Abstract
BackgroundNicotine 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.MethodsHuman 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.ResultsThe 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.ConclusionsNicotine 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.
Highlights
Cigarette smoking is the leading cause of preventable death in the industrialized world, and it is far ahead of other causes of preventable death, including alcohol, drug abuse, and motor vehicle accidents [1]
The primary antibodies used were anti-rabbit antibodies against extracellular signal-regulated kinase (ERK) (9102), phosphorylated ERK (p-ERK) (9101), c-Jun N-terminal kinase (JNK) (9258), phosphorylated c-Jun N-terminal kinase (p-JNK) (9251), p38 mitogen-activated protein kinase (MAPK) (8690), phosphorylated p38 MAPK (p-p38 MAPK) (4631), Bax (2772), Bcl-2 (2870), the nuclear factor-κB (NF-κB) p65 subunit (3034), cyclin B1 (4138), phosphorylated cdc2 (Tyr 15) (9111), phosphorylated histone H3 (Ser 10) (3377), and histone H3 (9715), all of which were obtained from Cell Signaling Technology, Inc. (Beverly, MA), and anti-rabbit antibodies against nicotinic acetylcholine receptors (nAChRs) α3 (NBP1-18793), nAChR α5 (NBP1-69122), and nAChR β1 (ANC-001), which were obtained from Novus Biochemicals (Littleton, CO) and Alomone Labs (Jerusalem, Israel)
Effects of nicotine on cell viability and reactive oxygen species generation 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)
Summary
Cigarette smoking is the leading cause of preventable death in the industrialized world, and it is far ahead of other causes of preventable death, including alcohol, drug abuse, and motor vehicle accidents [1]. The findings from recent experimental studies have shown that nicotine promotes mesangial cell proliferation and hypertrophy via non-neuronal nicotinic acetylcholine receptors (nAChRs) in rats with 5/6 nephrectomies [6], the mechanism by which cigarette smoking worsens renal function has not been clearly elucidated. Nicotine seems to play an important role in smoking-mediated renal dysfunction [6,7,8]. Apoptosis is the process of programmed cell death, and it plays a central role in the physiological processes underlying kidney growth and remodeling and in various renal diseases [14,15,16]. Given the widely recognized deleterious effect of nicotine on the progression of kidney disease, it is conceivable that nicotine may promote tubular injury in human renal tubular epithelial (HK-2) cells. Our study may help to determine the pathophysiology of nicotine-mediated renal dysfunction
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