Detection of urinary podocytes by flow cytometry in idiopathic membranous nephropathy

Abstract

Idiopathic membranous nephropathy (iMN) is considered an immune-mediated disease where circulating autoantibodies against podocyte targets (mainly the PLA2R) cause the deposition of in-situ subepithelial immune-complexes. The consequent podocyte damage may cause cell detachment in urine (Podocyturia-PdoU). PdoU has been assessed in different kidney diseases, but limited data are available in iMN. In this study all patients with a diagnosis of iMN between 15/12/1999–16/07/2014 were tested for PLA2R antibodies (Ab anti-PLA2R, ELISA kit) and PdoU by flow cytometry with anti-podocalyxin antibody. A semi-quantitative PdoU score was defined according to the percentage of podocalyxin positive cells normalized to the total volume of sample and set relative to the urine creatinine measured in the supernatant. PdoU was positive in 17/27 patients (63%; 1+ score in 6/27—22.2%, 2+ in 4/27—14.8%, 3+ in 2/27—7.4%, 4+ in 5/27—18.5%). Only 2/7 patients with complete remission showed a positive PdoU (1+) while all six patients without remission have significant PdoU. PdoU+ was statistically correlated with the absence of remission and Ab anti-PLA2R + (p < 0.05) but PdoU, analysed as a continuous variable, showed a non-linear correlation with proteinuria or PLA2R antibody levels also in the cohort of patients with two available PdoU tests. In conclusion, PdoU could be detected in iMN and seems to be associated with commonly considered markers of disease activity (proteinuria and Ab anti-PLA2R) with a non-linear correlation. Despite data should be confirmed in large and prospective cohorts, according to the podocyte depletion hypothesis PdoU may represent an early marker of immunological activation with potential prognostic utility.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

“…The general principle of quantifying urine-derived kidney epithelial cells is based on the identification of genes and proteins specific to these cells in fresh urine samples. These specific genes and proteins are mainly podocalyxin, nephrin, podocin and synaptopodin, which are markers of podocytes and aquaporin-1, megalin, cubilin and CD13, the markers of PTECs [6,25,[36][37][38][39].…”

“…There are several methods used for the quantification of urine-derived epithelial cells. These methods include: colorimetric quantitative methods [6,37], flow cytometry [36], Western Blot [25], mass spectrometry [40] and quantitative real-time polymerase chain reaction (qRT-PCR) [25,38].…”

“…In this technique, the epithelial cells are sorted by utilizing highly specific antibodies labelled with fluorescent conjugates [25,36]. Flow cytometry has been demonstrated to be a very reliable, sensitive and accountable method in assessing kidney pathologies [48].…”

Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells

“…Additionally, testing the combination of our preservation method with sample freezing at −80 °C for even longer time intervals could benefit long term storage possibilities. Lastly, more urinary cell types should be examined for compatibility with our protocol; especially podocytes, which have been reported as biomarkers previously (12) (11).…”

Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro

“…These findings give evidence to the morphologic and motility changes of podocytes and were consistent with the findings of proteomics data. Podocyte loss is an important component of disease progression in MN [ 26 ]. The potential causes of podocyte loss are podocyte detachment and apoptosis [ 27 ].…”