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Abstract: PO1399

Effects of Enzymatic Cross-Linking and Increased Stiffness on Glomerular Basement Membrane and Podocyte Function

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

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

The Glomerular basement membrane (GBM) is a critical component of the glomerular filtration barrier. Stiffening of the extracellular matrix is an important regulator of cellular function. How GBM stiffening affects podocyte function is not fully understood. This work aims to investigate the effect of GBM stiffening on molecular permeability and the podocyte function using a biomimetic in vitro model directly derived from kidney glomeruli.

Methods

Decellularized glomeruli isolated from porcine kidneys were pressure compacted on a Transwell membrane. GBM stiffness was tuned by crosslinking with transglutaminase (TG). The stiffness of the TG-crosslinked decellularized glomeruli was evaluated using a custom cantilever-based compression system Podocytes cultured on the TG crosslinked GBM were immunofluorescence stained with YAP, phalloidin and the nuclei were counterstained with DAPI. The diffusional molecular permeability was evaluated on native and TG crosslinked GBM with and without podocytes using FITC-Ficoll and AF488-BSA. Effects of GBM stiffening on gene expression of markers of podocyte differentation (NEPH1, WT1, Synaptopodin) were screened by qPCR.

Results

The stiffness of the decellularized glomeruli showed a dose-dependent increase after incubating with TG for 1 day and 4 days. On stiffer GBM, immunofluorescence imaging showed translocation of YAP to the podocyte nucleus. Passive molecular permeability of GBM was similar for native the TG crosslinked GBM. Podocytes cultured on both native and TG crosslinked GBM forms a stringent barrier to large molecules. qPCR results show an upregulation of differentiation markers as podocytes cultured on native and TG crosslinked GBM.

Conclusion

We developed a biomimetic in vitro model that fabricated directly from kidney tissue. mTG crosslinked glomeruli show a dose-dependent increase of stiffness. TG did not significantly effect the diffusive permeabilty of the GBM. GBM stiffness affects the YAP localization in the podocytes. The current in vitro model upregulates the gene expression of podocyte markers.

Funding

  • Other U.S. Government Support