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Kidney Week

Abstract: PO0207

Loss of HDAC8 Leads to γH2AX-Induced Cellular Repair and Decreased Epithelial-Mesenchymal Transition in Renal Tubule Epithelial Cells

Session Information

  • AKI Mechanisms - 2
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Crunk, Amanda, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Han, Hwa In, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • McDaniels, Michael D., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Hukriede, Neil A., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

Acute kidney injury (AKI) remains a significant worldwide problem. Our previous work has shown that hdac8-/- larval zebrafish model of AKI improved survival and increased repair and proliferation after AKI. However, these mechanisms have not been elucidated. AKI is known to induce double stranded DNA breaks (DSB), activating factors including the phosphorylation of the histone variant H2AX producing γH2AX. Damaged cells undergo a complex multifactorial fate determination leading to either DNA repair and cell survival or apoptosis.

Methods

hdac8sa14948/-and hdac8 sa14948/+mutant zebrafish were injected with gentamicin to induce AKI. Immunofluorescent microscopy, fluorescently activated cell sorting (FACS) of renal tubule epithelial cells (RTEC) were isolated for RNA-seq.

Results

We evaluated DNA DSB in RTEC through immunohistochemistry of γH2AX in WT and hdac8-/-zebrafish with and without AKI. There were a greater number γH2AX foci in RTEC of the hdac8-/- larvae with AKI than in either the uninjured hdac8-/- or WT AKI larvae. hdac8-/- zebrafish with AKI exhibited less apoptosis compared to WT zebrafish with AKI, and as expected there were undetected levels of TUNNEL positive cells in WT and hdac8-/-uninjured tubules. We observed more Pax2a positive cells in hdac8-/-larvae with AKI than WT larvae with AKI. Immunohistochemical analysis of Na+/K+-ATPase, a marker for epithelial polarization, in hdac8-/-zebrafish tubules maintain partial to full polarization during AKI, whereas WT larvae with AKI had complete mis-polarization of RTEC. RNA-Seq data analysis confirmed the epithelial polarization data demonstrating increased expression of mesenchymal genes and decreased epithelial gene expression in WT tubules with AKI compared to hdac8-/-in AKI injured zebrafish.

Conclusion

This study suggests a mechanism of repair and recovery after AKI in hdac8-/-zebrafish is mediated through increased γH2AX at sites of DNA damage. hdac8-/-mutant zebrafish preferentially uses mechanism of DDR for repair and proliferation, as opposed to apoptosis. In the absence of apoptosis hdac8-/-tubules can prevent depolarization of epithelial membranes and maintain their epithelial gene signatures, whereas the WT zebrafish lose their polarization and begin EMT. These data support our hypothesis that the loss of HDAC8 allows cells to repair after injury from AKI

Funding

  • NIDDK Support