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Abstract: PO1391

Molecular Changes Associated with Type IV Collagen Switching in 1-Day-Old Alport Murine Glomeruli

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Rana, Akanchaya, Toronto General Research Institute, Toronto, Ontario, Canada
  • Farkona, Sofia, Toronto General Research Institute, Toronto, Ontario, Canada
  • Scholey, James W., Toronto General Research Institute, Toronto, Ontario, Canada
  • Konvalinka, Ana, Toronto General Research Institute, Toronto, Ontario, Canada
  • Barua, Moumita, Toronto General Research Institute, Toronto, Ontario, Canada
Background

Alport syndrome (AS) is an inherited disorder caused by pathogenic variants in COL4A3, COL4A4 or COL4A5, which encodes proteins that comprise basement membranes of the ear, eye and kidney glomerulus. Type IV collagen chains assemble as heterotrimers and during glomerular development, α1α2α1 (IV) is replaced by α3α4α5 (IV) within the developing glomerular basement membrane (GBM). This “switching” defines the starting point of disease in AS. We aimed to identify the molecular changes at the time of disease initiation in the developing glomeruli of Alport murine kidneys.

Methods

Immunofluorescence (IF) staining was done to identify the GBM distribution of type IV collagen chains in 1 day old COL4A3 knockout (KO) and wildtype (WT) mice. Urine albumin to creatinine ratio (uACR) was also measured. Subsequently, glomeruli from 1 day old (P1) COL4A3 KO and WT mice were isolated by cardiac injection of magnetic dynabeads that embolized to glomerular capillaries enabling their extraction from surrounding tissue. Protein was isolated and subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis.

Results

IF localized type IV collagen α1 in the GBM, Bowman’s capsule and mesangial matrix in both P1 KO and WT mice. Type IV collagen α5 was present in short segments of some developing GBM in P1 WT but was absent in KO mice indicating switching. uACR was increased in P1 KO (423.61±396.13 mg/mmol) compared to WT (128.67±69.86 mg/mmol) with a p-value of 0.02. LC-MS/MS identified >4300 proteins from glomerular isolates. In males, 2 and 15 proteins were significantly upregulated and downregulated, respectively, in KO compared to WT mice. In females, 543 and 978 proteins were significantly upregulated and downregulated, respectively, in KO compared to WT mice. Pathway analysis revealed alteration in collagen metabolic process, collagen biosynthesis, collagen formation and extracellular matrix organization.

Conclusion

Increased uACR in P1 KO mice showed disease onset at the time of type IV collagen switching. LC-MS/MS analysis revealed dysregulation of matrix turnover pathways in P1 KO mice, identifying potential molecular targets.

Funding

  • Private Foundation Support