Human acute renal failure (ARF) is often caused by ischemic and nephrotoxic insults, commonly acting in combination. Whereas many agents are protective in animal models of renal ischemic-reperfusion injury (I/R) (1), only a few agents inhibit renal injury in the widely used mercuric chloride (HgCl2) model of nephrotoxic ARF. The agents that have been demonstrated to reduce HgCl2-induced renal injury include DMSO (2), dimethylthiourea (DMTU) (2), N-acetylcysteine (NAc) (3), spironolactone (4), hepatocyte growth factor (HGF) (5,6), superoxide dismutase (SOD) (7), and melatonin (8). Because it is often difficult clinically to differentiate ischemic from nephrotoxic ARF, it would be helpful to have agents that work in both ischemic and toxic injury. The anti-inflammatory agents α-melanocyte stimulating hormone (α-MSH) and IL-10 and antibodies to intracellular adhesion molecule-1, lymphocyte function-associated antigen 1 (LFA-1), and TNF-α inhibit renal injury after ischemia but are ineffective as treatments for HgCl2-induced renal failure (9,10). This is possibly because the inflammatory phase of the nephrotoxic model occurs late, after the renal injury is established. Only a few agents, such as HGF, NAc, and DMSO (2,11–13), protect in both types of renal injury.
Recent work has focused on the role of free radicals and oxidants in the renal injury induced by HgCl2. For example, HgCl2 increases the production of many endogenous oxidants such as hydrogen peroxide (14), depletes protective antioxidants such as glutathione (GSH), and reduces free radical scavenging systems such as SOD and GSH peroxidase (GPx) (15). Indeed, HgCl2-induced injury can be ameliorated by SOD or the antioxidants NAc and melatonin in some (3,7,8) but not all (2) studies. DMSO is an aprotic solvent with anti-ischemic, anti-inflammatory, and antioxidant properties [reviewed by Santos et al. (16)]. DMSO protects rats against I/R injury (13,17) and nephrotoxic injury when administered before HgCl2 (2). Another hydroxyl radical scavenger, DMTU, protected the kidney when administered in a mixture with HgCl2 (2). Late DMSO administration protects against chemically induced liver and kidney injury (18). Indeed, DMSO protected against chloroform-induced ARF when administered 10 h after the toxicant. However, the mechanism of action of DMSO has been questioned as it has the ability to form complexes with Hg (2). This DMSO-Hg complex formation may prevent Hg from binding to protein sulfhydryl groups. Also, several metal-oil agents, such as 2,3-dimercapto-1-propane sulfonate (DMPS), dimercaptosuccinic acid (DMSA), and the low molecular weight thiol NAc inhibit the renal tubular uptake of mercuric ion and enhance urinary excretion (19,20). Therefore, it has been suggested that DMSO may inhibit Hg-induced renal injury by reducing the intracellular availability of free Hg rather than by scavenging free radicals.
In this study, we determined the window of opportunity for DMSO protection of renal function in a mouse model of HgCl2 injury and investigated potential mechanisms of protection. Because it has been suggested that DMSO may prevent uptake of HgCl2 into cells by binding HgCl2, the effect of DMSO on the uptake of Hg2+ into cultured renal cells was also studied.
Materials and Methods
Animals
Male BALB/c mice, aged 6 to 7 wk, were purchased from the National Institutes of Health (NIH). All animals had free access to water and chow (NIH-07 Rodent Chow; Zeigler Bros., Gardners, PA). Animal care followed the criteria of the National Institutes of Health for the care and use of laboratory animals in research.
Experimental Design
The animals were given HgCl2 (5 mg/kg in normal saline) by subcutaneous injection, and DMSO (3.8 ml/kg, 40% in PBS) or vehicle (PBS) was administered by intraperitoneal injection at 0 and 24 h. Other groups of mice received DMSO treatment whereby the first dose was delayed for 3 and 5 h after HgCl2 injection. Animals were killed at 12, 24, and 48 h after HgCl2 injection. Blood was collected for measurement of serum creatinine, and both kidneys were harvested for histologic and other studies.
Histologic Examination
Four percent paraformaldehyde-fixed and paraffin-embedded kidney specimens were stained with periodic acid-Schiff reagent or Naphthol AS-D chloracetate esterase (kit no. 91-C; Sigma, St. Louis, MO). Histologic changes in the outer stripe of outer medulla were evaluated by semiquantitative measurement of tissue damage. Tubular damage was estimated semiquantitatively in eight high-power fields per section by using a scoring system based on the percentage of damaged tubules (i.e., necrotic) per high-power field (1, < 25%; 2, 25 to 50%; 3, 50 to 75%; 4, >75%). The mean score of each animal was compared. Leukocyte infiltration was evaluated by counting the number of esterase-positive cells in eight high-power fields in the outer stripe of outer medulla.
Measurement of Tissue GSH Level, GPx, SOD, and Catalase Activity
Renal cellular GSH levels were determined using a GSH colorimetric assay kit (Calbiochem, La Jolla, CA). Kidney tissue was homogenized in cold 5% (wt/vol) metaphosphoric acid (Aldrich, Milwaukee, WI), centrifuged at 3000 × g for 10 min at 4°C, and the supernatant was assayed for GSH according to instructions provided with the kit. The amount of GSH was expressed as μmol/g kidney (wet wt). The sensitivity of the assay was 0.25 μmol/g kidney.
Kidney GPx activity was measured indirectly by using a GPx assay kit (Calbiochem, La Jolla, CA). Briefly, kidney tissues were homogenized in ice-cold buffer that contained 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 1 mM 2-mercaptoethanol and was centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was added to a solution that contained 1 mM GSH, 4 U/ml GSH reductase, and 0.2 mM NADPH. The reaction was initiated by the addition of 0.22 mM tert-butyl-hydroperoxide. GPx activity was then quantified by measuring the rate of change in the absorbance at 340 nm caused by the oxidation of NADPH. Enzyme activity was expressed as U/mg protein. The sensitivity of the assay was 90 U/mg protein.
Total SOD activities were determined by a SOD assay kit (Calbiochem) that is based on the SOD-mediated increase in the rate of auto-oxidation of 5,6, 6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene in aqueous alkaline solution. The activity of total SOD was determined from the ratio of the auto-oxidation rates measured in the presence and in the absence of SOD. The activities were expressed as U/mg protein. The sensitivity of the assay was 0.2 U/mg protein.
Catalase activity was determined by a catalase assay kit (Calbiochem). Briefly, supernatant from a tissue homogenate was incubated with 10 mM H2O2 for 1 min, and the amount of H2O2 that remained in the reaction mixture was determined by an oxidative coupling reaction of chromagen in the presence of H2O2. The change in absorbance was read at 520 nm. Catalase activities were expressed as U/mg protein. The sensitivity of the assay was 2 U/mg protein.
In Vitro 203Hg Uptake Study
To examine the effect of DMSO on the uptake of Hg2+ into tubular cells, 203Hg uptake in the presence of 1% DMSO, or low molecular thiol, we examined NAc (20 μM) in hOAT1-transfected MDCK cells. hOAT1-transfected MDCK cells were generated as described previously (21,22). 203Hg was a gift from Dr. Delon Barfuss (Georgia State University) and Dr. Rudolfs K. Zalups (Mercer University) and was produced as described previously (21,23). Cells were plated in 24-well culture plates (5 × 105 cells/well) in EMEM supplemented with 10% FBS and 1 mM pyruvate. After 2 d of growth in a humidified incubator (5% CO2/95% air, 37°C), 203Hg2+ transport was assessed. Briefly, cells were washed three times with Hanks’ buffered saline solution with 10 mM HEPES (pH 7.4; transport buffer), and then transport buffer that contained 5 microM 203Hg2+ with or without 1% DMSO or 20 μM NAc was added to each well. After 1 h, wells were rinsed with cold (4°C) stop buffer (transport buffer that contained 1 mM DMPS and 200 μM probenecid). One milliliter of 1 N NaOH was then added to each well to lyse the cells. A total of 700 μl of cell lysate was then removed from each well and neutralized with 700 μl of 1 N HCl. This lysate mixture was then added to 15 ml of EcoLume (ICN Biomedical, Cleveland, OH) scintillation fluid, and sample radioactivity was determined by scintillation counting with a Tri-Carb 2900 TR Liquid Scintillation Analyzer (Packard, Meridan, CT). Fifty microliters of the remaining cell lysate was processed for Bradford protein assay, and data were normalized to the protein concentration.
Statistical Analyses
All data were expressed as means ± SEM. Differences between experimental groups were examined by t test (SigmaStat), and P < 0.05 was considered statistically significant.
Results
Effect of DMSO in HgCl2-Induced Renal Injury
To evaluate the effect of DMSO in HgCl2-induced renal injury, we examined serum creatinine and histologic changes at 48 h after injection. As expected, HgCl2 administration increased the serum creatinine (normal, 0.3 ± 0.0, versus HgCl2 + vehicle, 2.1 ± 0.2 mg/dl). DMSO, when injected simultaneously with HgCl2, significantly decreased serum creatinine compared with vehicle-treated mice (DMSO, 0.7 ± 0.2 mg/dl; P < 0.05; Figure 1). Similarly, extensive tubular damage and necrosis in vehicle-treated mice significantly decreased in DMSO-treated mice (vehicle, 3.7 ± 0.0, versus DMSO, 2.1 ± 0.1; P < 0.05; Figure 2).
;)
Figure 1. :
Effect of DMSO on serum creatinine level at 48 h after HgCl2 injection. Mice received a subcutaneous injection of 5 mg/kg HgCl2 and vehicle (PBS; ▪) or DMSO (3.8 ml/kg, 40% in PBS; ) by intraperitoneal injection at 0, 3, and 5 h after HgCl2, and an additional dose of DMSO was given 24 h later. □, normal mice; n = 10 animals/group.
Figure 2. :
Renal histology 48 h after HgCl2 administration. Mice received a subcutaneous injection of 5 mg/kg HgCl2 and an intraperitoneal injection of vehicle (A) or DMSO at 0 (B), 3 (C), or 5 h (D) after HgCl2 injection. An additional dose of DMSO was given at 24 h.
Delayed DMSO Administration
To investigate the therapeutic window of DMSO treatment for HgCl2-induced renal injury, we examined the effect of delayed DMSO treatment 3 or 5 h after HgCl2 administration. DMSO given 3 h after HgCl2 attenuated renal damage (creatinine, 0.7 ± 0.2 mg/dl; tubular damage score, 2.2 ± 0.1; both P < 0.05 versus vehicle), but 5-h delayed DMSO treatment had no significant effect (creatinine, 2.0 ± 0.1 mg/dl; tubular damage score, 2.9 ± 0.2; Figures 1 through 3). In a separate experiment, we found that lower doses of DMSO (1.9 and 0.95 ml/kg) administered with a 3-h delay also had significantly lower serum creatinine values (vehicle, 3.3 ± 0.2, 1.9 ml/kg, 1.4 ± 0.3, P < 0.05 versus vehicle; 0.95 ml/kg, 1.2 ± 0.2 mg/dl, P < 0.05 versus vehicle).
Figure 3. :
Effect of DMSO on semiquantitative renal histology at 48 h after HgCl
2 injection. Mice received a subcutaneous injection of 5 mg/kg HgCl
2 and vehicle (▪) or DMSO (). Data are mean scores that correspond to the percentage of damaged tubules in eight high-power fields per section (see
Figure 2; Materials and Methods), *
P<0.05
versusvehicle;
n = 7 to 8 animals/group.
Effect of DMSO on Oxidative Stress
Previous studies have shown that oxidative stress plays a key role in HgCl2-induced renal injury (8). Therefore, the effect of DMSO on various parameters of oxidative stress was investigated. HgCl2 induced depletion of renal GSH content at 24 and 48 h after injection (Figure 4). In contrast, DMSO treatment significantly increased the intracellular GSH level at both time points (normal, 0.46 ± 0.07, versus HgCl2 + vehicle 0.28 ± 0.02, versus HgCl2 + DMSO 0.83 ± 0.02 at 24 h; HgCl2 + vehicle 0.29 ± 0.01, versus HgCl2 + DMSO 0.73 ± 0.02 μmol/g tissue at 48 h).
GPx activity also decreased at 48 h after HgCl2 injection, and this decrease was partially reversed in DMSO-treated animals (Figure 5). The difference between groups was not significantly different at 24 h (normal, 700 ± 154, versus HgCl2 + vehicle, 232 ± 1 9, versus HgCl2 + DMSO, 379 ± 59 U/mg protein) but was significantly different at 48 h (HgCl2 + vehicle, 179 ± 29, versus HgCl2 + DMSO, 357 ± 7 U/mg protein).
Figure 4. :
Effect of DMSO on renal GSH content. Mice received a subcutaneous injection of 5 mg/kg HgCl2 and vehicle (▪) or DMSO () intraperitoneally. Mice were killed at 0, 6, 24, or 48 h. Data are mean ± SEM. *P < 0.05 versus normal; #P < 0.05 versus vehicle; n = 4 animals/group.
Figure 5. :
Effect of DMSO on renal glutathione peroxidase (GPx) activity. Mice received a subcutaneous injection of 5 mg/kg HgCl
2 and vehicle (▪) or DMSO (). Mice were killed at 24 and 48 h. Data are displayed as in
Figure 4;
n = 4 animals/group.
Total SOD activity was also markedly decreased at 24 h after mercuric chloride injection, and DMSO treatment restored SOD activity at 24 h (normal, 0.67 ± 0.02, versus HgCl2 + vehicle, 0.3 ± 0.06, versus HgCl2 + DMSO, 0.72 ± 0.07 U/mg protein at 24 h). At 48 h, SOD activity was not significantly different from normal (Figure 6).
Figure 6. :
Effect of DMSO on renal superoxide dismutase (SOD) activity. Mice received a subcutaneous injection of 5 mg/kg HgCl
2 and vehicle (▪) or DMSO (). Mice were killed at 24 and 48 h. Data are displayed as in
Figure 4;
n = 3 animals/group.
Renal catalase activity was not different at any time point after HgCl2 injection, and DMSO had no effect on its level (normal, 50.2 ± 3.6, versus HgCl2 + vehicle, 47.0 ± 4.1, versus HgCl2 + DMSO, 60.3 ± 10.0 at 24 h; HgCl2 + vehicle, 47.9 ± 8.5, versus HgCl2 + DMSO, 61.2 ± 8.1 U/mg protein; Figure 7).
Figure 7. :
Effect of DMSO on renal catalase activity. Mice received a subcutaneous injection of 5 mg/kg HgCl
2 and vehicle (▪) or DMSO (). Mice were killed at 24 and 48 h. Data are displayed as in
Figure 4;
n = 3 animals/group.
203Hg Uptake in MDCK Cells
A previous study by Paller (2) suggested that the beneficial effect of DMSO in mercuric chloride-induced ARF could be mediated through its ability to complex with Hg2+, thus protecting protein sulfhydryl groups. Several metal complexing agents, such as DMPS, DMSA, and the low molecular weight thiol NAc, inhibit injury and are thought to additionally inhibit the cellular uptake of mercuric ion and enhance urinary excretion (19,20). To determine whether DMSO complexes with Hg2+ and thus prevents its uptake into tubular cells, we measured the uptake of 203Hg2+in vitro using hOAT1-transfected MDCK cells. Co-incubation of 203Hg2+ with 20 μM NAc markedly decreased Hg2+ uptake to 5.8% of control (Figure 8). In contrast, co-incubation with 1% DMSO did not affect significantly Hg2+ uptake, indicating that DMSO did not have an effect on the uptake of Hg2+ into tubular cells. (Hg2+, 100, versus Hg2+ + DMSO, 87 ± 5, versus Hg2+ + NAc, 5.8 ± 0.02%).
Figure 8. :
Effect of DMSO and N-acetylcysteine (NAc) on the uptake of 203Hg2+ in hOAT1-transfected MDCK cells. Cells were treated in transport buffer that contained 5 μM 203Hg2+ alone (control), with 1% DMSO or 20 μM NAc. After 1 h, cells were washed and radioactivity was measured. Data were expressed as the percentage of 203Hg2+ uptake compared with control. *P < 0.01 versus normal; n = 3 replicates/group.
Discussion
This study demonstrates that late administration of DMSO inhibits nephrotoxic injury caused by HgCl2 even when DMSO is given 3 h after HgCl2. Whereas HgCl2 decreases the levels of GSH, GPx, and SOD, DMSO restores the level of GPx and SOD toward normal and results in supranormal levels of GSH. DMSO has no effect on Hg entry into cultured renal tubule cells.
Protection and Window of Opportunity
Renal damage was assessed by both changes in creatinine and renal histology. DMSO dramatically decreases the rise in serum creatinine and also decreases the histologic damage (Figures 1 through 3). This protection is more effective than seen in previous studies. Paller (2) saw only a 60% protection in serum creatinine, despite giving DMSO 10 min before HgCl2. As all previous studies had given DMSO either before or at the same time as HgCl2, it was surprising to find a 3-h window of opportunity for DMSO to attenuate renal injury (Figures 1 through 3). Another antioxidant, melatonin, works only if administered 30 min before HgCl2; administration at the time or 30 min after Hg is ineffective (8). The results with DMSO are particularly striking because mercury enters cells rapidly after administration (24) and renal damage can be detected early. Therefore, when given 3 h after HgCl2 injection, DMSO may be acting after the initiation of renal cellular injury.
Binding and Sequestration of HgCl2
Some therapeutics, such as spironolactone, have been shown to bind HgCl2 in the circulation. The resulting stable complex is secreted by the liver into the bile, thus bypassing the kidney (4). Liver secretion of Hg is further enhanced by the ability of spironolactone to induce drug-metabolizing enzymes. Other agents, such as NAc and DMSA, also bind Hg and increase urinary excretion. However, this study demonstrates that delayed DMSO is still effective (Figures 1 through 3) and that DMSO had no effect on Hg uptake into renal tubule cells (Figure 8). In contrast, NAc reduced the Hg uptake into renal tubule cells, as described previously (21). These data suggest that DMSO does not act via blocking HgCl2 entry into cells, although we cannot eliminate the possibility that HgCl2 could enter proximal tubule cells in vivo through a non-OAT1 transporter. Once HgCl2 has entered cells, it is still possible that DMSO could bind to unreacted HgCl2 intracellularly.
Mechanism of Action of DMSO
DMSO has a potentially complex mechanism of action as it has anti-ischemic, anti-inflammatory, and antioxidant properties (16). For example, DMSO prevents induction of inflammatory cascades (e.g., activation of NF-κB), adhesion molecules (e.g., intracellular adhesion molecule-1), and cytokines (e.g., IL-8). Previous studies of anti-inflammatory drugs (e.g., α-MSH, IL-10) failed to confer renal protection in animal models of mercury injury. This suggests that the mechanism of action of DMSO may be due to its antioxidant properties.
GSH represents an important cellular defense system against oxidative stress. Gstraunthaler et al. (25) showed that mercury caused oxidative damage in renal proximal tubule cells, which they demonstrated by a decrease in GSH content. The pathophysiologic importance of reductions in GSH was demonstrated by Girardi and Elias (26), who showed that modulation of tissue nonprotein sulfhydryl levels and GSH paralleled renal injury. Thus, reduction of GSH levels with diethylmaleate increased renal injury, whereas increasing GSH levels with NAc decreased renal injury. The data presented in this article confirm that HgCl2 decreases GSH levels and demonstrate that DMSO prevents the decrease in GSH levels (Figure 4).
The effect of DMSO on GPx was also studied, because this enzyme consumes free peroxides in the cell by oxidizing GSH. Some studies have demonstrated that HgCl2 inhibits the activity of renal GPx in vivo in a selenium-dependent manner (27,28). We found that HgCl2 decreased GPx levels, although the decrease was statistically significant only at 48 h (Figure 5). DMSO administration restored GPx levels toward normal.
Reactive oxygen species can be destroyed by SOD and catalase. Twenty-four hours after administration, HgCl2 administration significantly decreased (∼50%) kidney SOD activity, which was completely restored to normal by DMSO (Figure 6). Thus, DMSO reverses the harmful effects of HgCl2 on SOD, one of the major pathways for ROS destruction. Because approximately three fourths of the SOD activity in the mouse kidney is Cu,Zn SOD (29), this isoform accounts for at least part of the inhibition by HgCl2. Mn SOD activity is also inhibited in mice that receive an injection of HgCl2, which may act directly on the enzyme (30). Although HgCl2 decreases SOD activity after 24 h, by 48 h the SOD activity returns to normal levels. This restoration of SOD activity may be a result of induction of Mn SOD, which is increased in response to HgCl2 in the brain by a posttranscriptional and dexamethasone-sensitive mechanism (31). The importance of SOD in HgCl2-induced renal injury is supported by exogenous SOD treatment, which results in protection from HgCl2. In contrast, HgCl2 had no effect on catalase activity (Figure 7), in agreement with the findings of previous studies (32).
The ability of HgCl2 to impair antioxidant defense, as reflected in decreased GSH, GPx, and SOD, suggests that it causes renal injury via free radical damage. It has been suggested that HgCl2 binds to protein sulfhydryl groups and inhibits enzyme function, leading to oxidative stress in the renal cells (2). A recent study suggested a role for nitric oxide in HgCl2 nephrotoxicity in rats. The level of mRNA for inducible nitric oxide synthase (iNOS) was increased in the renal cortex and proximal tubule epithelium after HgCl2 exposure. The selective iNOS inhibitor aminoguanidine modestly reduced proximal tubule injury and the rise in creatinine induced by HgCl2 (33). Therefore, the oxidative stress induced by HgCl2 may be due, at least in part, to excess nitric oxide production from iNOS. That the antioxidant DMSO antagonizes the nephrotoxicity induced by HgCl2 further supports the hypothesis that HgCl2 is nephrotoxic as a result of free radical generation. DMSO does not affect entry of HgCl2 into cells in vitro, but the possibility that DMSO forms a complex with Hg intracellularly still exists. The formation of the complex may inhibit the ability of Hg to produce cellular toxicity.
In summary, treatment or, more critically, delayed treatment with DMSO markedly decreased inorganic mercury-induced nephrotoxicity with a 3-h window of opportunity, which is much wider than any other reported agent. Because of effects on GSH, GPx, and SOD, the mechanism of action of DMSO could be related to its antioxidant properties.
See related editorial, “Need to Intervene in Established Acute Renal Failure,” on pages 2756–2758.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Environmental Health Sciences.
203Hg was kindly provided by Dr. Delon Barfuss (Georgia State University) and Dr. Rudolfs K. Zalups (Mercer University).
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