Nitrix Oxide in Chronic Renal Disease

描述（由申请人提供）：一氧化氮（NO）缺乏发生在慢性肾脏疾病（CKD）中，与主要原因无关，并有助于心血管并发症和CKD的进展。CKD中NO缺乏的原因有很多；一个主要因素是内源性NOS抑制剂不对称二甲基精氨酸ADMA的积累，部分原因是二甲基精氨酸二甲基氨基水解酶（DDAHs）的失效。肾脏是DDAH活性的主要部位。另一个原因是一氧化氮合酶（NOS）蛋白和活性的丧失，我们的研究表明，在肾脏中，神经元(n)一氧化氮合酶是一个主要靶点，CKD会改变几种同种异构体。我们提出了两种不同的CKD模型，5/6肾团块消融/梗死（A/I）和慢性嘌呤霉素氨基核苷肾病（PAN）模型的体内和体外研究，以确定肾脏中不同NOS和DDAH酶的变化模式，以及这如何促进CKD的进展和高血压的发展。DDAH和NOS蛋白的丰度和活性都受到CKD高氧化应激的严重损害，我们将使用apocyanin抑制氧化剂的产生和饮食调节来增强氧化应激作为改变DDAH和NOS活性的方法。肾单位数量是CKD进展和高血压发展的主要决定因素，我们将确定肾单位数量减少如何影响肾脏NOS和DDAH酶。通过使用我们新开发的选择性肾转染感兴趣基因的方法，我们将操纵局部肾内NOS和DDAH酶，以确定不同的nNOS（和其他NOS）亚型和2种DDAH亚型在CKD进展中的作用。我们将在清醒的大鼠体内进行功能研究，并使用麻醉制剂进行肾小球微穿刺。我们还将进行一系列体外研究，以确定酶的位置、丰度和活性。研究将在Sprague Dawley大鼠（易患CKD和高血压的品种）和Wistar Furth（受多种类型CKD和高血压保护的品种）中进行，以深入了解进展机制。在美国，CKD的发病率正在迅速增加，这些患者的心血管发病率和死亡率也非常高，这显然与NO缺乏有关。这些拟议的研究将确定肾NO系统在CKD及其心血管并发症进展中的作用，这将导致新的治疗方法。

DESCRIPTION (provided by applicant): Nitric oxide (NO) deficiency occurs in chronic kidney disease (CKD) irrespective of the primary cause and contributes to the cardiovascular complications and progression of CKD. There are many reasons for NO deficiency in CKD; 1 major factor is accumulation of the endogenous NOS inhibitor, asymmetric dimethylarginine, ADMA, due partly to a failure of the hydrolyzing enzymes dimethylarginine dimethylamino-hydrolases (DDAHs). The kidney is a major site of DDAH activity. Another cause is loss of NO synthase (NOS) protein and activity and our work suggests that in kidney the neuronal (n)NOS is a primary target and that several isoforms are present and altered by CKD. We propose in vivo and in vitro studies in 2 separate models of CKD, the 5/6 renal mass ablation/infarction (A/I) and chronic puromycin aminonucleoside nephrosis (PAN) models to determine the pattern of change of the different NOS and DDAH enzymes in the kidney and how this contributes to the progression of CKD and development of hypertension. Both DDAH and NOS protein abundance and activity are severely compromised by oxidative stress, which is high in CKD and we will use apocyanin to inhibit oxidant production and dietary manipulation to enhance oxidative stress as methods of changing DDAH and NOS activity. Nephron number is a major determinant of progression of CKD and development of hypertension and we will determine how reductions in nephron number impact on the renal NOS and DDAH enzymes. By using our newly developed method of selective kidney transfection with genes of interest we will manipulate the local intrarenal NOS and DDAH enzymes to determine the role of different nNOS (and other NOS) isoforms and the 2 DDAH isoforms in progression of CKD. We will conduct in vivo functional studies in the conscious rat as well as glomerular micropuncture using anesthetized preparations. We will also conduct a range of in vitro studies to determine enzyme location, abundance and activity. Studies will be in the Sprague Dawley rat a strain vulnerable to CKD and hypertension and in the Wistar Furth which is protected from multiple types of CKD and hypertension to gain insights into mechanisms of progression. The incidence of CKD is rapidly increasing in the US and these patients also have a very high rate of cardiovascular morbidity and mortality, which is clearly linked to NO deficiency. These proposed studies will determine the role of the renal NO system in the progression of CKD and its cardiovascular complications which will lead to new therapeutic approaches.