Na+-independent phosphate transport in Caco2BBE cells

Na�-independent phosphate transport in Caco2BBE cells

Eduardo Candeal,1 Yupanqui A. Caldas,1,2 Natalia Guillén,1 Moshe Levi,2 and X Víctor Sorribas1

1Department of Toxicology, University of Zaragoza, Zaragoza, Spain; and 2Division of Renal Diseases and Hypertension,Department of Medicine, University of Colorado Denver, Aurora, Colorado

Submitted 16 July 2014; accepted in final form 1 October 2014

Candeal E, Caldas YA, Guillén N, Levi M, Sorribas V. Na�-independent phosphate transport in Caco2BBE cells. Am J PhysiolCell Physiol 307: C1113–C1122, 2014. First published October 8,2014; doi:10.1152/ajpcell.00251.2014.—Pi transport in epithelia hasboth Na�-dependent and Na�-independent components, but so faronly Na�-dependent transporters have been characterized in detailand molecularly identified. Consequently, in the present study, weinitiated the characterization and analysis of intestinal Na�-indepen-dent Pi transport using an in vitro model, Caco2BBE cells. OnlyNa�-independent Pi uptake was observed in these cells, and Pi uptakewas dramatically increased when cells were incubated in high-Pi

DMEM (4 mM) from 1 day to several days. No response to low-Pi

medium was observed. The increased Pi transport was mainly caused byVmax changes, and it was prevented by actinomycin D and cyclohexi-mide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased atpH � 7.5, and it was inhibited with proton ionophores. Pi transport incells incubated with 4 mM Pi increased with alkaline pH, suggesting apreference for divalent phosphate. Pi uptake in cells in 1 mM Pi wascompletely inhibited only by Pi and partially inhibited by phosphonofor-mate, oxalate, DIDS, SITS, SO4

2�, HCO3�, and arsenate. This inhibition

pattern suggests that more than one Pi transporter is active in cellsmaintained with 1 mM Pi. Phosphate transport from cells maintained at4 mM Pi was only partially inhibited by phosphonoformate, oxalate, andarsenate. Attempts to identify the responsible transporters showed thatmultifunctional anion exchangers of the Slc26 family as well as membersof Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropicretrovirus receptor 1 are not involved.

phosphate transport; Caco2BBE cells; Na�-independent Pi uptake;phosphate absorption; small intestine; inorganic phosphate

CONTROL of Pi homeostasis is mediated by the coordinated activityof a complex set of physiological mechanisms acting on the rateof intestinal absorption, rate of renal excretion, and eventualmobilization of the bone reservoir. These mechanisms consist ofhormones and nonhormonal events that act either acutely orslowly (chronically) to modulate the activity of plasma membranePi transporters (2, 4, 23). The precision of Pi homeostasis controlis critical not only because of the extreme relevance of thephysiological roles of Pi in the organism (pH buffer, energybonds, signal transduction, phospholipid composition, bone for-mation, etc.) but also because serious conditions can emerge whencontrol of Pi homeostasis is lost, resulting in either hypophos-phatemia (7) or hyperphosphatemia (20, 23).

While the kidney has historically been recognized as themajor checkpoint and regulator of Pi homeostasis, morerecently it has been revealed that the intestine is a relevantmodulator of Pi signaling and is now a target for pharma-cological interventions in phosphate disorders (23, 30, 31).The majority of Pi absorption takes place in the small

intestine, with important regional differences depending onthe animal species (22). With respect to the mechanisms ofPi absorption, the classical combination of both saturated(transcellular transport) and unsaturated (paracellular diffu-sion) components have been described (16, 21). A detailedkinetic characterization of the transport component wasachieved using brush-border membrane vesicles (BBMVs)of the small intestine, thereby showing the existence of bothNa�-dependent and Na�-independent components, with simi-lar characteristics and pH dependence (3, 5).

More recently, the molecular characterization of Na�-de-pendent Pi transport in the intestine revealed the complemen-tary participation of different Pi transporters: NaPi2b andretroviral receptors Pit-1 and Pit-2. These transporters aremainly located in the apical membrane of enterocytes, and theyexhibit different kinetic behavior, substrate specificity, and pHdependence (11, 12) as well as regulation by several agents,including vitamin D3 and changes in dietary Pi (14, 18, 22).

In addition to classical kinetic studies, mouse models haverevealed the relevance of the different transport componentsthrough NaPi2b gene deletion (26, 29). Studies with thesemodels revealed that whereas NaPi2b seemed to be responsiblefor most of the Na�-dependent Pi absorption, the Na�-inde-pendent component (including downhill transport and the para-cellular route) was still responsible for �50% of total Pi

absorption. However, the exact percentage of Na�-indepen-dent Pi absorption depends on the experimental setup (3, 5, 29),among other factors. The molecular identity of Na�-indepen-dent transport is still unknown, as is the basolateral exit of Pi

from the enterocyte into the blood. However, it has recentlybeen proposed that the basolateral step could be mediated byxenotropic and polytropic retrovirus receptor 1 (XPR1), an-other cell surface multipass retroviral receptor (13).

In the present study, we characterized Na�-independent Pi

transport in a human intestinal cell line (Caco2BBE cells) as apreliminary step toward molecular identification of the respon-sible transporter. In addition to physiological and regulatoryexperiments, we also assayed several candidate transportersaccording to the pattern of inhibition that we observed.

MATERIALS AND METHODS

Cell culture. All cell culture products were from Life Technolo-gies-GIBCO (Paisley, UK). Caco2BBE cells were obtained andgrown, as previously described (15), in high-glucose (4.5 mg/ml)DMEM supplemented with 20% FCS, penicillin, streptomycin, andL-glutamine at 37°C and 5% CO2. The incubation of cells at differentconcentrations of Pi was initiated at 100% confluence.

Opossum kidney (OK) cells were grown, as previously described(32), in DMEM-Ham’s F-12 supplemented with 10% FCS, penicillin,streptomycin, and L-glutamine at 37°C and 5% CO2. For experimentswith Caco2BBE or OK cells in which the Pi concentration was below1 mM, Pi-free DMEM was used (GIBCO).

Address for reprint requests and other correspondence: V. Sorribas, Univ. ofZaragoza, Calle Miguel Servet 177, Zaragoza E50013, Spain (e-mail: [email protected]).

Am J Physiol Cell Physiol 307: C1113–C1122, 2014.First published October 8, 2014; doi:10.1152/ajpcell.00251.2014.

0363-6143/14 Copyright © 2014 the American Physiological Societyhttp://www.ajpcell.org C1113

http://orcid.org/0000-0003-3457-323X

mailto:[email protected]

mailto:[email protected]

Animals. Male Wistar rats were purchased from Janvier SAS(Berthevin, France). Animals were cared for in accordance withEuropean legislation, and procedures were approved by the EthicalCommittee of the University of Zaragoza. Rodent fodder containing1.2% or 0.1% Pi was purchased from Provimi Kliba SA (Penthalaz,Switzerland).

Rats were fed with 0.1% Pi diet for 4 h every day (from 8:00 AMto 12:00 PM) over 5 days. On the day of the experiment, some of therats were fed with 1.2% Pi diet for 4 h to achieve acute adaptation tothe high-Pi diet. The remaining rats once again received 0.1% Pi dietfor 4 h.

Transport assays. These assays have been extensively explained inour previous publications, either for cells grown on plastic support(32) or using intestinal BBMVs according to the rapid filtrationmethod (14). In both cases, [32P]H3PO4 (Perkin-Elmer, Waltham,MA) was used as a radiotracer. D-[3H]glucose and D-[14C]fructosewere obtained from Amersham Biosciences (Buckinghamshire, UK).Fits to either Michaelis-Menten for the kinetic parameters of transportor to the sigmoidal curve for mean inhibitory concentration (IC50)determinations were performed by nonlinear regression using Graph-Pad Prism 5.0 software (San Diego, CA). For Pi saturation kinetics, anequation containing both saturable and nonsaturable (diffusion plusunspecific binding) components was used: v � [Vmax � Pi concen-tration/(Km � Pi concentration)] � (Kd � Pi concentration). For theestimation of IC50, the following equation was used: v � V0 � (V100 �V0)/[1 � 10e(logEC50 � Pi concentration)], where V100 is the velocityof transport at zero concentration of inhibitor and V0 is the minimaltransport rate obtained with the maximal concentration of inhibitor.

Pharmacological treatments. Caco2BBE cells were incubated witheither 1 or 4 mM Pi for 24 h in the presence or absence of 1 �g/mlactinomycin D (inhibitor of RNA transcription) or 100 �M cyclohex-imide (inhibitor of protein synthesis). To study the involvement ofproton gradients in Na�-independent Pi transport, the following com-pounds were used: 10 �M FCCP, 10 �M CCCP, 0.1 �M bafilomycinA1, and 2 mM furosemide. All drugs were dissolved in DMSOexcept for furosemide, bafilomycin A1, and FCCP, which weredissolved in ethanol. The same amount of solvent was added to thecorresponding control cells. All chemicals were from Sigma-Aldrich (St. Louis, MO).

RNA interference. Validated short interfering (si)RNA molecules(Silencer Select siRNAs) were obtained from Life Technologies-Ambion (Austin, TX). siRNAs were transfected using Lipofectamine2000 (Life Technologies) at a final concentration of 5 nM following

the manufacturer’s instructions. The maximal reduction of targetRNAs was observed after 48 h in Caco2BBE cells after real-timequantification. The corresponding negative controls with a similarbase composition were also used.

Real-time PCR. Total RNA was purified from Caco2BBE cellsusing the SV Total RNA Isolation Kit (Promega, Fitchburg, WI).After being treated with DNase I, total RNA was retrotranscribedwith a Transcriptor First Strand cDNA Synthesis Kit and amplifiedin a LightCycler 1.5 using a FastStart Master SYBR Green I kit (allfrom Roche, Mannheim, Germany). The primers used are shown inTable 1. Gene expression data were normalized to an endogenousreference (peptidylprolyl isomerase B, also known as cyclophilinB) and to a calibrator (a combination of identical amounts of RNAfrom cells incubated for 48 h in culture medium containing 1 or 4mM Pi) as previously described (24) and according to the manu-facturer’s instructions.

Statistics. With the exception of the animal experiments, whichwere confirmatory, all other experiments were repeated three timesusing triplicates per condition. GraphPad Prism 5.0 was also used forstatistical analysis, and significances of differences were determinedby one-way ANOVA and a Tukey posttest for multiple comparisons.P values are indicated in the figures when necessary. To compare thefits to the same model of Michaelian behavior, an extra sum-of-squares F-test was performed. To compare the specific parameters andto test whether the kinetic constant was shared among all data sets, aglobal fit was also performed.

RESULTS

Response to Pi concentration in rats and Caco2BBE cells. Inprevious experiments, rats chronically adapted to a low-Pi diet(0.1%) showed an increase of duodenal BBMV Pi uptake whenrats were acutely fed a high-Pi diet (1.2%) for 4 h (14). Beforewe studied Caco2BBE cells as a model of the intestinalabsorption of Pi, we repeated and extended the previous studyon rats by analyzing the duodenum, jejunum, and ileum in thepresence and absence of Na� (Fig. 1A). In this experiment,acute, 4-h feeding of a high-Pi diet (1.2%) significantly in-creased Pi uptake in the duodenum and jejunum. Interestingly,this increase was also significant in the absence of Na� in theduodenum and jejunum.

Table 1. Primers used in real-time PCR

Gene Name Protein Name Accession No.

SequencesAmplification,

bpForward Reverse

PPIB Peptidylprolyl isomeraseB/cyclophilin B

NM_000942 5=-CCTTAGCTACAGGAGAGAAAG-3= 5=-CCCTGGATCATGAAGTCCTTGA-3= 402–481

SLC26A1 Sat1 NM_022042 5=-GATGACCGGGCTTTACCAG-3= 5=-AGGTGTTTGAGCTGCGAG-3= 680–810SLC26A2 DTDST NM_000112 5=-CTATGCAATTATGGTTGGCAGC-3= 5=-AGGCATCTGAGAGGTAGACAG-3= 916–1022SLC26A3 DRA NM_000111 5=-TGGTATCAGCACAGGGATTG-3= 5=-ATATGTGTCTGGAAGTGCCG-3= 448–587SLC26A4 Pendrin NM_000441 5=-ACTGCGGGTGATTGTCAAAG-3= 5=-TCGTCAAAGAACCCGCATTG-3= 2249–2356SLC26A5 Prestin NM_198999 5=-ATCATGGGAGCAAGGAGAAAG-3= 5=-GCATCCTCTCCATCTACTTCTG-3= 1935–2047SLC26A6 PAT1 NM_022911 5=-CCTCTATAGCTCCTTCTACCCTG-3= 5=-ATCATGGAGTCGTTCAAGGC-3= 466–612SLC26A7 SUT2 NM_052832 5=-GCTTCCCAAGAGCAATGACT-3= 5=-GCAGGGTTTCACTGTCCATT-3= 1651–1737SLC26A8 TAT1 NM_052961 5=-GCCCAGGATTCTTTACACAGAG-3= 5=-TGAGACACGGACGATACTGT-3= 2144–1992SLC26A9 SLC26A9 NM_052934 5=-CACCCACTGCTTGTAAATGC-3= 5=-TGTCCGGTCCTTCTTCTCAA-3= 88–204SLC26A10 SLC26A10 NM_133489 5=-AATCCTGCGAGAAGCCTTAGA-3= 5=-TGATCTTCCTCAGACCCTTTCC-3= 167–2044SLC26A11 SUT1 NM_173626 5=-GTCTCCTTCTACACCTTCCATG-3= 5=-GGTGAAGCCTTTAATGACGGG-3= 603–743SLC17A1 NPT1/NaPi1 NM_005074 5=-TTTGTCCCGAATCAGTGGGT-3= 5=-ACCATTGTGAGGTTCAGGCA-3= 52–250SLC34A2 NaPi2b NM_001177999 5=-AGCCACTGTCCATGACTTCTTC-3= 5=-GAGCTTTGTGAAGGGCTTAGTG-3= 749–923SLC20A1 Pit-1 NM_005415 5=-TGGGAGCCAATGATGTAGCA-3= 5=-ACATCTCCACGTCAATCAAGCC-3= 655–822SLC20A2 Pit-2 NM_001257181 5=-ATTTCCTTTGGTGTCGCCCT-3= 5=-TTGGCACCTGGTAGCTCTTT-3= 1346–1527XPR1 Xenotropic and polytropic

retrovirus receptor 1NM_004736 5=-TTTGCAGCCCTTTACAGCAC-3= 5=-GAGGAAAGTGTTCTCTCCAGCA-3= 1666–1833

C1114 Na�-INDEPENDENT Pi TRANSPORT

AJP-Cell Physiol • doi:10.1152/ajpcell.00251.2014 • www.ajpcell.org

We then studied the response of Caco2BBE cells incubatedfrom 1 h to 4 days in high-Pi (4mM) culture medium (Fig. 1B).32Pi uptake was determined during 20 min at room temperatureand in the presence of Na�. The results revealed a significantincrease that started at 24 h and increased thereafter up to 4days.

Subsequently, we incubated Caco2BBE cells for 48 h with 4mM Pi in DMEM to compare their uptake of Pi with the uptake

of cells maintained in 1 mM Pi. Figure 1C shows that 32Pi

uptake of Caco2BBE cells incubated with 4 mM Pi increasedcompared with cells maintained in DMEM with 1 mM Pi. Onceagain, Na�-dependent Pi transport was not observed, i.e., theeffect and total uptake were similar in the presence and absenceof Na�. The maximal increase (�15 times) was observed at 10min of uptake time. Thereafter, the increase dropped progres-sively as a consequence of the saturation of Pi uptake in cellspreincubated with 4 mM Pi, and at 60 min the increase wasonly six times.

Kinetic characteristics of Pi transport in Caco2BBE cells.The absence of Na�-dependent Pi uptake was checked underdifferent conditions. First, it was assayed after the time ofconfluence, because the differentiation state could affect theexpression of Na�-dependent (NaPi2b) transporters. As shownin Fig. 2A, no Na�-coupled Pi uptake was observed above thelevel indicated by uptake in the absence of Na�, even after 17days postconfluence in Caco2BBE cells maintained in normalDMEM (1 mM Pi). However, uptake was drastically dimin-ished between 0 and 5 days of confluence (see DISCUSSION).

To further characterize Pi uptake in Caco2BBE cells, Mi-chaelis-Menten saturation kinetics were performed in cellspreincubated with either 1 or 4 mM Pi for 48 h (Fig. 2B). Theseassays were performed in the presence of Na� to guaranteemore physiological conditions. Fits revealed that the increaseduptake was mediated by an increase of 12 times Vmax: from0.073 to 0.849 nmol Pi·mg cell protein�1·min�1. The apparentKm value increased from 0.071 to 0.16 mM, but a global(shared) fit revealed that the difference was not statisticallysignificant. This was confirmed by a t-test using the differentaffinity constants (three Km per condition). To illustrate that theuptake change was mainly caused by an increase in capacity(Vmax) rather than affinity, the theoretical (i.e., saturable) trans-port components of the total uptake in Fig. 2B are shown inFig. 2C.

pH effect on Pi transport in Caco2BBE cells. The transportresponse to environmental pH changes is a hallmark of everyPi transporter. Therefore, we also studied this effect inCaco2BBE cells. In cells maintained in DMEM with 1 mM Pi,Pi transport was identical in the presence and absence of Na�

(Fig. 3A,1) at all pHs. From pH 6.0 to 7.5, Pi transportremained constant, and thereafter it decreased constantly at pH8.0 and 8.5, similar to Pi transport in the small intestine (seeDISCUSSION). Conversely, cells preincubated with 4 mM Pi (Fig.3A,2) exhibited transport in which, while still Na� indepen-dent, the behavior was similar to that of the proximal tubule ofthe kidney: Pi uptake increased steeply from pH 6.0 to 8.5. Asa control to confirm the accuracy of our determinations, wealso measured the well-known effect of pH on 50 �M Pi uptakein OK cells, a well-established cell line model of the renalproximal tubule obtained from a female American opossum,which exhibits strong type IIa Na�-dependent Pi transport.Figure 3A,3 shows that OK cells maintained in standard me-dium only express Na�-dependent Pi transport, which in-creases when the pH of the uptake medium also increases,similar to Na�-independent Pi uptake in Caco2BBE cellspreincubated with 4 mM Pi.

The effect of pH could be interpreted as a preference of thetwo Pi transports (mainly being expressed in cells maintainedin 1 vs. 4 mM Pi culture medium) for the two Pi species,H2PO4

� versus HPO42�. Regarding the transport in 1 mM

Fig. 1. Pi transport upregulation in response to a high Pi concentration. A: acute(4 h) intestinal response to a 1.2% Pi diet in rats fed with a diet of 0.1% Pi for5 days. D, duodenum; J, jejunum; I, ileum. ***P 0.001; **P 0.01.B: effect of hours of incubation at 4 mM Pi in cell culture on 32Pi uptake in thepresence of Na� in Caco2BBE cells. C: time course of Pi uptake in Caco2 cellspreincubated or not with 4 mM Pi for 24 h.

C1115Na�-INDEPENDENT Pi TRANSPORT


Pi-maintained cells, which exhibit higher Pi uptake at a low pH,this could also be interpreted as the use of a proton gradient asthe driving force. Therefore, we treated cells with two iono-phores (FCCP and CCCP) and a vacuolar-type H�-ATPaseinhibitor (bafilomycin A1), and Pi transport was measured instandard conditions (pH 7.5). FCCP and CCCP only reduced Pi

uptake by 50% in Caco2BBE cells maintained in 1 mM Pi (Fig.3B,1), but they failed to modify Pi uptake in cells preincubatedin 4 mM Pi (Fig. 3B,2). Similarly, bafilomycin A1 did not alterPi transport at either 1 or 4 mM Pi. This can be explained as theproton gradient being maintained by another type of pump or,most likely, as the experimental proton gradient created by thelow pH is sufficient to sustain Pi transport during the short,linear velocity time of uptake. As expected, furosemide, aNa�-ATPase inhibitor, did not modify Pi uptake in any con-dition. To confirm the specificity of the effect of FCCP andCCCP in 1 mM Pi, the absence of an effect by these drugs onNa�-independent uptake of D-glucose and D-fructose was alsotested (Fig. 3B,3).

The response of Caco2BBE cells to a high Pi concentrationis progressive. Caco2BBE cells were incubated for 48 h inDMEM containing 0.2, 1, 2, or 4 mM Pi, and 32Pi uptake wasthen assayed in the presence or absence of Na� and at differentpHs (6.0, 7.5, and 8.5; Fig. 4). The changes in Pi transport asa consequence of incubation with different concentrations of Pi

were not observed when uptake was assayed at pH 6.0. At pH7.5, however, a significant increase was already observed incells preincubated with 4 mM Pi, and at pH 8.5, even thetransport in cells preincubated with 2 mM Pi was significantlydifferent from that of cells maintained in 1 mM Pi. No changeswere observed with a low concentration of Pi (0.2 mM) at anypH. In Fig. 4, each graph represents a different experiment,meaning different cell cultures, and therefore results in A–Ccannot be compared in absolute terms with respect to the transportrate (according to the critical changes in transport rate dependingon confluence, as shown in Fig. 2A) but rather in relation to theexperimental conditions used in each assay or graph.

The response to a high Pi concentration is dependent on denovo RNA and protein syntheses. To ascertain whether theincrease in Pi uptake was dependent on the synthesis of newtransporters or activators, or conversely if it was caused by theposttranslational reorganization or modification of preexistingmolecules, Caco2BBE cells were incubated for 24 h in thepresence or absence of inhibitors of transcription (1 �g/mlactinomycin D) or translation (100 �M cycloheximide). Cellswere incubated for only 24 h to avoid toxic overexposure to thedrugs. Figure 5 shows that, in this particular experiment, cellsincubated with 4 mM Pi exhibited five times more Pi transportthan cells incubated with 1 mM Pi, yet when cells weresimultaneously treated with actinomycin D or cycloheximide,the increase was only twice as much. This means that both ofthe pharmacological agents prevented the increased Pi trans-port by 70%, and, therefore, the transcription and translation ofnew proteins is a requisite step for the response to 4 mM Pi.

Pattern of Pi transport inhibition on Caco2BBE cells. Inhi-bitions of Pi uptake using potential substrates were performedin Caco2BBE cells cultured in the presence of 1 or 4 mM Pi for48 h to gain knowledge of the likely transport systems involvedin this Na�-independent Pi transport. 32Pi uptake was assayedat 50 �M for 20 min in the absence or presence of theinhibitors shown in Fig. 6A. We mainly tested several inor-ganic and organic anions of biological relevance, whose trans-porters could also be involved in Na�-independent phosphateuptake. For example, sulfate is Na� independently transportedby Sat-1 and other members of the Slc26 family, includingoxalate, formate, bicarbonate, etc. DIDS and SITS are stilbenedisulphonate derivatives that inhibit most anion antiporters,

Fig. 2. Kinetic characteristics of Pi transport in Caco2BBE cells. A: total Pi

uptake in the presence and absence of Na� as a function of postconfluencedays in regular DMEM (1 mM Pi). B: nonlinear regression fits of total Pi

uptake to an equation containing a Michaelis-Menten component and anonsaturable component (see MATERIALS AND METHODS). Both fits were signif-icantly different, as a consequence of the Vmax increase, with P 0.0001. C:net, nonlinear regression of theoretical transport in the same cells, according tothe kinetic parameters of transport obtained in B. See text for further details.



whereas phosphonoformic acid (PFA) and arsenate are mainlyinhibitors of type II Pi transporters. The results showed that inboth cases (i.e., cells maintained at either 1 or 4 mM Pi inculture medium) no other inhibitor was as effective as 5 mMphosphate at inhibiting 32Pi uptake. In cells maintained at 1mM Pi, incubation with 10 mM alanine, glutamate, or formatedid not inhibit Pi transport, and only partial inhibition wasreached with 10 mM sodium sulfate, sodium oxalate, or so-dium bicarbonate, with 5 mM PFA, with 0.1 mM DIDS, with0.1 mM SITS, and with 5 mM sodium arsenate. Regardingcells incubated for 48 h with 4 mM Pi, the pattern of inhibitionwas more restricted, because Pi transport was only partiallyinhibited with oxalate, PFA, and arsenate.

The different patterns of inhibition in cells maintained in 1or 4 mM Pi suggest that the inhibition is not caused by toxiceffects such as metabolic alterations induced by an excess ofinhibitors. Nevertheless, to completely discard possible sideeffects induced by the inhibitors, we also treated Caco2BBEcells with the inhibitors but without 32Pi. After 20 min ofincubation, cells were washed twice, and 32Pi uptake was thenmeasured for an additional 20 min. The assay revealed nochanges induced by the inhibitors during the 20 min of expo-sure (Fig. 6A,3).

The inhibitions of Pi transport with PFA, oxalate, andarsenate were further characterized using dose-response rela-tionships. The results are shown in Fig. 6B: complete inhibi-

Fig. 3. Effect of pH and proton ionophores on 50 �M Pi uptake in two cell lines. A: effect on Caco2BBE cells incubated for 48 h in 1 mM Pi (1) or 4 mM Pi

(2) as well as the effect of pH on Pi uptake in opossum kidney (OK) cells incubated in standard conditions (DMEM-Ham’s F-12 medium; 3). B: effect ofionophores and ATPAse inhibitors on Pi uptake in 1 mM Pi-maintained cells (1) or 4 mM Pi-maintained cells (2) as well as the absence of an effect onNa�-independent uptake of D-glucose and D-fructose (3). Cho, choline; Bafilo A1, bafilomycin A1.



tions were barely achieved, and apparent IC50 values were, ingeneral, very high. Arsenate was a poor inhibitor of Pi transportin cells incubated with either 1 or 4 mM Pi, with IC50 values of�3 mM. Oxalate inhibited Pi transport with more intensity incells maintained at 1 mM Pi than at 4 mM Pi, whereas theopposite was observed for PFA, because a stronger inhibitionof Pi transport was observed in cells maintained at 4 mM Pi.

Toward the molecular identification of Na�-independent Pi

transport. The pattern of inhibition of the two Na�-indepen-dent Pi transport systems (i.e., in cells incubated with either 1or 4 mM Pi) suggested the involvement of some members ofthe Slc26 family of multifunctional anion transporters andchannels, among others. Previously known Pi transporters,such as Slc20 family members Pit-1 and Pit-2 (Slc20A1 andSlc20A2), Slc17A1 (NaPi-I), and Slc43a2 (NaPi-IIb), shouldnot be involved based on the respective Na� dependence. Wefirst analyzed the expressions of the corresponding RNAs byreal-time PCR using a combination of cDNAs from Caco2BBEcells grown in 1 or 4 mM Pi culture medium for 48 h (i.e., ourcalibrator for quantitative PCR; see MATERIALS AND METHODS).The primers used are shown in Table 1, and significant expres-sions are shown in Fig. 7A. The relative abundances are plottedin Fig. 7 with respect to the abundance of Slc26a8, which is theSlc26 family member with the lowest significant amplification.Very abundant transcripts corresponded to Slc26 family mem-bers 1, 2, 3, and 6. We also found low but significant expres-sion of Slc26A1 and Slc26A11 as well as Slc20a1 and Slc20a2.

Next, we compared the expressions of those transcripts incDNA prepared from cells grown at 1 versus 4 mM Pi for 48h (Fig. 7B). The Na�-independent Pi exporter XPR1 was alsoincluded in this assay. No significant differences were ob-served between 1 and 4 mM Pi for any transcript, even fortranscripts apparently inhibited at 4 mM Pi.

Even though the absence of differences in RNA expressionsuggest that the corresponding transporters are not involved in theincreased Na�-independent Pi transport observed in cells incu-bated with 4 mM Pi, we also knocked down the expression ofSlc26 family members with siRNA. Using this approach, theinvolvement of these members in the basal Pi transport observedin cells at 1 mM Pi could also be tested. As shown in Fig. 7C, theeffect by the reduced expression of the indicated members of theSlc26 family did not alter the Pi transport rate in Caco2BBE cells.Real-time PCR confirmed the success of the siRNA treatment,showing a reduction of at least 75% compared with scrambledsiRNA with a similar CG content (not shown).

Fig. 4. Progressive upregulation of Pi transport in Caco2BBE cells as aresponse to increasing Pi concentrations. The increased uptake effect is bluntedwhen measured at pH 6.0 (A), and it can only be observed at pH 7.5 and 8.5and at 2 and 4 mM Pi (B and C). Uptake is shown in the presence or absenceof Na�. Comparisons are only shown for conditions in the presence of Na�.The same significances were obtained in the absence of Na�. *P 0.05.

Fig. 5. The effect by the incubation of Caco2BBE cells in a culture medium of4 mM Pi for 24 h on Pi transport is mostly prevented by inhibitors of RNA andprotein synthesis, actinomycin D and cycloheximide. P 0.0001 by ANOVA.**P 0.01; ***P 0.001.



DISCUSSION

In this work, we have functionally characterized a Na�-independent Pi transport system in the Caco2BBE cell line.This is a well-known colon carcinoma cell line from a Cauca-

sian American male used as a model to study the enterocytephysiology of the small intestine, because it shares manycharacteristics of solute transport with the intestinal epithelium(27). However, contrary to a previous work (25), we did not

Fig. 6. Pattern of Pi transport inhibition. A: normalized graphs of Pi uptake inhibition with several substrates. 32Pi uptake was assayed at 50 �M for 20 min. Solidand shaded bars indicate significant (complete or partial, respectively) inhibition compared with the control (Con; open bars). Pi, phosphonoformic acid (PFA),and arsenate (AsV) were used at 5 mM; alanine (Ala), glutamate (Glu), formate (Form), sulfate (SO4), oxalate (Ox), and carbonate (HCO3) were used at 10 mM;DIDS and SITS were used at 0.1 mM. The dashed line indicates the level of inhibition with 5 mM Pi as an inhibitor. A: effect on 1 mM Pi-maintained Caco2BBEcells (1), effect on 4 mM Pi-preincubated Caco2BBE cells (2), and absence of metabolic/toxic effects of the inhibitors after 20 min of preincubation in the absenceof 32Pi (3). B: dose-response relationships of the indicated inhibitors using Caco2BBE cells incubated with 1 or 4 mM Pi. The corresponding mean inhibitoryconcentrations are indicated.



detect a Na�-dependent Pi transport system in Caco2BBEcells. This absence could be explained by the fact that theCaco2BBE cell line corresponds to a C2 subclone of theoriginal Caco-2 cells (28), whereas Mohrmann et al. (25) usedthe original cells.

Even though it is still Na� independent, Pi transport inCaco2BBE cells also decreases after confluence, similar tothe previous report (25). When epithelial cells, such asCaco2BBE cells, become confluent, they initiate a series ofgene expression changes that induces differentiation (10).The change in Pi transport rate upon confluence could berelated to this differentiation, e.g., it could be caused by theswitch from a transport system of proliferating cells to adifferent system, more characteristic of intestinal, epithelialcells. In addition, the Caco2BBE cell line is an in vitromodel cell line, and even if many authors have used it as amodel of small intestine, this should be proved for everystudy and experimental condition. Consequently, we do notknow yet whether the Pi transport that we measured inCaco2BBE cells is representative of either the small intes-tine or the colon. In this respect, not much is known aboutthe role of the colon in Pi absorption, but findings in theliterature point to the existence of, at least, a paracellularroute that could explain the observed enema-induced hyper-phosphatemia (17).

We also observed that Pi uptake is upregulated in Caco2BBEcells after cells were incubated with a high Pi concentration (4mM; Fig. 1B). This increase is also strictly Na� independent,it becomes significant at 24 h, and it increases even further withincubation time in a culture medium of 4 mM Pi (Fig. 1B). Thisfinding is notable, because it shows that Caco2BBE cellsbehave similarly to the small intestine with respect to the Na�-independent component of Pi transport observed in BBMVs fromthe duodenum and jejunum of rats fed with an acutely high-Pi diet(Fig. 1A) (12). However, incubation of Caco2BBE cells in a lowPi culture medium (0.2 mM Pi) did not significantly change thetransport rate of Pi, which is in contrast with the chronic intestinalresponse to a low-Pi diet (14).

In the present study, the characterization of Pi transport inCaco2BBE cells was always done under two conditions,namely, cells were maintained in a culture medium containingeither 1 or 4 mM Pi. The rationale was to mimic the highconcentrations of Pi that can be reached in the intestinal lumen(19). The subsequent results showed that the increased Pi

uptake observed after incubation with 4 mM Pi is not simply aconsequence of increased diffusion, unspecific uptake, or anyother side effect resulting from Pi toxicity or the precipitationof calcium phosphates in the culture medium (for example). Tothe contrary, this increased rate of uptake exhibits severalcharacteristics that are exclusive of transport mechanisms:saturation (Fig. 2, B and C), pH dependence (Fig. 3), inhibitionby putative substrates (Fig. 6), and prevention by inhibitors oftranscription and translation (Fig. 5). A summary of the dif-ferent characteristics of Pi transport is shown in Table 2.

The increased Pi uptake observed in cells incubated with 4mM Pi is caused by a significant change in Vmax (Fig. 2C). Thiscould be interpreted as a consequence of the incorporation/activation of more transporters of the same kind as thoseobserved in cells incubated with 1 mM Pi. However, othercharacteristics clearly differentiate between both transport sys-tems described in cells incubated with 1 and 4 mM Pi: differentpH dependence and pattern of inhibition. With respect to pHdependence, as shown in Fig. 3A, Pi transport in Caco2BBEcells incubated with 1 mM Pi remains unchanged from pH 6.0to 7.5, and thereafter Pi transport drops. Conversely, Pi trans-port in cells maintained in a medium of 4 mM Pi for 48 h

Fig. 7. Analysis of the involvement of different carriers on Na�-independent Pi

transport. A: real-time analysis of RNA expression of the indicated Slc26,Slc20, Slc17, and Slc34 family members. B: comparison of the relativeabundance of the indicated RNAs after treatment with 1 or 4 mM Pi for 48 h.C: Pi transport after 48 h of small interfering (si)RNA transfections against theindicated Slc26 family members in Caco2BBE cells incubated with 1 or 4 mMPi. C, nontransfected cells; C�, scrambled siRNA-transfected cells.



increases linearly to a maximum at pH 8.5, where 6.3 times theuptake of Pi at pH 6.0 is observed. Because Pi is a polyproticacid, one possibility is that the transport observed in cellsmaintained with 4 mM Pi corresponds to a transporter thatmainly carries divalent phosphate (HPO4

2�), whereas at 1 mMPi the major transport system would prefer H2PO4

� overHPO4

2�. pH dependence is a known characteristic of Pi trans-porters, because most of them transport preferentially monova-lent or divalent Pi. Type II Na�-Pi cotransporters (NaPi2a,PaPi2b, and NaPi2c), for example, preferentially transportdivalent Pi. Consequently, because Pi transport in the kidney(and OK cells) is mostly mediated by NaPi2a, the transport ofPi in the proximal tubule also increases with the pH, as shownwhen kidney cortex BBMVs are used (1, 33). The behavior ofthe small intestine, however, is opposite that of the kidney, i.e.,Pi transport decreases as pH increases (3). This is a paradoxbecause this behavior corresponds to type III Pi transporters,Pit-1 and Pit-2, despite the prominent expression of NaPi2b(6). In the case of Caco2BBE cells, only Na�-independent Pi

transport is observed, and, therefore, type II and III Pi trans-porters should not be involved. In cells maintained at 1 mM Pi,Pi transport decreases as pH increases, whereas cells preincu-bated with 4 mM Pi show Na�-independent Pi uptake thatincreases with pH. Therefore, the molecular identity of thetransport systems acting in cells maintained at either 1 or 4 mMPi should be different.

The response of Pi transport to pH changes in Caco2BBEcells maintained in 1 mM Pi medium is not identical to changesobserved in the intestine: the drop in transport does not initiateat pH 6.0 according to the decreasing concentration of H2PO4

�;rather, it remains constant up to pH 7.5 (Fig. 3A,1). Therefore,additional causes could be involved in pH dependence at 1 mMPi, such as the use of a proton gradient in microorganisms, asit has been recently suggested (9). The successful use of theionophores FCCP and CCCP to reduce Pi uptake in 1 mMPi-maintained cells strongly supports this view (Fig. 3B,1). Thepreference for HPO4

2� as a substrate of H�-mediated uptake at1 mM Pi (or no preference by Pi species, either for H2PO4

� orHPO4

2�) could explain the lack of inhibition of Pi transportbetween pH 6.0 and 7.5.

Finally, the pattern of inhibition with potential competitivesubstrates is also different in Caco2BBE cells maintained at 1or 4 mM Pi. In both cases, maximal inhibition was obtainedwith 5 mM Pi, whereas partial inhibition was observed with 5mM PFA and arsenate and with 10 mM oxalate (Fig. 6A). Inthe case of cells maintained in a medium of 1 mM Pi, Pi

transport was also partially inhibited with sulfate, bicarbonate,

DIDS, and SITS. These differences could once again be aconsequence of more than one transport system participating inthe Na�-independent uptake of Pi.

There are a large number of possible transporters that couldbe involved in the two Na�-independent Pi transport systemsof Caco2BBE cells (observed in cells incubated at 1 or 4 mMPi), according to the inhibitions shown in Fig. 6A. Thesetransporters could belong to the SLC26 gene family of multi-functional anion exchangers (which include anions such asbicarbonate, sulfate, oxalate, or formate and inhibitions withDIDS or SITS), among others. With the exception of formate,all substrates behaved as partial inhibitors of Pi transportexpressed in cells maintained in 1 mM Pi. In the transportobserved in cells maintained with 4 mM Pi, inhibition wasrestricted to oxalate, PFA, and arsenate (Fig. 6A). Whenconsidering the use of a proton gradient as the source of energyin 1 mM Pi-maintained cells, the possibilities are furtherreduced. For example, SLC25A3 is a H�-coupled mitochon-drial Pi carrier of the inner membrane, and several plasmamembrane H�-coupled transporters of the SLC36 family onlycarry several amino acids. The same can be said for SLC45A1,which seems to transport H�/sugars, and SLC46A1, whichseems to transport H�/folates.

Furthermore, we tried to identify potential transporters inseveral ways. For example, we analyzed RNA expressionchanges between cells maintained at 1 or 4 mM Pi or usingsiRNA-induced downregulation of specific transporters to pre-vent the increase of transport or to reduce basal Pi uptake (Fig.7). None of these approaches were successful, but they help toeliminate potential candidates.

The functional and molecular nature of Na�-independent Pi

transport remains elusive so far, and only a few classical workshave made initial characterizations, such as Pi-HCO3

� ex-change in capillaries of the blood-brain barrier, which is alsoinhibited by DIDS, SITS, sulfate, and other substrates (8). Ourwork represents an initial characterization of the intestinalNa�-independent Pi transport system, and the findings couldhelp to identify the specific carriers by using alternative ap-proaches. For example, current work in our laboratory includesPCR subtraction of cDNAs prepared from cells maintained in1 or 4 mM Pi as well as a proteomic strategy to identifydifferentially expressed proteins in cell lysates under the sameconditions. This should help to understand the complex phys-iological mechanism of intestinal absorption of phosphate,whose knowledge is presently restricted to the Na�-dependentcomponent of absorption.

GRANTS

This work was supported by Spanish Ministry of Economy and Competi-tiveness Research Grants SAF2012-33898 (to V. Sorribas) and PredoctoralFellowship BES-2010-029965 (to E. Candeal).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: E.C., Y.A.C., and N.G. performed experiments; E.C.,Y.A.C., N.G., and V.S. analyzed data; E.C., Y.A.C., N.G., M.L., and V.S.interpreted results of experiments; E.C., Y.A.C., N.G., M.L., and V.S. editedand revised manuscript; E.C., Y.A.C., N.G., M.L., and V.S. approved finalversion of manuscript; V.S. conception and design of research; V.S. preparedfigures; V.S. drafted manuscript.

Table 2. Characteristics of Pi transport in Caco2BBE cells

Parameter 1 mM Pi 4 mM Pi

Vmax, nmol Pi·mg cellprotein�1·min�1 0.073 0.017 0.849 0.11

Km (mM) 0.071 0.020 0.155 0.025pH correlation Negative PositiveNa� gradient Independent IndependentProton gradient Dependent IndependentPartial inhibitors PFA, arsenate, oxalate,

sulfate, bicarbonate,DIDS, SITS

PFA, arsenate, oxalate

1 and 4 mM Pi refers to Pi transport in Caco2BBE cells maintained for 48h in culture medium containing 1 or 4 mM Pi. PFA, phosphonoformate.



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Na+-independent phosphate transport in Caco2BBE cells

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