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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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Mature podocytes are highly resistant to oxidative stress. (a) Nondifferentiated and differentiated podocytes were exposed to H2O2 (0–500 μmol/L in HBSS) for 4 hours. Cell viability was assessed by MTT assay. (b) Nondifferentiated and differentiated podocytes were subjected to heme treatment (0–5 μmol/L in HBSS). After 1 hour heme was replaced by HBSS. Cell viability was measured by MTT assay after a 4-hour incubation period. (c) Nondifferentiated and differentiated podocytes were treated with heme (2.5 μmol/L in HBSS) or vehicle for 1 hour. After washing the heme away cells were exposed to H2O2 (125 μmol/L in HBSS) or vehicle for 4 hours. Cell viability was determined by MTT assay. Results are shown as mean values ± SEM of at least three independent experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001.
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fig4: Mature podocytes are highly resistant to oxidative stress. (a) Nondifferentiated and differentiated podocytes were exposed to H2O2 (0–500 μmol/L in HBSS) for 4 hours. Cell viability was assessed by MTT assay. (b) Nondifferentiated and differentiated podocytes were subjected to heme treatment (0–5 μmol/L in HBSS). After 1 hour heme was replaced by HBSS. Cell viability was measured by MTT assay after a 4-hour incubation period. (c) Nondifferentiated and differentiated podocytes were treated with heme (2.5 μmol/L in HBSS) or vehicle for 1 hour. After washing the heme away cells were exposed to H2O2 (125 μmol/L in HBSS) or vehicle for 4 hours. Cell viability was determined by MTT assay. Results are shown as mean values ± SEM of at least three independent experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001.

Mentions: To compare oxidative resistance of undifferentiated and mature podocytes, we tested the effect of H2O2 on cell viability. Nondifferentiated cells were sensitive to H2O2 that caused about 50% of cell death at doses between 125 and 500 μmol/L. In contrast, no cytotoxicity was observed in differentiated podocytes using the same doses of H2O2 (Figure 4(a)). Heme, as a prooxidant, can sensitize various cell types to oxidative stimuli, such as H2O2 [14] or inflammatory cytokines, such as TNF-alpha [23, 24]. We carried on checking the effect of heme alone or in combination with H2O2 on cell viability. As shown in Figure 4(b), heme alone caused death of nondifferentiated podocytes in a dose-dependent manner, while the same doses did not exert any cytotoxic effect to differentiated cells. Treatment of nondifferentiated podocytes with heme prior to H2O2 challenge led to decreased cell viability (53.3% versus 25.3%) (Figure 4(c)). In contrast, mature podocytes survived this lethal combination of heme and H2O2.


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)

Mature podocytes are highly resistant to oxidative stress. (a) Nondifferentiated and differentiated podocytes were exposed to H2O2 (0–500 μmol/L in HBSS) for 4 hours. Cell viability was assessed by MTT assay. (b) Nondifferentiated and differentiated podocytes were subjected to heme treatment (0–5 μmol/L in HBSS). After 1 hour heme was replaced by HBSS. Cell viability was measured by MTT assay after a 4-hour incubation period. (c) Nondifferentiated and differentiated podocytes were treated with heme (2.5 μmol/L in HBSS) or vehicle for 1 hour. After washing the heme away cells were exposed to H2O2 (125 μmol/L in HBSS) or vehicle for 4 hours. Cell viability was determined by MTT assay. Results are shown as mean values ± SEM of at least three independent experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109136&req=5

fig4: Mature podocytes are highly resistant to oxidative stress. (a) Nondifferentiated and differentiated podocytes were exposed to H2O2 (0–500 μmol/L in HBSS) for 4 hours. Cell viability was assessed by MTT assay. (b) Nondifferentiated and differentiated podocytes were subjected to heme treatment (0–5 μmol/L in HBSS). After 1 hour heme was replaced by HBSS. Cell viability was measured by MTT assay after a 4-hour incubation period. (c) Nondifferentiated and differentiated podocytes were treated with heme (2.5 μmol/L in HBSS) or vehicle for 1 hour. After washing the heme away cells were exposed to H2O2 (125 μmol/L in HBSS) or vehicle for 4 hours. Cell viability was determined by MTT assay. Results are shown as mean values ± SEM of at least three independent experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001.
Mentions: To compare oxidative resistance of undifferentiated and mature podocytes, we tested the effect of H2O2 on cell viability. Nondifferentiated cells were sensitive to H2O2 that caused about 50% of cell death at doses between 125 and 500 μmol/L. In contrast, no cytotoxicity was observed in differentiated podocytes using the same doses of H2O2 (Figure 4(a)). Heme, as a prooxidant, can sensitize various cell types to oxidative stimuli, such as H2O2 [14] or inflammatory cytokines, such as TNF-alpha [23, 24]. We carried on checking the effect of heme alone or in combination with H2O2 on cell viability. As shown in Figure 4(b), heme alone caused death of nondifferentiated podocytes in a dose-dependent manner, while the same doses did not exert any cytotoxic effect to differentiated cells. Treatment of nondifferentiated podocytes with heme prior to H2O2 challenge led to decreased cell viability (53.3% versus 25.3%) (Figure 4(c)). In contrast, mature podocytes survived this lethal combination of heme and H2O2.

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