Implication of reactive oxygen species, nitric oxide, and their imbalance in the pathogenesis of chronic kidney disease

内皮功能障碍是各种形式的肾损伤和肾脏老化中血管和肾脏并发症发生的一个公认的病理机制。本研究的目的是阐明肾损伤的发病机制，并探讨进行性肾损伤和肾衰老过程中全身性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）之间的不平衡与肾损伤和大血管功能障碍有关。我们已经成功地开发了一种新的方法，通过这种方法可以原位观察ROS和NO的生成。简单地说，在全身麻醉下，用37℃磷酸缓冲盐水（PBS）灌注泵灌注大鼠全身，流速5ml/min。取血后全身灌注含有0.01mmol/L二氨基膦胺-…More 4M AM （par -4M AM）、0.05mmol/L二氯双氢荧光素二乙酸酯（DCFH-DA）、0.1mmol/L L精氨酸和2mmol/L CaCl2的PBS，以3ml/min流速灌注10分钟。为了去除未反应试剂并固定组织，灌注后加入含有0.2mmol/L N□-硝基-L-精氨酸甲酯盐酸盐（L- name; Sigma-Aldrich Japan）的4%多聚甲醛，以5ml/min的流速浸泡30分钟。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。波长如下：DAR-4M AM， 560nm激发，575nm发射；DCFH-DA，激发波长490nm，发射波长530nm。我们发现在5/6肾切除大鼠肾组织中ROS和死baioavailable NO的生成增加。硝基酪氨酸的积累（一种亚硝化应激的痕迹）也在肾血管和主动脉中增加。我们还发现，在该模型中，NADPH氧化酶活性与ROS的产生有关。血管紧张素受体阻滞剂抑制ROS和NO的增加，显著降低亚硝化应激

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 N□-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