SLC26A6 and Nadc-1 Transporters Interact to Regulate Oxalate and Citrate Homeostasis

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SLC26A6 and NaDC-1 Transporters Interact to Regulate Oxalate and Citrate Homeostasis

† Ehud Ohana,* Nikolay Shcheynikov,* Orson W. Moe, and Shmuel Muallem*

*Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, and †Department of Internal Medicine, Department of Physiology, and Charles and Jane Pak Center of Mineral Metabolism, University of Texas Southwestern Medical Center, Dallas, Texas

ABSTRACT The combination of hyperoxaluria and hypocitraturia can trigger Ca2+-oxalate stone formation, even in the absence of hypercalciuria, but the molecular mechanisms that control urinary oxalate and citrate levels are not understood completely. Here, we examined the relationship between the oxalate transporter SLC26A6 and the citrate transporter NaDC-1 in citrate and oxalate homeostasis. Compared with wild- type mice, Slc26a6-null mice exhibited increased renal and intestinal sodium-dependent succinate uptake, as well as urinary hyperoxaluria and hypocitraturia, but no change in urinary pH, indicating enhanced transport activity of NaDC-1. When co-expressed in Xenopus oocytes, NaDC-1 enhanced Slc26a6 transport activity. In contrast, Slc26a6 inhibited NaDC-1 transport activity in an activity dependent manner to restricted tubular citrate absorption. Biochemical and physiologic analysis revealed that the STAS domain of Slc26a6 and the ﬁrst intracellular loop of NaDC-1 mediated both the physical and functional interactions of these transporters. These ﬁndings reveal a molecular pathway that senses and tightly regulates oxalate and citrate levels and may control Ca2+-oxalate stone formation.

J Am Soc Nephrol 24: 1617–1626, 2013. doi: 10.1681/ASN.2013010080

Formation of calcareous stones is a major health Dietary oxalate is an important source of exog- problem, mainly afﬂicting the kidney1 and salivary enous oxalate and is absorbed by the intestinal glands.2 Most stones are Ca2+-oxalate, with the mi- epithelium via the paracellular pathway.9 The liver nority being Ca2+-phosphate.1 Ca2+-oxalate stone is the main source of endogenous oxalate; however, formation can result from hyperoxaluria, hy- under physiologic conditions, a small fraction of percalciuria, or reduction in the major urinary the bodily oxalate is derived from hepatic pro- 2 2 Ca2+ buffer citrate.3 Ca2+-oxalate stones can form duction.10 Studies using slc26a6 / mice showed in the absence of hypercalciuria when hyper- that increased serum oxalate is the result of im- oxaluria is coupled with hypocitraturia. The anion paired intestinal excretion that, in turn, leads to in- transporter slc26a6 (National Center for Bio- creased ﬁltered renal oxalate load and formation of technology Information [NCBI] accession no. Ca2+-oxalate stones.8 However, under normal NM_134420) has a major role in controlling systemic oxalate metabolism.4 Slc26a6 is expressed at high levels in most epithelia, including the Received January 21, 2013. Accepted April 24, 2013. proximal intestine, renal proximal tubule, sali- E.O. and N.S. contributed equally to this work. vary glands, and pancreas.5 Slc26a6 functions as a 2 2 2 Published online ahead of print. Publication date available at 6 1Cl /2HCO3 or 1Cl /1oxalate and 1Cl /1for- www.jasn.org. mate exchanger.7 Its pivotal role in oxalate homeo- 2/2 Correspondence: Dr. Shmuel Muallem, National Institute of stasis was demonstrated in slc26a6 mice, where Dental and Craniofacial Research, National Institute of Health, the most prominent phenotype is increased serum Building 10, Room 1N-112, Bethesda, MD 20892. Email: shmuel. and urine oxalate that lead to Ca2+-oxalate kidney [email protected]. stones.8 Copyright © 2013 by the American Society of Nephrology

J Am Soc Nephrol 24: 1617–1626, 2013 ISSN : 1046-6673/2410-1617 1617 BASIC RESEARCH www.jasn.org physiologic conditions, two major factors prevent Ca2+-oxalate RESULTS stones formation. Slc26a6 mediates oxalate clearance via the intestine,9 and urinary citrate chelates the Ca2+ to reduce the Urine Oxalate, Citrate, pH, and Na+-Dependent 2 2 free Ca2+ available for binding to oxalate.11 Citrate binds Ca2+ Dicarboxylic Uptake in slc26a6 / Mice at a higher affinity than does oxalate;12 thus, in the presence of Knockout of slc26a6 was reported to have increased urinary 2 2 a high citrate concentration, Ca2+-oxalate does not reach the oxalate, resulting in kidney stones.8 The slc26a6 / mice super-saturation needed for stone formation. In addition, once developed in our laboratory also have massive formation of crystals are formed, citrate adsorbs to the crystal surfaces and Ca2+-oxalate stones in the kidney and stone accumulation in suppresses their growth and attachment to epithelia.13 By at- the urinary bladder (not shown). It was assumed that the hy- taching to crystal surfaces, citrate also amplifies the protective peroxaluria was the sole driver for lithogenesis in these ani- effect of other stone inhibitors, such as the Tamm-Horsfall mals, but a critical determinant in formation of Ca2+/oxalate protein14 and osteopontin.15 stone is urinary citrate. To determine the state of citrate in the 2 2 At a pH of 7.4, citrate is mostly in the form of a tricarboxylic slc26a6 / mice and monitor the effect of slc26a6 on urine acid, but it can be reabsorbed only by the proximal tubule citrate/oxalate homeostasis, we collected 24-hour urine 2 2 epithelium in its divalent form.16 The luminal pH at the prox- samples from slc26a6 / and wild-type (WT) mice over 3 imal tubule drops from 7.4 to 6.5,17 which favors the divalent consecutive days and performed a kidney stone profile (Table form of citrate and thereby allows citrate transport. In mam- 1).Figure1,A–C, shows about a four-fold increase in urine mals, the major luminal citrate transporter in both the oxalate, a prominent three-fold reduction in urinary citrate, 2 2 intestine and proximal tubule is the Na+-dependent dicar- and no significant change in urinary pH in slc26a6 / mice. boxylate co-transporter, NaDC-1 (NCBI accession no. Although the original study analyzing electrolytes in another 2 2 AY186579).18 NaDC-1 (Slc13a2), the second member of slc26a6 / mice line reported no change in urinary citrate,8 the SLC13 family, has 11 transmembrane domains, with subsequent analysis with the same mouse line revealed reduc- cytoplasmic N- and extracellular C-termini.18 In light of the tion in urinary citrate,19 as found here. The hypocitraturia importance of the citrate/oxalate balance in regulating free cannot be explained by the known causes, including K+ de- Ca2+ in bodily fluids, we hypothesized that citrate/oxalate ficiency, increased acid load, and frank metabolic acidosis. balance may be regulated by a molecular mechanism that This urine composition greatly increases the Ca2+-oxalate fine-tunes citrate and oxalate concentrations. Such a mech- stone risk.1 anism should involve the two major transporters, slc26a6 and Urinary citrate is determined by proximal tubule citrate NaDC-1. absorption, which is mediated by Na+-dicarboxylate co-trans- In the present work, we report on a new pathway that porter NaDC-1.20 Reduced urinary citrate suggests increased 2 2 involves interplay between slc26a6 and NaDC-1 that deter- citrate absorption and NaDC-1 activity in the slc26a6 / mines citrate/oxalate homeostasis. The pathway involves the mice. In preliminary experiments, the signal-to-noise ratio mutual but reciprocal regulation of slc26a6 (activation) and of Na+-dependent succinate transport in renal brush-border NaDC-1 (inhibition). The interaction between the oxalate membranes preparation from mice kidney was too small to and citrate transporters is mediated by the slc26a6 STAS resolve the effect of slc26a6 on the transport. Therefore, domain and the NaDC-1 first intracellular loop (ICL1), which we prepared kidney cortical slices (see Concise Methods sec- recapitulate the function of the full-length transporters. tion). Figure 1D shows that deletion of slc26a6 increased the These findings reveal a molecular pathway that tightly re- Na+-dependent succinate uptake by about 70%. A limitation of gulates oxalate and citrate to guard against Ca2+-oxalate stone the slice preparation is that altered succinate uptake could not formation. be assigned to the luminal membrane. However, because

2 2 Table 1. Electrolyte proﬁle of wild-type and slc26a6 / mice urine 2 2 Slc26a6+/+ Slc26a6 / Variable P Value Mean 6 SEM Urine Samples (n) Mean 6 SEM Urine Samples (n) Citrate (mg/24 hr) 0.4560.07 18 0.1460.03 18 ,0.05 Oxalate (mg/24 hr) 0.0660.01 18 0.1860.03 18 ,0.05 Total volume (ml/24 hr) 1.660.16 18 1.660.19 18 NS pH 6.260.07 18 6.0760.04 18 NS Creatinine (mg/24 hr) 0.7460.05 18 0.6560.06 18 NS Ca2+ (mg/24 hr) 0.1260.02 18 0.0660.01 18 ,0.05 Mg2+ (mg/24 hr) 0.3660.04 18 0.4660.04 17 NS Cl-(mEq/24 hr) 0.2660.02 17 0.2260.02 16 NS + NH4 (mEq/24 hr) 0.0860.01 18 0.0960.01 18 NS 22 SO4 (mmol/24 hr) 0.0560.01 18 0.0560.01 18 NS Urine samples were collected in 3 consecutive days from six mice from each group and analyzed for the listed electrolytes and metabolites. NS, not signiﬁcant.

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these conditions, succinate has one negative charge and coupling to 3Na+ generated the expected inward current. Figure 2, A and B, shows that slc26a6 markedly inhibits the activity of NaDC-1. The control experiments in Supplemental Figure 2 show that slc26a6 does not transport succinate and NaDC-1 does not transport oxalate. Importantly, Figure 2C shows that surface expression of NaDC-1 is not affected by coexpression with slc26a6, indicating that slc26a6 regulates transport by NaDC-1. On the other hand, slc26a6 and NaDC-1 can be readily co-immunoprecipitated (see Figure 2D and discussion below). Finally, inhibition of NaDC-1 was not restricted - 2 6,7 to slc26a6 because the 2Cl /1HCO3 exchanger slc26a3 similarly inhibited NaDC-1 activity (Figure 2B), although the 22 2 2 23 SO4 /OH /Cl exchanger slc26a2 did not (Supplemental Figure 3). An important question is whether NaDC-1 senses the activity of slc26a6: namely, whether the inhibition is more significant when slc26a6 is active. To address this question we compared the inhibition of NaDC-1 by active slc26a6 and by the inactive slc26a6(E357K) mutant, which lacks all forms of slc26a6 activity.7 Interestingly, Figure 3 shows that although both active and inactive slc26a6 inhibit NaDC-1, active slc26a6 inhibited NaDC-1 about two-fold more than did inactive slc26a6(E357K). These findings indicate that once interacting, slc26a6 inhibits NaDC-1, but when slc26a6 is active Figure 1. Deletion of slc26a6 in mice upregulates Na+-dependent it may resume a conformation that enhances interaction with dicarboxylate uptake. (A–C) Analysis of urine cittrate (A), oxalate (B), NaDC-1. This may function as a sensing mechanism whereby 2/2 and pH (C) in wild-type and slc26a6 mice collected during 3 inhibition of NaDC-1 becomes more effective when oxalate consecutive days. Notice the significant decreased citrate and in- 2 2 excretion takes place. creased oxalate with minimal change in urinary pH in slc26a6 / Inhibition of NaDC-1 by slc26a6 raised the question of mice urine. (D) Na+-dependent succinate uptake by kidney slices 2 2 obtained from wild-type and slc26a6 / mice. whether NaDC-1 reciprocally affects the activity of slc26a6. In Figure 4, A and B, we measured the effect of NaDC-1 on 2 slc26a6-mediated Cl /oxalate exchange in oocytes expressing slc26a6 is expressed in the luminal membrane, the increase slc26a6 alone or coexpressing slc26a6 and NaDC-1. NaDC-1 most likely reflects increased activity of NaDC-1. Since does not transport oxalate (Supplemental Figure 2). Addition 2 slc26a6 and NaDC-1 are expressed at a high level in the jeju- of 1 mM oxalate to oocytes bathed in Cl -free solution resul- 2 num,16,21,22 and slc26a6-mediated jejunal oxalate excretion ted in an outward current due to Cl /oxalate exchange.7 The has a major role in controlling systemic oxalate metabolism,4 slow reduction in the current is due to accumulation of 2 2 we also used jejuna sheets from slc26a6 / and WT mice and intracellular oxalate, which accounts for the overshoot in 2 measured the Na+-dependent succinate uptake. Supplemental the inward current upon readdition of Cl . Coexpression of Figure 1, A and B, shows that deletion of slc26a6 had no effect NaDC-1 increased Cl-/oxalate exchange by about 30% on the Na+-independent succinate uptake while increasing the (Figure 4B). Another important function mediated by + 2 2 Na -dependent uptake by about 35%. This increase reflects an slc26a6 is 1Cl /2HCO3 exchange. Figure 4, C and D, increased native NaDC-1 activity and can account for the re- shows that NaDC-1 similarly increased the slc26a6-mediated 2/2 2 2 duced urinary citrate in the slc26a6 mice. The next question 1Cl /2HCO3 exchange without affecting the stoichiome- we asked is: If the increased Na+-dependent succinate uptake is try of the exchange. This may function to increase the pH at mediated by NaDC-1, do slc26a6 and NaDC-1 interact bio- the NaDC-1/slc26a6 microdomain to limit access of divalent chemically and functionally? citrate to NaDC-1. Figure 4E shows that surface expression of slc26a6 is not increased by NaDC-1, indicating that Functional and Biochemical Interaction between NaDC-1 regulates slc26a6 activity rather than trafficking. slc26a6 and NaDC-1 The results in Figures 2–4 were obtained with opossum First, we asked whether slc26a6 regulates the activity of NaDC-1 that was used originally to characterize NaDC- NaDC-1. For this, we expressed NaDC-1 and slc26a6 separately 1.18 Supplemental Figure 4 shows that similar results were or together in Xenopus oocytes and monitored NaDC-1 activ- obtained with human NaDC-1 (NCBI accession no. ity as Na+-dependent succinate current at a pH of 7.5.20 Under BC096277). Taken together, the results in Figures 1–4

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The slc26a6 STAS Domain and NaDC- 1 ICL1 Mediate the Reciprocal Regulation Our next aim was to identify the molecular determinants that mediate the interaction between NaDC-1 and slc26a6. We have previously identified the SLC26 transporter STASdomainasaprotein-protein–interacting domain that participates in regulation of other transporters, such as cystic fibrosis transmembrane conductance regulator,24 and therefore tested whether it may mediate the slc26a6-NaDC1 interaction. Figure 5, A and B, shows that both the slc26a6 and slc26a3 STAS domains inhibit NaDC-1 to the same extent measured with the full- length transporters. In addition, the slc26a6 STAS domain and NaDC-1 can be co-immunoprecipitated (Figure 5C). These observations show that slc26a6 inhibits NaDC-1 via STAS domain-mediated + Figure 2. NaDC-1 mediated Na -succinate cotransport is inhibited by slc26a6. (A) interaction. Representative traces. (B) Summary of the current mediated by NaDC-1-dependent To locate the NaDC-1 site that interacts Na+-succinate cotransport in oocytes expressing NaDC-1 alone (black) or coexpressed with the STAS domain, we generated the with slc26a6 (gray) or slc26a3 (light gray). The transport was initiated by the addition of fi 1 mM succinate to the perfusate, as indicated. (C) Surface expression of NaDC-1(Flag) ve NaDC-1 fragments illustrated in Figure in the presence and absence of slc26a6. Note that the coexpression with slc26a6 has 6A, based on the 11 transmembrane do- 25 no effect on surface NaDC-1. (D) Reciprocal Co-IP of NaDC-1(Flag) and slc26a6(GFP). main topology model of NaDC-1 (Pajor Expression in HEK cells was used for surface expression and Co-IP. and Supplemental Figure 5). All fragments except NaDC-1-M453 interacted with slc26a6, suggesting that NaDC-1(45–118) that includes the first NaDC-1 ICL1 is probably the slc26a6 interacting site. To as- certainthatthelackofinteractionof NaDC-1(1–45) and the interaction of NaDC-1(1–118) was not promoted by the mCherry tag, we prepared hemagglutinin- tagged fragments and obtained the same results (Figure 6B). We also prepared NaDC-1 with truncated ICL1 (NaDC-1 [DICL1]) (Figure 6C). Unfortunately, this construct did not traffictotheplasma membrane, as evident from the presence of only the nonmature form of the protein (Figure 6C). This precluded determination of the effect of the truncation on NaDC-1 activity and co-regulation by slc26a6. Nev- Figure 3. Transport competent slc26a6 enhances inhibition of NaDC-1. (A) Repre- ertheless, we used this construct to test for sentative traces. (B) Summary of NaDC-1 current when expressed alone or together interaction with slc26a6, and as shown in with slc26a6 or the inactive slc26a6(E357K) mutant. Figure 6D, truncation of ICL1 markedly reduced interaction of NaDC-1 with slc26a6. Together,theresultsinFigure6indicate indicate that NaDC-1 and slc26a6 interact and reciprocally that NaDC-1 ICL1 mediates the interaction with slc26a6. regulate their activities; NaDC-1 activates slc26a6 while To further establish the role of ICL1 in the regulation of slc26a6 inhibits NaDC-1, and the inhibition is modulated slc26a6, we prepared a His-tagged ICL1 construct that includes by the activity of slc26a6. residues 35–119 of NaDC-1 and tested its interaction with and

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2 2 2 Figure 4. slc26a6-mediated Cl /oxalate and Cl /HCO3 exchange is augmented by interaction with NaDC-1. (A) Current was 2 measured in Xenopus oocytes expressing slc26a6 alone or slc26a6 and NaDC-1 to monitor slc26a6 Cl /oxalate exchange. The oocytes 2 were incubated in Ca2+-free solution and then exposed to 1 mM oxalate, as indicated by the bar. Re-addition of Cl and removal of oxalate resulted in the expected inward current. The summary (B) is given as mean 6 SEM, and the number of observations is listed on 2 2 2 the columns. (C) Measurement of slc26a6-mediated Cl /HCO3 exchange by the simultaneous measurements of [Cl ]i and pHi in oocytes expressing slc26a6 alone or with NaDC-1. (D) Mean 6 SEM of 7–9 experiments. (E) Lack of effect of NaDC-1 on surface expression of slc26a6(mKate) expressed in HEK cells. activation of slc26a6. Figure 7, A and B, shows that ICL1 ac- organs that express the oxalate transporter slc26a6 and the 2 tivated slc26a6-mediated Cl /oxalate exchange to the same citrate transporter NaDC-1, and that experience variable loads extent as NaDC-1. Furthermore, ICL1 interacts with both of these molecules. Biochemical and functional analyses reveal full-length slc26a6 and the slc26a6-STAS domain (Figure interaction between slc26a6 and NaDC-1 mediated by speciﬁc 7C). Hence, the interaction between NaDC-1 and slc26a6 modules. The STAS domains of several SLC26 transporters— occurs intracellularly and is mediated by the slc26a6 STAS slc26a3, slc26a6, and SLC26A924,26–28—were shown to func- domain and the NaDC-1 ICL1. tion as protein-protein–interacting domains to mediate the mutual regulation of the SLC26 transporters and CFTR. The slc26a6 STAS domain also interacts with carbonic anhydrase DISCUSSION II.29Here we show that the STAS domains of slc26a6 and slc26a3 interact with NaDC-1 and inhibit NaDC-1 activity, We describe a molecular mechanism via interaction of two similar to the full-length protein. It is of interest that slc26a3 transporters that can set the oxalate/citrate level in several and slc26a6 but not slc26a2 inhibited the activity of NaDC-1

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indicating that STAS-ICL1 domains interaction underlies the molecular mechanism of citrate/oxalate regulation by slc26a6 and NaDC-1. That ICL1 and the STAS domain recapitulate the interaction and mutual regulation of NaDC-1 and slc26a6 indicates that interaction between the transporters is required for the regulation. Indeed, the mutant slc26a6(E357K) that lacks all slc26a6 functional modes7 inhibited NaDC-1. However, slc26a6(E357K) was less effective than slc26a6 in inhibiting NaDC-1, suggesting two levels of regulation of NaDC-1 by slc26a6. The inhibition is mediated by direct interaction between the transporters and is augmented when slc26a6 is engaged in transport. The re- quirement for an active slc26a6 may reflect favoring of a STAS domain conformation that interacts better with ICL1, sensitivity of NaDC-1 to pH, the membrane potential, or all of the above. Our findings are not sufficient to firmly support any of these possibilities, although sensitivity of NaDC-1 to pH and the membrane poten- Figure 5. The SLC26 transporters STAS domain mediates NaDC-1 inhibition. (A) tial is well established (see below). The Example traces. (B) Mean 6 SEM of the indicated number of experiments showing that present findings raise the possibility that the slc26a6 (gray) and Slc26a3 (light gray) STAS domains inhibit NaDC-1 activity. (C) physiologically NaDC-1 activity is inhibi- Reciprocal co-immunoprecipitation of the slc26a6(myc) STAS domain and NaDC-1 ted mostly when slc26a6 is actively trans- (Flag) expressed in HEK cells. porting oxalate. The mutual and reciprocal regulation of slc26a6 and NaDC-1 suggests the senso- (Supplemental Figure 3). This may relate to the variability in regulatory molecular mechanism for oxalate/citrate homeo- the STAS domain. The highest variability and the most stasis proposed in Figure 8. Slc26a6-NaDC-1 exists in a complex prominent difference between the STAS domains of in which slc26a6 is active to excrete oxalate to the urine, in the SLC26 transporters is at a definedregionreferredtoas addition to the amount of oxalate that is already present in the intervening sequence or variable loop found between the the filtrate. Under these conditions, high urine citrate is first a-helix and third b-sheet.30 This region is predicted to required to chelate Ca2+ and prevent its binding to oxalate. be disordered in solution and contains many positively Inhibition of NaDC-1 by slc26a6 in the complex inhibits charged residues. Slc26a2 lacks the intervening sequence citrate absorption to increase luminal citrate, thus pro- loop. viding a safe haven to increase luminal oxalate. The physiologic The molecular identity of NaDC-1 has been known for relevance of the stimulation of slc26a6 activity by NaDC-1 is several years.20 Despite the low sequence similarity with mam- not known with certainty. However, it is possible that activation + - 2- malian NaDC-1, the bacterial Na /citrate symporter citS and of slc26a6 depolarizes the plasma membrane due to Cl out/Ox in NaDC-1 share many features. Both proteins are predicted to exchange to further reduce the 3Na+/dicarboxylate transport have 11 transmembrane domain spans, which is supported by by NaDC-1. In addition, NaDC-1 may be modulated by 2 25,31 - experimental data. This was further established recently the other slc26a6 function of Cl /2HCO3 exchanger. This by the resolved structure of the bacterial Na+-dicarboxylate can function as a negative feedback mechanism to limit 2 32 transporter VcINDY. NaDC-1 and VcINDY also mediate citrate uptake by increasing the HCO3 concentration in the Na+/citrate cotransport. Here we used the 11 transmembrane microenvironment around the NaDC-1/slc26a6 complex in domain topology of NaDC-1 (Supplemental Figure 5) to the tubular lumen and dampening NaDC-1 activity by reduc- identify ICL1 as the NaDC-1 module that interacts with the tion of substrate availability33 and direct pH gating.34 The STAS domain to enhance slc26a6 activity. Significantly, ICL1 result is further reduction in citrate absorption to increase tu- 2 2 is as effective as full-length NaDC-1 in the activation of slc26a6, bular citrate. The hypocitraturia in slc26a6 / mice cannot be

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functioning and the signiﬁcance of the slc26a6-NaDC-1 homeostatic system in multiple organs.

CONCISE METHODS

Animal Care and Metabolism Experiments All of the work on mice was conducted following the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and was approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center and the National Institute of Craniofacial and Dental Research, National Institutes of Health (NIH). Wild-type 2 2 and slc26a6 / mice were individually housed in MMC100 metabolic cages (Hatteras Instru- ments Inc.). All mice were on rodent diet NIH 31 and tap water during the experiments. After acclimatization, 24-hour urine samples were collected over the course of 3 consecutive days. The collected samples were analyzed for urine electrolytes, and the results are summarized in Supplemental Table 1. Figure 6. NaDC-1 ICL1 mediates interaction with slc26a6. (A and B) The indicated NaDC-1 truncation constructs (Flag or hemagglutinin-tagged, see illustration) were expressed in HEK cells with or without slc26a6 and were used to test co-immuno- Succinate Uptake precipitation with slc26a6. M453, which retains the first 45 residues of NaDC-1, is the For all experiments, mice were euthanized with only construct that does not interact with slc26a6, providing the first evidence that CO2. ICL1 is the interacting site. Although deletion of ICL1 (HA-NaDC-1ΔICL1) results in impaired maturation of NaDC-1 (C), deletion of ICL1 markedly reduces the interaction Kidney Slices of NaDC-1 with slc26a6 (D). The two kidneys were excised into PBS (pH, 7.4), and the capsules were removed by pinching. Fresh kidneys were embedded in 4% agarose accounted for by dietary difference in acid load (equivalent uri- blocks in PBS and polymerized at room temperature. The agarose- nary sulfate and ammonium excretion) or potassium status kidney block was cooled and 1-mm-thick slices were cut using (equivalent plasma and urinary potassium), strongly supporting Vibratome (Electron Microscopy Sciences model OTS-3000) (insert an intrinsic change in proximal tubule citrate reabsorption. in Supplemental Figure 6A). The kidney slices were separated from The slc26a6-NaDC-1 homeostatic system is likely tooperate the agarose, and the cortical section was cut out and placed in ice in several organs and to be disrupted in disease states. In the chilled PBS. Photo images on top of a graded paper used as scale were present studies we showed that deletion of slc26a6 increases taken for determining the kidney cortical area (Supplemental Figure intestinal NaDC-1 activity. Stones are very common in salivary 6A). glands2 and may result from malfunctioning or overwhelming of the slc26a6-NaDC-1 homeostatic system. Indeed, recent Intestinal Slices studies reported Ca2+-oxalate stones in salivary glands of sev- Themid-jejunawereremoved,andthetissuewastransferred eral patients with CKD.35 Intriguingly, a recent study found a immediately into ice-chilled PBS (pH, 7.4), cut along the mesenteric subgroup of stone formers with slight alkalinuria (pH .6) border, cleaned, and cut to small pieces of about 535mm.Thetissues that showed suboptimal citraturic response to standard alkali were placed in six-well dishes fully stretched with 27-gauge needles therapy. The authors speculated on dysregulation of NaDC-1 and photo images on top of a graded paper used as scale were taken as a possible reason for this condition.36 We show here that for determining the epithelium area (Supplemental Figure 6B). 2 2 slc26a6 / mice indeed have significantly reduced citrate and Image analysis of all tissues was performed with ImageJ v1.43u high oxalate. Maintenance of inhibition of NaDC-1 by slc26a6 software (NIH). The tissues were then incubated in a water bath and may be an important homeostatic mechanism that is deranged agitated (100 oscillations/min) for 5 minutes at 37°C in 24-well dishes instoneformers.Itwillbeofinteresttoexplorethe containing 500 ml of incubation solution (in mM): 5 KCl, 10 HEPES,

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Deletion mutations were generated using Quik- Change II XL Site-Directed Mutagenesis Kit (Agilent). All constructs were verified by se- quencing and Western blot analysis of the protein product. The genes cloned into vectors pCDNA 3.1(+),pCMV-SPORT6, pCMV6-AC- mKate, and pCMV-AC-myc-His were linearized using relevant restriction enzymes and tran- scribed in vitro with T7 or sp6 mMessage mMa- chineormMessagemMachineultrakits (Applied Biosystems). To prepare the STAS domains, human slc26a6 (NM_022911) residues 530–742 were amplified by PCR and subsequently cloned into pCMV-HA using XhoIand NotI restriction sites (pCMV-HA\STAS-A6). Mouse slc26a6 (NM_134420) residues 508– 718 were similarly cloned into pCMV-Myc vector (pCMV-Myc\STAS-A6). For Xenopus oocyte expression, pCMV-HA\STAS-A6 and pCMV-Myc\STAS-A6 inserts were subcloned into pCDNA 3.1(-) using flanking ApaIand Figure 7. NaDC-1 ICL1 activates slc26a6 by interaction with the STAS domain. (A) NotI restriction sites. pXBG-ev1\STAS-A3 (res- Example current traces. (B) is the mean 6 SEM of the indicated number of experiments idues 518–713) was also expressed in Xenopus 2 of slc26a6-mediated Cl /oxalate exchange activity from Xenopus oocytes expressing oocytes and the construct is reported by slc26a6 alone (black) or slc26a6 and NaDC1-ICL1. (C) Reciprocal co-immunoprecipi- Ko et al.24 tation of ICL1(His-myc) with full-length slc26a6 and with the slc26a6 STAS(HA) domain expressed in HEK cells. Preparation and Injection of Oocytes Oocytes were obtained by partial ovariectomy of female Xenopus laevis (Xenopus Express, Beverly 10 glucose, 140 NaCl or NMDG-Cl, pH of 7.4. Subsequently, the Hills, FL), as previously described.37 Briefly, animals were anesthe- tissues were transferred to wells containing uptake solution supple- tized and follicle cells were removed in OR-2 calcium-free medium. mented with 0.1 mM (kidney) or 0.5 mM (intestine) succinic acid Defolliculated oocytes were washed with OR-2 calcium-free medium. and 1 mCi 14C-succinic acid (ViTrax Inc.) per 0.12 mmol (kidney) or Healthy oocytes in stages V to VI were collected under a microscope 0.7 mmol (intestine) of cold succinate. Tissues were incubated by and maintained at 18°C overnight in ND96 solution. Oocytes were agitation for an additional 2 minutes (kidney) or 5 minutes (intes- injected with cRNA using a Nanoliter 2000 injector (World Precision tine) at 37°C. The samples were then washed in ice-chilled PBS for 2 Instruments Inc., Sarasota, FL). Oocytes were incubated at 18°C in minutes and transferred into scintillation veils containing 0.5 ml ND96 solution with pyruvate and antibiotics and were tested 48–96 NaOH (1 M) and incubated for 1 hour at room temperature with hours after cRNA injection. intermittent agitation to solubilize the tissues. After addition of 0.5 2 ml of HCl (1 M), radioactivity was determined by liquid scintillation Voltage, Current, pH, and Cl Measurement in oocytes 2 counting using a Beckman sc60001C counter (Beckman Instru- Current, voltage, pHi,and[Cl ]i recordings were performed with ments). Osmolarity of all solutions was adjusted to 300 mOsm with two-electrode voltage clamp as described elsewhere.6 In brief, current the major salt. was recorded using Warner Instrument Corp. amplifier model OC- 725C and digitized via an A/D converter (Digidata 1322A; Axon Plasmid Construction and cRNA Preparation Instruments Inc.). Slc26a6-dependent currents were recorded at a In the current study, we used the untagged opossum NaDC-1 (NCBI holding membrane potential of 0 mV and NaDC-1–dependent cur- accession no. AY186579) pCDNA 3.1(+)/opNaDC-1 clone and mouse rents at a membrane potential of 230 mV. Data were analyzed using slc26a6 (NCBI accession no. NM_134420) pCMV-SPORT6/ the Clampex 8.1 system (Axon Instruments Inc.). The electrode tips 2 mslc26a6. The open reading frame of opossum NaDC-1 was were filled with 0.5 mlofH+ or Cl exchanger resin, hydrogen ion- 2 subcloned into pRK5-HA and p3xFLAG-CMV-7.1/mCherry plas- ophore I, cocktail B (Sigma-Aldrich), and Cl -sensitive liquid ion mids. The open reading frame of mouse slc26a6 was subcloned into exchanger 477913 (Corning), respectively, and then backfilled with a pEGFP-C1 and pCMV6-AC-mKate plasmids. Human NaDC-1 3 M KCl solution. During measurements, two channels were used for (NCBI accession no. BC096277) was cloned into pCMV-AC-myc- ion-sensitive measurement and another was used to record or control 2 His. All NaDC-1 site-directed mutants were generated by PCR with the membrane potential. The pH and Cl signals were extracted by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). subtracting the membrane potential signal from the ion-selective

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cocktail. Total protein concentration was determined by the Laurie method. Biotinylated proteins were then isolated with avidin beads by overnight incubation with the lysates and recovered by heating in SDS sample buffer at 37°C for 30 minutes. Similar lysis buffer was used for co-immunoprecipitation, extracts were incubated O.N. with the indicated antibodies, and complexes were collected with protein A or G Sepharose beads by incubation for 4 hours at 4°C. Beads were collected by centrifugation and washed three times with lysis buffer; proteins were recovered by heating in SDS sample buffer at 37°C for 30 minutes. The samples were subjected to SDS- PAGE and subsequently transferred to polyvinylidene diﬂuoride membranes.

Statistical Analyses All results are given as mean 6 SEM, and signiﬁcance was analyzed by t test or ANOVA using Origin software 8.1.

Figure 8. A model for the molecular mechanism of slc26a6 and ACKNOWLEDGMENTS NaDC-1 reciprocal regulation in epithelia. The apical slc26a6 and NaDC-1 are present in a complex to allow their interaction. The We thank Dr. Danielle Springer, Robin Schwartzbeck, and the staff of interaction is mediated by the slc26a6 STAS domain and the the National Heart, Lung, and Blood Institute phenotyping core at the NaDC-1 ICL1. When interacting, slc26a6 inhibits NaDC-1, while National Institutes of Health for providing metabolic cages and as- NaDC-1 activates slc26a6. The result is retention of citrate in the sistance in mice urine collection. 2+ filtrate and perhaps other luminal fluids to chelate part of the Ca This work was supported by Intramural Research Program of the to allow safe oxalate excretion. When minimal oxalate excretion is NIH, National Institute of Craniofacial and Dental Research grant required, the interaction between slc26a6 and NaDC-1 relaxes to ZIA-DE000735. increase the activity of NaDC-1 and citrate absorption and salvage. Disruption of this regulation in disease states leads to Ca2+/oxalate stone formation. DISCLOSURES None. electrodes signal using Origin software (version 8.1; OriginLab). For 2 simultaneous measurement of pHi, and [Cl ]i in oocytes, a three- electrode method was used as described previously.6 The following REFERENCES solutions were used as indicated in the figures: Standard HEPES- buffered ND96 oocyte regular medium containing (in mM) 96 1. Moe OW: Kidney stones: Pathophysiology and medical management. 2 Lancet 367: 333–344, 2006 NaCl, 2 KCl, 1.8 CaCl2,1MgCl2,and5HEPES,pHof7.5.Cl free 2 2. Harrison JD: Causes, natural history, and incidence of salivary stones solutions for oocytes were prepared by replacing Cl with gluconate and obstructions. Otolaryngol Clin North Am 42: 927–947, 2009 2 and MgCl2 with MgSO4.HCO3 -containing solutions were prepared 3. Moe OW, Preisig PA: Dual role of citrate in mammalian urine. Curr Opin + Nephrol Hypertens 15: 419–424, 2006 by replacing 25 mM NaCl or Na -gluconate with 25 mM NaHCO3 4. Aronson PS: Role of SLC26A6-mediated Cl⁻-oxalate exchange in renal and were continually gassed with 5% CO2/95% O2. Na-Oxalate and – fi physiology and pathophysiology. JNephrol23[Suppl 16]: S158 S164, Na-succinate were added to the solutions as indicated in the gures. 2010 5. Lee MG, Ohana E, Park HW, Yang D, Muallem S: Molecular mechanism Surface Expression and Co-immunoprecipitation of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev Surface expression was analyzed by biotinylation. Because oocytes 92: 39–74, 2012 transport biotin, biotinylation assays are not reliable in oocytes. Thus, 6. Shcheynikov N, Wang Y, Park M, Ko SB, Dorwart M, Naruse S, Thomas PJ, Muallem S: Coupling modes and stoichiometry of Cl-/HCO3- ex- biotinylation was performed using HEK cells. HEK cells expressing change by slc26a3 and slc26a6. J Gen Physiol 127: 511–524, 2006 slc26a6 and/or NaDC-1 were incubated with 0.5 mg/ml EZ-LINK 7. Ohana E, Shcheynikov N, Yang D, So I, Muallem S: Determinants of Sulfo-NHS-LC-biotin (Thermo Scientific) for 30 minutes on ice, coupled transport and uncoupled current by the electrogenic SLC26 quenched by incubation with 50 mM glycine for 10 min and washed transporters. J Gen Physiol 137: 239–251, 2011 twice with PBS. Cells were collected by scraping and centrifugation for 8. Jiang Z, Asplin JR, Evan AP, Rajendran VM, Velazquez H, Nottoli TP, Binder HJ, Aronson PS: Calcium oxalate urolithiasis in mice lacking 20 minutes. Lysates were prepared by incubating the cells in ice-cold anion transporter Slc26a6. Nat Genet 38: 474–478, 2006 lysis buffer containing PBS, 10 mM Na-pyrophosphate, 50 mM NaF, 1 9. Knauf F, Ko N, Jiang Z, Robertson WG, Van Itallie CM, Anderson JM, mM Na-orthovanadate, 1% Triton X-100, and protease inhibitors Aronson PS: Net intestinal transport of oxalate reflects passive

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