Abstract: TH-OR087

Protein Kinase 2b (PTK2b): A Novel Marker That Controls Urinary Pacemaker Cell Function

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 401 Developmental Biology

Authors

  • Rosenblum, Norman D., The Hospital for Sick Children, Toronto, Ontario, Canada
  • Iskander, Samir M., The Hospital for Sick Children, Toronto, Ontario, Canada
Background

Congenital non-obstructive hydronephrosis occurs in 0.5-1% of pregnancies. Previously, we demonstrated that murine congenital non-obstructive hydronephrosis is associated with absent expression of HCN3 and cKIT, which mark distinct populations of urinary tract pacemaker cells (utPMCs) located in the pelvis-kidney junction and ureter, respectively (JCI, 2011). Further, we showed that utPMCs are derived from the Wnt1+ neural crest (NC) cell lineage (Feeney M, ASN, 2015). Elucidation of the ontogeny and function of utPMCs in health and disease are limited by the lack of known cellular markers expressed earlier than the onset of expression of cKIT/HCN3 (E15.5). The goal of these studies is to identify novel utPMC proteins that control their function.

Methods

HCN3+ and cKIT+ cells were isolated and purified using FACS. RNAs differentially expressed in HCN3+ and HCN- cells were identified by RNA sequencing (Seq) and validated by quantitative (q) PCR. Protein expression was analyzed by immunoflouresence in situ. Contractile function was analyzed in murine pyeloureteric explants with time-lapse imaging. NC derived-cells were identified by TOMATO expression in Wnt1-Cre;ROSAtdTomato+ mice.

Results

RNASeq of FACS-isolated murine E18.5 HCN3+ utPMCs versus adjacent HCN3- cells, identified Ptk2b, a regulator of Ca2+ signaling and ion channel activity, among seven other novel gene transcripts enriched in HCN3+ cells (P<0.0001, n=3/group). Validation of RNASeq results by qPCR revealed 11.5- and 2.5-fold higher Ptk2b mRNA in FACS-isolated HCN3+ and cKIT+ utPMCs, respectively, compared to their adjacent HCN3- and cKIT- cells (P<0.05, n=3). Co-localization with TOMATO in Wnt1-Cre;ROSAtdTomato+ mice demonstrated PTK2B expression in NC cells as early as E12.5. Immunofluorescence in situ showed colocalization of PTK2b with HCN3 and cKIT as early as E15.5 with sustained PTK2B expression in cKIT+ and HCN3+ cells until P8 and adulthood, respectively. Treatment of murine pyeloureteric explants with the PTK2b inhibitors, PF431396 and Leflunomide (associated with human fetal non-obstructive hydronephrosis), decreased contraction frequency by 50% compared to vehicle-treated controls (P<0.01, n=3).

Conclusion

PTK2b is a novel marker of utPMCs that: (i) is expressed in neural crest cells before the onset of and coincident with HCN3 and cKIT expression, and (ii) controls pelvic-ureteric contraction.

Funding

  • Government Support - Non-U.S.