Epigenetic Control of Kidney Fibrosis

摘要 发展新的和强大的再生疗法的前提是揭示其作用机制 控制干/祖细胞的增殖和分化。干细胞能够增殖 无限期(自我更新)和分化为一种或多种细胞类型(多能性)。祖先是 干细胞的早期后代，具有多潜能，但不能无限分裂。干/祖细胞 对动态平衡组织的维护和修复至关重要。然而，肾脏的身份、起源和作用 肾脏干细胞/祖细胞的再生仍然存在争议。活体血统追踪是一种强大的 在自然环境中发现干细胞/祖细胞的技术。用这项技术，几只小鼠的肾脏 干细胞/祖细胞标记已被确定，包括SIX2、Lgr5和Pax8。干细胞/祖细胞 表达这些标记可分化为各种类型的细胞，但不能分化为集合管细胞。因此， 集合管的干细胞/祖细胞仍然神秘，因为一个特定的血统追踪标记仍然存在。 不可用。集合管系统是肾脏的最后部分，影响身体的电解质、酸- 底座和流体平衡。它具有结构和功能上不同的主细胞(PC)、�-插入细胞(�- IC)和�-插层细胞(�-IC)。体外研究表明，�-IC是一种可能的干细胞，可产生�-IC。 IC和PC，而PC是终极分化的。然而，PI最近对集合管的体内研究- 特异组蛋白H3K79甲基转移酶DOT1L基因敲除小鼠(Dot1lAC)推翻了这一传统观点。 在没有DOT1L功能的情况下，表达PC标志物Aqp2的细胞同时产生�-和�-IC。 在这项提案中，国际和平研究所建议扩展和巩固这些新的发现。具体地说，国际和平研究所打算 发现从表达Aqp2的细胞中衍生出IC是自然发生的(即不需要DOT1L缺失)。这 将导致将Aqp2确定为缺失的集合管细胞前体标记(目标1)。《少年派》 还打算将DOT1L定义为集合管分化的关键新表观遗传调节因子(目标2)。 最后，PI建议发现DOT1L如何发挥其调节作用。在这方面，他将发掘HDAC2 作为DOT1L的新合作伙伴和负调控因子。DOT1L和HDAC2相互抑制其对手的功能 通过限制与DNA的联系。PI将直接检验HDAC2缺失是否拯救了假设 Dot1lAC表型(目标3)。所有必需的关键试剂，包括多个已发表和未发表的 PI的实验室已经为这个项目专门开发了双重和三重转基因小鼠模型。 细胞、分子、遗传学、肾生理、电生理、病理学和电子显微镜 将使用各种方法。这一建议确实具有很高的意义、影响力和新颖性，因为如果成功的话，它 将建立Aqp2作为新的肾脏集合管特异性祖细胞标记物，DOT1L是第一个 表观遗传在PC和IC分化中的作用，以及HDAC2作为DOT1L的新伙伴和调节因子。

Abstract A prerequisite to the development of novel and powerful regenerative therapeutics is to reveal the mechanisms controlling the proliferation and differentiation of stem/progenitor cells. Stem cells are capable of proliferating indefinitely (self-renewal) and differentiating into one or multiple cell types (pluripotentiality). Progenitors are early descendants of stem cells that have pluripotentiality, but cannot divide indefinitely. Stem/progenitor cells are critical for homeostatic tissue maintenance and repair. However, the identity, origin, and role in renal regeneration of kidney stem/progenitor cells remain controversial. In vivo lineage tracing is a powerful technique to discover stem/progenitor cells in their native context. With this technique, a few mouse kidney stem/progenitor cell markers have been identified, including Six2, Lgr5, and Pax8. The stem/progenitor cells expressing these markers differentiate into various cell types, but not the collecting duct cells. Therefore, the stem/progenitor cells of the collecting duct remain mysterious, because a specific lineage-tracing marker is still not available. The collecting duct system is the final part of the kidney to influence the body's electrolyte, acid- base, and fluid balance. It has structurally and functionally distinct principal cells (PC), �-intercalated cells (�- IC), and �-intercalated cells (�-IC). In vitro studies suggest that �-IC are putative stem cells and give rise to �- IC and PC, while PC are terminally differentiated. However, the PI's recent in vivo studies with collecting-duct- specific histone H3 K79 methyltransferase Dot1l knockout mice (Dot1lAC) overturned this traditional view. Without Dot1l function, the cells expressing Aqp2, a well established PC marker, give rise to both �-, and �-IC. In this proposal, the PI proposes to extend and solidify these novel findings. In particular, the PI intends to discover derivation of IC from Aqp2-expressing cells occurs naturally (i.e, without need of Dot1l deletion). This would lead to identification of Aqp2 as the missing progenitor marker of collecting duct cells (Aim 1). The PI also intends to define Dot1l as a critical novel epigenetic regulator of collecting duct differentiation (Aim 2). Finally, the PI proposes to discover how Dot1l plays its regulatory role. In this regard, he will unearth HDAC2 as a new partner and negative regulator of Dot1l. Dot1l and HDAC2 mutually inhibit their opponent's function by restricting association with DNA. The PI will directly test the hypothesis whether HDAC2 deletion rescues the Dot1lAC phenotype (Aim 3). All of the required key reagents including multiple published and unpublished double and triple transgenic mouse models have been exclusively developed in the PI's lab for this project. Cellular, molecular, genetic, reno-physiological, electro-physiological, pathological, and electron microscopic approaches will be used. This proposal truly has high significance, impact and novelty because if successful, it will establish Aqp2 as a novel progenitor cell marker specific for renal collecting duct, Dot1l as the first epigenetic player in PC and IC differentiation, and HDAC2 as a novel partner and regulator of Dot1l.