Abstract: FR-PO693
Altered Podocytes and Glomerular Stiffness in Alport Syndrome Occurs in Association With Increased SMPDL3b Expression
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Gurumani, Margaret Zvido, University of Miami School of Medicine, Miami, Florida, United States
- Mallela, Shamroop Kumar, University of Miami School of Medicine, Miami, Florida, United States
- Mitrofanova, Alla, University of Miami School of Medicine, Miami, Florida, United States
- Pressly, Jeffrey D., University of Miami School of Medicine, Miami, Florida, United States
- Moy, Vincent T., University of Miami School of Medicine, Miami, Florida, United States
- Merscher, Sandra M., University of Miami School of Medicine, Miami, Florida, United States
- Fornoni, Alessia, University of Miami School of Medicine, Miami, Florida, United States
Background
Alport Syndrome (AS) is a hereditary disease caused by mutations in different chains of collagen IV, a component of the Glomerular Basement Membrane (GBM). While alterations in the composition of GBM that occur during pathogenesis has been extensively studied, less is known about the immediate cellular consequences occurring in podocytes contacted with an altered GBM. Sphingolipids are essential plasma membrane (PM) constituents, and the PM sphingolipids composition regulates the mobility of integrins, thereby promoting cell adhesion. We, and others have identified sphingomyelin-phosphodiesterase-acidlike-3b (SMPDL3b), an enzyme expressed in lipid raft domains of podocytes, where it regulates receptor assembly in caveolin-rich PM domains and where it regulates the availability of active sphingolipids such as ceramide 1 phosphate (C1P). With this study, we test the hypothesis that increased SMPDL3b expression in podocytes in the context of AS contributes to altered glomerular/podocyte stiffness.
Methods
Human podocyte cell lines transfected with a thermosensitive SV40- T construct and AS podocytes established from AS and wild type (WT) control mice were used for analysis. Atomic force microscopy (AFM) was used to assess the stiffness of podocyte in vitro and of micro dissected glomeruli ex vivo. RT-PCR was used to determine SMPDL3b expression in sieved glomeruli. PM fluidity was determined using a membrane fluidity kit from ABCAM.
Results
The expression of SMPDL3b was increased in AS podocytes and glomeruli when compared to WT. AS podocytes and glomeruli are associated with a significant decrease in stiffness when compared to WT. However, no changes in PM fluidity were observed in SMPDL3b OE podocytes when compared to control. SMPDL3b OE podocytes are also associated with a decrease in cell stiffness. C1P replenishment restores cell stiffness in SMPDL3b OE podocytes.
Conclusion
Our data demonstrate that SMPDL3b expression is increased in AS podocytes and glomeruli. Both SMPDL3b OE and AS podocytes and glomeruli are characterized by decrease in stiffness. Further studies are required to determine if SMPDL3b mediates cells and glomerular stiffness in AS and to determine how changes in stiffness affect the interaction with other components of filtration barrier.
Funding
- NIDDK Support