Kidney Week

Abstract: PO0640

Constitutive Activation of Hedgehog Signaling in FOXD1+ Renal Stromal Cells Impairs Ureteric Epithelial Branching via CXCL12

Session Information

• Development, Stem Cells, and Regenerative Medicine
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 500 Development, Stem Cells, and Regenerative Medicine

Authors

• D'Cruz, Robert, The Hospital for Sick Children, Toronto, Ontario, Canada

Robert D'Cruz, MS

• Kim, Yun-Kyo, The Hospital for Sick Children, Toronto, Ontario, Canada

Yun-Kyo Kim,

• Rosenblum, Norman D., The Hospital for Sick Children, Toronto, Ontario, Canada

Norman D. Rosenblum,

Background

Renal dysplasia, characterized by disorganization of stromal tissue, malformation of collecting ducts, and decreased nephron number, is a major cause of renal failure in children. Nephrons and collecting ducts develop via reciprocal inductive interactions between two intermediate mesodermal tissue elements - the Osr1+ metanephric mesenchyme (MM), within which reside Six2+ nephrogenic and Foxd1+ stromal cell progenitors, and the Hoxb7+ ureteric bud, which gives rise to ureteric branches and their daughter collecting ducts. The key roles of expanded Foxd1+ stromal progenitors in the genesis of dysplastic tissue are largely undefined. Here, we identify a novel pathogenic stromal Hh-Cxcl12 signaling axis driving impaired ureteric epithelial branching.

Methods

Mice with deficiency of Ptch1 specific to FOXD1+ stromal cells (Foxd1Cre;Ptch1^loxP/-; referred to as stromal Ptch1-deficient) were analyzed by immunostaining, single-cell RNA sequencing, and bulk RNA sequencing. To investigate Cxcl12 as a downstream target of increased stromal Hh signaling, in vivo analysis of ureteric branching was assessed in Cxcl12 compound mutants (Foxd1Cre;Ptch1^loxP/-; Cxcl12^loxP/+).

Results

Stromal Ptch1-deficient kidneys exhibited renal dysplasia characterized by a 41% reduction in nephron number (P<0.01, n=4) at E18.5 and a 26% reduction in ureteric epithelial branch tips at E12.5 (P<0.01, n=6). Whole kidney qRT-PCR analysis at E13.5 further demonstrated a reduction in ureteric branching effectors Gdnf, Wnt11, Etv4, and Etv5 in stromal Ptch1-deficient mice (P<0.05, n=4). Bulk RNA sequencing of stromal Ptch1-deficient whole kidneys revealed a significant upregulation in numerous medullary stromal markers including the secreted stromal cell-derived factor Cxcl12 (P<0.01, n=3). Complementary single-cell RNA sequencing analysis further demonstrated upregulation of Cxcl12 in medullary stromal clusters of Ptch1-deficient kidneys relative to control samples. Analysis of branching morphogenesis in compound mutant mice heterozygous for Cxcl12 in a stromal Ptch1-deficient background revealed a complete rescue of ureteric epithelial branching defects (P<0.001, n=6).

Conclusion

A pathogenic increase in stromal Hh signaling in mice causes nephron deficiency and impaired ureteric epithelial branching due to increased Cxcl12 activation.

Funding

• Government Support – Non-U.S.