Epigenetic Control of Kidney Fibrosis

描述（由申请人提供）：肾纤维化是慢性肾病（CKD）的标志。尽管积极的管理，CKD往往进展为终末期肾病，这花费了美国超过400亿美元，每年死亡人数超过90，000。目前的主要治疗靶向肾素-血管紧张素-醛固酮（aldosterone，aldo）系统的药物，包括螺内酯往往延迟，但不能阻止进展。这也适用于所有其他药物，如内皮素1（ET 1）阻滞剂阿曲生坦。无效性和副作用，包括高钾血症和水肿，有必要确定新的治疗靶点，以开发更有效的治疗方法。从其主要作用部位连接小管/集合管（CNT/CD）调节aldo全局效应的因素可能证明是更好的靶点。然而，这种遗传和表观遗传因素仍然几乎未知，部分原因是临床研究的内在局限性。这些局限性包括缺乏肾脏活检， 验证疾病的状态，不可能在患者中进行遗传操作以建立因果关系，以及通过血液DNA突变分析来识别体细胞突变的不可行性，这在人类肾脏中以非典型的高比率发生。我们发表的和初步的数据表明，1）糖尿病肾病（DN）和CKD患者可能有组蛋白H3 K79甲基转移酶hDOT 1 L突变，并取消H3二甲基化（H3 m2 K79）在他们的肾活检; 2）Dot 1a（由Dot 1 l编码）抑制ET 1和其他aldo靶基因。Aldo通过多种机制缓解Dot 1a介导的抑制; 3）CNT/CD特异性消融Dot 1 lAC小鼠中的Dot 1 l导致H3 m2 K79的消除、ET 1的上调和整个肾脏的严重肾纤维化的发展。因此，在本建议中，我们将开发遗传标记来克服上述限制。为此，我们将使用来自DN和CKD患者的肾活检，我们的CNT/CD中携带完整或破坏的Dot 1 l和ET 1的小鼠模型，以及它们的CNT/CD原代细胞，结合尖端技术，包括激光捕获显微切割，下一代测序和体内谱系追踪。我们的具体目标是研究DN和CKD患者是否存在hDOT 1 L遗传缺陷（目的1），研究Dot 1 l缺失是否部分通过上调小鼠ET 1加速肾纤维化（目的2），以及研究Dot 1a和ET 1是否调节Aldo促纤维化作用的整体效应（目的3）。我们的研究可能将DOT 1 L鉴定为ET 1的新型阻遏物，从而鉴定为新的肾保护因子，确认DOT 1 L功能丧失，从而确认H3 m2 K79为CKD的表观遗传驱动因子，将螺内酯+阿曲生坦定义为CKD的新的有效组合疗法，并为新的基因检测奠定基础。如果Dot 1 l和ET 1与人类CKD有遗传联系，则可以利用它们来开发遗传筛查测试，以识别CKD高风险患者，并确定他们对各种Aldo和ET 1抑制剂的反应性。与ET 1一样，DOT 1 L也可以被认为是CKD潜在的新治疗靶点。

DESCRIPTION (provided by applicant): Kidney fibrosis is the hallmark of chronic kidney disease (CKD). Despite aggressive management, CKD often progresses to end-stage renal disease, which costs the US >$40 billion dollars and >90,000 deaths annually. The current main therapy targeting the renin-angiotensin-aldosterone (aldo) system with drugs including Spironolactone often delays, but does not stop the progression. This is also true for all other drugs such as endothelin 1 (ET1) blocker Atrasentan. The ineffectiveness and side effects including hyperkalemia and edema necessitate identification of novel therapeutic targets for the development of more effective treatments. Factors modulating the aldo global effect from its primary action site connecting tubule/collecting duct (CNT/CD) may prove better targets. However, such genetic and epigenetic factors remain virtually unknown, partially because of the intrinsic limitations of the clinical studies. These limitations include lack of kidney biopsies to verify the status of the disease, impossibility of genetic manipulation in patients to establish th causative relationship, and impracticability through mutational analyses with blood DNA to identify somatic mutations, which occur at atypical high rate in human kidney. Our published and preliminary data suggest that 1) Patients with diabetic nephropathy (DN) and CKD may have mutations in histone H3 K79 methyltransferase hDOT1L and abolished H3 dimethylation (H3m2K79) in their kidney biopsies; 2) Dot1a (encoded by Dot1l) represses ET1 and other aldo target genes. Aldo relieves Dot1a-mediated repression by multiple mechanisms; 3) CNT/CD-specific ablation of Dot1l in Dot1lAC mice causes abolition of H3m2K79, upregulation of ET1, and development of severe kidney fibrosis throughout the whole kidney. Accordingly, in this proposal, we will develop genetic markers to overcome the above limitations. To this end, we will use kidney biopsies from patients with DN and CKD, our mouse models bearing intact or disrupted Dot1l and ET1 in the CNT/CD, and their CNT/CD primary cells in combination of cutting-edge technologies including laser capture microdissection, next generation sequencing, and in vivo lineage tracing. Our specific aims are to study if DN and CKD patients have genetic defects in hDOT1L (Aim 1), study if Dot1l deletion accelerates kidney fibrosis in part by upregulating ET1 in mice (Aim 2), and study if Dot1a and ET1 modulate the global effect of aldo profibrotic action (Aim 3). Our studies may identify DOT1L as a novel repressor of ET1 and thus a new renoprotective factor, confirm loss of DOT1L function and thus H3m2K79 as an epigenetic driver of CKD, define Spironolactone + Atrasentan as a new effective combinational therapy of CKD, and lay the foundation of new genetic tests. If Dot1l and ET1 are genetically linked to CKD in humans, they may be exploited to develop genetic screening tests to identify patients at high risk of CKD and to determine their responsiveness to various aldo and ET1 inhibitors. Like ET1, DOT1L can also be considered as a potential new therapeutic target of CKD.