Novel Human Polymorphisms Define a Key Role for the SLC26A6-STAS Domain in Protection From Ca2+-Oxalate Lithogenesis

Abstract

Impaired homeostasis of the carboxylic acids oxalate and citrate, dramatically increases the risk for the formation of Ca²⁺-oxalate kidney stones, which is the most common form of kidney stones in humans. Renal homeostasis of oxalate and citrate is controlled by complex mechanisms including epithelial transport proteins such as the oxalate transporter, SLC26A6, and the citrate transporters, the SLC13's. These transporters interact via the SLC26A6-STAS domain in vitro, however, the role of the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain in Ca²⁺-oxalate stone formation was not investigated in humans. Here, we report two novel human SLC26A6 polymorphisms identified in the STAS domain of SLC26A6 in two heterozygous carriers. Intriguingly, these individuals have low urinary citrate, but different clinical manifestations. Our in vitro experiments indicate that the homolog mutations of SLC26A6(D23H/D673N) and SLC26A6(D673N) alone abolished the expression and function of SLC26A6, and impaired the regulation of SLC13-mediated citrate transport by SLC26A6. On the other hand, the SLC26A6(R621G) variant showed reduced SLC26A6 protein expression and membrane trafficking, retained full transport activity, but impaired the regulation of the citrate transporter. Accordingly, the human SLC26A6(D23H/D673N) carrier showed a dramatic reduction in urinary citrate concentrations which resulted in Ca²⁺-oxalate stones formation, as opposed to the carrier of SLC26A6(R621G). Our findings indicate that the human SLC26A6-STAS domain mutations differentially impair SLC26A6 expression, function, and regulation of citrate transporters. This interferes with citrate and oxalate homeostasis thus potentially predisposes to Ca²⁺-oxalate kidney stones.

Keywords: NaDC-1; SLC26A6; citrate; kidney stones; oxalate.

Figure 1

Two individuals carrying heterozygous SLC26A6-STAS domain polymorphisms show hypocitraturia. We monitored urinary oxalate (A) and citrate (B) concentrations in hyperoxaluric stone formers (SF) and non-stone formers (NSF) that carry the V206M, D23H/D673N, R621G polymorphisms, or WT. P values by ANOVA are indicated. It is noteworthy that both hyperoxaluria and hypocitraturia are risk factors in the general Ca²⁺-oxalate SF population but cohort who underwent genotyping was biased for hyperoxaluric patient and hence hypocitraturia was “selected out”.

Figure 2

Functional and regulatory properties of SLC26A6 are compromised by the sulfate transporter and anti-sigma factor antagonist (STAS) domain polymorphisms (D23H/D673N) and (R621G). (A) Representative traces and summary of the human SLC26A6 Cl⁻/HCO₃⁻ exchange activity monitored in cells transfected with either an empty vector (control), SLC26A6(WT), SLC26A6(D674N), SLC26(D23H/D674N), or SLC26A6 (R621G), as indicated. Transport activity was monitored as the fluorescence change from the new baseline (dashed line) to peak after prefusion with 0 Cl⁻. (B) NaDC-1-mediated succinate uptake was monitored using a radiolabeled ¹⁴C-succinate flux assay in cells expressing NaDC-1 in the presence or absence of WT or mutant SLC26A6, as indicated. The background signal monitored in control cells (transfected with and empty vector) was subtracted. *P < 0.05, **P < 0.01, ***P < 0.001, NS = P > 0.05.

Figure 3

The sulfate transporter and anti-sigma factor antagonist (STAS) domain mutations impair protein expression, trafficking to the plasma membrane but not the interaction with NaDC-1. (A) The expression of human SLC26A6 (hA6) monitored in lysates of HEK293T cells transfected with an empty vector SLC26A6(WT), SLC26A6(D674N), or SLC26A6 (R621G) compared to β-actin expression. (B) The membrane expression of the indicated proteins was monitored using a biotinylation assay after adjustment of the total protein levels as shown in the input blot. The interaction between NaDC-1, SLC26A6, and mutants was monitored using a co-immunoprecipitation (Co-IP) assay. The western blot analyses in (C, D) indicate that the SLC26A6-STAS domain mutations retain interaction with NaDC-1. *P < 0.05, **P < 0.01, ***P < 0.001, NS = P > 0.05.

Figure 4

The membrane expression of SLC26A6(D674N) is recovered by the mKate tag, but not by NaDC-1 expression. We monitored human SLC26A6 (hA6) trafficking to the plasma membrane by biotinylation using similar total protein concentrations (no adjustment). As shown in (A), in the presence of NaDC-1, the expression of slc26a6 was even lower than in the absence of NaDC-1. However, the mKate tagged SLC26A6(D674N)mKate protein showed membrane expression similar to that of SLC26A6(R621G)mKate (B). The images in (C) describe the cellular distribution of the indicated mKate tagged proteins compared to the cytoplasmic distribution of mKate alone.