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、肾小管间质瘢痕形成和慢性肾脏疾病进展的重要因素。先前的研究表明，一氧化氮(NO)的可获得性降低、四氢生物蝶呤(BH4)和谷胱甘肽(GSH)水平降低、氧化应激和过氧化产物积累是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合成酶进一步合成ECD。初步研究表明，ADMA水平升高可诱导内皮细胞出现促纤维化表型。一氧化氮合酶抑制导致体内和体外分化内皮细胞功能丧失和获得肌成纤维细胞表型。这项建议中有待测试和进一步发展的概念归因于NO缺乏和ECD诱导的内皮-间充质转分化的作用：1)最初的微循环功能障碍的触发作用；2)微血管退行性变的结构阶段--组织缺氧和进行性肾硬化的先决条件。这些目标将通过1)阐明体内一氧化氮合酶抑制的促纤维化后遗症；2)评估血管无灌注和结构退化对肾小球硬化和肾小管间质瘢痕形成的贡献；3)研究内皮细胞的转分化作为导致血管丢失的潜在重要因素；3)研究内皮细胞的转分化及其刺激和介质，如转化生长因子-β、血管抑素和内皮抑素；4)检测和鉴定亚硝基硫醇修饰缺陷的细胞内蛋白质，作为下游内皮-间充质转分化的原因；以及5)尝试使用一氧化氮合成的中间体-羟基L精氨酸来改善内皮-间充质的跨分化、血管退化和肾硬化症。这些研究将使用已建立的研究细胞生理学、肾脏形态、蛋白质组学和肾脏生理学的方法进行，所有这些都可供调查小组使用。预计拟议的一系列研究将提供关于慢性肾脏疾病血管退行性变的潜在重要机制-内皮-间充质转分化，它在肾硬化症中的作用，以及羟基-L-精氨酸的治疗作用，它可以改善NO缺乏，伴随着内皮-间质转分化和血管脱落，从而减缓慢性肾脏疾病的进展。

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.