Tonicity-Responsive Enhancer Binding Protein Is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells
Udo Hasler*,
Un Sil Jeon,
Jeong Ah Kim,
David Mordasini*,
H. Moo Kwon,
Eric Féraille* and
Pierre-Yves Martin*
* Service of Nephrology, Fondation pour Recherches Médicales, Geneva, Switzerland; and Department of Medicine, University of Maryland, Baltimore, Maryland
Address correspondence to: Dr. Udo Hasler, Service de Néphrologie, Fondation pour Recherches Médicales, 64 Avenue de la Roseraie, GE 1211, Genève 4, Switzerland. Phone: +41-22-382-3833; Fax: +41-22-347-5979; E-mail: udo.hasler{at}medecine.unige.ch
Received for publication December 19, 2005.
Accepted for publication March 30, 2006.
Tonicity-responsive enhancer binding protein (TonEBP) playsa key role in protecting renal cells from hypertonic stressby stimulating transcription of specific genes. Under hypertonicconditions, TonEBP activity is enhanced via increased nucleartranslocation, transactivation, and abundance. It was reportedpreviously that hypertonicity exerted a dual, time-dependenteffect on vasopressin-inducible aquaporin-2 (AQP2) expressionin immortalized mouse collecting duct principal cells (mpkCCDcl4).Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge,it increased after 24 h of hypertonic challenge. This studyinvestigated the role that TonEBP may play in these events bysubjecting mpkCCDcl4 cells to 3 or 24 h of hypertonic challenge.Hypertonic challenge increased TonEBP mRNA and protein contentand enhanced TonEBP activity as illustrated by both increasedTonEBP-dependent luciferase activity and mRNA expression ofseveral genes that are targeted by TonEBP. Irrespective of theabsence or presence of vasopressin, decreased TonEBP activityin cells that were transfected with either TonEBP small interferingRNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNAand protein content under iso-osmotic conditions and bluntedthe increase of AQP2 abundance that was induced after 24h ofhypertonic challenge. Conversely, decreased TonEBP activitydid not significantly alter reduced expression of AQP2 mRNAthat was induced by 3 h of hypertonic challenge. Mutation ofa TonE enhancer element located 489 bp upstream of the AQP2transcriptional start site abolished the hypertonicity-inducedincrease of luciferase activity in cells that expressed AQP2promoter-luciferase plasmid constructs, indicating that TonEBPinfluences AQP2 transcriptional activity at least partiallyby acting directly on the AQP2 promoter. These findings demonstratethat in collecting duct principal cells, TonEBP plays a centralrole in regulating AQP2 expression by enhancing AQP2 gene transcription.
On the basis of homologies in the DNA binding domain, tonicity-responsiveenhancer binding protein (TonEBP or NFAT5/OREBP) belongs tothe Rel family of transcription factors. TonEBP activity isupregulated by hypertonicity and plays a major role in protectingmammalian cells from hypertonic stress. Whereas TonEBP is partiallyactive under isotonic conditions (1), TonEBP activity is enhancedby hypertonicity through combined increased TonEBP nuclear localizationand transactivation (2–4). Longer periods of hypertonicstimulation additionally lead to increased abundance of TonEBPmRNA and protein (1,2). In the kidney medulla, the driving forcefor water reabsorption is provided by elevated concentrationsof urea and NaCl, which in turn give rise to exceptionally highlevels of osmolarity (>1200 and 4000 mOsmol/kg under conditionsof water restriction in human and mouse inner medulla, respectively).Renal cells adapt to the high levels of osmolarity partly byaccumulating intracellular osmolytes that reduce intracellularionic strength. By binding to TonE enhancer elements, TonEBPplays a key role in this process by stimulating transcriptionof aldose reductase (AR) (5), sodium-chloride-betaine co-transporter(BGT1) (2), sodium-myo-inositol co-transporter (SMIT) (6), andtaurine transporter (7), which mediate intracellular accumulationof sorbitol, betaine, myo-inositol, and taurine, respectively.In addition to these genes, TonEBP stimulates transcriptionof the antidiuretic hormone (8-arginine)vasopressin (AVP)-regulatedurea transporter (UT-A) (8) as well as heat-shock protein 70(9), which helps to protect cells from the damaging effectsof high urea concentration.
The high water permeability of renal tubule segments is duemostly to the presence of aquaporin (AQP) water channels. RegulatedAQP2 expression, restricted to the collecting system (connectingtubule and collecting duct [CD]), represents the final checkpoint of controlled water reabsorption. AVP plays a major rolein determining AQP2 abundance by binding to basolateral V2 receptorsthat are located in CD principal cells, leading to Gs/adenylylcyclase activation, increased intracellular cAMP concentration,and protein kinase A (PKA) activation (10). Several pieces ofevidence indicate that AQP2 abundance additionally is influencedto a large extent by extracellular tonicity. This has been illustratedby in vivo experiments in water-restricted animals treated withV2-receptor antagonists that display increased AQP2 content(11,12) and in water-loaded animals that display decreased AQP2content despite ongoing V2 receptor stimulation (11,13). Inaddition, cAMP-independent decreased AQP2 expression that occursin parallel with low levels of medullary osmolarity in senescentrats was reverted by raising medullary osmolarity (14,15). Invitro experiments using primary cultured inner medullary CDcells have shown further that extracellular hypertonicity stimulatesAQP2 expression independent of PKA activity (16,17).
The aim of this study was to investigate the role of TonEBPon AQP2 gene transcription in cultured mpkCCDcl4 cells thatwere derived from microdissected CD of a SVPK/Tag transgenicmouse (18). When grown on permeable filters, these cells developinto tight and highly differentiated epithelium (18–20)that displays low levels of native AQP2 mRNA and protein inthe absence of AVP. AQP2 expression levels are increased dramaticallywhen AVP is added to the basolateral medium (21–23). Werecently showed that extracellular hyperosmolarity affects AQP2mRNA and protein abundance in a time-dependent manner in mpkCCDcl4cells and that this is due to changes in AQP2 gene transcription(17). Whereas AQP2 abundance first decreased after 3 h of hyperosmoticchallenge, it increased after 24 h of hypertonic stimulation.In addition, the effects of hypertonicity on AQP2 mRNA and proteinabundance were similar in the absence and presence of AVP, indicatingthat hypertonicity regulates AQP2 abundance independent of AVP.Here, we investigated whether TonEBP mediates the decrease and/orincrease of AQP2 expression by subjecting mpkCCDcl4 cells thatexhibit reduced TonEBP activity to various conditions of hypertonicchallenge.
Cell Culture and Transfection
mpkCCDcl4 cells (passages 22 to 31) were seeded on permeablefilters (Transwell; Corning Costar, Cambridge, MA) and grownin medium supplemented with 2% FCS and as described previously(17). Iso-osmotic medium (300 mOsmol/kg) was made hypertonic(400 or 500 mOsmol/kg) by replacing a fraction of apical andbasal medium (75 to 150 µl/600 µl apical mediumand 150 to 300 µl/1200 µl basal medium) with NaCl-enrichedmedium. Medium osmolarity was checked using a Roebling osmometer.
Transfection was performed by electroporating cells as describedpreviously (24) in the presence of 1.2 nmol of small interferingRNA (siRNA); 8 pmol of plasmid that contained either eGFP orhuman dominant-negative TonEBP (DN-TonEBP) (2); or 8 pmol ofplasmid that contained luciferase constructs. Sense primersfor Stealth siRNA (Invitrogen, San Diego, CA) were the following:5'-GGUGUUGCAGGUAUUUGUGGGCAAU-3', 5'-GGAUUCUAUCAGGCCUGUAGAGUAA-3',and 5'-CCUAGUUCUCAAGAUCAGCAAGUAA-3'. That for scramble siRNA(corresponding to the third siRNA) was 5'-CCUCUUACUAGAACUACGGAAGUAA-3'.
Immunofluorescence
Confluent cells that were grown on filters were rinsed twicewith PBS, fixed with 4% (wt/vol) paraformaldehyde, and permeabilizedwith 0.1% Triton X-100. After blocking with 0.5% (wt/vol) BSAin PBS, cells were incubated with a polyclonal anti-TonEBP antibody(2) diluted 1:1000 in PBS/BSA and incubated with a secondaryanti-rabbit IgG Alexa 488–conjugated antibody (MolecularProbes, Eugene, OR; dilution 1:100). Filters were mounted onglass coverslips with polyvinyl alcohol solution, and fluorescenceimages were captured at 488 nm with an Axiovert 200M Zeiss microscope(Oberkochen, Germany).
Western Blot Analysis
After incubation, cells were homogenized and equal amounts ofprotein were separated by 10% SDS-PAGE and transferred to polyvinylidenedifluoride membranes (Immobilon-P; Millipore, Bedford, MA) asdescribed previously (21). TonEBP, AQP2, and Na,K-ATPase -subunitwere detected by Western blotting using polyclonal rabbit antibodies(2,25,26) at 1:2000, 1:20,000, and 1:20,000 dilutions, respectively.The antigen–antibody complexes were detected by the SuperSignal Substrate method (Pierce, Rockford, IL). Bands were quantifiedusing a video densitometer and ImageQuant software (MolecularDynamics, Sunnyvale, CA).
Real-Time PCR Analysis
RNA extraction, reverse transcription, and real-time PCR analysiswere performed as described previously (17). Primers that wereused for detection of mouse acidic ribosomal phosphoproteinP0 were 5'-AATCTCCAGAGGCACCATTG-3' and 5'-GTTCAGCATGTTCAGCAGTG-3',those for AQP2 were 5'-CTTCCTTCGAGCTGCCTTC-3' and 5'-CATTGTTGTGGAGAGCATTGAC-3',those for TonEBP were 5'-GCTTCAGCCCAAGGCATACA-3' and 5'-GTCCCGGGCTGTGAGATG-3',those for AR were 5'-AGTGCGCATTGCTGAGAACTT-3' and 5'-GTAGCTGAGTAGAGTGGCCATGTC-3',those for BGT1 were 5'-CTGGGAGAGACGGGTTTTGGGTATTACATC-3' and5'-GGACCCCAGGTCGTGGAT-3', and those for SMIT were 5'-CCGGGCGCTCTATGACCTGGG-3'and 5'-CAAACAGAGAGGCACCAATCG-3'. P0 was used as an internalstandard, and data were analyzed as described previously (27).
Luciferase Assay
Luciferase plasmid constructs that were used for transfectionwere made by cloning three copies of TonE in front of SV40 promoterand luciferase using a commercial vector pGL3-promoter (Promega)"TonE-driven luciferase," nucleotides –517 to +109 ofthe mouse AQP2 gene in front of luciferase using pGL3-Basic(Promega) "AQP2 0.6K," nucleotides –2043 to +109 of themouse AQP2 gene in front luciferase using pGL3-Basic "AQP2 2.1K,"and mutating TGGAA of TonE in AQP2 0.6K to TTTAA "AQP2 0.6KTonEmut." The numbering was based on the genomic sequence inthe National Center for Biotechnology Information. We confirmedthe transcription start site by primer extension and ribonucleaseprotection assay (data not shown). Luciferase activity was measuredusing the Luciferase Assay System (Promega) according to themanufacturer’s instructions. The light produced was measuredusing a Lumat LB 9507 luminometer (EG&G Berthold).
Statistical Analyses
Results are given as the mean ± SEM from n independentexperiments. Each experiment was performed on mpkCCDcl4 cellsfrom the same passage. Statistical differences were assessedusing the Mann-Whitney U test or the Kruskal-Wallis test forcomparison of two groups or more, respectively. P < 0.05was considered significant.
TonEBP Is Activated in Response to Increased Extracellular Tonicity in mpkCCDcl4 Cells
The effects of hypertonicity on TonEBP expression and activitywas evaluated in mpkCCDcl4 cells that were exposed to eitherisotonic or NaCl-supplemented hypertonic medium. Immunofluorescenceanalysis using a polyclonal anti-TonEBP antibody revealed thatTonEBP was localized predominantly in the nucleus in cells thatwere exposed to 300 mOsmol/kg medium with a small but discerniblesignal observed in the cytoplasm (Figure 1A). Increasing thetonicity to 400 mOsmol/kg further reduced TonEBP cytoplasmicexpression. These observations show that TonEBP is expressedmostly in the nucleus under isotonic (300 mOsmol/kg) conditions.However, hypertonic challenge dramatically enhanced TonEBP activityas revealed by an almost 200-fold increase of luciferase activityin cells that were transfected with TonE-driven luciferase reporterplasmid (Figure 1B) and increased AR, BGT1, and SMIT mRNA content,revealed by real-time PCR analysis (Figure 1C). These mRNA expressionlevels increased independent of AVP 10–10 M and were accompaniedby a transient increase of TonEBP mRNA content that occurredafter 3 h of hypertonic stimulation (Figure 1C). As shown byWestern blot analysis, hypertonic challenge increased TonEBPprotein abundance, revealed by an expected approximately 200-kDband (2) (Figure 3A, lanes 1 through 3). These results demonstratethe occurrence of major hallmarks of a hypertonicity-mediatedTonEBP response in mpkCCDcl4 cells, i.e., a transient increaseof TonEBP mRNA content, increased TonEBP protein abundance,increased TonEBP activity, and, to a lesser extent, increasedredistribution of TonEBP to the nucleus.
Figure 1. Tonicity-responsive enhancer binding protein (TonEBP) activity is increased in response to hypertonicity in mpkCCDcl4 cells. (A) Cultured mpkCCDcl4 cells that were grown on filters were incubated for 24 h in isotonic (300 mOsmol/kg) serum- and hormone-free defined medium (DM) and then for 24 h in either isotonic or NaCl-supplemented hypertonic medium (400 mOsmol/kg) before indirect immunofluorescence analysis using a polyclonal anti-TonEBP antibody. Representative images from one of three independent experiments are shown. (B) Cultured mpkCCDcl4 cells were transiently transfected with TonE-driven luciferase plasmid, seeded on filters, and grown in isotonic medium for 24 h. Cells then were exposed to isotonic serum- and hormone-free DM for 2 h and then for an additional 24 h to either the same isotonic medium or to NaCl-supplemented hypertonic medium (400 mOsmol/kg) before measurement of luciferase activity. Bars are mean ± SE from four independent experiments. *P < 0.05. (C) Cultured mpkCCDcl4 cells that were grown on filters were incubated for 24 h in isotonic (300 mOsmol/kg) serum- and hormone-free DM, then for 24 h in the absence or presence of (8-arginine)vasopressin (AVP) 10–10 M, and then for either 3 or 24 h in either isotonic or NaCl-supplemented hypertonic medium (400 mOsmol/kg) in the continuous absence or presence of AVP. Total RNA was extracted and reverse transcribed, and real-time PCR was performed using primers that are specific for aldose reductase (AR), sodium-chloride-betaine co-transporter (BGT1), sodium-myo-inositol co-transporter (SMIT), and TonEBP. Results are expressed relative to control values determined for each gene after 3 h of incubation in isotonic medium in the absence of AVP. Bars are mean ± SE from five independent experiments. *P < 0.05.
Figure 3. Increased aquaporin 2 (AQP2) transcription induced by long-term hypertonic challenge is blunted by TonEBP small interfering RNA (siRNA) knockdown. (A) Cultured mpkCCDcl4 cells were transiently transfected by electroporation with scramble (lanes 1 through 3) or TonEBP siRNA (lanes 4 through 6), seeded on filters, and grown in isotonic medium for 24 h. Cells then were exposed to isotonic serum- and hormone-free DM for 2 h and then for an additional 24 h to either the same isotonic medium or to NaCl-supplemented hypertonic medium (400 or 500 mOsmol/kg). 40 µg of protein was separated by 10% SDS-PAGE, and TonEBP was detected as an approximately 200-kD band. Several bands of smaller molecular weight but similar intensities also were apparent. A representative immunoblot is shown. ns, nonspecific bands. (B) Cultured mpkCCDcl4 cells were transiently transfected with TonE-driven luciferase plasmid together with either TonEBP scramble siRNA or TonEBP siRNA, seeded on filters, and grown in isotonic medium for 24 h. Cells then were exposed to isotonic serum- and hormone-free DM for 2 h and then for an additional 24 h to either the same isotonic medium or to NaCl-supplemented hypertonic medium (500 mOsmol/kg) before measurement of luciferase activity. Bars are mean ± SE from four independent experiments. *P < 0.05. (C and D) Cells transfected with either TonEBP scramble siRNA or TonEBP siRNA were grown in isotonic medium for 24 h. Cells then were exposed to isotonic serum- and hormone-free DM for 2 h and then for an additional 24 h to either the same isotonic medium or to NaCl-supplemented hypertonic medium (400 or 500 mOsmol/kg). For experiments performed with AVP 10–10 M, AVP was added 2 h after cells were exposed to isotonic serum and hormone-free DM and 2 h before hypertonic stimulation and real-time PCR was performed using primers specific for AR (C) and AQP2 (D). Results shown are expressed relative to control values determined in scramble siRNA-transfected cells after 24 h of incubation in isotonic DM in the absence of AVP (C and D, left) or presence of AVP (D, right). Bars are mean ± SE from four independent experiments. *P < 0.05. (E and F) Cells transfected with either TonEBP scramble siRNA or TonEBP siRNA were grown in isotonic medium for 48 h. Sufficient quantities of AQP2 protein necessary for Western blot quantification were obtained first by exposing cells to isotonic serum- and hormone-free DM for 2 h, then to AVP 10–9 M for 24 h, and then to the same isotonic medium or NaCl-supplemented hypertonic medium (500 mOsmol/kg) for an additional 24 h in the continuous presence of AVP. 90 µg of protein was separated by 10% SDS-PAGE. (E) A representative immunoblot is shown. AQP2 (top) was detected. AQP2 was revealed as a narrow 28-kD band and a more diffuse band of approximately 35 kD corresponding to the nonglycosylated and glycosylated forms, respectively. The Na,K-ATPase -subunit (bottom) was used as a loading control. (F) Densitometric quantification of AQP2 protein expressed as the ratio of optical density values measured at each experimental condition and that measured under isotonic conditions in cells that were transfected with TonEBP scramble siRNA. Bars are mean ± SE from three independent experiments. *P < 0.05.
TonEBP Is Required for Basal and AVP-Stimulated AQP2 Expression in mpkCCDcl4 Cells
We investigated the effects of reduced TonEBP activity on AQP2expression by transfecting mpkCCDcl4 cells with either siRNAtargeting TonEBP mRNA or with DN-TonEBP (2). The transfectionefficiency was estimated at approximately 70% by fluorescencemicroscopy (Figure 2) and FACS analysis (data not shown) ofmpkCCDcl4 cells that were electroporated with eGFP reportergene. Three different siRNA (Stealth siRNA; Invitrogen) thattargeted three different sites of TonEBP mRNA (correspondingto nucleotides 747 to 772, 796 to 821, and 3874 to 3899 of mouseTonEBP) were tested and produced similar results. Cells thatwere transfected with TonEBP siRNA displayed greatly reducedTonEBP protein (Figure 3A, lanes 4 through 6) and mRNA (three-to four-fold decrease as revealed by real-time PCR analysis;data not shown) abundance in both isotonic (300 mOsmol/kg) andhypertonic (400 and 500 mOsmol/kg) medium as compared with cellsthat were transfected with TonEBP scramble siRNA. Reduced TonEBPexpression was accompanied by decreased TonEBP activity as revealedby reduced TonE-driven luciferase activity (Figure 3B) and ARmRNA content (Figures 3C and 4A) in cells that were transfectedwith TonEBP siRNA under both isotonic and hypertonic conditions.TonEBP siRNA produced a large decrease of AQP2 mRNA (Figures3D and 4B) and significantly reduced AQP2 protein content (Figure 3, E and F)in cells that were maintained in isotonic medium as comparedwith cells that were transfected with TonEBP scramble siRNA.The extent of decreased AQP2 mRNA expression that was inducedby TonEBP siRNA under isotonic conditions was not affected byAVP. Microscopy analysis revealed indistinguishable morphologyand eGFP expression between cells transfected with eGFP plasmidand either TonEBP siRNA or TonEBP scramble siRNA (data not shown).In addition, total mRNA and protein content was not differentbetween cells transfected with either siRNA, and, contrary toAQP2, AQP4 mRNA expression was not affected by TonEBP siRNA(103.1 ± 7.3% AQP4 expression in cells transfected withTonEBP siRNA as compared with that of cells transfected withTonEBP scramble siRNA; n = 8). Finally, expression of Na,K-ATPase-subunit protein was not altered by TonEBP siRNA transfection(Figure 3E). Together, these observation clearly indicate thattransfection of mpkCCDcl4 cells with TonEBP siRNA does not affectcell viability and that, consequently, the reduced levels ofAQP2 expression that is induced by TonEBP siRNA transfectiondo not arise from cell loss or reduced cell viability. In theabsence of AVP, whereas 24 h of hypertonic challenge increasedAQP2 mRNA content in cells transfected with TonEBP scramblesiRNA, this increase was largely reduced by TonEBP siRNA (Figure 3D).AQP2 mRNA content increased by approximately 20-fold after 24h of AVP 10–10 M stimulation in mpkCCDcl4 cells transfectedwith TonEBP scramble siRNA. These expression levels were enhancedfurther by 24 h of hypertonic challenge, whereas hypertonicstimulation of AQP2 mRNA was almost entirely abolished in cellstransfected with TonEBP siRNA (Figure 3D). Similarly, increasedAQP2 protein expression that was induced by 24 h of hypertonicchallenge was significantly reduced in the presence of TonEBPsiRNA (Figure 3E, compare lanes 2 and 4).
Figure 2. Electroporation of mpkCCDcl4 cells yields high transfection efficiencies. Cultured mpkCCDcl4 cells were electroporated in the presence of 8 pmol of plasmid that contained eGFP reporter gene as described previously (24) and grown on a plastic support. Forty-eight hours after transfection, the transfection efficiency was estimated by comparing cells that were visualized under bright field (A) with the eGFP fluorescence signal emitted by the same cells (B). Transfection efficiency was estimated to be 70%. One of three similar experiments is shown.
Figure 4. Decreased AQP2 transcription induced by short-term hypertonic challenge is not altered by TonEBP siRNA knockdown. Cultured mpkCCDcl4 cells transiently transfected with either scramble or TonEBP siRNA were seeded on filters and grown in isotonic medium for 24 h, after which time the cells were exposed to isotonic serum- and hormone-free DM for 24 h and then for an additional 3 h to either the same isotonic medium or to NaCl-supplemented hypertonic medium (400 or 500 mOsmol/kg). For experiments performed with AVP 10–10 M, AVP was added 2 h after cells were exposed to isotonic serum and hormone-free DM and 24 h before hypertonic stimulation. Total RNA was extracted and reverse transcribed. (A) Real-time PCR was performed using primers specific for AR. Results shown are expressed relative to control values determined in scramble siRNA–transfected cells after 3 h of incubation in isotonic DM. Bars are mean ± SE from four independent experiments. *P < 0.05. (C) Real-time PCR was performed using primers specific for AQP2. Results are expressed relative to control values determined in scramble siRNA–transfected cells incubated for 3 h in isotonic DM in the absence (left) or presence (right) of AVP. Bars are mean ± SE from four independent experiments. *P < 0.05.
The effect of TonEBP on downregulated AQP2 expression that wasinduced after 3 h of hypertonic challenge was investigated bycomparison of the hypertonicity-induced decrease of AQP2 mRNAcontent in cells transfected with either TonEBP scramble orTonEBP siRNA. Independent of AVP stimulation, TonEBP siRNA hadno effect on the extent of decreased AQP2 mRNA expression thatwas induced by 3 h of hypertonic challenge (Figure 4B).
The consequences of downregulated TonEBP activity on AQP2 expressionobserved in mpkCCDcl4 cells transfected with TonEBP siRNA werecompared with those observed in cells transfected with DN-TonEBP.DN-TonEBP consists of the first 472 N-terminal amino acids (2)that contain the DNA-binding domain but not C-terminal transactivationdomains (28). Western blot analysis revealed an approximately70-kD band that corresponded to DN-TonEBP (9) along with a secondband of smaller size that most likely represents a DN-TonEBPdegradation product (Figure 5A, lanes 4 through 6). Expressionof full-length endogenous TonEBP protein (Figure 5A, 200-kDband) and mRNA (data not shown) of DN-TonEBP–transfectedcells was not different from that of eGFP-transfected cellsthat were incubated in either isotonic or hypertonic medium.Cells transfected with DN-TonEBP displayed two-fold lower TonE-dependentluciferase activity (Figure 5B) and slightly but significantlydecreased AR mRNA content (Figures 5C and 6A) than cells transfectedwith eGFP in both isotonic and hypertonic medium. Similar toTonEBP siRNA, DN-TonEBP produced a large decrease of AQP2 mRNA(Figures 5D and 6B) and significantly reduced AQP2 protein content(Figure 5, E and F) in cells maintained in isotonic medium ascompared with cells transfected with plasmid that containedeGFP. The extent of decreased AQP2 mRNA expression that wasinduced by DN-TonEBP under isotonic conditions was not affectedby AVP. These reduced levels of AQP2 expression did not resultfrom cell loss, because various observations (described abovefor TonEBP siRNA) clearly indicate that DN-TonEBP transfectiondid not affect cell viability. DN-TonEBP significantly reducedthe extent of increased AQP2 mRNA (Figure 5D) and protein content(Figure 5, E and F) that was induced after 24 h of hypertonicchallenge but did not affect the extent of decreased AQP2 mRNAcontent that was induced by 3 h of hypertonic challenge (Figure 6B).Taken together, these results reveal that reduced TonEBP activityleads to decreased AQP2 mRNA content under isotonic conditionsand decreased induction of AQP2 transcription after 24 h ofhypertonic challenge but has no effect on decreased AQP2 transcriptionthat occurs after 3 h of hypertonic challenge. These eventsare not influenced by AVP.
Figure 5. Increased AQP2 transcription induced by long-term hypertonic challenge is reduced by dominant-negative TonEBP (DN-TonEBP). Cultured mpkCCDcl4 cells were transiently transfected by electroporation with an expression plasmid that contained either eGFP or DN-TonEBP or contained the first 472 N-terminal amino acids (A, C, and D) or with TonE-driven luciferase plasmid together with eGFP or DN-TonEBP expression plasmids (B). Cells were subjected to 24 h of hypertonic and AVP stimulation (when indicated) as described in Figure 2. (A) Total protein was extracted, 40 µg protein was separated by 10% SDS-PAGE, and TonEBP was detected as an approximately 200-kD band in cells that were transfected with eGFP (lanes 1 through 3) or as an additional double band of approximately 70 kD in cells that were transfected with DN-TonEBP (lanes 4 through 6). A representative immunoblot is shown. ns, nonspecific. (B) Luciferase activity was measured. Bars are mean ± SE from four independent experiments. *P < 0.05. (C and D) Total RNA was extracted and reverse transcribed, and real-time PCR was performed using primers specific for AR (C) or AQP2 (D). Results shown are expressed relative to control values determined in eGFP-transfected cells after 24 h of incubation in isotonic DM in the absence of AVP (C and D, left) or presence of AVP (D, right). Bars are mean ± SE from four independent experiments. *P < 0.05. (E and F) Cells were transfected with plasmid containing either eGFP or DN-TonEBP and were grown in isotonic medium for 48 h. Cells then were exposed to AVP 10–9 M and hypertonic medium (500 mOsmol/kg) as described in Figure 3. Total protein was extracted, and 90 µg of protein was separated by 10% SDS-PAGE. (E) A representative immunoblot is shown. AQP2 (top) was detected. The Na,K-ATPase -subunit (bottom) was used as a loading control. (F) Densitometric quantification of AQP2 protein expressed as the ratio of optical density values measured at each experimental condition and that measured under isotonic conditions in cells transfected with plasmid containing eGFP. Bars are mean ± SE from three independent experiments. *P < 0.05.
Figure 6. Decreased AQP2 transcription induced by short-term hypertonic challenge is not altered by DN-TonEBP. Cultured mpkCCDcl4 cells were transiently transfected by electroporation with an expression plasmid that contained either eGFP or DN-TonEBP. Cells were subjected to hypertonic and AVP stimulation as described in Figure 3. Total RNA was extracted and reverse transcribed. (A) Real-time PCR was performed using primers that are specific for AR. Results shown are expressed relative to values determined in eGFP-transfected cells after 3 h of incubation in isotonic DM. Bars are mean ± SE from four independent experiments. *P < 0.05. (B) Real-time PCR was performed using primers specific for AQP2. Results are expressed relative to control values that were determined in eGFP-transfected cells incubated for 3 h in isotonic DM in the absence (left) or presence (right) of AVP. Bars are mean ± SE from four independent experiments. *P < 0.05.
Characterization of a TonE Element Located in the AQP2 Promoter
To establish whether the AQP2 gene represents a primary targetfor TonEBP, we investigated the functional influence of a highlyconserved TonE sequence located 489 bp upstream of the AQP2transcription start site by inserting the first 2043 or 517bp of mouse AQP2 promoter into a reporter construct that encodesfirefly luciferase. As shown in Figure 6, luciferase expressionof cells transfected with reporters that contain the first 2043or 517 bp of mouse AQP2 promoter was significantly increasedin response to hypertonicity. Co-transfection of cells withTonEBP siRNA reduced hypertonicity-induced luciferase activityby six-fold in cells that expressed TonE-driven luciferase reporterplasmid (data not shown) and abolished the hypertonicity-inducedincrease of luciferase activity in cells that expressed AQP2-luciferaseconstructs (Figure 7). In addition, the hypertonicity-inducedincrease of luciferase activity was abolished by mutation ofthe TonE sequence contained in AQP2 promoter-luciferase construct(Figure 7). These results indicate that the TonE sequence locatedat –489 bp of the AQP2 promoter participates in the TonEBP-mediatedincrease of AQP2 transcription after hypertonic challenge.
Figure 7. A TonE enhancer element located 489 bp upstream of the AQP2 transcription start site influences hypertonic stimulation of AQP2 transcriptional activity. Cultured mpkCCDcl4 cells were transiently transfected by electroporation with an expression plasmid that contained either luciferase reporter gene 5'-flanked by the first 2043 (AQP2 2.1K) or 517 (AQP2 0.6K) bp of mouse AQP2 promoter or with plasmid that contained the first 517 bp of mouse AQP2 promoter of which the TonE sequence was mutated (AQP2 0.6K TonE mut) or were co-transfected with TonEBP siRNA and with plasmid that contained luciferase reporter gene 5'-flanked by the first 517 bp of mouse AQP2 promoter. Transfected cells were seeded on filters, grown in isotonic medium for 24 h, exposed to isotonic serum- and hormone-free DM for 2 h, and then either subjected to hypertonic stimulation (500 mOsmol/kg) by NaCl supplementation for 24 h or incubated continuously in isotonic (300 mOsmol/kg) DM before measurement of luciferase activity from cell extracts. Results are expressed relative to control values that were determined in cells transfected with AQP2 2.1K (for cells transfected with AQP2 2.1K and subjected to hypertonic stimulation) or with AQP2 0.6K (for all other experimental points) and incubated for 24 h in isotonic DM. Bars are mean ± SE from five independent experiments. *P < 0.05.
Several studies performed on animals (11–15) and celllines (16,17,29) have documented increased AQP2 abundance inresponse to extracellular hypertonicity. Using cultured mpkCCDcl4cells, we showed previously that hyperosmolarity induces large,time-dependent variation of AQP2 mRNA and protein and that thesechanges are not due to changes in AQP2 protein turnover or mRNAstability but rather to altered AQP2 gene transcription (17).The results of this study from mpkCCDcl4 cells extend thesefindings by revealing that TonEBP transcription factor playsa key role in controlling both basal and hypertonicity-stimulatedAQP2 expression.
In the kidney, osmotic challenge is most important in the innermedulla and gradually decreases in more cortical segments. Importantchanges in tonicity-regulated gene expression were observedin mpkCCDcl4 cells that were derived from the cortical CD (18).Possibly, these cells may share some characteristics of medullaryCD cells. Hypertonic stress alternatively may trigger a similarregulatory response over the entire CD. This is supported bythe observation that although TonEBP is highly expressed inthe renal medulla, there is a significant level of TonEBP expressionin the cortex, especially in the cortical CD (30), an eventthat may reflect the >300 mOsmol/kg osmolality that prevailsin this segment of the CD (31). Consequently, the abundant expressionof TonEBP in mpkCCDcl4 cells reported here adds to the listof characteristics of these cells that are relevant to the CD.It should be emphasized that TonEBP, like AQP2, is expressedabundantly throughout the CD, from the cortex down to the innermedulla, supporting the idea that the observations reportedhere are relevant to the entire CD.
Our results are in good agreement with observations that weremade in mutant mice that lacked TonEBP activity, which displayan altered kidney phenotype associated with low levels of AQP2expression. The first study (32) was performed on TonEBP-nullmice, in which the DNA-binding domain of the TonEBP gene wasrendered functionally inactive. Although only 3.4% of TonEBP–/– mice survived to reach adulthood (postnatalday 21), those that did survive displayed impaired activationof several genes that are targeted by TonEBP under conditionsof hypertonic stress as well as markedly downregulated AQP2protein expression. However, because TonEBP knockout was associatedwith severe renal apoptotic atrophy, especially prevalent inregions of the kidney that endure the highest extents of extracellulartonicity, such as the renal medulla, i.e., high AQP2-expressingregions, it is difficult to determine whether the decreasedAQP2 expression levels in TonEBP –/– mice resultsdirectly from loss of TonEBP activity or arises indirectly fromthe general alteration of kidney structure and function. Thesecond study (33) was performed on mice that overexpressed DN-TonEBPspecifically in kidney CD. Like TonEBP –/– mice,transgenic mice that overexpressed DN-TonEBP displayed renalatrophy, although to variable extents and only at approximately8 wk of age. The authors of this study took care to investigatethe effects of inhibited TonEBP activity on mice that were 5wk of age, i.e., at a time that preceded the gross structuralabnormalities that were observed in the transgenic kidney thatdisplayed reduced levels of AQP2 expression. In addition, theauthors showed a large reduction of AQP2 expression in renalepithelial cell cultures that were derived from transgenic mice.However, the possibility that downregulated AQP2 expressionis manifested as an indirect consequence of the lack of TonEBPactivity still cannot be excluded because the differentiationstates of transgenic CD cells might be altered, an event thatarises from the incremental stress that is incurred on transgeniccells and ultimately leads to kidney atrophy. The phenotypeof CD principal cells is retained largely in cultured mpkCCDcl4cells that were used in our study, as demonstrated by AVP- andaldosterone-sensitive electrogenic Na+ transport. Moreover,this highly differentiated state is maintained in transfectedcells (24,34). In this respect, we believe that the changesthat were observed in cultured mpkCCDcl4 cells that were transientlytransfected with TonEBP siRNA or DN-TonEBP more accurately reflectdirect consequences of altered TonEBP activity than changesthat were observed in cells that displayed a reprogrammed phenotypeas a result of decreased TonEBP activity.
Short-term (3 h) hypertonic challenge was found to decreaseAQP2 mRNA abundance in mpkCCDcl4 cells to similar extents inboth the presence and the absence of AVP. Because the ratioof decreased AQP2 mRNA abundance was similar among eGFP-, TonEBPsiRNA–, and DN-TonEBP–transfected cells, we reasonablycan assume that TonEBP is not involved directly in this event.This is supported further by the observation that AQP2 mRNAand protein abundance also is reduced by elevated concentrationsof urea (17), a condition that does not induce TonEBP activation.However, TonEBP was found to play a major role in increasedAQP2 transcriptional activity that was induced by long-term(24 h) hypertonic challenge. This is supported by a bluntedincrease of AQP2 mRNA content in both TonEBP siRNA–andDN-TonEBP–transfected cells that displayed downregulatedTonEBP activity in response to 24 h of hypertonic challengeas compared with cells that displayed normal TonEBP activity.Taken together, the results of our study indicate that TonEBPplays a key role in regulating AQP2 transcriptional activityand that the initial decrease of AQP2 transcriptional activitythat is induced by hyperosmotic challenge would be eclipsedby TonEBP-mediated enhanced transcription of the AQP2 gene.
The results of this study further indicate that the highly conservedTonE sequence located 489 bp upstream of the AQP2 transcriptionstart site represents a target site for TonEBP. Luciferase activityonly slightly increased in response to hypertonicity in cellsthat expressed AQP2 promoter-luciferase constructs as comparedwith that of cells that expressed TonE-driven luciferase reporterplasmid. However, only a short sequence of AQP2 promoter wasinvestigated. Analysis of SMIT promoter revealed that at leastfive TonE sequences spread over 50-kb pairs participate in TonEBP-mediatedtranscriptional stimulation (6). Similarly, three TonE sequencesthat were identified in AR promoter may participate collectivelyin the increased transcriptional activity of this gene by hypertonicity(5). The influence that TonEBP exerts on AQP2 transcriptionthereby may result from the combined targeting of several putativeTonE elements that are present in the AQP2 promoter. In addition,transcriptional factors other than TonEBP that bind to the AQP2promoter may influence the transcriptional activity of TonEBPin response to hypertonicity. Indeed, the literature depictsa complex multifactorial process that governs AQP2 gene transcription.AVP-inducible phosphorylated cAMP response element-binding protein(pCREB), via binding to cAMP response element cis-element ofthe AQP2 promoter (35,36), plays an undisputed role in AQP2gene transcription, and several other cis-elements that eitherpromote or repress AQP2 gene transcription have been proposed(29,36–39). The ratio of trans-acting nuclear factorsthat are bound to the AQP2 promoter, that in turn is determinedby specific extracellular conditions, most likely would influencedifferentially the activities of each individual factor, suchas TonEBP, an event that would determine the overall transcriptionalactivity of the AQP2 gene.
Several observations indicate that TonEBP regulates AQP2 transcriptionalactivity independent of AVP and that the extent of influencethat is exerted by TonEBP on AQP2 transcription is comparableto that exerted by AVP. First, AQP2 mRNA content of mpkCCDcl4cells that were incubated in isotonic medium in the absenceof AVP was reduced by both TonEBP siRNA and DN-TonEBP transfection,indicating that baseline expression of AQP2 mRNA is highly dependenton TonEBP. Second, the relative effect of TonEBP on increasedAQP2 mRNA expression that was induced by long-term (24 h) hypertonicchallenge was the same in the presence and absence of AVP, indicatingthat AVP and TonEBP each influence AQP2 transcriptional activityvia independent mechanisms of regulation. Third, the extentof TonEBP-mediated increased AQP2 mRNA expression that was inducedby long-term (24 h) hypertonic challenge that occurred in theabsence of AVP was comparable to that produced by 24 h of AVPstimulation. Moreover, 24 h of hypertonic challenge increasedAQP2 expression regardless of the absence or presence of myristoylatedPKA inhibitor in AVP-pretreated mpkCCDcl4 cells (17). However,maximal induction of AQP2 transcription was achieved only incells that were incubated in hypertonic medium that containedAVP, indicating that maximal AQP2 gene transcription dependson both TonEBP-mediated hypertonic stimulation and plasma AVPconcentration. Such a dual influence would provide the cellwith at least two independent but complementary pathways tocontrol AQP2 transcriptional activity.
TonEBP plays at least two major roles in the kidney: It participatesin the generation of the corticopapillary osmotic gradient bycontributing to interstitial urea build-up via stimulation ofAVP-regulated urea transporter transcriptional activity, andit protects cells from the devastating effects of urea and hypertonicityby respectively inducing expression of heat-shock protein 70and accumulating organic osmolytes (40). Our study shows thatTonEBP additionally influences AQP2 gene transcription and thatTonEBP- and AVP-induced increase of AQP2 abundance and, therefore,CD water permeability cooperate to allow the organism to adaptto water restriction.
Acknowledgments
This work was supported by the Swiss National Foundation forScience grant 3100-067878.02 and a grant from the Carlos etElsie De Reuter Foundation to E.F., a grant from the NovartisFoundation to Udo Hasler, National Institutes Health grant DK42479to H.M.K., and the National Kidney Foundation Fellowship toJ.A.K.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
E.F. and P.-Y.M. contributed equally to this work.
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Abstract
Introduction
/adenylyl cyclase activation, increased intracellular cAMP concentration, and protein kinase A (PKA) activation (10). Several pieces of evidence indicate that AQP2 abundance additionally is influenced to a large extent by extracellular tonicity. This has been illustrated by in vivo experiments in water-restricted animals treated with V2-receptor antagonists that display increased AQP2 content (11,12) and in water-loaded animals that display decreased AQP2 content despite ongoing V2 receptor stimulation (11,13). In addition, cAMP-independent decreased AQP2 expression that occurs in parallel with low levels of medullary osmolarity in senescent rats was reverted by raising medullary osmolarity (14,15). In vitro experiments using primary cultured inner medullary CD cells have shown further that extracellular hypertonicity stimulates AQP2 expression independent of PKA activity (16,17). 
