Abstract: SA-PO551

Human Missense Variants in Integrin-Linked Kinase Abrogate Renal Branching Morphogenesis by Disrupting MAPK Signaling

Session Information

  • Developmental Biology
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Developmental Biology and Inherited Kidney Diseases

  • 401 Developmental Biology

Authors

  • Kablawi, Dana, The Hospital for Sick Children, Toronto, Ontario, Canada
  • Renkema, Kirsten Y., University Medical Center Utrecht, Utrecht, UTRECHT, Netherlands
  • Knoers, Nine V., University Medical Center Utrecht, Utrecht, UTRECHT, Netherlands
  • Rosenblum, Norman D., The Hospital for Sick Children, Toronto, Ontario, Canada
Background

Integrin-linked kinase (ILK) is required for murine renal branching morphogenesis and acts via p38MAPK/ATF2 signaling. Yet, its contribution to human congenital kidney-urinary tract anomalies (CAKUT) is undefined. The objective of these studies is to identify ILK variants in CAKUT and define their functional consequences.

Methods

Patients with CAKUT were analyzed by exome sequencing. Variants were verified by Sanger sequencing. Cells stably expressing human ILK variants were generated by lentivirus transduction and clonal selection. Signaling pathway activity and transcription factor activation were analyzed using immunoblotting. Subcellular localization of transcription factors was imaged by immunofluorescence. Gene expression and pathway enrichment were assayed using whole genome microarray and quantitative (q) PCR. Ex vivo assays were conducted using lentivirus-based transduction of mouse embryonic kidney explants.

Results

Exome sequencing of 208 CAKUT candidate genes in 453 probands with CAKUT identified ILKT173I (1 proband) and ILKN22S (4 probands), both of which are missense variants in ILK ankyrin repeat domains. Stimulation of mIMCD3 cells stably expressing ILKT173I (versus ILKWT) with EGF (15 minutes) revealed increased phosphorylation of each of JNK, C-Jun and AKT (P<0.05, n=3), as well as increased nuclear translocation of P-C-Jun (P<0.05, n=3). c-Jun RNA, a JNK signaling target, was increased 2.6-fold (P<0.05, n=6, qPCR). Pathway enrichment analysis of genome-wide RNA expression after 1 hour of EGF stimulation revealed dysregulation of AKT/MTOR target genes (n=3). In contrast to ILKT173I, EGF mediated stimulation of mIMCD3 cells expressing ILKN22S increased ERK phosphorylation (P<0.05, n=3). Treatment of E12.5 kidney explants with lentivirus expressing ILKT173I resulted in hypoplasia with a 45% decrease in ureteric bud (UB) tip number (P< 0.025, n=9 kidneys). Treatment with lentivirus expressing ILKN22S generated a spectrum of phenotypes including reduced number of UB tips (n=6 kidneys, P<0.009), increased (1.5-fold) number of UB tips (n=6 kidneys, P<0.048), and ectopic branching of the main ureter (n=3 kidneys).

Conclusion

ILK ankyrin-repeat domain missense variants associated with human CAKUT cause distinct abnormalities in MAPK signaling and variable patterns of disrupted kidney explant development.

Funding

  • Government Support - Non-U.S.