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Abstract: PO0617

Three-Dimensional Visualization of Neonatal Glomerulogenesis in the PodoTRAP Model by Simplified Tissue-Clearing Approach

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Preußner, Mathieu, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Hofmeister, Andreas, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Bedenbender, Simon, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Roth, Katrin Kr, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Steinhoff, Ulrich Johannes, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Grgic, Ivica, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
Background

The process of glomerulogenesis is complex and the dynamics and spatio-temporal coordination involved in the formation of the glomerular architecture are still poorly understood. Conventional histopathological methods and 2D-microscopy techniques allow only a limited visualization and reconstruction of processes in the developing kidney which can only be fully appreciated in a 3-dimensional context.

Methods

To specifically study the organisation, maturation and arrangement of podocytes during glomerulogenesis, we used neonatal kidneys from PodoTRAP transgenic animals (P0, P3, P7) in combination with a modified ethyl cinnamate (ECi)-based clearing approach for immunostaining and subsequent 2-photon microscopy. We used IMARIS for comprehensive morphometric analysis and 3D-reconstruction of podocytes and glomeruli during postnatal kidney development.

Results

Tissue clearing is a technique to render biological samples transparent, thereby allowing for high resolution 3D-microscopic imaging of structures deep within the tissue without the need for conventional tissue-sectioning. We used this technique for 3D-imaging, reconstruction and analysis of different glomerular developmental stages (renal vesicles, S-phase, capillary loop, maturing glomerulus) in transparent kidneys of P0, P3, P7 as well as adult PodoTRAP mice. Eci-clearing followed by 2-photon microscopy achieved significantly higher imaging depth compared to uncleared kidneys (~1600µm vs. ~150µm). GFP+ podocytes in ECi-treated PodoTRAP kidneys were readily identified due to robust cellular epifluorescence, with GFP signal intensities increasing as podocyte maturation progressed. Amongst others, we conducted comprehensive quantification of glomerular volume increases during postnatal kidney development.

Conclusion

The combination of Eci-clearing and 2-photon microscopy in the PodoTRAP model is well suited for high-resolution 3D-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 FSGS.