The involvement of ATP-dependent inhibition of ENaC in ARPKD cystogenesis

ENaC 的 ATP 依赖性抑制参与 ARPKD 囊肿发生

• 批准号: 9146873
• 负责人: Daria Ilatovskaya
• 金额: $ 8.82万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-21 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9146873
• 关键词: Address; Adenosine Triphosphate; Adolescence; Affect; Agonist; Animals; Attenuated; Autosomal Recessive Polycystic Kidney; Binding; Biochemistry; Biosensor; Calcium; Cells; Chronic; Chronic Kidney Failure; Confocal Microscopy; Coupled; Cyst; Cystic kidney; Data; Development; Disease; Disease Progression; Duct (organ) structure; Electrophysiology (science); Endoplasmic Reticulum; Epithelial; Epithelial Cells; Fluids and Secretions; G alpha q Protein; Genetic; Genetic study; Growth; Health; Hereditary Disease; Immunohistochemistry; Incidence; Infant; Inherited; Inositol; Intervention; Ion Channel; Kidney; Kidney Diseases; Kidney Transplantation; Liquid substance; Live Birth; Measurement; Mediating; Methods; Modeling; Molecular; Nephrons; P2Y2 receptor; Perinatal; Polycystic Kidney Diseases; Process; Production; Publishing; Purines; Purinoceptor; Rattus; Regulation; Role; Scheme; Second Messenger Systems; Signal Transduction; Sodium; Sodium Channel; Sprague-Dawley Rats; Testing; apical membrane; autocrine; benzamil; clinically relevant; driving force; epithelial Na+ channel; in vivo; inhibitor/antagonist; innovation; new therapeutic target; novel; paracrine; receptor; research study; second messenger; tripolyphosphate

 DESCRIPTION (provided by applicant): Polycystic kidney diseases (PKD) are a group of inherited nephropathies characterized by the formation of fluid-filled cysts along the nephron. Autosomal Recessive form of PKD (ARPKD) has an incidence of 1 in 20,000 live births; infants with this disease that survive beyond the perinatal period develop chronic renal failure by adolescence and eventually require kidney transplantation. This proposal focuses on the sodium transport regulation in the renal collecting ducts in ARPKD and potential means of pharmacological intervention with the cysts' progression. Specifically, we have determined that Epithelial Sodium Channels (ENaCs), which are expressed in the collecting ducts and represent the rate-limiting step of sodium reabsorption in this nephron segment, are involved into the process of cystogenesis in the ARPKD setting. Our preliminary data indicate that ENaC expression and activity are significantly lower in the cystic epithelial cells of a rat model of ARPKD, as assessed with immunohistochemistry and single channel analysis in isolated cysts; furthermore, chronic administration of the ENaC-specific inhibitor, benzamil, aggravated cyst formation. Thus, we propose that ENaC inhibition exacerbates PKD progression. Using a novel enzymatic microbiosensors approach we established that concentration of adenosine triphosphate (ATP) was significantly higher in PCK rat cortical cysts compared to control rats. ATP was shown to inhibit ENaC via signaling cascades initiated by binding to its receptors. Therefore, we hypothesize here that accumulation of excessive levels of ATP in the lumen of the dilated collecting ducts affects specific purinergic receptors in the cystic cells (primary candidates being P2Y2 or P2X7) and results in ENaC inhibition; this suppresses normal sodium reabsorption in these collecting ducts, and thus promotes fluid accumulation and cysts' expansion. The integrative experimental approach used in this study will include single nephron electrophysiology, in vivo animal studies, genetics, biochemistry, biosensors amperometry and confocal microscopy and will address the clinically relevant problem of cyst expansion in ARPKD. Specifically, this proposal will identify the involvement of the ATP- triggered signaling into regulation of sodium reabsorption in this setting. This proposal will address the following specific aims: 1. Determine the relationship between ENaC activity and cystogenesis in ARPKD; 2. Elucidate the cellular and molecular mechanism by which excessive levels of ATP modulate sodium transport, promoting cyst growth; and 3. Explore if P2 receptor agonists/antagonists and suppression of the ATP levels can affect cystogenesis.

 描述（由申请人提供）：多囊肾病（PKD）是一组遗传性肾病，其特征是沿肾单位形成充满液体的囊肿沿着。常染色体隐性PKD（ARPKD）的发病率为1/20，000活产婴儿;患有这种疾病的婴儿在围产期后存活，到青春期发展为慢性肾衰竭，最终需要肾移植。该建议的重点是ARPKD中肾集合管中的钠转运调节和囊肿进展的潜在药物干预手段。具体来说，我们已经确定，上皮钠通道（ENaCs），这是在集合管中表达，并代表在这个肾单位段钠重吸收的限速步骤，参与了在ARPKD设置的囊肿形成的过程。我们的初步数据表明，ENaC的表达和活性显着较低的囊性上皮细胞的大鼠模型的ARPKD，评估与免疫组化和单通道分析孤立的囊肿;此外，长期管理的ENaC特异性抑制剂，苯扎明，加剧囊肿形成。因此，我们认为ENaC抑制会加剧PKD的进展。使用一种新的酶微生物传感器的方法，我们建立了三磷酸腺苷（ATP）的浓度显着较高的PCK大鼠皮质囊肿相比，对照组大鼠。ATP显示通过与其受体结合引发的信号级联抑制ENaC。因此，我们假设在扩张的集合管管腔中积累过量的ATP会影响囊性细胞中的特异性嘌呤能受体（主要候选者是P2 Y2或P2 X7），并导致ENaC抑制;这会抑制这些集合管中正常的钠重吸收，从而促进液体积聚和囊肿扩张。在这项研究中使用的综合实验方法将包括单肾单位电生理学，在体内动物研究，遗传学，生物化学，生物传感器安培法和共聚焦显微镜，并将解决临床相关的问题，囊肿扩张在ARPKD。具体而言，该提议将确定ATP触发的信号传导在这种情况下参与钠重吸收的调节。本提案将针对以下具体目标：1.探讨ENaC活性与ARPKD囊肿形成的关系; 2.阐明细胞和分子机制，通过过量的ATP调节钠转运，促进囊肿生长;和3。探索P2受体激动剂/拮抗剂和ATP水平的抑制是否会影响膀胱生成。