Mechanisms of active renal repair: Single-cell trajectories of chromatin accessibility and gene expression changes in renal tubule cells

急性肾损伤(AKI)是一种肾脏功能的突然丧失，是一种与高发病率和高死亡率相关的日益严重的全球健康负担，迄今尚无针对性的治疗方法。AKI的常见原因是肾血流灌注不足、脓毒症和肾毒性。它们会导致急性肾小管损伤和肾功能丧失，同时触发肾脏的内在修复过程。在急性损伤中幸存下来的肾小管细胞重新进入细胞周期并增殖，以再生对恢复肾功能至关重要的上皮细胞。然而，令人惊讶的是，肾小管细胞也可以变得不适应，并通过持续的促纤维化信号而促进慢性肾脏疾病的发展。目前，由于对潜在分子机制的了解不足，阻止这种适应不良过程的治疗方法的发展受到阻碍。因此，该项目的目的是在单细胞分辨率下对急性肾损伤后肾脏修复的分子基础有一个全面的了解，并确定控制适应性和适应性不良修复过程的关键调节因素。我们将使用单核ATAC和单核RNA测序的新组合，在转基因小鼠轻度和重度AKI后的不同时间点，表征所有肾单位细胞染色质的可及性和相关基因的表达。此外，我们还将从适应不良的小管细胞中获得相同的数据，具体方法是根据标记基因的表达分离它们的核。总而言之，这些数据集将提供轻度和重度AKI后不同阶段肾单位的全面调控图。我们将使用最先进的生物信息学工具进一步分析这些数据，以重建导致适应不良和适应性肾小管细胞的单细胞轨迹。从这些轨迹中，我们可以确定决定修复和适应不良修复过程的关键调控因素。我们将使用其他方法，如RNAScope原位杂交和肾脏免疫染色来更详细地研究这些调节因素。最终，这项研究的目标是实现对肾脏修复过程的详细了解，以促进未来治疗AKI的靶向药物开发。

Acute kidney injury (AKI), an abrupt loss of kidney function, is an increasing global health burden associated with high morbidity and mortality, for which no targeted therapy exits to date. Common causes of AKI are renal hypoperfusion, sepsis and nephrotoxicity. They result in an acute tubular injury and loss of kidney function, while simultaneously triggering the intrinsic repair processes of the kidney. Tubule cells that survive the acute insult re-enter the cell cycle and proliferate to regenerate the epithelial cells that are essential for the recovery of kidney function. Yet, surprisingly, tubule cells can also become maladaptive and contribute to the development of chronic kidney disease by continuous profibrotic signaling. The development of therapeutics to prevent such maladaptive processes is currently hampered by an insufficient understanding of the underlying molecular mechanisms. Therefore, the aim of the proposed project is to obtain a comprehensive understanding of the molecular underpinnings of renal repair after AKI at a single-cell resolution and to identify key regulatory factors governing adaptive and maladaptive repair processes. We will characterize both chromatin accessibility and associated gene expression of all nephron cells at various time points after both mild and severe AKI in transgenic mice, using a novel combination of single-nucleus ATAC- and single-nucleus RNA-sequencing. In addition, we will acquire the same data from maladaptive tubule cells specifically by isolating their nuclei based on the expression of a marker gene. Together, these data sets will provide a comprehensive regulatory map of the nephron at various stages after mild and severe AKI. We will further analyze these data using state-of-the-art bioinformatic tools to reconstruct the single-cell trajectories that lead to both maladaptive and adaptive tubule cells. From these trajectories, we can identify the pivotal regulatory factors determining both reparative and maladaptive repair processes. We will employ additional methods like RNAScope in situ hybridization and immunostaining of the kidney to study these regulatory factors in even greater detail. Ultimately, the goal of this study is to achieve a detailed understanding of renal repair processes that could facilitate targeted drug development to treat AKI in the future.