Most kidney stones are composed of calcium oxalate, and minor changes in urine oxalate affect the stone risk. Obesity is a risk factor for kidney stones and a positive correlation of unknown etiology between increased body size, and elevated urinary oxalate excretion has been reported. Here, we used obese ob/ob (ob) mice to elucidate the pathogenesis of obesity-associated hyperoxaluria. These ob mice have significant hyperoxaluria (3.3-fold) compared with control mice, which is not due to overeating as shown by pair-feeding studies. Dietary oxalate removal greatly ameliorated this hyperoxaluria, confirming that it is largely enteric in origin. Transporter SLC26A6 (A6) plays an essential role in active transcellular intestinal oxalate secretion, and ob mice have significantly reduced jejunal A6 mRNA (- 80%) and total protein (- 62%) expression. While net oxalate secretion was observed in control jejunal tissues mounted in Ussing chambers, net absorption was seen in ob tissues, due to significantly reduced secretion. We hypothesized that the obesity-associated increase in intestinal and systemic inflammation, as reflected by elevated proinflammatory cytokines, suppresses A6-mediated intestinal oxalate secretion and contributes to obesity-associated hyperoxaluria. Indeed, proinflammatory cytokines (elevated in ob mice) significantly decreased intestinal oxalate transport in vitro by reducing A6 mRNA and total protein expression. Proinflammatory cytokines also significantly reduced active mouse jejunal oxalate secretion, converting oxalate transport from net secretion in vehicle-treated tissues to net absorption in proinflammatory cytokines-treated tissues. Thus, reduced active intestinal oxalate secretion, likely secondary to local and systemic inflammation, contributes to the pathogenesis of obesity-associated hyperoxaluria. Hence, proinflammatory cytokines represent potential therapeutic targets.

Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [¹⁴C]oxalate uptake in the presence of an outward Cl⁻ gradient as an assay of Cl⁻/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y₂ receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y₂ receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y₂ purinergic receptor, PLC, and PKC-δ.

Most kidney stones (KS) are composed of calcium oxalate and small increases in urine oxalate enhance the stone risk. Obesity is a risk factor for KS, and urinary oxalate excretion increases with increased body size. We previously established the obese ob/ob ( ob) mice as a model (3.3-fold higher urine oxalate) to define the pathogenesis of obesity-associated hyperoxaluria (OAH). The purpose of this study was to test the hypothesis that the obesity-associated enhanced small intestinal paracellular permeability contributes to OAH by increasing passive paracellular intestinal oxalate absorption. ob Mice have significantly higher jejunal (1.6-fold) and ileal (1.4-fold) paracellular oxalate absorption ex vivo and significantly higher (5-fold) urine [13C]oxalate following oral gavage with [13C]oxalate, indicating increased intestinal oxalate absorption in vivo. The observation of higher oxalate absorption in vivo compared with ex vivo suggests the possibility of increased paracellular permeability along the entire gut. Indeed, ob mice have significantly higher fractions of the administered sucrose (1.7-fold), lactulose (4.4-fold), and sucralose (3.1-fold) excreted in the urine, reflecting increased gastric, small intestinal, and colonic paracellular permeability, respectively. The ob mice have significantly reduced gastrointestinal occludin, zonula occludens-1, and claudins-1 and -3 mRNA and total protein expression. Proinflammatory cytokines and oxidative stress, which are elevated in obesity, significantly enhanced paracellular intestinal oxalate absorption in vitro and ex vivo. We conclude that obese mice have significantly higher intestinal oxalate absorption and enhanced gastrointestinal paracellular permeability in vivo, which would likely contribute to the pathogenesis of OAH, since there is a transepithelial oxalate concentration gradient to drive paracellular intestinal oxalate absorption. NEW & NOTEWORTHY This study shows that the obese ob/ob mice have significantly increased gastrointestinal paracellular oxalate absorption and remarkably enhanced paracellular permeability along the entire gut in vivo, which are likely mediated by the obesity-associated increased systemic and intestinal inflammation and oxidative stress. A transepithelial oxalate concentration gradient driving gastrointestinal paracellular oxalate absorption exists, and therefore, our novel findings likely contribute to the hyperoxaluria observed in the ob/ob mice and hence to the pathogenesis of obesity-associated hyperoxaluria.

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.

Site-directed mutants of the gene encoding wild-type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild-type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild-type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild-type VHb as determined by CO-difference spectra. Circular-dichroism spectra also showed the two mutants to be essentially the same as wild-type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7-1.9 Å , which are similar to that of the original wild-type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild-type VHb as well as in the wild-type VHb structure reported here. In the Tyr29Ala mutant, the five-membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wildtype structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen-binding pocket.