Abstract: SA-OR010

Dissociation-Induced Maff and Egr1 Upregulation Triggers Dedifferentiation of Podocytes

Session Information

  • A View on the Glomerulus
    November 04, 2017 | Location: Room 294, Morial Convention Center
    Abstract Time: 06:18 PM - 06:30 PM

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Okabe, Masahiro, Tokai University School of Medicine, Isehara, Japan
  • Motojima, Masaru, Tokai University School of Medicine, Isehara, Japan
  • Miyazaki, Yoichi, Jikei University School of Medicine, Tokyo, Japan
  • Yokoo, Takashi, Jikei University School of Medicine, Tokyo, Japan
  • Matsusaka, Taiji, Tokai University School of Medicine, Isehara, Japan
Background

Podocytes quickly lose their characteristics when they are cultured in vitro, suggesting that detachment from the glomerular basement membrane (GBM) may trigger dedifferentiation. We aimed to explore gene expression changes induced by podocyte dissociation from the GBM.

Methods

We obtained podocyte-specific RNAs by two methods. One method was to dissociate glomerular cells and purify RNAs from FACS-sorted podocytes that are transgenically labeled with fluorescent protein. The other was to utilize RiboTag transgenic mice, in which podocyte ribosomes are tagged with HA epitope, and podocyte RNAs were obtained by immunoprecipitation without dissociating glomerular cells. We then analyzed them (each n=4) with Agilent 8X66K array and compared the two profiles.

Results

Array and qRT-PCR analyses confirmed that known podocyte-specific mRNAs were similarly concentrated in both samples. Among 39,430 probes, 554 were downregulated (<0.125-fold) in dissociated podocytes, including Itga8 and Icam2 (0.019 and 0.12-fold, respectively). 1,013 probes were upregulated (>8-fold), including Fos and Fosb (720 and 2,000-fold, respectively).
We next searched for mRNAs that were commonly upregulated by both dissociation and injury. For this, we analyzed mRNA profiles of injured podocytes in NEP25 mice (n=4) 7 days after injection with podocyte-targeted immunotoxin.
Expression of 55 genes was increased more than 16-fold by both dissociation and injury. Among them, we focused on transcription factors, Maff and Egr1. MafF and MafB, as well as EGR-1 and WT-1, share the same DNA binding sequence, but not transcriptional activation domain. MafB and WT-1 are indispensable for maintaining podocyte differentiation state. In baseline primary cultured podocytes, Maff and Egr1 mRNAs were increased 15 and 4.2-fold compared to in vivo podocytes of RiboTag mice, respectively. Maff knockdown increased MafB target- Nphs2 mRNA (2.4-fold) without change of Mafb mRNA. Egr1 knockdown increased Wt-1 target- Ptpro mRNA (2.2-fold) without change of Wt1 mRNA.

Conclusion

These findings indicate that detachment from the GBM activates MafF and EGR-1 in podocytes, which actively facilitate dedifferentiation by competing with MafB and WT-1, respectively. These molecules may be novel drug targets for preventing progressive podocyte deterioration.

Funding

  • Government Support - Non-U.S.