, 12858–62.
Ma, K., Vattem, K. M., and Wek, R. C. (2002). Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J. Biol. Chem. 277, 18728–18735.
Maulik, N., Engelman, R. M., Wei, Z., Liu, X., Rousou, J. A., Flack, J. E., Deaton, D. W., and Das, D. K. (1995). Drug-induced heat-shock preconditioning improves postischemic ventricular recovery after cardiopulmonary bypass. Circulation 92(Suppl. II), 381–388.
Morimoto, R. I. (2002). Dynamic remodeling of transcription complexes by molecular chaperones. Cell 110, 281–284.
Nikawa, J., and Yamashita, S. (1992). IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae. Mol. Microbiol. 6, 1441–1446.
Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003). Regulation of cell death: The calcium-apoptosis link. Nat. Rev. Mol. Cell Biol. 4, 552–565.
Oyadomari, S., Araki, E., and Mori, M. (2002). Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 7, 335–345.
Park, K. M., Byun, J. Y., Kramers, C., Kim, J. I., Huang, P. L., and Bonventre, J. V. (2003). Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney. J. Biol. Chem. 278, 27256–27266.
Park, K. M., Chen, A., and Bonventre, J. V. (2001). Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J. Biol. Chem. 276, 11870–11876.
Park, K. M., Kramers, C., Vayssier-Taussat, M., Chen, A., and Bonventre, J. V. (2002). Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J. Biol. Chem. 277, 2040–2049.
Rao, R. V., Ellerby, H. M., and Bredesen, D. E. (2004). Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11, 372–380.
Ron, D. (2002). Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110, 1383–1388.
Rutkowski, D. T., and Kaufman, R. J. (2004). A trip to the ER: Coping with stress. Trends Cell Biol. 14, 20–28.
Salminen, W. F., Jr., Voellmy, R., and Roberts, S. M. (1997). Protection against hepatotoxicity by a single dose of amphetamine: The potential role of heat shock protein induction. Toxicol. Appl. Pharmacol. 147, 247–258.
Shen, J., Chen, X., Hendershot, L., and Prywes, R. (2002). ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell 3, 99–111.
Stevens, J. L., Hayden, P., and Taylor, G. (1986). Studies on the mechanism of S-cysteine conjugate metabolism and toxicity in rat liver, kidney, and a cell culture model. Adv. Exp. Med. Biol. 197, 381–390.
Takayama, S., Reed, J. C., and Homma, S. (2003). Heat-shock proteins as regulators of apoptosis. Oncogene 22, 9041–9047.
van de Water, B., Wang, Y., Asmellash, S., Liu, H., Zhan, Y., Miller, E., and Stevens, J. L. (1999). Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment. Chem. Res. Toxicol. 12, 943–951.
Wang, X. Z., Harding, H. P., Zhang, Y., Jolicoeur, E. M., Kuroda, M., and Ron, D. (1998). Cloning of mammalian Ire1 reveals diversity in the ER stress responses. Embo J. 17, 5708–5717.
Wang, Y., Shen, J., Arenzana, N., Tirasophon, W., Kaufman, R. J., and Prywes, R. (2000). Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275, 27013–27020.
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891.
Yoshida, H., Okada, T., Haze, K., Yanagi, H., Yura, T., Negishi, M., and Mori, K. (2000). ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol. Cell Biol. 20, 6755–6767.(Senait Asmellash, James L. Stevens and T)
Toxicology and Drug Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, Indiana 46146
Renal Division, Brigham and Women's Hospital, Boston, Massachusetts
Department of Medicine, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Agents that disrupt functions of the endoplasmic reticulum (ER) induce expression of ER stress-response genes including ER chaperones. Increased expression of the major ER chaperone, Grp78, protects cells, including renal epithelial cells, from chemically induced injury and death in vitro. In this study, we determined if pharmacological manipulation of the ER stress-response gene is an effective strategy to protect the kidney from chemical stress in vivo. Treatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), a novel inducer of ER stress proteins, stimulated a time-and dose-dependent increase in Grp78 expression in the kidney, but it did not cause detectable injury. Furthermore, prior treatment with DTTox protected the proximal tubular epithelium against a subsequent challenge with the nephrotoxicant S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC). In contrast, activating a heat shock response did not have a protective effect. Prior treatment with DTTox did not reduce covalent binding of radiolabeled reactive metabolites of 35S-TFEC to renal proteins, indicating that protection was not due to an effect on the metabolic activation of TFEC to the reactive metabolite(s) responsible for renal injury. Antisense grp78 expression in the renal epithelial cell line LLC-PK1 blocked the DTTox-induced Grp78 increase and ablated the protective effect against TFEC damage, indicating that the induction of grp78 expression and the ER stress response were critical for the protective effect of DTTox. These findings suggest that increased expression of Grp78 plays a major role in the protection of renal epithelial cells from reactive intermediate–induced chemical injury in vivo and that pharmacological manipulation is an effective strategy to prevent damage by some classes of nephrotoxicants.
Key Words: ER stress; Grp78; renal injury; nephrotoxicant; S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC); trans-4,5-dihydroxy-1,2-dithiane (DTTox).
INTRODUCTION
Physical and chemical stresses activate stress response genes in a wide range of cell types and tissues. This molecular stress response is important physiologically because it protects cells against injury and provides tolerance to subsequent toxic insults (Kaufman, 2002; Lee, 2001; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004). Among the many stress-inducible proteins are members of the heat shock proteins (Hsp) and glucose-regulated proteins (Grp), families defined by their induction by heat shock and glucose deprivation, respectively (Lee, 2001; Takayama et al., 2003). The exact mechanism by which these stress proteins confer protection is not entirely clear, but their ability to confer tolerance to injury is well documented (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). Genes, such as hsp70, hsp90, grp78, and grp94, are an important class of stress-inducible genes as they encode chaperone proteins that monitor and repair damage to the proteome by directing the refolding and/or degradation of denatured and damaged proteins (Lee, 2001; Takayama et al., 2003). Heat shock proteins mediate cytosolic protein folding, whereas the Grps are responsible for folding and assembly of proteins that traverse the endoplasmic reticulum and Golgi apparatus (Lee, 2001; Takayama et al., 2003). It is generally accepted that chaperones mediate cellular tolerance by preventing the accumulation of cytotoxic aggregates of damaged proteins, i.e., proteotoxic stress (Hightower and Ryan, 1997; Rutkowski and Kaufman, 2004; Takayama et al., 2003).
The transcriptional control of stress-gene expression is sometimes regulated by a direct feedback loop that balances the amount of stress protein available against the need for increased expression in response to a new insult. For example, heat shock factor (HSF) regulates hsp70 transcription through a feedback loop in which the demand for chaperones to fold nascent and damaged proteins is linked to HSF activation through negative regulation by free Hsps (Morimoto, 2002). This feedback regulation is a common theme in the response to proteotoxic stress.
Regulation of the ER stress response, also called the "unfolded protein response" (UPR) is more complex, but it also involves feedback regulation by chaperones; recent reviews summarize three sensing mechanisms that converge on a consensus CCAAT(N9)CCACG endoplasmic reticulum stress-response element (ERSE) in genes such as xbp-1, chop/gadd153, grp78, grp94, and calreticulin (Kaufman, 2002; Lee, 2001; Oyadomari et al., 2002; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004). The first sensing mechanism involves IRE1, a transmembrane protein with a serine-threonine kinase domain in the ER lumen and a cytoplasmic tail with site-specific endonuclease activity (Nikawa and Yamashita, 1992; Yoshida et al., 2001). Grp78 interacts with the IRE1 kinase domain, but increasing demand to fold client ER proteins releases Grp78 activating the IRE1 kinase and RNase activities (Wang et al., 1998, 2000). Unspliced X-box DNA binding protein-1 (XBP1) message is a target for active IRE1 RNase activity; cleavage results in a translation frame-shift producing a protein that binds to the opposite strand complement of the CCACG sequence in the ERSE. The second sensing mechanism requires ATF6, a 90 kDa transmembrane ER protein that is cleaved to release an active p50 DNA-binding protein that binds the CCACG in ERSE when the CCAAT motif is occupied by NF-Y/CBF (Yoshida et al., 2000). Glucose-regulated protein 78 binds p90 ATF6, preventing trafficking to the Golgi, where proteolytic processing to active ATF6 p50 occurs (Shen et al., 2002). Unfolded proteins in the ER bind Grp78, releasing ATF6 for proteolytic processing to active p50. The third mechanism involves a third ER transmembrane protein, the PKR-like kinase (PERK) (Harding et al., 2000; Ma et al., 2002). The PERK is also held in check by Grp78; upon release, the cytoplasmic serine-threonine kinase phosphorylates the translation initiation factor eIF2, blocking translation of most mRNAs. Thus, all three mechanisms may be controlled by a feedback loop that depends on the interaction of Grp78, and possibly other chaperones, with three proximal sensors, IRE1, ATF6, and PERK. Only the IRE1 pathway is replicated in yeast. The presence of multiple sensors in mammalian cells provides increased levels of complexity and flexibility in the mammalian UPR (Halleck et al., 1997; Hung et al., 2003; Kaufman, 2002; Lee, 2001; Liu et al., 1997,1998; Rao et al., 2004; Ron, 2002; Rutkowski and Kaufman, 2004; Takayama et al., 2003; van de Water et al., 1999).
Nephrotoxic cysteine conjugates (NCC) generate reactive acylating species in the proximal tubule epithelium and are a well-characterized model of chemically induced renal injury in vitro and in vivo. Prior treatment of LLC-PK1 cells with DTTox, a novel and nontoxic inducer of ER stress (Halleck et al., 1997) prevents toxicity caused by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), an NCC, and the penultimate toxic metabolites derived from glutathione conjugation of the nephrotoxic gas tetrafluoroethylene (Hayden and Stevens, 1990). Therefore we used TFEC to study the role of ER stress in protection against chemical damage to the kidney in vivo, and determined if DTTox was suitable as a pharmacological modulator of ER stress in the kidney. Amphetamine, which activates a heat shock response in vivo by increasing core temperature, was used to investigate the effect of the heat shock response on TFEC-induced renal injury. The results suggest that the ER stress response, but not the heat shock stress response, prevents NCC-induced renal injury in vivo.
MATERIALS AND METHODS
Animals and reagents.
All chemicals, unless otherwise stated, were purchased from Sigma Chemical Company (St. Louis, MO). Unlabeled and 35S-radiolabeled TFEC were synthesized and characterized as previously described (Hayden and Stevens, 1990). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were obtained from GIBCO (Grand Island, NY).
Male Fischer 344 rats were purchased from Taconic Farms (Germantown, NY). The animals were housed in temperature- and humidity-controlled animal quarters with a 12-h light/dark cycle with free access to food and water at all times. The rats were allowed to acclimatize for 2 weeks before any experimental procedures. All animal experiments were conducted according to approved Institutional Animal Care and Use Committee protocols.
Antibodies.
The following antibodies were purchased from the companies indicated: monoclonal antibodies against Grp78 (SPA-827, anti-KDEL; from StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) and Hsp70 (Hsp72/73, Ab-1; from Oncogene Sciences, Cambridge, MA); polyclonal antibody against Grp78 (PA1-014; from Affinity Bioreagents Inc., Golden, CO). Polyclonal antisera against the porcine Grp78 were produced as described below. Secondary goat anti-rabbit and anti-mouse antibodies were purchased from Amersham Biosciences (Piscataway, NJ).
Antibodies recognizing porcine Grp78 were raised using two peptides designated C13L and C18L. The peptides consist of the C-terminal 13 and 18 amino acids of pig Grp78 sequence (GenBank accession no. X92446) with an additional cysteine at the amino terminal. Peptides were synthesized by the solid phase method using an automated synthesizer (model 431A, Applied Biosystems, Foster City, CA). Synthesis was performed on the 0.1 mmole level using the Fmoc (flouren-9-methoxycarbonyl) protocol. The quality of the crude peptide preparations (data not shown) was evaluated by reverse phase high performance liquid chromatography using a C-18 column eluted with a mobile phase consisting of a gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid and electrospray spectrometry on a PerkinElmer Sciex AP1300 triple quadrupole mass spectrometer (Concord, Thornbill, Canada). The crude synthetic preparations contained the desired peptide as a major component and were used to generate specific anti-peptide polyclonal antisera. The KLH-peptide immunogens were prepared according to the standard protocol supplied by Pierce (Rockford, IL). Briefly, 2 mg of each peptide was mixed with 2 mg of the maleimide activated KLH in 0.5 ml of PBS, pH 7.2 and allowed to react at room temperature for 2 h. The KLH-peptide conjugates were purified by dialysis and gel filtration on a column provided in a SulfoLink kit. The KLH-peptide conjugates were emulsified in Freund's complete medium (0.2 mg/injection) and injected intradermally into several sites in New Zealand white rabbits. Initial booster injections in incomplete Freund's adjuvant were administered sc after 1 and 2 weeks and im after 4 weeks. The first bleed was collected 6 weeks after the first injection. Further booster injections were administered every 4 weeks.
Chemical treatments and stress protein expression.
To assess the effect of various treatments on stress protein level in rat kidney, animals weighing 200–250 g were injected ip with vehicle or various chemicals as described below. At the appropriate times, blood and kidney samples were collected from the animals under anesthesia (pentobarbital, ip; 60–75 mg/kg). After removal of the kidney, a 2–4-mm-thick piece of kidney tissue was excised by lateral slicing. The kidney slice was further dissected to obtain the "cortex," which included the outer cortex and the outer stripe of the medulla (OSOM), the medulla (section between papilla and OSOM), and the papilla as previously described (Ichimura et al., 1995). Remaining kidney samples were snap frozen in liquid nitrogen and stored at –70°C for later use.
TFEC, DTTox, and amphetamine were administered as follows: Rats were injected ip with vehicle or 20 mg/kg of TFEC in phosphate buffered saline (PBS). At 6, 12, 24, and 48 h blood and kidneys were collected. To determine the concentration-dependent effect of DTTox on Grp78 expression, rats were treated ip with 0, 100, 200, and 400 mg/kg DTTox, and kidneys were collected after 24 h for Western analysis. The time-dependent effect on Grp78 expression was also determined using cortex sections from rats treated with a single 200 mg/kg dose of DTTox. Amphetamine was used to induce a heat shock response in vivo. Rats were treated ip with 7.5 mg/kg of amphetamine. Kidneys were collected at 0, 6, and 24 h after treatment, and Hsp70 levels were determined by Western blot analysis.
To determine the effect of prior DTTox and amphetamine exposure on TFEC toxicity, rats were pretreated with a single ip injection of 200 mg/kg of DTTox or 7.5 mg/kg of amphetamine, followed by 20 mg/kg TFEC 24 h later. Forty-eight hours after TFEC injection, blood and kidney samples were collected. Kidney samples were prepared for Western analysis as described above, and a section of kidney tissue was collected from the contralateral kidney of each animal for histological evaluation.
Renal injury.
The extent of protection afforded by stress-response–inducing agents against TFEC-induced renal damage was determined both by histological examination and by measurement of blood urea nitrogen (BUN) content. For histopathology, a transverse section of the kidney was fixed in Carnoy's solution and processed for paraffin embedding. Sections measuring 5-μm in thickness were mounted and stained with hematoxylin and eosin for histopathological examination by light microscopy. The extent of TFEC-induced renal damage was graded according to the following criteria: score 1, no exfoliated cells or casts; score 2, few sloughed cells and no casts; score 3, sloughed cells mostly in cortical S3 segment (S3C), less damage (exfoliated cells) in medullary S3 segment (S3M) and minor cast involvement; score 4, damage to S3C and S3M, casts; score 5, severe damage in both S3C and S3M, casts in both the medullary and cortical regions, dilated collecting ducts. Blood collected from the vena cava was allowed to coagulate at room temperature. The serum fraction was separated and the BUN concentrations were measured with the diacetyl monoxime assay according to the procedure provided with the kit (535A; Sigma).
Covalent binding of TFEC to renal protein.
35S-labeled TFEC was used to measure the effect of DTTox pretreatment on the covalent binding of TFEC to renal proteins. Rats were injected (ip) with saline or 200 mg/kg of DTTox in saline followed by 20 μCi of 35S-TFEC in a dose of 20 mg/kg. At 6, 12, and 24 h after 35S-TFEC treatment, animals were anesthetized with 60 mg/kg of pentobarbital, and the kidneys were perfused with PBS. The kidneys were removed and homogenized in 5 ml of water. Protein was precipitated by addition of cold 20% trichloroacetic acid. After 15 min on ice, the protein pellet was collected by centrifugation and washed three times each with 5% trichloroacetic acid, methanol, and hexane. The final pellet was dried in a dessicator (overnight) and dissolved in 1 ml of 1 N potassium hydroxide at 80°C for 2 h. Ecoscint (10 ml) was added to 100 μ1 of the protein solution, neutralized with phosphoric acid, and left at room temperature overnight. The radioactivity in each sample was determined by liquid scintillation spectrometry (LKB Wallac, 1211 Rackbeta, Finland). The amount of 35S-TFEC bound is expressed as nanomoles 35S-label bound to protein per kidney.
Cell culture.
LLC-PK1 cells, an epithelial cell line derived from porcine kidney (Hull et al., 1976; Stevens et al., 1986), were purchased from American Type Culture Collection (Manassas, VA). LLC-PK1 cells were cultured as previously described (Liu et al., 1996) and maintained in DMEM supplemented with 10% fetal bovine serum (complete medium). pkASgrp78, and pkNEO clones generated from LLC-PK1 cells transfected with antisense grp78 mRNA or empty vector, respectively (Liu et al., 1997), were cultured in complete medium during the experimental procedures. The pkASgrp78, and pkNEO clones were seeded and grown to confluence in complete medium supplemented with 400 μg/ml of G418 for routine maintenance prior to the tolerance experiments.
To produce ER stress preconditioning, confluent cells were treated with DTTox (10 mM in EBSS, for 3 h) and thapsigargin (300 nM in complete medium, for 12 h) as described elsewhere (Halleck et al., 1997; Liu et al., 1997, 1998). Cells were then rinsed and processed for Western analysis as described below. Heat shock proteins were induced in confluent pkASgrp78 (clones 5, 8, and 10), and pkNEO (clones 2, 9, and 10) cells were grown in 6-well dishes as previously described (Liu et al., 1997). To determine if prior ER or heat shock protein induction would confer protection, chemically pretreated or heat shocked cells were subsequently exposed to 0.5 mM of TFEC (in EBSS) for 4 h, followed by 2 additional h in medium without TFEC (6 h total). DTTox-pretreated cells were allowed to recover in complete medium for 12 h before TFEC treatment, whereas thapsigargin-treated cells were dosed immediately after the EBSS wash step. Media aliquots were removed at 4 h, before the cells were washed free of TFEC, and again after the cells were returned to complete medium for 2 h. The samples were then combined, and cytotoxicity was assessed by measuring the amount of lactate dehydrogenase (LDH) released into the pooled medium according to the protocol described in Liu et al. (1996).
Immunoblot analysis.
Regions of the kidney prepared by microdissection were directly homogenized in sodium dodecyl sulfate (SDS) sample preparation buffer (Laemmli) to which a mixture of protease inhibitors containing 5 μg/ml each of leupeptin, aprotinin, and antipain and 1 mM phenylmethylsulfonyl fluoride (PMSF) was added. Protein concentrations were determined using the BioRad (BioRad Laboratories, Richmond, CA) protein assay kit with bovine immunoglobulin G (IgG) as the standard. The sample concentrations were kept low in the assay buffer to avoid interference from the SDS in the homogenization buffer. Equal amounts (10–25 μg) of each sample were resolved on 7.5% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose and immunostained with the appropriate antibodies. Bands were visualized by enhanced chemiluminescence (Amersham Biosciences) and quantitated by scanning densitometry with a Bio Image Densitometer (Bio Image Laboratories).
For Western analysis of cell culture samples, cells were washed with cold PBS followed by cold Tris/sucrose buffer (10 mM Tris/HCl, 250 mM sucrose, 1 mM EGTA; pH 7.4). Rinsed cells were scraped into Tris/sucrose buffer containing leupeptin (10 μg/ml), aprotinin (10 μg/ml) and dithiothreitol (1 mM) and PMSF (1 mM) and sonicated to break up the cells. Cell lysates were solubilized in Laemmli sample preparation buffer and Western blots were performed as described for the in vivo studies above.
Statistical analysis.
Data are presented as means ± standard deviation and were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keul's test when multiple means were compared with the exception of the data for TFEC-induced stress protein expression, which were compared by least significant differences using Student's t-test. A p value under 0.05 was considered significant. Letter designations are used to indicate significant difference. Means with the same letter designation are not different, whereas those with a different letter designation are significantly different from all other means with different letter designations. Means with two-letter designations are not different from groups with either letter designation.
RESULTS
TFEC-Induced Expression of Stress Proteins in Rat Kidney
Exposure to chemical stress can activate stress response genes in renal epithelial cell lines (Liu et al., 1996). TFEC is a well-characterized nephrotoxicant that damages proximal tubule epithelial cells in rat kidney (Hayden et al., 1991). To determine if TFEC treatment alone activated either the heat shock or ER stress responses in vivo, we measured Hsp70 and Grp78 protein expression in protein extracts from the outer medulla region of the kidney at various times after treating rats with a single 20 mg/kg dose of TFEC (Fig. 1). Hsp70 was present in low levels in kidneys from control animals (data not shown; see Fig. 3 below), but increase markedly by 12 h post-TFEC. Although results with the two different antibodies cannot be compared directly, Grp78 expression appeared to be more abundant than Hsp70 in control rat kidneys but also increased after TFEC treatment. Relative to Hsp70, there was a later increase in Grp78 (2–3-fold). These results showed that both cytosolic and ER stress response proteins levels increase after TFEC treatment.
Time Course and Concentration Dependence of DTTox Induction of Grp78
Activation of either the heat shock or ER stress response can protect cells from injury (Rutkowski and Kaufman, 2004; Takayama et al., 2003). Since DTTox can induce an ER stress response and prevent cell injury and has been reported to be well tolerated in mice, we determined if DTTox is a useful tool to activate an ER stress response and Grp78 expression in rat kidneys in vivo (Halleck et al., 1997). Rats were treated with a single dose of 200 mg/kg DTTox and kidney extract prepared 0, 6, 12, 24, 48, and 72 h later (Fig. 2A). There was a time-dependent increase in Grp78 expression with a maximum induction of about twofold, similar to the magnitude seen with TFEC (Fig. 1) 12 h to 24 h post-treatment. Grp78 levels returned to control levels by 48 h. The effect was dose dependent at 24 h over a range of 0–400 mg/kg DTTox (Fig. 2B). DTTox did not induce Hsp70 (Fig. 3). Thus, DTTox induces a reversible increase in Grp78, but not Hsp70, in rat kidney in vivo.
Induction of Hsp70 Expression by Amphetamine
Amphetamine treatment raises core temperature and is a noninvasive way to activate Hsp70 expression in rat liver (Cairo et al., 1985). Amphetamine pretreatment also protects against hepatotoxicants (Salminen et al., 1997). To determine if amphetamine increases Hsp70 expression in the kidney, we treated rats with amphetamine (7.5 mg/kg) and then measured Hsp70 expression in kidney extracts 6 and 24 h later (Fig. 3). There was a marked induction of Hsp70, comparable to that seen with TFEC treatment by 6 h (data not shown); the response was maintained through 24 h. Amphetamine did not induce Grp78 (Fig. 3). Thus, amphetamine treatment is an effective inducer of heat shock stress response but not an ER stress response in rat kidney.
DTTox, but Not Amphetamine Pretreatment Causes Tolerance to TFEC in Rat Kidney
To determine if inducing either an ER or a heat shock stress response produces tolerance to TFEC treatment, rats were pretreated with 200 mg/kg DTTox or 7.5 mg/kg amphetamine and then challenged with 20 mg/kg of TFEC 24 h later. The extent of damage was evident histologically as a typical coagulative necrosis as noted previously (data not shown; Hayden et al., 1991). Examination of kidney sections showed that DTTox pretreatment resulted in a substantial protection against TFEC-induced kidney damage (Table 1). DTTox pretreatment significantly reduced the TFEC-induced increase in BUN (Fig. 4). DTTox alone had no observable effect on histopathology or BUN. In contrast, amphetamine pretreatment did not confer proteBUN measurements.
Effect of DTTox on TFEC Binding
Although we suspected that DTTox treatment prevented renal injury by uncoupling injury from cell death, we could not exclude the possibility that DTTox pretreatment interfered with TFEC absorption, distribution, or metabolism. To exclude this possibility, rats were pretreated with DTTox or vehicle and then challenged with radiolabeled 35S-TFEC. The amount of radiolabel covalently bound to kidney macromolecules, a measure of the amount of reactive metabolite produced, was determined 6, 12, and 24 h later. DTTox pretreatment had no effect on covalent binding of 35S-radiolabeled TFEC at any time point (Table 2). Because inhibition of absorption, distribution, or metabolism would lead to a reduction in binding, we concluded that DTTox was not preventing TFEC activation but was, in fact, uncoupling damage caused by the reactive metabolite from cell death.
Blocking GRP78 Expression Prevents DTTox-Induced Protection
Although the data suggested a cause-and-effect relationship between Grp78 induction and protection in vivo, the association was only correlative. Previously, we have shown that stable expression of a 0.5-kb antisense grp78 fragment in LLC-PK1 cells—so called pkASgrp78 cells—disrupts the induction of Grp78 and the ability of ER stress to protect against the toxicity of iodoacetamide, t-butylhydroperoxide, and hydrogen peroxide (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). To determine the effect of the antisense grp78 on basal and inducible Grp78, we measured Grp78 content in pkASgrp78 and pkNEO cell lysates (pkNEO cells contain only the empty vector with the selectable marker). As shown in Figure 5, pkASgrp78 cells have normal, constitutive levels of Grp78 but are unable to increase expression after treatment with DTTox or thapsigargin, another classic Grp78 activator (Liu et al., 1997). However, induction of Hsp70 expression by heat shock remained intact (data not shown). Because pkASgrp78 cells were unable to increase Grp78 expression after DTTox treatment, we determined if they developed tolerance to TFEC. Three clones of the pkASgrp78 and the pkNeo lines were treated with DTTox and thapsigargin before a challenge with TFEC (Fig. 6). The pkASgrp78 cells were more sensitive to TFEC relative to pkNEO or nontransfected LLC-PK1 cells, as noted with other toxicants (Liu et al., 1997, 1998), suggesting that the inability to increase grp78 expression upon toxicant exposure sensitizes cells to injury. In addition to the enhanced sensitivity of pkASgrp78 cells to TFEC, DTTox and thapsigargin treatment prevented cell death only in pkNEO or LLC-PK1 but not in pkASgrp78 cells. The data suggest that the ability of DTTox to prevent TFEC toxicity and the cellular response to TFEC treatment depend on an increase in the expression of ER stress proteins. In contrast, heat shock conferred no protection against TFEC toxicity in pkASgrp78, pkNEO cells, or LLC-PK1 cells (data not shown), corroborating the results in vivo. These findings suggest that the induction of Grp78 after ER stress plays a major role in protecting proximal tubules from chemically induced cell damage in vitro and in vivo.
DISCUSSION
Heat shock protects cells against a wide variety of insults (Takayama et al., 2003), but less is known about the role of the ER stress response in cytoprotection and tolerance. Nonetheless, evidence is accumulating that the ER stress response is important in the cellular response to heavy metals, oxidative stress, ischemia, toxic prostaglandins, reactive chemical species, and excitotoxicity (Lee, 2001; Ron, 2002; Rutkowski and Kaufman, 2004). In addition, the ER stress response plays an important role in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, which are mediated by mutations in vital cellular proteins resulting in processing and/or functional defects (Kaufman, 2002). The kidney, and specifically the proximal tubular epithelium, is a major target for chemical injury; previous data suggest that the ER stress response prevents injury to renal epithelial cells in culture (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). In the LLC-PK1 renal epithelial cell line, treatment with the alkylating agent iodoacetamide increased expression of hsp70 and grp78 (Liu et al., 1996). Prior ER stress, but not heat shock, prevents cell death from a subsequent challenge with iodoacetamide, hydrogen peroxide or t-butylhydroperoxide and 11-deoxy-16,16-dimethyl prostaglandin E2 (DDM-PGE2) (Halleck et al., 1997; Hung et al., 2003; Jia et al., 2004; Liu et al., 1997, 1998; van de Water et al., 1999). Although ER stress can prevent injury to renal epithelial cells in culture, the importance of the ER stress pathway in vivo is less clear.
Previously, we had shown that ER stress prevents injury to renal epithelial cells from damage by chemical toxicants in vitro (Halleck et al., 1997; Hung et al., 2003; Liu et al., 1997, 1998; van de Water et al., 1999). We now show that DTTox is an effective inducer of an ER stress response in rat kidney in vivo. Prior exposure to DTTox prevented chemical injury in a manner consistent with a mechanism involving increased expression of grp78 and, perhaps, other ER stress proteins. It is interesting that this is not the first time that DTTox has been shown to protect against injury in vivo. Falconi et al. (1970) showed that DTTox protects mice against ultraviolet (UV) irradiation. Although it is possible that DTTox may prevent UV damage directly, or after reduction to DTT (Falconi et al., 1970; Halleck et al., 1997), our studies suggest that protection could also be due to induction of an ER stress response. Regardless, the fact that modulating the ER stress response provides protection from injury in vivo suggests that the ER stress response pathway should be considered a likely candidate as a general mechanism of cellular resistance to stress. A number of key points are worth additional consideration.
The mechanism by which ER stress protects against chemical damage in vivo is not entirely clear, but the accumulated data support a role for intracellular calcium. Studies using LLC-PK1 cells show that an increase in cellular calcium is an important contributor to cell death (Chen et al., 1994; Hung et al., 2003; Liu et al., 1997, 1998). Both alkylating agents and oxidants can disrupt the ability of cells to regulate intracellular calcium levels in renal epithelial cells. Moreover, prior ER stress prevents the increase in cellular calcium and oxidative stress (Hung et al., 2003; Liu et al., 1997, 1998). We have proposed that the ER with increased stress proteins may be better able to buffer intracellular calcium level during stress and thereby prevent calcium-mediated adverse consequences such as activation of proteases, caspases, and phosphorylation of JNKs (Hung et al., 2003). Better regulation of intracellular calcium protects mitochondrial function by preventing the accumulation of calcium in the mitochondria, which otherwise would disrupt mitochondrial function (Orrenius et al., 2003). Thus, it is plausible to propose that a similar mechanism of protection is active in protecting the TFEC treated-kidneys in vivo. Thus, it seems reasonable to propose, based on our own data (Hung et al., 2003; Liu et al., 1997, 1998), that an effect on intracellular calcium may underpin the protection noted in vivo.
The heat shock response has received considerable attention as a protective response (Goldberg, 2003; Takayama et al., 2003). Several studies have shown that amphetamine not only increases core temperature and induces expression of heat shock proteins but also protects against toxicant injury and cardiac ischemia–reperfusion injury (Maulik et al., 1995; Salminen et al., 1997). Although amphetamine treatment produced a heat shock response in rat kidney, it provided only modest protection as reflected in the small, but statistically significant, reduction in BUN levels in the amphetamine-pretreated TFEC group relative to the saline-pretreated TFEC-treated group. This is consistent with studies in LLC-PK1 cells showing that heat shock does not protect against iodoacetamide-induced (Liu et al., 1997) and t-butyl-hydroperoxide-induced (Liu et al., 1998) cell death. Nonetheless, Hsp70 was induced in rat kidney after TFEC treatment, suggesting that the heat shock response was induced. Although Hsp70 induction did not correlate with protection against chemically induced damage in vitro or in vivo, the results do not necessarily exclude a role for Hsp70. Indeed, unpublished work (B. van de Water and J. L. Stevens) indicates that when cells are protected by prior ER stress, one consequence is a more robust induction of heat shock proteins in response to an insult that would be lethal to nave cells. Thus, it is likely that these two stress responses play complementary but distinct roles in the cellular response to stress.
Thus, the induction of Grp78 by DTTox protects against chemically induced renal damage both in vitro and in vivo. Endoplasmic reticulum stress response as a cellular defense mechanism may serve as an important adaptive response accounting for the preconditioning response our lab (Park et al., 2001, 2002, 2003) and others have previously reported in the kidney. This relationship between an upregulated ER stress response and protection against nephrotoxin raises possibilities for therapeutic interventions directed toward these responses in the prophylaxis against nephrotoxic injury.
NOTES
2 Present Address: Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294.
ACKNOWLEDGMENTS
We acknowledge Dr. Joe Bonventre for critical reading of the manuscript and constructive comments. This work was supported in part by National Institutes of Health grant DK46267.
REFERENCES
Cairo, G., Bardella, L., Schiaffonati, L., and Bernelli-Zazzera, A. (1985). Synthesis of heat shock proteins in rat liver after ischemia and hyperthermia. Hepatology 5, 357–361.
Chen, Q., Jones, T. W., and Stevens, J. L. (1994). Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates. J. Cell. Physiol. 161, 293–302.
Falconi, C., Scotto, P., and De Franciscis, P. (1970). Radioprotection and recovery by dithiothreitol. Experientia 26, 172–3.
Goldberg, A. L. (2003). Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–9.
Halleck, M. M., Liu, H., North, J., and Stevens, J. L. (1997). Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells. J. Biol. Chem. 272, 21760–21766.
Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H., and Ron, D. (2000). Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904.
Hayden, P. J., Ichimura, T., McCann, D. J., Pohl, L. R., and Stevens, J. L. (1991). Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts. J. Biol. Chem. 266, 18415–18418.
Hayden, P. J., and Stevens, J. L. (1990). Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria. Mol. Pharmacol. 37, 468–476.
Hightower, L. E., and Ryan, J. A. (1997). Are stress proteins part of a cell's solution to toxicity or are they part of the problem Hepatology 25, 1279–1281.
Hull, R. N., Cherry, W. R., and Weaver, G. W. (1976). The origin and characteristics of a pig kidney cell strain, LLC-PK. In Vitro 12, 670–677.
Hung, C. C., Ichimura, T., Stevens, J. L., and Bonventre, J. V. (2003). Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J. Biol. Chem. 278, 29317–29326.
Ichimura, T., Maier, J. A.-M., Maciag, T., Zhang, G., and Stevens, J. L. (1995). Fibroblast growth factor-1 (acidic FGF) in normal and regenerating kidney: Expression in mononuclear, interstitial and regenerating epithelial cells. Am. J. Physiol. 269, F653–F662.
Jia, Z., Person, M. D., Dong, J., Shen, J., Hensley, S. C., Stevens, J. L., Monks, T. J., and Lau, S. S. (2004). Grp78 is essential for 11-deoxy-16,16-dimethyl PGE2-mediated cytoprotection in renal epithelial cells. Am. J. Physiol. Renal Physiol. 287, F1113–F1122.
Kaufman, R. J. (2002). Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 110, 1389–1398.
Lee, A. S. (2001). The glucose-regulated proteins: Stress induction and clinical applications. Trends Biochem. Sci. 26, 504–510.
Liu, H., Bowes, R. C., III, van de Water, B., Sillence, C., Nagelkerke, J. F., and Stevens, J. L. (1997). Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J. Biol. Chem. 272, 21751–21759.
Liu, H., Lightfoot, R., and Stevens, J. L. (1996). Activation of heat shock factor by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. J. Biol. Chem. 271, 4805–4812.
Liu, H., Miller, E., van de Water, B., and Stevens, J. L. (1998). Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death. J. Biol. Chem. 273, 12858–62.
Ma, K., Vattem, K. M., and Wek, R. C. (2002). Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J. Biol. Chem. 277, 18728–18735.
Maulik, N., Engelman, R. M., Wei, Z., Liu, X., Rousou, J. A., Flack, J. E., Deaton, D. W., and Das, D. K. (1995). Drug-induced heat-shock preconditioning improves postischemic ventricular recovery after cardiopulmonary bypass. Circulation 92(Suppl. II), 381–388.
Morimoto, R. I. (2002). Dynamic remodeling of transcription complexes by molecular chaperones. Cell 110, 281–284.
Nikawa, J., and Yamashita, S. (1992). IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae. Mol. Microbiol. 6, 1441–1446.
Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003). Regulation of cell death: The calcium-apoptosis link. Nat. Rev. Mol. Cell Biol. 4, 552–565.
Oyadomari, S., Araki, E., and Mori, M. (2002). Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 7, 335–345.
Park, K. M., Byun, J. Y., Kramers, C., Kim, J. I., Huang, P. L., and Bonventre, J. V. (2003). Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney. J. Biol. Chem. 278, 27256–27266.
Park, K. M., Chen, A., and Bonventre, J. V. (2001). Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J. Biol. Chem. 276, 11870–11876.
Park, K. M., Kramers, C., Vayssier-Taussat, M., Chen, A., and Bonventre, J. V. (2002). Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J. Biol. Chem. 277, 2040–2049.
Rao, R. V., Ellerby, H. M., and Bredesen, D. E. (2004). Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11, 372–380.
Ron, D. (2002). Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110, 1383–1388.
Rutkowski, D. T., and Kaufman, R. J. (2004). A trip to the ER: Coping with stress. Trends Cell Biol. 14, 20–28.
Salminen, W. F., Jr., Voellmy, R., and Roberts, S. M. (1997). Protection against hepatotoxicity by a single dose of amphetamine: The potential role of heat shock protein induction. Toxicol. Appl. Pharmacol. 147, 247–258.
Shen, J., Chen, X., Hendershot, L., and Prywes, R. (2002). ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell 3, 99–111.
Stevens, J. L., Hayden, P., and Taylor, G. (1986). Studies on the mechanism of S-cysteine conjugate metabolism and toxicity in rat liver, kidney, and a cell culture model. Adv. Exp. Med. Biol. 197, 381–390.
Takayama, S., Reed, J. C., and Homma, S. (2003). Heat-shock proteins as regulators of apoptosis. Oncogene 22, 9041–9047.
van de Water, B., Wang, Y., Asmellash, S., Liu, H., Zhan, Y., Miller, E., and Stevens, J. L. (1999). Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment. Chem. Res. Toxicol. 12, 943–951.
Wang, X. Z., Harding, H. P., Zhang, Y., Jolicoeur, E. M., Kuroda, M., and Ron, D. (1998). Cloning of mammalian Ire1 reveals diversity in the ER stress responses. Embo J. 17, 5708–5717.
Wang, Y., Shen, J., Arenzana, N., Tirasophon, W., Kaufman, R. J., and Prywes, R. (2000). Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275, 27013–27020.
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891.
Yoshida, H., Okada, T., Haze, K., Yanagi, H., Yura, T., Negishi, M., and Mori, K. (2000). ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol. Cell Biol. 20, 6755–6767.(Senait Asmellash, James L. Stevens and T)