Session: Session 1

Combined proteomic analyses of recurrent idiopathic nephrotic syndrome plasma and plasma-treated human podocytes

Cerina CHHUON¹, Luis Vicente HERRERA-MARCOS², Ines METATLA¹, Vincent AUDARD^2,3, Mario OLLERO^2,4, Ida Chiara GUERRERA¹

¹Necker Proteomics, Université Paris Cité, SFR Necker, INSERM US24, Paris, France
²Institut Mondor de Recherche Biomédicale, INSERM, U955, 94010 Créteil, France, Créteil, France
³AP-HP (Assistance Publique des Hôpitaux de Paris), Department of Nephrology and Renal Transplantation, Groupe Hospitalier Henri-Mondor, 94010 Créteil, France, Créteil, France
⁴Université Paris Est Créteil, 94010, Créteil, France, Créteil, France

Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to the deleterious effect of putative circulating factors on podocytes. This is suggested by the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS, one of the forms of INS) after renal transplant. To better understand the mechanisms involved in FSGS recurrence and to find potential predictors, we had two main objectives: (i) perform a differential analysis of soluble plasma proteins and of plasma extracellular vesicles (EV) from rFSGS patients and controls; and (ii) study the early events in podocytes in response to the same plasma.

For pilot plasma analysis and EV analysis, we collected first post-transplant plasma exchange fluid from rFSGS and non-INS patients, and blood plasma from healthy individuals (n=4 per group). We then enlarged the cohort with pre-transplant plasma from non-recurrent patients (nrFSGS, n=9) and rFSGS patients (n=15). We performed neat soluble plasma analysis and Proteograph workflow with Proteograph Assay (Seer). EV were enriched by centrifugations at 20,000g. We incubated human podocytes with plasma from patients and controls. For both analyses, proteins were digested using S-Trap columns. We performed DIA using a nElute-timsTOF HT and analysed the data using DIA-NN and Mass Dynamics softwares.

We could quantify 3856 PGs on average with the Proteograph Assay, 1130 PGs for neat plasma, and 2915 PGs from EV. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins was found decreased in the soluble set from rFSGS patients. Podocyte proteome analysis highlighted 71 modulated proteins that could be involved in alterations of podocyte function.

The combined approaches highlighted differential circulating proteins as potential rFSGS biomarkers, along with novel putative mechanisms of podocyte damage.