Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Abstract

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.