Nitrix Oxide in Chronic Renal Disease

说明(申请人提供)：慢性肾脏疾病(CKD)中存在一氧化氮(NO)缺乏，无论其主要原因如何，都会导致CKD的心血管并发症和进展。慢性肾脏病中NO缺乏的原因有很多：1主要原因是内源性一氧化氮合酶抑制物不对称二甲基精氨酸(ADMA)的积聚，部分原因是水解酶二甲基精氨酸二甲氨基水解酶(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，我们的工作表明，在肾脏中，神经元(N)NOS是主要的靶标，并且存在几种亚型并被CKD改变。我们在体内和体外研究了两种不同的CKD模型，即5/6肾肿块消融/梗死(A/I)模型和慢性嘌呤霉素氨基核苷肾病(PAN)模型，以确定肾脏中不同的NOS和DDAH酶的变化规律以及这在CKD的进展和高血压的发展中所起的作用。氧化应激严重损害了DDAH和NOS蛋白的丰度和活性，而氧化应激在CKD中含量很高，我们将使用载脂蛋白来抑制氧化剂的产生，并通过饮食调控来增强氧化应激，作为改变DDAH和NOS活性的方法。肾单位数目是慢性肾脏病进展和高血压发展的主要决定因素，我们将确定肾单位数目减少对肾脏一氧化氮合酶和DDAH酶的影响。通过我们新开发的选择性肾脏转染法，我们将操纵肾内局部的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。我们将在清醒大鼠中进行体内功能研究，以及使用麻醉制剂进行肾小球微穿刺术。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。研究将在易患慢性肾脏病和高血压的SpragueDawley大鼠和受到多种类型慢性肾脏病和高血压保护的Wistar Furth中进行，以深入了解进展机制。CKD在美国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脏NO系统在慢性肾脏病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.