Glomerular endothelial dysfunction in progressive renal diseases and aging kidney and development the novel therapeutic strategy

内皮功能障碍是各种形式的肾损伤和衰老肾脏中血管和肾脏并发症发展的一个公认的发病机制。本研究的目的是阐明肾损伤的发病机制，并探讨在肾损伤和肾脏衰老的进展形式中全身血管内皮功能障碍的可能机制。我们假设血管中活性氧（ROS）和一氧化氮（NO）的失衡与肾损伤和大血管功能障碍有关，并成功地发展了原位观察ROS和NO生成的新方法。简单地说，在全身麻醉下，用37℃磷酸盐缓冲液（PBS）以5 ml/min的流速通过输液泵灌注大鼠全身。一旦去除血液，用含0.01mmol/L二氨基罗丹明- ...更多信息 上午4个月用DAR-4 M AM、0.05mmol/L DCFH-DA、0.1mmol/L L-精氨酸、2 mmol/L CaCl_2以3 ml/min的流速灌注10分钟，后灌注加入含0.2mmol/L N-硝基-L-精氨酸甲酯盐酸盐的4%多聚甲醛用共聚焦激光扫描显微镜TCS-NT（Leica-Microsystems，Tokyo，Japan）获得ROS和NO的荧光图像。DAR-4 M AM，激发波长560 nm，发射波长575 nm; DCFH-DA，激发波长490 nm，发射波长530 nm。在肾血管和主动脉中，硝基酪氨酸的积累也增加，这是亚硝化应激的痕迹。我们还发现，在该模型中，NADPH氧化酶活性与ROS的产生有关。血管紧张素受体阻断剂抑制ROS和NO的产生，并显着降低亚硝化应激

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- … More 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