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Abstract: SA-PO029

High-Dimensional Imaging of Postnatal Glomerulogenesis in the PodocyteTRAP Model by Simplified Tissue-Clearing Approach

Session Information

  • Bioengineering
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Preußner, Mathieu, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Einloft, Jonas, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Bedenbender, Simon, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Hofmeister, Andreas, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Grgic, Ivica, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
Background

The development of glomeruli is complex. Its dynamics and spatio-temporal coordination during the formation of the glomerular architecture are still poorly understood. Classical histopathological methods and microscopy techniques yield a rather limited scope of visualization and reconstruction of evolving glomeruli that can only be fully comprehended in a 3-dimensional context.

Methods

To study the successive buildup and maturation of podocytes during glomerulogenesis, we used neonatal kidneys (P0-P7) from transgenic PodocyteTRAP mice in combination with an adjusted ethyl cinnamate (ECi)-based clearing approach for immunostaining and subsequent 2-photon and light-sheet microscopy. We used IMARIS for detailed morphometric analysis and 3D-reconstruction of podocytes and glomeruli during advanced stages of kidney development.

Results

Tissue clearing is a technique to render biological samples transparent, thereby allowing for high-dimensional 3D-imaging of structures deep within the tissue without the need for tissue-sectioning. We used this technique for 3D-visualization, reconstruction and analysis of different glomerular developmental stages (renal vesicles, S-phase, capillary loop, maturing glomerulus) in transparent kidneys of P0 to P7 and adult PodocyteTRAP mice. ECi-clearing followed by 2-photon and light-sheet microscopy allowed for 10-fold larger imaging depth compared to uncleared kidneys (~1500µm vs. ~150µm). EGFP-L10a+ podocytes in ECi-treated PodocyteTRAP kidneys were easily identified by their robust epifluorescence, with eGFP-L10a intensifying as podocyte maturation progressed. We conducted detailed analyses which included comprehensive quantifications of glomerular volume changes during postnatal kidney development.

Conclusion

The combination of ECi-tissue clearing with 2-photon and light-sheet microscopy in the PodocyteTRAP model is well suited for high-dimensional imaging of renal tissue including detailed morphometry of maturing glomeruli in whole neonatal mouse kidneys. Moreover, this approach could also be useful for holistic histopathological analyses and assessments in various glomerular disease models including experimental GN and FSGS.

Funding

  • Government Support – Non-U.S.