Kidney Week

Abstract: FR-PO369

Rac1 Promotes Epithelial Collecting Duct Repair by Maintaining Mitotic Morphology

Session Information

• Genetics, Development, Regeneration
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development‚ Stem Cells‚ and Regenerative Medicine

• 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

• Bock, Fabian, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Fabian Bock,

• Viquez, Olga, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Olga Viquez,

• Sha, Eric, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Eric Sha,

• Dong, Xinyu, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Xinyu Dong,

• Pozzi, Ambra, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Ambra Pozzi,

• Zent, Roy, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Roy Zent,

Background

The kidney collecting duct (CD) is a major source of inflammation and fibrosis after injury but how it repairs is not known. The actin cytoskeleton is critical to restore cellular morphology and for normal cell cycle progression during epithelial repair. We have recently shown that the small Rho GTPase Rac1 is a key regulator maintaining the CD actin cytoskeleton at baseline. We thus hypothesize that Rac1 is required for CD repair after injury.

Methods

We crossed Rac1^flox/flox with AQP2-Cre mice deleting Rac1 in the collecting system and performed reversible unilateral ureteral obstruction (rUUO). Furthermore, we performed cell cycle synchronization in vitro and investigated the role of Rac1 in regenerating epithelial monolayers, utilizing isolated Rac1 null CD cells.

Results

AQP2:Rac1^flox/flox mice show impaired fibrosis regression after reversal of obstruction and were unable to normally restore epithelial and actin cytoskeletal integrity and polarity. Rac1 deletion decreased post-injury CD cell turnover with less proliferation and more apoptosis. In vivo cell cycle characterization by flow cytometry revealed a G2/M cell cycle defect. Upon in vitro cell cycle synchronization using a double thymidine block we found abnormal mitotic entry, delayed mitotic progression and mitotic cell death in the Rac1 null CD cells. High-resolution confocal microscopy in vivo and in vitro demonstrated that Rac1 is required for normal metaphase rounding, a critical mitotic morphological transformation. Furthermore, Rac1 null CD cells were unable to maintain mechanical G2/M checkpoint integrity thus allowing morphologically abnormal cells with a disrupted actin cytoskeleton to enter mitosis.

Conclusion

We find that Rac1 promotes kidney CD repair and Rac1 is required for both, morphological reconstitution, and post-injury cell cycle progression. Specifically, Rac1 promotes normal mitotic morphology likely by maintaining mechanical G2/M checkpoint integrity. We thus propose that Rac1 promotes repair by preventing morphologically abnormal cells from entering cell division.

Funding

• NIDDK Support