BASIS FOR RENAL RESISTANCE TO ATRIAL NATRIURETIC PEPTIDE

肾对心房钠尿肽抵抗的基础

• 批准号: 6233033
• 负责人: MICHAEL H HUMPHREYS
• 金额: $ 21.28万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233033
• 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：病理性钠潴留和水肿形成的状态，例如 如充血性心力衰竭、肾病综合征和肝硬化 其特点是肾对心房利尿钠作用的抵抗 利尿钠肽（ANP）。这种异常现象被认为是 钠排泄受损的介质，导致正钠平衡 和水肿的发展。人们争论了许多机制 在这些情况下有助于肾对 ANP 的抵抗，包括 激活抗尿钠途径，例如肾素-血管紧张素系统和 交感神经活动，减少滤液对 ANP 反应的输送 内髓集合管（IMCD）中的位点，并且结合受损 ANP 作用于其肾脏受体。我们已经开发出证据表明另一种机制 有助于实验性肾病综合征中肾 ANP 抵抗， 肝硬化。该机制涉及特定的活动的增强 肾靶细胞中的磷酸二酯酶 (PDE) 发挥 ANP 作用，从而 ANF 细胞内第二信使环鸟苷-3',5'-单磷酸 (cGMP)，通常在 ANP 与其生物活性受体结合后形成， 在发挥其全部细胞作用之前被迅速分解代谢。心钠素 体外肾细胞的反应性和体内排尿钠的反应性得到恢复 通过对 PDES 选择性的药物抑制剂。我们将确定角色 在患有 ANP 的大鼠中观察到肾抵抗中 PDE5 活性升高 实验性肾病或肝硬化（1）测定cGMP率 从这些大鼠中分离出的肾小球和 IMCD 细胞匀浆的水解 在选择性 PDE 抑制剂存在下以及免疫沉淀后 PDE5； (2)定量肾小球和IMCD中PDES酶蛋白的量 通过西方分析，将其分布定位于这些动物的细胞 通过免疫组织化学检测肾单位，并确定是否磷酸化 有助于提高 PDE5 活性； (3) 测定PDE5基因表达量 通过 mRNA 定量分析肾病和肝硬化大鼠的肾小球和 IMCD 细胞 丰度，并通过原位定位表达 PDE5 mRNA 的肾单位位点 杂交； (4)确定肾神经的贡献和 内皮素通过增加 ANP 作用的肾脏靶标中的 PDE5 活性 测量肾病失神经肾脏中的 PDE5 活性和蛋白质水平 和肝硬化大鼠，研究内皮素受体拮抗剂的作用 ANP 体内和体外反应以及 PDE5 活性和定量 暴露于内皮素和内皮素的 IMCD 细胞培养物中的 PDE5 活性和蛋白质水平 aipha-肾上腺素能激动剂。这些实验将充分描述 肾 ANP 抵抗中 PDE5 活性增加，从而揭示了 水肿形成的发病机制。这反过来又会提出新的选择 治疗这种经常令人烦恼的临床病症。