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Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining   

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Di Wang Di Wang
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Original research article

A brief version of this protocol appeared in:
Cytopathology
Dec 2017

Abstract

In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.

Keywords: Podocytes, Parietal epithelial cells, Liquid-based cytology, WT1, Immunocytochemistry, Glomerular disease, Crescent formation, Urine

Background

Podocytes line the exterior of glomerular capillaries and thus face the Bowman’s capsule and primary urine. In glomerular injury, podocytes may detach from the glomerular basement membrane. The detachment of podocytes and their shedding in the urine have been implicated in the progression of glomerular diseases and crescent formation. Parietal epithelial cells (PECs) cover the inner aspect of the Bowman’s capsule. Similar to podocytes, it has been reported that PECs positive for WT1, proliferate and are shed in the urine during active glomerular disease (Zhang et al., 2012; Fujita et al., 2017).

Previous studies have shown a relationship between the number of urinary podocytes and PECs and various types of glomerular disease (Hara et al., 1998; Nakamura et al., 2000; Achenbach et al., 2008). The studies cited above used conventional methods (direct smears and cytospin) to prepare the urine samples. However, these conventional methods have common problems, including an air-drying effect and the possibility of significant cell loss during the cytocentrifugation and fixation processes. In addition, standardization and quantitative evaluation are difficult with the conventional methods. Although most previous studies have used immunofluorescence staining with the podocalyxin antibody, this method is difficult to perform in small- and medium-sized hospitals because of the specialty antigen and the requirement for a fluorescence microscope.

A useful urinary biomarker test should be easy to perform and have minimal requirements for standardized sample preparation and immunocytochemistry. Therefore, we have devised a method for detecting podocytes and PECs by combining liquid-based cytology, the WT1 antibody, and immunoenzyme staining. SurePathTM liquid-based cytology was developed as a replacement for the conventional sample preparation method because of its better cell preservation and higher cell recovery rate. SurePathTM allows the possibility of standardization and quantitative evaluation, and this system can be operated manually without machines. Furthermore, immunoenzyme staining using the WT1 antibody is performed in most pathological laboratories. Therefore, our method can be an inexpensive, simple, and internationally standardized method for the detection of urinary podocytes and PECs.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Ohsaki, H., Matsunaga, T., Fujita, T., Tokuhara, Y., Kamoshida, S. and Sofue, T. (2018). Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining. Bio-protocol 8(9): e2827. DOI: 10.21769/BioProtoc.2827.
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