Nitric oxide deficiency in chronic renal disease

说明(申请人提供)：慢性肾脏病患者存在一氧化氮(NO)缺乏，无论其主要原因如何，并导致心血管并发症的高发生率和慢性肾脏病的进展。慢性肾脏病的NO缺乏有许多可能的机制，本应用研究了3种可能的途径：内源性一氧化氮合酶抑制剂不对称二甲基精氨酸的清除障碍；由于肾脏L-精氨酸合成减少和内皮精氨酸运输减少而导致底物(L-精氨酸)利用率降低；肾皮质神经元型一氧化氮合酶亚型的改变，这容易导致慢性肾脏病的进展。我们有支持每个目标的初步数据。体内研究将使用两种CKD模型：5/6肾切除/梗死(A/I)和中度和重度慢性氨基核苷(PAN)诱导的肾脏损伤。5/6AI模型将在轻度(=CKD1-2)、中度(=CKD3)和重度(=CKD4)阶段进行研究。大多数研究将在“易患慢性肾脏病”的Spraogue-Dawley大鼠身上进行，一些研究将在抵抗慢性肾脏病的Wistar Furth身上进行。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ADMA的总清除量。肾脏选择性沉默负责ADMA代谢的两种酶将确定正常大鼠肾脏ADMA代谢的具体作用。在CKD动物中，将进行选择性的肾脏ADMA代谢酶的过度表达，以确定这是否减少ADMA并延缓进展和高血压的发展。在目标2中，我们将确定慢性肾脏病患者肾脏和整体(全身)对瓜氨酸的摄取以及L-精氨酸的合成与正常肾脏的差异。我们将在体外检测不同慢性肾脏病模型肾脏和血管内皮细胞中L-精氨酸转运体的丰度和活性，以及体内对L-精氨酸的摄取。在第三个目标中，我们将在两个不同的模型中检验假设，即肾皮质中nNOSalpha亚型的减少和/或nNOSbeta亚型的增加与CKD的进展存在因果关系。在每个目标中，我们将同时进行感兴趣的酶的丰度和活性的体外测量。我们还将确定氧化应激如何以及在哪里发展，以及这对NO产生的这些决定因素有何影响。CKD在美国的发病率正在迅速增加，这些研究将有助于确定CKD总体上缺乏NO的原因，并可能导致新的治疗方法。 公共卫生相关性：这项应用调查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。我们使用了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(SpragueDawley)和抵抗力(Wistar Furth)。我们认为，慢性肾脏疾病的进展是由于一氧化氮缺乏，肾脏损害的易感性与肾脏L-精氨酸合成减少，肾脏排泄/代谢减少导致的不对称二甲基精氨酸积聚，以及两种神经元型一氧化氮合酶的肾皮质丰度反转有关。

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.