Delon W. Barfuss

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

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

Pretreatment with p-aminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat.

The effects of intravenous pretreatment with the organic anion p-aminohippurate (PAH) on the disposition of intravenously administered inorganic mercury in the kidneys, liver and blood were evaluated in rats. In dose-response experiments, the renal uptake (and/or accumulation) of mercury, 1 h after the injection of a nontoxic 0.5 mumol/kg dose of mercuric chloride (HgCl2), was significantly reduced in rats when a 1.0, 3.3 or 10 mmol/kg dose of PAH was administered 5 min prior to the injection of HgCl2. This reduction was due to reduced uptake of mercury in both the renal cortex and outer stripe of the outer medulla. Near maximal inhibition appeared to be achieved with the 10 mmol/kg dose of PAH. Inhibition of the uptake (an/or accumulation) of mercury in the renal cortex and outer stripe of the outer medulla, 1 h after the injection of the nontoxic dose of HgCl2, was also detected in experiments where HgCl2 was injected 5, 30, 60 or 180 min after pretreatment with a 10 mmol/kg dose of PAH. The renal uptake of mercury was inhibited significantly when the nontoxic dose of inorganic mercury was administered 5, 30, or 60, but not 180 min after pretreatment with the 10 mmol/kg dose of PAH. In another experiment, the renal burden of mercury was significantly reduced for 24 h when pretreatment with a 10 mmol/kg dose of PAH was administered 5 min prior to the injection of HgCl2. Pretreatment with PAH did not have an effect on the hepatic disposition of mercury, but it did cause a significant increase in the fraction of mercury present in the plasma of blood. In summary, the findings in the present study indicate that pretreatment with PAH inhibits the renal uptake of injected inorganic mercury in a dose-dependent and time-dependent manner. In addition, the findings tend to indicate that some fraction of the mercury that enters into renal tubular epithelial cells is by a mechanism involving the organic anion transport system.

Nephrotoxicity of inorganic mercury co-administered with l-cysteine

In the present study, we tested the hypothesis that co-administration of low nephrotoxic doses of inorganic mercury (Hg++) with L-cysteine (in a 1:2 mol ratio of inorganic mercury to L-cysteine), alters significantly the nephropathy induced by inorganic mercury. In the first experiment, the effect of co-administering L-cysteine on the nephropathy induced by a 1.8 or 2.0 micromol/kg dose of inorganic mercury was evaluated in rats 24 h after the administration of inorganic mercury. According to histopathological assessment of sections of kidney and evaluation of the urinary excretion of lactate dehydrogenase, total protein and inorganic mercury (which were used as indices of renal injury), the severity of renal injury in rats co-administered the L-cysteine with the inorganic mercury was significantly greater than that in corresponding rats injected with only inorganic mercury. In a second experiment, the disposition of mercury was evaluated 1 h after the administration of 1.8 micromol inorganic mercury/kg with or without 3.6 micromol L-cysteine/kg. The renal accumulation of mercury, specifically in the cortex and outer stripe of the outer medulla, was significantly greater the rats co-administered the inorganic mercury and L-cysteine than in the rats given only inorganic mercury. In addition, the content of mercury in the blood and liver was significantly lower, and the fraction of mercury in the blood present in the plasma was significantly greater, in the rats co-administered inorganic mercury and L-cysteine than in the rats given only inorganic mercury. On the basis of the findings from this study, the nephropathy induced by low nephrotoxic doses of inorganic mercury is made more severe when the inorganic mercury is co-administered in a 1:2 mol ratio with L-cysteine. Moreover, it appears that the enhanced severity in the nephropathy induced by the co-administration of inorganic mercury and L-cysteine is linked to an increase in the tubular uptake of mercury in the cortex and outer stripe of the outer medulla.

Mercury-metallothionein and the renal accumulation and handling of mercury

In the present study, we evaluated the renal and hepatic accumulation of mercury, the intrarenal distribution of mercury and the urinary and fecal excretion of mercury in rats injected intravenously with a non-toxic 0.1 mumol/kg-dose of mercury in the form of mercuric chloride (HgCl2) or a complex of mercury-metallothionein (Hg-MT). Between 6 and 72 h after injection, the concentration of mercury in the kidneys of the rats injected with Hg-MT was significantly greater than that in the rats injected with HgCl2. The greatest difference in the renal concentration of mercury between the two groups of rats was detected 6 h after injection. In the kidneys of both experimental groups of rats, the cortex and the outer stripe of the outer medulla contained the highest concentrations of mercury, with the greatest concentrations found in the renal cortex and outer stripe of the outer medulla of the rats injected with Hg-MT. No differences were found between the two experimental groups with respect to the concentration of mercury in the renal inner stripe of the outer medulla and inner medulla throughout 72 h of study. The content of mercury in the blood and liver decreased over time in both groups of rats, but was always significantly greater in the blood and liver of rats injected with HgCl2. The rats injected with Hg-MT excreted more than eight times the amount of mercury in the urine than the corresponding rats injected with HgCl2 during 72 h. These data indicate that there may be decreased tubular reabsorption of filtered Hg-MT and/or tubular secretion of mercury in the rats injected with Hg-MT. In contrast, the rats injected with HgCl2 excreted significantly more mercury in the feces during the same period of time than the corresponding rats injected with Hg-MT. In conclusion, our data clearly indicate that the renal and hepatic uptake and accumulation of mercury, and the urinary and fecal excretion of mercury, are altered significantly when inorganic mercury is administered intravenously as a complex with metallothionein.

Relationships between alterations in glutathione metabolism and the disposition of inorganic mercury in rats: effects of biliary ligation and chemically induced modulation of glutathione status.

Influences of biliary ligation and systemic depletion of glutathione (GSH) or modulation of GSH status on the disposition of a low, non-nephrotoxic i.v. dose of inorganic mercury were evaluated in rats in the present study. Renal and hepatic disposition, and the urinary and fecal excretion, of inorganic mercury were assessed 24 h after the injection of a 0.5-micromol/kg dose of mercuric chloride in control rats and rats pretreated with acivicin (two 10-mg/kg i.p. doses in 2 ml/kg normal saline, 90 min apart, 60 min before mercuric chloride), buthionine sulfoximine (BSO; 2 mmol/kg i.v. in 4 ml/kg normal saline, 2 h before mercuric chloride) or diethylmaleate (DEM; 3.37 mmol/kg i.p. in 2 ml/kg corn oil, 2 h before mercuric chloride) that either underwent or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except in theacivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. The present findings indicate that biliary ligation combined with methods used to modulate GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. Additionally, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepato-biliary system.

Altered intrarenal accumulation of mercury in uninephrectomized rats treated with methylmercury chloride.

We tested the hypothesis that the intrarenal accumulation of mercury in rats treated with methylmercury is altered significantly as a result of unilateral nephrectomy and compensatory renal growth. Renal accumulation of mercury was evaluated by radioisotopic techniques in both uninephrectomized (NPX) and sham-operated (SO) rats 1, 2, and 7 days after the animals received a nonnephrotoxic intravenous dose of methylmercury chloride (5 mg/kg Hg). At all times studied after the injection of the dose of methylmercury, the renal accumulation of mercury (on a per gram kidney basis) was significantly greater in the NPX rats than that in the SO rats. The increased accumulation was due to a specific increase in the accumulation of mercury in the outer stripe of the outer medulla. Renal cortical accumulation of mercury was similar in both the NPX and SO rats. The percentage of the administered dose of mercury that was present in the total renal mass of the NPX and SO rats ranged between 5 and 15, depending on the day that the renal accumulation was studied. Approximately 40-50% of the total renal burden of mercury in both the NPX and SO rats was in the inorganic form. However, only less than 1% of the mercury in blood was in the inorganic form at the three times accumulation was studied. Very little mercury was excreted in the urine by either the NPX or SO rats. Only about 2 to 3% of the administered dose of mercury was excreted in the urine in 7 days. By contrast, the cumulative fecal excretion of mercury over 7 days was substantial in the NPX and SO rats, and significantly more mercury was excreted in the feces by the NPX rats (about 19% of the dose) than by that in the SO rats (about 16% of the dose). In conclusion, our findings indicate that unilateral nephrectomy and compensatory renal growth cause a significant increase in the accumulation of mercury in the renal outer stripe of the outer medulla in rats exposed to methylmercury. In addition, the findings indicate that the fecal excretion of mercury is also significantly increased.

Potential mechanisms involved in the absorptive transport of cadmium in isolated perfused rabbit renal proximal tubules

UNLABELLED Lumen-to-cell transport, cellular accumulation, and toxicity of cadmium as ionic cadmium (Cd(2+)) or as the L-cysteine (Cys) or D,L-homocysteine (Hcy) S-conjugate of cadmium (Cys-S-Cd-S-Cys, Hcy-S-Cd-S-Hcy) were studied in isolated, perfused rabbit proximal tubular segments. When Cd(2+) (0.73 microM) or Cys-S-Cd-S-Cys (0.73 microM) was perfused through the lumen of S(2) segments of the proximal tubule, no visual evidence of cellular pathological changes was detected during 30 min of study. Cd(2+)-transport was temperature-dependent and was inhibited by Fe(2+), Zn(2+), and elevated concentrations of Ca(2+). Luminal uptake of Cys-S-Cd-S-Cys was also temperature-dependent and was inhibited by the amino acids L-cystine and L-arginine, while stimulated by L-methionine. Neither L-aspartate, L-glutamate, the synthetic dipeptide, Gly-Sar nor Zn(2+) had any effect on the rate of Cys-S-Cd-S-Cys transport. CONCLUSIONS When delivered to the luminal compartment, Cd(2+) appears to be capable of utilizing certain transporter(s) of Zn(2+) and some transport systems sensitive to Ca(2+) and Fe(2+). In addition, Cys-S-Cd-S-Cys and Hcy-S-Cd-S-Hcy appear to be transportable substrates of one or more amino acid transporters participating in luminal absorption of the amino acid L-cystine (such as system b(0,+)). These findings indicate that multiple mechanisms could be involved in the luminal absorption of cadmium (Cd) in proximal tubular segments depending on its form. These findings provide a focus for future studies of Cd absorption in the proximal tubule.

Intrarenal distribution of inorganic mercury and albumin after coadministration.

The renal disposition and the intrarenal distribution of albumin and mercury were studied simultaneously in rats co-injected with a 0.5-mumol/kg dose of albumin and a 0.25-mumol/kg dose of inorganic mercury at 2, 5, 30, and 180 min after injection. These studies were carried out to test the hypothesis that one of the mechanisms involved in the renal tubular uptake of inorganic mercury is cotransport with albumin. By the end of the first 2 min after injection, the ratio of inorganic mercury to albumin in the renal cortex and outer stripe of the outer medulla was approximately 2.6 and 1.6, respectively. Both the cortex and outer stripe contain segments of the proximal tubule, and it is these segments that have been shown to be principally involved in the renal tubular uptake of both albumin and inorganic mercury. The ratio increased slightly in these two zones after 5 and 20 min after injection. These data demonstrate that there is a relatively close relationship in the renal content of inorganic mercury and albumin during the early minutes after coinjection of inorganic mercury and albumin. However, the ratios are significantly greater than the ratio of inorganic mercury to albumin in the injection solution, which was 0.5. After 180 min following co-injection, the ratio increased to about 38 in the cortex and 15 in the outer stripe. This increase in the ratio is probably related to the metabolism of albumin. Based on the ratios of inorganic mercury to albumin in the renal cortex and outer stripe of the outer medulla, it appears that some proximal tubular uptake of inorganic mercury occurs by mechanisms other than endocytotic cotransport of inorganic mercury with albumin. However, since the ratios were small during the early times after injection, cotransport of inorganic mercury with albumin cannot be excluded as one of the mechanisms involved in the proximal tubular uptake of inorganic mercury.

Lack of luminal or basolateral uptake and transepithelial transport of mercury in isolated perfused proximal tubules exposed to mercury‐metallothionein

The lumen-to-bath and bath-to-lumen transport, cellular uptake, and toxicity of inorganic mercury bound to metallothionein (203Hg-MT) were studied in isolated perfused S1, S2, and S3 segments of the renal proximal tubule of rabbits. Evidence of very mild toxicity was displayed in some of the segments perfused through the lumen with 18.4 microM inorganic mercury in the form of Hg-MT. The toxic response was restricted primarily to mild swelling of the epithelial cells localized at the end of the tubular segments where the perfusion pipette was inserted into the lumen. The cells in the proximal portions of perfused S2 segments appeared to be most severely affected in that a few blebs would on occasion come off the epithelial cells. Mild cellular swelling was also observed in some S2 and S3 segments that were exposed to 18.4 microM inorganic mercury in the form of Hg-MT in the bath. The swelling was more generalized, involving all the epithelial cells along the perfused segment. Very little, or no, measurable lumen-to-bath or bath-to-lumen transport of Hg as Hg-MT could be detected in any of the 3 perfused segments of the proximal tubule during 40-45 min of perfusion. The complex of Hg-MT appeared to behave in a manner similar to that of the volume marker [3H]-L-glucose. The lack of tubular transport of Hg as Hg-MT was confirmed by little or no measurable uptake and accumulation of inorganic mercury in the tubular epithelial cells. Thus, our findings indicate that the Hg-MT complex is not taken up avidly in isolated perfused S1, S2, or S3 segments of the proximal tubule.

LUMINAL TRANSPORT OF THIOL S-CONJUGATES OF METHYLMERCURY IN ISOLATED PERFUSED RABBIT RENAL PROXIMAL TUBULES

Lumen-to-cell transport, cellular accumulation, and toxicity of L-cysteine (Cys), glutathione (GSH) and N-acetylcysteine (NAC) S-conjugates of methylmercury (CH(3)Hg(+)) were evaluated in isolated, perfused rabbit proximal tubular segments. When these conjugates were perfused individually through the lumen of S(2) segments of the proximal tubule it was found that Cys-S-CH(3)Hg and GSH-S-CH(3)Hg were transported avidly, while NAC-S-CH(3)Hg was transported minimally. In addition, 95% of the (203)Hg taken up by the tubular cells was associated with precipitable proteins of the tubule, while very little was found in the acid-soluble cytosol. No visual cellular pathological changes were observed during 30min of study. Luminal uptake of Cys-S-CH(3)Hg was temperature-dependent and inhibited significantly by the amino acids L-methionine and l-cystine. Rates of luminal uptake of GSH-S-CH(3)Hg were twice as great as that of Cys-S-CH(3)Hg and uptake was inhibited significantly (74%) by the presence of acivicin. When 2,3-bis(sulfanyl)propane-1-sulfonate (DMPS) was added to the bathing or luminal fluid, luminal uptake of Cys-S-CH(3)Hg was diminished significantly. Overall, our data indicate that Cys-S-CH(3)Hg is likely a transportable substrate of one or more amino acid transporters (such as system B(0,+) and system b(0,+)) involved in luminal absorption of L-methionine and L-cystine along the renal proximal tubule. In addition, GSH-S-CH(3)Hg appears to be degraded enzymatically to Cys-S-CH(3)Hg, which can then be taken up at the luminal membrane. By contrast NAC-S-CH(3)Hg and Cys-S-CH(3)Hg (in the presence of DMPS) are not taken up avidly at the luminal membrane of proximal tubular cells, thus promoting the excretion of CH(3)Hg(+) into the urine.