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Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells.

Kim CS, Choi JS, Joo SY, Bae EH, Ma SK, Lee J, Kim SW - PLoS ONE (2016)

Bottom Line: Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression.While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.

ABSTRACT

Background: Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods: Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results: The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.

Conclusions: Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

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The effects of nicotine on cell viability and reactive oxygen species (ROS) generation in human tubular epithelial cells.(a) The HK-2 cells were treated with nicotine at different concentrations, namely, 0, 50, 100, 200, and 400 μM. Cell viability was assessed using the WST-1 assay after treatment with nicotine for 24 h. (b) The cells were incubated for 24 h with different nicotine concentrations, namely, 0, 50, 100, 200, and 400 μM. ROS generation was detected using the fluoroprobe, 2′,7′-dichlorodihydrofluorescein diacetate. *P < 0.05 or ** P < 0.01 compared with the controls. (c) ROS formation was detected using ROS-sensitive fluorescent dye. (d) The HK-2 cells were exposed to nicotine (200 μM for 24 h) with or without pretreatment with hexamethonium chloride (1 mM) for 3 h. **P < 0.01 compared with the controls. ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments.
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pone.0152591.g002: The effects of nicotine on cell viability and reactive oxygen species (ROS) generation in human tubular epithelial cells.(a) The HK-2 cells were treated with nicotine at different concentrations, namely, 0, 50, 100, 200, and 400 μM. Cell viability was assessed using the WST-1 assay after treatment with nicotine for 24 h. (b) The cells were incubated for 24 h with different nicotine concentrations, namely, 0, 50, 100, 200, and 400 μM. ROS generation was detected using the fluoroprobe, 2′,7′-dichlorodihydrofluorescein diacetate. *P < 0.05 or ** P < 0.01 compared with the controls. (c) ROS formation was detected using ROS-sensitive fluorescent dye. (d) The HK-2 cells were exposed to nicotine (200 μM for 24 h) with or without pretreatment with hexamethonium chloride (1 mM) for 3 h. **P < 0.01 compared with the controls. ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments.

Mentions: The treatment of the HK-2 cells with nicotine reduced their viability in a dose-dependent manner, which was determined by the WST-1 assay (Fig 2A). The generation of ROS was detected using the ROS-sensitive fluorescent dye, DCF-DA. After nicotine treatment for 24 h, the intensity of DCF-DA fluorescence increased in a dose-dependent manner (Fig 2B and 2C). Nicotine treatment significantly increased the intensity of DCF-DA fluorescence, and this increase was moderated by inhibiting the nAChRs with hexamethonium chloride (Fig 2D).


Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells.

Kim CS, Choi JS, Joo SY, Bae EH, Ma SK, Lee J, Kim SW - PLoS ONE (2016)

The effects of nicotine on cell viability and reactive oxygen species (ROS) generation in human tubular epithelial cells.(a) The HK-2 cells were treated with nicotine at different concentrations, namely, 0, 50, 100, 200, and 400 μM. Cell viability was assessed using the WST-1 assay after treatment with nicotine for 24 h. (b) The cells were incubated for 24 h with different nicotine concentrations, namely, 0, 50, 100, 200, and 400 μM. ROS generation was detected using the fluoroprobe, 2′,7′-dichlorodihydrofluorescein diacetate. *P < 0.05 or ** P < 0.01 compared with the controls. (c) ROS formation was detected using ROS-sensitive fluorescent dye. (d) The HK-2 cells were exposed to nicotine (200 μM for 24 h) with or without pretreatment with hexamethonium chloride (1 mM) for 3 h. **P < 0.01 compared with the controls. ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814027&req=5

pone.0152591.g002: The effects of nicotine on cell viability and reactive oxygen species (ROS) generation in human tubular epithelial cells.(a) The HK-2 cells were treated with nicotine at different concentrations, namely, 0, 50, 100, 200, and 400 μM. Cell viability was assessed using the WST-1 assay after treatment with nicotine for 24 h. (b) The cells were incubated for 24 h with different nicotine concentrations, namely, 0, 50, 100, 200, and 400 μM. ROS generation was detected using the fluoroprobe, 2′,7′-dichlorodihydrofluorescein diacetate. *P < 0.05 or ** P < 0.01 compared with the controls. (c) ROS formation was detected using ROS-sensitive fluorescent dye. (d) The HK-2 cells were exposed to nicotine (200 μM for 24 h) with or without pretreatment with hexamethonium chloride (1 mM) for 3 h. **P < 0.01 compared with the controls. ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments.
Mentions: The treatment of the HK-2 cells with nicotine reduced their viability in a dose-dependent manner, which was determined by the WST-1 assay (Fig 2A). The generation of ROS was detected using the ROS-sensitive fluorescent dye, DCF-DA. After nicotine treatment for 24 h, the intensity of DCF-DA fluorescence increased in a dose-dependent manner (Fig 2B and 2C). Nicotine treatment significantly increased the intensity of DCF-DA fluorescence, and this increase was moderated by inhibiting the nAChRs with hexamethonium chloride (Fig 2D).

Bottom Line: Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression.While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.

ABSTRACT

Background: Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods: Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results: The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.

Conclusions: Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

Show MeSH
Related in: MedlinePlus