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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 effect of nicotine on the expression of nuclear factor-κB (NF-κB) in human tubular epithelial cells.(a) The HK-2 cells were exposed to nicotine (200 μM) for 0.5, 1, and 3 h, then the levels of the expression of the NF-κB p65 subunit and the cytosol nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) proteins were determined. *P < 0.05 compared with the controls. (b) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment with 10 mM N-acetyl-L-cysteine for 1 h. (c) The cells were incubated with nicotine (200 μM for 1 h) after pretreatment for 1 h with PD98059, an extracellular signal-regulated kinase inhibitor, SP600125, a c-Jun N-terminal kinase inhibitor, or SB203580, or a p38 mitogen-activated protein kinases inhibitor. (d) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment for 1 h with Bay 11–7082, an NF-B inhibitor. *P < 0.05 compared with the controls. †P < 0.05 or ††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. (e, f, and g) Immunofluorescence of the NF-κB p65 subunit. Original magnification, 200 ×. Scale bar = 50 μm.
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pone.0152591.g004: The effect of nicotine on the expression of nuclear factor-κB (NF-κB) in human tubular epithelial cells.(a) The HK-2 cells were exposed to nicotine (200 μM) for 0.5, 1, and 3 h, then the levels of the expression of the NF-κB p65 subunit and the cytosol nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) proteins were determined. *P < 0.05 compared with the controls. (b) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment with 10 mM N-acetyl-L-cysteine for 1 h. (c) The cells were incubated with nicotine (200 μM for 1 h) after pretreatment for 1 h with PD98059, an extracellular signal-regulated kinase inhibitor, SP600125, a c-Jun N-terminal kinase inhibitor, or SB203580, or a p38 mitogen-activated protein kinases inhibitor. (d) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment for 1 h with Bay 11–7082, an NF-B inhibitor. *P < 0.05 compared with the controls. †P < 0.05 or ††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. (e, f, and g) Immunofluorescence of the NF-κB p65 subunit. Original magnification, 200 ×. Scale bar = 50 μm.

Mentions: We investigated the effects of nicotine on the expression of the NF-κB p65 subunit in nuclear extracts and on the expression of total nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) within the cytosol from HK-2 cells. After the incubation of the HK-2 cells with nicotine for 1 h, the cells exhibited an increase in the expression of the NF-κB p65 subunit compared with the control cells. The cytoplasmic expression of total IκBα began to decrease after 0.5 h and then it returned to the levels observed before treatment (Fig 4A). NAC pretreatment of the HK-2 cells reversed the nicotine-induced increase in the expression of the NF-κB p65 subunit (Fig 4B). We investigated the effects of specific inhibitors, including ERK (PD98059), JNK (SP600125), and p38 (SB203580) MAPK inhibitors, on the expression of the NF-κB p65 subunit to determine whether the MAPK pathway modulates NF-κB pathway signaling in nicotine-treated HK-2 cells. The ERK and JNK inhibitors attenuated the nicotine-induced increase in NF-κB p65 subunit expression, but the expression of the NF-κB p65 subunit was not affected by the p38 MAPK inhibitor (Fig 4C). The nicotine-induced increase in the expression of the NF-κB p65 subunit was also attenuated by Bay 11–7082, an NF-κB inhibitor (Fig 4D). Immunofluorescence analysis confirmed that nicotine treatment increased the nuclear translocation of the NF-κB p65 subunit and that this decreased following treatment with NAC, and the ERK and JNK inhibitors, but not after treatment with the p38 MAPK inhibitor (Fig 4E–4G).


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 effect of nicotine on the expression of nuclear factor-κB (NF-κB) in human tubular epithelial cells.(a) The HK-2 cells were exposed to nicotine (200 μM) for 0.5, 1, and 3 h, then the levels of the expression of the NF-κB p65 subunit and the cytosol nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) proteins were determined. *P < 0.05 compared with the controls. (b) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment with 10 mM N-acetyl-L-cysteine for 1 h. (c) The cells were incubated with nicotine (200 μM for 1 h) after pretreatment for 1 h with PD98059, an extracellular signal-regulated kinase inhibitor, SP600125, a c-Jun N-terminal kinase inhibitor, or SB203580, or a p38 mitogen-activated protein kinases inhibitor. (d) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment for 1 h with Bay 11–7082, an NF-B inhibitor. *P < 0.05 compared with the controls. †P < 0.05 or ††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. (e, f, and g) Immunofluorescence of the NF-κB p65 subunit. Original magnification, 200 ×. Scale bar = 50 μm.
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pone.0152591.g004: The effect of nicotine on the expression of nuclear factor-κB (NF-κB) in human tubular epithelial cells.(a) The HK-2 cells were exposed to nicotine (200 μM) for 0.5, 1, and 3 h, then the levels of the expression of the NF-κB p65 subunit and the cytosol nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) proteins were determined. *P < 0.05 compared with the controls. (b) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment with 10 mM N-acetyl-L-cysteine for 1 h. (c) The cells were incubated with nicotine (200 μM for 1 h) after pretreatment for 1 h with PD98059, an extracellular signal-regulated kinase inhibitor, SP600125, a c-Jun N-terminal kinase inhibitor, or SB203580, or a p38 mitogen-activated protein kinases inhibitor. (d) The cells were exposed to nicotine (200 μM for 1 h) with or without pretreatment for 1 h with Bay 11–7082, an NF-B inhibitor. *P < 0.05 compared with the controls. †P < 0.05 or ††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. (e, f, and g) Immunofluorescence of the NF-κB p65 subunit. Original magnification, 200 ×. Scale bar = 50 μm.
Mentions: We investigated the effects of nicotine on the expression of the NF-κB p65 subunit in nuclear extracts and on the expression of total nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) within the cytosol from HK-2 cells. After the incubation of the HK-2 cells with nicotine for 1 h, the cells exhibited an increase in the expression of the NF-κB p65 subunit compared with the control cells. The cytoplasmic expression of total IκBα began to decrease after 0.5 h and then it returned to the levels observed before treatment (Fig 4A). NAC pretreatment of the HK-2 cells reversed the nicotine-induced increase in the expression of the NF-κB p65 subunit (Fig 4B). We investigated the effects of specific inhibitors, including ERK (PD98059), JNK (SP600125), and p38 (SB203580) MAPK inhibitors, on the expression of the NF-κB p65 subunit to determine whether the MAPK pathway modulates NF-κB pathway signaling in nicotine-treated HK-2 cells. The ERK and JNK inhibitors attenuated the nicotine-induced increase in NF-κB p65 subunit expression, but the expression of the NF-κB p65 subunit was not affected by the p38 MAPK inhibitor (Fig 4C). The nicotine-induced increase in the expression of the NF-κB p65 subunit was also attenuated by Bay 11–7082, an NF-κB inhibitor (Fig 4D). Immunofluorescence analysis confirmed that nicotine treatment increased the nuclear translocation of the NF-κB p65 subunit and that this decreased following treatment with NAC, and the ERK and JNK inhibitors, but not after treatment with the p38 MAPK inhibitor (Fig 4E–4G).

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