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Abstract: PO0873

Constitutive Activation of Hedgehog Signaling Disrupts Nephrogenic and Stromal Differentiation

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • D'Cruz, Robert, Hospital for Sick Children, Toronto, Ontario, Canada
  • Kim, Yun-Kyo, Hospital for Sick Children, Toronto, Ontario, Canada
  • Mulder, Jaap, Hospital for Sick Children, Toronto, Ontario, Canada
  • Rosenblum, Norman D., Hospital for Sick Children, Toronto, Ontario, Canada
Background

Nephron progenitors (NPs) and stromal cells differentiate from a common Osr1+ progenitor. While maldifferentiation of nephrogenic and stromal tissue is a hallmark feature of renal dysplasia, signaling mechanisms regulating the genesis of stroma relative to NPs are largely undefined. We have shown that increased Hedgehog (Hh) signaling in murine Osr1+ cells in vivo causes urinary tract obstruction through abnormal stromal cell localization (Sheybani-Deloui et al., 2018). Here, we investigated mechanisms that function downstream of Hh to control NP and stromal cell differentiation using human induced pluripotent stem cell (hiPSC) kidney organoids and genetic mouse models.

Methods

Agonists of the Hh receptor, SMO, were added to hiPSCs differentiated into kidney organoids at the stage of cell aggregation. Mature organoids were analyzed by histology, light sheet fluorescence microscopy, and RNA microarray. Processes downstream of Hh signaling were investigated in mouse kidneys with deficiency of Ptch1 specific to FOXD1(+) stromal cells (FoxD1Cre;Ptch1loxP/-) using histology, RNAseq, and scRNAseq.

Results

Stimulation of Hh activity in kidney organoids with SAG (120 nM) or Purmorphamine (10 µM) resulted in a 26% increase in surface area compared to controls. Volumetric analysis using light sheet fluorescent imaging of WT1+ nephrogenic structures and CDH1+ tubular structures in SAG-treated organoids demonstrated an 88% (n=3, p<0.01) and 67% (n=3, p<0.05) reduction, respectively. In contrast, the mass of non-epithelial cells was increased by 79% (n=2, p<0.05). RNA microarray analysis of SAG-treated organoids (n=3) revealed elevated expression of medullary stromal markers TnC (2.60 fold-change [FC], p<0.01) and Pdgfrb (1.60 FC, p<0.01), and decreased expression of nephron markers Nphs1 (0.23 FC, p<0.01), Slc3a1 (0.32 FC, p<0.01), and Slc12a1 (0.08 FC, p<0.001). Mice with constitutive Hh activity in FOXD1+ stromal cells showed a 41% reduction in nephrons at E18.5 (n=4, p<0.05) and a 19.5% decrease in nephron intermediate structures at E15.5 (n=4, p<0.01). In contrast, RNAseq of E13.5 mutant kidney tissue demonstrated increased expression of medullary stroma genes TnC and Pdgfrb.

Conclusion

Increased Hh signaling in human and mouse increases differentiation of stroma compared to NPs, providing new insights into mechanisms that may underlie kidney dysplasia.

Funding

  • Government Support - Non-U.S.