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Abstract: FR-PO405

Evidence for NF-kB/Inflammatory Cytokine Signaling in New Nephron Formation After AKI in Adult Zebrafish

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • Schenk, Heiko Joachim, Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States
  • Kamei, Caramai Nanae, Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States
  • Hughes, Samuel M., Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States
  • Wolf, Amber M., Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States
  • Drummond, Iain A., Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, United States
Background

Adult progenitor cells in the mesonephric zebrafish kidneys are required during neo-nephrogenesis replacing injured tubules by forming new nephrons. Single-cell RNA transcriptomes of adult kidney progenitor cells point to components of NF-kB and inflammatory cytokine receptors that may initiate stem cell-based nephrogenesis. Here, we present evidence that gentamicin induces inflammation-associated injury, which potentially stimulates stem cell-based nephrogenesis, while the stimulatory effect on the progenitor cells to form new nephrons can be recapitulated by LPS injection.

Methods

Adult zebrafish were injected i.p. with gentamicin or LPS at day 0. NF-kB signaling was determined 4 days post-injection (dpi) by NF-kB:GFP detection of the NF-kB reporter line Tg(NF-kB:EGFP) and NF-kB-associated gene expression using qRTPCR. The requirement of NF-kB signaling during regeneration was evaluated by pharmacological NF-kB inhibition. Bulk RNAseq from positive selected GFP+ and mcherry+ single cells by FACS was performed from kidneys 7 dpi by gentamicin injection using Tg(lhx1a:EGFP;cdh17:mCherry) fish.

Results

Gentamicin-induced kidney injury leads to increased tubular NF-kB reporter expression at 4 dpi and is associated with an upregulation of NF-kB target gene expression detected by qRTPCR. Gentamicin also causes GH receptors mRNA upregulation at 7 dpi along with the kidney progenitor markers osr1 and eya4, while the formation of new nephron aggregates as marked by Tg(lhx1a:GFP) expression is increased. NF-kB pharmacological inhibition reduces mRNA expression of kidney progenitor markers, while LPS injection induces mRNA upregulation of kidney progenitor markers. Bulk RNAseq from positive selected GFP+ Lhx1a+ cells 7 dpi with gentamicin confirmed the induction of cytokine receptors in the kidney progenitor cells.

Conclusion

Multiple pathways may converge on adult kidney stem cells to activate new nephron formation. We conclude that DAMP and NF-kB signaling is required and sufficient to induce neo-nephrogenesis. Further experiments are required to determine whether cytokine stimulation of neo-nephrogenesis is a direct or indirect effect.

Funding

  • NIDDK Support