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Trehalose, an mTOR independent autophagy inducer, alleviates human podocyte injury after puromycin aminonucleoside treatment.

Kang YL, Saleem MA, Chan KW, Yung BY, Law HK - PLoS ONE (2014)

Bottom Line: Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect.Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility.These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hunghom, Hong Kong, China; Department of Nephrology and Rheumatology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

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Trehalose decreased PAN-induced apoptosis in human podocytes.Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 48 h. (A) Trehalose induced autophagy in PAN-treated human podocytes. The expression of LC3-II slightly increased after 48 h PAN treatment, while it dramatically up-regulated in Tre and PAN+ Tre groups. Representative immunoblot images were shown along with the statistical results. **p<0.01 versus CON, n = 6. (B–C) The findings of (A) were confirmed by LC3 immunostaining. Obvious elevated LC3-II bright green puncta (indicated by white arrows) were visualized in trehalose-treated groups (Tre and PAN+ Tre groups), the representative images and statistical results were shown. Nuclei were stained in blue. **p<0.01, ***p<0.001 versus CON, n = 6. (D) No significant changes were observed in podocyte necrosis. LDH in culture medium of 4 groups was measured, n = 4. (E) Elevated apoptosis in PAN-treated podocytes was decreased by trehalose. Apoptosis was measured by flow cytometry with YO-PRO-1/PI assay. Podocyte apoptosis was induced by PAN and decreased significantly by trehalose. *p<0.05, **p<0.01 versus CON, n = 8. (F) The findings of (E) were confirmed by the data of active caspase-3 measurement. The active caspase-3 positive podocytes were measured by flow cytometry. The changes pattern was similar to podocyte apoptosis measured by YO-PRO-1/PI assay. **p<0.01 versus CON, n = 7.
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pone-0113520-g004: Trehalose decreased PAN-induced apoptosis in human podocytes.Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 48 h. (A) Trehalose induced autophagy in PAN-treated human podocytes. The expression of LC3-II slightly increased after 48 h PAN treatment, while it dramatically up-regulated in Tre and PAN+ Tre groups. Representative immunoblot images were shown along with the statistical results. **p<0.01 versus CON, n = 6. (B–C) The findings of (A) were confirmed by LC3 immunostaining. Obvious elevated LC3-II bright green puncta (indicated by white arrows) were visualized in trehalose-treated groups (Tre and PAN+ Tre groups), the representative images and statistical results were shown. Nuclei were stained in blue. **p<0.01, ***p<0.001 versus CON, n = 6. (D) No significant changes were observed in podocyte necrosis. LDH in culture medium of 4 groups was measured, n = 4. (E) Elevated apoptosis in PAN-treated podocytes was decreased by trehalose. Apoptosis was measured by flow cytometry with YO-PRO-1/PI assay. Podocyte apoptosis was induced by PAN and decreased significantly by trehalose. *p<0.05, **p<0.01 versus CON, n = 8. (F) The findings of (E) were confirmed by the data of active caspase-3 measurement. The active caspase-3 positive podocytes were measured by flow cytometry. The changes pattern was similar to podocyte apoptosis measured by YO-PRO-1/PI assay. **p<0.01 versus CON, n = 7.

Mentions: Interplay exists between autophagy and apoptosis. It has been reported that autophagy suppresses cell apoptosis by inhibiting apoptotic proteins [28]. Since trehalose can induce autophagy in podocytes without increasing ROS, we hypothesized that trehalose decreases podocyte apoptosis. PAN was used to induce podocyte apoptosis in vitro. We found that trehalose up-regulated the expression of LC3-II in PAN-treated human podocytes (Figure 4A). The data of LC3 immunostaining confirmed this finding as LC3-II bright puncta were obviously presented in trehalose alone and PAN+ Tre groups (Figures 4B–4C). To confirm the protective effects of trehalose, LDH was measured to evaluate podocyte necrosis. As shown in Figure 4D, the changes in LDH level was negligible. However, PAN-induced apoptosis was significantly down-regulated after trehalose treatment, accompanying with the decrease in active caspase-3 positive cells (Figures 4E–4F).


Trehalose, an mTOR independent autophagy inducer, alleviates human podocyte injury after puromycin aminonucleoside treatment.

Kang YL, Saleem MA, Chan KW, Yung BY, Law HK - PLoS ONE (2014)

Trehalose decreased PAN-induced apoptosis in human podocytes.Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 48 h. (A) Trehalose induced autophagy in PAN-treated human podocytes. The expression of LC3-II slightly increased after 48 h PAN treatment, while it dramatically up-regulated in Tre and PAN+ Tre groups. Representative immunoblot images were shown along with the statistical results. **p<0.01 versus CON, n = 6. (B–C) The findings of (A) were confirmed by LC3 immunostaining. Obvious elevated LC3-II bright green puncta (indicated by white arrows) were visualized in trehalose-treated groups (Tre and PAN+ Tre groups), the representative images and statistical results were shown. Nuclei were stained in blue. **p<0.01, ***p<0.001 versus CON, n = 6. (D) No significant changes were observed in podocyte necrosis. LDH in culture medium of 4 groups was measured, n = 4. (E) Elevated apoptosis in PAN-treated podocytes was decreased by trehalose. Apoptosis was measured by flow cytometry with YO-PRO-1/PI assay. Podocyte apoptosis was induced by PAN and decreased significantly by trehalose. *p<0.05, **p<0.01 versus CON, n = 8. (F) The findings of (E) were confirmed by the data of active caspase-3 measurement. The active caspase-3 positive podocytes were measured by flow cytometry. The changes pattern was similar to podocyte apoptosis measured by YO-PRO-1/PI assay. **p<0.01 versus CON, n = 7.
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pone-0113520-g004: Trehalose decreased PAN-induced apoptosis in human podocytes.Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 48 h. (A) Trehalose induced autophagy in PAN-treated human podocytes. The expression of LC3-II slightly increased after 48 h PAN treatment, while it dramatically up-regulated in Tre and PAN+ Tre groups. Representative immunoblot images were shown along with the statistical results. **p<0.01 versus CON, n = 6. (B–C) The findings of (A) were confirmed by LC3 immunostaining. Obvious elevated LC3-II bright green puncta (indicated by white arrows) were visualized in trehalose-treated groups (Tre and PAN+ Tre groups), the representative images and statistical results were shown. Nuclei were stained in blue. **p<0.01, ***p<0.001 versus CON, n = 6. (D) No significant changes were observed in podocyte necrosis. LDH in culture medium of 4 groups was measured, n = 4. (E) Elevated apoptosis in PAN-treated podocytes was decreased by trehalose. Apoptosis was measured by flow cytometry with YO-PRO-1/PI assay. Podocyte apoptosis was induced by PAN and decreased significantly by trehalose. *p<0.05, **p<0.01 versus CON, n = 8. (F) The findings of (E) were confirmed by the data of active caspase-3 measurement. The active caspase-3 positive podocytes were measured by flow cytometry. The changes pattern was similar to podocyte apoptosis measured by YO-PRO-1/PI assay. **p<0.01 versus CON, n = 7.
Mentions: Interplay exists between autophagy and apoptosis. It has been reported that autophagy suppresses cell apoptosis by inhibiting apoptotic proteins [28]. Since trehalose can induce autophagy in podocytes without increasing ROS, we hypothesized that trehalose decreases podocyte apoptosis. PAN was used to induce podocyte apoptosis in vitro. We found that trehalose up-regulated the expression of LC3-II in PAN-treated human podocytes (Figure 4A). The data of LC3 immunostaining confirmed this finding as LC3-II bright puncta were obviously presented in trehalose alone and PAN+ Tre groups (Figures 4B–4C). To confirm the protective effects of trehalose, LDH was measured to evaluate podocyte necrosis. As shown in Figure 4D, the changes in LDH level was negligible. However, PAN-induced apoptosis was significantly down-regulated after trehalose treatment, accompanying with the decrease in active caspase-3 positive cells (Figures 4E–4F).

Bottom Line: Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect.Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility.These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hunghom, Hong Kong, China; Department of Nephrology and Rheumatology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

Show MeSH
Related in: MedlinePlus