Towards the Definition of the Molecular Hallmarks of Idiopathic Membranous Nephropathy in Serum Proteome: A DIA-PASEF Approach

Abstract

Idiopathic membranous nephropathy (IMN) is a pathologically defined disorder of the glomerulus, primarily responsible for nephrotic syndromes (NS) in nondiabetic adults. The underlying molecular mechanisms are still not completely clarified. To explore possible molecular and functional signatures, an optimised mass spectrometry (MS) method based on next-generation data-independent acquisition combined with ion-mobility was applied to serum of patients affected by IMN (n = 15) or by other glomerulopathies (PN) (n = 15). The statistical comparison highlighted a panel of 57 de-regulated proteins with a significant increase in lipoprotein-related proteins (APOC1, APOB, APOA1, APOL1 and LCAT) and a substantial quantitative alteration of key serpins (including A4, D1, A7, A6, F2, F1 and 1) possibly associated with IMN or NS and podocyte stress. A critical dysregulation in metabolisms of lipids (e.g., VLDL assembly and clearance) likely to be related to known hyperlipidemia in IMN, along with involvement of non-classical complement pathways and a putative enrolment of ficolin-2 in sustaining the activation of the lectin-mediated complement system have been pinpointed. Moreover, mannose receptor CD206 (MRC1-down in IMN) and biotinidase (BTD-up in IMN) are able alone to accurately distinguish IMN vs. PN. To conclude, our work provides key proteomic insights into the IMN complexity, opening the way to an efficient stratification of MN patients.

Keywords: DIA-PASEF; mass spectrometry; membranous nephropathies; proteomics; serum.

Figure 1

Volcano plot. Volcano plot showing the differentially expressed protein groups between IMN and PN groups. FC is set to 1.5 and p_adj-value at least < 0.05. Blue = downregulated proteins; red = upregulated proteins; grey = nonsignificant and/or unvaried proteins.

Figure 2

Summary of the most relevant deregulated pathways. This graph shows the most relevant processes which are upregulated in IMN (in red), in PN (in blue) and in both groups (in purple), with a particular focus on complement cascade involvement (g:Profiler [20]). The graph is illustrated following the hierarchical clusterisation reported in Reactome database.

Figure 3

Boxplot of FCN2. Boxplot of FCN2, which was found to be significantly upregulated in IMN (p_adj-value < 0.05).

Figure 4

Decision tree. The decision tree shows the proteins which are able to discriminate IMN patients from PN ones and their cut-off. In each box are specified the following values: the name of the more frequent class (IMN or PN) and the number of cases belonging to the two classes, on the left are IMN cases and on the right are the PN cases. Below, the boxplots of MRC1 and BTD for each group are shown. The 5 PN clusterised with IMN in the first step include PN10 (IgAN), PN14 (IgAN), PN2 (FGS), PN3 (MCD/FSGS) and PN7 (MPGN).

Figure 5

Lipoprotein-related proteins upregulated in IMN. Boxplots of the lipoprotein-related proteins (APOC1, APOB, APOC4, APOL1 and LCAT) found to be significantly upregulated (p_adj-value < 0.05) in IMN serum samples. Red = IMN; green = PN.

Figure 6

Serpins in IMN. Boxplots of the serpins found to be differentially expressed between IMN and PN groups using high (a) (p_adj-value < 0.05) or lower stringency (b) (p-value < 0.05). In particular, KAIN, HEP2, THBG, CBG, A2AP, PEDF, SPA11 and A1AT were found to be upregulated in IMN, whereas ILEU, IPSP and IC1 were downregulated. Red = IMN; green = PN.

Figure 7

Hypothesis of a possible activated pathway linked to FCN2. Hypothesis of possible activation of the lectin-mediated pathway alternatively sustained by FCN2 in IMN [29,31]. Red = IMN; green = PN. Created with BioRender.com accessed on 20 July 2023.