Activation of the PKA signaling pathway stimulates oxalate transport by human intestinal Caco2-BBE cells

Abstract

Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [¹⁴C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [¹⁴C]oxalate transport as a function of increasing [¹⁴C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in V_max and reduction (2-fold) in K_m. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced K_m. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.

INTRODUCTION

Nephrolithiasis is one of the most common urological diseases in Western societies. It is a major source of patient discomfort and disability, lost working days, and healthcare costs (~$10 billion annually). Hyperoxaluria is a major risk factor for kidney stones (KS), and ~80% of KS are composed of calcium oxalate (8). Urinary oxalate is an important determinant of supersaturation, and the risk for stone formation is affected by small changes in urine oxalate (9). Of note is that the risk of stone formation begins to rise in both men and women even at urinary oxalate levels traditionally considered to be within the normal range (25–30 mg/day) (9). Importantly, a history of a single KS is associated with a significantly increased risk of poor renal outcome, including advanced chronic kidney disease (CKD) and end-stage renal disease (ESRD) (1). Both CKD and ESRD are associated with significantly higher long-term morbidity and mortality as well as a substantial healthcare cost.

The mammalian intestine plays a critical role in oxalate homeostasis by serving as a site for dietary oxalate absorption as well as an avenue, together with the kidneys, for oxalate excretion (21). The amount of oxalate excreted in the urine is influenced by dietary oxalate intake, net intestinal absorption, endogenous production, and renal clearance. Intestinal oxalate absorption is predominantly passive through the paracellular pathway, while secretion is through the transcellular (transepithelial) pathway (15, 26, 30). The following examples reflect the crucial role of the intestine in oxalate homeostasis. Hyperoxaluria and a high incidence of KS are common in patients with inflammatory bowel disease (IBD) (7). Hyperoxaluria is a major complication (developing in ≥50% of patients) of malabsorptive bariatric surgery for obesity and small-bowel resection (37). In addition, anion exchanger SLC26A6 (A6)-null mice have a critical defect in intestinal oxalate secretion, leading to enhanced net absorption of ingested oxalate, hyperoxalemia, hyperoxaluria, and a high incidence of calcium oxalate KS (COKS) (15, 26), indicating that defects in the function or regulation of this key oxalate transporter are potential molecular mechanisms predisposing to COKS in humans. Collectively, understanding the molecular mechanisms regulating A6 and intestinal oxalate transport are potentially very important for the management of hyperoxaluria, hyperoxalemia, and COKS. Better understanding of such molecular regulatory mechanisms could yield valuable information that might lead to the design of novel therapeutic approaches for the prevention and/or treatment of hyperoxalemia, hyperoxaluria, and COKS. Unfortunately, hyperoxaluria remains without specific therapy.

As described above, A6 plays a crucial role in enteric transcellular oxalate secretion, thereby preventing hyperoxaluria and related COKS. Transcellular intestinal oxalate secretion requires oxalate influx into the enterocyte from blood (basolateral side), where anion exchanger SLC26A1 (A1) is possibly involved, and then its efflux from the luminal side by A6 [with or without other transporters, potentially including SLC26A2 (A2)] (12, 15, 26). Of note is that A6 mediates ≥49% of apical oxalate uptake by human intestinal Caco2-BBE (C2) cells (5, 16), and it functions in the direction of exchanging intracellular oxalate for mucosal Cl during the process of transepithelial intestinal oxalate secretion. However, A6 can operate in either direction (27), and we therefore measured its activity by the more convenient assay of cellular oxalate uptake. Worthy of mention is that we (4, 5, 28) had previously established C2 cells as a useful model to study the regulation of intestinal oxalate transport by purinergic and adenosinergic signaling, as well as by Oxalobacter-derived secreted bioactive factors.

We (5) previously reported that Oxalobacter-derived bioactive factors secreted in its culture-conditioned medium stimulate oxalate transport by intestinal cells by activating the PKA signaling pathway. We therefore evaluated whether intestinal oxalate transport is subject to direct regulation by activation of the PKA signaling pathway. We find that PKA activation with forskolin plus 3-isobutyl-1-methylxanthine (IBMX) (F/I) significantly stimulates oxalate transport by C2 cells through mechanisms including increased A6 surface protein expression and enhanced A6 and/or A2 transport activity.

MATERIALS AND METHODS

Cell culture.

Human intestinal C2 and T84 cells were grown and maintained (including monitoring of the transepithelial resistance) as we reported previously (4, 20). The oxalate flux and other studies described below were performed using confluent cells grown for 4–13 days postplating on 0.4-µm collagen-coated polystyrene transwell membrane filters (Corning, Inc., Corning, NY) in 12-mm inserts. The cells were switched from DMEM containing 8–10% fetal bovine serum (FBS) to DMEM with 0.1% FBS overnight before treatment with F/I.

Radioactive flux studies.

Apical [¹⁴C]oxalate flux studies in C2 and T84 cells were performed following our previously published methods (4, 20). After aspiration of the culture medium, the cells were incubated in an isotonic NaCl solution (in mM: NaCl 120, CaCl₂ 2, MgCl₂ 1, HEPES 20, glucose 5, titrated with Tris base to pH 7.4) at 37°C for 30 min. This solution was then aspirated and replaced with a Cl⁻-free solution (in mM: K-gluconate 130, glucose 5, HEPES 20, pH 7.4) containing 20 μM [¹⁴C]oxalate to impose an outward Cl gradient by removing extracellular Cl [Cl_i > Cl_o], and the influx of [¹⁴C]oxalate in exchange for intracellular Cl [i.e., apical Cl⁻-oxalate exchange activity] was measured over 6 min. This 6-min influx period was chosen because it falls within the linear range of oxalate uptake by these cells. Of note is that we (20) had previously shown that imposing an outward Cl gradient by removing extracellular Cl (Cl_i > Cl_o) significantly stimulates [¹⁴C]oxalate uptake, which is greatly inhibited (>80%) by external Cl (Cl_o > Cl_i), consistent with Cl⁻-oxalate exchange. In addition, we also showed that this apical Cl⁻-oxalate exchange activity is significantly inhibited (>91%) by the anion exchange inhibitor DIDS (100 µM; luminal) (4), indicating that the observed oxalate uptake is mediated by one or more of the involved anion exchanger(s) in C2 cells. Importantly, we (5) and others (16) have shown that A6 mediates ≥49% of the above described Cl⁻-oxalate exchange activity in C2 cells. The influx of [¹⁴C]oxalate was terminated by 2–3 rapid washes of the cell monolayers with ice-cold Cl⁻-free solution, and the transwells were then placed upside down and allowed to dry for several minutes. Membrane filters containing the cells were cut from the support and placed into vials with scintillation fluid (Opti-Fluor, Packard), and the radioactivity was measured by scintillation spectrometry following overnight solubilization.

A2 and A6 knockdown in C2 cells.

Using slight modification of a published protocol (16), we had previously utilized siRNA to knockdown A6 total protein expression by ~61% in C2 cells (5). Using the same protocol (including the same A6 and negative control siRNAs), we similarly knocked down A6 and A2 expression in C2 cells. Briefly, C2 cells were untransfected (Control) or transfected with the negative control siRNA (NC siRNA; cat. no. AM4611-3690528207, ThermoFisher Scientific), the A6-specific siRNA (A6 siRNA; cat. no. AM16708-3690520878, ThermoFisher Scientific), or the A2-specific siRNA (A2 siRNA; cat. no. AM16708-3667241045, ThermoFisher Scientific), and the relevant studies were then performed 2–4 days after transfection.

Immunocytochemistry.

For the immunocytochemistry studies, C2 cells grown on transwell inserts were serum starved as described above. Cells were treated with vehicle or F/I (625/500 μM) for 30 min in the serum-starved medium followed by washing once in PBS. Cells were fixed for 1 h in a solution containing 1% paraformaldehyde in PBS-1 mM calcium, followed by one wash in PBS. Quenching for 5 min three times in PBS containing 50 mM ammonium chloride–1 mM calcium. Cells were then blocked with blocking buffer (10% goat serum cat. no. G9023, Sigma-Aldrich) and 0.05% saponin (to permeabilize the cells) in PBS for 30 min at room temperature, and then incubated overnight at 4°C with primary antibody (anti-SLC26A6 antibody (Novus Biologicals; cat. no. H00065010-A01; RRID: AB_548168; 1:25 dilution in blocking buffer). Cells were washed six times in washing buffer (3% normal goat serum and 0.05% saponin) and then incubated for 1 h at room temperature with Alexa fluor 488 goat anti-mouse IgG (Invitrogen, 1:200 dilution in blocking buffer) protected from light. Cells were then washed six times in blocking buffer. Finally, sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA), and then visualized by laser scanning confocal microscope (Leica SP2) at Oil ×63 to capture the images.

The images were analyzed in ImageJ. Peak immunofluorescence intensity was determined in the z-direction from the average peak intensity of five different areas in each image.

Materials.

[¹⁴C]oxalate was purchased from Vitrax (specific activity 54 mCi/mmol). Forskolin, H89 dihydrochloride, and IBMX were purchased from Tocris, Selleck Chemicals, and ThermoFisher Scientific, respectively. Forskolin, IBMX, and H89 were dissolved in DMSO and stored at −20°C. Equivalent volumes of DMSO (0.1–0.3%) were added to control media.

Statistical analysis.

Experimental data are presented as means ± SE. Data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni or Student-Newman-Keuls post hoc test, or by Student’s t test for paired or unpaired samples when two groups were compared. P values < 0.05 were considered statistically significant.

RESULTS

The purpose of the current study was to evaluate whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, we used forskolin and IBMX (F/I) to activate the PKA signaling pathway. We assessed apical oxalate uptake by C2 cells, ≥49% of which is mediated by A6 (5, 16), by imposing an outward Cl gradient by removing extracellular Cl (Cl_i > Cl_o) and measuring DIDS-sensitive influx of radioactive [¹⁴C]oxalate in exchange for intracellular Cl (i.e., Cl⁻-oxalate exchange activity) as we previously reported (4, 20). As shown in Fig. 1, preincubation of C2 cells with F/I (625/500 µM for 30 min) caused significant stimulation (3.7-fold) of [¹⁴C]oxalate transport by C2 cells. [¹⁴C]oxalate transport by cells treated with vehicle (DMSO) was similar to uptake by untreated (UT) cells (UT, 3.19 ± 0.07; vehicle, 3.17 ± 0.07 pmol·cm⁻²·min⁻¹) and therefore is not shown. The F/I concentrations and preincubation periods were selected based on the F/I dose-response (Fig. 2) and time course (Fig. 3) described below. These results show that intestinal oxalate transport is likely subject to stimulatory regulation by the PKA signaling pathway.

Fig. 1.

Effect of forskolin/IBMX (F/I) on [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. C2 cells were untreated (UT) or were treated apically with F/I (625/500 µM) for 30 min in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 24 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by C2 cells (*P < 2.4E⁻¹¹, by unpaired t test; n = 24).

Fig. 2.

Effect of different concentrations of forskolin/IBMX (F/I) on [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. C2 cells were untreated (UT) or were treated apically with different concentrations of F/I (5/500 = F5/I500, 25/500 = F25/I500, 125/500 = F125/I500, and 625/500 = F625/I500 µM) for 30 min in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 3 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by C2 cells (*P < 0.05, < 0.01, < 0.001, and < 0.001 for UT vs. F5/I500, F25/I500, F125/I500, and F625/I500 µM, respectively; `P < 0.001 for F625/I500 vs. F5/I500, F25/500, and F125/I500; `P < 0.05 for F125/I500 vs. F5/I500, by ANOVA; n = 3).

Fig. 3.

Effect of different preincubation periods with forskolin/IBMX (F/I) on [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. To determine the time course over which the stimulatory effects of F/I occur, C2 cells were untreated (UT) or were treated apically with F/I (625/500 µM) for 5, 15, 30, and 60 min in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 3–4 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by C2 cells over the 15-, 30-, and 60-min time points (*P < 0.05, < 0.001, and < 0.001 for UT vs. 15, 30, and 60 min, respectively; `P < 0.001 and < 0.01 for 30 min vs. 5 and 30 min, respectively; `P < 0.01 and < 0.05 for 60 min vs. 5 and 30 min, respectively; `P < 0.05 for 15 min vs. 5 min, by ANOVA; n = 3–4).

To generate an F/I dose response, the effects of 5/500, 25/500, 125/500, and 625/500 µM F/I for 30 min on [¹⁴C]oxalate uptake by C2 cells were evaluated. As shown in Fig. 2, F/I significantly stimulated oxalate transport by C2 cells in a dose-dependent manner, with the 625/500 µM concentration giving the maximum stimulation of oxalate transport. Based on these results, the 625/500 µM concentration was selected for this study.

To determine the time course over which the stimulatory effects of F/I occurs, C2 cells were treated with F/I (625/500 µM) for 5, 15, 30, and 60 min before [¹⁴C]oxalate uptake was measured. F/I significantly stimulated oxalate transport by C2 cells ~1.4-, 2.3-, 3.7-, and 3.3-fold over 5, 15, 30, and 60 min, respectively (Fig. 3), with the highest stimulation observed over the 30-min preincubation period. There was no significant difference between the F/I-induced stimulation over 30 min compared with 60 min. Based on these results, the 30-min preincubation period was chosen for the study.

To ensure that the stimulatory regulation of intestinal oxalate transport by the PKA signaling pathway is not cell line specific, we similarly evaluated the effects of F/I on oxalate uptake by the human colonic cell line T84. Of note is that we (5, 20, 28) had previously established T84 cells as a useful model to study regulation of intestinal oxalate transport by cholinergic and adenosinergic signaling, as well as by Oxalobacter-derived secreted factors. We (20) had also shown that A6 mediates most of oxalate transport in T84 cells. Compared with UT cells, preincubation of T84 cells with F/I (625/500 µM for 30 min) significantly stimulated (~2.3-fold) oxalate transport by T84 cells (Fig. 4). These results indicate that the observed stimulatory regulation of oxalate transport by intestinal epithelial cells by the PKA signaling pathway is not cell line specific.

Fig. 4.

Effect of forskolin/IBMX (F/I) on [¹⁴C]oxalate uptake by T84 cells. T84 cells were untreated (UT) or were treated apically with F/I (625/500 µM) for 30 min in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 6 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by T84 cells (*P < 0.02 for F/I vs. UT, by unpaired t test; n = 6).

F/I could potentially have effects beyond activation of the PKA signaling pathway, and therefore, to provide further evidence that the observed F/I-induced stimulation of oxalate transport by C2 cells was mediated by activation of the PKA signaling pathway, we evaluated whether it is blocked by the PKA inhibitor H89. To this end, preincubation of C2 cells with the PKA inhibitor H89 (20 µM for 30 min) (5, 36) before incubation with F/I (625/500 µM for 30 min; F/I+H89) completely blocked the F/I-induced stimulation, whereas it had no effect on baseline transport (H89) (Fig. 5). These results support that conclusion that the observed F/I-induced stimulation of oxalate transport by C2 cells is indeed most likely mediated by activation of the PKA signaling pathway.

Fig. 5.

Effect of PKA inhibitor H89 on the forskolin/IBMX (F/I)-induced stimulation of [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. C2 cells were untreated (UT) or were treated apically with F/I (625/500 µM) for 30 min in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. C2 cells were also treated apically with PKA inhibitor H89 (20 µM) for 30 min followed by F/I (625/500 µM) for 30 min with continued presence of H89 (F/I+H89), or with H89 (20 µM) alone for 60 min, and then [¹⁴C]oxalate uptake was similarly measured. Values are means ± SE of 4 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by C2 cells, an effect completely blocked by H89 (*P < 0.001 for F/I vs. UT, F/I+H89, and H89, by ANOVA; n = 4).

As a first step in elucidating the molecular mechanism(s) behind the observed PKA-induced stimulation of oxalate transport by C2 cells, we examined whether the observed stimulatory regulation was due to a F/I-mediated increase in the surface membrane expression of the key oxalate transporter A6. To this end, we performed immunofluorescence confocal microscopy on cells treated without or with F/I. As shown in Fig. 6, we observed that F/I under the same conditions leading to stimulation of oxalate transport by C2 cells also led to significantly increased (2.5-fold) A6 surface protein expression assayed by quantification of peak immunofluorescence intensity. These results indicate that F/I stimulatory regulation of oxalate transport by C2 cells is mediated by mechanisms including F/I-induced enhanced A6 surface protein expression.

Fig. 6.

Effect of forskolin/IBMX (F/I) on SLC26A6 (A6) surface expression in Caco2-BBE (C2) cells assayed by immunocytochemistry. A: C2 cells were treated apically with vehicle (Control) or F/I (625/500 µM) for 30 min in NaCl buffer before immunofluorescence confocal microscopy on sections of paraformaldehyde-fixed C2 cells was performed, as described in materials and methods. Scale bar, 10 μm. F/I led to increased A6 surface protein expression. B: analysis of images in ImageJ. Peak immunofluorescence intensity was determined in the z direction from the average peak intensity of 5 different areas in each image. Values are means ± SE of 4 independent experiments. F/I significantly increased A6 surface protein expression (*P < 0.03, by paired t test; n = 4).

To examine whether the observed PKA stimulatory regulation affects oxalate transport kinetic characteristics [i.e., the apparent affinity for oxalate (K_m) and maximal velocity (V_max)], [¹⁴C]oxalate uptake as a function of increasing [¹⁴C]oxalate concentration in the flux medium (0.3, 1, 3, 10, 30, 100, and 225 µM) was assessed. As shown in Fig. 7, and compared with UT cells, F/I significantly stimulated (>2-fold) oxalate uptake (which is saturable in the presence of increasing oxalate concentrations, reflecting a carrier-mediated transport process) at each concentration. Analysis of these results with a nonlinear regression Michaelis-Menten fit yielded a K_m of 120.8 ± 69 and 59.7 ± 22.3 µM and a V_max of 16.8 ± 4.4 and 29.4 ± 4 pmol·cm⁻²·min⁻¹ for UT and F/I, respectively. These results indicate that the observed stimulation was due to the PKA-induced increase (~1.8-fold) in V_max (i.e., greater transport capacity) and reduction [>2-fold) in K_m, i.e., greater affinity for oxalate) of the involved oxalate transporter(s).

Fig. 7.

Effect of forskolin/IBMX (F/I) on kinetic characteristics of oxalate transport in Caco2-BBE (C2) cells. C2 cells were untreated (UT) or were treated apically with F/I (625/500 µM) for 30 min in NaCl buffer, and then [¹⁴C]oxalate uptake as a function of increasing [¹⁴C]oxalate concentration in the flux medium (0.3, 1, 3, 10, 30, 100, and 225 µM) was assessed. Values are means ± SE of 3–5 independent experiments, each done in triplicate. F/I significantly stimulated [¹⁴C]oxalate uptake by C2 cells at the 0.3, 1, 3, 10, 30, and 100 µM oxalate concentrations (*P < 0.02, < 0.007, < 0.00007, < 0.006, < 0.0008, and < 0.02 for F/I vs. UT at 0.3, 1, 3, 10, 30, and 100 µM [¹⁴C]oxalate concentrations, respectively, by unpaired t test; n = 3–5).

To directly evaluate the role of A6 in the observed stimulation, A6 expression in C2 cells was knocked down using siRNA. C2 cells were untransfected (Control) or transfected with a negative control siRNA (NC siRNA) or an A6-specific siRNA (A6 siRNA). Silencing A6 significantly reduced the F/I-induced stimulation of oxalate transport by C2 cells (Fig. 8). A6 siRNA knockdown also reduced baseline oxalate transport in C2 by ~57% (Fig. 8), which was similar to what we previously reported (5). F/I stimulated oxalate transport by ~3.4-fold and ~2.1-fold in Control and A6 siRNA cells, respectively, reflecting a 38% reduction in the F/I-induced stimulation of oxalate transport. Of note is that F/I stimulated oxalate transport by C2 cells transfected with the NC siRNA to a level similar to that of Control (data not shown). These results indicate that a significant component of the PKA-induced stimulation of oxalate transport by C2 cells was mediated by the key oxalate transporter A6.

Fig. 8.

Effect of SLC26A6 (A6) knockdown on forskolin/IBMX (F/I)-induced stimulation of [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. Control (untransfected) and A6 siRNA (transfected with the siRNA targeting A6) C2 cells were untreated (UT) or were treated apically with forskolin/IBMX (F/I; 625/500 µM) in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 10 independent experiments, each done in triplicate. Silencing A6 significantly reduced F/I-induced stimulation of oxalate transport by C2 cells [*P < 0.001 for F/I (Control) vs. UT (Control), UT (A6 siRNA), and F/I (A6 siRNA); #P < 0.001 for UT (Control) vs. UT (A6 siRNA), by Wilcoxon rank-sum test; n = 10].

To directly examine the role of the oxalate transporter A2 in the observed stimulation, A2 expression in C2 cells was similarly knocked down using siRNA. C2 cells were untransfected (Control) or transfected with a negative control siRNA (NC siRNA) or an A2-specific siRNA (A2 siRNA). Silencing A2 significantly reduced the F/I-induced stimulation of oxalate transport by C2 cells (Fig. 9). A2 siRNA knockdown also reduced baseline oxalate transport by ~23% (Fig. 9). F/I stimulated oxalate transport ~3.5- and ~2.3-fold in Control and A2 siRNA cells, respectively, reflecting a 36% reduction in the F/I-induced stimulation of oxalate transport. Of note is that F/I similarly stimulated oxalate transport by NC siRNA C2 cells (data not shown). These results indicate that a significant component of the PKA-induced stimulation of oxalate transport by C2 cells was also mediated by A2.

Fig. 9.

Effect of SLC26A2 (A2) knockdown on forskolin/IBMX (F/I)-induced stimulation of [¹⁴C]oxalate uptake by Caco2-BBE (C2) cells. Control (untransfected) and A2 siRNA (transfected with the siRNA targeting A2) C2 cells were untreated (UT) or were treated apically with forskolin/IBMX (F/I; 625/500 µM) in NaCl buffer, and then [¹⁴C]oxalate uptake was measured as described in materials and methods. Values are means ± SE of 15 independent experiments, each done in triplicate. Silencing A2 significantly reduced F/I-induced stimulation of oxalate transport by C2 cells [*P < 0.001 for F/I (Control) vs. UT (Control), UT (A2 siRNA), and F/I (A2 siRNA); #P < 0.007 for UT (Control) vs. UT (A2 siRNA), by Wilcoxon rank-sum test; n = 15].

DISCUSSION

In this study, we used the human intestinal epithelial cell line C2 to examine whether intestinal oxalate transport is subject to regulation by the PKA signaling pathway. We found that oxalate transport by C2 cells is positively regulated by activation of the PKA signaling pathway using F/I. F/I-induced stimulation of oxalate transport by C2 cells is completely blocked by the PKA inhibitor H89, indicating that it is indeed PKA dependent. F/I similarly stimulated oxalate transport by T84 cells, indicating that PKA stimulatory regulation of intestinal oxalate transport is not cell line specific. By use of immunofluorescence confocal microscopy, the observed stimulatory regulation was due to mechanisms including enhanced A6 surface protein expression. Assessing [¹⁴C]oxalate transport as a function of increasing [¹⁴C]oxalate concentration in the flux medium showed that that the observed stimulation was due to PKA-induced increase (1.8-fold) in V_max and reduction (2-fold) in K_m. siRNA knockdown studies showed that significant components of the observed stimulation were mediated by the oxalate transporters A2 and A6. Worthy of mentioning is that we had previously shown that A6 mediated >49% of apical oxalate transport by C2 cells (5), while here we are showing that A6 mediated >57% of apical oxalate transport by C2 cells (Fig. 8). The difference is likely due to siRNA lot-to-lot variability (we used a new batch/lot of A6 siRNA in this study).

A6 plays an essential role in mouse duodenal and ileal oxalate secretion, and A6-null mice develop significant hyperoxalemia, hyperoxaluria, and a high incidence of COKS (15, 26). A6 is also expressed apically in mouse colon (42), but its role in colonic oxalate transport awaits further investigations. It is possible that A6-null mice might have reduced active transcellular colonic oxalate secretion, which also potentially contributes to the reported phenotype of hyperoxaluria, hyperoxalemia, and COKS (15, 26). Knockdown studies showed that a significant component of the PKA-induced stimulation of oxalate transport by C2 cells is mediated by A6. Protein kinases can positively regulate a transporter by increasing the number of the transporters at the apical membrane. Protein kinases can also positively regulate a transporter by modifying the intrinsic activity (e.g., substrate kinetic parameters) of preexisting membrane transporters without changing the number of the transporters at the apical membrane. An increase in V_max indicates greater transport capacity, which could result from more apical membrane transporters as observed with A6. In addition, and in view of the reduced K_m (reflecting an increase in A6 affinity for the substrate oxalate in the setting of PKA activation), it is also possible that the observed stimulation is due to mechanisms including F/I-induced enhanced A6 transport activity resulting from an increase in the intrinsic activity of the preexisting A6 apical membrane transporters. Of note is that A6 is not predicted to have a PKA phosphorylation site. However, indirect PKA regulation of A6 is possible through its interaction with cystic fibrosis transmembrane conductance regulator (CFTR). CFTR stimulates SLC26A3 and A6 functions in cultured pancreatic duct cells (17). PKA-dependent phosphorylation of the CFTR R domain promotes its binding to SLC26 anion exchanger sulfate transporter and anti-Sigma factor antagonist (STAS) domain, resulting in significant mutual activation of CFTR and the SLC26 anion exchanger (32, 40). Therefore, A6 could be involved in the observed F/I-induced stimulation through mechanisms including PKA-dependent CFTR activation. It is also possible that the observed PKA-induced stimulation of intestinal oxalate transport could be directly mediated by a noncanonical PKA phosphorylation site(s), and future studies will attempt to identify such sites in A6.

A2 is apically expressed in enterocytes (colon > small intestine) (2, 19, 39) and it mediates DIDS-sensitive oxalate transport when expressed in heterologous systems (24). However, its role in intestinal oxalate transport remains unknown. The observed changes in K_m and V_max could also result from PKA-mediated increase in A2 total and/or surface protein expression and/or transport activity. Since transcellular oxalate secretion requires oxalate influx into the enterocyte from the blood side, where A1 might be involved (12), and then its efflux from the luminal side, it is possible that PKA-induced changes in A1 total and/or surfac expression and/or transport activity might also contribute to the previously reported stimulation of oxalate secretion in rabbit proximal and distal colonic tissues by cAMP (22, 23). However, since we are assessing apical oxalate transport in C2 cells, the basolateral A1 is unlikely to be involved in F/I-induced stimulation of oxalate transport by C2 cells. Of interest is that A1 is predicted to have two PKA phosphorylation sites (Ser¹²² and Ser⁴⁶⁷), while A2 is predicted to have one PKA phosphorylation site (Ser⁶⁹³). Ser⁴⁶⁷ and Ser⁶⁹³ are highly conserved, raising their functional significance and potential involvement in PKA regulation of these transporters. Due to lack of good antibodies, the effects of F/I on A1 and A2 total and surface protein expression were not assessed, and this will be evaluated in future studies once good antibodies become available. It should be noted that studies in A1-null mice in a mixed 129/JSv × CD1 background indicated that loss of A1-mediated intestinal oxalate secretion was responsible for the hyperoxaluria, hyperoxalemia, and urolithiasis observed in these mice (12). However, recent studies in the same A1-null mice after being transferred to a C57BL background show that these mice do not have hyperoxaluria or hyperoxaluria and that A1 does not play a role in duodenal, distal ileal, and distal colonic oxalate secretion (31, 43).

The PKA-mediated stimulatory regulation of oxalate transport by C2 and T84 cells fits well with previous studies showing that cAMP stimulates oxalate secretion in rabbit proximal and distal colonic tissues (22, 23). Of interest in this regard is that we (5) had previously demonstrated that Oxalobacter-derived bioactive factors secreted in its culture conditioned medium stimulate oxalate transport by C2 cells through a PKA-dependent pathway and activation of A6 transport activity. It will be of significant pathophysiological interest to evaluate in future studies whether cAMP-producing agonists/mediators such vasoactive intestinal peptide, prostaglandin E₂, and PTH directly regulate intestinal oxalate transport through activation of the PKA signaling pathway.

Eight to 50 percent of kidney stone formers have hyperoxaluria (6, 33, 35). IBD patients have a significantly increased risk of COKS from the associated enteric hyperoxaluria (7, 38). Hyperoxaluria is a major complication of malabsorptive bariatric surgery for obesity (13, 37), and obesity itself is associated with mild to moderate hyperoxaluria (3, 29, 34, 41). Primary hyperoxaluria (PH) is a rare genetic disease, in which high urinary oxalate cause recurrent COKS, CKD, and ESRD (10). Significant hyperoxalemia, to levels that could lead to systemic oxalosis, is seen in association with PH, CKD, and ESRD (11, 14, 18, 25). Medical treatment with pyridoxine is effective in lowering urine oxalate in less than 30% of PH I patients and ineffective in those with PH II and III. The only treatment known to fully correct the underlying metabolic defect PH is liver transplantation (10). Combined kidney-liver transplantation is the effective treatment once ESRD develops. Cardiovascular diseases are the leading cause of morbidity and mortality in ESRD patients, and emerging data show that the ESRD-associated hyperoxalemia may contribute to this increased risk (18), and therefore, lowering serum oxalate could improve cardiovascular outcomes in ESRD patients if these findings are confirmed. In addition, the CKD-associated hyperoxalemia might potentially contribute to CKD progression (14). Unfortunately, there are currently no FDA-approved drugs that specifically reduce urine and/or plasma oxalate levels. Dietary oxalate restriction is very difficult to achieve, since most foods contain oxalate, and it is ineffective in PH. The risk of recurrent COKS, nephrocalcinosis, oxalate nephropathy, CKD, ESRD, and systemic oxalosis remains substantial in the absence of treatment. Our finding that intestinal oxalate transport is subject to stimulatory regulation by the PKA signaling pathway has potential therapeutic relevance, since stimulation of active transcellular intestinal secretion is expected to result in reduced plasma and urine oxalate levels. Of interest in this regard is that we (5) previously showed that Oxalobacter-derived bioactive factors secreted in its culture conditioned medium significantly reduce urinary oxalate excretion in hyperoxaluric mice by stimulating distal colonic secretion. Importantly, we have identified Oxalobacter-derived small peptides that stimulate oxalate transport in C2 cells by activating PKA, and studies are in progress to evaluate their therapeutic potential for hyperoxaluria and hyperoxalemia. Future studies will also focus on identifying agonists capable of stimulating transcellular intestinal oxalate secretion through activation of the PKA signaling pathway.

The limitations of our study are summarized as follows. Absolute selectivity is always an issue with pharmacological agonists and antagonists, and the mixture of F/I could potentially have a variety of effects beyond activation of the PKA signaling pathway. Although our finding that the F/I-induced stimulation of oxalate transport by C2 cells is completely blocked by the PKA inhibitor H89 supports our conclusion that it is most likely PKA-dependent, we acknowledge the fact that H89 is not absolutely specific for PKA. In addition, due to lack of good antibodies, the effects of F/I on A1 and A2 total and surface protein expression were not assessed as described above.

In summary, we have shown that activation of the PKA signaling pathway stimulates oxalate transport by C2 cells through mechanisms including enhanced A6 surface protein expression and an increase in the intrinsic activity of preexisting A6 and/or A2 surface membrane transporters. These findings suggest that PKA-mediated stimulatory regulation of intestinal oxalate transport might play an important role in overall oxalate homeostasis and thereby could potentially reduce plasma and urine oxalate levels and the stone risk.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants K08 DK-067245 and K08 DK-067245-S1 (H. Hassan), and P30 DK-42086 (the Digestive Disease Research Center of the University of Chicago).

DISCLOSURES

H. Hassan is cofounder and Chief Scientific Officer for Oxalo Therapeutics. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

H.H. conceived and designed research; D.A., A.A., and M.B. performed experiments; D.A., A.A., M.B., C.W., and H.H. analyzed data; D.A., A.A., C.W., and H.H. interpreted results of experiments; H.H. prepared figures; H.H. drafted manuscript; D.A., C.W., and H.H. edited and revised manuscript; D.A., A.A., M.B., C.W., and H.H. approved final version of manuscript.