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BASIS FOR RENAL RESISTANCE TO ATRIAL NATRIURETIC PEPTIDE

肾对心房钠尿肽抵抗的基础

基本信息

批准号：
6708000
负责人：
MICHAEL H HUMPHREYS
金额：
$ 21.82万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-03-01 至 2006-01-31
项目状态：
已结题

项目摘要

DESCRIPTION: States of pathological sodium retention and edema formation such as congestive heart failure, nephrotic syndrome, and cirrhosis of the liver are characterized by renal resistance to the natriuretic action of atrial natriuretic peptide (ANP). This abnormality has been suggested to be the mediator of the impaired sodium excretion leading to positive sodium balance and the development of edema. A number of mechanisms have been argued to contribute to the renal resistance to ANP in these conditions, including activation of antinatriuretic pathways such as the renin-angiotensin system and sympathetic nerve activity, reduced delivery of filtrate to ANP-responsive sites in the inner medullary collecting duct (IMCD), and impaired binding of ANP to its renal receptors. We have developed evidence that another mechanism contributes to renal ANP resistance in experimental nephrotic syndrome and liver cirrhosis. This mechanism involves a heightened activity of a specific phosphodiesterase (PDE) enzyme in renal target cells for ANP action such that ANFs intracellular second messenger cyclic guanosine-3',5'-monophosphate (cGMP), normally formed after ANP binds to its biologically active receptors, is rapidly catabolized before it can exert its full cellular actions. ANP responsiveness of renal cells in vitro, and of natriuresis in vivo, is restored by pharmacologic inhibitors selective for PDES. We shall determine the role of heightened PDE5 activity in the renal resistance to ANP observed in rats with experimental nephrosis or liver cirrhosis by (1) measuring the rate of cGMP hydrolysis in homogenates of glomeruli and IMCD cells isolated from these rats in the presence of selective PDE inhibitors and after inimunoprecipitation of PDE5; (2) quantitating the amount of PDES enzyme protein in glomeruli and IMCD cells from these animals by Western analysis, localizing its distribution along the nephron by immunohistochemistry, and determining if phosphorylation contributes to heightened PDE5 activity; (3) measuring PDE5 gene expression in glomeruli and IMCD cells from nephrotic and cirrhotic rats by quantitating mRNA abundance, and localizing the nephron sites expressing PDE5 mRNA by in situ hybridization; and (4) determining the contributions of the renal nerves and endothelin to increased PDE5 activity in renal targets for ANP action by measuring PDE5 activity and protein level in denervated kidneys from nephrotic and cirrhotic rats, studying the effect of endothelin receptor antagonism on ANP responsiveness in vivo and in vitroand on PDE5 activity, and quantitating PDE5 activity and protein level in IMCD cell cultures exposed to endothelin and aipha-adrenergic agonists. These experiments will describe fully the role of increased PDE5 activity in renal ANP resistance, and thereby shed light on the pathogenesis of edema formation. This in turn will suggest new options for treatment of this often vexing clinical condition.

描述：病理性钠滞留和水肿形成的状态，如如充血性心力衰竭、肾病综合征和肝硬变以肾脏对心房利钠作用的抵抗为特征利钠肽(ANP)。这一反常现象被认为是钠排泄受损导致正钠平衡的介体和浮肿的发展。许多机制已经被证明是为了在这些情况下导致肾脏对心钠素的抵抗，包括肾素-血管紧张素系统和血管紧张素系统等抗利尿途径的激活交感神经活动，减少向ANP反应的滤液输送内髓集合管(IMCD)中的部位，以及结合受损 ANP与其肾脏受体结合。我们发现了另一种机制参与实验性肾病综合征的肾心钠素抵抗肝硬变。这种机制涉及一种特定的肾靶细胞中磷酸二酯酶(PDE)对心钠素作用的研究 ANF细胞内第二信使环鸟苷-3‘，5’-单磷酸 (CGMP)，通常是在ANP与其生物活性受体结合后形成的，在它发挥其全部细胞作用之前被迅速分解。ANP 体外肾细胞和体内钠尿的反应性被恢复通过对PDE有选择性的药物抑制剂。我们将确定大鼠肾脏对心钠素抵抗中PDE5活性升高的观察实验性肾病或肝硬变：(1)测定cGMP含量大鼠肾小球匀浆和IMCD细胞的水解液在选择性PDE抑制剂存在的情况下和在免疫共沉淀之后 PDE5；(2)肾小球和IMCD中PDEs酶蛋白的定量通过Western分析从这些动物中提取细胞，确定其沿通过免疫组织化学方法检测肾单位，并测定是否磷酸化有助于提高PDE5活性；(3)检测PDE5基因在肾病和肝硬变大鼠肾小球和IMCD细胞的mRNA定量丰度，原位定位表达PDE5基因的肾单位部位杂交；(4)测定肾神经和肾神经的贡献。内皮素通过增加肾靶向心钠素作用的PDE5活性肾病患者失神经肾组织中PDE5活性和蛋白水平的测定和肝硬变大鼠，研究内皮素受体拮抗剂对大鼠血管内皮功能的影响体内、外和体外ANP对PDE5活性的反应性及定量内皮素和低密度脂蛋白对IMCD细胞PDE5活性和蛋白水平的影响 α-肾上腺素能激动剂。这些实验将充分描述在肾型心钠素抵抗中PDE5活性增加，从而阐明水肿形成的发病机制。这反过来将提出新的选择治疗这一经常令人烦恼的临床状况。