Second‐order stereology and ultrastructural examination of the spatial arrangements of tissue compartments within glomeruli of normal and diabetic kidneys

The present study explores 3D spatial arrangements of compartments within the rat renal glomerulus and tests for differences after chemically‐induced diabetes. In particular, the arrangements of capillaries, podocytes, mesangium and urinary space are quantified and compared between (a) kidneys within groups and (b) kidneys from streptozotocin‐diabetic rats and age‐matched controls. The stereological tool employed is the pair correlation function which is estimated by counting linear dipole probes of different sizes superimposed on ultrathin sections so as to be random in position and orientation. Unbiased estimates of the volume density of each glomerular component were estimated by point counting. Thereafter, estimates of the covariance and pair correlation function were determined from corresponding dipole counts. Plots of covariance and pair correlation functions against dipole length were almost identical in control and diabetic groups, indicating that diabetes did not disturb the normal spatial arrangements within glomeruli. However, differences were detected between compartments within groups. Whilst volume elements within all compartments were clustered at distances below about 8 μm (the approximate size of the basic cellular or other structural unit), the cluster size varied between compartments. The pattern was one of progressively smaller clusters in the sequence capillaries, podocytes, urinary space, mesangium. Beyond a distance of 8 μm, all glomerular components (in both control and diabetic groups) were arranged as expected for a ‘random’ (meaning neither clustered nor repulsed) volume process. These studies re‐emphasize the relative invariance of biological organization and the value and limitations of covariance analysis for quantifying different levels of organization in different tissues and experimental groups.