Pannexin-1/P2X7 interaction promotes excessive ATP release in kidney cysts and ADPKD progression via reduced NaCl reabsorption

项目总结 常染色体显性遗传性多囊肾病是最常见的遗传性进行性疾病。 肾脏疾病在美国影响1：1000的活产儿。遗传易感性是多囊肾的必要条件 疾病(PKD)的启动，尽管囊肿需要其他未完全确定的下游过程 成长。它们的特征可能为临床干预提供一个独特的机会。其中一个糟糕的人 PKD的研究现象是囊内高含量的腺苷-3-磷酸(ATP)。作为一个强大的监管机构 上皮水-电解质转运，三磷酸腺苷可降低集合管系统的重吸收，其中~70% 的ADPKD囊性病变发生。特别是，作为旁分泌物质，ATP限制上皮钠通道的活动。 (ENAC)。ENaC活性降低被认为是细胞发生的关键因素。然而，这一机制 致病性的ATP释放及其在包囊发育过程中的Na+转运中的作用尚不清楚。 膜联蛋白是近年来发现的具有释放三磷酸腺苷功能的跨膜蛋白。 与P2X7受体的相互作用通过形成高传导通道促进pAnnexin-1(Panx1)的激活 起到了透三磷酸腺苷的作用。我们最近公布的和试点数据表明，PKD囊肿表现出独特的 当Panx1和P2X7在囊肿衬里细胞中表达异常高时的致病状态。 我们假设依赖于P2X7的pAnnexin-1到活性形式的转换是中心 三磷酸腺苷在包囊中的积累及其生长机制。我们的数据表明，两者的管腔表达 Panx1和P2X7蛋白在人ADPKD包囊和PKD啮齿动物模型中显著增加。 此外，药物和基因靶向的Panx1通道和P2X7受体可以减缓囊泡的生长。 在拟议的项目中，我们计划研究Panx1/P2X7相互作用是否有助于在 ADPKD亚型Pkd1RC/RC小鼠模型及其机制如何减少囊性重吸收 上皮组织。我们将研究Panx1和P2rx7在ADPKD发生发展过程中的表达。一系列实验 在细胞培养和自然囊泡中设计膜片钳来研究Panx1介导的ATP的调节 放手。由于嘌呤能信号对上皮运输很重要，我们将描述钙信号和 ENAC在新鲜分离的包囊和正常的集合管中起作用。选育Panx1突变系小鼠的研究 Pkd1RC/RC菌株测试pAnnexin-1的基因消融是否通过限制ATP的释放和 改善钠的重吸收。我们还计划研究丙磺舒的再利用潜力，这是一种已知的痛风 药物和pannin-1阻滞剂，用于ADPKD的治疗。

PROJECT SUMMARY ADPKD (autosomal dominant polycystic kidney disease) is the most prevalent inherited progressive kidney disease affecting 1:1000 live births in USA. Genetic predisposition is necessary for polycystic kidney disease (PKD) initiation, although other, incompletely identified downstream processes are required for cyst growth. Their characterization may provide a unique opportunity for clinical interventions. One of the poorly studied phenomena in PKD is high adenosine-3-phosphate (ATP) content in cysts. As a powerful regulator of epithelial water-electrolyte transport, ATP can decrease reabsorption in the collecting duct system where ~70% of ADPKD cysts develop. Particularly, as a paracrine agent, ATP limits activity of the epithelial sodium channel (ENaC). Reduced ENaC activity is recognized as pivotal factors of cytogenesis. However, the mechanism of the pathogenic ATP release and its role in Na+ transport during cyst development remains unclear. Pannexins are transmembrane proteins which were recently characterized to be capable of ATP release. Interaction with P2X7 receptors promotes activation of pannexin-1 (PANX1) by forming high-conductive channels serving as ATP-permeable pores. Our recently published and pilot data indicate that PKD cysts exhibit a unique pathogenic situation when abnormally high level of both PANX1 and P2X7 express in cyst-lining cells. We hypothesize that the P2X7-dependent switching of pannexin-1 to the active form is the central mechanism of ATP accumulation in the cysts and their growth. Our data suggest that luminal expression of both PANX1 and P2X7 proteins dramatically increase in the human ADPKD cysts and in rodent models of PKD. Moreover, pharmacological and genetic targeting of PANX1 channels and P2X7 receptors slows cyst growth. In the proposed project we plan to study if PANX1/P2X7 interaction contributes to ATP release in the hypomorphic Pkd1RC/RC mouse model of ADPKD and how this mechanism reduces reabsorption across the cystic epithelium. We will investigate Panx1 and P2rx7 expression during development of ADPKD. A set of experiments involving patch-clamp is designed in cell cultures and native cysts to study regulation of PANX1-mediated ATP release. As purinergic signaling is important for epithelial transport we will characterize calcium signaling and ENaC function in freshly isolated cysts and normal collecting ducts. Breeding of Panx1 mutanat mice with Pkd1RC/RC strain tests if genetic ablation of pannexin-1 inhibits cystogenesis by limiting ATP release and improving sodium reabsorption. We also plan to study the potential of repurposing probenecid, a known gout remedy and pannexin-1 blocker, for ADPKD treatment.