Nitric oxide deficiency in chronic renal disease

描述（由申请人提供）：一氧化氮（NO）缺乏症发生在CKD中，与主要原因无关，并导致心血管并发症和CKD进展的高发生率。CKD中NO缺乏有许多潜在的机制，本申请研究了3种可能的途径：内源性NO合酶（NOS）抑制剂不对称二甲基精氨酸（ADMA）的去除受损;由于肾L-Arg合成减少以及内皮精氨酸转运减少而导致的底物（L-Arg）可用性降低;肾皮质中神经元NOS（nNOS）亚型的改变易导致CKD进展。我们有初步的数据来支持每一个目标。 体内研究将涉及使用2种CKD模型; 5/6肾消融/梗死（A/I）和中度和重度慢性嘌呤霉素氨基糖苷（PAN）诱导的肾损伤。将在轻度（= CKD 1 -2）、中度（=CKD 3）和重度（=CKD 4）阶段研究5/6 AI模型。大多数研究将在“CKD易感”的Sprague-Dawley大鼠和一些CKD耐药的Wistar Furth中进行。在第一个目标中，我们将研究CKD如何改变ADMA的肾脏排泄和代谢以及肝脏和总ADMA清除率。将使用负责ADMA代谢的两种酶中的每一种的肾脏选择性沉默来确定肾ADMA代谢在正常大鼠中的特定作用。在CKD动物中，将进行ADMA代谢酶的选择性肾脏过表达，以确定这是否会降低ADMA并延缓进展和高血压发展。在目标2中，我们将确定CKD与正常肾脏对瓜氨酸的肾脏和总（全身）摄取以及L-Arg合成的差异。我们将在体外测量不同CKD模型中肾脏和血管内皮中L-Arg转运体的丰度和活性，以及体内L-Arg摄取。在第三个目标中，我们将在2种不同模型中检验肾皮质中nNOS α亚型减少和/或nNOS β亚型增加与CKD进展有因果关系的假设。在每个目标中，我们将进行平行的体外测量的丰度和活性的酶的兴趣。我们还将确定氧化应激如何以及在何处发展，以及如何影响NO产生的这些决定因素。CKD的发病率在美国迅速增加，这些研究将有助于确定CKD整体NO缺乏的原因，并可能导致新的治疗方法。 公共卫生相关性：本申请研究慢性肾脏疾病对一氧化氮产生的几个决定因素的影响，即底物（L-精氨酸）的可用性，内源性一氧化氮合酶抑制剂不对称二甲基精氨酸的清除，以及肾脏内特定一氧化氮合酶的变化如何导致肾内一氧化氮缺乏和肾脏疾病的进展。我们使用两种不同品系的完整动物，它们对肾脏疾病的进展表现出易感性（Sprague道利）和抗性（Wistar Furth）。我们认为，慢性肾脏疾病的进展是由于一氧化氮缺乏和肾损害的易感性是有关减少肾脏L-精氨酸合成，积累不对称二甲基精氨酸由于减少肾脏排泄/代谢，和不利的转变，在肾皮质丰富的两个神经元型一氧化氮合酶。

DESCRIPTION (provided by applicant): Nitric oxide (NO) deficiency occurs in CKD irrespective of primary cause and contributes to the high rate of cardiovascular complications and progression of CKD. There are many potential mechanisms for NO deficiency in CKD and this application examines 3 possible pathways: Impaired removal of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA); decreased substrate (L- Arg) availability due to reduction in renal L-Arg synthesis and also reductions in endothelial arginine transport; alterations in the neuronal NOS (nNOS) isoforms in the kidney cortex which predispose to CKD progression. We have preliminary data to support each aim. In vivo studies will involve use of 2 CKD models; 5/6 renal ablation/infarction (A/I) and moderate and severe chronic puromycin aminonucleoside (PAN)-induced kidney damage. The 5/6 AI model will be investigated at mild (=CKD1-2), moderate (=CKD 3) and severe (=CKD 4) stages. Most studies will be in the "CKD-vulnerable" Sprague-Dawley rat and some in the CKD-resistant Wistar Furth. In the first aim we will investigate how CKD alters renal excretion and metabolism of ADMA as well as hepatic and total ADMA clearance. The specific role of renal ADMA metabolism in normal rats will be determined using kidney selective silencing of each of the two enzymes responsible for ADMA metabolism. In CKD animals, selective renal over-expression of the ADMA metabolizing enzymes will be conducted to determine if this reduces ADMA and retards progression and hypertension development. In Aim 2 we will determine how renal and total (whole body) uptake of citrulline and synthesis of L-Arg differs in CKD vs normal kidneys. We will measure abundance and activity of L-Arg transporters in kidney and vascular endothelium in different CKD models in vitro, and L-Arg uptake in vivo. In the 3rd aim we will test the hypotheses that reduction in the nNOSalpha isoform and/or increases in the nNOSbeta isoform in renal cortex are causally related to the progression of CKD in 2 different models. In each aim we will conduct parallel in vitro measurements of abundance and activity of enzymes of interest. We will also determine how and where oxidative stress develops, and how this impacts on these determinants of NO production. The incidence of CKD is rapidly increasing in the US and these studies will help determine the reasons for the overall NO deficiency of CKD and could lead to new therapeutic approaches. PUBLIC HEALTH RELEVANCE: This application investigates the impact of chronic kidney disease on several determinants of nitric oxide production, namely substrate (L-arginine) availability, clearance of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine and how changes in specific nitric oxide synthases within the kidney, contribute to intrarenal nitric oxide deficiency and progression of renal disease. We use intact animals of two different strains which exhibit susceptibility (Sprague Dawley) and resistance (Wistar Furth) to progression of kidney disease. We suggest that progression of chronic kidney disease is due to nitric oxide deficiency and that susceptibility to kidney damage is related to reduce renal L-Arginine synthesis, accumulation of asymmetric dimethylarginine due to reduced renal excretion / metabolism, and an adverse shift in the renal cortical abundance of two neuronal nitric oxide synthases.