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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.

项目摘要多囊肾病（PKD）是一组遗传性肾病，其特征在于形成液体- 沿着肾单位沿着充满囊肿。常染色体隐性PKD（ARPKD）的发病率为1/20，000 出生;存活超过围产期的患有这种疾病的婴儿通过以下方式发展为慢性肾衰竭：青春期，最终需要肾移植。该提案侧重于钠转运 ARPKD中肾集合管的调节和药物干预的潜在方法囊肿的进展。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，并代表该肾单位中钠重吸收的限速步骤在ARPKD背景下，参与了囊肿发生的过程。我们的初步数据显示，在ARPKD大鼠模型的囊性上皮细胞中，ENaC表达和活性显着降低，因为在孤立囊肿中用免疫组织化学和单通道分析进行评估;此外， ENaC特异性抑制剂苯扎明的施用加重了囊肿形成。使用一种新的酶微生物传感器方法，我们建立了三磷酸腺苷（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通过信号级联反应抑制ENaC 通过与其受体结合而启动。因此，我们在这里假设，扩张集合管管腔中的ATP影响囊细胞中特异性嘌呤能受体，在ENaC抑制中;这抑制了这些集合管中正常的钠重吸收，并促进了液体积累和包囊扩张。本研究中使用的综合实验方法将包括单一的肾单位电生理学，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检查，并将解决临床相关的问题，囊肿扩张ARPKD。具体地说，这该提案将确定参与囊性细胞中嘌呤能信号传导的受体，并研究其相关性。钠重吸收依赖于饮食中的钠含量。该提案将解决具体目标如下：1。探讨ENaC活性与ARPKD囊肿形成的关系; 2. 阐明ATP水平过高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/拮抗剂和ATP水平抑制会影响膀胱生成