Current therapies for inorganic mercury (Hg 2ϩ ) intoxication include administration of a metal chelator, either 2,3-dimercaptopropane-1-sulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA). After exposure to either chelator, Hg 2ϩ is rapidly eliminated from the kidneys and excreted in the urine, presumably as an S-conjugate of DMPS or DMSA. The multidrug resistance protein 2 (Mrp2) has been implicated in this process. We hypothesize that Mrp2 mediates the secretion of DMPSor DMSA-S-conjugates of Hg 2ϩ from proximal tubular cells. . Twenty-four and 28 h later, rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h after HgCl 2 exposure. The renal and hepatic burden of Hg 2ϩ in the saline-injected TR Ϫ rats was greater than that of controls. In contrast, the amount of Hg 2ϩ excreted in urine and feces of TR Ϫ rats was less than that of controls. DMPS, but not DMSA, significantly reduced the renal and hepatic content of Hg 2ϩ in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of Hg 2ϩ

Cysteine (Cys) and homocysteine (Hcy)-S-conjugates of inorganic mercury (Hg2+) are transportable species of Hg2+ that are taken up readily by proximal tubular cells. The metal chelators, 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), have been used successfully to extract Hg2+ from these cells, presumably via the multidrug resistance protein (Mrp2). In the current study, we tested the hypothesis that Mrp2 is involved in the DMPS- and DMSA-mediated extraction of Hg2+ following administration of Hg2+ as an S-conjugate of Cys or Hcy. To test this hypothesis, control and TR(-) (Mrp2-deficient) rats were injected with 0.5 micromol/kg HgCl2 (containing 203Hg2+) conjugated to 1.25 micromol/kg Cys or Hcy. After 24 and 28 h, rats were treated with saline or 100 mg/kg DMPS or DMSA. Tissues were harvested 48 h after Hg2+ exposure. The renal and hepatic burden of Hg2+ was greater in saline-injected TR- rats than in corresponding controls. Accordingly, the content of Hg2+ in the urine and feces was less in TR- rats than in controls. Following treatment with DMPS or DMSA, the renal content of Hg2+ in both groups of rats was reduced significantly and the urinary excretion of Hg2+ was increased. In liver, the effect of each chelator appeared to be dependent upon the form in which Hg2+ was administered. In vitro experiments provide direct evidence indicating that DMPS and DMSA-S-conjugates of Hg2+ are substrates for Mrp2. Overall, these data support our hypothesis that Mrp2 is involved in the DMPS and DMSA-mediated extraction of the body burden of Hg2+.

Mercuric ions accumulate preferentially in renal tubular epithelial cells and bond with intracellular thiols. Certain metal-complexing agents have been shown to promote extraction of mercuric ions via the multidrug resistance-associated protein 2 (MRP2). Following exposure to a non-toxic dose of inorganic mercury (Hg 2+ ), in the absence of complexing agents, tubular cells are capable of exporting a small fraction of intracellular Hg 2+ through one or more undetermined mechanisms. We hypothesize that MRP2 plays a role in this export. To test this hypothesis, Wistar (control) and TR − rats were injected intravenously with a non-nephrotoxic dose of HgCl 2 (0.5 μmol/kg) or CH 3 HgCl (5 mg/kg), containing [ 203 Hg], in the presence or absence of cysteine (Cys; 1.25 μmol/ kg or 12.5 mg/kg, respectively). Animals were sacrificed 24 h after exposure to mercury and the content of [ 203 Hg] in blood, kidneys, liver, urine and feces was determined. In addition, uptake of Cys-S-conjugates of Hg 2+ and methylmercury (CH 3 Hg + ) was measured in inside-out membrane vesicles prepared from either control Sf9 cells or Sf9 cells transfected with human MRP2. The amount of mercury in the total renal mass and liver was significantly greater in TR − rats than in controls. In contrast, the amount of mercury in urine and feces was significantly lower in TR − rats than in controls. Data from membrane vesicles indicate that Cys-S-conjugates of Hg 2+ and CH 3 Hg + are transportable substrates of MRP2. Collectively, these data indicate that MRP2 plays a role in the physiological handling and elimination of mercuric ions from the kidney.

The role of the multi-resistance protein 2 (Mrp2) in the nephropathy induced by inorganic mercuric mercury (Hg(2+)) was studied in rats (TR(-)) and mice (Mrp2(-/-)), which lack functional Mrp2, and control animals. Animals were exposed to nephrotoxic doses of HgCl2. Forty-eight or 24 hours after exposure, tissues were harvested and analyzed for Hg content and markers of injury. Histological analyses revealed that the proximal tubular segments affected pathologically by Hg(2+) were significantly different between Mrp2-deficient animals and controls. In the absence of Mrp2, cellular injury localized almost exclusively in proximal tubular segments in the subcapsular (S1) to midcortical regions (early S2) of the kidney. In control animals, cellular death occurred mainly in the proximal tubular segments in the inner cortex (late S2) and outer stripe of the outer medulla (S3). These differences in renal pathology indicate that axial heterogeneity exists along the proximal tubule with respect to how mercuric ions are handled. Total renal and hepatic accumulation of mercury was also greater in animals lacking Mrp2 than in controls, indicating that Mrp2 normally plays a significant role in eliminating mercuric ions from within proximal tubular cells and hepatocytes. Analyses of plasma creatinine, BUN, and renal expression of Kim-1 and Ngal tend to support the severity of the nephropathies detected histologically. Collectively, our findings indicate that a fraction of mercuric ions is normally secreted by Mrp2 in early portions of proximal tubules into the lumen and then is absorbed downstream in straight portions, where mercuric species typically induce toxic effects.

Secretion of inorganic mercury (Hg2+) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg2+ was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg2+. To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg2+-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp−/−) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol • kg−1), a moderately nephrotoxic (1.5 μmol • kg−1) or a significantly nephrotoxic (2.0 μmol • kg−1) dose of HgCl2. In general, the accumulation of Hg2+ was greater in organs of bcrp−/− rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg2+ from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp−/− rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2.