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）是一种突然的肾功能丧失，是一种日益增加的全球健康负担，与高发病率和死亡率相关，迄今尚无靶向治疗方法。AKI的常见原因是肾灌注不足、败血症和肾毒性。它们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。在急性损伤中存活下来的小管细胞重新进入细胞周期并增殖再生上皮细胞，这对肾功能的恢复至关重要。然而，令人惊讶的是，小管细胞也可以变得不适应，并通过持续的纤维化信号传导促进慢性肾脏疾病的发展。目前，由于对潜在的分子机制了解不足，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全面了解AKI后肾脏修复的分子基础，并确定控制适应性和非适应性修复过程的关键调节因子。我们将利用单核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，对所有肾细胞的染色质可及性和相关基因表达进行表征。此外，我们将根据标记基因的表达特异性地分离其细胞核，从适应不良的小管细胞中获得相同的数据。总之，这些数据集将提供轻度和重度AKI后不同阶段肾元的综合调控图。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，以重建导致适应性不良和适应性小管细胞的单细胞轨迹。从这些轨迹中，我们可以识别出决定修复和不适应修复过程的关键调节因素。我们将采用其他方法，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子。最终，本研究的目标是实现对肾脏修复过程的详细了解，从而促进未来治疗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.