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Abstract: TH-PO803

Investigating Basement Membrane Assembly

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Koudis, Nikki-Maria, University of Manchester, Manchester, United Kingdom
  • Loewenhardt, William, University of Manchester , Manchester, United Kingdom
  • Randles, Michael J., De Montfort University, Leicester, United Kingdom
  • Lennon, Rachel, University of Manchester, Manchester, United Kingdom
  • Derby, Brian, University of Manchester , Manchester, United Kingdom
Background

The glomerular basement membrane (GBM) is a specialised condensed network of extracellular matrix. Monocultures of podocytes and endothelial cells synthesise and organise a complex matrix in vitro and this has significant overlap with the components of the GBM in vivo. Whilst these cultures assemble type IV collagen this is predominantly the α1α1α2 network and not the mature α3α4α5 network, which is required for the long-term integrity of the GBM. We hypothesised that the combination of glomerular cell coculture and flow would alter the composition of matrix and lead to a more mature phenotype with greater similarity to the GBM in vivo.

Methods

To generate flow we utilised the QV600 bioreactor (Kirkstall) and a custom-designed bioreactor constructed with the silicone-based polymer PDMS. Detachable podocyte and endothelial cell sheets were engineered and assembled into the bioreactors to create cocultures without the need for an intervening material for support. Cells and matrix were examined in static and flow conditions using light and electron microscopy and mass spectrometry (MS)-based proteomics. The function of this coculture system as a semi-permeable filtration barrier was examined using fluorescently labelled dextrans.

Results

Endothelial cells and podocytes readily formed cell sheets and could be assembled as cocultures into bioreactors. Both cell types were viable in coculture and in static and flow conditions. Electron microscopy revealed the presence of basement membrane-like structures between cocultured cells and functional analysis confirmed intact barrier function. Proteomic analysis of cells under flow conditions demonstrated an upregulation of basement membrane proteins including altered laminin and collagen IV isoforms compared to static conditions.

Conclusion

Podocyte and endothelial cells are viable in direct contact and under flow conditions and flow influences both the composition and assembly of matrix in our system suggesting roles for both cell-cell cross-talk and fluid flow in the assembly of basement membranes. This system has utility for understanding the basic biology of GBM formation and glomerular cell interaction with the GBM in addition to testing compounds that could repair a damaged GBM in the context of glomerular disease.

Funding

  • Commercial Support