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Abstract: PO1719

Podocyte Damage in Chimeric Kidney Organoids

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Matsusaka, Taiji, Tokai Daigaku Igakubu Daigakuin Igaku Kenkyuka, Isehara, Kanagawa, Japan
  • Udagawa, Tomohiro, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan
  • Osafune, Kenji, Kyoto Daigaku, Kyoto, Kyoto, Japan
  • Araoka, Toshikazu, Kyoto Daigaku, Kyoto, Kyoto, Japan
Background

We previously found that when a portion of podocytes are injured, other podocytes are secondarily injured in a mouse model, which may underlie the relentless progression of chronic renal failure. In the present study, we tested whether this phenomenon is observed in kidney organoids.

Methods

Nephron progenitor cells (NPCs) were established by culture-dependent purification (CDP) method from 12.5 dpc NEP25 and RiboTag mouse kidneys (Cell Stem Cell. 2016, 19, 516-29). Podocytes of NEP25 mice express hCD25 and can be injured by hCD25-targeted immunotoxin, LMB2. Podocytes of RiboTag mice express hemagglutinin (HA)-tagged ribosomal protein. Kidney organoids were generated by transient stimulation with FGF2 and CHIR99021 of NPC aggregates and subsequent 8-day culture. On day 8, LMB2 (20 nM) was added to induce injury in hCD25+ podocytes.

Results

We confirmed that podocytes in both organoids are stained for nephrin, podocin and WT1. Podocytes derived from NEP25 NPC expressed hCD25 and were injured by LMB2, and those from Ribotag NPC expressed HA and were resistant to LMB2. When two types of NPCs were mixed at 1:1 ratio, organoids showed a chimeric pattern containing hCD25+ and HA+ podocytes (Fig1). 2 days after LMB2 treatment, hCD25 staining completely disappeared accompanied by cleaved (c) lamin A staining, a cell death marker. HA staining was retained, but WT1 was diminished and podocin disappeared (Fig 2). Occasionally, c-lamin A was positive in HA+ cells. RNA of Ribotag podocytes can be obtained by immunoprecipitation with anti-HA antibody. Q-PCR revealed that Nphs1 (0.31), Nphs2 (0.04), Wt1 (0.37) were decreased to the indicated fold by LMB2 and that Gadd45b was increased to 4.82-fold.

Conclusion

Thus, podocyte damage damages podocytes in kidney organoids that lack glomerular filtration.

Funding

  • Government Support – Non-U.S.