Using Stereologic Techniques for Podocyte Counting in the Mouse: Shifting the Paradigm

Abstract

Background: The podocyte serves the important function of maintaining the glomerular filtration barrier, and many studies report a decrease in podocyte number relative to the development of proteinuric states. However, there is significant inconsistency in the number of podocytes counted, possibly due to different counting methods. We previously counted the three glomerular cell types in the mouse kidney and showed that the fractionator/disector method is a close approximation of the exhaustive count or the gold standard method. In this study, we compared the commonly used model-based approach with the design-based approach to count podocytes in the db/m and db/db mouse and illustrate that the design-based approach, which uses the fractionator/disector method, provides an accurate determination of podocyte number. Methods: In the design-based approach, toluidine blue-stained 1-µm-thick sections from glutaraldehyde perfusion-fixed kidneys were used (n = 15) with the fractionator/disector method. In the model-based approach, WT-1-immunolabeled podocyte nuclei in 3- to 4-µm-thick formalin-fixed paraffin-embedded sections of the same kidneys were counted with the Weibel-Gomez method. Glomerular volume was determined for each method. Results: We discovered that the fractionator/disector method counted 89 ± 10 podocytes compared to the Weibel-Gomez method, which counted 137 ± 38 podocytes and overestimated podocyte number by 54% (p < 0.05). In addition, glomerular volume (231 ± 52 × 10³ vs. 192 ± 64 × 10³ µm³) was significantly underestimated by 17% (p < 0.0002). Moreover, the model-based approach was more time consuming. Conclusion: We conclude that the fractionator/disector method offers an unbiased and efficient determination of podocyte counts.