It has been known for several centuries that nephrolithiasis (commonly referred to as kidney stones) is a significant health problem that may lead to loss of kidney function (1), and that it is associated with other morbidities such as hypertension and fractures (2,3). Nephrolithiasis is a complex multifactorial disease that is the result of interactions between environmental, dietary and genetic factors. Studies have shown that the lifetime risk of kidney stones can vary between 5–20%, and this is exhibiting an increasing trend (4,5). Whilst men are affected twice as much as women, in children, there is no bias towards one sex (6).

Ca²⁺ oxalate stones are the most prevalent type of kidney stones, and are responsible for 70–80% of cases of kidney stones in humans (7,8). Ca²⁺ oxalate stones are caused by elevated urinary Ca²⁺ and oxalate levels, and are termed hypercalciuria and hyperoxaluria, respectively (8). Hyperoxaluria is a major risk factor of Ca²⁺ oxalate stone formation, which leads to an increase in urinary saturation of Ca²⁺ to form Ca²⁺ oxalate stones (9). However, hyperoxaluria is primarily caused by three aspects, including enhanced absorption of oxalate by the intestine, internal production of oxalate by the liver and excretion of oxalate by the kidneys (10). Additionally, oxalate homeostasis is maintained by solute carrier family 26 member 6 (SLC26A6) in the intestinal and renal tubular epithelium, imbalances of which result in hyperoxaluria and hyperoxalemia, suggesting that oxalate secretion is dependent on the transcellular mechanisms of SLC26A6c (11).

Conversely, even in the absence of hypercalciuria, low concentrations of the Ca²⁺ chelator citrate in urine can promote the formation of Ca²⁺ stones, as urinary citrate can inhibit the crystallization and precipitation of Ca²⁺ in the renal calculi by chelating Ca²⁺ ions (8). In the vast majority of patients with Ca²⁺ kidney stones, they exhibit low urinary citrate excretion, and the incidence of hypocitraturia ranges from 19–60%. Therefore, sufficient urinary citrate concentration is also the key to preventing stone formation. Notably, Na⁺-dependent dicarboxylate-1 (NADC-1) reabsorbs most of the citrate in the proximal tubular apical membrane; thus, NADC-1 is one of the main determinants of renal calculi (12,13). This is consistent with another previous study, in which it was shown that SLC26A6 and NADC-1 transporters can function to prevent stone formation by dual method (14).

Similarly, succinate, an intermediate of the tricarboxylic acid cycle, is also absorbed by NADC-1 in the apical membrane of the proximal tubule (15). Previously, succinate was only regarded as an intermediate of the tricarboxylic acid cycle, but more recent data has suggested that it may function as a crucial extracellular signaling molecule, which is consistent with the discovery of the succinate-specific G-protein-coupled receptor succinate receptor 1 (SUCNR1), in the epithelium of several organs, such as the kidneys and intestines (16). Hyperperfusion studies and intravenous results suggest that succinate stimulates renin secretion from granular cells at the juxtaglomerular apparatus (17), confirming that an increase in blood pressure can be induced through the SUCNR1 signaling pathway (18,19), proving a novel direction for the association between NADC-1 and calculus-related hypertension.

The question as to how the formation of renal calculi and Ca²⁺ oxalate stones are associated with hypertension has not been fully addressed. The emergence of SLC26A6, and in particular, the synergistic function of SLC26A6 and NADC-1, has shed light on the current understanding of the mechanisms underlying the processes involved in the formation of kidney stones, as well as the association between nephrolithiasis and hypertension. In the present review, the family, structure and functional expression of the two proteins are first described in order to further understand the significance of SLC26A6 and NADC-1 in human physiology. Next, this review examined the results from studies on oxalate and citrate transport by the kidney tubule, highlighting areas where the transporters may be involved in the processes of Ca²⁺ oxalate formation, and summarized the reported molecular mechanisms of the synergistic action between SLC26A6 and NADC-1 in renal tubular epithelial cells in the literature. Additionally, a summary of the function of SLC26A6/NADC-1 in hypertension associated with Ca²⁺ oxalate kidney stones is provided, indicating the possible role of the two transporters in the formation of Ca²⁺ oxalate kidney stones and their implications for hypertension.

The phylogenetically ancient SLC26-sulfate transporter (SulP) gene family is a part of the adenomatous polyposis coli gene superfamily, encoding membrane proteins that exchange electroneutral or univalent and bivalent anionic substrates, and are of crucial importance in metabolic processes, pH regulation and electrolyte homeostasis. Notably, the SLC26 or SulP proteins are universally expressed in prokaryotes and eukaryotes (20–22). Bacterial SLC26-related SulP proteins and SLC26-related Sultr proteins are the major contributors to the marine carbon cycle and sulfate transport by yeast, algae and plants (20). In humans, the SLC26 family plays an important role as a multifunctional anion transporter in various physiological activities to maintain homeostasis in the body, including 11 proteins (SLC26A1-A11) (Table I), of which A10 is a pseudogene (23). Amongst these, the protein encoded by the gene SLC26A6 exhibits the most extensive exchange function of the SLC26 family members, particularly with regard to oxalate, where it has a high affinity (24).

The SLC26A6 gene was cloned on the basis of homology to the other two members of the SLC26 family, SLC26A3 and SLC26A4 (5). The SLC26A6 gene maps to chromosome 3p21.3-4, which consists of 21 relatively short exons interrupted by 20 intronic sequences (25). The SLC26A6 protein has a molecular mass of 82 kDa, functions as a secondary cytomembrane transporter and consists of 759 amino acids with a predicted topological structure of 14 transmembrane α-helices (the 3rd and 10th helices do not completely span the entire cytomembrane) and an intracellular -NH₂ and -COOH terminal (26) (Fig. 1). The -COOH terminal of SLC26A6 possesses a conserved domain, namely sulfate transporter and anti-sigma factor antagonist (STAS), which plays a vital role in regulating protein function and expression (21,27). Furthermore, the -COOH terminal of SLC26A6 contains a consensus PDZ interaction motif identical to that found in the cystic fibrosis transmembrane conductance regulator, which provides interaction sites for other interacting proteins and ultimately participates in the regulation of membrane protein function (27). There are also three alternative splicing variants of the SLC26A6 gene, termed SLC26A6A, SLC26A6C and SLC26A6D, which consist of 12, 8 and 12 transmembrane domains, respectively. SLC26A6A is primarily a spicing variant expressed in the small intestine and colon (28). SLC26A6D is primarily expressed in the kidney and pancreas, whereas SLC26A6C is faintly expressed in the human kidney (29), suggesting that various slc26a6 variants are tissue specific. Similarly, the SLC26A6 transporter is widely expressed in various organs, such as the salivary glands (30), heart (31), intestine (32,33), pancreas (34), kidney (35) and uterus (36), with the highest expression observed in the apical membrane of the kidney proximal tubule and small intestinal villi (25). Heterologous expression studies have demonstrated that mouse Slc26A6 and human Slc26A6 can function in multiple transport modes, including acting as a coupled ion channel to mediate the exchange of a cluster of anions, including HCO₃⁻, Cl⁻ and OXa²⁻ in epithelial cells, and can also act as an uncoupled ion channel to transport SNC⁻, NO₃⁻ and Cl⁻, amongst others (20,31,37–42). In the present review, a focus is placed on the function of SLC26A6 as an Cl⁻_(in)/OXa²⁻_(out) exchanger in maintaining the dynamic balance of oxalate equilibrium, as the deletion of the SLC26A6 gene can lead to a decrease of intestinal secretion, which will lead to hyperoxalemia and hyperoxaluria (43). Notably, SLC26A6 is intricately associated with renal Ca²⁺-oxalate stones (6).

The SLC13 gene family consists of five sequence-related members that have been identified in several animals, plants, yeast and bacteria. The proteins encoded by these genes are divided into two distinct groups: The Na⁺-sulphate co-transporters and the Na⁺-carboxylate co-transporters. Members of the SLC13 family include renal Na⁺-dependent inorganic sulphate transporter-1 (SLC13A1), Na⁺-dependent dicarboxylate transporters NADC-1/SDCT1 (SLC13A2), NADC-3/SDCT2 (SLC13A3), sulphate transporter-1 (SLC13A4) and Na⁺-coupled citrate transporter (SLC13A5) (Table II) (44).

Initially, the original SLC13 family members were isolated from Xenopus oocytes. The first member was Slc13a1, encoding a rat Na⁺-sulphate cotransporter (45), followed by Slc13a2, encoding a rabbit Na⁺-dicarboxylate cotransporter (13), the Xenopus Slc13a2 (46) and the winter flounder Slc13a3 (47). SLC13A2 has been isolated from five vertebrates: Humans (48), rabbits (13), mice (49), rats (50–52) and Xenopus (46). The human NADC-1 gene contains 12 exons consisting of 1953 base pairs, encoding 593 amino acids (48), and the gene is found on chromosome 17p11.1-q11.1 (53). NADC-1 possesses an 11-transmembrane α-helices topological structure, with an intracellular -NH₂ terminal and an extracellular -COOH terminal (Fig. 2). There is a conserved N-glycosylation site (Asn578) in the extracellular -COOH terminal, which is an important structure to control the function and expression of NADC-1 (54). In addition, there are two N-glycosylation sites in the -COOH terminal of mouse NADC-1, namely Asn584 and Asn580 (49). NADC-1 is widely expressed in various tissues, particularly in kidney and gastrointestinal epithelium. Western blotting showed that human NADC-1 was present in the kidneys and intestines (48), and rabbit NADC-1 was strongly expressed in the kidneys and jejunum, with weaker expression detected in the liver (13). Similarly, mouse and rat NADC-1 were also detected in the kidneys and intestines (49). In immunocytochemical experiments and in situ hybridization studies, rat NADC-1 protein was confirmed to be present in the outer stripe of the outer medulla and in the luminal membranes of the renal superficial cortex (51). As a Na⁺-coupled symporter, NADC-1 transporter exhibits strong cation selectivity for Na⁺, the coupling ratio of Na⁺ to anions is 3:1, and it has a preference for divalent anions, including tricarboxylic or Krebs cycle intermediates, such as succinate and citrate, with a high affinity for succinate and a lower affinity for citrate (44,54,55). It is notable that >65% of the intermediate products of the Krebs cycle excreted in the kidney are reabsorbed by NADC-1 in the proximal tubules for intracellular metabolism or exchange with organic anions in the process of organic anion secretion (55). In particular, NADC-1 can affect Ca²⁺ citrate chelates by regulating the concentration of citrate to prevent the formation of kidney stones, as citrate competes for oxalate to bind with ions with higher affinity, such that supersaturation of stones will not be achieved at high concentrations of citrate. Furthermore, it has been shown that ~50% of patients with nephrolithiasis exhibit hypocitraturia, consistent with the role of NADC-1 as a Ca²⁺ inhibitor (56).

As aforementioned, Ca²⁺ oxalate stones are the most prevalent type of renal stones, and are predominately determined by the high levels of urinary oxalate and urinary Ca²⁺, or the decrease in urinary citrate concentration (the major Ca²⁺ inhibitor) (8). There are two sources of oxalate in the human body, absorption through the intestinal exogenous paracellular pathway and endogenous liver production (57). Oxalate is primarily excreted by the intestines and kidneys, and >90% of oxalate is excreted via urine. Thus, the secretion of oxalate in the kidney plays a crucial role in the development of nephrolithiasis. Jiang et al (43) showed that the exchange of Cl⁻_(in)/OXa²⁻_(out) at the apical membrane of the proximal tubule is entirely mediated by SLC26A6, consistent with its expression on the brush border membrane of the renal proximal tubule cells (24). Similarly, in humans, previous studies suggested that >65% of citrate is reabsorbed in the renal tubule after glomerular filtration (56), whereas in vitro perfusion studies using rabbit nephrons showed that citrate is taken up exclusively in the proximal tubule (58). In the proximal tubule, the reabsorption of citrate and succinate in the apical membrane is predominantly mediated by NADC-1, via Na⁺ coupled electrogenic exchange (48,49,55).

There is an increasing body of studies that have suggested that even in the absence of hypercalciuria, the simultaneous occurrence of hyperoxaluria and hypocitraturia can trigger the formation of Ca²⁺-oxalate stones, which has increased widespread concern amongst researchers. Ohana et al (14) studied the molecular mechanisms involving the oxalate transporter SLC26A6 and citrate transporter NADC-1 in controlling the dynamic balance of urinary citrate and oxalate. In the study, NADC-1 and SLC26A6 were co-expressed in Xenopus laevis oocytes and the activity of the two exchangers, the Na⁺-dicarboxylate transporter and oxalate transporter, were monitored. The results showed that NADC-1 increased SLC26A6 activity, in turn increasing Cl⁻-oxalate exchange by 30% and similarly increasing 1Cl⁻−2HCO₃⁻ exchange, and that there were no changes in the stoichiometry of exchange (14). Conversely, the study indicated that SLC26A6 restricted the activity of NADC-1 and that the effect of SLC26A6 in the active state was more significant than that in the inactive state. Notably, other members of the SLC26 family also exhibit an inhibitory effect on NADC-1, such as SLC26A3 (59) (Fig. 3).

In addition, Khamaysi et al (60) recently showed that the SLC26A6/NADC-1 complex participates in hypertension by regulating local succinate levels (Fig. 3). The study additionally demonstrated the synergistic structural domain of the complex. It was concluded that the SLC26A6 and inositol triphosphate (IP₃) receptor-binding protein (IRBIT) inhibited NADC-1-mediated succinate transport by ~50%, with a superimposed effect that made the inhibition more potent. In turn, NADC-1 elevated SLC26A6 transporter activity and increased IRBIT release by transporting succinate to enrich the concentration of IP₃. In addition, the interaction between NADC-1/SLC26A6 is largely mediated through the amino acid K107 in the vcINDY H4c-like region of NADC-1 (61) and E613 in the SLC26A6-STAS domain, and the STAS domain of SLC26A6 has previously been shown to be the transport determining functional domain (60). Succinate transport by NADC-1 can activate phospholipase C β to increase Ca²⁺ and IP3 levels by stimulating SUCNR1 (62,63), whereas IRBIT competes with IP₃ for binding to the IP₃ receptor protein. When IP₃ levels increase, it triggers an increase in IRBIT release (64), and IRBIT can act on various transporters, such as activating the anion transporter SLC26A6 (65) and inhibiting the succinate transporter NADC-1 on the apical membrane of the lumen. In addition, IRBIT inhibits the NADC-3 transporter on the basolateral membrane of the proximal tubule, which mediates citrate/succinate influx from the interstitium into the epithelial cells (24), orchestrating the succinate inflow to control succinate absorption and metabolism. The organic anion transporters 1–3 that extrude succinate from the proximal tubule basement membrane are also significantly inhibited by IRBIT (66). If the regulation of the SLC26A6 and NADC-1 transporters becomes imbalanced, it can readily lead to an increase in serum succinate and calculus-related salt-independent hypertension. Although several hypotheses have been suggested to describe the association between kidney stones and hypertension, such as tubulointerstitial damage and altered renal handling of Ca²⁺, amongst others (2), succinate stimulates renin secretion and increases the risk of developing hypertension, making the mechanism suggested by Khamaysi et al (60), wherein NADC-1/SLC26A6 mediation of citrate and succinate contribute to the association between renal calculi and hypertension, more convincing.

Another association between NADC-1 and SLC26A6 is the acid-base balance. Immunohistochemistry has shown that patients with low pH in urine are more likely to exhibit higher NADC-1 expression (67), which is consistent with chronic acid intake-induced renal stone formation and upregulation of NADC-1 mRNA expression in a rat model. The reason behind this may be that citrate, rather than the succinate, only present in the form of tricarboxylic acid under alkaline conditions, is not reabsorbed in the proximal tubule. That is, citrate can only be reabsorbed by the NADC-1 transporter in the proximal renal tubule in its divalent form (51). Notably, in vitro microperfusion studies of proximal tubule segments in mice have shown that SLC26A6 also acts as a major HCO₃⁻/Cl⁻ exchanger (35), leading to the hypothesis that SLC26A6 can inhibit NADC-1 by increasing the pH of the urine, although this hypothesis remains to be confirmed.

The co-expression of NADC-1/SLC26A6 in Xenopus laevis oocytes and extensive in vitro experiments has further deepened the current understanding of the synergistic molecular mechanisms involved in the formation of Ca²⁺ oxalate stones and the associated hypertension, whereas the understanding of the molecular mechanism of Ca²⁺ oxalate stone formation by the secretion of oxalate from SLC26A6 has been vastly improved by numerous studies in mouse models (11,68,69). In order to improve the current understanding of the transporter function relevant to nephrolithiasis, a micro-perfusion study found that the renal function of Slc26a6-null mice did not change significantly, but the Cl⁻/oxalate exchange mediated by the SLC26A6 transporter was abolished completely (35), meaning that the Cl⁻/oxalate exchange in the apical membrane of the renal proximal tubule is entirety mediated by SLC26A6. Similarly, Jiang et al (43) and Freel et al (33) also established SLC26A6 null mice that demonstrated a 4-fold increase in urine oxalate excretion. A large amount of oxalate in urine can increase the protein expression of NADPH oxidase in the renal epithelial cells, which leads to oxidative stress in cells to promote the formation of renal stones (70). This is consistent with the high expression of A6 found by Jiang et al (71) in NRE-52 cells, which increased damage to the cells and resulted in increased crystal adhesion to the cells. Moreover, oxalate is also the most common type of kidney stone, specifically Ca²⁺ oxalate kidney stones (7), thus a large amount of oxalate in urine is a high risk factor for nephrolithiasis.

Several SLC26A6 variants were also found during the literature review, such as the SLC26A6 (V206M) and SLC26A6 (G539R) polymorphisms, which can generate the phenotypes of hyperoxaluria and hyperoxalemia to promote the formation of kidney stones (68,72,73). Conversely, research on NADC-1 is relatively limited, and only one related mutant has been identified. The variant I550V in the NADC-1 transporter is reported to decrease urinary citrate excretion, although it has a mild effect on the transporter function, resulting in a 20% decrease in transporter activity (74). Unexpectedly, the two variants were located in the region encoding the STAS domain, as found in SLC26A6 by Shimshilashvili et al (11), further demonstrating the crucial role of the SLC26A6/NADC-1 complex in maintaining the dynamic balance of citrate/succinate and oxalate to prevent kidney stones from forming. The two STAS domain polymorphisms SLC26A6 (R621G) and SLC26A6 (D673N or D674N) both decreased SLC26A6 expression, transport activity and mutual mediation with transporter NADC-1. Notably, the former variant resulted in a significantly lower concentration of urinary citrate and normal concentrations of urinary oxalate were sufficient to induce kidney stones. However, the latter variant had a high urinary oxalate concentration and a 50% higher citrate concentration than the former variant, but this did not successfully induce kidney stone formation. This demonstrates the importance of SLC26A6 in mediating urinary citrate concentration, that is, it emphasizes the role of SLC26A6 and NADC-1 in preventing the formation of kidney stones. Furthermore, partner proteins that form complexes in the membrane, as demonstrated for the cystic fibrosis transmembrane conductance regulator (CFTR) (75), can compensate for the weakening of the SLC26A6 (D674N) polymorphism transport function, which makes the SLC26A6-STAS domain a potential target for the treatment of diseases caused by transporter dysfunction.

In the present review, the association between the NADC-1/SLC26A6 transporter and nephrolithiasis and calculus-related hypertension was discussed. The roles of oxalate transporter SLC26A6 and citrate transporter NADC-1 in nephrolithiasis and calculus-related hypertension remain elusive, and the synergistic molecular mechanisms between these transporters require further investigation. Nevertheless, SLC26A6 and NADC-1 transporters may serve as a future direction in the study of kidney stones and calculus-related hypertension.

Various variants of SLC26A6 and NADC-1 have been shown to be involved in the formation of kidney stones. However, to the best of our knowledge, there are no studies on the synergistic region of the two transporters in nephrolithiasis and calculus-related hypertension. That is to say, the crucial role of SLC26A6/NADC-1 in kidney stones and calculus-related hypertension requires further study, perhaps with a particular focus on SLC26A6 and NADC-1 in the intestinal villus epithelium. Conversely, further verification is needed with regard to whether the SLC26A6 transporter can function as an HCO₃⁻/Cl⁻ exchanger to mediate the activity of NADC-1 transporter by adjusting the pH of urine. In the present review, discussion around the use of soluble polypeptides for management of transport disorders caused by the functional structural variations in the SLC26A6 transporter were discussed, highlighting a novel treatment direction in the management of kidney stones and calculus-related hypertension.

In conclusion, SLC26A6/NADC-1 is a promising target and potential marker for nephrolithiasis and calculus-related hypertension disease treatment in future. However, drugs targeting SLC26A6/NADC-1 need to be examined further in animal experiments and clinical studies.