Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway

Abstract

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex-mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD.

Figure 1. Representative light, immunofluorescence, and electron microscopy in a case of Dense Deposit Disease

(a) Periodic acid schiff (PAS)–stained section showing a membranoproliferative glomerulonephritis pattern of Dense Deposit Disease; (b) Immunofluorescence microscopy with C3 deposition in the mesangium and along glomerular capillary walls; (c) Electron microscopy resolving dense deposits in the mesangium and along the glomerular basement membranes (arrows).

Figure 2. Laser microdissection of a glomerulus

(a) Glomerulus to be microdissected; (b) Vacant space on slide following microdissection; (c) Fragments of the microdissected glomerulus in the microcentrifuge tube cap.

Figure 3. Protein identification by LCMS

(a) Representative scaffold readout of glomerular proteins for Case 4; (b) C5a identification by peptide analysis; (c) Scaffold readout of proteins of interest in all eight cases of dense deposit disease and one control sample.

Figure 3. Protein identification by LCMS

(a) Representative scaffold readout of glomerular proteins for Case 4; (b) C5a identification by peptide analysis; (c) Scaffold readout of proteins of interest in all eight cases of dense deposit disease and one control sample.

Figure 4. Scaffold readout of proteins of interest for all nine cases of immune complex-mediated membranoproliferative glomerulonephritis (MPGN)

Clinical history is as follows: patient 1 was a case of hepatitis B; patient 2 had cryptogenic cirrhosis; patient 3 had an autoimmune disease with positive antinuclear antibodies and rheumatoid factor; patients 4 and 5 had no significant history and likely had idiopathic MPGN type I; patient 6 had monoclonal gammopathy of unknown significance; patient 7 had cryoglobulins and a questionable autoimmune disease; patient 8 likely had idiopathic MPGN type I; and patient 9 had chronic bacterial infections.

Figure 5. Comparison of the SCRs of factor H with those of FHR1

FHR1 is comprised of five SCRs. The last three SCRs have nearly 100% identity with SCRs 18, 19, and 20 of factor H and are shown in blue. SCRs 1 and 2 of FHR1 have modest identity with SCRs 6 and 7 of factor H, hence the slightly different colors. SRC, short consensus repeat; FHR1, factor H-related protein 1; GBM, glomerular basement membrane.