Mechanisms Controlling Airway Surface Liquid pH

控制气道表面液体 pH 值的机制

• 批准号: 8716459
• 负责人: Viral Shailesh Shah
• 金额: $ 2.81万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716459
• 关键词: Accounting; Acidity; Acids; Affect; Animal Model; Anions; Apical; Bacteria; Bacterial Infections; Bicarbonates; Cell Culture Techniques; Chronic; Chronic Obstructive Airway Disease; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Defect; Disease; Drug Targeting; Electrophysiology (science); Epithelial Cells; Epithelium; Family suidae; Fellowship; Genes; Goals; H(+)-K(+)-Exchanging ATPase; Health; Host Defense; Human; In Vitro; Infection; Innovative Therapy; Ion Channel; Ion Transport; Laboratories; Lead; Liquid substance; Lung; Measurement; Measures; Mediating; Messenger RNA; Mission; Modeling; Morbidity - disease rate; Mutation; National Heart, Lung, and Blood Institute; Ouabain; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Physiology; Play; Proteins; Pulmonary Cystic Fibrosis; Quality of life; RNA Interference; Relative (related person); Reporting; Respiratory Tract Infections; Role; Small Interfering RNA; Stimulus; Stomach; Surface; Techniques; Technology; Testing; Therapeutic; Time; Titrations; Translating; Viscosity; Work; airway epithelium; airway surface liquid; antimicrobial; base; clinically relevant; cystic fibrosis airway; direct application; drug testing; expectation; fluorophore; in vivo; killings; knock-down; mortality; novel; novel strategies; pre-doctoral; public health relevance; vacuolar H+-ATPase; voltage clamp

Project Summary/Abstract Respiratory infections are a common cause of morbidity and mortality in many diseases including Cystic Fibrosis (CF). In spite of current therapies more than 90% of people with CF die from respiratory infections. CF is caused by mutations in the gene for cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. How defects in this anion channel led to respiratory infections is unclear. To better understand CF lung disease, we produced CF null pigs. CF pigs show host defense defects, similar to humans. We found that CF pigs have an abnormally acidic airway surface liquid (ASL), the thin layer of fluid lining the lung epithelia. Increasing the pH of the ASL in CF pigs rescues the host defense defect. Thus, we hypothesize that infections in CF are due to an acidic ASL pH, in part, by a dysfunctional CFTR, which normally secretes HCO3-. In addition to CFTR, a number of ion channels and transporters have been hypothesized to be involved in HCO3- secretion and H+ secretion. However, which ion channels and transporters primarily contribute to ASL pH is unclear. If the dominant pathways can be identified, the most efficacious pharmacological therapy to increase ASL pH and presumably decrease lung infections can be developed. To accomplish this, we propose the following specific aims: 1) Determine the channels and transporters that secrete HCO3- into the ASL 2) Determine the primary mechanism of H+ secretion into the ASL. To determine which HCO3--secreting and which H+-secreting proteins are most important, we will use small interfering RNA (siRNA) to selectively knock down specific mRNAs and pharmacologic agents to inhibit ion channels and transporters. We will measure the effect of inhibiting these proteins on ASL pH in airway epithelia. We will quantify this effect using three techniques: pH sensitive fluorophores, Ussing chamber electrophysiology, and pH stat. By dispersing a pH sensitive fluorophore into the ASL, we can measure the effects of on ASL pH directly. In Ussing Chambers, we will voltage-clamp cultures to measure electrogenic HCO3- transport generated by pharmacologic stimulus. To account for electro-neutral ion transport, we will use pH stat to pH-clamp and measure rates of H+ or HCO3- secretion. We expect that with these techniques, we will identify which proteins contribute most to HCO3- secretion and H+ secretion into the ASL. We will then test the effects on CF host defense defects in vitro by measuring bacterial killing and ASL viscosity. Knowing which proteins are the major contributors to ASL pH, is the first step towards our long-term goal of developing drugs to increase ASL pH. Using our access to CF pigs, we can test the drugs in vivo, and then develop them as therapies for humans. These drugs would have a major impact for not only CF, but also for other diseases like COPD, which also show acidic ASL and bacterial infections. This project is a direct application of understanding a physiology mechanism to better human health, the mission of NHLBI.

项目摘要/摘要 呼吸道感染是许多疾病（包括囊性）发病率和死亡率的常见原因 纤维化（CF）。尽管目前有超过90％的CF患者死于呼吸道感染。 CF 是由囊性纤维化跨膜电导调节剂（CFTR）的突变引起的，一种阴离子 渠道。该阴离子通道中的缺陷如何导致呼吸道感染。更好地了解CF 肺部疾病，我们产生了CF无效的猪。 CF猪表现出与人类类似的宿主防御缺陷。我们发现 CF猪具有异常的酸性气道表面液体（ASL），这是肺上皮衬里的薄层。 增加CF猪中ASL的pH值挽救了宿主防御缺陷。因此，我们假设感染 在CF中，是由于酸性ASL pH，部分原因是功能失调的CFTR，通常分泌HCO3-。在 除CFTR外，已经假设许多离子通道和转运蛋白参与了HCO3-- 分泌和H+分泌。但是，哪些离子通道和转运蛋白主要有助于ASL pH 不清楚。如果可以确定主要的途径，则最有效的药理疗法增加 ASL pH和大概可以减少肺部感染。为此，我们提出了 遵循特定目的： 1）确定将HCO3-分泌到ASL的通道和转运蛋白 2）确定H+分泌在ASL中的主要机制。 为了确定哪种HCO3-分泌和哪些H+分泌蛋白最重要，我们将使用小 干扰RNA（siRNA），有选择地击倒特定的mRNA和药理剂以抑制离子 通道和转运蛋白。我们将测量抑制这些蛋白在气道中ASL pH的效果 上皮。我们将使用三种技术来量化这种效果：pH敏感荧光团，使用室 电生理学和pH STAT。通过将pH敏感的荧光团分散到ASL中，我们可以测量 直接对ASL pH的影响。在使用室中，我们将电压钳培养物测量电源 HCO3-由药理刺激产生的转运。为了解释电离离子运输，我们将使用 pH stat to pH钳和H+或HCO3分泌的测量率。我们希望通过这些技术，我们 将确定哪种蛋白质对HCO3-分泌和H+分泌最大的作用。然后我们将测试 通过测量细菌杀伤和ASL粘度，对CF宿主防御缺陷的影响。 知道哪种蛋白质是ASL pH的主要因素，是朝着我们的长期目标迈出的第一步 开发药物以增加ASL pH。使用我们对CF猪的访问，我们可以在体内测试药物，然后 将它们作为人类的疗法发展。这些药物不仅会对CF产生重大影响，而且对 其他疾病（例如COPD）也显示出酸性ASL和细菌感染。这个项目是一个直接的 了解生理机制来改善人类健康的应用，NHLBI的使命。