BASIS FOR RENAL RESISTANCE TO ATRIAL NATRIURETIC PEPTIDE

肾对心房钠尿肽抵抗的基础

• 批准号: 6635330
• 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
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635330
• 关键词: alpha adrenergic receptor; atrial natriuretic peptide; biological signal transduction; cyclic GMP; denervation; dietary control; dietary sodium; edema; endothelin; enzyme activity; enzyme induction /repression; enzyme mechanism; immunocytochemistry; in situ hybridization; kidney cell; kidney function; laboratory rat; liver cirrhosis; metal metabolism; nephrosis; neurons; phosphodiesterases; phosphorylation; protein localization; renal glomerulus; renal medulla; sodium

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：病理钠保留和水肿形成状态 作为充血性心力衰竭，肾病综合征和肝硬化是 以对心房的肾功能作用为特征 亚钠肽（ANP）。这种异常被认为是 钠排泄受损的介体导致钠平衡 以及水肿的发展。已经争辩了许多机制 在这些条件下，包括 激活抗钠的途径，例如肾素 - 血管紧张素系统和 交感神经活动，滤液递送到ANP响应 内部髓质收集管（IMCD）中的位置，并结合受损 ANP的肾脏受体。我们已经开发了另一种机制的证据 在实验性肾病综合征中有助于肾脏ANP的耐药性和 肝肝硬化。这种机制涉及特定的活动的增强 肾脏靶细胞中的ANP作用的磷酸二酯酶（PDE）酶，以至于 ANFS细胞内第二信使循环鸟嘌呤-3'，5'-单磷酸盐 （CGMP），通常在ANP结合其生物活性受体后形成， 在迅速分解它可以发挥其完整的细胞作用之前。 ANP 肾细胞体外的响应性和体内纳特里雷SIS的响应能力已恢复 通过药理学抑制剂选择PDE。我们将确定 在大鼠中观察到的ANP的PDE5活性提高了 （1）测量CGMP速率 从这些大鼠分离的肾小球和IMCD细胞匀浆中的水解 在有选择性PDE抑制剂的情况下以及未经沉淀后 PDE5; （2）定量肾小球和IMCD中PDES酶蛋白的量 通过西方分析来自这些动物的细胞，将其分布沿 免疫组织化学的肾单位，并确定磷酸化是否磷酸化 有助于提高PDE5活性； （3）测量PDE5基因表达 通过定量mRNA，来自肾病和肝硬化大鼠的肾小球和IMCD细胞 丰度，并将其定位在原位表达PDE5 mRNA的肾单位位点 杂交； （4）确定肾神经的贡献 内皮素以增加肾脏靶标的PDE5活性，以实现ANP作用 测量肾病的未经肾脏的PDE5活性和蛋白质水平 和肝硬化大鼠，研究内皮素受体拮抗作用对 体内和体外的ANP反应性在PDE5活性上，并定量 IMCD细胞培养物的PDE5活性和蛋白质水平暴露于内皮素和 AIPHA-肾上腺素能激动剂。这些实验将充分描述 肾脏ANP抗性中的PDE5活性增加，从而阐明了 水肿形成的发病机理。反过来，这将为 治疗这种经常烦恼的临床状况。