Kidney Week

Abstract: SA-OR21

DNA Damage Sensor BRCA1 Potentiates Fibrosis by Inducing Proximal Tubule G2/M Arrest and Senescence

Session Information

• CKD: New Insights into Mechanisms and Treatment Strategies
  October 26, 2024 | Location: Room 24, Convention Center
  Abstract Time: 04:50 PM - 05:00 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, MS, PhD

• Akinfolarin, Akinwande A., Dallas Nephrology Associates, Dallas, Texas, United States

Akinwande A. Akinfolarin, MD

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

Joseph V. Bonventre, MD, PhD, FASN

Group or Team Name

• Bonventre Laboratory.

Background

DNA damage is a major contributing factor in the progression of fibrotic renal disease. Proximal tubular epithelial cells (PTECs) are the primary site of injury caused by toxins, ischemia, and obstructive injury in the kidney, leading to DNA breaks and triggering the DNA damage response (DDR). In response to DNA damage, the ATM-mediated phosphorylation of BRCA1^Ser1524 initiates the DDR. 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 and senescence.

Methods

We utilized human CKD kidney tissues, Slc34a1-Cre mice crossed with Brca1^flox/flox mice, yielding PTEC Brca1 exon 11 gene deletion. We performed bilateral ischemia/reperfusion (BIRI) or aristolochic acid (AA)-induced injury models in mice. Markers of DNA damage, cell cycle arrest, senescence, and fibrosis were evaluated by immunofluorescence staining and western blot analysis of tissue sections. We utilized in vitro models of patient-derived PTCs and HKC8 cells treated with AA or cisplatin. In addition, siRNA and shRNA-mediated knockdown, followed by cisplatin or AA treatment, was employed to study the role of BRCA1 in growth arrest and senescence.

Results

Human CKD kidneys showed a significant increased expression of BRCA1 protein expression compared to kidneys without CKD. Following BIRI or AA treatment in mice, the expression of Brca1 exon 11 was increased. Deletion of Brca1 from PTECs protected mice from the development of fibrosis, as shown by Sirius red staining, fibronectin, collagen 1, and a-smooth muscle actin following BIRI or AA. Following BIRI or AA treatment, PTECs-Brca1 depleted mice had reduced 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. siRNA and shRNA-induced reduction of BRCA1 in HKC8 and HK2 cells decreased cell viability and increased the gene expression of pro-fibrotic factors (Acta2, Fibronectin, and Collagen1).

Conclusion

BRCA1 in PTECs induces G2/M cell cycle arrest and cellular senescence in vivo and in vitro following injury. Transient inhibition of BRCA1 represents a novel treatment strategy to prevent the development of fibrosis after acute injury.

Funding

• NIDDK Support