Department of Pharmacology, Wayne State University School of
Medicine, Detroit, Michigan (L.H.L., D.A.P.), and
Division of Basic
Medical Sciences, Mercer University, School of Medicine, Macon, Georgia
(R.K.Z.)
Distribution of inorganic mercury (Hg) into both acid-soluble and
protein-bound fractions of proximal tubular (PT) cells from the rat
increased with increasing concentrations of Hg up to 10 µM. Little
correlation was found between subcellular distribution of Hg and dose
in distal tubular (DT) cells. Cellular accumulation of Hg was rapid,
reaching equilibrium values by 10 to 15 min. Cellular content of Hg was
significantly higher in PT cells than in DT cells at 1 µM Hg. To
assess the effect of extracellular thiols on the intracellular
accumulation of Hg, PT and DT cells were coincubated with Hg and
cysteine, glutathione (GSH), bovine serum albumin (BSA) or
2,3-dimercapto-1-propanesulfonic acid (DMPS) in a 4:1 thiol:Hg molar
ratio. Coexposure with Hg and cysteine increased intracellular
accumulation of Hg in PT cells at 0.1 µM Hg relative to exposure to
Hg alone, consistent with an Hg-cysteine conjugate being a transport
form of Hg. In contrast, coexposure with Hg and BSA or DMPS markedly
decreased accumulation of Hg relative to cells exposed to Hg alone in
both cell types. Coexposure with Hg and GSH also decreased accumulation
of Hg relative to exposure to Hg alone, but the decrease was less than
coexposure with either BSA or DMPS, suggesting that either an Hg-GSH
complex may be a transport form or that some of the Hg-GSH complexes
were degraded to Hg-cysteine by the action of brush-border membrane enzymes. These results demonstrate that extracellular thiols markedly alter the renal accumulation of Hg and suggest that some Hg-thiol conjugates may be important physiological transport forms of Hg in the
kidney.
 |
Introduction |
Hg
is a potent and specific nephrotoxicant in vivo that
accumulates predominantly in the kidneys (Zalups, 1993a
) and
selectively in PT cells (Zalups, 1991a, 1991b). Intrarenal content of
Hg correlates with the severity of Hg-induced nephropathy or cellular
injury only up to the point where injury occurs (Bohets et
al., 1995; Burton et al., 1995; Girardi and Elias,
1991; Houser et al., 1992; Johnson, 1982; Zalups and
Diamond, 1987; Zalups and Lash, 1990; Zalups et al., 1987).
Hence, delineation of mechanisms that determine transport and
intracellular accumulation of Hg in the target cells within the kidney
is critical to understanding Hg-induced nephropathy.
Study and characterization of Hg transport are difficult because of the
high binding affinity of Hg toward low-molecular weight thiols, such as
GSH and Cys (Rabenstein, 1989) or toward protein sulfhydryl groups,
such as those on serum albumin. There is evidence that most of the Hg
in plasma is bound to serum albumin (Friedman, 1957; Cember et
al., 1968; Foulkes, 1974; Lau and Sarkar, 1979). There are three
potential mechanisms by which the mercuric ion in Hg-albumin complexes
in plasma can be taken up by the kidneys: (1) exchange of the albumin
ligand with plasma GSH or Cys and transport of the Hg-GSH or Hg-Cys
complexes into renal cells, (2) exchange of the albumin ligand with
other protein sulfhydryl groups, such as those on cellular plasma
membranes, or (3) endocytosis of Hg with filtered albumin. However,
because there is a significant amount of free GSH and Cys in plasma
(Lash and Jones, 1985a), Hg bound to these low-molecular-weight thiols
are additional forms by which Hg may be translocated to the kidneys.
Due to the lability of Hg-S bonds in biological fluids (Oram et
al., 1996; Rabenstein, 1989), ligand exchange is likely to be an
important component of the handling of Hg complexes by the kidneys.
Several studies have provided evidence for a role for exogenous thiols
or sulfhydryl-containing compounds in the renal uptake and accumulation
of Hg. Studies in isolated PT segments from rabbit kidney (Zalups
et al., 1993) demonstrated protection from injury induced by
exposure to 10 µM Hg by the addition of 40 µM GSH, Cys or BSA to
the extracellular medium. This protection was associated with decreased
uptake of Hg. In two in vivo studies, however, the
simultaneous addition of GSH or Cys with Hg produced elevations in the
renal concentration of Hg relative to animals that were given Hg alone
(Zalups and Barfuss, 1995a, 1995b). There is greater association of Hg
with isolated brush-border membrane vesicles from rat renal cortex when
they are exposed to Hg-Cys complexes compared with mercuric complexes
with other thiols (Zalups and Lash, 1997), and coadministration to rats
of low, nephrotoxic doses of Hg with Cys resulted in a significant
increase in the content of Hg in renal cortex and outer stripe of the
outer medulla compared with that in rats administered Hg alone (Zalups
and Barfuss, 1996). Hence, it appears that an Hg-Cys complex may be a
transport form of Hg into renal PT cells. By contrast, the synthetic
dithiol and clinically used, heavy metal chelator DMPS binds Hg with
high affinity, significantly reduces renal accumulation and/or the renal burden of Hg and protects both in vivo and in
vitro from Hg-induced renal injury (Zalups, 1993b; Zalups et
al., 1991a). Current evidence from studies in renal brush-border
and basolateral membrane vesicles (Zalups and Lash, 1997) indicates
that complexes of Hg and DMPS are poorly, if at all, transported. This
contrasts with free DMPS, which is avidly transported into PT cells
(Klotzbach and Diamond, 1988).
The potential role of both intracellular and extracellular GSH in Hg
uptake and intracellular accumulation is even less clear. Johnson
(1982) decreased total renal nonprotein sulfhydryl content with diethyl
maleate pretreatment and observed a decrease in renal accumulation of
Hg. Rats that have undergone uninephrectomy and compensatory renal
growth exhibit both increased GSH content in the renal outer stripe of
the outer medulla and increased accumulation of Hg (both total content
and content normalized to either tissue weight or protein) when
administered Hg (Zalups and Lash, 1990). In contrast, Girardi and Elias
(1991, 1993) and Houser et al. (1992) observed an inverse
relationship between renal contents of GSH and Hg.
Interpretation of findings on the effects of extracellular GSH are
complicated by the potential degradation of GSH or Hg-GSH complexes to
Cys or Hg-Cys complexes, respectively, by GGT and dipeptidases on renal
brush-border membranes. Inhibition of GGT in mice (Tanaka et
al., 1990; Tanaka-Kagawa et al., 1993) or rats (Baggett
and Berndt, 1986; Berndt et al., 1985; de Ceaurriz et al., 1994; Zalups, 1995) with acivicin
[L-(
S,5S)--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] reduced renal accumulation of Hg compared with animals that were
not pretreated with acivicin. Hence, a portion of renal uptake and
accumulation of Hg occurs by a GGT-dependent mechanism.
Based on the above-mentioned studies, we hypothesize that under
appropriate conditions of time and Hg and/or thiol concentrations, extracellular thiols can alter significantly the intracellular accumulation of Hg in renal epithelial cells. We hypothesize further that coexposure with Hg and GSH or Cys can facilitate accumulation of
Hg. To test these hypotheses, we investigated the subcellular distribution of Hg and the influence of extracellular thiols on the
accumulation of Hg in PT and DT cells from rat kidney, which represent
a target and a nontarget renal cell population, respectively. The
results demonstrate differences between PT and DT cells in the
distribution of cell-associated Hg into protein-bound and soluble
fractions and effects of extracellular thiols on cellular accumulation
of Hg, supporting the hypothesis that complexes of Hg with GSH or Cys
may be physiological transport forms of Hg. A preliminary report of
this work has been presented (Lash et al., 1997).
| |
Experimental Procedures |
Materials.
Percoll, collagenase (type I), BSA (fraction V)
and DMPS were purchased from Sigma Chemical (St. Louis, MO).
203HgCl2 (specific
activity, 2.3 mCi/mg) was purchased from Buffalo Materials (Buffalo,
NY). All other chemicals were of the highest purity available and were
purchased from commercial sources.
Isolation of rat renal PT and DT cells.
Renal cortical cells
were isolated from the kidneys of male Sprague-Dawley rats (Harlan,
Indianapolis, IN; 200-300 g) by collagenase perfusion (Jones et
al., 1979). Animals were housed in the Wayne State University
vivarium, were allowed access to food and water ad libitum
and were kept in a room on a 12-hr light/dark cycle. Before surgery,
rats were anesthetized with intraperitoneal injections of sodium
pentobarbital (50 mg/kg b.wt.). Enriched populations of PT and DT cells
were then obtained by Percoll density-gradient centrifugation of
cortical cells (Lash and Tokarz, 1989). Briefly, cortical cells (5 ml,
5-8 × 106 cells/ml) were layered on 35 ml
of 45% (v/v) isosmotic Percoll solution in 50-ml polycarbonate
centrifuge tubes and were centrifuged at 4°C for 30 min at
20,000 × g in a Sorvall RC2B centrifuge in an SS34
rotor. Fractions were collected, and cell types of origin were
identified by the use of marker enzymes and cell type-specific respiratory responses (Lash and Tokarz, 1989). Based on enzymology and
morphology, the renal PT cell preparation contains cells derived from
both convoluted and straight segments and is estimated to be 
97%
pure; the renal DT cell preparation contains cells derived from the
distal convoluted tubule, the cortical collecting duct and connecting
tubules but not the thick ascending limbs and is estimated to have
<10% contamination from PT cells, with a purity of ~88%.
Before incubations, cells were diluted 5-fold with Krebs-Henseleit
buffer, pH 7.4, containing 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and metabolic
substrates (5 mM glucose, 5 mM glutamine), washed to remove Percoll and
resuspended in fresh buffer at a concentration of 1.2 × 106 cells/ml. Cell concentrations were determined
in the presence of 0.2% (w/v) trypan blue in a hemacytometer, and cell
viability was estimated by either trypan blue exclusion or the release
of lactate dehydrogenase activity from the cells (Lash and Tokarz, 1989). All buffers were equilibrated with 95%
O2/5% CO2, and cells were
stored on ice in 25-ml polyethylene Erlenmeyer flasks until used.
Incubations were performed in 1.5-ml polyethylene microcentrifuge tubes
in a 37°C Dubnoff metabolic shaking incubator (60 cycles/min).
Methods to study distribution of cell-associated Hg.
Due to
the limitations in our ability to differentiate between transport of Hg
into the soluble fraction of cells and direct binding of Hg to cellular
plasma membranes or transport followed by binding to cellular plasma or
intracellular membranes or intracellular macromolecules, we refer to Hg
recovered in cell extracts as "cell-associated" and the process by
which this occurs as "accumulation."
Renal PT and DT cells were incubated with
203HgCl2 (0.5, 1, 10 and
100 µM; 0.05 µCi per 0.5-ml sample plus cold
HgCl2 to the indicated concentration) for 1, 5, 10 or 60 min. At indicated times, aliquots of cells (0.5 ml) were
layered on 1 ml of 20% (v/v) Percoll in saline in 1.5-ml
microcentrifuge tubes to separate cellular contents from extracellular
media. Two fractions were obtained: a supernatant and a cell pellet.
The supernatant fraction (1.5 ml), which is equivalent to extracellular
media, was removed for gamma counting. The cell pellets were
resuspended in 0.5 ml of saline, 0.1 ml of 30% (w/v) TCA was added,
the suspension was mixed and the tubes were centrifuged to separate the
TCA-soluble (transported Hg that remains unbound or free within the
cell) and TCA pellet (membrane- or protein-bound Hg) fractions.
Effect of extracellular thiols on cellular accumulation of
Hg.
Incubation mixtures (1.0 ml) contained 0.75 ml of appropriate
HgCl2 stock solution containing 0.05 µCi of
203HgCl2 and 0.25 ml of
cells (1.2 × 106 cells/ml) and were placed
in 1.5-ml microcentrifuge tubes. Final concentrations of Hg were 0.1, 1, and 5 µM because previous studies (Lash and Zalups, 1992) showed
that these concentrations of Hg were below the threshold concentration
that produced PT cellular injury. Mixtures were incubated at 37°C
with shaking in a Dubnoff water bath for 1, 5, 10 or 60 min. At
indicated times, Hg accumulation was stopped by centrifugation for 30 sec at 13,000 × g. Supernatants were transferred to
2.0-ml cryovials with a 1.0-ml Hamilton gastight syringe. The syringe
was rinsed with 0.5 ml of saline, and the rinse was added to the
supernatant (total volume, 1.5 ml). Supernatants and cell pellets were
then counted in a gamma counter.
To assess the effects of extracellular thiols on cellular accumulation
of Hg, cells were incubated simultaneously with Hg and a 4-fold molar
excess of BSA, DMPS, GSH or Cys. This excess of thiols was used to
ensure that all Hg ions were bound to a ligand. Data are presented in
two formats: First, data for the amount of Hg in extracellular
supernatants and cell pellets are expressed in pmol; second, data on
the effects of thiols on Hg accumulation are expressed as percent of Hg
alone to make a direct comparison of the effects of the four thiols
easier. For coexposures with Cys, studies were performed with only 0.1 and 1 µM Hg due to limitations in the supply of radiolabel.
Determination of content of Hg in samples.
The radioactivity
of 203Hg in samples of cells or cell extracts was
determined by counting the samples in a 1282 Compugamma CS deep-well
gamma spectrometer (Pharmacia-LKB, Gaithersburg, MD) operating at a
counting efficiency of 50% for 203Hg. The actual
content of Hg in each sample was calculated by dividing the
radioactivity of 203Hg in the sample (dpm) by the
specific activity of 203Hg in the stock solution
(dpm/nmol). The amount of Hg in each sample was then expressed as
pmol/106 cells.
Statistical analyses.
Results are expressed as the mean ± S.E. of measurements from the indicated number of separate cell
preparations. Significant differences among selected means were first
assessed by a one-way or two-way analysis of variance. When significant
F values were obtained with the analysis of variance, the Fisher's
protected least significant difference t test was performed
to determine which means were significantly different from each other
with two-tail probabilities of <.05 considered significant. For
figures 1 through 3, results of statistical comparisons are described in the legends for clarity. Otherwise, significant differences are
indicated on the figures or in the tables.
| |
Results |
Cellular distribution of Hg in renal PT and DT cells.
Isolated
PT and DT cells were incubated with 0.5, 1, 10 or 100 µM Hg for up to
60 min, and cells were centrifuged through Percoll to separate
intracellular from extracellular contents. Cellular contents were then
fractionated into TCA-soluble and TCA-pellet fractions to assess the
time and concentration dependence of the distribution of Hg into
protein-free and protein-bound fractions (figs.
1-3). The
percentage of Hg found in the supernatant fraction (extracellular
medium) ranged from ~60% to 85% of total cpm over the 60-min time
course (fig. 1). Recovery of Hg in the supernatant fraction was nearly
maximal by the earliest time point, indicating rapid extraction of Hg
from the extracellular medium.
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
Distribution of Hg: Extracellular Hg-supernatant.
PT (A) and DT cells (B) from rat kidneys (1.2 × 106
cells/ml) were incubated with 203HgCl2 (0.5, 1, 10 or 100 µM; 0.05 µCi/0.5-ml sample plus cold HgCl2 to
the indicated concentration) for 1, 5, 10 and 60 min. At the indicated
times, aliquots of cells (0.5 ml) were layered onto 1 ml of 20% (v/v)
Percoll in 1.5-ml microcentrifuge tubes and centrifuged at 13,000 × g for 30 sec. The extracellular supernatants (1.5 ml)
were removed for gamma counting. The percent of total cpm from PT and
DT cells that was recovered in the extracellular supernatant is plotted
versus incubation time. Results are mean ± S.E. of measurements
from four separate cell preparations. Statistical analyses: The
following pairs ([Hg]/time) were significantly different (P < .05) from each other: For PT cells, 0.5/5 and 10/5, 0.5/10 and 10/10,
0.5/60 and 10/60, 0.5/60 and 100/60, 1/5 and 10/5, 1/10 and 10/10, 1/60
and 10/60, 10/5 and 100/5. For DT cells, 0.5/10 and 100/10, 10/5 and
10/60, 10/60 and 100/60. For PT cells vs. DT cells,
10/5.
|
|
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Distribution of Hg: Cellular Hg-TCA pellet. PT and
DT cells from rat kidneys (1.2 × 106 cells/ml) were
incubated with 203HgCl2 and processed as
described in the legend to figure 1. The cell pellets were resuspended
in 0.5 ml of saline, 0.1 ml of 30% (w/v) TCA was added, and the
suspension was mixed and was then centrifuged to separate the
TCA-soluble and TCA pellet fractions. TCA-pellet fractions were removed
for gamma counting. The percent of total cpm from PT and DT cells that
was recovered in the TCA-pellet fraction is plotted vs.
incubation time. Results are mean ± S.E. of measurements from
four separate cell preparations. Statistical analyses: The following
pairs ([Hg]/time) were significantly different (P < .05) from
each other: For PT cells, 0.5/1 and 100/1, 0.5/10 and 100/10, 0.5/60
and 100/60, 1/1 and 100/1, 1/10 and 100/10, 1/60 and 100/60, 10/60 and
100/60. For DT cells, 0.5/1 and 100/1, 0.5/5 and 100/5, 0.5/10 and
100/10, 0.5/60 and 100/60, 1/1 and 100/1, 1/5 and 100/5, 1/10 and
100/10, 1/60 and 100/60, 10/1 and 100/1, 10/5 and 100/5, 10/10 and
100/10, 10/60 and 100/60. For PT cells vs. DT cells,
none.
|
|
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Distribution of Hg: Cellular Hg-TCA supernatant. PT
and DT cells from rat kidneys (1.2 × 106 cells/ml)
were incubated with 203HgCl2 and processed as
described in the legend to figure 1. TCA-soluble and TCA pellet
fractions were separated as described in the legend to figure 2.
TCA-soluble fractions were removed for gamma counting. The percent of
total cpm from PT and DT cells that was recovered in the TCA-soluble
fraction is plotted versus incubation time. Results are mean ± S.E. of measurements from four separate cell preparations. Statistical
analyses: The following pairs ([Hg]/time) were significantly
different (P < .05) from each other: For PT cells, 0.5/1 and
100/1, 0.5/5 and 100/5, 0.5/10 and 100/10, 0.5/60 and 100/60, 1/1 and
100/1, 1/5 and 100/5, 1/10 and 100/10, 1/60 and 100/60, 10/10 and
100/10, 10/60 and 100/60, 100/1 and 100/60, 100/5 and 100/60. For DT
cells, none. For PT cells vs. DT cells, 0.5/60,
100/60.
|
|
Increased total cpm were recovered in the protein-bound fraction (TCA
pellet) with increasing time and concentration of Hg up to 10 µM Hg
(fig. 2). A more consistent increase in the fraction of Hg in the
TCA-pellet fraction was seen in the PT cells, where the amount of Hg
increased from ~12% to 32% within 10 min. In the DT cells, the
maximal percent of Hg recovered was somewhat lower, at ~25%. With
the 100 µM concentration of Hg, the percent of total cpm recovered
decreased to ~12% and 6% in PT cells and DT cells, respectively.
The percentage of total cpm recovered in the protein-free fraction of
cells (TCA-soluble) exhibited a similar pattern of distribution as in
the TCA-pellet fraction, but somewhat smaller increases occurred with
increasing Hg concentration and time (fig. 3). The major difference,
however, was that markedly higher amounts of Hg were recovered in the
TCA-soluble fraction of PT cells after exposure to 100 µM Hg than
after exposure to 10 µM Hg, which is the reverse pattern of that seen
in the TCA pellet fraction. There was no effect of time or
concentration of Hg on the recovery of total cpm in the TCA pellet
fraction of DT cells.
Influence of extracellular thiols on accumulation of Hg in renal PT
and DT cells exposed to 0.1 µM Hg.
The time course of cellular
accumulation of Hg in PT and DT cells exposed to .1 µM Hg shows rapid
accumulation of Hg in both cell types (fig.
4). No significant differences were
detected between the two cell types at corresponding times. Within the first minute, total cellular Hg content reached ~50% of the
approximate maximal level that was reached after 60 min of incubation.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Time course of renal PT and DT cellular
accumulation of 0.1 µM Hg. Incubation mixtures (1.0 ml) contained
0.75 ml of appropriate HgCl2 stock solution (0.05 µCi
203HgCl2, cold HgCl2 to give a
final Hg concentration of 0.1 µM) and 0.25 ml of PT or DT cells
(1.2 × 106 cells/ml) in 1.5-ml microcentrifuge tubes.
Mixtures were incubated at 37°C with shaking in a Dubnoff water bath
for 1, 5, 10 or 60 min. Hg accumulation was stopped by centrifugation
for 2 min at 13,000 × g. Supernatants were transferred to 2.0-ml
cryovials with a 1.0-ml Hamilton gastight syringe. The syringe was
rinsed with 0.5 ml of saline, and the rinse was added to the
supernatant (total volume, 1.5 ml). Radioactivity in supernatants and
cell pellets was then determined in a gamma counter. Results are the
mean ± S.E. of measurements from four or five separate cell
preparations. Significantly different (P < .05) pairs: a,
vs. 1-min time point in same cell type; b,
vs. 5-min time point in same cell type; c,
vs. 10-min time point in same cell type; d,
vs. 60-min time point in same cell type; e,
vs. DT cells at same time point; f, vs.
PT cells at same time point.
|
|
Total amounts of Hg in extracellular media and cell pellets for cells
incubated with 0.1 µM Hg alone ("buffer") or cells incubated with
0.1 µM Hg plus 0.4 µM of either BSA, DMPS, GSH or Cys are summarized in tables 1 and
2 for PT and DT cells, respectively. During 60 min of incubation with Hg alone, the amount of Hg in the
supernatants gradually decreased over time to ~37% of the theoretical initial supernatant amount of 100 pmol. Cellular
accumulation of Hg was most rapid during the first minute. Nearly 50%
of the total accumulation of Hg that occurred over the entire 60-min incubation was observed during the first minute. For both PT and DT
cells, coexposure to either Hg plus BSA or Hg plus DMPS resulted in the
lowest amount of accumulation of Hg relative to exposure to Hg alone.
On average, accumulation of Hg after exposure to Hg plus BSA or Hg plus
DMPS was ~33% to 60% lower than that after exposure to Hg alone.
Incubation of cells with Hg plus GSH also resulted in decreased Hg
accumulation, but this coexposure resulted in smaller decreases in Hg
accumulation than coexposure to either Hg plus BSA or Hg plus DMPS.
Cellular contents of Hg after exposure to Hg plus GSH were 40% to 50%
lower than those after exposure to Hg alone at the 1-min time point,
~30% lower after the 5-min time point, but were not significantly
different at later time points. In contrast to coexposure to Hg with
the other thiols, cell pellet contents of PT cells exposed to Hg plus
Cys were modestly, but significantly, elevated compared with those
detected after exposure to Hg alone, whereas those in DT cells
exhibited no significant differences from values after exposure to Hg
alone.
View this table:
[in this window]
[in a new window]
|
TABLE 1
Total extracellular and cellular Hg content in renal PT cells incubated
with 0.1 µM Hg: Effect of extracellular thiols
Incubation mixtures (1.0 ml) contained 0.75 ml of appropriate
HgCl2 stock solution (0.05 µCi of 203HgCl2,
cold HgCl2 to give a final Hg concentration of 0.1 µM and
buffer or the indicated thiol compound at a final concentration of 0.4 µM) and 0.25 ml of PT cells (1.2 × 106 cells/ml) in
1.5-ml microcentrifuge tubes. Mixtures were incubated at 37°C with
shaking in a Dubnoff water bath for 1, 5, 10 or 60 min. Hg accumulation
was stopped by centrifugation for 2 min at 13,000 × g.
Supernatants were transferred to 2.0-ml cryovials with a 1.0-ml
Hamilton gastight syringe. The syringe was rinsed with 0.5 ml of
saline, and the rinse was added to the supernatant (total volume, 1.5 ml). Radioactivity in supernatants and cell pellets was then determined
in a gamma counter. Results are the mean ± S.E. of measurements
from four or five separate cell preparations.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2
Total extracellular and cellular Hg content in renal DT cells incubated
with 0.1 µM Hg: Effect of extracellular thiols
Hg accumulation in renal DT cells was measured as described in the
legend to table 1. Results are the mean ± S.E. of measurements
from four or five separate cell preparations.
|
|
The relative effects of the four extracellular thiols on the
disposition of Hg are shown graphically in figure
5. Exposure of PT and DT cells to Hg plus
BSA or Hg plus DMPS exhibited the lowest amounts of cellular Hg
accumulation compared with those detected after exposure to Hg alone.
During exposure to Hg plus BSA or Hg plus DMPS in both cell types, Hg
accumulation was 40% to 70% lower than that which was detected after
exposure to Hg alone over the 60-min time course. By contrast, Hg
accumulation in both PT and DT cells incubated with Hg plus GSH was
only modestly lower (20-30%) than that in cells incubated with Hg
alone, and this occurred only at the first two time points. Hg
accumulation in PT cells exposed to Hg plus Cys was significantly
higher than that in PT cells exposed to Hg alone at the 5- and 10-min
time points. No significant differences in the cellular contents of Hg
between DT cells exposed to Hg plus Cys and Hg alone were detected at
any time point studied.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Modulation of renal PT and DT cellular accumulation
of 0.1 µM Hg by extracellular thiols. Cellular accumulation of 0.1 µM Hg in the presence of buffer or 0.4 µM thiol in PT cells (A) or
DT cells (B) was measured as described in the legend to figure 4.
Results are the mean ± S.E. of measurements from four or five
separate cell preparations. *Significantly different (P < .05)
from Hg added alone at same time point.
|
|
Influence of extracellular thiols on accumulation of Hg in renal PT
and DT cells exposed to 1 µM Hg.
The time course for cellular
accumulation of 1 µM Hg was also rapid, with maximal levels
essentially reached by 10 min (fig. 6).
In contrast to the results obtained after exposure to 0.1 µM Hg,
during exposure to 1 µM Hg, maximal content of Hg in PT cells was
~30% higher than that in DT cells.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Time course of renal PT and DT cellular
accumulation of 1 µM Hg. Cellular accumulation of 1 µM Hg was
measured as described in the legend to figure 4. Results are the
mean ± S.E. of measurements from four or five separate cell
preparations. Significantly different (P < .05) pairs: a,
vs. 1-min time point in same cell type; b,
vs. 5-min time point in same cell type; c,
vs. 10-min time point in same cell type; d,
vs. 60-min time point in same cell type; e,
vs. DT cells at same time point; f, vs.
PT cells at same time point.
|
|
Contents of Hg in extracellular supernatants in both PT cells (table
3) and DT cells (table
4) incubated with either Hg plus BSA, Hg
plus DMPS, Hg plus GSH or Hg plus Cys were significantly higher than in
cells incubated with Hg alone at all time points, indicating a marked
reduction in cellular extraction of Hg in the presence of extracellular
thiols. As with the case after exposure to 0.1 µM Hg, the smallest
amount of Hg accumulation was found after coexposure to Hg plus BSA or
Hg plus DMPS. In PT cells, Hg accumulation after coexposure to either
Hg plus BSA or Hg plus DMPS was >80% lower than that after exposure
to Hg alone. In DT cells, Hg accumulation after coexposure to Hg plus
BSA or Hg plus DMPS was reduced by 85% or >90%, respectively,
relative to that after exposure to Hg alone. Coexposure to Hg plus Cys
resulted in the highest amounts of Hg accumulation of all the Hg plus
thiol coexposures, whereas coexposure to Hg plus GSH resulted in
intermediate accumulation values. Cellular accumulation of Hg after
exposure to Hg plus Cys was greater in PT cells than in DT cells, which is consistent with the results with 0.1 µM Hg, in which coexposure with Hg plus Cys increased Hg accumulation relative to exposure to Hg
alone in PT cells only. Cellular accumulation of Hg after coexposure to
Hg plus GSH for 1 min was 82% and 75% lower in PT and DT cells,
respectively, than that after exposure to Hg alone. After 60 min,
however, Hg accumulation was only 57% and 71% lower in PT and DT
cells, respectively, than that after exposure to Hg alone.
View this table:
[in this window]
[in a new window]
|
TABLE 3
Total extracellular and cellular Hg content in renal PT cells incubated
with 1 µM Hg: Effect of extracellular thiols
Hg accumulation in renal PT cells was measured as described in the
legend to table 1 except that final Hg and thiol concentrations were 1 and 4 µM, respectively. Results are the mean ± S.E. of
measurements from four or five separate cell preparations.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 4
Total extracellular and cellular Hg content in renal DT cells incubated
with 1 µM Hg: Effect of extracellular thiols
Hg accumulation was measured in renal DT cells as described in the
legend to table 1 except that final Hg and thiol concentrations were 1 and 4 µM, respectively. Results are the mean ± S.E. of
measurements from four or five separate cell preparations.
|
|
Direct comparison of effects of the four thiols on Hg accumulation,
expressed as a percentage of Hg alone, in incubations with 1 µM Hg
(fig. 7) showed clearly that coexposure
with Hg plus DMPS produced the lowest amounts of Hg accumulation in
both PT and DT cells: Cellular Hg contents in the presence of Hg plus DMPS were <10% of those in the presence of Hg alone at all time points. At the 1 µM concentration of Hg, coexposure with 4 µM BSA
or 4 µM GSH resulted in approximately equivalent amounts of cellular
accumulation of Hg, with values ranging from 20% to 40% of those
after exposure to Hg alone in PT cells and from 18% to 30% of those
after exposure to Hg alone in DT cells. Coexposure with Cys resulted in
the highest amounts of Hg accumulation of the four thiol coexposures,
but this effect was greater in PT cells than in DT cells (maximal Hg
accumulation was ~65% and 45% of Hg alone, respectively). An
important difference in both cell types in the time course of Hg
accumulation after exposure to Hg plus Cys or Hg plus GSH as compared
with that after exposure to Hg plus BSA or Hg plus DMPS was that the
percentage of decrease in Hg accumulation relative to that after
exposure to Hg alone diminished with time of exposure to Hg plus Cys or
Hg plus GSH but increased or remained stable with time of exposure to
Hg plus BSA or Hg plus DMPS.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Modulation of renal PT and DT cellular accumulation
of 1 µM Hg by extracellular thiols. Cellular accumulation of 1 µM
Hg in the presence of buffer or 4 µM thiol in PT cells (A) or DT
cells (B) was measured as described in the legend to figure 4. Results
are the mean ± S.E. of measurements from four or five separate
cell preparations. *Significantly different (P < .05) from Hg
added alone at same time point.
|
|
Influence of extracellular thiols on accumulation of Hg in renal PT
and DT cells exposed to 5 µM Hg.
The time courses for
accumulation of Hg after exposure to 5 µM Hg were similarly rapid,
reaching equilibrium values in both cell types within 10 min (fig.
8). In contrast to the results with
exposure to 1 µM Hg, no significant differences in the cellular accumulation of Hg were observed between PT and DT cells after exposure
to 5 µM Hg.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Time course of renal PT and DT cellular
accumulation of 5 µM Hg. Cellular accumulation of 5 µM Hg was
measured as described in the legend to figure 4. Results are the
mean ± S.E. of measurements from four or five separate cell
preparations. Significantly different (P < .05) pairs: a,
vs. 1-min time point in same cell type; b,
vs. 5-min time point in same cell type; c,
vs. 10-min time point in same cell type; d,
vs. 60-min time point in same cell type; e,
vs. DT cells at same time point; f, vs.
PT cells at same time point.
|
|
The amount of Hg in the extracellular supernatant was greater when
cells were exposed to Hg plus BSA, Hg plus DMPS or Hg plus GSH compared
with Hg alone at 5 µM Hg (tables 5 and
6). Furthermore, the fraction of Hg that
was removed from the extracellular space was greatest with coexposure
to one of the three thiols with 5 µM Hg than with either of the two
lower concentrations of Hg that were tested. Amounts of Hg in the
supernatant fractions of both PT and DT cells after 60-min exposures
were >2-fold higher in cells exposed to Hg plus BSA than in those
exposed to Hg alone, >4-fold higher in cells exposed to Hg plus DMPS
and ~1.5-fold higher in cells exposed to Hg plus GSH. As with the
lower concentrations of Hg, Hg accumulation after exposure to 5 µM Hg
was lowest with coexposure with BSA or DMPS, whereas amounts of Hg in
cells incubated with Hg plus GSH were significantly higher than in
those incubated with either Hg plus BSA or Hg plus DMPS.
View this table:
[in this window]
[in a new window]
|
TABLE 5
Total extracellular and cellular Hg content in renal PT cells incubated
with 5 µM Hg: Effect of extracellular thiols
Hg accumulation in renal PT cells was measured as described in the
legend to table 1 except that final Hg and thiol concentrations were 5 µM and 20 µM, respectively. Results are the mean ± S.E. of
measurements from four or five separate cell preparations.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 6
Total extracellular and cellular Hg content in renal DT cells incubated
with 5 µM Hg: Effect of extracellular thiols
Hg accumulation in renal DT cells was measured as described in the
legend to table 1 except that final Hg and thiol concentrations were 5 and 20 µM, respectively. Results are the mean ± S.E. of
measurements from four or five separate cell preparations.
|
|
Direct comparison of Hg accumulation in cells exposed to Hg alone and
those exposed to Hg plus BSA, Hg plus DMPS or Hg plus GSH shows clearly
that the percent decrease of Hg accumulation was much greater at 5 µM
Hg than at either of the two lower concentrations studied (fig.
9). Another difference in the time course
with exposure to 5 µM Hg compared with exposure to 1 µM or 0.1 µM
Hg was that there was little if any change in the percent decrease of
Hg accumulation during the course of the 60-min incubation in cells
exposed to Hg plus GSH. Rather, the time courses for Hg accumulation in
each of the Hg plus thiol coexposures exhibited the same pattern in both PT and DT cells.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Modulation of renal PT and DT cellular accumulation
of 5 µM Hg by extracellular thiols. Cellular accumulation of 5 µM
Hg in the presence of buffer or 20 µM thiol in PT cells (A) or DT
cells (B) was measured as described in the legend to figure 4. Results
are the mean ± S.E. of measurements from four or five separate
cell preparations. *Significantly different (P < .05) from Hg
added alone at same time point.
|
|
| |
Discussion |
The high affinity of Hg for sulfhydryl groups plays a critical
role in its disposition and mechanism of toxicity (Zalups and Lash,
1994). Hence, alteration of either intracellular or extracellular thiol
status should affect the renal uptake and cellular accumulation of Hg.
Indeed, several studies have demonstrated that either alteration of
intracellular thiol content or coadministration of thiols with Hg can
have profound effects on the uptake and toxicity of Hg in the target
organ, the kidney. The relationship between intrarenal Hg content and
the severity of Hg-induced nephropathy, however, is a difficult one to
define mechanistically. On the one hand, a dose response exists for Hg,
although the curve is extremely steep, with almost an "all-or-none"
response often being observed. On the other hand, although higher doses
of Hg do lead to higher intrarenal contents of Hg, as doses are
increased from low levels, there is little correlation between uptake
and renal cellular accumulation of Hg once intoxication has occurred
(Zalups et al., 1988). An example of this is seen in the
present study, where exposure of renal PT and DT cells to
concentrations of Hg of >10 µM resulted in decreases in the
percentage of Hg recovered in the cell pellets and increases in the
percent of Hg recovered in the acid-soluble fraction of cells (compare
figs. 2 and 3). This agrees with the concentration dependence of
Hg-induced renal cellular injury in PT cells that we observed
previously (Lash and Zalups, 1992), where exposure of cells to Hg
concentrations above 10 µM produced marked increases in cell death.
Rapid accumulation of Hg in renal PT cells is expected because these
cells are the primary in vivo sites of Hg accumulation (Zalups, 1991a, 1991b; Zalups and Lash, 1994). In a study of Hg accumulation in microdissected nephron segments from rabbits given a
subtoxic dose of Hg, maximal accumulation of Hg was found in the
proximal tubular segments and no significant accumulation of Hg was
detected in nephron segments distal to the proximal tubule (Zalups and
Barfuss, 1990). Similar findings were obtained from nephron segments
isolated from rats treated with a subtoxic dose of Hg (Zalups, 1991a).
Based on these results, the isolated renal DT cells might not be
expected to accumulate significant amounts of Hg. However, depending on
the concentration of Hg administered, the accumulation of Hg in DT
cells was similar to or only modestly lower than values in PT cells
(compare figs. 2-4, 6 and 8). Differences in the nature of the
in vitro model systems used may contribute to these results.
With isolated PT and DT cells, the normal tubular polarity that
separates luminal from basolateral plasma membranes is lost and all
surfaces have equal access to compounds in the extracellular medium.
With isolated nephron segments, in contrast, intact polarity is
maintained. Another possible explanation is that the observed cellular
accumulation of Hg in the DT cell preparation may reflect that which is
occurring in contaminating PT cells. However, this explanation is not
likely to account for more than a small portion of the observed
accumulation of Hg since, based on enzymatic and functional markers,
the maximal amount of contamination of the DT cell preparation with PT
cells is only 5% to 10% (Lash and Tokarz, 1989). Hence, we conclude
that renal cells from the DT region have the capacity to accumulate Hg
but that in vivo, renal blood flow, the high affinity of Hg
transport and binding in the PT region, and other factors alter the
delivery of Hg so that minimal accumulation of Hg is observed in the DT
region. This is the first report describing accumulation of Hg in a
cell type derived from the distal nephron.
Although the general patterns of accumulation of Hg were the same in
the two cell populations, some differences were observed. Concentration-dependent increases in recovery of Hg in the TCA-soluble and TCA-pellet fractions were observed in PT cells but not in DT cells.
Comparison of the subcellular distribution patterns with the pattern of
Hg-induced cellular injury in isolated PT cells (Lash and Zalups, 1992)
suggests that the amount of Hg in the TCA-soluble fraction appears to
correlate with the degree of Hg-induced renal cellular injury.
The rapid accumulation of Hg in renal cells may be due in part to the
decrease in the extracellular concentrations of Hg during the
incubations. Although the ideal conditions would be to have constant
extracellular concentrations of Hg over the entire time course of
incubations, isolated PT fragments rapidly extract Hg, leading to the
observed decreases in extracellular concentrations of Hg (see
discussion in Zalups et al., 1993). It is difficult under
the current experimental conditions of media volume and cell
concentration to achieve these ideal conditions. Hence, there is a
significant decrease in extracellular concentrations of Hg over time
and as this occurs, the kinetics of Hg transport and/or binding change.
In vivo, one also has a similar situation because after a
given dose of Hg, there is a finite amount of Hg in plasma that is
avidly extracted by the kidneys, leading to a situation where the
concentration of Hg being presented to the kidneys decreases, thereby
altering elimination kinetics.
The results from this study confirm previous findings and support the
hypothesis that extracellular thiols modulate cellular accumulation of
Hg. BSA and DMPS, regardless of concentration, decreased Hg
accumulation in both cell populations. The extent of the decrease,
however, increased with increasing incubation time and concentration of
Hg, despite the constancy of the thiol:Hg ratio. Although DMPS itself
is avidly transported into renal PT cells (Klotzbach and Diamond,
1988), the present results showing minimal accumulation of an Hg-DMPS
complex in PT and DT cells are consistent with the in vitro
ability of DMPS to prevent uptake and binding of Hg in renal
brush-border membrane vesicles (Zalups and Lash, 1997) and to be an
extremely effective protective agent in renal PT cells against
Hg-induced renal cellular injury (Lash and Zalups, 1992). DMPS is also
a highly effective protective agent in vivo against
Hg-induced nephropathy (Zalups et al., 1991a). The mode of
in vivo protection by DMPS presumably involves extraction of
cellular Hg by intracellular chelation, whereas under the conditions of
the present study where DMPS was added simultaneously with Hg, the
decrease in cellular accumulation of Hg is due to prevention or
inhibition of Hg transport and/or binding in the cell. In a previous
study (Lash and Zalups, 1992), in contrast, isolated PT cells were
preloaded with DMPS and were completely protected from Hg-induced
cellular injury, suggesting that intracellular chelation was the
mechanism of protection.
The similarity in the ability of coexposure of renal cells with Hg plus
BSA or Hg plus DMPS to decrease Hg accumulation raises an important
question about the role of serum albumin as a physiological ligand for
Hg and the ability of Hg that is bound to albumin to be extracted by
the kidneys. A major difference exists, however, between conditions
in vivo and those with the in vitro exposures conducted in the present study. In the in vivo state, other
thiols, including GSH and Cys, are present when the Hg-albumin complex reaches the kidneys. Accordingly, exchange between albumin and these
low-molecular-weight thiols can occur, thereby facilitating renal
cellular uptake and accumulation of Hg. In the present study, in
contrast, cells were exposed to Hg plus BSA in the absence of other
competing thiols, so additional ligands to which Hg can bind were not
present. With these differences between in vivo and in
vitro exposure conditions in mind, as well as the known ability of
Hg bound to thiols to exchange with other ligands (Oram et
al., 1996; Rabenstein, 1989), the hypothesis that complexes of Hg
with albumin can function to deliver Hg to the target organ remains
viable.
In contrast to the similarities in effects of BSA and DMPS on Hg
accumulation, coexposure with a 4-fold molar excess of Cys exhibited
different effects at 0.1 and 1 µM Hg. Coexposure of PT cells, but not
DT cells, to 0.1 µM Hg and 0.4 µM Cys significantly enhanced Hg
accumulation relative to incubations with Hg alone. Coexposure of both
PT and DT cells to 1 µM Hg and 4 µM Cys, in contrast, significantly
decreased Hg accumulation relative to incubations with Hg alone at
early incubation times, and the extent of the decrease diminished with
increasing time. The extent of the decrease in Hg accumulation with
coexposure to Cys, however, was significantly less than that seen with
coexposure to either BSA or DMPS. Hence, the present results are
consistent with an Hg-Cys complex being transported into renal PT
cells. Furthermore, these studies agree with a recent study (Zalups and
Lash, 1997) that showed that coincubation of brush-border membrane
vesicles from rat renal cortex with Hg and Cys markedly enhanced uptake and binding of Hg compared with membrane vesicles incubated with Hg
alone.
At both 0.1 and 1 µM Hg and in both PT and DT cells, coexposure with
0.4 and 4 µM GSH, respectively, significantly decreased Hg
accumulation relative to incubations with Hg alone, and the extent of
the decrease diminished with incubation time. However, the accumulation
of Hg in exposures to Hg-GSH was significantly higher than that in
exposures to Hg-BSA or Hg-DMPS. In contrast, at 5 µM Hg in both PT
and DT cells, coexposure with 20 µM GSH decreased Hg accumulation
relative to incubations with Hg alone to a similar extent as did
coexposure with 20 µM BSA or 20 µM DMPS.
It is clear that BSA and DMPS are extremely effective inhibitors of Hg
accumulation in renal PT and DT cells, which corresponds with their
ability to provide virtually complete protection of renal cells from
Hg-induced injury (Lash and Zalups, 1992; Zalups, 1993b; Zalups
et al., 1991a, 1991b, 1993). The results with Cys are
consistent with an Hg-Cys complex functioning as a transport form of
Hg. However, due to the apparent transport and/or binding kinetics, the
Hg-Cys complex is transported more efficiently at low concentrations of
Hg (i.e., <1 µM Hg) than at higher
concentrations of Hg. Because renal PT cells have such excessively high
GGT activity, it is likely that degradation of GSH or an Hg-GSH
complex, producing, in combination with dipeptidase activity, Cys or an
Hg-Cys complex, respectively, can occur (Visarius et al.,
1996). Although it cannot be excluded that an Hg-GSH complex is a
transport form of Hg, it is likely that at least some of the Hg-GSH
complex is degraded to the corresponding Hg-Cys complex and that the
latter complex is the transport form. Clarkson (1993) has previously
suggested that thiol conjugates of Hg act like molecular mimics of
other thiol conjugates and are thus transported and metabolized by
analogous pathways. Transport of GSH and Cys conjugates of organic
compounds such as trichloroethylene into renal PT cells occurs by both
Na+-dependent pathways and the organic anion
transporter (Lash and Jones, 1985b; Lash and Anders, 1989). Conjugates
of Hg with GSH or Cys may mimic such conjugates and may be transported
into PT cells by the same pathways.
Another factor that should be taken into account is potential
competition for transport between the Hg-thiol conjugates and the
corresponding disulfides that may form from the thiols in solution due
to autoxidation. There is evidence that the dibasic amino acid carrier,
which transports cystine, may play a role in Hg-Cys transport across
the renal brush-border membrane (R.K. Zalups and D.W. Barfuss,
unpublished observations). This carrier, however, is a low-affinity
transporter. If all the unbound Cys was oxidized to cystine, then the
maximal concentration of cystine would be the same as that of Hg-Cys,
which suggests that competition would be minimal. Nevertheless, the
values for Hg accumulation with Hg-Cys may be underestimates because of
this potential competition. Similarly, GSSG uptake across renal plasma
membranes occurs by a low-affinity, low-capacity process (Lash and
Jones, 1984), suggesting that competition between GSSG and Hg-GSH for
transport would also be minimal.
It is critical to note that it is very unlikely that renal epithelial
cells will be exposed in vivo to free Hg ions. Rather, Hg
will be bound to a ligand (presumably albumin, GSH or Cys). Hence, the
most relevant comparisons of Hg accumulation values in the present
study are those among coexposures of renal cells to the various thiols
plus Hg. Considered in this light, then, the higher amounts of cellular
accumulation of Hg with exposures to Hg plus GSH or Hg plus Cys
compared with Hg plus BSA or Hg plus DMPS take on added significance
and provide stronger support for the hypothesis that complexes of Hg
with GSH or Cys are physiological transport forms of Hg in renal
epithelial cells.
Based on data from in vivo studies using ureteral ligation
(Zalups and Minor, 1995) and inhibition of renal GGT activity by acivicin pretreatment (de Ceaurriz et al., 1994; Tanaka
et al., 1990; Tanaka-Kagawa et al., 1993; Zalups,
1995) and in vitro studies using renal plasma membrane
vesicles (Zalups and Lash, 1997), transport of the putative Hg-Cys or
Hg-GSH complex can be localized to the brush-border membranes of PT
cells. Additionally, there is strong evidence for a role for the renal
organic anion transporter in the basolateral uptake of Hg (Zalups,
1995; Zalups and Barfuss, 1995c; Zalups and Minor, 1995). Hence, there
are clearly two mechanisms, one luminal and one basolateral, for the
renal cellular uptake and accumulation of Hg.
In conclusion, these studies have demonstrated that Hg is accumulated
by both isolated PT and DT cells and is differentially distributed into
soluble and protein-bound fractions. The hypothesis that extracellular
thiols significantly modulate cellular accumulation of Hg is supported,
confirming and extending findings with other in vitro
models. Furthermore, these studies show that Cys and/or GSH are
important physiological ligands for Hg that are involved in their
transport into renal cells.
BSA, bovine serum albumin;
Cys, cysteine;
DT, Distal tubular;
DMPS, 2,3-dimercapto-1-propanesulfonic
acid;
GGT, 
-glutamyltransferase;
GSH, glutathione;
Hg, inorganic
mercury;
PAH, p-aminohippurate;
PT, proximal tubular;
TCA, trichloroacetic acid.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
