Abstract
In glomerular diseases, podocytes are one type of cells involved in dysfunction of glomerular filtration. In these conditions, podocyte proteins expression change. Therefore, immunofluorescent staining of podocytes can be performed to visualize podocyte localization of proteins of interest. In this protocol, we detail a method to stain podocytes with a specific marker WT-1 (Wilms Tumor-1) for “in situ” podocyte analysis by immunofluorescent microscopy, more informative technique than other techniques (as Western blot).
Keywords: Immunofluorescence, Staining, Podocyte, Mouse, IF, WT-1, Podocalyxin, Kidney
Background
In vivo, mature podocytes are glomerular epithelial cells that are normally terminally differentiated (Pavenstadt et al., 2003). Under pathological conditions, podocytes may undergo mitosis and proliferate. For example, crescentic glomerulonephritis is an exceptional condition where podocytes undergo a dysregulation of their differentiated phenotype and start to proliferate and migrate, resulting in the so-called extracapillary glomerulopathy. Several studies have shown involvement of podocytes in crescentic glomerulonephritis (Moeller et al., 2004). Wilm’s tumor 1 (WT-1) is a zinc finger transcription factor present in nucleus of podocyte. Podocalyxin is a sialoglycoprotein constituting the glycocalyx of podocytes and endothelial cells in glomerulus. The expression of WT1 and podocalyxin are not decreased in early-stage of glomerular damage compared to proteins of slit diaphragm as podocin or nephrin. Moreover, WT1 is useful to evaluating podocyte number and density under different circumstances. The advantages to use podocalyxin staining are i) to analyze the glomerular morphology, ii) to quantify flocculus area to perform WT1+ cells / flocculus area ratio; iii) to ensure that WT1+ cells are podocytes and no other cells as parietal epithelial cells which could express WT1 in pathophysiological context. Our protocol can be used in crescentic glomerulonephritis (Henique et al., 2017) but also in studies on different glomerulopathies as hypertensive nephropathy.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
For an example of an immunofluorescent picture obtained with this protocol, see Figures 1 and 2. Figure 1. Negative control with secondary antibody only. The picture shows that the secondary antibodies do not cause any background. Scale bar: 50 µm. Figure 2. Immunofluorescent image of glomerulus with visualization of podocytes with markers. WT-1 (red) and podocalyxin (green). Nuclei are stained with DAPI (blue). A. Normal kidney from male mouse (C57Bl/6J, 12 weeks). B. Kidney from mouse with hypertension (male, C57Bl/6J). These pictures show a decrease in WT1 and podocalyxin staining suggesting a loss of podocytes and dilated capillaries. Scale bars: 50 µm.
Recipes
Acknowledgments
We thank members of Tharaux lab, as well as histology core facility at PARCC for helpful input. This work was supported by Inserm, the European Research Council-ERC Grant 107037 to P-L.T. and the Association des maladies d’un syndrome Néphrotique. We are grateful to the French National Agency for Research (ANR grant “SWITCHES” to P-L.T.) for supporting C. Henique.
Competing interests
The authors declare no competing financial interests.
References
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