Receptor Na/K-ATPase Antagonists As Novel Therapeutics For Renal/Cardiac Diseases

受体 Na/K-ATP 酶拮抗剂作为肾病/心脏病的新型疗法

• 批准号: 8159937
• 负责人: JOSEPH Isaac SHAPIRO
• 金额: $ 56.69万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-18 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8159937
• 关键词: 1-Phosphatidylinositol 3-Kinase; Abbreviations; Address; Affinity; Agonist; Angiotensin II; Animals; Antibodies; Attenuated; Binding; Biology; Blood Pressure; Cardiac; Cardiac Glycosides; Cardiomyopathies; Cell Culture Techniques; Cells; Chronic; Chronic Kidney Failure; Clinical; Complex; Congestive Heart Failure; Cultured Cells; Diet; Dominant-Negative Mutation; Effectiveness; Endocytosis; Fibrosis; Fluorescence; Functional disorder; Generations; Goals; Heart; Heart Diseases; Heart Hypertrophy; ITPR1 gene; In Vitro; Inositol; Ion Pumps; Kidney; Kidney Diseases; Kidney Failure; Lesion; Ligands; Mediating; Modeling; Mus; Muscle Cells; Na(+)-K(+)-Exchanging ATPase; Nephrectomy; Organ; Ouabain; Pathogenesis; Pathway interactions; Peptides; Play; Protein Kinase; Protein Kinase C; Proteins; Rattus; Reactive Oxygen Species; Receptor Activation; Regulation; Research; Resistance; Risk Factors; Role; Series; Signal Transduction; Sodium Chloride; Solid; Steroid Receptors; Stimulus; Stress; Testing; Therapeutic; Transgenic Mice; Transgenic Organisms; base; in vitro activity; in vivo; marinobufagenin; novel; novel therapeutics; prevent; receptor; receptor function; small molecule; tool; tripolyphosphate

DESCRIPTION (provided by applicant): We find that the Na/K-ATPase has an ion pumping-independent receptor function. Specifically, it associates with Src to form a receptor complex. Binding of cardiotonic steroids to this receptor complex activates Src, which, in turn, initiates a series of signaling cascades including the generation of reactive oxygen species (ROS) and the activation of PI3K/Akt pathways in renal and cardiac cells. Moreover, we demonstrate a significant increase in circulating CTS in rat model of uremic cardiomyopathy induced by 5/6 nephrectomy (PNx). Neutralization of this increase in CTS reduced ROS stress and diminished cardiomyopathy characterized by myocyte hypertrophy and cardiac fibrosis. Thus, we hypothesize that chronic stimulation of this newly appreciated receptor mechanism by elevated CTS may be the cause of the cardiac remodeling observed in uremic rats. Conversely, inhibition of the receptor function could attenuate the pathological changes in the target organs under this and other clinical conditions where CTS are elevated. Recently, we have developed several new tools that allow us to further test these hypotheses in vivo. First, we find that the N-terminus (NT) of a1 subunit acts as a dominant negative mutant capable of inhibiting CTS-induced signal transduction. Second, NT transgenic mice, in contrast to their wild-type littermates, are resistant to high salt diet-induced structural damages in the heart and in the kidney. Third, we have developed a peptide antagonist of receptor Na/K-ATPase/Src complex and demonstrated its effectiveness in vitro and in vivo. Finally, we have identified a novel class of small molecule antagonists that prevent ouabain from activating protein kinases in cell cultures. Therefore, we propose the following three specific aims. Aim 1 will test whether that chronic stimulation of the ion pumping-independent receptor function of Na/K-ATPase by CTS could result in pathological changes in the heart and the kidney. Aim 2 will address whether inhibition of Na/K-ATPase- mediated signal transduction by NT or pNaKtide attenuates high salt- and PNx-induced remodeling. In aim 3 we will further characterize the in vitro and in vivo activity of MB5 as a new class of CTS antagonists and test the in vivo effectiveness of MB5 in conferring resistance to CTS-induced organ damage. The completion of these three specific aims would significantly advance our understanding of endogenous CTS and Na/K- ATPase-mediated signal transduction in animal pathophysiology. Moreover, it would provide proof of the concept that antagonists of the receptor Na/K-ATPase can actually reduce renal and cardiac damages. PUBLIC HEALTH RELEVANCE: We have discovered a new receptor mechanism that plays an important role in the pathogenesis of renal and cardiac diseases. The proposed research will further advance our understanding of this receptor mechanism and explore the possibility of using the newly discovered receptor antagonists to prevent renal insufficiency and high salt diet-induced cardiac lesions.

描述（由申请人提供）：我们发现Na/K-ATP酶具有离子泵非依赖性受体功能。具体而言，它与Src结合形成受体复合物。强心类固醇与该受体复合物的结合激活Src，Src继而启动一系列信号级联，包括肾和心脏细胞中活性氧（ROS）的产生和PI 3 K/Akt通路的激活。此外，我们证明了在5/6肾切除术（PNx）诱导的尿毒症心肌病大鼠模型中循环CTS的显著增加。CTS的这种增加的中和作用降低了ROS应激并减少了以肌细胞肥大和心脏纤维化为特征的心肌病。因此，我们假设CTS升高对这种新认识的受体机制的慢性刺激可能是尿毒症大鼠中观察到的心脏重塑的原因。相反，在CTS升高的这种和其他临床条件下，受体功能的抑制可以减弱靶器官的病理变化。最近，我们开发了几种新的工具，使我们能够进一步测试这些假设在体内。首先，我们发现α 1亚基的N端（NT）作为一个显性负突变体，能够抑制CTS诱导的信号转导。第二，NT转基因小鼠，与野生型同窝出生的小鼠相比，对高盐饮食诱导的心脏和肾脏结构损伤具有抗性。第三，我们开发了一种Na/K-ATP酶/Src受体复合物的肽拮抗剂，并在体外和体内证明了其有效性。最后，我们已经确定了一类新的小分子拮抗剂，防止哇巴因激活细胞培养中的蛋白激酶。因此，我们提出以下三个具体目标。目的1探讨CTS对Na/K-ATP酶离子泵非依赖性受体功能的慢性刺激是否会导致心、肾的病理改变。目的2将探讨NT或pNaKtide抑制Na/K-ATP酶介导的信号转导是否减弱高盐和PNx诱导的重塑。在目标3中，我们将进一步表征MB 5作为一类新的CTS拮抗剂的体外和体内活性，并测试MB 5在赋予对CTS诱导的器官损伤的抗性中的体内有效性。这三个具体目标的完成将显着推进我们对动物病理生理学中内源性CTS和Na/K-ATP酶介导的信号转导的理解。此外，它将提供Na/K-ATP酶受体拮抗剂实际上可以减少肾脏和心脏损伤的概念的证据。 公共卫生相关性：我们发现了一种新的受体机制，在肾脏和心脏疾病的发病机制中发挥重要作用。这项研究将进一步推进我们对这种受体机制的理解，并探索使用新发现的受体拮抗剂预防肾功能不全和高盐饮食诱导的心脏病变的可能性。