Role of Tissue Repair in Survival from S-(1,2-Dichlorovinyl)-L-Cysteine–Induced Acute Renal Tubular Necrosis in the Mouse

Vishal S. Vaidya*, Kartik Shankar*, Edward A. Lock{dagger}, Thomas J. Bucci{ddagger} and Harihara M. Mehendale*,1

* Department of Toxicology, School of Pharmacy, The University of Louisiana at Monroe, 700 University Avenue, Sugar Hall, Room 306, Monroe, Louisiana 71209-0470; {dagger} Syngenta, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK 104TJ, United Kingdom; and {ddagger} Pathology Associates International, National Center for Toxicological Research, Jefferson, Arkansas 72079

Received February 3, 2003; accepted April 9, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a model nephrotoxicant in mice, causes acute tubular necrosis and death at high doses. Our earlier studies revealed that renal tissue repair was critical for survival in mice with DCVC nephrotoxicity. The objective of this study was to investigate if increasing renal tissue repair could protect mice from the lethal outcome of DCVC. Male Swiss Webster (SW) mice were administered a low dose of DCVC (15 mg/kg, ip) 72 h before injection of a normally lethal dose of DCVC (75 mg/kg, ip); this resulted in 100% protection against the lethal effect of DCVC. Because DCVC caused ~twofold decrease in cytosolic and mitochondrial ß-lyase activity, the possibility that DCVC protection may be caused by decreased bioactivation was examined. Mercuric chloride (HgCl2, 6 mg/kg), a nephrotoxicant with no effect on ß-lyase activity, was administered 96 h before a lethal dose of DCVC. This also resulted in 100% protection from the lethal effect of DCVC. In both studies total glutathione was unchanged at any time after the lethal dose of DCVC was administered, obviating the role of glutathione in protection. In both cases the augmented and sustained tissue repair induced by priming dose and documented by 3H-thymidine pulse labeling and immunocytochemistry for proliferating cell nuclear antigen resulted in 100% survival in spite of the extensive renal injury. These findings suggest that stimulation of renal tubular repair by the priming dose, through augmented cell division, and the resistance of new cells to mechanisms of progression of injury, underlies auto- and heteroprotection against DCVC. The molecular mechanisms may have potential application in pharmacotherapeutic intervention for treatment of acute renal failure.

Key Words: ß-lyase; DCVC; mercuric chloride; protection; renal injury; tissue repair.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protection afforded by prior administration of a small dose of a chemical against the toxic and lethal effects of a high dose of the same chemical is termed "autoprotection." The protection afforded by pretreatment with one compound against the lethal toxicity of a different compound is termed "heteroprotection."

Augmented and sustained cell proliferation, tissue repair, and resiliency of newly divided cells induced by protective low dose have been established as the mechanism of autoprotection in liver, lung, and hematotoxicity models (Barton et al., 2000Go; Mangipudy et al., 1995Go; Mehendale et al., 1994Go; Sivarao and Mehendale, 1995Go). Although auto- and heteroprotection have not been demonstrated in kidney using survival as the endpoint, existing literature reports suggest that this phenomenon may also exist for renal toxicants. In a study by Oken et al.(1975)Go, rats recovering from glycerol-induced acute renal failure (ARF) were protected from mercury-induced nephropathy. However, the hypothesized mechanism of renal renin depletion was found not to be responsible for the observed effect. Another study showed protection against nephrotoxic effects of HgCl2 by pretreatment with a small or nephrotoxic dose of mercuric chloride (Yoshikawa, 1970Go) or cadmium (Magos et al., 1974Go). The possible mechanisms for protection in this model were suggested to be involvement of metallothionein and delayed renal uptake of mercury. These possibilities were ruled out by Tandon et al.(1980)Go, who showed that pretreatment with sodium chromate, p-aminophenol, sodium maleate, and uranyl acetate (nephrotoxic agents that do not induce metallothionein) also afforded protection from HgCl2-induced nephrotoxicity. Pretreatment by lead nitrate has also been shown to reduce the nephrotoxic effects of HgCl2 in female mice (Ewald and Calabrese, 2001Go). In all of these studies, only renal injury was measured, and none of the studies investigated if animals receiving an ordinarily lethal dose of nephrotoxicant (HgCl2) could survive if pretreated with glycerol, cadmium, low dose of HgCl2, or lead nitrate. Only renal injury was measured at selected time points. The role of renal regeneration was postulated as the likely mechanism behind protection without providing any experimental evidence. Our studies with DCVC have also suggested that renal tissue repair is an important response to enable recovery from acute renal failure observed in male Swiss Webster (SW) mice (Vaidya et al., 2003Go). However, the mechanisms of protection remain obscure, and whether protection can be seen as survival from a lethal dose remains uninvestigated. A greater understanding of the mechanisms of protection afforded by tissue responses to injury may lead to strategies for application of these responses therapeutically to prevent/overcome nephrotoxic injury and possibly to avert death due to ARF.

The acute toxicity of DCVC is well characterized in various animal models including young calves, dogs, cats, rabbits, guinea pigs, turkeys, rats, and mice (Anders et al., 1987Go; Eyre et al., 1995Go; Hassall et al., 1983Go; Krejci et al., 1991Go; Terracini and Parker, 1965Go). With the possible exception of young calves, the primary target for DCVC is proximal tubules in the kidney (McKinney et al., 1959Go). DCVC, a metabolite of trichloroethylene (TCE) is a halogenated hydrocarbon, which selectively affects the proximal straight tubules (PST) following in vivo and in vitro exposure (Darnerud et al., 1988Go; Wolfgang et al., 1989Go). GSH conjugation of TCE has been demonstrated in vivo in rats and humans exposed to TCE by detection of two mercapturic acids of TCE in urine (Dekant et al., 1986Go, 1990Go). Cell-death is initiated by covalent binding of a sulfur-containing fragment of DCVC to cellular macromolecules (Stevens et al., 1986Go). The sulfur-containing fragment of DCVC arises from the bioactivation of DCVC by the renal enzyme, cysteine conjugate ß-lyase.

The primary aim of the present study was to determine whether a priming dose of DCVC or HgCl2 affords protection against a lethal dose of DCVC. We report here that a low priming dose of either chemical provides protection against a subsequently administered lethal dose of DCVC. Protection is characterized by (1) optimal time interval between the priming dose and the lethal dose suggesting that protection is unlikely to be due to chemical interaction, (2) restoration of renal function and structure, (3) a need for prompt and sustained renal tubular repair, and (4) lack or adequacy of any effect the priming dose may have on the activity of renal cysteine conjugate ß-lyase and GSH content of the kidney.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male SW mice (25–29 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and were maintained in our central animal facility under conditions of 21 ± 1°C and 50–80% relative humidity at all times in a 12 h light-dark cycle over wood chips free of any known chemical contaminants. The mice received commercial rodent chow (Teklad rodent diet # 7012) and water ad libitum and they were acclimated for one week prior to use. All animal maintenance and treatment protocols were in compliance with the Guide for Care and Use of Laboratory animals as adopted and promulgated by the U.S. National Institutes of Health and were approved by our Institutional Animal Care and Use Committee (IACUC).

Chemicals.
Unless stated otherwise, all chemicals and biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). DCVC was provided by Syngenta, Central Toxicology Laboratory, Macclesfield, Cheshire, U.K. and was >99.5% pure. Diethyl ether was purchased from EM Science (Gibbstown, NJ). Thymidine [3H-CH3] (3H-T, specific activity 2 Ci/mmol) was obtained from Moravek Biochemicals, Inc. (Brea, CA). The scintillation fluid (Scintiverse SX 1604) was purchased from Fisher Scientific (Pittsburgh, PA). All chemicals were highest quality available.

Toxicity Studies in "Autoprotection Model"
Mortality study.
Mice were divided into seven groups (n = 10 each). Group 1 (controls) received distilled water (10 ml/kg ip). Groups 2 and 3 were injected intraperitoneally (ip) with 15 and 75 mg DCVC/kg, respectively, dissolved in distilled water (10 ml/kg). Mice in groups 4, 5, 6, and 7 were injected ip with 15 mg DCVC/kg in distilled water (10 ml/kg) on day 0 and the same mice were injected with 75 mg DCVC/kg on days 1, 2, 3, and 4, respectively, after the injection of priming dose of DCVC. Survival/mortality were observed and recorded twice daily for 14 days.

Time-course of renal injury and repair in autoprotection.
Male Swiss Webster (SW) mice (n = 4 in each) were divided into three groups. Group 1 received 15 mg DCVC/kg ip in distilled water (10 ml/kg) on day 0 and the same mice received 10 ml distilled water (DW)/kg ip on day 3. Mice in group 2 were injected with 10 ml DW/kg ip on day 0 and 75 mg DCVC/kg ip on day 3. Group 3, the autoprotected group of mice, were treated with 15 mg DCVC/kg on day 0 and 75 mg DCVC/kg ip on day 3. Two h prior to termination the mice were given a single injection of 3H-T (50 µCi/mouse, ip). Mice (n = 4) were terminated under diethyl ether anesthesia at various time points from 0 to 13 days after the respective treatments. Blood was collected retroorbitally in heparinized tubes for blood urea nitrogen (BUN) estimation. Both the kidneys were taken for estimation of 3H-T S-phase stimulation measured as an index of renal tissue repair. Ninety percent of the mice in group 2 receiving 10 ml DW/kg ip on day 0 and 75 mg DCVC/kg ip on day 3 died between 4.5 to 5 days after the priming dose injection and therefore all the results from this group are recorded only until 4.5 days.

Another study was designed to obtain kidney sections for hematoxylin-eosin (H&E) staining and proliferating cell nuclear antigen (PCNA) assay. Male SW mice (n = 3 per time point) were divided into the same three groups as described above and each group received same treatments as described earlier. Mice from group I were terminated under diethyl ether anesthesia at 12, 24, 36, 48, 72 h, 5 and 13 days after the priming dose administration. Mice in group 2 (n = 3 per time point) were terminated under diethyl ether anesthesia at 3.5, 4, and 4.5 days after the priming dose (DW) administration. Ninety percent of the mice in this group died between 4.5 and 5 days and therefore all the results from this group are recorded only until 4.5 days. Mice in group 3 (n = 3 per time point) were terminated under diethyl ether anesthesia at 3.5, 4, 4.5, 5, 6, 8, and 13 days following the priming dose administration. The kidneys were surgically removed from mice under diethyl ether anesthesia, fixed, etc. and processed for histopathological examination and PCNA measurements (see later).

Toxicity Studies in "Heteroprotection Model"
Mortality study.
Mice were divided into five groups (n = 10 each; groups 8 to 12 of Table 1Go). Group 8 mice were injected ip with 6 mg HgCl2/kg in DW (10 ml/kg). Mice in groups 9, 10, 11, and 12 were injected ip with 6 mg HgCl2/kg in distilled water (10 ml/kg) on day 0 and the same mice were injected with 75 mg DCVC/kg on days 1, 2, 3, and 4, respectively, after injection of the priming dose of HgCl2. Survival/mortality were observed and recorded twice daily for 14 days.


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TABLE 1 Protection from Mortality of S-1,2-Dichlorovinyl-L-Cysteine (DCVC) as a Function of Time between the Priming and Lethal Doses in the Auto- and Heteroprotection Models
 
Time-course of renal injury and repair.
Mice were injected ip with 6 mg HgCl2/kg in DW (10 ml/kg) or same volume of DW alone. Another group of mice were administered with 6 mg HgCl2/kg and the same mice received 75 mg DCVC/kg on day 4 in DW (10 ml/kg). Two h prior to termination the mice were given a single injection of 3H-T (50 µCi/mouse, ip). Mice (n = 4) were terminated under diethyl ether anesthesia from 0 to 14 days after the respective treatments. Blood was collected retroorbitally in heparinized tubes for BUN estimation. Both the kidneys were taken for quantitative estimation of 3H-T as an index of renal tissue repair. Ninety percent of the mice in group receiving 10 ml DW/kg ip on day 0 and 75 mg DCVC/kg ip on day 3 died between 4.5 to 5 days after the priming dose injection and therefore all the results from this group are recorded only until 4.5 days.

Another study was designed to obtain kidney sections for H & E staining and PCNA assay. Male SW mice (n = 3) were injected ip with 6 mg HgCl2/kg and were terminated under diethyl ether anesthesia at 0 h, days 1, 2, 3, 5, and 10. Another group of mice (n = 3) were injected with 6 mg HgCl2/kg + 75 mg DCVC/kg on day 4 in DW (10 ml/kg) and were terminated under diethyl ether anesthesia on days 4, 5, 6, 7, 9, and 14. The kidneys were surgically removed from mice under diethyl ether anesthesia, fixed, etc. and processed for histopathological examination and PCNA measurements (see later).

Plasma enzymes.
Plasma was separated by centrifugation. BUN (Sigma cat. # 63–25; procedure # 63-UV) was measured as a biomarker for renal failure and damage using a commercially available kit from Sigma Chemical Co. (St. Louis, MO).

3H-T pulse labeling study.
S-phase DNA synthesis was estimated by 3H-T incorporation into the renal nuclear DNA as described by Chang and Looney (1965)Go. Because increased 3H-T incorporation in DNA may also occur due to DNA repair rather than S-phase DNA synthesis, data obtained through these pulse-labeling studies were confirmed by proliferating cell nuclear antigen analysis (PCNA, see below). Both kidneys from each mouse were removed, minced using scissors, and homogenized in ice-cold 2.2 M sucrose (10 ml) using a Teflon homogenizer. The homogenate was further centrifuged at 40,000 x g for 1 h to isolate the nuclei by differential cetrifugation. The isolated nuclei were suspended in ice-cold 0.25 mM sucrose (1 ml), mixed in tubes on ice with 0.5 ml of 0.6 N perchloric acid (PCA), and centrifuged at 10,000 x g for 20 min. The pellets were suspended in 0.2 N PCA (2 ml), centrifuged, and the washing with 0.2 N PCA was repeated one more time. The pellets were suspended in 0.5 N PCA (6 ml), heated at 70–80°C for 20 min, and centrifuged at 10,000 x g for 20 min. DNA and tritiated thymidine (3H-T) were estimated in the supernatant. DNA was determined by the diphenylamine reaction as described by Burton (1956)Go using calf thymus DNA as standard. 3H-T incorporated in DNA was determined by liquid scintillation analyzer (Packard Instrument Co., Meriden, CT).

Urinalysis.
Mice were divided into five groups (n = 6 each) and injected ip with 15 mg DCVC/kg, 75 mg DCVC/kg, 15 + 75 mg DCVC/kg three days apart, 6 mg HgCl2/kg, or 6 mg HgCl2/kg + 75 mg DCVC/kg four days apart, respectively, in DW (10 ml/kg). Controls received only the same volume of DW. Urine samples were collected from control and treated mice with three mice kept in individual Nalgene® mouse metabolic cages (Fisher Scientific, Pittsburgh, PA). Urine was collected daily and stored at -80°C until analysis. Urine volume (expressed as ml/mouse/day), pH, and specific gravity were measured by conventional methods at various time points (0 to 10 days). Urine glucose (cat. # 17 UV; expressed as mg/dl) was measured using a commercially available kit from Sigma Chemical Company.

ß-Lyase estimation.
Male SW mice were injected ip with 15 mg DCVC/kg, 75 mg DCVC/kg, 6 mg HgCl2/kg in DW (10 ml/kg), or same volume of DW alone. Mice (n = 5) were terminated under diethyl ether anesthesia at 0 and 24 h following the preceding treatments. Another group of mice given 15 mg DCVC/kg ip were terminated at 72 h under diethyl ether anesthesia. Blood was collected retroorbitally in heparinized tubes, centrifuged and plasma was stored at -80°C for future analysis. Both the kidneys (weighing approximately 0.5 g) were surgically removed, washed in phosphate buffered saline and were homogenized in 2.5 ml of 0.25 M sucrose. The volume was adjusted to 4 ml by 0.25 M sucrose and cytosol and mitochondrial fractions were isolated as described by Johnson and Lardy (1967)Go. The cytosolic samples were further purified by transferring into Ehrlenmeyer flasks and agitating in a water bath maintained at 60°C until the temperature of contents in tubes equilibrated with that in water bath for about 10 min. After standing for another 2 min in the water bath the suspension was cooled to 4°C in ice-chilled water and centrifuged at 9000 x g for 20 min to remove the inactive precipitate. Protein was estimated in the purified cytosolic and mitochondrial fractions by the Bradford method, using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The ß-lyase assay was performed in cytosolic and mitochondrial fractions by the colorimetric assay of Greenberg et al.(1964)Go after the addition of 2,4-dinitrophenylhydrazine (DNP) as described by Dohn and Anders (1982)Go. Briefly, to 200 µl sample of cytosol, 1 ml of 75 mM ß-chloroalanine was added and this mixture was incubated at 37°C for 30 min. One ml of DNP was added to this and incubated at room temperature for 20 min. Then 10 ml of 0.4 N NaOH was added to each tube and after 5 min the absorbance was read at 505 nm. Standard curves were made by adding known amounts of pyruvate into the enzyme solution followed by the derivatization reaction with DNP. The enzyme activity was calculated taking into account the 25 min incubation time and protein concentration for every sample and was expressed as nmoles of pyruvate/mg protein/min.

Glutathione estimation.
Male SW mice were injected ip with 15 or 75 mg DCVC/kg or 6 mg HgCl2/kg in DW (10 ml/kg) or the same volume of DW alone. Mice (n = 4) were terminated under diethyl ether anesthesia at 0, 6, 12, 24, 36, 48, 72, and 96 h following the above treatment. Blood was collected retroorbitally in heparinized tubes, centrifuged, and plasma was stored at -80°C for future analysis. Both the kidneys (weighing approximately 0.5 g) were surgically removed, washed in phosphate buffered saline and were homogenized in 1.5 ml of cold buffer (i.e., 50 mM 2-(n-morpholino) ethanesulphonic acid [MES], pH 6 to 7 containing 1 mM EDTA). The homogenate was further centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was deproteinized by adding to it an equal volume of metaphosphoric acid and then centrifuging it at ~2000 x g for 2 min. The supernatant was carefully collected for renal glutathione (reduced + oxidized) estimation using the commercially available glutathione assay kit (cat. # 703002) from Cayman Chemical Company (Ann Arbor, MI).

Histopathology.
Kidneys samples from control and treated mice were washed with ice-cold normal saline (0.9 % NaCl), cut sagitally into thin slices, and then fixed into 10% phosphate-buffered formaldehyde for 48 h. The tissues were then transferred into 70% ethyl alcohol, processed, and embedded in paraffin wax. Kidney sections (5 µm thin) were stained with H&E for histological examination under a light microscope. Unstained kidney sections were prepared for proliferating cell nuclear antigen immunocytochemistry.

PCNA.
S-phase stimulation and cell cycle progression were assessed by immunocytochemical analysis of PCNA expression as described by Greenwell et al. (1991)Go. Briefly, the kidney sections mounted on glass slides were first blocked with casein and then reacted with mouse monoclonal antibody against PCNA (Dako Corporation, Carpinteria, CA). The antibody was then linked with biotinylated goat anti-mouse IgG antibody (Boehringer/Mannheim, Indianapolis, IN), which was then labeled with streptavidin-conjugated peroxidase (Jackson Immunoresearch, West Grove, PA). Color was developed by exposing the peroxidase-labeled streptavidin to diaminobenzidine, which forms a brown reaction product at the site of the antibody. The sections were then counterstained with Gill’s hematoxylin. Observed under light microscopy, G0 cells were blue and did not take the PCNA stain; G1 cell nuclei were light brown while S-phase cell nuclei were stained dark brown; G2 cells had light cytoplasmic staining with or without positive speckling in the nucleus; M cells had diffuse positive cytoplasmic stain and deep blue (negative) chromosomal staining. In each section 10 fields were observed under microscope (magnification x200) and the number of positively stained cells in S-Phase was counted in the area from cortex to the outer stripe of outer medulla. In the control kidney sections ~ 500 nuclei were counted in the region from cortex to the outer stripe of outer medulla under x200 magnification. In the necrosed sections, the number of S-phase cells was counted out of the undamaged nuclei and all the results are expressed as a percentage of total number of cells counted (data not shown). PCNA studies were conducted to confirm the stimulation of cell division and cell cycle progression suggested by the 3H-T pulse labeling studies. Because DNA synthesis is required for cell division, stimulated DNA synthesis measured as increased 3H-T incorporation in nuclear DNA is determined as an estimate of cell division.

Statistical analysis.
Data were expressed as means ± SE. Statistical differences were determined by one-way analysis of variance followed by Tukey’s HSD and Duncan’s multiple range tests to determine which means were significantly different from each other or from controls using SPSS, ©SPSS Inc. (Chicago, IL). In all cases, p ≤ 0.05 was used as the statistical criterion to determine significant differences.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autoprotection Studies
In this series of experiments mice were given a priming dose of 15 mg/kg DCVC by ip administration, and then at selected times this was followed by a lethal dose of 75 mg/kg DCVC. The priming dose of DCVC was shown to alter renal function with a marked azotemia, which peaked about 1.5 days after dosing and then returned to normal (Fig. 1AGo). Histological examination of kidney confirmed that this dose caused a mild renal proximal tubular necrosis that peaked at 1.5 days (Fig. 1BGo) and had recovered by day 13 (Fig. 1CGo). This dose of DCVC also produced marked diuresis and glucosuria within the first 24 h of dosing that gradually returned to normal about five days after dosing (Figs. 2AGo and 2BGo). This priming dose of DCVC was not fatal to mice (Table 1Go). In contrast, increasing the dose of DCVC to 75 mg/kg caused a 90% mortality (Table 1Go) within 36 to 48 h of dosing. These mice had marked and sustained azotemia (Fig. 1DGo) and glucosuria without any significant effect on urine output (Figs. 2CGo and 2DGo). Histopathological examination of the kidney showed extensive proximal renal tubular necrosis at 24 h (Fig. 1EGo), which spread throughout the kidney damaging almost 80 to 90% of the nephrons (Fig. 1FGo). In mice given 15 mg/kg DCVC there was clear evidence of renal tubule regeneration (Fig. 1CGo), as shown by renal tubule basophilia. This stimulated tissue repair response was evident in incorporation of 3H-T into renal DNA. Because higher 3H-T incorporation in nuclear DNA measured by pulse-labeling studies occurs during DNA repair, stimulation of S-phase DNA synthesis was also confirmed independently by PCNA analysis. Immunocytochemical assessment of renal PCNA confirmed the findings of 3H-T pulse-labeling studies. A priming dose of 15 mg/kg DCVC increased 3H-T incorporation into renal DNA. This response was increased 1.5 days after dosing and peaked around day 3 (Figs. 3AGo and 3BGo) and then gradually returned to normal by day 10 (Figs. 3AGo and 3CGo). In agreement with this, PCNA analysis indicated an increase in the number of S-phase cells during this time period (Figs. 3BGo and 3CGo). In contrast, with the high dose of DCVC no increase in 3H-T incorporation was seen at any time (Fig. 3DGo) nor any increase in PCNA staining (Figs. 3EGo and 3FGo), indicating that renal tubule repair was inhibited at this high dose.



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FIG. 1. Plasma blood urea nitrogen (A, D, and G) and renal histopathology (B, C, E, F, H, and I) over a time course in the autoprotection model. Treatment details—(A, B, and C) Male Swiss Webster (SW) mice (n = 4) were injected with 15 mg DCVC/kg ip in distilled water (10 ml/kg) on day 0 and the same mice received DW (10 ml/kg, ip) as a vehicle on day 3. (D, E, and F) Male SW mice (n = 4) were injected ip with 10 ml/kg of DW on day 0 and the same mice received 75 mg DCVC/kg ip in distilled water (10 ml/kg) on day 3. (G, H, and I) Male SW mice (n = 4) were injected with 15 mg DCVC/kg ip in distilled water (10 ml/kg) on day 0 and same mice received 75 mg DCVC/kg ip in distilled water on day 3. (A, D, and G) Plasma blood urea nitrogen (BUN) was measured using a kit from Sigma chemical company. Unfilled arrow indicates the time of injection of distilled water (DW). Stippled arrow indicates the time of injection of 15 mg DCVC/kg. Filled arrow indicates the time of injection of a normally lethal dose of DCVC. *Statistically higher than respective 0 h controls. !Statistically lower than 75 mg DCVC/kg at that particular time point. p ≤ 0.05. (B, C, E, F, H, and I) Representative photomicrographs of H & E stained kidney sections from mice with respective ip treatments: (B) 36 h after 15 mg DCVC/kg; (C) 13th day after 15 mg DCVC/kg; (E) 24 h after 75 mg DCVC/kg; (F) 36 h after 75 mg DCVC/kg; (H) 4th day after receiving the priming dose of the autoprotection protocol (15 + 75 mg DCVC/kg). (I) 13th day after receiving the priming dose of the autoprotection protocol (15 + 75 mg DCVC/kg ip). All fields were chosen from outer stripe of outer medulla (OSOM). N, areas of necrosis. Original magnification x200.

 


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FIG. 2. Urine output (A, C, and E) and glycosuria (B, D, and F) in the autoprotection model. Male Swiss Webster (SW) mice (n = 6) were administered ip with (A and B) 15 mg DCVC/kg + DW, (C and D) DW + 75 mg DCVC/kg, and (E and F) 15 + 75 mg DCVC/kg. Treatment details are same as described in Figure 1Go. Three mice were kept in each individual metabolic cage and were acclimatized for three days before respective treatment. Unfilled arrow indicates the time of injection of DW. Stippled arrow indicates the time of injection of 15 mg DCVC/kg. Filled arrow indicates the time of injection of a normally lethal dose of DCVC. *Statistically higher than respective 0 h controls. !Statistically higher than DW + 75 mg DCVC/kg at that particular time point. p ≤ 0.05.

 


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FIG. 3. 3H-Thymidine (3H-T) incorporation in renal nuclear DNA (A, D, and G) and proliferating cell nuclear antigen (PCNA) immunocytochemistry (B, C, E, F, H, and I) in the autoprotection model. (A, D, and G) The mice used in the autoprotection experiment (see Fig. 1Go) received 50 mCi 3H-T ip 2 h prior to the time points indicated in Figure 3Go. At post mortem both kidneys were taken to estimate 3H-T incorporation in renal nuclear DNA. Unfilled arrow indicates the time of injection of DW. Stippled arrow indicates the time of injection of 15 mg DCVC/kg. Filled arrow indicates the time of injection of a normally lethal dose of DCVC. *Statistically higher than respective 0 h controls. !Statistically higher than 75 mg DCVC/kg at that particular time point. p ≤ 0.05. (B, C, E, F, H, and I) Representative photomicrographs of PCNA stained kidney sections from mice with respective treatments: (B) 72 h after 15 mg DCVC/kg ip treatment; (C) 13th day after 15 mg DCVC/kg ip; (E) 24 h after 75 mg DCVC/kg ip; (F) 36 h after 75 mg DCVC/kg ip; (H) 4th day after receiving the priming dose of the autoprotection protocol (15 + 75 mg DCVC/kg ip). (I) 13th day after receiving the priming dose of the autoprotection protocol (15 + 75 mg DCVC/kg ip). Treatment details are as in Figure 1Go. All fields were chosen from outer stripe of outer medulla (OSOM). S, cells in the S-phase of the cell cycle stained with dark-brown nuclear staining. Original magnification x400.

 
Prior administration of DCVC (15 mg/kg) was shown to afford protection against a lethal dose of DCVC (75 mg/kg) when given three or four days later (Table 1Go), while partial protection was seen at two days and no protection when given one day after the priming dose (Table 1Go). Renal function was markedly affected as evidenced by azotemia, glucosuria, and diuresis over a more extended time period, only returning to normal 10 days after the high dose of DCVC (Figs. 1GGo, 2EGo, and 2FGo). Renal tubule necrosis was confirmed by the finding of histological damage as early as the fourth day after the priming dose administration (Fig. 1HGo). Histology of the kidney revealed considerable re-epithelialization of the proximal tubules, with marked basophilia (Fig 1IGo) and renal structural restoration was evident by the 13th day (Fig. 1IGo). Renal repair as judged by 3H-T incorporation into renal DNA showed a similar profile, with an initial peak about three days after the priming dose and then a more marked and sustained phase after the high dose of DCVC (Fig. 3GGo). PCNA immunocytochemistry showed a similar temporal response with a lot of positively stained S-phase nuclei as early as the fourth day (Fig. 3HGo). Most of the S phase cells were observed in the outer stripe of outer medulla where the maximal damage occurred but overall these cells were scattered all over the cortex and the medulla (Figs. 3HGo and 3IGo) with the least number present in the papilla.

Heteroprotection Studies
Administration of an ordinarily lethal dose of DCVC (75 mg DCVC/kg) on days 1 or 2 after injection of a priming dose of HgCl2 (6 mg/kg, ip) did not yield any protection from DCVC-induced mortality, although when given on day 2, the time to death was delayed (Table 1Go). When the priming dose was given on day 3 before administration of 75 mg DCVC/kg, a 70% protection was evident (Table 1Go). Even though 100% protection was not evident by increasing the interval between the priming dose of HgCl2 and lethal dose of DCVC to 72 h, 30% mortality that did occur was considerably delayed (Table 1Go). Increasing the time between the priming dose and lethal dose to four days afforded complete protection (Table 1Go).

Administration of HgCl2 (6 mg/kg, ip) alone caused only a transient increase in BUN at 24 h (Fig. 4AGo). Although there was no significant increase in the urine volume (Fig. 5AGo), urine glucose levels increased five- to sixfold on day 1 and remained elevated until day 4 after 6 mg HgCl2/kg administration (Fig. 5BGo). Renal functional impairment was confirmed by histopathology that showed renal proximal tubule necrosis 24 h after dosing (Fig. 4CGo). The necrosis was mainly restricted to the proximal tubules in the outer medulla and the cortex without damaging the inner stripe of medulla and papilla. By the tenth day the renal tubule injury had repaired, with evidence of regenerated tubules (Fig. 4DGo) and restoration of renal function (Figs. 5AGo and 5BGo). Marked 3H-T incorporation into renal DNA was evident at day 2, after the priming dose of HgCl2, which remained elevated compared to vehicle treated controls until day 6 and declined thereafter showing only a modest and transient increase on day 10 (Fig. 6AGo). The percent of S-phase stained nuclei was low on day 2, but then increased markedly on days 3, 4 (Fig. 6CGo), and 5 indicating regenerative activity. S-Phase had returned to almost normal by day 10 (Fig. 6DGo). PCNA analysis confirmed positively staining S-phase nuclei on day 4 (Fig. 6CGo), supporting the use of this time point as being optimal for protection against a lethal dose of DCVC.



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FIG. 4. Plasma blood urea nitrogen (A and B) and renal histopathology (C, D, E, and F) over a time course in the heteroprotection model. Treatment details—(A, C, and D) Male SW mice (n = 4) were administered 6 mg HgCl2/kg ip in distilled water (10 ml/kg) and controls (0 day time point) received equal volume of distilled water (DW) alone as vehicle. (B, E, and F) Male SW mice were divided into two groups (n = 4). On day 0, mice in first group received 10 ml/kg of distilled water ip as vehicle and second group received 6 mg HgCl2/kg ip in distilled water. On day 4 both the groups were injected with 75 mg DCVC/kg ip in DW. (A and B) Blood samples were collected retroorbitally under diethyl ether anesthesia at various time points after respective treatment as mentioned above. BUN was measured using a kit from Sigma chemical company. Unfilled arrow indicates the time of injection of DW. Stippled arrow indicates the time of injection of 6 mg HgCl2/kg. Filled arrow indicates the time of injection of 75 mg/kg of DCVC. *Statistically higher than respective 0 h controls. !Statistically lower than DW + 75 mg DCVC/kg at that particular time point. p ≤ 0.05. (C, D, E, and F) Representative photomicrographs of H & E stained kidney sections from mice with respective ip treatments: (C) 6 mg HgCl2/kg at 24 h; (D) 6 mg HgCl2/kg on 10 day; (E) 5th day after receiving the priming dose of the heteroprotection protocol (6 mg HgCl2/kg + 75 mg DCVC/kg ip); (F) 14th day after receiving the priming dose of the heteroprotection protocol (6 mg HgCl2/kg + 75 mg DCVC/kg ip). All fields were chosen from OSOM region. N, areas of necrosis. Original magnification x200.

 


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FIG. 5. Urine output (A and C) and glycosuria (B and D) in the heteroprotection model. Male Swiss Webster (SW) mice (n = 6) were administered ip with (A and B) 6 mg HgCl2/kg + DW, (C and D) DW + 75 mg DCVC/kg and 6 mg HgCl2/kg + 75 mg DCVC/kg. Treatment details are same as described in Figure 4Go. Three mice were kept in each individual metabolic cage and were acclimatized for three days before respective treatment. Unfilled arrow indicates the time of injection of DW. Stippled arrow indicates the time of injection of 15 mg DCVC/kg. Filled arrow indicates the time of injection of a normally lethal dose of DCVC. *Statistically higher than respective 0 h controls. !Statistically different than DW + 75 mg DCVC/kg at that particular time point. p ≤ 0.05.

 


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FIG. 6. 3H-Thymidine (3H–T) incorporation in renal nuclear DNA (A and B) and proliferating cell nuclear antigen (PCNA) immunocytochemistry (C, D, E, and F) in heteroprotection model. Male Swiss Webster (SW) mice (n = 6) were administered ip with (A, C, and D) 6 mg HgCl2kg + DW, (B, E, and F) DW + 75 mg DCVC/kg and 6 mg HgCl2kg + 75 mg DCVC/kg at 96 h in distilled water (10 ml/kg). Experimental details are same as in Figure 4Go.(A and B). The same mice used in the experiments mentioned in Figure 4Go were used for pulse labeling studies with 3H-T. Two h prior to the time points indicated in Figure 6Go all mice received 50 µCi 3H-T ip. At post mortem both kidneys were taken to estimate 3H-T incorporation in renal nuclear DNA. Unfilled arrow indicates the time of injection of DW. Stippled arrow indicates the time of injection of 6 mg HgCl2/kg. Filled arrow indicates the time of injection of a normally lethal dose of DCVC. *Statistically higher than respective 0 h controls. !Statistically higher than DW + 75 mg DCVC/kg at that particular time point. p ≤ 0.05. (C, D, E, and F) Representative photomicrographs of PCNA stained kidney sections from mice with respective ip treatments: (C) 6 mg HgCl2/kg at 96 h; (D) 6 mg HgCl2/kg on day 10; (E) fifth day after receiving the priming dose of the heteroprotection protocol (6 mg HgCl2/kg + 75 mg DCVC/kg ip); (F) 14th day after receiving the priming dose of the heteroprotection protocol (6 mg HgCl2/kg + 75 mg DCVC/kg ip). All fields were chosen from outer stripe of outer medulla (OSOM). S, cells in the S-phase of the cell cycle stained with dark-brown nuclear staining. Original magnification x400.

 
Increasing the interval between the priming dose (6 mg HgCl2/kg) and 75 mg DCVC/kg to four days yielded 100% protection from the lethal outcome of 75 mg DCVC/kg (Table 1Go). This interval was chosen for detailed time course studies. Renal injury at early time points (4.5 and 5 days) in the heteroprotection group (6 mg HgCl2/kg + 75 mg DCVC/kg on the fourth day) was similar to that observed in mice receiving only the high dose of DCVC (Fig. 4BGo). BUN peaked on day 5 and then remained elevated until day 7 gradually declining to normal by day 14 (Fig. 4BGo). Significant polyuria was observed from 6 to 9 days in the heteroprotection group (Fig. 5CGo), while a marked glucosuria was sustained until day 7 thereafter returning to normal (Fig. 5DGo). Renal proximal tubule injury, as indicated by the azotemia and glucosuria, was confirmed by histopathology (Figs. 4E and FGo). In the heteroprotection group extensive renal proximal tubule necrosis was observed on day 5 (Fig. 4EGo). Some focal necrosis was still evident on the 14th day (Fig. 4FGo) but most of the debris had been cleared and the majority of damaged proximal tubules had regenerated. As expected the 3H-T incorporation into renal DNA was stimulated following the priming dose of HgCl2 (Fig. 6BGo). Upon challenge with 75 mg DCVC/kg, there was an initial decrease in 3H-T incorporation into renal DNA on day 4.5, which then increased on day 5, peaked on day 6, with much higher levels of 3H-T incorporation into renal DNA than seen with the priming dose and then gradually declined with time (Fig. 6BGo). These findings were supported by PCNA immunocytochemistry, which showed active S-phase stimulation on days 5 and 6 (Fig. 6EGo) with cell cycle progression. The percent of S phase cells remained high throughout the time course till day 14 (Fig. 6FGo) when re-epithelialization was almost complete.

Effect of DCVC and HgCl2 Treatment on Renal ß-Lyase Activity and GSH Content
Because the nephrotoxicity of DCVC is mediated by a reactive metabolite, we examined the possibility that the priming dose of either DCVC or HgCl2 could have destroyed the activating enzyme ß-lyase. In addition, we have investigated whether a priming dose of DCVC or HgCl2 could have increased the renal content of GSH, thereby enabling more of the reactive metabolite to be scavenged.

ß-Lyase Activity
Renal cytosolic and mitochondrial ß-lyase activities were measured, because it is known to be the predominant enzyme responsible for renal bioactivation of DCVC. Renal ß-lyase activity was higher in the mitochondrial fraction than in the cytosol in control mice. Cytosolic ß-lyase activity was decreased by 50% 1 and 3 days after 15 mg DCVC/kg administration (Table 2Go) and by about 85% one day after 75 mg/kg of DCVC (Table 2Go). Mitochondrial ß-lyase activity was only decreased by about 40% on days 1 and 3 after dosing with 15 mg DCVC/kg and 65% after the high dose of DCVC (Table 2Go). Treatment with HgCl2 did not affect the activity of ß-lyase in either cellular compartment (Table 2Go).


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TABLE 2 Renal Cytosolic and Mitochondrial ß-Lyase Activity following DCVC or HgCl2 Administration
 
Total Renal Glutathione (GSH) Levels (Reduced + Oxidized)
A small but not statistically significant decrease in renal GSH was seen after the priming dose of DCVC (15 mg/kg), with a small rebound above control values 48 and 72 h after dosing (Table 3Go). A more marked decrease was seen after the high dose of DCVC (Table 3Go) with about 70% loss at 6 h after dosing, which did not recover, still being about 50% at the time of the animal’s death. In contrast, HgCl2 increased renal GSH 24 h after dosing which was still present, although less marked at 96 h after dosing (Table 3Go).


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TABLE 3 Renal Glutathione (GSH µmol/g Wet Weight of Kidney) Content following DCVC or HgCl2 Administration
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report for the first time that prior treatment of mice with a low nephrotoxic dose of either DCVC or HgCl2 affords protection against a lethal dose of DCVC when given 72 to 96 h after the priming dose. Our findings suggest that stimulation of renal tubule regeneration prior to administration of a lethal dose of DCVC is an important factor in protecting the mice.

Previous dose-response and time-course studies with DCVC in the mouse (Vaidya et al., 2003Go), suggested that renal tissue repair was an important response leading to full recovery from low to moderate doses of DCVC. At high doses, DCVC prevented or delayed tissue repair, resulting in progression of injury, renal failure, and death.

DCVC-induced nephrotoxicity is well documented (Beuter et al., 1989Go; Lash and Anders, 1986Go), but little is known about potential mechanisms to prevent acute tubular necrosis and death. Our studies indicate that priming renal tissue repair with either DCVC or HgCl2 can obviate fatal ARF from a high (normally lethal) dose of DCVC. Other groups have previously reported that prior administration of a low dose of a nephrotoxin can protect against the nephrotoxicity produced by a larger dose (Magos et al., 1974Go; Oken et al., 1975Go; Tandon et al., 1980Go; Yoshikawa, 1970Go). These studies indicated that renal renin depletion, induction of metallothionen, or reduced delivery of the chemical to the kidney could not explain the protection. We have examined three potential mechanisms that may provide some insight into the basis for the auto- and heteroprotection observed: (1) stimulation of tissue repair, (2) inhibition/destruction of ß-lyase, and (3) increased renal GSH content, which will be discussed below.

Role of Tissue Repair Stimulated by the Priming Dose
For maximal autoprotection a minimum of 72 h was required between the priming dose and the lethal dose (Table 1Go). At three days sustained S-phase stimulation (Fig. 3AGo) and cell cycle progression (Fig. 3BGo) were evoked by the priming dose. Therefore, the renal cells in the autoprotected mice were already primed to divide, resulting in an enhanced and sustained tissue repair from days 4 to 8 in response to the high dose of DCVC (Fig. 3GGo). This suggests that cells primed to divide rapidly by a low dose of DCVC are able to cope with a large ordinarily lethal dose of DCVC. This autoprotection phenomenon seen with DCVC in kidney is similar to that seen with carbon tetrachloride, thioacetamide, and acetaminophen in the liver, where stimulation of hepatocellular regeneration by the protective dose is the underlying mechanism (Chanda et al., 1995Go; Mangipudy et al., 1995Go; Mehendale et al., 1994Go).

If tissue repair stimulated by a priming dose of DCVC is an important mechanism in the observed autoprotection, it should not matter which toxicant is used to provide the priming stimulus. HgCl2, a chemically and mechanistically different nephrotoxicant from DCVC was chosen as the tissue repair-priming agent. A priming dose of 6 mg HgCl2/kg ip stimulated renal tubule cell proliferation, as evident from days 2 to 6 as increased DNA synthesis and by PCNA (Figs. 6AGo, 6CGo, and 6DGo). Further, mice primed with 6 mg HgCl2/kg ip four days prior to administration of 75 mg DCVC/kg were completely protected from ARF and death (Table 1Go). The initial renal injury (at four to five days) as assessed by azotemia, glucosuria, and histopathology in the heteroprotected mice, was similar to that seen in the group not given the priming dose (Figs. 4BGo, 5CGo, and 5DGo), and to that seen in the autoprotection model, ruling out decreased DCVC bioactivation as a likely mechanism to explain the protection against a lethal dose of DCVC. In the heretoprotected mice the rate of stimulated cell division remained elevated until the 14th day, actively replacing cells lost by necrosis (Fig. 6FGo).

It should be noted that renal injury is the same or nearly the same in mice receiving the normally lethal dose of DCVC, regardless of whether they receive a priming dose. A preponderance of experimental evidence indicates that protection against ARF and death is not due to diminished bioactivation-based nephrotoxic injury. How can the dramatic regression of nephrotoxic injury be surmised? The newly divided cells bring two distinct advantages that add to recovery. First, new cells are available to replace the dead or dying cells. Second, if the newly divided cells stimulated by the priming dose are resistant to or only partly susceptible to the mechanisms that cause progression of injury a dramatic recovery can be surmised. Progression of injury is known to be mediated by death proteins such as calpain and cytosolic phospholipase A2 (cPLA2) leaking out of the dying or dead cells (Limaye et al., 2002Go). When newly divided cells replace the dead cells, injury no longer progresses (Limaye et al., in pressGo; Masaki et al., 1994Go). Newly divided cells are known to overexpress inhibitors of death proteins such as calpain, cPLA2, such as calpastatin, annexins, etc., thereby preventing the spread of injury (Limaye et al., 2002Go; Masaki et al., 1994Go; Yoshida et al., 2002Go). The priming dose stimulates cells to divide, and the presence of newly divided cells prevents the progression of injury paving the way for regression and recovery from even the massive and normally lethal level of renal injury.

Inhibition of ß-Lyase by the Priming Dose
ß-Lyase-catalyzed cleavage of DCVC yields a reactive thiol, S-(1,2-dichlorovinyl) thiol (DCVSH), which is a metabolite of short life. This thiol is chemically unstable and rearranges to a reactive species that can form covalent adducts with cellular nucleophiles, including proteins (Elfarra et al., 1987Go; Lash et al., 1994Go). MacFarlane et al.(1993)Go, using the mercapturic acid metabolite of hexachloro-1,3-butadiene (HCBD) N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (Nac-PCBD), and Kim et al.(1997)Go using HCBD showed that pretreatment of rats with low or subtoxic doses produced a modest (1.5- to 3-fold) induction of ß-lyase in renal cytosol. MacFarlane et al.(1993)Go reported that the ß-lyase activity, protein, and mRNA were induced by a non-nephrotoxic dose of 3 mg/kg ip. However, ß-lyase activity was inhibited by a higher dose of 10 mg/kg Nac-PCBD at 24 h. We considered the possibility that the reactive thiol moiety generated by the 15 mg DCVC/kg could destroy ß-lyase, and thereby inhibit bioactivation of the 75 mg DCVC/kg given 3 days later. Cytosolic and mitochondrial ß-lyase activities were decreased by about 50% at day 1 and 3 after the priming dose (Table 2Go). However, this reduction in ß-lyase activity did not translate into lower bioactivation of 75 mg DCVC administration at day 3 as judged by the extent of azotemia, glucosuria, and histopathology, which suggested that renal proximal tubule necrosis was not reduced in primed versus nonprimed mice (Figs. 1Go and 2Go). Moreover, although mitochondrial and cytosolic ß-lyase activities were equally reduced at day 1 and 3 after the 15 mg/kg priming dose of DCVC, mice were not autoprotected at 24 h whereas at 72 h they were 100% protected. Therefore, it appears that in spite of the decreased ß-lyase activity, the remaining ß-lyase was adequate to bioactivate DCVC to inflict uncompromised nephrotoxicity. The possibility that decreased ß-lyase activity may play a role in nephroprotection is further weakened by the heteroprotection studies. Heteroprotection by HgCl2 was accompanied by neither decreased mitochondrial nor cytosolic ß-lyase activity (Table 2Go).

GSH as the Mechanism of Auto- and Heteroprotection
Both in vitro and in vivo studies suggest that DCVC and/or its glutathione conjugates are metabolized in the renal cortex to a reactive species that causes a significant depletion of GSH in mice, rabbits, and rat kidney cells (Beuter et al., 1989Go; Hassall et al., 1983Go; Lash and Anders, 1986Go). Thus, modulation of renal GSH levels could potentially affect the nephrotoxicity of DCVC. The priming dose of DCVC did not significantly alter renal GSH at any time, the marginal increase at 72 h (Table 3Go) was not considered sufficient for autoprotection. Several reports, using both in vivo and in vitro systems, have shown increases in GSH in renal tubular epithelial cells after administration of low, subtoxic, or toxic doses of HgCl2 (Lash and Zalups, 1996Go; Zalups and Veltman, 1988Go). We observed elevated GSH content in kidney homogenates 24 h after injection of 6 mg HgCl2/kg ip (Table 3Go). However, GSH was neither diminished nor elevated at 96 h, the interval needed for optimal heteroprotection, suggesting that modulation of GSH by the priming dose of HgCl2 is unlikely to be a key factor in protection from DCVC-induced mortality. McGuire et al.(1997)Go have reported a marked (fourfold) increase in glutathione S transferases (mGSTA1/A2) in kidney cytosol four days following a dose of 3 mg HgCl2/kg. Induction of GSTs has been suggested to be a general mechanism for cell protection against a wide variety of toxicants through detoxication. If induction of glutathione S transferase (GST) were to be a mechanism for protection in the hetero- and autoprotection models then one would have observed attenuated renal injury upon administration of the high dose of DCVC to mice primed with HgCl2 or DCVC. However, our results suggest that there was a marked increase in renal injury in both the models following the high dose of DCVC making it unlikely that GST is involved in the observed protection.

In summary, our studies with the auto- and heteroprotection models suggest that stimulation of renal tubule regeneration is an important response enabling protection against DCVC-induced toxicity. The molecular mechanisms behind this paradigm are not fully understood and other factors such as the balance between metabolic activation and detoxification of DCVC as well as understanding the reasons for the resistance of newly synthesized renal tubule cells to toxic insult require further study. Understanding the molecular mechanisms involved in the auto- and heteroprotection models may direct future research into novel avenues for treatment of ARF.


    ACKNOWLEDGMENTS
 
We thank Dr. Adnan Elfarra for his helpful discussion and valuable suggestions concerning the ß-lyase assay. These studies were made possible through a National Institutes of Health grant from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) DK-61650. This study was partly supported by the Louisiana Board of Regents Fund through the University of Louisiana at Monroe, Kitty DeGree Endowed Chair in Toxicology.


    NOTES
 
The studies reported in this article are part of the Ph.D. dissertation of Vishal S. Vaidya and were presented at the 41st annual meeting of the Society of Toxicology, March 2002, Nashville, TN (The Toxicologist 66, 880, March 2002).

1 To whom correspondence should be addressed. Fax: (318) 342-1686. E-mail: mehendale{at}ulm.edu. Back


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