ENDOTHELIAL DYSFUNCTION, NITRIC OXIDE AND RENAL FAILURE

描述（由申请人提供）：内皮细胞功能障碍（ECD）、过早衰老和细胞凋亡是毛细血管脱落、肾小球硬化、肾小管间质疤痕和慢性肾病进展的重要因素。先前的研究表明，一氧化氮 (NO) 可用性下降、四氢生物蝶呤 (BH4) 和谷胱甘肽 (GSH) 水平下降、氧化应激和过氧化物积累是 ECD 的主要原因。不对称二甲基精氨酸 (ADMA) 水平升高通过抑制 NO 合成酶进一步复合 ECD。初步研究表明，ADMA 水平升高会诱导内皮细胞出现促纤维化表型。 NOS 抑制导致体外和体内表现的分化内皮细胞功能丧失和肌成纤维细胞表型的获得。本提案中待测试和进一步发展的概念将 NO 缺乏和 ECD 诱导的内皮间质转分化作为触发因素的作用：1）微循环的初始功能不足，随后是 2）微血管退化的结构阶段 - 组织缺氧和进行性肾硬化的先决条件。这些目标将通过以下方式实现：1) 阐明体内 NOS 抑制的促纤维化后遗症； 2) 评估血管非灌注和结构退化对肾小球硬化和肾小管间质疤痕的影响； 3）研究内皮细胞的转分化作为血管脱落的潜在重要因素；它的刺激剂和介质，如 TGF-β、血管抑制素和内皮抑素； 4) 检测和鉴定缺乏亚硝基硫醇修饰的细胞内蛋白质，这是下游内皮-间质转分化的原因； 5)尝试使用NO合成中间体羟基-L-精氨酸来改善内皮-间质转分化、血管退化和肾硬化，它结合了超氧化物清除剂和NO供体的特性。这些研究将使用细胞生理学、肾脏形态学、蛋白质组学和肾脏生理学研究的既定方法进行，所有这些都可供研究团队使用。预计所提出的一系列研究应为慢性肾脏病血管消退的潜在重要机制——内皮-间质转分化、其对肾硬化的贡献以及羟基-L-精氨酸的治疗作用提供广泛的视角，羟基-L-精氨酸可以改善NO缺乏，伴随内皮-间质转分化和血管脱落，从而减少慢性肾脏病的进展。

DESCRIPTION (provided by applicant): Endothelial cell dysfunction (ECD), premature senescence and apoptosis are important contributors to capillary drop-out, glomeruosclerosis, tubulointerstitial scarring and progression of chronic renal disease. Previous studies showed decreased availability of nitric oxide (NO), decreased levels of tetrahydrobiopterin (BH4) and glutathione (GSH), oxidative stress, and accumulation of peroxides as leading causes of ECD. Elevated levels of asymmetric dimethylarginine (ADMA) further compound ECD by inhibiting NO synthease. Preliminary Studies showed induction of a profibrotic phenotype of endothelial cells subjected to elevated levels of ADMA. NOS inhibition resulted in the in vitro and in vivo manifestations of the loss of differentiated endothelial cell functions and acquisition of myofibroblast phenotype. The concept to be tested and further developed in this proposal ascribes to NO deficiency and ECD-induced endothelial-mesenchymal transdifferentiation the role of a trigger of 1) initial functional insufficiency of microcirculation followed by 2) structural phase of microvascular regression - prerequisites of tissue hypoxia and progressive nephrosclerosis. These goals will be accomplished through 1) elucidating profibrotic sequelae of NOS inhibition in vivo; 2) assessing the contribution of vascular non-perfusion and structural regression to glomerulosclerosis and tubulointerstitial scarring; 3) investigations into transdifferentiation of endothelial cells as a potentially important contributor to vascular drop-out; its stimulators and mediators, like TGF-beta, angiostatin and endostatin; 4) detection and identification of intracellular proteins deficient in nitrosothiol modification, as a cause of the downstream endothelial-mesenchymal transdifferentiation; and 5) attempts to ameliorate endothelial-mesenchymal transdifferentiation, vascular regression and nephrosclerosis using an intermediate in NO synthesis, hydroxy-L-arginine, which combines the properties of superoxide scavenger and NO donor. These studies will be performed using established approaches for studies into cell physiology, renal morphology, proteomics, and renal physiology, all available to the investigative team. It is anticipated that proposed set of studies should provide a broad view on the potentially important mechanism of vascular regression in chronic kidney disease - endothelial-mesencymal transdifferentiation, its contribution to nephrosclerosis and therapeutic actions of hydroxy-L-arginine, which can ameliorate NO deficiency, accompanied by endothelial-mesenchymal transdifferentiation and vascular drop-out, thus reducing progression of chronic renal diseases.