Related Experiment Videos
Reactive oxygen species induce apoptosis in cultured human mesangial cells
H Sugiyama1, N Kashihara, H Makino
1Third Department of Internal Medicine, Okayama University Medical School, Japan.
Abstract:
Apoptosis is a distinct form of cell death that is observed under various physiologic and pathologic conditions, and it is thought to be important in regulating the number of glomerular cells. This study investigated the possible role of reactive oxygen species in the induction of apoptosis in cultured human mesangial cells. Fragmented nuclei with condensed chromatin, a morphologic characteristic of apoptosis, were observed by electron microscopy in mesangial cells exposed to 0.02 mM hydrogen peroxide for 4 h. Nuclear DNA extracted from mesangial cells that had been incubated with hydrogen peroxide (2 to 20 mM) or with xanthine (0.05 mM) and xanthine oxidase (5 to 100 mU/mL) showed the ladder pattern on electrophoresis that is a biochemical marker for apoptosis. Hydrogen peroxide (0.02 to 20 mM) decreased the number of viable cells, as determined by trypan blue exclusion, in a dose-dependent manner. Hydrogen peroxide or xanthine and xanthine oxidase increased the lactate dehydrogenase release from mesangial cells in a dose- and time-dependent manners. The release of lactate dehydrogenase was prevented by treatment with a free radical scavenger, catalase. Hydrogen peroxide (2 mM) also significantly increased the number of mesangial cells with fragmented DNA as detected by in situ nick end-labeling Results indicate that reactive oxygen species induce apoptosis in cultured human mesangial cells. Furthermore, apoptosis of mesangial cells induced by reactive oxygen species may contribute to the loss of such cells observed in glomerular disease.
Insights
Reactive oxygen species induce apoptosis, a form of programmed cell death, in human mesangial cells. This finding suggests a role for oxidative stress in glomerular diseases.
Area of Science:
- Cell Biology
- Nephrology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial for regulating cell numbers in the glomerulus.
- Glomerular diseases often involve the loss of mesangial cells.
Purpose of the Study:
- To investigate the role of reactive oxygen species (ROS) in inducing apoptosis in cultured human mesangial cells.
- To understand the potential contribution of ROS-mediated mesangial cell apoptosis to glomerular disease pathogenesis.
Main Methods:
- Human mesangial cells were exposed to hydrogen peroxide or xanthine/xanthine oxidase to generate ROS.
- Morphological changes (condensed chromatin, fragmented nuclei) and DNA fragmentation (ladder pattern) were assessed.
- Cell viability (trypan blue exclusion) and lactate dehydrogenase release were measured.
- The effect of the free radical scavenger catalase was evaluated.
Main Results:
- Hydrogen peroxide exposure induced characteristic apoptotic morphology and DNA fragmentation in mesangial cells.
- ROS significantly reduced cell viability and increased lactate dehydrogenase release in a dose- and time-dependent manner.
- Catalase treatment prevented lactate dehydrogenase release, confirming the role of ROS.
Conclusions:
- Reactive oxygen species are potent inducers of apoptosis in cultured human mesangial cells.
- ROS-mediated apoptosis of mesangial cells may be a significant factor in the pathogenesis of glomerular diseases.