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The podocyte is a terminally differentiated cell of the renal glomerulus that, by wrapping around the basement membrane through its foot processes, controls the selective permeability of the glomerular filtration barrier.
In diabetic nephropathy and other chronic glomerular diseases, a gradual decrease in intraglomerular podocytes, paralleled by increased urinary excretion of these same cells (up to 60,000 daily) has been demonstrated,
Whether urinary podocytes of healthy individuals consist of dedifferentiating cells and, if so, whether the dedifferentiation process precedes or follows detachment from the basement membrane presently is unknown. To clarify this, we collected sterile spot urine samples from 20 healthy individuals (mean age, 64.5 ± 4.1 [SD] years) and fragments of normal cortical tissue (derived from 15 adult kidneys removed for renal or urothelial carcinoma or from donor kidneys not suitable for transplantation) and searched for immature podocytes. Clinical characteristics of participants are provided in Table S1. The study was approved by the Ethics Committee of the San Raffaele Scientific Institute, and written informed consent was obtained from all participants.
Immature podocytes were defined as cells co-expressing the podocyte-specific antigens podocin, nephrin, and podocalyxin (Podxl)
Analysis of RNA extracted from urine sediments showed expression of the podocyte and embryonic markers (Fig 1A). In experiments run in parallel, we identified (in cytospins of all 20 spot urine samples) viable podocytes, evident as round, mono- or binucleated, propidium iodide–negative, Podxl-positive cells (0.20 ± 0.02 viable podocytes per milligram of creatinine, equivalent to ∼300 podocytes per day; Fig 1B). We detected Nanog in 96% of viable podocytes, in line with their being immature cells (Fig 1B). These urinary podocytes also coexpressed nephrin and podocin with Nanog (Fig 1C), as well as Oct3/4 and Sox2 (not shown). Synaptopodin was undetectable. When we seeded urinary sediments into culture medium, clusters of immature podocytes coexpressing Nanog and Podxl could be detected within a few days (Fig 1D).
In immunofluorescence studies of glomerular sections, we found cell clusters coexpressing Nanog with podocin (Fig 2A) and Podxl (Fig 2B). We also observed colocalization of Nanog with Oct3/4 (Fig 2C) and Sox2 (not shown). Consistent with these cells being immature, we detected no colocalization of Nanog and synaptopodin (Fig 2D). Clusters of immature cells were detected in ∼60% of glomeruli considered, independent of donor age.
These study results show that viable podocyturia, as detectable in urine of healthy individuals, consists of dedifferentiating cells coexpressing mature podocyte antigens (with the exception of postmitotic synaptopodin) along with immaturity markers. The presence of immature podocytes inside the glomerulus alternatively could be interpreted as an ongoing differentiative process allowing a still unknown stem cell pool to replace dying or detaching podocytes. Although this hypothesis cannot be excluded, it nonetheless is not in line with the data in Fig 2, which show that no niches of undifferentiated stem cells expressing embryonic transcription factors in absence of podocyte markers could be detected inside the glomerulus. Our study also demonstrates that the process of podocyte dedifferentiation precedes detachment from the basement membrane because clusters of immature podocytes were detected inside normal glomerular sections. Of course, this evidence does not exclude the possibility that some podocytes may detach from the basement membrane when still differentiated. To confirm the relationship between intraglomerular immature podocytes and urinary immature podocytes, lineage tracking experiments in an appropriate mouse model will be required.
Our findings suggest in vivo podocyte dedifferentiation as the origin of viable podocyturia in healthy individuals. Whether podocyturia in healthy individuals may represent a “side effect” of physiologic podocyte turnover (based on a dedifferentiation-redifferentiation process
) and, if so, by what means chronic glomerular diseases may unbalance this scenario (leading, in some podocytopathies, to huge numbers of podocytes being lost in urine), remains to be clarified.
Part of this work was carried out at Alembic (Advanced Light and Electron Microscopy Bio-Imaging Centre) of San Raffaele Scientific Institute. Dr Maestroni conducted this study in partial fulfillment of her PhD in Molecular Medicine (Program in Cellular and Molecular Biology, San Raffaele University). We are indebted to medical and technical members of the Pathology Unit. We thank the Comitato Carlo Brambilla Onlus for the generous donation of a fluorescence microscope. This article was written in memory of our late colleagues Francesca Podestà and Dora Ruggieri.
Support: This work was funded by a grant from the Italian Ministry of Instruction, University and Research (Cofin 2005-2005068145_004 ). The funders did not have any role in study design; collection, analysis, and interpretation of data; writing the report; and the decision to submit the report for publication.
Financial Disclosure: The authors declare that they have no relevant financial interests.
Contributions: Research idea and study design: GZ, SM, GDA; data acquisition: SM, AM, DG, ST, AS; data analysis/interpretation: GM, GC, SM, GZ, GDA; statistical analysis: GZ; supervision or mentorship: GZ, GC. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. GZ takes responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.