title: 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-μm 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.
authors.affiliation: Departments of Internal Medicine.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Venkatareddy
authors.affiliation: Departments of Internal Medicine.
authors.forename: Su
authors.initials: S
authors.lastname: Wang
authors.affiliation: Departments of Internal Medicine.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Departments of Internal Medicine.
authors.forename: Sanjeevkumar
authors.initials: S
authors.lastname: Patel
authors.affiliation: Pediatrics and Communicable Diseases, and.
authors.forename: Larysa
authors.initials: L
authors.lastname: Wickman
authors.affiliation: Departments of Internal Medicine.
authors.forename: Ryuzoh
authors.initials: R
authors.lastname: Nishizono
authors.affiliation: Departments of Internal Medicine.
authors.forename: Mahboob
authors.initials: M
authors.lastname: Chowdhury
authors.affiliation: Pathology, University of Michigan, Ann Arbor, Michigan; and.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hodgin
authors.affiliation: Department of Physics and Department of Bioengineering, University of Washington, Seattle, Washington.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Wiggins
authors.affiliation: Departments of Internal Medicine, rwiggins@umich.edu.
authors.forename: Roger C
authors.initials: RC
authors.lastname: Wiggins
mesh_terms: D000818:Animals; D001706:Biopsy; D002452:Cell Count; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis / Q000276:immunology; D050199:Podocytes / Q000166:cytology*; D051381:Rats; D012015:Reference Standards; D012097:Repressor Proteins / Q000032:analysis / Q000276:immunology; D013048:Specimen Handling; D025721:WT1 Proteins / Q000032:analysis / Q000276:immunology
chemical_list: D009687:Nuclear Proteins; D012097:Repressor Proteins; C465817:TLE4 protein, human; D025721:WT1 Proteins