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Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

Bányai E, Balogh E, Fagyas M, Arosio P, Hendrik Z, Király G, Nagy G, Tánczos B, Pócsi I, Balla G, Balla J, Bánfalvi G, Jeney V - Oxid Med Cell Longev (2014)

Bottom Line: We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges.Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression.Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Debrecen, Debrecen 4032, Hungary.

ABSTRACT
Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte differentiation are accompanied by changes in oxidative resistance in a manner that could support their long-term survival. We used a conditionally immortalized human podocyte cell line to study the morphological and functional changes during differentiation. We followed the differentiation process for 14 days by time-lapse microscopy. During this period nondifferentiated podocytes gradually transformed into large, nonproliferating, frequently multinucleated cells, with enlarged nuclei and opened chromatin structure. We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges. Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression. Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

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Podocyte differentiation under nonpermissive (37°C) condition. At 50–60% confluence, nondifferentiated cells were placed from 33°C to 37°C to induce differentiation which was followed by video microscopy for 14 days. (a) On selected time-lapse phase contrast images change in the shape and size of podocytes is indicated by broken outlines. Black arrows show shorter and rounded projections of differentiating podocytes. White arrows indicate longer and spindle like projections of arborizing podocytes. Mitotic cells, marked with red double sharps, become detached from the surface of the vessel for a while before the daughter cells adhere again to the bottom. Red asterisks denote apoptotic cells. (b) Modified Giemsa staining was performed on parallel cell cultures at different time points. Static visualization of the cell culture supports the findings of dynamic assessment by time-lapse monitoring. Cytoplasmic staining (pink), nuclear staining (blue). Magnification 10x. (c) Light microscopy and immunofluorescent microscopy images of cell cultures demonstrate characteristic features of human podocytes. Left panels represent nondifferentiated podocytes grown under permissive condition; right panels represent differentiated podocytes after 10 days of culturing under nonpermissive condition. (i) Light microscopy images, scale bars: 300 μm, each. Nondifferentiated cells are small in size and show a cobble-stone like morphology, whilst differentiated cells have enlarged cell bodies with large nucleus, bulky cytoplasm, and ramifying processes. (ii) Synaptopodin-Cy3 (red) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Synaptopodin expression is present only in differentiated cells and shows filamentous staining. (iii) F-actin (phalloidin-FITC, green) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Images are representative of three experiments with similar results.
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fig2: Podocyte differentiation under nonpermissive (37°C) condition. At 50–60% confluence, nondifferentiated cells were placed from 33°C to 37°C to induce differentiation which was followed by video microscopy for 14 days. (a) On selected time-lapse phase contrast images change in the shape and size of podocytes is indicated by broken outlines. Black arrows show shorter and rounded projections of differentiating podocytes. White arrows indicate longer and spindle like projections of arborizing podocytes. Mitotic cells, marked with red double sharps, become detached from the surface of the vessel for a while before the daughter cells adhere again to the bottom. Red asterisks denote apoptotic cells. (b) Modified Giemsa staining was performed on parallel cell cultures at different time points. Static visualization of the cell culture supports the findings of dynamic assessment by time-lapse monitoring. Cytoplasmic staining (pink), nuclear staining (blue). Magnification 10x. (c) Light microscopy and immunofluorescent microscopy images of cell cultures demonstrate characteristic features of human podocytes. Left panels represent nondifferentiated podocytes grown under permissive condition; right panels represent differentiated podocytes after 10 days of culturing under nonpermissive condition. (i) Light microscopy images, scale bars: 300 μm, each. Nondifferentiated cells are small in size and show a cobble-stone like morphology, whilst differentiated cells have enlarged cell bodies with large nucleus, bulky cytoplasm, and ramifying processes. (ii) Synaptopodin-Cy3 (red) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Synaptopodin expression is present only in differentiated cells and shows filamentous staining. (iii) F-actin (phalloidin-FITC, green) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Images are representative of three experiments with similar results.

Mentions: We placed nondifferentiated human podocytes expressing the thermosensitive A58 T antigen at 50–60% confluency from 33°C to 37°C to induce differentiation. We followed changes in cell number and morphology by video microscopy for 14 days. By analysing the growth curve we observed that the initial exponential growth phase lasted for 9 days (Figure 1). After cell number reached its peak between days 9 and 10, we saw a slight decrease in cell number followed by stabilization of the culture (days 10–14) when we could hardly detect any mitotic event. When the temperature was shifted to 37°C the morphology of nondifferentiated podocytes gradually transformed into that of differentiated cells (Figures 1 and 2). At permissive temperature (33°C) nondifferentiated podocytes showed typical epithelial cobblestone morphology. In contrast, differentiated podocytes are characterized by enlarged cell bodies with an irregular shape (Figure 2(a), broken outlines) and the formation of processes involving shorter and rounded (Figure 2(a), white arrows) as well as longer and spindle-like projections (Figure 2(a), black arrows). Formations of spindle-like projections were often preceded by partial retraction of cytoplasmatic protrusions. Many differentiated cells developed cellular hypertrophy, some of the cells died (Figure 2(a), red asterisks), and others started to arborize. We detected numerous binucleated but nonproliferating cells. After day 9 we registered a reduction of cell number, but the motility of cells remained high even after differentiation, and the culture proved capability of migration until all bare fields of the culturing vessel were completely covered.


Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

Bányai E, Balogh E, Fagyas M, Arosio P, Hendrik Z, Király G, Nagy G, Tánczos B, Pócsi I, Balla G, Balla J, Bánfalvi G, Jeney V - Oxid Med Cell Longev (2014)

Podocyte differentiation under nonpermissive (37°C) condition. At 50–60% confluence, nondifferentiated cells were placed from 33°C to 37°C to induce differentiation which was followed by video microscopy for 14 days. (a) On selected time-lapse phase contrast images change in the shape and size of podocytes is indicated by broken outlines. Black arrows show shorter and rounded projections of differentiating podocytes. White arrows indicate longer and spindle like projections of arborizing podocytes. Mitotic cells, marked with red double sharps, become detached from the surface of the vessel for a while before the daughter cells adhere again to the bottom. Red asterisks denote apoptotic cells. (b) Modified Giemsa staining was performed on parallel cell cultures at different time points. Static visualization of the cell culture supports the findings of dynamic assessment by time-lapse monitoring. Cytoplasmic staining (pink), nuclear staining (blue). Magnification 10x. (c) Light microscopy and immunofluorescent microscopy images of cell cultures demonstrate characteristic features of human podocytes. Left panels represent nondifferentiated podocytes grown under permissive condition; right panels represent differentiated podocytes after 10 days of culturing under nonpermissive condition. (i) Light microscopy images, scale bars: 300 μm, each. Nondifferentiated cells are small in size and show a cobble-stone like morphology, whilst differentiated cells have enlarged cell bodies with large nucleus, bulky cytoplasm, and ramifying processes. (ii) Synaptopodin-Cy3 (red) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Synaptopodin expression is present only in differentiated cells and shows filamentous staining. (iii) F-actin (phalloidin-FITC, green) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Images are representative of three experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Podocyte differentiation under nonpermissive (37°C) condition. At 50–60% confluence, nondifferentiated cells were placed from 33°C to 37°C to induce differentiation which was followed by video microscopy for 14 days. (a) On selected time-lapse phase contrast images change in the shape and size of podocytes is indicated by broken outlines. Black arrows show shorter and rounded projections of differentiating podocytes. White arrows indicate longer and spindle like projections of arborizing podocytes. Mitotic cells, marked with red double sharps, become detached from the surface of the vessel for a while before the daughter cells adhere again to the bottom. Red asterisks denote apoptotic cells. (b) Modified Giemsa staining was performed on parallel cell cultures at different time points. Static visualization of the cell culture supports the findings of dynamic assessment by time-lapse monitoring. Cytoplasmic staining (pink), nuclear staining (blue). Magnification 10x. (c) Light microscopy and immunofluorescent microscopy images of cell cultures demonstrate characteristic features of human podocytes. Left panels represent nondifferentiated podocytes grown under permissive condition; right panels represent differentiated podocytes after 10 days of culturing under nonpermissive condition. (i) Light microscopy images, scale bars: 300 μm, each. Nondifferentiated cells are small in size and show a cobble-stone like morphology, whilst differentiated cells have enlarged cell bodies with large nucleus, bulky cytoplasm, and ramifying processes. (ii) Synaptopodin-Cy3 (red) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Synaptopodin expression is present only in differentiated cells and shows filamentous staining. (iii) F-actin (phalloidin-FITC, green) and DNA (DAPI, blue) are shown, scale bars: 300 μm, each. Images are representative of three experiments with similar results.
Mentions: We placed nondifferentiated human podocytes expressing the thermosensitive A58 T antigen at 50–60% confluency from 33°C to 37°C to induce differentiation. We followed changes in cell number and morphology by video microscopy for 14 days. By analysing the growth curve we observed that the initial exponential growth phase lasted for 9 days (Figure 1). After cell number reached its peak between days 9 and 10, we saw a slight decrease in cell number followed by stabilization of the culture (days 10–14) when we could hardly detect any mitotic event. When the temperature was shifted to 37°C the morphology of nondifferentiated podocytes gradually transformed into that of differentiated cells (Figures 1 and 2). At permissive temperature (33°C) nondifferentiated podocytes showed typical epithelial cobblestone morphology. In contrast, differentiated podocytes are characterized by enlarged cell bodies with an irregular shape (Figure 2(a), broken outlines) and the formation of processes involving shorter and rounded (Figure 2(a), white arrows) as well as longer and spindle-like projections (Figure 2(a), black arrows). Formations of spindle-like projections were often preceded by partial retraction of cytoplasmatic protrusions. Many differentiated cells developed cellular hypertrophy, some of the cells died (Figure 2(a), red asterisks), and others started to arborize. We detected numerous binucleated but nonproliferating cells. After day 9 we registered a reduction of cell number, but the motility of cells remained high even after differentiation, and the culture proved capability of migration until all bare fields of the culturing vessel were completely covered.

Bottom Line: We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges.Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression.Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Debrecen, Debrecen 4032, Hungary.

ABSTRACT
Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte differentiation are accompanied by changes in oxidative resistance in a manner that could support their long-term survival. We used a conditionally immortalized human podocyte cell line to study the morphological and functional changes during differentiation. We followed the differentiation process for 14 days by time-lapse microscopy. During this period nondifferentiated podocytes gradually transformed into large, nonproliferating, frequently multinucleated cells, with enlarged nuclei and opened chromatin structure. We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges. Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression. Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

Show MeSH
Related in: MedlinePlus