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 （PC1和PC2）的改变引起。ADPKD的特征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 - atp酶在肾脏中盐和水的矢量运动中具有重要作用，因此一直是研究ADPKD病理生理的重点。然而，Na， k - atp酶在ADPKD中的确切作用尚不清楚。有趣的是，除了作为离子转运体的活性外，Na， k - atp酶还具有沃巴因受体的功能。瓦巴因是一种具有良好特征的激素，已知可触发信号事件，诱导几种细胞类型的代谢、运动和生长变化。令人惊讶的是，我们发现ADPKD患者肾囊肿上皮细胞（ADPKD细胞）的Na， k - atp酶对瓦巴因的亲和力异常增加。此外，我们还发现，瓦巴因的量与血液循环中的量相似，可以刺激ADPKD细胞的细胞生长。这种作用在正常的人肾上皮细胞（NHK细胞）中没有发现，这些细胞对激素的亲和力较低。此外，瓦巴因促进ADPKD细胞凋亡，其方式与细胞增殖的增加并不平行；降低细胞的粘附性能；并增强其在培养中形成囊肿的能力。所有这些事件都是ADPKD囊发生的标志。因此，我们发现，在体外和体内，瓦巴因都加剧了ADPKD小鼠模型中肾囊肿的发展。总之，这些结果表明，瓦巴因是一种促进肾囊肿形成和进展的新因素。目前，瓦苦因在ADPKD细胞中介导囊肿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-ouabain信号如何影响ADPKD。我们的中心假设是，由于ADPKD细胞的高瓦巴因敏感性表型，它们更容易受到循环激素水平的影响；异常刺激Na， k - atp酶信号，导致细胞增生，液体分泌和粘附特性和运动性的变化。实验旨在确定在ADPKD细胞囊肿形成过程中，瓦巴因刺激的Na， k - atp酶信号传导机制，细胞内负责瓦巴因作用于细胞的途径，以及ADPKD细胞异常的瓦巴因亲和力的决定因素。这些结果将具有重要的积极影响，因为它们将有助于确定ADPKD囊肿发生和进展的分子机制，并将有助于未来开发针对Na， k - atp酶信号传导的方法来控制疾病。

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.