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Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo
Environmental Toxicology and Pharmacology 7 (1999) 179 – 187
Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo Shino Homma-Takeda a,*, Yasuhiro Takenaka b, Yoshito Kumagai a, Nobuhiro Shimojo a a
Department of En6ironmental Medicine, Institute of Community Medicine, Uni6ersity of Tsukuba, 1 -1 -1 Tennodai, Tsukuba, Ibaraki 305 -8575, Japan b Master’s Program in En6ironmental Sciences, Uni6ersity of Tsukuba, 1 -1 -1 Tennodai, Tsukuba, Ibaraki 305 -8572, Japan Received 31 August 1998; received in revised form 24 February 1999; accepted 1 March 1999
Abstract A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl2). A single injection of HgCl2 to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl2 were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; DNA fragmentation; Synchrotron radiation X-ray fluorescence analysis; Mercuric chloride; Kidney; Rat
1. Introduction Apoptosis and necrosis are quite distinct forms of cell death with different morphology and implications for the surrounding tissue (Kerr et al., 1972; Whyllie et al., 1992; Corcoran et al., 1994). Necrosis, a passive or accidental cell death caused by noxious stimuli, is typified by cell and organelle swelling with spillage of the intracellular contents as well as loss of functional and structural integrity of the cell membrane and mitochondria, leading to injury of the surrounding tissue. In
Presented in part at the 4th International Symposium on Metal Ions in Biology and Medicine, Barcelona, Spain, 19–22 May 1996. * Corresponding author. Present address: Graduate School Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: + 81-298-53-3297, fax: +81-297-52-4015. E-mail address: [email protected] (S. Homma-Takeda)
contrast, apoptosis is a physiological mode of cell death designed to dispose of unwanted cells promptly. Nuclear shrinkage associated with nuclear condensation and enzymatic DNA cleavage into oligonucleosomesize fragments (DNA ladders) without changes of cell membrane and mitochondria is characteristic of apoptosis. Consequently, the resulting apoptotic bodies are phagocytosed by neighboring cells to prevent inflammation and damage to the surrounding tissue (Savill et al., 1993). A notion of metal-induced cell death has been currently changed because some reports suggested that cells from toxic-target organs can undergo apoptosis by a variety of toxic metals (i.e. cadmium, mercury, platinum, and lead) which are thought to cause necrosis (Kunimoto, 1994; Ishido et al., 1995; Duncan-Achanzar et al., 1996; Lieberthal et al., 1996; Oberto et al., 1996). From several observations with cultured cells the cell
1382-6689/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 9 9 ) 0 0 0 1 2 - 5
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death, whether via either apoptosis or necrosis, has been considered to depend on the concentration of toxicant: High concentration is necrosis and low concentration is apoptosis (Duncan-Achanzar et al., 1996; Lieberthal et al., 1996). However, little information about metal-promoted apoptosis in vivo has been reported (Nagashima et al., 1996; Xu et al., 1996; Liu et al., 1998), and the question of how apoptotic and necrotic phenomena occur during exposure of animals to mercury remains open. Thus, we show here evidence for the induction of apoptosis in the kidney in mercuric chloride (HgCl2)-treated rats, which is well recognized as a nephrotoxic model. A dose – response relationship in the induction of apoptosis and site-specificity of apoptosis and necrosis caused by mercury are also discussed.
2. Material and methods
2.1. Chemicals Chemicals were obtained as follows: HgCl2, nitric acid, sulfuric acid, potassium permanganate, stannous chloride, PAS reagent, and methylgreen from Wako Pure Chemical Industries, (Osaka, Japan); 3,3%-diaminobenzidine tetrahydro chloride (DAB) from Dojindo Laboratories (Kumamoto, Japan); Agarose, RNase A, proteinase K, and bisbenzamide from Sigma (St. Louis, MO, USA); Size markers for DNA agarose electrophoresis (HindIII-digested l-DNA and HaeIIIdigested fX174-DNA) from TOYOBO (Tokyo, Japan). All other chemicals used were of the highest grade commercially available.
2.2. Animals and treatments Male Wistar rats (7 weeks old) were obtained from Clea Japan, (Tokyo, Japan). The animals were allowed to acclimate in an environment of controlled temperature, humidity, and light/dark cycle for 1 week before study. All animal procedures received prior approval from the University Laboratory Animal Care and Use Committee and met the current local regulations. HgCl2 was administered to the rats by s.c. injection at doses of 0.5, 1, 2, or 4 mg/kg and the animals were housed in individual metabolic cages. For control, rats without any administration were used. Three, 7, 12, 16, 24, and 48 h after HgCl2 administration, the rats were killed by withdrawing approximately 7 ml of blood from the heart under ether anesthesia and the kidneys were removed. Urine samples were collected for 7, 12, 16, and 24 (0– 24 and 24 – 48) h after administration.
2.3. Measurements of mercury le6el in the kidney Kidney samples (200 mg) were digested with nitric acid, potassium permanganate, and sulfuric acid at room temperature. Then the mercury concentrations were measured as reported previously (Shinyashiki et al., 1998).
2.4. Synchrotron radiation X-ray fluorescence (SR-XRF) imaging of mercury in the kidney The two dimensional distribution of mercury in the kidney was clarified by SR-XRF analysis according to the established method as reported previously (Shimojo et al., 1991; Homma et al., 1993, 1995). The renal specimens were fixed in 10% buffered formalin (pH 7.4, 4°C), embedded in paraffin, cut to a thickness of 2 mm by a microtome (Yamato Kohki, Tokyo, Japan), and then subjected to SR-XRF analysis. XRF measurements for mercury was made at the Photon Factory of the Institute of Materials Structure Science (KEK-PF), Tsukuba, Japan, utilizing an energy dispersive SR-XRF system with monochromatic X-rays obtained by a Si(111) double crystal monochromator. Hg (La) was excited with 16 keV X-rays (beam size, 300 × 300 mm2). The X-ray intensity data of the Hg La fluorescence lines (counting times, 10 s/point) of each point obtained by step-scanning (200 mm/step) on the samples were processed with a personal computer and the results were shown as a tone from black to white classified into 14° from maximum to minimum, that is linearly proportional to the 14 levels of the element concentrations.
2.5. E6aluation of nephrotoxicity Alkaline phosphatase (ALP) activity in the urine was measured using an ALP assay kit (Wako, Osaka, Japan) by the method of Bessey et al. (1946). For histological examination, renal specimens (4 mm) were stained with periodic acid Schiff (PAS) reaction according to the method of Thompson (1966).
2.6. DNA fragmentation analysis Quantitation of DNA fragmentation was performed by the method of Ray et al. (1992) with modification. Briefly, the frozen kidney was homogenized with 5 volumes of a ice-cold lysis buffer (5 mM Tris–HCl (pH 8.0)–20 mM EDTA–0.5% Triton X-100). (The efficiency of DNA extraction from the tissues in the present method was improved 50 times over the method for our preliminary experiment; the extraction was performed by shaking the tissues in the lysis buffer for 1 h at 4°C (Homma-Takeda et al., 1997a).) The homogenates were centrifuged at 16 000× g for 20 min to separate intact chromatin and fragmented DNA. The
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pellets obtained (intact chromatin) were suspended in 0.5 N perchloric acid, heated at 80°C for 15 min, and then centrifuged at 16 000×g for 15 min to remove the protein. The resulting supernatants were used for DNA assay by a coloring reaction with diphenylamine (Burton, 1956) with calf DNA as the standard. Absorbance was measured at 600 nm with a Shimadzu UV-1600 double beam spectrophotometer (Kyoto, Japan). For the supernatants (fragmented DNA), substitution of phenol–chloroform (1:1, v/v) for perchloric acid to remove the protein resolved the problem of unidentified materials interfering with the assay. DNA extracted with phenol–chloroform were precipitated in isopropanol (overnight at − 80°C). The DNA was washed with 70% ethanol, redissolved in 0.5 N perchloric acid, and heated at 80°C for 15 min. Then resulting DNA sample was subjected to DNA assay. The recoveries of intact chromatin and fragmented DNA by the present method were 95.591.8 and 70.99 3.7%, respectively, (n= 4). Isolation of the DNA for agarose gel electrophoresis was performed by the same procedure described above. After precipitation in isopropanol, followed by washing with 70% ethanol, the pellets obtained were suspended in 10 mM Tris–HCl (pH 7.4) – 20 mM EDTA and then treated with RNase A (final concentration 2 mg/ml) for 2.5 h at 37°C. The sample was electrophresed on a 1.2% agarose gel containing ethidium bromide (0.5 mg/ml). Electrophoresis gels were illuminated with 312 nm light (Funakoshi, Tokyo, Japan) and a photographic record of the DNA ladders was made with instant film (Fuji Photofilm, Tokyo, Japan).
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medullary tubules in the specimen of positive control (staining of the medullary tubules were stronger than those of the glomeruli and cortical tubules). The renal section in the absence of exogenous terminal deoxynucleotidyl transferase (negative control) had no positive staining, suggesting the specific color reaction in the experimental condition (data not shown).
2.8. Chromatin condensation PAS-stained renal sections (4 mm) were treated with 0.5% Triton X-100 in PBS and incubated with 5 mM bisbenzamide for 10 min at room temperature. The nuclei were visualized using a fluorescence microscopy (LEITZ DMR, Leica Mikroskopie und Systeme, Germany).
2.9. Combination analysis of SR-XRF imaging and TUNEL staining TUNEL-stained renal sections (10 mm) was attached to mylar film and then subjected to SR-XRF analysis. The XRF measurements were performed with monochromatic a X-ray microbeam obtained with a multilayer monochromator and K-B type focusing optics (Iida and Noma, 1993) for Hg imaging under the following conditions: energy of X-rays, 14.38 keV; beam size, 5×5 mm2; step size, 5 mm/step; and counting time, 10 s/step. After SR-XRF, the specimens were counter-stained with Carrazzi’s hematoxylin. Minimal effect of TUNEL staining on Hg La detection was observed (data not shown).
2.7. Identification of apoptotic cells in the kidney
2.10. Statistics
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method (Gavrieli et al., 1992) was employed for renal sections with slight modification using a in situ apoptosis detection kit (Oncor, Gaitherburg, Germany). Sections (4 mm) were deparaffinized, rinsed with PBS, treated with proteinase K (20 mg/ml) to strip the nuclear proteins, and then quenched in endogenous peroxidases with 2% H2O2 in phosphate buffered saline (PBS). DNA 3%OHends were labeled with digoxigenin-conjugated dUTP. Bound digoxigenin was detected by incubation with peroxidase-conjugated antibody against digoxigenin at 37°C for 2 h and then developed with 50 mM Tris –HCl (pH 7.4)–0.1% DAB – 0.02% H2O2. After TUNEL staining, the specimens were counter-stained with 0.1 M sodium acetate–2% methylgreen (pH 4.0). For negative and positive controls of TUNEL staining, renal sections of control rats exposed to DNase (at room temperature for 10 min) in vitro were used. Intensive staining was seen in the nucli of all the type of renal cells, such as the glomeruli, and the cortical or
Data are expressed as the means9 S.D.s for each group. A t-test and one way analysis of variance (ANOVA) were carried out on a personal computer using a biomedical program (HALBAU, Japan). When statistically significant F-values (PB 0.05) were obtained with the ANOVA, and Bonferroni’s correction was used.
3. Results
3.1. Mercury distribution in kidney After s.c. injection of HgCl2 (2 and 4 mg/kg) to rats, accumulation of mercury in the kidney reached a maximum level at 7 h (2 mg/kg, 68.391.7; 4 mg/kg, 79.19 1.6), and then decreased gradually. These concentrations of mercury at 48 h were about half of those at 7 h (Fig. 1). The two-dimensional distributions of mercury in the kidney are shown in Fig. 2. Mercury distributed was higher in the renal cortex than in the
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Fig. 1. Time course of Hg concentration in kidney of the rats exposed to HgCl2. Rats were treated with HgCl2 (2 mg/kg () or 4 mg/kg ( ) by s.c. administration) and kidney mercury levels were measured from 3 to 48 h after administration. Zero h represents the control group. Each point is the mean 9 S.D. of four animals.
medulla. Upon further analysis, the renal distribution of mercury varied with time. For example, the mercury localized around the boundary between the cortex and the medulla at 48 h after HgCl2 administration (4 mg/kg) (Fig. 2D), while the metal accumulated all over the cortex at 16 h (Fig. 2B).
Fig. 3. Release of ALP in urine after exposure to HgCl2. d, 2 mg/kg; b, 4 mg/kg. ALP activities in urine samples collected for 7, 12, 16, and 24 (0 – 24 and 24 – 48) h after administration were measured. Data are expressed as percent of control obtained from each experimental period. Each point is the mean 9S.D. of four animals. * Significantly different from control, P B0.05.
3.2. Nephrotoxicity by mercury Renal damage caused by HgCl2 was inferred from the morphological changes in renal sections and the urinary excretion of ALP which is a marker enzyme of manifest proximal tubular injury by mercury (Stroo and Hook, 1997). No appreciable urinary excretion of ALP was seen after injection of HgCl2 at lower doses such as 0.5 or 1 mg/kg (data not shown). By contrast, exposure of the rats to HgCl2 at a dose of 2 mg/kg resulted in a significant leakage of ALP into the urine
Fig. 2. Mercury distribution in rat kidney. Renal specimens obtained at 16 (A) or 48 h (C) after injection of HgCl2 (4 mg/kg) were employed for SR-XRF imaging (B, D). The sample scanning condition was 40 × 48 (B, at 16 h) and 39 × 48 (D, at 48 h) steps with 200 mm/step. The X-ray intensity data of the Hg La fluorescence lines of each point are shown as a tone from black to white classified into 14 degrees from maximum to minimum. The mercury concentration range of the imaging was calculated using the mercury concentration in the kidneys determined by FAAS (see Fig. 1) as described previously (Homma-Takeda et al., 1998). Bar =2 mm.
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at 24 h after injection (Fig. 3). Increasing the dose of HgCl2 from 2 to 4 mg/kg made the occurrence of the urinary ALP excretion accelerate, indicating a dose-dependent renal injury caused by HgCl2. The morphological changes, such as a loss of brush border and cell loss, occurred at the inner zone of the cortex at 12 h after HgCl2 administration (4 mg/kg) as shown in Fig. 4E and F. At 48 h, severe injured tubules were noted particularly in the outer stripe as well as in many areas of the cortex (Fig. 4J and K).
3.3. Induction of apoptosis by mercury in the kidney The rate of fragmented DNA versus total DNA (percentage of DNA fragmentation) in the kidney of control rats was 0.3090.04% (n =4). After HgCl2 (4 mg/kg) was injected into the rats, the renal fragmented DNA increased at 7 h (1.4-fold the control level; DNA fragmentation, 0.4190.03%) (Fig. 5A). The apoptosis assessed by the fragmented DNA peaked at 16 h (12fold the control level; DNA fragmentation, 3.589 0.54%), and then decreased (48 h; DNA fragmentation, 1.439 0.12%). In the case of the lower dose (1 mg/kg), however, a significant induction of apoptosis by HgCl2 was found at 48 h. A good dose – response relationship was observed in the inorganic mercury-induced DNA fragmentation (0.5–4 mg/kg). DNA fragmentation was also confirmed by electrophoretic analysis (Fig. 5B). In spite of minimal leakage of ALP into the urine and morphological alterations, obvious DNA cleavage into oligonucleosome-size fragment was observed at 7 h after injection of HgCl2 (4 mg/kg). Such DNA ladders caused by HgCl2 was maintained for at least 48 h. When the rats were exposed to lower doses of HgCl2, the formation of DNA ladders was observed at 12 h (2 mg/kg), at 16 h (1 mg/kg), and at 48 h (0.5 mg/kg), respectively (data not shown). To detect apoptotic cells associated with the DNA fragmentation in situ, TUNEL staining was employed for the renal sections. In the specimen at 12 h after injection of HgCl2 (4 mg/kg), the proximal tubules, a toxic target region for mercury, contained apoptotic cells whereas the distal tubules, glomeruli, or medullary tubules did not (Fig. 4D and G). Examination of the nuclear morphology in the proximal tubules of the HgCl2-treated rat also gave a further characterization of apoptosis, chromatin condensation as shown in Fig. 6. The time course of the histochemical examination was parallel with that of DNA fragmentation as shown in Fig. 5A. Almost no renal apoptotic cells were found in the control rats (Fig. 4A). A similar phenomenon was also observed at 3 h after injection of HgCl2 (4 mg/kg, data not shown). Although there were a few TUNEL positive cells at 7 h after administration (data not shown), the apoptotic cells increased with time
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(Fig. 4D and H). Moreover, the TUNEL positive area was detected in the inner zone of the cortex (Fig. 4D), where tubular damage was also observed (Fig. 4E and F), and spread over the cortex with time (Fig. 4H). At 48 h the outer stripe showing necrotic damage (Fig. 4J and K) and was predominantly stained by TUNEL (Fig. 4I).
3.4. Mercury accumulation and apoptosis To attempt simultaneous determination of mercury accumulation and apoptotic cells in the same specimen of kidney after injection of HgCl2 (4 mg/kg) into the rats, the combination analysis of SR-XRF and TUNEL staining was performed (Fig. 7). Mercury accumulated more in the proximal tubules than in the glomeruli or the distal tubules. The mercury distribution was varied among the proximal tubules, but TUNEL-positive proximal tubules associated with induction of apoptosis were found to have higher mercury levels compared with the TUNEL-negative proximal tubules.
4. Discussion The present results indicated that a single s.c. injection of HgCl2 (2 or 4 mg/kg) into rats caused induction of not only necrosis but also apoptosis in the kidney in vivo in a dose-dependent fashion. Renal DNA fragmentation was detected earlier than urinary excretion of ALP, when the rats were treated with HgCl2 at a dose of 4 mg/kg. The time lag between induction of DNA fragmentation and leakage of ALP in the urine was longer in the case of the lower dose (2 mg/kg) than in the case of the higher dose (4 mg/kg). Although the occurrence of necrosis by a toxicant is generally recognized to be irreversible (Corcoran and Ray, 1992), the HgCl2-mediated apoptotic phenomena assessed by nuclear condensation and DNA fragmentation were transient exhibiting that fragmented DNA peaked at 16 h and then decreased. Minimal morphological changes were also observed at the beginning of the induction of DNA fragmentation, which is thought to be a later event of the apoptotic death process (Schwartzman and Cidlowski, 1993; Oberhammer et al., 1993) (see Fig. 4D–F and Fig. 5). Again, it was shown that necrotic renal tubules were found at least 8 h after the peak of the apoptotic event. From these results it seems likely that apoptosis caused by HgCl2 in vivo would be an early event compared with the necrotic phenomenon. Inorganic Hg is known to cause renal tubular damage mainly in the S3 segment of the proximal tubules, which is localized in the renal cortex and in the outer stripe of the outer medulla (Magos et al., 1984; Burfuss
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Fig. 4.
Fig. 7.
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Fig. 5. Formation of DNA fragmentation in kidney after mercury exposure. (A) quantitation of DNA fragmentation. DNA fragmentation is as expressed the percentage of fragmented DNA vs. total DNA (fragmented DNA +intact DNA). , 0.5 mg/kg; , 1 mg/kg; , 2 mg/kg; , 4 mg/kg. Zero h represents the control group. Each point is the mean 9 S.D. of four to five animals. Significant differences are indicated by the following letters; a, control vs. 4 mg/kg (PB 0.05); b, control vs. 2 mg/kg (PB 0.05); c, control vs. 1 mg/kg (P B0.05); d, control vs. 0.5 mg/kg (P B0.05). (B) DNA electrophoretic analysis. Fragmented DNA extracted from renal tissue (20 mg) of rats treated with HgCl2 (4 mg/kg) was loaded in agarose gel and stained with ethidium bromide. Lane 1, HindIII-digested l-DNA and HaeIII-digested fX174-DNA as molecular size marker; lanes: 2, control; 3, 3 h; 4, 7 h; 5, 12 h; 6, 16 h; 7, 24 h; and 8, 48 h.
Fig. 6. Effect of HgCl2 administration on morphological changes associated with apoptosis in the proximal tubules. Renal sections were stained with PAS reagent and bisbenzamide. DNA-fluorescence images (C, D) were photographed under fluorescence microscopy. Corresponding light microscopy of PAS staining (A, B) are also shown. (A) and (C) Control kidney. (B, D) At 12 h after HgCl2 administration (4 mg/kg). (D) The proximal tubules, identified by PAS-positive brush border (B), contain condensed nuclei (arrows). Bar =10 mm. Fig. 4. Light microscopy of renal sections. Serial tissue sections were stained with either TUNEL (A, D, G – I) or PAS reagent (B, E, F, J, K); photomicrographs were taken from same areas illustrated in C. (A and B) Control kidney. Almost no TUNEL-positive cells were found. (D–G) At 12 h after administration of HgCl2 (4 mg/kg). Corresponding to the results of histochemical staining with illustration of renal section (C), yellow-stained apoptotic cells (D, G) and morphological changes (E, F) were observed in the inner zone of the cortex. (H) At 24 h. Apoptotic cells were also found in the outer zone of the cortex. (I–K) At 48 h. Apoptotic cells localized at the inner stripe (I), where tubular injury was particularly noted (J, K); G, glomerulus; D, distal tubule; *, brush border-lost proximal tubules. Bar = 250 mm (A, D, E, H – J) and 50 mm (B, F, G, K). Fig. 7. Distribution of apoptotic cells and mercury in the kidney of the rat exposed to HgCl2. A renal section from the HgCl2-treated rat (4 mg/kg) at 12 h after injection was employed for the combined method of TUNEL staining (A) with SR-XRF imaging (B). Arrow heads, apoptotic cells; G, glomerulus; S, space, not tissue; arrow, distal tubule. (B) Mercury imaging by step-scanning (5 mm/step) on the renal section after TUNEL staining. Mercury concentration range for the XRF imaging with a tone from red to blue classified into 14 degrees was calculated using mercury concentration in the kidney determined by FAAS (see Fig. 1). Bar =50 mm.
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et al., 1990; Nouwen and De Broe, 1994). The data obtained here demonstrated that the area triggered apoptosis and the site of renal injury induced in the kidney after HgCl2 administration to rats were found to be the same (Fig. 4), suggesting that these cell death forms are brought about within identical cell proximal tubules despite distinct morphological characteristics. Similar results were also obtained in the case of cadmium (Homma-Takeda et al., 1997b). It has been, however, reported that induction of renal apoptosis by sclerosis and hypoxia was observed in the glomeruli (Sugiyama et al., 1996) and in the medullary tubules (Beeri et al., 1995). Taken together, it was suggested that the induction site of renal apoptotic phenomenon seems to be stimuli-specific. Furthermore, we have found that the mercury administered is higher in the damaged proximal tubules than the undamaged proximal tubules, the distal tubules, and glomeruli in the kidney of rats exposed to HgCl2 (Homma-Takeda et al., 1998). Mercury-induced renal apoptosis also occurred in the site where the mercury was highly distributed (see Figs. 2, 4 and 7). In our preliminary study we observed that HgCl2 (4 mg/kg) also caused DNA fragmentation in the liver while showing minimal mercury accumulation, but the frequency of apoptotic cells in the liver was quite low compared to the kidney (Homma-Takeda, unpublished observation). Therefore, the site-specific induction of apoptosis and renal injury by mercury may be attributable to accumulation of the metal. In conclusion, apoptosis has been emphasized in studies of renal tubular injury during various nephropathy (Lieberthal and Levine, 1996; Savill, 1996; Ueda et al., 1997). Impairment of the cell membrane and mitochondrial dysfunction have been considered to be an important nephrotoxic action for mercury-induced renal injury (Weinberg et al., 1982; Goldstein and Schnellmann, 1994). Nevertheless, the present data suggest that the mechanistic details of apoptosis caused by inorganic mercury are required to understand the involvement of physiological alterations in metal-promoted renal damage.
Acknowledgements This work was supported by a Scientific Research Grant-in-Aid c 08770248 from the Ministry of Education, Science and Culture of Japan and by funding (University Research Project) from the University of Tsukuba. This study was performed under the approval of the Photon Factory Advisory Council (Proposal Nos. 96G095 and 96G081). We are grateful to Dr A. Iida, the Photon Factory, Institute of Material Structure Science, for his kind advice during the experiments at the Photon Factory.
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S. Homma-Takeda et al. / En6ironmental Toxicology and Pharmacology 7 (1999) 179–187 Liu, J., Liu, Y., Habeebu, S.S.M., Klaassen, C.D., 1998. Metallothionein (MT)-null mice are sensitive to cisplatin-induced hepatotoxicity. Toxicol. Appl. Pharmacol. 149, 24. Magos, L., Sparrow, S., Snowden, R.T., 1984. Effect of prolonged saline loading on HgCl2-induced renal tubular damage. Br. J. Exp. Path. 65, 567. Nagashima, K., Fujii, Y., Tsukamoto, T., et al., 1996. Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats. Acta Neuropathol. 91, 72. Nouwen, E.J., De Broe, M.E., 1994. Human intestinal versus tissuenonspecific alkaline phosphatase as complementary urinary markers for the proximal tubule. Kidney Int. 46, 43. Oberhammer, F., Wilson, J.W., Dive, C., et al., 1993. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679. Oberto, A., Marks, N., Evans, H.L., Guidotti, A., 1996. Lead (Pb + + ) promotes apoptosis in newborn rat cerebellar neurons: pathological implications. J. Pharmacol. Exp. Ther. 279, 435. Ray, S.D., Sorge, C.L., Kamendulis, L.M., Corcoran, G.B., 1992. Ca + + -activated DNA fragmentation and dimethylnitrosamineinduced hepatic necrosis: effects of Ca + + -endonuclease and poly (ADP-ribose) polymerase inhibitors in mice. J. Pharmacol. Exp. Ther. 263, 387. Savill, J., 1996. Apoptosis and renal scarring. Kidney Int. 49, 14. Savill, J., Fadok, V., Henson, P., Haslett, C., 1993. Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14, 131.
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Schwartzman, R.A., Cidlowski, J.A., 1993. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr. Rev. 14, 133. Shimojo, N., Homma, S., Nakai, I., Iida, A., 1991. Nondestructive synchrotron radiation X-ray fluorescence imaging of trace elements on methylmercury and selenium administered guinea pigs. Anal. Lett. 24, 1767. Shinyashiki, M., Kumagai, Y., Nakajima, H., et al., 1998. Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury. Brain Res. 798, 147. Stroo, W.E., Hook, J.B., 1997. Enzymes of renal origin in urine as indicators of nephrotoxicity. Toxicol. Appl. Pharmacol. 39, 423. Sugiyama, H., Kashihara, N., Makino, H., Yamasaki, Y., Ota, Z., 1996. Apoptosis in glomerular scleosis. Kidney Int. 49, 103. Thompson, S.W., 1966. Periodic acid – Schiff reaction. In: Thompson, S.W. (Ed.), Selected Histochemical and Histopathological Methods. Thomas, Springfield, IL, p. 480. Ueda, N., Kaushaland, G.P., Shah, S.V., 1997. Recent advances in understanding mechanisms of renal tubular injury. Adv. Ren. Replace. Ther. 4, 17. Xu, C., Johnson, J.E., Singh, P.K., Jones, M.M., Yan, H., Carter, C.E., 1996. In vivo studies of cadmium-induced apoptosis in testicular tissue of the rat and its modulation by a chelating agent. Toxicology 107, 1. Weinberg, J.M., Harding, P.G., Humes, H.D., 1982. Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. J. Biol. Chem. 257, 60. Whyllie, A.H., Kerr, J.F.R., Currie, A.R., 1992. Cell Death: the significance of apoptosis. Int. Rev. Cytol. 68, 251.
Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo Shino Homma-Takeda a,*, Yasuhiro Takenaka b, Yoshito Kumagai a, Nobuhiro Shimojo a a
Department of En6ironmental Medicine, Institute of Community Medicine, Uni6ersity of Tsukuba, 1 -1 -1 Tennodai, Tsukuba, Ibaraki 305 -8575, Japan b Master’s Program in En6ironmental Sciences, Uni6ersity of Tsukuba, 1 -1 -1 Tennodai, Tsukuba, Ibaraki 305 -8572, Japan Received 31 August 1998; received in revised form 24 February 1999; accepted 1 March 1999
Abstract A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl2). A single injection of HgCl2 to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl2 were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; DNA fragmentation; Synchrotron radiation X-ray fluorescence analysis; Mercuric chloride; Kidney; Rat
1. Introduction Apoptosis and necrosis are quite distinct forms of cell death with different morphology and implications for the surrounding tissue (Kerr et al., 1972; Whyllie et al., 1992; Corcoran et al., 1994). Necrosis, a passive or accidental cell death caused by noxious stimuli, is typified by cell and organelle swelling with spillage of the intracellular contents as well as loss of functional and structural integrity of the cell membrane and mitochondria, leading to injury of the surrounding tissue. In
Presented in part at the 4th International Symposium on Metal Ions in Biology and Medicine, Barcelona, Spain, 19–22 May 1996. * Corresponding author. Present address: Graduate School Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: + 81-298-53-3297, fax: +81-297-52-4015. E-mail address: [email protected] (S. Homma-Takeda)
contrast, apoptosis is a physiological mode of cell death designed to dispose of unwanted cells promptly. Nuclear shrinkage associated with nuclear condensation and enzymatic DNA cleavage into oligonucleosomesize fragments (DNA ladders) without changes of cell membrane and mitochondria is characteristic of apoptosis. Consequently, the resulting apoptotic bodies are phagocytosed by neighboring cells to prevent inflammation and damage to the surrounding tissue (Savill et al., 1993). A notion of metal-induced cell death has been currently changed because some reports suggested that cells from toxic-target organs can undergo apoptosis by a variety of toxic metals (i.e. cadmium, mercury, platinum, and lead) which are thought to cause necrosis (Kunimoto, 1994; Ishido et al., 1995; Duncan-Achanzar et al., 1996; Lieberthal et al., 1996; Oberto et al., 1996). From several observations with cultured cells the cell
1382-6689/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 9 9 ) 0 0 0 1 2 - 5
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death, whether via either apoptosis or necrosis, has been considered to depend on the concentration of toxicant: High concentration is necrosis and low concentration is apoptosis (Duncan-Achanzar et al., 1996; Lieberthal et al., 1996). However, little information about metal-promoted apoptosis in vivo has been reported (Nagashima et al., 1996; Xu et al., 1996; Liu et al., 1998), and the question of how apoptotic and necrotic phenomena occur during exposure of animals to mercury remains open. Thus, we show here evidence for the induction of apoptosis in the kidney in mercuric chloride (HgCl2)-treated rats, which is well recognized as a nephrotoxic model. A dose – response relationship in the induction of apoptosis and site-specificity of apoptosis and necrosis caused by mercury are also discussed.
2. Material and methods
2.1. Chemicals Chemicals were obtained as follows: HgCl2, nitric acid, sulfuric acid, potassium permanganate, stannous chloride, PAS reagent, and methylgreen from Wako Pure Chemical Industries, (Osaka, Japan); 3,3%-diaminobenzidine tetrahydro chloride (DAB) from Dojindo Laboratories (Kumamoto, Japan); Agarose, RNase A, proteinase K, and bisbenzamide from Sigma (St. Louis, MO, USA); Size markers for DNA agarose electrophoresis (HindIII-digested l-DNA and HaeIIIdigested fX174-DNA) from TOYOBO (Tokyo, Japan). All other chemicals used were of the highest grade commercially available.
2.2. Animals and treatments Male Wistar rats (7 weeks old) were obtained from Clea Japan, (Tokyo, Japan). The animals were allowed to acclimate in an environment of controlled temperature, humidity, and light/dark cycle for 1 week before study. All animal procedures received prior approval from the University Laboratory Animal Care and Use Committee and met the current local regulations. HgCl2 was administered to the rats by s.c. injection at doses of 0.5, 1, 2, or 4 mg/kg and the animals were housed in individual metabolic cages. For control, rats without any administration were used. Three, 7, 12, 16, 24, and 48 h after HgCl2 administration, the rats were killed by withdrawing approximately 7 ml of blood from the heart under ether anesthesia and the kidneys were removed. Urine samples were collected for 7, 12, 16, and 24 (0– 24 and 24 – 48) h after administration.
2.3. Measurements of mercury le6el in the kidney Kidney samples (200 mg) were digested with nitric acid, potassium permanganate, and sulfuric acid at room temperature. Then the mercury concentrations were measured as reported previously (Shinyashiki et al., 1998).
2.4. Synchrotron radiation X-ray fluorescence (SR-XRF) imaging of mercury in the kidney The two dimensional distribution of mercury in the kidney was clarified by SR-XRF analysis according to the established method as reported previously (Shimojo et al., 1991; Homma et al., 1993, 1995). The renal specimens were fixed in 10% buffered formalin (pH 7.4, 4°C), embedded in paraffin, cut to a thickness of 2 mm by a microtome (Yamato Kohki, Tokyo, Japan), and then subjected to SR-XRF analysis. XRF measurements for mercury was made at the Photon Factory of the Institute of Materials Structure Science (KEK-PF), Tsukuba, Japan, utilizing an energy dispersive SR-XRF system with monochromatic X-rays obtained by a Si(111) double crystal monochromator. Hg (La) was excited with 16 keV X-rays (beam size, 300 × 300 mm2). The X-ray intensity data of the Hg La fluorescence lines (counting times, 10 s/point) of each point obtained by step-scanning (200 mm/step) on the samples were processed with a personal computer and the results were shown as a tone from black to white classified into 14° from maximum to minimum, that is linearly proportional to the 14 levels of the element concentrations.
2.5. E6aluation of nephrotoxicity Alkaline phosphatase (ALP) activity in the urine was measured using an ALP assay kit (Wako, Osaka, Japan) by the method of Bessey et al. (1946). For histological examination, renal specimens (4 mm) were stained with periodic acid Schiff (PAS) reaction according to the method of Thompson (1966).
2.6. DNA fragmentation analysis Quantitation of DNA fragmentation was performed by the method of Ray et al. (1992) with modification. Briefly, the frozen kidney was homogenized with 5 volumes of a ice-cold lysis buffer (5 mM Tris–HCl (pH 8.0)–20 mM EDTA–0.5% Triton X-100). (The efficiency of DNA extraction from the tissues in the present method was improved 50 times over the method for our preliminary experiment; the extraction was performed by shaking the tissues in the lysis buffer for 1 h at 4°C (Homma-Takeda et al., 1997a).) The homogenates were centrifuged at 16 000× g for 20 min to separate intact chromatin and fragmented DNA. The
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pellets obtained (intact chromatin) were suspended in 0.5 N perchloric acid, heated at 80°C for 15 min, and then centrifuged at 16 000×g for 15 min to remove the protein. The resulting supernatants were used for DNA assay by a coloring reaction with diphenylamine (Burton, 1956) with calf DNA as the standard. Absorbance was measured at 600 nm with a Shimadzu UV-1600 double beam spectrophotometer (Kyoto, Japan). For the supernatants (fragmented DNA), substitution of phenol–chloroform (1:1, v/v) for perchloric acid to remove the protein resolved the problem of unidentified materials interfering with the assay. DNA extracted with phenol–chloroform were precipitated in isopropanol (overnight at − 80°C). The DNA was washed with 70% ethanol, redissolved in 0.5 N perchloric acid, and heated at 80°C for 15 min. Then resulting DNA sample was subjected to DNA assay. The recoveries of intact chromatin and fragmented DNA by the present method were 95.591.8 and 70.99 3.7%, respectively, (n= 4). Isolation of the DNA for agarose gel electrophoresis was performed by the same procedure described above. After precipitation in isopropanol, followed by washing with 70% ethanol, the pellets obtained were suspended in 10 mM Tris–HCl (pH 7.4) – 20 mM EDTA and then treated with RNase A (final concentration 2 mg/ml) for 2.5 h at 37°C. The sample was electrophresed on a 1.2% agarose gel containing ethidium bromide (0.5 mg/ml). Electrophoresis gels were illuminated with 312 nm light (Funakoshi, Tokyo, Japan) and a photographic record of the DNA ladders was made with instant film (Fuji Photofilm, Tokyo, Japan).
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medullary tubules in the specimen of positive control (staining of the medullary tubules were stronger than those of the glomeruli and cortical tubules). The renal section in the absence of exogenous terminal deoxynucleotidyl transferase (negative control) had no positive staining, suggesting the specific color reaction in the experimental condition (data not shown).
2.8. Chromatin condensation PAS-stained renal sections (4 mm) were treated with 0.5% Triton X-100 in PBS and incubated with 5 mM bisbenzamide for 10 min at room temperature. The nuclei were visualized using a fluorescence microscopy (LEITZ DMR, Leica Mikroskopie und Systeme, Germany).
2.9. Combination analysis of SR-XRF imaging and TUNEL staining TUNEL-stained renal sections (10 mm) was attached to mylar film and then subjected to SR-XRF analysis. The XRF measurements were performed with monochromatic a X-ray microbeam obtained with a multilayer monochromator and K-B type focusing optics (Iida and Noma, 1993) for Hg imaging under the following conditions: energy of X-rays, 14.38 keV; beam size, 5×5 mm2; step size, 5 mm/step; and counting time, 10 s/step. After SR-XRF, the specimens were counter-stained with Carrazzi’s hematoxylin. Minimal effect of TUNEL staining on Hg La detection was observed (data not shown).
2.7. Identification of apoptotic cells in the kidney
2.10. Statistics
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method (Gavrieli et al., 1992) was employed for renal sections with slight modification using a in situ apoptosis detection kit (Oncor, Gaitherburg, Germany). Sections (4 mm) were deparaffinized, rinsed with PBS, treated with proteinase K (20 mg/ml) to strip the nuclear proteins, and then quenched in endogenous peroxidases with 2% H2O2 in phosphate buffered saline (PBS). DNA 3%OHends were labeled with digoxigenin-conjugated dUTP. Bound digoxigenin was detected by incubation with peroxidase-conjugated antibody against digoxigenin at 37°C for 2 h and then developed with 50 mM Tris –HCl (pH 7.4)–0.1% DAB – 0.02% H2O2. After TUNEL staining, the specimens were counter-stained with 0.1 M sodium acetate–2% methylgreen (pH 4.0). For negative and positive controls of TUNEL staining, renal sections of control rats exposed to DNase (at room temperature for 10 min) in vitro were used. Intensive staining was seen in the nucli of all the type of renal cells, such as the glomeruli, and the cortical or
Data are expressed as the means9 S.D.s for each group. A t-test and one way analysis of variance (ANOVA) were carried out on a personal computer using a biomedical program (HALBAU, Japan). When statistically significant F-values (PB 0.05) were obtained with the ANOVA, and Bonferroni’s correction was used.
3. Results
3.1. Mercury distribution in kidney After s.c. injection of HgCl2 (2 and 4 mg/kg) to rats, accumulation of mercury in the kidney reached a maximum level at 7 h (2 mg/kg, 68.391.7; 4 mg/kg, 79.19 1.6), and then decreased gradually. These concentrations of mercury at 48 h were about half of those at 7 h (Fig. 1). The two-dimensional distributions of mercury in the kidney are shown in Fig. 2. Mercury distributed was higher in the renal cortex than in the
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Fig. 1. Time course of Hg concentration in kidney of the rats exposed to HgCl2. Rats were treated with HgCl2 (2 mg/kg () or 4 mg/kg ( ) by s.c. administration) and kidney mercury levels were measured from 3 to 48 h after administration. Zero h represents the control group. Each point is the mean 9 S.D. of four animals.
medulla. Upon further analysis, the renal distribution of mercury varied with time. For example, the mercury localized around the boundary between the cortex and the medulla at 48 h after HgCl2 administration (4 mg/kg) (Fig. 2D), while the metal accumulated all over the cortex at 16 h (Fig. 2B).
Fig. 3. Release of ALP in urine after exposure to HgCl2. d, 2 mg/kg; b, 4 mg/kg. ALP activities in urine samples collected for 7, 12, 16, and 24 (0 – 24 and 24 – 48) h after administration were measured. Data are expressed as percent of control obtained from each experimental period. Each point is the mean 9S.D. of four animals. * Significantly different from control, P B0.05.
3.2. Nephrotoxicity by mercury Renal damage caused by HgCl2 was inferred from the morphological changes in renal sections and the urinary excretion of ALP which is a marker enzyme of manifest proximal tubular injury by mercury (Stroo and Hook, 1997). No appreciable urinary excretion of ALP was seen after injection of HgCl2 at lower doses such as 0.5 or 1 mg/kg (data not shown). By contrast, exposure of the rats to HgCl2 at a dose of 2 mg/kg resulted in a significant leakage of ALP into the urine
Fig. 2. Mercury distribution in rat kidney. Renal specimens obtained at 16 (A) or 48 h (C) after injection of HgCl2 (4 mg/kg) were employed for SR-XRF imaging (B, D). The sample scanning condition was 40 × 48 (B, at 16 h) and 39 × 48 (D, at 48 h) steps with 200 mm/step. The X-ray intensity data of the Hg La fluorescence lines of each point are shown as a tone from black to white classified into 14 degrees from maximum to minimum. The mercury concentration range of the imaging was calculated using the mercury concentration in the kidneys determined by FAAS (see Fig. 1) as described previously (Homma-Takeda et al., 1998). Bar =2 mm.
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at 24 h after injection (Fig. 3). Increasing the dose of HgCl2 from 2 to 4 mg/kg made the occurrence of the urinary ALP excretion accelerate, indicating a dose-dependent renal injury caused by HgCl2. The morphological changes, such as a loss of brush border and cell loss, occurred at the inner zone of the cortex at 12 h after HgCl2 administration (4 mg/kg) as shown in Fig. 4E and F. At 48 h, severe injured tubules were noted particularly in the outer stripe as well as in many areas of the cortex (Fig. 4J and K).
3.3. Induction of apoptosis by mercury in the kidney The rate of fragmented DNA versus total DNA (percentage of DNA fragmentation) in the kidney of control rats was 0.3090.04% (n =4). After HgCl2 (4 mg/kg) was injected into the rats, the renal fragmented DNA increased at 7 h (1.4-fold the control level; DNA fragmentation, 0.4190.03%) (Fig. 5A). The apoptosis assessed by the fragmented DNA peaked at 16 h (12fold the control level; DNA fragmentation, 3.589 0.54%), and then decreased (48 h; DNA fragmentation, 1.439 0.12%). In the case of the lower dose (1 mg/kg), however, a significant induction of apoptosis by HgCl2 was found at 48 h. A good dose – response relationship was observed in the inorganic mercury-induced DNA fragmentation (0.5–4 mg/kg). DNA fragmentation was also confirmed by electrophoretic analysis (Fig. 5B). In spite of minimal leakage of ALP into the urine and morphological alterations, obvious DNA cleavage into oligonucleosome-size fragment was observed at 7 h after injection of HgCl2 (4 mg/kg). Such DNA ladders caused by HgCl2 was maintained for at least 48 h. When the rats were exposed to lower doses of HgCl2, the formation of DNA ladders was observed at 12 h (2 mg/kg), at 16 h (1 mg/kg), and at 48 h (0.5 mg/kg), respectively (data not shown). To detect apoptotic cells associated with the DNA fragmentation in situ, TUNEL staining was employed for the renal sections. In the specimen at 12 h after injection of HgCl2 (4 mg/kg), the proximal tubules, a toxic target region for mercury, contained apoptotic cells whereas the distal tubules, glomeruli, or medullary tubules did not (Fig. 4D and G). Examination of the nuclear morphology in the proximal tubules of the HgCl2-treated rat also gave a further characterization of apoptosis, chromatin condensation as shown in Fig. 6. The time course of the histochemical examination was parallel with that of DNA fragmentation as shown in Fig. 5A. Almost no renal apoptotic cells were found in the control rats (Fig. 4A). A similar phenomenon was also observed at 3 h after injection of HgCl2 (4 mg/kg, data not shown). Although there were a few TUNEL positive cells at 7 h after administration (data not shown), the apoptotic cells increased with time
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(Fig. 4D and H). Moreover, the TUNEL positive area was detected in the inner zone of the cortex (Fig. 4D), where tubular damage was also observed (Fig. 4E and F), and spread over the cortex with time (Fig. 4H). At 48 h the outer stripe showing necrotic damage (Fig. 4J and K) and was predominantly stained by TUNEL (Fig. 4I).
3.4. Mercury accumulation and apoptosis To attempt simultaneous determination of mercury accumulation and apoptotic cells in the same specimen of kidney after injection of HgCl2 (4 mg/kg) into the rats, the combination analysis of SR-XRF and TUNEL staining was performed (Fig. 7). Mercury accumulated more in the proximal tubules than in the glomeruli or the distal tubules. The mercury distribution was varied among the proximal tubules, but TUNEL-positive proximal tubules associated with induction of apoptosis were found to have higher mercury levels compared with the TUNEL-negative proximal tubules.
4. Discussion The present results indicated that a single s.c. injection of HgCl2 (2 or 4 mg/kg) into rats caused induction of not only necrosis but also apoptosis in the kidney in vivo in a dose-dependent fashion. Renal DNA fragmentation was detected earlier than urinary excretion of ALP, when the rats were treated with HgCl2 at a dose of 4 mg/kg. The time lag between induction of DNA fragmentation and leakage of ALP in the urine was longer in the case of the lower dose (2 mg/kg) than in the case of the higher dose (4 mg/kg). Although the occurrence of necrosis by a toxicant is generally recognized to be irreversible (Corcoran and Ray, 1992), the HgCl2-mediated apoptotic phenomena assessed by nuclear condensation and DNA fragmentation were transient exhibiting that fragmented DNA peaked at 16 h and then decreased. Minimal morphological changes were also observed at the beginning of the induction of DNA fragmentation, which is thought to be a later event of the apoptotic death process (Schwartzman and Cidlowski, 1993; Oberhammer et al., 1993) (see Fig. 4D–F and Fig. 5). Again, it was shown that necrotic renal tubules were found at least 8 h after the peak of the apoptotic event. From these results it seems likely that apoptosis caused by HgCl2 in vivo would be an early event compared with the necrotic phenomenon. Inorganic Hg is known to cause renal tubular damage mainly in the S3 segment of the proximal tubules, which is localized in the renal cortex and in the outer stripe of the outer medulla (Magos et al., 1984; Burfuss
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Fig. 4.
Fig. 7.
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Fig. 5. Formation of DNA fragmentation in kidney after mercury exposure. (A) quantitation of DNA fragmentation. DNA fragmentation is as expressed the percentage of fragmented DNA vs. total DNA (fragmented DNA +intact DNA). , 0.5 mg/kg; , 1 mg/kg; , 2 mg/kg; , 4 mg/kg. Zero h represents the control group. Each point is the mean 9 S.D. of four to five animals. Significant differences are indicated by the following letters; a, control vs. 4 mg/kg (PB 0.05); b, control vs. 2 mg/kg (PB 0.05); c, control vs. 1 mg/kg (P B0.05); d, control vs. 0.5 mg/kg (P B0.05). (B) DNA electrophoretic analysis. Fragmented DNA extracted from renal tissue (20 mg) of rats treated with HgCl2 (4 mg/kg) was loaded in agarose gel and stained with ethidium bromide. Lane 1, HindIII-digested l-DNA and HaeIII-digested fX174-DNA as molecular size marker; lanes: 2, control; 3, 3 h; 4, 7 h; 5, 12 h; 6, 16 h; 7, 24 h; and 8, 48 h.
Fig. 6. Effect of HgCl2 administration on morphological changes associated with apoptosis in the proximal tubules. Renal sections were stained with PAS reagent and bisbenzamide. DNA-fluorescence images (C, D) were photographed under fluorescence microscopy. Corresponding light microscopy of PAS staining (A, B) are also shown. (A) and (C) Control kidney. (B, D) At 12 h after HgCl2 administration (4 mg/kg). (D) The proximal tubules, identified by PAS-positive brush border (B), contain condensed nuclei (arrows). Bar =10 mm. Fig. 4. Light microscopy of renal sections. Serial tissue sections were stained with either TUNEL (A, D, G – I) or PAS reagent (B, E, F, J, K); photomicrographs were taken from same areas illustrated in C. (A and B) Control kidney. Almost no TUNEL-positive cells were found. (D–G) At 12 h after administration of HgCl2 (4 mg/kg). Corresponding to the results of histochemical staining with illustration of renal section (C), yellow-stained apoptotic cells (D, G) and morphological changes (E, F) were observed in the inner zone of the cortex. (H) At 24 h. Apoptotic cells were also found in the outer zone of the cortex. (I–K) At 48 h. Apoptotic cells localized at the inner stripe (I), where tubular injury was particularly noted (J, K); G, glomerulus; D, distal tubule; *, brush border-lost proximal tubules. Bar = 250 mm (A, D, E, H – J) and 50 mm (B, F, G, K). Fig. 7. Distribution of apoptotic cells and mercury in the kidney of the rat exposed to HgCl2. A renal section from the HgCl2-treated rat (4 mg/kg) at 12 h after injection was employed for the combined method of TUNEL staining (A) with SR-XRF imaging (B). Arrow heads, apoptotic cells; G, glomerulus; S, space, not tissue; arrow, distal tubule. (B) Mercury imaging by step-scanning (5 mm/step) on the renal section after TUNEL staining. Mercury concentration range for the XRF imaging with a tone from red to blue classified into 14 degrees was calculated using mercury concentration in the kidney determined by FAAS (see Fig. 1). Bar =50 mm.
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et al., 1990; Nouwen and De Broe, 1994). The data obtained here demonstrated that the area triggered apoptosis and the site of renal injury induced in the kidney after HgCl2 administration to rats were found to be the same (Fig. 4), suggesting that these cell death forms are brought about within identical cell proximal tubules despite distinct morphological characteristics. Similar results were also obtained in the case of cadmium (Homma-Takeda et al., 1997b). It has been, however, reported that induction of renal apoptosis by sclerosis and hypoxia was observed in the glomeruli (Sugiyama et al., 1996) and in the medullary tubules (Beeri et al., 1995). Taken together, it was suggested that the induction site of renal apoptotic phenomenon seems to be stimuli-specific. Furthermore, we have found that the mercury administered is higher in the damaged proximal tubules than the undamaged proximal tubules, the distal tubules, and glomeruli in the kidney of rats exposed to HgCl2 (Homma-Takeda et al., 1998). Mercury-induced renal apoptosis also occurred in the site where the mercury was highly distributed (see Figs. 2, 4 and 7). In our preliminary study we observed that HgCl2 (4 mg/kg) also caused DNA fragmentation in the liver while showing minimal mercury accumulation, but the frequency of apoptotic cells in the liver was quite low compared to the kidney (Homma-Takeda, unpublished observation). Therefore, the site-specific induction of apoptosis and renal injury by mercury may be attributable to accumulation of the metal. In conclusion, apoptosis has been emphasized in studies of renal tubular injury during various nephropathy (Lieberthal and Levine, 1996; Savill, 1996; Ueda et al., 1997). Impairment of the cell membrane and mitochondrial dysfunction have been considered to be an important nephrotoxic action for mercury-induced renal injury (Weinberg et al., 1982; Goldstein and Schnellmann, 1994). Nevertheless, the present data suggest that the mechanistic details of apoptosis caused by inorganic mercury are required to understand the involvement of physiological alterations in metal-promoted renal damage.
Acknowledgements This work was supported by a Scientific Research Grant-in-Aid c 08770248 from the Ministry of Education, Science and Culture of Japan and by funding (University Research Project) from the University of Tsukuba. This study was performed under the approval of the Photon Factory Advisory Council (Proposal Nos. 96G095 and 96G081). We are grateful to Dr A. Iida, the Photon Factory, Institute of Material Structure Science, for his kind advice during the experiments at the Photon Factory.
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