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

内皮功能障碍是一种公认的致病机制，在各种形式的肾损伤和衰老的肾脏中，血管和肾脏并发症的发生是潜在的。本研究的目的是阐明肾脏损伤的发病机制，并探讨进行性肾损伤和肾脏衰老过程中血管内皮细胞功能障碍的可能机制。我们假设血管中的ROS和NO之间的失衡与肾脏损伤和大血管功能障碍有关。我们成功地开发了一种新的方法，可以在原位观察ROS和NO的产生。简言之，在全身麻醉下，用输液泵以5ml/℃的速度向大鼠全身灌流37min的磷酸盐缓冲液。取血后，全身注入含0.01 mmol/L二氨基若丹明-…的PBS。加入4M AM(DAR-4M AM)，0.05 mmol/L二氯二氢荧光素二乙酸酯(DCFH-DA)，0.1 mmol/L L-精氨酸，2 mmol/L CaCl2，以3ml/min的流速静置10min。为去除未反应试剂并固定组织，后灌流加入含0.2 mmol/L N-硝基-L-精氨酸甲酯盐酸盐(L-NAME；Sigma-Aldrich Japan)的4%多聚甲醛30分钟，流速为5ml/min。用共聚焦激光扫描显微镜TCS-NT(Leica-MicroSystems，日本东京)获得ROS和NO的荧光图像。DCFH-DA为DCFH-DA，激发波长为490 nm，发射波长为530 nm。我们发现5/6肾切除大鼠肾组织中ROS生成增加，有效NO生成减少。亚硝化应激的微量硝基酪氨酸在肾血管和主动脉中的积聚也增加。我们还发现NADPH氧化酶活性与该模型中ROS的产生有关。血管紧张素受体阻滞剂抑制ROS和NO的产生，并显著减轻亚硝化应激

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