Mitochondrial Metabolism in Primary Hyperoxaluria

描述（由申请人提供）：原发性高尿酸血症（PH）是一种罕见的遗传性疾病，其特征是尿草酸盐排泄增加，形成草酸钙肾结石，严重时发生肾衰竭。在最极端的情况下，有些人在婴儿时期患上肾钙质沉着症和肾衰竭，生存前景很差。我们的研究表明，羟脯氨酸代谢作出了重大贡献，增加草酸合成发生在PH。这种代谢发生在前列腺素，是异常的，在最近确定的形式的疾病，3型，其中的酶，4-羟基-2-酮戊二酸醛缩酶（HOGA），降解途径的一个组成部分的活性，是缺乏的。另一种线粒体酶乙醛酸还原酶的缺乏与2型疾病相关。在1型疾病中，当羟脯氨酸代谢在线粒体中产生的乙醇酸在过氧化物酶体中氧化为乙醛酸并由于AGT的缺乏而在细胞质中还原回乙醇酸时，在肝脏中发生乙醇酸-乙醛酸循环。我们推测，过氧化氢产生的这种循环有助于线粒体功能障碍，由于增加产生的活性氧。我们进一步假设，在这些类型的PH代谢的改变可能会导致改变浓度的草酸盐，乙醛酸和乙醇酸在线粒体和细胞质。离子水平的变化，特别是草酸盐和钙，可以进一步改变线粒体的特性。在这个提议中，我们将使用转基因小鼠来确定与PH相关的酶组成的变化如何改变肝细胞和肾近端小管细胞（RPTC）中的线粒体特性。第一个具体目标将确定Hoga 1基因敲除小鼠的表型，并检查HOGA缺失时底物HOG如何分裂。我们假设，一种替代的醛缩酶能够分裂HOG时，其浓度增加到足够的。第二个具体目标将使用XF分析仪检查正常和转基因小鼠的完整肝细胞和RTPC中发生的线粒体特性和代谢变化。还将在小鼠模型的肝脏和肾脏组织以及接受移植的PH患者的肝脏组织中评估线粒体质量。将研究线粒体特异性药物（如MitoQ）是否可以抵消任何不良变化。第三个具体目标将确定从这些小鼠中分离的线粒体在代谢羟脯氨酸和乙醛酸时发生的变化。这些实验将阐明与PH中发生的草酸盐合成增加相关的代谢，突出线粒体在疾病过程中发挥的作用，并说明肝脏和肾脏线粒体之间的重要差异。这项研究应该导致新的方法来减少草酸盐的过度合成和修改PH中的线粒体功能障碍。

DESCRIPTION (provided by applicant): Primary Hyperoxaluria (PH) is a rare, genetic disorder that is characterized by an increased urinary oxalate excretion, the formation of calcium oxalate kidney stones, and in severe cases renal failure. In the most extreme cases, some develop nephrocalcinosis and renal failure as infants with a poor survival outlook. Our research suggests that hydroxyproline metabolism makes a major contribution to the increased oxalate synthesis that occurs in PH. This metabolism occurs in the mitochondrion and is aberrant in a recently identified form of the disease, Type 3, where the activity of the enzyme, 4-hydroxy-2-oxoglutarate aldolase (HOGA), a component of the degradation pathway, is deficient. A deficiency in another mitochondrial enzyme, glyoxylate reductase, is associated with Type 2 disease. In Type 1 disease, glycolate-glyoxylate cycling occurs in the liver when glycolate produced in mitochondria from hydroxyproline metabolism is oxidized in peroxisomes to glyoxylate and reduced back to glycolate in the cytoplasm because of the absence of AGT. We hypothesize that the hydrogen peroxide produced with this cycling contributes to mitochondrial dysfunction due to an increased generation of reactive oxygen species. We further hypothesize that the altered metabolism in these types of PH may result in an altered concentration of oxalate, glyoxylate and glycolate in mitochondria and the cytosol. Changes in ion levels, particularly oxalate and calcium, could further modify mitochondrial properties. In this proposal, we will use genetically modified mice to determine how the changes in enzyme composition associated with PH alter mitochondrial properties in hepatocytes and renal proximal tubule cells (RPTC). The first specific aim will determine the phenotype of Hoga1 knock-out mice and examine how the substrate, HOG, is split when HOGA is absent. We hypothesize that an alternative aldolase is able to split HOG when its concentration increases sufficiently. The second specific aim will examine mitochondrial properties and metabolic changes that occur in intact hepatocytes and RTPC from normal and genetically modified mice using an XF-analyzer. Mitochondrial quality will also be assessed in liver and kidney tissue of the mouse models and in liver tissue from PH patients receiving a transplant. Whether a mitochondrial specific drug such as MitoQ can offset any adverse changes will be investigated. The third specific aim will identify changes that occur in mitochondria isolated from these mice when they metabolize hydroxyproline and glyoxylate. These experiments will illuminate the metabolism associated with the increased oxalate synthesis that occurs in PH, highlight the role played by mitochondria in the disease process, and illustrate important differences between liver and kidney mitochondria. This research should lead to novel approaches to decrease excessive oxalate synthesis and modify mitochondrial dysfunction in PH.