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Abstract: PO1981

Atypical Caspase 3-Dependent Death Process in Podocytes

Session Information

  • Podocyte Biology
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

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, Japan
Background

Apoptosis of podocytes has been widely reported in many in vitro studies, but definitive apoptosis has been rarely documented in vivo podocytes. To elucidate this discrepancy, we analyzed dying process in podocytes in vitro and in vivo.

Methods

Primary mouse podocytes were transiently transfected with hCD25 and EGFP expression plasmids and treated with a hCD25-targeting immunotoxin, LMB2 (1nM). 24 hours later, EGFP, cleaved-caspase 3 (cCasp3) and TUNEL staining were imaged. In in vivo experiments, podocyte injury was induced by injecting LMB2 into NEP25 mice, which express hCD25 in podocytes. In some experiments, NEP25 mice carrying another transgene expressing EGFP in podocytes were used.

Results

In in vitro studies, administration of LMB2 caused leakage of co-introduced EGFP in 56.8±13.6% of hCD25-transfected cells, incorporation of propidium iodide in 13.6±2.5%, activation of caspase 3 in 19.6±2.6% and TUNEL staining in 4.5±1.3%. However, LDH activity in the culture medium did not significantly increase. These phenomena were not observed in cells without hCD25 or without LMB2. Ac-DEVD-CHO (10µM), a caspase-3 inhibitor, attenuated the leakage of EGFP by 38.2%, while inhibitors for caspase-1, necroptosis or autophagy did not. These indicate that LMB2 induced typical caspase-3 dependent apoptosis in podocytes in vitro.
In in vivo studies, injection of LMB2 (25ng/g BW) frequently induced leakage of EGFP from podocytes. In separate six NEP25 mice, 7 days after injection of LMB2 (1.25ng/gBW), 41.8±5.1% of glomeruli were found to contain cCasp3-positive cells, but no TUNEL-positive cell was observed. The urinary sediments contained podocalyxin-positive podocytes (2.5±0.3/µl). Among these, 39.1±3.7% were stained for cCasp3 and 21.7±5.5% were stained for TUNEL. EM analysis showed no apoptotic body, but occasionally rupture of plasma membrane of podocytes. To evaluate the effect of glomerular filtration, three NEP25 mice were similarly injected with LMB2 and subjected to UUO 1 day before sacrifice. Detaching podocyte cell bodies were frequently observed in the contralateral kidney by SEM analysis, but never in the obstructed kidney.

Conclusion

These collectively indicate that podocytes dying dependently on caspase 3 are quickly lost by detachment or plasma membrane rupture before completing full apoptotic processes. Glomerular filtration facilitates detachment of dying podocytes.