Kidney Week

Abstract: FR-PO755

A Hedgehog (Hh)-TGFb Signaling Axis Controls Murine Stromal Cell Development and Ureteropelvic Junction Patency

Session Information

• Development and Organoid Models
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 500 Development, Stem Cells, and Regenerative Medicine

Authors

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

Norman D. Rosenblum,

• Stronks, Katryna, Hospital for Sick Children, Toronto, Ontario, Canada

Katryna Stronks,

• Sheybani-Deloui, Sepideh, Hospital for Sick Children, Toronto, Ontario, Canada

Sepideh Sheybani-Deloui,

• Mulder, Jaap, The Hospital for Sick Children, Toronto, Ontario, Canada

Jaap Mulder,

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

Robert D'Cruz,

Background

Signaling mechanisms that control stromal differentiation from Osr1+ progenitors during normal and malformed renal development are largely undefined. Hh proteins bind cell surface PTCH1, thereby disabling PTCH1-mediated inhibition of SMO and increasing Hh-mediated transcriptional activity. We showed that Cre-mediated Ptch1 deletion in Osr1+ progenitor cells at mouse E9.5 increased Hh signaling and induced formation of an ectopic population of Foxd1+Raldh2+ cells that block the ureteropelvic junction (UPJ) and are present in obstructing tissue in human UPJO (Shebani-Deloui et al, 2018). Here, we investigated molecular mechanisms that function downstream of Hh in this developmental disease context.

Methods

Hh and TGFb signaling was investigated in mice with Tamoxifen (TM)-induced deficiency of either Ptch1, Tgfbr2, or both. EGFP+Osr1+ cells were isolated by flow sorting and analyzed by RNASeq. Kidney tissue was analyzed by histology, immunostaining, and light sheet fluorescence imaging. TGFbRII deficiency was induced with ITD-1, which causes proteosomal degradation of TGFbRII (Willems et al, 2012), at the stage of cell aggregation in human kidney organoid tissue.

Results

RNASeq of Osr1+ cells isolated from urogenital ridge of Osr1-EGFP-Cre^ERt2;Ptch1^loxP/- mice at E11.5, 2 days post TM, demonstrated increased Tgfb2 expression compared to controls (P=0.02). Yet, TM-dependent Cre-mediated Tgfbr2 deficiency induced in Osr1+ cells at E9.5 revealed normal renal structural development and cell differentiation. In contrast, promotion of TGFbR2 degredation with ITD-1 in BiPS-derived human kidney organoid tissue treated from the stage of aggregation inhibited organoid growth, a finding consistent with nephron deficiency in mutant mice with Tgfbr2 deficiency in both Foxd1+ and Six2+ cell (Rowan et al, Development, 2018). Analysis of mice at E18.5 with both Tgfbr2 and Ptch1-deficiency (Osr1-EGFP-Cre^ERt2;Ptch1^loxP/-;Tgfbr2^loxP/-), induced at E9.5, demonstrated a marked rescue of hydronephrosis (5/11 Ptch1;Tgfbr2-deficient vs. 13/15 Ptch1-deficient mice; P=0.02 ) and a major decrease in ectopic peri-ureteric RADH2-positive stromal cells.

Conclusion

Increased Hh signaling in Osr1+ cells increases TGFB signaling that controls formation of ectopic Raldh1+ stromal cells and obstruction of the UPJ.

Funding

• Government Support - Non-U.S.