Abstract: TH-OR119

Interaction of SHROOM3 with FYN Impacts Phosphorylation of Nephrin Causing Proteinuria with Foot Process Effacement

Session Information

Category: Transplantation

  • 1701 Transplantation: Basic and Experimental

Authors

  • Menon, Madhav C., Mount Sinai School of Medicine, Forest Hills, New York, United States
  • Philippe, Nimrod, Mount Sinai Hospital, New York, New York, United States
  • Wei, Chengguo, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Liu, Ruijie, Mount Sinai Medical School, New York, New York, United States
  • Wong, Jenny, Mount Sinai School of Medicine, Forest Hills, New York, United States
  • Yi, Zhengzi, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Zhang, Weijia, Mount Sinai School of Medicine, Forest Hills, New York, United States
  • He, John C., Mount Sinai School of Medicine, Forest Hills, New York, United States
  • Murphy, Barbara T., Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background

In the GoCAR study, we identified that a CKD-associated SHROOM3-SNP, and tubular Shroom3 expression correlated with the development of renal fibrosis post-transplant. We showed that SHROOM3 facilitated TGF-B signaling suggesting its potential role as a therapeutic target. However, recent data suggest a protective role for SHROOM3 in glomerular development.

Methods

To study the role of SHROOM3 in adult glomeruli, we used doxycycline-inducible (DOX), shRNA-mediated SHROOM3 knockdown, Podocin- and tubular-specific (PAX8)-RTTA mice, comparing these to non-transgenic DOX-fed littermates.

Results

Adult Podo-RTTA mice, but not PAX8-RTTA, developed significant albuminuria compared to littermates with DOX. Albuminuria was reversible on DOX-withdrawal, and reappeared on re-initiation. EM revealed diffuse foot process effacement (Fig1). Glomerular RNA-seq identifed downregulated intracellular signaling/ actin-cytoskeleton among Gene-ontology terms in knockdown mice. We performed mass spectrometry on protein lysates of 293-T cells overexpressing SHROOM3 immunoprecipitated (IP) with either anti-V5, -SHROOM3 or IgG. Among 491 unique interactions, we identified FYN – a src-kinase – as a top ranking candidate. Podocyte FYN is crucial for NPHS1-phosphorylation, and FYN-deficient mice show a proteinuria phenotype. In human podocytes, we confirmed the interaction of endogenous SHROOM3 and FYN by IP. Glomerular protein extracts of Shroom3-knockdown mice showed decreased phosphorylation of FYN, and NPHS1. In human allografts from GoCAR, we identifed a corresponding reduced albuminuria (>1year post-transplant) associated with homozygosity of the risk allele in the donor.

Conclusion

In summary, Podocyte-specific SHROOM3 knockdown causes a reversible proteinuria phenotype in adult mice, by interacting with FYN, a mechanism distinct from its effect on renal fibrosis in allografts.