Estimating Podocyte Number and Density Using a Single Histologic Section

Abstract

The reduction in podocyte density to levels below a threshold value drives glomerulosclerosis and progression to ESRD. However, technical demands prohibit high-throughput application of conventional morphometry for estimating podocyte density. We evaluated a method for estimating podocyte density using single paraffin-embedded formalin-fixed sections. Podocyte nuclei were imaged using indirect immunofluorescence detection of antibodies against Wilms' tumor-1 or transducin-like enhancer of split 4. To account for the large size of podocyte nuclei in relation to section thickness, we derived a correction factor given by the equation CF=1/(D/T+1), where T is the tissue section thickness and D is the mean caliper diameter of podocyte nuclei. Normal values for D were directly measured in thick tissue sections and in 3-to 5-mm sections using calibrated imaging software. D values were larger for human podocyte nuclei than for rat or mouse nuclei (P,0.01). In addition, D did not vary significantly between human kidney biopsies at the time of transplantation, 3-6 months after transplantation, or with podocyte depletion associated with transplant glomerulopathy. In rat models, D values also did not vary with podocyte depletion, but increased approximately 10% with old age and in postnephrectomy kidney hypertrophy. A spreadsheet with embedded formulas was created to facilitate individualized podocyte density estimation upon input of measured values. The correction factor method was validated by comparison with other methods, and provided data comparable with prior data for normal human kidney transplant donors. This method for estimating podocyte density is applicable to high-throughput laboratory and clinical use. Pagtalunan et al. used the term podocyte density to describe the key relationship between podocyte number and glomerular tuft volume. 1 Model systems have proven the causative relationship between podocyte depletion (resulting from reduced podocyte number or dysfunction and/or glomerular enlargement) and glomerulosclerosis and progression to ESRD. 2-9 Groundbreaking kidney morphometric biopsy reports from type 1 and 2 diabetes, IgA nephropathy, and hypertensive kidney biopsies in humans support the concept that reduced podocyte number and density is associated with development of glomerulosclerosis and progression, 1,10-15 and strongly imply that podocyte density estimation could help guide clinical decision making.The importance of avoiding simplistic podocyte counting strategies and using appropriate stereologic considerations for estimating podocyte number and density have recently been re-emphasized. [16][17][18][19][20] Optimal research methods for estimating podocyte density, such as the disector/fractionator approach, are too technically demanding for high-throughput

The Two Kidney to One Kidney Transition and Transplant Glomerulopathy

“…18 Regular formalinfixed paraffin-embedded histologic sections available as unstained slides from routine pathologic analysis were deparaffinized in fresh xylene, rehydrated, and permeabilized with 0.1% Triton-X100 in PBS for 10 minutes at room temperature. Slides were then incubated at 92°C for 2 hours in Retrieve-All 1 solution (Signet Laboratories, Dedham, MA) for antigen unmasking.…”

“…relating section thickness (T), apparent mean caliper diameter (d), and a podocyte nuclear shape coefficient (k=0.72). 18 The appropriate correction factor (CF) required for each individual slide was then calculated using the equation CF=1/(D/T+1), where T is the section thickness (in micrometers) and D is the true mean podocyte nuclear caliper diameter (in micrometers). The observed podocyte nuclear number was then multiplied by the CF to obtain the true podocyte number present in each glomerular profile from the biopsy.…”

“…Podocyte nuclear density is estimated by imaging software using TLE4 antibodies to identify podocyte nuclei and measure their mean caliper diameter (D) and thereby estimate their density in the glomerular tuft. 18 Podocyte cell area density is then estimated in the same section using Glepp1 immunoperoxidase histochemistry. Correlation between these parameters is shown in Figure 2.…”

“…If ,8 tuft profiles were present in the biopsy, then glomerular volume estimates were not made because there are insufficient profiles to reliably make the estimate as previously described. 18 Globally sclerotic glomeruli were excluded from analysis.…”

“…16 We recently adapted methodologies for quantitating podocytes in urine and kidney biopsies for use in humans. 17,18 Therefore, tools are available to begin to understand what happens to podocytes in the transition from a two-kidney (2K) state to a 1K state as well as in glomerulopathies. Human kidney transplantation offers a scenario in which these questions can be examined.…”