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

Abstract

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-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) 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 UO(2)(PO(4))(-) and UO(2)(HPO(4))(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.

Données nouvelles sur la néphrotoxicité de l’uranium

“…La relation entre l'entrée de l'uranium dans la cellule et sa toxicité a été étudiée sur des cellules du TCP (ex vivo et in vitro). Plusieurs auteurs proposent que la cytotoxicité de l'uranium est directement liée à son entrée cellulaire alors que d'autres le démentent (Leggett, 1989 ;Muller et al, 2006, Carriere et al, 2008Ghadially et al, 1982 ;L'Azou et al, 2002 ;Mirto et al, 1999).…”

“…D'après l'analyse aux rayons X, ces précipités seraient composés de potassium, de phosphate, de calcium et de soufre complexés à l'uranium (Carriere et al, 2008 ;Ghadially et al, 1982 ;Mirto et al, 1999 ;Muller et al, 2006). Néanmoins, l'apparition de ces précipités est décrite pour des concentrations d'uranium élevées et supérieures aux concentrations minimales induisant une mort cellulaire.…”

“…La seconde hypothèse est que l'uranium entrerait dans la cellule par l'intermédiaire de transporteurs. Le rôle d'un transport de l'uranium via les transporteurs de type NaPi-IIa (co-transporteur de phosphate dépendant du sodium) a été mis en évidence (Muller et al, 2006(Muller et al, , 2008.…”

In vivo screening of proteins likely to bind uranium in exposed rat kidney

“…blood mainly studied, phosphate or carbonate species mainly dominate [12]. In kidneys, uranyl complexes filtered through glomeruli are mostly present as carbonate and citrate complexes but also as phosphate species [19]. These low molecular weight complexes then should behave as chemical from support electrolyte, i.e.…”

Interaction of Eu(III) with mammalian cells: Cytotoxicity, uptake, and speciation as a function of Eu(III) concentration and nutrient composition

“…Nevertheless, up to now, knowledge concerning the speciation of long-lived radionuclides in biological systems and its impact on their chemo-and radiotoxicity is limited. Most actinide studies were carried out with actinides in higher oxidation states, especially with uranium(VI) (Carrière et al, 2004(Carrière et al, , 2005aMilgram et al, 2008aMilgram et al, , 2007Milgram et al, , 2008bMirto et al, 1999a,b;Muller et al, 2006;Rouas et al, 2010), for which the relationship between its speciation and its cytotoxicity onto mammalian cells is comprehensively studied. A second, well studied actinide is plutonium(IV), whose uptake and binding was studied with various cell lines Fouillit et al, 2004;Gorman-Lewis et al, 2011;Jensen et al, 2012Jensen et al, , 2011Planas-Bohne and Duffield, 1988;Planas-Bohne et al, 1985Planas-Bohne and Rau, 1990;Schuler and Taylor, 1987).…”