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

基本信息

批准号：
10419229
负责人：
Daria Ilatovskaya
金额：
$ 2.11万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-15 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10419229
关键词：
involvement ATP dependent inhibition ENaC

项目摘要

PROJECT SUMMARY 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. 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 hypothesized here that accumulation of excessive levels of ATP in the lumen of the dilated collecting ducts affects specific purinergic receptors in the cystic cells and results in ENaC inhibition; this suppresses normal sodium reabsorption in these collecting ducts, and 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 receptors involved in the purinergic signaling in the cystic cells, and study the relevance of ATP signaling to sodium reabsorption dependent on sodium content in the diet. 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.

项目摘要多囊肾脏疾病（PKD）是一组遗传性肾病，其特征是形成液体沿肾单位填充的囊肿。 PKD（ARPKD）的常染色体隐性形式有20,000分之1 出生；患有这种疾病的婴儿在围产期超出围产期生存青少年，最终需要肾脏移植。该提案重点介绍钠运输 ARPKD中肾脏收集管道的调节和药物干预的潜在手段囊肿的进展。具体而言，我们已经确定上皮钠通道（ENAC）是在收集管道中表达，代表该肾单位中钠重吸收的速率限制步骤段，参与ARPKD设置中的囊肿过程。我们的初步数据表明在ARPKD大鼠模型的囊性上皮细胞中，ENAC表达和活性显着降低，AS 通过免疫组织化学和单个通道分析评估；此外，慢性施用ENAC特异性抑制剂苯甲胺，总囊肿形成。使用新型酶促微生物传感器方法我们确定三磷酸腺苷的浓度显着与对照大鼠相比，PCK大鼠皮质囊肿的更高。 ATP显示通过信号级联抑制ENAC 通过与其接收器结合起来。因此，我们在这里假设，过量水平的积累扩张的收集导管管腔中的ATP影响囊性细胞中的特定嘌呤能受体，结果在ENAC抑制中；这抑制了这些收集管道中正常的钠重吸收，并促进液体积累和囊肿的扩展。本研究中使用的综合实验方法将包括单一肾单位电生理学，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，将解决ARPKD中囊肿扩展的临床相关问题。具体来说，这是提案将确定囊性细胞中嘌呤能信号传导涉及的接收器，并研究相关性饮食中钠含量取决于钠含量的ATP信号传导。该建议将解决以下特定目的：1。确定ARPKD中的ENAC活性与囊肿之间的关系； 2。阐明过量ATP调节钠转运的细胞和分子机制，促进囊肿生长；和3。探索P2受体激动剂/拮抗剂以及ATP水平的抑制会影响囊生。