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Imaging of hydroperoxides in a rat glomerulus stimulated by puromycin aminonucleoside
K Aoyagi1, K Akiyama, C Tomida
1Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan. aoyagi-k@md.tsukuba.ac.jp
Background:
To determine the locus of the increased oxidation induced by puromycin aminonucleoside (PAN), we imaged hydroperoxides in glomeruli stimulated by PAN in vivo and in vitro.
Methods:
Dichlorofluorescein diacetate (DCFH-DA) in cells makes dichlorofluorescein, a substance that fluoresces when reacted with hydroperoxides. Fluorescence was detected using a photon detection video camera connected to a microscope. Two kinds of isolated glomeruli of Wistar rats were examined. One was the glomerulus obtained from rats on the seventh day following the injection of PAN. In this case, glomeruli were incubated in a buffer containing 5 mM DCFH-DA. Another was the glomerulus collected at 30 minutes after a large amount of DCFH-DA was intravenously injected. These glomeruli were incubated with either PAN or phorbol myristate acetate (PMA) in Krebs-Henseleite bicarbonate buffer.
Results:
The images from the glomeruli treated by PAN in vivo resemble pictures of a galaxy by telescope. When the glomeruli were treated by PAN in vitro, two localized points appeared in each glomerulus after 15 minutes of incubation with PAN, and after 75 minutes of incubation, the fluorescence spread throughout the glomerulus. When glomeruli were incubated with PMA, two points that gave a very strong fluorescence were observed in each glomerulus, but they did not spread throughout the glomeruli. In both experiments, glomeruli without stimulants did not fluoresce.
Conclusion:
Increases in hydroperoxides were observed in the glomeruli from rats made nephrotic by exposure to PAN, and were also observed in glomeruli following 15 minutes of incubation with PAN in vitro.
Insights
Puromycin aminonucleoside (PAN) causes increased oxidation in rat glomeruli. This study visualized hydroperoxide formation in glomeruli, identifying specific locations of oxidative stress.
Area of Science:
- Nephrology
- Oxidative Stress Research
- Cellular Imaging
Background:
- Puromycin aminonucleoside (PAN) is known to induce nephrotoxicity.
- The precise location of PAN-induced oxidative damage within glomeruli requires further elucidation.
Purpose of the Study:
- To pinpoint the locus of increased oxidation induced by puromycin aminonucleoside (PAN).
- To visualize hydroperoxide formation in glomeruli stimulated by PAN in vivo and in vitro.
Main Methods:
- Utilized dichlorofluorescein diacetate (DCFH-DA) to detect hydroperoxides via fluorescence microscopy.
- Examined isolated glomeruli from Wistar rats treated with PAN or phorbol myristate acetate (PMA).
- Conducted both in vivo and in vitro imaging experiments.
Main Results:
- PAN-induced oxidation in vivo created a 'galaxy-like' fluorescence pattern in glomeruli.
- In vitro, PAN treatment initially localized fluorescence to two points within 15 minutes, spreading later.
- Phorbol myristate acetate (PMA) also induced strong, localized fluorescence in two points but without widespread distribution.
- Control glomeruli without stimulants showed no fluorescence.
Conclusions:
- Increased hydroperoxides were confirmed in glomeruli of PAN-nephrotic rats.
- PAN exposure in vitro rapidly induced localized hydroperoxide formation in glomeruli.