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

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3− and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little 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−, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl−/oxalate exchange. Of note, both Cl−/oxalate exchange and Cl−/HCO3− exchange were significantly 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 acinar cells, where it mediates Cl−/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

SLC26A6 has cytoplasmic N and C termini that flank an integral membrane domain containing 10 to 14 transmembrane segments as well as a sulfate transporter and anti-factor antagonist (STAS) 4 domain located in the C terminus. The STAS domain interacts with other ion transport proteins to form transport metabolons (1, 6 -8). One proposed example of such an interaction is the mutual activation of CFTR and SLC26A6 in pancreatic duct cells, which depends on the physical association of the STAS domain of SLC26A6 with the R domain of CFTR (7). CFTR and SLC26A6 activation is apparently enhanced by protein kinase A (PKA)-mediated phosphorylation of the R domain (9). SLC26A6 expression is ubiquitous, but it is highly expressed in the pancreas, small intestine, and kidney (10,11). Transcriptional profiling also found that Slc26a6 is highly expressed in murine salivary glands (12). Targeted disruption of mouse Slc26a6 inhibited HCO 3 Ϫ and fluid secretion in the pancreas (13), whereas oxalate secretion in the small intestine and reabsorption by the kidney were markedly reduced (14 -17). These results suggest that Slc26a6 may play several crucial roles in mammalian physiology, e.g. secretion of fluid and HCO 3 Ϫ by the pancreas to neutralize stomach acid and secretion of oxalate by the intestine to regulate plasma oxalate levels and prevent kidney stone formation (15, 18 -20).
The function of Slc26a6 in salivary glands is unclear, although it has been suggested that Slc26a6 contributes to fluid and HCO 3 Ϫ secretion, as demonstrated in the pancreas (21)(22)(23)(24). Alternatively, Slc26a6 in salivary glands may play a role in the secretion of oxalate, as in the small intestine (16). In fact, oxalate 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 predominantly as a Cl Ϫ /HCO 3 Ϫ exchanger and contributes to HCO 3 Ϫ secretion and/or Slc26a6 largely functions in Cl Ϫ /oxalate exchange mode and thus plays a critical role in oxalate secretion. Our results suggest, in contrast to the pancreas, that Slc26a6 does not target to the apical membrane of duct cells, nor does it promote HCO 3 Ϫ secretion. Instead, Slc26a6 localizes to the apical membrane of salivary gland acinar cells, where it appears to play a major role in oxalate secretion.

Slc26a6 mRNA is expressed in murine salivary glands
RNA-seq data were acquired and described previously (12), and the full data sets were deposited in the Gene Expression Omnibus (GEO accession number GSE96747). Further analysis of these data revealed Slc26a6 transcript expression in the major salivary glands, the parotid gland (PG), submandibular gland (SMG), and sublingual (SLG) gland. SLG Slc26a6 expression exceeded the levels found in the PG and SMG by ϳ33and 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 Ϯ S.E. ϭ 403.5 Ϯ 27.3, 503.5 Ϯ 93.3, and 553.3 Ϯ 82.6, respectively; n ϭ 6 for each gland). This was also confirmed by qPCR analysis, where the Cq values for ␤-actin were 20.8 Ϯ 0.5, 19.4 Ϯ 0.6, and 19.1 Ϯ 0.3 for the PG, SMG, and SLG, respectively (n ϭ 6 for each gland).

Slc26a6 is modified by N-linked glycosylation
To examine Slc26a6 protein expression across the three major murine salivary glands, 40 g of plasma membrane protein 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 ϳ39and 7-fold higher than in the PG and SMG, respectively (Fig. 2, A  and B). Little Slc26a6 protein was detectable above background 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 ϳ10 -15 kDa smaller compared with the plasma membrane protein isolated expression acquired by RNA-seq analysis for mouse PGs, SMGs, and SLGs are displayed as FPKM per 40 million mapped reads. Data from individual glands are displayed as circles (n ϭ 6 for PG, SMG, and SLG). B, Slc26a6 mRNA expression levels were confirmed by qPCR and normalized to ␤-actin (Actb) (n ϭ 6 for PG, SMG, and SLG). C, PCR band size and product amount for slc26a6 (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 Ͻ 0.01.

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) to Slc26a6 and ␤-actin. To more clearly visualize Slc26a6 protein 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 ϭ 4). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; *, p Ͻ 0.002, **, p Ͻ 0.01. C, SMG (40 g) and SLG (20 g) plasma membrane proteins from Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice and from Slc26a6-transfected CHO cells (20 g) were treated without (Ϫ) or with (ϩ) PNGaseF (PNG). The same membrane as in A was stripped and immunoblotted with an anti-␤-actin antibody (n ϭ 3).

Slc26a6 facilitates oxalate secretion in saliva
from the SMG and from Slc26a6 cDNA-transfected CHO cells (Fig. 2C). It was reported previously that human and mouse SLC26A6 are heavily N-glycosylated in a tissue-specific manner (27). Correspondingly, treatment with the N-linkage-specific glycosidase PNGaseF resulted in a shift to a similar molecular mass of ϳ70 -80 kDa in SMG, SLG and Slc26a6 pcDNA transfected CHO cells, suggesting that the smaller apparent size of Slc26a6 in the SLG is caused by less N-glycosylation modification (Fig. 2C) and not gland-specific differences in alternative splicing of the Slc26a6 transcript. Indeed, a comparison of the salivary gland transcriptomes revealed that the 18 exons comprising the Slc26a6 gene, including predicted N-glycosylation sites at asparagine 151 and 303 (http://www.cbs.dtu.dk/services/ NetNGlyc/), 5 are expressed in the PG, SMG, and SLG (12). Note that the predicted unmodified protein molecular mass of mouse Slc26a6 is ϳ83 kDa (UniportKB Q8CIW6-1).
Furthermore, comparison of SMG and SLG transcriptomes failed to detect major differences in the expression of genes involved in N-glycosylation of Slc26a6 (28), e.g. the 12 members of the Alg gene family of glucosyltransferases, except for Alg8. Although called significantly different by bioinformatics analysis (Cufflinks/Cuffdiff 2.2.1), Alg8 expression in the SMG was only about 40% less than that detected in the SLG (FPKM ϭ 5.76 and 9.79, respectively; q Ͻ 0.026; n ϭ 6 for each gland type). 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 differences in expression of genes that add other moieties to the oligosaccharide structure and/or signaling pathways that regulate translation of Alg mRNAs and glucosyltransferase activity.

Slc26a6 localizes to the apical membrane of salivary gland acinar cells
Previous reports suggest that Slc26a6 is localized to the apical 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 HCO 3 Ϫ secretion and intestinal and renal oxalate transport (20,32,33). Thus, immunohistochemistry was performed to better understand the functional targeting of Slc26a6 in the SMG, SLG, and PG of WT mice (Slc26a6 ϩ/ϩ ). Strong immunostaining 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 negative in Slc26a6 Ϫ/Ϫ mice, verifying the specificity of the anti-Slc26a6 antibody (Fig. 3, C and F), whereas Nkcc1 staining (Fig.  3, filled red arrow) was unaffected in the basolateral membranes of acinar cells in Slc26a6 Ϫ/Ϫ mice.

Slc26a6 exchanges Cl ؊ for oxalate in transfected CHO-K1 cells
Slc26a6 (also known as CFEX, chloride/formate exchanger) was initially reported to exchange Cl Ϫ for formate (5), but Slc26a6 was subsequently recognized to also transport other anions, such as oxalate and HCO 3 Ϫ (4). The above results demonstrated that Slc26a6 is exclusively expressed in salivary gland acinar cells; however, these cells also express the anion exchangers Ae2 (Slc4a2) and Ae4 (Slc4a9) (34,35), which complicates the interpretation of studies to quantify Slc26a6-mediated anion exchange. Consequently, to verify that Slc26a6 oxalate transport activity can be isolated in salivary gland acinar cells, mouse Slc26a6, Slc4a2, and Slc4a9 cDNAs were expressed in CHO-K1 cells, and Cl Ϫ /oxalate exchange was monitored.

Slc26a6 mediates Cl ؊ /oxalate exchange in salivary gland acinar cells
Mouse SMG acinar cells were treated as above (Fig. 4) to induce Slc26a6-mediated Cl Ϫ /oxalate anion exchange. To further isolate the Slc26a6-mediated Cl Ϫ /oxalate exchange activity, Cl Ϫ fluxes via the Ca 2ϩ -activated anion channel Tmem16A and the Na ϩ /K ϩ /2Cl Ϫ cotransporter Nkcc1 were prevented 5 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party-hosted site.

Slc26a6 facilitates oxalate secretion in saliva
with the specific inhibitors T16Ainh-A01 (A01) and bumetanide, respectively, whereas Cl Ϫ /HCO 3 Ϫ exchanger activity was eliminated with the carbonic anhydrase inhibitor ethoxzolamide (EZA) under HCO 3 Ϫ -free conditions. No Cl Ϫ uptake was observed in the absence of oxalate in Slc26a6 ϩ/ϩ SMG acinar cells, confirming that Cl Ϫ uptake under these conditions represents Cl Ϫ /oxalate exchange activity (Fig. 5A). Furthermore, Cl Ϫ uptake was abolished in Slc26a6 Ϫ/Ϫ mice (Fig. 5B), indicating that Slc26a6 is the primary mediator of Cl Ϫ /oxalate exchange across the plasma membrane of SMG acinar cells. The results shown in Fig. 5, A and B, are summarized in Fig. 5C.

Slc26a6-dependent oxalate secretion in saliva
The above results demonstrate that Slc26a6 is localized to the apical membrane of salivary gland acinar cells, where it exchanges Cl Ϫ 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 stimulated by perfusion with the ␤-adrenergic receptor agonist isoproterenol (IPR, 1 M) under oxalate-containing (1 mM) and oxalate-free conditions. Fig. 6A shows that the salivary flow rate was unaffected by disruption of Slc26a6 (filled circles, Slc26a6 Ϫ/Ϫ ) or by the presence of oxalate in the perfusate. In contrast, the SMG of Slc26a6 Ϫ/Ϫ mice secreted significantly less oxalate compared with Slc26a6 ϩ/ϩ mice in the presence of oxalate, whereas there was no difference in the oxalate concentration of saliva from Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice in the oxalate-free perfusion solution (Fig. 6B). A summary of the net oxalate transport (oxalate concentration in oxalate-containing solution minus oxalate-free solution) for each gland is shown in 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 oxalate 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 saliva samples collected from Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice were then assayed for oxalate content by both assay methods (Slc26a6 ϩ/ϩ , colorimetric ϭ 0.21 Ϯ 0.01 mM, ion exchange chromatography ϭ 0.19 Ϯ 0.02 mM; Slc26a6 Ϫ/Ϫ , colorimetric ϭ 0.17 Ϯ 0.04 mM, ion exchange chromatography ϭ 0.21 Ϯ 0.02 mM; n ϭ 8 for each group). There were no significant differences between assay methods or animal groups. Note that this experiment was performed in the absence of oxalate, indicating that salivary glands secrete endogenously generated oxalate. Moreover, there was no difference between Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice, suggesting the existence of additional unknown oxalate transport pathways.

Slc26a6 Disruption fails to alter HCO 3 ؊ secretion
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 HCO 3 Ϫ and fluid secretion (13,36), and that 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 HCO 3 Ϫ secretion, we monitored the Cl Ϫ /HCO 3 Ϫ exchange activity of SMG acinar cells loaded with the pH-sensitive indicator BCECF. Fig. 7A shows that Cl Ϫ /HCO 3 Ϫ exchange activity was significantly reduced in acinar cells from Slc26a6 Ϫ/Ϫ mice. Fig. 7B summarizes the alkalization rates for the results shown in Fig. 7A (Bϩ) and also demonstrates that the alkalization induced by removal of extracellular Cl Ϫ was HCO 3 Ϫ -dependent (BϪ). In contrast to Cl Ϫ /HCO 3 Ϫ exchange, the kinetics and magnitude of fluid secretion in response to the ␤-adrenergic receptor agonist (1 M isoproterenol, IPR) were not altered in the SMG of Slc26a6 Ϫ/Ϫ mice (Fig. 7C, similar to Fig. 6A). Moreover, the HCO 3 Ϫ concentration of saliva was comparable in Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice (Fig. 7D), suggesting that Slc26a6-mediated Cl Ϫ /HCO 3 Ϫ exchange does not appreciably contribute to HCO 3 Ϫ secretion by the SMG. Note that Slc26a6 mediated both Cl Ϫ /oxalate and Cl Ϫ / HCO 3 Ϫ exchange in SMG acinar cells. Consequently, it seems incongruous that Slc26a6 does not contribute to HCO 3 Ϫ secretion, whereas oxalate secretion was markedly reduced in Slc26a6 Ϫ/Ϫ mice. However, Slc26a6 is the dominant oxalate transporter in SMG acinar cells, whereas it is but one of several HCO 3 Ϫ transport pathways in salivary glands. Indeed, in response to IPR stimulation, the SMG secretes Յ0.5 mM oxalate in contrast to about 80 mM HCO 3 Ϫ . We speculate that the contribution of Slc26a6-mediated Cl Ϫ /HCO 3 Ϫ exchange to total HCO 3 Ϫ secretion is likely to be inconsequential compared with Na ϩ /HCO 3 Ϫ cotransporters, HCO 3 Ϫ -permeant anion channels,

Slc26a6 facilitates oxalate secretion in saliva
and the other Cl Ϫ /HCO 3 Ϫ exchangers expressed in salivary gland acinar and duct cells, which both contribute to HCO 3 Ϫ secretion.

Discussion
The function of Slc26a6 in salivary glands is unknown, but Slc26a6 can function in multiple ion exchange modes, including Cl Ϫ /oxalate exchange and Cl Ϫ /HCO 3 Ϫ exchange in various tissues, such as kidney proximal tubule and small intestine (17). In the kidney proximal convoluted tubule, encompassing S1 and S2 segments, Slc26a6 predominantly functions in Cl Ϫ / oxalate exchange mode (39), which, in parallel with SO 4 2Ϫ / oxalate exchange and Na ϩ -SO 4 2Ϫ cotransport, recycles the 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 Ϫ /HCO 3 Ϫ exchange mode (39).
In the apical membrane of pancreatic duct cells, Slc26a6 acts as a Cl Ϫ /HCO 3 Ϫ exchanger, where it co-expresses and is physically and functionally linked with the cAMP-activated Cl Ϫ channel Cftr to drive HCO 3 Ϫ and fluid secretion (13). However, 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

Slc26a6 facilitates oxalate secretion in saliva
glands are quite different from the exocrine pancreas (21,29). Indeed, Slc26a6 seems to contribute little, if at all, to either HCO 3 Ϫ or fluid secretion in salivary glands. So what is the function of Slc26a6 in salivary glands? The role 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 Ϫ /HCO 3 Ϫ exchanger (39), whereas in the 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 Ϫ /oxalate exchange and plays a critical role in systemic oxalate homeostasis by exchanging the intracellular oxalate for luminal chloride (16). The net oxalate transport in the ileum favors oxalate secretion under baseline conditions (secretion Ͼ absorption); deletion of Slc26a6 abrogates oxalate secretion, thus leaving the oxalate absorption pathway uncontested in the intestine, resulting in increased blood oxalate concentration with subsequent enhanced urine oxalate excretion (16) and kidney oxalate stone formation (19). It has been suggested 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 Ϫ /oxalate exchanger activity in the apical membrane of salivary gland acinar cells was eliminated in Slc26a6 Ϫ/Ϫ mice. Consistent with acinar Slc26a6 playing a major role in the secretion of salivary oxalate, the concentration of oxalate in the saliva of Slc26a6 Ϫ/Ϫ mice was markedly reduced.  Ϫ (open circles, Slc26a6 ϩ/ϩ ; closed circles, Slc26a6 Ϫ/Ϫ ). B, summary of the alkalization rates from the data in A (Bϩ) and in HCO 3 Ϫ -free (BϪ) solutions. The alkalization rate in the presence of HCO 3 Ϫ was significantly reduced in Slc26a6 Ϫ/Ϫ SMG acinar cells (Slc26a6 ϩ/ϩ , Bϩ n ϭ 17; Slc26a6 Ϫ/Ϫ , Bϩ n ϭ 9), whereas alkalization was essentially eliminated under HCO 3 Ϫ -free conditions (Slc26a6 ϩ/ϩ , BϪ n ϭ 16; Slc26a6 Ϫ/Ϫ , BϪ n ϭ 11). C, an ex vivo SMG was perfused with a 25 mM HCO 3 Ϫ -containing solution and stimulated with the ␤-adrenergic receptor agonist IPR (1.0 M). Flow rate and flow amount (microliters) were comparable in Slc26a6 ϩ/ϩ (n ϭ 22) and Slc26a6 Ϫ/Ϫ (n ϭ 23) mice. D, bicarbonate concentration in saliva from Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ mice were comparable (Slc26a6 ϩ/ϩ , n ϭ 14; Slc26a6 Ϫ/Ϫ , n ϭ 13). Unpaired Student's t test was performed; *, p Ͻ 0.05. Results are given as mean Ϯ S.E.

Slc26a6 facilitates oxalate secretion in saliva
The functional importance of oxalate in saliva remains to be determined, but one possibility is that insoluble calcium oxalate 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 role on tooth enamel, it is noteworthy that oxalate is detectable in salivary stones (25,26), suggesting that, under some conditions, Slc26a6-mediated oxalate secretion may contribute to sialolithiasis.

Materials and animals
Gene targeting and genotyping protocols for Slc26a6 Ϫ/Ϫ mice were as described previously (39). Slc26a6 Ϫ/Ϫ mice were backcrossed onto the FVB/NJ mouse background (The Jackson Laboratory). Mice were housed in micro-isolator cages with ad libitum access to laboratory chow and water during 12-h light/ dark cycles. Slc26a6 ϩ/ϩ 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% CO 2 , followed by cervical dislocation. All animal procedures were approved 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 otherwise indicated.

RNA sequencing analysis
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 ϳ40 million reads. The data were put out as fragments per kilobase of transcript per million mapped reads (FPKM) values.

Validation of RNA-seq by quantitative PCR (qPCR)
The PG, SMG, and SLG from Slc26a6 ϩ/ϩ and Slc26a6 Ϫ/Ϫ littermates (three males and three females for each group, 8 -12 weeks old) were removed with the aid of a dissecting microscope, immediately frozen in liquid nitrogen, and stored at Ϫ80°C until shipping to MyOmicsDx (Towson, MD) for processing. Total RNA from each tissue was isolated using the MyGene TM -RNA-EX kit, and 1 g was reverse-transcribed to synthesize cDNA (MyGene TM -RNA-RT kit). For negative controls, all components, except the reverse transcriptase, were included in the reaction mixtures. Real-time PCR was performed using the Bio-Rad CFX96 Touch TM real-time PCR detection system with specific primers and the MyGene TM qPCR master kit. PCR specificity was verified by the dissociation curve from a single peak, which was run following the realtime PCR reaction. The 2 (Ϫ⌬⌬Cq) (Livak) method was used to 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 5Ј-AGA-TCTTCCTTGCGTCTGC-3Ј and reverse 5Ј-GCCTTTCCA-CATGGTAGTCTC-3Ј, amplicon length 149 bp; Actb mRNA, exon 3-4, forward 5Ј-ACCTTCTACAATGAGCTGCG-3Ј and reverse 5Ј-CTGGATGGCTACGTACATGG-3Ј, amplicon length 147bp. The PCR protocol was 1 min at 95°C, 40 cycles of 15 s at 95°C, and 30 s at 65°C. Amplified products were visualized in 1.6% agarose gels in TAE (Tris-acetate buffer and EDTA). qPCR reactions were repeated three times for each sample and averaged to determine the expression -fold change.

Protein isolation and Western blot analysis
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 incubation at 37°C. The dispersed cells were subsequently passed through a 150-m cell strainer (Thermo Fisher), centrifuged in a clinical tabletop centrifuge for 1 min, and resuspended in lysate buffer containing 0.5% Igepal CA-630, 10 mM HEPES (pH 7.2 for the PG and SMG and pH 6.8 for the SLG), 0.3 M sucrose, 2 mM EDTA, 0.2 mM EGTA, 1 mM PMSF, and phosphatase and protease inhibitor mixtures (Roche). Lysed cells were centrifuged at 4000 ϫ g for 10 min, and the supernatant was centrifuged at 22,000 ϫ g for 20 min. The pellet was resuspended in lysate buffer and centrifuged at 46,000 ϫ g. The resultant pellet was resuspended in the lysate buffer as a crude plasma membrane 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 radioimmune precipitation assay buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktails. The dissolved cells were spun at 14,000 ϫ g for 20 min at 4°C, and the resultant 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 Scientific), separated in 4 -12% SDS-PAGE BisTris gels, and transferred to a polyvinylidene difluoride membrane using the iBlot 2 Dry blotting system according to the manufacturer's instructions (Thermo Fisher Scientific). The transferred membrane was blocked for 1 h at 20°C in 5% dry milk (Bio-Rad) in Trisbuffered saline supplemented with 0.1% Tween 20 (TTBS) and then incubated with mouse monoclonal anti-SLC26A6 antibody (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. Following 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

Slc26a6 facilitates oxalate secretion in saliva
according to the manufacturer's denaturing protocol (New England Bio-Rad).

Mouse submandibular gland acinar cell isolation
Submandibular glands were surgically removed and enzymatically dispersed as before (47). Briefly, cells were incubated in BME containing 1.0 mg/ml collagenase 2 (Worthington) and digested by two steps of 25-min incubation each at 37°C with continuous gassing with 95% O 2 and 5% CO 2 .

Intracellular Cl ؊ and pH measurements
Salivary gland acinar cells were loaded with the Cl Ϫ -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 resuspended in 5 ml of BME at 37°C. Plasmid-transfected CHO-K1 cells were loaded with SPQ using a hypotonic loading buffer, as described previously (48). In brief, cells attached to a coverglass were incubated in a 150 mOsm hypotonic solution containing 5 mM SPQ for 4 min at 23°C.
Cells loaded with the Cl Ϫ -sensitive dye SPQ were transferred to a perfusion chamber mounted on the stage of an inverted microscope (Nikon, Eclipse TE 300) equipped with an Optoscan Imaging System (CAIRN Instruments) coupled to a digital CMOS camera (Hamamatsu, C11440). SPQ fluorescence was excited at 340 nm, and emissions were collected at 510 nm.
Mouse salivary gland acinar cells express three distinct types of Cl Ϫ flux pathways, including Tmem16a Ca 2ϩ -activated chloride channels, Nkcc1 Na ϩ /K ϩ /2Cl Ϫ cotransporters, and the anion exchangers Ae2, Ae4, and Slc26a6. Consequently, the following strategy was designed to isolate Slc26a6-mediated Cl Ϫ /oxalate anion exchange in isolated acinar cells. First, SPQloaded acinar cells were simultaneously depleted of intracellular Cl Ϫ and loaded with oxalate by exposure to a low Cl Ϫ (4 mM), oxalate-containing (25 mM) solution and then returned to a high Cl Ϫ (153.3 mM), oxalate-free solution (Na 2 oxalate was replaced with NaCl) to induce Cl Ϫ /oxalate anion exchange in the presence of 10 M T16Ainh-A01, 80 M bumetanide, and 30 M EZA to inhibit Tmem16a, Nkcc1, and carbonic anhydrases, respectively. All solutions were HCO 3 Ϫ -free and gassed with 100% O 2 to prevent Cl Ϫ /HCO 3 Ϫ exchanger activity. The intracellular pH of isolated SMG acinar cells was monitored 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% O 2 and 5% CO 2 . Cl Ϫ /HCO 3 Ϫ exchange activity was induced in BCECF-loaded SMG cells by exposure to a low Cl Ϫ solution. Solutions were as follows. High Cl Ϫ solution (Bϩ) contained 4.3 mM KCl, 120 mM NaCl, 25 mM NaHCO 3 Ϫ , 5 mM glucose, 10 mM HEPES, 1 mM CaCl 2 , and 1 mM MgCl 2 . Low Cl Ϫ solution contained 4.3 mM potassium gluconate, 120 mM sodium gluconate, 25 mM NaHCO 3 Ϫ , 5 mM glucose, 10 mM HEPES, 1 mM CaCl 2 , and 1 mM MgCl 2 . HCO 3 Ϫ -containing solutions were gassed with 95% O 2 , 5% CO 2 for at least 30 min. The HCO 3 Ϫ -free, high Cl Ϫ and HCO 3 Ϫ -free, low Cl Ϫ -solutions (BϪ) were made by substituting NaHCO 3 Ϫ with NaCl or sodium gluconate, respectively. The HCO 3 Ϫ -free solutions were gassed with 100% O 2 . Solutions had a pH level of 7.4. The initial rates of Cl Ϫ uptake and intracellular alkalization were estimated from the linear portion of the [Cl Ϫ ] i and pH i increases divided by the corresponding time periods (ϳ40 s) and expressed as the Cl Ϫ uptake and alkalization rates (10 Ϫ3 s Ϫ1 ), respectively.

Ex vivo perfusion of submandibular glands
Mice were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg of body weight), and the SMG was surgically 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 Slc26a6 facilitates oxalate secretion in saliva the flow rate and total amount of collected saliva were measured by recording the progression of saliva through a capillary tube at 1-min intervals. The secreted saliva was stored at Ϫ20°C until further analysis. The bicarbonate-containing perfusion solution was composed of 4.3 mM KCl, 120 mM NaCl, 25 mM NaHCO 3 Ϫ , 5 mM glucose, 10 mM HEPES, 1 mM CaCl 2 , and 1 mM MgCl 2 (pH 7.4, gassed with 95% O 2 /5% CO 2 ). The oxalatecontaining solution was the same as the bicarbonate-containing solution with the addition of 1 mM Na 2 Oxalate.

Ion composition of saliva
The HCO 3 Ϫ concentration in saliva was determined using a 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).

Statistical analysis
Results are presented as the mean Ϯ S.E. Statistical significance 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 from three or more mice for each condition. For RNA sequencing analysis (12), the FPKM values acquired from the three major salivary glands were compared using one-way ANOVA followed by Bonferroni's post hoc test, and p Ͻ 0.05 was considered to be statistically significantly different.