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

尽管急性肾损伤(AKI)是住院患者中最常见的疾病之一，但人类肾脏损伤后的再生机制尚不完全清楚。研究表明，肾脏损伤后发育基因如PAX2重新激活。PAX2在健康条件下无法在成熟的肾小管和肾小球中检测到，已知通过组蛋白甲基化和明确的肾脏染色质修饰在表观遗传学规范中起着关键作用。如果这些机制在AKI后被重新激活，目前还不能回答。肾脏的发育和修复是相同的过程，还是不同环境下相似的基因表达被误读仍有待阐明。我们的假设是，发育基因重新激活可能是一种独特的细胞事件，可以区分慢性肾脏疾病的纤维化后果(如瘢痕形成)和急性肾损伤后的修复损伤反应。我们认为，肾小管细胞先前损伤的表观遗传修饰决定了转化的肾小管上皮细胞的分化方向AKI中的细胞(TEC)。由于在小鼠身上进行的AKI研究在得出适用于人类的结论方面存在严重限制，我们将利用人类多能干细胞(HPSC)衍生的有机化合物，在这个项目中，我们将阐明从AKI不同阶段患者分离的TECs中组蛋白标记和PAX2结合位点在基因组中的占有率。这些发现在更大的回溯性患者样本队列中得到验证，以全面识别AKI中受Pax2调控的前5个候选者。此外，在确定了PAX2的顶端结合部位后，将产生患者TECs和人类多能干细胞株中PAX2和PAX2转录靶标的转录抑制和激活。最终，我们将从命名的CRISPR/Cas9工程化株中建立肾脏细胞毒性的生理学体内模型，以评估Pax2和PAX2-top转录靶标在人类AKI中的可能作用。该项目将回答肾脏修复和修复领域中的紧迫问题，并为PAX2在AKI病理和进展中的作用提供新的见解。我们将辨别AKI过程中的表观遗传修饰和再生反应是Pax2表观遗传学变化的原因还是结果。最终，我们将能够提供一种工具来擦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.