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Kidney Week

Abstract: PO0302

An In Vitro Model of the Glomerular Filtration Barrier Using Tissue-Derived Glomerular Basement Membrane

Session Information

  • Bioengineering
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Wang, Dan, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Sant, Snehal, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Ferrell, Nicholas J., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background

The glomerular filtration barrier consists of the glomerular basement membrane (GBM), podocytes and endothelial cells that regulate kidney permeability to macromolecules. Damage to podocytes increases albumin permeability, resulting in proteinuria. Interactions between podocytes and the GBM are important for regulating glomerular permeability and are not captured in standard in vitro cell culture systems. This work aims to investigate molecular permeability of the GBM and podocytes using a novel in vitro model that incorporates decellularized GBM.

Methods

GBM substrates were made by pressure compacting decellularized glomeruli from porcine kidneys against a Transwell membrane in a stirred cell. GBM was evaluated by immunofluorescence staining of decellularized glomeruli. Mouse podocytes were plated on the GBM at low and high concentrations. Transepithelial electrical resistance (TEER) was measured before molecular permeability measurements. Podocytes on GBM were imaged by staining with phalloidin and DAPI. Permeability of the GBM with and without podocytes were analyzed by measuring FITC-BSA and FITC-Ficoll diffusion through the filtration barrier.

Results

GBM characterization showed that cells are efficiently removed from the glomeruli, and the GBM retains laminin and collagen IV after decellularization. GBM alone provided a stringent barrier to diffusion of both albumin and Ficoll. Podocytes attached and spread on the GBM to further restricted albumin diffusion. TEER showed an increased resistance of GBM with podocyte compared to GBM alone. Podocytes resulted in slightly lower permeability at high seeding concentration than low concentration.

Conclusion

Interactions between the GBM and podocytes are important for regulating the permeability of the glomerulus. We developed a new in vitro model of the glomerular filtration barrier that incorporates tissue derived GBM to support podocyte culture. GBM alone restricted albumin and Ficoll diffusion and incorporation of podocytes further restricted albumin diffusion. Future work will focus on the co-culture of podocytes and endothelial cells on both sides of the GBM for evaluating the permeability of the filtration barrier and evaluate how podocyte and endothelial injury regulate permeability.

Funding

  • NIDDK Support