Human proximal tubule cells form functional microtissues


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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Abbott A (2003) Cell culture: biology’s new dimension. Nature 424:870–872

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Ahn DW, Chung JM, Kim JY et al (2005) Inhibition of renal Na+/H+ exchange in cadmium-intoxicated rats. Toxicol Appl Pharmacol 204:91–98

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Ali BH (1995) Gentamicin nephrotoxicity in humans and animals: some recent research. Gen Pharmacol 26:1477–1487

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Araki M, Takano T, Uemonsa T et al (2002) Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation. Dev Biol 244:358–371

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Boehnke K, Mirancea N, Pavesio A et al (2007) Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents. Eur J Cell Biol 86:731–746

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Christensen EI, Birn H, Storm T et al (2012) Endocytic receptors in the renal proximal tubule. Physiology (Bethesda) 27:223–236

    CAS  Article  Google Scholar 

  7. 7.

    Christensen EI, Devuyst O, Dom G et al (2003) Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc Natl Acad Sci U S A 100:8472–8477

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Devuyst O, Luciani A (2015) Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule. J Physiol 593:4151–4164

  9. 9.

    Drewitz M, Helbling M, Fried N et al (2011) Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues. Biotechnol J 6:1488–1496

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Eckardt KU, Coresh J, Devuyst O et al (2013) Evolving importance of kidney disease: from subspecialty to global health burden. Lancet 382:158–69

    Article  PubMed  Google Scholar 

  11. 11.

    El Ghalbzouri A, Ponec M (2004) Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components. Wound Repair Regen 12:359–367

    Article  PubMed  Google Scholar 

  12. 12.

    Gena P, Calamita G, Guggino WB (2010) Cadmium impairs albumin reabsorption by down-regulating megalin and ClC5 channels in renal proximal tubule cells. Environ Health Perspect 118:1551–1556

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Giles RH, Ajzenberg H, Jackson PK (2014) 3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders. Nat Protoc 9:2725–2731. doi:10.1038/nprot.2014.181

  14. 14.

    Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392–401

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Kelm JM, Moritz W, Schmidt D et al (2007) In vitro vascularization of human connective microtissues. Methods Mol Med 140:153–166

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Kobayashi K, Nomoto Y, Suzuki T et al (2006) Effect of fibroblasts on tracheal epithelial regeneration in vitro. Tissue Eng 12:2619–2628

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Lancaster MA, Knoblich JA (2014) Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345:1247125

    Article  PubMed  Google Scholar 

  18. 18.

    Lima WR, Parreira KS, Devuyst O et al (2010) ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells. J Am Soc Nephrol 21:478–488

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lote CJ (2012) In: principles of renal physiology, 5th edn. Springer, New York, p 25

    Book  Google Scholar 

  20. 20.

    Meli L, Barbosa HS, Hickey AM et al (2014) Three dimensional cellular microarray platform for human neural stem cell differentiation and toxicology. Stem Cell Res 13:36–47

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Monteil C, Leclere C, Fillastre JP et al (1993) Characterization of gentamicin-induced dysfunctions in vitro: the use of optimized primary cultures of rabbit proximal tubule cells. Ren Fail 15:475–483

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Pampaloni F, Stelzer EH, Masotti A (2009) Three-dimensional tissue models for drug discovery and toxicology. Recent Pat Biotechnol 3:103–117

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Pfaller W, Gstraunthaler G (1998) Nephrotoxicity testing in vitro—what we know and what we need to know. Environ Health Perspect 106(Suppl 2):559–569

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Raggi C, Fujiwara K, Leal T et al (2011) Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent’s disease. Pflugers Arch 462:851–860

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Raggi C, Luciani A, Nevo N et al (2014) Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis. Hum Mol Genet 23:2266–2278

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Reed AA, Loh NY, Terryn S et al (2010) CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent’s disease. Am J Physiol Renal Physiol 298:F365–380

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Regec AL, Trifillis AL, Trump BF (1986) The effect of gentamicin on human renal proximal tubular cells. Toxicol Pathol 14:238–241

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Schmidt SL, Carter LL (1990) ATP is required for receptor-mediated endocytosis in intact cells. JCB 111:2307–2318

    Article  Google Scholar 

  30. 30.

    Silverblatt FJ, Kuehn C (1979) Autoradiography of gentamicin uptake by the rat proximal tubule cell. Kidney Int 15:335–345

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Takasato M, Er PX, Becroft M et al (2014) Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 16:118–126

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Takasato M, Er PX, Chiu HS et al (2015) Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526:564–568

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Tanaka K, Terryn S, Geffers L et al (2010) The transcription factor HNF1α regulates expression of chloride-proton exchanger ClC-5 in the renal proximal tubule. Am J Physiol Renal Physiol 299:F1339–F1347

  34. 34.

    Terryn S, Jouret F, Vandenabeele F et al (2007) A primary culture of mouse proximal tubular cells, established on collagen-coated membranes. Am J Physiol Renal Physiol 293:F476–485

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Thevenod F (2003) Nephrotoxicity and the proximal tubule. Insights from cadmium. Nephron Physiol 93:p87–93

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    van Meerloo J, Kaspers GJ, Cloos J (2011) Cell sensitivity assays: the MTT assay. Methods Mol Biol 731:237–245

    Article  PubMed  Google Scholar 

  37. 37.

    Vandewalle A, Farman N, Morin JP et al (1981) Gentamicin incorporation along the nephron: autoradiographic study on isolated tubules. Kidney Int 19:529–539

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Wohlwend A, Montesano R, Vassalli JD, Orci L (1985) LLC-PK1 cysts: a model for the study of epithelial polarity. J Cell Physiol 125:533–539

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Zalups RK, Ahmad S (2003) Molecular handling of cadmium in transporting epithelia. Toxicol Appl Pharmacol 186:163–188

    CAS  Article  PubMed  Google Scholar 

Download references


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.

Author information



Corresponding author

Correspondence to Olivier Devuyst.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material


(PPTX 3369 kb)


(MP4 3323 kb)


(MP4 3172 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation


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