Limitations of previous methods
Podocytes are key cellular components of the glomerular filtration barrier in the kidney1. However, they are terminally differentiated and thus incapable of completing successful cell division under normal conditions. As a result, podocyte depletion (reduction in total number per glomerulus or density per glomerulus), is a significant indicator of the development of progressive kidney disease2. Podocyte depletion has, in fact, become a unifying principle of glomerular disease, and to date is routinely assessed by counting podocyte nuclei per glomerular cross-section in histological sections3. Recent studies have, highlighted the limitations of this approach, which include low levels of accuracy and precision, and a high degree of biological variability (particularly in studies in mice).
Stereological methods, both “model-based” and “design-based” (unbiased) methods, produce estimates of podocyte number determined from sampled sections. Model-based methods require knowledge or assumptions of podocyte nuclear size and shape. Generally, these values are assumed, and to the extent these assumptions differ from true values, the estimates obtained are biased. Design-based stereological methods used to estimate podocyte number are extremely time-consuming and have not been adopted by more than a handful of laboratories. Alternative methods for podocyte number estimation using serial histological sections4 (“exhaustive enumeration”) are extremely tedious and laborious.
The combination of tissue clearing and optical sectioning enable new approaches
Fortunately, the advent of new tissue clearing techniques5 in combination with optical sectioning, achievable through confocal, multiphoton or lightsheet microscopy, now make exhaustive enumeration not only attractive but a significant improvement for podocyte quantification, and any other cell of interest, especially those that play central roles in the development and progression of disease.
We have adopted this approach, and developed a novel, time-efficient method to study podocyte depletion using a combination of immunofluorescence, optical clearing, confocal microscopy and 3D analysis6. The technique has been used to quantify features of podocyte depletion in transgenic mice expressing the human diphtheria toxin (DT) receptor constitutively in podocytes, which allows conditional and dose-dependent podocyte death.
Our quantitative morphological tool significantly improves the assessment of podocyte depletion, and allows the study of whole glomeruli across the renal cortex. Differences in podocyte depletion between glomeruli in individual kidneys can be detected, a significant advance in the study of focal glomerular diseases.
Development of a novel method for imaging and quantifying podocyte depletion
Formalin-fixed kidneys are sliced to 800µm thickness, and the slices then subjected to 1hr antigen retrieval using a modified immunofluorescence protocol. Optical clearing is achieved by embedding the slices in agarose which are then dehydrated through changes in methanol, and cleared by several changes of benzyl alcohol, benzyl benzoate (BABB).
By gluing the edges of the dehydrated agarose to a glass petri dish, we immobilise the thick kidney slices on the bottom of the dish, making the specimens amenable to imaging on an upright Leica TCS SP8 MP confocal/multiphoton microscope equipped with the specialised Leica 20x/0.95 NA BABB immersion objective. The BABB mixture used has a refractive index of 1.56, which also permits higher resolution imaging using x40 and x63 oil objectives to be carried out on an inverted microscope when required.