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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.

项目概要多囊肾病（PKD）是一组遗传性肾病，其特征是形成液体- 沿肾单位充满囊肿。常染色体隐性 PKD (ARPKD) 的发病率为每 20,000 人中就有 1 人出生；患有这种疾病的婴儿在围产期之后存活下来，会发展为慢性肾功能衰竭青春期并最终需要肾移植。该提案重点关注钠转运 ARPKD 肾集合管的调节以及药物干预的潜在方法囊肿的进展。具体来说，我们确定了上皮钠通道（ENaC），它们是在集合管中表达，代表该肾单位钠重吸收的限速步骤段，参与 ARPKD 环境中的囊肿发生过程。我们的初步数据表明 ARPKD 大鼠模型的囊性上皮细胞中 ENaC 的表达和活性显着降低，如对分离的囊肿进行免疫组织化学和单通道分析评估；此外，慢性给予 ENaC 特异性抑制剂苯扎米尔，会加剧囊肿形成。使用新型酶通过微生物传感器方法，我们确定三磷酸腺苷 (ATP) 的浓度显着与对照大鼠相比，PCK 大鼠皮质囊肿中的含量更高。 ATP 被证明可以通过信号级联抑制 ENaC 通过与其受体结合而启动。因此，我们在这里假设过量水平的积累扩张集合管管腔中的 ATP 影响囊性细胞中的特定嘌呤能受体并产生结果 ENaC 抑制；这会抑制这些集合管中正常的钠重吸收，并促进液体积累和囊肿扩张。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、生物传感器电流分析法和共聚焦显微镜检查并将解决 ARPKD 囊肿扩张的临床相关问题。具体来说，这该提案将鉴定囊性细胞中参与嘌呤能信号传导的受体，并研究其相关性 ATP 信号对钠重吸收的影响取决于饮食中的钠含量。该提案将解决具体目标如下： 1. 确定 ARPKD 中 ENaC 活性与囊肿发生之间的关系； 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； 3. 探讨 P2 受体激动剂/拮抗剂是否能抑制 ATP 水平可影响囊肿发生。