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

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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.