Abstract: SA-OR001

Novel Imaging Technique Reveals Changes in the Podocyte Actin Network after Injury

Session Information

  • A View on the Glomerulus
    November 04, 2017 | Location: Room 294, Morial Convention Center
    Abstract Time: 04:30 PM - 04:42 PM

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Suleiman, Hani, Washington University, Saint Louis, Missouri, United States
  • Shaw, Andrey S., Genentech, South San Francisco, California, United States
  • Miner, Jeffrey H., Washington University School of Medicine, St. Louis, Missouri, United States
Background

Actin stress fibers are abundant structures in cultured cells, including podocytes, yet no clearly equivalent structures have been observed in vivo. This discrepancy has been thought to be the result of current tissue preparation methods that do not preserve actin structures in their intact forms. Here we developed a new method that allows us to view actin stress fibers in their native state in the whole kidney glomerulus using focused ion beam scanning electron microscopy (FIB-SEM).

Methods

Kidney glomeruli from wild-type and Col4a3-/- (Alport) mice were isolated, the cytoskeleton was stabilized, and at the same time all cell membranes were extracted with detergent. Samples were processed and imaged using FIB-SEM using the serial block-face imaging mode.

Results

Block-face imaging of membrane-extracted healthy glomeruli showed all nuclei and basement membranes as electron dense material, similar to their appearance by transmission electron microscopy. In contrast, the cellular edges were completely gone, leaving behind only the contours of the cytoskeleton. This method allowed us to view the cytoskeleton of the foot processes (FPs), which formed a continuous electron dense sheet incorporating the slit diaphragms (SDs) and covering the glomerular basement membrance (GBM). Tracking this dense material across serial sections in the FPs revealed continuity with the central actin cables in individual FPs that fuse together to form thick actin bundles in the podocyte’s major processes (MP). These bundles, in turn, appear to anchor at the periphery of podocyte cell bodies. In contrast, analysis of Alport glomeruli revealed thick actin bundles running in different directions juxtaposed to the GBM as part of the cytoskeletal transformation that accompanies podocyte injury.

Conclusion

Here, we imaged the complete cytoskeleton of the podocytes in both health and disease. Our data indicate that in the normal podocyte, actin filaments in the FPs are connected to the SDs to form one continuous network enclosing the capillary wall, like a wire mesh, suggesting that podocyte FPs and the SDs work as one unit to form the outer structure of the glomerular filtration barrier. Our data also indicate that in injured podocytes, foot process effacement changes the orientation of the actin cables and affects the SDs, thus disrupting the integrity of the wire mesh, leading to proteinuria.

Funding

  • NIDDK Support