| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
We previously reported that adrenomedullin (AM), a vasoactive peptide belonging to the calcitonin-gene related peptide (CGRP) family, was expressed in murine podocyte cell lines (MPC) and was upregulated by various stimuli including puromycin (PAN). In this study, we examined the role of AM and oxidative stress in the development of PAN-induced podocyte injury. AM mRNA expression was analyzed by real-time PCR and oxidative stress was determined by ELISA for 8-OHdG, Western blot for nitrotyrosine and fluorescence study using CM-H_2DCFDA for reactive oxygen species (ROS) production. Apoptotic cells were detected by Hoechst33342 and caspase-3 staining. First, we showed that PAN caused an enhancement of oxidative stress markers and also an increase in apoptotic cell number, which was inhibited by antioxidants antimycin and diphenyleneiodium chloride (DPI), indicating that the PAN-induced apoptosis was mediated by enhanced oxidative stress. Because AM was shown to be induced by PAN, we next examined if AM had a protective or pathogenetic role in this condition. The result was that the PAN-induced oxidative stress and apoptosis were inhibited by addition of AM to the culture medium, and moreover, both were clearly exacerbated by blockade of AM action by treatment with an AM antagonist CGRP8-37. In summary, AM was induced by PAN in MPC via oxidative stress, causing an inhibitory effect on PAN-induced oxidative stress and apoptosis. These results suggest that AM may play a protective role as an endogenous regulator with anti-oxidative and anti-apoptotic action, mitigating the cell injury caused by PAN.
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