The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells - PDF Document

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  1. cro ARTICLE The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells Receivedforpublication,February9,2018,andinrevisedform,March8,2018 Published,PapersinPress,March12,2018,DOI10.1074/jbc.RA118.002378 Taro Mukaibo‡§1, Takashi Munemasa‡§1, Alvin T. George‡, Duy T. Tran¶2, Xin Gao‡?, Jesse L. Herche‡, Chihiro Masaki§, Gary E. Shull**, Manoocher Soleimani‡‡, and James E. Melvin‡3 Fromthe‡SecretoryMechanismsandDysfunctionsSectionand¶BiologicalChemistrySection,NIDCR,NationalInstitutesof Health,Bethesda,Maryland20892,the§DepartmentofOralReconstructionandRehabilitation,KyushuDentalUniversity, Kitakyushu,Fukuoka803-8580,Japan,the?JointInstituteforFoodSafetyandAppliedNutrition,UniversityofMaryland,College Park,Maryland20742,andtheDepartmentsof‡‡Medicineand**MolecularGenetics,Biochemistry,andMicrobiology,University ofCincinnatiCollegeofMedicine,Cincinnati, Ohio 45267 Edited by Mike Shipston The solute carrier family 26 (SLC26) gene family encodes at least10differentanionexchangers.SLC26member6(SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane con- ductance regulator (CFTR) co-localize to the apical membrane ofpancreaticductcells,wheretheyactinconcerttodriveHCO3 and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, lit- tle is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing–based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl?for oxalate and HCO3 expressedinsalivaryglandacinarcells,Slc4a4andSlc4a9,medi- ated little, if any, Cl?/oxalate exchange. Of note, both Cl?/oxa- late exchange and Cl?/HCO3 reduced in acinar cells isolated from the submandibular glands of Slc26a6?/?mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6?/?mice, but HCO3 secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinarcells,whereitmediatesCl?/oxalateexchangeandplaysa critical role in the secretion of oxalate into saliva. may be a pseudogene in humans (1). SLC26 proteins transport numerous anions, such as HCO3 late,andsulfate(2–4).AlthoughsomemembersoftheSLC26A family transport a limited number of specific substrates, SLC26A6 displays little anion selectivity (2, 5). SLC26A6hascytoplasmicNandCterminithatflankaninte- gral membrane domain containing 10 to 14 transmembrane segments as well as a sulfate transporter and anti-? factor antagonist (STAS)4domain located in the C terminus. The STAS domain interacts with other ion transport proteins to formtransportmetabolons(1,6–8).Oneproposedexampleof such an interaction is the mutual activation of CFTR and SLC26A6 in pancreatic duct cells, which depends on the phys- ical association of the STAS domain of SLC26A6 with the R domain of CFTR (7). CFTR and SLC26A6 activation is appar- entlyenhancedbyproteinkinaseA(PKA)–mediatedphosphor- ylation of the R domain (9). SLC26A6 expression is ubiquitous, but it is highly expressed in the pancreas, small intestine, and kidney (10, 11). Transcrip- tional profiling also found that Slc26a6 is highly expressed in murine salivary glands (12). Targeted disruption of mouse Slc26a6 inhibited HCO3 (13), whereas oxalate secretion in the small intestine and reab- sorption by the kidney were markedly reduced (14–17). These results suggest that Slc26a6 may play several crucial roles in mammalianphysiology,e.g.secretionoffluidandHCO3 pancreastoneutralizestomachacidandsecretionofoxalateby the intestine to regulate plasma oxalate levels and prevent kid- ney stone formation (15, 18–20). The function of Slc26a6 in salivary glands is unclear, althoughithasbeensuggestedthatSlc26a6contributestofluid andHCO3 Alternatively, Slc26a6 in salivary glands may play a role in the ?, OH?, Cl?, I?, formate, oxa- Downloaded from ? ?, whereas two other anion exchangers known to be by guest on May 4, 2020 ?exchange were significantly ? ?and fluid secretion in the pancreas ?bythe The SLC26 gene family encodes at least ten functionally diverse anion exchangers (SLC26A1–11), including SAT-1 (SLC26A1), DTDST (SLC26A2), DRA/CLD (SLC26A3), PDS/ Pendrin (SLC26A4), PRES/Prestin (SLC26A5), CFEX/PAT-1 (SLC26A6), SUT2 (SLC26A7), SPGF3 (SLC26A8), and SLC26A9 and SLC26A11, and there is suggestive evidence that SLC26A10 ?secretion,asdemonstratedinthepancreas(21–24). 4The abbreviations used are: STAS, sulfate transporter and anti-? factor antagonist; CFTR, cystic fibrosis transmembrane conductance regulator; RNA-seq, RNA sequencing; PG, parotid gland; SMG, submandibular gland; SLG, sublingual gland; qPCR, quantitative PCR; FPKM, fragments per kilo- base of transcript per million mapped reads; cDNA, complementary DNA; CHO, Chinese hamster ovary; CFEX, chloride/formate exchanger; BCECF, 2?,7?-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; AM, acetoxym- ethylester;SPQ,6-methoxy-N-(3-sulfopropyl)quinolimium;A01,T16Ainh- A01; EZA, ethoxzolamide; IPR, isoproterenol; BME, basal medium Eagle; ANOVA, analysis of variance; NMDG, N-methyl-D-glucamine. This work was supported by National Institutes of Health Grant 1-ZIA- DE000738 (to J. E. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon- sibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviews of the National Institutes of Health. 1Both authors contributed equally to this work. 2SupportedbyGrant1-ZIA-DE000739(toLawrenceA.Tabak)fromtheIntra- mural Research Program of the NIDCR, National Institutes of Health. 3To whom correspondence should be addressed. Tel.: 301-402-1706; E-mail: J. Biol. Chem. (2018) 293(17) 6259–6268 6259 Published in the U.S.A.

  2. Slc26a6facilitatesoxalatesecretioninsaliva Figure 1. Slc26a6 mRNA expression in mouse salivary glands. A, Slc26a6 expression acquired by RNA-seq analysis for mouse PGs, SMGs, and SLGs are displayedasFPKMper40millionmappedreads.Datafromindividualglands aredisplayedascircles(n?6forPG,SMG,andSLG).B,Slc26a6mRNAexpres- sion levels were confirmed by qPCR and normalized to ?-actin (Actb) (n ? 6 forPG,SMG,andSLG).C,PCRbandsizeandproductamountforslc26a6(149 bp) and Actb (147 bp) after PCR 27 cycles of cDNA amplification (100 ng). One-way ANOVA followed by Bonferroni’s post hoc test was performed for statistical analysis; **, p ? 0.01. Downloaded from secretion of oxalate, as in the small intestine (16). In fact, oxa- late has been detected in human saliva and sialolithes (25, 26), where it might contribute to salivary gland stone formation. Thus, the aim of this study is to test, in murine salivary glands, two major hypotheses: Slc26a6 functions predomi- nantly as a Cl?/HCO3 secretion and/or Slc26a6 largely functions in Cl?/oxalate exchange mode and thus plays a critical role in oxalate secre- tion. Our results suggest, in contrast to the pancreas, that Slc26a6 does not target to the apical membrane of duct cells, nordoesitpromoteHCO3 to the apical membrane of salivary gland acinar cells, where it appears to play a major role in oxalate secretion. by guest on May 4, 2020 ?exchanger and contributes to HCO3 ? Figure 2. Slc26a6 protein expression in mouse salivary glands. A, crude plasma membrane was isolated from PGs, SMGs, and SLGs. Each lane was loaded with 40 ?g of protein and immunoblotted with mouse monoclonal antibodies(Ab)toSlc26a6and?-actin.TomoreclearlyvisualizeSlc26a6pro- tein expression in PGs, the blot was developed for 40 s (first lane). B, band intensities from A were quantified using ImageJ software and normalized to ?-actin (n ? 4). One-way ANOVA followed by Bonferroni’s post hoc test was performedforstatisticalanalysis;*,p?0.002,**,p?0.01.C,SMG(40?g)and SLG (20 ?g) plasma membrane proteins from Slc26a6?/?and Slc26a6?/? mice and from Slc26a6-transfected CHO cells (20 ?g) were treated without (?)orwith(?)PNGaseF(PNG).ThesamemembraneasinAwasstrippedand immunoblotted with an anti-?-actin antibody (n ? 3). ?secretion.Instead,Slc26a6localizes Results Slc26a6mRNAisexpressedinmurinesalivaryglands RNA-seq data were acquired and described previously (12), and the full data sets were deposited in the Gene Expression Omnibus(GEOaccessionnumberGSE96747).Furtheranalysis of these data revealed Slc26a6 transcript expression in the major salivary glands, the parotid gland (PG), submandibular gland(SMG),andsublingual(SLG)gland.SLGSlc26a6expres- sionexceededthelevelsfoundinthePGandSMGby?33-and 9-fold, respectively (Fig. 1A). Validation by qPCR analysis showed that the relative expression of Slc26a6 mRNA in the SLG was 22- and 9-fold greater than in the PG and SMG, respectively (Fig. 1B), comparable with the RNA-seq data. Slc26a6 mRNA expression was normalized to the ?-actin housekeeping gene Actb (Fig. 1C), whose expression, as resolved by RNA-seq, was not significantly different in the PG, SMG, and SLG (mean FPKM ? S.E. ? 403.5 ? 27.3, 503.5 ? 93.3, and 553.3 ? 82.6, respectively; n ? 6 for each gland). This was also confirmed by qPCR analysis, where the Cq values for ?-actin were 20.8 ? 0.5, 19.4 ? 0.6, and 19.1 ? 0.3 for the PG, SMG, and SLG, respectively (n ? 6 for each gland). Slc26a6ismodifiedbyN-linkedglycosylation To examine Slc26a6 protein expression across the three major murine salivary glands, 40 ?g of plasma membrane pro- tein from each gland was separated by SDS-PAGE followed by Western blotting. Consistent with Slc26a6 mRNA expression levels, the protein expression level in the SLG was ?39- and 7-fold higher than in the PG and SMG, respectively (Fig. 2, A andB).LittleSlc26a6proteinwasdetectableabovebackground in the PG lane when the exposure time was only 5 s, but it was clearly visible when developed for 40 s (Fig. 2A, first lane). The protein band detected in the SLG lane ran ?10–15 kDa smaller compared with the plasma membrane protein isolated 6260 J. Biol. Chem. (2018) 293(17) 6259–6268

  3. Slc26a6facilitatesoxalatesecretioninsaliva from the SMG and from Slc26a6 cDNA-transfected CHO cells (Fig. 2C). It was reported previously that human and mouse SLC26A6areheavilyN-glycosylatedinatissue-specificmanner (27). Correspondingly, treatment with the N-linkage–specific glycosidase PNGaseF resulted in a shift to a similar molecular mass of ?70–80 kDa in SMG, SLG and Slc26a6 pcDNA trans- fected CHO cells, suggesting that the smaller apparent size of Slc26a6 in the SLG is caused by less N-glycosylation modifica- tion (Fig. 2C) and not gland-specific differences in alternative splicing of the Slc26a6 transcript. Indeed, a comparison of thesalivaryglandtranscriptomesrevealedthatthe18exonscom- prisingtheSlc26a6gene,includingpredictedN-glycosylationsites at asparagine 151 and 303 ( NetNGlyc/),5areexpressedinthePG,SMG,andSLG(12).Note that the predicted unmodified protein molecular mass of mouse Slc26a6 is ?83 kDa (UniportKB Q8CIW6-1). Furthermore, comparison of SMG and SLG transcriptomes failed to detect major differences in the expression of genes involvedinN-glycosylationofSlc26a6(28),e.g.the12members of the Alg gene family of glucosyltransferases, except for Alg8. Although called significantly different by bioinformatics analy- sis (Cufflinks/Cuffdiff 2.2.1), Alg8 expression in the SMG was only about 40% less than that detected in the SLG (FPKM ? 5.76and9.79,respectively;q?0.026;n?6foreachglandtype). Thus, the expression patterns of glucosyltransferase genes do not easily explain the large differences in N-glycosylation of Slc26a6 in the SMG and SLG, suggesting that it likely involves differencesinexpressionofgenesthataddothermoietiestothe oligosaccharide structure and/or signaling pathways that regu- late translation of Alg mRNAs and glucosyltransferase activity. Figure 3. Slc26a6 localizes to the apical membranes of mouse salivary gland acinar cells. Shown is immunofluorescent staining of Slc26a6 (cyan), Cftr (green), and Nkcc1 (red). Nuclei were stained with 4?,6-diamidino-2-phe- nylindole (blue). A, Slc26a6 localized at the apical membrane of SMG acinar cells from Slc26a6?/?mice, with no overlap of apical duct Cftr (filled white arrow) or basolateral acinar Nkcc1 (filled red arrow) staining. B, high magnifi- cation image of Slc26a6 staining of the acinar cell apical membrane (open white arrow) from A. C, Slc26a6 was not detected in the SMGs of Slc26a6?/? mice, whereas Nkcc1 staining was unchanged. D and E, Slc26a6 localized at theapicalmembraneofSLGacinarcellsfromSlc26a6?/?mice,withnoCftror Nkcc1 overlap. F, Slc26a6 was not detected in the SMGs of Slc26a6?/?mice, whereasNkcc1stainingwasunchanged.Representativeimagesofthreeinde- pendent experiments are shown. Scale bars ? 10 ?m. Downloaded from Slc26a6exchangesCl?foroxalateintransfectedCHO-K1cells Slc26a6 (also known as CFEX, chloride/formate exchanger) was initially reported to exchange Cl?for formate (5), but Slc26a6 was subsequently recognized to also transport other anions, such as oxalate and HCO3 onstratedthatSlc26a6isexclusivelyexpressedinsalivarygland acinar cells; however, these cells also express the anion exchangersAe2(Slc4a2)andAe4(Slc4a9)(34,35),whichcom- plicates the interpretation of studies to quantify Slc26a6-medi- ated anion exchange. Consequently, to verify that Slc26a6 oxa- late transport activity can be isolated in salivary gland acinar cells, mouse Slc26a6, Slc4a2, and Slc4a9 cDNAs were expressed in CHO-K1 cells, and Cl?/oxalate exchange was monitored. Oxalate-dependent Cl?uptake (anion exchanger activity) wasmonitoredinCHO-K1cellswiththeintracellularCl?indi- cator SPQ under HCO3 “Experimental procedures.” Slc26a6-expressing CHO-K1 cells mediatedCl?/oxalateexchange(Fig.4A,resultssummarizedin Fig. 4B). In contrast, Slc4a2- and Slc4a9-expressing CHO-K1 cellsdisplayedlittle,ifany,oxalate-dependentCl?uptake(Cl?/ oxalate exchanger activity). Thus, Cl?/oxalate exchange can isolate Slc26a6 anion transport activity from the other known anion exchangers expressed in salivary gland acinar cells. ?(4). The above results dem- by guest on May 4, 2020 Slc26a6localizestotheapicalmembraneofsalivarygland acinarcells Previous reports suggest that Slc26a6 is localized to the api- cal membrane of epithelial tissues, including the duct cells of the pancreas (21, 29), small intestine, and kidney proximal tubules (11, 30, 31), where it is thought to regulate pancreatic HCO3 32, 33). Thus, immunohistochemistry was performed to better understand the functional targeting of Slc26a6 in the SMG, SLG, and PG of WT mice (Slc26a6?/?). Strong immuno- staining of Slc26a6 (Fig. 3, cyan, open white arrows) was detected at the apical membranes of SMG and SLG acinar cells (Fig. 3, A and B, and Fig. 3, D and E, respectively), whereas Nkcc1-specific antibody (Fig. 3, red, filled red arrows) labeled the basolateral membranes of acinar cells and Cftr-specific antibody (Fig. 3, green, filled white arrows) stained the apical membranes of duct cells. In contrast, considerably less Slc26a6 staining was detected in the PG of WT mice, consistent with lower expression of Slc26a6 in this gland (data not shown). Immunostaining of Slc26a6 in the SMG and SLG was nega- tive in Slc26a6?/?mice, verifying the specificity of the anti- Slc26a6antibody(Fig.3,CandF),whereasNkcc1staining(Fig. 3,filledredarrow)wasunaffectedinthebasolateralmembranes of acinar cells in Slc26a6?/?mice. ?secretion and intestinal and renal oxalate transport (20, ?-free conditions as described under Slc26a6mediatesCl?/oxalateexchangeinsalivarygland acinarcells Mouse SMG acinar cells were treated as above (Fig. 4) to induce Slc26a6-mediated Cl?/oxalate anion exchange. To fur- ther isolate the Slc26a6-mediated Cl?/oxalate exchange activ- ity,Cl?fluxesviatheCa2?-activatedanionchannelTmem16A and the Na?/K?/2Cl?cotransporter Nkcc1 were prevented 5Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party–hosted site. J. Biol. Chem. (2018) 293(17) 6259–6268 6261

  4. Slc26a6facilitatesoxalatesecretioninsaliva solutionminusoxalate-freesolution)foreachglandisshownin Fig. 6C. These results show that Slc26a6 does not support fluid secretion but that Slc26a6 significantly contributes to oxalate secretion in the intact mouse SMG organ system. To confirm the results obtained using the colorimetric oxa- late assay, ion exchange chromatography was performed. SMG saliva was stimulated by IPR in the ex vivo perfusion system under oxalate-free conditions as describe above. The SMG salivasamplescollectedfromSlc26a6?/?andSlc26a6?/?mice were then assayed for oxalate content by both assay methods (Slc26a6?/?, colorimetric ? 0.21 ? 0.01 mM, ion exchange chromatography ? 0.19 ? 0.02 mM; Slc26a6?/?, colorimet- ric ? 0.17 ? 0.04 mM, ion exchange chromatography ? 0.21 ? 0.02 mM; n ? 8 for each group). There were no significant dif- ferences between assay methods or animal groups. Note that this experiment was performed in the absence of oxalate, indi- catingthatsalivaryglandssecreteendogenouslygeneratedoxa- late. Moreover, there was no difference between Slc26a6?/? and Slc26a6?/?mice, suggesting the existence of additional unknown oxalate transport pathways. Figure 4. Oxalate-dependent Cl?uptake by mouse Slc26a6 in trans- fected CHO-K1 cells. CHO-K1 cells were transfected with plasmids contain- ingeithermouseSlc26a6,Ae2(Slc4a2),orAe4(Slc4a9)cDNA,andtheintracel- lular Cl?concentration was measured using the Cl?-sensitive indicator SPQ. Intracellular Cl?was initially depleting by exposure to a low Cl?external solution,andthenoxalate-dependentCl?uptakewasinducedbyreintroduc- tion of a high Cl?solution. A, the high Cl?solution induced little, if any, oxalate-dependent Cl?uptake in Ae2-expressing (black circles, n ? 26) or Ae4-expressing (gray circles, n ? 18) CHO-K1 cells, whereas Slc26a6-trans- fected (gray squares, n ? 26) CHO-K1 cells exhibited oxalate-dependent Cl? uptake.Emptyvector–transfectedCHOcelldidnotinduceCl?uptake(Empty, white circles, n ? 15). B, summary of Cl?uptake rates in the presence of oxa- late.Dataarepresentedasthemean?S.E.ofatleastfivecellsperexperiment fromatleastthreedifferenttransfections.One-wayANOVAfollowedbyBon- ferroni’s post hoc test was performed for statistical analysis; **, p ? 0.01. Downloaded from ?secretion Slc26a6DisruptionfailstoalterHCO3 It has been reported that Slc26a6 and Cftr co-express in the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3 this interaction may also be important in salivary gland ducts (37, 38). However, Fig. 3 clearly demonstrates that Slc26a6 is expressed in mouse salivary gland acinar cells, whereas Cftr is found in the ducts. Moreover, disruption of Slc26a6 had no effect on fluid secretion in the SMG (Fig. 6A). To explore whether Slc26a6 contributes to salivary gland HCO3 activity of SMG acinar cells loaded with the pH-sensitive indi- cator BCECF. Fig. 7A shows that Cl?/HCO3 wassignificantlyreducedinacinarcellsfromSlc26a6?/?mice. Fig. 7B summarizes the alkalization rates for the results shown in Fig. 7A (B?) and also demonstrates that the alkalization inducedbyremovalofextracellularCl?wasHCO3 (B?). In contrast to Cl?/HCO3 magnitude of fluid secretion in response to the ?-adrenergic receptor agonist (1 ?M isoproterenol, IPR) were not altered in theSMGofSlc26a6?/?mice(Fig.7C,similartoFig.6A).More- over, the HCO3 Slc26a6?/?and Slc26a6?/?mice (Fig. 7D), suggesting that Slc26a6-mediated Cl?/HCO3 contribute to HCO3 Note that Slc26a6 mediated both Cl?/oxalate and Cl?/ HCO3 incongruous that Slc26a6 does not contribute to HCO3 tion, whereas oxalate secretion was markedly reduced in Slc26a6?/?mice. However, Slc26a6 is the dominant oxalate transporter in SMG acinar cells, whereas it is but one of several HCO3 responsetoIPRstimulation,theSMGsecretes?0.5mMoxalate in contrast to about 80 mM HCO3 tribution of Slc26a6-mediated Cl?/HCO3 HCO3 Na?/HCO3 ?and fluid secretion (13, 36), and that with the specific inhibitors T16Ainh-A01 (A01) and bumet- anide,respectively,whereasCl?/HCO3 eliminated with the carbonic anhydrase inhibitor ethoxzol- amide (EZA) under HCO3 No Cl?uptake was observed in the absence of oxalate in Slc26a6?/?SMG acinar cells, confirming that Cl?uptake under these conditions represents Cl?/oxalate exchange activ- ity (Fig. 5A). Furthermore, Cl?uptake was abolished in Slc26a6?/?mice (Fig. 5B), indicating that Slc26a6 is the pri- mary mediator of Cl?/oxalate exchange across the plasma membrane of SMG acinar cells. The results shown in Fig. 5, A and B, are summarized in Fig. 5C. ?exchangeractivitywas by guest on May 4, 2020 ?secretion, we monitored the Cl?/HCO3 ?exchange ?-free conditions. ?exchange activity ?-dependent ?exchange, the kinetics and Slc26a6-dependentoxalatesecretioninsaliva The above results demonstrate that Slc26a6 is localized to the apical membrane of salivary gland acinar cells, where it exchanges Cl?for oxalate. We next tested whether Slc26a6 transport activity contributes to secretion of oxalate in saliva using the ex vivo mouse SMG. Submandibular glands were stimulatedbyperfusionwiththe?-adrenergicreceptoragonist isoproterenol(IPR,1?M)underoxalate-containing(1mM)and oxalate-free conditions. Fig. 6A shows that the salivary flow rate was unaffected by disruption of Slc26a6 (filled circles, Slc26a6?/?) or by the presence of oxalate in the perfusate. In contrast, the SMG of Slc26a6?/?mice secreted significantly less oxalate compared with Slc26a6?/?mice in the presence of oxalate,whereastherewasnodifferenceintheoxalateconcen- tration of saliva from Slc26a6?/?and Slc26a6?/?mice in the oxalate-free perfusion solution (Fig. 6B). A summary of the net oxalate transport (oxalate concentration in oxalate-containing ?concentration of saliva was comparable in ?exchange does not appreciably ?secretion by the SMG. ?exchange in SMG acinar cells. Consequently, it seems ?secre- ?transport pathways in salivary glands. Indeed, in ?. We speculate that the con- ?exchange to total ?secretion is likely to be inconsequential compared with ?cotransporters, HCO3 ?-permeant anion channels, 6262 J. Biol. Chem. (2018) 293(17) 6259–6268

  5. Slc26a6facilitatesoxalatesecretioninsaliva Downloaded from by guest on May 4, 2020 Figure5.Slc26a6mediatesCl?/oxalateexchangeinmouseSMGacinarcells.AcinarcellsisolatedfromSlc26a6?/?andSlc26a6?/?submandibularglands wereloadedwithSPQtomonitorCl?uptake.Cl?depletionwasinducedbyincubationinalowCl?solutionintheabsence(?Oxalate)orpresence(?Oxalate) of 25 mM oxalate, and then Cl?uptake was stimulated by reintroduction of high extracellular Cl?in the absence of oxalate. Experiments were performed in bicarbonate-free solutions containing ethoxyzolamide (30 ?M B?/EZA) to eliminate Cl?/HCO3 (Bumet,80?M)toinhibitTmem16aandNkcc1,respectively.A,oxalate-dependentCl?uptakewasobservedinSlc26a6?/?SMGacinarcells,butnoCl?uptake was observed when oxalate was absent (? Oxalate, open circles, n ? 15; ? Oxalate, closed circles, n ? 15). B, Cl?/oxalate exchange activity was absent in SMG acinarcellsfromSlc26a6?/?mice(?Oxalate,opencircles,n?14;?Oxalate,closedcircles,n?16).C,summaryoftheCl?uptakeexperimentsshowninAand B.Dataarepresentedasthemean?S.E.ofcellsisolatedfromatleastfourdifferentmicepergroup.Statisticalanalysiswasperformedusingunpairedttest;**, p ? 0.01. ?exchange and T16Ainh-01 (10 ?M A01) and bumetanide and the other Cl?/HCO3 gland acinar and duct cells, which both contribute to HCO3 secretion. ?exchangers expressed in salivary oxalate back, thus allowing a small concentration of oxalate to spur (drive up) chloride reabsorption (40). In the straight segment of the kidney proximal tubule (also known as the S3 segment), Slc26a6 predominantly functions in Cl?/HCO3 exchange mode (39). Intheapicalmembraneofpancreaticductcells,Slc26a6acts as a Cl?/HCO3 cally and functionally linked with the cAMP-activated Cl? channel Cftr to drive HCO3 immunofluorescent staining showed that Slc26a6 targets to the apical membrane of salivary gland acinar cells, and thus, Slc26a6 clearly does not co-localize with Cftr in salivary gland duct cells. Functionally, these results demonstrate that salivary ? ? Discussion The function of Slc26a6 in salivary glands is unknown, but Slc26a6 can function in multiple ion exchange modes, includ- ing Cl?/oxalate exchange and Cl?/HCO3 tissues,suchaskidneyproximaltubuleandsmallintestine(17). In the kidney proximal convoluted tubule, encompassing S1 and S2 segments, Slc26a6 predominantly functions in Cl?/ oxalate exchange mode (39), which, in parallel with SO4 oxalate exchange and Na?-SO4 ?exchanger, where it co-expresses and is physi- ?exchange in various ?and fluid secretion (13). However, 2?/ 2?cotransport, recycles the J. Biol. Chem. (2018) 293(17) 6259–6268 6263

  6. Slc26a6facilitatesoxalatesecretioninsaliva Downloaded from by guest on May 4, 2020 Figure 7. Acinar Cl?/HCO3 ducedHCO3 lization was induced by Cl?/HCO3 thepHindicatorBCECFbyshiftingfromahighCl?toalowCl?bathsolution in the presence of HCO3 B,summaryofthealkalizationratesfromthedatainA(B?)andinHCO3 (B?)solutions.ThealkalizationrateinthepresenceofHCO3 reduced in Slc26a6?/?SMG acinar cells (Slc26a6?/?, B? n ? 17; Slc26a6?/?, B? n ? 9), whereas alkalization was essentially eliminated under HCO3 conditions(Slc26a6?/?,B?n?16;Slc26a6?/?,B?n?11).C,anexvivoSMG was perfused with a 25 mM HCO3 the ?-adrenergic receptor agonist IPR (1.0 ?M). Flow rate and flow amount (microliters)werecomparableinSlc26a6?/?(n?22)andSlc26a6?/?(n?23) mice.D,bicarbonateconcentrationinsalivafromSlc26a6?/?andSlc26a6?/? mice were comparable (Slc26a6?/?, n ? 14; Slc26a6?/?, n ? 13). Unpaired Student’s t test was performed; *, p ? 0.05. Results are given as mean ? S.E. ?exchanger activity and isoproterenol-in- ?secretioninSMGsfromSlc26a6?/?mice.A,intracellularalka- ?exchange in SMG acinar cells loaded with Figure 6. Slc26a6 mediates oxalate secretion in response to ?-adrener- gic receptor stimulation. The ex vivo SMG was perfused in the presence (1 mM) or absence of oxalate and subsequently stimulated with the ?-adrener- gic receptor agonist IPR (1.0 ?M) to induce saliva secretion. A, flow rate and flow amount (microliters) were comparable in Slc26a6?/?(n ? 26) and Slc26a6?/?(n?23)mice.B,theoxalateconcentrationinsalivainducedinthe presence of oxalate was significantly less in Slc26a6?/?mice, whereas the oxalate concentration in the oxalate-free solution was comparable for Slc26a6?/?(n ? 17) and Slc26a6?/?(n ? 16) mice. C, net oxalate concentra- tion ? (oxalate concentration in oxalate-containing solution) minus (oxalate concentrationinoxalate-freesolution),subtractedfromconsecutivestimula- tionswithinanindividualgland.PairedStudent’sttestwasperformed;**,p? 0.001. Results are given as the mean ? S.E. ?(open circles, Slc26a6?/?; closed circles, Slc26a6?/?). ?-free ?wassignificantly ?-free ?- containing solution and stimulated with oxalate for luminal chloride (16). The net oxalate transport in the ileum favors oxalate secretion under baseline conditions (secretion ? absorption); deletion of Slc26a6 abrogates oxalate secretion, thus leaving the oxalate absorption pathway uncon- testedintheintestine,resultinginincreasedbloodoxalatecon- centration with subsequent enhanced urine oxalate excretion (16) and kidney oxalate stone formation (19). It has been sug- gested that urolithiasis and sialolithes may be linked (42, 43). Indeed, oxalate is secreted in human saliva (44) and is detected in sialolithes (25, 26). Accordingly, we found that Cl?/oxalate exchanger activity in the apical membrane of salivary gland acinarcellswaseliminatedinSlc26a6?/?mice.Consistentwith acinar Slc26a6 playing a major role in the secretion of salivary oxalate,theconcentrationofoxalateinthesalivaofSlc26a6?/? mice was markedly reduced. glands are quite different from the exocrine pancreas (21, 29). Indeed, Slc26a6 seems to contribute little, if at all, to either HCO3 SowhatisthefunctionofSlc26a6insalivaryglands?Therole of Slc26a6 in salivary glands appears to mimic its role in the ileum with respect to oxalate. Slc26a6 shows strong expression along the length of small intestine, including the duodenum, jejunum, and ileum (11). In the duodenum, Slc26a6 functions predominantly as a Cl?/HCO3 jejunum, Slc26a6, along with Slc26a3, plays critical roles in salt absorption, with Slc26a6 exhibiting enhanced activation by luminal fructose (41). In the ileum, Slc26a6 predominantly functions in Cl?/oxalate exchange and plays a critical role in systemic oxalate homeostasis by exchanging the intracellular ?or fluid secretion in salivary glands. ?exchanger (39), whereas in the 6264 J. Biol. Chem. (2018) 293(17) 6259–6268

  7. Slc26a6facilitatesoxalatesecretioninsaliva The functional importance of oxalate in saliva remains to be determined,butonepossibilityisthatinsolublecalciumoxalate crystals deposit on the surface of the teeth, where it protects tooth enamel from the acid formed by oral bacteria following carbohydrate ingestion. Consistent with this model, oxalate is found on tooth enamel in various animals (44), whereas oxalic acid applied to teeth forms crystals and acts as a sealant on the enamel surface of the teeth (45). In addition to its physiological roleontoothenamel,itisnoteworthythatoxalateisdetectable in salivary stones (25, 26), suggesting that, under some condi- tions, Slc26a6-mediated oxalate secretion may contribute to sialolithiasis. analyze the expression -fold change, and message levels were normalized to the abundance of ?-actin messages. The primer sets were as follows: Slc26a6 mRNA, exon 1–2, forward 5?-AGA- TCTTCCTTGCGTCTGC-3? and reverse 5?-GCCTTTCCA- CATGGTAGTCTC-3?, amplicon length 149 bp; Actb mRNA, exon3–4,forward5?-ACCTTCTACAATGAGCTGCG-3?and reverse 5?-CTGGATGGCTACGTACATGG-3?, length147bp.ThePCRprotocolwas1minat95 °C,40cyclesof 15 s at 95 °C, and 30 s at 65 °C. Amplified products were visu- alized in 1.6% agarose gels in TAE (Tris-acetate buffer and EDTA). qPCR reactions were repeated three times for each sampleandaveragedtodeterminetheexpression-foldchange. amplicon ProteinisolationandWesternblotanalysis The crude plasma membrane protein fraction was isolated essentially as described previously (46). Briefly, mouse PGs, SMGs, and SLGs were removed, minced with fine scissors, and incubated in basal medium Eagle (BME) containing 1.0 mg/ml collagenase 2 (Worthington) with two steps of 25-min incuba- tion at 37 °C. The dispersed cells were subsequently passed througha150-?mcellstrainer(ThermoFisher),centrifugedin a clinical tabletop centrifuge for 1 min, and resuspended in lysatebuffercontaining0.5%IgepalCA-630,10mMHEPES(pH 7.2forthePGandSMGandpH6.8fortheSLG),0.3 Msucrose, 2 mM EDTA, 0.2 mM EGTA, 1 mM PMSF, and phosphatase and protease inhibitor mixtures (Roche). Lysed cells were centri- fuged at 4000 ? g for 10 min, and the supernatant was centri- fuged at 22,000 ? g for 20 min. The pellet was resuspended in lysatebufferandcentrifugedat46,000 ?g.Theresultantpellet was resuspended in the lysate buffer as a crude plasma mem- brane fraction. All procedures were performed at 4 °C. CHO-K1 cells expressing Slc26a6 were scraped off dishes 20 h after the transfection and incubated for 30 min in radio- immune precipitation assay buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktails. The dissolved cells were spun at 14,000 ? g for 20 min at 4 °C, and the result- ant supernatant was used as whole-cell lysate. For Western blot analysis, the protein concentrations of the crude plasma membrane from salivary glands and whole-lysate from Slc26a6-transfected CHO-K1 cells were determined using the bicinchoninic acid method (Thermo Fisher Scien- tific), separated in 4–12% SDS-PAGE BisTris gels, and trans- ferred to a polyvinylidene difluoride membrane using the iBlot 2 Dry blotting system according to the manufacturer’s instruc- tions (Thermo Fisher Scientific). The transferred membrane was blocked for 1 h at 20 °C in 5% dry milk (Bio-Rad) in Tris- buffered saline supplemented with 0.1% Tween 20 (TTBS) and then incubated with mouse monoclonal anti-SLC26A6 anti- body (Santa Cruz Biotechnology) at 1:100 overnight at 4 °C or with peroxidase-conjugated mouse monoclonal anti-?-actin antibody (Sigma) at 1: 25,000 for 1 h at room temperature. Fol- lowing three washes with TTBS, the membrane was incubated with rabbit peroxidase-conjugated anti-mouse antibody at 1:1000 for 1 h at room temperature. The signal was developed using Femto SuperSignal according to the manufacturer’s instructions (Thermo Fisher Scientific), and images were acquired using ImageJ software. For the glycosidase treatment, N-linked glycosylated proteins were digested with PNGaseF Experimental procedures Materialsandanimals Gene targeting and genotyping protocols for Slc26a6?/? mice were as described previously (39). Slc26a6?/?mice were backcrossedontotheFVB/NJmousebackground(TheJackson Laboratory). Mice were housed in micro-isolator cages with ad libitum access to laboratory chow and water during 12-h light/ dark cycles. Slc26a6?/?littermates, or in some cases FVB/NJ mice (The Jackson Laboratory, stock no. 001800), were used as WT controls. Experiments were performed on approximately equal numbers of 2- to 4-month-old male and female mice. Mice were euthanized by inhalation of 100% CO2, followed bycervicaldislocation.Allanimalprocedureswereapproved by the Animal Care and Use Committee of the NIDCR, National Institutes of Health (ASP 16-802). Reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless oth- erwise indicated. Downloaded from by guest on May 4, 2020 RNAsequencinganalysis RNA sequencing data were acquired as described previously (12). In brief, the PG, SMG, and SLG were surgically removed from six adult mice. The total RNA of the 18 glands was extracted, followed by cDNA synthesis and fragmentation to make Illumina libraries. The libraries were applied to Illumina HiSeq2500 sequencing, and the 18 sample libraries each had ?40 million reads. The data were put out as fragments per kilobaseoftranscriptpermillionmappedreads(FPKM)values. ValidationofRNA-seqbyquantitativePCR(qPCR) The PG, SMG, and SLG from Slc26a6?/?and Slc26a6?/? littermates(threemalesandthreefemalesforeachgroup,8–12 weeks old) were removed with the aid of a dissecting micro- scope, immediately frozen in liquid nitrogen, and stored at ?80 °C until shipping to MyOmicsDx (Towson, MD) for pro- cessing. Total RNA from each tissue was isolated using the MyGeneTM-RNA-EX kit, and 1 ?g was reverse-transcribed to synthesizecDNA(MyGeneTM-RNA-RTkit).Fornegativecon- trols, all components, except the reverse transcriptase, were included in the reaction mixtures. Real-time PCR was per- formed using the Bio-Rad CFX96 TouchTMreal-time PCR detection system with specific primers and the MyGeneTM qPCR master kit. PCR specificity was verified by the dissocia- tioncurvefromasinglepeak,whichwasrunfollowingthereal- time PCR reaction. The 2(???Cq)(Livak) method was used to J. Biol. Chem. (2018) 293(17) 6259–6268 6265

  8. Slc26a6facilitatesoxalatesecretioninsaliva according to the manufacturer’s denaturing protocol (New England Bio-Rad). CMOS camera (Hamamatsu, C11440). SPQ fluorescence was excited at 340 nm, and emissions were collected at 510 nm. Anion exchanger activity was monitored in GFP-positive CHO-K1cellsandisolatedSMGacinarcellsusingthefollowing solutions: for Cl?/oxalate exchanger activity, oxalate-contain- ing,highCl?solutioncomposedof4.3mMKCl,95mMNaCl,27 mMNMDG-Cl,5mMglucose,10mMHEPES,1mMMgCl2,and 25 mM Na2oxalate; and oxalate-containing, low Cl?solution composed of 4.3 mM potassium gluconate, 95 mM sodium gluconate, 2 mM NMDG-Cl, 25 mM mannitol, 5 mM glucose, 10 mM HEPES, 1 mM MgCl2, and 25 mM Na2oxalate. The solution pH and osmolality were adjusted to 7.4 and 300–310 mOsm H2O/kg with UltraPure Tris (Invitrogen) and sucrose, respectively. Mouse salivary gland acinar cells express three distinct types ofCl?fluxpathways,includingTmem16aCa2?-activatedchlo- ride channels, Nkcc1 Na?/K?/2Cl?cotransporters, and the anion exchangers Ae2, Ae4, and Slc26a6. Consequently, the following strategy was designed to isolate Slc26a6-mediated Cl?/oxalate anion exchange in isolated acinar cells. First, SPQ- loaded acinar cells were simultaneously depleted of intracellu- lar Cl?and loaded with oxalate by exposure to a low Cl?(4 mM),oxalate-containing(25mM)solutionandthenreturnedto a high Cl?(153.3 mM), oxalate-free solution (Na2oxalate was replaced with NaCl) to induce Cl?/oxalate anion exchange in the presence of 10 ?M T16Ainh-A01, 80 ?M bumetanide, and 30 ?M EZA to inhibit Tmem16a, Nkcc1, and carbonic anhy- drases, respectively. All solutions were HCO3 with 100% O2to prevent Cl?/HCO3 The intracellular pH of isolated SMG acinar cells was moni- tored as described previously (35). Briefly, cells were incubated with 2 ?M BCECF/AM for 15 min at 37 °C in an incubator gassed with 95% O2and 5% CO2. Cl?/HCO3 was induced in BCECF-loaded SMG cells by exposure to a low Cl?solution.Solutionswereasfollows.HighCl?solution(B?) contained 4.3 mM KCl, 120 mM NaCl, 25 mM NaHCO3 glucose,10mMHEPES,1mMCaCl2,and1mMMgCl2.LowCl? solution contained 4.3 mM potassium gluconate, 120 mM sodium gluconate, 25 mM NaHCO3 HEPES,1mMCaCl2,and1mMMgCl2.HCO3 tions were gassed with 95% O2, 5% CO2for at least 30 min. The HCO3 were made by substituting NaHCO3 conate, respectively. The HCO3 with100%O2.SolutionshadapHlevelof7.4.Theinitialratesof Cl?uptake and intracellular alkalization were estimated from thelinearportionofthe[Cl?]iandpHiincreasesdividedbythe corresponding time periods (?40 s) and expressed as the Cl? uptake and alkalization rates (10?3s?1), respectively. Immunohistochemistry PGs, SMGs, and SLGs from Slc26a6?/?and Slc26a6?/? mice were fixed with 4% paraformaldehyde and paraffin-em- bedded as described previously (47). The 5-?m-thick sections were deparaffinized and rehydrated and, before immuno- staining, underwent antigen retrieval in Tris-EDTA–buffered saline(pH9.0)for10minwithapressurecooker.Sectionswere blocked in 20% donkey serum for 30 min at room temperature andincubatedwithrabbitanti-Slc26a6antibody(11)at1:50,rat anti-Cftr antibody (CFTR Folding Consortium) at 1:100, and goat anti-NKCC1 antibody (Santa Cruz Biotechnology) at 1:100 overnight at 4 °C. For the secondary antibody, donkey anti-rabbit (Alexa 647,), donkey anti-rat (Alexa 488), and don- keyanti-goat(Alexa555)wereappliedin1:200,1:200,and1:400 dilutions, respectively (Invitrogen). Slides were mounted with FluoroshieldTMcontaining4?,6-diamidino-2-phenylindoleand visualized on a Nikon A1R? confocal microscope with a Plan Fluor ?40/1.3 numerical aperture oil immersion objective. Downloaded from Cellcultureandtransfections CHO-K1cells(Sigma-Aldrich)weremaintainedasdescribed previously (48). Plasmids encoding Mus musculus Slc26a6 (GenBank accession no. NM_134420), Slc4a2 (Ae2, GenBank accessionno.BC_054102),Slc4a9(Ae4,GenBankaccessionno. NM-172830.2), and pCMV-Entry as an empty vector control were obtained from OriGene. Cells were electroporated (Nucleofector II, Amaxa) with 6 ?g of each plasmid along with 6 ?g of pmax GFP (Amaxa) using Nucleofector kit V (Lonza) according to the manufacturer’s instructions and seeded onto 5-mm-diameter coverslips (Warner Instrument). Exchanger activity was determined 18–20 h after electroporation in cells that co-expressed GFP signal. ?-free and gassed ?exchanger activity. by guest on May 4, 2020 ?exchange activity ?, 5 mM Mousesubmandibularglandacinarcellisolation Submandibular glands were surgically removed and enzy- matically dispersed as before (47). Briefly, cells were incubated inBMEcontaining1.0mg/mlcollagenase2(Worthington)and digested by two steps of 25-min incubation each at 37 °C with continuous gassing with 95% O2and 5% CO2. ?, 5 mM glucose, 10 mM ?-containingsolu- ?-free,highCl?andHCO3 ?-free,lowCl?-solutions(B?) ?with NaCl or sodium glu- ?-free solutions were gassed IntracellularCl?andpHmeasurements SalivaryglandacinarcellswereloadedwiththeCl?-sensitive dye 6-methoxy-N-(3-sulfopropyl) quinolimium (SPQ, 5 mM) for 25 min during the second collagenase digestion step. After dispersion and dye loading, acinar cells were rinsed and resus- pended in 5 ml of BME at 37 °C. Plasmid-transfected CHO-K1 cellswereloadedwithSPQusingahypotonicloadingbuffer,as describedpreviously(48).Inbrief,cellsattachedtoacoverglass wereincubatedina150mOsmhypotonicsolutioncontaining5 mM SPQ for 4 min at 23 °C. CellsloadedwiththeCl?-sensitivedyeSPQweretransferred to a perfusion chamber mounted on the stage of an inverted microscope (Nikon, Eclipse TE 300) equipped with an Opto- scanImagingSystem(CAIRNInstruments)coupledtoadigital Exvivoperfusionofsubmandibularglands Mice were anesthetized by intraperitoneal injection of chlo- ral hydrate (400 mg/kg of body weight), and the SMG was sur- gically removed for ex vivo perfusion as described previously (49). Briefly, the isolated SMG was transferred to a perfusion chamber and perfused at 37 °C through the common carotid artery with experimental solutions. Salivation was induced by addition of a ?-adrenergic agonist (isoproterenol, 1.0 ?M), and 6266 J. Biol. Chem. (2018) 293(17) 6259–6268

  9. Slc26a6facilitatesoxalatesecretioninsaliva the flow rate and total amount of collected saliva were mea- sured by recording the progression of saliva through a capillary tube at 1-min intervals. The secreted saliva was stored at ?20 °C until further analysis. The bicarbonate-containing per- fusion solution was composed of 4.3 mM KCl, 120 mM NaCl, 25 mMNaHCO3 mM MgCl2(pH 7.4, gassed with 95% O2/5% CO2). The oxalate- containing solution was the same as the bicarbonate-contain- ing solution with the addition of 1 mM Na2Oxalate. 3. Whittamore,J.M.,andHatch,M.(2017)LossoftheanionexchangerDRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis. Am. J. Physiol. Gastrointest. Liver Physiol. 313, G166–G179 CrossRef Medline 4. Xie, Q., Welch, R., Mercado, A., Romero, M. F., and Mount, D. B. (2002) Molecular characterization of the murine Slc26a6 anion exchanger: func- tional comparison with Slc26a1. Am. J. Physiol. Renal Physiol. 283, F826–F838 CrossRef Medline 5. Knauf, F., Yang, C. L., Thomson, R. B., Mentone, S. A., Giebisch, G., and Aronson, P. S. (2001) Identification of a chloride-formate exchanger ex- pressed on the brush border membrane of renal proximal tubule cells. Proc. Natl. Acad. Sci. U.S.A. 98, 9425–9430 CrossRef Medline 6. Chernova,M.N.,Jiang,L.,Friedman,D.J.,Darman,R.B.,Lohi,H.,Kere,J., Vandorpe, D. H., and Alper, S. L. (2005) Functional comparison of mouse slc26a6 anion exchanger with human SLC26A6 polypeptide variants: dif- ferencesinanionselectivity,regulation,andelectrogenicity.J.Biol.Chem. 280, 8564–8580 CrossRef Medline 7. Ko, S. B., Zeng, W., Dorwart, M. R., Luo, X., Kim, K. H., Millen, L., Goto, H., Naruse, S., Soyombo, A., Thomas, P. J., and Muallem, S. (2004) Gating of CFTR by the STAS domain of SLC26 transporters. Nat. Cell Biol. 6, 343–350 CrossRef Medline 8. Ohana,E.,Shcheynikov,N.,Moe,O.W.,andMuallem,S.(2013)SLC26A6 and NaDC-1 transporters interact to regulate oxalate and citrate homeo- stasis. J. Am. Soc. Nephrol. 24, 1617–1626 CrossRef Medline 9. Dorwart, M. R., Shcheynikov, N., Yang, D., and Muallem, S. (2008) The solute carrier 26 family of proteins in epithelial ion transport. Physiology 23, 104–114 CrossRef Medline 10. Lohi, H., Lamprecht, G., Markovich, D., Heil, A., Kujala, M., Seidler, U., and Kere, J. (2003) Isoforms of SLC26A6 mediate anion transport and havefunctionalPDZinteractiondomains.Am.J.Physiol.CellPhysiol.284, C769–C779 CrossRef Medline 11. Wang,Z.,Petrovic,S.,Mann,E.,andSoleimani,M.(2002)Identificationof an apical Cl?/HCO3 Gastrointest. Liver Physiol. 282, G573–G579 CrossRef Medline 12. Gao, X., Oei, M. S., Ovitt, C. E., Sincan, M., and Melvin, J. E. (2018) Tran- scriptional profiling reveals gland-specific differential expression in the three major salivary glands of the adult mouse. Physiol. 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Corbetta, S., Eller-Vainicher, C., Frigerio, M., Valaperta, R., Costa, E., Vi- centini,L.,Baccarelli,A.,Beck-Peccoz,P.,andSpada,A.(2009)Analysisof the 206M polymorphic variant of the SLC26A6 gene encoding a Cl?ox- alate transporter in patients with primary hyperparathyroidism. Eur. J. Endocrinol. 160, 283–288 Medline 19. Jiang,Z.,Asplin,J.R.,Evan,A.P.,Rajendran,V.M.,Velazquez,H.,Nottoli, T. P., Binder, H. J., and Aronson, P. S. (2006) Calcium oxalate urolithiasis in mice lacking anion transporter Slc26a6. Nat. Genet. 38, 474–478 CrossRef Medline 20. Monico, C. G., Weinstein, A., Jiang, Z., Rohlinger, A. L., Cogal, A. G., Bjornson, B. B., Olson, J. B., Bergstralh, E. J., Milliner, D. S., and Aronson, P. S. (2008) Phenotypic and functional analysis of human SLC26A6 vari- ants in patients with familial hyperoxaluria and calcium oxalate nephroli- thiasis. Am. J. Kidney Dis. 52, 1096–1103 CrossRef Medline ?,5mMglucose,10mMHEPES,1mMCaCl2,and1 Ioncompositionofsaliva The HCO3 colorimetric assay as described by the manufacturer (Diazyme Laboratories, DZ122A-K). Oxalate concentration in saliva was measured using a colorimetric oxalate assay kit (Abnova) and plate reader (Bio-Rad, model 680). The colorimetric oxalate assay results were confirmed by ion exchange chromatography (Proteomics and Mass Spectrometry Facility, Danforth Plant Science Center). ?concentration in saliva was determined using a Statisticalanalysis Results are presented as the mean ? S.E. Statistical signifi- cance was determined using Student’s t test or one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons (Origin 7.0 Software, OriginLab, Northampton, MA). p values of less than 0.05 were considered statistically significant. Experiments were performed using preparations fromthreeormoremiceforeachcondition.ForRNAsequenc- ing analysis (12), the FPKM values acquired from the three major salivary glands were compared using one-way ANOVA followedbyBonferroni’sposthoctest,andp?0.05wasconsid- ered to be statistically significantly different. Downloaded from ?exchanger in the small intestine. Am. J. Physiol. by guest on May 4, 2020 Author contributions—T. Mukaibo, A. T. G., D. T. T., X. G., C. M., G. E. S., M. S., and J. E. M. conceptualization; T. Mukaibo, T. Mun- emasa, A. T. G., D. T. T., X. G., and J. E. M. formal analysis; T. MukaiboandJ. E. M.validation;T.Mukaibo,T.Munemasa,A. T. G., D. T. T., J. H., G. E. S., M. S., and J. E. M. investigation; T. Mukaibo, T. Munemasa, D. T. T., X. G., and J. E. M. visualization; T. Mukaibo, T. Munemasa, A. T. G., D. T. T., X. G., J. H., C. M., G. E. S., M. S., and J. E. M. methodology; T. Mukaibo, X. G., and J. E. M. writing- original draft; T. Mukaibo and J. E. M. project administration; T. Mukaibo,T.Munemasa,A. T. G.,D. T. T.,X. G.,J. H.,C. M.,G. E. S., M. S., and J. E. M. writing-review and editing; X. G. and J. E. M. data curation; X. G. software; J. E. M. resources; J. E. M. supervision; J. E. M. funding acquisition. ?secretion:relevanceto Acknowledgments—WethankYasnaJaramilloandJaideepHonavar for technical assistance and Dr. Keitaro Satoh for generous advice regarding protein isolation. The Veterinary Research Core was sup- ported by Core Grant 1-ZIG-DE000740 from the NIDCR, National Institutes of Health. References 1. Alper,S.L.,andSharma,A.K.(2013)TheSLC26genefamilyofaniontrans- porters and channels. Mol. Aspects Med. 34, 494–515 CrossRefCrossRef MedlineMedline 2. Jiang, Z., Grichtchenko, I. I., Boron, W. F., and Aronson, P. S. (2002) Specificity of anion exchange mediated by mouse Slc26a6. J. Biol. Chem. 277, 33963–33967 CrossRef Medline J. Biol. Chem. (2018) 293(17) 6259–6268 6267

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  11. The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells Taro Mukaibo, Takashi Munemasa, Alvin T. George, Duy T. Tran, Xin Gao, Jesse L. Herche, Chihiro Masaki, Gary E. Shull, Manoocher Soleimani and James E. Melvin J. Biol. Chem. 2018, 293:6259-6268. doi: 10.1074/jbc.RA118.002378 originally published online March 12, 2018 Access the most updated version of this article at doi: 10.1074/jbc.RA118.002378 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Downloaded from This article cites 49 references, 11 of which can be accessed free at by guest on May 4, 2020