| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Endothelial dysfunction is a well-established pathogenetic mechanism underlying the development of vascular and renal complications in the various forms of renal injuries and aging kidney as well. The aim of this study is to elucidate the pathogenetic mechanism of renal injury and explore the possible mechanism that could underlie endothelial dysfunction in the systemic vasculature in the progressive forms of renal injuries and senescent kidney. We hypothesized that an imbalance between reactive oxygen species (ROS) and nitric oxide (NO) in the vasculature is implicated in both renal injury and macrovascular dysfunction.
We have successfully developed the novel method by which one could observe the generation of ROS and NO in situ. Briefly, under general anesthesia, The rat whole body was perfused by the infusion pump with 37℃ phosphate buffer saline (PBS) at a flow rate of 5ml/min. Once blood had been removed, the whole body was perfused with PBS containing 0.01mmol/L diaminorhodamine-
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4M AM (DAR-4M AM), 0.05mmol/L dichlorodihydrofluorescin diaceate (DCFH-DA), 0.1mmol/L L-Arginine, and 2mmol/L CaCl2 for 10 minutes at a flow rate of 3ml/min. To remove the unreacted reagent and fix tissues, a postperfusion was added with 4% paraformaldehyde containing 0.2mmol/L Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME ; Sigma-Aldrich Japan) for 30 minutes at a flow rate of 5ml/min. Fluorescent images of ROS and NO were obtained with a confocal laser-scanning microscopy TCS-NT (Leica-Microsystems, Tokyo, Japan). The wavelength was as follow ; DAR-4M AM, excitation at 560nm and emission at 575nm ; DCFH-DA, excitation at 490nm and emission at 530nm.
We have discovered increased generation of ROS and deceased baioavailable NO in the renal tissue in the 5/6 nephrectomized rats. Accumulation of nitrotyrosine, a trace of nitrosative stress, was also increased in the renal vasculature and aorta We also found NADPH oxidase activity was implicated in the generation of ROS in this model. Angiotensin receptor blocker inhibited increased productions of ROS and NO, and significantly reduced nitrosative stress Less
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