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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 annexin V/propidium iodide and 4′-6-diamidino-2-phenylindole staining assay.(a) The HK-2 cells were treated with nicotine at 200 or 400 μM for 24 h. (b) The cells were exposed to nicotine (200 μM) for 24 h with or without pretreatment with Bay 11–7082 for 1 h, a nuclear factor-κB inhibitor, and the percentages of the cells residing in the lower right regions of the scatter plots of annexin V-fluorescein isothiocyanate staining, which represented the apoptotic cells, were determined. (c) Chromatin condensation and apoptotic bodies were stained bright blue (arrow) in the HK-2 cells that had been treated with nicotine (200 or 400 μM for 24 h), with or without pretreatment with Bay 11–7082. The nuclear morphologies were examined using fluorescence microscopy. Original magnification, 200 ×. Scale bar = 50 μm.
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pone.0152591.g007: The annexin V/propidium iodide and 4′-6-diamidino-2-phenylindole staining assay.(a) The HK-2 cells were treated with nicotine at 200 or 400 μM for 24 h. (b) The cells were exposed to nicotine (200 μM) for 24 h with or without pretreatment with Bay 11–7082 for 1 h, a nuclear factor-κB inhibitor, and the percentages of the cells residing in the lower right regions of the scatter plots of annexin V-fluorescein isothiocyanate staining, which represented the apoptotic cells, were determined. (c) Chromatin condensation and apoptotic bodies were stained bright blue (arrow) in the HK-2 cells that had been treated with nicotine (200 or 400 μM for 24 h), with or without pretreatment with Bay 11–7082. The nuclear morphologies were examined using fluorescence microscopy. Original magnification, 200 ×. Scale bar = 50 μm.

Mentions: We confirmed the apoptotic effect of nicotine on HK-2 cells using annexin-V binding and PI staining. HK-2 cells treated with 200 μM or 400 μM nicotine for 24 h showed a significant progressive increase with respect to annexin V-positive/PI-negative staining compared with the control cells. Pretreatment with Bay 11–7082 reduced the numbers of apoptotic cells (Fig 7A and 7B). We also detected bright blue apoptotic nuclei, which contained condensed chromatin, and apoptotic bodies in HK-2 cells that had been treated with nicotine; these effects were attenuated when the cells were pretreated with Bay 11–7082 (Fig 7C).


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 annexin V/propidium iodide and 4′-6-diamidino-2-phenylindole staining assay.(a) The HK-2 cells were treated with nicotine at 200 or 400 μM for 24 h. (b) The cells were exposed to nicotine (200 μM) for 24 h with or without pretreatment with Bay 11–7082 for 1 h, a nuclear factor-κB inhibitor, and the percentages of the cells residing in the lower right regions of the scatter plots of annexin V-fluorescein isothiocyanate staining, which represented the apoptotic cells, were determined. (c) Chromatin condensation and apoptotic bodies were stained bright blue (arrow) in the HK-2 cells that had been treated with nicotine (200 or 400 μM for 24 h), with or without pretreatment with Bay 11–7082. The nuclear morphologies were examined using fluorescence microscopy. Original magnification, 200 ×. Scale bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152591.g007: The annexin V/propidium iodide and 4′-6-diamidino-2-phenylindole staining assay.(a) The HK-2 cells were treated with nicotine at 200 or 400 μM for 24 h. (b) The cells were exposed to nicotine (200 μM) for 24 h with or without pretreatment with Bay 11–7082 for 1 h, a nuclear factor-κB inhibitor, and the percentages of the cells residing in the lower right regions of the scatter plots of annexin V-fluorescein isothiocyanate staining, which represented the apoptotic cells, were determined. (c) Chromatin condensation and apoptotic bodies were stained bright blue (arrow) in the HK-2 cells that had been treated with nicotine (200 or 400 μM for 24 h), with or without pretreatment with Bay 11–7082. The nuclear morphologies were examined using fluorescence microscopy. Original magnification, 200 ×. Scale bar = 50 μm.
Mentions: We confirmed the apoptotic effect of nicotine on HK-2 cells using annexin-V binding and PI staining. HK-2 cells treated with 200 μM or 400 μM nicotine for 24 h showed a significant progressive increase with respect to annexin V-positive/PI-negative staining compared with the control cells. Pretreatment with Bay 11–7082 reduced the numbers of apoptotic cells (Fig 7A and 7B). We also detected bright blue apoptotic nuclei, which contained condensed chromatin, and apoptotic bodies in HK-2 cells that had been treated with nicotine; these effects were attenuated when the cells were pretreated with Bay 11–7082 (Fig 7C).

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