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Abstract: FR-PO770

Kidney Organoid Model of Selective Podocyte Injury

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Udagawa, Tomohiro, Tokai University School of Medicine, Isehara, Kanagawa, Japan
  • Tanaka, Keiko, Tokai University School of Medicine, Isehara, Kanagawa, Japan
  • Araoka, Toshikazu, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
  • Osafune, Kenji, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
  • Matsusaka, Taiji, Tokai University School of Medicine, Isehara, Kanagawa, Japan
Background

Podocyte injury triggers progressive loss of kidney function. Determining the mechanisms involved in the progression of podocyte damage is critical to developing renoprotective therapies. We previously generated a transgenic mouse line, NEP25, which expresses human (h) CD25 on podocytes. Selective podocyte injury can be induced by the hCD25-targeted immunotoxin, LMB2. In addition, we analyzed podocyte-specific gene expression profiles utilizing RiboTag mice, which selectively express hemagglutinin (HA)-tagged ribosomal protein in podocytes. In the present study, to efficiently investigate the function of altered genes during podocyte injury,we aimed to generate NEP25/RiboTag kidney organoids and establish an in vitro model of podocyte injury.

Methods

Nephron progenitor cells (NPCs) were established by culture-dependent purification (CDP) method from 12.5 dpc NEP25/RiboTag mouse kidneys (Cell Stem Cell. 2016, 19, 516-29). Kidney organoids were generated by transient stimulation with FGF2 and CHIR99021 of NPC aggregates and subsequent 6–8 day culture. Podocyte-specific polysomes were obtained via immunoprecipitation using an anti-HA antibody.

Results

We confirmed that the organoids show glomerulus-like and tubule-like structures. Immunostaining revealed that the former expressed nephrin, WT1, podcalyxin, and synaptopodin and the latter expressed LTL and megalin. Nephrin-positive podocytes also expressed hCD25 and HA. Q-PCR confirmed that Nphs1 (33.3), Nphs2 (21.2), Wt1 (24.9), and Dach1 (6.2) were concentrated in HA-immunoprecipitated RNA by the indicated fold. When organoids were incubated with LMB2 (20 nM) for 4 days, podocalyxin and WT1 staining disappeared. Nphs1 and Nphs2 mRNAs became undetectable, Wt1 (0.05 fold) and Dach1 (0.37) decreased, and Cxcl1 (4.2) and Epha8 (55.6) increased, thus reproducing in vivo injured podocytes.

Conclusion

We established kidney organoids in which podocytes can be selectively injured and podocyte mRNA can be selectively obtained. This organoid system will be a powerful tool for investigating mechanisms underlying podocyte injury.

Funding

  • Government Support - Non-U.S.