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Kidney Week

Abstract: PO0311

Stiffening of Decellularized Tubular Basement Membrane Regulates Renal Tubular Epithelial Cell Function

Session Information

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

Category: Bioengineering

  • 300 Bioengineering

Authors

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

Damage to renal proximal tubular epithelial cells (RPTEC) plays an important role in chronic kidney disease. Epithelial cells are supported by a specialized extracellular matrix called the basement membrane (BM). The structure of the BM is altered in various kidney diseases such as diabetic nephropathy and may result in increased BM stiffness. We have developed a novel cell culture model that utilizes tissue derived tubular basement membrane (TBM) with tunable stiffness as a culture substrate for RPTEC. The aim of this study was to determine if TBM stiffening promotes activation of pro-fibrotic signaling pathways and/or regulates RPTEC differentiation.

Methods

TBM was isolated from decellularized porcine kidneys. TBM cell culture substrates were made by pressure compacting the TBM on Tranwell inserts. Conditionally immortalized mouse RPTEC were grown TBM substrates. To induce stiffening, TBM was treated with the chemical crosslinker genipin. Decellularized TBM was characterized by western blot and immunofluorescence staining. Viability and morphology of RPTEC were performed on TBM of varying stiffnesses. Real-time PCR was performed on RPTEC to evaluate the effect of stiffness on multiple genes related to kidney fibrosis and RPTEC differentiation.

Results

Western blot analysis of decellularized TBM showed the presence of laminin and collagen IV and absence of lamin B1 showing proper decellularization of TBM. Genipin treatments (0.05% and 0.5%) resulted in average stiffness of 2 kPa and 3.2 kPa respectively, compared to 0.5 kPa for untreated TBM. Neither decellularization nor genipin modification had a significant effect on cell viability. Pro-fibrotic downstream targets of YAP activation (CTGF, AREG, and ANKRD1) were upregulated on stiff TBM substrates. Additionally, stiffness regulated expression of cell-cell junction markers E-cadherin and N-cadherin.

Conclusion

A new cell culture model was developed using tissue derived TBM as a culture substrate for tubular epithelial cells. Stiffness of the TBM was tuned using genipin. Increased TBM stiffness upregulated pro-fibrotic targets of YAP activation and altered RTPEC cell differentiation. These data show that stiffness significantly affects renal tubular cell function and suggest that TBM stiffening in chronic kidney disease may play a role in disease progression.

Funding

  • NIDDK Support