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Gap Junction-mediated Regulation of the V-type H+-ATPase in a Renal Epithelium

间隙连接介导的肾上皮细胞中 V 型 H-ATP 酶的调节

基本信息

批准号：
8290440
负责人：
Peter M Piermarini
金额：
$ 7.55万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8290440
关键词：
Acids Aedes Anions Apical Applications Grants Bicarbonates Biological Assay Biological Models Cells Connexins Coupled Coupling Culicidae Data Defect Disease Disease Vectors Diuretics Electrodes Epithelial Fluids and Secretions Functional disorder Gap Junctions Genes Genetic Goals Grant Health Heart Human Insecta Intercalated Cell Kidney Malpighian Tubules Mammalian Cell Mammals Measures Mediating Mentored Research Scientist Development Award Mentorship Metabolic Methods Microelectrodes Mitochondria Modeling Molecular Oocytes Orthologous Gene Osteoporosis Parents Pathology Peptides Physiological Play Proton Pump Proton-Translocating ATPases Publications Pump RNA Interference Regulation Renal function Renal tubule structure Research Research Support Role Sterility Stilbenes Study models Testing Universities base basolateral membrane carbonate dehydratase career career development chloride-cotransporter potassium deafness inhibitor/antagonist insight interest knock-down male novel prevent renal epithelium stellate cell voltage clamp

项目摘要

DESCRIPTION (provided by applicant): The present R03 resubmission is motivated by progress made by the PI (Piermarini) during the first two years of his K01 award under the mentorship of Prof. Klaus W. Beyenbach at Cornell University. The proposed research is an extension of the PI's on-going molecular and physiological studies of the renal (Malpighian) tubules of the mosquito Aedes aegypti, which are a valuable model for studying transepithelial transport that is powered exclusively by the vacuolar (V)-type H+ATPase. The proposed R03 research will enhance the PI's career development by allowing him to 1) maintain the momentum of his on-going K01 research on Aedes Malpighian tubules, and 2) collect preliminary data for a subsequent R01 application. The proposed studies will also provide insights into mammalian cells that are energized by the V-type H+ATPase. Such insights are of significant biomedical interest, because several important pathologies are now known to be associated with defects or dysfunction of the V-type H+ATPase. Lastly, the proposed research has the potential to 1) shift the current paradigm of renal function in mosquitoes, which are important vectors of diseases that afflict humans, and 2) provide the first example of gap junction- mediated (intercellular) regulation of intracellular pH (pHi) in a renal epithelium. The research goal of the parent K01 award is to decipher the molecular mechanisms of acid-base transport in the 'principal' cells of Aedes Malpighian tubules. These cells resemble acid-secreting (a) intercalated cells of the mammalian renal collecting tubule in that they are mitochondrion-rich and express an apical V-type H+ ATPase. Based on these similarities, the PI hypothesized that principal cells express a basolateral Cl/HCO3 anion exchanger (AE), as is found in mammalian a-intercalated cells. The PI has now shown that the Aedes AE (AeAE) is expressed in the basolateral membrane of the neighboring 'stellate' cells, which intercalate between principal cells. Furthermore, inhibiting AeAE in Malpighian tubules disrupts transepithelial transport in a manner consistent with inhibition of the V-type H+ATPase in principal cells. Thus, the activity of AeAE in stellate cells appears to be coupled to the apical V-type H+ATPase in principal cells. The goal of the present R03 grant application is to determine if gap junctions mediate the putative functional coupling between principal and stellate cells. In aim 1, I will use a pharmacological approach to test the hypothesis that the 1) activity of the V-type H+ATPase in principal cells, 2) regulation of pHi in principal cells, and 3) rates of transepithelial fluid secretion mediated by principal cells, are functionally coupled to the AeAE in stellate cells via gap junctions. In aim 2, I will use a functional genetic approach (RNA interference) to test the hypothesis that innexins-the gap-junction proteins of insects-are the molecular entities that couple principal cells to stellate cells.

描述（由申请人提供）：本次R 03重新提交的动机是PI（Piermarini）在Klaus W教授的指导下，在K 01奖的前两年取得的进展。Beyenbach在康奈尔大学。拟议的研究是PI正在进行的埃及伊蚊肾（马氏管）小管分子和生理学研究的延伸，这是研究仅由空泡（V）型H+ ATP酶提供动力的跨上皮转运的有价值模型。拟议的R 03研究将促进主要研究者的职业发展，使他能够1）保持他正在进行的关于马氏伊蚊小管的K 01研究的势头，以及2）为随后的R 01申请收集初步数据。拟议的研究还将提供对由V型H+ ATP酶供能的哺乳动物细胞的见解。这些见解具有重要的生物医学意义，因为现在已知几种重要的病理与V型H+ ATP酶的缺陷或功能障碍有关。最后，所提出的研究有可能1）改变蚊子中肾功能的当前范例，蚊子是折磨人类的疾病的重要媒介，以及2）提供肾上皮中细胞内pH（pHi）的间隙连接介导的（细胞间）调节的第一个实例。 K 01奖的研究目标是破译伊蚊马氏管“主”细胞中酸碱转运的分子机制。这些细胞类似于哺乳动物肾集合管的酸分泌（a）插入细胞，因为它们富含钙离子并表达顶端V型H+ ATP酶。基于这些相似性，PI假设主细胞表达基底外侧Cl/HCO 3阴离子交换剂（AE），如在哺乳动物α-嵌入细胞中发现的。PI现在已经表明伊蚊AE（AeAE）在相邻的“星状”细胞的基底外侧膜中表达，所述星状细胞插入主细胞之间。此外，抑制马氏管中的AeAE以与抑制主细胞中的V型H+ ATP酶一致的方式破坏跨上皮转运。因此，AeAE在星状细胞中的活性似乎与主细胞中的顶端V型H+ ATP酶偶联。目前R 03基金申请的目的是确定缝隙连接是否介导了主细胞和星状细胞之间的假定功能偶联。在目的1中，我将使用药理学方法来检验以下假设：1）主细胞中V型H+ ATP酶的活性，2）主细胞中pHi的调节，以及3）由主细胞介导的跨上皮液体分泌速率，通过间隙连接与星状细胞中的AeAE功能性偶联。在目标2中，我将使用一种功能遗传学方法（RNA干扰）来验证这样一个假设，即连接蛋白（昆虫的间隙连接蛋白）是将主细胞与星状细胞偶联的分子实体。