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Kidney Week

Abstract: FR-PO747

Angiomotin Knockout Causing Glomerulotubular Nephropathy May Be via an Activated Hippo Signalling Pathway

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Zhang, Yaochun, National University of Singapore, Singapore, Singapore
  • Binti Ahmad, Nurul Jannah, National University of Singapore, Singapore, Singapore
  • Lin, Qingsong, National University of Singapore, Singapore, Singapore
  • Ward, Jerrold m., Global VetPathology, Montgomery Village, Maryland, United States
  • Sun, Zi Jin, National University of Singapore, Singapore, Singapore
  • Yap, Hui Kim, National University Hospital, Singapore, Singapore
  • Ng, Kar Hui, National University of Singapore, Singapore, Singapore

Group or Team Name

  • pae/imm
Background

Angiomotin (Amot) is an angiostatin binding protein involved in endothelial cell migration. Using CRISPR/Cas9, we created Amot knockout (KO) rats that developed cysts from 1 month and proteinuria by 6 months. Histology revealed dilated tubules, podocyte hypertrophy, foot process effacement, thick glomerular and tubular basement membranes. We aimed to elucidate the pathomechanism of Amot KO with iTRAQ (isobaric tags for relative and absolute quantitation) based quantitative proteomics.

Methods

Kidney cortex from 1-mth rats were homogenized and labelled with iTRAQ. Quantitative proteomic analysis was performed using 2D-nLC-MS/MS. Peptide identification and quantification was carried out on ProteinPilot 5.0 software using Paragon database search algorithm (5.0.0.0.4767) and integrated false discovery rate(FDR) analysis function. Data were searched against UniProtKB protein sequence databases. Statistically significant differences were based on fold change ≥1.5 and p-value <0.05. Rat glomeruli were isolated for ex vivo podocyte culture or total RNA extraction. Immunofluorescence(IMF) and western blot(WB) were performed on podocytes for Yap (Yes-associated protein) nucleus translocation analysis. Real-time PCR were performed for Yap target genes.

Results

5030 proteins were quantified with confidence corresponding to peptide and protein FDR <0.01 and with ≥2 unique peptides per protein. We identified 101 up-regulated and 209 down-regulated proteins in KO compared to WT rats. Expression of TEAD1, a major YAP target transcription factor, was significantly decreased. IMF and WB showed increased Yap nucleus deposition. Transcriptional expression of YAP target genes (Ankrd1, Cyr61, Diaph3, Anln and Ctgf) were increased.

Conclusion

In vivo Amot KO caused Yap nuclei translocation and activated the Hippo/Yap pathway.

Funding

  • Government Support - Non-U.S.