Abstract: SA-OR085

Endocycle-Mediated Hypertrophy and Progenitor Proliferation as Central Mechanisms of Response to AKI

Session Information

Category: Acute Kidney Injury

  • 002 AKI: Repair and Regeneration

Authors

  • Lazzeri, Elena, University of Florence, Florence, Italy
  • Angelotti, Maria Lucia, University of Florence, Florence, Italy
  • Peired, Anna Julie, University of Florence, Florence, Italy
  • Conte, Carolina, University of Florence, Florence, Italy
  • Lasagni, Laura, University of Florence, Florence, Italy
  • Romagnani, Paola, University of Florence, Florence, Italy
Background


Acute kidney injury (AKI) is considered a reversible disease because of the capacity of all tubular cells to proliferate as demonstrated by proliferation markers. As AKI can be followed by chronic kidney disease (CKD), we questioned the high intrinsic regenerative capacity of tubules.

Methods

To investigate the proliferative capacity of tubules after AKI, we developed 2 conditional transgenic mouse models: 1. Pax8.rtTA;TetO.Cre;R26.FUCCI2aR (Pax8/FUCCI2aR) to track all tubular cells; 2. Pax2.rtTA;TetO.Cre;R26.FUCCI2aR (Pax2/FUCCI2aR) to track a putative scattered tubular progenitor population. After doxycycline administration, we investigated the reporter expression in Pax8 and Pax2 cells (cells in G1 phase are mCherry+, in S/G2/M phase are mVenus+) in healthy and at 30 days after unilateral ischemia reperfusion injury (IRI).

Results

Confocal analysis of Pax8/FUCCI2aR mice demonstrated that immunostaining for proliferation markers (KI-67, PCNA and p-H3) didn’t mirror exactly cell-cycle phase as indicated by FUCCI2aR reporter. This suggests that cell-cycle markers indicate cell-cycle activation but not cell division after IRI. We, therefore, explored aberrant cell-cycles such as endocycle, where G1 and S proceed repeatedly wihout mitosis, generating cells arrested in G1 phase with a doubled DNA content and an increased size. To detect these cells, we combined FUCCI2aR expression and DNA content assessment by flow-cytometry. This analysis revealed that 11.5±0.8% of Pax8+tubular cells underwent endocycles at day 30 after IRI. Most of endocycling cells were in S1-S2 segments of the cortex with a higher cell surface area in comparison to cells in G1 phase. Accordingly, tubular cell hypertrophy via endocycle is the dominant feature in the cortex of human biopsies with CKD after AKI. By contrast, Pax2+tubular cells didn’t undergo endocycle but rather complete mitosis at day 30 after IRI. Automatic quantification analysis by flow-cytometry demonstrated that while Pax8+tubular cells are irreversibly lost, Pax2+tubular cells are the only proliferating cells, generating new tubular cells after AKI.

Conclusion

These results demonstrate that, although limited regeneration occurs via Pax2 cell proliferation, persistent tubular cell loss and tubular cell hypertrophy via endocycle are the dominant features after AKI.