INFLUENCE DES CONDITIONS DE NUTRITION SUR LA QUALITÉ ET LA QUANTITÉ DE L'URINE DU RAT

International audienc

Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK1) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK1 cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-d-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO2(PO4)- and UO2(HPO4)aq. Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system. © 2006 Elsevier Inc. All rights reserved

Role of the sodium-dependent phosphate cotransporters and absorptive endocytosis in the uptake of low concentrations of uranium and its toxicity at higher concentrations in LLC-PK1 cells

It has been suggested that uranium uptake and toxicity could be mediated by endocytosis and/or the type IIa sodium-dependent phosphate cotransporter (NaPi-IIa). The aim of this study was therefore to characterize in vitro the role of these two cellular mechanisms in the uptake and toxicity of low (200-3200nM) and high (0.5 and 0.8mM) concentrations of uranium, respectively. At low concentrations, uranium uptake in LLC-PK1 cells was saturable (Vmax = 3.09 ± 0.22 ng/mg protein) and characterized by a K0.5 of 1022 ± 63nM and a Hill coefficient of 3.0 ± 0.4. The potential involvement of endocytosis and NaPi-IIa in the uptake of uranium was assessed by the use of various drugs and culture conditions known to alter their relative activity, and 233uranium uptake was monitored. Interestingly, the inhibitory effect of colchicine, cytochalasin D, phorbol 12-myristate 13-acetate, and chlorpromazine on endocytosis was highly correlated with their effect on uranium uptake, a relationship that was not true when the NaPi-IIa transport system was studied. Whereas the competitive inhibition of the NaPi-IIa by phosphonoformic acid (PFA) significantly decreased uranium uptake, this effect was not reproduced when NaPi-IIa inhibition was mediated by the replacement of extracellular Na+ with N-methyl-D-glucamine. Uranium uptake was also not significantly altered when NaPi-IIa expression was stimulated in MDCK cells. More surprisingly, we observed by transmission electron microscopy that uranium cytotoxicity was dependent upon the extent of its intracellular precipitation, but not on its intracellular content, and was suppressed by PFA. In conclusion, our results suggest that low-dose uranium uptake is mainly mediated by absorptive endocytosis, and we propose PFA as a potential uranium chelator. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved