Characteristics of Complement Protein Deposition in Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposition

Abstract

Background: Hypocomplementemia and complement co-deposition with monoclonal immunoglobulins in glomeruli are not rare in proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). Deposition of monoclonal immunoglobulins in glomeruli has been suggested to activate complement and cause kidney injury. However, the profiles of complement activation in PGNMID and their clinical and pathologic significance need to be clarified.

Methods: Forty-six patients with PGNMID were enrolled. Proteomic analysis of glomeruli using laser microdissection and mass spectrometry was performed for ten patients with PGNMID to determine the composition of glomerular deposits. Kidney deposition of complement components was detected by immunohistochemistry and immunofluorescence. Urinary and plasma levels of complement components were measured by an enzyme-linked immunosorbent assay. Group differences were assessed using t tests or Mann-Whitney U tests depending on the distribution. Correlation analysis was performed using Spearman rank correlation or Pearson correlation.

Results: Laser microdissection and mass spectrometry-based proteomic analysis showed that complement components were the most enriched proteins deposited in the glomeruli of patients with PGNMID. Glomerular deposition of C3c, C4d, and C5b-9 was detected in most patients. Levels of urinary and plasma C3a, C5a, soluble C5b-9, C4d, Bb, and C1q as well as urinary mannose-binding lectin were significantly higher in patients with PGNMID compared with healthy controls. The intensity of C3c and C4d deposition in glomeruli correlated with serum creatinine and the percentage of crescents, respectively. Furthermore, levels of urinary complement components correlated positively with serum creatinine, urinary protein excretion, percentage of crescents, and global glomerulosclerosis in kidney biopsies, whereas plasma levels of most complement components did not show a significant correlation with clinicopathologic parameters. In multivariable analysis, a higher level of urinary C4d was identified as an independent risk factor of kidney failure.

Conclusions: The complement system was found to be overactivated in PGNMID, and levels of urinary complements correlated with disease severity. A higher level of urinary C4d was identified as an independent risk factor of kidney failure.

Graphical abstract

Figure 1

Differentially expressed proteins between patients with PGNMID and healthy controls. (A) Heatmap of glomerular complement protein and complement regulatory protein abundance in patients with PGNMID and healthy controls. Each column represents an individual patient with PGNMID (P1–P10) or healthy control (C1–C4). Each row represents protein abundance. Red represents higher protein expression, and blue represents lower protein expression. Scale −2 to 2 reflects relative protein expression rescaled within each protein by centering at 0 and dividing by the SD. (B) Scatterplot for the top 20 pathways in KEGG enrichment of differentially expressed proteins between patients with PGNMID and healthy controls sorted by the −log₁₀ P value. The enrichment score was calculated according to the number of annotated genes and that of all annotated genes in this pathway term (see Methods in detail). Lower P values indicate higher pathway enrichment. (C) Top ten gene ontology terms in the biological process of differentially expressed proteins between patients with PGNMID and healthy controls sorted by the −log₁₀ P value. COVID-19, coronavirus disease 2019; KEGG, Kyoto Encyclopedia of Genes and Genomes; PGNMID, proliferative glomerulonephritis with monoclonal immunoglobulin deposits.

Figure 2

Staining for complement components in glomeruli detected by IHC and IF. Granular staining for C5b-9 (A) and C4d (B) along glomerular capillary walls and mesangial areas identified by IHC. Strong positive staining of C3c (C), C1q (D), and Bb (F) but weak positive staining of MBL (E) along glomerular capillary walls and mesangial areas were detected by IF. (A–F) Original magnification, ×400. IF, immunofluorescence; IHC, immunohistochemistry; MBL, mannose-binding lectin. Figure 2 can be viewed in color online at www.cjasn.org.

Figure 3

Levels of urinary and plasma complement components in patients with PGNMID. (A) Urinary C3a. (B) Urinary C5a. (C) Urinary sC5b-9. (D) Urinary C4d. (E) Urinary C1q. (F) Urinary MBL. (G) Urinary Bb. (H) Plasma C3a. (I) Plasma C5a. (J) Plasma sC5b-9. (K) Plasma C4d. (L) Plasma C1q. (M) Plasma MBL. (N) Plasma Bb. Cr, creatinine; sC5b-9, soluble C5b-9. Figure 3 can be viewed in color online at www.cjasn.org.

Figure 4

Kaplan–Meier curves demonstrating differences in kidney survival. (A) Kidney survival according to serum creatinine (P = 0.001). (B) Kidney survival according to the percentage of global glomerulosclerosis (P < 0.001). (C) Kidney survival according to the level of urinary C4d/creatinine (P = 0.001). Figure 4 can be viewed in color online at www.cjasn.org.