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Abstract: FR-OR029

ATR Deletion Drives TOR-Autophagy Spatial Coupling Compartment (TASCC) Formation and Kidney Fibrosis

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Brooks, Craig R., Vanderbilt University Medical Center/Division of Nephrology & Hpertension, Nashville, Tennessee, United States
  • Kishi, Seiji, Kawasaki Medical School, Kurashiki, Japan
  • Taguchi, Kensei, Department of Nephrology and Hypertension, Nashville, Tennessee, United States
  • Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background

Acute kidney injury (AKI) occurs in ~20% of hospitalized patients. While the kidney can functionally recover from AKI, AKI predisposes patients to subsequent chronic kidney disease (CKD), which effects 13% of the global population. Our lab, and others, have shown the AKI-CKD transition involves maladaptive repair leading to G2/M arrest regulated by DNA damage response (DDR) genes. Recently, we have identified target of rapamycin–autophagy spatial coupling compartments (TASCCs) as components of the secretory response in G2/M arrested cells. To determine how the DDR modulates AKI-CKD transition, we tested if the loss of ataxia telangiectasia and Rad3-related (ATR) regulates TASCC formation and fibrosis.

Methods

1: ATR flox/flox mice were breed to SLC34a1-Cre-ERT2 mice to generate proximal tubular cell (PTC) specific ATR deletion (ATRRPTC-/-) upon tamoxifen injection. ATR floxed mice lacking the Cre acted as control (ATRCtrl). ATRCtrl and ATRRPTC-/- received unilateral ureteral obstruction (UUO). Kidneys were taken at day 7 and analyzed for fibrosis, G2/M arrest markers, injury markers and TASCC formation by immunostaining. TASCCs were identified by super-resolution microscopy. 2: Fucci2a mice were breed to γGT-Cre mice to generate PTC specific Fucci2a expression. The PTCs were then isolated and treated with aristolochic acid (AA), with/without the ATR inhibitor VE-821. Cells were analyzed for increased connective tissue growth factor (CTGF) by western blot.

Results

Inhibition of ATR in PTCs resulted in increased numbers of G2/M arrested cells following AA treatment, as measured by the Fucci cell cycle reporter, and greater production of CTGF, in vitro. In vivo, ATRRPTC-/- mice had more sever and rapidly progressing fibrosis compared to ATRCtrl mice. Deletion of ATR from PTCs resulted in increased tubular cell injury, caspase 3 staining, and G2/M arrest in response to UUO. G2/M arrest was associated with formation of TASCCs in PTCs. ATRRPTC-/- mice had greater numbers of TASCC/cell compared to controls.

Conclusion

ATR deletion sensitized PTCs to injury and G2/M arrest, which drove TASCC formation and production of CTGF. These data indicate the DDR modulates the AKI-CKD transition through a pathway involving G2/M arrest, TASCC formation and profibrotic factor secretion, as well as identifying novel targets for therapeutic intervention.

Funding

  • NIDDK Support