BASIS FOR RENAL RESISTANCE TO ATRIAL NATRIURETIC PEPTIDE

描述：病理性钠潴留和水肿形成的状态， 如充血性心力衰竭、肾病综合征和肝硬化， 以肾抵抗心房利钠作用为特征 利钠肽（ANP）。这种异常被认为是 导致正钠平衡的钠排泄受损的介质 和水肿的发展。有许多机制被认为 在这些情况下，有助于肾抵抗ANP，包括 激活抗心房利尿通路，如肾素-血管紧张素系统， 交感神经活性，滤液向ANP反应性的递送减少 内髓集合管（IMCD）中的位点， 心钠素与其肾受体。我们已经有证据表明另一种机制 在实验性肾病综合征中有助于肾ANP抵抗， 肝硬化患者这种机制涉及一种特定的 磷酸二酯酶（PDE）在肾靶细胞中对ANP起作用， ANFs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就被迅速分解代谢。ANP 恢复了体外肾细胞和体内尿钠排泄的反应性 对PDES有选择性的药物抑制剂。我们将确定 在大鼠中观察到对ANP的肾抵抗中PDE 5活性升高， 实验性肾病或肝硬化，通过（1）测定cGMP 从这些大鼠分离的肾小球和IMCD细胞匀浆中的水解 在选择性PDE抑制剂存在下， （2）测定肾小球和IMCD中PDES酶蛋白的含量 通过Western分析，定位其分布沿着 免疫组化检测肾单位的磷酸化程度， 有助于提高PDE 5活性;（3）测量PDE 5基因表达， 用mRNA定量法测定肾病和肾小球疾病大鼠肾小球和IMCD细胞 丰度，并通过原位杂交定位表达PDE 5 mRNA的肾单位位点， 杂交;和（4）确定肾神经的贡献， 内皮素增加肾脏靶点的PDE 5活性， 肾病综合征患者失神经支配肾脏中PDE 5活性和蛋白水平的测定 内皮素受体拮抗剂对内皮素受体的影响， ANP反应性在体内和体外和PDE 5活性，并定量 暴露于内皮素的IMCD细胞培养物中的PDE 5活性和蛋白水平， α-肾上腺素能激动剂。这些实验将充分描述 增加肾脏ANP抵抗中的PDE 5活性，从而阐明 水肿形成的发病机制。这反过来又将为以下方面提出新的选择： 治疗这种经常令人烦恼的临床状况。

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.