A Parathyroid Hormone-Stimulated Transport Process in Avian Renal Proximal Tubule Cells. Characterization of a Novel Hormone Response.

禽肾近端小管细胞中甲状旁腺激素刺激的转运过程。

• 批准号: 0343478
• 负责人: Gary Laverty
• 金额: $ 19.49万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0343478&HistoricalAwards=false
• 关键词: Parathyroid; Hormone; Stimulated; Transport; Process

The comparative physiology approach has provided many examples of discoveries of novel epithelial transport systems or of models in which known systems are more easily studied. In this proposal experiments will be done on a primary cell culture model of the chick renal proximal tubule (PT). In this model renal cells are grown on rigid, microporous filters, altering the tissue architecture from tubules to flat monolayers. The monolayers reach a confluent, polarized state and display transport properties consistent with the PT. An electrophysiological approach is used to measure ion transport currents under varying conditions and in the presence or absence of agonists or antagonists. Initial studies have revealed a novel ion current response to parathyroid hormone (PTH), a calcium-regulating hormone with known actions on the PT. The novel current response appears to be chloride ion secretion (transport into the apical space) and to be dependent on two separate, but linked transport systems, a cystic fibrosis transmembrane regulator (CFTR)-like chloride channel and a new, chloride-dependent member of the sodium hydrogen exchanger (NHE) family, recently cloned from rat colon. The hypothesis of this study is that this PTH response is part of an orchestrated change in the overall pattern of proximal tubule transport, and that, although never previously reported, it also exists in mammalian proximal tubules at much lower expression levels. Thus, the chick culture system may represent an ideal model for further investigating this PTH effect and the role of CFTR in the proximal tubule, both of which seem to be highly expressed in this particular system. A combination of molecular studies (PCR and immunoblotting), isotopic tracers, fluorescent assays of intracellular pH (an assay for NHE activity) and electrophysiological methods will be used to determine whether the PTH effect can also be detected in native (non-cultured) chick kidney and whether it is found in rat proximal tubule cultures. Potential physiological roles for the PTH response will also be examined, including effects on fluid and urate secretion in this segment. The potential role of CFTR as a regulator of other transport systems will also be specifically addressed, using known blockers and antibodies to the CFTR protein. This study will provide significant new information about the role of PTH and CFTR in the kidney and will be of interest to the fields of comparative, renal and epithelial transport physiology.

比较生理学方法提供了许多发现新的上皮转运系统或模型的例子，其中已知的系统更容易研究。本实验采用鸡肾近曲小管原代细胞培养模型。在该模型中，肾细胞生长在刚性微孔过滤器上，将组织结构从小管变为扁平单层。单层达到汇合，极化状态和显示运输性能与PT一致。电生理学方法用于在不同条件下以及在存在或不存在激动剂或拮抗剂的情况下测量离子转运电流。初步研究揭示了一种新的离子电流对甲状旁腺激素（PTH）的反应，PTH是一种钙调节激素，已知对PT有作用。新的电流反应似乎是氯离子分泌（运输到顶端空间），并依赖于两个独立的，但连接的运输系统，囊性纤维化跨膜调节因子（CFTR）样氯离子通道和一个新的，氯离子依赖性成员的钠氢交换器（NHE）家庭，最近克隆从大鼠结肠。本研究的假设是，这种PTH反应是近端小管转运的整体模式中的协调变化的一部分，并且尽管以前从未报道过，但它也以低得多的表达水平存在于哺乳动物近端小管中。因此，鸡培养系统可能代表了一个理想的模型，用于进一步研究这种PTH的影响和CFTR在近端小管中的作用，这两者似乎都在这个特定的系统中高度表达。将使用分子研究（PCR和免疫印迹）、同位素示踪剂、细胞内pH的荧光测定（NHE活性测定）和电生理学方法的组合来确定是否也可以在天然（非培养）鸡肾中检测到PTH效应，以及是否在大鼠近端小管培养物中发现PTH效应。还将检查PTH反应的潜在生理作用，包括对该部分中液体和尿酸盐分泌的影响。CFTR作为其他运输系统的调节剂的潜在作用也将使用已知的CFTR蛋白的阻断剂和抗体来具体解决。这项研究将提供有关PTH和CFTR在肾脏中的作用的重要新信息，并将对比较，肾脏和上皮运输生理学领域感兴趣。