Abstract: SA-OR003

Super Resolution Microscopy and Automatized Digital Image Processing as a Novel and Rapid Diagnostic Tool for Podocyte Foot Process Effacement

Session Information

  • A View on the Glomerulus
    November 04, 2017 | Location: Room 294, Morial Convention Center
    Abstract Time: 04:54 PM - 05:06 PM

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Endlich, Nicole, University Medicine Greifswald, Greifswald, Germany
  • Siegerist, Florian, University Medicine Greifswald, Greifswald, Germany
  • Ribback, Silvia, University Medicine Greifswald, Greifswald, Germany
  • Dombrowski, Frank, University Medicine Greifswald, Greifswald, Germany
  • Amann, Kerstin U., University Medicine Erlangen, Erlangen, Germany
  • Endlich, Karlhans, University Medicine Greifswald, Greifswald, Germany
Background

Podocyte foot process morphology plays an essential role for proper glomerular filtration. In glomerular diseases like minimal change disease, interdigitating foot processes are replaced by flattened cellular protrusions. Since foot processes are around 200-300nm in width, morphometrics could only be performed by time consuming electron microscopy. Recently, super resolution microscopy techniques have been developed which allow lightmicroscopic imaging beyond Abbe’s optical resolution limit of ~200nm. Using one of those techniques, structured illumination microscopy (SIM), it is now possible to double the optical resolution limit. We hypothesized that SIM would allow the analysis of podocyte morphology and to diagnose foot process effacement.

Methods

4µm sections were obtained from formalin fixed and paraffin embedded renal tissue of human and murine origin and stained with an antibody against the slit diaphragm protein nephrin. Morphometrics of SIM-reconstructed podocyte foot processes were manually measured using ImageJ. For automatic evaluation of the slit diaphragm density, meaning slit diaphragm length / glomerular capillary area, we programmed a specific ImageJ plugin. The plugin automatically segments the nephrin positive slit diaphragm and measures the total length and the glomerular capilarry area.

Results

In human samples, we measured a mean foot process width of 0.249±0.68µm in healthy, and 0.675±0.246µm in MCD patients. By the use of our custom made software we measured a slit diaphragm density of 3.099±0.268µm/µm2 in healthy compared to 1.825±0.493µm/µm2 in MCD patients. Using both methods we found statistically significant differences between the MCD patients in comparison to the healthy control subjects. As we found out that the both results highly correlate (R2=0.91) we show that the techniques can be used equivalently. Furthermore, we show that we can image and measure foot process morphology in murine kidneys.

Conclusion

Taken together, we have established a novel method which allows quick analysis of kidney sections for foot process effacement in an automated fashion. Including this technique into the diagnostic routine could effectively shorten the time until diagnosis of podocyte foot process effacement in patients and in animal models.