C3 glomerulonephritis: clinicopathological findings, complement abnormalities, glomerular proteomic profile, treatment, and follow-up

Abstract

C3 glomerulonephritis (C3GN) is a recently described disorder that typically results from abnormalities in the alternative pathway (AP) of complement. Here, we describe the clinical features, kidney biopsy findings, AP abnormalities, glomerular proteomic profile, and follow-up in 12 cases of C3GN. This disorder equally affected all ages, both genders, and typically presented with hematuria and proteinuria. In both the short and long term, renal function remained stable in the majority of patients with native kidney disease. In two patients, C3GN recurred within 1 year of transplantation and resulted in a decline in allograft function. Kidney biopsy mainly showed a membranoproliferative pattern, although both mesangial proliferative and diffuse endocapillary proliferative glomerulonephritis were noted. AP abnormalities were heterogeneous, both acquired and genetic. The most common acquired abnormality was the presence of C3 nephritic factors, while the most common genetic finding was the presence of H402 and V62 alleles of Factor H. In addition to these risk factors, other abnormalities included Factor H autoantibodies and mutations in CFH, CFI, and CFHR genes. Laser dissection and mass spectrometry of glomeruli from patients with C3GN showed accumulation of AP and terminal complement complex proteins. Thus, C3GN results from diverse abnormalities of the alternative complement pathway leading to subsequent glomerular injury.

Figure 1

Representative light, immunofluorescence, and electron microscopy in C3GN. A, B, C. Light microscopy showing different pattern of injury of the 3 different cases of C3GN. (A) shows a predominantly mesangial proliferative glomerulonephritis (PAS 20x), (B) shows a membranoproliferative glomerulonephritis (PAS 40x), and (C) shows a diffuse endocapillary proliferative glomerulonephritis with numerous infiltrating neutrophils within the glomerular capillaries (PAS 40x). D, E, F. Three different cases of C3GN showing bright C3 in the mesangium and/or along capillary walls (40x). G, H, I. Three different cases of C3GN showing large mesangial (black arrow), subendothelial deposits (thick black arrow), and subepithelial deposits (white arrow) on electron microscopy.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 2

Laser microdissection and mass spectrometry analysis of glomerular proteins in 8 patients of C3GN and 1 patent of Dense Deposit Disease (DDD). (A) Glomeruli marked prior to dissection in patient 5, and (B) empty space following microdissection. (C) Representative scaffold readout of proteins of interest for 8 patients of C3GN and 1 patient of DDD (last column). The proteomic data show extensive accumulation of proteins of AP including C3, C9, C8, C5, C7 and C6 in order of abundance, with >95% probability. CFHR-1, CFHR-5, Vitronectin, Apolipoprotein E, clusterin are also present in relative abundance, with >95% probability. Yellow stars indicate proteins of interest, while red stars indicate protein ambiguity when two proteins share conserved regions. (D, E) Sequence coverage for C3 and C9 in all patients showing the number of peptides, number of unique peptides, number of spectra and percentage of coverage of peptide sequence for C3 (Figure 2 D) and C9 (Figure 2E). (F) Analysis of C3 in one sample showing 21 unique peptides, 26 unique spectra and 77 total spectra, all of which result in 16% peptide coverage with 100% probability for C3. (G) Analysis of C9 in one sample showing 9 unique peptides, 10 unique spectra and 22 total spectra, all of which result in 21% peptide coverage with 100% probability for C9. The yellow highlighted areas in F and G shows the actual peptides detected by the mass spectrometry, and the green highlight shows oxidized or methylated amino acids.

Figure 3

Serum creatinine at presentation and follow-up (in months) of all patients. Patient 5 was on dialysis soon after presentation. * Patient 8 follow-up is for 23 years, with stable kidney function.