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Kidney Week

Abstract: PO0941

Novel Analysis Approach for Intravital Single Nephron GFR Measurement in Mice

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Kroeger, Hannah, University Hospital Dresden Center of Internal Medicine - Nephrology, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
  • Kessel, Friederike, University Hospital Dresden Center of Internal Medicine - Nephrology, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
  • Sradnick, Jan, University Hospital Dresden Center of Internal Medicine - Nephrology, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
  • Gerlach, Michael, University Hospital Dresden, Core Facility Cellular Imaging, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
  • Todorov, Vladimir T., University Hospital Dresden Center of Internal Medicine - Nephrology, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
  • Hugo, Christian, University Hospital Dresden Center of Internal Medicine - Nephrology, Universitatsklinikum Carl Gustav Carus, Dresden, Dresden, Sachsen, Germany
Background

Intravital microscopy in animals is an emerging technique with advanced applications in kidney research. Particularily, the measurement of single nephron (SN) GFR in mice comprises a method to assess a key parameter of kidney disease. Filtration in single glomeruli is measured by two-photon microsopy in a time series after intravenous injection of a freely filtered fluorescent dye. From the intraglomerular capillaries to the connected proximal tubulus (PT) the glomerular filtration is observed and the intratubular dye intensity shift is measured. However, existing methods for the analysis of the image data in rats (Kang et al. 2006) had limited robustness in mice, due to smaller size, higher tubular curvature and therefore smaller acquisition distances.

Methods

By continuous, rather than punctual measurement of signal intensity along the PT in the time series we extended the published workflow. To further increase reliability and objectivity, we replaced the inaccurate estimation of the tubular volume by additional modelling in a 3D dataset. After normalizing the shift of the intensity position along the PT over time against the exact tubular volume, the filtrated volume per second is calculated by linear regression.

Results

With the method published by Kang the results were highly variable in our hands. After repeated analysis of image material (10 glomeruli in 5 animals, analyzed 5 times by one person), the GFR varied by a mean relative SD of 41%. By reducing overall user interaction with our method, this SD could be decreased to 14%. When applying the analysis to image data acquired in healthy and diabetic C57BL/6J mice, we detected a 4-fold increase in SN GFR in diabetic mice. Administration of the ACE inhibitor enalapril for three days ameliorated this effect in diabetic mice by 50%.

Conclusion

To increase the reliabilty of SN GFR measurements by intravital microscopy in mice, we extended an existing workflow by continuous measurement, 3D-modelling and sophisticated data analysis while reducing manual interaction. Application to microscopy data acquired in diabetic and healthy mice prove the general applicability and high reliability of this novel analysis approach. The clinical relevance is apparant in the context of monitoring disease progression as well as effects of medical intervention.