ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: FR-PO766

Fibrotic Kidney Organoids and Human CKD Samples Demonstrate Loss of Homologous Recombination as a Critical Mediator of Maladaptive Repair

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Gupta, Navin R., Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Hiratsuka, Ken, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Miyoshi, Tomoya, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Susa, Koichiro, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Tatsumoto, Narihito, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Yamashita, Michifumi, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Morizane, Ryuji, Brigham and Women's Hospital, Boston, Massachusetts, United States
Background

Myofibroblasts are a hallmark of maladaptive repair that have been demonstrated in injured kidney organoids and kidney tissue in vivo. Tubular epithelial DNA damage potentiates myofibroblast generation. Mechanisms for DNA repair include genome-conserving homologous recombination (HR) and mutation-generating non-homologous end joining (NHEJ). Here we hypothesize that DNA damage repair between HR and NHEJ in tubular epithelial cells governs maladaptive repair, which could be examined using kidney organoids and human kidney tissue.

Methods

Kidney organoids were generated from hPSCs by our established protocol. Organoids were treated with repetitive cisplatin to induce tubular epithelial DNA damage, ascertained by γH2AX positivity. Stromal cells were monitored for the advent of the myofibroblast marker, αSMA. DNA damage repair was modulated to inhibit either HR or NHEJ in cisplatin treated organoids. FANCD2, a crucial HR element, was used as a surrogate for HR activity. HR and NHEJ activities were evaluated by qPCR for multiple genes of DNA repair. Findings in organoids were confirmed in patient biopsy samples of CKD and minimal change disease (MCD) with tubular injury.

Results

Cisplatin treatment induced DNA damage to tubular epithelial cells. HR activity increased in injured tubules, yet repetitive cisplatin treatment downregulated HR-related genes wheras NHEJ activity persisted. Suppression of HR activity by RAD51 inhibition hastened nephron loss and the evolution of myofibroblasts, while inhibition of the critical NHEJ enzyme, DNA ligase IV, delayed maladaptive responses. Human CKD patient samples with moderate to severe parenchymal scarring demonstrated little HR activity in injured tubules, whereas non-fibrotic MCD patient samples with tubular injury displayed activation of HR.

Conclusion

Human kidney organoids model maladaptive repair upon recurrent DNA damage in tubular epithelial cells. Mechanistic analysis of maladaptive repair in kidney organoids revealed loss of HR as a pathologic process. As opposed to non-fibrotic human kidney tissue, CKD patient samples demonstrated little HR activity in DNA damaged tubular epithelial cells. Human kidney organoids represent a novel model to decipher pathophysiologic processes in human kidney injury and repair.

Funding

  • NIDDK Support