Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant.
BACKGROUND
We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes.


METHODS
The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H(2)DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining.


RESULTS
PAN (100 microg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10(-6) M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli.


CONCLUSIONS
This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.
Oba
Shigeyoshi
S
Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Hino
Masayo
M
Fujita
Toshiro
T
D053607:Adrenomedullin / Q000627:therapeutic use*; D000818:Animals; D000975:Antioxidants / Q000627:therapeutic use*; D002478:Cells, Cultured; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects*; D050199:Podocytes / Q000187:drug effects*; D011692:Puromycin Aminonucleoside / Q000494:pharmacology
D000975:Antioxidants; D053607:Adrenomedullin; D011692:Puromycin Aminonucleoside