The mammalian intestine sits at the center of oxalate homeostasis, serving both as a site of oxalate absorption from the diet and, together with the kidney, as a site of oxalate excretion into the stool. Physiologists have long known that intestinal oxalate absorption is predominantly passive, moving through the paracellular pathway between cells, while secretion is active and transcellular, requiring specific transport proteins. The SLC26 family of anion exchangers has emerged as the critical group of transporters governing these processes. Mouse studies have shown that SLC26A6 null mice have a critical defect in small intestinal transcellular oxalate secretion, leading to enhanced absorption of ingested oxalate, high blood oxalate, high urinary oxalate, and calcium oxalate kidney stones. Meanwhile, SLC26A3 null mice show decreased transcellular ileal, cecal, and distal colonic oxalate absorption and a 66 percent reduction in urinary oxalate excretion, underscoring its role in transcellular intestinal oxalate absorption.

A new study published in Physiological Reports has now dissected the relative contributions of four members of this transporter family, SLC26A1, SLC26A2, SLC26A3, and SLC26A6, to oxalate movement across human intestinal Caco2-BBE cells, a widely used human intestinal cell line. Using siRNA knockdown to silence each transporter individually and in combination, combined with radioactive oxalate flux assays, the researchers quantified exactly how much of the oxalate crossing these cells depends on each exchanger. The results provide the most complete picture to date of how the human gut epithelium handles oxalate at the molecular level, and they validate the cell model as a screening platform for drugs designed to lower oxalate levels in the body.

The experimental approach was technically rigorous. Caco2-BBE cells were grown as confluent monolayers on Transwell membrane filters, allowing the apical and basolateral sides to be studied separately. Cells were transfected with siRNAs targeting each transporter, or with a negative control siRNA, and knockdown efficiency was verified by real-time quantitative PCR for mRNA expression and by Western blot analysis for total protein abundance. Functional consequences were measured using radioactive carbon-14 labeled oxalate uptake assays performed in chloride-free solutions, with a six-minute incubation period chosen because it falls within the linear range of oxalate uptake by these cells. Statistical significance was assessed by one-way analysis of variance followed by Tukey post hoc testing.

The findings on the apical side, where oxalate exits the enterocyte into the gut lumen during secretion, were decisive. Silencing SLC26A2, at 74 percent reduced mRNA expression, reduced apical oxalate transport by 31.1 percent. Silencing SLC26A6, at 64 percent reduced total protein abundance, reduced apical oxalate transport by 57 percent. Most strikingly, combined silencing of both SLC26A2 and SLC26A6 in the same batches of cells reduced apical oxalate transport by 78 percent. These numbers support the hypothesis that most, if not all, apical oxalate transport in these cells is mediated by the combined action of SLC26A2 and SLC26A6, with SLC26A6 making the considerably larger contribution. The negative control siRNA had no significant effect on any of these measurements, confirming that the reductions were specific to the targeted transporters rather than a general consequence of the silencing procedure.

On the basolateral side, where oxalate enters the enterocyte from the blood before being secreted into the lumen, the study identified SLC26A1 as a significant player. Silencing SLC26A1 reduced its mRNA expression by 68.4 percent and its total protein abundance by 59.5 percent, and functionally reduced basolateral oxalate transport by 30.9 percent. This provides direct evidence for SLC26A1 as a basolateral oxalate transporter working in series with the apical exchangers to complete transcellular oxalate secretion. The finding is consistent with mouse studies showing that SLC26A1 null mice have hyperoxalemia, hyperoxaluria, and calcium oxalate kidney stones, likely due to decreased intestinal oxalate secretion, and with clinical association studies linking SLC26A1 variants to nephrolithiasis susceptibility.

In contrast, SLC26A3, despite being knocked down to 78.7 percent reduced total protein abundance, showed no significant contribution to apical oxalate transport in these cells, with only a statistically insignificant 13 percent reduction in oxalate uptake. This result is intriguing because SLC26A3 null mice show decreased transcellular intestinal oxalate absorption and reduced urinary oxalate excretion, and a small molecule SLC26A3 inhibitor decreased oxalate absorption by 70 percent in mouse colonic loops. However, previous in vitro studies have shown that human SLC26A3 transports oxalate poorly even at very high oxalate concentrations when expressed in Xenopus oocytes, suggesting that chloride-bicarbonate exchange is its main physiological function. The discrepancy between the in vitro and in vivo data may reflect differences in transporter behavior between settings, where SLC26A3 transport activity could be modulated by regulatory factors such as microbiota and dietary factors, or by the prevailing electrochemical gradients and interactions among multiple transport pathways in the intact animal. It is possible that SLC26A3 deletion results in loss of counter-ion driver gradients, leading to reduced transcellular oxalate absorption by unknown oxalate transporters rather than being directly mediated by SLC26A3 itself.

The study also addressed the in vivo relevance of the cell model. SLC26A6 is highly expressed in mouse small intestine with much lower expression in the colon, yet its expression is more than 1.8-fold higher in human colon compared to human small intestine, suggesting that SLC26A6 likely plays an important role in human transcellular colonic oxalate secretion. Previous work by the same group showed that factors derived from Oxalobacter formigenes, an oxalate-degrading gut bacterium, stimulate oxalate transport in Caco2-BBE cells through SLC26A6 and SLC26A2, and that rectal administration of these bacterial culture factors reduced urinary oxalate excretion in hyperoxaluric mice by stimulating transcellular distal colonic oxalate secretion. Because the knockdown studies for SLC26A2, SLC26A6, and the combined double knockdown were performed simultaneously in the same batches of cells, the researchers could directly compare their relative contributions, leading them to speculate that SLC26A6 mediates about 65 to 75 percent of the total apical oxalate transport while SLC26A2 accounts for the remaining 25 to 35 percent.

Therapeutically, the implications are substantial. Enhancing transcellular intestinal oxalate secretion or reducing transcellular oxalate absorption has great potential to lower both plasma and urinary oxalate levels, which could slow chronic kidney disease progression and improve cardiovascular and renal transplant outcomes. SLC26A2 and SLC26A6 are attractive targets for enhancing oxalate secretion, and the study establishes Caco2-BBE cells as a valid human intestinal model for investigating the molecular mechanisms regulating these transporters and for screening novel agents targeting them. The results also establish the model as a reasonable platform for studying SLC26A1 regulation, since its activation has the potential to enhance transepithelial oxalate secretion. The researchers had previously identified positive hits as potential SLC26A6 activators through a virtual screen of a large compound library, with several hits stimulating apical oxalate uptake in these cells by more than twofold.

A limitation acknowledged by the authors is that while they confirmed the impact of siRNA knockdown on SLC26A1, SLC26A3, and SLC26A6 total protein abundance, they could not do so for SLC26A2 because none of the tested antibodies yielded reliable signals for immunoblotting. However, they consider the observed reduced functional oxalate transport as indirect evidence that the decreased SLC26A2 mRNA expression was translated into some degree of reduced protein abundance. Taken together, the study confirms that SLC26A2 and SLC26A6 account for most apical oxalate transport in human intestinal cells, identifies SLC26A1 as a significant basolateral oxalate transporter, and rules out a major direct role for SLC26A3 in apical oxalate transport in this model. These findings open the door to screening and testing novel drugs, including small molecule activators and positive allosteric modulators, targeting SLC26A1, SLC26A2, and SLC26A6, with significant therapeutic potential for hyperoxaluria, hyperoxalemia, and related disorders.

Subject of Research: Intestinal oxalate transport mediated by SLC26 anion exchangers in human Caco2-BBE cells

Article Title: Contributions of the anion exchangers SLC26A1, SLC26A2, SLC26A3, and SLC26A6 to intestinal oxalate transport in human Caco2‐BBE cells

Article References: Abdelhalim, K. A., Arvans, D., Wang, Y., Bashir, M. E. H., & Hassan, H. A. (2026). Contributions of the anion exchangers SLC26A1 , SLC26A2 , SLC26A3 , and SLC26A6 to intestinal oxalate transport in human Caco2‐ BBE cells. Physiological Reports, 14(19), Article e71125. https://doi.org/10.14814/phy2.71125

Image Credits: AI Generated

DOI: 10.14814/phy2.71125

Keywords: oxalate transport, SLC26A6, SLC26A2, SLC26A1, SLC26A3, Caco2-BBE cells, kidney stones, hyperoxaluria, anion exchangers, siRNA knockdown, intestinal epithelium, Physiological Reports