Regulation of cyclooxygenase-2 (COX-2) in rat renalcortex by
adrenal glucocorticoidsand mineralocorticoidsMing-Zhi Zhang*,
Raymond C. Harris*, and James A. McKanna**George M. OBrien Center
for Research in Kidney and Urologic Diseases, andDepartment of Cell
Biology, andDepartment of Medicine, VanderbiltUniversity School of
Medicine, Nashville, TN 37232Communicated by Stanley Cohen,
Vanderbilt University School of Medicine, Nashville, TN, October
15, 1999 (received for review June 7, 1999)Productionof
prostaglandins involvedinrenal salt andwaterhomeostasis is
modulated by regulated expression of the inducibleformof
cyclooxygenase-2 (COX-2) at restricted sites in the rat
renalcortex. Because inammatory COX-2 is suppressed by
glucocorti-coids, andprostaglandinlevels
inthekidneyaresensitivetosteroids, the sensitivity of COX
expression to adrenalectomy (ADX)wasinvestigated.
By2weeksafterADXinmaturerats,
corticalCOX-2immunoreactivityincreased10-foldinthecortical
thickascending limb and macula densa. The constitutive isoform,
COX-1,was unchanged. The magnitude of the changes and specicity
ofCOX-2immunoreactivitywerevalidatedbyinsituhybridizationhistochemistryof
COX-2mRNAandWesternblot analysis. In-creasedCOX-2 activity
(>5-fold) was documentedby usingaspecic COX-2 inhibitor. The
COX-2 up-regulation in ADX rats wasreversedbyreplacement
therapywitheither corticosteroneordeoxycorticosterone acetate. In
normal rats, inhibition of
glucocor-ticoidreceptorswithRU486ormineralocorticoidreceptorswithspironolactone
caused up-regulation of renal cortical COX-2. Theseresults indicate
that COX-2 expression in situ is tonically inhibitedby adrenal
steroids, and COX-2 is regulated by mineralocorticoidsas well as
glucocorticoids.Prostaglandins,
cyclicoxygenatedderivativesofarachidonicacid,
mediatesignalingininflammationandmanynormalbiological processes.
Individual prostanoid species are generatedbyspecificsynthases, but
initial
commonprecursorsarepro-ducedbytherate-limitingenzymeprostaglandinG2H2syn-thase,
also known as cyclooxygenase (COX). In the late 1980s,it became
apparent that basal levels of COX activity could
bedistinguishedfromdynamic levels inducedby cytokines andendotoxins
and suppressed by glucocorticoids (1). Shortly there-after,
twodistinct COXgenesweredescribed: constitutiveCOX-1 encoding a
2.7- to2.9-kbtranscript andinducible,glucocorticoid-sensitive COX-2
encoding a 4.0- to 4.5-kb tran-script (2,
3).WhereasmuchresearchhasfocusedoncharacterizationofCOX-2regulationinculturedandorinflammatorycells,
ex-ceptionstotransientinducibilityandsteroidsuppressionhavebeendetectedincontrol
animalsatrestrictedepithelial sites.Sustained high levels of COX-2
expression were demonstrated inimmature rat kidney (4) and adult
rat vas deferens (5). At thesesites, sustained COX-2 expression was
not suppressed even byexcess exogenous steroids; in fact, continued
expression ofCOX-2 in the vas deferens was testosterone
dependent.In mammalian kidneys, prostaglandins are known to
regulaterenalhemodynamicsandsaltwaterhomeostasis. StudieswithCOX
inhibitors such as aspirin and indomethacin demonstratedimportant
roles for prostaglandins in regulating the
renin-angiotensinsystemthroughsignals generatedat the maculadensa,
but these data remained enigmatic because the predom-inant isoform,
COX-1, had been localized elsewhere; i.e., to
thepapillaryandcortical collectingducts, arteries,
andarterioles(6). Byusingimprovedmethodsof antigenpreservation,
ourlaboratoriesdetectedCOX-2immunoreactivity(COX-2-ir)incells of
themaculadensaandcortical thickascendinglimb(cTAL)of Henlesloop(7),
anddemonstratedup-regulationafter dietarysalt restriction.
Continuingstudies showedthatrenal cortical COX-2 expression was
up-regulated by otherphysiologicexperimentsinsitu; e.g., partial
renal ablation(8)and disruption of angiotensin signaling (9). Other
investigatorshave shown that COX-2 is less apparent in the macula
densa ofimmaturerats(10)but appearstoincreaseinthecTALandmacula
densa of human kidneys with aging
(11).Becauseglucocorticoidregulationwasahallmarkof
manyearlystudiesofCOX-2expression, itwasnaturaltopostulatethat
glucocorticoids could regulate renal COX-2; our
pilotstudiesshowingup-regulationafteradrenalectomy(ADX)in-dicated
that under control conditions, renal cortical COX-2 wastonically
suppressedby glucocorticoids as hadbeendemon-strated for
macrophages in vivo (12). However, additional com-plexity was
possiblebecausethekidney is sensitivetobothglucocorticoids and
mineralocortcoids, and both are eliminatedby ADX. In cultured cells
of renal origin, either
glucocorticoidsormineralocorticoidsdown-regulatedCOX-2expression(13),butotherstudiesfoundnoevidenceforsteroidregulationofrenal
COX-2 in situ (10, 14).To resolve apparent contradictions and
investigate the differ-ential contributions of glucocorticoids and
mineralocorticoids,we further studied the effects of maturity and
steroids. Exper-imental replacement of individual steroids after
ADX, andinhibition of steroid receptors in control intact animals,
revealedthat mineralocorticoids play predominant roles in
regulation ofrenal cortical COX-2 expression in mature adult
rats.Materials and MethodsAnimals. Male rats of Sprague-Dawley and
Long-Evans strains, aswell as F1 hybrids (LE-SD), were used. Under
sterile conditionsandnembutalanesthesia,
bilateralADXwasperformedviaasingle dorsal incision. After surgery,
ADX and sham-operatedcontrol rats received 1% NaCl in tap water ad
libitum to preventvolume depletion. Glucocorticoid or
mineralocorticoid replace-ment was achieved with subcutaneous
pellets (50% cholesterol)of deoxycorticosterone acetate (DOCA) or
corticosterone (CS),or daily injections toachieveadoseof 15mgkgday.
Theglucocorticoidreceptor(GR)antagonist, RU486,
andminer-alocorticoidreceptor (MR) antagonist, spironolactone,
weregiven at the dose of 7.5 and 20 mgkgday, respectively, eitherby
daily injections or subcutaneous pellets.Abbreviations: COX,
cyclooxygenase; COX-2-ir, COX-2immunoreactivity; cTAL,
corticalthick ascending limb; ADX, adrenalectomy; DOCA,
deoxycorticosterone acetate; GR, glu-cocorticoid receptor; MR,
mineralocorticoid receptor; PGE2, prostaglandin E2; CS,
cortico-sterone.To whom reprint requests should be addressed.
E-mail: [email protected] publication costs of
this article were defrayed in part by page charge payment.
Thisarticle must therefore be hereby marked advertisement in
accordance with 18 U.S.C.1734 solely to indicate this
fact.1528015285 PNAS December 21, 1999 vol. 96 no.
26Immunohistochemistry. Ingeneral, attheterminationofanex-periment,
one kidney of each rat was removed for Western
blotanalysis;theotherwasperfusedwithfixativeinsituforhisto-logical
examination. Under deep anesthesia with nembutal (70mgkg, i.p.),
rats were exsanguinated with 50 ml100 g
heparin-izedsaline(0.9%NaCl2units/ml heparin0.02%sodiumni-trite)
through a transcardial aortic cannula and fixed
withglutaraldehyde-periodate-acetate-saline as described (15).
Glu-taraldehyde-periodate-acetate-saline provides excellent
preser-vation of tissue structure, COX-2 antigenicity, and mRNA.
Thefixed kidney was dehydrated through a graded series of
ethanols,embedded in paraffin, sectioned (4m for
immunohistochem-istry, 10Mforinsituhybridization),
andmountedonglassslides. COX-2-ir was immunolocalizedwithrabbit
polyclonalanti-murine COX-2 antibody (Cayman Chemicals, Ann
Arbor,MI) at a 2.5 gml dilution. The primary antibodies
werelocalized by using Vectastain ABC-Elite (Vector, Burlingame,CA)
with diaminobenzidine as chromogen, followed by a lightcounterstain
with toluidine blue. The specificity of our
COX-2immunolocalizationwas confirmedbytwofundamental tests(16).
Staining was eliminated by preabsorption of the
primaryserumwithCOX-2proteinpurifiedfromthe rat distal vasdeferens
epithelium(5); COX-2-ir colocalized with COX-2mRNA detected by in
situ hybridization.Immunoblotting. Homogenates of
kidneycortex(10%wtvol)were prepared in 20 mM TrisCl, pH 8.01 mM
EGTA1 mMEDTA1 mM PMSF with proteinase inhibitor mixture
(Boehr-inger Mannheim). After a 10-min centrifugation at 10,000
g,thesupernatantwascentrifugedfor60minat100,000gtopreparemicrosomes
as described(7). Themicrosomes wereresuspended in homogenizing
buffer, mixed with an equalvolumeof2SDSsamplebuffer,
andboiledfor5min. Theproteins wereseparatedona10%SDSgel under
reducingconditions and transferred to Immobilon-P transfer
membranes(Millipore). The blots were blockedovernight with20
mMTrisCl pH 7.4500 mM NaCl5% nonfat milk0.05%
Tween-20,followedbyincubationfor3hatroomtemperaturewiththerabbit
polyclonal antiserum raised against murine COX-2 (Cay-man
Chemicals) at a 2.5 gml dilution. The primary antibodieswere
detected with goat anti-rabbit IgG-horseradish peroxidase(Santa
Cruz Biotechnology) and exposed on filmby usingenhanced
chemiluminescence (Amersham International).In Situ Hybridization.
Beginning with the saline exsanguination, allsolutions for this
protocol were prepared with deionized auto-claved water containing
0.1% diethyl pyrocarbonate.
Glutaral-dehyde-periodate-acetate-saline, as described above, was
asgood or better than all other fixatives examined. Before
hybrid-ization, sections were deparaffinized, treated with
proteinase K(5gml) for 20 min at room temperature, washed with
PBS,refixed in 3.7% formaldehyde, and treated with 0.1 M
trietha-nolamine, pH8.0, plusaceticanhydride(0.25%volvol), andthen
dehydrated through a graded series of ethanols.The sense and
antisense probes were synthesized by linearizingthe 1.3-kb 3-UTR
rat COX-2 fragment ligated into pBSK()and transcribing from the
flanking T7 or T3 promoters in thepresenceofdigoxigenin-UTP.
Theprobeswerehybridizedtosections at 50C for 18 h as described (4).
No signal was detectedin parallel hybridizations with sense
RNA.MicrosomeCOXActivityAssay. Kidneymicrosomes,
purifiedasdescribed above, were preincubated at 37C in the presence
of 4M hematin with COX inhibitors at appropriate concentrationsas
determined (8, 17, 18). After 15 min, an equal volumecontaining
100M arachidonic acid was added to initiate thereaction. After
10minat 37C, thereactionwas stoppedbyboiling 1 min. Production of
prostaglandin E2(PGE2)
wasdeterminedimmediatelybyenzymeimmunoassay(AmershamInternational).Micrography.
Bright-field images from the Leitz Orthoplan mi-croscope with DCV
digital RGB video camera were digitized bythe BIOQUANT TCW image
analysis system and saved as computerfiles. Contrastandcolor-level
adjustment(AdobePhotoshop)were performed for the entire image;
i.e., no region- or object-specific editing or enhancements were
performed.ResultsCOX-2 Expression After ADX. Under control
conditions with intactadrenal glands, kidneys from mature adult
rats (males 250 g)immunostained to localize COX-2 were
indistinguishable fromthoseillustratedinFig. 1AandB(7).
IntenseCOX-2-irwasapparentinoneortwoisolatedcellsinsomecTALnearthemacula
densa but rarely in the macula densa itself. COX-2-ir alsowas
detectedininterstitial macrophages that accountedfor1020% of the
total cortical COX-2 in normal specimens.Two weeks after ADX,
widespread increases in COX-2-ir wereapparentinthecTAL(Fig.
1C)andmaculadensa(Fig. 1D).However, the histological pattern of
COX-2 cells was differentin these two loci. In the cTAL, groups of
unstained cells wereinterspersedwithstainedcells(Fig. 1E); i.e.,
COX-2wasex-pressedinsomecells but was not
generalizedtothecTALepithelium. At the macula densa, intense
COX-2-ir generally wasexhibited by all of the macula densa cells
(Fig. 1E).To examine COX-2 up-regulation at the level of
transcriptionandconfirmthespecificityoftheincreasedimmunoreactivityafter
ADX, insituhybridizationwas usedtodetect COX-2mRNA. In control
rats, COX-2 mRNAexhibited a sparsedistribution in renal cortex
similar to that seen for COX-2-ir (notillustrated). Two weeks after
ADX, up-regulation was apparentin the cTAL and strong signals were
detected consistently in themacula densa (Fig.
1F).Subcellularfractionspreparedfromrenal
cortexweresub-jectedtoWesternblotanalysis;
pilotexperimentsdeterminedthat virtually all COX-2 was contained in
the microsome frac-tion. Incontrolspecimens,
theCOX-2-irbandat73kDawasweak (Fig. 2, lane 1); 1 week after ADX,
the COX-2 band hadincreased moderately (lane 2), and by 2 weeks
after ADX, theCOX-2signalwasmaximal(lane3).
WesternblotsofCOX-2customarily display a doublet representing
variable posttransla-tional modifications (7); we have been unable
to correlateconsistent changes in the ratio of these bands with
ADX.Identicalsamplesimmunoblottedfortheconstitutiveisoform,COX-1,
showed no changes in response to ADX(not illustrated).Hormone
Replacements. Glucocorticoids, the adrenal steroids
thathavebeenshowntosuppressCOX-2up-regulationininflam-matory cells,
are eliminated by ADX. To test for similar effectsin renal cortex,
CS, the predominant glucocorticoid in rats,
wasadministeredtoADXratsviadailyinjectionorsubcutaneouspellets.
Identical results wereobtainedfor bothmethods ofadministration. In
a representative experiment shown in Fig. 2,five of six sibling
male rats were adrenalectomized on the sameday and maintained for 2
weeks on normal food supplementedwith 1% salt water to prevent
volume depletion. Kidneys fromthe ADX specimen that received no
additional treatment exhib-ited maximal COX-2 up-regulation as
described above (lane 3).CSwasadministeredaccordingtotwoschedules.
Todeter-mine whether CS prevented COX-2 up-regulation, one
ratreceived CS replacement from the time of ADX through the 2weeks
survival (lane 4). Cortical COX-2 remained down-regulated,
approximating control levels. To determine whetherup-regulated
COX-2 could be down-regulated by exogenous
CS,anotherrat(lane5)receivednosteroidduringthefirstweekfollowingADX.
Presumably, duringthisperiod, renalcorticalZhang et al. PNAS
December 21, 1999 vol. 96 no. 26 15281PHYSIOLOGYFig. 1. (Legend
appears at the bottom of the opposite page.)15282 www.pnas.org
Zhang et al.COX-2 in this rat was up-regulated to levels shown for
the 1 weekADX specimen (lane 2). On receiving CS replacement
duringthe second week of its 2 weeks ADX, cortical COX-2
expressioninthis rat hadreturnedtocontrol levels (lane 5).
Parallelhistologic data were obtained by perfusing one kidney from
eachratforimmunohistochemicalstaining.ADXkidneysreceivingsteroidreplacementexhibitedsparsescatteredCOX-2cellsinthecTALnearthemaculadensa(Fig.
1AandB)andwereindistinguishable from controls.In experiments
originally conceived as negative controls, themineralocorticoid
DOCA, a long-lived aldosterone analog, wasadministered to ADX rats.
Surprisingly, DOCA also preventedup-regulationof COX-2intherenal
cortex(lane6) andordown-regulated COX-2 after up-regulation (not
illustrated).Thus, COX-2 expression in renal cortex was suppressed
by ADXreplacement therapy withmineralocorticoids as well as
glu-cocorticoids. Furthermore, because glucocorticoids can
alsostimulate MR, these data raised the possibility that
predominantregulation of cortical COX-2 was through the MR
pathway.Receptor Antagonists. To determine whether renal
corticalCOX-2expressionnormally is suppressedby basal levels
ofsteroids acting through the MR andor GR pathways, compet-itive
inhibitors of the steroid receptors were administered dailyfor 2
weeks to mature rats with intact adrenals. Compared withcontrol
(Fig. 3, lane 1), some up-regulationof COX-2 wasapparent after
treatment with RU486, a putative GR antagonist(lane 2). Strong
up-regulation of COX-2 (equivalent to ADX)was induced by treatment
with the MR antagonist spironolac-tone (lane 3), andnofurther
increase inCOX-2 couldbeappreciated with RU486 and spironolactone
together (lane 4).Histologically, the COX-2 up-regulation induced
by the inhibi-tors was focused at the macula densa. In the
specimens treatedwith RU486, COX-2-ir was observed at approximately
30% ofthe macula densa (Fig. 1G); in spironolactone specimens,
COX-2-ir was observed in nearly every macula densa and was
moreintense (Fig. 1H). In comparison with ADX rats, fewer
COX-2cells were observed in cTAL after inhibitor treatment.Further
experiments wereundertakentotest whether theinhibitor effects could
be overwhelmed by exogenous steroids.Normal
ratstreatedwitheitherRU486orspironolactoneasabove also received
either CS or DOCA at doses adequate tosuppress COX-2 up-regulation
in ADX. Exogenous DOCAstrongly inhibited the induction of COX-2 by
spironolactone andtotallyabolishedtheeffects of RU486, whereas
CSpartiallyinhibited the effects of spironolactone but had less
influence onRU486.COX Activity. To determine whether increases in
immunoreactiveCOX-2 protein represented increased enzymatic
activity, PGE2production was assayed in vitro. Total COX activity
was assayedas PGE2productionintheabsenceof COXinhibitors;
thisactivity was totally inhibited by the nonspecific COX
inhibitor,indomethacin (104M). SC-58236, a COX inhibitor that
report-edlydisplays1,700-foldselectivityforCOX-2COX-1,wasused at
105M to block COX-2 activity with negligible
effectsonCOX-1activity.
COX-1activitywasassayedasPGE2pro-ducedinthepresenceof SC-58236;
COX-2activitywas theamount of PGE2 production suppressed by
SC-58236. In com-parison with control rats, total renal cortical
COXactivityincreased 2-fold 2 weeks after ADX (6.85 0.3 vs. 13.50
1.29PGE2 ngminmg); COX-1 activity did not change
appreciably(5.600.57vs. 6.601.07PGE2ngminmg), but COX-2activity
increased6-fold(1.25 0.57 vs. 6.90 1.07 PGE2ngminmg) (Fig. 4).
Thus, the increases in renal cortical COXactivityafter 2weeks
ADXweredueprimarilytoincreasedCOX-2, but not COX-1, activity. The
enzymatic assays comple-mented immunohistochemical, Western blot,
and in situ hybrid-ization data to demonstrate that COX-2 in renal
cortex increasedsignificantly after 2 weeks ADX.DiscussionThe
present studies demonstrated that COX-2 expression in therenal
cortexof maturerats is regulatedbyadrenal steroids.Although little
COX-2 was apparent in control rats, substantialincreases were noted
after ADX. Both mRNA and protein wereup-regulated in the cTAL and
macula densa, and COX-2 activityin cortical microsome fractions
increased 6-fold. The effects ofADX were prevented or reversed by
steroid replacement, andmimickedincontrol rats bytreatment
withsteroidreceptorantagonists. This report identifies factors that
regulate COX-2expressioninthemaculadensa, anddemonstratesthat
renalcortical COX-2 is under tonic regulation by circulating
adrenalcorticosteroids. Furthermore, thedatasuggest that
mineralo-corticoids are the dominant adrenal steroids regulating
expres-sion of COX-2 in the renal cortex.In considering the roles
of renal COX-2, we are reminded thatprostaglandins serveas
feedbackmessengers controllingtheFig. 2. Effects of ADXand steroid
replacement therapy on COX-2 expressioninrat kidneycortex.
Immunoblots of microsomes: lane1, faint signal insham-operated
control rat; lane 2, increased signal after ADXfor 1 week; lane3,
maximum signal after 2 weeks ADX; lane 4, 2 weeks ADX with
concurrentCS replacement; lane 5, 2 weeks ADX with CS replacement
after 1 week; andlane 6, 2 weeks ADX with concurrent DOCA
replacement.Fig. 3. Effects of steroid receptor inhibitors on renal
cortical COX-2 expres-sion. Immunoblots of microsomes after a
2-week treatment: lane 1, control;lane 2, RU486; lane 3,
spironolactone; and lane 4, RU486 spironolactone.Fig. 1. (On the
opposite page.) Histological sections of kidney cortex fromadult
rats (250 g). (Aand B) After ADXcombined with CS replacement for 2
weeks,intense COX-2-ir is observed in macrophages () and a very few
scattered cTAL cells, but generally is absent from the macula densa
(). These images areindistinguishable from controls. (C and D) ADX
littermate without CS replacement shows intense COX-2-ir in some
cTAL () and nearly all macula densa (). (E)At higher magnication,
it is apparent that COX-2-ir lls the cytoplasmof macula densa cells
() as well as cTAL cells in the opposite wall. (F) In situ
hybridizationofADXkidneydemonstratesCOX-2mRNAatsitesidenticaltoCOX-2-ir.(GandH)COX-2expressionisobservedatthemaculadensa()incontrolratsadministered
RU486 (G) or spironolactone (H) to block steroid receptors (GR and
MR, respectively). (Figure widths: A and C, 2.4 mm; B, D, and F,
600 m; E, G,and H 180 m.)Zhang et al. PNAS December 21, 1999 vol.
96 no. 26 15283PHYSIOLOGYrenin-angiotensin-aldosterone system in
the kidney. Renin, thespecific proteinase that generates the
peptide angiotensin thatstimulates aldosterone release from the
adrenal, is synthesizedand stored in granule cells at the vascular
pole of the glomerulusand released in response to signals fromthe
macula densa. Reninrelease was previously shown to be suppressed by
nonselectiveCOX inhibitors such as aspirin or indomethacin, and
stimulatedby exogenous prostaglandins (19).Detection of COX-2
protein and mRNA at the macula densaunder conditions of increased
renin expression and releasesuggested a role for macula densa COX-2
in mediation of reninrelease(7, 2022). AlthoughCOX-2-ir is
rarelyobservedinmacula densa cells under control conditions, it is
possible thatadequate COX-2 activity normally is below the limits
of immu-nohistochemicaldetection.
Recentexperimentsdemonstratingthat specific COX-2 inhibitors
suppress renin levels in the plasmaand kidney (8, 9, 23) further
support the concept that COX-2 atthe macula densa plays a role in
regulating renin synthesis andsecretion.The present studies
confirmed original reports of Harris et al.(7)that
COX-2-irinmatureratsundercontrol conditionsisdetectedinasmall
populationof cellslocatedmostlyinthecTAL with a minor component in
the macula densa. Two weeksafter ADX, an increased number of COX-2
cells was apparentin the cTAL, but the most dramatic changes
involved the maculadensa. COX-2 was up-regulated in the macula
densa of nearlyevery nephron; the COX-2-positive cells were tightly
packed andincluded most of the macula itself. Based on this
evidence, wepostulatedthatundercontrolconditions,
physiologiclevelsofadrenal steroids tonically suppressed COX-2
expression in thecTALandmaculadensa. This hypothesis was
supportedbyexperiments demonstrating that COX-2 up-regulation in
ADXrats was inlarge part preventedby replacement of
missingglucocorticoids with CS via subcutaneous pellets. If the
replace-ment therapy was delayedfor 2weeks after ADXtoallowmaximal
up-regulation of COX-2, treatment with exogenous
CSfor2subsequentweeksdown-regulatedrenal cortical
COX-2nearlytocontrol levels. Itiswell
knownthatglucocorticoidssuppress COX-2 expression in inflammatory
cells (14,
2427),andtheresultsofourADXandCSreplacementexperimentswere
consistent with that pattern.The kidney is a major target of
aldosterone, the highlyconservedmineralocorticoidthat stimulates
salt retentioninnearlyall vertebrates(28, 29);
asdemonstratedintransgenicmice, the MRs are essential for survival
(30) The kidney is wellendowed with MR; they have been localized to
all segments ofthe nephron except the proximal tubules (31).
However, unlikeGR,
MRsarenonselectiveandequivalentlyactivatedbyglu-cocorticoids.
Specificity of MRaction is achieved throughinactivation of
glucocorticoids by 11-hydroxysteroid dehydro-genase type II
(11-HSD2). High levels of 11-HSD2 found inthe cortical collecting
duct imply that MRs in the collecting
ductarestrictlysensitivetoaldosterone. Ontheotherhand, lowerlevels
of 11-HSD2 are detected in the cTAL and macula densa,suggesting
that glucocorticoids might stimulate the MR at thesesites(31).
Thus, thehighcirculatinglevelsofCSachievedinreplacement
therapycouldalsohaveeffectedCOX-2down-regulationby activating the
MRpathway inthe cTALandmacula densa. Therefore, we postulated that
renal COX-2regulation might involve mineralocorticoids and the
MR.Our laboratories previously reported COX-2 up-regulation inthe
macula densa and cTAL in response to angiotensin-converting enzyme
inhibitors and angiotensin receptor antago-nists that interfere
with production or activation of angiotensinII (9). One consequence
of these inhibitors is to prevent stim-ulation of aldosterone
release from the adrenal cortex withoutinterfering with
glucocorticoid production. Thus, when aldoste-rone activation of MR
was compromised, normal tonic levels ofglucocorticoids were
insufficient to suppress COX-2 expressionin the macula densa.
Therefore, in addition to any direct effectsof angiotensin II and
the AT1 receptors on regulation of COX-2expression, inhibition of
circulating mineralocorticoids may havecontributed to the increases
in COX-2 seen in these studies.MR involvement was tested directly
by administration of thealdosterone analog, DOCA, to ADX rats.
Renal cortical COX-2was suppressed to undetectable levels. Because
DOCA has verylow GR agonist activity, these data indicated that MR
activationalone was sufficient to down-regulate COX-2. The
importance ofMR was supported further by experiments with steroid
receptorantagonists. Incontrol ratswithintactadrenals,
inhibitionofMRwith spironolactone produced strong up-regulation
ofCOX-2 in renal cortical microsomes analyzed by Western
blotsandintenseCOX-2-irinnearlyeverymaculadensaexaminedhistologically.
Spironolactone effects were strongly inhibited byexogenous DOCA and
weakly inhibited by exogenous CS. Over-all, the spironolactone data
are consistent with a strong influ-ence of MR on COX-2
regulation.Experiments with the GR antagonist RU486 produced
similarbutlesspronouncedeffects. Incontrolrats,
RU486producedsignificantbutsubmaximalCOX-2up-regulation.
TheRU486effects were abolished by exogenous DOCA but affected
mini-mally by exogenous CS. The response was not dose
dependent;i.e.,
doublingthedoseofRU486didnotincreasetheCOX-2expression.
Furthermore, the spironolactone and RU486 effectswere not additive;
the response to spironolactone was
maximal.Thus,maculadensaCOX-2expressionisdown-regulatedtoagreater
degree by mineralocorticoids than glucocorticoids.In summary, our
studies demonstrate that COX-2 in the cTALand macula densa is under
tonic regulation by adrenal steroids,and it is up-regulated under
stimuli that call for increased reninsynthesis andrelease. It will
beof interest inthefuturetoinvestigate signaling pathways that
regulate COX-2 by
directlyinfluencingthesteroidsandtheirreceptors.Detailsregardingthetranscriptional
regulationandroles of
COX-2inkidneyfunctionmayassumeincreasingrelevanceas
specificCOX-2inhibitors are employed as clinical anti-inflammatory
agents.WethankDr. LarryMarnettforhiscollegialcritique.
Thisworkwasperformed in the George M. OBrien Center for Kidney and
UrologicDiseases, which is supported by National Institutes of
Health Grant DK39261 and funds from the Department of Veterans
Affairs.Fig. 4. COX-2activityofrenal cortical
microsomeswasassayedasPGE2producedby equivalent samples. Twoweeks
after ADX, total activity doubled;COX-1activity (determinedas
PGE2producedinthepresenceof specic COX-2inhibitor) remained
unchanged; COX-2 activity (the difference between Totaland COX-1
activities) increased 6-fold.15284 www.pnas.org Zhang et al.1. Raz,
A., Wyche, A., Fagan, D. & Needleman, P. (1989) Adv. Exp. Med.
Biol.259, 121.2. Kujubu, D. A., Fletcher, B. S., Varnum, B. C.,
Lim, R. W. & Herschman, H. R.(1991) J. Biol. Chem. 266,
1286612872.3. OBanion, M. K., Sadowski, H. B., Winn, V. &
Young, D. A. (1991) J. Biol.Chem. 266, 2326123267.4. Zhang, M. Z.,
Wang, J. L., Cheng, H. F., Harris, R. C. & McKanna, J. A.
(1997)Am. J. Physiol. 273, F994F1002.5. McKanna, J. A., Zhang, M.
Z., Wang, J. L., Cheng, H. & Harris, R. C. (1998)Am. J.
Physiol. 275, R227R233.6. Smith, W. L. & Bell, T. G. (1978) Am.
J. Physiol. 235, F451F457.7. Harris, R. C., McKanna, J. A., Akai,
Y., Jacobson, H. R., DuBois, R. N. &Breyer, M. D. (1994) J.
Clin. Invest. 94, 25042510.8. Wang, J. L., Cheng, H. F., Zhang, M.
Z., McKanna, J. A. & Harris, R. C. (1998)Am. J. Physiol. 275,
F613F622.9. Cheng, H.-F., Wang, J.-L., Zhang, M.-Z., Miyazaki, Y.,
Ichikawa, I., McKanna,J. A. & Harris, R. C. (1999) J. Clin.
Invest. 103, 110.10. Vio, C. P., Cespedes, C., Gallardo, P. &
Masferrer, J. L. (1997) Hypertension30, 687692.11. Nantel, F.,
Meadows, E., Denis, D., Connolly, B., Metters, K. M. & Giaid,
A.(1999) FEBS Lett. 457, 475477.12. Masferrer, J. L., Reddy, S. T.,
Zweifel, B. S., Seibert, K., Needleman, P.,Gilbert, R. S.
&Herschman, H. R. (1994) J. Pharmacol. Exp. Ther.
270,13401344.13. Schaefers, H. J. &Goppelt-Struebe, M. (1996)
Biochem. Pharmacol. 52,14151421.14. Masferrer, J. L., Seibert, K.,
Zweifel, B. & Needleman, P. (1992) Proc. Natl.Acad. Sci. USA
89, 39173921.15. McKanna, J. A. & Zhang, M. Z. (1997) J.
Histochem. Cytochem. 45, 527538.16. Larsson, L. I. (1988)
Immunocytochemistry: Theory and Practice (CRC, BocaRaton, FL), pp.
636.17. Capdevila, J. H., Morrow, J. D., Belosludtsev, Y. Y.,
Beauchamp, D. R.,DuBois, R. N. & Falck, J. R. (1995)
Biochemistry 34, 33253337.18. Penning, T. D., Talley, J. J.,
Bertenshaw, S. R., Carter, J. S., Collins, P. W.,Docter, S.,
Graneto, M. J., Lee, L. F., Malecha, J. W., Miyashiro, J. M., et
al.(1997) J. Med. Chem. 40, 13471365.19. Schnermann, J.
&Briggs, J. P. (1992)inTheKidney:PhysiologyandPatho-physiology,
eds. Seldin, D. W. & Giebisch, G. (Raven, New York),
pp.12491289.20. Jensen, B. L. & Kurtz, A. (1997) Kidney Int.
52, 12421249.21. Yang, T., Singh, I., Pham, H., Sun, D., Smart, A.,
Schnermann, J. B. & Briggs,J. P. (1998) Am. J. Physiol. 274,
F481F489.22. Hartner, A., Goppelt-Struebe, M. &Hilgers, K. F.
(1998)Hypertension31,201205.23. Harding, P., Sigmon, D. H., Alfie,
M. E., Huang, P. L., Fishman, M. C.,Beierwaltes, W. H. &
Carretero, O. A. (1997) Hypertension 29, 297302.24. Kujubu, D. A.
& Herschman, H. R. (1992) J. Biol. Chem. 267, 79917994.25.
OBanion, M. K., Winn, V. D. &Young, D. A. (1992) Proc. Natl.
Acad. Sci. USA89, 48884892.26. Newman, S. P., Flower, R. J. &
Croxtall, J. D. (1994) Biochem. Biophys. Res.Commun. 202,
931939.27. Needleman, P. & Isakson, P. C. (1997) J. Rheumatol.
24, Suppl. 49, 68.28. Rossier, B. C. & Palmer, G. (1992) in The
Kidney: Physiology and Pathophys-iology, eds. Seldin, D. W. &
Giebisch, G. (Raven, New York), pp.15591560.29. Funder, J. W.,
Krozowski, Z., Myles, K., Sato, A., Sheppard, K. E. & Young,M.
(1997) Recent Prog. Horm. Res. 52, 247260.30. Berger, S., Bleich,
M., Schmid, W., Cole, T. J., Peters, J., Watanabe, H., Kriz,W.,
Warth, R., Greger, R. & Schutz, G. (1998) Proc. Natl. Acad.
Sci. USA 95,94249429.31. Bostonjoglo, M., Reeves, W. B., Reilly, R.
F., Velazquez, H., Robertson, N.,Litwack, G., Morsing, P., Dorup,
J., Bachmann, S. &Ellison, D. H. (1998)J. Am. Soc. Nephrol. 9,
13471358.Zhang et al. PNAS December 21, 1999 vol. 96 no. 26
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