Species differences in Cl- affinity and in electrogenicity of SLC26A6-mediated oxalate/Cl- exchange correlate with the distinct human and mouse susceptibilities to nephrolithiasis

Abstract

The mouse is refractory to lithogenic agents active in rats and humans, and so has been traditionally considered a poor experimental model for nephrolithiasis. However, recent studies have identified slc26a6 as an oxalate nephrolithiasis gene in the mouse. Here we extend our earlier demonstration of different anion selectivities of the orthologous mouse and human SLC26A6 polypeptides to investigate the correlation between species-specific differences in SLC26A6 oxalate/anion exchange properties as expressed in Xenopus oocytes and in reported nephrolithiasis susceptibility. We find that human SLC26A6 mediates minimal rates of Cl(-) exchange for Cl(-), sulphate or formate, but rates of oxalate/Cl(-) exchange roughly equivalent to those of mouse slc2a6. Both transporters exhibit highly cooperative dependence of oxalate efflux rate on extracellular [Cl(-)], but whereas the K(1/2) for extracellular [Cl(-)] is only 8 mM for mouse slc26a6, that for human SLC26A6 is 62 mM. This latter value approximates the reported mean luminal [Cl(-)] of postprandial human jejunal chyme, and reflects contributions from both transmembrane and C-terminal cytoplasmic domains of human SLC26A6. Human SLC26A6 variant V185M exhibits altered [Cl(-)] dependence and reduced rates of oxalate/Cl(-) exchange. Whereas mouse slc26a6 mediates bidirectional electrogenic oxalate/Cl(-) exchange, human SLC26A6-mediated oxalate transport appears to be electroneutral. We hypothesize that the low extracellular Cl(-) affinity and apparent electroneutrality of oxalate efflux characterizing human SLC26A6 may partially explain the high human susceptibility to nephrolithiasis relative to that of mouse. SLC26A6 sequence variant(s) are candidate risk modifiers for nephrolithiasis.

Figure 1. Relative rates of tracer anion efflux by SLC26A6 chimeras, normalized to rate constants of wild-type mouse slc26a6

A, schematic diagram of mouse slc26a6/human SLC26A6 chimeras tested. B, normalized ³⁶Cl⁻ efflux rate constants measured in Cl⁻ bath (with 100% value assigned to mouse slc26a6). Human SLC26A6 mediated minimal Cl⁻ efflux, whereas chimera mh exhibited nearly 70% of the activity displayed by mouse slc26a6 (P < 0.05). C, normalized [¹⁴C]oxalate efflux rate constants measured in Cl⁻ bath differed minimally between human SLC26A6 and mouse slc26a6 (P < 0.05). Rate constants for other constructs equalled or exceeded that of mouse slc26a6, except for mhh and hmm (P < 0.03). D, normalized [¹⁴C]formate efflux rate constants measured in Cl⁻ bath. Human SLC26A6-mediated formate efflux was only 25%, whereas chimera mh-mediated efflux was nearly 50% that of mouse slc26a6 (P < 0.05). The formate rate constant for construct mhh did not differ from that in DIDS (P = 0.4). Water-corrected values are means ±s.e.m. for (n) oocytes. Mouse slc26a6 efflux rate constants were 0.133 ± 0.017 for ³⁶Cl⁻, 0.091 ± 0.07 for [¹⁴C]oxalate, and 0.026 ± 0.005 for [¹⁴C]formate. Efflux differences among constructs were significant by ANOVA for each substrate anion (P < 0.05). All oocytes were injected with 10 ng of the indicated cRNA. ³⁶Cl⁻ rate constants for m, h, mh and hm are reproduced from Chernova et al. (2005).

Figure 5. Functional effect of human SLC26A6 V185M polymorphism

A, [¹⁴C]oxalate uptake by oocytes expressing human wild-type SLC26A6 or the variant V185M, or by oocytes previously injected with water. *P < 0.005 compared to wild-type. B, ³⁶Cl⁻ uptake by oocytes expressing human WT SLC26A6 or the variant V185M, or by oocytes previously injected with water, measured immediately after injection of 50 nl of water or oxalate. Values in panels A and B are means ±s.e.m. for (n) oocytes. *P < 0.002 compared to wild-type. C, bath [Cl⁻] dependence of [¹⁴C]oxalate efflux rate constant in oocytes expressing human SLC26A6 variant V185M (t, n = 18–30). Curve is a Hill fit to the data, which are compared with bath [Cl⁻] dependence of human WT SLC26A6 (•, n = 6–33) or mouse slc26a6 (○, n = 11–47) reproduced from Fig. 4C.

Figure 4. Mouse slc26a6 and human SLC26A6 exhibit distinct affinities for extracellular Cl⁻

A, effect of increasing bath [Cl⁻] from 0 to 3 to 96 mm on [¹⁴C]oxalate uptake by oocytes expressing mouse slc26a6, human SLC26A6, or by oocytes previously injected with water. Values in panels are means ±s.e.m. for (n) oocytes. **P < 10⁻⁶ compared to 0 mm[Cl⁻]; *P < 0.005 compared to 3 mm[Cl⁻]. B, postprandial luminal [Cl⁻] (left) and [Na⁺] in the human stomach, duodenum, and upper jejunumn (reproduced with permission from Fordtran & Locklear, 1966). C, bath [Cl⁻] dependence of [¹⁴C]oxalate efflux rate constant in oocytes expressing human SLC26A6 (•, n = 6–33) compared with oocytes expressing mouse slc26a6 (○, n = 11–47, reproduced from Fig. 2D). D, bath [Cl⁻] dependence of [¹⁴C]oxalate efflux rate constant in oocytes expressing chimeric SLC26A6 polypeptides ‘mh’ (○, n = 6–23) or ‘hm’ (•, 6–18) as schematized in Fig. 1A. Curves are Hill fits to the data.

Figure 2. Clo- concentration dependence of ³⁶Cl⁻ efflux and [¹⁴C]oxalate efflux by mouse slc26a6

A, ³⁶Cl⁻ efflux traces from 6 individual oocytes expressing mouse slc26a6 and subjected in a single experiment to sequentially increasing bath [Cl⁻]. B, [Cl⁻]_o dependence of mouse slc26a6-mediated Cl⁻/Cl⁻ exchange measured as in panel A. Symbols represent mean rate constants ±s.e.m. for n = 11–30 oocytes. C, [¹⁴C]oxalate efflux traces from 6 individual oocytes expressing mouse slc26a6 and subjected in a single experiment to sequentially increasing bath [Cl⁻]. D, [Cl⁻]_o dependence curve for mouse slc26a6-mediated oxalate/Cl⁻ exchange measured as in panel C. Symbols represent mean rate constants ±s.e.m. for n = 11–47 oocytes. Curves in B and D are fitted by the 4-parameter Hill equation (SigmaPlot). Oocytes were injected with 10 ng mouse slc26a6 cRNA.

Figure 3. Oxalate_o concentration dependence of mouse slc26a6 and human SLC26A6 activities

A, bath [oxalate] dependence of ³⁶Cl⁻ efflux rate constant in oocytes expressing mouse slc26a6 (•, n = 18) or human SLC26A6 (○, n = 18). B, bath [oxalate] dependence of [¹⁴C]oxalate uptake by oocytes expressing mouse slc26a6 (•, n = 25–50), human SLC26A6 (○, n = 25–54), or by oocytes previously injected with water (t, n = 16–44). Inset: y-axis magnification of uptake data for human SLC26A6 and water. Curves in panels A and B are fitted by the 4-parameter Hill equation. C, effect of overnight Cl⁻ depletion on oxalate uptake by oocytes expressing mouse slc26a6 or human SLC26A6, or by oocytes previously injected with water. D, effect of acute oxalate injection on Cl⁻ uptake by oocytes expressing mouse slc26a6 or human SLC26A6, or by oocytes previously injected with water. Values in panels C and D are means ±s.e.m. for (n) oocytes. (*P < 0.01; **P < 10⁻⁶).

Figure 6. Exchange of bath oxalate for intracellular anion is electrogenic when mediated by mouse slc26a6, but electroneutral when mediated by human SLC26A6

A, I–V relationship measured in oocytes expressing mouse slc26a6 (open symbols, n = 47) or human SLC26A6 (filled symbols, n = 9) exposed first to NaCl bath, then to sodium gluconate (circles), and subsequently gluconate with 5 mm oxalate (triangles). Lines are least squares linear fits to the data. Data recorded in Cl⁻ bath (not shown for clarity) was superposable with that in gluconate bath (circles). B, bath oxalate concentration dependence of current measured at +40 mV holding potential in 5 oocytes expressing mouse slc26a6. Data are fitted with the Hill equation. C, bath oxalate difference current measured at +40 mV in 12 oocytes (means ±s.e.m.) expressing mouse slc26a6 (m), human SLC26A6 (h) or the chimeric polypeptides mh or hm. *P < 0.05 compared to CFEX. Inset: normalized [¹⁴C]oxalate uptake measured on the same days in oocytes from the same two frogs (means ±s.e.m. for (n) oocytes).

Figure 7. Exchange of bath Cl⁻ for intracellular oxalate depolarizes oocytes expressing mouse slc26a6 but not oocytes expressing human SLC26A6

V_m recorded from representative individual oocytes expressing mouse slc26a6 (A, B) or human SLC26A6 (C, D) in the absence (A, C) or presence of 100 μM DIDS (B, D). In each panel the initial hyperpolarization reflects insertion of the recording pipette. Insertion of the injection pipette (black circle) is followed by oxalate injection (arrow) while the oocyte remains in sulfamate bath. The sulfamate bath is then replaced with Cl⁻ bath.

Figure 8. Exchange of bath Cl⁻ for intracellular oxalate by mouse slc26a6 (A) is electrogenic, but exchange by human SLC26A6 (B) is electroneutral

Oocyte I–V curves were measured in NMDG sulfamate bath before (○) or after (•) injection of oxalate, then after bath change to NMDG chloride (▵) and finally, after addition of 100 μm DIDS (▴). Values are means ±s.e.m. for n = 5 oocytes in each panel. Lines are least squares linear fits to the data.

Figure 9. Tests of explanations for electroneutrality of human SLC26A6-mediated oxalate/Cl⁻ exchange

A, [¹⁴C]oxalate efflux rate constants for oocytes expressing human SLC26A6 or mouse slc26a6 and exposed sequentially to Cl⁻-free cyclamate bath without or containing sulphate, phosphate or oxalate, with a final addition of 100 μm DIDS. *P < 0.005; *P < 10⁻⁶ compared to cyclamate. B, extracellular pH dependence of oxalate uptake into oocytes expressing human SLC26A6 or mouse slc26a6. *P < 0.05 compared to pH 7.4; **P < 10⁻⁶ compared to pH 6. C, intracellular pH dependence of [¹⁴C]oxalate efflux rate constants in oocytes expressing human SLC26A6 or mouse slc26a6, measured as the response to bath butyrate removal. *P < 0.05 compared to presence of butyrate. Values are means ±s.e.m. for (n) oocytes.