Abstract: PO1343
A Human Missense Integrin-Linked Kinase Variant Negatively Regulates Murine Renal Branching Morphogenesis via mTOR Signaling
Session Information
- Genetic Diseases of the Kidneys: Non-Cystic - II
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Hu, Xiangyue, Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
- Kablawi, Dana, Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
- Renkema, Kirsten Y., Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, Utrecht, Netherlands
- Knoers, Nine V., Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, Utrecht, Netherlands
- Rosenblum, Norman D., Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
Background
Branching morphogenesis is critical to kidney development and the pathogenesis of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). Identification of gene variants via genomic sequencing aims to elucidate molecular mechanisms underlying CAKUT. The pathogenic contributions of such variants are largely unknown; functional analyses are required to identify pathogenic mechanisms. Here, we identify pathogenic effects of a CAKUT-associated human missense variant of Integrin-Linked Kinase (ILK), a key regulator of renal branching morphogenesis, on ureteric branching.
Methods
Targeted gene panel sequencing was performed to identify gene variants. ILK-T173I function was investigated in mouse inner medullary collecting duct (mIMCD3) cells and mouse embryonic kidney explants transduced with lentivirus expressing ILK-T173I. Gene expression was analyzed by RNA microarray and validated by qPCR and Western analysis. Mutant mice with a ILK-c.518C>T point mutation were generated using CRISPR/Cas9. Morphogenic effects of ILK-T173I on ureteric branching were visualized using Hoxb7-driven fluorescent marker (MyrVenus) and quantitated by counts of ureteric bud tips and nephrons.
Results
An ILK missense variant, ILK-T173I, was identified in a CAKUT patient and her mother by targeted gene panel sequencing and verified by Sanger sequencing. mIMCD3 cells expressing ILK-T173I demonstrated dysregulated expression of AKT/mTOR target mRNAs, identified by RNA microarray and qPCR, and elevated levels of phospho-p70-S6Kinase, a mTOR target (n=3, P=0.03). Overexpression of ILK-T173I in embryonic kidney explants increased phospho-p70-S6Kinase expression (n=3, P=0.03) and decreased ureteric tip number by 50% (n=15, P=0.003), both of which were rescued by treatment with Rapamycin, an mTOR inhibitor (n=4, P=0.04). Knock-in mice in which ILK-T173I replaces the ILK-WT allele were characterized by low nephron number (n=6, P=0.04) and decreased ureteric branching (n=5, P=0.006), and increased expression of phospho-p70-s6Kinase (n=3, P=0.014). Treatment of mutant cultured embryonic kidney explants with Rapamycin rescued ureteric branching to levels observed in Ilk-WT mice.
Conclusion
Human Ilk-T173I variant decreases branching morphogenesis in a mTOR-dependent manner. Increased mTOR signaling disrupts mouse kidney development.
Funding
- Government Support – Non-U.S.