Laser Microdissection and Mass Spectrometry Analysis of Complement in Membranous Nephropathy (MN)

Background: Complement activation is well described in PLA2R-associated MN. Kidney biopsy shows IgG and C3 along the GBM. However, the exact components of complement factors in PLA2R-associated MN are not known. 'New' target antigens including EXT1/EXT2, NELL1, NCAM1, FAT1, NDNF and PCDH7 have recently been discovered. The complement proteins and complement pathways in the 'new' antigen-associated MN are not known. Methods: We used laser microdissection and mass spectrometry (LMD/MS) to determine the complement proteomic profile in PLA2R- (n=7), NELL- (n=5), EXT1/EXT2- (n=6), TSHD7A- (n=7), NCAM1- (n=5), FAT1- (n=5), and PCDH7-associated MN (n=7). The MS results are expressed as total spectral counts (TSC) which also represent the relative abundance of the given protein. Results: LMD/MS show high TSC of C3, C4A, C4B, C5 and C9 complement proteins and CFH, CFHR1, and CFHR5 complement regulating proteins in all MN compared to control cases. However, complement proteins and complement regulating proteins were significantly higher in PLA2R-, EXT1/EXT2- and NCAM1- associated MN compared to NELL1-, THSD7A-, FAT1-, NDNF- and PCDH7-associated MN (2-5-fold increase, p<0.05). The increase in complement proteins did not correspond to the IgG subtype. i.e. IgG subtype was IgG4 in PLA2R-, and IgG1 in EXT1/EXT2- and NCAM1-associated MN). In addition, C1q was highest in NDNF-, EXT1/EXT2-, and NCAM1-associated MN compared to remaining antigen-associated MN. Conclusion: Complement activation occurs in MN with accumulation of complement factors and complement regulating proteins. All complement pathways appear to be involved. The highest complement activation occurs in PLA2R-, EXT1/EXT2- and NCAM1-associated MN. C1q-based classical complement pathway likely plays role in NDNF-, EXT1/EXT2-, and NCAM1-associated MN.LMD/MS of complement in MN.