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（常染色体显性多囊肾病）是最常见的遗传性进行性肾病， 肾脏疾病影响美国1：1000活产婴儿。多囊肾的病因有哪些 疾病（PKD）的启动，虽然其他的，不完全确定的下游过程是需要囊肿 增长它们的表征可能为临床干预提供独特的机会。一个可怜的 PKD中研究的现象是囊肿中的高腺苷-3-磷酸（ATP）含量。作为一个强大的监管机构， 上皮水电解质转运，ATP可以减少集合管系统中的重吸收，其中约70% ADPKD囊肿的发展。特别地，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， 病原性ATP释放及其在包囊发育过程中Na+转运中的作用尚不清楚。 泛联蛋白是一种跨膜蛋白，最近被鉴定为能够释放ATP。 与P2 X7受体的相互作用通过形成高传导通道促进泛连接蛋白-1（PANX 1）的活化 用作ATP可渗透的孔。我们最近发表的试验数据表明，PKD囊肿表现出独特的 当囊肿衬里细胞中PANX 1和P2 X7均异常高水平表达时的致病情况。 我们假设P2 X7依赖性泛连接蛋白-1转换为活性形式是 ATP在孢囊中积累的机制及其生长。我们的数据表明，管腔表达的两个 PANX 1和P2 X7蛋白在人类ADPKD囊肿和PKD啮齿动物模型中显著增加。 此外，PANX 1通道和P2 X7受体的药理学和遗传靶向减缓了囊肿生长。 在拟议的项目中，我们计划研究PANX 1/P2 X7相互作用是否有助于ATP释放。 ADPKD的亚型Pkd 1 RC/RC小鼠模型以及这种机制如何减少跨囊的重吸收 上皮我们将研究Panx 1和P2 rx 7在ADPKD发展过程中的表达。一组实验 在细胞培养物和天然包囊中设计涉及膜片钳的方法来研究PANX 1介导的ATP的调节 release.由于嘌呤能信号传导对于上皮转运很重要，我们将表征钙信号传导， ENaC在新鲜分离的囊肿和正常集合管中起作用。Panx 1突变小鼠的选育 Pkd 1 RC/RC菌株测试泛连接蛋白-1的基因消融是否通过限制ATP释放来抑制囊肿发生， 提高钠重吸收。我们还计划研究丙磺舒（一种已知的痛风药物）的再利用潜力。 药物和泛连接蛋白-1阻滞剂，用于ADPKD治疗。