Epigenetic Control of Kidney Fibrosis

描述(申请人提供)：肾脏纤维化是慢性肾脏疾病(CKD)的标志。尽管采取了积极的治疗措施，慢性肾脏病往往进展为终末期肾病，每年给美国造成400亿美元的损失和9万人死亡。目前针对肾素-血管紧张素-醛固酮(ALDO)系统的主要治疗方法包括螺内酯，通常会延迟，但并不能阻止进展。所有其他药物也是如此，如内皮素1(ET1)阻滞剂阿特拉森坦。无效和副作用，包括高钾血症和水肿，需要确定新的治疗靶点，以开发更有效的治疗方法。从连接小管/集合管的主要作用部位(CNT/CD)调节Aldo整体效应的因素可能被证明是更好的靶点。然而，这种遗传和表观遗传因素实际上仍然未知，部分原因是临床研究的内在局限性。这些限制包括缺乏肾脏活检以 通过对血液DNA进行突变分析，以确定在人类肾脏中以非典型高发率发生的体细胞突变，来验证疾病的状态、患者无法通过基因操作来建立因果关系以及不切实际。我们已发表的和初步的数据表明，1)糖尿病肾病和慢性肾脏病患者的肾脏活检组织中可能存在组蛋白H3K79甲基转移酶hDOT1L突变和取消H3m2K79甲基化；2)Dot1a(由DOT1L编码)抑制ET1和其他Aldo靶基因。ALDO通过多种机制缓解Dot1a介导的抑制；3)在Dot1lAC小鼠体内，CNT/CD特异性的DOT1L消融导致H3m2K79消失，ET1上调，并在整个肾脏发展为严重的肾脏纤维化。因此，在这项建议中，我们将开发遗传标记来克服上述限制。为此，我们将使用来自糖尿病肾病和慢性肾脏病患者的肾脏活检组织，我们的小鼠模型在CNT/CD中携带完整或破坏的DOT1L和ET1，以及他们的CNT/CD原代细胞，结合包括激光捕获显微解剖、下一代测序和体内谱系追踪在内的尖端技术。我们的具体目标是研究糖尿病肾病和慢性肾脏病患者是否存在hDOT1L基因缺陷(目标1)，研究DOT1L缺失是否部分通过上调ET1而加速小鼠肾脏纤维化(目标2)，以及研究Dot1a和ET1是否调节Aldo促纤维化作用的整体效应(目标3)。我们的研究可能确认DOT1L是一种新的ET1抑制因子，从而可能是一种新的肾脏保护因子，证实DOT1L功能的丧失从而证实H3m2K79是CKD的表观遗传驱动因素，确定螺内酯+阿特拉森坦为治疗CKD的新的有效联合疗法，并为新的基因测试奠定基础。如果DOT1L和ET1在基因上与人类CKD相关，则可以利用它们来开发基因筛查测试，以识别CKD的高危患者，并确定他们对各种ALDO和ET1抑制剂的反应性。与ET1一样，DOT1L也可被认为是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.