Pathways of Proximal Tubular Cell Death in Bismuth Nephrotoxicity

Toxicology and Applied Pharmacology 180, 100 –109 (2002) doi:10.1006/taap.2002.9379, available online at http://www.idealibrary.com on

Pathways of Proximal Tubular Cell Death in Bismuth Nephrotoxicity Berend T. Leussink,* ,† J. Fred Nagelkerke,‡ Bob van de Water,‡ Anja Slikkerveer,* ,§ Gijsbert B. van der Voet,* Anu Srinivasan,储 Jan A. Bruijn,† Frederik A. de Wolff,* and Emile de Heer† *Toxicology Laboratory and †Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; ‡Leiden Amsterdam Centre for Drug Research, Leiden, The Netherlands; §Yamanouchi Europe BV, Research Laboratories, Leiderdorp, The Netherlands; and 储Idun Pharmaceuticals, San Diego, California, 92121 Received October 22, 2001; accepted February 7, 2002

Pathways of Proximal Tubular Cell Death in Bismuth Nephrotoxicity. Leussink, B. T., Nagelkerke, J. F., van de Water, B., Slikkerveer, A., van der Voet, G. B., Srinivasan, A., Bruijn, J. A., de Wolff, F. A., and de Heer, E. (2002). Toxicol. Appl. Pharmacol. 180, 100 –109. Colloidal bismuth subcitrate (CBS), a drug for treatment of peptic ulcers, has been reported in the literature to be nephrotoxic in humans when taken in high overdoses. To investigate the mechanism of bismuth nephropathy, we developed an animal model by feeding rats single doses of CBS containing 3.0 mmol Bi/kg body weight. Terminal deoxyribonucleotidyl transferasemediated dUTP-digoxigenin nick end labeling assay, immunostaining for active caspase-3, and electron microscopy showed that proximal tubular epithelial cells die by necrosis and not by apoptosis within 3 h after CBS administration. Exposure of the renal epithelial cell lines NRK-52E and LLC-PK1 to Bi 3ⴙ in citrate buffer served as an in vitro model of bismuth nephropathy. NRK52E cells exposed to 100 ␮M Bi 3ⴙ or more died by necrosis, as was demonstrated by nuclear staining with Hoechst 33258 and flow cytometry using Alexa 488-labeled Annexin-V and the vital nuclear dye TOPRO-3. Bismuth-induced cell death of NRK-52E cells was not prevented by the caspase-3 inhibitor z-VAD-fmk, whereas this inhibitor did prevent cisplatinum-induced apoptosis. Mitochondrial dysfunction and induction of free radicals were shown not to be involved in bismuth nephrotoxicity. The early time point of damage induction in vitro as well as in vivo and the early displacement of N-cadherin, as found in previous studies, suggest that bismuth induces cell death by destabilizing the cell membrane. In conclusion, we showed that high overdose of bismuth induced cell death by necrosis in vivo as well as in vitro, possibly by destabilization of the cell membrane. © 2002 Elsevier Science (USA) Key Words: bismuth; kidney; proximal tubule; cell death; nephrotoxicity.

Bismuth may affect several target organs, depending on speciation, dose, and duration of exposure. Encephalopathy is the most life-threatening consequence of long-term oral exposure to some bismuth compounds, causing 72 fatalities during a toxic epidemic in France in the 1970s (Martin-Bouyer et al., 1980). An acute overdose with colloidal bismuth subcitrate 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

(CBS) results in nephrotoxicity because it induces cell death in proximal tubular epithelial cells (Leussink et al., 2001b). The aims of the experiments presented here are to investigate whether proximal tubular epithelial cells die by apoptosis or necrosis after bismuth exposure and to study the mechanism of bismuth-induced cell death. CBS is used to treat peptic ulcers and as part of a combination therapy to eradicate Helicobacter pylori, an important cause of peptic ulcers (Slikkerveer and de Wolff, 1989; Malfertheimer and Miehlke, 1997). Seven patients suffering from nephrotoxicity after a single oral overdose of CBS, varying from 7 to 25 times a daily therapeutic dose, have been reported in the literature (Islek et al., 2001; Akpolat et al., 1996; Stevens et al., 1995; Huwez et al., 1992; Taylor and Klenerman, 1990; Hudson and Mowat, 1989; Schweiberer and Vogelgesang, 1993). All patients except one (Huwez et al., 1992) needed transient hemodialysis. One patient died due to the complications of a duodenal ulcer (Taylor and Klenerman, 1990), whereas the other patients recovered without permanent loss of renal function. The mechanism of bismuth-induced nephropathy is largely unexplored. We therefore developed an animal model by feeding a single large CBS dose to rats, resulting in a severe but temporary reduction of the kidney function (Leussink et al., 2001b). Histological examinations showed that bismuth nephrotoxicity is caused by injury to and subsequent death of proximal tubular epithelial cells. Cell death was observed 3 h after CBS administration in the S3 segment and 12 h after CBS administration in the S1/S2 segment of the proximal tubule (Leussink et al., 2000). In the glomerulus and the more distal parts of the tubule, no cell death was observed. Further research showed that bismuth-induced proximal tubular epithelial cell death was preceded both by loss of homotypic cell– cell binding and relocation from the cell membrane to the cytoplasm of N-cadherin, a protein involved in intercellular adhesion. By contrast, expression patterns of other proteins playing a role in cell– cell adhesion, e.g., E-cadherin, ZO-1, and Desmoplakin, did not change under the influence of bismuth, suggesting that N-cadherin plays a specific role in bismuth nephrotoxity (Leussink et al., 2001a). In the current study, an in vitro model of bismuth nephrop-

100

BISMUTH NEPHROTOXICITY

101

FIG. 1. Kidney sections of rats with bismuth nephropathy stain positive with TUNEL and negative for active caspase-3. Paraffin-embedded adjacent tissue sections were stained with TUNEL (A–C) or with CM1, a rabbit polyclonal antibody specific for active caspase-3 (D–F), and counterstained with hematoxylin. In rat prostates obtained 3 days after testectomy (A and D) and ureter-obstructed rat kidneys obtained 9 days after unilateral ureter obstruction (B and E), cells positive for both TUNEL and CM1 were observed (arrowheads). Proximal tubular epithelial cells in the S3 segment of rats treated with an oral dose of CBS containing 3.0 mmol Bi/kg 3 h before, stain only with TUNEL (C) and not with CM1 (F). Magnification 400⫻.

athy was used in addition to the in vivo model described earlier to further investigate the mechanism of bismuth-induced tubular cell death. The in vitro model consisted of monolayers from the renal tubular epithelial cell lines NRK-52E and LLC-PK1, which were exposed to culture medium supplemented with Bi 3⫹ in citrate buffer. We show that bismuth induces cell death in proximal tubular epithelial cells through a necrotic pathway and not by apoptosis. Additional in vitro experiments show that deterioration of the mitochondrial function and the formation of free radicals are not involved in the mechanism of bismuthinduced cell death. MATERIALS AND METHODS Antibodies and Reagents Colloidal bismuth subcitrate, containing 35.4% (w/w) of the element bismuth, was donated by Yamanouchi Europe (Leiderdorp, The Netherlands). The CM1 rabbit polyclonal antibody against active caspase-3 was described earlier (Srinivasan, 1998). Biotinilated swine anti-rabbit IgG and peroxidaselabeled SABcomplex were provided by DAKO (Glostrup, Denmark). Finally, the sections were treated with peroxidase-labeled SABcomplex (DAKO). Alexa 488-labeled Annexin-V was prepared as described by Blom et al. (1999). Phosphate-buffered formaldehyde (3.8% w/v) at pH 7.4 was provided by Mallinckrodt Baker (Deventer, The Netherlands). The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay kit was supplied by Boehringer (Mannheim, Germany). Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were provided by Life Technologies (Paisley, Scotland). Streptomycin, penicillin, and glutamate were from ICN Biomedicals (Costa Mesa, CA); BiCl 3 and EPON812 were from Fluka (Neu-Ulm, Germany). HCl (37%), paraformaldehyde, glutarealdehyde, methyl butane, EDTA, NaCl, KCl, MgCl 2, CaCl 2, and

Triton X-100 were provided by Merck (Darmstadt, Germany). Vectashield was provided by Vector Laboratories (Burlingame, CA); TOPRO-3 iodide and z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-fmk) was from Molecular Probes (Eugene, OR); Hank’s buffer, N-2-hydroxyethylpiperazine-N⬘-2-ehanesulfonic acid, nicotinamide hypoxanthine dinucleotide, bovine serum albumin, diaminobenzidine (DAB), Hoechst 33258, cis-platinum(II)-diammine dichloride (CDDP), t-butylhydroperoxide (TBHP), Rhodamin 123, and N,N⬘-diphenyl-p-phenylenediamine (DPPD) were provided by Sigma (St. Louis, MO); glucose was from BDH (Poole, Dorset, England); trypsin was from BioWhittaker (Walkersville, MD); and glass cover slips (15 mm diameter) were provded by Omnilabo (Breda, The Netherlands). In Vivo Experiments Animals. Inbred female Wistar rats were obtained from the breeding facilities of the Department of Pathology at the Leiden University Medical Center. The animals were housed under a 12/12-h light/dark cycle in climatecontrolled chambers and had free access to tap water and rat chow (Hope Farms, Woerden, The Netherlands). The experiment was approved by the Animal Experiments Committee of Leiden University. At the start of the experiment, the animals were 11–12 weeks old and weighed 150 –200 g. Experimental design. Bismuth nephrotoxicity was induced by administrating a single CBS dose, freshly dissolved in 0.5 ml saline, by oral gavage. The dose contained 3.0 mmol Bi/kg body wt, which is comparable to 100 times the daily therapeutic dose for humans. This is the lowest dose that induces a considerable amount of renal damage in these rats (Leussink et al., 2001b). From 18 h before administration until the moment of euthanasia, the animals had access to water only. The animals were killed by puncture of the abdominal aorta with a heparinized syringe under anesthesia with a mixture of ketamin, fluanison, fentanyl, and droperidol. Both kidneys were perfused with phosphate-buffered saline. For cryostat sectioning, tissue samples were snap-frozen in methyl butane and stored at ⫺80°C. For paraffin sectioning, the kidney was perfused and fixed with phosphate-buffered formaldehyde (3.8%) at pH 7.4. Tissue samples were

102

LEUSSINK ET AL.

subsequently immersion fixed with formaldehyde for 24 h, transferred to 70% ethanol, and embedded in paraffin. Tissue for ultrathin sectioning for electron microscopy (EM) was obtained by perfusing the kidney with 1.0% glutarealdehyde in 0.1 M cacodylate buffer, pH 7.4, overnight immersion fixation with the same fixative, and then embedding in EPON812. Detection of cell death by TUNEL. TUNEL assay was performed on formalin-fixed paraffin-embedded kidney sections with the In Situ Cell Death Detection Kit of Boehringer according to the instructions of the manufacturer. Briefly, deparaffinized kidney sections were treated with 0.4% H 2O 2 in methanol for 20 min to block endogenous peroxidase. After washing with 100% ethanol, rehydrating, and washing with MilliQ, the sections were incubated in 0.01 M citrate buffer, pH 6.0, at 70°C for 10 min and then allowed to cool to 30°C. After washing with PBS, the sections were treated with a 10 ␮g/L proteinase K solution for 10 min before being washed with PBS again. TUNEL reagent, consisting of terminal deoxynucleotydyl transferase and fluoresceinlabeled nucleotides, was administrated on the sections for 1 h. Sections treated with TUNEL mix lacking the enzyme served as negative controls. The sections were washed with PBS and developed with DAB after incubation with peroxidase-labeled anti-fluorescein antibody Fab fragments for 1 h. The sections were counterstained with hematoxylin. Detection of apoptosis by active caspase-3 immunostaining. Active caspase-3 was detected in paraffin-embedded 3-␮m tissue sections using the CM1 rabbit polyclonal antibody (Srinivasan, 1998). Antigen retrieval was performed by boiling the deparaffinized sections for 10 min in 0.01 M citrate buffer at pH 6.0. Overnight incubation with the primary antibody, diluted at 1:2000, was followed after washing by a 1-h incubation with 1:400 diluted biotinilated swine anti-rabbit IgG. Finally, the sections were treated with peroxidase-labeled SABcomplex, with DAB as a chromogen. Negative control sections were treated similarly with omission of the primary antibody. Positive controls were prepared from sections of tissues known to contain apoptotic cells, such as prostrates of rats castrated 3 days before death (Perlman et al., 1999) and ureter-obstructed kidneys of rats that had undergone a unilateral ureter obstruction 9 days before death (Kennedy et al., 1997). Autometallography. The presence of bismuth in cryostat sections of the kidney was demonstrated with autometallography according to Danscher et al. (Danscher et al., 1997; Danscher and Moller-Madsen, 1985). This method is based on the ability of submicroscopic bismuth crystallites to cause precipitation of silver from a silver nitrate solution. The results were photographed by reflection contrast microscopy (RCM) as described by Prins et al. (1996). In Vitro Experiments NRK-52E cells, a cell line of rat renal tubular origin (De Larco and Todaro, 1978), were obtained from the American Type Culture Collection (ATCC, Rockville, MD). NRK-52E cells have been characterized as proximal tubular cells because they have a profile of collagen production consistent with that of proximal tubular cells, they secrete C-type natriuretic peptide, and they express receptors for epidermal growth factor (EGF) without expressing EGF itself (Walker et al., 1991; Creely et al., 1992; Suzuki et al., 1997). LLC-PK1, a cell line of porcine renal tubular origin (Hull et al., 1976), was also obtained from the ATCC. LLC-PK1 cells have also been characterized as proximal tubular cells, because they express the endocytosis receptor megalin (Nielsen et al., 1998). NRK-52E cells were used in passages 20 to 35 and LLC-PK1 cells were used in passages 206 to 230. Both cell lines were cultured in DMEM supplemented with 50 ␮g/ml streptomycin, 50 U/ml penicillin, and 2 mM glutamate. FCS (5%) was added for NRK-52E cells and 10% FCS for LLC-PK1 cells. FCS was heat-inactivated by incubating it for 30 min at 56°C. Cells were grown at 37°C in a humid atmosphere containing 5% CO 2. Before the start of an experiment, monolayers of LLC-PK1 or NRK-52E cells were growth arrested by incubating them for 24 h in DMEM without FCS. During all experiments, DMEM supplemented with streptomycin, penicillin, and glutamate but not with FCS was used as incubation medium. Bismuth-containing medium was prepared by diluting a 1:100 stock with DMEM immediately

FIG. 2. Autometallography on kidney sections. Autometallography according to the method of Danscher (Danscher et al., 1997; Danscher and Moller-Madsen, 1985) was performed on a cryostat kidney section obtained from a rat 3 h after oral administration of either 0.5 ml PBS (A) or a nephrotoxic dose of CBS containing 3.0 mmol Bi/kg body wt (B). White shining grains of silver (B, arrows), indicating the presence of bismuth, were observed in the proximal tubular epithelial cells of bismuth-treated rats (B), whereas they were absent in control rats (A). The sections were counterstained with hematoxylin and photographed with RCM. Magnification 1000⫻.

before the start of the experiment. The 1:100 stock was made freshly by diluting 2 M BiCl 3 in 37% HCl with 1 M citrate buffer, pH 6.3, and water until a concentration of 100 times the final Bi 3⫹ concentration was reached in 160 mM citrate buffer. Citrate buffer was needed to prevent the immediate precipitation of bismuth. Nuclear morphology. To investigate the effect of Bi 3⫹ on nuclear morphology, monolayers of NRK-52E or LLC-PK1 cells grown on glass coverslips (15 mm diameter) were fixed by a 10-min incubation with 4% paraformaldehyde, washed three times with PBS, and stained with Hoechst 33258 DNA dye (10 ␮g/ml in PBS) for 10 min. After washing three times with PBS again, the coverslips were mounted on an object slide using Vectashield. A

BISMUTH NEPHROTOXICITY

103

FIG. 3. EM of kidney sections of bismuth-treated rats and control rats. Kidneys were obtained from either control rats (A and B) or rats that had received a nephrotoxic CBS dose containing 3.0 mmol Bi/kg body wt 1 h before (C and D) and were prepared for EM as described under Materials and Methods. The rectangle in A indicates the area shown enlarged in B. The rectangle in D indicates the area shown enlarged in the inset at the lower left corner. One hour after CBS administration, vacuoles had started to form at the basal side of the cell. No abnormalities were seen in the nucleus, in the mitochondria (D, inset, arrows point to the normal-appearing cristae), and at homotypic cell– cell adhesion sites at this time point. Magnifications A, 900⫻; B, 8100⫻; C, 900⫻; D, 8100⫻; inset in D, 14,200⫻.

fragmented nucleus was considered representative for apoptosis; a shrunken nucleus was considered representative for necrosis. Treatment of the cells with CDDP, an apoptosis-inducing platina-containing cytostatic pharmaceutical (Lieberthal et al., 1996), was used as a positive control. Caspase 3 inhibition studies. To further investigate whether apoptosis was responsible for bismuth-induced cell death, NRK-52E cells cultured on glass slides were exposed to 1 ␮M z-VAD-fmk, an inhibitor of caspase-3, a protease involved in all known apoptotic pathways (Wolf and Green, 1999). z-VAD-fmk exposure started 30 min before exposure to Bi 3⫹ or CDDP and lasted until the end of the experiment. Vital, necrotic, and apoptotic cells were scored after nuclear staining with Hoechst 33258. Flow cytometry. Apoptosis was detected by using Alexa 488-labeled Annexin-V, TOPRO-3, and a fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, CA). Cells were washed twice with Hank’s buffer before being trypsinized. The culture medium and the washes were collected in the same tube as the trypsinized cells. After trypsinization, cells were left to recover for 30 min at 37°C in DMEM containing 5% FCS. Afterward, the cells were washed twice with PBS containing 1 mM EDTA. The cells were taken up

in Annexin-V binding buffer (10 mM N-2-hydroxyethylpiperazine-N⬘-2ehanesulfonic acid, 145 mM NaCl, 5 mM KCl, 1 mM MgCl 2, and 1.8 mM CaCl 2). Alexa 488-labeled Annexin-V was added 10 to 15 min before measurement and the vital dye TOPRO-3 iodide was added 5 min before measurement. Cells excluding both Annexin-V and TOPRO-3 iodide were regarded as vital cells, cells positive for Annexin-V but excluding TOPRO-3 iodide were regarded as apoptotic cells, and cells positive for both Annexin-V and TOPRO-3 iodide were regarded as necrotic cells. LD release. To determine the effect of Bi 3⫹ on the integrity of the cellular membrane, the release of the cytoplasmic enzyme lactate dehydrogenase (LD) into the culture medium was measured according to Bergmeyer et al. (1986). NRK-52E or LLC-PK1 cells (8 ⫻ 10 3) were seeded in each well of a 96-well culture dish and grown until they reached near-confluence. The cells were growth arrested for 24 h by removing the FCS from the culture medium. Bismuth was added afterward as described previously. At the end of the experiment, LD activity was determined in the culture medium by measuring the decrease of NAPH with a spectrophotometric method using an ELISA plate reader. LD activity was expressed as a percentage of the maximum LD activity

104

LEUSSINK ET AL.

FIG. 4. Bismuth induces necrotic cell death of the renal epithelial cell line NRK-52E. NRK-52E cells were exposed to 0 ␮M Bi 3⫹(A and D), 400 ␮M Bi 3⫹(B and E), or 200 ␮M CDDP (C and F) for 24 h either with (D–F) or without (A–C) 1 ␮M z-VAD-fmk, an inhibitor of the protease caspase-3, which is involved in every apoptotic pathway. The nuclear morphology was visualized with Hoechst 33258. Exposure to 400 ␮M Bi 3⫹ induced shrinkage of the nuclei, indicating necrotic cell death (B), whereas incubation with 200 ␮M CDDP resulted in the fragmentation of the nucleus, a hallmark of apoptosis (C). Addition of z-VAD-fmk to the culture medium 1 h before incubation with Bi 3⫹ or CDDP started did not affect cells treated with 0 ␮M Bi 3⫹ (D) and had no effect on the cell death induced by exposure to 400 ␮M Bi 3⫹ (E), whereas cisplatin-induced apoptosis was prevented (F). Magnification 1000⫻. as provoked by adding 0.1% (final concentration) Triton X-100 detergent to the medium. Mitochondrial function. The effect of Bi 3⫹ exposure on the mitochondrial function of NRK-52E was determined by measuring the ability of the cells to accumulate the fluorescent dye Rhodamin-123 by flow cytometry. NRK-52E cells cultured in six-well culture dishes were trypsinized and taken up in Hank’s buffer supplemented with 5 mM glucose, 0.2% (w/v) bovine serum albumin, 1 ␮M Rhodamin-123, and 10 nM TOPRO-3 iodide. After incubation at 37°C for 15 min, the trypsinized cells were pelletted and resuspended in Hank’s buffer supplemented with 5 mM glucose, 0.2% (w/v) bovine serum albumin, 0.2 ␮M Rhodamin-123, and 10 nM TOPRO-3 iodide. The resuspended cells were analyzed by flow cytometry. The mean Rhodamin-123 uptake by TOPRO-3 iodide negative cells was used as a measure for mitochondrial function. Antioxidants. In order to investigate whether the deleterious effects of Bi 3⫹ were mediated by free radicals, NRK-52E cells were exposed to 0, 100, or 400 ␮M Bi 3⫹ in the absence or presence of 20 ␮M DPPD. A 6-h incubation with 0.5 mM TBHP was used as a positive control (Kakkar et al., 1996). The effect on the survival of the cells was determined by flow cytometry. Calculations and statistics. All data are expressed as means ⫾ SD. A statistical analysis was carried out using SPSS 7.5.2 (SPSS, Chicago, IL). Means of parameters were compared with a paired or unpaired Student’s t test. A p value ⬍ 0.05 was considered to indicate statistical significance.

RESULTS

Bismuth-Induced Cell Death in Vivo Prostates of castrated rats (Fig. 1A), kidneys of ureterobstructed rats (Fig. 1B), and kidneys of rats that had received

a nephrotoxic dose of CBS orally (Fig. 1C) all stained positive with the TUNEL method, indicating DNA strand breaks. Only prostates of castrated rats and kidneys of ureter-obstructed rats also stained positive with CM1 (Figs. 1D and 1E), an antibody specific for active caspase-3, whereas the kidneys of CBStreated rats did not (Fig. 1F). Bismuth Accumulation in Renal Tubular Epithelium With autometallography and RCM, white shining silver grains were detected in the S3 segment of proximal tubules of rats 3 h after CBS administration (Fig. 2B). No grains were present in kidney sections of control rats (Fig. 2A). Silver grains were already visible 1 h after CBS administration (not shown). Despite the positive autometallography results, no bismuth could be demonstrated in ultrathin sections obtained from the same kidney using microanalysis (not shown). Further analysis by EM showed vacuolation at the basal side of the proximal tubular epithelial cell 1 h after CBS administration (Fig. 3C). In addition, normal mitochondria (Fig. 3D, inset), condensed chromatin (Fig. 3D), and damaged brush borders (Fig. 3C) were observed. Electron-dense particles and typical ultrastructural signs of apoptosis, such as fragmented nuclei and apoptotic bodies, were absent.

BISMUTH NEPHROTOXICITY

105

TABLE 1 The Caspase-3 Inhibitor z-VAD-fmk Does Not Diminish Bismuth-Induced Cell Death Percent of total no. of cells

Vital

Necrotic

Apoptotic

0 ␮M Bi 3⫹ 400 ␮M Bi 3⫹ 200 ␮M CDDP 0 ␮M Bi 3⫹ 400 ␮M Bi 3⫹ 200 ␮M CDDP 0 ␮M Bi 3⫹ 400 ␮M Bi 3⫹ 200 ␮M CDDP

0 ␮M z-VAD-fmk

SD

1 ␮M z-VAD-fmk

SD

98.9 3.9 75.3 0.2 85.3 0.0 0.9 10.8 24.7

0.6 2.4 4.2 0.3 2.1 0.0 0.3 1.7 4.2

99.8 4.9 69.6 0.2 95.1 30.4 0.0 0.0 0.0

0.3 3.3 2.9 0.3 3.3 2.9 0.0 0.0 0.0

Note. NRK-52E cells were exposed to 0 ␮M Bi 3⫹, 400 ␮M Bi 3⫹, or 200 ␮M CDDP for 24 h with or without 1 ␮M z-VAD-fmk. Nuclei were visualized by staining them with Hoechst 33258 and counted in three microscopic fields at 400⫻ magnification. The number of cells was expressed as a percentage of the total number of cells ⫾ SD. No apoptotic cells, i.e., cells with fragmented nuclei, were observed after treatment with z-VAD-fmk. The percentage of necrotic-appearing cells, i.e., cells with a shrunken nucleus, observed after treatment with 400 ␮M Bi 3⫹ was not diminished by z-VAD-fmk and must therefore consist of truly necrotic cells.

In Vitro Results Exploration of the cell death pathway by exposing renal epithelial cell lines to Bi 3⫹. Exposing NRK-52E cells to 400 ␮M Bi 3⫹ for 24 h resulted in shrunken nuclei, a phenomenon indicative of necrotic cell death (Fig. 4B). Treatment for 24 h with 200 ␮M CDDP, a cytostatic pharmaceutical that induces apoptosis (Lieberthal et al., 1996), resulted in fragmented nuclei (Fig. 4D), indicating that NRK-52E cells were able to undergo apoptosis. Coincubation with z-VAD-fmk, an inhibitor of caspase-3 that is involved in every apoptotic pathway, did not prevent bismuth-induced cell death (Fig. 4E), whereas cisplatin-induced apoptosis was inhibited (Fig. 4F). Similar results were obtained with LLC-PK1 cells (not shown). Quantitative measurement of the effect of z-VAD-fmk on bismuth- and CDDP-induced cell death confirmed that caspase-3 inhibition could not diminish bismuth-induced cell death of NRK-52E cells (Table 1). Flow cytometry of bismuth-exposed NRK-52E cells incubated with Alexa488-labeled Annexin-V and the vital dye TOPRO-3 iodide showed that only cells that had taken up TOPRO-3 iodide, and thus had lost their membrane integrity, could bind Annexin-V (Fig. 5). Effect of exposure time and concentration on bismuth-induced cell death. Bismuth concentrations of 66 ␮M or more were able to raise the percentage of dead cells above the control level, whereas the number of dead cells observed at 33 ␮M Bi 3⫹ or less did not differ from that at 0 ␮M Bi 3⫹ (Fig. 6). Bismuth exposures lasting 9 h did not induce cell death in NRK-52E cells above the control level, whereas exposures

FIG. 5. The effect of bismuth on the integrity and Annexin-V-binding capacity of NRK-52E cells. Typical FACS results of trypsinized NRK-52E cells after exposure to 0 ␮M Bi 3⫹ (A) or 100 ␮M Bi 3⫹ (B) for 18 h. Each dot represents a cell. The ability of a single cell to bind Alexa 488-labeled Annexin-V is displayed on the horizontal axis and its ability to take up the vital dye TOPRO-3 iodide is displayed on the vertical axis. Two populations can be discerned after bismuth exposure: vital cells (lower left) and dead cells that take up TOPRO-3 and bind Annexin-5 (upper right).

lasting 12 h or longer at concentrations of 100 and 400 ␮M resulted in the death of extra NRK-52E cells (Fig. 7). Effect of Bi 3⫹ exposure on destabilization of the cell membrane of LLC-PK1 and NRK-52E cells. The exposure of NRK-52E cells to 50 ␮M Bi 3⫹ or more for 41 h resulted in a significant increase in the amount of LD in the medium, indicating a partial loss of membrane integrity (Fig. 8). After 12 h of exposure, LD release was significantly less than in the control setting. We checked whether this was caused by an inhibitory effect of Bi 3⫹ on the enzyme activity of LD, but this appeared not to be the case (data not shown). The same experiment performed with LLC-PK1 cells showed similar results (not shown). Effect of bismuth exposure on the mitochondrial function. The possible effect of Bi 3⫹ on the mitochondrial function was monitored by its ability to take up the fluorescent dye Rho-

FIG. 6. Bismuth-induced cell death of of NRK-52E cells is concentration dependent. After 24 h of growth arrest, monolayers of NRK-52E cells were exposed to 0 – 400 ␮M Bi 3⫹ for 24 h. Flow cytometry was performed on trypsinized cells after incubation with Alexa 488-labeled Annexin-V and TOPRO-3. Cells taking up TOPRO-3 and binding Annexin-V were regarded as dead cells. Exposure to 66 ␮M Bi 3⫹ or more resulted in an increase of the percentage of dead cells above the control level.

106

LEUSSINK ET AL.

FIG. 7. Bismuth-induced cell death of NRK-52E cells is time dependent. After 24 h of growth arrest, monolayers of NRK-52E cells were exposed to 0, 100, or 400 ␮M bismuth for the indicated number of hours. Flow cytometry was performed on trypsinized cells after incubation with Alexa 488-labeled Annexin-V and TOPRO-3 iodide as described under Materials and Methods. Cells binding Annexin-V and taking up TOPRO-3 iodide were regarded as dead cells. The bars represent the percentage of dead cells. After 12 h of incubation, the number of dead cells had increased in bismuth-exposed cells compared to nonexposed cells, an effect that had increased after 18 h of exposure.

damin-123. This dye can easily enter the cell and accumulates in functioning mitochondria because of their positive membrane potential. Exposure to 10 mM of the chemical anoxiainducing agent sodium azide for 30 min diminished Rhodamin123 uptake by 18% as measured by flow cytometry (Fig. 9). Bismuth exposure did not decrease Rhodamin-123 uptake. Role of free radicals in the toxicity of bismuth. The ability of bismuth to stimulate the generation of free radicals was assessed in vitro by making use of the antioxidant DPPD.

FIG. 9. Effect of bismuth exposure on the mitochondrial function of NRK-52E cells. Monolayers of NRK-52E cells were grown in six-well plates. After reaching confluency, the monolayers were growth arrested for 24 h and subsequently exposed to 0, 10, 100, or 400 ␮M Bi 3⫹ for 24 h. Trypsinized cells were incubated with Hank’s buffer containing 5 mM glucose, 0.2% BSA, and 1 ␮M Rhodamin-123 (R-123), a dye accumulating in functional mitochondria, for 15 min at 37°C. After pelleting, the cells were taken up in the same buffer but with 0.2 ␮M R-123 and 10 mM TOPRO-3 iodide and subjected to flow cytometry. The bars represent the mean ⫹ SD of the R-123 uptake of TOPRO-3-excluding cells. The experiment was performed in triplicate. Cells treated with 10 mM NaN 3 at 37°C for 30 min to induce chemical anoxia served as positive controls. The bismuth concentration had no effect on the uptake of R-123 by surviving cells.

DPPD was able to prevent cell death caused by TBHP, which is mediated by free radicals (Kakkar et al., 1996), but did not diminish bismuth-induced cell death of NRK-52E cells (Fig. 10). DISCUSSION

The aim of these experiments was to study whether proximal tubular epithelial cells die by apoptosis or necrosis after bismuth exposure and to further elucidate the mechanism of

FIG. 8. Effect of bismuth exposure on LD release by NRK-52E cells. Monolayers of NRK-52E cells were grown in 96-well plates. After reaching confluency, the monolayers were growth arrested for 24 h and subsequently exposed for 12, 24, or 41 h to 0, 10, 50, 100, or 400 ␮M Bi 3⫹. The release of the cytoplasmic enzyme LD was measured enzymatically as described under Materials and Methods. The experiment was performed in triplicate. The bars represent the mean ⫹ SD of the LD activity in the medium as a percentage of the amount of LD activity measured after adding 0.1% Triton X-100 to the medium. [staro]Values testing significantly different from the 0 ␮M Bi 3⫹ value of the same incubation period with the Student’s t test. Exposure to 50 ␮M Bi 3⫹ or more increased the LD release significantly after 41 h of exposure but not after 24 h or less.

FIG. 10. Role of free radicals in bismuth-induced cell death. Monolayers of NRK-52E cells were grown in six-well plates. After reaching confluency, the monolayers were growth arrested for 24 h and subsequently exposed to 0, 100, or 400 ␮M Bi 3⫹ for 12 h or to 0.5 mM TBHP for 6 h. The incubations were performed either with or without 20 ␮M DPPD as an antioxidant. Flow cytometry was performed on trypsinized cells after incubation with Alexa 488labeled Annexin-V and TOPRO-3 iodide as described under Materials and Methods. Cells positive for Annexin-V and TOPRO-3 iodide were regarded as dead cells. The bars represent the percentage of dead cells. DPPD could only decrease the percentage of cell death induced by TBHP incubation and not that induced by Bi 3⫹ incubation.

BISMUTH NEPHROTOXICITY

bismuth-induced cell death. Necrotic cell death is known to result in loss of cell membrane integrity and subsequent leakage of cellular contents, such as LD, into the direct environment, attracting inflammatory cells that may contribute to additional tissue damage. Cells can also terminate their existence by apoptosis (Green, 1998). In this case, the cell dissolves into small vesicles that are absorbed by neighboring cells. The membrane of apoptotic cells initially remains intact, which prevents it from uptaking vital dyes, such as TOPRO-3 iodide or propidium iodide. Bismuth-Induced Cell Death in Proximal Tubular Epithelial Cells Apoptotic cell death is accompanied by intranucleosomal DNA strand breaks, which can be demonstrated with the TUNEL method. Tissues known to contain apoptotic cells, such as prostrates of rats 3 days after testectomy (Perlman et al., 1999) and ureter-obstructed kidneys of rats that had undergone a unilateral ureter obstruction 9 days before death (Kennedy et al., 1997), stained positive with the TUNEL method. Proximal tubular epithelial cells in the kidneys of rats exposed to an oral nephrotoxic dose of CBS 3 h before death were also TUNEL positive, suggesting that the cells had died by apoptosis (Fig. 1). However, it was recently found that necrotic as well as apoptotic cells contain DNA strand breaks and that they are therefore both TUNEL positive (GraslKraupp et al., 1995; Labat-Moleur et al., 1998; Taimor et al., 1999; Ferrer, 1999). This means that, although the TUNEL method can be used to demonstrate cell death, it cannot be used to distinguish apoptosis from necrosis. Antibodies specific for the active form of caspase-3 can be used to discriminate apoptotic cells immunohistochemically in histologic tissue sections (Srinivasan, 1998). Caspase-3 is a member of a family of proteases, called caspases, that cleave proteins after aspartate residues and that play an indispensable role in apoptosis. Caspases can be subdivided in initiator and effector caspases. Caspase-3 is an effector caspase (Wolf and Green, 1999). Its inactive form is present in every cell; its active form is present only in apoptotic cells. Proximal tubular epithelial cells of rat kidneys with bismuth nephropathy did not stain positive with CM1, an antibody specific for the activated form of caspase-3, whereas tissues known to contain apoptotic cells (prostrates of castrated rats and ureter-obstructed kidneys) stained positive with this antibody, confirming the specificity of the CM1 antibody for apoptotic cells. The absence of activated caspase-3 in the kidneys of bismuth-treated rats suggested that bismuth nephropathy is the result of necrosis and not of apoptosis of proximal tubular epithelial cells. The EM results confirmed this conclusion because signs of apoptotic cell death, such as fragmented nuclei and swollen mitochondria, were lacking in proximal tubular epithelial cells of rats with bismuth nephropathy (Fig. 3). The capability of proximal tubular epithelial cells to undergo apoptosis was demonstrated

107

in experiments in which rats were treated with a nephrotoxic dose of CDDP, a platinum-containing cytostatic (Lieberthal et al., 1996). This justifies the conclusion that bismuth can induce necrotic cell death of proximal tubular epithelial cells in a specific way that is not the result of the incapability of the cells to undergo apoptosis. Bismuth-Induced Cell Death in Cell Lines of Renal Epithelial Origin To elucidate the mechanism of bismuth-induced cell death, we developed an in vitro model of bismuth-induced cell death by exposing two renal epithelial cell lines, NRK-52E and LLC-PK1, to Bi 3⫹. Because experiments with both cell lines produced similar results, only the results of experiments with the NRK-52E cell line will be discussed. Nuclear morphology is generally accepted as the gold standard to demonstrate apoptosis. NRK-52E cells exposed to toxic concentrations of Bi 3⫹ did not show nuclear fragmentation after staining with the DNA-binding dye Hoechst 33258 (Fig. 4); therefore, we concluded that the cells had died through necrosis. This conclusion was supported by the fact that z-VAD-fmk, an inhibitor of caspase-3, did not prevent bismuth-induced cell death (Table 1, Fig. 4). Apoptotic cells can also be distinguished from other cells by the appearance of phosphatidylserine residues at the outside of the cellular membrane (Fadok et al., 1992). This feature is used to label apoptotic cells in flow cytometric experiments by Alexa 488- labeled Annexin-V, a protein capable of binding specifically to phosphatidylserine. Apoptotic cells differ from necrotic cells in their ability to bind Annexin-V and to exclude vital dyes at the same time. Because necrotic cells have lost their membrane integrity, they cannot bind Annexin-V without staining for vital dyes. Flow cytometry experiments also provided evidence that bismuth-treated cells died through necrosis because no cells excluding the vital dye TOPRO-3 and binding Alexa 488-labeled Annexin-V were observed after bismuth exposure (Fig. 5), indicating that phosphatidyl serine was absent in the outer leaflet of the cellular membrane, a hallmark of apoptotic cells. These findings taken together justify the conclusion that bismuth induces necrosis in vitro as well as in vivo. Mechanism of Bismuth-Induced Tubular Cell Death After having demonstrated that bismuth induces necrosis in vitro and in vivo, we wanted to know whether the accumulation of bismuth in certain cell organelles could explain the destructive effect of bismuth on proximal tubular epithelial cells. Autometallography makes bismuth microcrystallites in tissues visible as silver grains. The number of grains observed in the kidneys of animals that had received a single oral dose of CBS containing 3.0 mmol Bi/kg 3 h before death (Fig. 2) was relatively low compared to the number of grains found in morphologically normal kidneys of mice exposed subchroni-

108

LEUSSINK ET AL.

cally to the metallic form of bismuth for 9 weeks by implanting five bismuth shotgun pellets into their abdominal cavities (Pamphlett et al., 2000). Unfortunately, the grains were not confined to recognizable cell organelles. The relatively small number of grains found after acute bismuth intoxication seems to contradict the considerable tubular damage and the accumulation of bismuth demonstrated in a previous study using atomic absorption spectrometry (Leussink et al., 2000). This paradox may be solved by the assumption that acute CBS intoxication results only in a moderate accumulation of bismuth or that the main chemical form of the accumulation is not detected by autometallography. Bismuth could not be demonstrated by microanalysis of ultrathin sections of necrotic tubules from bismuthtreated rats, a method that successfully demonstrated bismuth in the kidney after subcutaneous injections of bismuth subnitrate (Ghadially, 1979). We were unable, however, to detect in which cell organelle bismuth accumulated after acute bismuth exposure, perhaps because bismuth had already left the cell or its remains after exerting its harmful effects. The question may be raised whether there is a direct relation between the intracellular amount of bismuth and the renal damage in bismuth nephropathy. For example, the passing of bismuth through the lumen of the tubule may be enough to lethally harm the cellular membrane of proximal tubular cells. This assumption is supported by the fact that the amount of bismuth that passes the kidney during the first 6 h after CBS intoxication correlates much better than the bismuth content of the kidney with renal function (Leussink et al., 2001b). Release of LD in the culture medium is a measure for membrane integrity and thus for cell death. Surprisingly, bismuth was shown to decrease LD release from NRK-52E cells after 12 h of exposure to 50 ␮M Bi 3⫹ or more (Fig. 8). An in vitro test showed that this effect could not be attributed to the inhibition of LD activity by bismuth. We have no explanations for this as yet unclarified phenomenon. LD release increased after 41 h of exposure to 50 ␮M Bi 3⫹ or more (Fig. 8). This is much later than the 12 h predicted by flow cytometric results (Fig. 7). This difference may be explained by assuming that bismuth-treated cells suffer much more from the necessary trypsinization than untreated cells, causing LH release, of which the measurement does not require trypsinization, to occur later than the loss of membrane integrity detected by flow cytometry. The minimum concentrations of bismuth needed to lower the percentage of surviving cells in flow cytometry and to increase the amount of released LD were essentially the same (66 and 50 ␮M, respectively). The fact that undamaged cells found in flow cytometry experiments do not release LD supports the hypothesis that cells treated with a cytotoxic dose of CBS are easily damaged by trypsinization. Bismuth did not affect mitochondrial function of renal tubular epithelial cell lines (Fig. 9), a result that is in agreement with the normal ultrastructural appearance of mitochondria in

proximal tubular epithelial cells of rats with bismuth nephropathy (Fig. 3). Bismuth did not increase the amount of free radicals present in renal tubular epithelial cell lines (Fig. 10). This result was to be expected because bismuth, not being a transition metal, is unable to induce the formation of free radicals. Bismuth also seems to be unable to displace transition metal ions from their intracellular storage places, since this would also result in free radical damage. Short exposure (3 h or less) of NRK-52E cells to 100 ␮M Bi 3⫹ or more is enough to induce cell death 9 h after the start of the exposure (results not shown). This suggests that the initial toxic events take place directly, or at least within 3 h, after the start of bismuth exposure, leading to cell death more than 9 h later. The cells cannot be rescued by removing bismuth from the medium. This phenomenon may be explained by assuming that Bi 3⫹ precipitates from the bismuth citrate solution on the cells, causing damage to the membrane. In vivo, the early displacement of N-cadherin may also indicate that bismuth exposure destabilizes the cellular membrane leading to cell death (Leussink et al., 2001a). This proposed mechanism of bismuth-induced cell death would explain why the amount of bismuth flowing through the kidney is much more related to renal damage than the amount retained in renal tissues (Leussink et al., 2001b). In conclusion, the experiments presented here demonstrate that high overdose of bismuth induces cell death by necrosis and not by apoptosis, possibly by destabilizing the cellular membrane. Further research is needed to unravel the exact mechanism of bismuth-induced cell death. ACKNOWLEDGMENTS The authors thank Dr. Wim Corver for assistance with the FACS experiments, Frans Prins and Hans Vermeulen for EM and microanalysis, Klaas van der Ham for his graphical expertise, Dr. Ingeborg Bajema for critical reading of the manscript, and Annemiek van der Wal for (immuno)histochemistry.

REFERENCES Akpolat, I., Kahraman, H., Arik, N., Akpolat, T., Kandemir, B., and Cengiz, K. (1996). Acute renal failure due to overdose of colloidal bismuth. Nephrol. Dial. Transplant. 11, 1890 –1891. Bergmeyer, H. U., Horder, M., and Rej, R. (1986). Approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. J. Clin. Chem. Clin. Biochem. 24, 497–510. Blom, W. M., de Bont, H. J. G. M., Meijerman, I., Mulder, G. J., and Nagelkerke, J. F. (1999). Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors. Biochem. Pharmacol. 58, 1891–1898. Creely, J. J., Dimari, S. J., Howe, A. M., and Haralson, M. A. (1992). Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells. Am. J. Pathol. 140, 45–55. Danscher, G., Jensen, K. B., Kraft, J., and Stoltenberg, M. (1997). Autometallographic silver enhancement of submicroscopic metal containing catalytic crystalites: A histochemical tool for detection of gold, silver, bismuth, mercury and zinc. Cell Vision 4, 375–386.

BISMUTH NEPHROTOXICITY Danscher, G., and Moller-Madsen, B. (1985). Silver amplification of mercury sulfide and selenide: A histochemical method for light and electron microscopic localization of mercury in tissue. J. Histochem. Cytochem. 33, 219 – 228. De Larco, J. E., and Todaro, G. J. (1978). Epitheliod and fibroblastic rat kidney cell clones: Epidermal growth factor (EGF) receptors and virus transformation. J. Cell. Physiol. 94, 335–342. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216. Ferrer, I. (1999). Nuclear DNA fragmentation in Creutzfeldt–Jakob disease: Does a mere positive in situ nuclear end-labeling indicate apoptosis. Acta Neuropathol. 97, 5–12. Ghadially, F. N. (1979). Ultrastructural localization and in situ analysis of iron, bismuth and gold inclusions. CRC Crit. Rev. Toxicol. 6, 303–350. Grasl-Kraupp, B., Ruttkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W., and Schulte-Hermann, R. (1995). In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology 21, 1465–1468. Green, D. R. (1998). Apoptotic pathways: The roads to ruin. Cell 94, 695– 698. Hudson, M., and Mowat, N. A. G. (1989). Reversible toxicity in poisoning with colloidal bismuth subcitrate. Br. Med. J. 299, 159. Hull, R. N., Cherry, W. R., and Weaver, G. W. (1976). The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro 12, 670 – 677. Huwez, F., Pall, A., Lyons, D., and Stewart, M. J. (1992). Acute renal failure after overdose of colloidal bismuth subcitrate. Lancet 340, 1298. Islek, I., Uysal, S., Gok, F., Dundaroz, R., and Kucukoduk, S. (2001). Reversible nephrotoxicity after overdose of colloidal bismuth subcitrate. Pediatr. Nephrol. 16, 510 –514. Kakkar, P., Mehrotra, S., and Viswanathan, P. N. (1996). tBHP induced in vitro swelling of rat liver mitochondria. Mol. Cell. Biochem. 154, 39 – 45. Kennedy, W. A. I., Buttyan, R., Garcia-Montes, E. G., D’Agati, V., Olsson, C. A., and Sawczuk, I. S. (1997). Epidermal growth factor supresses renal tubular apoptosis following ureteral obstruction. Urology 49, 973–980. Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C., Lantuejoul, S., Brambilla, E., and Negoescu, A. (1998). TUNEL apoptotic cell detection in tissue sections: Critical evaluation and improvement. J. Histochem. Cytochem. 46, 327–334. Leussink, B. T., Litvinov, S. V., de Heer, E., Slikkerveer, A., van der Voet, G. B., Bruijn, J. A., and de Wolff, F. A. (2001a). Loss of homotypic epithelial cell adhesion by selective N-cadherin displacement in bismuth nephrotoxicity. Toxicol. Appl. Pharmacol. 175, 54 –59. Leussink, B. T., Slikkerveer, A., Engelbrecht, M. R. W., van der Voet, G. B., Nouwen, E. J., de Heer, E., de Broe, M. E., de Wolff, F. A., and Bruijn, J. A. (2001b). Bismuth overdosing-induced reversible nephropathy in rats. Arch. Toxicol. 74, 745–754.

109

Leussink, B. T., Slikkerveer, A., Krauwinkel, W. J., van der Voet, G. B., de Heer, E., de Wolff, F. A., and Bruijn, J. A. (2000). Bismuth biokinetics and kidney histopathology after bismuth overdose in rats. Arch. Toxicol. 74, 349 –355. Lieberthal, W., Triaca, V., and Levine, J. (1996). Mechanisms of death induced by cisplatin in proximal tubular epithelial cells. Am J. Physiol. 270, F700 – F708. Malfertheimer, P., and Miehlke, S. (1997). Helicobacter pylori infection in ulcer pathogenesis. Digestion(Suppl. 1), 17–20. Martin-Bouyer, G., Foulon, B., Guerbois, H., and Barin, C. (1980). Aspects e´ pide´ miologiques des ence´ phalopaties apre`s administration de bismuth par voie orale. Therapie 35, 307–313. Nielsen, R., Birn, H., Moestrup, S., Nielsen, M., Verroust, P., and Christensen, E. I. (1998). Characterization of a kidney proximal tubule cell line, LLCPK1, expressing endocytotic active megalin. J. Am. Soc. Nephrol. 9, 1767– 1776. Pamphlett, R., Danscher, G., Rungby, J., and Stoltenberg, M. (2000). Tissue uptake of bismuth from shotgun pellets. Environ. Res. Sect. A 82, 258 –262. Perlman, H., Zhang, X., Chen, M. W., Walsh, K., and Buttyan, R. (1999). An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death Differ. 6, 48 –54. Prins, F. A., Bruijn, J. A., and de Heer, E. (1996). Applications in renal immunopathology of reflection contrast microscopy, a novel superior light microscopial technique. Kidney Int. 49, 261–266. Schweiberer, G., and Vogelgesang, W. (1993). Akutes Nierversagen bei Wismutintoxikation. Nieren Hochdruck 22, 381–385. Slikkerveer, A., and de Wolff, F. A. (1989). Pharmacokinetics and toxicity of bismuth compounds. Med. Toxicol. Exp. Drug 4, 303–323. Srinivasan, A. (1998). In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ. 5, 1004 –1016. Stevens, P. E., Moore, D. F., House, I. M., Volans, G. N., and Rainford, D. J. (1995). Significant elimination of bismuth by haemodialysis with a new heavy metal chelating agent. Nephrol. Dial. Transplant. 10, 696 – 698. Suzuki, E., Hirata, Y., Hayakawa, H., Omata, M., Kangawa, K., Minamino, N., and Matsuo, H. (1997). Evidence for C-type natriuric peptide production in the rat kidney. Biochem. Biophys. Res. Commun. 192, 532–538. Taimor, G., Lorenz, H., Hofstaetter, B., Schlu¨ ter, K. D., and Piper, H. M. (1999). Induction of necrosis but not apoptosis anoxia and reoxygenation in isolated adult cardiomyocytes of rat. Cardiovasc. Res. 41, 147–156. Taylor, E. G., and Klenerman, P. (1990). Acute renal failure after colloidal bismuth subcitrate overdose. Lancet 335, 670 – 671. Walker, C., Everitt, J., Freed, J. J., Knudson, J. A. G., and Whitely, L. O. (1991). Altered expression of transforming growth factor-alpha in hereditary rat renal cell carcinoma. Cancer Res. 51, 2973–2978. Wolf, B. B., and Green, D. R. (1999). Suicidal tendencies: Apoptotic cell death by caspase family proteinases. J. Biol. Chem. 274, 20049 –20052.