Evaluation of a Thick and Thin Section Method for Estimation of Podocyte Number, Glomerular Volume, and Glomerular Volume Per Podocyte in Rat Kidney with Wilms’ Tumor-1 Protein Used as a Podocyte Nuclear Marker

Abstract

Abstract. Podocyte loss and glomerular hypertrophy are associated with development of glomerulosclerosis, suggesting that there may be a maximal area for each podocyte in terms of its capacity to support and maintain the glomerular filter. This study hypothesized that exceeding this maximal threshold will result in mesangial expansion and glomerulosclerosis. It may therefore be useful to measure podocyte number, glomerular volume, and glomerular volume per podocyte in clinical biopsysamples. An approach that uses thick and thin histologic sections cut from paraffin-embedded tissue to measure Wilms' tumor-1 protein-positive podocyte nuclear number and glomerular tuft area was studied. A rat model of aging has been used to track changes in glomerular podocyte number, glomerular volume per podocyte, and glomerular volume. Implications for clinical use of these variables are discussed.There is increasing evidence from human biopsy material and experimental models that podocyte injury and reduction in the number of podocytes in a glomerular tuft may be an important factor determining the propensity for glomerulosclerosis . If this is true, then it may be useful to routinely quantitate the number of podocytes in renal biopsy material and in experimental models to predict susceptibility to development of glomerulosclerosis and efficacy of treatment. Measurement of podocyte number has proved quite difficult because of the complexity of both podocyte and glomerular structure (18 -26).Although podocyte number appears to be a key variable, it is likely that podocyte number in relation to the glomerular basement membrane (GBM) area to be served by the podocyte may be more important. The relationship between GBM area and the podocyte can be approximated by relating glomerular tuft volume to podocyte number, and has been previously expressed as the "density" of podocytes or the glomerular volume per podocyte (GV/P) (21-28). There are extensive data to support the relationship between large glomerular tuft size and glomerulosclerosis (reviewed in [29]), an observation that could well be related to a relative deficiency of podocytes.We have attempted to develop a method that might be applicable for regular clinical use in renal biopsy material and experimental models of glomerular disease. We then used the method to examine changes in glomerular volume, podocyte number, and GV/P in the renal cortex of Brown Norway rats during normal maturation and aging. Materials and Methods Experimental Animal ModelRats at a range of ages were obtained from the Aging Rodent Colony maintained by the National Institute for Aging. For these experiments, we used Brown Norway rats killed at 2 mo (adolescent), 5 to 7 mo (young adult), 15 to 17 mo (midage), and 32 mo (old). Rats were anesthetized by injection of xylazine and ketamine before perfusion of kidneys with phosphate-buffered saline for 2 min, followed by 2% PLP paraformaldehyde, lysine, and periodate in PBS for 8 min at a perfusion pressure of 125 mmHg. After perfusion, kidneys were quickl...

Urinary Podocyte Loss Is a More Specific Marker of Ongoing Glomerular Damage than Proteinuria

“…The number of WT-1-positive cells per glomerular cell volume was calculated using the thin and thick section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte as described (23). In brief, mean glomerular volume in a given tissue was calculated by measuring 50 glomerular diameters and calculating the mean glomerular diameter.…”

DGKE Variants Cause a Glomerular Microangiopathy That Mimics Membranoproliferative GN

“…17 Diacylglycerol kinases (DGKs) are intracellular lipid kinases that are devoted to phosphorylate diacylglycerol (DAG) to phosphatidic acid 18 (Supplemental Figure 4). We determined by immunofluorescence confocal microscopy that Dgk« is expressed in adult mouse and rat kidneys and colocalizes with the podocyte marker WT1 19 but not with the endothelial marker CD31 (Supplemental Figure 5A). Saturation of the antibody with molar excess of an unrelated Dgk« peptide proved the specificity of the immune reaction (Supplemental There were also secondary focal and segmental sclerotic glomeruli, which could be seen in the advanced stage of any glomerulopathy…”

Diminished Met Signaling in Podocytes Contributes to the Development of Podocytopenia in Transplant Glomerulopathy

“…47 Thick sections were cut at 8 m, thin sections at 4 m. Both thick and thin sections were stained in a Bond III automatic immunostainer according to the manufacturer's instructions using a mouse monoclonal primary antibody (WT1-562-L-CE; Leica Microsystems, Wetzlar, Germany). Representative micrographs are displayed in Figure 1.…”

“…Mean glomerular volume, mean podocyte number per glomerulus, and podocyte volume density were calculated as described previously. 47 Calculations of the glomerular area and the different podocyte counts in the thick and the thin sections gave the podocyte volume density. Multiplication of the mean glomerular volume with the podocyte volume density yielded the mean podocyte number per glomerulus.…”

“…Multiplication of the mean glomerular volume with the podocyte volume density yielded the mean podocyte number per glomerulus. 47 …”