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

Pannexin-1/P2X7 相互作用通过减少 NaCl 重吸收促进肾囊肿中 ATP 过度释放和 ADPKD 进展

• 批准号: 10614647
• 负责人: Tengis S Pavlov
• 金额: $ 39.88万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10614647
• 关键词: Ablation; Adenosine; Affect; Aging; Aldosterone; Autosomal Dominant Polycystic Kidney; Autosomal Recessive Polycystic Kidney; Breeding; Calcium; Calcium Signaling; Cell Culture Techniques; Cell Line; Cell membrane; Clinical; Clinical Research; Complex; Cyst; Cyst Fluid; Cystic kidney; Data; Development; Disease Progression; Duct (organ) structure; Electrolytes; Epithelium; Excretory function; Exhibits; Experimental Models; Funding; Genetic; Genetic Predisposition to Disease; Gout; Growth; Human; Image; Impairment; Inherited; Integral Membrane Protein; Intervention; Ion Transport; Kidney Diseases; Kidney Failure; Knock-out; Knockout Mice; Knowledge; Liquid substance; Live Birth; Mammalian Cell; Measurement; Mediating; Membrane; Modeling; Molecular Conformation; Monitor; Mus; Nephrons; Normal tissue morphology; P2X-receptor; Pathogenicity; Pathologic; Patients; Permeability; Polycystic Kidney Diseases; Probenecid; Process; Protein Family; Proteins; Publishing; Purinoceptor; Rattus; Regulation; Renal function; Reporting; Rodent Model; Role; Signal Transduction; Sodium; Stimulus; Study models; System; Testing; Water; absorption; base; design; epithelial Na+ channel; experimental study; improved; in vivo; inorganic phosphate; monolayer; mouse model; paracrine; patch clamp; pharmacologic; prevent; promoter; receptor

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.

项目摘要 ADPKD（常染色体显性多囊性肾脏疾病）是最普遍的遗传性渐进性 肾脏疾病影响美国1：1000个活产。遗传易感性是多囊性肾脏所必需的 疾病（PKD）的启动，尽管其他未完全鉴定出的下游过程是囊肿的 生长。它们的表征可以为临床干预提供独特的机会。不好的人之一 PKD中研究的现象是囊肿中的高腺苷-3-磷酸（ATP）含量。作为强大的监管者 上皮水电解质转运，ATP可以降低收集管系统中的重吸收，其中约70％ ADPKD囊肿的发展。特别是作为旁分泌剂，ATP限制了上皮钠通道的活性 （ENAC）。降低的ENAC活性被认为是细胞生成的关键因素。但是， 致病性ATP释放及其在囊肿发育过程中Na+转运中的作用尚不清楚。 Pannexins是跨膜蛋白，最近被表征为ATP释放。 与P2X7受体的相互作用通过形成高导通道促进Pannexin-1（Panx1）的激活 用作ATP渗透的毛孔。我们最近发表的数据和试点数据表明，PKD囊肿表现出独特的 当囊肿细胞中panx1和p2x7表达异常高水平的致病情况。 我们假设pannexin-1向活性形式的p2x7依赖性切换是中央 ATP在囊肿中积累的机制及其生长。我们的数据表明两者的腔表达 PANX1和P2X7蛋白在人ADPKD囊肿和PKD啮齿动物模型中急剧增加。 此外，PANX1通道和P2X7受体的药理和遗传靶向减慢了囊肿的生长。 在拟议的项目中，我们计划研究panx1/p2x7的相互作用是否有助于ATP释放 ADPKD的低态PKD1RC/RC小鼠模型以及该机制如何降低整个囊性的重吸收 上皮。我们将在ADPKD开发过程中研究PANX1和P2RX7的表达。一组实验 涉及贴片钳的涉及细胞培养物和天然囊肿，以研究PANX1介导的ATP的调节 发布。由于嘌呤能信号对于上皮运输很重要，因此我们将表征钙信号传导和 ENAC在新鲜分离的囊肿和正常收集管道中的功能。 panx1 mutanat小鼠的育种 PKD1RC/RC菌株测试是否通过限制ATP释放和 改善钠的重吸收。我们还计划研究重新利用ProbEnecid的潜力，这是一种已知的痛风 疗法和pannexin-1阻滞剂，用于ADPKD治疗。