Oxalate secretion is stimulated by a cAMP-dependent pathway in the mouse cecum.

Elevated levels of the intracellular second messenger cAMP can stimulate intestinal oxalate secretion however the membrane transporters responsible are unclear. Oxalate transport by the chloride/bicarbonate (Cl-/HCO3-) exchanger Slc26a6 or PAT-1 (Putative Anion Transporter 1), is regulated via cAMP when expressed in Xenopus oocytes and cultured cells but whether this translates to the native epithelia is unknown. This study investigated the regulation of oxalate transport by the mouse intestine focusing on transport at the apical membrane hypothesizing PAT-1 is the target of a cAMP-dependent signaling pathway. Adopting the Ussing chamber technique we measured unidirectional 14C-oxalate and 36Cl- flux ([Formula: see text] and [Formula: see text]) across distal ileum, cecum and distal colon, employing forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) to trigger cAMP production. FSK/IBMX initiated a robust secretory response by all segments but the stimulation of net oxalate secretion was confined to the cecum only involving activation of [Formula: see text] and distinct from net Cl- secretion produced by inhibiting [Formula: see text]. Using the PAT-1 knockout (KO) mouse we determined cAMP-stimulated [Formula: see text] was not directly dependent on PAT-1, but it was sensitive to mucosal DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), although unlikely to be another Cl-/HCO3- exchanger given the lack of trans-stimulation or cis-inhibition by luminal Cl- or HCO3-. The cAMP-activated oxalate efflux was reliant on CFTR (Cystic Fibrosis Transmembrane conductance Regulator) activity, but only in the presence of PAT-1, leading to speculation on the involvement of a multi-transporter regulatory complex. Further investigations at the cellular and molecular level are necessary to define the mechanism and transporter(s) responsible.

Oxalate Flux Across the Intestine: Contributions from Membrane Transporters.

Epithelial oxalate transport is fundamental to the role occupied by the gastrointestinal (GI) tract in oxalate homeostasis. The absorption of dietary oxalate, together with its secretion into the intestine, and degradation by the gut microbiota, can all influence the excretion of this nonfunctional terminal metabolite in the urine. Knowledge of the transport mechanisms is relevant to understanding the pathophysiology of hyperoxaluria, a risk factor in kidney stone formation, for which the intestine also offers a potential means of treatment. The following discussion presents an expansive review of intestinal oxalate transport. We begin with an overview of the fate of oxalate, focusing on the sources, rates, and locations of absorption and secretion along the GI tract. We then consider the mechanisms and pathways of transport across the epithelial barrier, discussing the transcellular, and paracellular components. There is an emphasis on the membrane-bound anion transporters, in particular, those belonging to the large multifunctional Slc26 gene family, many of which are expressed throughout the GI tract, and we summarize what is currently known about their participation in oxalate transport. In the final section, we examine the physiological stimuli proposed to be involved in regulating some of these pathways, encompassing intestinal adaptations in response to chronic kidney disease, metabolic acid-base disorders, obesity, and following gastric bypass surgery. There is also an update on research into the probiotic, Oxalobacter formigenes, and the basis of its unique interaction with the gut epithelium. © 2021 American Physiological Society. Compr Physiol 11:1-41, 2021.

The role of NHE3 (Slc9a3) in oxalate and sodium transport by mouse intestine and regulation by cAMP.

Intestinal oxalate transport involves Cl- /HCO3- exchangers but how this transport is regulated is not currently known. NHE3 (Slc9a3), an apical Na+ /H+ exchanger, is an established target for regulation of electroneutral NaCl absorption working in concert with Cl- /HCO3- exchangers. To test whether NHE3 could be involved in regulation of intestinal oxalate transport and renal oxalate handling we compared urinary oxalate excretion rates and intestinal transepithelial fluxes of 14 C-oxalate and 22 Na+ between NHE3 KO and wild-type (WT) mice. NHE3 KO kidneys had lower creatinine clearance suggesting reduced GFR, but urinary oxalate excretion rates (µmol/24 h) were similar compared to the WT but doubled when expressed as a ratio of creatinine. Intestinal transepithelial fluxes of 14 C-oxalate and 22 Na+ were measured in the distal ileum, cecum, and distal colon. The absence of NHE3 did not affect basal net transport rates of oxalate or sodium across any intestinal section examined. Stimulation of intracellular cAMP with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) led to an increase in net oxalate secretion in the WT distal ileum and cecum and inhibition of sodium absorption in the cecum and distal colon. In NHE3 KO cecum, cAMP stimulation of oxalate secretion was impaired suggesting the possibility of a role for NHE3 in this process. Although, there is little evidence for a role of NHE3 in basal intestinal oxalate fluxes, NHE3 may be important for cAMP stimulation of oxalate in the cecum and for renal handling of oxalate.

The anion exchanger PAT-1 (Slc26a6) does not participate in oxalate or chloride transport by mouse large intestine.

The membrane-bound transport proteins responsible for oxalate secretion across the large intestine remain unidentified. The apical chloride/bicarbonate (Cl-/HCO3-) exchanger encoded by Slc26a6, known as PAT-1 (putative anion transporter 1), is a potential candidate. In the small intestine, PAT-1 makes a major contribution to oxalate secretion but whether this role extends into the large intestine has not been directly tested. Using the PAT-1 knockout (KO) mouse, we compared the unidirectional absorptive ([Formula: see text]) and secretory ([Formula: see text]) flux of oxalate and Cl- across cecum, proximal colon, and distal colon from wild-type (WT) and KO mice in vitro. We also utilized the non-specific inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) to confirm a role for PAT-1 in WT large intestine and (in KO tissues) highlight any other apical anion exchangers involved. Under symmetrical, short-circuit conditions the cecum and proximal colon did not transport oxalate on a net basis, whereas the distal colon supported net secretion. We found no evidence for the participation of PAT-1, or indeed any other DIDS-sensitive transport mechanism, in oxalate or Cl- by the large intestine. Most unexpectedly, mucosal DIDS concurrently stimulated [Formula: see text] and [Formula: see text] by 25-68% across each segment without impacting net transport. For the colon, these changes were directly proportional to increased transepithelial conductance suggesting this response was the result of bidirectional paracellular flux. In conclusion, PAT-1 does not contribute to oxalate or Cl- transport by the large intestine, and we urge caution when using DIDS with mouse colonic epithelium.

The teleost fish intestine is a major oxalate-secreting epithelium.

Oxalate is a common constituent of kidney stones, but the mechanism of its transport across epithelia is not well understood. With prior research on the role of the intestine focused on mammals, the present study considered oxalate handling by teleost fish. Given the osmotic challenge of seawater (SW), marine teleosts have limited scope for urinary oxalate excretion relative to freshwater (FW) taxa. The marine teleost intestine was hypothesized as the principal route for oxalate elimination, thus demanding epithelial secretion. To test this, intestinal 14C-oxalate flux was compared between FW- and SW-acclimated sailfin molly (Poecilia latipinna). In SW, oxalate was secreted at remarkable rates (367.90±22.95 pmol cm-2 h-1), which were similar following FW transfer (387.59±27.82 pmol cm-2 h-1), implying no regulation by salinity. Nevertheless, this ability to secrete oxalate at rates 15-19 times higher than the mammalian small intestine supports this proposal of the teleost gut as a major, previously unrecognized excretory pathway.

125 Iodide as a surrogate tracer for epithelial chloride transport by the mouse large intestine in vitro.

NEW FINDINGS: What is the central question of this study? The tracer 36 Cl- , currently used to measure transepithelial Cl- fluxes, has become prohibitively expensive, threatening its future use. 125 Iodide, previously validated alongside 36 Cl- as a tracer of Cl- efflux by cells, has not been tested as a surrogate for 36 Cl- across epithelia. What is the main finding and its importance? We demonstrate that 125 I- can serve as an inexpensive replacement for measuring Cl- transport across mouse large intestine, tracking Cl- transport in response to cAMP stimulation (inducing Cl- secretion) in the presence and absence of the main gastrointestinal Cl- -HCO3- exchanger, DRA. ABSTRACT: Chloride transport is important for driving fluid secretion and absorption by the large intestine, with dysregulation resulting in diarrhoea-associated pathologies. The radioisotope 36 Cl- has long been used as a tracer to measure epithelial Cl- transport but is prohibitively expensive. 125 Iodide has been used as an alternative to 36 Cl- in some transport assays but has never been validated as an alternative for tracing bidirectional transepithelial Cl- fluxes. The goal of this study was to validate 125 I- as an alternative to 36 Cl- for measurement of Cl- transport by the intestine. Simultaneous fluxes of 36 Cl- and 125 I- were measured across the mouse caecum and distal colon. Net Cl- secretion was induced by the stimulation of cAMP with a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Unidirectional fluxes of 125 I- correlated well with 36 Cl- fluxes after cAMP-induced net Cl- secretion, occurring predominantly through a reduction in the absorptive mucosal-to-serosal Cl- flux rather than by stimulation of the secretory serosal-to-mucosal Cl- flux. Correlations between 125 I- fluxes and 36 Cl- fluxes were maintained in epithelia from mice lacking DRA (Slc26a3), the main Cl- -HCO3- exchanger responsible for Cl- absorption by the large intestine. Lower rates of Cl- and I- absorption in the DRA knockout intestine suggest that DRA might have a previously unrecognized role in iodide uptake. This study validates that 125 I- traces transepithelial Cl- fluxes across the mouse large intestine, provides insights into the mechanism of net Cl- secretion and suggests that DRA might be involved in intestinal iodide absorption.

Oxalate transport by the mouse intestine in vitro is not affected by chronic challenges to systemic acid-base homeostasis.

In rats, we recently showed how a chronic metabolic acidosis simultaneously reduced urinary oxalate excretion and promoted oxalate secretion by the distal colon leading to the proposition that acid-base disturbances may trigger changes to renal and intestinal oxalate handling. The present study sought to reproduce and extend these observations using the mouse model, where the availability of targeted gene knockouts (KOs) would offer future opportunities to reveal some of the underlying transporters and mechanisms involved. Mice were provided with a sustained load of acid (NH4Cl), base (NaHCO3) or the carbonic anhydrase inhibitor acetazolamide (ATZ) for 7 days after which time the impacts on urinary oxalate excretion and its transport by the intestine were evaluated. Mice consuming NH4Cl developed a metabolic acidosis but urinary oxalate was only reduced 46% and not statistically different from the control group, while provision of NaHCO3 provoked a significant 2.6-fold increase in oxalate excretion. For mice receiving ATZ, the rate of urinary oxalate excretion did not change significantly. Critically, none of these treatments altered the fluxes of oxalate (or chloride) across the distal ileum, cecum or distal colon. Hence, we were unable to produce the same effects of a metabolic acidosis in mice that we had previously found in rats, failing to find any evidence of the 'gut-kidney axis' influencing oxalate handling in response to various acid-base challenges. Despite the potential advantages offered by KO mice, this model species is not suitable for exploring how acid-base status regulates oxalate handling between the kidney and intestine.

Absence of the sulfate transporter SAT-1 has no impact on oxalate handling by mouse intestine and does not cause hyperoxaluria or hyperoxalemia.

The anion exchanger SAT-1 [sulfate anion transporter 1 (Slc26a1)] is considered an important regulator of oxalate and sulfate homeostasis, but the mechanistic basis of these critical roles remain undetermined. Previously, characterization of the SAT-1-knockout (KO) mouse suggested that the loss of SAT-1-mediated oxalate secretion by the intestine was responsible for the hyperoxaluria, hyperoxalemia, and calcium oxalate urolithiasis reportedly displayed by this model. To test this hypothesis, we compared the transepithelial fluxes of 14C-oxalate, 35SO42- , and 36Cl- across isolated, short-circuited segments of the distal ileum, cecum, and distal colon from wild-type (WT) and SAT-1-KO mice. The absence of SAT-1 did not impact the transport of these anions by any part of the intestine examined. Additionally, SAT-1-KO mice were neither hyperoxaluric nor hyperoxalemic. Instead, 24-h urinary oxalate excretion was almost 50% lower than in WT mice. With no contribution from the intestine, we suggest that this may reflect the loss of SAT-1-mediated oxalate efflux from the liver. SAT-1-KO mice were, however, profoundly hyposulfatemic, even though there were no changes to intestinal sulfate handling, and the renal clearances of sulfate and creatinine indicated diminished rates of sulfate reabsorption by the proximal tubule. Aside from this distinct sulfate phenotype, we were unable to reproduce the hyperoxaluria, hyperoxalemia, and urolithiasis of the original SAT-1-KO model. In conclusion, oxalate and sulfate transport by the intestine were not dependent on SAT-1, and we found no evidence supporting the long-standing hypothesis that intestinal SAT-1 contributes to oxalate and sulfate homeostasis. NEW & NOTEWORTHY SAT-1 is a membrane-bound transport protein expressed in the intestine, liver, and kidney, where it is widely considered essential for the excretion of oxalate, a potentially toxic waste metabolite. Previously, calcium oxalate kidney stone formation by the SAT-1-knockout mouse generated the hypothesis that SAT-1 has a major role in oxalate excretion via the intestine. We definitively tested this proposal and found no evidence for SAT-1 as an intestinal anion transporter contributing to oxalate homeostasis.

Loss of the anion exchanger DRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis.

The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial 35[Formula: see text] and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm-2·h-1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.

The role of intestinal oxalate transport in hyperoxaluria and the formation of kidney stones in animals and man.

The intestine exerts a considerable influence over urinary oxalate in two ways, through the absorption of dietary oxalate and by serving as an adaptive extra-renal pathway for elimination of this waste metabolite. Knowledge of the mechanisms responsible for oxalate absorption and secretion by the intestine therefore have significant implications for understanding the etiology of hyperoxaluria, as well as offering potential targets for future treatment strategies for calcium oxalate kidney stone disease. In this review, we present the recent developments and advances in this area over the past 10 years, and put to the test some of the new ideas that have emerged during this time, using human and mouse models. A key focus for our discussion are the membrane-bound anion exchangers, belonging to the SLC26 gene family, some of which have been shown to participate in transcellular oxalate absorption and secretion. This has offered the opportunity to not only examine the roles of these specific transporters, revealing their importance to oxalate homeostasis, but to also probe the relative contributions made by the active transcellular and passive paracellular components of oxalate transport across the intestine. We also discuss some of the various physiological stimuli and signaling pathways which have been suggested to participate in the adaptation and regulation of intestinal oxalate transport. Finally, we offer an update on research into Oxalobacter formigenes, alongside recent investigations of other oxalate-degrading gut bacteria, in both laboratory animals and humans.

Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker.

The gut sac is a long-standing, widely used in vitro preparation for studying solute and water transport, and calculation of these fluxes requires an accurate assessment of volume. This is commonly determined gravimetrically by measuring the change in mass over time. While convenient this likely under-estimates actual net water flux (Jv) due to tissue edema. We evaluated whether the popular in vivo volume marker [(14)C]-PEG 4000, offers a more representative measure of Jvin vitro. We directly compared these two methods in five teleost species (toadfish, flounder, rainbow trout, killifish and tilapia). Net fluid absorption by the toadfish intestine based on PEG was significantly higher, by almost 4-fold, compared to gravimetric measurements, compatible with the latter under-estimating Jv. Despite this, PEG proved inconsistent for all of the other species frequently resulting in calculation of net secretion, in contrast to absorption seen gravimetrically. Such poor parallelism could not be explained by the absorption of [(14)C]-PEG (typically <1%). We identified a number of factors impacting the effectiveness of PEG. One was adsorption to the surface of sample tubes. While it was possible to circumvent this using unlabelled PEG 4000, this had a deleterious effect on PEG-based Jv. We also found sequestration of PEG within the intestinal mucus. In conclusion, the short-comings associated with the accurate representation of Jv by gut sac preparations are not overcome by [(14)C]-PEG. The gravimetric method therefore remains the most reliable measure of Jv and we urge caution in the use of PEG as a volume marker.

Chronic metabolic acidosis reduces urinary oxalate excretion and promotes intestinal oxalate secretion in the rat.

Urinary oxalate excretion is reduced in rats during a chronic metabolic acidosis, but how this is achieved is not clear. In this report, we re-examine our prior work on the effects of a metabolic acidosis on urinary oxalate handling [Green et al., Am J Physiol Ren Physiol 289(3):F536-F543, 2005], offering a more detailed analysis and interpretation of the data, together with new, previously unpublished observations revealing a marked impact on intestinal oxalate transport. Sprague-Dawley rats were provided with 0.28 M ammonium chloride in their drinking water for either 4 or 14 days followed by 24 h urine collections, blood-gas and serum ion analysis, and measurements of (14)C-oxalate fluxes across isolated segments of the distal colon. Urinary oxalate excretion was significantly reduced by 75% after just 4 days compared to control rats, and this was similarly sustained at 14 days. Oxalate:creatinine clearance ratios indicated enhanced net re-absorption of oxalate by the kidney during a metabolic acidosis, but this was not associated with any substantive changes to serum oxalate levels. In the distal colon, oxalate transport was dramatically altered from net absorption in controls (6.20 ± 0.63 pmol cm(-2) h(-1)), to net secretion in rats with a metabolic acidosis (-5.19 ± 1.18 and -2.07 ± 1.05 pmol cm(-2) h(-1) at 4 and 14 days, respectively). Although we cannot rule out modifications to bi-directional oxalate movements along the proximal tubule, these findings support a gut-kidney axis in the management of oxalate homeostasis, where this shift in renal handling during a metabolic acidosis is associated with compensatory adaptations by the intestine.

Effects of acid-base variables and the role of carbonic anhydrase on oxalate secretion by the mouse intestine in vitro.

Hyperoxaluria is a major risk factor for calcium oxalate kidney stones and the intestine is recognized as an important extra-renal pathway for eliminating oxalate. The membrane-bound chloride/bicarbonate (Cl(-)/) exchangers are involved in the transcellular movement of oxalate, but little is understood about how they might be regulated. , CO2, and pH are established modulators of intestinal NaCl cotransport, involving Na(+)/H(+) and Cl(-)/ exchange, but their influence on oxalate transport is unknown. Measuring (14)C-oxalate and (36)Cl fluxes across isolated, short-circuited segments of the mouse distal ileum and distal colon we examined the role of these acid-base variables and carbonic anhydrase (CA) in oxalate and Cl(-) transport. In standard buffer both segments performed net oxalate secretion (and Cl(-) absorption), but only the colon, and the secretory pathway were responsive to and CO2. Ethoxzolamide abolished net oxalate secretion by the distal colon, and when used in tandem with an impermeant CA inhibitor, signaled an intracellular CA isozyme was required for secretion. There was a clear dependence on as their removal eliminated secretion, while at 42 mmol/L was also decreased and eradicated. Independent of pH, raising Pco2 from 28 to 64 mmHg acutely stimulated net oxalate secretion 41%. In summary, oxalate secretion by the distal colon was dependent on , CA and specifically modulated by CO2, whereas the ileum was remarkably unresponsive. These findings highlight the distinct segmental heterogeneity along the intestine, providing new insights into the oxalate transport mechanism and how it might be regulated.

Bifidobacterium animalis subsp. lactis decreases urinary oxalate excretion in a mouse model of primary hyperoxaluria.

Hyperoxaluria significantly increases the risk of calcium oxalate kidney stone formation. Since several bacteria have been shown to metabolize oxalate in vitro, including probiotic bifidobacteria, we focused on the efficiency and possible mechanisms by which bifidobacteria can influence oxalate handling in vivo, especially in the intestines, and compared these results with the reported effects of Oxalobacter formigenes. Bifidobacterium animalis subsp. lactis DSM 10140 and B. adolescentis ATCC 15703 were administered to wild-type (WT) mice and to mice deficient in the hepatic enzyme alanine-glyoxylate aminotransferase (Agxt(-/-), a mouse model of Primary Hyperoxaluria) that were fed an oxalate-supplemented diet. The administration of B. animalis subsp. lactis led to a significant decrease in urinary oxalate excretion in WT and Agxt(-/-) mice when compared to treatment with B. adolescentis. Detection of B. animalis subsp. lactis in feces revealed that 3 weeks after oral gavage with the bacteria 64% of WT mice, but only 37% of Agxt(-/-) mice were colonized. Examining intestinal oxalate fluxes showed there were no significant changes to net oxalate secretion in colonized animals and were therefore not associated with the changes in urinary oxalate excretion. These results indicate that colonization with B. animalis subsp. lactis decreased urinary oxalate excretion by degrading dietary oxalate thus limiting its absorption across the intestine but it did not promote enteric oxalate excretion as reported for O. formigenes. Preventive or therapeutic administration of B. animalis subsp. lactis appears to have some potential to beneficially influence dietary hyperoxaluria in mice.

Transcellular oxalate and Cl- absorption in mouse intestine is mediated by the DRA anion exchanger Slc26a3, and DRA deletion decreases urinary oxalate.

Active transcellular oxalate transport in the mammalian intestine contributes to the homeostasis of this important lithogenic anion. Several members of the Slc26a gene family of anion exchangers have a measurable oxalate affinity and are expressed along the gut, apically and basolaterally. Mouse Slc26a6 (PAT1) targets to the apical membrane of enterocytes in the small intestine, and its deletion results in net oxalate absorption and hyperoxaluria. Apical exchangers of the Slc26a family that mediate oxalate absorption have not been established, yet the Slc26a3 [downregulated in adenoma (DRA)] protein is a candidate mediator of oxalate uptake. We evaluated the role of DRA in intestinal oxalate and Cl(-) transport by comparing unidirectional and net ion fluxes across short-circuited segments of small (ileum) and large (cecum and distal colon) intestine from wild-type (WT) and DRA knockout (KO) mice. In WT mice, all segments demonstrated net oxalate and Cl(-) absorption to varying degrees. In KO mice, however, all segments exhibited net anion secretion, which was consistently, and solely, due to a significant reduction in the absorptive unidirectional fluxes. In KO mice, daily urinary oxalate excretion was reduced 66% compared with that in WT mice, while urinary creatinine excretion was unchanged. We conclude that DRA mediates a predominance of the apical uptake of oxalate and Cl(-) absorbed in the small and large intestine of mice under short-circuit conditions. The large reductions in urinary oxalate excretion underscore the importance of transcellular intestinal oxalate absorption, in general, and, more specifically, the importance of the DRA exchanger in oxalate homeostasis.

Sulfate secretion and chloride absorption are mediated by the anion exchanger DRA (Slc26a3) in the mouse cecum.

Inorganic sulfate (SO4²â») is essential for a multitude of physiological processes. The specific molecular pathway has been identified for uptake from the small intestine but is virtually unknown for the large bowel, although there is evidence for absorption involving Na⁺-independent anion exchange. A leading candidate is the apical chloride/bicarbonate (Cl⁻/HCO3⁻) exchanger DRA (down-regulated in adenoma; Slc26a3), primarily linked to the Cl⁻ transporting defect in congenital chloride diarrhea. The present study set out to characterize transepithelial ³5SO4²â» and ³6Cl⁻ fluxes across the isolated, short-circuited cecum from wild-type (WT) and knockout (KO) mice and subsequently to define the contribution of DRA. The cecum demonstrated simultaneous net SO4²â» secretion (-8.39 ± 0.88 nmol·cm⁻²·h⁻¹) and Cl⁻ absorption (10.85 ± 1.41 µmol·cm⁻²·h⁻¹). In DRA-KO mice, SO4²â» secretion was reversed to net absorption via a 60% reduction in serosal to mucosal SO4²â» flux. Similarly, net Cl⁻ absorption was abolished and replaced by secretion, indicating that DRA represents a major pathway for transcellular SO4²â» secretion and Cl⁻ absorption. Further experiments including the application of DIDS (500 µM), bumetanide (100 µM), and substitutions of extracellular Cl⁻ or HCO3⁻/CO2 helped to identify specific ion dependencies and driving forces and suggested that additional anion exchangers were operating at both apical and basolateral membranes supporting SO4²â» transport. In conclusion, DRA contributes to SO4²â» secretion via DIDS-sensitive HCO3⁻/SO4²â» exchange, in addition to being the principal DIDS-resistant Cl⁻/HCO3⁻ exchanger. With DRA linked to the pathogenesis of other gastrointestinal diseases extending its functional characterization offers a more complete picture of its role in the intestine.

Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish.

For teleost fish living in seawater, drinking the surrounding medium is necessary to avoid dehydration. This is a key component of their osmoregulatory strategy presenting the challenge of excreting excess salts while achieving a net retention of water. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid is crucial to compensating for water losses to the hyperosmotic environment. Despite this, the potential for the teleost intestine to serve as a comparative model for detailed, integrative experimental studies on epithelial water transport has so far gone largely untapped. The following review aims to present an assessment of the teleost intestine as a fluid-transporting epithelium. Beginning with a brief overview of marine teleost osmoregulation, emphasis shifts to the processing of ingested seawater by the gastrointestinal tract and the characteristics of intestinal ion and fluid transport. Particular attention is given to acid-base transfers by the intestine, specifically bicarbonate secretion, which creates the distinctly alkaline gut fluids responsible for the formation of solid calcium carbonate precipitates. The respective contributions of these unique features to intestinal fluid absorption, alongside other recognised ion transport processes, are then subsequently considered within the wider context of the classic physiological problem of epithelial water transport.

The influence of 17ß-estradiol on intestinal calcium carbonate precipitation and osmoregulation in seawater-acclimated rainbow trout (Oncorhynchus mykiss).

The intestine of marine teleosts produces carbonate precipitates from ingested calcium as part of their osmoregulatory strategy in seawater. The potential for estrogens to control the production of intestinal calcium carbonate and so influence osmoregulation was investigated in seawater-acclimated rainbow trout following intraperitoneal implantation of 17ß-estradiol (E2) at two doses (0.1 and 10 µg E2 g(-1)). Levels of plasma vitellogenin provided an indicator of estrogenic effect, increasing significantly by three and four orders of magnitude at the low and high doses, respectively. Plasma osmolality and muscle water content were unaffected, whereas E2-treated fish maintained lower plasma [Na(+)] and [Cl(-)]. Plasma [Ca(2+)] and [Mg(2+)] and muscle [Ca(2+)] increased with vitellogenin induction, whereas the intestinal excretion of calcium carbonate was reduced. This suggests that elevated levels of circulating E2 may enhance Ca(2+) uptake via the gut and simultaneously reduce CaCO(3) formation, which normally limits intestinal availability of Ca(2+). Increasing E2 caused an elevation of [Na(+)] and [Cl(-)] and a reduction of [HCO(3(-))] in intestinal fluid. We speculate that E2 may influence a number of intestinal ion transport processes that ultimately may influence water absorption: (1) reduced NaCl cotransport, (2) reduced Cl(-) uptake via Cl(-)/HCO(3(-)) exchange and (3) reduced precipitation of Ca(2+) and Mg(2+) carbonates. Despite these effects on intestinal ion and water transport, overall osmoregulatory status was not compromised in E2-treated fish, suggesting the possibility of compensation by other organs.

HCO (3)(-) secretion and CaCO3 precipitation play major roles in intestinal water absorption in marine teleost fish in vivo.

The intestine of marine teleosts must effectively absorb fluid from ingested seawater to avoid dehydration. This fluid transport has been almost exclusively characterized as driven by NaCl absorption. However, an additional feature of the osmoregulatory role of the intestine is substantial net HCO(3)(-) secretion. This is suggested to drive additional fluid absorption directly (via Cl(-)/HCO(3)(-) exchange) and indirectly by precipitating ingested Ca(2+) as CaCO(3), thus creating the osmotic gradient for additional fluid absorption. The present study tested this hypothesis by perfusing the intestine of the European flounder in vivo with varying [Ca(2+)]: 10 (control), 40, and 90 mM. Fractional fluid absorption increased from 47% (control) to 73% (90 mM Ca(2+)), where almost all secreted HCO(3)(-) was excreted as CaCO(3). This additional fluid absorption could not be explained by NaCl cotransport. Instead, a significant positive relationship between Na(+)-independent fluid absorption and total HCO(3)(-) secretion was consistent with the predicted roles for anion exchange and CaCO(3) precipitation. Further analysis suggested that Na(+)-independent fluid absorption could be accounted for by net Cl(-) and H(+) absorption (from Cl(-)/HCO(3)(-) exchange and CO(2) hydration, respectively). There was no evidence to suggest that CaCO(3) alone was responsible for driving fluid absorption. However, by preventing the accumulation of luminal Ca(2+) it played a vital role by dynamically maintaining a favorable osmotic gradient all along the intestine, which permits substantially higher rates of solute-linked fluid absorption. To overcome the resulting hyperosmotic and highly acidic absorbate, it is proposed that plasma HCO(3)(-) buffers the absorbed H(+) (from HCO(3)(-) production), and consequently reduces the osmolarity of the absorbed fluid entering the body.

Ca2+-driven intestinal HCO(3)(-) secretion and CaCO3 precipitation in the European flounder in vivo: influences on acid-base regulation and blood gas transport.

Marine teleost fish continuously ingest seawater to prevent dehydration and their intestines absorb fluid by mechanisms linked to three separate driving forces: 1) cotransport of NaCl from the gut fluid; 2) bicarbonate (HCO(3)(-)) secretion and Cl(-) absorption via Cl(-)/HCO(3)(-) exchange fueled by metabolic CO(2); and 3) alkaline precipitation of Ca(2+) as insoluble CaCO(3), which aids H(2)O absorption). The latter two processes involve high rates of epithelial HCO(3)(-) secretion stimulated by intestinal Ca(2+) and can drive a major portion of water absorption. At higher salinities and ambient Ca(2+) concentrations the osmoregulatory role of intestinal HCO(3)(-) secretion is amplified, but this has repercussions for other physiological processes, in particular, respiratory gas transport (as it is fueled by metabolic CO(2)) and acid-base regulation (as intestinal cells must export H(+) into the blood to balance apical HCO(3)(-) secretion). The flounder intestine was perfused in vivo with salines containing 10, 40, or 90 mM Ca(2+). Increasing the luminal Ca(2+) concentration caused a large elevation in intestinal HCO(3)(-) production and excretion. Additionally, blood pH decreased (-0.13 pH units) and plasma partial pressure of CO(2) (Pco(2)) levels were elevated (+1.16 mmHg) at the highest Ca perfusate level after 3 days of perfusion. Increasing the perfusate [Ca(2+)] also produced proportional increases in net acid excretion via the gills. When the net intestinal flux of all ions across the intestine was calculated, there was a greater absorption of anions than cations. This missing cation flux was assumed to be protons, which vary with an almost 1:1 relationship with net acid excretion via the gill. This study illustrates the intimate link between intestinal HCO(3)(-) production and osmoregulation with acid-base balance and respiratory gas exchange and the specific controlling role of ingested Ca(2+) independent of any other ion or overall osmolality in marine teleost fish.