I. Introduction

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Among metals, mercury is unique in that it is found in the environment in several physical and chemical forms. At room temperature, elemental (or metallic) mercury exists as a liquid. As a result of its high vapor pressure, this form of mercury is released into the environment as mercury vapor. Mercury also exists as a cation with an oxidation state of 1+ (mercurous) or 2+ (mercuric). In occupational and environmental settings, the most common cationic form of mercury encountered is the mercuric form, which may have a valence of 1+ or 2+, depending on whether the mercuric ion is covalently bonded to a carbon atom of an organic side group, such as an alkyl group. With respect to organic forms of mercury, methylmercury is the most frequently encountered organic mercuric compound in the environment. It forms mainly as the result of methylation of inorganic (mercuric) forms of mercury by microorganisms in soil and water.

Due to industrialization and changes in the environment during the twentieth century, humans and animals are exposed to numerous chemical forms of mercury, including elemental mercury vapor (Hg⁰), inorganic mercurous (Hg⁺) and mercuric (Hg²⁺) compounds, and organic mercuric (R-Hg⁺ or R-Hg-R; where R represents any organic ligand) compounds (Fitzgerald and Clarkson, 1991). Inasmuch as mercury is ubiquitous in the environment, it is nearly impossible for most humans to avoid exposure to some form or forms of mercury on a regular basis.

All forms of mercury cause toxic effects in a number of tissues and organs, depending on the chemical form of mercury, the level of exposure, the duration of exposure, and the route of exposure. The kidneys are the primary target organ where inorganic mercury is taken up, accumulated, and expresses toxicity. Organic mercuric compounds are also nephrotoxic but to a lesser degree than inorganic mercurous or mercuric compounds. Systemic distributions of organic mercury are more diffuse than inorganic forms, and they affect other target organs, including hematopoietic and neural tissues (Clarkson, 1972; World Health Organization, 1991; Agency for Toxic Substance and Disease Registry, 1999). Differences in the mechanisms involved in the transport and metabolism of inorganic and organic forms of mercury (in the various compartments of the body) are likely responsible for the disparity in their distribution in tissues and organs, pattern of biological effect, and toxicity (Zalups and Lash, 1994).

When considering the biological activity of mercuric ions in humans or other mammals, one must take into account the bonding characteristics of these ions. Although mercuric ions will bind to numerous nucleophilic groups on molecules, they have a greater predilection to bond to reduced sulfur atoms, especially those on endogenous thiol-containing molecules, such as glutathione, cysteine, homocysteine, N-acetylcysteine, metallothionein, and albumin. The affinity constant for mercury bonding to thiolate anions is on the order of 10¹⁵ to 10²⁰. In contrast, the affinity constants for mercury bonding to oxygen- or nitrogen-containing ligands (e.g., carbonyl or amino groups) are about 10 orders of magnitude lower. Hence, it is reasonable, in most cases, to consider the biological effects of inorganic or organic mercury in terms of their interactions with sulfhydryl-containing residues.

In the presence of an excess of a low-molecular-weight thiol-containing molecule, mercuric ions have a high propensity toward linear II coordination with two of these molecules. For example, in a situation in which there are twice as many molecules of glutathione as inorganic mercuric ions in aqueous solution (at room temperature), there will be a strong tendency for each mercuric ion to form a linear II coordinate covalent complex with two molecules of glutathione by bonding to the sulfur atom on the cysteinyl residue of each of those two molecules (Fuhr and Rabenstein, 1973; Rabenstein, 1989). Organic mercurials, such as methylmercury, tend to form 1:1 complexes with thiol-containing molecules. Despite the thermodynamic stability of the (linear I or II) coordinate covalent bonds formed between mercuric ions and various thiol-containing molecules in aqueous solution, the bonding characteristics between mercuric ions and these thiol-containing molecules appear to be more labile within the living organism (Rabenstein, 1989). Complex factors such as thiol- and/or other nucleophilic competition and exchange are likely the most cogent explanation for the perceived labile nature of bonding that occurs between mercuric ions and certain thiol-containing molecules in particular tissue and cellular compartments. For example, most of the mercuric ions present in plasma (shortly after exposure to inorganic mercury) are bound to sulfhydryl-containing proteins, such as albumin (Friedman, 1957; Mussini, 1958; Cember et al., 1968; Lau and Sarkar, 1979). However, these mercuric ions do not remain bound to these proteins for very long. During the initial hours after exposure to inorganic mercury, there is a rapid decrease in the plasma burden of mercury concurrent with a rapid rate of uptake of inorganic mercury in the kidneys and liver. Because current evidence (to be discussed) indicates that mercuric S-conjugates of small endogenous thiols (e.g., glutathione and cysteine) are likely the primary transportable forms of mercury in the kidneys, it must be that mercuric ions are transferred from the plasma proteins to these low-molecular-weight thiols by some form of currently undefined complex ligand-exchange mechanism or mechanisms. Moreover, the effectiveness of thiol-containing pharmacological agents, such as penicillamine, N-acetylpenicillamine, meso-2,3-dimercaptosuccinic acid (DMSA),² 2,3-dimercapto-1-propanesulfonic acid (DMPS), dithioerythritol, and dithiothreitol, in reversal of or protection against toxic effects of mercury-containing compounds is fundamentally premised on, and best explained by, the ability of these agents to remove inorganic and organic mercuric ions from endogenous ligands via nucleophilic competition and exchange, thereby forming new thiol-mercury complexes.

Dose-response relationships for the toxicity of inorganic mercury are extremely steep in a variety of renal systems, including in vivo treatment of rats and rabbits (Zalups and Diamond, 1987b; Zalups et al., 1988; Zalups and Lash, 1990; Zalups, 1991c), renal cortical slices (Ruegg et al., 1987) and isolated segments of proximal tubules from rabbits (Barfuss et al., 1990; Zalups et al., 1993a), freshly isolated proximal tubular cells from rats (Lash and Zalups, 1992), and primary cultures of renal cortical cells from rats (Smith et al., 1986; Lash et al., 1999). In all of these various renal systems, a threshold effect is generally observed, in that no cellular necrosis (death) is observed up to a certain dose. Above that dose, however, cellular death progresses rapidly, and in some systems an all-or-none response is observed. This does not mean that subtoxic doses of mercury do not have biochemical or physiological effects. One possible explanation for the threshold effect and the subsequent steep dose-response curve is that endogenous ligands, such as glutathione, bind mercury and may act as a buffer to prevent functional changes from occurring. Above a certain dose or concentration of mercury, the buffer becomes depleted, and mercuric or mercurous ions can bind more readily to critical nucleophilic groups in the cell, thereby causing functional impairment. Intracellular sulfhydryl-containing proteins such as metallothionein or low-molecular-weight thiols, in particular glutathione, likely function in such a capacity.

To understand the nephropathy induced by mercury and to find therapeutic regimens to treat this nephropathy, it is essential to understand the mechanisms involved in the uptake, intracellular binding, and cellular elimination of mercury in the target cells, namely the epithelial cells lining the proximal tubule. In addition to seeking a better understanding of the chemical properties of mercury-containing compounds and the intracellular buffering capacity of both target and nontarget organs, other factors must be considered to define more precisely the biochemical and molecular mechanisms of action of mercury-containing compounds in the kidney. Particular attention must be paid to the potential role of "molecular mimicry" and the species of mercury involved in the renal (proximal) tubular uptake and transport of mercuric ions. Susceptibility to the injurious effects of mercury may be modified by a number of intracellular and extracellular factors. These very factors are the theoretical basis for most of the currently used therapeutic strategies. Physiological or pathological alterations in cellular function, particularly in the kidney and liver, may also play important roles in modifying susceptibility to mercury-induced renal injury. Consideration of these factors can provide clues that will aid in understanding the basic mechanisms of mercury-induced renal cellular injury.

	II. Renal Disposition and Transport of Mercury

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In humans and other mammals, the kidneys are the primary targets where mercuric ions accumulate after exposure to elemental or inorganic forms of mercury (Adam, 1951; Ashe et al., 1953; Friberg, 1956, 1959; Rothstein and Hayes, 1960; Berlin and Gibson, 1963; Clarkson and Magos, 1967; Swensson and Ulfvarson, 1968; Cherian and Clarkson, 1976; Zalups and Diamond, 1987a,b; Hahn et al., 1989, 1990; Zalups and Barfuss, 1990; Zalups, 1991a,b,c, 1993a). Renal uptake and accumulation of mercury in vivo are very rapid. As much as 50% of a low (0.5 µmol/kg) dose of inorganic mercury has been shown to be present in the kidneys of rats within a few hours after exposure (Zalups, 1993a). Significant amounts of mercury also accumulate in the kidneys after exposure to organic forms of mercury (Prickett et al., 1950; Friberg, 1959; Norseth and Clarkson, 1970a,b; Magos and Butler, 1976; Magos et al., 1981, 1985; McNeil et al., 1988; Zalups et al., 1992). However, the level of accumulation is much less than that which occurs after exposure to inorganic or elemental forms of mercury. For example, only about 10% of the administered dose of mercury has been shown to be present in the combined renal mass of rats 24 h after the administration of a non-nephrotoxic (5 mg/kg) dose of methylmercury (Zalups et al., 1992).

Within the kidneys, both inorganic and organic forms of mercury have been shown to accumulate primarily in the cortex and outer stripe of the outer medulla (Friberg et al., 1957; Bergstrand et al., 1959; Berlin, 1963; Berlin and Ullberg, 1963a,b; Taugner et al., 1966; Zalups and Barfuss, 1990; Zalups and Lash, 1990; Zalups, 1991a,b,c, 1993; Zalups and Cherian, 1992a,b). Until relatively recently, however, very little was known about which segments of the nephron take up and accumulate the various forms of mercury. This prompted numerous studies to determine where inorganic and organic forms of mercury are taken up and accumulated along the nephron. Histochemical and autoradiographic data from studies in mice and rats (Taugner et al., 1966; Hultman et al., 1985; Magos et al., 1985; Hultman and Enestrom, 1986, 1992; Rodier et al., 1988; Zalups, 1991a) and tubular microdissection data from studies in rats and rabbits (Zalups and Barfuss, 1990; Zalups, 1991b) indicate that the accumulation of inorganic mercury in the renal cortex and outer stripe of the outer medulla occurs mainly along the convoluted and straight segments of the proximal tubule. Deposits of mercury have also been localized in the renal proximal tubule of monkeys exposed to elemental mercury from dental amalgams (Danscher et al., 1990). It should be stressed, however, that although the segments of the proximal tubule appear to be the primary sites where mercuric ions are taken up and accumulated, there are currently insufficient data to exclude the possibility that other segments of the nephron and/or collecting duct may also, to a minor extent, take up, accumulate, and transport inorganic and/or organic forms of mercury.

It is interesting that deposits of presumed inorganic mercury have also been found along segments of proximal tubules in the kidneys of rats and mice treated with organic forms of mercury (Magos et al., 1985; Rodier et al., 1988). Additional findings indicate that a significant fraction of the mercury in the kidneys of animals exposed to methylmercury is in the inorganic form (Gage, 1964; Norseth and Clarkson, 1970a,b; Omata et al., 1980; Zalups et al., 1992), suggesting that organic mercury is oxidized to inorganic mercury before and/or after it enters the renal tubular epithelial cells. Furthermore, there is evidence that intracellular conversion of methylmercury to inorganic mercury can occur (Dunn and Clarkson, 1980). However, the mechanism for this conversion is currently unknown.

Numerous theories and postulates regarding the mechanisms by which inorganic and organic forms of mercury gain entry into renal tubular epithelial cells have been put forth during the past two decades. In 1980, Madsen, and then later Zalups and Barfuss (1993b), put forth the hypothesis that a mechanism by which some mercuric ions gain entry into proximal tubular cells is through endocytosis of filtered mercury-albumin complexes. Albumin is by far the most abundant protein in plasma, and it has a free sulfhydryl group on a terminal cysteinyl residue (Brown and Shockley, 1982), to which mercuric ions can bind. Previous data indicate that the largest percentage of mercury in the plasma is bound to acid-precipitable proteins, such as albumin (Friedman, 1957; Mussini, 1958; Cember et al., 1968; Lau and Sarkar, 1979). Despite the fact that the sieving coefficient for albumin is low, significant amounts of protein, mainly albumin, are filtered during each day. Thus, the notion of albumin-mercury complexes being filtered at the glomerulus is a reasonable one. In fact, Madsen (1980) showed that when rats were made proteinuric by treatment with the proximal tubular toxicant gentamicin, inorganic mercury was excreted in the urine primarily as a conjugate of albumin. Assuming that the proteinuria (induced by gentamicin) was not due to increased glomerular permeability, these data suggest that a significant fraction of inorganic mercury that is filtered into the proximal tubular lumen is bound to albumin. Zalups and Barfuss (1993b) attempted to implicate a mercuric conjugate of albumin in the luminal uptake of inorganic mercury by simultaneously evaluating the renal disposition of inorganic mercury and albumin after administering mercuric conjugates of albumin containing both ¹²⁵I-albumin and ²⁰³Hg²⁺. Although their data provided some interesting new insights, there was insufficient evidence to implicate the transport of a mercuric conjugate of albumin as a primary mechanism involved in the luminal uptake of inorganic mercury. Conversely, there were insufficient data to exclude endocytosis of a mercuric conjugate of albumin as a minor mechanism.

A series of recent studies have provided much more definitive evidence on the mechanisms involved in the proximal tubular uptake of mercury. Data from these studies indicate that there are at least two distinct primary mechanisms involved in the uptake of mercuric ions by proximal tubular epithelial cells. One of the mechanisms is localized at the luminal membrane (Zalups et al., 1991, 1998; Zalups and Barfuss, 1993a, 1998b; Zalups, 1995, 1997, 1998b,c; Zalups and Minor, 1995; Zalups and Lash, 1997a) and the other is localized at the basolateral membrane (Zalups and Barfuss, 1993a, 1995a, 1998b; Zalups, 1995, 1997, 1998b; Zalups and Minor, 1995; Zalups and Lash, 1997a).

C. Mechanisms of Luminal Uptake of Mercury

1. Role of -Glutamyltransferase. There is a strong body of evidence linking the luminal uptake of inorganic mercury and, to a lesser extent, organic forms of mercury to the activity of -glutamyltransferase (-GT). In the kidney, this enzyme is localized predominantly in the luminal (brush-border) membrane of proximal tubular epithelial cells. The function of the enzyme is to cleave the -glutamylcysteine bond in molecules of glutathione (which are present in the proximal tubular lumen). Much of the evidence implicating the activity of the enzyme in the renal tubular uptake of mercury comes from in vivo experiments in which inhibition of renal (and hepatic) -GT, by pretreatment with L-(S,5S)--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), has been shown to have profound effects on the renal disposition of administered mercury. More specifically, pretreatment with acivicin has been shown to cause significant decreases in the renal uptake and/or accumulation of mercury and significant increases in the urinary excretion of mercury in mice (Tanaka et al., 1990; Tanaka-Kagawa et al., 1993) and rats (Berndt et al., 1985; de Ceaurriz et al., 1994; Zalups, 1995) treated with inorganic mercury or in mice administered methylmercury (Tanaka-Kagawa et al., 1993) or exposed to mercury vapor (Kim et al., 1995). Enhanced urinary excretion of glutathione has also been documented in acivicin-pretreated rats that were subsequently injected with inorganic mercury (Berndt et al., 1985). Cannon et al. (1998a, 2000) recently provided direct evidence, from isolated perfused S2 segments of the rabbit proximal tubule, that inhibition of -GT (by the direct application of acivicin to the luminal plasma membrane) causes significant reductions in the luminal uptake (disappearance flux, J_D) and cellular accumulation of mercuric ions when they are in the form of mercuric conjugates of glutathione. Collectively, the in vivo and in vitro data described earlier indicate strongly that a significant fraction of the mercuric ions taken up by proximal tubular epithelial cells is accomplished by a luminal absorptive mechanism dependent on the actions of -GT.

2. Presence and Formation of Mercuric Conjugates in Proximal Tubular Lumen. A major implication of the data obtained during in vivo inhibition of -GT is that some pool of mercuric ions present in the lumen of the proximal tubule exists in the form of a mercuric S-conjugate of glutathione before being taken up. Although it is not known exactly where these mercuric conjugates of glutathione are formed before arriving in the lumen of the proximal tubule, one must consider the possibility that some of them are formed outside the kidneys and then enter into the lumen of the proximal tubule via glomerular filtration. There are a few reasons to suspect that this may occur. First, the formation of mercuric conjugates of glutathione in the plasma (after exposure to mercuric compounds) is theoretically possible because the concentration of this thiol-containing molecule in plasma (of rats) has been estimated to be approximately 10 µM (Lash and Jones, 1985a), which provides a sufficiently large pool of glutathione to form conjugates with mercuric ions in plasma. Second, the liver is a major source for glutathione in the body, and mercuric conjugates of glutathione have been shown to form in hepatocytes. Once formed, these conjugates may enter into systemic circulation along with glutathione, where they can then be delivered to the kidneys. Third, the size and shape of these conjugates are such that they can, and should, pass through the glomerular filtration barrier unimpeded.

3. Cleavage Products of Mercuric Conjugates of Glutathione as Transportable Forms of Mercury at Luminal Plasma Membrane. Considering that luminal uptake of mercuric ions by proximal tubular cells is linked to the activity of -GT and the presence of mercuric S-conjugates of glutathione in the tubular lumen, the actual luminal uptake of mercuric ions would appear to involve the transport of some product formed by the actions of the -GT. One such product might be a mercuric conjugate of cysteinylglycine, which could be transported potentially by one of the small peptide transport systems in the luminal plasma membrane (Silbernagl, 1992). However, because of the high level of activity of luminal membrane dehydropeptidases (e.g., cysteinylglycinase), one would predict that if there is transport of this mercuric conjugate along the proximal tubule in vivo, the rate of transport would be very low. Based on the high activities of both -GT and cysteinylglycinase, it is most likely that the actual, or primary, species of mercury transported at the luminal membrane is a mercuric conjugate of L-cysteine, via one or more of the amino acid transport systems. It should be stressed that there is in vitro evidence indicating that sequential enzymatic degradation of glutathione to cysteinylglycine, and then to cysteine, is possible while a mercuric ion remains bound to the cysteinyl residue (at the site of the ---SH group) of the molecules of glutathione that are being degraded (Naganuma et al. 1988).

4. Role of Cysteinylglycinase. Potential luminal transport of mercuric conjugates of cysteinylglycine was investigated recently in isolated perfused S2 segments of the rabbit proximal tubule by Cannon et al. (1998a, 2000). They demonstrated that near-complete inhibition of cysteinylglycinase, with the dehydropeptidase-1 inhibitor cilastatin, caused significant reductions in the luminal uptake of inorganic mercury when it was in the form of a mercuric S-conjugate of cysteinylglycine. These findings support the hypothesis that when inorganic mercury is conjugated to cysteinylglycine, much of the luminal absorption of mercury is linked to the actions of the dehydropeptidase-1 (cysteinylglycinase) that cleaves the peptide bond in molecules of cysteinylglycine. Cannon et al. (2000) discovered, however, that inhibition of luminal dehydropeptidases did not completely prevent the luminal uptake of mercury when it was in the form of a mercuric conjugate of cysteinylglycine. These findings tend to indicate that at least in isolated perfused proximal tubular segments, some level of transport of mercuric conjugates of cysteinylglycine may actually occur at the luminal membrane while luminal dehydropeptidases are inhibited. However, before one can make any definitive conclusions about potential transport of mercuric conjugates of cysteinylglycine in the proximal tubule in vivo, one needs to consider additional factors, such as potential heterogeneity in the handling of glutathione, cysteinylglycine, and mercuric conjugates of glutathione and cysteinylglycine along the entire proximal tubule. In fact, there are recent findings indicating there is significant heterogeneity in the synthesis, secretion, and/or transport of glutathione along the length of the rabbit proximal tubule (Parks et al., 1998, 2000).

5. Mercuric Conjugates of Cysteine as Primary Transportable Form of Mercury at Luminal Plasma Membrane. Numerous sets of recent findings indicate that mercuric conjugates of cysteine, such as the dicysteinylmercury complex, are likely the primary species of inorganic mercury transported at the luminal membrane of proximal tubular cells. For example, there are in vivo data showing that the renal uptake and accumulation of inorganic mercury (Zalups and Barfuss, 1995b, 1998b) and the level of renal tubular injury induced by inorganic mercury (Zalups and Barfuss, 1996b) were increased in animals when the inorganic mercury was administered as a mercuric conjugate of cysteine. In addition, there are in vitro data showing that mercuric ions gained entry into brush-border membrane vesicles far more readily when they were in the form of mercuric conjugates of cysteine than when they were in the form of mercuric conjugates of glutathione or even mercuric chloride (Zalups and Lash, 1997a). By far, the most convincing evidence for the luminal transport of a mercuric conjugate of cysteine comes from the isolated perfused tubule studies of Cannon et al. (1998a,b, 1999, 2000). These investigators demonstrated that the rates of luminal uptake (disappearance flux) of mercuric ions in isolated perfused proximal tubular segments were approximately 2-fold or more greater when mercuric conjugates of cysteine (103 ± 4 fmol/min/mm tubule) were present in the luminal compartment than when mercuric conjugates of either glutathione (39 ± 1 fmol/min/mm tubule) or cysteinylglycine (53 ± 3 fmol/min/mm tubule) were present in the lumen. Their findings also show that mercuric conjugates of cysteine, presumably in the form of a single mercuric ion bonded to the sulfur atoms of two molecules of cysteine (in a linear II coordinate covalent complex), are taken up at the luminal membrane of proximal tubular cells by known amino acid transporters (Cannon et al., 1999). These investigators also provide data indicating that the luminal uptake of these mercuric conjugates involves at least two separate amino acid transport systems, with one being sodium-dependent and the other being sodium-independent. Another set of their data indicates that one or more of the same transport systems involved in the luminal uptake of the amino acid cystine may be involved in the luminal uptake of mercuric conjugates of cysteine (Cannon et al., 2000). These data show that the addition of 3 mM L-lysine to a perfusate containing 20 µM inorganic mercury and 80 µM cysteine caused an approximate 50% reduction in the net rate of luminal uptake of inorganic mercury in isolated perfused S2 segments of the rabbit proximal tubule. To put these findings into context, Schafer and Watkins (1984) had established previously in isolated perfused S2 segments that L-lysine (3 mM) inhibits the luminal uptake of cystine (300 µM) by approximately 50%. Their findings suggest that some component of the luminal absorption of cystine occurs through a transporter shared by the dibasic amino acid lysine. Overall, it appears that some fractions of the luminal uptake of both cystine and dicysteinylmercury occur via the same transport system. A diagrammatic summary of the known and putative mechanisms involved in the luminal transport of inorganic mercury is presented in Figs. 1 and 2.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Diagrammatic representation of mechanisms involved in the luminal uptake of inorganic mercury in proximal tubular epithelial cells. The presented scheme is premised on having mercuric conjugates of glutathione (GSH-Hg-GSH) and cysteine (CYS-Hg-CYS) present in the proximal tubular lumen. At present, it is not clear where these conjugates form, but filtration, secretion, and/or formation in the tubular lumen all must be considered. Experimental evidence indicates that the primary mechanism involved in the luminal uptake of inorganic mercury along proximal tubular segments involves the actions of the brush-border enzyme, -GT. When there is near-complete inhibition of the enzyme (with acivicin), there are significant decreases in the luminal uptake of inorganic mercury and enhanced urinary excretion of mercury and glutathione (GSH). According to the scheme presented, the mechanism of action of the -glutamyltranspeptidase in the luminal uptake of mercury involves the catalytic cleavage of the -glutamylcysteine bond on molecules of GSH bonded (via the sulfur atom of the cysteinyl residue) to mercuric ions. There also is the possibility that mixed mercuric conjugates may be present in the tubular lumen, but these have not been included in this figure. According to the presented scheme, after -GT has cleaved the -glutamylcysteine bond on molecules of GSH bonded to a mercuric ion, the resulting mercuric conjugate of cysteinylglycine can potentially enter two pathways. The most likely pathway involves the catalytic cleavage of the cysteinylglycinyl bond on molecules of cysteinylglycine bonded to a mercuric ion by the dehydropeptidase-1 cysteinylglycinase located on the luminal membrane. The second pathway might involve the transport of a mercuric S-conjugate of cysteinylglycine into the proximal tubular cell by one of the sodium-dependent peptide transport systems. This pathway is shown as a dotted line attached to an arrowhead, just above the depiction of cysteinylglycinase. Due to the abundance of peptidase activity on the luminal membrane of proximal tubular epithelial cells, it seems unlikely, at present, that any appreciable amount of transport of a mercuric conjugate of cysteinylglycine would occur under normal circumstances. After the actions of cysteinylglycinase, a mercuric conjugate of cysteine remains, presumably as dicysteinylmercury (CYS-Hg-CYS). This resulting mercuric S-conjugate of cysteine then appears to enter proximal tubular epithelial cells via amino transporters in the luminal plasma membrane. Current evidence indicates that there are both sodium-dependent and sodium-independent amino transport systems involved. One cannot exclude, however, the possibility that some type of a mercuric conjugate of GSH is also taken up intact by one of the luminal transport systems. This is shown using a dotted line attached to an arrowhead near the top of the figure. This scheme also shows the potential for endocytosis of a mercuric conjugate of albumin. Moreover, the scheme shows that the GSH secreted into the tubular lumen of proximal tubular segments may preferentially compete for the mercuric ions carried into the tubular lumen by albumin or other plasma proteins. Finally, this scheme shows that mercuric conjugates of cysteine can be filtered into the tubular lumen, bypass the actions of -GT and cysteinylglycinase, and enter the proximal tubular cells via one of the amino acid transporters.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   This figure provides a few details on the potential mechanisms involved in the luminal uptake of the mercuric conjugate of cysteine, dicysteinylmercury (Cys-Hg-Cys), in proximal tubular segments. At least two amino acid transporters appear to be involved. One of these is a sodium-dependent transporter, and at least one of them is a sodium-independent transporter. A likely candidate for the sodium-dependent transporter involved in the uptake of dicysteinylmercury is System ASC. Among the transporters that are sodium-independent, System L or b0,+ are potential candidates. One of the current hypotheses regarding the mechanism by which some dicysteinylmercury gains entry into the proximal tubular epithelial cells is by a mechanism of molecular homology, which some refer to as "mimicry." The hypothesis states that the dicysteinylmercury complex is functionally homologous to the amino acid cystine and enters through one the transport systems involved in the absorption of cystine. Current molecular biological evidence from studies on the gene responsible for cystinuria indicate that System b0,+ is one transport system involved in the luminal transport of cystine. However, there is controversy on what other systems might be involved in the uptake of cystine along the various segments of the proximal tubule. Data from Cannon et al. (1999a) also tend to implicate System L in the luminal uptake of dicysteinylmercury.

6. Role of Molecular Homology. Based on experimental findings of Cannon et al. (2000) and Schafer and Watkins (1984) and the similarity in structure of cystine and the dicysteinylmercury complex (Fig. 3), researchers at the laboratories of Zalups and Barfuss hypothesized recently that some component of the absorptive luminal transport of dicysteinylmercury occurs by a mechanism involving molecular homology (or "mimicry"). They postulate that dicysteinylmercury may act as a molecular homolog, or "mimic," of the amino acid cystine at the site of one or more transporter responsible for the luminal uptake of cystine (Cannon et al., 2000).

View larger version (65K): [in this window] [in a new window]   Fig. 3.   A three-dimensional space-fill rendering of the molecule cystine and the complex formed by two molecules of cysteine bonded to an inorganic mercuric ion. This figure shows some of the homology that exists between these two molecules. A current hypothesis expounded to explain a significant component of the luminal uptake of dicysteinylmercury (along the proximal tubule) is one involving molecular homology. The dicysteinylmercury complex is thought to "mimic" or be functionally homologous to the amino acid cystine at the site of the transporters involved in the luminal uptake of cystine.

D. Mechanisms of Basolateral Uptake of Mercury

1. Role of Organic Anion Transport System. In addition to the large body of evidence indicating that mercuric ions are taken up at the luminal membrane of proximal tubular cells, there is substantial evidence indicating that mercuric ions are also taken up at the basolateral membrane of these cells. Approximately 40% of the dose of inorganic mercury is normally taken by the total renal mass of rats during the initial hour after the i.v. injection of a nontoxic dose of mercuric chloride (Zalups and Diamond, 1987a; Zalups and Lash, 1994; Zalups and Barfuss, 1995a, 1998a,b; Zalups, 1996, 1997). Current evidence indicates that approximately 40 to 60% of this renal burden of mercury can be attributed to a basolateral mechanism (Zalups, 1995, 1997, 1998b,c; Zalups and Barfuss, 1995, 1998a,b; Zalups and Minor, 1995). It should be stressed that this applies only to doses of inorganic mercury that are non-nephrotoxic. Under conditions where the dose is increased to levels that induce renal tubular injury, the percentage of the dose found in the kidneys (at various times after exposure) decreases. This is due in part to necrosis of tubular epithelial cells and the subsequent release and excretion of cytosolic mercury. (Zalups and Diamond, 1987b; Zalups et al., 1988).

2. Role of Dicarboxylate Transporter. In an early study, Clarkson and Magos (1967) demonstrated that pretreatment with the dicarboxylate maleate caused dose-dependent reductions in the net renal accumulation of inorganic mercury when it was given as a cysteine-mercury complex (100 µg Hg/kg). Unfortunately, it is not clear from this study whether the changes in the renal disposition of mercury were due to the inhibitory effects of maleate on renal cellular metabolism (Rogulski and Angielski, 1963) or whether they were due to direct effects at the site of a transporter of mercury. Interestingly, they found that fumarate (an isomer of maleate) did not have the same effects as maleate, which suggests isomer specificity.

View larger version (61K): [in this window] [in a new window]   Fig. 4.   Diagram outlining the putative roles of both the organic anion and dicarboxylic acid transport systems in the basolateral uptake of inorganic mercury along the proximal tubule. On the basis of the current line of evidence regarding the organic anion transport system, intracellular generation of -ketoglutarate (as a result of normal metabolic processes) creates a chemical gradient facilitating the movement of this dicarboxylate out of the cell. When intracellular concentrations of -ketoglutarate are sufficiently high, it exits proximal tubular cells at the basolateral membrane by exchanging with organic anions. After it is transported out of proximal tubular cells, -ketoglutarate is taken back up into the cell across the basolateral membrane via a sym-port involving the cotransport of sodium. This sym-port is driven by the sodium-gradient generated by the Na+,K+-ATPase localized in the basolateral membrane. According to the scheme presented, inorganic mercury enters proximal tubular epithelial cells (presumably as a conjugate of glutathione (GSH) and/or cysteine (CYS)) via the organic anion transport system in exchange for intracellular -ketoglutarate. The most likely species of inorganic mercury taken up at basolateral membrane by the organic anion exchanger include mercuric conjugates of GSH (GSH-Hg-GSH), CYS (CYS-Hg-CYS), and other small molecules possessing a negative charge (R-Hg-R), such as N-acetylcysteine. Support for this notion comes from the fact that the basolateral uptake of mercury can be inhibited by para-aminohippurate (PAH) and probenecid and dicarboxylic acids. The scheme presented also shows that succinate, glutarate, and adipate compete with -ketoglutarate at the site of the dicarboxylic acid transporter. It appears that both glutarate and adipate, but not succinate, can influence the basolateral uptake of mercury by theoretically acting at two sites. Evidence indicates that these two dicarboxylic acids can compete for the uptake of substrates transported by the organic anion transporter and other dicarboxylic acids (e.g., -ketoglutarate) transported by the sodium-dicarboxylic acid sym-port system. Succinate, on the other hand, appears to influence the basolateral uptake of mercury by competing only at the site of the dicarboxylic acid transporter. Preferential uptake of succinate over -ketoglutarate would result in a decrease in the intracellular concentration of -ketoglutarate, which would decrease the driving force behind the activity of the organic anion transporter. The scheme also shows the transport of newly synthesized GSH from within proximal tubular cells into the blood. There is evidence from isolated perfused proximal tubular segments demonstrating that approximately 25 to 30% of the GSH synthesized by proximal tubular epithelial is secreted into the basal compartment surrounding the tubule. This could provide a substantial pool of GSH at the basolateral membrane to interact with other molecules bonded to mercuric ions, resulting in the formation of mercuric conjugates of GSH, which could then be transported into the proximal tubular cells.

3. Possible Ligands and Conjugates Involved in Basolateral Uptake of Mercury. As mentioned earlier, the majority of the mercury that is present in plasma is bound to albumin and other large proteins. It is quite certain that the organic anion transport system does not transport mercuric conjugates of proteins into proximal tubular epithelial cells. At present, it appears that mercuric conjugates of low-molecular-weight ligands are the most likely species of mercury taken up at the basolateral membrane by the organic anion transporter. Two of the conjugates that have been implicated in the basolateral transport of mercury are mercuric conjugates of glutathione and/or cysteine (Zalups, 1998b).

4. Mercuric Conjugates of Glutathione as Transportable Forms of Mercury at Basolateral Membrane. Molecules of glutathione have a net negative charge at physiological pH. Because of this charge and its size, glutathione has been postulated to be substrate at the site of the organic anion transporter. Support for this comes in part from the studies of Lash and Jones (1983, 1984), who demonstrated transport of glutathione (as an intact tripeptide) in basolateral membrane vesicles (isolated from the renal cortex of rats) via a mechanism that was sodium-dependent and that could be blocked by probenecid. They also demonstrated basolateral transport of certain organic S-conjugates of glutathione, such as S-(1,2-dichlorovinyl)glutathione, into proximal tubular epithelial cells by a probenecid-sensitive mechanism (Lash and Jones, 1985b).

5. Mercuric Conjugates of Cysteine as Transportable Forms of Mercury at Basolateral Membrane. Despite the fact that cysteine has a net neutral charge at physiological pH, it has become highly relevant to consider that inorganic or organic mercuric conjugates of cysteine are transportable species at the site of the organic anion transporter. The relevance for this consideration comes from studies in which organic S-conjugates of cysteine have been shown to be taken up at the basolateral membrane of proximal tubular cells by a mechanism consistent with the activity of the organic anion transporter. For example, Lash and Anders (1989) demonstrated that organic S-conjugates of cysteine [e.g., S-(1,2-dichlorovinyl)-L-cysteine] were taken up by isolated proximal tubular epithelial cells from rats by a sodium-dependent and probenecid- and para-aminohippurate-sensitive transport system. More recently, Dantzler et al. (1995), using isolated proximal tubules from rabbits, also demonstrated that certain organic S-conjugates of cysteine were taken up at the basolateral membrane by a probenecid- and p-aminohippurate-sensitive transport mechanism.

6. Other Mercuric Conjugates as Transportable Forms of Mercury at Basolateral Membrane. Although current experimental evidence tends to point to mercuric conjugates of cysteine and glutathione being primarily involved in the luminal and basolateral uptake of inorganic mercury along the proximal tubule (after exposure to mercuric chloride), it is clear that other thiols, especially homologues of cysteine, such as homocysteine and N-acetylcysteine, can significantly influence the manner in which inorganic mercury is being handled in the kidneys (Zalups, 1998c; Zalups and Barfuss, 1998b). This point is exemplified in the recent studies of Zalups and Barfuss (1998b) and Zalups (1998c), who studied and compared in rats the mechanisms involved in the renal tubular uptake of inorganic mercury when it was coadministered with cysteine, homocysteine, or N-acetylcysteine. When inorganic mercury was administered with cysteine or as mercuric chloride, the levels of luminal and basolateral uptake of mercury in the kidneys were similar. In contrast to this pattern of uptake, when inorganic mercury was administered with homocysteine, a much lower level of uptake of mercury occurred at the luminal membrane relative to that which occurred at the basolateral membrane. Even greater differences in the levels of luminal uptake versus basolateral uptake of mercury were detected when rats were treated with inorganic mercury and N-acetylcysteine. When inorganic mercury was administered with this negatively charged molecule, virtually all of the renal tubular uptake of mercury occurred at the basolateral membrane, and the majority of this uptake could be inhibited by pretreatment with para-aminohippurate. In fact, regardless of how inorganic mercury was administered, the majority of the basolateral uptake of mercury was inhibited by pretreatment with para-aminohippurate, which implicates the activity of the organic anion transport system in the basolateral uptake of inorganic mercury under all of the experimental conditions studied.

It appears that some aspects of hepatic function play a role in at least a component of the renal uptake and transport of mercury. Evidence for this hypothesis comes from recent dispositional studies. In one study, specific depletion of hepatic glutathione with 1,2-dichloro-4-nitrobenzene before the administration of inorganic mercury was shown to cause a significant diminution in the renal uptake and/or accumulation of inorganic mercury in mice (Tanaka et al., 1990). In other studies, it has been demonstrated that biliary ligation or cannulation before the administration of inorganic mercury caused a decrease in the renal tubular uptake and accumulation of inorganic mercury in rats (Zalups and Barfuss, 1996a, Zalups, 1998a; Zalups et al., 1999a,b,c). Taken together, these findings indicate that some aspects of hepatic function are linked to a component in the renal tubular uptake and/or accumulation of inorganic mercury. Hepatic synthesis and secretion of glutathione represent a possible candidate. Additional studies are necessary to better determine the role of the liver in the renal tubular uptake of mercury.

Once inorganic mercuric ions gain entry in proximal tubular cells, it appears that they distribute throughout all intracellular pools (Madsen, 1980; Omata et al., 1980; Baggett and Berndt, 1985; Houser and Berndt, 1988). Cellular fractionation studies using the renal cortex from rats treated acutely or chronically with mercuric chloride indicate that mercury distributes in nuclear, lysosomal, mitochondrial, brush-border, and supernatant fractions, with the nuclear fraction containing the greatest amount of mercury among the organelle fractions (Madsen, 1980; Madsen and Hansen, 1980). Similar findings have also been obtained in other studies using homogenates of the renal cortex from normal and uninephrectomized rats treated with mercuric chloride (Baggett and Berndt, 1985; Houser and Berndt, 1988). In these studies, however, the cytosolic fraction was found to contain the greatest content of mercury.

Interestingly, the relative specific content of mercury was shown to increase to the greatest extent in the lysosomal fraction when rats were made proteinuric with an aminoglycoside (Madsen, 1980) or when rats were treated chronically with mercuric chloride (Madsen and Hansen, 1980). Increases in the lysosomal content of mercury may reflect the fusion of primary lysosomes with endocytotic or cytosolic vesicles containing complexes of inorganic mercury bound to proteins.

	III. Urinary Excretion of Mercury

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Urinary and fecal excretion of mercury are the principal means by which humans and other mammals eliminate the different forms of mercury from the body. Under most circumstances, a greater fraction of a dose of mercury is excreted in the feces than in the urine early after exposure (Rothstein and Hayes, 1960; Magos and Clarkson, 1977; Zalups et al., 1987, 1988, 1991a, 1992, 1993; World Health Organization, 1991; Zalups et al., 1987, 1988, 1991a, 1992, 1993; Zalups and Lash, 1994). In rats, it has been shown that more than twice as much inorganic mercury is excreted in the feces than in the urine during the initial days after exposure to a non-nephrotoxic dose of mercuric chloride (Rothstein and Hayes, 1960; Zalups et al., 1987, 1988; Zalups and Lash, 1994). Less than 10% of the administered dose is excreted in the urine during this time. In one study, rats injected i.v. with a non-nephrotoxic dose of mercuric chloride had excreted about 20% of the dose in the urine and 30% of the dose in the feces during the initial 54 days after injection (Rothstein and Hayes, 1960). The low level in the urinary excretion of mercury is due to two principal factors, the avid uptake of mercuric ions and the retention of accumulated mercuric ions, in proximal tubular segments.

After exposure to organic forms of mercury, even less mercury is excreted in the urine than after exposure to inorganic mercury. For example, it was demonstrated recently that both normal and uninephrectomized rats excreted only about 3% of the dose of mercury in the urine by the end of the initial 7 days after the i.v. injection of a low dose (5 mg/kg) of methylmercury (Zalups et al., 1992). By contrast, more than 15% of the administered dose was excreted in the feces during the same period of time. In a recent study in which seven adult men received a tracer amount of ²⁰³Hg-labeled methylmercury i.v., the cumulative fecal excretion of mercury over 70 days was much greater than the cumulative urinary excretion of mercury (Smith et al., 1994). More specifically, about 30% of the dose was excreted in the feces, whereas only about 4% of the dose was excreted in the urine.

Early reports (Mambourg and Raynaud, 1965; Vostal, 1966) had claimed that mercury appeared in the urine before inulin (which is filtered and not absorbed or secreted along the nephron). This was interpreted by some (Clarkson and Magos, 1967) to indicate that urinary mercury represented a pool of mercury that had been secreted from the blood into the tubular lumen by a transepithelial mechanism. This was a reasonable view considering there was a published report claiming that approximately 99% of the mercury in plasma was not filterable (Berlin and Gibson, 1963). Based on recent data, however, it appears that much more than 1% on the mercury in plasma is filtered into the proximal tubule lumen (Madsen, 1980; Zalups and Minor, 1995; Zalups, 1997, 1998b,c; Zalups and Barfuss, 1998a,b) and that the mechanisms involved in the urinary excretion of mercury are less clear than once thought.

It should be emphasized that although 95 to 99% (depending on animal species and experimental conditions) of the mercury in plasma is bound to albumin (and other plasma proteins), a significant fraction of albumin is filtered at the glomerulus. Thus, substantial amounts of mercury could theoretically gain access to the luminal compartment of proximal tubules by filtration of a mercury-albumin complex. There is some indirect in vivo evidence supporting this notion. Madsen (1980) demonstrated in rats made proteinuric by gentamicin (presumably by decreasing the absorptive capacity of the proximal tubular epithelium by cellular necrosis) that much of the administered mercury excreted in the urine was associated with albumin. A fundamental assumption in with these findings, however, is that the preponderance of the albumin associated with the mercury in the urine came from glomerular filtration rather than intercellular leak. In contrast to the findings of Madsen (1980), Clarkson and Magos (1967) found that about 70% of the mercury excreted in urine by rats treated with sodium maleate, subsequent to the exposure of inorganic mercury, was not bound to protein. This finding is actually not that surprising, because much of the mercury excreted in the urine probably originated from cellular stores, and thus was likely bound to low-molecular-weight thiols, such as glutathione.

Some insight into mechanisms involved in the urinary excretion of mercury has been gained through experimental maneuvers that cause the urinary excretion of mercury to increase. In most cases, the increased urinary excretion of mercury is associated with decreased luminal absorption of mercury and/or the luminal elimination or extraction of accumulated mercury along the proximal tubule (and/or other segments of the nephron). Some examples of these maneuvers are listed below.

In an early study by Clarkson and Magos (1967), pretreatment of female rats with sodium maleate, before the injection of a low 100 µg/kg dose of mercury in the form of mercuric chloride or a mercury-cysteine complex, was shown to cause the urinary excretion of mercury to increase and the renal accumulation of mercury to decrease. Sodium maleate was used because it caused "profound metabolic disturbances in renal cells." The authors also found that the administration of sodium maleate after treatment with mercury caused the renal content of mercury to decrease and the urinary excretion of mercury to increase.

As mentioned earlier, the urinary excretion of mercury also increases dramatically when renal -GT is inhibited before the administration of inorganic mercury (Berndt et al., 1985; Zalups, 1995; Zalups et al., 1999b,c). Much of the mercury excreted in urine after the inhibition of -GT appears to be associated with glutathione, which implicates the presence of mercuric conjugates of glutathione in the proximal tubular lumen (Baggett and Berndt, 1986). Current evidence indicates that the increased urinary excretion of mercury associated with the inhibition -GT is due mainly to decreased luminal absorption and transport of mercury along the proximal tubule (Berndt et al., 1985; Tanaka et al., 1990; Tanaka-Kagawa et al., 1993; de Ceaurriz et al., 1994; Kim et al., 1995; Zalups, 1995; Cannon et al., 2000).

When inorganic mercury is applied to the luminal membrane of proximal tubular epithelial cells as a mercuric conjugate of N-acetylcysteine (Zalups and Barfuss, 1998b), DMPS (Zalups et al., 1998), DMSA (Zalups, 1993c), or metallothionein (Zalups et al., 1993a), urinary excretion of mercury increases greatly due to the lack of luminal uptake of these mercuric conjugates. In general, it appears that when mercuric ions are bound to organic ligands possessing a net negative charge, the mercuric conjugates of these molecules are not taken up readily at the luminal membrane and in turn are excreted in the urine. When DMPS is administered after exposure to mercury, the urinary excretion of mercury also increases greatly (Zalups, 1993c). Recent evidence (obtained from isolated perfused proximal tubular segments) indicates that the increased urinary excretion of mercury that occurs under these conditions results from unidirectional extraction of mercury from within or on proximal tubular epithelial cells into the tubular lumen (Z