title: Pathways of proximal tubular cell death in bismuth nephrotoxicity.
abstract: 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 transferase-mediated 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(3+) in citrate buffer served as an in vitro model of bismuth nephropathy. NRK-52E cells exposed to 100 microM Bi(3+) 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.
authors.affiliation: Toxicology Laboratory, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Berend T
authors.initials: BT
authors.lastname: Leussink
authors.forename: J Fred
authors.initials: JF
authors.lastname: Nagelkerke
authors.forename: Bob
authors.initials: B
authors.lastname: van de Water
authors.forename: Anja
authors.initials: A
authors.lastname: Slikkerveer
authors.forename: Gijsbert B
authors.initials: GB
authors.lastname: van der Voet
authors.forename: Anu
authors.initials: A
authors.lastname: Srinivasan
authors.forename: Jan A
authors.initials: JA
authors.lastname: Bruijn
authors.forename: Frederik A
authors.initials: FA
authors.lastname: de Wolff
authors.forename: Emile
authors.initials: E
authors.lastname: de Heer
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000897:Anti-Ulcer Agents / Q000378:metabolism / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis / Q000096:biosynthesis; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D007674:Kidney Diseases / Q000139:chemically induced* / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018374:LLC-PK1 Cells; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009942:Organometallic Compounds / Q000378:metabolism / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000897:Anti-Ulcer Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009942:Organometallic Compounds; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C002791:bismuth tripotassium dicitrate