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

Abstract

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 ($J_{ms}^{ion}$ and $J_{sm}^{ion}$) 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 $J_{sm}^{Ox}$ and distinct from net Cl⁻ secretion produced by inhibiting $J_{ms}^{Cl}$. Using the PAT-1 knockout (KO) mouse we determined cAMP-stimulated $J_{sm}^{Ox}$ 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.

INTRODUCTION

Oxalate is the salt-forming anion of oxalic acid (C₂O₄H₂), a normal end-product of metabolism which is also regularly consumed as part of the diet. With no recognized function in mammals this non-metabolizable waste compound is primarily eliminated in the urine. Although, in excessive amounts (hyperoxaluria) it presents a considerable risk for calcium oxalate crystal formation, one of the most common constituents of kidney stones [5,81,56]. Animal models have also revealed a small but significant proportion (<10 %) of endogenous oxalate is excreted via the gut [14], and the intestinal epithelium itself can be induced to actively secrete oxalate in response to diminished kidney function [34,29], systemic oxalate loading [30], or colonization with the symbiotic oxalate-degrading gut bacterium Oxalobacter formigenes [26,8,32,27,36]. Taken together, this implies oxalate excretion may be coordinated between renal and enteric routes [28,31]. Identifying the membrane transport proteins responsible for secretion by the intestinal epithelium and how they are regulated will extend our understanding of how the gastrointestinal (GI) tract contributes to oxalate homeostasis. In turn, this may offer insights into the pathophysiology of hyperoxaluria and whether these secretory pathways might serve as potential therapeutic targets.

Oxalate secretion by the intestinal epithelium has been characterized as secondary active. The anion exchanger PAT-1 (Putative Anion Transporter 1), encoded by gene Slc26a6, is a chloride/bicarbonate (Cl⁻/HCO₃⁻) exchanger located at the apical membrane of the intestinal epithelium with an established role in oxalate secretion. PAT-1 knockout (KO) mice were found to be hyperoxaluric, hyperoxalemic, and more susceptible to calcium oxalate kidney stone formation due to the excessive absorption of dietary oxalate from the small intestine [19,41], emphasizing the significance of this transporter to GI oxalate secretion. Another related apical Cl⁻/HCO₃⁻ exchanger, DRA (Down-Regulated in Adenoma; Slc26a3), has been shown to contribute to oxalate absorption by the mouse intestine [22]. The functions of DRA and PAT-1 are subject to acute regulation by cell signaling cascades involving the ubiquitous intracellular second messenger, cyclic adenosine monophosphate (cAMP) [46,47,49,43]. Elevated levels of cAMP stimulate transcellular Cl⁻ secretion and inhibit NaCl absorption resulting in overall net fluid secretion by the intestinal epithelium. This Cl⁻ secretory response relies on the coordinated actions of the basolateral sodium-potassium-chloride (Na⁺-K⁺-Cl⁻) cotransporter, NKCC1 (Slc12a2), to import Cl⁻ into the enterocyte which then exits through the apical anion channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator; ABCC7) [23,44]. The inhibition of electroneutral NaCl absorption specifically involves withdrawal of the Na⁺/H⁺ exchanger, NHE3 (Slc9a3) and DRA from the apical membrane [49,43].

Initial studies demonstrated oxalate secretion by rabbit distal ileum [20] and colon [33,35] could also be stimulated by cAMP through a mechanism closely resembling electrogenic Cl⁻ secretion, i.e. involving basolateral NKCC1 and an apical anion conductance similar to the CFTR. However, despite relatively broad selectivity, the (human) CFTR does not directly transport oxalate [18]. Rather, the CFTR may regulate oxalate secretion by the intestinal epithelium through its interaction with PAT-1 [45]. This could occur directly through phosphorylation of the CFTR by cAMP-dependent protein kinase A (PKA) which facilitates its physical binding to and subsequent initiation of PAT-1 transport activity [47]. For example, using Xenopus oocytes expressing (human) PAT-1, Cl⁻/oxalate exchange was stimulated by cAMP and could be significantly enhanced by co-expression with the CFTR [45]. Additionally, increased Cl⁻-driven oxalate uptake by human Caco-2 monolayers following incubation with O. formigenes-conditioned media has been attributed, in large part, to the upregulation of PAT-1 activity through a signaling pathway involving PKA [4]. In this same cultured cell model, activation of cAMP and PKA using a cocktail of forskolin (FSK), an agonist of the adenylyl cyclases, along with phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), acutely stimulated oxalate uptake by PAT-1 [3].

Oxalate transport by PAT-1 thus appears to be regulated via cAMP but how these results in oocytes and cultured cells translate to the native intestine is unclear. An initial report using mouse distal ileum in vitro found PAT-1 mediated oxalate secretion was unresponsive to dibutyryl-cAMP [19]. However, we recently showed that net secretion by this segment, and also the cecum, could be stimulated by FSK/IBMX [69]. Even though PAT-1 does not contribute to baseline oxalate secretion by mouse large intestine [80], the question remains of whether it is activated by a cAMP-dependent signaling pathway. The following study therefore set out to test the hypothesis that PAT-1 supports cAMP-stimulated oxalate secretion by the native intestinal epithelium. Adopting the classical Ussing chamber technique, we began by surveying how oxalate transport across three different segments of the mouse intestine (distal ileum, cecum, and distal colon) responded to FSK/IBMX. We proceeded to focus on the cecum as the only region where oxalate secretion was successfully stimulated. Subsequent experiments thus attempted to distinguish the role of PAT-1 by comparing the cecum from wild-type (WT) and PAT-1 KO mice. We also utilized DIDS, a non-specific inhibitor of anion transport (including PAT-1), CFTR_inh-172 to block the CFTR, and employed individual substitutions of Cl⁻ and HCO₃⁻ from the mucosal (luminal) bath to probe the apical transport mechanisms involved. Since Cl⁻ and oxalate share some of the same cAMP-regulated transporters and pathways [33,35,20,19,22], we simultaneously measured unidirectional Cl⁻ flux alongside oxalate.

MATERIALS AND METHODS

Experimental animals.

PAT-1 KO (Slc26a6 −/−) mice were obtained from breeding pairs maintained on a C57BL background and housed at the University of Florida. Information on the generation of this model and its genotyping have been described previously [77]. Mice were given free access to standard chow (diet 7912; Harlan Teklad, Indianapolis, IN) and sterile drinking water. A total of 78 mice (n = 39 WT, n = 39 PAT-1 KO) of both sexes, ranging in age from 2 to 7 months old, with a mean ± SEM body mass of 24.7 ± 0.7 g (WT) and 23.8 ± 0.5 g (PAT-1 KO), were used. All animal experimentation was approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were euthanized by inhalation of 100 % CO₂ followed by cervical dislocation or exsanguination via cardiac puncture.

Transepithelial flux experiments.

Portions of the distal ileum, cecum and distal colon were placed in modified Ussing chambers (Physiologic Instruments, San Diego, CA) for measuring the transepithelial unidirectional flux of oxalate and Cl⁻ under symmetrical, short-circuit conditions. Following euthanasia, the lower portion of the intestinal tract (ileum to distal colon) was removed to ice-cold buffered saline. After removing external connective tissue, a pair of adjacent tissues from the distal ileum (2 × 2 cm lengths immediately proximal to the ileo-cecal valve) and distal colon (2 × 2 cm lengths immediately proximal to the peritoneal border, representing the lower 30 % of the colon) were opened longitudinally along the mesenteric line and any contents removed. The cecum was opened along its minor curvature from the ampulla to the apex and cleaned of its contents, with a tissue pair created from the corpus ceci. Each individual tissue was mounted flat and intact (without stripping the outer muscle layers) on a slider (P2304, Physiologic Instruments), exposing a gross surface area of 0.3 cm², and secured between the two halves of the chamber (P2300, Physiologic Instruments). Tissues were bathed on both sides by 4 mL of buffered saline and maintained at 37 °C by a water jacket while being simultaneously gassed and stirred with a humidified 95 % O₂/5 % CO₂ gas mixture. Each preparation was continuously voltage-clamped to zero (VCC6, Physiologic Instruments), with the mucosal bath serving as ground. Approximately 10-15 min after tissues were mounted, 0.27 μCi ¹⁴C-oxalate (specific activity 115 mCi/mmol) and 0.09 μCi ³⁶Cl⁻ (specific activity 571 μCi/mmol) were added to either the mucosal (M) or serosal (S) half-chamber which was then designated as the “hot side”. Sodium oxalate (1 mmol/L) was used to achieve the desired final concentration of 1.5 μmol/L oxalate in each half-chamber. To calculate the specific activity (dpm/mmol) of each isotope, 50 μL samples were collected from the “hot side” at the beginning and end of each experiment. After 15 min, and for every subsequent 15-min interval up to either 105 or 165 min, the appearance of ¹⁴C-oxalate and ³⁶Cl⁻ were detected in 1 mL samples from the opposing “cold side”, which were immediately replaced with 1 mL of appropriate, warmed buffer. Transepithelial potential difference (mV) and short-circuit current, I_sc, (μA) were also recorded at each 15-min sampling interval. Experiments investigating the response of oxalate and Cl⁻ transport to a cocktail of FSK/IBMX followed a paired design and were divided into time periods, the first consisting of an initial “control” period (Period I, 0–45 min), followed by a second “experimental” period (Period II, 60 to 105 min), and sometimes extending to a third (Period III, 120 to 165 min). The activity of ¹⁴C-oxalate and ³⁶Cl⁻ in each sample was determined by liquid scintillation spectrophotometry (Beckman LS6500, Beckman-Coulter Inc., Fullerton, CA) with quench correction following the addition of 5 mL scintillation cocktail (Ecoscint A, National Diagnostics, Atlanta, GA). A series of external standards established the validity of counting dual-labeled samples, thus allowing the individual activities of ¹⁴C-oxalate and ³⁶Cl⁻ to be calculated based on their relative counting efficiencies after minimizing and accounting for overlap in their energy spectra [80].

Chemicals, buffers and reagents.

The standard bicarbonate-buffered saline contained the following (in mmol/L): 139.4 Na⁺, 122.2 Cl⁻, 21 HCO₃⁻, 5.4 K⁺, 2.4 HPO₄²⁻, 1.2 Ca²⁺, 1.2 Mg²⁺, 0.6 H₂PO₄⁻, 0.5 SO₄²⁻, 10 D-glucose (serosal only), 10 D-mannitol (mucosal only), and was adjusted to pH 7.4 after equilibrating with 95 % O₂/5 % CO₂. For experiments with Cl⁻-free buffer, NaCl was replaced by Na⁺-isethionate while CaCl₂ and MgCl₂ were substituted for their respective gluconate salts. For HCO₃⁻/CO₂-free buffer, HCO₃⁻ was replaced by HEPES (specifically, 14 mmol/L HEPES free-acid and 7 mmol/L HEPES Na⁺-salt), and gassed with 100 % O₂. D-mannitol (9 mmol/L) was also added to achieve an osmolality comparable with the standard bicarbonate buffer. To inhibit spontaneous endogenous prostanoid production all buffers contained 5 μmol/L indomethacin. The radioisotope ¹⁴C-oxalate was a custom preparation from ViTrax Radiochemicals (Placentia, CA) and ³⁶Cl⁻ was purchased as H³⁶Cl from Amersham Biosciences (Piscataway, NJ). A cocktail of FSK and IBMX in DMSO was added to both sides of the epithelium at final concentrations of 10 μmol/L and 100 μmol/L, respectively. The anion transport inhibitor, DIDS (Molecular Probes, Eugene, OR) was dissolved in DMSO and added to the mucosal half-chamber for a final concentration of 500 μmol/L. The CFTR inhibitor, CFTR_inh-172 in DMSO was added to the mucosal half-chamber for a final concentration of 20 μmol/L. Stock solutions of FSK/IBMX, DIDS and CFTR_inh-172 were made fresh on the day of each experiment. All chemicals and reagents were sourced from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.

Calculations and statistical analyses.

The flux of oxalate and Cl⁻ in the absorptive, mucosal-to-serosal ($J_{ms}^{ion}$) direction and secretory, serosal-to-mucosal ($J_{sm}^{ion}$) direction were calculated from the change in activity of ¹⁴C-oxalate and ³⁶Cl⁻ detected on the “cold side” at each 15 min sampling point, having corrected for dilution by replacement buffer between samples. The flux of each anion was expressed per cm² of tissue per hour. The recordings of short-circuit current (I_sc, μA/cm²) and potential difference (mV) were used to calculate transepithelial conductance (G_T, mS/cm²) using Ohm’s Law. Net flux of each ion was calculated as: $J_{net}^{ion}=J_{ms}^{ion}-J_{sm}^{ion}$ for pairs of tissues matched based on G_T (no greater than a ± 25% difference in G_T between pairs from the cecum and distal colon, and ± 15 % between pairs of distal ilea). The following data are presented as mean ± SEM. A repeated measures one-way ANOVA was used to evaluate the epithelial responses to FSK/IBMX, mucosal DIDS and CFTR_inh-172 at each 15 min time point compared to the mean value during the preceding control period (Period I). Significant differences were distinguished by multiple comparisons using Holm-Sidak post-hoc tests. Where data failed to meet the assumptions of approximate normality and equality of variance the corresponding non-parametric test was performed. The results of all tests were accepted as significant at P ≤ 0.05, or the appropriately corrected P-value following post-hoc analysis. The figures were drawn, and statistical analysis performed, using SigmaPlot v14.0 (Systat Software Inc. San Jose, CA).

RESULTS

Effects of cAMP stimulation on mouse intestine.

Figure 1 illustrates the distinct segmental heterogeneity of oxalate and Cl⁻ transport across the mouse intestine under symmetrical short-circuit conditions in vitro. The unstimulated distal ileum (Fig. 1A) and distal colon (Fig. 1C) spontaneously secreted oxalate on a net basis and neither were subsequently enhanced by FSK/IBMX. For the cecum, there was no initial net transport of oxalate ($J_{net}^{Ox}$ = −1.86 ± 2.16 pmol/cm²·h), but $J_{sm}^{Ox}$ progressively increased during Period II resulting in overall net secretion averaging −11.18 ± 2.54 pmol/cm²·h (Fig. 1B). In terms of net Cl⁻ transport, the distal ileum secreted Cl⁻ under control conditions ($J_{net}^{Cl}$ = −2.60 ± 1.18 μmol/cm²·h) and FSK/IBMX significantly increased this net secretion to an average of −8.23 ± 1.55 μmol/cm²·h through a sustained reduction in $J_{ms}^{Cl}$ and a transient rise in $J_{sm}^{Cl}$ (Fig. 1D). FSK/IBMX also produced a short-lived increase in $J_{sm}^{Cl}$ in the cecum, but overall net Cl⁻ secretion by the large intestine was exclusively through the inhibition of $J_{ms}^{Cl}$ for both cecum (Fig. 1E) and distal colon (Fig. 1F). In the distal ileum, G_T steadily declined following application of FSK/IBMX, from a mean of 27.95 ± 0.89 mS/cm² in Period I to 21.23 ± 1.50 mS/cm² in Period II (Fig. 1G), whereas it remained unchanged in the cecum (Fig. 1H) and increased modestly in the distal colon (Fig. 1I). Even though the stimulation of oxalate secretion by cAMP was restricted to the cecum, FSK/IBMX consistently produced overall net electrogenic anion secretion by each segment as judged from the change to I_sc (Figs. 1G-I). To gain some insight into the nature and characteristics of the apical transporter(s) responsible for cAMP-stimulated oxalate secretion, in particular the role of PAT-1, subsequent experiments focused on the cecum.

Fig. 1. Raising intracellular cAMP stimulates oxalate secretion by mouse cecum, but not distal ileum or distal colon.

Unidirectional flux of oxalate, J^Ox (Panels A-C), and chloride, J^Cl (Panels D-F) were measured simultaneously across isolated short-circuited segments of distal ileum, cecum, and distal colon from wild-type (WT) mice prior to and following application of a cocktail of forskolin (10 μmol/L) and IBMX (100 μmol/L). Panels G-I compare the responses of short-circuit current (I_sc) and transepithelial conductance (G_T) for each intestinal segment. M, mucosal; S, serosal. Each data point represents mean ± SE of n = 6 (distal ileum), n = 7 (cecum), and n = 8 (distal colon) tissue pairs from n = 8 mice. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

Effect of cAMP stimulation on PAT-1 KO cecum.

Our hypothesis predicted cAMP-stimulated oxalate secretion would be dependent on PAT-1, however the effect of FSK/IBMX on $J_{sm}^{Ox}$ by the PAT-1 KO cecum (Fig. 2A) was identical to the WT (Fig. 1B). There were also no differences in unidirectional Cl⁻ flux which was remarkably consistent between WT (Fig. 1E) and PAT-1 KO cecum (Fig. 2B) before and after the addition of FSK/IBMX. The response of I_sc was indistinguishable too with maximal stimulation at 75 min. Relative to Period I, I_sc increased on average by 6.88 ± 0.77 μeq/cm²·h (WT) and 7.04 ± 0.63 μeq/cm²·h (PAT-1 KO), hence overall cAMP-stimulated net electrogenic anion secretion was not impacted by the loss of PAT-1. Table 1 presents a comparison of net transport between WT (Fig. 1) and PAT-1 KO (Fig. 2) cecum confirming there were no major differences between WT and PAT-1 KO cecum either under control conditions (Period I) or in response to cAMP (Period II). The sole exception was basal G_T, which was 15 % lower in the absence of PAT-1 (15.28 ± 0.76 mS/cm²). Based these findings oxalate and Cl⁻ transport by mouse cecum were not dependent on PAT-1, either under resting conditions or after the stimulation of cAMP.

Fig. 2. cAMP stimulates oxalate secretion by mouse cecum in the absence of PAT-1.

The unidirectional flux of oxalate, J^Ox (Panel A) and chloride, J^Cl (Panel B) measured simultaneously across isolated short-circuited segments of cecum from PAT-1 KO mice following application of a cocktail of forskolin (10 μmol/L) and IBMX (100 μmol/L). Panel C compares the responses of short-circuit current (I_sc) and transepithelial conductance (G_T). M, mucosal; S, serosal. Each data point represents mean ± SE of n = 6 tissue pairs from n = 8 PAT-1 KO mice. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

Table 1.

A comparison of net oxalate (${\mathit{J}}_{\mathit{Net}}^{\mathit{Ox}}$) and chloride (${\mathit{J}}_{\mathit{Net}}^{\mathit{Cl}}$) flux across the cecum from WT and PAT-1 KO mice during the initial control (CON) experimental period and following activation of intracellular cAMP (FSK/IBMX) under symmetrical, short-circuit conditions in vitro. I_sc = short-circuit current and G_T = transepithelial conductance. For each time period, differences between WT and KO tissues were determined by independent t-test and significance is indicated with an asterisk. Data were calculated from unidirectional flux in Figures 1 and 2 and are presented as mean ± SEM. The numbers in parentheses denote associated sample size.

Period	Genotype	${\mathit{J}}_{\mathit{Net}}^{\mathit{Ox}}$ (pmol/cm2·h)	${\mathit{J}}_{\mathit{Net}}^{\mathit{Cl}}$ (μmol/cm2·h)	Isc (μeq/cm2·h)	GT (mS/cm2)
CON(0-45 min)	WT	−1.86 ± 2.16 (7)	9.38 ± 2.06 (7)	−0.87 ± 0.26 (14)	18.11 ± 0.98 (14)
	PAT-1 KO	−2.30 ± 2.10 (6)	9.60 ± 1.87 (6)	−0.98 ± 0.27 (12)	15.28 ± 0.76* (12)
P-value (t-test)		0.945	0.938	0.760	0.036
FSK/IBMX (60-105 min)	WT	−11.18 ± 2.54 (7)	−9.70 ± 1.86 (7)	−7.75 ± 0.61 (14)	18.17 ± 0.89 (14)
	PAT-1 KO	−9.57 ± 2.43 (6)	−8.08 ± 1.47 (6)	−7.94 ± 0.57 (12)	17.07 ± 0.71 (12)
P-value (t-test)		0.660	0.519	0.821	0.346

Mucosal DIDS.

The disulfonic stilbene DIDS (500 μmol/L) attempted to determine the involvement of an alternative anion transporter(s). Figure 3 shows mucosal DIDS did not affect resting oxalate flux in either WT (Fig. 3A) or PAT-1 KO (Fig. 3B) ceca during Period II. However, applying FSK/IBMX in the presence of DIDS (Period III) was unsuccessful for both sets of tissues suggesting the mechanism responsible for cAMP-stimulated $J_{sm}^{Ox}$ had been inhibited. In terms of Cl⁻ transport (Figs. 3C and 3D), mucosal DIDS brought about a small, but significant, 13-14 % reduction in $J_{ms}^{Cl}$ by WT and PAT-1 KO ceca, while $J_{sm}^{Cl}$ was unaffected. This did not mute the subsequent response to FSK/IBMX, and the abrupt reduction in absorption, and to a lesser extent $J_{sm}^{Cl}$. The mean cAMP-stimulated $J_{net}^{Cl}$ was −10.01 ± 1.60 μmol/cm²·h for WT and −9.69 ± 1.51 μmol/cm²·h for PAT-1 KO cecum, comparable with prior experiments summarized in Table 1, suggesting the presence of mucosal DIDS does not interfere with the production of net Cl⁻ secretion. Even at this relatively high concentration (500 μmol/L), I_sc was not significantly impacted during Period II (Fig. 3E). The subsequent magnitude of cAMP-stimulated ΔI_sc (i.e., Period III – Period II) in WT tissues was 5.24 ± 0.61 μeq/cm²·h, which is slightly lower than without DIDS in Figure 1H (ΔI_sc = 6.88 ± 0.77 μeq/cm²·h). Similarly, for PAT-1 KO ceca, the ΔI_sc in response to FSK/IBMX was 5.57 ± 0.38 μeq/cm²·h in the presence of DIDS (Fig. 3E), relative to 7.04 ± 0.63 μeq/cm²·h (Fig. 2C). G_T was not affected by mucosal DIDS or following the subsequent application of FSK/IBMX (Fig. 3F). The transport mechanism responsible for the stimulation of oxalate secretion via cAMP was therefore considered DIDS-sensitive.

Fig. 3. Pre-treatment with mucosal DIDS blocks cAMP-stimulated oxalate secretion by WT and PAT-1 KO mouse cecum.

Unidirectional flux of oxalate, J^Ox (Panels A-B) and chloride, J^Cl (Panels C-D) were measured simultaneously across isolated short-circuited segments of cecum from WT and PAT-1 KO mice following the addition of DIDS (500 μmol/L) to the mucosal bath (Period II) and subsequent bilateral application of forskolin (10 μmol/L) and IBMX (100 μmol/L) (Period III). Panel E compares the responses of short-circuit current (I_sc) and Panel F transepithelial conductance (G_T). M, mucosal; S, serosal. Each data point represents mean ± SE of n = 6 tissue pairs. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

Blocking the CFTR.

To investigate whether cAMP-stimulated oxalate secretion by the cecum involved a functioning CFTR we applied the specific inhibitor CFTR_inh-172 to the mucosal bath. On its own, CFTR_inh-172 did not significantly affect baseline oxalate transport by the WT cecum (Fig. 4A). For PAT-1 KO cecum, $J_{sm}^{Ox}$ was 27 % higher, on average, during Period II relative to the preceding control period (Fig. 4B), although this was not statistically significant (P = 0.385). The subsequent application of FSK/IBMX to WT cecum in the presence of CFTR_inh-172 failed to stimulate $J_{sm}^{Ox}$ (Fig. 4A), however, this secretory flux was significantly enhanced in PAT-1 KO cecum (Fig. 4B). Nevertheless, similar rates of mean net oxalate secretion were achieved for both WT (−8.87 ± 1.87 pmol/cm²·h) and PAT-1 KO (−12.37 ± 3.76 pmol/cm²·h) tissues (P = 0.419), involving a pronounced (but non-significant) reduction in $J_{ms}^{Ox}$. CFTR_inh-172 did not affect $J_{sm}^{Cl}$ in the resting state but, similar to mucosal DIDS, caused a modest fall in $J_{ms}^{Cl}$ prior to the addition of FSK/IBMX (Figs. 4C and 4D). During Period III, this pre-exposure to CFTR_inh-172 did not affect the sharp decline in $J_{ms}^{Cl}$ and appeared to produce substantial rates of net Cl⁻ secretion (WT = −12.13 ± 2.89 μmol/cm²·h and PAT-1 KO = −14.97 ± 2.02 μmol/cm²·h), relative to prior observations (Table 1). However, we note that baseline $J_{sm}^{Cl}$ was slightly elevated in this series with CFTR_inh-172 (~25 μmol/cm²·h), compared to experiments in Figures 1E and 2B (~22 μmol/cm²·h), thus giving the impression of higher net Cl⁻ secretion. In Period II, I_sc across the unstimulated cecum was not impacted by CFTR_inh-172 but, in response to FSK/IBMX, the average ΔI_sc (i.e., Period III – Period II) was limited to −4.10 ± 0.62 μeq/cm²·h for WT and −4.38 ± 0.71 μeq/cm²·h for PAT-1 KO (Fig. 4E), approximating 60 % of the corresponding ΔI_sc calculated from Figures 1H and 2C, respectively. There were no changes to G_T in response to the sequential additions of CFTR_inh-172 and FSK/IBMX across experimental periods (Fig. 4F). Inhibiting the CFTR prevented the cAMP-mediated stimulation of $J_{sm}^{Ox}$, but only when PAT-1 was present, and it did not forestall net oxalate and Cl⁻ secretion by either WT or PAT-1 KO cecum.

Fig. 4. Blocking the CFTR does prevent net cAMP-stimulated oxalate secretion by WT or PAT-1 KO mouse cecum.

Unidirectional flux of oxalate, J^Ox (Panels A-B) and chloride, J^Cl (Panels C-D) were measured simultaneously across isolated short-circuited segments of cecum from WT and PAT-1 KO mice following the addition of CFTR_inh-172 (20 μmol/L) to the mucosal bath (Period II) and subsequent bilateral application of forskolin (10 μmol/L) and IBMX (100 μmol/L) (Period III). Panel E compares the responses of short-circuit current (I_sc) and Panel F transepithelial conductance (G_T). M, mucosal; S, serosal. Each data point represents mean ± SE of n = 8 tissue pairs. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

Replacing mucosal Cl⁻.

If cAMP stimulates oxalate secretion through apical Cl⁻/HCO₃⁻(oxalate) exchange, then the response of $J_{sm}^{Ox}$ to FSK/IBMX will be dependent on the presence of luminal Cl⁻. Despite the diffusion potentials generated by replacing Cl⁻ in the mucosal buffer with isethionate and gluconate, the epithelium was still voltage clamped to 0 mV. The measurements of unidirectional flux in asymmetric buffer conditions are therefore comparable with other experiments since they were measured under short-circuit conditions, although baseline I_sc (Fig. 5C) and G_T (Fig. 5D) will not represent true net electrogenic ion transport. Replacing mucosal Cl⁻ failed to significantly alter resting $J_{ms}^{Ox}$ and $J_{sm}^{Ox}$ for either WT or PAT-1 KO cecum compared to standard buffer (Table 2), and consequently net transport remained indistinguishable from zero for both WT (P = 0.138) and PAT-1 KO (P = 0.074) tissues under these conditions. After stimulating cAMP with FSK/IBMX, $J_{sm}^{Ox}$ increased significantly but this response was delayed until 90 min (Fig. 5A), compared to 75 min previously (Fig. 1B), and $J_{ms}^{Ox}$ also began to increase in parallel following an earlier decline, which contributed to a somewhat muted (though still significant) net secretion (−3.12 ± 1.43 pmol/cm²·h compared to 3.31 ± 1.97 pmol/cm²·h in Period I; T_8,8 = 4.96, P = 0.002) (Fig. 5A). An almost identical pattern was recorded for oxalate flux across PAT-1 KO cecum (Fig. 5B), where $J_{net}^{Ox}$ reached a similar modest value (−3.36 ± 1.11 pmol/cm²·h compared to 2.44 ± 1.16 pmol/cm²·h in Period I; T_8,8 = 6.81, P = <0.001). Substituting mucosal Cl⁻ was anticipated to limit apical Cl⁻/HCO₃⁻ exchange thus the observed stimulation of I_sc by FSK/IBMX most likely represents contributions from net electrogenic Cl⁻ and (Cl⁻-independent) HCO₃⁻ secretion (Fig. 5C). Compared to prior observations in standard buffer (Figs. 1H and 2C), FSK/IBMX led to a noticeable reduction in G_T by approximately 25 % (Fig. 5D), possibly reflecting the lower permeability of the larger isethionate and gluconate ions used to replace Cl⁻ in the mucosal bath. While the ability of cAMP to initiate net oxalate secretion was hampered, there was no overwhelming evidence for $J_{sm}^{Ox}$ being specifically dependent on mucosal Cl⁻ and serving as a counter ion for apical Cl⁻/oxalate exchange mechanism.

Fig. 5. Net cAMP-stimulated oxalate secretion by mouse cecum is dependent on mucosal chloride.

Unidirectional flux of oxalate, J^Ox measured across isolated short-circuited segments of cecum from WT (Panel A) and PAT-1 KO (Panel B) mice prior to and following the bilateral application of forskolin (10 μmol/L) and IBMX (100 μmol/L) to raise intracellular cAMP. Panels C and D compare the responses of short-circuit current (I_sc) and transepithelial conductance (G_T), respectively. M, mucosal; S, serosal. Each data point represents mean ± SE of n = 8 tissue pairs. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

Table 2. A comparison of basal, unstimulated oxalate and chloride flux by the cecum from WT and PAT-1 KO mice in standard (Std.) buffer and following asymmetrical substitution of mucosal chloride or bicarbonate.

Transepithelial oxalate and chloride flux were measured simultaneously across pairs of isolated, short-circuited segments of cecum. Within each genotype, values labelled with a different letter indicate a significant difference (P <0.05) between buffer arrangements as determined by one-way ANOVA following multiple comparisons using the Holm-Sidak method (or unpaired t-test for J^Cl). Data are mean ± SEM during the initial control experimental period (0-45 min) for all experiments and numbers in parentheses denote associated sample size.

	Buffer		JOx (pmol/cm2·h)			JCl (μmol/cm2·h)
Genotype	Mucosal	Serosal	M-S	S-M	Net	M-S	S-M	Net
WT	Std.	Std.	11.81 ± 0.89 (21)	14.60 ± 0.67a (21)	−2.79 ± 1.05a (21)	30.75 ± 1.18a (21)	22.47 ± 0.61 (21)	8.28 ± 1.27a (21)
	Cl− free	Std.	13.90 ± 1.16 (8)	10.59 ± 1.26b (8)	3.31 ± 1.97b (8)	-	-	-
	HCO3− free	Std.	12.27 ± 0.79 (8)	15.35 ± 1.16a (8)	−3.08 ± 1.02a (8)	23.23 ± 1.82b (8)	21.43 ± 1.16 (8)	1.80 ± 1.88b (8)
PAT-1 KO	Std.	Std.	11.61 ± 0.79 (20)	15.30 ± 1.37 (20)	−3.70 ± 1.56 (20)	31.21 ± 1.02a (20)	23.65 ± 0.87a (20)	7.56 ± 1.16 (20)
	Cl− free	Std.	12.98 ± 0.52 (8)	10.54 ± 1.05 (8)	2.44 ± 1.16 (8)	-	-	-
	HCO3− free	Std.	11.14 ± 0.76 (6)	12.95 ± 0.90 (6)	−1.81 ± 0.93 (6)	22.51 ± 2.86b (6)	19.43 ± 0.78b (6)	3.08 ± 2.97 (6)

Replacing mucosal HCO₃⁻/CO₂.

The substitution of HCO₃⁻ (and CO₂) from the mucosal buffer was adopted to help determine whether cAMP-stimulated $J_{sm}^{Ox}$ involved apical HCO₃⁻/oxalate exchange. Additionally, imposing an outwardly directed HCO₃⁻ gradient across the apical membrane would be anticipated to favor Cl⁻(oxalate)/HCO₃⁻ exchange thus potentially enhancing both Cl⁻ and oxalate absorption. For the initial control period, substituting mucosal HCO₃⁻/CO₂ did not significantly impact either unidirectional oxalate flux in WT or PAT-1 KO ceca, relative to symmetrical standard HCO₃⁻-containing buffer (Table 2). In Figure 6A, subsequent application of FSK/IBMX to WT cecum did cause $J_{sm}^{Ox}$ to trend upward, albeit non-significantly, resulting in significant net oxalate secretion (−9.71 ± 2.59 pmol/cm²·h compared to −3.08 ± 1.02 pmol/cm²·h in Period I; T_8,8 = 2.51, P = 0.040) through a pronounced decline in $J_{ms}^{Ox}$. In the absence of PAT-1, there was a very similar response except the increase in $J_{sm}^{Ox}$ was significant (Fig. 6B), and even though the decline in $J_{ms}^{Ox}$ rebounded by 105 min there was, on average, net oxalate secretion by the PAT-1 KO cecum in response to cAMP (−7.93 ± 3.02 pmol/cm²·h compared to −1.81 ± 0.93 pmol/cm²·h in Period I; T_6,6 = 2.42, P = 0.075). Despite the outwardly directed HCO₃⁻ gradient, $J_{ms}^{Cl}$ by WT and PAT-1 KO ceca was substantially reduced, net Cl⁻ absorption was effectively abolished and indistinguishable from zero (Table 2). Following the addition of FSK/IBMX, $J_{ms}^{Cl}$ and $J_{sm}^{Cl}$ responded as in earlier experiments, leading to robust net Cl⁻ secretion by both genotypes (Figs 6C and 6D). On average, the change in I_sc during Period II was 3.05 ± 0.50 μeq/cm²·h for WT cecum, similar to 3.81 ± 0.85 μeq/cm²·h for the PAT-1 KO (Fig. 6E). The WT, but not PAT-1 KO, cecum responded with a 16 % decline in G_T after stimulating cAMP production (Fig. 6F).

Fig. 6. Net cAMP-stimulated oxalate secretion by WT (but not PAT-1 KO) mouse cecum is dependent on mucosal bicarbonate.

Unidirectional flux of oxalate, J^Ox measured across isolated short-circuited segments of cecum from WT (Panel A) and PAT-1 KO (Panel B) mice prior to and following the bilateral application of forskolin (10 μmol/L) and IBMX (100 μmol/L) to raise intracellular cAMP. Panels C and D compare the responses of short-circuit current (I_sc) and transepithelial conductance (G_T), respectively, between WT and PAT-1 KO tissues. M, mucosal; S, serosal. Each data point represents mean ± SE of n = 8 (WT) and n = 6 (KO) tissue pairs. *Statistically significant change from the preceding control period (Period I, 0-45 min) determined by Holm-Sidak multiple comparisons following a one-way repeated measures ANOVA

DISCUSSION

The absorption and secretion of oxalate by the GI tract can profoundly influence its urinary excretion. Identifying the membrane transport proteins involved in moving oxalate across the intestinal epithelium and how they are regulated is important for understanding the pathophysiology of hyperoxaluria and kidney stone risk. Intestinal oxalate secretion can be stimulated by raising intracellular cAMP [69], via a mechanism resembling electrogenic Cl⁻ secretion [20,33,35]. The apical anion exchanger PAT-1 has an established role in oxalate secretion by the small intestine [19,41], and has been shown to be activated through a cAMP-dependent pathway when expressed in oocytes [45] and cultured cells [4,3], but whether this translates to the native intestinal epithelium is uncertain. The objective of this study was to investigate the apical transport mechanisms responsible for cAMP-stimulated oxalate secretion by the native intestine and to specifically test the hypothesis that PAT-1 is a target of this cell signaling pathway. We exposed mouse distal ileum, cecum and distal colon to a cocktail of FSK/IBMX to stimulate intracellular cAMP production. Each segment responded with robust net electrogenic anion secretion, reflected by the characteristic change to I_sc and abrupt initiation of net Cl⁻ secretion, but an accompanying stimulation of oxalate secretion was confined to the cecum only. The latter was driven by a significant increase in $J_{sm}^{Ox}$ and, contrary to earlier investigations, was distinct from the mechanism responsible for Cl⁻ secretion. Focusing on the cecum, subsequent experiments using the PAT-1 KO mouse observed an identical response of $J_{sm}^{Ox}$ to FSK/IBMX, indicating this anion exchanger was not required for cAMP-stimulated oxalate secretion. The transport mechanism responsible was sensitive to mucosal DIDS yet, with no evidence for trans-stimulation or cis-inhibition by luminal Cl⁻ or HCO₃⁻, it was unlikely to be an apical Cl⁻/HCO₃⁻ exchanger and did not contribute to cAMP-stimulated I_sc. Even though we excluded a direct role for the CFTR, the increase in $J_{sm}^{Ox}$ could be blocked by CFTR_inh-172 suggesting the possible (indirect) involvement of a functional CFTR, but only in the presence of PAT-1, and this nevertheless did not ablate overall net secretion. Further investigations are therefore necessary to identify the transporter(s) responsible and define the mechanisms regulating their function.

The effects of cAMP on oxalate and Cl⁻ transport by the native intestinal epithelium.

Net anion secretion by mouse distal ileum, cecum and distal colon was uniformly activated by FSK/IBMX based on I_sc, alongside net Cl⁻ secretion, but the stimulation of oxalate secretion was confined to the cecum (Fig. 1B). This contrasts with the rabbit model where oxalate secretion was initiated by elevated intracellular cAMP in distal ileum [20], proximal colon [33] and distal colon [35]. Hence, there are species- and (for the mouse at least) segment-specific differences in the signaling pathways regulating oxalate transport. Simultaneously measuring Cl⁻ flux also revealed these two anions do not share the same cAMP-dependent secretory pathway, as previously described for the rabbit [33,35,20], and the mechanisms producing net Cl⁻ secretion differ between mouse small and large intestine. For distal ileum, net Cl⁻ secretion was achieved through increased $J_{sm}^{Cl}$ and a corresponding decline in $J_{ms}^{Cl}$ (Fig. 1D), similar to mouse jejunum [13,59,75], reflecting the characteristic epithelial response to cAMP (i.e. activation of Cl⁻ secretion and inhibition of NaCl absorption). In the large intestine, however, net Cl⁻ secretion was achieved exclusively by reducing $J_{ms}^{Cl}$ (Figs. 1E and 1F), as observed previously [68]. Since $J_{ms}^{Cl}$ by mouse cecum and mid-distal colon is overwhelmingly DRA-mediated [1,78,22,68,37], we speculate this may represent withdrawal of this Cl⁻/HCO₃⁻ exchanger from the apical membrane [53,49]. In the cecum, such a large-scale endocytosis could also explain the modest decrease in $J_{ms}^{Ox}$ (Fig. 1B), given almost 60 % of this flux has been attributed to DRA [22]. Although a similar response of $J_{ms}^{Cl}$ was evident for distal ileum (Fig. 1A) and colon (Fig. 1C), there were no accompanying changes in oxalate absorption, even though DRA constitutes approximately 40 % of $J_{ms}^{Ox}$ across each of these segments [22].

cAMP-stimulated oxalate secretion by the mouse cecum does not require PAT-1.

Recently, we concluded PAT-1 does not support basal oxalate or Cl⁻ transport by any portion of mouse large intestine [80], and we have subsequently confirmed this for the cecum (Table 1). Nevertheless, since it is expressed in this segment [36,70], and PAT-1 mediated oxalate transport is upregulated by FSK/IBMX in oocytes [45], and cultured cells [3], we sought to determine whether PAT-1 was also being recruited via a cAMP-dependent pathway and responsible for the increased $J_{sm}^{Ox}$ in Figure 1B. After finding an identical stimulation of oxalate and Cl⁻ secretion, and I_sc by FSK/IBMX in the PAT-1 KO cecum (Fig. 2 and Table 1), we conclude PAT-1 was not an essential contributor to any of these responses. Prior studies with mouse intestine similarly determined PAT-1 does not participate in FSK-stimulated anion secretion by the duodenum [77,74,63] and jejunum [59,75]. Relative to the involvement of PAT-1 in epithelial ion and fluid transport, and acid-base homeostasis along the small intestine [19,41,74,61,62,59,75,64,65,82,79,11], where it is more abundantly expressed [77,42], the role of PAT-1 in the large intestine remains obscure. In the cecum, there appears to be a minor contribution to sulfate secretion [78], and PAT-1 expression in this segment can be up-regulated by the gut flora [70], and short-chain fatty acids [50]. Despite these connections to the microbiota, the oxalate-degrading symbiont O. formigenes has no absolute requirement for PAT-1 in order to elicit oxalate secretion by mouse large intestine [36,26]. Whether O. formigenes engages with a cAMP-dependent signaling pathway in host enterocytes, as indicated from studies with Caco-2 monolayers [4], remains to be seen.

cAMP-stimulated oxalate secretion involves apical DIDS-sensitive transport.

Having found cAMP-stimulated oxalate secretion was independent of PAT-1 there must be another apical anion transporter involved. To probe this possibility, we utilized the classical stilbene inhibitor, DIDS. Unlike PAT-1, DRA is relatively insensitive to DIDS [52,40,10,48,6,71,78,79], evident from the very modest 13-14 % reduction in DRA-mediated $J_{ms}^{Cl}$ (Figs. 3C and 3D). Thus, using mucosal DIDS in conjunction with the PAT-1 KO cecum was anticipated to help reveal other participating transporters. Applying 500 μmol/L DIDS had no effect on baseline oxalate flux, contrary to our findings at a lower concentration (200 μmol/L) where $J_{ms}^{Ox}$ and $J_{sm}^{Ox}$ increased 43 % and 25 %, respectively, which was attributed to increasing bidirectional paracellular flux [80]. The presence of DIDS, however, prevented the subsequent stimulation of oxalate secretion by FSK/IBMX for WT (Fig. 3A) and PAT-1 KO (Fig. 3B) cecum. The apical transport pathway responsible is therefore DIDS-sensitive and appears inactive until triggered by cAMP. This could signify recruitment of an anion exchanger, although lack of clear dependence on either mucosal Cl⁻ (Fig. 5), or HCO₃⁻ (Fig. 6), does not offer convincing evidence for a dedicated Cl⁻/HCO₃⁻ exchanger. One intriguing candidate is the sulfate transporter, DTDST (Diastrophic Dysplasia Sulfate Transporter; Slc26a2), which is a component of resting and FSK/IBMX-stimulated Cl⁻-driven oxalate uptake by Caco-2 monolayers, alongside PAT-1 [3]. DTDST has been characterized as a DIDS-sensitive sulfate/anion exchanger which counts oxalate, Cl⁻, and possibly HCO₃⁻, among its transported substrates [57,38,54]. Prominently localized at the apical membrane in human colon [24], DTDST is also abundantly expressed in the mouse colon [55,2], although whether this includes the cecum has not been reported. DTDST has an essential role in sulfate uptake by chondrocytes, fibroblasts and osteoblasts [25,17,55], and while we have speculated upon its contribution to sulfate transport by the cecum [78], the function of DTDST in the intestine has not been examined. Alternatively, this oxalate efflux pathway could be a DIDS-sensitive, channel-like conductance, similar to the one defined using brush-border membrane vesicles from rabbit ileum [20]. DIDS is capable of blocking a variety of anion channels [7], with the notable exception of the CFTR, which is characteristically unresponsive to extracellular DIDS [58]. Almost the entire cAMP-stimulated I_sc across mouse cecum requires a functional CFTR [12,15,66]. The ΔI_sc (i.e. Period III – Period II) brought about by FSK/IBMX was not significantly affected by 500 μmol/L DIDS (Fig. 7), or even as much as 1 mmol/L [39]. We can thus infer the CFTR was operating normally and the DIDS-sensitive transporter responsible for $J_{sm}^{Ox}$ was also not a major contributor to I_sc. Furthermore, we can exclude the possibility that the mouse CFTR was directly mediating oxalate secretion, consistent with investigations of the human CFTR which demonstrated no selectivity for oxalate when expressed in oocytes and activated by FSK/IBMX [18,45]. More detailed cellular and membrane-level studies are therefore necessary to distinguish the nature of this DIDS-sensitive apical oxalate transport mechanism and to help uncover the molecular identity of this pathway, whether it is another anion exchanger such as the DTDST, or perhaps a cAMP-activated conductance.

Fig. 7. A comparison of the change in short circuit current (ΔI_sc) across WT and PAT-1 KO mouse cecum in response to the stimulation of cAMP by a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX).

The ΔI_sc was calculated as the difference between the average I_sc measured during the 45 min time period following application of FSK/IBMX subtracted from the average I_sc in the preceding 45 min based on data presented in Figures 1-6. A two-way ANOVA determined there were significant differences in the ΔI_sc between experiments (F_4,131 = 16.62, P <0.001), but not WT and PAT-1 KO tissues (P = 0.598), and these differences were not dependent on genotype (P = 0.912). Multiple comparisons were made to the ‘FSK/IBMX only’ set of experiments using the Holm-Sidak method with differences indicated by an asterisk

Role of the CFTR in cAMP-stimulated oxalate secretion by the cecum.

We have deduced the CFTR is unlikely to be directly responsible for oxalate efflux, yet it remains relevant since co-expression with PAT-1 in oocytes enhanced FSK-stimulated oxalate transport by the latter [45], in accordance with the regulatory interactions exhibited between the CFTR and Slc26 anion transporter family [46,47,9,16]. We therefore utilized CFTR_inh-172 to specifically block CFTR activity without impeding anion exchange [51,83,73]. Upon mucosal application of CFTR_inh-172 to WT cecum there was no impact on basal oxalate flux (Fig. 4A), consistent with Knauf et al. [45] who previously showed $J_{sm}^{Ox}$ across mouse duodenum was similarly unresponsive to this inhibitor. Therefore, a functional CFTR is not necessary to support baseline rates of oxalate secretion, for example by providing a shunt or “leak” path allowing Cl⁻ to recycle across the apical membrane in support of Cl⁻/oxalate exchange. CFTR_inh-172 did, however, block the stimulation of $J_{sm}^{Ox}$ by FSK/IBMX (Fig. 4A), indicating participation of a functional CFTR. Conversely, repeating this experiment with the PAT-1 KO cecum found $J_{sm}^{Ox}$ could be significantly increased by FSK/IBMX (Fig. 4B). The stimulation of $J_{sm}^{Ox}$ via cAMP would therefore appear dependent on a functional CFTR, but only in the presence of PAT-1. Conceivably, PAT-1 may function as part of a multi-transporter complex by regulating the ability of the CFTR to activate the (DIDS-sensitive) transporter responsible for oxalate secretion. A similar interplay has been proposed to explain how HCO₃⁻ secretion is controlled at the luminal membrane of mouse pancreatic ducts, where PAT-1 regulates CFTR activity which, in turn, augments the function of a separate Cl⁻/HCO₃⁻ exchanger [76]. However, it is notable this apparent influence of PAT-1 on $J_{sm}^{Ox}$ does not extend to FSK-stimulated net Cl⁻ secretion (Figs. 4C and 4D) or I_sc (Fig. 4E) which were indistinguishable between WT and PAT-1 KO tissues, suggesting PAT-1 is not a major partner for the CFTR in the cecum. Regardless of mechanism, blocking the CFTR whether in the presence or absence of PAT-1 was not a barrier to achieving net oxalate secretion in response to FSK/IBMX (Fig. 8A), via decreased $J_{ms}^{Ox}$, which may be DRA-related [22] given the timing was coincident with the abrupt inhibition of $J_{ms}^{Cl}$ (Figs. 4C and 4D). There was evidence hinting at a potential functional interaction between DRA and the CFTR in mouse cecum since CFTR_inh-172 brought about a gradual, steady decline in $J_{ms}^{Cl}$ by both WT and PAT-1 KO tissues during Period II (Figs. 4C and 4D), similar to mucosal DIDS (Figs. 3C and 3D). Under resting conditions, this could reflect the CFTR allowing Cl⁻ to recycle back into the lumen in support of apical (DRA-mediated) Cl⁻/HCO₃⁻ exchange, as suggested from prior studies of the duodenum [67,60,64]. Alternatively, we cannot exclude the possibility of CFTR_inh-172 acting directly on DRA. For example, while CFTR_inh-172 did not affect (human) DRA activity in a number of different cell systems [73], it reduced Cl⁻ uptake into oocytes expressing (guinea pig) DRA by 45 % [71].

Fig. 8. A comparison of the change in net oxalate ($\Delta {\mathit{J}}_{\mathit{net}}^{\mathit{Ox}}$) and chloride ($\Delta {\mathit{J}}_{\mathit{net}}^{\mathit{Cl}}$) transport across WT and PAT-1 KO mouse cecum in response to the stimulation of cAMP by a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX).

For both anions, the $\Delta J_{net}^{ion}$ was calculated as the difference between the average $J_{net}^{ion}$ measured during the 45 min time period following application of FSK/IBMX subtracted from the $J_{net}^{ion}$ in the preceding 45 min based on data presented in Figures 1-6. For $\Delta J_{net}^{Ox}$ in Panel A, a two-way ANOVA determined there were significant differences between experiments (F_4,61 = 3.08, P = 0.022), but not WT and PAT-1 KO tissues (P = 0.746), and these differences in $\Delta J_{net}^{Ox}$ were not dependent on genotype (P = 0.919). For $\Delta J_{net}^{Cl}$ in Panel B, a two-way ANOVA determined there were significant differences between experiments (F_3,47 = 2.99, P = 0.040), but not WT and PAT-1 KO tissues (P = 0.632), and these differences in $\Delta J_{net}^{Ox}$ were not dependent on genotype (P = 0.862). Multiple comparisons were made to the ‘FSK/IBMX only’ set of experiments using the Holm-Sidak method with differences indicated by an asterisk. n.d. = not determined

Mucosal anion substitutions.

Cl⁻ and HCO₃⁻ exert a sizeable influence on J^Ox [33,35,19,21], on account of oxalate being a substrate for the Cl⁻/HCO₃⁻ exchangers where it can compete and/or be counter-transported with these two major extracellular anions. The classical maneuver of substituting these anions out of the bathing solution can therefore reveal evidence for involvement of an anion exchange mechanism. For example, substituting Cl⁻ from the mucosal bath reduced $J_{sm}^{Ox}$ by distal ileum from WT but not the PAT-1 KO mouse, implying PAT-1 operates as an apical Cl⁻/oxalate exchanger [19]. For the adjacent cecum, however, a similar transport mechanism was not apparent (Table 2). While FSK/IBMX activated Cl⁻/oxalate exchange by PAT-1 and DTDST in Caco-2 monolayers [3], restricting luminal Cl⁻ failed to impede the ability of FSK/IBMX to stimulate $J_{sm}^{Ox}$ (Figs. 5A and 5B) or net oxalate secretion (Fig. 8A), by both the WT and PAT-1 KO cecum. The relatively low concentrations of luminal Cl⁻ (~20 mmol/L) measured within the rodent cecum in vivo [72,37], are unlikely to impede secondary-active cAMP-dependent oxalate secretion in vivo. The contribution of DRA to $J_{ms}^{Ox}$ across mouse intestine was proposed to occur via Cl⁻(oxalate)/HCO₃⁻ exchange [22]. By eliminating Cl⁻ as a competing anion we predicted oxalate absorption would enhance baseline $J_{ms}^{Ox}$, however, this did not change significantly (Table 2).

We also substituted mucosal HCO₃⁻ and CO₂, leaving carbonic anhydrase (CA) intact. PAT-1 can function as a base importer in mouse small intestine, operating as a HCO₃⁻/anion exchanger [62,82], making absorption via HCO₃⁻(oxalate)/anion exchange or secretion by HCO₃⁻/oxalate exchange conceivable modes of transport. However, neither competition with, nor dependence on, extracellular HCO₃⁻ were revealed under basal, non-stimulated conditions for WT and PAT-1 KO cecum (Table 2). In response to FSK/IBMX, the absence of luminal HCO₃⁻/CO₂ failed to restrict net oxalate secretion (Fig. 8A), suggesting cAMP was not recruiting a HCO₃⁻/oxalate exchanger. Furthermore, with no drastic changes to $J_{sm}^{Cl}$ in either WT (Fig. 6C) or PAT-1 KO (Fig. 6D) cecum, it would appear that (PAT-1 mediated) HCO₃⁻/Cl⁻ exchange is not a prominent transport process in this segment [80], unlike the jejunum [82]. With this buffer arrangement, the gradient for intracellular HCO₃⁻ exit across the apical membrane will be maximized potentially driving DRA-mediated Cl⁻(oxalate)/HCO₃⁻ exchange. On the contrary, $J_{ms}^{Ox}$ was not affected and $J_{ms}^{Cl}$ substantially reduced, effectively abolishing net Cl⁻ absorption (Table 2). We previously reported that bilateral substitution of HCO₃⁻/CO₂ similarly attenuated Cl⁻ absorption via DRA in mouse cecum, which was ascribed to the loss of basolateral HCO₃⁻ import from the serosal bath thus limiting its exchange with luminal Cl⁻ [78]. However, this cannot explain why, in the present work, Cl⁻ absorption was suppressed despite maintaining the supply of serosal HCO₃⁻/CO₂ and tissue CA activity (Table 2). For mouse jejunum, $J_{ms}^{Cl}$ was almost 50 % lower in the absence of luminal HCO₃⁻ compared to bilateral HCO₃⁻-containing buffer [75]. How Cl⁻ absorption is specifically inhibited by removing mucosal HCO₃⁻/CO₂ is unclear.

Summary.

The objective of this study was to investigate the cAMP-dependent regulation of oxalate transport across the native intestinal epithelium. Employing the in vitro Ussing chamber technique with the mouse model we focused on trying to distinguish the apical transport mechanisms involved, specifically testing the hypothesis that oxalate secretion by the anion exchanger PAT-1 (Slc26a6) was the target of this cell signaling pathway. Our main findings can be summarized as follows:

1) Contrary to the earlier rabbit model, the regulation of oxalate secretion by a cAMP-dependent signaling pathway was not a feature shared by all regions of the mouse intestine and the underlying oxalate transport mechanisms were clearly distinct from those responsible for net Cl⁻ secretion.

2) Of the three intestinal segments examined, acutely elevating intracellular cAMP activated oxalate secretion by the cecum only, which occurred via increased $J_{sm}^{Ox}$, but was without effect on existing net secretion by distal ileum and distal colon.

3) cAMP-stimulated $J_{sm}^{Ox}$ by the cecum was not directly dependent on PAT-1 but could be inhibited by mucosal DIDS suggesting a related anion exchange mechanism. However, this was unlikely to be another apical Cl⁻/HCO₃⁻ exchanger per se, since there was no evidence of trans-stimulation or cis-inhibition by luminal Cl⁻ or HCO₃⁻, and this unidentified transporter was also not a major contributor to I_sc.

4) This DIDS-sensitive apical efflux pathway could also be represented by a channel-like conductance. Despite no evidence of a direct role for the CFTR, specifically inhibiting this cAMP-activated anion channel prevented the stimulation of $J_{sm}^{Ox}$, but only in the presence of PAT-1. We suggest the apical oxalate transporter responsible for $J_{sm}^{Ox}$ was reliant on a functional CFTR and the latter regulated by PAT-1 as part of a multi-protein complex.