Endogenous Oxalate Synthesis

内源性草酸合成在特发性草酸钙结石和原发性高草酸尿症中起重要作用。尽管经过了几十年的研究，人们对这种合成背后的途径知之甚少。在上一个资助周期中，我们确定了氨基酸和糖代谢，以前被认为是主要的贡献，只产生有限的草酸盐。在这个应用中，我们建议研究乙二醛是否是草酸盐合成的重要来源。我们的初步研究表明乙二醛可以转化为草酸盐。乙二醛主要通过与谷胱甘肽和乙二醛酶系统的相互作用转化为乙醇酸。这些途径的活性取决于谷胱甘肽和NADPH。任何减少这些成分的氧化应激都可能加速草酸盐的合成。已经制定了三个具体目标。特异性目的1：将在培养的肝癌（HepG2）细胞中使用纯化的重组酶和siRNA敲低来评估可能催化乙二醛转化为草酸盐的酶。与乙二醛生成和乙二醛酶系统的相互关系将用谷胱甘肽耗竭来检验。葡萄糖和亚油酸将被评估为人类红细胞和HepG2细胞生成乙二醛和草酸的潜在来源。13c同位素的使用和13c标记的草酸盐、乙醇酸盐和乙二醛的定量，离子色谱耦合到质量检测（IC/MS）或液相色谱耦合到质量检测（LC/MS），将允许跟踪碳的通量。特异性目标2将评估修改乙二醛酶系统是否会影响草酸盐合成。据推测，减少这一体系将增加草酸盐的合成。人红细胞、Glyoxalase-1 （GLO-1）缺陷小鼠红细胞和野生型小鼠红细胞会暴露于乙二醛、谷胱甘肽耗竭和氧化应激。预计这些操作将增加草酸盐合成，特别是在GLO-1缺陷细胞中。HepG2细胞、GLO-1缺陷和野生型小鼠也将受到氧化应激、谷胱甘肽耗竭和抗氧化剂的影响，以评估乙二醛酶系统的影响。在特定目标3中，将评估2型糖尿病与内源性草酸盐合成增加相关的假设。正常成人、伴有或不伴有2型糖尿病的草酸钙结石患者以及无肾结石史的糖尿病患者将被列为特征。对内源性草酸合成的贡献将通过将参与者置于严格控制的低草酸饮食中来估计。尿草酸和乙醇酸排泄与氧化应激和乙二醛生成之间的关系将被确定。Zucker大鼠是一种胰岛素抵抗动物，最近发现草酸排泄量增加，将用于确定抗氧化治疗是否可以减少乙二醛的产生以及尿中乙醇酸和草酸。所提出的实验应该对草酸盐如何合成产生新的见解，并应该揭示抗氧化治疗是否是防止草酸钙结石形成的潜在治疗方法。

DESCRIPTION: Endogenous oxalate synthesis plays an important role in idiopathic calcium oxalate stone disease and primary hyperoxaluria. The pathways that underlie this synthesis are poorly understood despite decades of research. In the last funding cycle we established that amino acid and sugar metabolism, previously believed to make major contributions, produce only a limited amount of oxalate. In this application, we propose investigating whether glyoxal is a significant source of oxalate synthesis. Our preliminary studies have shown that glyoxal is converted to oxalate. Glyoxal is primarily converted to glycolate by its interaction with glutathione and the glyoxalase system. Activity of these pathways is contingent on glutathione and NADPH. Any oxidant stress reducing these components could potentially accelerate oxalate synthesis. Three specific aims have been developed. Specific Aim 1: The enzyme(s) potentially catalyzing the conversion of glyoxal to oxalate will be assessed using purified recombinant enzymes and siRNA knockdown in cultured hepatoma (HepG2) cells. The inter-relationship with glyoxal generation and the glyoxalase system will be examined using glutathione depletion. Glucose and linoleate will be assessed as potential sources of glyoxal and oxalate generation using human red cells and HepG2 cells. The use of 13C-isotopes and the quantification of 13C-labelled oxalate, glycolate and glyoxal with ion chromatography coupled to mass detection (IC/MS) or liquid chromatography coupled to mass detection (LC/MS), will allow the flux of carbon to be tracked. Specific Aim 2 will assess if modifying the glyoxalase system impacts oxalate synthesis. It is hypothesized that reducing this system will increase oxalate synthesis. Human erythrocytes and those from Glyoxalase-1 (GLO-1) deficient and wild type mice will be exposed to glyoxal, glutathione depletion and oxidative stress. It is anticipated that these maneuvers will increase oxalate synthesis especially in GLO-1 deficient cells. HepG2 cells, GLO-1 deficient and wild type mice will also be subjected to oxidative stress, glutathione depletion and antioxidants to assess the impact of the glyoxalase system. In Specific Aim 3 the hypothesis that type 2 diabetes is associated with increased endogenous oxalate synthesis will be assessed. Normal adults, those with calcium oxalate stones with and without type 2 diabetes, and diabetics without a history of kidney stones will be characterized. The contribution to endogenous oxalate synthesis will be estimated by placing the participants on tightly controlled low oxalate diets. The relationships between urinary oxalate and glycolate excretion to oxidative stress and glyoxal production will be determined. The Zucker rat, an animal with insulin resistance and recently identified increased oxalate excretion, will be utilized to determine if antioxidant therapy can decrease glyoxal production and urinary glycolate and oxalate. The proposed experiments should produce novel insights into how oxalate is synthesized and should reveal whether antioxidant therapy is a potential therapy to prevent calcium oxalate stone formation. PUBLIC HEALTH RELEVANCE: This project examines how oxalate, a major component of the most common type of kidney stone (calcium oxalate), is made in the body. Understanding how this occurs will help implement treatments, to decrease the amount of oxalate made, and thereby decrease calcium oxalate kidney stone formation.