Role of Fumarate and Nrf2 response in the pathogenesis of Autosomal Dominant Polycystic Kidney Disease

常染色体显性遗传性多囊肾病(ADPKD)是一种多系统破坏性疾病， 以双侧多发肾囊肿、肾脏并发症和进展为终末期肾脏疾病为特征。 异常的上皮细胞增殖是PKD的一个显著特征，是囊肿形成和增大的基础。 因此，识别已知的促进细胞增殖的细胞机制中的失调代表 这是进行治疗干预的重要机会。核因子(红系衍生的2)样2(NRF2)是一种 一种转录因子，通过调节表达来调节细胞对应激和生存的保护 抗氧化性蛋白质。去甲二氢愈创木酸(NDGA)等酚类化合物干扰NRF2 泛素化，有利于其转录激活。已知NDGA可导致非PKD大鼠的囊变。 富马酸是柠檬酸循环(TCA)和尿素循环的中间产物，也可以调节Nrf2泛素化。 富马酸水合酶(FH)中富马酸的增加与膀胱癌和肾癌有关 缺乏症。在初步研究中，我们发现PKD缺陷细胞中富马酸水平增加。 此外，我们对早期PKD大鼠(PCK)的研究发现，尿液和肾脏组织增加 富马酸与野生型(WT)大鼠比较。此外，尿液和肾组织中的富马酸水平在 Pkd1RC/RC小鼠与对照组相比，并且与疾病严重程度(囊性指数和纤维化)呈正相关。 值得注意的是，与对照组相比，Pkd1RC/RC小鼠肾脏核Nrf2的表达更高。最后，尿液 与正常志愿者相比，年轻ADPKD患者的富马酸水平升高。然而， 在PKD的背景下，富马酸水平的增加是否有助于Nrf2反应的失调， 归根结底，促进囊变的作用从未被探索过。这一提议背后的假设是 ADPKD导致富马酸水平升高，这种增加导致Nrf2信号上调 导致细胞增殖并促进囊变。因此，确定增长的来源 富马酸将发现ADPKD中改变的代谢途径，有助于识别新的疾病 生物标志物和开发有针对性的治疗干预措施。为了检验这一假设，我们将利用 基因工程啮齿动物模型，我们先前开发的ADPKD的成像分类，以及 ART光谱技术以及独特的稳定同位素代谢组学和光谱成像 技巧。将追求三个具体目标：具体目标1将检验假设，即在 ADPKD的同源模型伴随着代谢组学的改变和富马酸水平的升高 导致Nrf2信号的上调和囊变。《特定目标2》将检验富马酸 ADPKD患者的血清水平和Nrf2反应增加，且与疾病严重程度相关。特定的 目标3将检验这一假设，即患者和啮齿动物肾脏和尿液中富马酸水平的增加 ADPKD模型是由于TCA循环、谷氨酰胺代谢或尿素循环的失调和 富马酸水合酶活性。成功的研究将具有重要的临床意义 了解该病的病理生理学，识别新的早期生物标志物，并强调 其他代谢途径，可作为治疗干预的目标疾病，没有特定的 提供治疗。

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multisystem devastating disease, characterized by multiple bilateral renal cysts, renal complications, and progression to end-stage renal disease. Abnormal epithelial cell proliferation, a distinctive feature in PKD, underlies cyst formation and enlargement. Therefore, identifying dysregulations in the cellular mechanisms known to promote cell proliferation represents a major opportunity for therapeutic interventions. The Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that regulates cellular protection against stress and survival by modulating the expression of antioxidant proteins. Phenolic compounds like nordihydroguaiaretic acid (NDGA) interfere with Nrf2 ubiquitination, favoring its transcriptional activation. NDGA is known to cause cystogenesis in non-PKD rats. Fumarate is a citric acid cycle (TCA) and urea cycle intermediate that can also modulate Nrf2 ubiquitination. Increased fumarate has been associated with cystogenesis and renal cell cancer in fumarate hydratase (FH) deficiency. In preliminary studies we have discovered an increase in fumarate levels in PKD deficient cells. What is more, our studies in early stage PKD rats (PCK) discovered increased urinary and renal tissue fumarate compared to wild-type (WT) rats. In addition, urinary and renal tissue fumarate levels were higher in Pkd1RC/RC mice compared to controls, and positively correlated with disease severity (cystic index and fibrosis). Notably, renal expression of nuclear Nrf2 was higher in Pkd1RC/RC mice compared to controls. Finally, urine from young patients with ADPKD had increased levels of fumarate compared to normal volunteers. However, whether increased fumarate levels in the context of PKD contribute to a dysregulation in the Nrf2 response, ultimately promoting cystogenesis has never been explored. The hypothesis underlying this proposal is that ADPKD results in increased levels of fumarate and that this increase results in upregulation of Nrf2 signaling leading to cellular proliferation and contributing to cystogenesis. Hence, determining the origin of the increase in fumarate would uncover metabolic pathways altered in ADPKD that could help identifying novel disease biomarkers and developing targeted therapeutic interventions. To test this hypothesis we will take advantage of genetically engineered rodent models, our previously developed imaging classification of ADPKD, and state of the art spectroscopic techniques as well as unique stable isotope metabolomics and spectroscopic imaging techniques. Three specific aims will be pursued: Specific Aim 1 will test the hypothesis that cystogenesis in orthologous models of ADPKD is accompanied by altered metabolomics and increased levels of fumarate that leads to up regulation of Nrf2 signaling and cystogenesis. Specific Aim 2 will test the hypothesis that fumarate levels and Nrf2 response are increased in patients with ADPKD and correlate with disease severity. Specific Aim 3 will test the hypothesis that the increased levels of fumarate in kidneys and urines of patients and rodent models of ADPKD result from dysregulation of TCA cycle, glutamine metabolism, or the urea cycle and fumarate hydratase activity. Successful studies will have important clinical implications by advancing understanding of the pathophysiology of the disease, identifying novel early biomarkers, and highlighting additional metabolic pathways that could be targeted for therapeutic intervention in a disease with no specific treatment available.