Abstract: FR-PO251
RAD51 Inhibition Exacerbates Injury in a Human Pluripotent Stem Cell-Derived Nephron Organoid Model in Which Repetitive DNA Damage Models Acute Injury and Transition to Fibrosis
Session Information
- Stem Cells
November 03, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Developmental Biology and Inherited Kidney Diseases
- 402 Stem Cells
Authors
- Gupta, Navin R., Brigham and Women's Hospital, Cambridge, Massachusetts, United States
- Garcia, Edgar, Brigham and Women's Hospital, Cambridge, Massachusetts, United States
- Miyoshi, Tomoya, Brigham and Women's Hospital, Cambridge, Massachusetts, United States
- Susa, Koichiro, Brigham and Women's Hospital, Cambridge, Massachusetts, United States
- Morizane, Ryuji, Brigham and Women's Hospital, Cambridge, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Cambridge, Massachusetts, United States
Background
Tubular DNA damage has been demonstrated to play a central role in tubulointerstitial crosstalk, implicated in the pathogenesis of kidney injury and fibrosis. Unlike traditional cell culture techniques, human pluripotent stem cell (hPSC)-derived nephron organoids contain multiple compartments representing glomeruli, proximal and distal tubules, and an interstitium. Following interstitial characterization, the organoid’s response to cisplatin, a known nephrotoxicant that induces proximal tubular (PT) DNA damage, may enable the study of DNA damage response (DDR) and interactions between the tubular and interstitial compartments in vitro.
Methods
Nephron organoids were treated with twice weekly, 24 hour periods of cisplatin (5μM) for 5 treatments. Following each treatment, samples and controls were gathered for immunostaining and qRT-PCR. Flow cytometric gating on LTL-fluorescein determined the preponderance of PTs in nephron organoids. B02 was used at 5μM for continuous inhibition of RAD51, a DDR element for double strand break (DSB) repair.
Results
PDGFR-β+ desmin+ cells (pericytes) and PDGFR- β+ desmin- cells (fibroblasts) comprise the interstitium of nephron organoids, in a similar distribution and ratio as in our human nephrectomy samples. Following acute injury with cisplatin, LTL+ PT cells manifested preferential DSBs, entered the cell cycle, expressed luminal KIM-1, underwent mesenchymal dedifferentiation, and were associated with PTEN+NCAM+ peritubular interstitial cells. Following repeated cisplatin injury, LTL+ cells had a reduced brush border, arrested at the G2/M DNA damage checkpoint, and pro-inflammatory cytokines were upregulated on organoid lysates. With further injury, peritubular interstitial cells transdifferentiated into myofibroblasts and fibronectin and collagen I accumulated in the interstitial space. B02, a RAD51 inhibitor, exacerbated proximal tubular G2/M arrest and interstitial fibrosis.
Conclusion
Inhibition of RAD51 augmented injury in nephron organoids treated with repetitive cisplatin, implicating DDR in the pathogenesis of kidney fibrosis. As an effective human model system in vitro, nephron organoids can be exploited to understand mechanisms of injury and repair and screen anti-fibrotic therapeutics.
Funding
- Other NIH Support