Dataset Information

Laser microdissection and mass spectrometry shows loss of podocyte proteins in minimal change disease and focal segmental glomerulosclerosis.

ABSTRACT: Minimal change disease and primary focal segmental glomerulosclerosis (FSGS) are common causes of nephrotic syndrome. Both diseases present with massive proteinuria and widespread foot process effacement on electron microscopy examination. On the other hand, secondary FSGS can also present with significant proteinuria, although the extent of foot process effacement can be variable. In this study, we performed laser microdissection of glomeruli followed by mass spectrometry to study podocyte proteins and compare the differences between the 3 diseases. Our study shows significant decrease in total spectral counts of nephrin, podocin, CD2-associated protein, alpha actinin 4, inverted formin 2 and dystroglycan 1 in all 3 diseases compared to time zero kidney transplant controls and membranous nephropathy biopsies (p <0.001). On the other hand, the decrease in the podocyte proteins in minimal change disease, primary FSGS and secondary FSGS was comparable and there was no significant difference. Surprisingly, there was no significant decrease in the podocyte proteins in membranous nephropathy compared to time zero kidney transplant control biopsies. In conclusion, our study shows significant decrease in major podocyte proteins in minimal change disease, primary and secondary FSGS, but not in time zero kidney transplant controls and in membranous nephropathy.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Benjamin Madden

LAB HEAD: Sanjeev Sethi

PROVIDER: PXD057165 | Pride | 2025-12-29

REPOSITORIES: Pride

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Mass Spectrometry of Minimal Change Disease and Focal Segmental Glomerulosclerosis.

Fervenza Fernando C FC Vargas-Brochero Maria J MJ Debiec Hanna H Madden Benjamin B Andrade Luciana L Zand Ladan L Ronco Pierre P Sethi Sanjeev S

Kidney international reports 20251011 12

<h4>Introduction</h4>The relation between minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS) (pFSGS) remains debatable, and the recent discovery of antinephrin and antislit antibodies, supports the hypothesis of a continuum disease. However, the overall expression of podocyte proteins in these disease states is not known.<h4>Methods</h4>In this observational proteomic study, we used laser microdissection and mass spectrometry (LMD/MS) to determine the proteomic pr ...[more]

PMID: 41426026

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Project description:Background: In recent years, an innovative strategy using laser microdissection and mass spectrometry markedly expanded the landscape of antigens associated with membranous nephropathy (MN). Specific associations with phenotypes, diseases and sometimes reversible triggers led to a novel antigen-based classification of MN, paving the way for precision medicine and stressing the need for more routine use of proteomics in MN. Methods: To explore the proteomic landscape of human glomeruli and identify podocyte antigens and disease mechanisms in MN, we expanded the original technique to an integrative approach combining laser capture microdissection, next-generation mass spectrometry and computational analysis. Next to conventional data-dependent acquisition (DDA), we used and assessed the diagnostic yield of the more comprehensive data-independent acquisition (DIA) mass spectrometry, which enables the detection and quantification of every peptide in a sample irrespective of its level of abundance or m/z value. Our proteomic pipeline was applied to residual material from kidney biopsies in 64 individuals, including 31 healthy controls; 5 disease controls; 5 PLA2R-associated MN; and 23 PLA2R-negative MN. Results: Unbiased analyses confirmed the significant enrichment in PLA2R, IgG4 and complement proteins in glomeruli from patients with PLA2R-MN compared with healthy and disease controls, while molecular characterization of complement fragments provided evidence for complement activation in PLA2R-MN. Compared to DDA, DIA mass spectrometry increased the number of glomerular proteins (~3800 vs. ~1200) identified in healthy glomeruli; allowed the detection all known antigens except NELL1 in normal glomeruli; and increased the detection rate of podocyte antigens from 50% to >80% in PLA2R-negative MN. Conclusions: This proof-of-concept study suggests that an integrative approach combining laser microdissection, DIA mass spectrometry and computational biology is a powerful tool, with translational potential, to identify podocyte antigens and unravel disease mechanisms in MN.

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).

			Action	DRS
	MCD_01.mgf	Mgf
	MCD_01.raw	Raw
	MCD_02.mgf	Mgf
	MCD_02.raw	Raw
	MCD_03.mgf	Mgf