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

描述（由申请人提供）：常染色体显性多囊肾病（ADPKD）是最常见的肾脏遗传性疾病，由多囊蛋白1和2（PC 1和PC 2）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，导致慢性肾衰竭。由于Na，K-ATP酶在肾脏中盐和水的矢量运动中起重要作用，因此它一直是研究ADPKD病理生理学的焦点。然而，Na，K-ATP酶在ADPKD中的确切作用仍不清楚。有趣的是，除了作为离子转运体的活性外，Na，K-ATP酶还作为哇巴因的受体发挥作用。哇巴因是一种特征良好的激素，已知可触发信号传导事件，诱导多种细胞类型的代谢、运动和生长变化。令人惊讶的是，我们已经发现，来自患有ADPKD的患者的肾囊肿的上皮细胞（ADPKD细胞）的Na，K-ATP酶表现出对哇巴因的亲和力的异常增加。此外，我们发现哇巴因的量与血液中循环的量相似，可刺激ADPKD细胞的细胞生长。这种效应在正常人肾上皮细胞（NHK细胞）中没有发现，NHK细胞对激素的亲和力较低。此外，哇巴因增强ADPKD细胞凋亡，以不平行于细胞增殖增加的方式;降低细胞的粘附特性;并增强其在培养物中形成包囊的能力。所有这些事件都是ADPKD囊肿形成的标志。因此，我们已经发现哇巴因在体内和体外都加剧了ADPKD小鼠模型中肾囊肿的发展。总之，这些结果表明，哇巴因是一种新的因素，促进肾囊肿的形成和发展。目前，哇巴因介导ADPKD细胞中囊肿形成的作用和机制尚不清楚。本研究的总体目标是了解Na，K-ATP酶-哇巴因信号通路如何影响ADPKD。我们的中心假设是，由于它们的高哇巴因敏感表型，ADPKD细胞对激素的循环水平更敏感;这异常地刺激Na，K-ATP酶信号传导，导致增生性生长，液体分泌以及细胞粘附特性和运动性的变化。本实验旨在探讨哇巴因刺激Na，K-ATP酶信号通路在ADPKD细胞包囊形成中的作用机制，哇巴因作用于ADPKD细胞的细胞内通路，以及ADPKD细胞异常哇巴因亲和力的决定因素。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囊肿发展和进展的分子机制，并将有助于未来开发靶向Na，K-ATP酶信号传导的方法来控制疾病。 公共卫生相关性：Na，K-ATP酶是细胞中具有重要离子转运和信号传导功能的必需分子。本申请提出研究Na，K-ATP酶作为激素哇巴因在来自常染色体显性多囊肾病（ADPKD）患者的肾的肾上皮小管细胞的增殖、液体分泌和囊肿形成中的作用的受体和信号转导物的作用和作用机制。这对于确定ADPKD囊肿发展和进展的分子机制非常重要，并将有助于未来开发靶向Na，K-ATP酶信号传导以控制疾病的方法。

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.