Volume of Glomerular Components: Podocyte, Mesangium, Capillary Lumen, Other

Using the same ultramicrotome fitted with an Ultra diamond knife (Diatome US), a 1-μm thick scout section was cut from an epoxy block and used to identify complete glomerular profiles at least one large glomerular diameter from the edge of the block. Silver-grey sections were then cut, placed on formvar coated slot grids (1 × 2 mm slot) and stained with uranyl acetate and lead citrate. These sections were observed with a JEM 1200-EX electron microscope (JEOL USA, Inc., Peabody, MA) fitted with a digital camera and DigitalMicrographs software (Gatan, Inc., Pleasanton, CA). At an initial magnification of 2500X the complete glomerular profile was imaged. Small profiles needed a single image, while larger profiles needed up to four images that were fitted together using the Photoshop software to make a montage of the complete glomerular profile. An average of 5.0, range 4–6 glomerular profiles were analyzed per mouse.

Using the Polygonal Lasso tool of Photoshop, a minimum string polygon was drawn around the tuft defining the limits of the glomerular profile. The glomeruli were divided into four components: podocyte, mesangium, capillary lumen (including endothelial and circulating cells) and other (including Bowman's space and glomerular basement membrane) (Figure 2, Left Panel). To measure the areal density of each of the four components, a grid of points was superimposed over the glomerular profile using the Layers function of Photoshop and the number of points falling on each of the components was counted (28, 30) (Figure 2, Right Panel). The areal density of a component per glomerular profile was calculated using the equation:

Areal density of glomerular components. Left Panel. On a low magnification EM image the glomerulus was defined by drawing a minimum string polygon around the tuft. Four components were defined: podocyte (POD), mesangium (MES), capillary lumen (CL), and remainder which included Bowman's space (BS) and GBM. Right Panel. A counting grid was randomly placed over the image and the number of points falling on each component is counted. The areal density of component X was calculated by dividing the number of grid points falling on component X by the total number of points falling on all four components.

where ∑P_componentX represents the sum of points falling on Component X, either podocyte, mesangium, capillary lumen or other for all the glomerular profiles from a kidney, and ∑P_{fourcomponents} is the sum of points falling on all the components from all glomerular profiles from a kidney. Because the areal densities were measured on random profiles from the glomeruli the measured areal densities equal the volume densities for each component according to the Delesse Principle (28):

The volume density does not determine the volumes of the components but only the ratio between the component volume and the glomerular volume. The individual component volumes were calculated using the equation: