Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.
- regulation of gene expression
- ROMK channel
- immunoblotting analysis
- quantitative reverse transcriptase-polymerase chain reaction
- isolated tubules
in the thick ascending limb (TAL) of the nephron, potassium that enters the cell by the activity of the apical Na+-K+(NH4 +)-2Cl−cotransporter is largely recycled back into the lumen through potassium channels (5, 17, 32). This K+ recycling has a major role in NaCl reabsorption by the TAL by providing a potassium supply to the cotransporter and establishing the lumen-positive transepithelial potential difference that provides the driving force for sodium reabsorption through the paracellular pathway. ROMK channels are believed to constitute the major K+ secretory pathway in the distal nephron (15, 28). Indeed, the ROMK protein has been localized by antibodies at the apical membrane of cells of the part of the renal tubule extending from the beginning of the TAL to the initial portion of the inner medullary collecting duct (19, 25,33). The essential role of ROMK channels in TAL transport functions and thus in the renal regulation of sodium and water balance is demonstrated by the fact that mutations in the ROMK gene cause Bartter's syndrome, which is characterized by severe salt wasting and impaired urinary concentrating ability (14, 18, 30).
It is well established that the activity or the membrane density of ROMK 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 and protein-tyrosine kinase (26), and interactions with associated proteins (31). In contrast, little is known about the chronic regulation of ROMK expression in the TAL. Ecelbarger et al. (13) showed that the abundance of ROMK protein in the rat outer medulla is augmented by 1-deamino-(8-d-arginine)-vasopressin (dDAVP), restriction of water intake, and high levels of sodium intake, and decreased by low levels of sodium intake. However, the direct stimuli and cellular mechanisms of these chronic regulations of ROMK expression in the TAL are unknown. Possible candidate mechanisms include direct effects of cAMP-dependent pathways and the variations in the osmolality of the surrounding medullary interstitium that accompany the states of water diuresis and antidiuresis. In addition, beside adenylyl cyclase-coupled receptors, the TAL possesses specific glucocorticoid receptors (GR), the activation of which exerts important actions on TAL transport functions. Indeed, the glucocorticoid dexamethasone has been shown to stimulate Na+-K+-ATPase activity within a few hours (12, 29). More recently, we showed that dexamethasone increases both the expression and activity of the Na+-K+(NH4 +)-2Cl−cotransporter BSC1/NKCC2 of MTALs incubated in vitro (4). Of relevance to these observations, glucocorticoids have long been known to contribute to the renal urinary concentrating ability, at least in part, through the maintenance of medullary hyperosmolality (11).
These considerations prompted us to assess directly in vitro the possible regulation by osmolality, cAMP, and glucocorticoids of the expression 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 results establish that ROMK mRNA and protein abundance are regulated directly by the osmolality of the incubation medium and glucocorticoids not by dDAVP or cAMP. We also show that in vivo dexamethasone administration increases the abundance of ROMK mRNA and protein in the MTAL.
MATERIALS AND METHODS
In Vivo Studies
Male Sprague-Dawley rats (250–300 g) were used and had free access to standard rat chow and drinking solution until the time of the experiment. Rats were adrenalectomized (ADX) under light ether anesthesia and given, as drinking solution, distilled water containing 0.9% NaCl for 6 days before the experiment. A microosmotic pump (Alza, Palo Alto, CA) was implanted subcutaneously in the nape of some of the ADX rats, through which we delivered 1.2 μg · 100 g body wt−1 · day−1 of the glucocorticoid hormone dexamethasone, a dose that is known to restore normal glucocorticoid activity, for 6 days (ADX + Dexa). These rats also had access to 0.9% NaCl in distilled water. Control rats were sham operated under ether anesthesia and had access to water or to distilled water containing 0.9% of NaCl. After pentobarbital sodium anesthesia, the kidneys were rapidly removed and cut into thin slices along the corticopapillary axis and, under a dissecting microscope, the inner stripe of outer medulla of each slice was excised and cut into uniform small pieces that were used for immunoblotting of membrane proteins and mRNA determinations.
In Vitro Studies
Suspension of rat MTAL tubules.
The method used to isolate MTAL fragments in suspension has been previously described (1). We established by light and electron microscopy that this suspension was made almost exclusively of MTALs (≥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 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 supplemented with 5 mM heptanoic acid, 5 mMl-leucin, 0.1 g/l bovine serum albumin, 200 IU/ml penicillin G, 250 μg/ml streptomycin, 10 μg/ml minocyclin, 15 mM HEPES, 10 mM Tris, and 25 mM NaHCO3, pH 7.35, when gassed with 95% O2-5% CO2 (HDMEM). The MTALs were then 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% O2-5% CO2 gas mixture. According to results obtained in a previous study from this laboratory (2), the MTAL shake suspension was allowed to stabilize during the first 9 h of incubation. Then, samples of MTALs were further incubated for 7 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 four preliminary experiments that ROMK mRNA abundance was stable during this experimental time under control conditions (0.96 ± 0.17 vs. 0.85 ± 0.16 amol/100 ng RNAtot; not significant; the quantitative RT-PCR is described below).
Crude Membrane Preparation
Tissues from inner stripe of outer medulla dissection or from MTAL suspensions were homogenized in a medium composed of 150 mM sucrose, 12 mM Trizma (Tris base, pH 7.4), 0.1 mM 4-(2-amino-ethyl)-benzenesulfonyl fluoride, and 5 μg/ml leupeptin. These homogenates were centrifuged at 1,000 g for 5 min in a Beckman GS-6KR centrifuge with a G-H3.7 rotor, and the supernatants were further centrifuged at 200,000 g for 60 min in a Beckman L-70 Ultracentrifuge with a 70 TI rotor. The membrane pellets were suspended in the above medium and stored at −80°C until use.
Electrophoresis and Immunoblotting of Membrane Proteins
Semiquantification of membrane protein amounts was performed by immunoblotting after SDS-PAGE. Membranes were solubilized first at ambient temperature for 20 min in Laemmli medium containing 62.5 mM Tris · 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 lanes of 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 an affinity-purified polyclonal anti-ROMK rabbit antibody (APC001, Alomone Labs, Jerusalem, Israel) diluted 1:150. This antibody has been previously documented to reveal both native and heterologously expressed ROMK protein as a ∼45-kDa band (24), which appeared 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-antigen complexes 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 also reveals a band of unknown identity of 90 kDa. Both the ROMK 42- to 45-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 be shown below, it does not respond like ROMK to the various experimental conditions.
RNA Extraction, Reverse Transcription, and PCR
Total RNA (RNAtot) was extracted from aliquots of kidney MTAL with use of the SV Total RNA Isolation System kit (Promega). To obtain competitor RNAs (RNAc) that differed from the wild-type ROMK1, 2, 3 mRNAs, deletions of 99 bp, located in the core exon common to all ROMK isoforms, of the ROMK1, 2, and 3 plasmids were obtained by digestion with Bgl II and Msc I restriction enzymes (from bp 692 to 790 of ROMK1), followed by blunt-end ligation of the cut plasmids. The deleted ROMK plasmids were subcloned and linearized with NotI restriction enzyme. In vitro transcription was then performed with the use of T7 RNA polymerase (mCAP RNA Capping kit, Stratagene, La Jolla, CA) and [32P]UTP. The amounts of transcribed RNAcwere determined by the measure of its optical density at 260 nm corrected for the ratio of TCA-precipitated RNAc to total RNAc determined by liquid scintillation spectroscopy. We thus obtained three RNAc that gave identical results in the RT-PCR described below.
The primers (GIBCO BRL) for cDNA synthesis and PCR amplification were chosen from the published ROMK1, 2, and 3 sequences with the help of Oligo 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 sense primer was directed against a segment common to ROMK1, 2, 3, and 6 but located within the intron retained in the ROMK core exon. This retained intron is subject to low-frequency alternative splicing that generates a set of truncated hydrophilic ROMK isoforms of unknown function, which thus were not amplified by our method. The primers used in the present study yielded, as expected, only one PCR product common to ROMK1, 2, and 3 of 510 bp from MTAL RNAtot and the ROMK cDNA plasmids and of 411 bp from the RNAc and the deleted ROMK plasmids, respectively. After 30 PCR cycles, PCR products resulting from nonspecific hybridization were never observed. The identities of the PCR products were confirmed by digestion with SalI, which generated two bands of 74 and 436 bp from the wild DNA and of 74 and 337 bp from the competitive DNA as expected.
cDNAs were synthesized from MTAL RNAtot and RNAc by reverse transcription at 37°C for 60 min with 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies), 30 pmol of antisense primer, 4 μg of yeast transfer RNA, 1 mM of each deoxyribonucleotide (dNTP), 10 mM DTT, 2 U of ribonuclease inhibitor (GIBCO BRL), and RT buffer in a final volume of 22 μl. Reverse transcriptase was then inactivated at 95°C for 5 min. Each reaction was performed in parallel with an otherwise identical one that contained no reverse transcriptase.
For PCR, 10 μl of the cDNA solution were supplemented with PCR buffer, 5.4 mM MgCl2, 30 pmol of sense and antisense primers, 1 mM of each dNTP, and 5 U of Taq DNA polymerase (Life Technologies) in a final volume of 50 μl. Samples were denatured at 94°C for 5 min, which were followed by cycles consisting of denaturation at 94°C (1 min), annealing at 50°C (1 min), and extension at 72°C (1.5 min). PCR was completed by a final extension step at 72°C for 10 min. Quantitative PCR was performed using 27 cycles of amplification of cDNAs simultaneously obtained from a fixed amount of MTAL RNAtot (25 to 100 ng, as appropriate) and 0.27 to 2 amol of RNAc. Under these PCR conditions, heteroduplexes of PCR amplicons were never observed. The PCR amplicons were resolved by 1.8% agarose gel electrophoresis and stained with ethidium bromide. The bands were digitized, and quantification was performed by densitometry with use of NIH Image 1.62 software. To correct for differences in molecular weight, the densitometry values of the competitive DNA bands were multiplied by the 510/411-bp ratio. We checked 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 that the amounts of amplicons obtained after 27 PCR cycles were well within the exponential phase of amplification. The ROMK mRNA abundance was calculated from the linear log-log scale plot of the ratio of the fluorescence intensities of RNAc to RNAtot PCR products against the known amount of RNAc added in each reaction tube; r values for these linear plots were all >0.99. Results are expressed in attomoles of ROMK mRNA per 100 ng RNAtot.
Results are expressed as means ± SE. Statistical significance between experimental groups was assessed by Student's paired or unpaired t-test or by one-way ANOVA completed by at-test using the within-groups residual variance of ANOVA, as appropriate.
In Vitro Studies
For the present experiments, tubule fragments were incubated in experimental media for 7 h. The MTAL is surrounded in vivo by the interstitial medium of variable osmolality of the inner stripe of the outer medulla of the kidney depending on the state of water diuresis or antidiuresis and is subjected to tonic influences by cAMP-generating peptide hormones such as AVP, glucagon, and calcitonin (27). Thus, we first assessed possible effects on ROMK expression by hyperosmolality and dDAVP/8-bromo-cAMP. The hyperosmotic HDMEM was obtained by adding 50 mM NaCl and 50 mM urea to normal HDMEM as physiologically occurs during antidiuresis. As shown in Fig.1, hyperosmolality increased ROMK mRNA abundance from 0.31 ± 0.03 amol/100 ng RNAtot in control isosmotic medium to 0.55 ± 0.12 (P < 0.04). ROMK protein abundance was also increased moderately by ∼28% but very significantly (P < 0.006) by hyperosmolality, whereas the 90-kDa band was not affected (Fig.2). By contrast, the V2receptor-specific analog dDAVP (10−9 M) or 0.5 mM 8-bromo-cAMP had no effect on ROMK mRNA or protein abundance in both the isosmotic and hyperosmotic medium (Table1). To gain some insight into the hyperosmolality effect, the following experiments were performed. As shown in Fig. 3, a 75 mM increase in medium NaCl concentration augmented ROMK mRNA abundance from 0.40 ± 0.04 amol/100 ng RNAtot in control isoosmotic medium to 0.65 ± 0.03 (P < 0.006). Such an effect was not seen with 150 mM urea or mannitol (Fig. 3). The dose-response curve depicted in Fig. 4 shows that ROMK mRNA abundance was regulated between 170 and 205 mM medium NaCl concentration. Thus these results establish that the hyperosmolality effect was mediated via the increase in the NaCl concentration specifically.
We previously showed that glucocorticoids regulate the expression of the Na+-K+(NH4 +)-2Cl−cotransporter BSC1/NKCC2 in the MTAL through interactions with cAMP-dependent factors (22). Accordingly, we assessed possible effects of glucocorticoids on ROMK expression. Dexamethasone (10 nM) increased the abundance of ROMK mRNA from 0.36 ± 0.04 in the control isosmotic medium to 0.67 ± 0.11 amol/100 ng RNAtot (P < 0.02; Fig.5). This dexamethasone-induced increase in ROMK mRNA was abolished in the additional presence of 8-bromo-cAMP (0.47 ± 0.09 amol/100 ng RNAtot with dexamethasone plus 8-bromo-cAMP; not significant compared with control and P < 0.004 compared with dexamethasone alone; Fig. 5). The abundance of ROMK protein or the 90-kDa band was not significantly affected under any of these experimental conditions (Fig. 6).
Thus both dexamethasone and hyperosmolality increased ROMK mRNA compared with the control isosmotic experimental condition. We thus assessed whether the effects of hyperosmolality and glucocorticoids were additive. As shown in Fig. 7, the effects on ROMK mRNA abundance of hyperosmolality and dexamethasone were not additive. However, in contrast to what was observed in the isosmotic medium, the presence of 8-bromo-cAMP in addition to dexamethasone in the hyperosmotic medium did not alter the stimulating effect of these latter agents on ROMK mRNA abundance (Fig. 7). ROMK protein abundance was augmented ∼33% by hyperosmolality but not significantly in this experimental series compared by ANOVA with the level seen in the isosmotic medium (Fig.8). However, hyperosmolality plus dexamethasone and hyperosmolality plus dexamethasone plus cAMP significantly augmented by ∼45 (P < 0.04) and ∼64% (P < 0.01), respectively, the ROMK protein abundance. The 90-kDa band abundance was affected by none of these experimental conditions (Fig. 8).
In Vivo Studies
To assess the physiological significance of the effects of dexamethasone observed in vitro, we performed the following in vivo studies. As shown in Table 2, there was no significant difference in the abundance of ROMK protein and mRNA between ADX and control rats that drank normal water or 0.9% NaCl. However, adrenalectomy is a complex condition in which several factors may have had opposing effects on ROMK expression, as discussed below. Thus, in another experimental series, results obtained from five ADX rats were compared with those obtained from five ADX + Dexa rats. As shown in Fig. 9, dexamethasone administration increased ROMK protein abundance in crude membranes of the inner stripe of the outer medulla by ∼61% (161 ± 16 arbitrary units in ADX + Dexa vs. 100 ± 8 in ADX;P < 0.004). It may be noted that the 90-kDa band was decreased by dexamethasone administration (Fig. 9) and increased by adrenalectomy when control rats drank 0.9% NaCl (Table 2), as opposed to what was observed for ROMK. The dexamethasone-induced increase in ROMK protein abundance was accompanied by a ∼67% increase in ROMK mRNA abundance (9.2 ± 0.5 amol/100 ng RNAtot in ADX + Dexa vs. 5.5 ± 0.5 in ADX; P < 0.002; Fig.10). These results establish that glucocorticoid administration enhances ROMK mRNA and protein expression in the MTAL.
This study is the first, to our knowledge, to assess the direct in vitro effects of glucocorticoids, dDAVP and cAMP, and hyperosmolality on ROMK expression in rat MTAL fragments. The main observations were that 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 and glucocorticoids on mRNA abundance were not additive; 3) an increase in ROMK protein abundance was seen after 7 h of incubation in a hyperosmotic medium; 4) the hyperosmolality effect was mediated by the increase in medium NaCl concentration, but not by urea or mannitol, and ROMK mRNA abundance was regulated by NaCl concentrations between 170 and 205 mM; 5) in vivo administration of dexamethasone over 6 days increased ROMK mRNA and protein abundance in the MTAL; and 6) finally, dDAVP or 8-bromo-cAMP alone had no significant effect per se on ROMK mRNA or protein abundance but may modulate the effects of hyperosmolality and glucocorticoids.
The effects on ROMK protein expression in the MTAL of changes in sodium chloride intake, water restriction, and dDAVP administration were previously assessed in vivo (13) but not those of glucocorticoid administration. We thus performed in vivo studies using ADX rats and ADX rats supplemented with dexamethasone. Comparing the level 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 impaired cardiac function (8), and dDAVP administration and water restriction that stimulates the endogenous AVP secretion have been shown to strongly stimulate ROMK expression in the MTAL (13). In addition, NaCl administration, which was used in ADX rats to minimize urinary NaCl losses, has also been shown to stimulate ROMK expression (13). With these issues in mind, we observed that the abundance of ROMK mRNA or protein in the inner stripe of outer medulla was not significantly different after adrenalectomy from that in control rats drinking normal water or 0.9% NaCl. On the other hand, supplementing ADX rats with dexamethasone appears as a better means of assessing the effects of glucocorticoids on ROMK expression. Glucocorticoid administration to ADX rats strongly stimulated ROMK mRNA and protein expression in the inner stripe of outer medulla compared with ADX rats. These results must reflect changes of ROMK expression in the MTAL because the level of ROMK expression in this segment is much higher than in the outer medullary collecting duct (OMCD) (7, 25, 33) and because the MTAL mass of tissue is approximately sixfold that of the OMCD. Thus these results establish that glucocorticoids enhance ROMK mRNA and protein expression in the MTAL when administered in vivo. Furthermore, when 10 nM dexamethasone was directly applied to MTALs incubated in an isosmotic medium, ROMK mRNA expression was stimulated. Thus glucocorticoids directly regulate ROMK mRNA expression in the MTAL in vitro. The present finding that glucocorticoids physiologically stimulate ROMK expression would be consistent with the observations that dexamethasone also stimulates BSC1/NKCC2 expression and activity (4) and Na+-K+-ATPase activity in the MTAL (12, 29). Thus glucocorticoids coordinately stimulate basolateral Na+-K+-ATPase and apical BSC1/NKCC2 and ROMK to increase NaCl absorption by the MTAL, which explains at least in part the role of glucocorticoids in the ability of the kidney to maximally concentrate or dilute the urine.
In previous in vivo studies, rats that were water restricted for 7 days, which stimulates the endogenous AVP secretion, or that were administered dDAVP for 7 days exhibited an enhanced abundance of ROMK protein in the MTAL compared with control rats, as assessed by immunolocalization and immunoblotting analysis (13). The mechanisms of the latter regulations were not investigated in this previous work (13) but results obtained in vitro in the present study indicate that AVP and cAMP-dependent pathways per se are not directly responsible for the increased ROMK expression. Indeed, both dDAVP and 8-bromo-cAMP had no effect on ROMK mRNA and protein abundance after 7 h of incubation. However, increases in medium NaCl concentration did increase ROMK mRNA and protein expression in the present study. These observations taken together thus suggest that chronic water restriction and dDAVP administration stimulate ROMK expression indirectly, at least in part, through the increase in the medullary NaCl concentration that occurs under these conditions. Note that a high-sodium diet increased and a low-sodium diet decreased ROMK protein expression in a previous study (13), which also may have been due to the changes in the medullary NaCl concentration that follow these high- and low-sodium intakes. Thus the medullary NaCl concentration directly regulates ROMK expression in the MTAL. Note that the NaCl effect seems very specific since ROMK expression was not enhanced by urea or mannitol, which can affect gene expression by various mechanisms (9, 10, 16).
The intracellular mechanisms by which hyperosmolality caused by NaCl and glucocorticoids enhanced ROMK mRNA and protein expression in the MTAL were not investigated in the present study. However, it must be emphasized that the promoter region 5′ of exon 1 in the human ROMK gene, KCNJ1, contains both a glucocorticoid response element and a sequence 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 the transcription 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 to differences between cultured and fresh cells. Otherwise, other intracellular events, such as altered ROMK mRNA decay and ROMK protein synthesis and/or degradation, may have combined to explain our results. Regulation of intermediate protein(s) expression may also have occurred. Further work is needed to address these issues.
In summary, ROMK expression in the MTAL is regulated in vivo by changes in 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 as direct regulators of ROMK expression. As stated above, luminal K+ recycling through ROMK channels has a major role in NaCl reabsorption by the TAL by providing a potassium supply to the cotransporter BSC1/NKCC2 and establishing the lumen-positive transepithelial potential difference that provides the driving force for sodium reabsorption through the paracellular pathway. Present and previous (4) findings show that BSC1/NKCC2 and ROMK mRNA and protein expressions in the MTAL are coordinately regulated by various mechanisms including glucocorticoids, cAMP-dependent factors, and medium NaCl concentration to set MTAL NaCl absorption at a level appropriate to the renal regulation of sodium and water balance. Furthermore, the effects of glucocorticoids on MTAL ROMK and BSC1/NKCC2 described in the present and previous (4) studies may explain, at least in part, the well-known inability of the kidney to maximally concentrate or dilute the urine during adrenal insufficiency.
This study was supported by grants from the Institut National de la Recherche Médicale and the Université Paris 7. Amel Attmane-Elakeb is supported by a grant from La Fondation pour la Recherche Médicale; David B. Mount is supported by National Institutes of Health (NIH) Grant RO1-DK-57708 and by an Advanced Career Development Award from the Veterans Administration; and Steven C. Hebert is supported by NIH Grant DK-54999.
Address for reprint requests and other correspondence: M. Bichara, INSERM U.426/IFR 02 Claude Bernard, B.P. 416-16, rue Henri Huchard, 75870 Paris Cedex 18, France (E-mail:).
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First published January 21, 2003;10.1152/ajprenal.00255.2002
- Copyright © 2003 the American Physiological Society