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

比较生理学方法提供了许多发现新的上皮运输系统或模型的例子，在这些模型中已知的系统更容易被研究。在这项建议中，实验将在鸡肾近端小管(PT)的原代细胞培养模型上进行。在这个模型中，肾细胞生长在坚硬的微孔过滤器上，改变了组织结构，从小管到扁平的单层。单分子膜达到融合的偏振状态，并显示与PT一致的传输特性。电生理学方法用于测量不同条件下的离子传输电流，以及在存在或不存在激动剂或拮抗剂的情况下。初步研究揭示了甲状旁腺激素(PTH)的一种新的离子电流反应，PTH是一种钙调节激素，已知对PT有作用。这种新的电流反应似乎是氯离子分泌(转运到根尖间隙)，并依赖于两个独立但相互关联的运输系统，一个是囊性纤维化跨膜调节器(CFTR)样氯离子通道，另一个是最近从大鼠结肠克隆的钠氢交换器(NHE)家族中一个新的氯离子依赖成员。这项研究的假设是，这种甲状旁腺素反应是近端小管运输整体模式的精心安排的一部分，尽管以前从未报道过，但它也存在于哺乳动物近端小管中，表达水平要低得多。因此，鸡的培养系统可能是一个理想的模型，进一步研究这种甲状旁腺素效应和CFTR在近端小管中的作用，这两个似乎都在这个特定的系统中高度表达。结合分子研究(聚合酶链式反应和免疫印迹)、同位素示踪剂、细胞内pH的荧光分析(NHE活性的测定)和电生理方法，将确定甲状旁腺素效应是否也可以在天然(未培养的)鸡肾中检测到，以及是否在大鼠近端小管培养中发现。还将研究甲状旁腺素反应的潜在生理作用，包括对这一部分液体和尿酸盐分泌的影响。还将利用已知的CFTR蛋白阻滞剂和抗体，具体说明CFTR作为其他运输系统的调节器的潜在作用。这项研究将为甲状旁腺激素和CFTR在肾脏中的作用提供重要的新信息，并将对比较、肾脏和上皮运输生理学领域产生兴趣。

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.