Proteomic Analysis of Complement Proteins in Membranous Nephropathy

Abstract

Introduction: Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)- and exostosin 1 (EXT1)/exostosin 2 (EXT2)-associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN.

Methods: We used laser microdissection and mass spectrometry (MS/MS) to dissect glomeruli and identify glomerular complement proteins in PLA2R-associated (n = 7), EXT1/EXT2-associated MN (n = 21), and 11 control cases (time 0 transplant biopsies).

Results: MS/MS identified high total spectral counts for PLA2R and EXT1/EXT2 in corresponding cases of PLA2R- and EXT1/EXT2-positive MN. Both PLA2R- and EXT1/EXT2-associated MN had high spectral counts of complement proteins C3, C4, C5, C6, C7, C8, and C9. Complement protein C1 was present in low spectral counts in EXT1/EXT2-associated MN. Regulators of complement activation that were detected in MN included higher spectral counts of FH, FHR-1, FHR-5, clusterin, vitronectin and lower spectral counts of FHR-3, FHR-4, and CD59. Low spectral counts of FB and properdin, key components of the alternative pathway, also were detected. IgG4 and IgG1 were the most abundant IgG subclasses in PLA2R- and EXT1/EXT2-associated MN. Lower spectral counts for C3, C4, and C5 were detected in control cases when compared with MN.

Conclusion: Significant complement activation is present in MN as evidenced by large spectral counts of complement proteins from C3- and C4-based pathways, including regulatory proteins of complement pathways. These data suggest that anticomplement drugs may be effective in treatment for MN.

Keywords: complement; laser microdissection; mass spectrometry; membranous nephropathy.

Graphical abstract

Figure 1

(a) Proteomic identification of complement proteins in phospholipase A2 receptor (PLA2R)–associated membranous nephropathy (MN). Complement proteins identified in 7 cases of PLA2R-associated MN is shown. Numbers in green boxes represent spectral counts of tandem mass spectrometry (MS/MS) matches to a respective protein. Protein grouping ambiguity (red star) indicates shared amino acid sequences for certain proteins. (b) Proteomic identification of complement proteins in exostosin (EXT)1/EXT2–associated MN. Complement proteins identified in 21 cases of EXT1/EXT2-associated MN are shown. Numbers in green boxes represent spectral counts of MS/MS matches to a respective protein. Protein grouping ambiguity (red star) indicates shared amino acid sequences for certain proteins.

Figure 1

(a) Proteomic identification of complement proteins in phospholipase A2 receptor (PLA2R)–associated membranous nephropathy (MN). Complement proteins identified in 7 cases of PLA2R-associated MN is shown. Numbers in green boxes represent spectral counts of tandem mass spectrometry (MS/MS) matches to a respective protein. Protein grouping ambiguity (red star) indicates shared amino acid sequences for certain proteins. (b) Proteomic identification of complement proteins in exostosin (EXT)1/EXT2–associated MN. Complement proteins identified in 21 cases of EXT1/EXT2-associated MN are shown. Numbers in green boxes represent spectral counts of MS/MS matches to a respective protein. Protein grouping ambiguity (red star) indicates shared amino acid sequences for certain proteins.

Figure 2

(a) Proteomic identification of complement-regulating proteins in in phospholipase A2 receptor (PLA2R)–associated membranous nephropathy (MN). Complement-regulating proteins identified in 7 cases of PLA2R-associated MN is shown. Numbers in green boxes represent spectral counts of tandem mass spectrometry (MS/MS) matches to a respective protein. (b) Proteomic identification of complement-regulating proteins in exostosin (EXT)1/EXT2–associated MN. Complement-regulating proteins identified in 21 cases of EXT1/EXT2-associated MN are shown. Numbers in green boxes represent spectral counts of MS/MS matches to a respective protein.

Figure 3

(a) Histogram comparing the complement proteins and Igs in controls, phospholipase A2 receptor (PLA2R)–associated membranous nephropathy (MN) and exostosin (EXT)1/EXT2–associated MN. The average spectral counts (± SD, indicated by 1-sided vertical error bars) are shown on the y-axis and the complement proteins and Igs on the x-axis. (b) Histogram comparing the complement-regulating proteins in controls, PLA2R-associated MN and EXT1/EXT2-associated MN. The average spectral counts (± SD, indicated by 1-sided vertical error bars) are shown on the y-axis and the complement-regulating proteins on the x-axis.