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 Vmax and reduction (2-fold) in Km. 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 Km. 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.

SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine

“…An earlier study in intestinal Caco-2 and T84 cells suggested that PAT1 activity might be subject to regulation by secondary messengers such as cAMP ( 12 ). To test whether PAT1 is subject to direct regulation by cAMP, cGMP, or Ca 2+ , PAT1-mediated Cl – /I – exchange was measured in the PAT1-expressing FRT cells following incubations with forskolin plus IBMX, 8-Br-cGMP, or ATP.…”

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

“…However, we note that successful cAMP ‐stimulated endocytosis of DRA has been observed in NHE3 KO jejuna (Musch et al, 2009). We cannot exclude the possibility that PAT1 may be involved in the cAMP ‐stimulated oxalate secretion since there is evidence PAT‐1 participates in basal sulfate secretion by the mouse cecum (Whittamore et al, 2013), and recent studies have shown FSK/IBMX can stimulate Cl − ‐driven oxalate uptake in Caco‐2‐BBE cells through protein kinase A, a response that involves recruitment of PAT1 (slc26a6), and to a lesser degree DTDST (Diastrophic Dysplasia Sulfate Transporter, Slc26a2) (Arvans et al, 2020). DTDST is present in mouse large intestine (Park et al, 2014) but it is not known whether it is specifically expressed in the cecum.…”

“…A variety of neurohumoral inputs modify and coordinate Na + /H + and Cl − /HCO 3 − exchange in a segment‐specific manner mediated through the intracellular secondary messengers cAMP, cGMP and Ca 2+ (Chen et al, 2010; Field, 2003; Kato & Romero, 2011). cAMP stimulates oxalate secretion in the rabbit's small and large intestine (Freel et al, 1998; Hatch, Freel, & Vaziri, 1993, 1994b), and recently, stimulation of cAMP production was shown to dramatically enhance apical PAT‐1‐mediated oxalate uptake by human Caco‐2 cell monolayers (Arvans et al, 2020). NHE3 is a well‐established target of cAMP, inhibited through changes in phosphorylation (Moe, 1999) and endocytosis to reduce sodium absorption with a parallel increase in anion secretion (Donowitz & Li, 2007; Xu et al, 2018), but the same cannot be said of Cl − /HCO 3 − exchangers directly.…”

Oxalate as a potent promoter of kidney stone formation

“…Furthermore, SLC26A6 is regulated through the PKA activation signal. After activating PKA, the expression of intestinal SLC26A6 was significantly increased, and its transport activity was enhanced ( 52 , 111 ). CFTR can activate PKA ( 79 ) and SLC26A6 through signaling pathways such as PKA, but the exact mechanism requires further study.…”