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Abstract: SA-PO378

P2X7-ATP May Mediate Propagation of Podocyte Damage

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Yamamoto, Kazuyoshi, The Jikei University School of Medicine, Tokyo, Japan
  • Okabe, Masahiro, The Jikei University School of Medicine, Tokyo, Japan
  • Matsusaka, Taiji, Tokai University School of Medicine, Isehara, KANAGAWA, Japan
  • Yokoo, Takashi, The Jikei University School of Medicine, Tokyo, Japan
Background

We previously generated a mosaic mouse model in which a fraction of podocytes express hCD25 and can be injured by hCD25-directed immunotoxin, LMB2. After injection with LMB2, not only hCD25(+) podocytes but also hCD25(-) podocytes were injured. A possible mechanism is that damage-associated molecular patterns (DAMPs) released from the damaged podocytes may secondarily injure the hCD25(-) podocytes. In the present study, we aim to find a candidate molecule that mediates spreading of podocyte damage.

Methods

Mosaic mice (n=4, each time point) were injected with 25 ng/g BW of LMB2, hCD25(+) and (-) podocytes were harvested by FACS sorting, and RNAs were analyzed with Agilent’s 8X66K microarray. Functional assay was performed in primary cultured mouse podocytes by transiently transfecting expression plasmid.

Results

Among DAMPs and their receptors, we found that P2X7 mRNA was markedly increased both in hCD25(+) podocytes (>3000 folds) and in hCD25(-) cells (59 folds) 4 days after LMB2, which were confirmed by qRT-PCR analysis (>40,000 folds, 53 folds). P2X7 is a receptor for extracellular ATP and triggers signaling pathways leading to inflammation and cell death in immune cells.
To test the role of P2X7-ATP in podocytes, we transfected primary cultured podocytes with P2X7 expressing plasmid. By 2 hours after administration of ATP (1 mM), cell number decreased by 53.4%, with 18.6% of cells incorporating propidium iodide. This is contrasting to P2X7 expressing cells without ATP, or mock-transfected cells with or without ATP, in which cell number decrease was <5% and propidium iodide incorporation was <2%. 6.7% of P2X7 expressing podocytes were stained with Annexin V one hour after ATP, whereas 2.6% of mock + ATP cells and 0% of P2X7 without ATP were Annexin V +.

Conclusion

These suggest that podocytes in damaged glomeruli express P2X7, respond to ATP released from adjacent dying podocytes, and are secondarily damaged. Thus, P2X7-ATP may mediate propagation of podocyte damage.

Funding

  • Government Support - Non-U.S.