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Kidney Week

Abstract: FR-PO1003

Human NOS1AP Recessive Mutations Impair Podocyte Filopodia Formation and Cell Migration and Cause Steroid-Resistant Nephrotic Syndrome Through CDC42 Dysregulation

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States
  • Klambt, Verena, Boston Children's Hospital, Boston, Massachusetts, United States
  • Schneider, Ronen, Boston Children's Hospital, Boston, Massachusetts, United States
  • Kitzler, Thomas, Boston Children's Hospital, Boston, Massachusetts, United States
  • Widmeier, Eugen, Boston Children's Hospital, Boston, Massachusetts, United States
  • Braun, Daniela A., Boston Children's Hospital, Boston, Massachusetts, United States
  • Shril, Shirlee, Boston Children's Hospital, Boston, Massachusetts, United States
  • Soliman, Neveen, Cairo University, Cairo, Egypt
  • Aufricht, Christoph, Medical University of Vienna, Vienna, Austria
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
Background

Steroid resistant nephrotic syndrome (SRNS) is the second leading cause of chronic kidney disease in the first three decades of life. Mutations in >39 genes provide a monogenic cause in up to 29.5% of SRNS cases (JASN 26:1279, 2015) with defined patho-mechanisms (NDT 31:1802, 2016) including dysregulation of the small GTPase CDC42 (Ashraf Nat Commun 2018, in press).

Methods

Whole exome sequencing (WES) was performed in ~500 SRNS subjects to discover a novel genetic cause. IF was performed on rat kidney sections. In a human podocyte cell line, we performed active-CDC42 ELISA, live cell imaging as well as shRNA-mediated downregulation and cDNA over-expression of NOS1AP.

Results

We identified homozygous recessive mutations in 2 unrelated children with SRNS in NOS1AP (c.428G>A; p.C143Y and c.345-3T>G; p.I116Afs*4). NOS1AP encodes an adaptor protein that interacts with nitric oxide synthase (NOS) and with NOS effectors through its phospho-tyrosine binding domain (PTB) (Neuron 28:183, 2000). The C143 residue within the PTB is conserved to C. elegans and in 85/101 human PTB sequences and registered strong in silico conservation scores. The c.345-3T>G coding change is predicted to reduce splicing, cause skipping of exon 5, and result in the truncation p.I116Afs*4. Both mutations are not observed, even heterozygously, in the gnomAD Genome Aggregation Database. NOS1AP is expressed selectively in podocytes, not endothelial or mesangial cells, of rat kidney glomeruli. Transfection of wild-type NOS1AP induced filopodia in 58% of immortalized human podocytes, while NOS1AP constructs containing SRNS subject mutations failed to generate filopodia. NOS1AP shRNA-mediated knockdown reduced podocyte migration. This was rescued by wild-type NOS1AP over-expression but not by NOS1AP mutants. Active CDC42 levels, which promote filopodia and migration (Curr Opin Cell Biol 36:103, 2015), were induced by wild-type NOS1AP but not by patients’ mutants. Induction of filopodia by NOS1AP over-expression was inhibited by CDC42 chemical inhibitor CASIN.

Conclusion

We discovered recessive NOS1AP mutations as a novel monogenic cause of SRNS, leading to podocyte dysfunction through CDC42 dysregulation.

Funding

  • NIDDK Support