Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells

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.

Fig 1. Expression of the nicotine acetylcholine receptors’ (nAChRs) messenger ribonucleic acid (mRNA) in human tubular epithelial cells.

(a) The mRNA expression associated with the α1–7 and β1–4 nAChR subunits in HK-2 cells was analyzed using the reverse transcription-polymerase chain reaction. (b) Western blot analysis determined the expression of the proteins for the α3, α5, and β1 subunits in HK-2 cells.

Fig 2. 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.

Fig 3. The effect of nicotine on the expression of mitogen-activated protein kinases (MAPKs) in human tubular epithelial cells.

(a) The HK-2 cells were treated with 200 μM nicotine for 0.5, 1, 2, and 3 h, then the levels of expression of the phosphorylated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK proteins were determined. *P < 0.05 or **P < 0.01 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, then the levels of expression of the phosphorylated ERK, JNK, and p38 MAPK proteins were determined. *P < 0.05 compared with the controls. †P < 0.05 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.

Fig 4. 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.

Fig 5. The effect of nicotine on cell cycle arrest in human tubular epithelial cells.

The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. *P < 0.05 or **P < 0.01 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. (c) To elucidate the distribution of the cell cycle phases, cell cycle analysis was performed using flow cytometry. The HK-2 cells were treated with nicotine (0, 200, and 400 μM for 16 h).

Fig 6. The effect of nicotine on apoptosis in human tubular epithelial cells.

(a) The HK-2 cells were exposed to nicotine (200 μM) for 1, 3, 6, and 10 h, then the expression of the Bax and Bcl-2 proteins was determined. *P < 0.05 or **P < 0.01 compared with the controls. (b) The cells were pretreated with hexamethonium chloride for 3 h at different concentrations, namely, 0, 0.25, 0.5, 1, and 3 mM, then they were incubated with nicotine (200 μM for 10 h). (c) The cells were exposed to nicotine (200 μM for 10 h) with or without pretreatment with 10 mM N-acetyl-L-cysteine for 1 h. (d) The cells were exposed to nicotine (200 μM for 10 h) with or without pretreatment with Bay 11–7082, which is an NF-B inhibitor, for 1 h. **P < 0.01 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. (e) Bax expression (red) was examined in the HK-2 cells after treatment with nicotine (200 μM for 10 h) and pretreatment with hexamethonium chloride (1 mM for 3 h). Original magnification, 200 ×. Scale bar = 50 μm. HC, hexamethonium chloride. The data are representative of at least three independent experiments.

Fig 7. 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.