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Originally published In Press as doi:10.1074/jbc.M502430200 on July 28, 2005 Originally published In Press as doi:10.1074/jbc.M502430200 on July 22, 2005 Originally published In Press as doi:10.1074/jbc.M502430200 on July 17, 2005

J. Biol. Chem., Vol. 280, Issue 41, 34966-34973, October 14, 2005
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Hypertonic Induction of COX-2 in Collecting Duct Cells by Reactive Oxygen Species of Mitochondrial Origin*

Tianxin Yang{ddagger}1, Aihua Zhang{ddagger}, Matthew Honeggar{ddagger}, Donald E. Kohan{ddagger}, Diane Mizel§, Karl Sanders{ddagger}, John R. Hoidal{ddagger}, Josephine P. Briggs§, and Jurgen B. Schnermann§

From the {ddagger}Department of Internal Medicine, University of Utah and Veterans Affairs Medical Center, Salt Lake City, Utah 84148 and §NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, March 4, 2005 , and in revised form, June 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells (Yang, T., Huang, Y., Heasley, L. E., Berl, T., Schnermann, J. B., and Briggs, J. P. (2000) J. Biol. Chem. 275, 23281-23286). The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E2 release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 µM). The general antioxidant N-acetyl-L-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mM) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E2 release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-L-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91phox or p47phox, nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, the renal medulla is one of the few tissues that is constantly exposed to hypertonicity. Furthermore, urinary osmolality in rodents increases to levels >3000 mosmol/kg following water deprivation. Survival of renal medullary cells in this hypertonic environment depends on the accumulation of compatible organic osmolytes (1, 2). This is accomplished by activation of a cluster of osmolyte-related genes, including aldose reductase and the transporters for betaine, taurine, and myo-inositol. Most of these genes are under the control of the tonicity-responsive enhancer-binding protein transcription factor TonEBP/NFAT5, which interacts with the tonicity-responsive enhancer/osmotic response element (TonE/ORE) (3, 4).

Cyclooxygenase (COX),2 also called prostaglandin H synthase, is the rate-limiting enzyme catalyzing the metabolism of arachidonic acid to prostaglandins. COX exists in two major isoforms, the inducible form, COX-2 and the constitutive form, COX-1 (5-8). COX-1 is expressed in a wide variety of tissues, and its expression level does not appear to change significantly; it is implicated in the regulation of housekeeping functions such as platelet aggregation and cytoprotective function in the gastrointestinal tract. In contrast, COX-2 is much more restricted in its expression to certain cell types, and its expression undergoes robust changes in response to growth factors and inflammatory stimuli. In addition to its role as an inducible generator of prostaglandin H in the inflammatory response, evidence is emerging to suggest that COX-2 also plays an important role in the regulation of physiological processes, including those in the kidney. Within the kidney, COX-2 is abundantly expressed in the inner medulla and is further induced by water restriction (9, 10) and chronic salt loading (11). In cultured renal medullary epithelial and interstitial cells (10, 12), as well as in liver macrophages (13), hypertonicity exerts a direct stimulatory effect on COX-2 expression. COX-2 activity is required for osmolyte accumulation and adaptation to hypertonic stress (14).

Previous studies from this laboratory have identified multiple members of the mitogen-activated protein kinase (MAPK) family, namely ERK1/2, p38, and c-Jun N-terminal kinase (JNK), as factors mediating tonicity-induced induction of COX-2 in cultured renal medullary epithelial cells (12). In these experiments, COX-2 induction by hypertonicity was partially inhibited by a blockade of each of these MAPKs and was completely blocked by simultaneous blockade of ERK1/2 and p38. The hypertonic induction of COX-2 expression in IMCD3 cells requires transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase (15). In cultured renal medullary interstitial cells, the COX-2 induction is dependent on NF{kappa}B (10).

The present study was performed to further define the transduction pathway responsible for osmotic regulation of COX-2 expression. Reactive oxygen species (ROSs) which are highly reactive by carrying one or more unpaired electrons in the outer orbits, are typically generated as a byproduct of oxygen metabolism. Large amounts of ROSs produced in phagocytes serve to kill invading microorganisms, whereas small amounts of ROSs produced in non-phagocytic tissues participate in signaling pathways. Increased formation of ROSs occurs when cells receive stress signaling. Because some of the best known targets of ROSs include MAPK as well as NF{kappa}B (16), we examined whether ROSs participate in the mediation of the hypertonic induction of COX-2 expression via MAPK in cultured renal medullary epithelial cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—The gp91phox null mutant mice were a gift from Mary C. Dinauer (University of Indiana) (17), and the p47phox null mutant mice were provided by Steve Holland (National Institutes of Health) (18).

Materials—Cell culture media and serum were from Invitrogen. PD-98059 and SB-203580 were from New England Biolabs and Upstate%20Biotechnology">Upstate Biotechnology, respectively. Cytochrome c, SOD, genistein, TEMPO, xanthine, xanthine oxidase, rotenone (ROT), carbonyl cyanide m-chlorophenylhydrazone (CCCP), thenoyltrifluoroacetone (TTFA), and indomethacin were from Sigma. The murine COX-2 polyclonal antibody and the PGE2 enzyme immunoassay kit were from Cayman Chemical. Polyclonal antibodies against phosphorylated p44/42 and phosphorylated p38 were from Cell Signaling Technology.

Cell Culture—mIMCD-K2 is an established mouse inner medullary collecting duct cell line provided by Dr. Bruce Stanton (14). The cells were routinely propagated in an Opti medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Primary cultures of kidney inner medullary cells were generated with modifications of previously described protocols (19). In brief, mice were anesthetized by ketamine/xylazine, and kidneys were quickly removed under sterile conditions. The renal inner medulla was dissected, minced, and digested for 45 min in 10 ml of medium (Dulbecco's modified Eagle's medium/Ham's F12, 1:1) (v/v) containing 0.2% collagenase and 0.2% hyaluronidase (w/v) at 37 °C with a stirrer. After incubation, 20 ml of distilled water was added to lyse cells other than collecting duct cells by osmotic shock (100 mosmol/kg). Cells were then centrifuged at 150 x g for 10 min, the supernatant was discarded, and the pellet was resuspended in the modified DM medium (DMEM/Ham's F12, 1:1 (v/v), 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-L-thyronine, 1 nM sodium selenate, 10 ng/ml epidermal growth factor, 5 mg/liter transferrin, 2 mM L-glutamine, 100 units/ml penicillin G, 100 units/ml streptomycin sulfate, and 10% fetal bovine serum (v/v). Cells were kept in a 6-well plate for at least 10 days until confluence. At least 48 h before experiments, the medium of the incubating cells was replaced with a minimum medium that contained no drugs or hormones.



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  		FIGURE 1.
Detection of hypertonicity-stimulated induction of ROSs by DCF fluorescence. Top, ROS production in response to osmotic stress; confluent mIMCD-K2 cells in chamber slides were exposed to isotonic or hypertonic (600 mosmol/kg by adding NaCl) medium in the presence of DCF. A, isotonic medium exposure for 2 min; B, isotonic medium exposure for 5 min; C, hypertonic exposure for 2 min; D, hypertonic exposure for 5 min. Middle, time course of ROS induction following the hypertonic treatment. Confluent mIMCD-K2 cells in 24-well plates were exposed to isotonic (300 mosmol/kg) or hypertonic (600 mosmol/kg by adding NaCl) medium in the presence of DCF. At the indicated time points, fluorescence was quantified using FLUOstar OPTIMA. #, p < 0.01; *, p < 0.05; compared with the corresponding isotonic group. Bottom, effects of genistein. Cells were treated with isotonic or hypertonic (600 mosmol/kg by adding NaCl) medium in presence or absence of 50 µM genistein. Results are expressed as fold increases over untreated cells.

 
2',7'-Dichlorodihydrofluorescence Diacetate (DCFDA) Fluorescence Measurement of ROSs—The fluorogenic substrate 2',7'-dichlorofluorescein diacetate (DCFH-DA) is a cell-permeable dye that is oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by H2O2 and can therefore be used to monitor intracellular generation of ROSs. For measurement of ROSs, cells were grown onto glass cover slides. When the cells reached confluence, they were washed twice with phosphate-buffered saline and incubated for 30 min with 50 µM 2',7'-dichlorofluorescein diacetate diluted in Opti-minimum essential medium with 10% fetal calf serum. Then hypertonic medium was added. At the end of the incubation period, the cells were again washed twice with phosphate-buffered saline and imaged by confocal laser microscopy. To quantitate ROS levels, cells were seeded to 96-well plate and treated as described above. Relative fluorescence was measured by a fluorescence plate reader (FLUOstar OPTIMA) at excitation and emission wavelengths of 485 and 528 nm, respectively, three times at 90-s intervals. Relative fluorescence units were expressed as fold increase over untreated cells.

Cytochrome c Reduction-based Superoxide Assay—The extracellular superoxide production by hypertonicity-treated cells seeded in 24-well plates was determined from the SOD-inhibitable reduction of cytochrome c (20). Confluent cells were treated with isotonic or hypertonic medium (550 mosmol/kg by adding NaCl) in the presence or absence of 50 µM genistein. 160 µM cytochrome c was added to all wells, and 100 units/ml SOD was added to a second well for each sample. Absorbance was measured in a plate reader at 550 nm. Superoxide production was calculated from the difference in the absorbance in the absence and presence of SOD.

Western Blotting for COX-2—mIMCD-K2 cells were lysed and subsequently sonicated in phosphate-buffered saline containing 1% TX-100, 250 µM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 5 mM dithiothreitol (pH 7.5). Protein concentration was determined by Coomassie reagent. 40 µg of protein from whole cell lysates was denatured in boiling water for 10 min, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline followed by incubation for 1 h with rabbit anti-murine polyclonal antiserum to COX-2 at a dilution of 1:1000. After washing with Tris-buffered saline, blots were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody and visualized with ECL kits (Amersham Biosciences).



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  		FIGURE 2.
Detection of hypertonicity induction of ROSs by the cytochrome c method. Confluent mIMCD-K2 cells in 24-well plates were treated with isotonic or hypertonic medium (550 mosmol/kg by adding NaCl) in the presence or absence of 50 µM genistein. 160 µM cytochrome c was added to all wells, and 100 units/ml SOD was added to a second well for each sample. Absorbance was measured in a plate reader at 550 nm. Shown are SOD-inhibitable absorbencies.

 



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  		FIGURE 3.
The contribution of mitochondria or NADPH oxidase to the hypertonicity-induced generation of ROSs. A, effects of the mitochondrial inhibitors ROT, CCCP, and TTFA on ROS production in mIMCD-K2 cells. Confluent mIMCD-K2 cells in 96-well plates were treated for 30 min with isotonic or hypertonic medium (550 mosmol/kg by adding NaCl) in the presence or absence of 5 µM ROT, 10 µM TTFA, or 0.5 µM CCCP and then subjected to the ROS assay. #, p < 0.01 compared with the isotonic group. B, ROS generation in response to osmotic stress in IMCD cells derived from gp91phox-/- and p47phox-/- mice and from wild type control mice. *, p < 0.01 compared with the corresponding isotonic group; {symbol}, p > 0.05 compared with the hypertonicity-treated wild type group.

 
Phosphorylation of MAP Kinases—mIMCD-K2 cells grown in a 6-well plate were lysed by sonication for 10 s in 300 µl of 1x Laemmli sample buffer containing 10 mM Tris, 1.4% SDS, and 40 mM dithiothreitol (pH 6.8). The protein samples were heated at 60 °C for 15 min, and electrophoresis was performed as described above. The blots were blocked in 5% nonfat dry milk for 1 h and incubated overnight at 4 °C with the primary antibodies against phospho-ERK1/2 and phospho-p38 at a dilution of 1:1000. The secondary antibody and ECL reaction were the same as described above.

COX-2 Promoter Activity Assay—2.7 kb of the 5'-flanking region of the mouse COX-2 gene was amplified by PCR and subcloned into the pGL3-Basic vector (Promega) upstream of the luciferase reporter gene. Transfection of mIMCD-K2 cells was performed using FuGENE6 according to the instruction from the manufacturer (Roche Applied Science). Luciferase activity was determined using a luminescence/fluorescence plate reader (FLUOstar OPTIMA).

PGE2 Enzyme Immunoassay—PGE2 in the culture media was measured with an enzyme immunoassay kit. The assay was performed according to the manufacturer's instructions. Briefly, 25 or 50 µl of the medium, along with a serial dilution of PGE2 standard samples, were mixed with appropriate amounts of acetylcholinesterase-labeled tracer and PGE2 antiserum and incubated at room temperature for 18 h. After the wells were emptied and rinsed with wash buffer, 200 µl of Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)) containing a substrate for acetylcholinesterase was added.



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  		FIGURE 4.
Effects of antioxidants on hypertonicity-stimulated COX-2 expression in mIMCD-K2 Cells. A-D, confluent mIMCD-K2 cells were exposed to isotonic or hypertonic medium for 16 h in the presence or absence of ROT, CCCP, and TTFA (A), DPI (B), NAC (C), or TEMPO (D). Hypertonic medium (550 mosmol/kg) was made by adding NaCl to isotonic medium. COX-2 protein expression was determined by immunoblotting. Shown are representative results from 2-3 separate experiments.

 
Statistical Analysis—Values shown represent means ± S.E. Statistical analysis was performed by analysis of variance and Bonferroni tests with a p value of <0.05 being considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Hypertonicity on ROSs—ROS levels in mIMCD-K2 cells exposed to isotonic or hypertonic medium were determined using dichlorodihydrofluorescence diacetate and cytochrome c. Osmolality in the medium was raised by adding NaCl. Exposure of the cells to hypertonicity markedly increased ROS levels as determined by the two independent methods (Figs. 1 and 2). The stimulation of ROSs was time-dependent, being detected at 2 min, gradually increasing with time, and peaking at 30 min following hypertonic treatment (Fig. 1, bottom). The increases in ROS levels were prevented by 50 µM genistein (Figs. 1 and 2).



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  		FIGURE 5.
Effects of antioxidants on hypertonicity-stimulated PGE2 release in mIMCD-K2 Cells. Confluent mIMCD-K2 cells were exposed to isotonic or hypertonic medium for 16 h in the presence or absence of DPI or NAC. Medium PGE2 concentration was determined by enzyme immunoassay.

 
The Source of Increased ROSs in Response to Hypertonicity—Two potential sources of the ROSs, e.g. mitochondria and NADPH oxidase, were examined. A 30-min exposure of mIMCD-K2 cells to hypertonic medium induced a >2-fold increase in DCF fluorescence that was completely abolished by 5 µM rotenone, an inhibitor of complex I, 10 µM TTFA, an inhibitor of mitochondrial electron transport chain complex II, or 0.5 µM CCCP, an uncoupler of oxidative phosphorylation (Fig. 3A). To examine NADPH oxidase as the source of the ROSs, DCF fluorescence was determined in cells derived from the renal inner medulla from gp91phox-/- and p47phox-/- and wild type control mice. The majority of cells exhibited epithelial cell-like morphology (data not shown) and were considered to be IMCD cells, although contamination of other cell types such as interstitial cells was inevitable. Hypertonic treatment induced an ~2-fold increase in DCF fluorescence in primary cultures of both wild type and in mIMCD-K2 cells (Fig. 3B). However, the increases in DCF fluorescence were not significantly altered in gp91phox-/- or p47phox-/- cells as compared with wild type controls (wild type, 1.03 ± 0.04 versus 2.06 ± 0.12, p < 0.05; gp91phox-/- 1.14 ± 0.07 versus 1.81 ± 0.07, p < 0.05; p47-/-, 1.21 ± 0.07 versus 1.87 ± 0.04, p < 0.05; n = 8 in each group).



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  		FIGURE 6.
COX-2 expression in primary cultures of control and NADPH oxidase-deficient IMCD cells in response to hypertonicity. A and B, IMCD cells from gp91phox-/- (A) and p47phox-/- (B) mice were isolated using standard methods. Confluent cells were treated for 16 h with isotonic or hypertonic medium. Hypertonic medium (550 mosmol/kg) was made by adding NaCl. COX-2 expression was determined by immunoblotting. Shown are representative results from three separate experiments.

 
Role of ROSs in Mediation of Hypertonicity Stimulation of COX-2 Expression—We examined the effects of antioxidants on the stimulation of COX-2 expression by hypertonicity in cultured mIMCD-K2 cells. To interfere with different steps in metabolism, four classes of antioxidants, namely mitochondrial inhibitors (ROT, CCCP, and TTFA), the NADPH oxidase inhibitor DPI, the synthetic SOD mimetic TEMPO, and the general antioxidant NAC, were used. When mIMCD-K2 cells were exposed to media made hypertonic (550 mosmol/kg) by the addition of NaCl for 16 h, a >5-fold increase in COX-2 protein expression was noted that was remarkably inhibited or abolished by 5 µM ROT, 10 µM TTFA, 0.5 µM CCCP, or 25-50 µM DPI. NAC (6 mM) or TEMPO (2.0 mM) reduced COX-2 levels by 75.6 and 79.8%, respectively (Fig. 4). The inhibition of COX-2 expression was paralleled by complete blockade of hypertonicity-stimulated PGE2 release (Fig. 5).



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  		FIGURE 7.
Effect of antioxidants on hypertonicity-induced COX-2 promoter activity. mIMCD-K2 cells were transiently transfected with the mouse COX-2 promoter-luciferase construct. Forty-eight hours later, the cells were treated for 16 h with hypertonic medium (550 mosmol/kg by adding NaCl) in the presence or absence of 25 µM DPI or 3 mM NAC. Luciferase activity was assayed and expressed as relative luciferase units/mg protein.

 
To examine the role of NADPH oxidase in mediation of the hypertonicitydependent stimulation of COX-2 expression, cells from the renal inner medulla from gp91phox-/- and p47phox-/- mice and wild type controls were exposed to hypertonicity for 16 h, and COX-2 expression was examined by immunoblotting. The magnitude of COX-2 stimulation in response to hypertonic treatment was comparable between gp91phox-/- and p47phox-/- cells and their wild type controls (Fig. 6).

To examine the effect of antioxidants on the hypertonicity-induced COX-2 transcriptional activity, mIMCD-K2 cells were transiently transfected with a reporter construct containing the luciferase gene driven by a 2.7-kb COX-2 mouse promoter. Hypertonic treatment for 16 h markedly induced COX-2 promoter activity that was abolished by 25 µM DPI and significantly inhibited by 3 mM NAC (Fig. 7). NAC and TEMPO were not toxic to the cells in the doses and durations used, whereas modest toxicity was noticed with 50 µM but not 25 µM DPI.

We examined whether oxidative stress generated by the xanthine/xanthine oxidase system had a direct effect on COX-2 expression. mIMCD-K2 cells were treated for 16 h with vehicle, xanthine alone, or xanthine plus xanthine oxidase. COX-2 expression was not affected by xanthine alone but was markedly stimulated by xanthine plus xanthine oxidase (Fig. 8A). The expression of COX-2 correlated well with PGE2 levels (Fig. 8B).

COX-2 expression can be regulated in a feed-forward process by its own prostaglandins, and there is a possibility that the effects observed with the antioxidants might be due to effects on COX activity. To rule out this possibility, we examined the effects of non-selective and selective non-steroidal anti-inflammatory agents on COX-2 expression. As shown in Fig. 9, neither indomethacin nor SC-58635 caused significant changes of hypertonicity-induced COX-2 expression. Thus, it is unlikely that in our model system COX-2 expression is regulated in a feed-forward process by its own products.

Role of ROSs in Mediation of the Activation of ERK1/2 and p38 by Hypertonicity—Based on the observation that MAPK mediates the hypertonicity-induced stimulation of COX-2 expression, we hypothesized that ROSs may act via MAPK. To test this hypothesis, we determined the effects of antioxidant treatment on the activation of MAPK by hypertonicity. Phosphorylation of ERK1/2 and p38 was determined by immunoblotting using phosphorylation-specific antibodies. Exposure of mIMCD-K2 cells to hypertonicity for 20 min markedly increased the abundance of the phosphorylated forms of both ERK1/2 (p44/42) and p38. The activation of both ERK1/2 and p38 by hypertonicity was prevented by NAC (Fig. 10).



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  		FIGURE 8.
Effect of xanthine/xanthine oxidase on COX-2 expression. Confluent mIMCD-K2 cells were exposed to vehicle, xanthine (160 µM) alone, or xanthine in combination with xanthine oxidase (0.6 units/ml) for 16 h. A, immunoblotting analysis of COX-2 protein expression. B, enzyme immunoassay for PGE2 concentration.

 



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  		FIGURE 9.
Effects of non-selective and selective COX inhibitors on COX-2 expression. Confluent mIMCD-K2 cells were treated for 16 h with hypertonic medium (550 mosmol/kg by adding NaCl) in the presence or absence of 10 µM indomethacin or 10 µM SC-58635. Shown are representative results from three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study attempts to extend our previous observation that MAPK mediates the stimulation of COX-2 expression by hypertonicity in cultured renal medullary epithelial cells. In this study we identified ROSs as upstream mediators of the activation of MAPK in response to hypertonic stress. We found that exposure of mIMCD-K2 cells to hypertonic stress induced rapid release of ROSs that were derived from mitochondria, but not NADPH oxidase. The notion of a causal relationship between ROS generation and COX-2 induction is based on the observation that treatment of mIMCD-K2 cells with exogenous generated by the xanthine/xanthine oxidase system mimicked the hypertonicity effect on COX-2 expression and PGE2 release. To interfere with intracellular production of ROSs, we employed structurally unrelated antioxidants, namely the NADPH oxidase inhibitor DPI, the superoxide scavenger TEMPO, and the general antioxidant NAC. The induction of COX-2 expression was effectively blocked by all three classes of antioxidants. Commensurate with the protein expression data, antioxidants also effectively blocked PGE2 release. Furthermore, the hypertonicity-induced phosphorylation of ERK1/2 and p38 was completely blocked by antioxidant treatment. These observations reveal the existence of a novel ROS/MAPK/COX-2 pathway in the osmotic response of collecting duct cells.

The present study determined ROS levels in mIMCD-K2 cells exposed to hypertonic NaCl treatment with the use of two independent methods involving the use of cytochrome c and dichlorodihydrofluorescence diacetate. Both methods were able to detect increases in ROS levels as early as 2 min following exposure to a hypertonic environment, indicating that oxidative stress is an early event in the osmosensing pathway. This finding agreed with the study of Zhang et al. (21), who observed a similar phenomenon in cultured IMCD3 cells.

It is of great significance to identify the source of increased ROSs in response to hypertonic treatment. NADPH oxidase is best characterized as a major superoxide-generating enzyme in phagocytes. It is a multicomponent enzyme comprised of five subunits, namely p40phox, p47phox, p67phox, p22phox, and gp91phox (22). In resting cells the membrane portion of enzyme, also known as cytochrome b558 and consisting of gp91phox and p22phox, is dissociated from the cytosolic portion that consists of p40phox phox, p47phox, and p67phox. When the cells are exposed to stimuli, p47phox is usually heavily phosphorylated, leading to translocation of the whole cytosolic complex to the membrane, where the enzyme is reassembled into an active oxidase. Among the five subunits, gp91phox and p47phox are of particular significance, as the former is the catalytic subunit and the latter is regulated by phosphorylation. Among the five subunits of NADPH oxidase detected in the kidney, only gp91phox and p47phox were regulated by sodium intake (23). However, neither the ROS formation nor the COX-2 stimulation in response to hypertonic treatment were measurably affected in gp91phox- or p47phox-deficient cells. These data virtually exclude gp91phox or p47phox as a source of hypertonicity-induced increases in ROS production.

We subsequently examined the possibility that mitochondria might be a source of the ROSs. Indeed, the hypertonicity-induced increases in ROSs were abolished by various mitochondrial inhibitors, including ROT, an inhibitor of complex I, TTFA, an inhibitor of mitochondrial electron transport chain complex II, and CCCP, an uncoupler of oxidative phosphorylation. Furthermore, all of these inhibitors consistently blocked hypertonicity-induced COX-2 expression. Taken together, these observations suggest that mitochondria, but not NADPH oxidase, contribute to the hypertonicity-induced ROS production and COX-2 expression.



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  		FIGURE 10.
Effect of antioxidants on the hypertonicity activation of p44/42 and p38. Confluent mIMCD-K2 cells were exposed to isotonic or hypertonic medium for 20 min in the presence or absence of NAC at 6 mM. Prior to the hypertonic treatment, cells were pretreated by vehicle or NAC for 24 h. Phosphorylation of p44/42 and p38 was determined by immunoblotting using phosphorylation-specific antibodies. Shown are representative results from three separate experiments.

 
We provide further evidence for the involvement of tyrosine kinases in the ROS production by showing that the hypertonicity-related induction of ROSs was completely blocked by genistein, a non-selective tyrosine kinase inhibitor. The data were validated by two independent methods of determining ROS levels, the DCF fluorescence method and cytochrome c method. In line with these finding, a number of tyrosine kinases, including receptor tyrosine kinase and epidermal growth factor receptor tyrosine kinase (24), as well as non-receptor tyrosine kinases such as Src (25), focal adhesion kinase (26), and Fyn (27), are activated by osmotic stress. Both non-selective inhibition of tyrosine kinase with genistein and selective inhibition of EGF receptor tyrosine kinase with AG-1478 and PD-153035 significantly blocked the tonicity-dependent induction of COX-2 expression (9, 15). It is possible that tyrosine kinases may represent an upstream component in the hypertonicity-initiated cascade leading to oxidative stress. Future studies will be needed to determine the identity of the kinases and the nature of their relationship with mitochondria.

A major contribution of this study is the identification of the ROS/MAPK/COX-2 pathway in the osmotic response. A significant role of this pathway is suggested by previous evidence implicating MAPK and COX-2 in the osmotic response. There is an impressive body of work supporting the cytoprotective role of MAPK, especially of p38, against osmotic stress. In yeast, deletion of HOG1, a yeast homolog of p38, is lethal under hypertonic conditions, and the lethality is rescued by over-expression of wild type mammalian p38 (28) or JNK (29). HOG1 is directly responsible for the induction of glycerol-3-phosphate dehydrogenase, the enzyme essential for production of glycerol, the major organic osmolyte in yeast (30, 31). In mammalian cells, hypertonicity activates multiple MAPKs including ERK1/2, JNK, and p38 in IMCD cells (32). Dominant negative inactivation of JNK2 sensitizes renal IMCD cells to hypertonicity-induced cell death (33).

There is also increasing evidence supporting a cytoprotective role of COX-2 in the osmotic response. Our previous study showed that inhibition of COX-2 increases cell death in mIMCD-K2 cells in hypertonic, but not isotonic, conditions (12). A similar phenomenon has been observed in cultured renal medullary interstitial cells (RMICs) in which COX-2 activity is required for osmolyte accumulation (14). In rodents, expression of renal medullary COX-2 is variably found in either RMICs (34, 35) or in the collecting duct (36, 37). COX-2 inhibition induced apoptosis in the RMICs of water-deprived rabbits (10) but in the collecting duct of water-deprived rats (38). In a recent study of 53 normal human nephrectomy specimens, renal medullary COX-2 was constitutively expressed in vasa recta endothelial cells and in the collecting duct, but not in RMICs (39). It is unclear whether the osmosensing pathway identified by the present study will apply to RMICs or/and the collecting duct or other cell types in the renal medulla in vivo.

Whether osmolyte-related genes are directly under the control of MAPK is still unclear because of conflicting reports. Pharmacological inhibition of p38 in monocytes and renal epithelial cells blocked the induction of mRNAs for betaine and myo-inositol transporters and for aldose reductase, as well as osmotic response element-driven reporter gene expression (40-42). Ko et al. (27) showed that inhibition of p38 by either a pharmacological approach or dominant negative mutation of p38{alpha} (27) significantly blocked osmotic response element reporter activity. Inconsistent with these observations, however, is the finding showing that a dominant negative mutation of MAPK kinase 3, an upstream activator of p38, did not affect the hypertonicity induced osmotic response element activity, nor did a mutation of JNK (43). Furthermore, hypertonicity-induced phosphorylation of tonicity-responsive enhancer-binding protein was not affected by the p38 inhibitor SB-203580. It seems possible that the ROS/MAPK/COX-2 pathway might be distinct from the conventional tonicity-responsive enhancer-binding protein-mediated pathway in the osmotic response.

In summary, the present study describes a novel hypertonicity-activated pathway in which hypertonicity induces a rapid release of ROSs from mitochondria that activate both ERK1/2 and p38, leading to induction of COX-2 expression. The ROS/MAPK/COX-2 pathway is expected to play a distinct role in the osmotic response.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO-1 HL079453, RO-1 DK066592, R21 DK069490, and KO-1 DK064981 (to T. Y.). Support also came from intramural funds from the NIDDK, National Institutes of Health (to J. B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: University of Utah and Veterans Affairs Medical Center, Bldg. 2, Research Service (151 E), 500 Foothill Dr., Salt Lake City, UT 84148. Tel.: 801-582-1565 (ext. 4334); Fax: 301-584-5658; E-mail: tianxin.yang{at}hsc.utah.edu.

2 The abbreviations used are: COX, cyclooxygenase; CCCP, cyanide m-chlorophenylhydrazone; DCF, 2',7'-dichlorofluorescein; DPI, diphenyleneiodonium; ERK, extracellular signal-regulated kinase; IMCD, inner medullary collecting duct; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAC, N-acetyl-L-cysteine; PGE2, prostaglandin E2; RMIC, renal medullar interstitial cell; ROS, reactive oxygen species; ROT, rotenone; SOD, superoxide dismutase; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; TTFA, thenoyltrifluoroacetone. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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