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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.


Related in: MedlinePlus

Cellular organelles and surface characteristics.(A) The morphology of organelles in matured ciPTEC cultured on HFM (H) was investigated by transmission electron microscopy and intact cells were detected. Next to numerous well-developed mitochondria, the nucleus (N) containing the chromatin (Ch), the endoplasmatic reticulum (E), ribosomes (R) and the Golgi apparatus could be nicely detected. (B) The surface of matured ciPTEC was visualized using scanning electron microscopy. At the apical membrane microvilli were observed, as expected for proximal tubule epithelial cells, though a heterogeneous distribution was detected.
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f3: Cellular organelles and surface characteristics.(A) The morphology of organelles in matured ciPTEC cultured on HFM (H) was investigated by transmission electron microscopy and intact cells were detected. Next to numerous well-developed mitochondria, the nucleus (N) containing the chromatin (Ch), the endoplasmatic reticulum (E), ribosomes (R) and the Golgi apparatus could be nicely detected. (B) The surface of matured ciPTEC was visualized using scanning electron microscopy. At the apical membrane microvilli were observed, as expected for proximal tubule epithelial cells, though a heterogeneous distribution was detected.

Mentions: Matured ciPTEC cultured on HFM retained intact cellular organelle morphology as observed by TEM (Fig. 3A). The presence of numerous mitochondria indicated that the cell monolayer consists of viable and metabolically active PTEC35. Moreover, other essential cellular components, including the Golgi apparatus, endoplasmatic reticulum, ribosomes, the nucleus and its chromatin, could be determined. The brush border membrane of ciPTEC is reasonably well-developed, containing many microvilli to enlarge the apical surface and to stimulate intensive reabsorption, as observed with SEM (Fig. 3B).


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)

Cellular organelles and surface characteristics.(A) The morphology of organelles in matured ciPTEC cultured on HFM (H) was investigated by transmission electron microscopy and intact cells were detected. Next to numerous well-developed mitochondria, the nucleus (N) containing the chromatin (Ch), the endoplasmatic reticulum (E), ribosomes (R) and the Golgi apparatus could be nicely detected. (B) The surface of matured ciPTEC was visualized using scanning electron microscopy. At the apical membrane microvilli were observed, as expected for proximal tubule epithelial cells, though a heterogeneous distribution was detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Cellular organelles and surface characteristics.(A) The morphology of organelles in matured ciPTEC cultured on HFM (H) was investigated by transmission electron microscopy and intact cells were detected. Next to numerous well-developed mitochondria, the nucleus (N) containing the chromatin (Ch), the endoplasmatic reticulum (E), ribosomes (R) and the Golgi apparatus could be nicely detected. (B) The surface of matured ciPTEC was visualized using scanning electron microscopy. At the apical membrane microvilli were observed, as expected for proximal tubule epithelial cells, though a heterogeneous distribution was detected.
Mentions: Matured ciPTEC cultured on HFM retained intact cellular organelle morphology as observed by TEM (Fig. 3A). The presence of numerous mitochondria indicated that the cell monolayer consists of viable and metabolically active PTEC35. Moreover, other essential cellular components, including the Golgi apparatus, endoplasmatic reticulum, ribosomes, the nucleus and its chromatin, could be determined. The brush border membrane of ciPTEC is reasonably well-developed, containing many microvilli to enlarge the apical surface and to stimulate intensive reabsorption, as observed with SEM (Fig. 3B).

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.


Related in: MedlinePlus