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Idiopathic focal segmental glomerulosclerosis plasmas increase lipid droplet formation and perilipin-2 expression in human podocytes

ABSTRACT: Background: Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but there is no consensus about the identity of CPFs in FSGS. We aimed to find proteins involved in the mechanism of action of the CPF(s) and/or potential biomarkers for the presence of CPF(s). Methods: Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s), or from (disease) controls. Podocyte proteomes were analyzed by mass spectrometry and differentially expressed proteins were validated using flow cytometry, RT-PCR, and immunofluorescence. Changes in podocyte granularity were examined using flow cytometry, electron microscopy imaging and BODIPY staining. Results: Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, which was related to disease state and capacity of plasma to induce granularity in podocytes. Elevated perilipin-2 levels were confirmed at protein and mRNA level. The granules observed in podocytes actually are lipid droplets. Importantly, increased perilipin-2 staining was also detected in glomeruli of the FSGS patients whose active disease plasmas induced lipid droplets in podocytes. Conclusions: Our study demonstrates that presumably CPF-containing plasma from FSGS patients alter podocyte lipid metabolism and increase perilipin-2 protein and lipid droplet accumulation. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism in the context of the pathogenesis of (recurrent) FSGS, which ultimately may result in novel leads for treatment.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Guido van Mierlo

LAB HEAD: Michiel Vermeulen

PROVIDER: PXD028536 | Pride | 2022-12-22

REPOSITORIES: Pride

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Project description:Recurrence of focal segmental glomerulosclerosis (rFSGS) after kidney transplantation is a cause of early and accelerated graft loss. Immuneadsorption can alleviate renal dysfunction and suggests that circulating antibodies (Ab) are likely implicated in disease pathogenesis. To evaluate pathogenic Ab in rFSGS, we processed 141 unique serum samples from patients with and without primary rFSGS (n=64) and 34 non-FSGS control, transplanted at five (US and EU) hospitals. 9000 antigens were screened in pre-transplant sera by protein arrays and 10 Ab targeting glomerular antigens were selected for ELISA validation. A panel of 7 Ab (CD40, PTPRO, CGB-5, FAS, P2RY11, SNRPB2 and APOL2) could predict post-transplant FSGS recurrence with 92% accuracy. Pre-transplant elevation of anti-CD40 Ab levels alone had a substantial impact (78% accuracy) on the identification of rFSGS risk after transplantation. Epitope mapping of CD40 with customized peptide arrays and rFSGS sera demonstrated altered immunogenicity of the extracellular CD40 domain in rFSGS. Immunohistochemistry of CD40 demonstrated a differential expression of these antigens in FSGS compared to non-FSGS. Anti-CD40 Ab purified from rFSGS patients were uniquely pathogenic in human podocyte cultures; injection of these Ab resulted in heightened proteinuria, independently and in combination with suPAR in a rodent model, abrogated by injection of monoclonal Ab to CD40. In conclusion, a panel of 7 Ab can identify primary FSGS patients at high risk of recurrence prior to transplantation, allowing for customized therapies and improved patient selection for transplant. Intra-renal CD40 is an important axis of disease pathogenesis, and human trials of anti-CD40 therapies are warranted to evaluate their efficacy in preventing rFSGS and improving graft survival. The purpose of the study was to identify potential auto-Abs associated with rFSGS. We used a discovery set of pre-transplant sera from 20 unique patients with biopsy confirmed diagnosis of primary FSGS as their cause of ESRD, of which 10 had progressed to rFSGS within the first post-transplant year and 10 did not have recurrence of proteinuria or histological disease after transplantation (nrFSGS).

Project description:Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syn-drome (INS), a glomerulopathy of presumable immune origin and attributed to extrarenal path-ogenic circulating factors. FSGS recurrence (rFSGS) after transplant occurs in 30 to 50% of cases. Direct analysis of patient plasma proteome has been scarcely addressed to date, mainly due to methodological difficulties associated with plasma complexity and dynamic range. In this study, firstly we compared different methods of plasma preparation, secondly we compared the plasma proteome of rFSGS and controls using two preparation methods, and thirdly we analyzed the early proximal signaling events in podocytes subjected to patient plasma, by 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 pro-teins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n=4). 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, three aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS. Next, we incubated human podocytes for 30min with 10% plasma from both groups of patients. Phosphoproteomics and raftomics of podocytes revealed profound differences in 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 high-light some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes.

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Idiopathic focal segmental glomerulosclerosis plasmas increase lipid droplet formation and perilipin-2 expression in human podocytes

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