Abstract: FR-PO716

3D Analysis of Optically Cleared Kidney Slices Reveals Focal Podocyte Loss in Crescentic Nephritis

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Puelles, Victor G., University Hospital RWTH Aachen, Aachen, Germany
  • Fleck, David, RWTH Aachen University, Aachen, Germany
  • Vogt, Michael, RWTH Aachen University, Aachen, Germany
  • Papadouri, Stella, University Hospital RWTH Aachen, Aachen, Germany
  • Strieder, Thiago, University Hospital RWTH Aachen, Aachen, Germany
  • Saritas, Turgay, University Hospital RWTH Aachen, Aachen, Germany
  • Nikolic-Paterson, David J., Monash Medical Centre, Clayton, Victoria, Australia
  • Spehr, Marc, RWTH Aachen University, Aachen, Germany
  • Moeller, Marcus J., University Hospital RWTH Aachen, Aachen, Germany
Background

Podocyte depletion is a common feature of glomerulosclerosis (FSGS), but its role in crescentic nephritis remains unclear. This study combined genetic tagging of podocytes with three different optical clearing techniques to determine podocyte depletion in whole glomeruli from mice with crescentic nephritis.

Methods

Podocyte nuclei were labeled by eGFP-histone in of adult male Pod-rtTA/H2B-eGFP mice by oral doxycycline, followed by a 7-day wash out period, and a single intra-peritoneal injection of nephrotoxic serum (NTS; 5mg/g). Experimental mice were killed 10 days after NTS injection, and compared to age-matched controls. Kidney slices were optically cleared with SCALE-A4, CLARITY and Ethyl Cinnamate (ECi). High-resolution serial optical images were obtained by confocal and two-photon microscopy.

Results

Mean podocyte number per mouse showed very low variability within controls (1-5% variability, P>0.05) and within NTS-injected mice (1-9% variability, P>0.05) independent of the clearing technique. In NTS-injected mice, a similar degree of average podocyte depletion per mouse was identified with all clearing methods (60-63%, P<0.001). The technical (dis-)advantages of each clearing protocol were also analysed, including optimal penetration depth and resolution, compatibility with immunofluorescence, microscopy set-ups, and cost-efficiency.
Importantly, total podocyte number per glomerulus showed great variability: controls (mean: 78,81; ranging from 49 to 128 podocytes per glomerulus) and in NTS-injected mice (mean: 29,99; ranging from 1 to 95 podocytes per glomerulus). Using the lowest value for podocyte number in controls as a cut-off reference, only 78% of analysed glomeruli (141 of 180) from NTS-injected mice had a certain degree of podocyte depletion. While deposition of the NTS within glomeruli occurred in a global and homogeneous fashion, podocyte loss was focal.

Conclusion

This study has identified early focal podocyte depletion in mice with crescentic nephritis suggesting a so far unrecognized role of podocyte depletion in the development of the focal crescentic lesions. The combination of lineage tracing and optical clearing provides a powerful new tool for analysis of podocyte depletion in large tissue samples.