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Abstract: TH-PO1099

P2X7 Expressed in Injured Podocytes May Spread the Kidney Injury Through Caspase 3

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

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

We previously generated a mosaic mouse model in which a fraction of podocytes express hCD25 and can be injured by a hCD25-directed immunotoxin, LMB2. After injection with LMB2, not only hCD25(+) but also hCD25(-) podocytes were injured along with dramatic induction of P2X7 mRNA in both types of podocytes. P2X7 is a receptor of extracellular ATP and is known to activate inflammation and induce cell death in immune cells. In the present study, we aim to analyze P2X7 protein expression in the mosaic mouse model and investigate the role of P2X7 in podocyte injury.

Methods

Kidneys were harvested from mosaic mice before or 2 weeks after injection with LMB2 (25ng/gBW). Immunofluorescence staining was performed with primary antibodies against P2X7 and cleaved-caspase 3. For functional study, primary cultured mouse podocytes were transiently transfected by electroporation with P2X7-expression or mock plasmid together with EGFP or tdTomato expression plasmid. Before or 1-2 hours after administration of ATP (0 or 2 mM), the same visual fields were photographed.

Results

No P2X7 staining was observed in the kidney without LMB2. In the kidneys injured by LMB2, which developed FSGS, 69.2±9.2% of glomeruli were positive for P2X7 staining. Some P2X7 staining was observed in GFP-labeled hCD25(-) podocytes, which indicated that indirect injury activated P2X7 expression. Cleaved-caspase 3 staining was also positive in 12.4±3.1% of glomeruli of LMB2-damaged kidneys, but not in those without LMB2.
In in vitro studies, administration of ATP caused leakage of co-introduced EGFP in 51.7±1.4% of P2X7-transfected cells, incorporation of propidium iodide in 18.1±0.9%, and activation of caspase 3 in 17.9±2.8%. However, increase in LDH activity in the medium remained minimum, corresponding to only 3.0±1.7% of cell death. These phenomena were not observed in mock-transfected cells treated with ATP or P2X7-transfected cells without ATP administration. Caspase-3 inhibitor significantly attenuated the leakage of EGFP induced by ATP (26.4±2.6 vs 37.9±3.3%), whereas Caspase-1 inhibitor did not (37.0±3.0%).

Conclusion

These results indicate that injured podocytes express P2X7, which may further augment injury by inducing caspase-3 dependent apoptosis.