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Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced
Atsushi Ueda1, Sohji Nagase, Hidekatsu Yokoyama
1Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan.
Abstract:
The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.
Insights
Puromycin aminonucleoside (PAN) nephrosis in rats significantly reduces kidney reducing activity. Electron paramagnetic resonance (EPR) studies show longer TEMPOL half-lives in PAN-treated rats, indicating decreased in vivo and mitochondrial function.
Area of Science:
- Biochemistry
- Nephrology
- Medical Imaging
Background:
- Puromycin aminonucleoside (PAN) nephrosis is a model for studying kidney damage.
- Electron paramagnetic resonance (EPR) using nitroxide radicals like TEMPOL can assess biological reducing activity.
- Kidney function and redox status are crucial in nephrotic conditions.
Purpose of the Study:
- To investigate the temporal changes in kidney reducing activity in a rat model of PAN nephrosis.
- To evaluate in vivo and mitochondrial reducing capacity using electron paramagnetic resonance (EPR) and TEMPOL.
- To correlate changes in TEMPOL half-life with the progression of PAN nephrosis.
Main Methods:
- Induction of PAN nephrosis in rats via intraperitoneal injection of PAN (75 mg/kg).
- Measurement of in vivo renal half-lives of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) via EPR after intravenous injection.
- Determination of mitochondrial half-lives of TEMPOL by incubating kidney mitochondrial fractions with TEMPOL solution.
Main Results:
- In vivo renal half-lives of TEMPOL were significantly longer in PAN nephrosis rats from 7 to 14 days post-administration compared to controls.
- Mitochondrial half-lives of TEMPOL showed a remarkable prolongation on day 9 after PAN administration (676 +/- 183 s vs. 378 +/- 69 s in controls, p < .01).
- These prolonged half-lives indicate a marked decrease in both in vivo and mitochondrial reducing activity in the kidneys of PAN-treated rats.
Conclusions:
- TEMPOL's altered half-life in the kidney serves as a reliable indicator of renal reducing activity.
- PAN nephrosis is associated with a significant decline in the kidney's overall reducing capacity.
- These findings highlight the utility of EPR spectroscopy in assessing metabolic dysfunction in kidney disease models.