Renal Oxalate Secretion

描述（由申请人提供）：高草酸尿是肾结石病因学中的主要危险因素之一，在原发性高草酸尿等高草酸尿疾病中，肾脏排泄途径不断受到草酸盐负荷排泄的挑战。虽然长期以来人们都认为肾脏会主动分泌草酸，并且已经确定了许多可能运输草酸阴离子的膜转运蛋白，但值得注意的是，这些研究中没有出现分泌的载体转运机制。本提案中描述的研究目的旨在扩展我们对高血氧大鼠肾小管经上皮草酸分泌的机制和调控的理解，其中草酸排泄量升高。植入大鼠体内的草酸盐负载渗透泵产生的高草酸尿将被用于评估（使用差异基因表达技术）运输系统的性质和程度，这些运输系统可能参与高草酸血症状态下沿肾元的运输。我们之前关于肠道草酸分泌的研究，以及关于肾单位氯化物分泌的新发现，使我们验证了机制相似的camp依赖通路可以介导沿着肾小管的草酸分泌的假设。这一假设将通过测量来自近端小管和内髓集管的细胞系的汇合单层的放射性同位素标记草酸通量来评估。此外，将利用高血氧动物肾皮质的膜囊来评估草酸盐依赖性分泌机制的一个特定方面（即草酸盐的顶膜传导途径的存在）。最后，通过CFTR基因产物在非洲爪蟾卵母细胞中的异源表达，明确验证草酸盐渗透氯离子通道的假设。在没有其他草酸转运途径的情况下，可以使用标记草酸的外排以及对照和表达CFTR的卵母细胞的电生理测量来检测草酸通过CFTR的通量。本课题所建立的高血氧/渗透泵动物模型、新的转运视角和新的实验方法将为肾脏草酸盐分泌提供具体的答案，并为未来肾脏草酸盐的研究提供基础和方向。

DESCRIPTION (provided by applicant): Hyperoxaluria is one of the principal risk factors in the etiology of renal stone disease and in hyperoxaluric diseases like Primary Hyperoxaluria the renal excretory pathways are continuously challenged to excrete the oxalate load. While it has long been agreed that the kidney actively secretes oxalate and a number of membrane transport proteins have been identified that may transport the oxalate anion, it is remarkable that no vectorial transport mechanism for secretion has emerged from these studies. The research aims described in this proposal are directed at extending our understanding of the mechanisms and regulation of transepithelial oxalate secretion by the renal tubule in hyperoxaluric rats where oxalate excretion is elevated. Hyperoxaluria generated by oxalate-loaded osmotic pumps implanted in rats will be used to assess (using differential gene expression techniques) the nature and degree of up-regulation of transport systems that may participate in oxalate transport along the nephron in the hyperoxaluric state. Our previous studies concerning intestinal oxalate secretion, together with new findings regarding chloride secretion in the nephron, have led us to test the hypothesis that mechanistically similar, cAMP-dependent pathways can mediate oxalate secretion along the renal tubule. This hypothesis will be evaluated by measuring camp stimulated, radioisotope-labeled oxalate fluxes across confluent monolayers of cell lines derived from the proximal tubule and the inner medullary collecting duct. Additionally, a specific aspect of the cAMPdependent secretory mechanism (i.e., presence of an apical membrane conductive pathway for oxalate) will be assessed using membrane vesicles derived from the renal cortex of the hyperoxaluric animals. Finally, the hypothesis that oxalate permeates chloride channels will be explicitly tested by using the heterologous expression of CFTR gene product in Xenopus laevis oocytes. Oxalate flux through CFTR can be examined in the nominal absence of other oxalate transport pathways using efflux of labeled oxalate together with electrophysiological measurements in control and CFTR expressing oocytes. The hyperoxaluric/osmotic pump animal model, the novel transport perspectives, and the new experimental approaches that will be developed and explored in this proposal will provide specific answers regarding renal oxalate secretion and they will offer a foundation and a direction for future studies of renal oxalate.