Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A_2B adenosine receptor

Abstract

Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate; thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured C-oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, {greater than or equal to} 49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8-SPT. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A adenosine receptor (A AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A AR in IBD, our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

Genetic Prioritization, Therapeutic Repositioning and Cross-Disease Comparisons Reveal Inflammatory Targets Tractable for Kidney Stone Disease

“…To complement the knowledge obtained from individual pathway prioritizations, we next exploited interaction information merged from all KEGG pathways to identify a pathway crosstalk that contains highly ranked and interconnecting genes ( p =3.2×10 −99 on permutation test; Figure 3A and Table S4 ). Genes in this crosstalk include those previously reported to be of functional relevance to nephrolithiasis, such as protein kinase C ( PRKCA , PRKCB , and PRKCZ ) ( 43 ), markers ( CD40 and TLR3 ) ( 44 ), and autophagy ( MTOR and SQSTM1 ) ( 13 ), thus validating our genetic prioritization at the gene and pathway level. Based on pathways significantly over-represented in the crosstalk ( Figure S5 ), we further constructed crosstalk at the pathway level, with edges estimated by the extent to which member genes are shared between two endpoints ( Figure 3B ).…”

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

“…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.…”

“…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.…”

Pathophysiology and Management of Hyperoxaluria and Oxalate Nephropathy: A Review

“…Cholinergic regulation inhibits oxalate uptake through reduced expression of SCL26A6 in human cell lines (8)(9)(10). A purinergic signaling system also regulates J o u r n a l P r e -p r o o f oxalate transport across digestive epithelia (11)(12). Lastly, in murine chronic kidney disease (CKD) models, Slc26a6-mediated enteric oxalate secretion is critical in decreasing the body burden of oxalate (13).…”