Proteomics of Plasma and Plasma-Treated Podocytes: Application to Focal and Segmental Glomerulosclerosis

Abstract

Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the 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 to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.

Neat plasma proteomics: getting the best out of the worst

“…To compare the proteome of the EV fraction with the total plasma proteome, we prepared the EV fraction through multiple centrifugation steps, as previously described [ 20 , 21 ]. We also processed neat plasma from the same samples.…”

“…Third, we demonstrate that the extracellular vesicles (EVs) plasma fraction can be used as a repertoire of the total content of neat plasma. We purified the EVs fraction by conventional multiple centrifugation, as described previously [ 20 ]. We confirmed the enrichment of EVs by the presence of EV markers and organ-specific proteins.…”

“…Extracellular vesicles were obtained by a centrifugation method based on the protocol published by Geiger’s laboratory [ 20 , 21 ]. All the steps were carried out at 4◦C.…”

Minimal Change Disease: Pathogenetic Insights from Glomerular Proteomics

“…Tissue proteomics from in vivo-induced podocyte disease data derive from the study of animal models of FSGS in which intracellular signaling pathways such as Jak-Stat, TCA cycle, mTOR pathway, mechanical stress responses (e.g., involving filamin B) and other cytoskeleton components are modified [ 33 , 34 , 35 ]. Data on MCD from in vivo models are nevertheless lacking.…”