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The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule

  • Ion channels, Receptors and Transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates dl-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.

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Abbreviations

OAT:

Organic anion transporter

OCT:

Organic cation transporter

ABC:

ATP-binding cassette

MDR:

Multidrug resistance

MRP:

Multidrug resistance protein

BCRP:

Breast cancer resistance protein

MCT:

Monocarboxylate transporter

SMCT:

Sodium-dependent monocarboxylate transporter

H33342:

Hoechst 33342

CMFDA:

5-Chloromethylfluorescein diacetate

GSMF:

Glutathione methylfluorescein

PAH:

para-Aminohippurate

CSA:

Cyclosporin A

PGE1 :

Prostaglandin E1

OATP:

Organic anion-transporting polypeptide

MATE:

Multidrug and toxic compound extrusion

References

  1. Bathula CS, Garrett SH, Zhou XD, Sens MA, Sens DA, Somji S (2008) Cadmium, vectorial active transport, and MT-3-dependent regulation of cadherin expression in human proximal tubular cells. Toxicol Sci 102:310–318

    Article  PubMed  CAS  Google Scholar 

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  3. Brown CDA, Sayer R, Windass AS, Haslam IS, De Broe ME (2008) Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling. Toxicol App Pharmacol 233:428–438

    Article  CAS  Google Scholar 

  4. Draper MP, Martell RL, Levy SB (1997) Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein. Br J Cancer 75:810–815

    Article  PubMed  CAS  Google Scholar 

  5. Forster F, Volz A, Fricker G (2008) Compound profiling for ABCC2 (MRP2) using a fluorescent microplate assay system. Eur J Pharm Biopharm 69:396–403

    Article  PubMed  Google Scholar 

  6. Gekeler V, Ise W, Sanders KH, Ulrich WR, Beck J (1995) The leukotriene LTD4 receptor agonist MK571 specifically modulates MRP associated drug resistance. Biochem Biophys Res Commun 208:345–352

    Article  PubMed  CAS  Google Scholar 

  7. Gopal E, Fei Y, Sugawara M, Miyauchi S, Zhuang L, Martin P, Smith SB, Prasad PD, Ganapathy V (2004) Expression of slc5a8 in kidney and its role in Na+-coupled transport of lactate. J Biol Chem 279:44522–44532

    Article  PubMed  CAS  Google Scholar 

  8. Gopal E, Umapathy NS, Martin PM, Ananth S, Gnana-Prakasam JP, Becker H, Wagner CA, Ganapathy V, Prasad PD (2007) Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney. Biochim Biophys Acta 1768:2690–2697

    Article  PubMed  CAS  Google Scholar 

  9. Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, Baumann C, Lang F, Busch AE, Koepsell H (1997) Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 16:871–881

    Article  PubMed  CAS  Google Scholar 

  10. Gstraunthaler G, Pfaller W, Kotanko P (1985) Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK). Am J Physiol 248:F536–F544

    PubMed  CAS  Google Scholar 

  11. Gunness P, Aleksa K, Kosuge K, Ito S, Koren G (2010) Comparison of the novel HK-2 human renal proximal tubular cell line with the standard LLC-PK1 cell line in studying drug-induced nephrotoxicity. Can J Physiol Pharmacol 88:448–455

    Article  PubMed  CAS  Google Scholar 

  12. Gutmann H, Fricker G, Torok M, Michael S, Beglinger C, Drewe J (1999) Evidence for ABC-transporters in Caco-2 cells modulating drug uptake. Pharm Res 16:402–407

    Article  PubMed  CAS  Google Scholar 

  13. Halestrap AP, Price NT (1999) The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343:281–299

    Article  PubMed  CAS  Google Scholar 

  14. Hilgendorf C, Ahlin G, Seithel A, Artursson P, Ungell A, Karlsson J (2007) Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug Metab Dispos 35:1333–1340

    Article  PubMed  CAS  Google Scholar 

  15. Hosoyamada M, Sekine T, Kanai Y, Endou H (1999) Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol Ren Physiol 276:F122–F128

    CAS  Google Scholar 

  16. Huls M, Brown CDA, Windass AS, Sayer R, van den Heuvel JJMW, Heemskerk S, Russel FGM, Masereeuw R (2008) The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane. Kidney Int 73:220–225

    Article  PubMed  CAS  Google Scholar 

  17. Ito K (2008) ABCC2/Abcc2 transport property in different species and its modulation by heterogeneous factors. Drug Metab Pharmacokinet 23:394–405

    Article  PubMed  CAS  Google Scholar 

  18. Koyama H, Goodpasture C, Miller MM, Teplitz RL, Riggs AD (1978) Establishment and characterization of a cell line from the American opossum (Didelphys virginiana). In Vitro 14:239–246

    Article  PubMed  CAS  Google Scholar 

  19. Lash LH, Putt DA, Cai H (2006) Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228:200–218

    Article  PubMed  CAS  Google Scholar 

  20. Launay-Vacher V, Izzedine H, Karie S, Baumelou A, Deray G (2006) Renal tubular drug transporters. Nephron Physiol 103:97–106

    Article  Google Scholar 

  21. Miyamoto Y, Watanabe H, Noguchi T, Kotani S, Nakajima M, Kadowaki D, Otagiri M, Maruyama T (2011) Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney. Nephrol Dial Transplant 26:2498–2502

    Article  PubMed  CAS  Google Scholar 

  22. Mutsaers HAM, Wilmer MJG, van den Heuvel LP, Hoenderop JG, Masereeuw R (2011) Letter to the editor. Nephrol Dial Transplant 26:4149

    Article  PubMed  CAS  Google Scholar 

  23. Nooter K, Westerman AM, Flens MJ, Zaman GJ, Scheper RJ, van Wingerden KE, Burger H, Oostrum R, Boersma T, Sonneveld P (1995) Expression of the multidrug resistance-associated protein (MRP) gene in human cancers. Clin Cancer Res 1:1301–1310

    PubMed  CAS  Google Scholar 

  24. Peng K, Cluzeaud F, Bens M, Van Huyen J-PD, Wioland MA, Lacave R, Vandewalle A (1999) Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney. J Histochem Cytochem 47:757–767

    Article  PubMed  CAS  Google Scholar 

  25. Race JE, Grassl SM, Williams JW, Holtzman EJ (1999) Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3). Biochem Biophys Res Commun 255:508–514

    Article  PubMed  CAS  Google Scholar 

  26. Reid G, Wielinga P, Zelcer N, van der Heijden I, Kuil A, de Haas M, Wijnholds J, Borst P (2003) The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A 100:9244–9249

    Article  PubMed  CAS  Google Scholar 

  27. Romiti N, Tramonti G, Chieli E (2002) Influence of different chemicals on MDR-1 P-glycoprotein expression and activity in the HK-2 proximal tubular cell line. Toxicol Appl Pharmacol 183:83–91

    Article  PubMed  CAS  Google Scholar 

  28. Ryan MJ, Johnson G, Kiiu J, Fuerstenburg SM, Zager RA, Torok-Storb B (1994) HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int 45:48–57

    Article  PubMed  CAS  Google Scholar 

  29. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A 84:7735–7738

    Article  PubMed  CAS  Google Scholar 

  30. Tramonti G, Romiti N, Norpoth M, Chieli E (2001) P-glycoprotein in HK-2 proximal tubule cell line. Ren Fail 23:331–337

    Article  PubMed  CAS  Google Scholar 

  31. van de Water FM, Masereeuw R, Russel FGM (2005) Function and regulation of multi-drug resistance proteins (MRPs) in renal elimination of organic anions. Drug Metab Rev 37:443–471

    Article  PubMed  Google Scholar 

  32. Wang Q, Lu Y, Morris ME (2007) Monocarboxylate transporter (MCT) mediates the transport of gamma-hydroxybutyrate in human kidney HK-2 cells. Pharm Res 24:1067–1078

    Article  PubMed  CAS  Google Scholar 

  33. Wang Q, Lu Y, Yuan M, Darling IM, Repasky EA, Morris ME (2006) Characterization of monocarboxylate transport in human kidney HK-2 cell. Mol Pharm 3:675–685

    Article  PubMed  CAS  Google Scholar 

  34. Wang Q, Strab R, Kardos P, Ferguson C, Li J, Owen A, Hidalgo IJ (2008) Application and limitation of inhibitors in drug–transporter interactions studies. Int J Pharm 356:12–18

    Article  PubMed  CAS  Google Scholar 

  35. Wieser M, Stadler G, Jennings P, Streubel B, Pfaller W, Ambros P, Riedl C, Katinger H, Grillari J, Grillari-Voglauer R (2008) hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am J Physiol Ren Physiol 295:F1365–F1375

    Article  CAS  Google Scholar 

  36. Wilmer MJ, Saleem MA, Masereeuw R, Ni L, van der Velden TJ, Russel FG, Mathieson PW, Monnens LA, van den Heuvel LP, Levtchenko EN (2010) Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters. Cell Tissue Res 339:449–457

    Article  PubMed  Google Scholar 

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Acknowledgments

We would like to thank Professor John Kirby, Newcastle University, for the generous gift of the HK-2 cell line.

Ethical standards

Ethical approval for using human tissue samples was from Newcastle Hospitals NHS Foundation Trust NRES Research Ethics Committee.

Conflict of interest

The authors declare that they have no conflict of interests.

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Authors and Affiliations

  1. Epithelial Cell Research Group, Institute for Cell and Molecular Biosciences, 1st Floor Cookson Building, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

    Sarah E. Jenkinson, Git W. Chung, Ellen van Loon, Nur S. Bakar, Abigail M. Dalzell & Colin D. A. Brown

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  1. Sarah E. Jenkinson
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  2. Git W. Chung
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Correspondence to Sarah E. Jenkinson.

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Jenkinson, S.E., Chung, G.W., van Loon, E. et al. The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule. Pflugers Arch - Eur J Physiol 464, 601–611 (2012). https://doi.org/10.1007/s00424-012-1163-2

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  • DOI: https://doi.org/10.1007/s00424-012-1163-2

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