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Kidney Week

Abstract: SA-PO972

BRCA1 Deletion Protects Mice From Kidney Fibrosis by Reducing G2/M Cell Cycle Arrest

Session Information

  • CKD: Pathobiology - II
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Ajay, Amrendra Kumar, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Akinfolarin, Akinwande A., Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Sabbisetti, Venkata, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background

Injury to proximal tubular (PT) epithelial cells leads to fibrotic kidney disease. Toxic, ischemic, and obstructive kidney injuries lead to replication fork arrest and DNA double strand breaks, triggering the DNA damage repair mechanisms. Breast Cancer gene 1 (Brca1) is upregulated in response to DNA damage to help repair the damaged DNA. The function of Brca1 is well investigated in cancer, recruiting RAD51 to the damaged DNA. We proposed that its effect to arrest the cell cycle might implicate it in fibrotic kidney disease.

Methods

Slc34a1 Cre mice were crossed with Brca1 floxed mice yielding mouse models with PT Brca1 exon 11 gene deletion. After tamoxifen-induced Cre induction, mice were subjected to bilateral ischemia/reperfusion (BIRI), or aristolochic acid (AA)-induced fibrosis. The deletion of Brca1 exon 11 was confirmed by fluorescence in situ hybridization (FISH). Markers of DNA damage, cell cycle arrest, and senescence were evaluated by immunofluorescence staining. Kidney tissue lysates were evaluated using western blot, real-time PCR, and PicroSirius (PS) Red staining and markers of DNA damage, senescence, and interstitial fibrosis. HKC8 and HK-2 human PT epithelial cells (HPTECs) were transfected with either shRNA or siRNA and treated with either cisplatin or AA to investigate the injury associated with cell cycle stages, apoptosis, and senescence.

Results

BRCA1 protein expression was increased in human CKD kidneys. The expression of Brca1 exon 11 was increased following BIRI or AA. Brca1 deletion protected mice from interstitial fibrosis when compared to control mice as shown by PS staining, fibronectin, collagen 1, and a-smooth muscle actin (a-SMA) following BIRI and AA. Western blot analysis of whole kidney cortex revealed reduction in CTGF, fibrogenic markers, collagen1, fibronectin and a-SMA. PT Brca1 depleted mouse had fewer pH3+ cells, a marker of G2/M cell cycle phase and reduced S-β-Gal, a marker of senescence. shRNA or siRNA-induced reduction in BRCA1 in HPTECs reduced secretion of the profibrogenic inflammatory cytokine IL-6 and sonic hedgehog following cisplatin or AA treatment.

Conclusion

BRCA1 induces interstitial fibrosis following tubular injury by inducing G2/M cell cycle arrest, cellular senescence, and secretion of profibrotic mediators.

Funding

  • NIDDK Support