Effect of Transglutaminase-Induced Stiffening of Glomerular Basement Membrane on Podocyte Function
- Glomerular Diseases: Podocyte Biology - II
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1403 Podocyte Biology
- Wang, Dan, The Ohio State University Wexner Medical Center, Columbus, Ohio, United States
- Madhavan, Sethu M., The Ohio State University Wexner Medical Center, Columbus, Ohio, United States
- Ferrell, Nicholas J., The Ohio State University Wexner Medical Center, Columbus, Ohio, United States
Glomerular basement membrane (GBM) and podocytes are important components of the glomerular filtration barrier. Podocytes are terminally differentiated cells that develop an intricate 3D architecture in vivo that is difficult to maintain in vitro. New approaches to maintaining podocyte differentiation may improve in vitro studies. Matrix stiffening is closely related to cellular function and GBM stiffness is altered in chronic kidney disease. How GBM mechanical properties affect molecular permeability and podocyte function is not fully understood. This work aims to investigate podocyte differentiation on native and transglutaminase crosslinked GBM and evaluate its effects on molecular transport.
Glomeruli were isolated from pig kidneys by sieving. The stiffness of native and transglutaminase (TG) crosslinked glomeruli was evaluated by a customized compression system. Decellularized and lyophilized glomerular matrix was rehydrated in PBS and coated on a 12-well plate. Immortalized human podocytes were plated on sterilized native GBM, TG treated GBM, and Matrigel coated plates. Podocytes were proliferated at 33°C, followed by transferring to 37°C for 14 days differentiation. Podocyte specific markers (nephrin, WT-1, and synaptopodin) and tight junction markers (ZO-1) were evaluated by immunofluorescence. Glomerular matrix was pressure compacted to Transwell membranes under pressure in a stirred cell to evaluate barrier function.
Crosslinking GBM with transglutaminase increases its stiffness. Podocytes express nephrin and WT-1 after 14 days differentiation in culture on native and crosslinked GBM, and Matrigel coated coverslips. There is no synaptopodin expression in undifferentiated podocytes, but it has strong expression in differentiated podocytes. Podocytes cultured on native GBM show clear ZO-1 staining but weak staining on the TG treated GBM. Diffusive permeability measured on GBM with cultured podocytes is significantly reduced after 14 days differentiation compared to 7 days differentiation. This suggests podocyte differentiation contributes to barrier permeability.
We provide an easy method to fabricate biomimetic GBM which can support podocyte differentiation during long-term culture. Stiffening of GBM affects podocyte differentiation and junction marker that may contribute to loss of filtration barrier integrity.
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