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作者：MorganGallazzini, AmelAttmane-Elakeb, David B.Mount, Steven C.Hebert, MauriceBichara作者单位：1 Institut National de la Santé et de la RechercheMédicale U.42 Institut Fédératif RégionalClaude Bernard, Faculté de Médecine Xavier Bichat,Université Paris 75018 Paris, France; RenalDivision, West Roxbury Veterans Administration Medical Cent
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【摘要】5 t q0 n8 J3 R2 O- W
Mechanismsof regulation of ROMK channel mRNA and protein expression in medullarythick ascending limb (MTAL) were assessed in rat MTAL fragmentsincubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCRand ROMK protein by immunoblotting analysis of crude membranes. Mediumhyperosmolality (450 mosmol/kgH 2 O; NaCl plus urea added toisoosmotic medium) increased ROMK mRNA ( P P increased ROMK mRNA ( P had no additive effects on ROMK mRNA. NaCl alone, but noturea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8- D -arginine) vasopressin (1 nM) or 0.5 mM8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However,8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMKmRNA in the isoosmotic but not in the hyperosmotic medium( P In in vivo studies, the abundance of ROMKprotein and mRNA increased in adrenalectomized (ADX) rats infused withdexamethasone compared with ADX rats ( P and medium NaCl concentration asdirect regulators of MTAL ROMK mRNA and protein expression, which maybe modulated by cAMP-dependent factors.
m3 x+ s' N8 ?. ~5 } 【关键词】 regulation of gene expression ROMK channel immunoblottinganalysis quantitative reverse transcriptasepolymerase chain reaction isolated tubules
+ v* k0 }" V1 r. \& _3 ^: N INTRODUCTION
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# d6 n) P, H& L: q8 z! cIN THE THICK ASCENDING LIMB (TAL) of the nephron, potassium that enters the cell by theactivity of the apicalNa -K (NH 4 )-2Cl cotransporter is largely recycled back into the lumen through potassiumchannels ( 5, 17, 32 ). This K recycling has amajor role in NaCl reabsorption by the TAL by providing a potassiumsupply to the cotransporter and establishing the lumen-positivetransepithelial potential difference that provides the driving forcefor sodium reabsorption through the paracellular pathway. ROMK channelsare believed to constitute the major K secretory pathwayin the distal nephron ( 15, 28 ). Indeed, the ROMK proteinhas been localized by antibodies at the apical membrane of cells of thepart of the renal tubule extending from the beginning of the TAL to theinitial portion of the inner medullary collecting duct ( 19, 25, 33 ). The essential role of ROMK channels in TAL transportfunctions and thus in the renal regulation of sodium and water balanceis demonstrated by the fact that mutations in the ROMK gene causeBartter's syndrome, which is characterized by severe salt wasting andimpaired urinary concentrating ability ( 14, 18, 30 ).% E" B/ j2 j/ J3 Y/ u' A$ u
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It is well established that the activity or the membrane density ofROMK channels expressed in Xenopus laevis oocytes or in HEK293 cells is acutely regulated by cAMP-dependent protein kinase ( 20, 23, 34 ), arachidonic acid and protein kinase C( 21, 22 ), protein-tyrosine phosphatase andprotein-tyrosine kinase ( 26 ), and interactions withassociated proteins ( 31 ). In contrast, little is knownabout the chronic regulation of ROMK expression in the TAL. Ecelbargeret al. ( 13 ) showed that the abundance of ROMK protein inthe rat outer medulla is augmented by1-deamino-(8- D -arginine)-vasopressin (dDAVP), restrictionof water intake, and high levels of sodium intake, and decreased by lowlevels of sodium intake. However, the direct stimuli and cellularmechanisms of these chronic regulations of ROMK expression in the TALare unknown. Possible candidate mechanisms include direct effects ofcAMP-dependent pathways and the variations in the osmolality of thesurrounding medullary interstitium that accompany the states of waterdiuresis and antidiuresis. In addition, beside adenylyl cyclase-coupledreceptors, the TAL possesses specific glucocorticoid receptors (GR),the activation of which exerts important actions on TAL transportfunctions. Indeed, the glucocorticoid dexamethasone has been shown tostimulate Na -K -ATPase activity within a fewhours ( 12, 29 ). More recently, we showed thatdexamethasone increases both the expression and activity of theNa -K (NH 4 )-2Cl cotransporter BSC1/NKCC2 of MTALs incubated in vitro ( 4 ). Of relevance to these observations, glucocorticoids have long beenknown to contribute to the renal urinary concentrating ability, atleast in part, through the maintenance of medullary hyperosmolality ( 11 ).
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These considerations prompted us to assess directly in vitro thepossible regulation by osmolality, cAMP, and glucocorticoids of theexpression of ROMK mRNA and protein in fragments of medullary TAL(MTAL) in suspension. To this end, we used the MTAL "shake" suspension previously described ( 2, 3 ). The resultsestablish that ROMK mRNA and protein abundance are regulated directlyby the osmolality of the incubation medium and glucocorticoids not bydDAVP or cAMP. We also show that in vivo dexamethasone administration increases the abundance of ROMK mRNA and protein in the MTAL.8 S& ~8 ?7 @ y( K n
! M; A X# E- _, r S1 [MATERIALS AND METHODS
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In Vivo Studies
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6 B/ b ]7 H. R- A# v6 jMale Sprague-Dawley rats (250-300 g) were used and had freeaccess to standard rat chow and drinking solution until the time of theexperiment. Rats were adrenalectomized (ADX) under light etheranesthesia and given, as drinking solution, distilled water containing0.9% NaCl for 6 days before the experiment. A microosmotic pump (Alza,Palo Alto, CA) was implanted subcutaneously in the nape of some of theADX rats, through which we delivered 1.2 µg · 100 g bodywt 1 · day 1 of the glucocorticoidhormone dexamethasone, a dose that is known to restore normalglucocorticoid activity, for 6 days (ADX Dexa). These rats alsohad access to 0.9% NaCl in distilled water. Control rats were shamoperated under ether anesthesia and had access to water or to distilledwater containing 0.9% of NaCl. After pentobarbital sodium anesthesia,the kidneys were rapidly removed and cut into thin slices along thecorticopapillary axis and, under a dissecting microscope, the innerstripe of outer medulla of each slice was excised and cut into uniformsmall pieces that were used for immunoblotting of membrane proteins andmRNA determinations.; S# o1 O! M7 u8 W9 P% Z; I2 F
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In Vitro Studies
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" @" P( |/ M2 D) t% D/ H7 N% iSuspension of rat MTAL tubules. The method used to isolate MTAL fragments in suspension has beenpreviously described ( 1 ). We established by light and electron microscopy that this suspension was made almost exclusively ofMTALs ( 95%), occasional thin limbs, and rare outer medullary collecting tubules, with no isolated cells or proximal tubules ( 1-3 ). The MTAL fragments were washed in a 1:1mixture of Dulbecco's modified Eagle's medium and Ham's nutrientmixture F-12 supplemented with 5 mM heptanoic acid, 5 mM L -leucin, 0.1 g/l bovine serum albumin, 200 IU/mlpenicillin G, 250 µg/ml streptomycin, 10 µg/ml minocyclin, 15 mMHEPES, 10 mM Tris, and 25 mM NaHCO 3, pH 7.35, when gassedwith 95% O 2 -5% CO 2 (HDMEM). The MTALs werethen suspended in 60 ml of this medium, placed in a rotary (100 rpm)shaking water bath at 37°C, and gassed with a humidified 95%O 2 -5% CO 2 gas mixture. According to resultsobtained in a previous study from this laboratory ( 2 ), theMTAL shake suspension was allowed to stabilize during the first 9 h of incubation. Then, samples of MTALs were further incubated for7 h in the presence or absence of 10 nM dexamethasone, 1 nM dDAVP,or 0.5 mM 8-bromo-cAMP in is- or hyperosmotic media. We checked in fourpreliminary experiments that ROMK mRNA abundance was stable during thisexperimental time under control conditions (0.96 ± 0.17 vs.0.85 ± 0.16 amol/100 ng RNAtot; not significant; the quantitativeRT-PCR is described below).
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Crude Membrane Preparation
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- C/ K. d0 s$ H% [Tissues from inner stripe of outer medulla dissection or fromMTAL suspensions were homogenized in a medium composed of 150 mMsucrose, 12 mM Trizma (Tris base, pH 7.4), 0.1 mM4-(2-amino-ethyl)-benzenesulfonyl fluoride, and 5 µg/ml leupeptin.These homogenates were centrifuged at 1,000 g for 5 min in aBeckman GS-6KR centrifuge with a G-H3.7 rotor, and the supernatantswere further centrifuged at 200,000 g for 60 min in aBeckman L-70 Ultracentrifuge with a 70 TI rotor. The membrane pelletswere suspended in the above medium and stored at 80°C until use.
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7 C7 f( v3 F* z' A4 S8 }# bElectrophoresis and Immunoblotting of Membrane Proteins
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( F5 i+ }$ b, q; G% @Semiquantification of membrane protein amounts was performed byimmunoblotting after SDS-PAGE. Membranes were solubilized first atambient temperature for 20 min in Laemmli medium containing 62.5 mMTris · HCl (pH 6.8), 5% SDS, 100 mM dithiothreitol, and 10%glycerol, then at 65°C for 10 min in the same medium. Samples containing 7 to 20 µg of proteins were loaded into individual lanesof 10% polyacrylamide minigels (Bio-Rad). Proteins were electrophoretically transferred from the gels to nitrocellulose membranes (Bio-Rad). Equal loading and transfer efficiency were systematically checked by Ponceau red staining of the nitrocellulose membranes. After 1 h of blocking at 37°C with TBS/T containing 5% nonfat milk powder, membranes were exposed overnight at 4°C to anaffinity-purified polyclonal anti-ROMK rabbit antibody (APC001, AlomoneLabs, Jerusalem, Israel) diluted 1:150. This antibody has beenpreviously documented to reveal both native and heterologously expressed ROMK protein as a ~45-kDa band ( 24 ), whichappeared as a 42- to 45-kDa doublet in the present study in MTAL total membranes. The nitrocellulose membranes were then exposed to a horseradish peroxydase-linked anti-rabbit Ig secondary antibody (Bio-Rad) for 1.5 h at ambient temperature. Antibody-antigencomplexes were detected using luminol-based enhanced chemiluminescence(Amersham-Pharmacia Biotech) before exposure to X-ray film (Fujifilm).As indicated by the manufacturer, the APC001 anti-ROMK antibody alsoreveals a band of unknown identity of 90 kDa. Both the ROMK 42- to45-kDa doublet and the 90-kDa bands were analyzed by densitometry with use of public domain National Institutes of Health (NIH) Image 1.62 software. The 90-kDa band was used as a control because, as will beshown below, it does not respond like ROMK to the various experimental conditions.) K! G- h6 ~! @7 Z5 h
2 e( j" B- r5 [1 |* d+ CRNA Extraction, Reverse Transcription, and PCR, a! S0 S! O4 l
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Total RNA (RNA tot ) was extracted from aliquots ofkidney MTAL with use of the SV Total RNA Isolation System kit(Promega). To obtain competitor RNAs (RNA c ) that differedfrom the wild-type ROMK1, 2, 3 mRNAs, deletions of 99 bp, located inthe core exon common to all ROMK isoforms, of the ROMK1, 2, and 3 plasmids were obtained by digestion with Bgl II and Msc I restrictionenzymes (from bp 692 to 790 of ROMK1), followed by blunt-end ligation of the cut plasmids. The deleted ROMK plasmids were subcloned andlinearized with Not I restriction enzyme. In vitrotranscription was then performed with the use of T7 RNA polymerase(mCAP RNA Capping kit, Stratagene, La Jolla, CA) and[ 32 P]UTP. The amounts of transcribed RNA c were determined by the measure of its optical density at 260 nmcorrected for the ratio of TCA-precipitated RNA c to totalRNA c determined by liquid scintillation spectroscopy. Wethus obtained three RNA c that gave identical results in theRT-PCR described below." Y! W$ k. ]( ?' j9 i3 X" S
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The primers (GIBCO BRL) for cDNA synthesis and PCR amplification werechosen from the published ROMK1, 2, and 3 sequences with the help ofOligo 4.04 Primer Analysis software (National Biosciences, Plymouth,MN). The sequences of the primers were 5'-GAC CTC CCA GAG TTC TAC-3'(sense) and 5'-AGG GCT GTT GTG GTC AAT AA-3' (antisense). The senseprimer was directed against a segment common to ROMK1, 2, 3, and 6 butlocated within the intron retained in the ROMK core exon. This retainedintron is subject to low-frequency alternative splicing that generatesa set of truncated hydrophilic ROMK isoforms of unknown function, which thus were not amplified by our method. The primers used in the presentstudy yielded, as expected, only one PCR product common to ROMK1, 2, and 3 of 510 bp from MTAL RNA tot and the ROMK cDNA plasmidsand of 411 bp from the RNA c and the deleted ROMK plasmids, respectively. After 30 PCR cycles, PCR products resulting from nonspecific hybridization were never observed. The identities of thePCR products were confirmed by digestion with Sal I, which generated two bands of 74 and 436 bp from the wild DNA and of 74 and337 bp from the competitive DNA as expected.
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cDNAs were synthesized from MTAL RNA tot andRNA c by reverse transcription at 37°C for 60 min with 200 U Moloney murine leukemia virus reverse transcriptase (LifeTechnologies), 30 pmol of antisense primer, 4 µg of yeast transferRNA, 1 mM of each deoxyribonucleotide (dNTP), 10 mM DTT, 2 U ofribonuclease inhibitor (GIBCO BRL), and RT buffer in a final volume of22 µl. Reverse transcriptase was then inactivated at 95°C for 5 min. Each reaction was performed in parallel with an otherwiseidentical one that contained no reverse transcriptase.
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: t# D6 Z4 [8 I: O' E9 I: h) S3 QFor PCR, 10 µl of the cDNA solution were supplemented with PCRbuffer, 5.4 mM MgCl 2, 30 pmol of sense and antisenseprimers, 1 mM of each dNTP, and 5 U of Taq DNA polymerase(Life Technologies) in a final volume of 50 µl. Samples weredenatured at 94°C for 5 min, which were followed by cycles consistingof denaturation at 94°C (1 min), annealing at 50°C (1 min), andextension at 72°C (1.5 min). PCR was completed by a final extensionstep at 72°C for 10 min. Quantitative PCR was performed using 27 cycles of amplification of cDNAs simultaneously obtained from a fixedamount of MTAL RNA tot (25 to 100 ng, as appropriate) and0.27 to 2 amol of RNA c. Under these PCR conditions,heteroduplexes of PCR amplicons were never observed. The PCR ampliconswere resolved by 1.8% agarose gel electrophoresis and stained withethidium bromide. The bands were digitized, and quantification wasperformed by densitometry with use of NIH Image 1.62 software. Tocorrect for differences in molecular weight, the densitometry values ofthe competitive DNA bands were multiplied by the 510/411-bp ratio. Wechecked that the amplification efficiencies of the wild and competitive DNAs were identical with up to 28 PCR cycles (0.41 ± 0.04 vs. 0.42 ± 0.04 per cycle, n = 4 for both) and thatthe amounts of amplicons obtained after 27 PCR cycles were well withinthe exponential phase of amplification. The ROMK mRNA abundance wascalculated from the linear log-log scale plot of the ratio of thefluorescence intensities of RNA c to RNA tot PCRproducts against the known amount of RNA c added in eachreaction tube; r values for these linear 0.99. Results are expressed in attomoles of ROMK mRNA per 100 ngRNA tot.1 A" w" v- b" p
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Results are expressed as means ± SE. Statisticalsignificance between experimental groups was assessed by Student'spaired or unpaired t -test or by one-way ANOVA completed by a t -test using the within-groups residual variance of ANOVA,as appropriate.# K! b3 T7 X, x( ^# l) r" {
' p# V+ C3 T, {2 JRESULTS
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In Vitro Studies& W% i! x8 K2 M6 x% \" `
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For the present experiments, tubule fragments were incubated inexperimental media for 7 h. The MTAL is surrounded in vivo by theinterstitial medium of variable osmolality of the inner stripe of theouter medulla of the kidney depending on the state of water diuresis orantidiuresis and is subjected to tonic influences by cAMP-generatingpeptide hormones such as AVP, glucagon, and calcitonin( 27 ). Thus, we first assessed possible effects on ROMKexpression by hyperosmolality and dDAVP/8-bromo-cAMP. The hyperosmoticHDMEM was obtained by adding 50 mM NaCl and 50 mM urea to normal HDMEMas physiologically occurs during antidiuresis. As shown in Fig. 1, hyperosmolality increased ROMK mRNAabundance from 0.31 ± 0.03 amol/100 ng RNAtot in controlisosmotic medium to 0.55 ± 0.12 ( P moderately by ~28% but verysignificantly ( P whereasthe 90-kDa band was not affected (Fig. 2 ). By contrast, the V 2 receptor-specific analog dDAVP (10 9 M) or 0.5 mM8-bromo-cAMP had no effect on ROMK mRNA or protein abundance in boththe isosmotic and hyperosmotic medium (Table 1 ). To gain some insight into thehyperosmolality effect, the following experiments were performed. Asshown in Fig. 3, a 75 mM increase inmedium NaCl concentration augmented ROMK mRNA abundance from 0.40 ± 0.04 amol/100 ng RNA tot in control isoosmotic medium to0.65 ± 0.03 ( P mM urea or mannitol (Fig. 3 ). The dose-response curvedepicted in Fig. 4 shows that ROMK mRNAabundance was regulated between 170 and 205 mM medium NaClconcentration. Thus these results establish that the hyperosmolalityeffect was mediated via the increase in the NaCl concentrationspecifically.
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: Y# l: W3 H) ^3 v( u" A6 X$ h6 hFig. 1. Effect of medium hyperosmolality (450 mosmol/kgH 2 O) on ROMK mRNA abundance after 7 h ofincubation of medullary thick ascending limb (MTAL) fragments. Linesconnect results obtained in the same experiment." K) n7 i( q- J# a
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Fig. 2. Effect of medium hyperosmolality (Hyperosmo.; 450 mosmol/kgH 2 O) on ROMK protein (45 kDa) and on 90-kDa bandabundance after 7 h of incubation of MTAL fragments. A :representative immunoblot in crude membranes from MTAL fragments. B : band densities (arbitrary units) of immunoblots made induplicate in 6 independent experiments. The augmentation of ROMK bands( 28 ± 12%) is significantly higher than that of the 90-kDa band( 9 ± 9%; P! k- D+ ?8 F; d
: {6 `* u6 a5 d7 [Table 1. Effects of dDAVP and 8-bromo-cAMP on ROMK protein and mRNA in MTALfragments after 7 h of incubation
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Fig. 3. Effect of hyperosmolality caused by 150 mM urea andmannitol and by 75 mM NaCl on ROMK mRNA abundance. Each bar representsmeans ± SE of 3 measurements.: k# U# _+ S) X V* O; @: L
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Fig. 4. Dose-response curve of the effect of medium NaCl concentration onROMK mRNA abundance. Each point represents means ± SE of at least3 measurements.
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! A+ P- w2 ^$ K: M, j& L0 w) W' P- LWe previously showed that glucocorticoids regulate the expression oftheNa -K (NH 4 )-2Cl cotransporter BSC1/NKCC2 in the MTAL through interactions with cAMP-dependent factors ( 22 ). Accordingly, we assessedpossible effects of glucocorticoids on ROMK expression. Dexamethasone(10 nM) increased the abundance of ROMK mRNA from 0.36 ± 0.04 inthe control isosmotic medium to 0.67 ± 0.11 amol/100 ngRNA tot ( P 5 ). This dexamethasone-induced increasein ROMK mRNA was abolished in the additional presence of8-bromo-cAMP (0.47 ± 0.09 amol/100 ng RNA tot withdexamethasone plus 8-bromo-cAMP; not significant compared withcontrol and P dexamethasonealone; Fig. 5 ). The abundance of ROMK protein or the 90-kDa band wasnot significantly affected under any of these experimental conditions(Fig. 6 ).
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Fig. 5. Effect of 10 nM dexamathasone (DEX) and DEX 0.5 mM8-bromo-cAMP (cAMP) on ROMK mRNA abundance after 7 h of incubationof MTAL fragments in isosmotic medium. Lines connect results obtainedin the same experiment.: v* f7 `. G- e3 P* s2 o
" T2 v* a) S* g- O+ V2 W' oFig. 6. Effect of 10 nM DEX (D) and DEX cAMP on ROMKprotein (45 kDa) and on 90-kDa band abundance after 7 h ofincubation of MTAL fragments in isosmotic medium. A :representative immunoblot in crude membranes from MTAL fragments. B : band densities (arbitrary units) of immunoblots made induplicate in 3 independent experiments. Bars represent means ± SE. Numbers within bars are the numbers of measurements. C, control.( u) B' y9 z* F$ _
# ]" M7 s+ k+ ^2 H% W9 AThus both dexamethasone and hyperosmolality increased ROMK mRNAcompared with the control isosmotic experimental condition. We thusassessed whether the effects of hyperosmolality and glucocorticoids were additive. As shown in Fig. 7, theeffects on ROMK mRNA abundance of hyperosmolality and dexamethasonewere not additive. However, in contrast to what was observed in theisosmotic medium, the presence of 8-bromo-cAMP in addition todexamethasone in the hyperosmotic medium did not alter the stimulatingeffect of these latter agents on ROMK mRNA abundance (Fig. 7 ). ROMKprotein abundance was augmented ~33% by hyperosmolality but notsignificantly in this experimental series compared by ANOVA with thelevel seen in the isosmotic medium (Fig. 8 ). However, hyperosmolality plusdexamethasone and hyperosmolality plus dexamethasone plus cAMPsignificantly augmented by ~45 ( P P proteinabundance. The 90-kDa band abundance was affected by none of theseexperimental conditions (Fig. 8 ).
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Fig. 7. Effect of hyperosmotic medium (450 mosmol/kgH 2 O; H), hyperosmotic medium 10 nM DEX(H D), and hyperosmotic medium 10 nM DEX cAMP(H D cAMP) on ROMK mRNA abundance after 7 h ofincubation of MTAL fragments.
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$ m7 y- H) M1 P- w2 H( M6 H( j% fFig. 8. Effect of H (450 mosmol/kgH 2 O), H D,and H D cAMP on ROMK protein (45 kDa) and on 90-kDa bandabundance after 7 h of incubation of MTAL fragments. A :representative immunoblot in crude membranes from MTAL fragments. B : band densities (arbitrary units) of immunoblots made induplicate in 4 independent experiments. C, control isoosmotic medium.Bars represent means ± SE. Numbers within bars are the numbers ofmeasurements.1 k; Z" `# M1 o4 v! E8 k
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To assess the physiological significance of the effects ofdexamethasone observed in vitro, we performed the following in vivostudies. As shown in Table 2, there wasno significant difference in the abundance of ROMK protein and mRNAbetween ADX and control rats that drank normal water or 0.9% NaCl.However, adrenalectomy is a complex condition in which several factorsmay have had opposing effects on ROMK expression, as discussed below.Thus, in another experimental series, results obtained from five ADXrats were compared with those obtained from five ADX Dexa rats.As shown in Fig. 9, dexamethasoneadministration increased ROMK protein abundance in crude membranes ofthe inner stripe of the outer medulla by ~61% (161 ± 16 arbitrary units in ADX Dexa vs. 100 ± 8 in ADX; P decreased by dexamethasone administration (Fig. 9 ) and increased byadrenalectomy when control rats drank 0.9% NaCl (Table 2 ), as opposedto what was observed for ROMK. The dexamethasone-induced increase inROMK protein abundance was accompanied by a ~67% increase in ROMKmRNA abundance (9.2 ± 0.5 amol/100 ng RNAtot in ADX Dexavs. 5.5 ± 0.5 in ADX; P 10 ). These results establish thatglucocorticoid administration enhances ROMK mRNA and protein expressionin the MTAL.
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Table 2. Quantification of ROMK protein and mRNA abundance in inner stripe ofouter medulla of control and ADX rats
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4 P6 s7 q5 L) A- V. m2 ]) ^" m9 yFig. 9. A : immunoblot of ROMK protein (45 kDa) and 90-kDa bandin crude membranes from inner stripe of outer medulla in 5 adrenalectomized (ADX) and 5 ADX DEX (Dexa) rats. B :band densities (arbitrary units) of immunoblots made in duplicate inADX and ADX Dexa rats. Bars represent means ± SE.9 q( h6 z; x& i* @' G Q
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Fig. 10. Determination of ROMK mRNA abundance in inner stripe ofouter medulla of 5 ADX and 5 ADX Dexa rats.# B/ W. u* O# q& d( i n
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DISCUSSION& h# Y1 `% o3 T: c
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This study is the first, to our knowledge, to assess the direct invitro effects of glucocorticoids, dDAVP and cAMP, and hyperosmolality on ROMK expression in rat MTAL fragments. The main observations werethat 1 ) ROMK mRNA abundance was increased when MTALs were incubated in a hyperosmotic medium or in the presence of 10 nM dexamethasone; 2 ) the effects of hyperosmolality andglucocorticoids on mRNA abundance were not additive; 3 ) anincrease in ROMK protein abundance was seen after 7 h ofincubation in a hyperosmotic medium; 4 ) the hyperosmolalityeffect was mediated by the increase in medium NaCl concentration, butnot by urea or mannitol, and ROMK mRNA abundance was regulated by NaClconcentrations between 170 and 205 mM; 5 ) in vivoadministration of dexamethasone over 6 days increased ROMK mRNA andprotein abundance in the MTAL; and 6 ) finally, dDAVP or8-bromo-cAMP alone had no significant effect per se on ROMK mRNA orprotein abundance but may modulate the effects of hyperosmolality and glucocorticoids.1 w0 H. x P) ~! M
5 G% [5 f9 b( |" u1 p) C9 o2 {' ~The effects on ROMK protein expression in the MTAL of changes in sodiumchloride intake, water restriction, and dDAVP administration werepreviously assessed in vivo ( 13 ) but not those ofglucocorticoid administration. We thus performed in vivo studies usingADX rats and ADX rats supplemented with dexamethasone. Comparing thelevel of ROMK expression in ADX rats to that in normal rats to estimate the effects of glucocorticoid deficiency can hardly be achieved satisfactorily because adrenalectomy is a complex condition with respect to ROMK expression. For example, adrenalectomy is associated with increased circulating AVP concentrations probably due to impairedcardiac function ( 8 ), and dDAVP administration and waterrestriction that stimulates the endogenous AVP secretion have beenshown to strongly stimulate ROMK expression in the MTAL ( 13 ). In addition, NaCl administration, which was used inADX rats to minimize urinary NaCl losses, has also been shown tostimulate ROMK expression ( 13 ). With these issues in mind,we observed that the abundance of ROMK mRNA or protein in the innerstripe of outer medulla was not significantly different afteradrenalectomy from that in control rats drinking normal water or 0.9%NaCl. On the other hand, supplementing ADX rats with dexamethasoneappears as a better means of assessing the effects of glucocorticoids on ROMK expression. Glucocorticoid administration to ADX rats stronglystimulated ROMK mRNA and protein expression in the inner stripe ofouter medulla compared with ADX rats. These results must reflectchanges of ROMK expression in the MTAL because the level of ROMKexpression in this segment is much higher than in the outer medullarycollecting duct (OMCD) ( 7, 25, 33 ) and because the MTALmass of tissue is approximately sixfold that of the OMCD. Thus theseresults establish that glucocorticoids enhance ROMK mRNA and proteinexpression in the MTAL when administered in vivo. Furthermore, when 10 nM dexamethasone was directly applied to MTALs incubated in anisosmotic medium, ROMK mRNA expression was stimulated. Thusglucocorticoids directly regulate ROMK mRNA expression in the MTAL invitro. The present finding that glucocorticoids physiologicallystimulate ROMK expression would be consistent with the observationsthat dexamethasone also stimulates BSC1/NKCC2 expression and activity( 4 ) and Na -K -ATPase activity inthe MTAL ( 12, 29 ). Thus glucocorticoids coordinatelystimulate basolateral Na -K -ATPase and apicalBSC1/NKCC2 and ROMK to increase NaCl absorption by the MTAL, whichexplains at least in part the role of glucocorticoids in the ability ofthe kidney to maximally concentrate or dilute the urine.- H) C2 O( ]' [8 Z& c6 f! W1 v
: d5 x# V3 j8 K5 u8 h4 o7 hIn previous in vivo studies, rats that were water restricted for 7 days, which stimulates the endogenous AVP secretion, or that wereadministered dDAVP for 7 days exhibited an enhanced abundance of ROMKprotein in the MTAL compared with control rats, as assessed byimmunolocalization and immunoblotting analysis ( 13 ). Themechanisms of the latter regulations were not investigated in thisprevious work ( 13 ) but results obtained in vitro in thepresent study indicate that AVP and cAMP-dependent pathways per se arenot directly responsible for the increased ROMK expression. Indeed,both dDAVP and 8-bromo-cAMP had no effect on ROMK mRNA and proteinabundance after 7 h of incubation. However, increases in mediumNaCl concentration did increase ROMK mRNA and protein expression in thepresent study. These observations taken together thus suggest thatchronic water restriction and dDAVP administration stimulate ROMKexpression indirectly, at least in part, through the increase in themedullary NaCl concentration that occurs under these conditions. Notethat a high-sodium diet increased and a low-sodium diet decreased ROMKprotein expression in a previous study ( 13 ), which alsomay have been due to the changes in the medullary NaCl concentrationthat follow these high- and low-sodium intakes. Thus the medullary NaClconcentration directly regulates ROMK expression in the MTAL. Note thatthe NaCl effect seems very specific since ROMK expression was notenhanced by urea or mannitol, which can affect gene expression byvarious mechanisms ( 9, 10, 16 ).+ p4 k9 s5 O( b4 R! j3 ?
* u# C& O4 g- Q/ }3 `# T8 SThe intracellular mechanisms by which hyperosmolality caused by NaCland glucocorticoids enhanced ROMK mRNA and protein expression in theMTAL were not investigated in the present study. However, it must beemphasized that the promoter region 5' of exon 1 in the human ROMKgene, KCNJ1, contains both a glucocorticoid response element and asequence 91% identical to the tonicity-responsive enhancer(TonE)/osmotic response element consensus [TGGAAANNYNY ( 9, 10, 16 )] that are located 27 and 358 bp, respectively, 5' of thetranscription start point of exon 1 ( 10 ). This suggests that high medium NaCl concentration and glucocorticoids may stimulate the ROMK gene transcription rate through activation of TonE binding protein ( 9 ) and GR, respectively. However, it may be noted that TonE is also usually activated by mannitol in cultured cells, whereas mannitol had no effect on ROMK mRNA expression in freshly harvested MTALs in the present study, which might have been due todifferences between cultured and fresh cells. Otherwise, other intracellular events, such as altered ROMK mRNA decay and ROMK proteinsynthesis and/or degradation, may have combined to explain our results.Regulation of intermediate protein(s) expression may also haveoccurred. Further work is needed to address these issues.
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In summary, ROMK expression in the MTAL is regulated in vivo by changesin sodium and water intake ( 13 ) and by glucocorticoid administration (present study). Results obtained in vitro in the present work establish medium NaCl concentration and glucocorticoids asdirect regulators of ROMK expression. As stated above, luminal K recycling through ROMK channels has a major role in NaClreabsorption by the TAL by providing a potassium supply to thecotransporter BSC1/NKCC2 and establishing the lumen-positivetransepithelial potential difference that provides the driving forcefor sodium reabsorption through the paracellular pathway. Present andprevious ( 4 ) findings show that BSC1/NKCC2 and ROMK mRNAand protein expressions in the MTAL are coordinately regulated byvarious mechanisms including glucocorticoids, cAMP-dependent factors, and medium NaCl concentration to set MTAL NaCl absorption at a levelappropriate to the renal regulation of sodium and water balance.Furthermore, the effects of glucocorticoids on MTAL ROMK and BSC1/NKCC2described in the present and previous ( 4 ) studies mayexplain, at least in part, the well-known inability of the kidney tomaximally concentrate or dilute the urine during adrenal insufficiency.0 A4 r, \0 ^$ [2 R& M, i
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ACKNOWLEDGEMENTS
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" i& T; ?% o) `& L2 KThis study was supported by grants from the Institut National de laRecherche Médicale and the Université Paris 7. Amel Attmane-Elakeb is supported by a grant from La Fondation pour laRecherche Médicale; David B. Mount is supported by National Institutes of Health (NIH) Grant RO1-DK-57708 and by an Advanced CareerDevelopment Award from the Veterans Administration; and Steven C. Hebert is supported by NIH Grant DK-54999.
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