ARTICLES J Am Soc Nephrol 10: 2261–2271, 1999

NS-398 Upregulates Constitutive Cyclooxygenase-2 Expression in the M-1 Cortical Collecting Duct Cell Line

SHAWN FERGUSON,* RICHARD L. HEBERT,*´ † and ODETTE LANEUVILLE‡ Departments of *Cellular and Molecular Medicine, †Medicine, and ‡Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.

Abstract. The cortical collecting duct (CCD) is a major site of Western blot analysis, COX-2 expression was significantly intrarenal prostaglandin E2 (PGE2) synthesis. This study ex- upregulated by incubation with either indomethacin or NS-398. amines the expression and regulation of the prostaglandin These drugs did not affect COX-1 protein expression. Evalu- synthesizing enzymes cyclooxygenase-1 (COX-1) and -2 in the ation of COX-2 mRNA expression by Northern blot analysis CCD. By indirect immunofluorescence using isoform-specific after NS-398 treatment demonstrated that the COX-2 protein antibodies, COX-1 and -2 immunoreactivity was localized to upregulation occurred independently of any change in COX-2 all cell types of the murine M-1 CCD cell line. By immuno- mRNA expression. These studies have for the first time local- histochemistry, both COX-1 and COX-2 were localized to ized COX-2 to the CCD and provided evidence that the inter- intercalated cells of the CCD on paraffin-embedded mouse calated cells of the CCD express both COX-1 and COX-2. The kidney sections. When COX enzyme activity was measured in results also demonstrate that constitutively expressed COX-2 is the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the major COX isoform contributing to PGE2 synthesis by the the specific COX-2 inhibitor NS-398 effectively blocked PGE2 M-1 CCD cell line. Inhibition of COX-2 activity in the M-1 synthesis. These results demonstrate that COX-2 is the major cell line results in an upregulation of COX-2 protein expres- contributor to the pool of PGE2 synthesized by the CCD. By sion.

Nonsteroidal anti-inflammatory drugs (NSAID) are used ex- modulating these and other renal processes is well documented tensively for their anti-inflammatory, antipyretic, and analgesic (4–11). effects (1). These therapeutic effects stem from the ability of The two COX isoforms are derived from two different genes these drugs to inhibit the activity of the prostaglandin-forming and differ significantly in their regulation (12). The COX-1 enzymes termed cyclooxygenase-1 (COX-1) and -2, also enzyme is constitutively expressed in most cell types, where it known as prostaglandin endoperoxide H synthase-1 and -2. plays a housekeeping role. COX-2 has been termed the induc- After the release of arachidonic acid (AA) from membrane ible cyclooxygenase because of its rapid upregulation in re- phospholipids by cytosolic phospholipase A2 (cPLA2), COX sponse to a variety of stimuli, including growth factors, cyto- isoforms convert AA to prostaglandin G2 (PGG2) (cyclooxy- kines, and phorbol esters (13–16). Early observations of genase activity) and then in a separate active site, PGG2 is different expression and regulation of the COX isoforms cou- reduced to PGH2 (peroxidase activity). PGH2 is then trans- pled with the observation that the two COX isoforms were formed to a biologically active prostanoid such as PGE2, PGI2, differently inhibited by some NSAID (17) have led to the or thromboxane A2 by specific synthases (2). Each prostag- development of COX-2-specific inhibitors (18,19). The ratio- landin-producing cell type synthesizes one major prostanoid. nale for their development was the association of COX-1 with For example, PGE2 is the major prostanoid produced in the physiologically necessary prostanoids such as those involved kidney cortical collecting duct (CCD) (3). The collecting duct in gastric cytoprotection and COX-2 with the production of represents an important segment of the nephron for the fine prostanoids in inflammatory pathologic states such as rheuma- regulation of salt and water reabsorption. The role of PGE2 in toid arthritis. Surprisingly, studies on COX-1 null mice have revealed that the phenotypes of these mice are relatively nor- mal, and the lack of COX-1 does not result in gastric ulcers or Received October 29, 1998. Accepted May 11, 1999. This work was presented in part at the 31st annual meeting of the American abnormal renal function (20). Although the COX-1 knockout Society of Nephrology and has been published in abstract form (JAmSoc mouse studies suggested a less important role for COX-1 than Nephrol 9: 412A, 1998). what previous evidence suggested, with respect to COX-2, the Correspondence to Dr. Odette Laneuville, Department of Biochemistry, Mi- knockout study demonstrated that COX-2 produces prosta- crobiology, and Immunology, 451 Smyth Road, University of Ottawa, Ottawa, noids, which play a crucial role in kidney development (21). Ontario, Canada K1H 8M5. Phone: 613-562-5800, extension 8402; Fax: 613- 562-5440; E-mail: [email protected] The kidneys of COX-2-disrupted mice were normal at birth; 1046-6673/1011-2261 however, they did not proceed to develop properly after this Journal of the American Society of Nephrology point. This aberrant renal development was the major abnormal Copyright © 1999 by the American Society of Nephrology phenotype observed in these COX-2 knockout mice. In addi- 2262 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2261–2271, 1999 tion to inflammatory and developmental states, it is now versity, East Lansing, MI). Two COX-2 antibodies raised against known that some cell types constitutively express the COX-2 different peptides from the murine COX-2 protein were purchased isoform under normal physiologic conditions. Within the kid- from Cayman Chemical, and both were effective at detecting murine ϩ ney, constitutive COX-2 expression has been observed in the COX-2 by our methodology. The coverslips were washed with PBS macula densa (22), cortical thick ascending limb (23), medul- 0.2% saponin before application of the secondary antibody. The secondary antibody, CY3-conjugated donkey anti-rabbit IgG (Jackson lary interstitial cells, and inner medullary collecting ducts (24). Immunoresearch Laboratories, Bio/Can Scientific, Mississauga, On- Although this renal COX-2 expression is constitutive, it can tario, Canada), was diluted 1:75 in PBS ϩ 10% FBS ϩ 0.2% saponin, also be upregulated. For example, in rats dietary salt restriction applied to the coverslips, and incubated at 37°C for 30 min. The upregulates COX-2 in the cortex (22,24,25), while a high salt coverslips were washed with PBS ϩ 0.2% saponin before being diet increases medullary COX-2 expression. mounted on slides with PermaFluor fade-resistant mounting medium These recent findings concerning renal COX-2 expression (Immunon Lipshaw, Pittsburgh, PA). The specificity of the COX -2 have prompted us to investigate the effects of the new class of antibodies was tested by preincubation with the cognate peptide COX-2-specific NSAID on COX expression and activity in the (Cayman Chemical). The COX-1 detection was controlled for by collecting duct. The M-1 mouse CCD cell line represents a omission of the primary antibody. The results were captured under an powerful tool for the study of the interaction between NSAID Axiophot™ microscope (Carl Zeiss, Thornwood, NY), using North- and the COX isoforms. The M-1 cell line was derived from ern Eclipse 5.0 imaging software (Carl Zeiss). collecting duct cells from the kidney of an SV40 transgenic mouse, and it has been characterized functionally, morpholog- Immunohistochemical Localization of COX-1 and ically, and immunologically to reflect the intact CCD (26). COX-2 on Mouse Kidney Slices This cell line has also been shown to constitutively express Mice (CD-1, 25 to 30 g body wt, 10 to 12 wk old) from Charles COX-2 (24). The availability of COX isoform-specific inhib- River were sacrificed and the kidneys were immediately removed. itors has allowed us to evaluate the effect of specific isoform The kidney was rinsed in PBS, decapsulated, sliced transversely, and inhibition on M-1 CCD cell PGE2 synthesis. Indomethacin was placed in fixative. The fixative consisted of 2% paraformaldehyde and chosen as a potent non-isoform-selective NSAID (27). NS-398 0.2% picric acid in PBS, pH 7.4, and the fixation lasted 18 h at 4°C. and resveratrol were chosen as COX-2- and COX-1-specific The kidneys were paraffin-embedded, and 4-␮m microtome sections inhibitors, respectively (18,28,29). Unlike traditional NSAID, were mounted onto SuperFrost Plus slides (VWR Canlab, Missis- which target the cyclooxygenase activity of COX, resveratrol sauga, Ontario, Canada). The sections were rehydrated and incubated ϩ preferentially inhibits both the cyclooxygenase and peroxidase in methanol 0.3% H2O2 for 30 min at room temperature. The cells ϩ activities of COX-1. The ability of these cells to compensate were permeabilized for 15 min in PBS 0.3% Triton X-100 and blocked for 30 min in PBS ϩ 1% skim milk powder. The COX-1 or for COX inhibition was assessed by measuring COX isoform COX-2 primary antibody (Cayman Chemical) was diluted in PBS ϩ protein and mRNA levels after NSAID treatment. 0.1% Triton X-100 ϩ 1% bovine serum albumin. For detection of the CCD via aquaporin-2 (AQP-2) labeling, a specific antibody obtained Materials and Methods from Dr. Mark A. Knepper (National Institutes of Health, Bethesda, Cell Culture MD) was diluted to 40 ng/ml (30,31). The sections were incubated M-1 mouse CCD cells (CRL-2038; American Type Culture Col- with these antibody solutions for 18 h at 4°C. The secondary antibody lection, Rockville, MD) passages 18 to 30 were grown at 37°C in was biotinylated anti-rabbit IgG (Amersham Canada, Oakville, On- ϩ 100-mm dishes in a medium consisting of Dulbecco’s minimal es- tario, Canada), which was diluted 1:100 in PBS 1% bovine serum ϩ sential medium/F12 supplemented with 5% fetal bovine serum (FBS) albumin 0.1% Triton X-100; this incubation lasted 30 min at 37°C. and 1% penicillin, streptomycin, and Fungizone antibiotic-antimy- After 10 min in 3% H2O2/PBS, the slides were incubated with cotic solution (Life Technologies, Burlington, Ontario, Canada) in a streptavidin-linked horseradish peroxidase (HRP) diluted 1:50 in PBS. Diaminobenzidine (DAB) substrate (Sigma Chemical) was used to humidified atmosphere containing 5% CO2. To evaluate the effect of various NSAID on COX isoform expression, the drugs indomethacin visualize the signals. The slides were counterstained with Mayer’s (Sigma Chemical Co., Mississauga, Ontario, Canada), NS-398 (Cay- hematoxylin (VWR Canlab) before dehydration and mounting with man Chemical, Ann Arbor, MI), and resveratrol (Sigma Chemical) Permount (Fisher Scientific, Nepean, Ontario, Canada). For double were added to the media dissolved in DMSO. Drug concentration was labeling of COX-1/AQP-2 and COX-2/AQP-2, the COX isoform in each case 10Ϫ5 M, and DMSO concentration was 0.02% (vol/vol). labeling was performed first as described above, up to and including the visualization with DAB. After this step, HRP activity was ϩ quenched by incubating the slides in PBS 3% H2O2 for 10 min at Immunofluorescence Localization of COX-1 and room temperature. The slides were rinsed with PBS and incubated for COX-2 in M-1 Cells 30 min with an unconjugated donkey anti-rabbit IgG Fab fragment M-1 cells were grown to confluence on glass coverslips, rinsed (Jackson Immunoresearch Laboratories) to block further detection of with phosphate-buffered saline (PBS), and fixed for 30 min at room the COX isoform primary antibody. The AQP-2 antibody incubations temperature with 1:1 (vol/vol) PBS:10% phosphate-buffered formalin. were carried out as described above. For detection of the AQP-2 The fixative was washed off, and the primary antibodies were applied localization, metal-enhanced DAB (Sigma Chemical) was used. This diluted 1:50 in PBS ϩ 10% FBS ϩ 0.2% saponin (Sigma Chemical). reagent allowed for the rapid detection of AQP-2 via the development The cells were incubated with the primary antibody solution at 37°C of an insoluble blue precipitate that is easily discernible from the DAB for 1 h. The COX-1 antibody was raised in a rabbit against a peptide (brown color) used for the COX-1 and COX-2 localization. After the corresponding to ovine COX-1 amino acids L274 to A288 and was AQP-2 detection, the slides were rinsed in H2O, dehydrated, and provided by Drs. W. L. Smith and D. DeWitt (Michigan State Uni- mounted with Permount. The results were analyzed and captured J Am Soc Nephrol 10: 2261–2271, 1999 NS-398 Upregulates COX-2 in M-1 Cells 2263 using an Axiophot™ microscope (Carl Zeiss) and Northern Eclipse purchased from Cayman Chemical. The specificity of the COX iso- 5.0 imaging software. form-specific antibodies was tested by Western blotting using 50 ng of purified COX-1 and -2 electrophoresis standards per lane (Cayman Enzyme Activity Chemical). In addition, the antibodies were tested after preincubation with their respective immunizing peptides. After washes with TBS-T, Confluent dishes of M-1 cells were harvested and resuspended in the membranes were incubated with HRP-conjugated goat anti-rabbit Dulbecco’s minimal essential medium/F12 media. The NSAID to be IgG (Promega, Madison, WI) diluted 1:2000 in TBS-T for1hatroom assessed were added dissolved in DMSO to give a final NSAID Ϫ5 temperature. Excess secondary antibody was washed away with concentration of 10 M and a DMSO concentration of 0.02% (vol/ TBS-T. The results were visualized after developing with the Amer- vol). The following drugs were studied: indomethacin, NS-398, res- sham-enhanced chemiluminescence (ECL) reagents according to the veratrol, and the combination of NS-398 ϩ resveratrol. The cells were 14 manufacturer’s instructions (Amersham Canada). To ensure equal incubated with the respective drugs for 30 min at 37°C before [1- C]- protein loading, the membranes were stripped as per the Amersham arachidonic acid (AA) (50 mCi/mmol) (Amersham Canada) was protocol, blocked for3hatroom temperature in TBS-T-10% milk, added to give a final concentration of 10 ␮ M, and the cells were rinsed with TBS-T, and incubated with anti-mouse ␤-actin antibody further incubated at 37°C for 40 min. At the end of the incubation, the (Sigma Chemical) diluted 1:5000. After rinsing the membranes, the ϫ samples were centrifuged (5 min, 2000 g), and the supernatant was HRP-conjugated anti-mouse IgG secondary antibody (Amersham retained for measurement of prostaglandin content. Protein samples Canada) was diluted 1:2000, and incubation took place for 30 min at from the cellular pellet were measured by the Bradford method to room temperature. After thorough washing, the results were visual- normalize each sample for cell number. The lipid fraction was ex- ized by the Amersham ECL protocol. Scanning densitometry was tracted from the supernatant with diethyl ether/methanol/0.2 M citric performed using the Image 1.47 program, and COX signal density acid solution (30:4:1 vol/vol/vol). This prostanoid and unreacted was normalized to ␤-actin density. AA-containing fraction was separated on Silica Gel 60 thin-layer chromatography plates (VWR Canlab), using the organic phase of an Northern Blotting ethyl acetate/2,2,4-trimethyl pentane/acetic acid/H2O (11:5:2:10 vol/ vol/vol/vol) solvent system. The presence of radiolabeled products Total RNA was isolated via the TriZol method (Life Technologies). ϩ was evaluated by autoradiography using Kodak BioMax MR film. Poly(A) RNA isolated from 100 ␮g of total RNA with Qiagen

Cold PGE2 standard (Cayman Chemical) visualized with iodine vapor Oligotex™ columns was loaded onto 1% denaturing agarose gels and was used to confirm the identity of the major prostaglandin (PGE2) electrophoresed. The RNA was transferred to nylon membranes produced by the M-1 cells. Scanning densitometry was performed (Boehringer Mannheim, Laval, Quebec, Canada) and fixed by baking using the Image 1.47 program, and PGE2 signal density was normal- at 80°C under vacuum. A probe of 1.3 kb (25 ng) complementary to ized to total protein content. the portion of the mouse COX-2 cDNA between the XmnI restriction site at 492 nucleotides and the 3Ј end was labeled with 32P-CTP (50 ␮Ci) by random priming with the Prime-It kit (Stratagene Cloning Western Blotting Systems, La Jolla, CA). After a 2-h prehybridization at 42°C, the NSAID were added to the media when the cells were plated and the labeled probe was added, and the membrane was allowed to hybridize cells were grown to confluence (approximately 72 h). Dishes of for 24 h at 42°C. After hybridization and high stringency washing, the confluent cells were rinsed with cold PBS and harvested in PBS by radioactive signal was detected by autoradiography with Kodak X- scraping with a cell lifter. The harvested cells were centrifuged, the OMAT AR film. The COX-2 probe was stripped from the membrane supernatant was aspirated, and the cell pellet was resuspended in 100 by boiling for 3 min in diethylpyrocarbonate-treated water. To control mM Tris, pH 7.4, containing 1 mM ethylenediaminetetra-acetic acid for the quantity of mRNA loaded per lane, ␣-tubulin mRNA levels ␤ Ј and 1 mM ethyleneglycol-bis( -aminoethyl ether)-N,N -tetra-acetic were measured using a 32P-CTP (50 ␮Ci)-labeled probe prepared and acid. The resulting solution was sonicated for 5 s with an Ultrasonics hybridized as for the COX-2 probe. Band intensity was quantified by ϫ cell disrupter to lyse the cells. The cell lysates were spun at 10,000 scanning densitometry using the Image 1.47 program. g for 10 min to pellet the nuclei and insoluble cytoskeleton. The supernatants were removed and assayed for protein content by the Bradford method (Bio-Rad Laboratories, Mississauga, Ontario, Can- Statistical Analyses Ϯ ada). Twenty-five micrograms of protein from each sample was Data are presented as mean SEM. Statistical analyses were denatured in boiling Laemmli buffer for 5 min and resolved by performed using the Prism Graphpad 2.01 software and consisted of sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a poly- ANOVA followed by the Bonferroni post test. Differences were Ͻ acrylamide gel consisting of a 4% stacking and a 10% resolving layer considered statistically significant at P 0.05. using a Mini-PROTEAN II apparatus (Bio-Rad Laboratories). After electrophoresis, the proteins were transferred to Hybond ECL nitro- Results cellulose membranes (Amersham Canada) with the Mini-Trans blot All M-1 Cell Types Express both COX Isoforms system (Bio-Rad). The membranes were blocked overnight at 4°C in The M-1 cell line reflects the intact collecting duct in that it Tris-buffered saline-0.1% Tween 20 (TBS-T) supplemented with 5 or is a heterogeneous population with characteristics of principal 10% fat-free dried milk for COX-1 and COX-2 detection, respec- and intercalated cells. Each of these cell types has been char- tively. After rinsing away the blocking solution with TBS-T, the acterized with respect to their individual roles in tubular trans- membranes were incubated with primary antibody diluted 1:2000 in TBS-T-2% milk for 90 min at room temperature. The COX-1 anti- port (26). Immunofluorescence experiments with COX iso- body was raised in a rabbit immunized with a peptide corresponding form-specific antibodies were performed to investigate the to amino acids L274 to A288 of mouse COX-1 (antibody and immu- distribution of the two isoforms in the M-1 cell line. Figure 1A nizing peptide were provided by Drs. W. L. Smith and D. DeWitt, is representative of the pattern of fluorescence associated with Michigan State University). The COX-2 primary antibodies were COX-1 immunoreactivity in a monolayer of confluent M-1 2264 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2261–2271, 1999

Figure 1. Indirect immunofluorescence demonstrating the pattern of cyclooxygenase (COX) isoform expression in cultured M-1 cells. (A) Using a COX-1 isoform-specific primary antibody, a perinuclear pattern of fluorescence was observed for COX-1 that is compatible with localization to the endoplasmic reticulum and nuclear envelope. (B) Omission of the COX-1 primary antibody results in complete loss of this labeling. (C) COX-2 was found to be most densely localized within the nuclear envelope, with less intense fluorescence associated with the endoplasmic reticulum. (D) Preincubation of the COX-2 primary antibody with the immunizing peptide abolishes all COX-2-related fluorescence. Magnification, ϫ1250. cells. The bright perinuclear/endoplasmic reticulum pattern of COX-2 expression by the M-1 cell line is not an artifact of the fluorescence is strikingly apparent in this micrograph. Figure M-1 cell line immortalization or culture conditions, COX-1, 1B demonstrates the lack of fluorescence when the COX-1 AQP-2, and COX-2 immunoreactivity was localized on three primary antibody is omitted. Like COX-1, COX-2 is also serial 4-␮m paraffin-embedded transverse sections of mouse expressed in all cell types (Figure 1C). Each cell is positive for kidney (Figure 2). Figure 2A illustrates COX-1 immunoreac- COX-2, and the bright perinuclear ring reflects the increased tivity in a subpopulation of cells in the cortex compatible with content of COX-2 protein in the nuclear envelope and is localization within CCD (indicated by arrows). The APQ-2 consistent with previously published observations describing water channel has been well characterized as the mediator of the intracellular location of this enzyme (32). Figure 1D estab- vasopressin-induced principal cell apical membrane water per- lishes the specificity of the COX-2 immunofluorescence. The meability (30,31). We have used AQP-2 as a marker of col- COX-2 signal is abolished when the primary antibody is pre- lecting ducts to clearly establish the identity of the COX incubated with the immunizing peptide against which it was isoform-positive tubules. By labeling three serial sections with raised. COX-1, AQP-2, and COX-2 antibodies, it was possible to confirm the localization of both COX-1and COX-2 to the In the Mouse Kidney Cortex, both COX-1 and COX-2 CCD. The AQP-2 immunohistochemistry result displayed in Isoforms Are Detectable in the Intercalated Cells of the Figure 2B was obtained on a section serial to that presented in CCD Figure 2A for COX-1 (the arrows have been added to indicate The distribution of COX-2 in the mouse kidney has not been AQP-2-positive tubules). The glomerulus on the right side of characterized. Therefore, to demonstrate that the constitutive the picture serves as a reference point between these two J Am Soc Nephrol 10: 2261–2271, 1999 NS-398 Upregulates COX-2 in M-1 Cells 2265

Figure 2. Immunohistochemical localization of COX-1 and COX-2 expression to the intercalated cells of the cortical collecting duct (CCD). Panels A, B, and C depict a series of three serial 4-␮m mouse kidney sections. (A) COX-1 localization to a subpopulation of CCD cells. (B) The identity of these CCD has been confirmed by detection of aquaporin-2 (AQP-2) in the same tubules on the next 4-␮m section. (C) The subsequent serial section has been labeled for COX-2, demonstrating the expression of COX-2 in a subpopulation of CCD cells. (D) Double immunohistochemistry localization of COX-1 (brown, arrows) and AQP-2 (blue) reveals the exclusive localization of COX-1 to intercalated cells. (E) As for COX-1, double immunohistochemical localization of COX-2 (brown, arrows) and AQP-2 (blue) demonstrates that COX-2 is exclusively and consistently expressed in intercalated cells. Magnification: ϫ500 in A through C; ϫ1250 in D and E. sections as well as the following section where COX-2 has subpopulation of the CCD cells expressed the COX-1 and -2 been localized. Figure 2C clearly demonstrates that in addition isoforms, we sought to determine the identity of these cells to COX-1, COX-2 is also expressed in the CCD. Since only a through double labeling for AQP-2 with each of the COX 2266 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2261–2271, 1999

isoforms. In Figure 2, D and E, we have localized both COX-1 and COX-2 respectively to the intercalated cells of the CCD. In each of these photos, the COX isoform was detected with DAB as the chromogen (indicated by arrows), whereas AQP-2 was visualized using metal-enhanced DAB as the chromogen (blue- colored cells). Two important observations can be made from Panels D and E. First, all CCD cells label for either AQP-2 or the respective COX isoform. Second, no cells label for both AQP-2 and a COX isoform. From these two observations, we conclude that because there are no unlabeled CCD cells, both COX isoforms are expressed by the ␣- and ␤-intercalated cells. The lack of COX/AQP-2 colocalization demonstrates the ab- sence of expression of both COX isoforms in the principal cells. Because there is no AQP-2/COX isoform colocalization, we can also conclude that there is no significant cross reactivity between the two detection systems that we have used. The evidence provided in Figure 2 presents for the first time the finding that the intercalated cells of the CCD constitutively express both COX-1 and COX-2.

COX-2 Is the Major Isoform Contributing to PGE2 Production by the M-1 Cell Line The immunofluorescence micrographs (Figure 1) demon- strate that both COX isoforms are expressed by M-1 cells; however, it is also relevant to determine how much each of these isoforms contributes to the total prostanoid pool pro- duced by these cells. This was addressed by measuring the activity of each isoform as evaluated by the conversion of 14 14 [1- C]-AA to C-PGE2 in the presence of the specific inhib- itors. The lipid fraction isolated from M-1 cells was separated by thin-layer chromatography, and the resulting signal was

detected by autoradiography. PGE2 was the only major prosta- noid produced by this CCD cell line (Figure 3). Therefore,

quantifying PGE2 synthesis gives a measurement of COX enzyme activity. The localization of the PGE2 band was per- formed by visualizing cold PGE2 on lane 1 using iodine vapor. Ϯ The Rf value calculated for PGE2 was 0.14 0.004. Indo- methacin was clearly effective at inhibiting both COX isoforms Ϯ Ͻ as it reduced PGE2 production to 13.7 3.2% of control (P ϭ 0.01, n 3). NS-398 effectively lowered PGE2 synthesis to 18.5 Ϯ 5.6% of control (P Ͻ 0.01, n ϭ 3), indicating that COX-2 is the major prostaglandin-producing enzyme in these cells. Meanwhile, resveratrol treatment lowered M-1 cell COX activity to 60.0 Ϯ 7.3% of control (P Ͼ 0.05, n ϭ 3). This

reduction in PGE2 synthesis reflects the inhibition of COX-1 activity by resveratrol. The combination of inhibition with both NS-398 and resveratrol yielded a reduction in COX enzyme activity to 10.3 Ϯ 2.7% of control (P Ͻ 0.01, n ϭ 3). The method of combining the two isoform-specific inhibitors to

Figure 3. Effect of nonsteriodal anti-inflammatory drugs (NSAID) on COX isoform activity in M-1 cells. (A) Representative autoradiograph of a thin-layer chromatography plate demonstrating radiolabeled pros- (10Ϫ5 M); lane 5, resveratrol (10Ϫ5 M); lane 6, NS-398 (10Ϫ5 M) ϩ 14 Ϫ5 taglandin E2 (PGE2) production from C-arachidonic acid (AA) resveratrol (10 M). (B) Bar graph depicting quantification of PGE2 substrate following preincubation with respective NSAID. Lane 1, production by densitometry. Bars 1 through 5 are representative of 14 cold PGE2 standard and C-arachidonic acid (no M-1 cells); lane 2, lanes 2 through 6 on the autoradiograph in Panel A. Results are control (no NSAID); lane 3, indomethacin (10Ϫ5 M); lane 4, NS-398 means Ϯ SEM (n ϭ 3). *P Ͻ 0.01 versus control. J Am Soc Nephrol 10: 2261–2271, 1999 NS-398 Upregulates COX-2 in M-1 Cells 2267

inhibit PGE2 production, as much as does indomethacin, indi- cates that each COX isoform is maximally inhibited by this concentration of the respective specific inhibitors.

Inhibition of COX-2 Activity Leads to an Increase in COX-2 Enzyme Expression M-1 cells constitutively express COX-1 and COX-2 protein (Figures 1, 4, and 5). To assess the ability of these cells to compensate for reductions in prostaglandin concentrations, the M-1 cells were grown in the presence of nonspecific (indo- methacin 10Ϫ5 M) and COX isoform-specific inhibitors (NS- 398 10Ϫ5 M and resveratrol 10Ϫ5 M). Confluent cells were harvested, and expression of COX isoforms was evaluated by Western blotting using COX isoform-specific antibodies as described in Materials and Methods. After culture in the pres- ence of COX-2 inhibition, there was an upregulation in the

Figure 5. Effect of NSAID administration on M-1 cell COX-1 ex- pression. (A) Representative autoradiograph of a Western blot detect- ing COX-1. (B) The corresponding bands for ␤-actin protein expres- sion were used to control for variations in loading the gels. Lane 1, control (no NSAID); lane 2, indomethacin (10Ϫ5 M); lane 3, NS-398 (10Ϫ5 M); lane 4, resveratrol (10Ϫ5 M). (C) Bar graph depicting quantification of COX-1 Western blot results by densitometry. Results are means Ϯ SEM (n ϭ 4). *P Ͻ 0.05 versus control.

expression of COX-2 protein (Figure 4). The extent of this COX-2 upregulation was 2.55 Ϯ 0.16- and 2.14 Ϯ 0.12-fold for indomethacin and NS-398, respectively (P Ͻ 0.001 versus control, n ϭ 4). Although there was a trend for a greater effect in the presence of indomethacin (inhibits both isoforms), the difference between the effect of indomethacin and NS-398 (specific COX-2 inhibitor) was not statistically significant. The COX-1-specific inhibitor resveratrol did not have a statistically significant effect on COX-2 expression (0.53 Ϯ 0.13-fold of Figure 4. Effect of NSAID administration on M-1 cell COX-2 ex- control, P Ͼ 0.05). The increase in COX-2 protein expression pression. (A) Representative autoradiograph of a Western blot detect- associated with COX-2 inhibition was measured and found not ing COX-2. (B) The corresponding bands for ␤-actin protein expres- sion were used to normalize for variations in loading the gels. Lane 1, to be significantly different at time points from 8 to 72 h of Ϫ Ͼ control (no NSAID); lane 2, indomethacin (10 5 M); lane 3, NS-398 NSAID exposure (data not shown, P 0.05). The quantifica- (10Ϫ5 M); lane 4, resveratrol (10Ϫ5 M). (C) Bar graph depicting tion of COX-2 protein expression as presented in Figure 4C quantification of COX-2 Western blot results by densitometry. Results was performed by scanning densitometry. In each lane, the are means Ϯ SEM (n ϭ 4). *P Ͻ 0.001 versus control. COX-2 level was normalized to the ␤-actin signal (COX-2 2268 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2261–2271, 1999 signal/␤-actin signal). COX-2 upregulation was then expressed The Increase in COX-2 Protein Expression Is Not as a fold increase of the control values. Correlated with an Increase in COX-2 mRNA Levels Identical results were obtained with both COX-2 antibodies Given the preceding observations regarding the upregulation obtained from Cayman Chemical. Each of these antibodies was of COX-2 protein by the inhibition of COX-2 activity, it was of raised against different peptides from the murine COX-2 pro- interest to study the effect of NS-398 treatment on levels of tein. We have found that neither of these antibodies detects the COX-2 mRNA expression. To assess whether the increase in Cayman Chemical COX-1 standard, whereas both detect the COX-2 protein is dependant on an increase in COX-2 mRNA COX-2 electrophoresis standard sold by Cayman Chemical synthesis or stability, Northern blot analysis was performed. (data not shown). Additionally, the COX-2 signal detected by Using a 32P-labeled 1.3-kb fragment of the murine COX-2 these antibodies can be abolished by preincubating the anti- cDNA as a probe, it was possible to detect a 4.2-kb signal body with the immunizing peptide (Figure 6). These observa- matching the known size of the COX-2 mRNA (Figure 7A). In tions ensure that the COX-2 antibodies used in our experiments addition to the time points depicted in Figure 3, COX-2 mRNA are specific for this COX isoform. levels were measured 2 h after addition of NS-398 to the culture media (data not shown). At no time point was there a M-1 CCD Cell COX-1 Expression Is Not Affected by significant increase in COX-2 mRNA expression compared COX Isoform Inhibition with the control (P Ͼ 0.05 versus control). In the histogram Following the same 72-h NSAID treatment described for (Figure 7C), COX-2 mRNA levels have been normalized to the COX-2 Western blot analysis, it was found that COX-1 iso- corresponding ␣-tubulin band. ␣-Tubulin levels were mea- form expression is not affected by the presence of the NSAID studied. Figure 5 demonstrates that although M-1 cells do express COX-1 protein constitutively, the level of expression of this COX isoform is not regulated by culture in the presence of the NSAID tested. The values for COX-1 expression ex- pressed as a fold increase of the control that were obtained for M-1 culture in the presence of indomethacin, NS-398, and resveratrol were 0.85 Ϯ 0.07, 1.07 Ϯ 0.20, and 0.85 Ϯ 0.23, respectively (P Ͼ 0.05 versus control). This COX-1 antibody raised against a peptide corresponding to mouse COX-1 amino acids L274 to A288 does not cross-react with the COX-2 standard (Cayman Chemical). The lack of any effect of indo- methacin or NS-398 on the band intensity for COX-1 further illustrates an absence of cross reactivity toward COX-2.

Figure 6. Preincubation of COX-2 antibody with the immunizing Figure 7. Time course for the regulation of M-1 cell COX-2 mRNA peptide completely blocks the signal for COX-2. This figure demon- expression by the COX-2-specific inhibitor NS-398 (10Ϫ5 M). (A) strates the specificity of the COX-2 antibody used in our experiments. Lane 1, control (no NS-398); lane 2, 8 h; lane 3, 18 h; lane 4, 30 h; Both lanes were loaded with 25 ␮g of M-1 cell lysate prepared as lane 5, 54 h. (B) Corresponding signals for ␣-tubulin mRNA detec- described in Materials and Methods. As opposed to lane 1, before tion. (C) Histogram depicting quantification of COX-2 mRNA signal detection, the antibody in lane 2 was preincubated with an excess (by by densitometry followed by normalizing with the ␣-tubulin signal. weight) of the immunizing peptide. Results are means Ϯ SEM (n ϭ 3). *P Ͻ 0.05 versus control. J Am Soc Nephrol 10: 2261–2271, 1999 NS-398 Upregulates COX-2 in M-1 Cells 2269 sured to control for variability in the efficiency of isolating These factors include: the use of exogenous versus endogenous poly(A)ϩ RNA from one sample to the next as well as to AA as substrate, purified enzyme versus broken cells versus control for variability in sample loading. intact cells as a source of COX enzymes, instantaneous versus time-dependant inhibition, and the study of endogenously ex- Discussion pressed COX versus vector-driven expression systems. Re- In 1978, Smith and Bell localized COX immunoreactivity to gardless of these limitations, the observations we make con- the CCD in the rabbit, cow, guinea pig, rat, and sheep (33–35). cerning COX isoform activity within the system we have used This antibody did not detect COX in the macula densa, sug- are significant. The novel finding in our results is that both gesting that it was more specific for COX-1 than COX-2. constitutively expressed COX isoforms in the CCD are func-

Additional studies that measured PGE2 synthesis by the mi- tional and make significant contributions to PGE2 synthesis by crodissected CCD demonstrated the prostaglandin-synthesiz- this nephron segment. ing ability of the CCD (3). That the COX-1 isoform is ex- Our finding that COX-2 inhibition results in an upregulation pressed in the rat and human CCD has been confirmed with in COX-2 expression is interesting in that it is the opposite of isoform-specific antibodies (22,34). However, with respect to what was observed with respect to compensatory upregulation COX-2, the human and rat studies diverge with respect to the of the remaining COX isoform in cultured fibroblast cells from renal localization. The M-1 cell line has been well character- COX null mice (39). In addition to the compensatory COX ized by Stoos et al. (26). These cells exhibit an amiloride- isoform upregulation, there is also cPLA2 protein overexpres- ϩ ϩ sensitive Na transport and K secretion characteristic of sion. In our experiments, cPLA2 overexpression was not de- principle cells as well as electrogenic Hϩ secretion character- tected by immunoblotting in the M-1 cells following COX-2 istic of immunocytochemistry. The COX isoform signal in the inhibition (S. Ferguson, R. Hébert, O. Laneuville, unpublished M-1 CCD cell line matches the bright nuclear envelope/endo- results). Also, our study found that COX-2 inhibition was plasmic reticulum pattern of fluorescence that is characteristic necessary for stimulation of COX-2 upregulation. Although of the intracellular localization of this enzyme as reported by not statistically significant, the trend is for COX-2 expression Morita et al. (32). Each of the COX isoforms is expressed in all to decrease in response to resveratrol treatment (COX-1 inhi- CCD cell types as represented by the M-1 CCD cell line bition).The comparison between these two studies suggests (Figure 1). that interactions between COX isoform expression and prosta- Our present study has demonstrated for the first time that in glandin production is cell type-specific. This idea is supported addition to COX-1, COX-2 is also expressed in the CCD. The by the observation in COX-1Ϫ/Ϫ mice that there is no com- demonstration of constitutively expressed COX-1 and COX-2 pensatory COX-2 upregulation in the stomach (20). immunoreactivity in only a portion of the cells of the native The NSAID-induced upregulation of COX-2 protein was not CCD (Figure 2) is not unlike the situation in the macula densa, accompanied by any effect on COX-2 mRNA expression. This where only a small subpopulation of the cells are COX-2- is in sharp contrast to the effect of inflammatory agents such as positive (22). However, because the CCD is composed of cytokines and phorbol esters, which stimulate increased principal, ␣-, and ␤-intercalated cell types, we sought to de- COX-2 protein and mRNA expression (40). For example, termine whether the subpopulation of COX isoform-positive interleukin-1␣ was shown to both stimulate COX-2 mRNA cells corresponded to a specific cell type. The AQP-2/COX transcription and prolong the half-life of this message in human isoform double-labeling evidence we have presented (Figure 2, umbilical vein endothelial cells and the human ECV324 cell D and E) reveals that in the native CCD under normal condi- line (41). The mechanism responsible for the COX-2 regula- tions, COX-1 and COX-2 are expressed in the intercalated tion we have documented remains unknown but is most likely cells. Full comprehension of the role of COX-1 and COX-2 in posttranscriptional. Evidence is available that allows for spec- the CCD will require an understanding of the localization and ulation on this subject. With the murine MC3T3-E1 osteoblast regulation of the PGE2 receptors in this segment of the cell line, NS-398 was able to block cytokine-induced upregu- nephron. The effects of PGE2 are mediated by a distinct class lation of COX-2 protein (42). This blockade was reversed by of receptors referred to as E-prostanoid (EP) receptors (36). the addition of exogenous PGE2. However, PGE2 did not The EP1,EP3, and EP4 receptor subtypes have been localized upregulate COX-2 protein in the absence of cytokine stimula- in the CCD (37). Functional studies examining the effects of tion. The M-1 cell line expresses COX-2 constitutively, thus it

PGE2 on salt and water transport have suggested that these is possible to study COX-2 regulation without any confounding receptors are present on the principal cells (4). Our results, effects induced by cytokine stimulation. Unlike the previously which localize both COX isoforms to the intercalated cells, mentioned study, the NS-398-stimulated COX-2 upregulation indicate that the effects exerted by PGE2 on principal cell salt that we observed is associated with an inhibition of PGE2 and water reabsorption represent a paracrine interaction be- synthesis. tween intercalated and principal cells. The interactions be- NSAID are a powerful tool in controlling inflammation; tween the EP receptors, the regulation of COX isoform expres- however, their clinical usefulness is limited by their negative sion, and PGE2 synthesis in this segment of the nephron side effects: primarily gastric ulcers and secondarily kidney represent an interesting field of study. impairment. The kidney impairment that follows NSAID ther- The comparison of studies of COX activity and inhibition in apy may be divided into several categories. The hemodynamic different cell types is complicated by several factors (38). effects of prostaglandins have been well studied. The vasodi- 2270 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2261–2271, 1999

latory effects of PGE2 and PGI2 are important in counteracting Acknowledgments the effects of vasoconstricting peptides such as angiotensin II This study was funded in part by grants from the Medical Research and endothelin I and renal sympathetic nerve activity (5–10). Council of Canada (MT-14103, to Dr. He´bert) and by a Kidney This is especially important in volume-depleted states in which Foundation of Canada grant to both Drs. He´bert and Laneuville. the vasodilatory prostaglandins are critical for the maintenance of renal blood flow and GFR. Electrolyte imbalances can occur References subsequent to vasoconstrictor-mediated reductions in GFR 1. DeWitt D, Smith WL: Yes, but do they still get headaches? Cell 83: 345–348, 1995 when NSAID inhibit PGE and PGI production in mesangial 2 2 2. Smith WL, Laneuville O: Cyclooxygenase and lipoxygenase cells. COX-2 has also been implicated in the regulation of renal pathways of arachidonic acid metabolism. In: Eicosanoids and renin levels. 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