ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

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: SA-PO522

Injury-Induced DNA Re-Replication Leads to Tubular Cell Polyploidization in FAN1-Deficient Kidneys

Session Information

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

Category: Genetic Diseases of the Kidneys

  • 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Airik, Merlin, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Huynh, Amy B., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Airik, Rannar, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

Karyomegalic interstitial nephritis (KIN) is a biopsy-based diagnosis in which the nuclei of the kidney proximal tubule cells (PTECs) appear abnormally enlarged or karyomegalic. Nuclear enlargement in KIN results from increased chromosomal numbers due to a mitotic failure. However, the molecular mechanism responsible for polyploidization has not been identified. Here we show that karyomegaly is caused in FAN1-deficient kidney tubular cells by aberrant DNA re-replication induced by failed DNA repair in the preceding S-phase.

Methods

KIN was induced in Fan1 KO kidneys by low dose cisplatin administration (5 weekly injections of 2 mg/kg). Histological analysis was performed using HE and PAS staining. RNA-seq analysis was performed on cisplatin-treated kidneys to identify molecular changes. FAN1 KO human PTECs were used to monitor changes in cell cycle progression with FUCCI(SA) and FUCCI(CA) probes, and to perform biochemical studies with the p21 inhibitor UC2288.

Results

Chronic treatment with low dose cisplatin induced tubular karyomegaly (KIN) in Fan1 KO kidneys but not in control mice. Marker analysis showed that the karyomegalic cells were arrested in G2. This finding was independently confirmed by RNAseq analysis, which revealed overrepresentation of G2/M checkpoint genes and upregulation of Cdkn1a in injured Fan1 KO kidneys. Unexpectedly, the G2-arrested cells displayed increased DNA replication activity and upregulation of DNA replication licensing factors (RLFs) CDT1 and CDC6 in KIN. In vitro assays in PTECs showed that CDT1/CDC6 upregulation and stabilization was triggered by p21 accumulation in cells with DNA damage. Treatment with UC2288 abolished CDT1/CDC6 accumulation in FAN1 KO PTECs and prevented their polyploidization. Cell cycle analysis with FUCCI probes revealed that karyomegaly leads to irreversible G2 cell cycle exit.

Conclusion

Our data show that karyomegaly arises in FAN1-deficient tubular epithelial cells through aberrant polyploidization due to DNA re-replication and coincides with p21 upregulation and G2 arrest. Inhibiting p21 blocks upregulation of RLFs and nuclear enlargement, suggesting that p21 may be a therapeutic target to mitigate KIN.

Funding

  • NIDDK Support