MO692COULD THE RESIDUAL RENAL FUNCTION REMAIN A KEY DETERMINANT OF PLASMA OXALIC ACID CONCENTRATION IN PERITONEAL DIALYSIS PATIENTS?

Background and Aims Under physiological conditions, the bulk of circulating oxalate (90% to 95%) is ultimately excreted by the kidneys. Under uremic and/or anuric conditions, dialysis is considered to be the main method of oxalate removal. Nevertheless, little evidence is available on oxalate balance in peritoneal dialysis (PD) patients. The present study aimed to evaluate the separate contribution of residual renal and peritoneal oxalate clearances to oxalate balance in PD patients. Method We performed a cross-sectional observational study involving 62 PD patients with the average age of 50.5±13.5 years and PD vintage of 37±24 months. Plasma oxalate (POx) concentration, levels of daily urinary (UOx) and peritoneal dialysis effluent oxalate (PDEOx) excretion were evaluated. POx concentration was measured spectrophotometrically using MAK315 kit (Sigma, Spain); UOx and PDEOx concentrations were determined using an oxalate oxidase/peroxidase reagent (BioSystems, Spain). In addition, oxalate transport status (4-hour D/P oxalate ratio), renal oxalate clearance (ROxCL) and peritoneal oxalate clearance (PerOxCL) were calculated. Results Among the examined PD patients were 41 (66%) patients with preserved diuresis and 21 (34%) patients with anuria. The anuric PD patients had lower PerOxCL and, accordingly, peritoneal and overall oxalate removal levels compared with the patients with preserved diuresis (Table 1). Conclusion The results of our research demonstrated an important role of the residual renal function in oxalate balance in PD patients. However, the decline in RRF could partially (but not completely) contribute to the increase in POx in PD patients. Thus, PerOxCL but not ROxCL could significantly affect oxalate balance in PD patients.

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

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.

Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor

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 [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥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- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B 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 A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

N-glycosylation critically regulates function of oxalate transporter SLC26A6

The brush border Cl−-oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.