Kidney Week

Abstract: FR-PO1065

BRCA1 Potentiates Maladaptive Repair and Fibrosis Through Induction of Proximal Tubule G2/M Arrest and Senescence

Session Information

• CKD Mechanisms: Progression, Fibrosis, and Beyond
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

• 2303 CKD (Non-Dialysis): Mechanisms

Authors

• Ajay, Amrendra Kumar, Brigham and Women's Hospital, Boston, Massachusetts, United States

Amrendra Kumar Ajay,

• Gifford, Cody Charles, Brigham and Women's Hospital, Boston, Massachusetts, United States

Cody Charles Gifford,

• Akinfolarin, Akinwande A., Brigham and Women's Hospital, Boston, Massachusetts, United States

Akinwande A. Akinfolarin,

• Sabbisetti, Venkata, Brigham and Women's Hospital, Boston, Massachusetts, United States

Venkata Sabbisetti,

• Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States

Joseph V. Bonventre,

Group or Team Name

• Bonventre Lab.

Background

DNA damage is a major contributing factor in the progression of fibrotic renal disease. Proximal tubular (PT) epithelial cells are particularly vulnerable to toxic, ischemic, and obstructive injury in the kidney, leading to replication fork arrest and DNA double-strand breaks (DSBs), triggering the DNA damage response. Subsequent to DSBs, ATM-mediated phosphorylation of BRCA1^Ser1524 initiates the DDR, a protective adaptation in many pathologies, notably in the prevention of carcinogenesis. Until recently, the role of BRCA1 in renal fibrosis has been largely unexplored. We hypothesized that the effect of BRCA1 on arresting the cell cycle would exacerbate maladaptive repair through the initiation of G2/M cell cycle arrest.

Methods

Slc34a1-Cre mice were crossed with Brca1^flox/flox mice yielding mice with PT Brca1 exon 11 gene deletion. Mice were subjected to bilateral ischemia/reperfusion (BIRI) or aristolochic acid (AA)-induced injury. Markers of DNA damage, cell cycle arrest, senescence and fibrosis were evaluated by immunofluorescence staining and western blot analysis of tissue sections and patient-derived PTCs subjected to AA or cisplatin. HKC8 cells were transfected with shRNA and treated with either cisplatin or AA to investigate the role of BRCA1 in injury-associated apoptosis, growth arrest and senescence.

Results

BRCA1 protein expression was increased in human CKD kidneys. The expression of Brca1 exon 11 was increased following BIRI or AA. PT Brca1 deletion protected mice from fibrosis, as shown by Sirius red staining, fibronectin, collagen 1, and a-smooth muscle actin (a-SMA) following BIRI or AA. PT-Brca1 depleted mice had fewer pH3+ cells, a G2/M cell cycle phase marker and reduced S-β-Gal, a senescence marker. Primary PTCs displayed increased p-BRCA1^Ser1524 after 48h AA or cisplatin treatment, with increased activation of growth arrest genes p53 and p21. shRNA-induced reduction of BRCA1 in HKC8s decreased the percentage of cells in the G2/M cell cycle phase and significantly reduced cell viability.

Conclusion

BRCA1 induces fibrosis after tubular injury. Loss of BRCA1 from PTs reduces G2/M cell cycle arrest, cellular senescence, and secretion of profibrotic mediators in vivo and in vitro. Thus, transient inhibition of BRCA1 can be beneficial for preventing AKI-CKD transition.

Funding

• NIDDK Support