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Effect of Epsilon Toxin–GFP on MDCK Cells and Renal Tubules In Vivo

Alex Soler-Jover, Juan Blasi, Inma Gómez de Aranda, Piedad Navarro, Maryse Gibert, Michel R. Popoff and Mireia Martín-Satué

Departament de Biologia Cel·lular i Anatomia Patològica, Campus de Bellvitge, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain (AS-J,JB,IGdA,PN,MM-S), and CNR Anaérobies, Institut Pasteur, Paris, France (MG,MRP)

Correspondence to: Dr. Mireia Martín-Satué, Dpt. Biologia Cel·lular i Anatomia Patològica, Lab 4145, 4^a Planta, Pavelló de Govern, Facultat de Medicina Campus de Bellvitge, C/Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Barcelona, Spain. E-mail: martin{at}medicina.ub.es

Epsilon toxin (-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant -toxin–green fluorescence protein (-toxin–GFP) and -prototoxin–GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant -toxin–GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. -Toxin–GFP and -prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with -toxin–GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that -toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an -toxin receptor is expressed on renal distal tubules of mammalian species, including human. (J Histochem Cytochem 52:931–942, 2004)

Key Words: clostridial toxins • epsilon toxin • MDCK cells • renal tubules • pulpy kidney disease

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