Rudolfs K. Zalups

Molecular handling of cadmium in transporting epithelia

Cadmium (Cd) is an industrial and environmental pollutant that affects adversely a number of organs in humans and other mammals, including the kidneys, liver, lungs, pancreas, testis, and placenta. The liver and kidneys, which are the primary organs involved in the elimination of systemic Cd, are especially sensitive to the toxic effects of Cd. Because Cd ions possess a high affinity for sulfhydryl groups and thiolate anions, the cellular and molecular mechanisms involved in the handling and toxicity of Cd in target organs can be defined largely by the molecular interactions that occur between Cd ions and various sulfhydryl-containing molecules that are present in both the intracellular and extracellular compartments. A great deal of scientific data have been collected over the years to better define the toxic effects of Cd in the primary target organs. Notwithstanding all of the new developments made and information gathered, it is surprising that very little is known about the cellular and molecular mechanisms involved in the uptake, retention, and elimination of Cd in target epithelial cells. Therefore, the primary purpose of this review is to summarize and put into perspective some of the more salient current findings, assertions, and hypotheses pertaining to the transport and handling of Cd in the epithelial cells of target organs. Particular attention has been placed on the molecular mechanisms involved in the absorption, retention, and secretion of Cd in small intestinal enterocytes, hepatocytes, and tubular epithelial cells lining both proximal and distal portions of the nephron. The purpose of this review is not only to provide a summary of published findings but also to provide speculations and testable hypotheses based on contemporary findings made in other areas of research, with the hope that they may promote and serve as the impetus for future investigations designed to define more precisely the cellular mechanisms involved in the transport and handling of Cd within the body.

TRANSPORT OF INORGANIC MERCURY AND METHYLMERCURY IN TARGET TISSUES AND ORGANS

Owing to the prevalence of mercury in the environment, the risk of human exposure to this toxic metal continues to increase. Following exposure to mercury, this metal accumulates in numerous organs, including brain, intestine, kidneys, liver, and placenta. Although a number of mechanisms for the transport of mercuric ions into target organs were proposed in recent years, these mechanisms have not been characterized completely. This review summarizes the current literature related to the transport of inorganic and organic forms of mercury in various tissues and organs. This review identifies known mechanisms of mercury transport and provides information on additional mechanisms that may potentially play a role in the transport of mercuric ions into target cells.

Dissociation of glomerular hypertrophy, cell proliferation, and glomerulosclerosis in mouse strains heterozygous for a mutation (Os) which induces a 50% reduction in nephron number.

We reported that the Os mutation in ROP mice induced a 50% reduction in nephron number, glomerular hypertrophy, and severe glomerulosclerosis. We examined two mouse strains with the Os mutation, ROP Os/+ and C57 Os/+ mice, to determine whether the genetic background influenced the development of glomerulosclerosis. Nephron number was decreased by 50% in both ROP Os/+ and C57 Os/+ mice, and a glomerular volume and labeling index were two- to threefold increased in both. Whereas glomerulosclerosis was severe in ROP Os/+ mice, it was absent or minimal in C57 Os/+ mice. ROP Os/+ glomeruli had two- to threefold more type IV collagen, laminin, and tenascin than C57 Os/+ by immunofluorescence microscopy. Glomerular alpha 1IV collagen and tenascin mRNA levels were increased (2.8- and 1.7-fold) in ROP Os/+ and in C57 Os/+ (1.7- and 1.4-fold) mice. Both ROP Os/+ and C57 Os/+ mice had a slight increase (1.5- and 1.7-fold) in 72-kD collagenase mRNA levels. Whereas laminin B1 mRNA levels were twofold higher in ROP +/+ than in C57 +/+ mice, there was no further change in the presence of the Os mutation. Thus, the response to the Os mutation depended on the mouse strain, since severe glomerulosclerosis occurred only in ROP Os/+ mice, even though cell proliferation and glomerular hypertrophy also were present in C57 Os/+ mice.

Understanding Renal Toxicity of Heavy Metals

The mechanisms by which metals induce renal injury are, in general, poorly understood. Characteristic features of metal nephrotoxicity are lesions that tend to predominate in specific regions of the nephron within specific cell types. This suggests that certain regions of the nephron are selectively sensitive to specific metals. Regional variability in sensitivity could result from the localization of molecular targets in certain cell populations and/or the localization of transport and binding ligands that deliver metals to targets within the nephron. Significant progress has been made in identifying various extracellular, membrane, and intracellular ligands that are important in the expression of the nephrotoxicity of metals. As an example, mercuric chloride induces a nephropathy that, at the lowest effective doses, is restricted primarily to the S3 segment of the proximal tubule, with involvement of the S2 and S1 segments at higher doses. This specificity appears to be derived, at least in part, from the distribution of enzymes and transport proteins important for the uptake of mercury into proximal tubule cells: apical γ-glutamyltranspeptidase and the basolateral organic anion transport system. Regional distributions of transport mechanisms for binding proteins appear to be important in the expression of nephrotoxicity of metals. These and other new research developments are reviewed.

Homocysteine and the Renal Epithelial Transport and Toxicity of Inorganic Mercury: Role of Basolateral Transporter Organic Anion Transporter 1

The epithelial cells that line the renal proximal tubule have been shown to be the primary cellular targets where mercuric ions gain entry, accumulate, and induce pathologic effects in vivo. Recent data have implicated at least one of the organic anion transport systems in the basolateral uptake of inorganic mercury (Hg). With the use of a line of type II MDCK cells transfected stably with the human organic anion transporter 1 (hOAT1), the hypothesis that hOAT1 can transport mercuric conjugates of homocysteine (Hcy) was tested. Indeed, MDCK II cells expressing a functional form of hOAT1 gained the ability to transport the mercuric conjugate 2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl) butyric acid (Hcy-S-Hg-S-Hcy). In addition, p-aminohippurate and the dicarboxylates adipate and glutarate (but not succinate or malonate) inhibited individually the uptake of Hcy-S-Hg-S-Hcy in a concentration-dependent manner. Furthermore, a direct relationship between the uptake of Hcy-S-Hg-S-Hcy and the induction of cellular injury and death was demonstrated in the hOAT1-expressing MDCK II cells only. These data represent the first line of direct evidence implicating one of the organic anion transporters in the uptake of a mercuric conjugate of Hcy in a mammalian cell. Thus, mercuric conjugates of Hcy are potential transportable substrates of OAT1. More important, the findings from the present study implicate the activity of OAT1 in the uptake and toxicity of Hg (when in the form of Hcy-S-Hg-S-Hcy in the extracellular compartment) in proximal tubular epithelial cells in vivo.

Autometallographic localization of inorganic mercury in the kidneys of rats: effect of unilateral nephrectomy and compensatory renal growth.

The histochemical technique of autometallography was used in the present study to demonstrate the zonal and tubular localization of inorganic mercury in the kidneys of unilaterally nephrectomized (NPX) and sham-operated (SO) rats given either a nontoxic 0.5 mumol/kg or a toxic 2.5 mumol/kg dose of mercuric chloride 10 days after surgery. Deposits were found in the cortex and outer stripe of the outer medulla in both groups of rats given either dose of mercuric chloride. The deposits were localized exclusively in the convoluted and straight portion of the proximal tubule. Forty eight hours after the administration of the 0.5 mumol/kg dose of mercuric chloride, there were significantly more deposits in the renal outer stripe of the NPX rats than in the renal outer stripe of the SO rats. The number of deposits in the renal outer stripe of the NPX and SO rats given the 2.5 mumol/kg dose of mercuric chloride was similar after 24 hr, but was greater than the corresponding rats given the nontoxic dose. These findings suggest that the proximal tubule (particularly the pars recta) is the primary site for the accumulation of inorganic mercury in the kidney. They also suggest that, in the rat, there is enhanced accumulation of inorganic mercury in the pars recta of proximal tubules in the outer stripe of the renal outer medulla when a nontoxic dose of inorganic mercury is given after unilateral nephrectomy or when a toxic dose of mercuric chloride is administered.

Mercuric Conjugates of Cysteine Are Transported by the Amino Acid Transporter System b0,+: Implications of Molecular Mimicry

Humans and other mammals continue to be exposed to various forms of mercury in the environment. The kidneys, specifically the epithelial cells lining the proximal tubules, are the primary targets where mercuric ions accumulate and exert their toxic effects. Although the actual mechanisms involved in the transport of mercuric ions along the proximal tubule have not been defined, current evidence implicates mercuric conjugates of cysteine, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys), as the most likely transportable species of inorganic mercury (Hg(2+)). Because Cys-S-Hg-S-Cys and the amino acid cystine (Cys-S-S-Cys) are structurally similar, it was hypothesized that Cys-S-Hg-S-Cys might act as a molecular mimic of cystine at one or more of the amino acid transporters involved in the luminal absorption of this amino acid. One such candidate is the Na(+)-independent heterodimeric transporter system b(0,+). Therefore, the transport of Cys-S-Hg-S-Cys and cystine was studied in MDCK II cells that were or were not stably transfected with b(0,+)AT-rBAT. Transport of Cys-S-Hg-S-Cys and cystine across the luminal plasma membrane was similar in the transfected cells, indicating that Cys-S-Hg-S-Cys can behave as a molecular mimic of cystine at the site of system b(0,+). Moreover, only the b(0,+)AT-rBAT transfectants became selectively intoxicated during exposure to Cys-S-Hg-S-Cys. These findings indicate that system b(0,+) likely contributes to the nephropathy induced by Hg(2+) in vivo. These data represent the first direct molecular evidence for the participation of a specific transporter in the luminal uptake of a large divalent metal cation in proximal tubular cells.

Accumulation of inorganic mercury along the renal proximal tubule of the rabbit.

The purpose of the present study is to characterize the accumulation of inorganic mercury along the proximal tubule of the rabbit. New Zealand white rabbits were given a 0.5 mumol/kg dose of mercuric chloride along with 150 microCi of 203Hg. Forty-eight hours after the animals had been treated, individual segments of the nephron were obtained by microdissection. The segments of the nephron were measured in length and then were counted in a gamma counter to determine the percentage of the administered dose of inorganic mercury that had accumulated in them. There was significant accumulation of mercury along the proximal tubule during the 48 hr after the dose of mercuric chloride was administered. The S1 segment of the proximal tubule accumulated 0.000226 +/- 0.000031% (mean +/- SE) of the administered dose of inorganic mercury per millimeter tubule. The amount of mercury that accumulated in the S2 segment of the proximal tubule was similar to that in the S1 segment. By contrast, only half as much inorganic mercury accumulated in each millimeter of the S3 segment of the proximal tubule. No significant accumulation of inorganic mercury could be detected in pooled samples of various segments of the distal nephron. The findings in the present study indicate that the renal accumulation of inorganic mercury in the rabbit occurs mainly as a result of the accumulation of the metal in the proximal tubule, with the accumulation predominating in the S1 and S2 segments.

Homocysteine, system b0,+ and the renal epithelial transport and toxicity of inorganic mercury.

Proximal tubular epithelial cells are major sites of homocysteine (Hcy) metabolism and are the primary sites for the accumulation and intoxication of inorganic mercury (Hg(2+)). Previous in vivo data from our laboratory have demonstrated that mercuric conjugates of Hcy are transported into these cells by unknown mechanisms. Recently, we established that the mercuric conjugate of cysteine [2-amino-3-(2-amino-2-carboxy-ethylsulfanylmercuricsulfanyl)propionic acid; Cys-S-Hg-S-Cys], is transported by the luminal, amino acid transporter, system b(0,+). As Cys-S-Hg-S-Cys and the mercuric conjugate of Hcy (2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl)butyric acid; Hcy-S-Hg-S-Hcy) are similar structurally, we hypothesized that Hcy-S-Hg-S-Hcy is a substrate for system b(0,+). To test this hypothesis, we analyzed the saturation kinetics, time dependence, temperature dependence, and substrate specificity of Hcy-S-Hg-S-Hcy transport in Madin-Darby canine kidney (MDCK) cells stably transfected with system b(0,+). MDCK cells are good models in which to study this transport because they do not express system b(0,+). Uptake of Hg(2+) was twofold greater in the transfectants than in wild-type cells. Moreover, the transfectants were more susceptible to the toxic effects of Hcy-S-Hg-S-Hcy than wild-type cells. Accordingly, our data indicate that Hcy-S-Hg-S-Hcy is transported by system b(0,+) and that this transporter likely plays a role in the nephropathy induced after exposure to Hg(2+). These data are the first to implicate a specific, luminal membrane transporter in the uptake and toxicity of mercuric conjugates of Hcy in any epithelial cell.

Multidrug Resistance Proteins and the Renal Elimination of Inorganic Mercury Mediated by 2,3-Dimercaptopropane-1-Sulfonic Acid and Meso-2,3-dimercaptosuccinic Acid

Current therapies for inorganic mercury (Hg2+) 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, Hg2+ 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 DMPS- or DMSA-S-conjugates of Hg2+ from proximal tubular cells. To test this hypothesis, the disposition of Hg2+ was examined in control and Mrp2-deficient TR- rats. Rats were injected i.v. with 0.5 μmol/kg HgCl2 containing 203Hg2+. Twenty-four and 28 h later, rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h after HgCl2 exposure. The renal and hepatic burden of Hg2+ in the saline-injected TR- rats was greater than that of controls. In contrast, the amount of Hg2+ excreted in urine and feces of TR- rats was less than that of controls. DMPS, but not DMSA, significantly reduced the renal and hepatic content of Hg2+ in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of Hg2+, which was greater in the controls, was also observed following DMPS treatment. Experiments utilizing inside-out membrane vesicles expressing MRP2 support these observations by demonstrating that DMPS- and DMSA-S-conjugates of Hg2+ are transportable substrates of MRP2. Collectively, these data support a role for Mrp2 in the DMPS- and DMSA-mediated elimination of Hg2+ from the kidney.