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Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

Jansen J, De Napoli IE, Fedecostante M, Schophuizen CM, Chevtchik NV, Wilmer MJ, van Asbeck AH, Croes HJ, Pertijs JC, Wetzels JF, Hilbrands LB, van den Heuvel LP, Hoenderop JG, Stamatialis D, Masereeuw R - Sci Rep (2015)

Bottom Line: A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin.Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine.In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT
The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

No MeSH data available.


ZO-1 and OCT2 protein expression.Representative confocal images of ZO-1 (green), OCT-2 (red) and nuclei (blue) in matured ciPTEC cultured on HFM are shown in x-y and y-z planes. (A–C,E) The ZO-1 expression was abundantly present along the cell boundaries within a tight and homogenous cell monolayer. (D,E) The endogenous OCT-2 protein expression in ciPTEC was observed heterogeneously in the membranes, moreover, some signal was detected in the cytoplasm.
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f4: ZO-1 and OCT2 protein expression.Representative confocal images of ZO-1 (green), OCT-2 (red) and nuclei (blue) in matured ciPTEC cultured on HFM are shown in x-y and y-z planes. (A–C,E) The ZO-1 expression was abundantly present along the cell boundaries within a tight and homogenous cell monolayer. (D,E) The endogenous OCT-2 protein expression in ciPTEC was observed heterogeneously in the membranes, moreover, some signal was detected in the cytoplasm.

Mentions: The presence of the tight junction protein ZO-1 in cell monolayers emphasizes cell polarity and monolayer tightness. In addition, tight junction proteins contribute to fluid and ion homeostasis mediated by paracellular transport36. A tight homogeneous cell monolayer was observed for ciPTEC on HFM with ZO-1 abundantly expressed along the cell boundaries as shown in representative z-scans (Fig. 4A,C). Moreover, the endogenous expression of the influx transporter OCT2 was demonstrated to be expressed basolaterally (Fig. 4D), although some cytoplasmic staining was visible as well, suggesting ongoing posttranslational OCT2 modifications as previously suggested37. The OCT2 antibody was validated in paraffin embedded human kidney tissue using immunohistochemistry and positive staining at the basolateral membrane of PTEC was observed (data not shown).


Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

Jansen J, De Napoli IE, Fedecostante M, Schophuizen CM, Chevtchik NV, Wilmer MJ, van Asbeck AH, Croes HJ, Pertijs JC, Wetzels JF, Hilbrands LB, van den Heuvel LP, Hoenderop JG, Stamatialis D, Masereeuw R - Sci Rep (2015)

ZO-1 and OCT2 protein expression.Representative confocal images of ZO-1 (green), OCT-2 (red) and nuclei (blue) in matured ciPTEC cultured on HFM are shown in x-y and y-z planes. (A–C,E) The ZO-1 expression was abundantly present along the cell boundaries within a tight and homogenous cell monolayer. (D,E) The endogenous OCT-2 protein expression in ciPTEC was observed heterogeneously in the membranes, moreover, some signal was detected in the cytoplasm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644946&req=5

f4: ZO-1 and OCT2 protein expression.Representative confocal images of ZO-1 (green), OCT-2 (red) and nuclei (blue) in matured ciPTEC cultured on HFM are shown in x-y and y-z planes. (A–C,E) The ZO-1 expression was abundantly present along the cell boundaries within a tight and homogenous cell monolayer. (D,E) The endogenous OCT-2 protein expression in ciPTEC was observed heterogeneously in the membranes, moreover, some signal was detected in the cytoplasm.
Mentions: The presence of the tight junction protein ZO-1 in cell monolayers emphasizes cell polarity and monolayer tightness. In addition, tight junction proteins contribute to fluid and ion homeostasis mediated by paracellular transport36. A tight homogeneous cell monolayer was observed for ciPTEC on HFM with ZO-1 abundantly expressed along the cell boundaries as shown in representative z-scans (Fig. 4A,C). Moreover, the endogenous expression of the influx transporter OCT2 was demonstrated to be expressed basolaterally (Fig. 4D), although some cytoplasmic staining was visible as well, suggesting ongoing posttranslational OCT2 modifications as previously suggested37. The OCT2 antibody was validated in paraffin embedded human kidney tissue using immunohistochemistry and positive staining at the basolateral membrane of PTEC was observed (data not shown).

Bottom Line: A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin.Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine.In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT
The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

No MeSH data available.