In situ Visualization of C3/C5 Convertases to Differentiate Complement Activation

Abstract

Deregulated complement activation contributes to or drives the pathogenesis of various kidney diseases. Targeting complement is a therapeutic option.1Thurman J.M. Complement in kidney disease: core curriculum 2015.Am J Kidney Dis. 2015; 65: 156-168Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,2Zipfel P.F. Skerka C. Chen Q. et al.The role of complement in C3 glomerulopathy.Mol Immunol. 2015; 67: 21-30Crossref PubMed Scopus (58) Google Scholar Inhibition on the level of C5 is an established therapy in atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria, and myasthenia gravis, and novel drugs targeting other components of the complement cascade are being developed.3Reddy Y.N.V. Siedlecki A.M. Francis J.M. Breaking down the complement system : a review and update on novel therapies.Curr Opin Nephrol Hypertens. 2017; 26: 123-128PubMed Google Scholar,4Ricklin D. Mastellos D.C. Reis E.S. et al.The renaissance of complement therapeutics.Nat Rev Nephrol. 2018; 14: 26-47Crossref PubMed Scopus (192) Google Scholar Currently, the diagnosis of complement activation in kidney diseases is based primarily on immunohistochemical detection of deposited complement activation products in tissue and the detection of consumption of complement components in plasma.5Thurman J.M. Nester C.M. All Things Complement.Clin J Am Soc Nephrol. 2016; 11: 1856-1866Crossref PubMed Scopus (43) Google Scholar To date, a method to directly identify, localize, quantify, and differentiate complement convertases in tissue has been lacking. We therefore established an in situ method to detect and localize assembled C3/C5 convertases of the classical/lectin and alternative pathways (Figure 1; Supplementary Methods; Supplementary Figures S1–S4). We utilized a bright field proximity ligation assay6Söderberg O. Gullberg M. Jarvius M. et al.Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1587) Google Scholar,7Hnasko R.M. Methods in molecular biology ELISA methods and protocols.in: ELISA Methods and Protocols. Methods In Molecular Biology series. Vol 1318. 1. Humana Press, New York, NY2015: 149-159Google Scholar because (i) the microanatomic context is preserved, allowing, for example, selection of preserved glomeruli and the exact localization of the signals, and (ii) the signals are stable and easily quantifiable. Close proximity of C2 and C4b was used to identify assembled classical/lectin C3/C5 convertases, and of C3b and Bb, the fragment of factor B, to identify the alternative C3/C5 convertase. As expected, cases with immune-complex glomerulonephritis in patients with systemic lupus erythematosus revealed higher densities of classical/lectin convertases within the glomerular mesangium and around capillary walls (median, 7685 signals/mm2), as compared to biopsies from patients with aHUS (median, 393 signals/mm2) and to normal control biopsies (median, 207 signals/mm2). The difference between aHUS versus normal controls was not statistically significant. In contrast, aHUS cases showed a predominance of alternative convertases (median, 3032 signals/mm2), as compared to systemic lupus erythematosus and normal control biopsies (median, 1329 signals/mm2 and 1418 signals/mm2, respectively; Figure 2), with most signals being located within the glomerular capillary lumen. Formation of the classical/lectin pathway and alternative pathway C3/C5 convertases is an important step in the activation of the complement cascade.1Thurman J.M. Complement in kidney disease: core curriculum 2015.Am J Kidney Dis. 2015; 65: 156-168Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The presented technique allows the detection of direct proximity of proteins of the classical/lectin pathway (C4b and C2b) and the alternative pathway (C3b and Bb fragment of factor B) forming the C3/C5 convertases, indicating convertase formation in the tissue. As expected, in aHUS, there is a predominance of the alternative convertase, and in systemic lupus erythematosus, the classical/lectin convertases dominate. Unexpectedly, in systemic lupus erythematosus biopsies, the alternative convertase (likely as amplification loop) level is not higher than in normal controls. The reason for this finding is not clear; unknown negative feedback mechanisms could play a role. Detection, quantification, and discrimination of the specific complement convertases in tissue allows monitoring of complement-inhibiting therapies, providing an additional tool for future individualized medicine. Such discrimination can be important for the heterogeneous group of C3 glomerulopathies, which have various genetic and autoimmune causes. Mutant complement proteins and autoantibodies modify the activation of the complement system, systemically and locally.8Smith R.J.H. Appel G.B. Blom A.M. et al.C3 glomerulopathy—understanding a rare complement-driven renal disease.Nat Rev Nephrol. 2019; 15: 129-143Crossref PubMed Scopus (78) Google Scholar Patients with immune complex–mediated membranoproliferative glomerulonephritis, or C3 glomerulonephritis, show variable response to eculizumab therapy; persistently reduced proteinuria is achieved in just a subgroup.9Ruggenenti P. Daina E. Gennarini A. et al.C5 convertase blockade in membranoproliferative glomerulonephritis: a single-arm clinical trial.Am J Kidney Dis. 2019; 74: 224-238Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Several other inhibitors of the complement system are either currently in development or undergoing clinical trials.4Ricklin D. Mastellos D.C. Reis E.S. et al.The renaissance of complement therapeutics.Nat Rev Nephrol. 2018; 14: 26-47Crossref PubMed Scopus (192) Google Scholar Complement activation plays an important role in other glomerular diseases also, including membranous nephropathy,S1,S2 IgA nephropathy,S3 and ANCA-associated necrotizing glomerulonephritis.S4 However, complement is involved in not only renal diseases but also other disorders, such as neoplastic diseases. Several studies indicate that the complement system is playing a dichotomous role in the formation of cancer, activating pro-survival pathways while attacking tumor cells.S5 Therefore, it is beneficial to detect the formed C3/C5 convertases in situ at the primary site of activation and damage in target tissue. The application of the bright field visualization protocol enables the depiction of the convertases in their histopathologic context, allowing direct correlation to the different types of tissue damage. We introduce the first methodological workflow for the visualization, differentiation, and quantification of classical/lectin and alternative C3/C5 convertases directly within a tissue specimen. This new approach represents a promising tool to discriminate complement pathways in tissue and show the dynamics of activation, enhancing diagnosis and potentially allowing future monitoring of efficacy during individualized therapy.S6 All the authors declared no competing interests. We thank Ulrike Langbehn for excellent technical assistance. This work is supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 1192, Project B6 to TW and PFZ and Kidneeds [PFZ, CS]). TW, PFZ, FP, and TP designed the research. SW, FP, TP, and FB performed histopathologic and immunohistochemical analysis. JO provided clinical data. FP, TW, CS, PFZ, SB, and WF wrote the manuscript. All the authors approved the manuscript. Download .pdf (2.81 MB) Help with pdf files Supplementary File (PDF)