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Kidney Week

Abstract: FR-PO786

Nos1ap-/- Mice Replicate the Human Nephrotic Syndrome Phenotype Potentially via a CDC42-Diaphanous-Related Mechanism

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Buerger, Florian, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Majmundar, Amar J., Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Klambt, Verena, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Schneider, Ronen, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Deutsch, Konstantin, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Onuchic-Whitford, Ana C., Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Kitzler, Thomas Michael, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Mao, Youying, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Ashraf, Shazia, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Shril, Shirlee, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Harvard Medical School, 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. We previously identified causative recessive mutations in NOS1AP in two SRNS families and demonstrated that NOS1AP mutations impaired CDC42 activation, podocyte migration rate (PMR) and filopodia formation (Majmundar, JASN 29:682, 2018). To further delineate the pathogenesis of SRNS due to NOS1AP loss-of-function, we generated a mouse model and performed additional cell culture studies.

Methods

Homozygous Nos1apex3-/ex3- C57Bl/6 mice, lacking Nos1ap exon 3 (Int Heart J 57:341, 2016), were screened monthly for proteinuria, and urine albumin-creatinine-ratios, BUN and serum creatinine levels were determined. We conducted live cell imaging in a human podocyte cell line under shRNA-mediated downregulation of NOS1AP and cDNA over-expression of NOS1AP, CDC42 or DIAPH3 as well as pharmacologic inhibition of DIAPH proteins (by SMIFH2).

Results

We examined Nos1apex3-/ex3- mice carrying biallelic Nos1ap Exon 3 deletion alleles for albuminuria. Mice developed albuminuria starting at the age of 5 months when compared to their wild-type and monoallelic littermates but had not developed renal failure by the age of 12 months. In a podocyte cell culture system we further studied the role of NOS1AP within the signaling pathway of CDC42 and its downstream effectors, the diaphanous proteins. Filopodia formation upon NOS1AP cDNA overexpression was assessed in the presence of the DIAPH protein inhibitor SMIFH2 and reduced in a dose-dependent manner similarly to prior CDC42 inhibiton by CASIN. Reversely, defective PMR, an established intermediate phenotype of SRNS, in NOS1AP shRNA-mediated knock-down podocytes was rescued by overexpression of DIAPH3 cDNA and constitutively active CDC42.

Conclusion

We show that the human SRNS phenotype due to recessive NOS1AP mutations is replicated in Nos1apex3-/ex3- mice. We demonstrate that DIAPH function is part of the CDC42 mediated pathogenesis of SRNS due to NOS1AP loss-of-function.

Funding

  • NIDDK Support