Human proximal tubule cells form functional microtissues

Abstract

The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.

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Acknowledgments

We are grateful to Claudia Meyer-Gresele (Institute of Anatomy, UZH, Zurich) and Ilka Edenhofer (Institute of Physiology, UZH) for their support in tissue processing and staining. We thank the Center for Microscopy and Image Analysis, Zurich, for their continuous help in transmission electron microscopy. We acknowledge Renata Kosiraki and Pierre Verroust for the generous gift of the anti-megalin and anti-cubilin antibodies as well as Alessandro Luciani for help with the confocal microscopy analyses.

This work was supported by the Commission of Technology and Innovation (CTI 13739.1), an Action de Recherche Concertée (Communauté Française de Belgique), the Fonds National de la Recherche Scientifique and the Fonds de la Recherche Scientifique Médicale (Brussels, Belgium), the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no 305608 (EURenOmics), the Cystinosis Research Foundation (Irvine, CA, USA), the KFSP Molecular Imaging Network Zurich (MINZ) of the University of Zurich, and the Swiss National Science Foundation project grant 310030_146490 (OD). Further support was received by Fundação Pesquisa e Desenvolvimento Humanitário and the Else Kröner-Fresenius Stiftung to Stephan Segerer.

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Correspondence to Olivier Devuyst.

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Prange, J.A., Bieri, M., Segerer, S. et al. Human proximal tubule cells form functional microtissues. Pflugers Arch - Eur J Physiol 468, 739–750 (2016). https://doi.org/10.1007/s00424-015-1771-8

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Keywords

  • Primary culture
  • Epithelial cells
  • Endocytosis
  • Albumin
  • Drug toxicity