Nitric oxide deficiency in chronic renal disease

描述（由申请人提供）：一氧化氮（NO）缺乏发生在CKD中，与主要原因无关，并导致心血管并发症和CKD进展的高发率。CKD中NO缺乏的潜在机制有很多，本应用研究了3种可能的途径：内源性NO合成酶（NOS）抑制剂不对称二甲基精氨酸（ADMA）的去除受损；由于肾脏L-精氨酸合成减少和内皮精氨酸运输减少，底物（L-精氨酸）可用性降低；肾皮质神经元NOS （nNOS）亚型的改变易导致CKD进展。我们有支持每个目标的初步数据。体内研究将涉及使用2种CKD模型；5/6肾消融/梗死（A/I）和中重度慢性嘌呤霉素氨基核苷（PAN）所致肾损害。5/6 AI模型将在轻度（=CKD1-2）、中度（= ckd3）和重度（=CKD 4）阶段进行研究。大多数研究将在“ckd易感”的Sprague-Dawley大鼠和一些ckd耐药的Wistar forth中进行。在第一个目标中，我们将研究CKD如何改变肾脏排泄和ADMA代谢以及肝脏和总ADMA清除。正常大鼠肾脏ADMA代谢的具体作用将通过肾脏选择性沉默负责ADMA代谢的两种酶来确定。在CKD动物中，将对ADMA代谢酶的选择性肾过度表达进行研究，以确定这是否会降低ADMA并延缓其进展和高血压的发展。在目标2中，我们将确定CKD与正常肾脏中瓜氨酸和l -精氨酸合成的肾脏和总（全身）摄取的差异。我们将测量不同CKD模型肾脏和血管内皮中l -精氨酸转运体的丰度和活性，以及体内l -精氨酸的摄取。在第三个目标中，我们将在两种不同的模型中检验nNOSalpha亚型的减少和/或肾皮质nNOSbeta亚型的增加与CKD进展的因果关系的假设。在每个目标，我们将进行平行的丰度和酶的活性感兴趣的体外测量。我们还将确定氧化应激是如何以及在哪里发展的，以及它如何影响这些NO生产的决定因素。在美国，CKD的发病率正在迅速增加，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。

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.