Inter-laboratory comparison of human renal proximal tubule (HK-2) transcriptome alterations due to Cyclosporine A exposure and medium exhaustion Paul Jennings a, * ,1 , Sonia Aydin a,1 , Jason Bennett b,1 , Rachael McBride c,1 , Christina Weiland d,1 , Niamh Tuite b,1 , Leonhard N. Gruber a , Paul Perco f , Peadar Ó Gaora c , Heidrun Ellinger-Ziegelbauer d , Hans-Juergen Ahr d , Cees Van Kooten e , Mohamed R. Daha e , Pilar Prieto g , Michael P. Ryan b , Walter Pfaller a , Tara McMorrow b a Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl Strasse 3, 6020 Innsbruck, Austria b UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland c UCD School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland d Bayer HealthCare AG, GDD-GED-GTOX, Aprather Weg 18a, 42096 Wuppertal, Germany e Department of Nephrology, C3-P25, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands f Institute for Theoretical Chemistry, University of Vienna, Waehringer Strasse 17, 1090 Vienna, Austria g ECVAM, Institute for Health and Consumer Protection, European Joint Research Centre, 21020 Ispra (VA), Italy article info Article history: Received 22 August 2008 Accepted 23 December 2008 Available online 31 December 2008 Keywords: Renal Proximal tubule DNA microarray Transcriptomics CsA Cyclosporine A Cell culture medium HK-2 Glucose Lactate Epithelial abstract There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework project ‘‘PREDICTOMICS” was established in order to contribute to the further development of in vitro toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray technology is being used more frequently in the in vitro field, however, only very few studies have assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised con- ditions, each laboratory treated HK-2 cells with 5 lM CsA for 12 and 48 h. RNA was isolated and hybri- dised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes responsible for this separate clustering were removed all laboratories showed similar expression profiles. There was a major impact of time since feed, due to medium exhaustion in the 48 h arrays compared to the 12 h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, amino acid metabolism, amino acid transport and amino acid biosynthesis were over-represented due to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-repre- sented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Addition- ally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as med- ium exhaustion must also be considered in such analyses. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction The evaluation of the toxic potential of chemicals and pharma- ceuticals is an integral part of new compound discovery and will ultimately decide the fate of lead compounds. Thus model systems used to assess toxicological hazard should be of the highest 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.12.023 * Corresponding author. E-mail address: paul.jennings@i-med.ac.at (P. Jennings). 1 These authors contributed equally to this work. Toxicology in Vitro 23 (2009) 486–499 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit