Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

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Abstract

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.

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Extension of life-span by introduction of telomerase into normal human cells.

A. G. Bodnar, M. Ouellette, M Frolkis … (1998)

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

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Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.

I Wilson, T Orton, F Pognan … (2000)

We show here the identity of Alamar Blue as resazurin. The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.

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Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.

Ann O’Hare, Jasvinder Singh, Peter W. Mathieson … (2006)

Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.

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Author and article information

Contributors

: +31-24-3618912 , +31-24-3618900 , m.wilmer@cukz.umcn.nl

Journal

Journal ID (nlm-ta): Cell Tissue Res

Title: Cell and Tissue Research

Publisher: Springer-Verlag (Berlin/Heidelberg )

ISSN (Print): 0302-766X

ISSN (Electronic): 1432-0878

Publication date (Electronic): 10 November 2009

Publication date PMC-release: 10 November 2009

Publication date (Print): February 2010

Volume: 339

Issue: 2

Pages: 449-457

Affiliations

[1 ]Laboratory of Pediatrics and Neurology (656), Radboud University Nijmegen Medical Center, P.O. 9101, 6500 HB Nijmegen, The Netherlands

[2 ]Academic Renal Unit, Southmead Hospital, Bristol, UK

[3 ]Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands

[4 ]Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands

[5 ]Department of Pediatrics, University Hospitals Leuven, Leuven, Belgium

Article

Publisher ID: 882

DOI: 10.1007/s00441-009-0882-y

PMC ID: 2817082

PubMed ID: 19902259

SO-VID: c170f531-1add-4dbf-a78d-6345d9e811f1

History

Date received : 9 March 2009

Date accepted : 9 September 2009

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ScienceOpen disciplines: Molecular medicine

Keywords: proximal tubule cell,albumin endocytosis,phosphate transport,organic cation transport,p-glycoprotein,human

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ScienceOpen disciplines: Molecular medicine

Keywords: proximal tubule cell, albumin endocytosis, phosphate transport, organic cation transport, p-glycoprotein, human

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

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Abstract

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Most cited references 26

Extension of life-span by introduction of telomerase into normal human cells.

Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.

Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.

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