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Abstract: TH-OR038

Application of a Newly Engineered Podocyte Culture System to Study Intracellular Complement Production and Activation

Session Information

  • Bioengineering
    November 07, 2019 | Location: 146 A/B, Walter E. Washington Convention Center
    Abstract Time: 05:54 PM - 06:06 PM

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Satyam, Abhigyan, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Bhargava, Rhea, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Tsokos, Maria, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Tsokos, George C., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
Background

Current technologies do not support long-term cell viability, differentiation and maintenance of podocytes. We developed a biophysical approach, termed macromolecular crowding(MMC), to create extracellular matrix(ECM)-rich tissue equivalents and decellularization. This approach generates decellularized grafts that scaffold podocytes to grow in an environment similar to native conditions. To show a potential application of this newly designed culture system we studied complement(C) activation in podocytes exposed to IgG from individuals with lupus nephritis(LN).

Methods

Human skin fibroblasts were cultured under MMC and then decellularized. Human immortalized podocytes were cultured on the decellularized matrix(DCM) at 33°C for 7 days and subsequently at 37°C for 14days. ECM deposition in the DCM-coated dishes was analyzed by SDS-PAGE, immunofluorescence(IF) and scanning EM and expression of podocyte markers by western blotting(WB) and IF. Podocytes were then exposed to IgG from patients with LN and C production and activation was studied.

Results

We found that DCM-coated dishes contained all major ECM molecules(laminin, fibronectin, collagen I & IV) and podocytes survived and differentiated on DCM-coated plates significantly better than on noncoated plates, as shown by development of interdigitating foot process (fig.a) and increased expression of nephrin and synaptopodin. Podocytes exposed to LN IgG displayed increased levels of C factors(C3, C4, C5, C5b9) and C3 activation products(fig.b).

Conclusion

Engineering in vitro microenvironment with DCM enhances podocyte viability, native physiology and morphology. This novel system enabled us to demonstrate increased C factor production by podocytes exposed to LN IgG and intracellular complement activation.

(a) Better podocyte morphology with interdigitating foot process on decellularized culture dishes (b) Lupus IgG enhances production of activated C3 molecules

Funding

  • Other NIH Support