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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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FtH is highly expressed in differentiated podocytes. (a) Nondifferentiated and differentiated podocytes were treated with heme (0–5 μmol/L in HBSS) and harvested after 4 hours. Equal amounts of total protein (25 μg) were applied to nonreducing gel for western blotting. Western blot showed that FtH is inducible and already present in both cell types at baseline; however, basic FtH expression is more robust in differentiated cells. Induction of FtH by heme treatment is more pronounced in nondifferentiated podocytes compared to differentiated cells. (b) FtH (brown) and WT-1 (purple) staining of native human kidney section are shown. Magnification demonstrates the lumen of a glomerular capillary (∗) and the cell body of a glomerular podocyte with its pedicles (POD) embracing the capillary wall in cross-section. Strong brown staining indicates the presence of FtH in the cytoplasm of podocytes. Scale bar: 10 μm. Representative image of 3 with similar result.
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fig7: FtH is highly expressed in differentiated podocytes. (a) Nondifferentiated and differentiated podocytes were treated with heme (0–5 μmol/L in HBSS) and harvested after 4 hours. Equal amounts of total protein (25 μg) were applied to nonreducing gel for western blotting. Western blot showed that FtH is inducible and already present in both cell types at baseline; however, basic FtH expression is more robust in differentiated cells. Induction of FtH by heme treatment is more pronounced in nondifferentiated podocytes compared to differentiated cells. (b) FtH (brown) and WT-1 (purple) staining of native human kidney section are shown. Magnification demonstrates the lumen of a glomerular capillary (∗) and the cell body of a glomerular podocyte with its pedicles (POD) embracing the capillary wall in cross-section. Strong brown staining indicates the presence of FtH in the cytoplasm of podocytes. Scale bar: 10 μm. Representative image of 3 with similar result.

Mentions: To explore the underlying molecular mechanism of high oxidative resistance of differentiated podocytes, we compared the expression of FtH in nondifferentiated and differentiated podocytes in the absence or presence of heme. Because of its iron content, heme is a strong inducer of ferritin. First, by comparing basal FtH expressions, we found that differentiated podocytes express about 4 times more FtH than nondifferentiated cells (Figure 7(a)). Upon heme treatment, nondifferentiated podocytes upregulated FtH expression dose dependently up to a 4-fold increase at the highest dose of heme applied (5 μmol/L). In contrast, in differentiated podocytes, which show high FtH expression at basal level, low doses of heme (1.2 and 2.5 μmol/L) failed to induce FtH expression further more. Higher doses of heme triggered the induction of FtH suggesting that these differentiated podocytes remained responsive to this challenge but their resistance is shifted. Next, we examined whether FtH is expressed in significant amount in human kidney podocytes. Adult human kidney sections were stained with WT-1 antibody to visualize podocytes (purple) structurally in the glomerulus. For functional assessment, the sections were counterstained with FtH antibody. Glomeruli in the cortex contained several podocytes with strong FtH staining (brown), which supports our in vitro findings of high FtH content and iron sequestering capacity of resting podocytes (Figure 7(b)).


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)

FtH is highly expressed in differentiated podocytes. (a) Nondifferentiated and differentiated podocytes were treated with heme (0–5 μmol/L in HBSS) and harvested after 4 hours. Equal amounts of total protein (25 μg) were applied to nonreducing gel for western blotting. Western blot showed that FtH is inducible and already present in both cell types at baseline; however, basic FtH expression is more robust in differentiated cells. Induction of FtH by heme treatment is more pronounced in nondifferentiated podocytes compared to differentiated cells. (b) FtH (brown) and WT-1 (purple) staining of native human kidney section are shown. Magnification demonstrates the lumen of a glomerular capillary (∗) and the cell body of a glomerular podocyte with its pedicles (POD) embracing the capillary wall in cross-section. Strong brown staining indicates the presence of FtH in the cytoplasm of podocytes. Scale bar: 10 μm. Representative image of 3 with similar result.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7: FtH is highly expressed in differentiated podocytes. (a) Nondifferentiated and differentiated podocytes were treated with heme (0–5 μmol/L in HBSS) and harvested after 4 hours. Equal amounts of total protein (25 μg) were applied to nonreducing gel for western blotting. Western blot showed that FtH is inducible and already present in both cell types at baseline; however, basic FtH expression is more robust in differentiated cells. Induction of FtH by heme treatment is more pronounced in nondifferentiated podocytes compared to differentiated cells. (b) FtH (brown) and WT-1 (purple) staining of native human kidney section are shown. Magnification demonstrates the lumen of a glomerular capillary (∗) and the cell body of a glomerular podocyte with its pedicles (POD) embracing the capillary wall in cross-section. Strong brown staining indicates the presence of FtH in the cytoplasm of podocytes. Scale bar: 10 μm. Representative image of 3 with similar result.
Mentions: To explore the underlying molecular mechanism of high oxidative resistance of differentiated podocytes, we compared the expression of FtH in nondifferentiated and differentiated podocytes in the absence or presence of heme. Because of its iron content, heme is a strong inducer of ferritin. First, by comparing basal FtH expressions, we found that differentiated podocytes express about 4 times more FtH than nondifferentiated cells (Figure 7(a)). Upon heme treatment, nondifferentiated podocytes upregulated FtH expression dose dependently up to a 4-fold increase at the highest dose of heme applied (5 μmol/L). In contrast, in differentiated podocytes, which show high FtH expression at basal level, low doses of heme (1.2 and 2.5 μmol/L) failed to induce FtH expression further more. Higher doses of heme triggered the induction of FtH suggesting that these differentiated podocytes remained responsive to this challenge but their resistance is shifted. Next, we examined whether FtH is expressed in significant amount in human kidney podocytes. Adult human kidney sections were stained with WT-1 antibody to visualize podocytes (purple) structurally in the glomerulus. For functional assessment, the sections were counterstained with FtH antibody. Glomeruli in the cortex contained several podocytes with strong FtH staining (brown), which supports our in vitro findings of high FtH content and iron sequestering capacity of resting podocytes (Figure 7(b)).

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