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Abstract: TH-PO949

Three Dimensional Super-Resolution Microscopy and Automatic Quantification of Podocyte Foot Processes in Humans, Rats, and Mice

Session Information

Category: CKD (Non-Dialysis)

  • 1903 CKD (Non-Dialysis): Mechanisms

Authors

  • Artelt, Nadine, University Medicine Greifswald, Greifswald, Germany
  • Siegerist, Florian, University Medicine Greifswald, Greifswald, Germany
  • Ritter, Alina M., University Medicine Greifswald, Greifswald, Germany
  • Grisk, Olaf, University Medicine Greifswald, Karlsburg, Germany
  • Schlueter, Rabea, University of Greifswald, Greifswald, Germany
  • Endlich, Karlhans, University Medicine Greifswald, Greifswald, Germany
  • Endlich, Nicole, University Medicine Greifswald, Greifswald, Germany
Background

Until today, laborious and time-consuming transmission electron microscopy has been used to assess podocyte effacement in human and rodents. Recently, our group has shown that super-resolution 3D-structured illumination microscopy (3D-SIM) can be applied to visualize individual foot processes in human kidney biopsies. Furthermore, we have developed a software-based approach to quantify the structure of podocyte foot processes named podocyte exact morphology measurement procedure (PEMP). PEMP allows to quantify podocyte injury using filtration slit density (FSD), i.e. filtration slit length per capillary surface area, as the surrogate parameter for podocyte effacement in patients suffering from minimal change disease. As in basic science rodent models are widely distributed, we applied PEMP to mice and rats, the most used rodent models.

Methods

We co-stained 4 µm formalin-fixed and paraffin-embedded sections of healthy human, rat and mouse kidney tissue with antibodies targeted against the slit diaphragm protein nephrin and the actin-associated protein synaptopodin detected by Cy3 and Alexa 488-labeled secondary antibodies, respectively. Subsequently, we imaged these sections by wide-field microscopy, confocal laser scanning microscopy and 3D-SIM. PEMP was performed by FIJI utilizing a custom-built macro that semi-automatically detects the filtration slit and calculates FSD.

Results

In a comparative analysis, we show that only 3D-SIM is able to clearly visualize the nephrin-stained slit diaphragm between synaptopodin-stained foot processes in all three species. Using PEMP, we determined values for FSD in healthy individuals from all three species. The FSD in humans was 3.106 ± 0.024 µm-1 (mean ± SEM). The FSD was significantly higher in rats and mice with a mean value of 3.364 ± 0.049 µm-1 and 3.829 ± 0.032 µm-1, respectively. Since foot process width is inversely related to FSD, the width of foot processes continuously increases from mice to rats and humans.

Conclusion

Taken together, we present a method for basic research which allows fast evaluation and quantification of podocyte foot process morphology in rodents by using 3D-SIM on standardized histological material and tissue processing.

Funding

  • Government Support - Non-U.S.