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Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury.

Chandrika BB, Yang C, Ou Y, Feng X, Muhoza D, Holmes AF, Theus S, Deshmukh S, Haun RS, Kaushal GP - PLoS ONE (2015)

Bottom Line: Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy.Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A.These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACT
We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase-3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus

Tunicamycin treatment does not impair autophagic flux in renal cells and kidney.A. Kidney tissue obtained from mice treated with 1 mg/kg b.w. tunicamycin alone, 50 mg/kg (b. w.)/day chloroquine alone or tunicamycin + chloroquine for 2d, 3d, and 4d or vehicle (30% DMSO + 70% saline) for 4d was homogenized with lysis buffer containing protease inhibitors. Tissue lysate (50 μg protein) was subjected to western blot analysis using specific antibodies to p62, LC3, and β-actin. β-actin was used as a loading control. The results shown are representative of three independent experiments. B. LLC-PK1 cells were treated with 1μg/ml tunicamycin (TN), chloroquine (CHL), and tunicamycin + chloroquine for 8 h, 16h, and 24 h. Cell lysates (50 μg protein) were subjected to western blot using specific antibodies to LC3 and β-actin. β-actin was served as a loading control. The results shown are representative of three independent experiments.
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pone.0140025.g006: Tunicamycin treatment does not impair autophagic flux in renal cells and kidney.A. Kidney tissue obtained from mice treated with 1 mg/kg b.w. tunicamycin alone, 50 mg/kg (b. w.)/day chloroquine alone or tunicamycin + chloroquine for 2d, 3d, and 4d or vehicle (30% DMSO + 70% saline) for 4d was homogenized with lysis buffer containing protease inhibitors. Tissue lysate (50 μg protein) was subjected to western blot analysis using specific antibodies to p62, LC3, and β-actin. β-actin was used as a loading control. The results shown are representative of three independent experiments. B. LLC-PK1 cells were treated with 1μg/ml tunicamycin (TN), chloroquine (CHL), and tunicamycin + chloroquine for 8 h, 16h, and 24 h. Cell lysates (50 μg protein) were subjected to western blot using specific antibodies to LC3 and β-actin. β-actin was served as a loading control. The results shown are representative of three independent experiments.

Mentions: Complete autophagic activity with degradation and clearance of autophagic cargo is referred to as autophagic flux. To determine that autophagic flux was not impaired by ER stress in the kidney, mice were administered the lysosomotropic agent chloroquine in the presence and absence of tunicamycin. Chloroquine inhibits lysosomal function (by increasing intralysosomal pH), blocks autophagosomal clearance, and impairs autophagic flux [35]. During impaired autophagic flux, LC3-II and p62 that are present in the inner membrane of the autophagosome are not degraded and are accumulated [35]. As shown in Fig 6A, chloroquine treatment increased tunicamycin-induced LC3-II and p62 levels in the kidney compared to tunicamycin treated mice. Tunicamycin alone did not increase p62 and LC3-II (Fig 6A) levels suggesting that tunicamycin does not impair the flux of the autophagy pathway. Similarly, in LLC-PK1 cells, chloroquine-treatment markedly increased the production of LC3-II compared to tunicamycin alone (Fig 6B). These findings indicated that tunicamycin-induced autophagic activity proceeds to completion and is not impaired.


Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury.

Chandrika BB, Yang C, Ou Y, Feng X, Muhoza D, Holmes AF, Theus S, Deshmukh S, Haun RS, Kaushal GP - PLoS ONE (2015)

Tunicamycin treatment does not impair autophagic flux in renal cells and kidney.A. Kidney tissue obtained from mice treated with 1 mg/kg b.w. tunicamycin alone, 50 mg/kg (b. w.)/day chloroquine alone or tunicamycin + chloroquine for 2d, 3d, and 4d or vehicle (30% DMSO + 70% saline) for 4d was homogenized with lysis buffer containing protease inhibitors. Tissue lysate (50 μg protein) was subjected to western blot analysis using specific antibodies to p62, LC3, and β-actin. β-actin was used as a loading control. The results shown are representative of three independent experiments. B. LLC-PK1 cells were treated with 1μg/ml tunicamycin (TN), chloroquine (CHL), and tunicamycin + chloroquine for 8 h, 16h, and 24 h. Cell lysates (50 μg protein) were subjected to western blot using specific antibodies to LC3 and β-actin. β-actin was served as a loading control. The results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4596863&req=5

pone.0140025.g006: Tunicamycin treatment does not impair autophagic flux in renal cells and kidney.A. Kidney tissue obtained from mice treated with 1 mg/kg b.w. tunicamycin alone, 50 mg/kg (b. w.)/day chloroquine alone or tunicamycin + chloroquine for 2d, 3d, and 4d or vehicle (30% DMSO + 70% saline) for 4d was homogenized with lysis buffer containing protease inhibitors. Tissue lysate (50 μg protein) was subjected to western blot analysis using specific antibodies to p62, LC3, and β-actin. β-actin was used as a loading control. The results shown are representative of three independent experiments. B. LLC-PK1 cells were treated with 1μg/ml tunicamycin (TN), chloroquine (CHL), and tunicamycin + chloroquine for 8 h, 16h, and 24 h. Cell lysates (50 μg protein) were subjected to western blot using specific antibodies to LC3 and β-actin. β-actin was served as a loading control. The results shown are representative of three independent experiments.
Mentions: Complete autophagic activity with degradation and clearance of autophagic cargo is referred to as autophagic flux. To determine that autophagic flux was not impaired by ER stress in the kidney, mice were administered the lysosomotropic agent chloroquine in the presence and absence of tunicamycin. Chloroquine inhibits lysosomal function (by increasing intralysosomal pH), blocks autophagosomal clearance, and impairs autophagic flux [35]. During impaired autophagic flux, LC3-II and p62 that are present in the inner membrane of the autophagosome are not degraded and are accumulated [35]. As shown in Fig 6A, chloroquine treatment increased tunicamycin-induced LC3-II and p62 levels in the kidney compared to tunicamycin treated mice. Tunicamycin alone did not increase p62 and LC3-II (Fig 6A) levels suggesting that tunicamycin does not impair the flux of the autophagy pathway. Similarly, in LLC-PK1 cells, chloroquine-treatment markedly increased the production of LC3-II compared to tunicamycin alone (Fig 6B). These findings indicated that tunicamycin-induced autophagic activity proceeds to completion and is not impaired.

Bottom Line: Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy.Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A.These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACT
We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase-3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus