FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. 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, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.