Mechanisms of developmental gene activation in the regeneration and repair of acute kidney injury

尽管急性肾损伤（acute kidney injury，阿基）是住院患者中最常见的疾病之一，但人类肾脏在损伤后的再生机制尚未完全了解。研究表明，在肾损伤后，发育基因PAX 2重新激活。PAX 2在健康条件下不能在成熟的肾小管和肾小球中检测到，已知AKI通过组蛋白甲基化和染色质修饰在表观遗传学特化中起关键作用。如果这些机制在AKI后重新激活，还不能回答。肾脏的发育和修复是否是AKI的关键。我们的假设是，发育基因的再激活可能是一个独特的细胞事件，它可以区分慢性肾脏疾病中的纤维化后果（如疤痕形成）我们假设损伤前肾小管细胞中存在的表观遗传修饰决定了细胞的分化方向，由于小鼠中关于阿基的研究在获得人类结论方面具有严重的局限性，我们将利用人类多能干细胞（hPSC）衍生的类器官，他们表现出与体内等同物接近的生理功能，并且可以重现与空间和时间组织相关的几个生物过程，如我们的模型中所示，该模型在转录上与妊娠中期相匹配。妊娠肾。在这个项目中，我们将阐明组蛋白标记和PAX 2结合位点在从阿基的不同阶段的患者中分离的TEC中的全基因组占有率。这些发现在更大的回顾性患者样本队列中得到验证，以全面鉴定阿基时受Pax 2调节的前5个候选者。此外，在鉴定了前PAX 2结合位点工程化的等基因CRISPR/Cas9敲除系和多西环素诱导型启动子的等基因系之后，在患者TEC和人多能干细胞系中PAX 2和PAX 2转录靶的增强子转录抑制和激活将生成。最终，我们将开发一种肾细胞毒性的生理学体内模型，该模型来源于所产生的命名的CRISPR/Cas9工程系，以评估Pax 2和PAX 2的假定作用。人类阿基中最重要的转录靶点。该项目将回答肾脏修复和修复领域的紧迫问题，并为PAX 2在阿基病理和进展中的作用提供新的见解。我们将辨别，阿基期间的表观遗传修饰和再生反应是Pax 2表观遗传变化的原因或结果。最终，我们将能够提供一种工具来消除或重写转化的肾小管细胞中的表观遗传修饰，以便能够将它们重定向到再生类型。

Despite acute kidney injury (AKI) being one of the most prevalent disorders in hospitalized patients, the regenerative mechanisms of the human kidney upon insult are not fully understood.Studies have revealed the reactivation of developmental genes as PAX2 upon kidney injury.PAX2,which cannot be detected in mature tubuli and glomeruli under healthy conditions, is known to have a pivotal role in the epigenetic specification through histone methylation and chromatin modification of the definitive kidney.If these mechanisms are reactivated upon AKI could not be answered yet.Whether development and repair in the kidney are the same processes or the similar expression of genes under different circumstances are misread remains to be elucidated.Our hypothesis is that developmental gene reactivation could be a unique cellular event that distinguishes a fibrogenic consequence in chronic kidney disease (as scar formation) from a reparative injury response after acute insult as in AKI.We assume that existing epigenetic modifications in tubular cells prior injury dictate the differentiating direction of transformed tubular epithelial cells (TEC) in AKI.As studies in mice on AKI have severe limitations in getting conclusions for humans we will take advantage of human pluripotent stem cells (hPSC)-derived organoids, who exhibit physiological functions close to their in vivo equivalents and can recapitulate several biological processes related to spatial and temporal organization as in our model which transcriptionally matches a second trimester gestational kidney.In this project we will elucidate the occupancy of histone marks and PAX2 binding sites genome-wide in TECs isolated from patients at different stages of AKI.These findings are validated in a larger cohort of retrospective patient samples for the overall identification of the top 5 candidates regulated by Pax2 upon AKI.Furthermore, after identifying the top PAX2 binding sites engineered isogenic CRISPR/Cas9 knock out lines and isogenic lines for doxycycline-inducible promoter/enhancer transcriptional repression and activation for both PAX2 and PAX2 transcriptional targets in patient TECs and human pluripotent stem cell lines will be generated. And eventually we will develop a physiological in vivo model of renal cell toxicity derived from the named CRISPR/Cas9 engineered lines generated in order to assess the putative role of Pax2 and PAX2-top transcriptional targets in human AKI.This project will answer urgent questions in the field of the reparation and repair in the kidney and offer new insights into the role of PAX2 in AKI pathology and progression.We will discern if the epigenetic modifications and regenerative responses during AKI are a cause or consequence of Pax2 epigenetic changes. Eventually we will be able provide a tool to erase or re-write epigenetic modifications in transformed tubular cells in order to be able to redirect them to a regenerative type.