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.

Figure 1

Correlation between Ab anti-PLA₂R and proteinuria. Ab anti-PLA₂R and proteinuria shows a linear correlation (R² = 0.6891, p < 0.005).

Figure 2

Correlation between podocyturia (semiquantitative score) and proteinuria values. Podocytura and proteinuria seem to not be linearly correlated (p = NS).

Figure 3

Correlation between podocyturia (semiquantitative score) and Ab anti-PLA₂R. As for proteinuria, podocytura and Ab anti-PLA₂R seem to not be linearly correlated (p = NS).

Figure 4

Graphical schematization of idiopathic membranous nephropathy in the context of podocyte depletion hypothesis. (1) In the initial stage, Ab anti-PLA₂R caused immunocomplexes-deposition and podocyte damage after interaction with their podocyte target; (2) after the initial damage, podocytura and proteinuria both increase; (3) in case of persisting immunological damage, the reduction of podocyte mass progressively leads to end-stage renal disease (ESRD) with podocyturia decrease and, per contrast, further increase in proteinuria. This figure is created with BioRender.com.

Figure 5

PdoU analysis (flow cytometry). (a) isotype negative control (b) podocalyxin positive cells (PDX PE) on the R3-gate.