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Abstract: FR-PO698

Combining Multiphoton Microscopy With Super-Resolution Microscopy to Investigate Podocyte Injury in Acute Murine Kidney Slices

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology

Authors

  • Wiesner, Eva, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Binz, Julia, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Hackl, Agnes, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Unnersjö-Jess, David, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Benzing, Thomas, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
  • Hackl, Matthias, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
Background

Maintenance of low intracellular calcium levels is important for podocyte health, as impairments in its homeostasis lead to kidney disease. However, it remains unclear what effects increasing calcium signals have on the actin cytoskeleton and the slit diaphragm. To combine functional measurements with a high-resolution structural readout, we established a novel co-imaging approach using two distinct microscopy techniques: multiphoton and STED microscopy, in an acute murine kidney slices (AKS) model.

Methods

For disease induction, nephrotoxic serum was injected into 5-week old C57BL6 mice expressing GCaMP3 under the Pod:Cre promoter exclusively in podocytes. 5 days after injection the animals were sacrificed. The kidneys were removed and cut into slices of 300 μm thickness. The first imaging step was conducted at a multiphoton microscope to measure the fluorescence intensity of the GCaMP3 sensor. During acquisition, the slice was burned at several points in the kidney interstitium, later used as hallmarks. The AKS was fixed and prepared by anti-nephrin staining for follow-up STED microscopy. During STED imaging previously recorded glomeruli were identified by the burning points and high-resolution images of the slit diaphragm were acquired.

Results

Imaging of control animals showed low intracellular calcium levels in podocytes and an intact slit diaphragm architecture. In animals after induction of a nephrotoxic serum nephritis, the combination of multiphoton with STED microscopy revealed higher intracellular calcium levels in podocytes and a destruction of the slit diaphragm and demonstrated a correlation between intracellular calcium levels of podocytes and changes in the slit diaphragm morphology of different glomeruli in a single animal.

Conclusion

This new co-imaging approach combines the technical advantages of the individual imaging techniques and enables us to image the same glomerulus sequentially to correlate intracellular calcium levels with ultrastructural impairments of the slit diaphragm in a podocyte disease model. Our established co-imaging protocol using AKS can be applied to several research questions, providing both functional and ultrastructural information.

Funding

  • Government Support – Non-U.S.