? DESCRIPTION (provided by applicant): Tubule atrophy and fibrosis often develop after acute kidney injury (AKI) favoring transition to chronic kidney disease (CKD). The AKI-CKD transition has major implications, but poorly understood. We found that tubules regenerating after AKI often fail to differentiate. Such tubules are growth arrested and atrophic, and exhibit signaling that drives fibrosis. The cause of this pathology is unknown. We obtained three lines of evidence that could explain why recovering tubules become atrophic and profibrotic. First, tubules showed damage to DNA and DNA damage repair responses (DDR) during ischemic AKI, and then, instead of subsiding, DNA damage and DDRs persisted into later stages of tubule atrophy and fibrosis. Altered gene expression caused by DNA damage may explain why tubules fail to recover normally. Chronic hypoxia in kidneys recovering from AKI could inhibit oxygen dependent ribonucleotide reductase (RNR) in tubules causing depletion of deoxynucleotide triphosphates (dNTPs) and thereby produce DNA damage. Second, dNTPs declined in hypoxic cultured tubule cells, producing DNA damage and DDRs. This was accompanied by growth arrest and a dedifferentiated abnormally signaling profibrotic phenotype similar to that seen during tubule atrophy after AKI in vivo. Provision of dNTP precursors ameliorated the hypoxic DNA damage and reversed the growth arrest. Third, by RNAseq transcriptional profiling of hypoxic cells, we identified increased expression of genes related to inflammation, atrophy and fibrosis. Our hypothesis is that following ischemic AKI, chronic hypoxia inhibits ribonucleotide reductase (RNR) in regenerating tubules, depletes dNTPs, and, thereby, causes DNA replication stress and DNA damage. Because DNA damage is chronic, the DNA damage repair response (DDR) also persists, giving rise to inflammation, atrophy and fibrosis. To test this hypothesis, we have three Specific Aims. In Aim 1 we will explore the relationships between DNA damage and development of tubule atrophy using immunohistochemistry and morphometry, a transgenic reporter DNA damage, and inducible deletion of critical DDR genes to investigate if the intervention modifies late pathology after AKI In Aim 2, we will investigate the relationships of hypoxic inhibition of RNR, dNTP depletion, DNA damage/DDR and cell pathology. We will determine if infusions of dNTP precursors in vivo ameliorate tubule pathology after AKI. In Aim 3, we will use RNAseq and proteomics to selectively identify hypoxic alterations specific to dNTP depletion as apart from other hypoxic effects. Thus, we hope to provide critically needed information relating to basic aspects in the pathogenesis of a major health problem, chronic kidney disease.
|Effective start/end date||1/1/16 → 12/31/19|