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Puromycin aminonucleoside modulates p130Cas of podocytes
Tae-Sun Ha1, Ji-Young Choi, Hye-Young Park
1Department of Pediatrics, Chungbuk National University College of Medicine, Cheongju, Korea. ; Postgraduate School, Chungbuk National University College of Medicine, Cheongju, Korea.
Purpose:
Puromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro.
Methods:
We observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction.
Results:
In the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants.
Conclusion:
These findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.
Insights
Puromycin aminonucleoside (PAN) damages podocytes, altering p130Cas protein expression and distribution. Antioxidants like EGCG and vitamin C reversed these PAN-induced changes, suggesting a role for oxidative stress in podocyte dysfunction.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Puromycin aminonucleoside (PAN) induces podocyte injury, characterized by foot process effacement and altered protein localization.
- p130Cas is a crucial docking protein linking the actin cytoskeleton to the glomerular basement membrane in podocytes.
Purpose of the Study:
- To investigate alterations in p130Cas expression and distribution within podocytes following PAN-induced injury in vitro.
- To assess the potential protective effects of antioxidants against PAN-induced changes in p130Cas.
Main Methods:
- Cultured rat glomerular epithelial cells and mouse podocytes were treated with PAN and various antioxidants (probucol, EGCG, vitamin C).
- Changes in p130Cas expression were analyzed using confocal immunofluorescence, Western blotting, and RT-PCR.
Main Results:
- PAN administration caused dose-dependent internalization and altered distribution of p130Cas in podocytes.
- PAN decreased p130Cas protein and mRNA levels, effects reversed by EGCG and vitamin C.
- p130Cas colocalized with P-cadherin, and its distribution shifted from diffuse to granular upon PAN treatment.
Conclusions:
- PAN induces quantitative and distributional changes in podocyte p130Cas, likely mediated by oxidative stress.
- Antioxidants demonstrate a protective effect, restoring normal p130Cas expression and distribution.
- These findings highlight the role of p130Cas in PAN-induced podocyte dysfunction and suggest therapeutic potential for antioxidants.
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