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Abstract: TH-PO775

Glomerular Basement Membrane Composition Controls Podocyte Morphology and Downstream Signaling

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Lausecker, Franziska, University of Manchester, Manchester, United Kingdom
  • Randles, Michael J., The University of Manchester, Chester, United Kingdom
  • Hamidi, Hellyeh, University of Turku, Turku, Finland
  • Humphries, Jon, University of Manchester, Manchester, United Kingdom
  • Byron, Adam, University of Edinburgh, Edinburgh, United Kingdom
  • Miner, Jeffrey H., Washington University School of Medicine, St. Louis, Missouri, United States
  • Ballestrem, Christoph, University of Manchester, Manchester, United Kingdom
  • Zent, Roy, Vanderbilt Medical Center, Nashville, Tennessee, United States
  • Humphries, Martin J., University of Manchester, Manchester, United Kingdom
  • Lennon, Rachel, University of Manchester, Manchester, United Kingdom
Background

Type IV collagen α3,4,5 networks are specific to the basement membranes in the eye, ear and the kidney. Absence of the heterotrimer leads to Alport syndrome, characterized by basement membrane defects and in the glomerulus by podocyte foot process effacement and detachment. Previous studies have shown that podocytes in Alport syndrome are exposed to ectopic matrix ligands as type IV collagen α1,1,2. Here, we investigated how exposure of podocytes to distinct matrix ligands influences molecular signaling events.

Methods

Human podocytes were spread on type IV collagen or laminins (511 or 521), and cellular morphology was assayed using light microscopy. Podocyte adhesion complexes were isolated and we analysed global protein levels on the different ligands using mass spectrometry-based proteomics. We used laser-based micropatterning to restrict cellular morphology and dissect the links between cell geometry and matrix ligand signalling and to determine how these factors regulate intracellular signaling in time and space.

Results

Matrix ligands determined distinct podocyte morphologies: podocytes on type IV collagen were rounded, compared to podocytes on laminin isoforms, which were more elongated. Proteomic analysis revealed ligand-dependent adhesion complex composition, with upregulation of PKCa in laminin-spread podocytes. Ratiometric imaging revealed increased focal adhesion signaling in podocytes spread on type IV collagen, associated with increased Rac1 signaling. Interestingly, when podocytes were restricted to micropatterned laminin circles or collagen IV lines, there was a partial rescue of the focal adhesion-signaling phenotype.

Conclusion

We show that exposure to different basement membrane ligands results in altered downstream signaling and podocyte morphology. Furthermore, we suggest that podocyte morphology controls downstream signaling independently of the basement membrane ligand. Loss of the mature type IV collagen α3,4,5 network exposes podocytes to a range of different matrix ligands. Our data suggest that altered basement membrane composition may contribute to podocyte effacement in Alport syndrome through changes in podocyte morphology and signaling. Rescuing these defects in podocyte morphology could represent a new therapeutic strategy.