Na,K-ATPase mediated ouabain effects in polycystic kidney disease

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)是最常见的肾脏遗传性疾病，由多囊蛋白1和2(PC1和PC2)的改变引起。ADPKD的特点是多个充满液体的囊肿，影响肾脏的结构和功能，导致慢性肾功能衰竭。由于Na，K-ATPase在肾脏盐和水的矢量运动中起着至关重要的作用，因此一直是研究ADPKD病理生理学的重点。然而，Na，K-ATPase在ADPKD中的确切作用仍不清楚。有趣的是，Na，K-ATPase除了作为离子转运体的活性外，还作为哇巴因的受体发挥作用。哇巴因是一种特性良好的激素，已知可以触发信号事件，导致几种细胞类型的新陈代谢、运动性和生长发生变化。令人惊讶的是，我们发现ADPKD患者肾囊肿上皮细胞(ADPKD细胞)对哇巴因的亲和力异常增加。此外，我们发现哇巴因的量与血液中循环的量相似，可以刺激ADPKD细胞的细胞生长。在正常的人肾上皮细胞(NHK细胞)中没有发现这种效应，因为NHK细胞对激素的亲和力较低。此外，哇巴因可促进ADPKD细胞的凋亡，其方式不能与细胞增殖的增加相提并论；降低细胞的粘附性；并增强其在培养中形成包囊的能力。所有这些事件都是ADPKD囊变的特征。因此，我们发现哇巴因在体外和体内都会加剧ADPKD小鼠模型的肾囊肿发展。综上所述，这些结果表明哇巴因是一种促进肾囊肿形成和进展的新因素。目前，哇巴因在ADPKD细胞中介导包囊形成的作用和机制尚不清楚。我们在这项研究中的总体目标是了解Na，K-ATPase-哇巴因信号如何影响ADPKD。我们的中心假设是，由于其高哇巴因敏感表型，ADPKD细胞更容易受到循环中激素水平的影响，这种激素异常地刺激Na，K-ATPase信号，导致细胞增殖、液体分泌以及细胞黏附特性和运动性的变化。本实验旨在探讨哇巴因刺激的Na，K-ATPase信号在ADPKD细胞包囊形成中的作用机制、哇巴因对细胞作用的细胞内途径以及哇巴因亲和力异常的决定因素。这些结果将产生重要的积极影响，因为它们将有助于确定ADPKD囊性发育和进展所涉及的分子机制，并将有助于未来开发以Na，K-ATPase信号为靶点的方法来控制疾病。 与公共健康相关：Na，K-ATPase是一种基本分子，在细胞中具有重要的离子运输和信号功能。本申请旨在探讨Na，K-ATPase作为激素哇巴因对常染色体显性遗传性多囊肾病患者肾小管上皮细胞增殖、液体分泌和囊腔形成影响的受体和信号转导作用的作用和机制(S)。这对于确定ADPKD囊性发育和进展所涉及的分子机制是重要的，并将有助于在未来开发针对Na，K-ATPase信号来控制疾病的方法。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney, caused by alterations of polycystin 1 and 2 (PC1 and PC2). ADPKD is characterized by multiple fluid-filled cysts that affect the structure and function of the kidney, leading to chronic renal failure. Because of its essential function in the vectorial movement of salt and water in the kidney, the Na,K-ATPase has been the focus of investigation to understand the pathophysiology of ADPKD. However, the precise role of the Na,K-ATPase in ADPKD remains unknown. Interestingly, besides its activity as an ion transporter, the Na,K-ATPase also functions as the receptor for ouabain. Ouabain is a well characterized hormone known to trigger signaling events that induce changes in metabolism, motility and growth in several cell types. Surprisingly, we have found that the Na,K-ATPase of epithelial cells from kidney cysts of patients with ADPKD (ADPKD cells) exhibit an abnormal increase in the affinity for ouabain. Moreover, we have found that ouabain, in amounts similar to those circulating in blood, stimulates cell growth in ADPKD cells. This effect is not found in normal human kidney epithelial cells (NHK cells), which have a lower affinity for the hormone. Furthermore, ouabain enhances ADPKD cell apoptosis, in a manner that does not parallel the increase in cell proliferation; decreases the adhesion properties of the cells; and enhances their ability to form cysts in culture. All these events are hallmarks of ADPKD cystogenesis. Accordingly, we have found that ouabain exacerbates renal cyst development in a mouse model of ADPKD, both in vitro and in vivo. Altogether, these results suggest that ouabain is a novel factor that promotes renal cyst formation and progression. At present, the role and mechanisms by which ouabain mediates cyst formation in ADPKD cells are unknown. Our overall goal in this research is to understand how Na,K-ATPase-ouabain signaling affects ADPKD. Our central hypothesis is that, due to their high ouabain sensitive phenotype, ADPKD cells are more susceptible to circulating levels of the hormone; which abnormally stimulates Na,K-ATPase signaling, to cause hyperplasic growth, fluid secretion and changes in the adhesion properties and motility of the cells. Experiments are designed to determine the mechanisms of ouabain stimulated Na,K-ATPase signaling in cyst formation of ADPKD cells, the intracellular pathways responsible for ouabain action on the cells, and the determinants of the abnormal ouabain affinity of ADPKD cells. The results will have an important positive impact, because they will help define the molecular mechanisms involved in cyst development and progression in ADPKD, and will contribute in the future to develop approaches that will target Na,K-ATPase signaling to control the disease. PUBLIC HEALTH RELEVANCE: The Na,K-ATPase is an essential molecule with important ion transport and signaling functions in the cell. The present application proposes to investigate the role and mechanism(s) of action of the Na,K-ATPase as a receptor and signal transducer of the effects of the hormone ouabain in the proliferation, fluid secretion and cyst formation of renal epithelial tubular cells from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD). This is important to define the molecular mechanisms involved in cyst development and progression in ADPKD, and will contribute in the future to develop approaches that will target Na,K-ATPase signaling to control the disease.