Life Sciences 93 (2013) 435–440

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Identification of SLC26A transporters involved in the Cl−/HCO3− exchange in proximal

tubular cells from WKY and SHR

Sónia Simão, Pedro Gomes, Maria João Pinho, Patrício Soares-da-Silva ⁎Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

Abbreviations: BCECF-AM, acetoxymethyl ester of 2′,boxyfluorescein; EDTA, ethylenediamine tetraacetic acid;piperazineethanesulfonic acid; NHE, Na+/H+ exchanger; PPT, proximal tubule; PTE, proximal tubular epithelial; RT-siRNA, small interfering RNA; SHR, spontaneously hyperten⁎ Corresponding author. Tel.: +351 22 5513642; fax: +

E-mail address: pss@med.up.pt (P. Soares-da-Silva).

0024-3205/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.lfs.2013.07.026

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 27 November 2012Accepted 29 July 2013

Keywords:slc26a familyCl−/HCO3

− exchangerKidneyProximal tubular epithelial cellsSHR

Aims: slc26a proteins are responsible for a large number of functions either in normal physiology or in humandisease. We have previously reported that proximal tubular epithelial (PTE) cells immortalized from spontane-ously hypertensive rats (SHRs) were endowedwith increased Cl−/HCO3

− exchanger activity and slc26a6 proteinexpression compared with PTE cells immortalized from normotensive Wistar Kyoto (WKY) rats. The aim of thepresent studywas to identify slc26amembers responsible for the Cl−/HCO3

− exchange inWKY and SHR PTE cells.Main methods: Cl−/HCO3

− exchanger activity was assessed as the initial rate of pHi recovery after removal ofHCO3

− or after removal of Cl−. The presence of slc26a genes was evaluated by means of reverse transcriptase-PCR (RT-PCR) in WKY and SHR PTE cell lines and in the kidney of WKY and SHR. Transcript abundance wasmeasured by quantitative real-time polymerase chain reaction (PCR).

Key findings: We detected slc26a4, slc26a6, slc26a7 and slc26a9 transcripts in the rat kidney of WKY and SHR.In WKY and SHR PTE cell lines we detected slc26a4, slc26a6 and slc26a9 transcripts, which were, respectively,12-, 4- and 15-fold upregulated in SHR cells. Gene silencing with small interfering RNAs (siRNAs) targetingslc26a4, slc26a6 and slc26a9 reduced Cl−/HCO3

− exchanger activity in both cell lines.Significance: These results indicate that Cl−/HCO3

− exchanger activity is mediated by, at least in part, slc26a4,slc26a6 and slc26a9 in cultured WKY and SHR cells. The overexpression of these slc26a members in SHR cellsmay correspond to an adaptive process to cope with the sustained increase in proximal tubular sodiumreabsorption.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Essential hypertension is a complex disease with several underlyingcauses. One reason for the development andmaintenance of this formofhypertension is the defective capacity of the kidneys to excrete waterand salt in appropriate relation to the intake. Regulation of renal NaClreabsorption is critical in the control of extracellular volume and bloodpressure (Zeng et al., 2004). The majority of the filtered NaCl isreabsorbed by the proximal tubule (PT) cells (Aronson and Giebisch,1997) and the main transporters responsible for this reabsorption arethe apical Na+/H+ exchanger subtype 3 (NHE3) and the Cl−/HCO3

−

exchanger, which are essential for intracellular pH and cell volumeregulation (Petrovic et al., 2003).

The solute carrier 26 (SLC26)proteins are a family of anion exchangersresponsible for a large number of functions in normal physiology andin human diseases (Mount and Romero, 2004; Ohana et al., 2009;

7′-bis (carboxyethyl)-5(6)-car-HEPES, 4-(2-hydroxyethyl)-1-CR, polymerase chain reaction;PCR, reverse transcriptase-PCR;sive rat;WKY,Wistar Kyoto rat.351 22 5513643.

ghts reserved.

Soleimani and Xu, 2006). Several rare recessive human pathologies, inparticular diastrophic dysplasia, congenital chloride diarrhea andPendredsyndrome, are associated with mutations in SLC26A2, SLC26A3 andSLC26A4, respectively. The SLC26 family comprises 10 different genes re-sponsible for the transport/exchange of a great variety ofmonovalent anddivalent ions, including chloride, iodide, sulphate andbicarbonate (Mountand Romero, 2004; Ohana et al., 2009; Soleimani and Xu, 2006). SomeSLC26 members show very specific tissue distribution while others arewidely expressed. SLC26 familymembers are expressed inmany epitheliaand play a central role in anion secretion and absorption. Our grouprecently demonstrated the presence of an apical Cl−/HCO3

− exchanger,the slc26a6 protein, in proximal tubular epithelial (PTE) cell lines fromWistar Kyoto (WKY) and spontaneously hypertensive rats (SHR)(Simão et al., 2008). An increased activity of the Cl−/HCO3

− exchanger inPTE cells from SHR when compared with WKY cells was also reported(Simão et al., 2008).

Recent studies in slc26a6-null mice suggest that this protein medi-ates only a part of apical proximal tubule Cl− transport (Wang et al.,2005), indicating that other transporters may also be involved in thisprocess. The purpose of this study was to identify the presence ofslc26a members in the kidney and in immortalized PTE cells of WKYand SHR and to determine their relative contribution to the Cl−/HCO3

−

exchange in both cell lines.

436 S. Simão et al. / Life Sciences 93 (2013) 435–440

Materials and methods

Cell culture

Immortalized renal PTE cells were obtained from primary culturesfrom S1 segments of proximal tubules of 4- to 8-week-old WKY andSHR (Woost et al., 1996). These cell lines formed polarized monolayerswith apical microvilli, tight junctional complexes and convolutions ofthe basolateral plasma membrane. WKY and SHR cell lines express aproximal tubular phenotype and are morphologically and functionallysimilar to primary cultures (Woost et al., 1996). Cells were maintainedin a humidified atmosphere of 5% CO2–95% air at 37 °C. WKY andSHR PTE cells were grown in Dulbecco's modified Eagle's mediumnutrient mixture F-12 Ham (Sigma Chemical Company, St. Louis,MO) supplemented with 100 U/ml penicillin G (Sigma), 0.25 μg/mlamphotericin B (Sigma), 100 μg/ml streptomycin (Sigma), 4 μg/mldexamethasone (Sigma), 5 μg/ml transferrin (Sigma), 5 μg/ml insulin(Sigma), 5 ng/ml selenium (Sigma), 10 ng/ml epidermal growthfactor (Sigma), 5% fetal bovine serum (Sigma) and 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanosulfonic acid (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); Sigma). For subculturing, thecells were dissociated with 0.10% trypsin–ethylenediamine tetraaceticacid (EDTA) (Sigma), split 1:8 and cultured in Costar plates with21-cm2 growth areas (Costar, Badhoevedorp, The Netherlands). Forintracellular pH (pHi) measurement experiments, cells were grown in10-mm diameter collagen-coated glass coverslips or in 96-well plates(Costar) and for polymerase chain reaction (PCR) cells were grown in6-well plates (Costar). The cell medium was changed every 2 days, andthe cells reached confluence after 3–5 days of incubation. For approxi-mately 2 h prior to each experiment, the cells were maintained in fetalbovine serum-free medium. Experiments were performed 4 days afterthe initial seeding; each cm2 contained about 50 μg of cell protein.

pHi measurements

In pHi measurement experiments, WKY and SHR PTE cells weregrown in 10-mm diameter collagen-coated glass coverslips or in96-well plates. pHi was measured as previously described (Pedrosaet al., 2004b). Briefly, 4 days after seeding glass coverslipswere incubat-ed with 10 μMof acetoxymethyl ester of 2′, 7′-bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) at 37 °C for 30 min. Coverslips werethen washed with pre-warmed modified Krebs buffer before initiationof the fluorescence recordings. The Krebs medium had the followingcomposition (in mM): 115 NaCl, 25 NaHCO3, 5.4 KCl, 2.8 CaCl2, 1.2MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 HEPES, and 5 glucose, at pH 7.4(adjusted with Tris base). Coverslips were mounted diagonally in anacrylic fluorometric cuvette that was inserted in a PerkinElmer cuvetteholder (model LS 50) and subsequently placed in the sample compart-ment of a FluoroMax-2 spectrofluorometer (Jobin Yvon-SPEX, Edison,NJ). The cuvette volume of 3.0 ml was constantly stirred and perfusedat 5.0 ml/min with modified Krebs buffer pre-warmed to 37 °C. Underthese conditions, the cuvette medium was replaced within 150 s. Aftera stabilization period of 5 min, fluorescence was measured every 5 salternating between 440- and 490-nm excitation (1-nm slit size) and525-nm emission (3-nm slit size). In the experiments performed inWKY and SHR cells cultured in 96-well plates, pHi measurementswere performed after the cells were loaded with 10 μM BCECF-AM at37 °C for 30 min. Cells were placed in the sample compartment of adual-scanning microplate spectrofluorometer (Spectramax Gemini XS,Molecular Devices, Sunnyvale, CA), and fluorescence was measuredevery 17 s alternating between 440- and 490-nm excitation and535-nmemissionwith a cutofffilter of 530 nm. The ratio of intracellularBCECF fluorescence at 490 nm and 440 nm was converted to pHi bycomparison with values from an intracellular calibration curve usingnigericin 10 μM in a high-K+ solution (in mM:15 NaCl, 130 KCl, 0.3

KH2PO4, 0.3 NaH2PO4, 5 glucose, 1.2 MgSO4, 2.8 CaCl2 and 10 HEPES)with pH ranging from 6.6 to 7.8.

Cl−/HCO3− exchanger activity

Cl−/HCO3− exchanger activity can be assessed by different ap-

proaches, i.e., Cl−/HCO3− exchanger activity can be defined as the

initial rate of pHi recovery after removal of HCO3− or after addition

of Cl−. The Na+-independent HCO3− transport system activity was

assayed as the initial rate of pHi recovery after an alkaline load(CO2/HCO3 removal), in the absence of Na+, as previously described(Pedrosa et al., 2004b). Briefly, cells were loaded in serum-free me-dium with 10 μM BCECF-AM, for 30 min at 37 °C in 5% CO2–95% airatmosphere. The cells were washed free of dye and loaded withKrebs–Henseleit solution (25 mM NaHCO3). Then, extracellularsolution was replaced by a Krebs–Henseleit NaHCO3-free solution.In the NaHCO3-free bathing solution, NaHCO3 was replaced by anequimolar concentration of choline. Cells were placed in the samplecompartment of a FluoroMax-2 spectrofluorometer or in a dual-scanning microplate Spectramax Gemini spectrofluorometer and fluo-rescencewasmonitored. Additionally, the Cl−-dependent transport sys-tem activity was assayed as the initial rate of intracellular alkalinizationwhen PTE cells were incubated alternately with Ringer's buffer (inmM: 5 glucose, 5 potassium gluconate, 1 CaCO3, 1 MgSO4, 2.5NaH2PO4, 25 NaHCO3, 10 Hepes, pH 7.4) containing either 140 mMNaCl or 140 mM sodium gluconate. Cells were placed in the samplecompartment of a FluoroMax-2 spectrofluorometer and fluorescencewas monitored as described above.

RNA extraction

Total RNA was isolated from kidney cortexes of 12-week-old WKYand SHR and from WKY and SHR PTE cell monolayers using TRIZOL(Invitrogen) according tomanufacturer's instructions. The RNA obtainedwas dissolved in diethylpyrocarbonate (DEPC)-treated water and quan-tified by spectrophotometry at 260 nm. The total RNA extracted wastreated with DNase (Ambion, USA), to eliminate potential genomicDNA contamination.

Reverse transcriptase-PCR (RT-PCR)

Approximately 250 ng of total RNA was reverse transcribed tocDNA with SuperScript III First Strand Synthesis SuperMix for RT-PCR(Invitrogen) using 50 ng/μl random hexamers as primers at 50 °C,according to the manufacturer's instructions. The cDNA was amplifiedby PCR using one set of rat specific primers for slc26a4 (forward: 5′-GTG GGG TCC GTT GTT C-3′ and reverse: 5′-CCG TTG TAG TTT TTGGTT GAG-3′), slc26a6 (forward: 5′-CGG GAG GCA ACA CGC AGA T-3′and reverse: 5′-GGT GGC TGA GGA ACG GAA GAC-3′), slc26a7(forward: 5′-ATC ATT GCT GCC TCA TTT GCT-3′ and reverse: 5′-TTTGCC CCC GTG CTG T-3′) and slc26a9 (forward: 5′-GCA AAA ACC TCCCCC ACA CCA-3′ and reverse: 5′-GAC TTCC CTC CAG CCC CAT CC-3′).PCR was performed with Platinum TaqPCRx DNA Polymerase(Invitrogene) with 1× enhancer. Amplification conditions were asfollows: hot start of 2 min at 95 °C; 30 cycles of denaturing (95 °C for30 s), annealing (54 °C for 30 s) and extension (68 °C for 1 min); anda final extension of 7 min at 68 °C. The PCR products were separatedby electrophoresis in a 1.5% agarose gel and visualized under UV lightin the presence of ethidium bromide. The obtained PCR productswere purified and sequenced in both directions, by GATC Biotech AG(Konstanz, Germany), using specific primers described above. Nucleotidehomology searching was performed against nonredundant and dbESTusing basic local alignment tool (BLAST) via online connection to the Na-tional Center for Biotechnology Information (NCBI, Bethesda, MD, USA).Multiple nucleotide sequence comparisons were done with CLUSTALW

437S. Simão et al. / Life Sciences 93 (2013) 435–440

via online connection to the European Bioinformatics Institute (EMBL-EBI).

Quantitative real-time PCR

Real-time PCR was carried out using a LightCycler (Roche,Switzerland). Briefly, each real-time PCR reaction mixture (20 μl) in-cluded reverse transcription products corresponding to 50 ng oftotal RNA or standard DNA, 1× SYBR Green I master mix (LightCyclerFastStart DNA MasterPLUS SYBR Green I, Roche) and 0.5 μM of eachforward and reverse primers (slc26a4 forward: 5′-CAA AAT ACC GAGTCA AGG AAT-3′ and reverse: 5′-TGG GGA AAA AGG CAG AG-3′;slc26a6 forward: 5′-CTG GGC CAC TGT CCG TTA TCT-3′ and reverse:5′-ACC ACT CCT GCC ACG ATT G-3′; slc26a9 forward: 5′-CAG GAGAAG AGG ATG GCA GTG-3′ and reverse: 5′-GGT TGT TGT TGG GGTCTG TG-3′). Cycling conditions were as follows: denaturation (95 °Cfor 1 min), amplification and quantification (95 °C for 10 s, 60 °C for7 s, 72 °C for 10 s, with a single fluorescence measurement at the endof the 72 °C for 5 s segment) repeated 35 times, a melting curveprogram (65–95 °C with a heating rate of 0.1 °C/s and continuousfluorescent measurement) and a cooling step to 40 °C. Amplificationspecificity was checked using melting curves following themanufacturer's instructions. Results were analyzed with LightCyclerSoftware version 3.5 (Roche) using the second derivate maximummethod. Quantification was performed using standard curves. Datawere normalized to the expression of the constitutively expressedgene GAPDH.

Small interfering RNA (siRNA)

For siRNA experiments WKY and SHR PTE cells were cultured in96-well plates. Briefly, siRNAs (2.5 nM final concentration) against dif-ferent isoforms of slc26a members were spotted in each 96-well. TheHiperFect Reagent was diluted in serum-free culture medium andadded to the prespotted siRNA. The mixture was incubated for 10 minat room temperature for complex formation. Then, cells were seededin culture medium (containing serum and antibiotic) into the well, onthe top of the transfection complexes. Cells were incubated with thesiRNAs for 12 h, under normal growth conditions. Cl−/HCO3

− exchangeractivity was measured 24 h after transfection.

Drugs

Forward and reverse primers for RT-PCR and for real-time quantita-tive RT-PCR were obtained from Sigma Chemical Company (St. Louis,MO). BCECF-AM and nigericin were obtained from Molecular Probes.slc26a4, slc26a6 and slc26a9 siRNAs, AllStars Negative Control siRNAand HiperFect Transfection Reagent were obtained fromQiagen (Hilden,Germany).

Data analysis

Arithmetic means are given with standard error of the mean (SEM).Statistical analysis was performed by one-way analysis of variance(ANOVA) followed by the Newman–Keuls test for multiple compari-sons. A P-value less than 0.05 was assumed to denote a significantdifference.

Results

The activity of the Cl−/HCO3− exchanger was assayed as the initial

rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in theabsence of sodium to avoid the contribution of Na+-dependent trans-porters, such as the NHE3 (Fig. 1A). In a HCO3

− containing medium,removal of CO2/HCO3 caused an initial cell alkalinization, as a result ofCO2 loss from the cell with subsequent return of pHi towards basal

values (Fig. 1A). The Na+-independent Cl−/HCO3− exchanger mediates

the pHi recovery process after the alkalinization. As shown in Fig. 1A,the slope of HCO3

−-dependent pHi recovery in SHR PTE cellswas steeperthan that observed inWKY PTE cells. Cl−/HCO3

− exchanger activity wasalso assessed by incubating cells alternately with NaCl and NaGluconate(Fig. 1B). Switching PTE cells to a Cl−-free solution resulted in cell alka-linization due to reversal of the Cl−/HCO3

− exchanger. Upon pHi stabili-zation in Cl−-free medium, cells were returned to the Cl− containingsolution. This procedure results in the return of pHi to basal levels dueto activation of the Cl−/HCO3

− exchanger in the forward mode. Asshown in Fig. 1B, the slope of Cl−-dependent pHi recovery in SHR PTEcells was greater than that observed in WKY PTE cells.

We next evaluated the presence of slc26a members potentially in-volved inmediating proximal tubule Cl−/HCO3

− exchange. The presenceof slc26a4, slc26a6, slc26a7 and slc26a9 transcripts in 12-week-oldkidney cortex from WKY and SHR and in WKY and SHR PTE cell lineswas evaluated by RT-PCR (Fig. 2). Fragments with the expected sizewere identified in total RNA from WKY and SHR kidney cortex(Fig. 2A) and from WKY and SHR PTE cell lines (Fig. 2B). The identityof the amplified products was confirmed by sequencing. The slc26a4,slc26a6 and slc26a9 fragments showed a nucleotide sequence identityof 99% with GenBank accession #NM_019214, #NM_001143817 and#NM_001107172, respectively (data not shown). Transcript abundanceof slc26a4, slc26a6 and slc26a9 inWKYand SHRPTE cells wasmeasuredby quantitative real-time PCR. As shown in Fig. 3, mRNA expression ofslc26a4, slc26a6 and slc26a9 was increased in SHR PTE cells, comparedwith WKY cells (12-, 4-, and 15-fold, respectively).

In the next series of experiments,WKY and SHR PTE cells were treat-ed with siRNAs targeted against specific sequences of slc26a4, slc26a6and slc26a9 genes or a mismatch sequence (siRNA−) (Fig. 4). slc26a4,slc26a6 and slc26a9 siRNA treatment significantly reduced Cl−/HCO3

−

exchanger activity in WKY and SHR PTE cells (20–30% inhibition)(Fig. 4). The siRNA negative control as well as the transfection reagentdid not significantly change the exchanger activity in both cell lines.The efficiency of siRNA silencing was evaluated through quantitativereal-time PCR and the results are depicted in Fig. 5. As shown, treatmentwith the siRNAs significantly reduced themRNA expression of the threeslc26a members in both WKY and SHR PTE cells (10–50% reduction)(Fig. 5).

Discussion

The present study was designed to identify slc26amembers presentin WKY and SHR PTE cells and also to determine the relative contribu-tion of each member to the Cl−/HCO3

− exchanger activity in these celllines. We found that the activity of Cl−/HCO3

− exchanger is mediatedby slc26a4, slc26a6 and slc26a9 in both WKY and SHR PTE cells. Addi-tionally, we demonstrated that SHR PTE cells overexpress these slc26agenes, which we suggest to be related to the increased renal NaClreabsorption associated with hypertension.

In addition to the procedure previously described to evaluate theactivity of Cl−/HCO3

− exchanger (initial rate of HCO3−-dependent pHi

recovery) (Simão et al., 2008) we also measured the exchanger activityby assessing the initial rate of Cl−-dependent intracellular alkalinizationin both WKY and SHR PTE cells. These two procedures yielded similarresults, demonstrating that both are appropriate to measure the activ-ity of this exchanger. In both methodologies the activity of Cl−/HCO3

− exchanger was shown to be consistently higher in SHR than inWKY PTE cells, which fits well with the overexpression of slc26a mem-bers in the kidney of the SHR (Simão et al., 2008).

We have previously demonstrated the presence of the slc26a6 pro-tein in kidney cortexes from WKY and SHR rats, as well as in WKY andSHR PTE cell lines (Simão et al., 2008). slc26a6 has been described asthemain candidate for apical Cl−/base exchange of brush border mem-branes in the renal PT and likely encodes the apical Cl− entry site in-volved in NaCl reabsorption (Mount and Romero, 2004). However,

Cl- Cl- free Cl-

WKY

SHR

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

delta

pH

i

A B

WKY

SHR

HCO3- free HCO3

- freeHCO3-

0 1000 2000 3000

Time (s)

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

delta

pH

i

0 1000 2000 3000

Time (s)

Fig. 1. (A) Intracellular pH inWKY and SHR PTE cells perfusedwith Krebs–Henseleit buffer containing either NaHCO3 or ClCholine. The activity of Cl−/HCO3− exchanger corresponds to the

initial rate of pHi recovery after removal of HCO3−. Representative traces of three experiments per group. (B) Intracellular pH in WKY and SHR PTE cells perfused with Ringer's buffer

containing either NaCl or NaGluconate. The activity of Cl−/HCO3− exchanger corresponds to the initial rate of pHi recovery after addition of Cl−. Representative traces of six experiments

per group.

438 S. Simão et al. / Life Sciences 93 (2013) 435–440

recent studies in slc26a6-null mice indicate that slc26a6 protein medi-ates only a fraction of apical PT Cl− transport (Wang et al., 2005),suggesting that other transporters may also be involved in this process.Although the slc26a family is composed by 10 different members, onlyslc26a3, 4, 6, 7 and 9 are recognized as bicarbonate transporters(Bonar and Casey, 2008). slc26a3 is one of the few slc26a membersthat is not expressed in the kidney (Bonar and Casey, 2008; Sindicet al., 2007). With the purpose of identifying members of the slc26afamily in cultured PTE cells and in the kidney from SHR and WKY ratsthat might mediate the Cl−/HCO3

− exchange, the expression of sometransporters of this family was evaluated. To the best of our knowledge,the presence of slc26a4, slc26a7 and slc26a9 mRNA in kidney cortexesfrom WKY and SHR rats has not been previously reported. In WKYand SHR PTE cell lines the presence of slc26a4 and slc26a9 mRNA wasalso detected. The absence of slc26a7 transcript from WKY and SHRPTE cells suggests that slc26a7 exchanger might be expressed in othersegments of the kidney cortexes of WKY and SHR, such as the distal tu-bule, rather than the PT. Similar to what we previously showed forslc26a6 protein (Simão et al., 2008), in the present study we also ob-served enhanced expression of slc26a4 and slc26a9 mRNA in SHR PTEcells. The overexpression of these transporters in the SHR is in linewith the findings that NHE3 activity and expression in renal PT (immor-talized and freshly isolated) from the SHR are greater than in WKY(Hayashi et al., 1997; Pedrosa et al., 2004a; Zeng et al., 2003). In fact, asustained NHE3 activation is only possible in the presence of a parallelincrease of an acidifying pathway, such as the slc26a, because sustainedNHE activity will alkalinize the cell, which in turn inactivates NHEthrough a cytosolic modifier site (Counillon and Pouyssegur, 2000). InPT cells, the parallel increase in NHE3 and Cl−/HCO3

− activity justifiesthe role of the Cl−/HCO3

− exchanger in apical membrane and doesnot conflict with the role of the renal proximal tubules in HCO3

−

Fig. 2. (A) Detection of slc26a family members in total RNA extracted from kidney cortexes ofrepresentative agarose gel of 3 independent experiments.

reabsorption. In fact, apical Cl−/HCO3− exchange results in the secretion

of HCO3− and absorption of Cl− that are crucial to Na+ absorption

through NHE3 activity.To determine the relative contribution of slc26a4, slc26a6 and

slc26a9 transporters in mediating Cl−/HCO3− exchange in WKY and

SHR PTE cells, both cell lines were transfected with siRNAs againstthese genes. The results reported here indicate that at least thesethree slc26a transporters mediate the Cl−/HCO3

− exchange in bothWKY and SHR PTE cells. The fact that the activity of the Cl−/HCO3

− ex-changer was not completely inhibited in the presence of each siRNAsuggests that more than one slc26amember contribute to the exchang-er activity inWKY and SHR PTE cells and are functioning to compensatethe inhibition induced by siRNA treatment. One possibility is that thedifferent exchangers may work in an integrated manner to providefull capacity to the cell on ion exchange. It is suggested that Cl−/HCO3

−

exchanger activity may be mediated by several exchangers from theslc26a family or from a different family of exchangers. Another possibil-ity for the absence of complete inhibition of the exchanger activity upontransfection with the siRNAs may concern the efficiency of gene silenc-ing achieved with the siRNA treatment.

Previous studies on slc26a4 described a role for this transporter insalt sensitive hypertension; slc26a4 is very susceptible to tubular deliv-ery of Cl− and plays an essential role in vascular volume homeostasisunder conditions of NaCl restriction (Quentin et al., 2004). Until recent-ly, slc26a4 mRNA expression has only been found in the corticalcollecting duct, with lower levels of expression in the PT. slc26a9 isone of the recently identified slc26a transporters, though some contro-versy has been raised in the literature. Initial studies suggested thatslc26a9 functions as a Cl− channel, with very little permeability toHCO3

− (Loriol et al., 2008). By contrast, more recent studies revealedthat slc26a9 also functions as Cl−/HCO3

− exchanger (Chang et al.,

12-week old WKY and SHR rats and from (B) WKY and SHR PTE cell lines. The image is a

Fig. 3. (A) Transcript abundance of slc26a4, (B) slc26a6 and (C) slc26a9 in WKY andSHR PTE cell lines. Results are expressed as ratio to GAPDH, as determined by quantitativereal-time PCR. Each column represents mean of 4 independent experiments; vertical linesindicate SEM. Significantly different fromWKY values (*P b 0.05).

Fig. 4. Cl−/HCO3− exchanger activity in the presence of siRNAs targeting slc26a4 (A4),

slc26a6 (A6), slc26a9 (A9) or a mismatch sequence (si−) in WKY and in SHR PTE cells.Each column represents mean of 4–8 independent experiments; vertical lines indicateSEM. Significantly different from control values (*P b 0.05).

Fig. 5. Transcript abundance of slc26a4 (A4), slc26a6 (A6) and slc26a9 (A9) in WKY andSHR PTE cell lines treated with the respective siRNA. Results are expressed as ratio toGAPDH, as determined by quantitative real-time PCR. Each column represents mean of 4independent experiments; vertical lines indicate SEM. Significantly different from controlvalues (*P b 0.05).

439S. Simão et al. / Life Sciences 93 (2013) 435–440

2009). Our results reinforce the view that slc26a9 operates as an ex-changer. Since we have not addressed the role of slc26a9 as a Cl− chan-nel inWKY and SHRPTE cells its action as a channel in these cells cannotbe excluded. It is suggested that if slc26a9 functions as a Cl− channel inWKY and SHR cells then its upregulationmight be important in the con-text of hypertension. The increased chloride reabsorption characteristicof the SHR would be compensated by means of these channels, whichwould lead to enhanced chloride excretion.

Conclusions

It is concluded that slc26a4, slc26a6 and slc26a9 genes are presentinWKY and SHR PTE cells as well as in the kidney from both rat strains.Cl−/HCO3

− exchanger activity is mediated, at least in part, by slc26a4,slc26a6 and slc26a9 in cultured WKY and SHR PTE cells. In a broaderperspective, the overexpression of these slc26a members in SHR PTEcells may correspond to an adaptive process to cope with the sustainedincrease in proximal tubular sodium absorption in the hypertensivekidney.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by Fundação para a Ciência e a Tecnologia(FCT), POCI, FEDER and Programa Comunitário de Apoio (PIC/IC/83204/2007).

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