Renal Oxalate Secretion

描述(申请人提供)：高草酸尿是肾结石病因学的主要危险因素之一，在高草酸尿疾病中，如原发性高草酸尿，肾脏排泄途径不断受到挑战，以排泄草酸负荷。虽然长期以来人们都认为肾脏活跃地分泌草酸，并且已经确定了一些可能转运草酸阴离子的膜转运蛋白，但值得注意的是，这些研究没有出现分泌的载体转运机制。这项建议中描述的研究目的是扩大我们对草酸排泄增加的高草酸尿大鼠肾小管跨皮细胞草酸分泌的机制和调节的理解。通过植入大鼠体内的草酸盐渗透泵产生的高草酸尿症将被用来评估(使用差异基因表达技术)可能参与高草酸状态下沿肾单位草酸转运的运输系统的性质和上调程度。我们之前关于肠道草酸分泌的研究，以及关于肾单位氯离子分泌的新发现，使我们检验了这样一种假设，即机械上类似的cAMP依赖的途径可以沿着肾小管介导草酸分泌。这一假说将通过测量cAMP刺激的、放射性同位素标记的草酸盐通量通过源自近端小管和内髓集合管的汇合单层细胞系来评估。此外，依赖cAMP的分泌机制的一个特定方面(即，草酸的顶端膜传导途径的存在)将使用来自高草酸动物肾皮质的膜小泡来评估。最后，利用CFTR基因产物在非洲爪哇卵母细胞中的异源表达，验证了草酸通过氯离子通道的假说。在标称没有其他草酸转运途径的情况下，可以使用标记草酸的外流结合电生理测量在对照和表达CFTR的卵母细胞中检测通过CFTR的草酸通量。高草酸/渗透泵的动物模型、新的转运角度和新的实验方法将为肾脏草酸分泌提供具体的答案，并将为未来肾脏草酸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.