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.

项目概要 ADPKD（常染色体显性多囊肾病）是最常见的遗传性进展性疾病 肾脏疾病影响美国 1:1000 的活产。多囊肾需要遗传倾向 疾病（PKD）的启动，尽管囊肿需要其他未完全确定的下游过程 生长。它们的表征可能为临床干预提供独特的机会。贫困者之一 研究的 PKD 现象是囊肿中高腺苷 3-磷酸 (ATP) 含量。作为强有力的监管者 上皮水电解质转运，ATP 可以减少集合管系统的重吸收，其中约 70% ADPKD 囊肿形成。特别是，作为旁分泌剂，ATP 限制上皮钠通道的活性 （ENaC）。 ENaC 活性降低被认为是细胞发生的关键因素。然而，该机制的 病原性 ATP 释放及其在包囊发育过程中 Na+ 转运中的作用仍不清楚。 Pannexins 是跨膜蛋白，最近被鉴定为能够释放 ATP。 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 充当 ATP 可渗透的毛孔。我们最近发布的试点数据表明 PKD 囊肿表现出独特的 当囊壁细胞中 PANX1 和 P2X7 表达异常高水平时的致病情况。 我们假设 P2X7 依赖性 pannexin-1 向活性形式的转换是核心 囊肿中 ATP 积累及其生长的机制。我们的数据表明，两者的管腔表达 PANX1 和 P2X7 蛋白在人类 ADPKD 囊肿和 PKD 啮齿动物模型中显着增加。 此外，PANX1 通道和 P2X7 受体的药理学和遗传靶向可减缓囊肿的生长。 在拟议的项目中，我们计划研究 PANX1/P2X7 相互作用是否有助于 ATP 释放 ADPKD 亚形性 Pkd1RC/RC 小鼠模型以及该机制如何减少囊性囊肿的重吸收 上皮。我们将研究 ADPKD 发展过程中 Panx1 和 P2rx7 的表达。一组实验 涉及膜片钳的设计在细胞培养物和天然囊肿中研究 PANX1 介导的 ATP 的调节 发布。由于嘌呤能信号传导对于上皮运输很重要，我们将描述钙信号传导和 ENaC 在新鲜分离的囊肿和正常集合管中发挥作用。 Panx1突变小鼠的选育 Pkd1RC/RC 菌株测试 pannexin-1 的基因消除是否通过限制 ATP 释放来抑制囊肿发生 改善钠的重吸收。我们还计划研究重新利用丙磺舒（一种已知的痛风药物）的潜力 补救措施和 pannexin-1 阻滞剂，用于 ADPKD 治疗。