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Kidney International, Vol. 67 (2005), pp. 1410–1419

ION CHANNELS – MEMBRANE TRANSPORT – INTEGRATIVE

Circadian clock genes directly regulate expression of the + + Na /H exchanger NHE3 in the kidney

MOHAMMAD SAIFUR ROHMAN,1 NORIAKI EMOTO,HIDEMI NONAKA,RYUSUKE OKURA, MASATAKA NISHIMURA,KAZUHIRO YAGITA,GIJSBERTUS T.J. VAN DER HORST,MASAFUMI MATSUO, HITOSHI OKAMURA, and MITSUHIRO YOKOYAMA

Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School, Kobe, Japan; Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan; and MGC, Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands

Circadian clock genes directly regulate expression of the Circadian rhythmicity is observed in many aspects Na+/H+ exchanger NHE3 in the kidney. of cellular function, including membrane excitation, en- Background. Daily rhythms in mammalian physiology are ergy metabolism, and cell division [1–3]. In mammals, generated by a /translation feedback loop orches- trated by a set of clock genes. However, little is known about the clock system is composed of a central clock in the the molecular cascade from the clock gene oscillators to cellular which generates the standard function. time of the body at systemic levels, and peripheral clocks Methods. The mRNA expression profiles of NHE3 and clock which perform the effector processes of the circadian genes were examined in mice and rat kidneys. First, luciferase rhythms with the aid of systemic suprachiasmatic nucleus assays followed by a site directed mutagenesis of an E-box se- quence were used to assess the CLOCK:BMAL1-transactivated signals in the variety of organs [4, 5]. NHE3 promoter activity. A direct binding of CLOCK:BMAL1 In both central and peripheral clock systems, circa- heterodimers to an E-box sequences of NHE3 promoter was dian rhythmicity is generated at the cellular level by the confirmed by electrophoretic mobility shift assay (EMSA). circadian core oscillator composed of an autoregulatory Results. We present evidence that renal tubular NHE3, the + + transcription-(post)translation-based feedback loop in- Na /H exchanger critical for systemic electrolyte and acid- base homeostasis, is a clock-controlled gene regulated directly volving a set of clock genes. In this molecular loop, two by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA transcriptional activator genes, CLOCK and BMAL1, level in rat kidney displayed circadian kinetics, and this circa- regulate gene expression by binding to specific enhancer dian expression was severely blunted in homozygous CRY1/2 elements, termed E boxes (CTCGTG). Target genes of double-deficient mice, suggesting that the transcriptional ma- these activators include several repressor proteins, in- chinery of peripheral clocks in renal tubular cells directly reg- ulates the circadian expression of NHE3.Byanalyzing the 5 cluding PER1, PER2, PER3, CRY1, and CRY2, which upstream region of the NHE3 gene, we found an E box critical function to inhibit the CLOCK:BMAL1 complex, thus for the transcription of NHE3 via the CLOCK:BMAL1-driven generating a circadian oscillation in their own transcrip- circadian oscillator. The circadian expression of NHE3 mRNA tion [6–8]. + was reflected by oscillating protein levels in the proximal tubules In the kidney, plasma Na concentration and renal of the rat kidney. + Conclusion. NHE3 should represent an output gene of the Na excretion are known to display a significant diur- peripheral oscillators in kidney, which is regulated directly by nal variation in both animals and humans [9–13]. The CLOCK:BMAL1 heterodimers. mechanisms involving these diurnal changes are related to the regulation of renal blood flow and renal cellular functions, though the underlying molecular mechanism 1The current address for Dr. Rohman is Faculty of Medicine, Brawijaya University, Malang, Indonesia. remains unclear. Since a number of genes in the kidney show diurnal variation, we searched for genes likely in- + + volved in these processes. NHE3 is one of the Na /H Keywords: NHE3, clock controlled gene, peripheral clock, circadian, acid-base homeostasis. exchangers (NHEs) that catalyze the electroneutral ex- + + change of one extracellular Na for one intracellular H Received for publication July 22, 2004 across the plasma membrane, to mediate bulk reabsorp- and in revised form October 9, 2004 + Accepted for publication October 25, 2004 tion of filtered Na in the proximal convoluted tubule [14–16]. Gene knockout experiments in mice have been C 2005 by the International Society of Nephrology suggested that NHE3 is required for maintenance of

1410 Rohman et al: Circadian regulation of NHE3 by peripheral clocks 1411

+ normal set point for Na fluid volume balance. In ad- transcriptase-polymerase chain reaction (RT-PCR) dition, NHE3 knockout mice shown a decreased blood using primers 5 -GGTCAATGTGGACTTCAGCAC- pressure, an elevated plasma and renin 3 and 5 -GGGGAGAACACAGGATTATCAAT-3 mRNA in the kidney [17], thus supporting the view that (GeneBank, accession number M.85300.1). The clock + the major renal transporter mediating Na reabsorption gene probes were generated as previously described [21]. plays a central role in long-term control of arterial blood The probes were labeled with [a 32P] deoxycytidimine pressure [18, 19]. triphosphate (dCTP) using random priming after se- In the present study, we investigated whether the ex- quence confirmations, and were exposed to the imaging pression of NHE3 has diurnal variations, and whether it plates of a Fuji-Bio Imaging Analyzer BAS 2000 (Fuji is regulated by the circadian clock system. We demon- Photo Film, Kanagawa, Japan). strate here that NHE3 mRNA and protein display circadian expression in the rodent kidney. Circadian ex- Construction of plasmids pression of NHE3 is mediated by the direct control of The promoter of rNHE3 was constructed by amplifi- CLOCK:BMAL1 heterodimers through an E box in the cation of a 1.36 kb fragment of the 5 flanking region of promoter of the NHE3 gene. This conclusion is further rNHE3 gene from rat genomic DNA using the primers supported by genetic studies. In homozygous CRY1/2 5 -TCCAGTTCCTTACCCAGTCAGTCTC-3 and 5 - double-deficient mice, circadian expression of NHE3 GCTCCAGGAGCCGACACGCATAC-3 .The ampli- mRNA is significantly reduced. Immunohistochemistry fied fragment was digested with BamHI followed by blunt experiments clearly showed the colocalization of NHE3 end generation at one end and cut with KpnIatthe other. and PER2 in the proximal tubule cells in kidney. Taken This fragment was directionally cloned into SmaI-KpnI together, these results strongly suggest that the transcrip- digested picagene basic vector (PGV-B) luciferase re- tional regulation of NHE3 is controlled by the core oscil- porter (Tokyo, Japan), resulting in a −1360/+58 bp (rela- lator, and that NHE3 represents a clock-controlled gene tive to the transcriptional initiation sites) promoter con- (Ccg)inthe kidney. struct. The constructs spanning −605/+58 bp, −484/+58 bp, −315/+58 bp, and −17/+58 bp were generated by METHODS digesting the 1.36 kb fragment of BamHI-KpnI5 flank- Materials ing region of rNHE3 with EcoRI, NcoI, SalI, and SmaI, respectively. These fragments were subcloned into the Enzymes used in molecular cloning were obtained SmaI/KpnI sites of PGV-B and used after confirma- from Roche Molecular Biochemicals or from New Eng- tion by enzymatic digestion. Deletion of −141/−18 bp land Biolabs (Beverly, MA, USA). fragment was done with BstXI and SmaItoremove the putative promoter region, whereas removal of the Animals and procedures −17/+58 bp fragment was accomplished by digestion of Sprague-Dawley rats purchased 5 weeks postpartum the −1360/+58 bp promoter construct with SmaI and were exposed to 2 weeks of 12-hour light (fluorescent KpnI. Clock gene expression constructs were obtained by light, 300 lux)/dark cycles and then kept in complete dark- RT-PCR from the coding regions of mPer2 (AF035830), ness (dark/dark) for 2 days as a continuation of the dark mCry1 (AB000777), hBMAL1 (AB000813), and hClock phase of the last cycle. Three- to 5-week-old Balb-c and (AB002332), as previously described [22, 23]. mCry1−/− mCry2−/− mice [20] were housed under the same conditions as Sprague-Dawley rats. The mRNA ex- Mutagenesis of E box pression profiles of NHE3 and clock genes were exam- Mutant construct was made by site directed muta- ined in the second dark-dark cycle every 4 hours, starting genesis [24] using the Muta-Gene Phagemid In Vitro at the beginning of the light cycle. The care and use of Mutagenesis Kit, version 2 (Bio-Rad Laboratories, Her- the animals strictly followed the guidelines of the Ani- cules, CA, USA) as described by the manufacturer. All mal Research Committee of Kobe University Graduate constructs were verified by sequencing the final plasmids. School of Medicine. Transcriptional assay Northern blot analysis Opossum kidney (OK) cells were grown in Dulbecco’s Ten micrograms of total RNA was electrophoresed in modified Eagle’s medium (DMEM) with 2 mmol/L a 1.2% denaturing agarose gel containing formaldehyde L-glutamine, Earle’s balanced salt solution (BSS), and and transferred onto nylon membranes. The blots 0.1 mmol/L nonessential amino acids supplemented with were hybridized in QuickHyb hybridization solution 10% fetal bovine serum (FBS), 1.5 g/L sodium bicarbon- (Stratagene, La Jolla, CA, USA) at 68◦Corina50% ate, and 1.0 mmol/L sodium pyruvate (Sigma Chemical formamide-containing hybridization solution at 42◦C. Co., St. Louis, MO, USA). OK cells passages 4 to 10 were To obtain a specific probe for rat NHE3 (rNHE3),a plated at 70% to 80% confluency in 60 mm dishes 24 fragment of the rNHE3 cDNA was amplified by reverse hours before transfection. The cells were transfected with 1412 Rohman et al: Circadian regulation of NHE3 by peripheral clocks

3 lg (total) of expression plasmids with the indicated in- natants were centrifuged at 100,000 × g for 30 minutes sert in pcDNA3 (Invitrogen, Carlsbad, CA, USA) and 1.7 at 4◦C, and the pellets containing membrane proteins lgofreporter plasmid using Lipofectamine Plus (Invit- were resuspended in homogenization buffer. The pro- rogen) according to the manufacturer’s instructions. To tein was assayed by a Multiskan JX Thermo Labsystem. correct for variation in transfection efficiency, pcDNA3 Membrane proteins were solubilized in sample buffer vector was adjusted to the total amount of DNA per dish. and separated by sodium dodecyl sulfate-polyacrylamide Cell extracts were prepared 23 to 24 hours after transfec- gel electrophoresis (SDS-PAGE) using 7.5% polyacry- tion by a lysis buffer. Twenty microliters of the extract lamide gels. Immunoblots were incubated in primary anti- was taken for luciferase assay using a luminometer as NHE3 monoclonal antibody (Chemicon Int., Temecula, described by the manufacturer (Pikkajin). For statistical CA, USA) (MAB3134) at a 1:750 dilution in the same analysis, a two sample t test was applied. buffer for overnight at 4◦C. The blots were then washed in washing buffer and incubated in secondary antimouse Gel shift assay IgG antibody for 1 hour. The signals were visualized as described [24]. Nuclear extracts were prepared as described [25]. Briefly, CLOCK:BMAL1-transfected or untransfected OK cells were washed with 5 mL 1 × Tris-buffered Immunohistochemistry saline (TBS) and pelleted by centrifugation at 1500g for Sections of rat kidney were analyzed at CT8 and 5 minutes. The pellet was resuspended in 1 mL TBS, CT20 by immunohistochemistry (N = 3ateach time transferred into an Eppendorf tube, and followed by point) for expression of NHE3 protein. Rats were deeply spinning for 5 seconds. The supernatant was removed anesthetized with ether and intracardially perfused with and the pellet was dissolved in 40 lL cold buffer A [10 ice-cold saline for 2 minutes, followed by a fixative con- mmol/L Hepes, pH 7.9; 10 mmol/L KCl; 0.1 mmol/L taining 4% paraformaldehyde in 0.1 mol/L phosphate ethylenediaminetetraacetic acid (EDTA); 0.1 mmol/L buffer for 5 minutes. The kidneys were removed, post- etheylenglycol tetraacetate (EGTA); 1 mmol/L dithio- fixed, embedded in paraffin, and cut into 5 lm thick sec- threitol (DTT); and 0.5 mnol/L phenylmethylsulfonyl tions. Sections were incubated in anti-NHE-3 antibody fluoride (PMSF)]. The cells were allowed to swell on ice (Chemicon Int.) (mouse monoclonal, 1:1000) for 2 days at for 15 minutes, after which 25 lLofa10% NP-40 was 4◦C, then treated with fluorescein isothiocyanate (FITC)- added. The tubes were vigorously vortexed for 10 seconds conjugated antimouse IgG (1:100) (Vector Laborato- and centrifuged for 30 seconds at 15,000 rpm. The nuclear ries, Burlingame, CA, USA), and observed with confo- pellet was dissolved in 50 lL ice- cold buffer containing cal fluorescent microscopy (Carl Zeiss AG, Oberkochen, 20 mmol/L Hepes, pH 7.9; 0.4 mol/L NaCl; 1 mmol/L Germany). For double labeling, sections were treated EDTA; 1 mmol/L EGTA; 1 mmol/L DTT; and 1 mmol/L with rabbit anti-PER2 antibody and mouse anti-NHE3 PMSF. The nuclear extract was centrifuged at 3000 rpm antibody followed by cyanine-3 (Cy3)-conjugated don- for 5 minutes, and 7 lLofthe supernatant was used for key antirabbit IgG and FITC-conjugated donkey anti- the gel shift assay. Electrophoretic mobility shift assay mouse IgG. Slices were washed, mounted with glycerol (EMSA) was carried out according to the manufacturer’s on a cover glass, and observed under a confocal fluores- protocol (Promega, Madison, WI, USA) using a double- cence microscope. stranded oligonucleotide: 5-ACGCAGCAGACTGTTT GCATTCACGTGCGC-3 , including the E-box binding Data analysis and statistics site derived from the rNHE3 promoter. The oligonu- The significance of differences between groups and cleotide was labeled with c adenosine triphosphate (ATP) among time points was determined by two-way or one- using T4 polynucleotide kinase. For the competition ex- way analysis of variance (ANOVA). Values are consid- periment, a 50-fold excess of double stranded unlabeled ered significantly different if P < 0.05. A test was calcu- nucleotide 5-ACGCAGCAGACTGTTTGCATTCAC lated using Statview software. GTGCGC-3 or probe containing a mutated E-box site 5-ACGCAGCAGACTGTTTGCATTGGATCCCGC- 3 was used. RESULTS NHE3 transcripts display a circadian rhythmic expression Immunoblotting in kidney Kidneys were homogenized in homogenization buffer We observed rhythmic expression of the rat NHE3 + + (50 mmol/L Tris, pH 7.5; 1 mmol/L EGTA; 250 mmol/L (rNHE3) gene, a predominant Na /H exchanger in the sucrose; 1 mmol/L DTT; 0.1 mmol/L leupeptin; 1 lmol/L rat kidney. As shown in Figure 1, the amount of rNHE3 pepstatin A; and 0.25 mmol/L PMSF), followed by cen- mRNA in the rat kidney began to increase at CT4 (circa- trifugation at 1000 × g for 10 minutes at 4◦C. The super- dian time used for assessing biologic time in a complete Rohman et al: Circadian regulation of NHE3 by peripheral clocks 1413

CT 048121620 ney (Fig. 2C). These results suggest that the circadian rNHE3 rhythmic expression of the mNHE3 gene is blunted in the GAPDH absence of clock gene rhythmicity in Cry1/Cry2-double mutant mice, and thus must depend on the presence of 100 an intact circadian oscillator.

80 The rNHE3 promoter contains an E box 60 To dissect the molecular mechanism behind the rhyth- mic expression of NHE3 at the transcriptional level, we 40 first performed a series of assays to determine the im- portant regions of the rNHE3 promoter for transcrip- tional activity. Constructs containing various lengths of

Relative mRNA level Relative 20 the rNHE3 5 flanking regions inserted upstream of the 0 firely luciferase gene were transiently transfected into 048121620 OK cells as described in the Methods section. As shown in Figure 3A, a −315/+58 bp construct showed compara- CT ble promoter activity to those containing up to 1360 bp Fig. 1. Circadian oscillation of rNHE3 mRNA. Rats were kept in of 5 sequence. By contrast, the −17/+58 bp SmaI-KpnI light/dark cycles as described in the Methods section. TotalRNA (10 lg) construct displayed substantially decreased promoter ac- from kidney was used for Northern blot analysis, and the signals were quantified and normalized to glyceraldehyde-3-phosphate dehydroge- tivity exhibiting only a threefold activation of luciferase nase (GAPDH). Representative Northern blots (upper panel). Normal- activity compared to the promoterless PGV-B vector. = ized mRNA levels (mean ± SEM) (N 6) (lower panel). Peak mRNA These results suggested that the fragment from −315 to expression was set at 100. One-way analysis of variance (ANOVA) − (P < 0.0001). CT is circadian time used for assessing biologic time in a 18 bp is important for basal transcription of rNHE3.To dark/dark cycle; CT0 is lights on and CT12 is lights off. confirm that this promoter region was directly involved in rNHE3 gene expression, the full-length (−1360/+58) reporter construct was digested with BstXI and SmaIto darkness cycle, as described in experimental procedures), delete bp −141 to −18. Deletion of this region resulted increasing steadily to the point of highest accumulation in an 87% decrease in promoter activity as compared to at CT16. rNHE3 mRNA levels subsequently decreased that of the −315/+58 bp construct, suggesting that se- and stayed low until CT4. The maximum rNHE3 mRNA quences between bp −141 to –18 are essential for pro- level was 2.5-fold higher than the minimal level. moter activity of rNHE3 in OK cells. We then examined whether regions further downstream might play an im- Circadian expression of clock gene and NHE3 mRNA portant role in rNHE3 promoter activity by removing the − + is severely blunted in Cry1/Cry2-double mutant mice 17/ 58 bp fragment. The transcriptional assay showed that deletion of this fragment significantly decreased pro- Next, we examined whether clock genes may control moter activity, indicating that the −17 to +58 fragment the rhythmic pattern of NHE3 transcription. First, we was required for optimal rNHE3 basal activity even if checked the circadian expression profiles of clock genes it was not sufficient to show high levels of promoter ac- in the kidney of wild-type and arrhythmic mCry mutant −/− −/− tivity when expressed alone. Taken together, the tran- mice (mCry1 and mCry2 ). Northern blot analysis scriptional assay suggested that the region encompassing revealed that mPer2, the main oscillator gene in the mPer –141/+58 bp is important for rNHE3 promoter activity in family, was rhythmic at the mRNA level in the kidney of OK cells. We next analyzed the sequence of the −141/+58 wild-type mice with a peak at CT12-16 and a trough at bp promoter region of rNHE3 and found that an E-box CT0-4. As expected, mPer2 mRNA no longer cycles in element (CACGTG), which is the consensus binding site Cry1/Cry2-deficient mice and expression levels are con- of CLOCK and BMAL1, is located at bp −86 to −81 stantly high (Fig. 2A). mBMAL1 mRNA showed an op- (Fig. 3B). The rat and mouse NHE3 genes are 92.7% posite rhythmic expression profile to mPer2 in wild-type identical in the –107/+58 bp regions with the E-box el- mice, and was constantly low in the kidney of Cry1/Cry2- ements of rat and mouse NHE3 genes being completely deficient mice (Fig. 2B). conserved. Next, we performed Northern blot analysis to de- termine whether the daily rhythm of mouse NHE3 (mNHE3)isabolished in the absence of the circadian CLOCK and BMAL1 transactivate the rNHE3 promoter pacemaker. In wild-type kidney, the accumulation of through the E-box site mNHE3 fluctuated during the day. In marked contrast The presence of an E box at the promoter region, and to wild-type mice, Cry1/Cry2 double-mutant mice failed the resemblance of the rNHE3 gene expression profile to to show significant mNHE3 mRNA cycling in the kid- that of previously reported CLOCK/BMAL1-mediated 1414 Rohman et al: Circadian regulation of NHE3 by peripheral clocks

A CT 048121620 CT 048121620 mPer2 mPer2 GAPDH GAPDH

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0 0 048121620 048121620 CT CT

B CT 048121620 CT 048121620 mBMAL1 mBMAL1 GAPDH GAPDH

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40 40 Relative mRNA level Relative Relative mRNA level Relative 20 20

0 0 04 8121620 048121620 CT CT

C CT 048121620 CT 048121620 mNHE3 mNHE3 GAPDH GAPDH

100 100 Fig. 2. Circadian rhythms of clock genes and NHE3 mRNA expression are blunted 80 80 in mCry1−/− mCry2−/−. Northern blot analysis of mPer2 (A), mBMAL1 (B), and mNHE3 (C)inwild-type mice (left panel) 60 60 −/− /− and mCry1 mCry2 (right panel). The peak values observed in wild-type mice were 40 40 adjusted to 100 after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are expressed as means ± 20 20 = Relative mRNA level Relative Relative mRNA level Relative SE (N 3). The mice were housed under 12- hour light ()/12-hour dark (), Light/dark cy- 0 0 cles lasted for at least 2 weeks. CT is circa- 048121620 048121620 dian time used for assessing biologic time in a dark/dark cycle; CT0 is lights on and CT12 is CT CT lights off. Rohman et al: Circadian regulation of NHE3 by peripheral clocks 1415

A +1 −1360 Luc −605 Luc

−484 Luc Luc −315 Luc −17 −1360 (∆ − 141 ∼ −18) Luc Luc

Luc −1360 (∆ − 17 ∼ +58) Basic Luc BamHI Ncol BstXI Kpnl 0102030405060 EcoRI Sa/I SmaI Luciferase activity +58

B −141

+1 +58

Fig. 3. The NHE3 promoter contains an E box. (A)Therat NHE3 promoter activity in opossum kidney (OK) cells. Luciferase reporter plasmids containing various lengths of the rat NHE3 5 flanking region were generated and transiently transfected into OK cells as described in the Methods section. Deletion of BstXI-SmaI and SmaI-KpnI fragments, corresponding to bp −141 to −18 and −17 to +58, respectively, resulted in an 87% decrease in promoter activity compared to that of the −314/+58 bp construct, suggesting the –141 to +58 bp fragment is essential for promoter activity of rat NHE3 in OK cells. Luciferase activity is represented as the ratio (mean ± SEM) of the indicated rat NHE3 5 flanking region value to the promoterless picagene basic vector (PGV-B) value. (B)Therat NHE3 promoter region contains an E-box element (CACGTG), the consensus binding site of CLOCK and BMAL1. The E box located at bp −86 to −81 of the rat NHE3 promoter. The rat and mouse NHE3 genes are 92.7% identical in the –107/+58 bp regions with the E-box elements of rat and mouse NHE3 are completely conserved. +1 represents the transcriptional initiation site of the rat NHE3 promoter sequences.

E box-regulated genes (e.g., dbp, mPer1, and vasopres- ment is important for CLOCK:BMAL1 transactivation sion) suggest the possibility that the circadian oscilla- of the rNHE3 promoter. tion of the rNHE3 gene is regulated through this E box. To assess whether PER and CRY, the components of To investigate this possibility, we performed a transient the negative limb of the circadian feedback loop, could transfection experiment using a 1360 bp fragment of the inhibit CLOCK:BMAL1-driven rNHE3 expression, we 5 flanking region of the rNHE3 promoter/luciferase re- cotransfected PER2 and CRY1 with CLOCK-BMAL1 porter plasmids. Transient transfection of BMAL1 alone in OK cells. We observed that PER2 alone partially in- resulted in decreased rNHE3 promoter by 35% of basal hibited CLOCK:BMAL1 activation of rNHE3, whereas activity. However, transfection of CLOCK alone did not cotransfection of CRY1 alone abolished the induction of significantly change rNHE3 promoter activity. Cotrans- the rNHE3 promoter activity (Fig. 5). These data suggest fection of CLOCK and BMAL1 increased rNHE3 pro- that PER2 and CRY1 can suppress CLOCK:BMAL1 me- moter activity by five- to sixfold (Fig. 4). Deleting bp diated transactivation of rNHE3 gene expression. −141 to –18 containing an E box of rNHE3 promoter (−1360 −141 ∼−18) markedly diminished this induc- tion by CLOCK and BMAL1 (data not shown). We then The E box in the rNHE3 promoter directly interacts generated a point mutation of this E box to determine with the CLOCK:BMAL1 heterodimer whether it is important for the transactivation of the To examine whether transcriptional activation of rNHE3 promoter by CLOCK and BMAL1. Mutation of NHE3 by CLOCK:BMAL1 is due to direct binding of the E box completely abolished the CLOCK and BMAL1 CLOCK:BMAL1 to the rNHE3 promoter, we performed mediated transactivation (Fig. 4). Thus, the E-box ele- a gel shift assay (Fig. 6). When a radiolabeled 30 bp probe, 1416 Rohman et al: Circadian regulation of NHE3 by peripheral clocks

6 * 6 *

5 5 4 4 3 3

old induction 2 F old induction

# F 2 1 # * 1 0 # NHE3 construct + +++ − 0 ∆ NHE3 construct − −−− + NHE3 construct + +++ hClock − +−+ + hClock − +++ hBMAL1 − −++ + hBMAL1 − +++ Fig. 4. CLOCK and BMAL1 transactivates the rat NHE3 promoter mPer2 − −+− through an E-box site. Transfection of 0.5 lg hCLOCK or hBMAL1 mCry1 − −−+ into opossum kidney (OK) cells with the rat NHE3 promoter reporter construct did not increase NHE3 transcriptional activity. Cotransfec- Fig. 5. PER2 or CRY1 inhibits transactivation of the NHE3 promoter tion of hCLOCK and hBMAL1 increased promoter activity by five by CLOCK:BMAL1. Cotransfection of 0.3 lg mPER2 or mCRY1 with ∼ sixfold. Mutation of the E box completely abolished the hCLOCK hCLOCK and hBMAL1 into opossum kidney (OK) cells abolished the and hBMAL1-mediated transactivation. The E box at bp –86 to –81 induction of the NHE3 promoter. Fold induction represents the ratio was mutated from CACGTG to GGATCC. The results are the mean (mean ± SE) (N = 3) of luciferase activity in cells transfected with of three independent experiments. Fold Induction represents the ratio the expression plasmid to that of cells transfected with empty vector of luciferase activity in cells transfected with expression plasmids to (pcDNA3) (mean ± SEM). ∗P < 0.01 vs. NHE3 construct only; #P < ± that of cells transfected with empty vector (pcDNA3) (mean SEM). 0.01 vs. NHE3 construct cotransfected with hCLOCk and hBMAL1 The NHE3 construct is 1.36 kb of the rat NHE3 promoter/luciferase into OK cells. plasmids. is the rat NHE3 promoter reporter construct containing an E-box mutation. ∗P < 0.01 vs. NHE3 construct only: #P < 0.01 vs. NHE3 construct cotransfected with hCLOCK and hBMAL1 into OK cells. the circadian expression of NHE3 is observed at the pro- tein level as well as the transcriptional level. encompassing the putative E box binding site, was in- NHE3 protein colocalizes with PER2 in the kidney cubated with a nuclear extract from untransfected OK NHE3 protein expression was examined in the kid- cells, only a weak band was observed, which is believed ney by immunohistochemistry (Fig. 8A). Similar to pre- to be due to binding to endogenous proteins. In con- vious reports [26, 27], the NHE3 protein was expressed trast, nuclear extracts from OK cells transfected with in the epithelial brush border of the proximal convo- CLOCK and BMAL1 expression vector gave a clear sin- luted tubules in the renal cortex, and the thick ascend- gle band. This intense band was abolished by addition ing limbs of Henle in the renal medulla. We found that of a 50-fold excess of an unlabeled competitor probe the immunoreactivity in the brush border was higher at containing the E-box consensus sequence, whereas ad- CT20 than at CT8, although the NHE3 immunoreactiv- dition of a 50-fold excess of competitor probe contain- ity in the renal medulla was not altered. To test whether ing a mutated E-box sequence had no effect. Taken to- NHE3 and the core clock protein PER2 coexpressed in gether, these results suggest that the intense band rep- the same cells, we performed double-labeling histochem- resents a specific CLOCK:BMAL1-E-box complex, and ical experiments. As shown by Figure 8B, NHE3 staining that CLOCK:BMAL1 transactivates NHE3 through di- on the tubular surface and PER2 staining in the nucleus rect binding of CLOCK:BMAL1 to the E box of the are present in the same cells of the proximal tubule of NHE3 promoter. the kidney. This in vivo study therefore complements our in vitro results that NHE3 is under the direct control of peripheral clock genes, where the CLOCK:BMAL1 het- Circadian expression of NHE3 protein in the kidney erodimer transactivates the promoter and PER2 inhibits To investigate whether the circadian oscillation of this transactivation. NHE3 is observed at the protein level, we performed immunoblot analysis on the samples obtained from rat kidney (Fig. 7). NHE3 protein levels reached a peak at DISCUSSION CT20-24 (0) and a trough at CT8, 8-hour delayed of In the present study, we have provided several lines rNHE3 mRNA levels. These findings demonstrate that of evidence showing that NHE3, a well-characterized Rohman et al: Circadian regulation of NHE3 by peripheral clocks 1417

NHE3 promoter probe + + + + CT 048121620 CLOCK/BMAL1 transfected − + + + rNHE3 Unlabelled probe − − + − Competitor [ − − − + E-box mutant 100

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20 Relative protein level Relative

0 Free 048121620 CT

Fig. 7. Circadian oscillation of rNHE3 protein levels. Peak protein ex- pression was set at 100. Representative Western blots (upper panel). Fig. 6. CLOCK and BMAL1 directly binds the E box of the ± = rat NHE3 promoter. A gel shift assay with a probe encompass- Normalized protein levels (mean SEM) (N 6) (lower panel). ing the putative CLOCK:BMAL1 heterodimer E-box binding site, Peak protein expression was set at 100. One-way analysis of variance =   5-ACGCAGCAGACTGTTTGCATTCACGTGCGC-3, were per- (ANOVA)(P 0.003). Light phase ( ). Dark phase ( ). CT is circadian formed as described in the Methods section. For the competition exper- time used for assessing biologic time in a darl/dark cycle; CT0 is lights iments, a 50-fold excess of unlabeled double-stranded oligonucleotide on and CT12 is lights off. probe or the double-stranded oligonucleotide probe containing a mu- tated E box (5 -ACGCAGCAGACTGTTTGCATTGGATCCCGC- protein expression is rhythmic in the renal cortex, but 3) was added. An arrow indicates the band representing the CLOCK:BMAL1 and probe complex. Arrowheads point to the free arrhythmic in the renal medulla (data not shown), circa- probes. dian changes of NHE3 protein in the renal cortex may be under the direct control of the negative limbs of the gene with both physiologic and pathologic functions, clock genes. Since the amount of NHE3 in the thick as- is a clock controlled gene (Ccg) directly regulated by cending limb of Henle is very small as compared to that the CLOCK:BMAL1-driven circadian oscillator in the in the proximal tubule [26, 27], the changes of NHE3 pro- kidney. First, NHE3 transcripts exhibit an apparent cir- tein expression in whole kidney observed by Western blot cadian oscillation in the kidney. Second, the rhythmic likely reflect NHE3 expression in the proximal tubule in expression of NHE3 mRNA was blunted in the Cry dou- the renal cortex. ble mutant mice that are known to have lost the biolog- Based on a recent study with a DNA array demonstrat- ical clock [20, 28]. Third, luciferase-based transcription ing that only a small subset of Ccgs are directly controlled assays demonstrated that the NHE3 gene is activated by by the core feedback loop in peripheral tissues, NHE3 CLOCK:BMAL1 heterodimers and repressed by PER2 represents a rare example of Ccg [29]. In mammals, the and CRY1 proteins. Fourth, the gel shift assay revealed most established mode of regulation of Ccgs involves the that CLOCK:BMAL1 heterodimers transactivate NHE3 direct control by the core feedback loop of the circa- through direct binding to the E box in the promoter re- dian oscillators through CLOCK:BMAL1 heterodimers gion. Fifth, immunohistochemical analysis showed that (so-called first-order Ccgs) [30]. Examples of such Ccgs NHE3 and PER2 proteins are coexpressed in the same include arginine (AVP),which encodes a neu- cells within the renal cortex. Finally, the rhythmic expres- ropeptide that modulates the firing-rate rhythm from sion of NHE3 was observed at the protein level. Con- the suprachiasmatic nucleus. The rhythmic expression of sidering the fact that NHE3 is a membrane transporter, AVP mRNA in the suprachiasmatic nucleus appeared NHE3 should qualify as an output gene that can be di- to be under the direct control of CLOCK:BMAL1 het- rectly linked to the peripheral clockwork in the kidney. erodimers through an E box in its promoter region [31]. It is already known that circadian gene expression is It is important to note that the expression of this gene tissue specific and participates in the principal function in other parts of the hypothalamus (i.e., a subpopulation of individual organs. However, is it also possible that dif- of neurons of the paraventricular and supraoptic nuclei) ferential regulation within a particular organ might occur is not regulated in a circadian manner, indicating AVP to reflect regionally specific functions? In this respect, it is a Ccg in the suprachiasmatic nucleus neurons but not is interesting to notice that in contrast to renal cortex, in cells in other regions. To our knowledge, NHE3 is the NHE3 immunoreactivity did not significantly change be- first membrane protein established as a first order Ccg in tween CT8 and CT20 in the renal medulla. Since PER2 peripheral tissues. 1418 Rohman et al: Circadian regulation of NHE3 by peripheral clocks

AB

Fig. 8. Expression of NHE3 and PER2 protein in the kidney. (A) Immunohistochemistry of NHE3 at CT8 and CT20. NHE3 protein was expressed in the epithelial brush border of the proximal convoluted tubules (pct), but not in the glomerulus (gl). The immunoreactivity in the brush border was highest at CT20, although the NHE3 immunoreactivity in renal medulla was not altered. Immunohistochemistry was performed as described in the Methods section. Bar indicates 10 lm. (B) Colocalization of NHE3 and PER2. Imunohistochemical localization of NHE3 and PER2 protein in a section of paraformaldehyde-fixed rat kidney embedded in paraffin. Sections were double labeled with antibodies against NHE3 (green) and PER2 (red) as described in the Methods section. Bar indicates 10 lm. Note that nuclear PER2-positive cells also show NHE3 immunoreactivity. CT is circadian time used for assessing biologic time in a dark/dark cycle; CT8 is lights on and CT20 is lights off.

Cloning and characterization of the rat NHE3 gene such as NHE3, is by altering the number of available revealed a variety of modes in its transcriptional reg- molecules at the plasma membrane where NHE3 func- ulation. Sequence analysis of the 5 flanking promoter tionally active [32]. In the present study, we have shown region demonstrated the presence of putative cis-acting that the amount of plasma membrane NHE3 was circa- elements recognized by various transcription factors, in- dian changed throughout the day. These results were con- cluding activating protein (AP)-1, AP-2, C/EBP, gluco- firmed by immunohistochemistry. Given that hundreds of corticoid, and thyroid hormone receptors. [32]. In this genes are circadian regulated in the rate-limiting steps for regard, in the present study, we cannot rule out the pos- the principal functions of an organ, including components sibility that the apparent circadian change of NHE3 ex- of oxidative phosphorylation and intracellular trafficking, pression in vivo may be due to changes in these factors. In it is possible that post-translational processes in the reg- fact, diurnal expression of glucocorticoid appears to be ulation of NHE3 activity may also be controlled by the controlled by the internal pacemaker, and glucocorticoid circadian clock system [29]. Whether NHE3 activity ex- is known to regulate the expression of NHE3. However, hibits a circadian variation remains to be investigated. based on the fact that the regulation of NHE3 expres- What is the physiologic relevance for the direct con- sion by glucocorticoid is a rather chronic process [32], it trol of NHE3 by the circadian clock system, and hence is unlikely that in vivo changes in NHE3 expression is by the peripheral core oscillator system? The circadian mediated by glucocorticoid. Nevertheless, the transcrip- clock system is thought to provide a selective advantage tional regulation of NHE3 expression has been exten- for “prediction” or “anticipation” enabling the organ to sively studied. However, little attention has been paid to prepare for environmental changes, eventually optimiz- the diurnal variations of its expression. Our study exposes ing the appropriate response [36]. Since NHE3 plays an a novel dynamic system of transcriptional regulation of important role in the basic homeostasis of the organ- + the NHE3 gene in the kidney. ism through the regulation of pH, Na , and water bal- In addition to altered expression of NHE3 mRNA and ance, fine control of the NHE3 activity by the internal protein, the various factors act in union to achieve the clock should allow adaptation to both acute and chronic fine regulation of NHE3 activity by controlling phos- changes in environment. For instance, peripheral clocks phorylation of the protein and/or accessory factors, al- are synchronized mainly by the photoperiod through the terations in the surface expression of the exchangers, and suprachiasmatic nucleus clock [29]. However, when feed- changes mediated through the cytoskeleton. For instance, ing is restricted to the light phase in nocturnal mice NHE3, in addition to being present at the surface, is de- and rats, peripheral clocks may escape suprachiasmatic tectable in intracellular vesicles [33–35]. A fast and effec- nucleus control and be reset by food availability [37]. tive way to control the function of cell surface protein, In such a case, direct control of NHE3 by peripheral Rohman et al: Circadian regulation of NHE3 by peripheral clocks 1419 oscillators has particular value because it enables the lo- of serum electrolytes in physiological aging. Funct Neurol 18:77–81, cal dominant cue (i.e., food availability) to be utilized 2003 14. CHOI JY, SHAH M, LEE MG, et al: Novel amiloride-sensitive sodium- immediately. dependent proton secretion in the mouse proximal convoluted tubule. J Clin Invest 105:1141–1146, 2000 15. WANG T, YANG CL, ABBIATI T, et al:Mechanism of proximal tubule CONCLUSION bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277:F298–F302, 1999 We have provided evidence demonstrating that NHE3 16. WU MS, BIEMESDERFER D, GIEBISCH G, ARONSON PS: Role of NHE3 + + is a first order Ccg in the peripheral organs. NHE3 ap- in mediating renal brush border Na -H exchange. J Biol Chem pears to be a critical mediator of circadian information 271:32749–32752, 1996 17. SCHULTHEIS PJ, CLARKE LL, MENETON P, et al: Renal and intestinal since it is regulated in the most direct way through which absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. the core feedback loop could transduce its signal to down- Nature Genetics 19:282–285, 1998 18. GUYTON AC: Blood pressure control-special role of the kidneys and stream effectors. Therefore, we predict that NHE3 will be body fluids. Science 252:1813–1816, 1991 useful as a marker gene for studying temporal regulations 19. LIFTON RP: Molecular genetics of human blood pressure variation. of physiology and behavior by clock genes. 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