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Integrated genomic, transcriptomic and metabolomic analysis reveals MDH2 mutation-induced metabolic disorder in recurrent focal segmental glomerulosclerosis

ABSTRACT: Focal segmental glomerulosclerosis (FSGS) has an over 30% risk of recurrence after kidney transplantation (Ktx) and is associated with an extremely high risk of graft loss. However, mechanisms remain largely unclear. Thus, this study identifies novel genes related to the recurrence of FSGS (rFSGS). Whole genome-wide sequencing and next-generation RNA sequencing were used to identify the candidate mutant genes associated with rFSGS in peripheral blood mononuclear cells (PBMCs) from patients with biopsy-confirmed rFSGS after KTx. To confirm the functional role of the identified gene with the MDH2 c.26C >T mutation, a homozygous MDH2 c.26C >T mutation in HMy2.CIR cell line was induced by CRISPR/Cas9 and co-cultured with podocytes, mesangial cells, or HK2 cells, respectively, to detect the potential pathogenicity of the c.26C >T variant in MDH2. A total of 32 nonsynonymous single nucleotide polymorphisms (SNPs) and 610 differentially expressed genes (DEGs) related to rFSGS were identified. DEGs are mainly enriched in the immune and metabolomic-related pathways. A variant in MDH2, c.26C >T, was found in all patients with rFSGS, which was also accompanied by lower levels of mRNA expression in PBMCs from relapsed patients compared with patients with remission after KTx. Functionally, co-cultures of HMy2.CIR cells overexpressing the mutant MDH2 significantly inhibited the expression of synaptopodin, podocin, and F-actin by podocytes compared with those co- cultured with WT HMy2.CIR cells or podocytes alone. We identified that MDH2 is a novel rFSGS susceptibility gene in patients with recurrence of FSGS after KTx. Mutation of the MDH2 c.26C >T variant may contribute to progressive podocyte injury in rFSGS patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE212610 | GEO | 2022/09/02

REPOSITORIES: GEO

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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.

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).

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Integrated genomic, transcriptomic and metabolomic analysis reveals MDH2 mutation-induced metabolic disorder in recurrent focal segmental glomerulosclerosis

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