Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli

Abstract

Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.

Keywords: glomerular disease; podocyte; renal morphology.

Figure 1.

Transgenic mouse model of conditional podocyte depletion. (A) Development of Pod^CreiDTR mice to induce conditional podocyte depletion after DT injection. (B) Immunofluorescence expression of the human DT receptor, also known as heparin-binding EGF (HB-EGF), exclusively in glomerular podocytes, which was confirmed by double labeling of the HB-EGF (green) and a podocyte-specific marker (p57; red). (C) Mild podocyte depletion was achieved by the injection of 0.05 μg/kg DT, (D) inducing reversible albuminuria but no histologic signs of glomerular disease. (E) Moderate podocyte depletion was achieved by the injection of 0.1 μg/kg DT, (F) inducing persistent albuminuria and in some cases, the development of glomerulosclerosis (red arrows). Bars (orange, mild depletion; red, moderate depletion; white, controls) represent means±SDs.

Figure 2.

Optical clearing, confocal microscopy, and 3D rendering of whole glomeruli. Optical clearing is performed in sequential steps. (A) Agarose-embedded tissue is placed in (B) 100% methanol, which dehydrates both the tissue and the agarose, and (C) BABB allows efficient tissue clearing (white arrow shows the location of the tissue). (D) A 3D reconstruction of a whole-mouse glomerulus showing all nuclei (DAPI; magenta) and podocyte nuclei (p57; green), which are shown as surfaces in E, highlighting the potential for both manual and semiautomated quantification. (F) A 3D reconstruction of a whole-mouse glomerulus showing podocyte nuclei (p57; red) and cytoplasm (SNP; green). (G) Podocyte nuclear and cytoplasmic surfaces are shown, highlighting the capability for semiautomated quantification of podocyte nuclear and cytoplasmic volumes. (H) Glomerular volume obtained as a volumetric dilation on the basis of podocyte cytoplasmic surfaces. All analyses were performed using Imaris (Bitplane AG).

Figure 3.

Absolute podocyte depletion in whole glomeruli. (A) A 3D reconstruction of one control glomerulus using p57 (red) and SNP (green). (B) A 3D reconstruction of three glomeruli with variable degrees of podocyte depletion using p57 (red) and SNP (green). (C) Total podocyte number per glomerulus by mouse and depletion group; each column represents one mouse with 20 analyzed glomeruli. (D) Total podocyte number per glomerulus by depletion group; each column represents one depletion group, and each circle represents the average of 20 glomeruli per mouse. White circles represent controls, orange circles represent mild depletion, and red circles represent moderate depletion. (E) Distributions of total podocyte number per glomerulus (relative frequencies) by depletion group. Error lines and bars represent means±SDs. ****P<0.001.

Figure 4.

Numbers of podocytes in the context of glomerular volume. (A) IGV by depletion group. (B) Podocyte density per glomerulus by depletion group. P values represent differences between depletion category and controls. *P<0.05; **P<0.01. (C) Percentage of podocyte volume-to-glomerular volume ratio (PV/GV) by depletion group. (D) Relative frequency of IGV by depletion group. (E) Relative frequency of podocyte density per glomerulus by depletion group. (F) Relative frequency of PV/GV by depletion group.

Figure 5.

Quantitative analysis of podocyte hypertrophy. (A) Average podocyte volume by depletion group. (B) Average podocyte cytoplasmic volume by depletion group. (C) Average podocyte nuclear volume by depletion group. P values represent differences between depletion category and controls. *P<0.05; **P<0.01. (D) Average cytoplasmic volume-to-average nuclear volume ratio (aCV/aNV) by depletion group. (E) Relative frequency of average podocyte volume by depletion group. (F) Relative frequency of average cytoplasmic volume by depletion group. (G) Relative frequency of average nuclear volume by depletion group. (H) Relative frequency of aCV/aNV by depletion group.

Figure 6.

The use of associations for the assessment of podocyte depletion. (A) Associations between total podocyte number and IGV by depletion group. (B) Associations between average podocyte volume and podocyte density per glomerulus by depletion group. (C) Associations between average podocyte nuclear and cytoplasmic volumes by depletion group; lines of best fit with 95% confidence intervals are provided per depletion group.

Figure 7.

From 3D to 2D: what have we learned? (A) Two optical sections from the same glomerulus at 20-μm intervals showing that cross-sections with a similar area from the same glomerulus provide very different numbers of podocytes using p57 (red) and DAPI (blue). (B) Two optical sections from the same glomerulus at 10-μm intervals showing that cross-sections with a similar area from the same glomerulus provide different numbers of podocytes using p57 (red) and SNP (green). (C) Total podocyte number per glomerulus by depletion group. The dotted box highlights the difference between mild and moderate depletion groups. (D) Podocytes per glomerular cross-section by depletion group. The dotted box highlights the similarity between mild and moderate depletion groups. (E) Association between total podocyte number per glomerulus and podocytes per glomerular cross-section. (F) Podocyte density per glomerulus by depletion group. (G) Podocytes per glomerular area by depletion group. (H) Association between podocyte density per glomerulus and podocytes per glomerular area. (I) Average podocyte cytoplasmic volume by depletion group. (J) Average podocyte cytoplasmic area by depletion group. (K) Association between average podocyte cytoplasmic volume and area. Distribution box plots show control (white), mild depletion (orange), and moderate depletion (red) groups; each box contains average values from ≥10 observations per mouse (n=4 mice per group).