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Abstract: TH-PO733

A Podocyte-Specific Injury Mouse Model with Inducible Yamanaka Factors

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Lemberg, Katharina, Boston Children's Hospital, Boston, Massachusetts, United States
  • Hölzel, Selina, Boston Children's Hospital, Boston, Massachusetts, United States
  • Buerger, Florian, Boston Children's Hospital, Boston, Massachusetts, United States
  • Franken, Gijs AC, Boston Children's Hospital, Boston, Massachusetts, United States
  • Kolvenbach, Caroline Maria, Boston Children's Hospital, Boston, Massachusetts, United States
  • Saida, Ken, Boston Children's Hospital, Boston, Massachusetts, United States
  • Salmanullah, Daanya, Boston Children's Hospital, Boston, Massachusetts, United States
  • Deutsch, Konstantin, Boston Children's Hospital, Boston, Massachusetts, United States
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
Background

Ectopic expression of the Yamanaka transcription factors Oct4, Klf4, c-Myc and Sox2 (OKMS) can reprogram somatic cells into pluripotent stem cells in vitro (Takahashi Cell 126, 2006). As shown recently for different tissues, in vivo expression of those factors can enhance regeneration and improve outcome after injury through partial cellular re-programming (Hishida Cell Rep. 39, 2022), even in non-mitotic cells (Lu Nature 588, 2020). In order to apply this approach to podocytes and examine effects of OKMS on podocyte regeneration, we employed a podocyte-specific injury model (Guo J Am Soc Nephrol 23, 2012) and crossed it with a conditional and inducible OKMS mouse model, thereby generating podocyte-specific expression of the Yamanaka factors.

Methods

We crossed the previously published inducible diphtheria toxin receptor-mouse (Buch Nat Methods 2, 2005) with a Podocin-Cre recombinase mouse (Moeller Genesis 35, 2003), and further with the ROSA26-rtTA(neo); OKMSCh250-mouse (JAX stock #031012). We induced OKMS-expression in vitro in extracted primary podocytes, as well as in vivo by 7-day application of doxycycline via drinking water. Furthermore, we determined urine albumin/creatinine ratios (ACR) and obtained histological samples to evaluate the extent of kidney injury after intraperitoneal diphtheria toxin (DT) injection in 5 different doses.

Results

To determine if OKMS can be induced, expression was first tested in vitro. Primary podocytes from OKMStg/WT; rtTAfl/WT; Podocin-Cre+ - mice showed increasing Oct4 and Sox2 expression over time, but no expression of Nanog after 6 days and no decrease in podocyte-specific markers, whereas primary podocytes from a OKMStg/tg; rtTAfl/WT; Podocin-Cre+ - mouse exhibited expression of Nanog, as well as a decrease of Nphs1 and Nphs2, but an increase in WT1. In vivo, administration of 10 μg/kg and 7.5 μg/kg DT led to proteinuria with ACR levels over 50 g/g after 4 days as well as glomerulosclerosis and tubular casts on light microscopy after 4 weeks. Administration of 5 μg/kg, 2.5 μg/kg and 1.25 μg/kg showed later onset (d6), lower levels and, after 14 days, a reduction of proteinuria.

Conclusion

We, hereby, provide a suitable mouse model to study the effect of Yamanaka-expression in a podocyte-specific injury model.

Funding

  • NIDDK Support