A Human Missense Integrin-Linked Kinase Variant Negatively Regulates Murine Renal Branching Morphogenesis via mTOR Signaling
Bibliographic record
Abstract
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. Conclusions: 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".