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Abstract: SA-PO336

Increased mTOR Signaling Secondary to a Human Integrin-Linked Kinase (ILK) Missense Variant Inhibits Nephrogenesis with Decreased ATP Generation

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine

Authors

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

Signaling pathways that control nephrogenesis are critical to our understanding of kidney health and disease. Previously, we demonstrated increased mTOR signaling in vitro by overexpression of a CAKUT-associated human missense variant (T173I) of Integrin-Linked Kinase (ILK), a key regulator of renal development. Here, we investigate the hypothesis that ILK-T173I disrupts renal development in vivo via dysregulation of mTOR signaling.

Methods

mTOR signaling was analyzed in Ilk-T173I knock-in mice with Ilk-T173I replacing the WT Ilk allele. Morphogenic effects of Ilk-T173I on renal development were quantitated. Ureteric and non-ureteric cell populations were isolated by FAC sorting. Gene expression was analyzed by whole genome RNA microarray and qPCR. Cell proliferation and nephron maturation were evaluated by immunostaining and specific markers. Energy metabolism was characterized using the Seahorse assay.

Results

Homozygous Ilk-T173I knock-in mice were characterized by a 13.6% decrease in nephron number (n=6, P=0.04), a 35.2% decrease in ureteric branching (n=5, P=0.006) and an 88% increase in mTOR signaling (n=3, P=0.01). Rapamycin treatment of Ilk-T173I-knock-in embryonic kidney explants rescued ureteric branching to normal levels. Genome-wide RNA expression analysis in Ilk-T173I-knock-in ureteric and non-ureteric cell populations demonstrated elevated mTOR signaling in non-ureteric cells only. Ilk-T173I-knock-in non-ureteric populations expressed a 431% increase in Osr1 (n=3, P=0.002), a mesenchymal cell progenitor marker, and a 54% decrease in Lhx1 (n=3, P=0.04), a marker for maturing nephrons. Ilk-T173I knock-in embryonic kidneys demonstrated a 2075% increase in phospho-Histone H3-labeled cells (n=3, P=0.0005), an indicator of cell proliferation, in the ureteric, nephrogenic and stromal lineages, and a 16.1% decrease in the percentage of S-shaped bodies (n=4, P=0.01), the mature form of nephrogenic intermediates. Metabolic profile of Ilk-T173I-knock-in, non-ureteric cells exhibited a 17% decrease in both oxidative ATP production (n=3, P=0.04) and total ATP production (n=3, P=0.02).

Conclusion

These data show that human Ilk-T173I variant impairs renal development in a mTOR-dependent manner, specifically acting within the non-ureteric cell population by increasing proliferation and disrupting maturation and cell metabolism.

Funding

  • Government Support – Non-U.S.