title: A screen for apoptotic synergism between clinical relevant nephrotoxicant and the cytokine TNF-α.
abstract: Nephrotoxicity remains one of the main reasons for post-market drug withdrawal. Tumour necrosis factor α (TNF-α) secretion has been shown to underlie the nephrotoxicity induced by some of these drugs. Yet, there is currently no reliable and sensitive in vitro assay available to screen for nephrotoxicants of which toxicity largely depends on TNF-α secretion. Therefore, we developed and applied a sensitive fluorescence-based in vitro assay for TNF-α-mediated nephrotoxicity screening using mouse immortalized proximal tubular epithelial cells (IM-PTECs). Our assay allows rapid evaluation of TNF-α-mediated toxicant-induced apoptosis and necrosis using fixed endpoint and live cell measurements. To evaluate our assay, sixteen nephrotoxicants and two control non-nephrotoxicants were used. Out of the sixteen nephrotoxicants, eight induced cell death, of which five induced apoptosis as well as necrosis. Moreover, TNF-α significantly enhanced apoptotic cell death induced by cisplatin, cyclosporine A, tacrolimus and azidothymidine. These nephrotoxicants are known to induce inflammation in vivo which has been linked to an enhancement of nephrotoxicity for cisplatin, cyclosporine A and tacrolimus, confirming the functionality of our assay. Overall, our assay allows rapid and sensitive measurement of apoptosis and necrosis induced by a combination of nephrotoxicants and inflammatory components such as TNF-α and can be used as an alternative assay for nephrotoxicity prediction in vitro.
authors.affiliation: Division of Toxicology, Leiden Academic Center for Drug Research, Leiden University, The Netherlands.
authors.forename: Giulia
authors.initials: G
authors.lastname: Benedetti
authors.forename: Sreenivasa
authors.initials: S
authors.lastname: Ramaiahgaris
authors.forename: Bram
authors.initials: B
authors.lastname: Herpers
authors.forename: Bob
authors.initials: B
authors.lastname: van de Water
authors.forename: Leo S
authors.initials: LS
authors.lastname: Price
authors.forename: Marjo
authors.initials: M
authors.lastname: de Graauw
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay; D002478:Cells, Cultured; D064420:Drug-Related Side Effects and Adverse Reactions; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D051379:Mice; D009336:Necrosis / Q000139:chemically induced; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D014409:Tumor Necrosis Factor-alpha
keywords: In vitro cytotoxicity assay; Mouse immortalized proximal tubular epithelial cells; Nephrotoxicity; Synergistic apoptosis; TNF-α