Elucidating the cellular mechanisms of a pro-regenerative drug therapy for acute kidney injury

摘要 急性肾损伤(AKI)是一个主要的健康问题，每年在美国有150万患者受到影响。Aki是一位 是慢性肾脏疾病的前驱症状，通常进展为肾衰竭。目前还没有有效的 AKI的治疗和治疗是迫切需要的。肾脏具有先天的再生能力。 在损伤后，增加了为AKI开发促再生疗法的可能性。肾 再生被认为是由经历去分化的存活的肾小管上皮细胞(RTECs)发生的 达到类祖细胞状态，随后进行增殖和再分化。此外，M1/‘KILL’(经典/专业- 炎性)和M2/‘修复’(替代/促进修复)型巨噬细胞在促进肾小管上皮细胞方面扮演相反的角色 而这些亚型的平衡对于健康的修复至关重要。对于贫穷的人 了解原因，再生过程可能会停滞，RTECs滞留在细胞的G2/M期 循环并产生促纤维化的细胞因子。因此，寻找促进肾小管修复和减少肾小管损伤的药物 纤维化将对临床产生巨大的影响。为此，我们培育了斑马鱼，并诱导 多能干细胞(IPSC)来源的人肾有机类物质作为研究AKI的模型，我们已经确定 新型促再生化合物。我们发现了一类新的组蛋白脱乙酰酶(HDAC)抑制剂 (HDI)，苯硫代丁酸酯(PTBA)，它能特异性地抑制已知的维甲酸调节剂HDAC8 酸(RA)信号。我们已经证明PTBA通过促进增殖来促进肾脏再生。 减少斑马鱼和小鼠RTECs的G2/M期停滞，减少小鼠AKI后的纤维化。我们 发现这些促再生活性依赖于AKI过程中的RA信号。研究表明 RA作用于RTEC和巨噬细胞，改变M1/M2转换，减少RTEC G2/M期滞留。 基于这些数据，我们假设PTBA通过抑制HDAC8促进肾脏再生，从而 增强RA信号，进而诱导RTEC增殖，改变M1/M2巨噬细胞转换。 为了验证这些假说，我们提出了以下目标：目标1.阐明维甲酸和维甲酸的作用 HDAC8对PtBA疗效的驱动作用。重点是确定HDAC8为PtBA的体内靶点，探索 PtBA对RA途径的影响，并进行无偏的RNA-Seq筛选以确定受影响的RTEC基因 经PtBA治疗。目的2.研究PtBA与斑马鱼免疫应答的关系。 我们的数据表明，RA信号驱动M2/“修复”极化，我们倾向于PtBA增强的模型 巨噬细胞中的RA信号，从而促进M2/“修复”介导的肾脏修复。目标3.建立人 肾脏有机化合物作为AKI的模型和临床前药物试验。我们已经开发出一种简单的生物反应器- 用于从IPSCs批量生成用于模拟AKI的人肾器官的方法。

Abstract Acute kidney injury (AKI) is a major health problem, affecting >1.5 million patients in the US each year. AKI is a precursor to chronic kidney disease and often progresses to kidney failure. Currently there are no effective treatments for AKI and therapies are urgently needed. The kidney has an inherent ability to regenerate following injury, raising the possibility that pro-regenerative therapies for AKI can be developed. Renal regeneration is thought to occur by surviving renal tubular epithelial cells (RTECs) undergoing dedifferentiation to a progenitor-like state followed by proliferation and re-differentiation. In addition, M1/'kill' (classic/pro- inflammatory) and M2/'heal' (alternative/pro-repair) type macrophages play opposing roles in promoting tubular regeneration, respectively, and the balance of these subtypes is critical for healthy repair. For poorly understood reasons, the regenerative process can stall, with RTECs arresting in the G2/M phase of the cell cycle and producing pro-fibrotic cytokines. Therefore, finding drugs that promote tubular repair and reduce fibrosis will have a tremendous clinical impact. Towards this goal, we have developed zebrafish and induced pluripotent stem cell (iPSC)-derived human kidney organoids as models to study AKI and we have identified novel pro-regenerative compounds. We discovered a new class of histone deacetylase (HDAC) inhibitors (HDIs), the phenylthiobutanoates (PTBAs), which specifically inhibit HDAC8, a known modulator of retinoic acid (RA) signaling. We have demonstrated that PTBAs promote renal regeneration by increasing proliferation and decreasing G2/M arrest of zebrafish and mouse RTECs, and reduce post-AKI fibrosis in mice. We discovered that these pro-regenerative activities are dependent on RA signaling during AKI. Studies suggest that RA acts on RTECs as well as macrophages to alter M1/M2 switching and reduce RTEC G2/M arrest. Based on these data, we hypothesize that PTBAs promote renal regeneration by inhibiting HDAC8, thereby enhancing RA signaling, which in turn, induces RTEC proliferation and alters M1/M2 macrophage switching. To test these hypotheses, we propose the following Aims: Aim 1. Elucidate the role of Retinoic Acid and HDAC8 in driving PTBA efficacy. The focus is to confirm HDAC8 as the in vivo target of PTBA, explore the effect of PTBA on the RA pathway, and perform an unbiased RNA-Seq screen to identify RTEC genes affected by PTBA treatment. Aim 2. Examine the relationship between PTBA and the immune response in zebrafish. Our data suggests that RA signaling drives M2/'heal' polarization, we favor a model in which PTBA enhances RA signaling in macrophages, thereby promoting M2/'heal'-mediated renal repair. Aim 3. Establish human kidney organoids as a model of AKI and pre-clinical drug testing. We have developed a simple bioreactor- based method for generating, in bulk, human kidney organoids from iPSCs for modeling AKI.