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Abstract: PO1703

Atypical Caspase 3-Dependent Death in Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Yamamoto, Kazuyoshi, Tokyo Jikeikai Ika Daigaku, Minato-ku, Tokyo, Japan
  • Okabe, Masahiro, Tokyo Jikeikai Ika Daigaku, Minato-ku, Tokyo, Japan
  • Matsusaka, Taiji, Tokai Daigaku, Hiratsuka, Kanagawa, Japan
  • Yokoo, Takashi, Tokyo Jikeikai Ika Daigaku, Minato-ku, Tokyo, Japan
Background

Apoptosis of podocytes has been widely reported in many in vitro studies, but definitive apoptosis has never been 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), and observed 1 day later. In some experiments, the cultured podocytes were transfected with Bak1 or Bax siRNA before treatment with LMB2. In in vivo experiments, podocyte injury was induced by injecting LMB2 (1.25ng/gBW) into NEP25 mice, which express hCD25 in podocytes, and analyzed 7 days later.

Results

In in vitro studies, administration of LMB2 caused loss of co-introduced EGFP in 56.8±13.6%, incorporation of propidium iodide in 13.6±2.5%, activation of caspase 3 (Casp3) in 19.6±2.6% and TUNEL staining in 4.5±1.3% without significant increase in LDH activity in the culture medium. These phenomena were not observed in cells without hCD25 or without LMB2. Ac-DEVD-CHO (10µM), a Casp3 inhibitor, attenuated the loss of EGFP by 38.2%. Inhibition of Bak1 and Bax using siRNAs attenuated EGFP loss by 77.6% and 28.4%, respectively. These indicate that LMB2 induced the typical Casp3 dependent intrinsic apoptosis in podocytes in vitro.
In in vivo studies, kidneys of NEP25 mice contained podocytes positive for cleaved (c) Casp3 and those for cLaminA, a product of Casp3, but no TUNEL+ podocytes. EM analysis showed no apoptotic body, but occasionally rupture of plasma membrane of podocytes. 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. To evaluate the effect of glomerular filtration, NEP25 mice were similarly injected with LMB2 and subjected to UUO 1 day before sacrifice. The obstructed kidney contained significantly more cLaminA+ podocytes than the contralateral kidney. In addition, detaching podocyte cell bodies were frequently observed in the contralateral kidney by SEM analysis, but never in the obstructed kidney.

Conclusion

Thus, due to physical force of glomerular filtration, podocytes doomed to Casp3 dependent death are quickly lost by detachment or plasma membrane rupture before completing full apoptotic processes. This accounts for the absence of podocyte apoptosis in vivo.