Calcyclin Is an Early Vasopressin-induced Gene in the Renal Collecting Duct ROLE IN THE LONG TERM REGULATION OF ION TRANSPORT*

Long-term effects of arginine vasopressin (AVP) in the kidney involve the transcription of unidentified genes. By subtractive hybridization experiments performed on the RCCD 1 cortical collecting duct cell line, we identi- fied calcyclin as an early AVP-induced gene (1 h). Calcyclin is a calcium-binding protein involved in the transduction of intracellular signals. In the kidney, calcyclin was localized at the mRNA level in the glomerulus, all along the collecting duct, and in the epithelium lining the papilla. In RCCD 1 cells and in m-IMCD 3 inner medullary collecting duct cells, calcyclin was evidenced in the cytoplasm. Calcyclin mRNA levels were progressively increased by AVP treatment in RCCD 1 (1.7-fold at 4 h) and m-IMCD 3 (2-fold at 7.5 h) cells. In RCCD 1 cells, calcyclin protein levels were increased b y 4 h of AVP treatment. In vivo , treatment of genetically vasopressin-deficient Brattleboro rats with AVP for 4 days induced an increase in both calcyclin and aquaporin-2 mRNA expression. Finally, introduction of anti-calcyclin of AVP on calcyclin these experiments, AVP a AVP ng/day rats pentobarbital PCR-based Suppression/Subtractive Hybridization— subtractive hybridization on cells (cid:4) AVP. RCCD up-regulated two of cDNA cells Adaptors hybridizations cDNA linkers first PCR amplification using suppression PCR exponentially differentially differentially expressed cDNA fragments. “Forward” subtraction was performed when linkers were added to cDNA fragments obtained from hormone-treated cells, whereas “reverse” subtraction was performed when linkers were added to cDNA fragments obtained from control cells. PCR products were cloned into the pT-Adv vector using the AdvanTAge TM PCR cloning kit (CLONTECH). Differential screening of the subtracted library was performed to eliminate false positives by hybridization with 32 P-labeled probes prepared from forward and reverse subtracted cDNAs (PCR-Select Differential Screening kit, CLON- TECH). Clones showing signal ratios of (cid:5) 5:1 (forward versus reverse subtracted probe) were further analyzed by DNA sequencing. In Situ Hybridization— For in situ hybridization studies, kidneys from Sprague-Dawley rats were fixed for 15 min in 4% paraformalde- hyde with 5 m M MgCl 2 and then kept at 4 °C in 70% ethanol. In situ hybridization experiments were performed as previously described (18). At the end of the experiment, sections were covered with an autora-diography emulsion (NTB2, Eastman Kodak Co.) and exposed at (cid:4) 20 °C for 10 days before development (Kodak D19 film). Cells were then stained with toluidine blue and examined under bright-field using a Zeiss Axioplan microscope. Calcyclin cDNA (nucleotides 51–327) was subcloned into the Eco RI site of the Bluescript KS vector. Sense and antisense cRNAs were synthesized after linearization using 35 S-labeled UTP (specific activity of 1000 Ci/mmol; Amersham Biosciences) and the Riboprobe ® Combination System T3/T7 kit (Promega). Other reagents used for the experiments (adenosine, guanosine, cytosine 5 (cid:6) -triphos- phate, ribonucleasin, dithiothreitol, and RNA polymerases) were from Promega.

Arginine vasopressin (AVP) 1 is a polypeptide hormone involved in the regulation of renal water and ion transport. In the collecting duct, AVP coordinately increases water and NaCl reabsorption by a two-step mechanism. The first mechanism is responsible for the short-term effects of AVP. These effects consist of the translocation of aquaporin-2 (AQP2) water channels and amiloride-sensitive sodium channels from intracellular pools to the apical membrane, promoting an increase in both sodium and water entry (1)(2)(3)(4). This increase induces, in turn, a coordinate rise in transepithelial water and sodium reabsorption (5)(6)(7)(8)(9)(10). These effects are rapid and transient because the effect is down-regulated after ϳ1 h in the rat collecting duct (11). In addition to this short-term effect, AVP induces a long-term effect. This effect consists of a late increase in transepithelial water, sodium, and chloride transport after several hours. This late effect is mediated through a transcriptional/translational mechanism and involves an increase in the mRNA and de novo synthesis of different proteins such as AQP2; the ␤ and ␥ (but not ␣) subunits of ENaC (epithelial Na ϩ channel); the ␣ 1 (but not ␤ 1 ) subunit of Na ϩ /K ϩ -ATPase; and CFTR (cystic fibrosis transmembrane conductance regulator) (12)(13)(14)(15). This long-term effect may involve the genomic pathway by activation of cAMP-responsive elements in the promoter region of these genes and may ensure a sustained increase in sodium, chloride, and water transport in this segment of the nephron. A recent study has focused on the effects of 4 h of treatment with vasopressin on the transcriptome of a mouse kidney cortical collecting duct cell line (16). Statistical comparison of the SAGE (serial analysis of gene expression) libraries revealed 48 vasopressin-induced transcripts and 11 vasopressinrepressed transcripts. A selection of the differentially expressed vasopressin-specific transcripts has been validated by Northern blot hybridization and by reverse transcription. Hepatocyte nuclear transcription factor-3␣ (VIT39 (vasopressin-induced transcript)) and receptor activity-modifying protein-3 (VIT48) have been suggested to be candidate proteins playing a role in physiological responses to vasopressin.
In this study, we have searched for mRNA induced by AVP treatment using the subtractive hybridization technique in the vasopressin-responsive RCCD 1 cell line. Results show that the calcium-binding protein calcyclin is an AVP-induced protein.
Interestingly, in the kidney, calcyclin is mainly expressed in the collecting duct and appears to be regulated by AVP in coordination with AQP2. Finally, calcyclin appears to be indispensable in allowing the long-term (but not short-term) response of the hormone in RCCD 1 cells.
Animals-Male Sprague-Dawley rats (250 -300 g) were used for in situ hybridization experiments. In addition, 16 male Brattleboro rats (250 -300 g) were used for Northern blot experiments aimed at deter-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 33-1-44-85-6325; Fax: 33-1-42-29-1644, E-mail: chabaud@bichat.inserm.fr. mining the effects of AVP on calcyclin mRNA expression. In these experiments, animals were treated with AVP (Sigma) using different protocols: either with a single intramuscular injection of AVP (2 g in 0.9% NaCl) 3 h before killing or with an osmotic minipump (500 ng/day in 0.9% NaCl) for 4 days. Control rats were treated similarly, except that only 0.9% NaCl was used. Animals had free access to tap water and were anesthetized with pentobarbital before killing.
PCR-based Suppression/Subtractive Hybridization-To determine the mRNAs differentially expressed after vasopressin treatment in the cortical collecting duct, subtractive hybridization was performed on RCCD 1 cells grown on Transwell filters in defined culture medium for 4 days, incubated overnight in minimum medium (Ham's F-12/Dulbecco's modified Eagle's medium (1:1), 14 mM NaHCO 3 , 2 mM glutamine, 10 units/ml penicillin/streptomycin, and 20 mM HEPES, pH 7.4), and then treated or not for 1 h basolaterally with 10 Ϫ8 M AVP. Poly(A) mRNAs were extracted from RCCD 1 cells treated or not with AVP using oligo(dT) 25 covalently bound to magnetic beads (Dynal, Oslo, Norway). Double-stranded cDNA was then synthesized and digested with RsaI. To identify up-regulated sequences, these two pools of cDNA fragments derived from control and AVP-treated cells were submitted to PCRbased subtractive hybridization and suppression PCR (PCR-Select TM cDNA Subtraction kit, CLONTECH) according to the manufacturer's protocol. Adaptors were linked to the AVP-treated cDNA pool, and two successive hybridizations followed by extension to fill in ends were performed in the presence of an excess of cDNA without linkers from the untreated cells. A first PCR amplification using suppression PCR amplified exponentially only differentially expressed sequences. A second PCR amplification reduced the background and further enriched differentially expressed cDNA fragments. "Forward" subtraction was performed when linkers were added to cDNA fragments obtained from hormone-treated cells, whereas "reverse" subtraction was performed when linkers were added to cDNA fragments obtained from control cells. PCR products were cloned into the pT-Adv vector using the AdvanTAge TM PCR cloning kit (CLONTECH). Differential screening of the subtracted library was performed to eliminate false positives by hybridization with 32 P-labeled probes prepared from forward and reverse subtracted cDNAs (PCR-Select Differential Screening kit, CLON-TECH). Clones showing signal ratios of Ͼ5:1 (forward versus reverse subtracted probe) were further analyzed by DNA sequencing.
In Situ Hybridization-For in situ hybridization studies, kidneys from Sprague-Dawley rats were fixed for 15 min in 4% paraformaldehyde with 5 mM MgCl 2 and then kept at 4°C in 70% ethanol. In situ hybridization experiments were performed as previously described (18). At the end of the experiment, sections were covered with an autoradiography emulsion (NTB2, Eastman Kodak Co.) and exposed at Ϫ20°C for 10 days before development (Kodak D19 film). Cells were then stained with toluidine blue and examined under bright-field using a Zeiss Axioplan microscope. Calcyclin cDNA (nucleotides 51-327) was subcloned into the EcoRI site of the Bluescript KS vector. Sense and antisense cRNAs were synthesized after linearization using 35 S-labeled UTP (specific activity of 1000 Ci/mmol; Amersham Biosciences) and the Riboprobe ® Combination System T3/T7 kit (Promega). Other reagents used for the experiments (adenosine, guanosine, cytosine 5Ј-triphosphate, ribonucleasin, dithiothreitol, and RNA polymerases) were from Promega.
Immunocytochemistry-Immunocytochemical experiments were performed with a rabbit polyclonal anti-calcyclin antibody (Swiss Swant Laboratories, Bellinzona, Switzerland) diluted 1:200. RCCD 1 and m-IMCD 3 cells were grown to confluence on collagen-coated Transwell filters (12-mm diameter) and then fixed in paraformaldehyde for 30 min at room temperature, washed, and incubated at room temperature for 1 h with the anti-calcyclin antibody. A secondary antibody (goat antirabbit Fab fraction (1:200); Jackson ImmunoResearch Laboratories, Inc.) coupled to the fluorochrome Cy3 (red fluorescence) was used to visualize the signal. In these experiments, the nucleus was stained with Sytox (green fluorescence; Molecular Probes, Inc.). xz sections of cells were realized by confocal laser scanning microscopy (Leica TCS4D apparatus).
Western Blot Experiments-Cells were plated on 24-mm Transwell filters and cultured for 3 days in defined culture medium before incubation overnight in minimum medium. Cells were then treated or not basolaterally for 4 h at 37°C with 10 Ϫ8 M AVP. Cells from each filter were rinsed and scraped at 4°C in phosphate-buffered saline before addition of lysis buffer (0.15 M NaCl, 5 mM EDTA, 1% Nonidet P-40, 50 mM Tris, pH 7.5, 10 g/ml protease inhibitor mixture (Sigma), and 0.5 mM phenylmethylsulfonyl fluoride) and incubation for 30 min at 4°C. Supernatants (after centrifugation at 12,000 ϫ g) corresponding to each filter were submitted to SDS-PAGE (15%) using the Laemmli buffer system before transfer onto a polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was then pretreated for 1 h at room temperature with 5% milk in Tris-buffered saline plus 0.1% Tween 20 and incubated with the anti-calcyclin antibody (1:250) overnight at 4°C, followed by incubation with a secondary antibody conjugated to peroxidase (1:20,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Proteins were visualized using the ECL or ECL Plus detection kit (Amersham Biosciences). Results were normalized to the signal obtained by Western blotting of ␤-actin in the same cell samples. To this end, the membrane was stripped and incubated overnight at 4°C with 5% milk in Tris-buffered saline plus 0.1% Tween 20 and then for 1 h at room temperature with an anti-␤-actin antibody (1/10,000; Santa Cruz Biotechnology, Inc.) before incubation with the peroxidaseconjugated secondary antibody (1:20,000) for 1 h at room temperature and ECL visualization.
Cell Permeabilization and Electrophysiological Studies-To examine the influence of calcyclin on the response of RCCD 1 cells to AVP, anti-calcyclin antibodies were introduced into the cells by permeabilizing their plasma membrane, and the effect of the hormone on transepithelial transport was examined by short-circuit current experiments. Indeed, in a previous study (20), it was shown that anti-calcyclin antibodies could block the activity of the protein and that introduction of these antibodies into the cells prevented the transduction of cellular processes in response to a hormonal stimulus. In our experiments, RCCD 1 cells were grown on Snapwell filters coated with collagen. The development of confluence was monitored by measuring the transepithelial voltage (mV) and the transepithelial resistance (ohms/cm 2 ) across the filters using a World Precision Instruments epithelial voltohm meter connected to sterile electrodes. When high transepithelial voltage and transepithelial resistance were recorded (5-8 days after seeding), filters were incubated overnight in minimum medium. The short-circuit current (I sc ) was then determined on these cells, which had been treated or not for two different times with AVP (15 min and 7.5 h), after permeabilization of the cells in the presence of the anti-calcyclin antibody or nonspecific IgG.
Two different protocols for cell membrane permeabilization were used. Cell membranes were permeabilized either by a freeze/thaw procedure as already described for RCCD 1 cells (14) or with digitonin (21). In both protocols, the apical medium of the cells (500 l) was removed FIG. 1. Localization of calcyclin mRNA in the rat kidney by in situ hybridization. The expression of calcyclin mRNA was evidenced by in situ hybridization in the different regions of the kidney. A 35 Slabeled antisense probe corresponding to nucleotides 51-327 of the mRNA was used, and control experiments were performed with a sense probe (the labeling was equivalent to the background without tissue) (data not shown). In the cortex, a signal was observed in the glomerulus (A, arrow) and in the collecting duct cells (B, arrows). In the medulla, the only structure that was labeled was the collecting duct (C, arrows). Finally, in the papilla (D), the collecting duct cells and the epithelium lining the papilla (arrows) strongly expressed calcyclin mRNA. and replaced with the same volume of either minimum medium supplemented with 5 l of anti-calcyclin antibody (freeze/thaw procedure) or phosphate-buffered saline supplemented with 40 M digitonin and 5 l of anti-calcyclin antibody (digitonin procedure). In the freeze/thaw procedure, filters were placed on a bath of ethanol at -30°C, and after rapid freezing of the cells, they were rapidly thawed by incubation at 37°C. In the digitonin procedure, cells were incubated for 5 min at 4°C with the digitonin medium. The medium was then removed and replaced with minimum medium. Preliminary experiments using trypan blue as a marker of cell permeabilization showed that Ͼ95% of the cells were permeabilized under these conditions. In control experiments, the same protocol was performed, except that rabbit IgG was used in place of the anti-calcyclin antibody.
To study the short-term effects of AVP (15 min), cells were permeabilized after incubation in minimum medium, incubated for 4.5 h at 37°C to allow membrane resealing, and then mounted in the voltageclamp system (Costar Corp. and World Precision Instruments, Inc.). To study the long-term effects of AVP (7.5 h), cells were first treated with AVP. After 3 h of incubation, cells were permeabilized and then further incubated for 4.5 h, always in the presence of AVP, at 37°C before mounting in the voltage-clamp system. In this system, cells were bathed on each side with 8 ml of minimum medium that was thermostatted at 37°C and that was circulated by a gas lift (95% O 2 and 5% CO 2 mixture). I sc (A/cm 2 ) was measured by clamping the transepithelial voltage to 0 mV for 1 s. To study the short-term effects of AVP, I sc was determined before and after 15 min of incubation with 10 Ϫ8 M AVP. To study the long-term effects of AVP, I sc was determined in cells treated or not with 10 Ϫ8 M AVP for 7.5 h.
Statistical Analysis-Results are expressed as means Ϯ S.E. Statistical analysis was performed using Student's t test for unpaired data according to the experiments.

Calcyclin Is a Differentially Expressed Gene in RCCD 1 Cells
Treated with Vasopressin-PCR-based subtractive hybridization was used to establish a library of cDNAs representing early (1 h) vasopressin-regulated mRNAs in RCCD 1 cells. To this end, mRNAs corresponding to 24-mm diameter filters treated or not for 1 h with 10 Ϫ8 M AVP were prepared with the poly(A) isolation kit and used in a PCR-based suppression/ subtractive hybridization experiment (see "Experimental Pro-  cedures"). The selected clones were sequenced and analyzed by homology searches using the BLAST program. Among these sequences, we have identified a clone highly homologous to calcyclin (GenBank TM /EBI accession Number AJ132717), which was further studied.
In the Nephron, Calcyclin Is Expressed in Collecting Duct Cells-To examine the expression of calcyclin in the kidney, in situ hybridization experiments were performed on kidneys from Sprague-Dawley rats. The results are presented in Fig. 1. Calcyclin mRNA was expressed all along the collecting duct and also in the glomerulus and in the epithelium lining the papilla. In the collecting duct, calcyclin mRNA was expressed at low levels in the cortical part, with a progressive increase along the medulla and the papilla.
Immunocytochemical Localization of Calcyclin in RCCD 1 and m-IMCD 3 Cells-The expression of calcyclin was examined in two different models of collecting duct cells: the RCCD 1 cell line, corresponding to cortical collecting duct cells, and the m-IMCD 3 cell line, corresponding to papillary collecting duct cells. Calcyclin was expressed in both models and appeared to be present essentially in the cytoplasmic compartment (Figs. 2 and 3, respectively). Fig. 4A shows a representative Northern blot experiment aimed at determining the time course of calcyclin mRNA induction by 10 Ϫ8 M AVP in RCCD 1 cells. Whereas GAPDH mRNA expression was not modified, calcyclin mRNA expression increased progressively with the time of AVP exposure. Fig. 4B illustrates the mean values of five different experiments. Calcyclin mRNA induction significantly increased as soon as 1 h after AVP addition, with a maximal effect after 4 h (ϳ70% increase). Thereafter, calcyclin mRNA expression returned to the control level (at 24 h). Fig. 5 shows the time course of calcyclin mRNA induction by 10 Ϫ8 M AVP in m-IMCD 3 cells as measured by Northern blotting. As noted for RCCD 1 cells, GAPDH mRNA expression was not modified, and calcyclin mRNA expression progressively increased after AVP exposure. Fig. 5B shows the mean values of four different experiments. The calcyclin mRNA induction was significant after 30 min of AVP exposure. A 100% increase was observed at 7.5 h of treatment with the hormone. Thereafter, calcyclin mRNA expression returned to the control level at 24 h.

Calcyclin mRNA Expression Is Increased by AVP Treatment in the m-IMCD 3 Inner Medullary Collecting Duct Cell Line-
Calcyclin Protein Level Is Increased by AVP Treatment in RCCD 1 Cells- Fig. 6A shows a representative Western blot experiment aimed at determining the effect on calcyclin expression of 4 h of treatment with 10 Ϫ8 M AVP in RCCD 1 cells. Calcyclin protein expression was largely increased by AVP treatment. Fig. 6B illustrates the mean values of three different experiments, taking into account the expression of ␤-actin in each sample. Calcyclin expression was significantly increased by 4 h of treatment with 10 Ϫ8 M AVP.

Calcyclin mRNA Expression Is Increased in Parallel with AQP2 mRNA by Chronic AVP Treatment in Brattleboro Rat
Kidney-To examine the in vivo effect of AVP on the expression of renal calcyclin mRNA, Northern blot experiments were performed with RNA obtained from Brattleboro rat whole kidneys treated or not for 3 h or 4 days with AVP (see "Experimental Procedures"). The expression of AQP2 mRNA was also examined as a positive control, and the effects were normalized to GAPDH mRNA expression. Whereas no significant effect was observed at 3 h on either AQP2 or calcyclin mRNA expression (data not shown), a clear effect was observed after 4 days of treatment. Fig. 7A shows a representative experiment using four different animals. Treatment of the animals for 4 days with AVP using osmotic minipumps resulted in a significant increase in mRNA encoding both AQP2 and calcyclin. Fig. 7B shows the quantification of the increase in AQP2 and calcyclin mRNA expression in four AVP-treated Brattleboro rats compared with that in four control Brattleboro rats.
Anti-calcyclin Antibodies Block the Long-term but Not Shortterm AVP Effect on Ion Transport in RCCD 1 Cells-Entry of an antibody inside the cell can be yielded by transient permeabilization procedures; thereafter, the cells recover and develop electrical properties similar to those of non-permeabilized cells (14,(21)(22)(23)(24)(25). To test the effect of anti-calcyclin antibodies on the AVP-induced increase in ion transport, two different cell permeabilization techniques were used to ensure antibody entry into the cells. Both the short-and long-term effects of AVP were examined by the short-circuit current (I sc ) technique in the presence of anti-calcyclin antibodies or rabbit IgG. The results are illustrated in Fig. 8. Anti-calcyclin antibodies introduced into the cells by the freeze/thaw procedure (Fig. 8A) blunted the long-term AVP-induced increase in I sc . The permeabilization procedure per se did not alter the effect of AVP on I sc . When the digitonin procedure was used (Fig. 8B), the same effect was observed, which was not reproduced by nonspecific rabbit IgG. In addition, experiments showed that the short-term effect of AVP on I sc was not blocked by anti-calcyclin antibodies or by rabbit IgG.

DISCUSSION
The short-term effects of AVP on renal ion and water transport have been widely documented (5-10). By contrast, very limited information is available on the long-term effects of this hormone. In addition to the long-term effects of AVP on water transport associated with an increase in AQP2 synthesis (12,26), we have recently shown that AVP exerts a delayed stimulation of sodium and chloride transport in collecting duct cells (13,14). This effect depends on transcription of several transporters of sodium and chloride, in particular ENaC, Na-K-ATPase, and CFTR. As a matter of fact, the cAMP and intracellular Ca 2ϩ pathways have been shown to exert transcriptional effects in different systems, in addition to their short-term actions as second messenger on several cell functions. In particular, the cAMP-or intracellular Ca 2ϩ -induced FIG. 7. Effect of chronic AVP treatment on calcyclin and AQP 2 mRNA expression in Brattleboro rat whole kidneys. The amount of mRNA encoding calcyclin (in parallel with AQP2 mRNA expression) in kidneys from Brattleboro rats treated or not with AVP was determined by Northern blotting. GAPDH was used as an internal control. Four rats were continuously treated with AVP at 500 ng/day for 4 days (4d) using an osmotic minipump. Four control rats were treated in a similar fashion, except that only diluent (0.9% NaCl) was used. A representative experiment of five different experiments is shown in A (each lane corresponds to one animal), and the quantified data are given in B. Both calcyclin and AQP2 mRNAs were significantly increased by AVP. *, p Ͻ 0.05; ***, p Ͻ 0.001 (AVP versus control).

FIG. 8. Effect of anti-calcyclin antibodies on the short-and long-term AVP-induced increases in I sc in permeabilized RCCD 1 cells.
The effect of anti-calcyclin antibodies was tested on AVP-induced I sc after two different cell permeabilization (P) procedures (see "Experimental Procedures"). A, results obtained with the freeze/thaw procedure. The increase in I sc observed after 7.5 h of incubation with AVP was blocked by preincubating cells with the anti-calcyclin antibody (Ab; 1:100) after cell permeabilization (see "Experimental Procedures"). The anti-calcyclin antibody was added after 3 h of treatment with AVP. B, results obtained with the digitonin permeabilization procedure. As described for A, introduction of anti-calcyclin antibodies prevented the long-term effect of AVP on I sc (7.5 h). In addition, the results show that the short-term effect of AVP (15 min) was not modified. The blocking effect of the antibody was not reproduced by unrelated antibodies from the same isotype (rabbit IgG). Each bar is the mean value of 9 -12 filters from four (A) and six (B) experiments. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 (AVP versus control (C)). phosphorylation of nuclear proteins such as the cAMP-responsive element-binding protein and the cAMP-responsive element modulator is responsible for transcriptional effects via cAMP-or Ca 2ϩ -responsive elements present in the promoter of genes (27). In this study, we have searched for proteins induced by 1 h of AVP treatment in a rat cortical collecting duct cell line. Using the subtractive hybridization technique, we have identified calcyclin as an early AVP response gene, and we have shown that AVP increased both the level of mRNA encoding calcyclin and calcyclin expression in RCCD 1 cells. Altogether, these results and those reported by Robert-Nicoud et al. (16) obtained by SAGE analysis of AVP-induced genes show that, in addition to the previously described effects on AQP2, ENaC, Na-K-ATPase, and CFTR, the long-term functional response to vasopressin depends on transcription and translation of numerous proteins in the cortical collecting duct.
Calcyclin is a 10.5-kDa protein that belongs to the family of calcium-binding proteins (28 -31). Calcium-binding proteins are divided into two groups: the first group is constituted by annexins, and the second group by EF-hand proteins such as calmodulin. Calcyclin belongs to this second group. It was first identified as a cell cycle-dependent protein highly induced by growth conditions (28), but its precise role remains unknown. Different studies have shown that calcyclin is expressed only in fibroblasts and epithelial cells and that it can associate with other proteins, in particular annexins (annexin-2, -6, and -11) (32,33). Calcyclin, as annexins, may be involved in exocytosis phenomena. In this way, it has been shown in numerous studies that modulation of intracellular calcium plays an important role in the regulation of exocytosis and that calcium-binding proteins can act as transducing proteins in coupling stimulus to exocytosis. It has also been shown that, in vitro, calcyclin can bind actin-binding proteins such as caldesmon, tropomyosin, and calponin (34 -36).
Our data obtained by in situ hybridization show that calcyclin was localized mainly in the collecting duct cells in the kidney, with additional staining in the glomerulus and in the epithelium lining the papilla. In a previous study, Lewington et al. (37) showed that calcyclin is present in glomeruli and distal tubules. Our results are compatible with those data. At the cellular level, it has been suggested that calcyclin could be localized in the cytoplasmic compartment of the cells under basal conditions and that it could be targeted to two different membranous compartments, the plasma membrane and the nuclear membrane, in response to an increase in intracellular calcium (38). In our experiments performed in RCCD 1 and m-IMCD 3 cells incubated under basal conditions (Figs. 2 and  3), calcyclin was localized in the cytoplasmic compartment. Further studies will be necessary to examine whether AVP treatment or modifications of the intracellular Ca 2ϩ concentration modify this localization.
Only a few studies concerning the hormonal regulation of calcyclin have been reported. The promoter of calcyclin has been reported to contain a serum-responsive element (39), and calcyclin is generally considered to be a growth-regulated gene (28). In the rat kidney, it has been shown that calcyclin is induced after ischemic injury (37). In this study, we have shown that AVP increased the amount of mRNA encoding calcyclin in RCCD 1 cells, in m-IMCD 3 cells, and in Brattleboro rat kidney. In addition, we have shown that calcyclin protein expression was increased in RCCD 1 cells after 4 h of treatment with 10 Ϫ8 M AVP. Interestingly, the time course of the phenomenon appears to be different in the two cell lines and in the rat kidney. In RCCD 1 cells derived from the cortical collecting duct, as in m-IMCD 3 cells derived from the inner medullary collecting duct, calcyclin mRNA was rapidly and transiently increased. A significant effect was observed as soon as 0.5-1 h after AVP treatment (Figs. 4 and 5). This result is in accordance with the fact that calcyclin mRNA was evidenced as an AVP-induced gene in RCCD 1 cells by subtractive hybridization after 1 h of AVP treatment. The time course shows that the maximal increase was observed after 4 -7.5 h of treatment and then declined at 24 h. After 24 h of treatment with AVP, the amount of mRNA encoding calcyclin was not different between control and AVP-treated cells. In contrast, the results obtained with Brattleboro rat whole kidneys showed no increase in calcyclin mRNA 3 h after injection of AVP, but a clear effect after 4 days of AVP treatment (Fig. 7). In the same way, note that AQP2 mRNA was not significantly increased after 3 h of AVP treatment, but largely increased after 4 days. The differences observed in the time course and in the magnitude of the AVP effect between cell models and Brattleboro rat kidneys might be related to differences between in vitro and in vivo models. The complexity of animal models, the delay in the response to the hormone when administrated intramuscularly, and the presence of several complementary hormonal regulatory systems might explain a delayed effect on calcyclin and AQP2 mRNA expression in the rat. In this way, it should be noted that hormonal effects on the amount of mRNA encoding newly synthesized proteins are often delayed when studied in animal models rather than in cell lines.
The precise role of calcyclin in epithelial cells is unknown. In a previous work (20), calcyclin has been described to be involved in the Ca 2ϩ -dependent secretion of insulin in pancreatic cells. Interestingly, it was shown that introduction into pancreatic beta-cells of anti-calcyclin antibodies by permeabilizing the plasma membrane prevented insulin secretion, a result that clearly defined calcyclin as a key protein in the exocytosis process. In our study, introduction of anti-calcyclin antibodies into permeabilized RCCD 1 cells supported a specific role of calcyclin in the long-term regulation of ion transport (Fig. 8). Indeed, anti-calcyclin antibodies prevented the AVP-induced increase in I sc observed after 7.5 h of treatment, whereas it was without effect on the short-term effect of the hormone (15 min). The localization of calcyclin in the cytoplasmic compartment of RCCD 1 cells and the possibility that it could move to the nucleus upon certain stimuli as described in other studies (38) suggest different hypotheses concerning its role. In the first hypothesis, calcyclin could be implicated in the process leading to the transcription of other genes (transcriptional role). In this way, by binding calcium, calcyclin could play a role in the Ca 2ϩ -induced activation of Ca 2ϩ -responsive elements through nuclear kinases such as Ca 2ϩ /calmodulin-dependent protein kinase (40). Alternatively, or additively, the established interaction of calcyclin with the calcium signaling pathways suggests that calcyclin could be involved in the delivery of newly synthesized proteins (due to the transcriptional effect of AVP) to the apical and/or the basolateral membrane of the cells (exocytotic role) (41). Further experiments will be necessary to test these hypotheses.
In conclusion, we have identified calcyclin as a new AVPinduced gene in the collecting duct. In addition, our experiments suggest that calcyclin could play an important role in the longterm response of the hormone in transepithelial ion transport.