Increased reactive oxygen species contribute to high NaCl-induced activation of the osmoregulatory transcription factor TonEBP/OREBP
http://www.100md.com
《美国生理学杂志》
Division of Nephrology, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Division of Nephrology and Hypertension, University of Alabama at Birmingham, Alabama
ABSTRACT
The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/OREBP transactivation. NG-monomethyl-L-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation.
superoxides; nitric oxide; ouabain; transactivation; nuclear translocation
ELEVATION OF NaCl above the normal systemic level of 290 mosmol/kgH2O can cause cell cycle delay (31), oxidative stress (51), DNA damage (11), and apoptosis (10). Interstitial NaCl concentration normally is very high in the renal medulla and varies with urine concentration. Nevertheless, renal medullary cells evidently survive and function. Tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) (also known as NFAT5) is a transcription factor that, when activated by high NaCl and other hypertonic stresses, increases transcription of osmoprotective genes (28, 35, 49). As a result, the cells are protected by an accumulation of compatible organic osmolytes (1) and by increased expression of heat shock proteins (42, 49). The organic osmolytes in renal medullary cells include glycine betaine, which accumulates because of TonEBP/OREBP-dependent increased transcription of the betaine/-aminobutyric acid transporter, BGT1 (28, 35, 49).
High NaCl also induces oxidative stress in renal medullary cells in culture (51) and in isolated, perfused rat livers (41). Oxidative stress is associated with increased reactive oxygen species (ROS). ROS include superoxides, hydrogen peroxide and its metabolytes, and hydroxyl radicals (17). Free radicals are produced during normal mitochondrial metabolism and also by the action of nonmitochondrial oxidases (17). Superoxides, formed by one-electron reduction of oxygen, are dismutated into hydrogen peroxide, either spontaneously or catalyzed by superoxide dismutase (SOD). Catalase and peroxidase decompose hydrogen peroxide into water and oxygen.
It is pertinent that ROS transduce messages originated by hormones, cytokines, and ions to effectors including transcription factors (17). For example, high NaCl-induced tyrosine phosphorylation of Syk kinase is involved in signaling by ROS (39). Furthermore, superoxide anions and hydrogen peroxide regulate the activity of transcription factors, including AP-1 and NF-B (27, 30). The present studies were undertaken to see whether ROS also contribute to high NaCl-induced activation of the transcription factor, TonEBP/OREBP. We confirm that high NaCl increases ROS, including superoxides, and find that the ROS are necessary for full high NaCl-induced activation of TonEBP/OREBP.
MATERIALS AND METHODS
Cells, cell culture, and chemicals. Human embryonic kidney 293 (HEK293) cells, purchased from ATCC (Manassas, VA), were incubated in Eagle's minimal essential medium plus 10% fetal bovine serum in 5% CO2-95% air at 37°C. The osmolality of this control "isotonic" medium was 300 mosmol/kgH2O. MnTBAP was purchased from Calbiochem (San Diego, CA), dihydroethidium (DHE) from Molecular Probes (Eugene, OR), and all other chemicals from Sigma (St. Louis, MO). All antioxidants and probes were freshly prepared. Cells overexpressing human catalase were from the same clone used in previous studies (8).
Measurement of ROS. 2',7'-Dichlorofluorescin diacetate (DCFH-DA) permeates cell membranes. Within cells it is cleaved to produce DCFH, which is trapped because it is poorly permeating. DCFH is oxidized by free radicals, producing fluorescence. HEK293 cells were detached by trypsinization, preloaded with 20 μM DCFH-DA in phenol red-free culture medium for 30 min, then transferred either to control culture medium or hypertonic medium (osmolality raised to 500 mosmol/kgH2O by adding NaCl) for the periods indicated. The intensity of fluorescence was analyzed by flow cytometry (FACSCalibur, Becton Dickinson) with excitation at 488 nm and emission at 535 nm. A gate was set to exclude signals from debris and aggregates. Results are expressed as fold change compared with corresponding controls. Ten thousand cells were analyzed in each assay and assays were run in duplicate.
Measurement of superoxide anions. Dihydroethidium (DHE) enters cells freely. Superoxides oxidize DHE into ethidium or a structurally similar product, which intercalates into DNA, producing fluorescence (5). One to 1.2 x 106 cells were grown overnight on the optical glass bottom of a dish (MatTek, Ashland, MA) and preincubated with 30 μM DHE for 30 min. Then, the medium was replaced with a dye-free one at 300 mosmol/kgH2O or at 450 mosmol/kgH2O (NaCl added) for 45 min. The fluorophore was excited at 488 nm. Fluorescent emission at 525 nm was recorded by confocal microscopy (LSM510 Meta, Zeiss, Thornwood, NY) and analyzed with MetaMorph software.
Measurement of TonEBP/OREBP transcriptional activity. The reporter, –1233/–1105 IL2min-GL3, was constructed by inserting nucleotides –1233 to –1105 of the 5'-flanking region of the human aldose reductase (AR) gene into MluI/NheI sites upstream of the human IL-2 minimal promoter (47). The parent vector, containing an IL-2 minimal promoter upstream of the Photinus pyralis luciferase gene, was a gift from Dr. S. N. Ho (University of California, San Diego, CA). This portion of the AR gene contains potentiation elements one (tggaaaaatat) and two (AAATTTTTCCA), an osmotic response element (ORE; TGGAAAATCAC) and an AP-1 binding site (TGAGTCAC) (13). An otherwise identical reporter to which binding of TonEBP/OREBP is prevented by mutation of the potentiating and ORE elements and the adjacent AP-1 site was used to control for specificity to TonEBP/OREBP. In the mutated version, the nucleotide sequences are changed to tgtctaaatat, AAATTTAGACA, TGTCTAATCAC, and TGCTGCAC, respectively. Mutations were performed using a QuikChange site mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol. Accuracy of the mutations was confirmed by sequencing.
The reporter, ORE-X IL2min-GL3, was constructed by inserting two tandem copies of the human ORE-X sequence (15) in the XhoI site of the human IL-2 minimal promoter (47). ORE and ORE-mutated constructs were modified to express the blasticidin resistance gene by insertion of nucleotides 1934 to 3204 of pCMV/Bsd (Invitrogen, Carlsbad, CA) into XmnI/BsaI sites. Stable reporter cell lines were established by transfection with Effectene (Qiagen, Valencia, CA), blasticidin selection, and screening for luciferase activity to select clones with the highest expression; 3 x 104 cells per well were seeded in 96-well white view plates (Packard, Wellesley, MA) and incubated for 24 h at 300 mosmol/kgH2O, then the medium was changed for 16 h to one still at 300 mosmol/kgH2O or elevated to 500 mosmol/kgH2O by adding NaCl. Luciferase activity was measured with Bright-Glo substrate (Promega, Madison, WI) in a Victor3 luminometer (Perkin Elmer, Wellesley, MA). Cell protein was determined with the BCA reagent (Pierce, Rockford, IL).
Measurement of BGT1 mRNA expression. This was performed as previously described (16); 2.5 x 105 cells/well were plated in a six-well dish, then incubated at the indicated osmolality for 16 h. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Amplicons were detected with an ABI Prism 7900HT detection system (Applied Biosystems). Primers for the human BGT1 mRNA were 5'-TGTTCAGCTCCTTCACCTCTGA-3' and 5'-GCAATGCTCTGTGTTCCAAAAG-3'. The 6-carboxyfluorescein (FAM)-labeled probe was 5'-CTGCCCTGGACGACCTGCAACAA-3'. As a control for reverse transcription efficiency and loading, 18S RNA was measured at the same time, using primers and a probe from Applied Biosystems. Fold differences in RNA abundance between conditions (F) was calculated, as F=E(Ct1–Ct2). E (= 1.98 ± 0.056, n = 18, means ± SE) is the efficiency of the reaction determined from the results of reactions containing 8 or 80 ng of cDNA template. Ct1 and Ct2 are the numbers of cycles required to reach the threshold of amplicon abundance in respective experimental conditions (16).
Measurements of TonEBP/OREBP transactivation. This was measured using a yeast binary GAL4 reporter assay system, as previously described (16). Briefly, the assay system comprises cotransfection of an expression vector containing the TonEBP/OREBP transactivation domain (TAD), GAL4dbd-TAD, and a reporter plasmid (GAL4UAS-GL3). GAL4dbd-TAD contains in-frame insertion of cDNA coding for amino acids 548–1531 of TonEBP/OREBP in a vector containing the neomycin resistance gene (pFA-CMV, Stratagene). An otherwise identical construct, in which the GAL4dbd construct did not contain a TAD, was used as a control for nonspecific effects. GAL4UAS-GL3 contains five tandem repeats of the yeast GAL4 binding site (upstream activating sequence) and a minimal promoter (TATATA) derived from pFR-Luc (Stratagene) and inserted into the NheI/HindIII sites of pGL3 (Promega) upstream of the P. pyralis luciferase gene. GAL4UAS-GL3 was further modified for blasticidin resistance as described above. Stable reporter cell lines were established by blasticidin and neomycin selection and used as described above for assays of TonEBP/OREBP transcriptional activity.
Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed by paired t-test or repeated ANOVA, as appropriate. Post hoc comparison was made by Dunnett's test. P < 0.05 is considered significant.
RESULTS
High NaCl increases ROS, including superoxides. Hypertonicity was previously found to activate TonEBP in HEK293 cells, which are renal epithelial cells of human origin (44). To examine whether hypertonicity increases ROS in HEK293 cells, we raised the osmolality from 300 to 500 mosmol/kgH2O by adding NaCl and measured ROS by flow cytometry, using DCFH-DA as a probe. High NaCl raises the fluorescence intensity of DCFH within 30 min, indicating increased ROS (Fig. 1A). To examine whether high NaCl increases formation of superoxides, we measured superoxide activity by confocal microscopy, using DHE as a probe. High NaCl increases DHE fluorescence by 1.37-fold (Fig. 1B), indicating that high NaCl induces generation of superoxides. The effect of high NaCl on superoxide activity is reduced by N-acetylcysteine (NAC; 5 mM), a general antioxidant (Fig. 1B). Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; 40 μM), a SOD mimetic (3), and catalase (400 U/ml) do not significantly lower high NaCl-induced superoxide generation. MnTBAP increases superoxide activity in cells kept at 300 mosmol/kgH2O (Fig. 1B).
Antioxidants attenuate high NaCl-induced TonEBP/OREBP transcriptional activity. We measured transcriptional activity of TonEBP/OREBP in HEK293 cells stably expressing an ORE luciferase reporter. High NaCl increases ORE reporter activity 96- to 106-fold (Fig. 2A). NAC and MnTBAP, but not exogenously added catalase, significantly reduce high NaCl-induced ORE reporter activity (38 and 29%, respectively; Fig. 2A). To examine whether this effect is specifically mediated by binding of TonEBP/OREBP to OREs, we used an otherwise identical reporter in which the ORE sites and the adjacent AP-1 site are mutated. This prevents both the binding of TonEBP/OREBP and high NaCl-induced increase of reporter activity (Fig. 2B). NAC and MnTBAP have no significant effect on activity of the mutated ORE reporter (Fig. 2B). We conclude that the full high NaCl-induced increase in TonEBP/OREBP transcriptional activity requires increased ROS, mainly superoxides.
The reporter that we used includes the AP-1 site adjacent to the OREs in the AR gene. Mutation of the AP-1 site reduces high NaCl-induced reporter activity (13). AP-1 is regulated by ROS (27, 30). To test whether antioxidants reduce ORE reporter activity by inhibiting AP-1 activity, we examined the effect of NAC on a different reporter, ORE-X, that does not include that AP-1 site. NAC reduces high NaCl-induced ORE-X activity by 41% (from 308 ± 124-fold increase, comparing 500 with 300 mosmol/kgH2O, to 181 ± 68-fold increase, P < 0.05, n = 5), even in the absence of the AP-1 site, excluding the possibility that the effect of NAC is directly mediated by AP-1.
BGT1 is a transcriptional target of TonEBP/OREBP (21). Activation of TonEBP/OREBP by high NaCl elevates BGT1 mRNA abundance (21). To further examine the effects of antioxidants on TonEBP/OREBP activity, we measured the effect of NAC and MnTBAP on the high NaCl-induced increase in BGT1 mRNA expression, using real-time RT-PCR. NAC and MnTBAP, but not catalase, inhibit high NaCl-induced BGT1 mRNA expression by 30 and 20%, respectively (Fig. 3A).
Exogenously added catalase does not affect ORE reporter activity (Fig. 2A) or BGT1 mRNA expression (Fig. 3A). Because it seemed possible that the exogenous catalase might not effectively reduce intracellular H2O2, we examined the effect of high NaCl on BGT1 mRNA expression in a clone of HEK293 cells that overexpresses human catalase (8). Overexpression of catalase does not reduce tonicity-dependent expression of BGT1 mRNA (Fig. 3B). The effectiveness of the overexpressed catalase in these cells is attested by resistance of the cells to H2O2-mediated cell death induced by FK506 (53).
Reduction of ROS suppresses high NaCl-induced TonEBP/OREBP transactivation. TonEBP/OREBP contains a TAD, whose activity is increased by high NaCl (14, 16, 26). We used HEK293 cells stably transfected with a binary GAL4 transactivation reporter system to test whether the ROS-induced increase of TonEBP/OREBP transcriptional activity is mediated by increased transactivation. Raising osmolality from 300 to 500 mosmol/kgH2O by adding NaCl increases transactivation about 10-fold (Fig. 4A). NAC and MnTBAP, but not catalase, reduce high NaCl-induced transactivation by 20 and 40%, respectively (Fig. 4A). We used an otherwise identical reporter that does not contain the TAD of TonEBP/OREBP to exclude a general effect on transactivation. NAC and MnTBAP have no significant effect on the control reporter activity (Fig. 4B). We tentatively conclude that ROS contribute to the activation of TonEBP/OREBP by transactivating it.
Effects of high NaCl on TonEBP/OREBP activity do not involve nitric oxide. Renal medullary cells can generate nitric oxide (29, 50). Water deprivation increases nitric oxide synthase mRNA expression in the renal medulla (43). Superoxides spontaneously react with nitric oxide to form peroxynitrite, which can induce gene expression in response to stress (24). For example, in human neuroblastoma cells, peroxynitrite induces DNA damage-inducible (Gadd) proteins 34, 45, 153 mRNA expression (37). NG-monomethyl-L-arginine (L-NMMA) is a general inhibitor of nitric oxide synthases (4). To test whether nitric oxide is involved in high NaCl-induced activation of TonEPB/OREBP, we examined the effect of 100 μM L-NMMA. L-NMMA has no significant effect on high NaCl-dependent increase in superoxides (Fig. 5A), TonEBP/OREBP transcriptional activity (Fig. 5B), BGT1 mRNA (Fig. 5C), or TonEBP/OREBP transactivation (Fig. 5D). We conclude that high NaCl-induced activation of TonEBP/OREBP does not involve nitric oxide.
Increase of ROS in the absence of high NaCl is not sufficient to activate TonEBP/OREBP. We elevated ROS at 300 mosmol/kgH2O by adding 1) xanthine oxidase (6 mU/ml) plus hypoxanthine (500 μM) to directly elevate ROS (23), 2) L-buthionine-[S,R]-sulfoximine (BSO; 100 and 400 μM) to deplete the reduced form of glutathione (19), or 3) diethyldithiocarbamate (100 μM and 1 mM) to elevate superoxide by inhibiting SOD (9). Although xanthine oxidase plus hypoxanthine increases superoxide (data not shown), ORE-X activity is unaffected at either 300 or 500 mosmol/kgH2O (Fig. 6A). BSO (Fig. 6B) and diethyldithiocarbamate (data not shown) also do not affect ORE-X activity at either osmolality. We conclude that increase in ROS in the absence of other high NaCl-induced signals is not sufficient to activate TonEBP/OREBP.
Ouabain inhibits both high NaCl-induced superoxide activity and activation of TonEBP/OREBP. Ouabain is an inhibitor of Na-K-ATPase (18). In Madin-Darby canine kidney cells, ouabain reduces high NaCl-dependent increase of AR, SMIT and BGT1 mRNA abundance and of TonEBP/OREBP protein abundance (36). Those effects were attributed to cell swelling caused by ouabain and were proposed as evidence that TonEBP/OREBP activity is regulated by cell volume, as well as by intracellular ionic strength (36). However, ouabain also reduces the high NaCl-induced increase in superoxide in rat medullary thick ascending limbs (33). We reasoned that if ouabain should also reduce high NaCl-induced superoxide formation in renal epithelial cells, that could explain how ouabain reduces TonEBP/OREBP activity without invoking changes in cell volume. Therefore, we tested the effect of a low concentration of ouabain (12 nM) on high NaCl-induced superoxide level and TonEBP/OREBP activity in HEK293 cells. Ouabain decreases superoxide (Fig. 7A), TonEBP/OREBP transcriptional activity (Fig. 7B), and TonEBP/OREBP transactivation (Fig. 7D). As controls for specificity, the effect on transcription does not occur in the absence of functional TonEBP/OREBP binding elements (Fig. 7C) nor on transactivation in the absence of the TonEBP/OREBP TAD (Fig. 7E). We conclude that ouabain-induced decrease in superoxide contributes to reduction by ouabain of the high NaCl-induced increase in TonEBP/OREBP activity.
DISCUSSION
Measurements of oxidized glutathione (41) and the effects of antioxidants (39, 52) originally indicated that osmotic stress causes oxidative stress. Subsequently, it was found that high NaCl increases ROS production and carbonylate proteins (indicative of oxidative damage to the proteins) in mIMCD3 cells and that protein carbonylation is abundant in normal renal medulla, associated with the normally high interstitial NaCl concentration in that tissue (51). In the present studies of HEK293 cells, we confirm by two independent methods, namely flow cytometry with DCFH as a probe (Fig. 1A) and confocal microscopy with DHE as an indicator (Fig. 1B), that high NaCl elevates ROS and superoxides. DCFH is oxidized to form DCF, which fluoresces. This was initially thought to require hydrogen peroxide (45). However, subsequent studies showed that DCFH can also be oxidized by enzymatic reactions and by other oxidants, including peroxynitrite and hypochlorous acid (45). DHE is oxidized by superoxides, but not by hydrogen peroxide, peroxynitrite, or hypochlorous acid (5), providing a specific probe for measuring superoxide activity (45).
ROS can serve as signaling molecules (17). In most cases, superoxides are the initial ROS produced. Examples include that, in vascular smooth muscle cells, IL-1 stimulates superoxide production, ERK activation, and matrix metalloproteinase-9 gene expression. Overexpression of SOD or addition of NAC inhibits all these effects (20). Similarly, ANG II activates NADPH oxidase, resulting in generation of ROS that, in turn, activate multiple transcription factors through the Raf-1-MAPK pathway, leading to increased vascular tone (54).
We used the antioxidants, NAC and MnTBAP, to study the role of ROS in high NaCl-induced activation of TonEBP/OREBP. These antioxidants reduce high NaCl-induced increase of ROS (Fig. 1B), TonEBP/OREBP transcriptional activity (Fig. 2, A and C), and the mRNA expression of TonEBP/OREBP's transciptional target, BGT1 (Fig. 3A). NAC, a membrane-permeating form of cysteine, is a precursor of glutathione. It serves as a general antioxidant by raising cellular reduced glutathione content, which alters redox balance, inhibiting formation of ROS (7). MnTBAP acts like SOD, dismutating superoxide to H2O2 (3).
Using these antioxidants, we found that ROS contribute to high NaCl-induced increase of TonEBP/OREBP transactivation (Fig. 4). At this point, we can only speculate on the mechanism. High NaCl increases phosphorylation of the TonEBP/OREBP TAD (14). Activation of a number of kinases, including PKAc, p38, and Fyn, is required for full high NaCl-induced increase in TonEBP/OREBP transactivation (14, 25). ROS can activate p38 (40). Thus ROS could transactivate TonEBP/OREBP through increased p38 kinase activity. An example of ROS affecting transactivation via phosphorylation by a MAP kinase is provided by the effect of PDGF. Activation of PDGF increases ROS, which contributes to activity of the transcription factor, AP-1 (32), mediated by ERK which increases phosphorylation of a TAD located at the COOH-terminal of c-Fos, a partner in AP-1 heterodimers (32). Phosphorylation of p38 by MEKK3 increases its activity (48) and dephosphorylation by protein tyrosine phosphatases (PTPs) can decrease it (34). Thiol groups of active site cysteines in PTPs are vulnerable to oxidation (17). Free radicals inhibit PTPs through either direct oxidation of the thiol groups or glutathionylation due to increased oxidized glutathione level (17). Hypertonicity inhibits the activity of PTPs, which can be prevented by NAC (40). Thus ROS may contribute to the activation of p38, and thereby of TonEBP/OREBP, by inhibiting PTPs.
We did not find any effect on TonEBP/OREBP transcriptional activity at 300 mosmol/kgH2O of exogenous agents that elevate ROS nor did the agents further increase the effect of high NaCl (Fig. 6). Thus, although increased ROS is necessary for full high NaCl-induced activation of TonEBP/OREBP, isolated elevation of ROS does not activate TonEBP/OREBP per se. This result is consistent with the previous conclusion that multiple signaling inputs are necessary for tonicity-induced activation of TonEBP/OREBP but that no one of the signals is sufficient by itself (22). A cogent previous example is provided by the DNA damage-inducible kinase, ATM (22). High NaCl damages DNA (11). Full high NaCl-dependent activation of TonEBP/OREBP requires the activation ATM (22). However, other agents such as high urea and radiation also activate ATM, but they do not increase TonEBP/OREBP transcriptional activity like high NaCl does (22) and high urea actually reduces tonicity-dependent activation of TonEBP/OREBP (46).
Hypertonicity shrinks cells by osmosis, resulting in increased concentration of all intracellular components, including intracellular ions (36) and macromolecules (6). Decrease in cell volume (12), increase in intracellular ionic strength (36), and increased macromolecular crowding (6) are all candidates to be sensors that trigger cellular responses to hypertonicity. Their relative importance remains controversial. Under conditions in which changes in cell volume are similar, TonEBP/OREBP activity correlates with the intracellular ionic strength regardless of the external tonicity (36). On the other hand, inhibition of Na-K-ATPase by ouabain leads to decreased activity of TonEBP/OREBP despite a marked increase in the intracellular ionic strength. It was suggested that, because isotonic swelling is known to occur under these conditions, dilution of the cytoplasmic constituents inhibits the activity of TonEBP/OREBP (36). However, the present study affords an alternative explanation as the effect of ouabain on TonEBP/OREBP is associated with suppression of ROS formation (Fig. 7). Given that ROS contribute to the activation of TonEBP/ OREBP, it is plausible that it is inhibition of ROS generation by ouabain that attenuates the activation of TonEBP/OREBP, rather than increased cellular water content.
It is interesting to speculate on how ouabain reduces ROS in cells stressed by high NaCl (Fig. 7). High NaCl increases Na-K-ATPase activity (38), presumably to energize increased Na and K transport. Production of ATP must increase to compensate for its greater consumption, which can entail greater mitochondrial oxidative metabolism. Ouabain reduces oxygen consumption by reducing Na and K transport and the associated consumption of ATP (2). Oxidative metabolism by mitochondria is associated with production of ROS (2). Putting all of this together, we propose that ouabain reduces high NaCl-induced increase in ROS by decreasing utilization of ATP by the Na-K-ATPase, thus reducing oxidative metabolism and the accompanying production of ROS. A similar process may be occuring in isolated rat medullary thick ascending limbs, where the Na transport inhibitors, dimethylamiloride and ouabain, blunt the effect of high NaCl-induced superoxide activity (33).
GRANTS
This study was supported in part by a Grant-in-Aid from National Kidney Foundation/National Capital Area (to X. Zhou).
ACKNOWLEDGMENTS
The authors thank Dr. J. Handler for thoughtful discussions during the course of the study, Dr. C. Combs for expert help with confocal microscopy, and A. Williams for assistance with flow cytometry.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Bagnasco S, Balaban R, Fales HM, Yang YM, and Burg M. Predominant osmotically active organic solutes in rat and rabbit renal medullas. J Biol Chem 261: 5872–5877, 1986.
Balaban RS and Mandel LJ. Coupling of aerobic metabolism to active ion transport in the kidney. J Physiol 304: 331–348, 1980.
Batinic-Haberle I, Benov L, Spasojevic I, and Fridovich I. The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic. J Biol Chem 273: 24521–24528, 1998.
Belenky SN, Robbins RA, and Rubinstein I. Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro. J Leukoc Biol 53: 498–503, 1993.
Benov L, Sztejnberg L, and Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25: 826–831, 1998.
Burg MB. Macromolecular crowding as a cell volume sensor. Cell Physiol Biochem 10: 251–256, 2000.
Cotgreave IA. N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv Pharmacol 38: 205–227, 1997.
Davis CA, Nick HS, and Agarwal A. Manganese superoxide dismutase attenuates cisplatin-induced renal injury: importance of superoxide. J Am Soc Nephrol 12: 2683–2690, 2001.
Didion SP, Ryan MJ, Baumbach GL, Sigmund CD, and Faraci FM. Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen. Am J Physiol Heart Circ Physiol 283: H1569–H1576, 2002.
Dmitrieva N, Kultz D, Michea L, Ferraris J, and Burg M. Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. J Biol Chem 275: 18243–18247, 2000.
Dmitrieva NI, Cai Q, and Burg MB. Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo. Proc Natl Acad Sci USA 101: 2317–2322, 2004.
Ericson AC and Spring KR. Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl–-HCO3– exchange. Am J Physiol Cell Physiol 243: C146–C150, 1982.
Ferraris J and Garcia-Perez A. Osmotically responsive genes: the mammalian osmotic response element (ORE). Am Zool 41: 734–742, 2001.
Ferraris JD, Persaud P, Williams CK, Chen Y, and Burg MB. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proc Natl Acad Sci USA 99: 16800–16805, 2002.
Ferraris JD, Williams CK, Ohtaka A, and Garcia-Perez A. Functional consensus for mammalian osmotic response elements. Am J Physiol Cell Physiol 276: C667–C673, 1999.
Ferraris JD, Williams CK, Persaud P, Zhang Z, Chen Y, and Burg MB. Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration. Proc Natl Acad Sci USA 99: 739–744, 2002.
Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol 15: 247–254, 2003.
Fujita M, Nagano K, Mizuno N, Tashima Y, and Nakao T. Ouabain-sensitive Mg2+-ATPase, K+-ATPase and Na+-ATPase activities accompanying a highly specific Na+-K+-ATPase preparation. J Biochem (Tokyo) 61: 473–477, 1967.
Gaetjens EC, Chen P, and Broome JD. L1210(A) mouse lymphoma cells depleted of glutathione with L-buthionine-S-R-sulfoximine proliferate in tissue culture. Biochem Biophys Res Commun 123: 626–632, 1984.
Gurjar MV, Deleon J, Sharma RV, and Bhalla RC. Role of reactive oxygen species in IL-1-stimulated sustained ERK activation and MMP-9 induction. Am J Physiol Heart Circ Physiol 281: H2568–H2574, 2001.
Handler JS and Kwon HM. Transcriptional regulation by changes in tonicity. Kidney Int 60: 408–411, 2001.
Irarrazabal CE, Liu JC, Burg MB, and Ferraris JD. ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci USA 101: 8809–8814, 2004.
Kellogg EW III and Fridovich I. Superoxide, hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J Biol Chem 250: 8812–8817, 1975.
Klotz LO, Schroeder P, and Sies H. Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways. Free Radic Biol Med 33: 737–743, 2002.
Ko BC, Lam AK, Kapus A, Fan L, Chung SK, and Chung SS. Fyn and p38 signaling are both required for maximal hypertonic activation of the OREBP/TonEBP. J Biol Chem 277: 46085–46092, 2002.
Lee SD, Colla E, Sheen MR, Na KY, and Kwon HM. Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity. J Biol Chem 278: 47571–47577, 2003.
Li JJ, Oberley LW, Fan M, and Colburn NH. Inhibition of AP-1 and NF-B by manganese-containing superoxide dismutase in human breast cancer cells. FASEB J 12: 1713–1723, 1998.
Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, and Olson EN. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 101: 2392–2397, 2004.
Mattson DL and Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688–692, 1996.
Meyer M, Schreck R, and Baeuerle PA. H2O2 and antioxidants have opposite effects on activation of NF-B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12: 2005–2015, 1993.
Michea L, Ferguson DR, Peters EM, Andrews PM, Kirby MR, and Burg MB. Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells. Am J Physiol Renal Physiol 278: F209–F218, 2000.
Monje P, Marinissen MJ, and Gutkind JS. Phosphorylation of the carboxyl-terminal transactivation domain of c-Fos by extracellular signal-regulated kinase mediates the transcriptional activation of AP-1 and cellular transformation induced by platelet-derived growth factor. Mol Cell Biol 23: 7030–7043, 2003.
Mori T and Cowley AW Jr. Renal oxidative stress in medullary thick ascending limbs produced by elevated NaCl and glucose. Hypertension 43: 341–346, 2004.
Munoz JJ, Tarrega C, Blanco-Aparicio C, and Pulido R. Differential interaction of the tyrosine phosphatases PTP-SL, STEP and HePTP with the mitogen-activated protein kinases ERK1/2 and p38 is determined by a kinase specificity sequence and influenced by reducing agents. Biochem J 372: 193–201, 2003.
Na KY, Woo SK, Lee SD, and Kwon HM. Silencing of TonEBP/NFAT5 transcriptional activator by RNA interference. J Am Soc Nephrol 14: 283–288, 2003.
Neuhofer W, Woo SK, Na KY, Grunbein R, Park WK, Nahm O, Beck FX, and Kwon HM. Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity. Am J Physiol Cell Physiol 283: C1604–C1611, 2002.
Oh-Hashi K, Maruyama W, and Isobe K. Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells. Free Radic Biol Med 30: 213–221, 2001.
Ohtaka A, Muto S, Nemoto J, Kawakami K, Nagano K, and Asano Y. Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 270: F728–F738, 1996.
Qin S, Ding J, Takano T, and Yamamura H. Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells. Biochem Biophys Res Commun 262: 231–236, 1999.
Robinson KA, Stewart CA, Pye QN, Nguyen X, Kenney L, Salzman S, Floyd RA, and Hensley K. Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture. J Neurosci Res 55: 724–732, 1999.
Saha N, Stoll B, Lang F, and Haussinger D. Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver. Eur J Biochem 209: 437–444, 1992.
Santos BC, Pullman JM, Chevaile A, Welch WJ, and Gullans SR. Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol Renal Physiol 284: F564–F574, 2003.
Shin SJ, Lai FJ, Wen JD, Lin SR, Hsieh MC, Hsiao PJ, and Tsai JH. Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats. Kidney Int 56: 2191–2202, 1999.
Simmons NL. A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp Physiol 75: 309–319, 1990.
Tarpey MM, Wink DA, and Grisham MB. Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol 286: R431–R444, 2004.
Tian W and Cohen DM. Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol Renal Physiol 280: F904–F912, 2001.
Trama J, Lu Q, Hawley RG, and Ho SN. The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. J Immunol 165: 4884–4894, 2000.
Uhlik MT, Abell AN, Johnson NL, Sun W, Cuevas BD, Lobel-Rice KE, Horne EA, Dell'Acqua ML, and Johnson GL. Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 5: 1104–1110, 2003.
Woo SK, Lee SD, Na KY, Park WK, and Kwon HM. TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity. Mol Cell Biol 22: 5753–5760, 2002.
Wu F, Park F, Cowley AW Jr, and Mattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol Renal Physiol 276: F874–F881, 1999.
Zhang Z, Dmitrieva NI, Park JH, Levine RL, and Burg MB. High urea and NaCl carbonylate proteins in renal cells in culture and in vivo, and high urea causes 8-oxoguanine lesions in their DNA. Proc Natl Acad Sci USA 101: 9491–9496, 2004.
Zhang Z, Yang XY, and Cohen DM. Urea-associated oxidative stress and Gadd153/CHOP induction. Am J Physiol Renal Physiol 276: F786–F793, 1999.
Zhou X, Yang G, Davis CA, Doi SQ, Hirszel P, Wingo CS, and Agarwal A. Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells. Kidney Int 65: 139–147, 2004.
Zimmerman MC and Davisson RL. Redox signaling in central neural regulation of cardiovascular function. Prog Biophys Mol Biol 84: 125–149, 2004.(Xiaoming Zhou, Joan D. Fe)
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Division of Nephrology and Hypertension, University of Alabama at Birmingham, Alabama
ABSTRACT
The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/OREBP transactivation. NG-monomethyl-L-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation.
superoxides; nitric oxide; ouabain; transactivation; nuclear translocation
ELEVATION OF NaCl above the normal systemic level of 290 mosmol/kgH2O can cause cell cycle delay (31), oxidative stress (51), DNA damage (11), and apoptosis (10). Interstitial NaCl concentration normally is very high in the renal medulla and varies with urine concentration. Nevertheless, renal medullary cells evidently survive and function. Tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) (also known as NFAT5) is a transcription factor that, when activated by high NaCl and other hypertonic stresses, increases transcription of osmoprotective genes (28, 35, 49). As a result, the cells are protected by an accumulation of compatible organic osmolytes (1) and by increased expression of heat shock proteins (42, 49). The organic osmolytes in renal medullary cells include glycine betaine, which accumulates because of TonEBP/OREBP-dependent increased transcription of the betaine/-aminobutyric acid transporter, BGT1 (28, 35, 49).
High NaCl also induces oxidative stress in renal medullary cells in culture (51) and in isolated, perfused rat livers (41). Oxidative stress is associated with increased reactive oxygen species (ROS). ROS include superoxides, hydrogen peroxide and its metabolytes, and hydroxyl radicals (17). Free radicals are produced during normal mitochondrial metabolism and also by the action of nonmitochondrial oxidases (17). Superoxides, formed by one-electron reduction of oxygen, are dismutated into hydrogen peroxide, either spontaneously or catalyzed by superoxide dismutase (SOD). Catalase and peroxidase decompose hydrogen peroxide into water and oxygen.
It is pertinent that ROS transduce messages originated by hormones, cytokines, and ions to effectors including transcription factors (17). For example, high NaCl-induced tyrosine phosphorylation of Syk kinase is involved in signaling by ROS (39). Furthermore, superoxide anions and hydrogen peroxide regulate the activity of transcription factors, including AP-1 and NF-B (27, 30). The present studies were undertaken to see whether ROS also contribute to high NaCl-induced activation of the transcription factor, TonEBP/OREBP. We confirm that high NaCl increases ROS, including superoxides, and find that the ROS are necessary for full high NaCl-induced activation of TonEBP/OREBP.
MATERIALS AND METHODS
Cells, cell culture, and chemicals. Human embryonic kidney 293 (HEK293) cells, purchased from ATCC (Manassas, VA), were incubated in Eagle's minimal essential medium plus 10% fetal bovine serum in 5% CO2-95% air at 37°C. The osmolality of this control "isotonic" medium was 300 mosmol/kgH2O. MnTBAP was purchased from Calbiochem (San Diego, CA), dihydroethidium (DHE) from Molecular Probes (Eugene, OR), and all other chemicals from Sigma (St. Louis, MO). All antioxidants and probes were freshly prepared. Cells overexpressing human catalase were from the same clone used in previous studies (8).
Measurement of ROS. 2',7'-Dichlorofluorescin diacetate (DCFH-DA) permeates cell membranes. Within cells it is cleaved to produce DCFH, which is trapped because it is poorly permeating. DCFH is oxidized by free radicals, producing fluorescence. HEK293 cells were detached by trypsinization, preloaded with 20 μM DCFH-DA in phenol red-free culture medium for 30 min, then transferred either to control culture medium or hypertonic medium (osmolality raised to 500 mosmol/kgH2O by adding NaCl) for the periods indicated. The intensity of fluorescence was analyzed by flow cytometry (FACSCalibur, Becton Dickinson) with excitation at 488 nm and emission at 535 nm. A gate was set to exclude signals from debris and aggregates. Results are expressed as fold change compared with corresponding controls. Ten thousand cells were analyzed in each assay and assays were run in duplicate.
Measurement of superoxide anions. Dihydroethidium (DHE) enters cells freely. Superoxides oxidize DHE into ethidium or a structurally similar product, which intercalates into DNA, producing fluorescence (5). One to 1.2 x 106 cells were grown overnight on the optical glass bottom of a dish (MatTek, Ashland, MA) and preincubated with 30 μM DHE for 30 min. Then, the medium was replaced with a dye-free one at 300 mosmol/kgH2O or at 450 mosmol/kgH2O (NaCl added) for 45 min. The fluorophore was excited at 488 nm. Fluorescent emission at 525 nm was recorded by confocal microscopy (LSM510 Meta, Zeiss, Thornwood, NY) and analyzed with MetaMorph software.
Measurement of TonEBP/OREBP transcriptional activity. The reporter, –1233/–1105 IL2min-GL3, was constructed by inserting nucleotides –1233 to –1105 of the 5'-flanking region of the human aldose reductase (AR) gene into MluI/NheI sites upstream of the human IL-2 minimal promoter (47). The parent vector, containing an IL-2 minimal promoter upstream of the Photinus pyralis luciferase gene, was a gift from Dr. S. N. Ho (University of California, San Diego, CA). This portion of the AR gene contains potentiation elements one (tggaaaaatat) and two (AAATTTTTCCA), an osmotic response element (ORE; TGGAAAATCAC) and an AP-1 binding site (TGAGTCAC) (13). An otherwise identical reporter to which binding of TonEBP/OREBP is prevented by mutation of the potentiating and ORE elements and the adjacent AP-1 site was used to control for specificity to TonEBP/OREBP. In the mutated version, the nucleotide sequences are changed to tgtctaaatat, AAATTTAGACA, TGTCTAATCAC, and TGCTGCAC, respectively. Mutations were performed using a QuikChange site mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol. Accuracy of the mutations was confirmed by sequencing.
The reporter, ORE-X IL2min-GL3, was constructed by inserting two tandem copies of the human ORE-X sequence (15) in the XhoI site of the human IL-2 minimal promoter (47). ORE and ORE-mutated constructs were modified to express the blasticidin resistance gene by insertion of nucleotides 1934 to 3204 of pCMV/Bsd (Invitrogen, Carlsbad, CA) into XmnI/BsaI sites. Stable reporter cell lines were established by transfection with Effectene (Qiagen, Valencia, CA), blasticidin selection, and screening for luciferase activity to select clones with the highest expression; 3 x 104 cells per well were seeded in 96-well white view plates (Packard, Wellesley, MA) and incubated for 24 h at 300 mosmol/kgH2O, then the medium was changed for 16 h to one still at 300 mosmol/kgH2O or elevated to 500 mosmol/kgH2O by adding NaCl. Luciferase activity was measured with Bright-Glo substrate (Promega, Madison, WI) in a Victor3 luminometer (Perkin Elmer, Wellesley, MA). Cell protein was determined with the BCA reagent (Pierce, Rockford, IL).
Measurement of BGT1 mRNA expression. This was performed as previously described (16); 2.5 x 105 cells/well were plated in a six-well dish, then incubated at the indicated osmolality for 16 h. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Amplicons were detected with an ABI Prism 7900HT detection system (Applied Biosystems). Primers for the human BGT1 mRNA were 5'-TGTTCAGCTCCTTCACCTCTGA-3' and 5'-GCAATGCTCTGTGTTCCAAAAG-3'. The 6-carboxyfluorescein (FAM)-labeled probe was 5'-CTGCCCTGGACGACCTGCAACAA-3'. As a control for reverse transcription efficiency and loading, 18S RNA was measured at the same time, using primers and a probe from Applied Biosystems. Fold differences in RNA abundance between conditions (F) was calculated, as F=E(Ct1–Ct2). E (= 1.98 ± 0.056, n = 18, means ± SE) is the efficiency of the reaction determined from the results of reactions containing 8 or 80 ng of cDNA template. Ct1 and Ct2 are the numbers of cycles required to reach the threshold of amplicon abundance in respective experimental conditions (16).
Measurements of TonEBP/OREBP transactivation. This was measured using a yeast binary GAL4 reporter assay system, as previously described (16). Briefly, the assay system comprises cotransfection of an expression vector containing the TonEBP/OREBP transactivation domain (TAD), GAL4dbd-TAD, and a reporter plasmid (GAL4UAS-GL3). GAL4dbd-TAD contains in-frame insertion of cDNA coding for amino acids 548–1531 of TonEBP/OREBP in a vector containing the neomycin resistance gene (pFA-CMV, Stratagene). An otherwise identical construct, in which the GAL4dbd construct did not contain a TAD, was used as a control for nonspecific effects. GAL4UAS-GL3 contains five tandem repeats of the yeast GAL4 binding site (upstream activating sequence) and a minimal promoter (TATATA) derived from pFR-Luc (Stratagene) and inserted into the NheI/HindIII sites of pGL3 (Promega) upstream of the P. pyralis luciferase gene. GAL4UAS-GL3 was further modified for blasticidin resistance as described above. Stable reporter cell lines were established by blasticidin and neomycin selection and used as described above for assays of TonEBP/OREBP transcriptional activity.
Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed by paired t-test or repeated ANOVA, as appropriate. Post hoc comparison was made by Dunnett's test. P < 0.05 is considered significant.
RESULTS
High NaCl increases ROS, including superoxides. Hypertonicity was previously found to activate TonEBP in HEK293 cells, which are renal epithelial cells of human origin (44). To examine whether hypertonicity increases ROS in HEK293 cells, we raised the osmolality from 300 to 500 mosmol/kgH2O by adding NaCl and measured ROS by flow cytometry, using DCFH-DA as a probe. High NaCl raises the fluorescence intensity of DCFH within 30 min, indicating increased ROS (Fig. 1A). To examine whether high NaCl increases formation of superoxides, we measured superoxide activity by confocal microscopy, using DHE as a probe. High NaCl increases DHE fluorescence by 1.37-fold (Fig. 1B), indicating that high NaCl induces generation of superoxides. The effect of high NaCl on superoxide activity is reduced by N-acetylcysteine (NAC; 5 mM), a general antioxidant (Fig. 1B). Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; 40 μM), a SOD mimetic (3), and catalase (400 U/ml) do not significantly lower high NaCl-induced superoxide generation. MnTBAP increases superoxide activity in cells kept at 300 mosmol/kgH2O (Fig. 1B).
Antioxidants attenuate high NaCl-induced TonEBP/OREBP transcriptional activity. We measured transcriptional activity of TonEBP/OREBP in HEK293 cells stably expressing an ORE luciferase reporter. High NaCl increases ORE reporter activity 96- to 106-fold (Fig. 2A). NAC and MnTBAP, but not exogenously added catalase, significantly reduce high NaCl-induced ORE reporter activity (38 and 29%, respectively; Fig. 2A). To examine whether this effect is specifically mediated by binding of TonEBP/OREBP to OREs, we used an otherwise identical reporter in which the ORE sites and the adjacent AP-1 site are mutated. This prevents both the binding of TonEBP/OREBP and high NaCl-induced increase of reporter activity (Fig. 2B). NAC and MnTBAP have no significant effect on activity of the mutated ORE reporter (Fig. 2B). We conclude that the full high NaCl-induced increase in TonEBP/OREBP transcriptional activity requires increased ROS, mainly superoxides.
The reporter that we used includes the AP-1 site adjacent to the OREs in the AR gene. Mutation of the AP-1 site reduces high NaCl-induced reporter activity (13). AP-1 is regulated by ROS (27, 30). To test whether antioxidants reduce ORE reporter activity by inhibiting AP-1 activity, we examined the effect of NAC on a different reporter, ORE-X, that does not include that AP-1 site. NAC reduces high NaCl-induced ORE-X activity by 41% (from 308 ± 124-fold increase, comparing 500 with 300 mosmol/kgH2O, to 181 ± 68-fold increase, P < 0.05, n = 5), even in the absence of the AP-1 site, excluding the possibility that the effect of NAC is directly mediated by AP-1.
BGT1 is a transcriptional target of TonEBP/OREBP (21). Activation of TonEBP/OREBP by high NaCl elevates BGT1 mRNA abundance (21). To further examine the effects of antioxidants on TonEBP/OREBP activity, we measured the effect of NAC and MnTBAP on the high NaCl-induced increase in BGT1 mRNA expression, using real-time RT-PCR. NAC and MnTBAP, but not catalase, inhibit high NaCl-induced BGT1 mRNA expression by 30 and 20%, respectively (Fig. 3A).
Exogenously added catalase does not affect ORE reporter activity (Fig. 2A) or BGT1 mRNA expression (Fig. 3A). Because it seemed possible that the exogenous catalase might not effectively reduce intracellular H2O2, we examined the effect of high NaCl on BGT1 mRNA expression in a clone of HEK293 cells that overexpresses human catalase (8). Overexpression of catalase does not reduce tonicity-dependent expression of BGT1 mRNA (Fig. 3B). The effectiveness of the overexpressed catalase in these cells is attested by resistance of the cells to H2O2-mediated cell death induced by FK506 (53).
Reduction of ROS suppresses high NaCl-induced TonEBP/OREBP transactivation. TonEBP/OREBP contains a TAD, whose activity is increased by high NaCl (14, 16, 26). We used HEK293 cells stably transfected with a binary GAL4 transactivation reporter system to test whether the ROS-induced increase of TonEBP/OREBP transcriptional activity is mediated by increased transactivation. Raising osmolality from 300 to 500 mosmol/kgH2O by adding NaCl increases transactivation about 10-fold (Fig. 4A). NAC and MnTBAP, but not catalase, reduce high NaCl-induced transactivation by 20 and 40%, respectively (Fig. 4A). We used an otherwise identical reporter that does not contain the TAD of TonEBP/OREBP to exclude a general effect on transactivation. NAC and MnTBAP have no significant effect on the control reporter activity (Fig. 4B). We tentatively conclude that ROS contribute to the activation of TonEBP/OREBP by transactivating it.
Effects of high NaCl on TonEBP/OREBP activity do not involve nitric oxide. Renal medullary cells can generate nitric oxide (29, 50). Water deprivation increases nitric oxide synthase mRNA expression in the renal medulla (43). Superoxides spontaneously react with nitric oxide to form peroxynitrite, which can induce gene expression in response to stress (24). For example, in human neuroblastoma cells, peroxynitrite induces DNA damage-inducible (Gadd) proteins 34, 45, 153 mRNA expression (37). NG-monomethyl-L-arginine (L-NMMA) is a general inhibitor of nitric oxide synthases (4). To test whether nitric oxide is involved in high NaCl-induced activation of TonEPB/OREBP, we examined the effect of 100 μM L-NMMA. L-NMMA has no significant effect on high NaCl-dependent increase in superoxides (Fig. 5A), TonEBP/OREBP transcriptional activity (Fig. 5B), BGT1 mRNA (Fig. 5C), or TonEBP/OREBP transactivation (Fig. 5D). We conclude that high NaCl-induced activation of TonEBP/OREBP does not involve nitric oxide.
Increase of ROS in the absence of high NaCl is not sufficient to activate TonEBP/OREBP. We elevated ROS at 300 mosmol/kgH2O by adding 1) xanthine oxidase (6 mU/ml) plus hypoxanthine (500 μM) to directly elevate ROS (23), 2) L-buthionine-[S,R]-sulfoximine (BSO; 100 and 400 μM) to deplete the reduced form of glutathione (19), or 3) diethyldithiocarbamate (100 μM and 1 mM) to elevate superoxide by inhibiting SOD (9). Although xanthine oxidase plus hypoxanthine increases superoxide (data not shown), ORE-X activity is unaffected at either 300 or 500 mosmol/kgH2O (Fig. 6A). BSO (Fig. 6B) and diethyldithiocarbamate (data not shown) also do not affect ORE-X activity at either osmolality. We conclude that increase in ROS in the absence of other high NaCl-induced signals is not sufficient to activate TonEBP/OREBP.
Ouabain inhibits both high NaCl-induced superoxide activity and activation of TonEBP/OREBP. Ouabain is an inhibitor of Na-K-ATPase (18). In Madin-Darby canine kidney cells, ouabain reduces high NaCl-dependent increase of AR, SMIT and BGT1 mRNA abundance and of TonEBP/OREBP protein abundance (36). Those effects were attributed to cell swelling caused by ouabain and were proposed as evidence that TonEBP/OREBP activity is regulated by cell volume, as well as by intracellular ionic strength (36). However, ouabain also reduces the high NaCl-induced increase in superoxide in rat medullary thick ascending limbs (33). We reasoned that if ouabain should also reduce high NaCl-induced superoxide formation in renal epithelial cells, that could explain how ouabain reduces TonEBP/OREBP activity without invoking changes in cell volume. Therefore, we tested the effect of a low concentration of ouabain (12 nM) on high NaCl-induced superoxide level and TonEBP/OREBP activity in HEK293 cells. Ouabain decreases superoxide (Fig. 7A), TonEBP/OREBP transcriptional activity (Fig. 7B), and TonEBP/OREBP transactivation (Fig. 7D). As controls for specificity, the effect on transcription does not occur in the absence of functional TonEBP/OREBP binding elements (Fig. 7C) nor on transactivation in the absence of the TonEBP/OREBP TAD (Fig. 7E). We conclude that ouabain-induced decrease in superoxide contributes to reduction by ouabain of the high NaCl-induced increase in TonEBP/OREBP activity.
DISCUSSION
Measurements of oxidized glutathione (41) and the effects of antioxidants (39, 52) originally indicated that osmotic stress causes oxidative stress. Subsequently, it was found that high NaCl increases ROS production and carbonylate proteins (indicative of oxidative damage to the proteins) in mIMCD3 cells and that protein carbonylation is abundant in normal renal medulla, associated with the normally high interstitial NaCl concentration in that tissue (51). In the present studies of HEK293 cells, we confirm by two independent methods, namely flow cytometry with DCFH as a probe (Fig. 1A) and confocal microscopy with DHE as an indicator (Fig. 1B), that high NaCl elevates ROS and superoxides. DCFH is oxidized to form DCF, which fluoresces. This was initially thought to require hydrogen peroxide (45). However, subsequent studies showed that DCFH can also be oxidized by enzymatic reactions and by other oxidants, including peroxynitrite and hypochlorous acid (45). DHE is oxidized by superoxides, but not by hydrogen peroxide, peroxynitrite, or hypochlorous acid (5), providing a specific probe for measuring superoxide activity (45).
ROS can serve as signaling molecules (17). In most cases, superoxides are the initial ROS produced. Examples include that, in vascular smooth muscle cells, IL-1 stimulates superoxide production, ERK activation, and matrix metalloproteinase-9 gene expression. Overexpression of SOD or addition of NAC inhibits all these effects (20). Similarly, ANG II activates NADPH oxidase, resulting in generation of ROS that, in turn, activate multiple transcription factors through the Raf-1-MAPK pathway, leading to increased vascular tone (54).
We used the antioxidants, NAC and MnTBAP, to study the role of ROS in high NaCl-induced activation of TonEBP/OREBP. These antioxidants reduce high NaCl-induced increase of ROS (Fig. 1B), TonEBP/OREBP transcriptional activity (Fig. 2, A and C), and the mRNA expression of TonEBP/OREBP's transciptional target, BGT1 (Fig. 3A). NAC, a membrane-permeating form of cysteine, is a precursor of glutathione. It serves as a general antioxidant by raising cellular reduced glutathione content, which alters redox balance, inhibiting formation of ROS (7). MnTBAP acts like SOD, dismutating superoxide to H2O2 (3).
Using these antioxidants, we found that ROS contribute to high NaCl-induced increase of TonEBP/OREBP transactivation (Fig. 4). At this point, we can only speculate on the mechanism. High NaCl increases phosphorylation of the TonEBP/OREBP TAD (14). Activation of a number of kinases, including PKAc, p38, and Fyn, is required for full high NaCl-induced increase in TonEBP/OREBP transactivation (14, 25). ROS can activate p38 (40). Thus ROS could transactivate TonEBP/OREBP through increased p38 kinase activity. An example of ROS affecting transactivation via phosphorylation by a MAP kinase is provided by the effect of PDGF. Activation of PDGF increases ROS, which contributes to activity of the transcription factor, AP-1 (32), mediated by ERK which increases phosphorylation of a TAD located at the COOH-terminal of c-Fos, a partner in AP-1 heterodimers (32). Phosphorylation of p38 by MEKK3 increases its activity (48) and dephosphorylation by protein tyrosine phosphatases (PTPs) can decrease it (34). Thiol groups of active site cysteines in PTPs are vulnerable to oxidation (17). Free radicals inhibit PTPs through either direct oxidation of the thiol groups or glutathionylation due to increased oxidized glutathione level (17). Hypertonicity inhibits the activity of PTPs, which can be prevented by NAC (40). Thus ROS may contribute to the activation of p38, and thereby of TonEBP/OREBP, by inhibiting PTPs.
We did not find any effect on TonEBP/OREBP transcriptional activity at 300 mosmol/kgH2O of exogenous agents that elevate ROS nor did the agents further increase the effect of high NaCl (Fig. 6). Thus, although increased ROS is necessary for full high NaCl-induced activation of TonEBP/OREBP, isolated elevation of ROS does not activate TonEBP/OREBP per se. This result is consistent with the previous conclusion that multiple signaling inputs are necessary for tonicity-induced activation of TonEBP/OREBP but that no one of the signals is sufficient by itself (22). A cogent previous example is provided by the DNA damage-inducible kinase, ATM (22). High NaCl damages DNA (11). Full high NaCl-dependent activation of TonEBP/OREBP requires the activation ATM (22). However, other agents such as high urea and radiation also activate ATM, but they do not increase TonEBP/OREBP transcriptional activity like high NaCl does (22) and high urea actually reduces tonicity-dependent activation of TonEBP/OREBP (46).
Hypertonicity shrinks cells by osmosis, resulting in increased concentration of all intracellular components, including intracellular ions (36) and macromolecules (6). Decrease in cell volume (12), increase in intracellular ionic strength (36), and increased macromolecular crowding (6) are all candidates to be sensors that trigger cellular responses to hypertonicity. Their relative importance remains controversial. Under conditions in which changes in cell volume are similar, TonEBP/OREBP activity correlates with the intracellular ionic strength regardless of the external tonicity (36). On the other hand, inhibition of Na-K-ATPase by ouabain leads to decreased activity of TonEBP/OREBP despite a marked increase in the intracellular ionic strength. It was suggested that, because isotonic swelling is known to occur under these conditions, dilution of the cytoplasmic constituents inhibits the activity of TonEBP/OREBP (36). However, the present study affords an alternative explanation as the effect of ouabain on TonEBP/OREBP is associated with suppression of ROS formation (Fig. 7). Given that ROS contribute to the activation of TonEBP/ OREBP, it is plausible that it is inhibition of ROS generation by ouabain that attenuates the activation of TonEBP/OREBP, rather than increased cellular water content.
It is interesting to speculate on how ouabain reduces ROS in cells stressed by high NaCl (Fig. 7). High NaCl increases Na-K-ATPase activity (38), presumably to energize increased Na and K transport. Production of ATP must increase to compensate for its greater consumption, which can entail greater mitochondrial oxidative metabolism. Ouabain reduces oxygen consumption by reducing Na and K transport and the associated consumption of ATP (2). Oxidative metabolism by mitochondria is associated with production of ROS (2). Putting all of this together, we propose that ouabain reduces high NaCl-induced increase in ROS by decreasing utilization of ATP by the Na-K-ATPase, thus reducing oxidative metabolism and the accompanying production of ROS. A similar process may be occuring in isolated rat medullary thick ascending limbs, where the Na transport inhibitors, dimethylamiloride and ouabain, blunt the effect of high NaCl-induced superoxide activity (33).
GRANTS
This study was supported in part by a Grant-in-Aid from National Kidney Foundation/National Capital Area (to X. Zhou).
ACKNOWLEDGMENTS
The authors thank Dr. J. Handler for thoughtful discussions during the course of the study, Dr. C. Combs for expert help with confocal microscopy, and A. Williams for assistance with flow cytometry.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Bagnasco S, Balaban R, Fales HM, Yang YM, and Burg M. Predominant osmotically active organic solutes in rat and rabbit renal medullas. J Biol Chem 261: 5872–5877, 1986.
Balaban RS and Mandel LJ. Coupling of aerobic metabolism to active ion transport in the kidney. J Physiol 304: 331–348, 1980.
Batinic-Haberle I, Benov L, Spasojevic I, and Fridovich I. The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic. J Biol Chem 273: 24521–24528, 1998.
Belenky SN, Robbins RA, and Rubinstein I. Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro. J Leukoc Biol 53: 498–503, 1993.
Benov L, Sztejnberg L, and Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25: 826–831, 1998.
Burg MB. Macromolecular crowding as a cell volume sensor. Cell Physiol Biochem 10: 251–256, 2000.
Cotgreave IA. N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv Pharmacol 38: 205–227, 1997.
Davis CA, Nick HS, and Agarwal A. Manganese superoxide dismutase attenuates cisplatin-induced renal injury: importance of superoxide. J Am Soc Nephrol 12: 2683–2690, 2001.
Didion SP, Ryan MJ, Baumbach GL, Sigmund CD, and Faraci FM. Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen. Am J Physiol Heart Circ Physiol 283: H1569–H1576, 2002.
Dmitrieva N, Kultz D, Michea L, Ferraris J, and Burg M. Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. J Biol Chem 275: 18243–18247, 2000.
Dmitrieva NI, Cai Q, and Burg MB. Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo. Proc Natl Acad Sci USA 101: 2317–2322, 2004.
Ericson AC and Spring KR. Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl–-HCO3– exchange. Am J Physiol Cell Physiol 243: C146–C150, 1982.
Ferraris J and Garcia-Perez A. Osmotically responsive genes: the mammalian osmotic response element (ORE). Am Zool 41: 734–742, 2001.
Ferraris JD, Persaud P, Williams CK, Chen Y, and Burg MB. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proc Natl Acad Sci USA 99: 16800–16805, 2002.
Ferraris JD, Williams CK, Ohtaka A, and Garcia-Perez A. Functional consensus for mammalian osmotic response elements. Am J Physiol Cell Physiol 276: C667–C673, 1999.
Ferraris JD, Williams CK, Persaud P, Zhang Z, Chen Y, and Burg MB. Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration. Proc Natl Acad Sci USA 99: 739–744, 2002.
Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol 15: 247–254, 2003.
Fujita M, Nagano K, Mizuno N, Tashima Y, and Nakao T. Ouabain-sensitive Mg2+-ATPase, K+-ATPase and Na+-ATPase activities accompanying a highly specific Na+-K+-ATPase preparation. J Biochem (Tokyo) 61: 473–477, 1967.
Gaetjens EC, Chen P, and Broome JD. L1210(A) mouse lymphoma cells depleted of glutathione with L-buthionine-S-R-sulfoximine proliferate in tissue culture. Biochem Biophys Res Commun 123: 626–632, 1984.
Gurjar MV, Deleon J, Sharma RV, and Bhalla RC. Role of reactive oxygen species in IL-1-stimulated sustained ERK activation and MMP-9 induction. Am J Physiol Heart Circ Physiol 281: H2568–H2574, 2001.
Handler JS and Kwon HM. Transcriptional regulation by changes in tonicity. Kidney Int 60: 408–411, 2001.
Irarrazabal CE, Liu JC, Burg MB, and Ferraris JD. ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci USA 101: 8809–8814, 2004.
Kellogg EW III and Fridovich I. Superoxide, hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J Biol Chem 250: 8812–8817, 1975.
Klotz LO, Schroeder P, and Sies H. Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways. Free Radic Biol Med 33: 737–743, 2002.
Ko BC, Lam AK, Kapus A, Fan L, Chung SK, and Chung SS. Fyn and p38 signaling are both required for maximal hypertonic activation of the OREBP/TonEBP. J Biol Chem 277: 46085–46092, 2002.
Lee SD, Colla E, Sheen MR, Na KY, and Kwon HM. Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity. J Biol Chem 278: 47571–47577, 2003.
Li JJ, Oberley LW, Fan M, and Colburn NH. Inhibition of AP-1 and NF-B by manganese-containing superoxide dismutase in human breast cancer cells. FASEB J 12: 1713–1723, 1998.
Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, and Olson EN. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 101: 2392–2397, 2004.
Mattson DL and Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688–692, 1996.
Meyer M, Schreck R, and Baeuerle PA. H2O2 and antioxidants have opposite effects on activation of NF-B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12: 2005–2015, 1993.
Michea L, Ferguson DR, Peters EM, Andrews PM, Kirby MR, and Burg MB. Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells. Am J Physiol Renal Physiol 278: F209–F218, 2000.
Monje P, Marinissen MJ, and Gutkind JS. Phosphorylation of the carboxyl-terminal transactivation domain of c-Fos by extracellular signal-regulated kinase mediates the transcriptional activation of AP-1 and cellular transformation induced by platelet-derived growth factor. Mol Cell Biol 23: 7030–7043, 2003.
Mori T and Cowley AW Jr. Renal oxidative stress in medullary thick ascending limbs produced by elevated NaCl and glucose. Hypertension 43: 341–346, 2004.
Munoz JJ, Tarrega C, Blanco-Aparicio C, and Pulido R. Differential interaction of the tyrosine phosphatases PTP-SL, STEP and HePTP with the mitogen-activated protein kinases ERK1/2 and p38 is determined by a kinase specificity sequence and influenced by reducing agents. Biochem J 372: 193–201, 2003.
Na KY, Woo SK, Lee SD, and Kwon HM. Silencing of TonEBP/NFAT5 transcriptional activator by RNA interference. J Am Soc Nephrol 14: 283–288, 2003.
Neuhofer W, Woo SK, Na KY, Grunbein R, Park WK, Nahm O, Beck FX, and Kwon HM. Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity. Am J Physiol Cell Physiol 283: C1604–C1611, 2002.
Oh-Hashi K, Maruyama W, and Isobe K. Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells. Free Radic Biol Med 30: 213–221, 2001.
Ohtaka A, Muto S, Nemoto J, Kawakami K, Nagano K, and Asano Y. Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 270: F728–F738, 1996.
Qin S, Ding J, Takano T, and Yamamura H. Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells. Biochem Biophys Res Commun 262: 231–236, 1999.
Robinson KA, Stewart CA, Pye QN, Nguyen X, Kenney L, Salzman S, Floyd RA, and Hensley K. Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture. J Neurosci Res 55: 724–732, 1999.
Saha N, Stoll B, Lang F, and Haussinger D. Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver. Eur J Biochem 209: 437–444, 1992.
Santos BC, Pullman JM, Chevaile A, Welch WJ, and Gullans SR. Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol Renal Physiol 284: F564–F574, 2003.
Shin SJ, Lai FJ, Wen JD, Lin SR, Hsieh MC, Hsiao PJ, and Tsai JH. Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats. Kidney Int 56: 2191–2202, 1999.
Simmons NL. A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp Physiol 75: 309–319, 1990.
Tarpey MM, Wink DA, and Grisham MB. Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol 286: R431–R444, 2004.
Tian W and Cohen DM. Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol Renal Physiol 280: F904–F912, 2001.
Trama J, Lu Q, Hawley RG, and Ho SN. The NFAT-related protein NFATL1 (TonEBP/NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. J Immunol 165: 4884–4894, 2000.
Uhlik MT, Abell AN, Johnson NL, Sun W, Cuevas BD, Lobel-Rice KE, Horne EA, Dell'Acqua ML, and Johnson GL. Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 5: 1104–1110, 2003.
Woo SK, Lee SD, Na KY, Park WK, and Kwon HM. TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity. Mol Cell Biol 22: 5753–5760, 2002.
Wu F, Park F, Cowley AW Jr, and Mattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol Renal Physiol 276: F874–F881, 1999.
Zhang Z, Dmitrieva NI, Park JH, Levine RL, and Burg MB. High urea and NaCl carbonylate proteins in renal cells in culture and in vivo, and high urea causes 8-oxoguanine lesions in their DNA. Proc Natl Acad Sci USA 101: 9491–9496, 2004.
Zhang Z, Yang XY, and Cohen DM. Urea-associated oxidative stress and Gadd153/CHOP induction. Am J Physiol Renal Physiol 276: F786–F793, 1999.
Zhou X, Yang G, Davis CA, Doi SQ, Hirszel P, Wingo CS, and Agarwal A. Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells. Kidney Int 65: 139–147, 2004.
Zimmerman MC and Davisson RL. Redox signaling in central neural regulation of cardiovascular function. Prog Biophys Mol Biol 84: 125–149, 2004.(Xiaoming Zhou, Joan D. Fe)