Complement C3 (MW: 183 kDa) is one of the most abundant plasma proteins. It is a pivotal component of complement and is central to the activation cascades. The mature protein is composed of two disulfide-bound polypeptide chains (C3α and C3β). The three complement activation pathways (alternative, classical, lectin) converge at the stage of C3 cleavage to generate the activated form of C3, which is C3b. C3b generates new C3 convertases by interacting with factors B, D and properdin. In the presence of abundant C3b, C5 convertases are formed.

Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two ultra-rare renal diseases characterized by fluid-phase dysregulation of C3 and C5 convertases that can lead to partial or complete consumption of circulating complement components, including C3. Consumption of C3 is consistent with activation of the alternative pathway of complement (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

C3 levels are also reduced in 30% to 50% of patients with atypical hemolytic uremic syndrome (aHUS) carrying Factor H mutations, and 20% to 30% of patients carrying Factor I mutations, a finding consistent with complement-dependent disease (Loirat & Frémeaux-Bacchi, 2011). Concentrations of factors H and I can clarify the mechanism of C3 consumption. In ~60% of aHUS patients, C3, Factor B, Factor H and Factor I levels are normal. In these patients, the type of complement-associated defect cannot be predicted by measuring plasma protein levels but may be discoverable by genetic analysis.

Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome Complement C4 (MW: 188 kDa), a central complement component in the classical and lectin pathways, is required to generate C4b2a, the C3 convertase of the classical pathway. C4 is cleaved to C4a (anaphylatoxin; 8 kDa) and C4b (180 kDa). C4b binds C2, and after removal of non-catalytic domain on C2, the C4b2a complex is formed. Unlike plasma C3, plasma C4 levels are typically normal in aHUS and C3G, however this test helps to rule out other complement-mediated renal diseases.

Factor B (MW: 93 kDa) is a complement protein unique to the alternative pathway (AP). In the presence of C3b, FB binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) to generate the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is the catalytically active site of the C3bBb C3 convertase complex and cleaves new C3 to C3a and C3b. If C3bBb recruits additional available C3b, the C5 convertase, C3bBbC3b, forms launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. In both DDD and C3GN, patients often have lower levels of factor B as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

Factor D (FD), also known as adipsin, is a 24 kDa serine protease predominantly produced by adipocytes. Its sole function is to cleave its natural substrate, C3b-bound factor B (FB), thereby generating C3bBb and activating the amplification loop of the alternative pathway (AP) of complement. Plasma FD levels in healthy individuals can vary but under normal conditions are within the range of 1–2 μg/mL. Due to its small size, FD is filtered through the glomerulus and then reabsorbed within the renal tubules. In patients with chronic kidney disease secondary to C3G and aHUS, factor D levels can be elevated up to 30-fold above normal. FD concentration can impact the activity of the AP and its elevation

Complement component 5 (C5) is a plasma glycoprotein (MW: 195 kDa) synthesized in the liver, monocytes and lymphocytes. The mature protein is composed of two disulfide-bound polypeptide chains (C5α and C5β). Upon activation, C5 is cleaved into C5a (MW: 11 kDa) and C5b (MW: 185 kDa) by C5 convertase. C5a is a potent anaphylatoxin that facilitates acute inflammatory responses; C5b initiates the sequential activation of the terminal pathway (C6 to C9) resulting in formation of the terminal complement complex (C5b-9).

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Plasma C5 levels are reduced in both DDD and C3GN as compared to controls (p<0.001 for both diseases) (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

Properdin is a single chain plasma glycoprotein (MW: 58 kDa) mainly secreted by white blood cells including monocytes, lymphocytes and neutrophils. It is present in the circulation as a mixture of head-to-tail dimers, trimers and tetramers, with a preponderance of trimers.

Properdin is a positive regulator of the alternative pathway (AP) of complement. In addition to initiating the AP, properdin binds to and stabilizes the C3 convertase, C3bBb increasing its half-life approximately 10 fold. The increased half-life enhances amplification of C3bBb formation, which eventually leads to the formation of the C5 convertase, C3bBbC3b, and initiation of the terminal pathway of complement.

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. While properdin plasma levels are often reduced in both DDD and C3GN, properdin levels are generally lower in C3GN as compared to DDD (p<0.01) (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome Complement Factor H (FH; MW: 155 kDa) is an important fluid-phase and cell-surface regulator of alternative pathway (AP) activity. Patients with FH mutations or FH autoantibodies may have reduced plasma FH levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.

Atypical Hemolytic Uremic Syndrome
Complement Factor I (FI; MW: 88 kDa) is an important regulator of complement activity triggered through the classical and alternative pathways. FI limits complement activation by cleaving surface-bound and fluid-phase C3b and C4b, preventing the assembly of the C3 and C5 convertases. Patients with FI mutations may have reduced plasma FI levels and/or function, and are at-risk to develop atypical hemolytic uremic syndrome or C3 glomerulopathy.

Dense Deposit Disease and C3 Glomerulonephritis
Complement activation and inactivation generate the degradation products of complement component 3 (C3; MW: 195 kDa). C3 convertases (C4b2a generated by the classical or lectin pathways, or C3bBb generated by the alternative pathway) cleave C3 into C3a (MW: 9 kDa) and C3b (MW: 185 kDa). Factor I and its co-factors, including factor H, inactivate C3b forming iC3b (MW: 183 kDa) and C3f (MW: 2 kDa). iC3b is further degraded into C3c (MW: 140 kDa), C3d (MW: 35 kDa) and C3g (MW: 7 kDa). The plasma concentration of C3d is reflective of this activity.

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP complement cascade. Consumption of AP complement components is dependent on the degree of dysregulation of both the C3 and C5 convertases. Plasma C3c levels are elevated in both DDD and C3GN as compared to controls (p<0.001).

Dense Deposit Disease and C3 Glomerulonephritis
Alternative pathway complement activation generates the active complement breakdown fragment Ba. In the presence of C3b, Factor B (MW: 93 kDa) binds to C3b to form the pre-convertase (C3bB). Factor D cleaves FB, generating the Ba fragment (MW: 33 kDa) and the proteolytic enzyme Bb (MW: 66 kDa). Bb is the catalytically active site of the C3bBb complex (C3 convertase) and is capable of cleaving new C3 to C3a and C3b. C3bBb also recruits available C3b to form the C5 convertase (C3bBbC3b) launching terminal pathway activation. The plasma concentration of Ba is reflective of this activity.

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP complement cascade. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Plasma levels of Ba are elevated in both DDD and C3GN as compared to controls (p<0.001), consistent with consumption of C3 in both diseases.

The activation of alternative pathway (AP) of complement generates the active proteolytic enzyme Bb. In the presence of C3b, factor B (MW: 93 kDa) binds to C3b to form the pre-convertase (C3bB). Factor D cleaves factor B releasing Ba (MW: 33 kDa) and generating the active proteolytic enzyme Bb (MW: 66kDa). The Bb subunit is catalytically active and cleaves new C3 to C3a and C3b. C3bBb recruits additional available C3b to form the C5 convertase, C3bBbC3b, launching terminal pathway activation. C3 convertase can be dissociated by spontaneous decay or complement regulators (factor H, CR1). It can also be inactivated by factor I-mediated C3b cleavage in presence of cofactors.

The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Plasma levels of Bb are elevated in both DDD and C3GN as compared to controls (p<0.001) consistent with dysregulation of the C3 convertase in both diseases (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

The complement system consists of three initiating pathways - the classical, alternative and lectin - activation of which leads to the generation of C3 and C5 convertases. The latter initiates the sequential activation of the terminal pathway (C5 to C9) resulting in the formation of pore-forming membrane attack complex C5b-9 (MAC), a stable complex that mediates irreversible cell lysis. Fluid-phase C5b-9 complexes (soluble C5b-9) may also form in complex with regulatory proteins like protein S.

C3 Glomerulopathy (C3G): The common pathophysiological basis of both Dense Deposit Disease (DDD) and C3 Glomerulonephritis (C3GN) is dysregulation of the AP. Consumption of AP complement components is dependent on the degree of dysregulation of the C3 and C5 convertases. Soluble C5b-9 is elevated in both DDD and C3GN, but only the difference between C3GN and controls reaches statistical significance (p<0.001 for only C3GN) (see Zhang et al., Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014).

Atypical Hemolytic Uremic Syndrome: Soluble C5b-9 levels are elevated in the majority of patients with active atypical hemolytic uremic syndrome but less frequently when the disease is in remission. Soluble C5b-9 may be useful as a biomarker to monitor activity of terminal pathway of complement (Bu et al., Soluble C5b-9 as a biomarker for complement activation in atypical hemolytic uremic syndrome, AJKD 2015).

Dense Deposit Disease; C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome CH50 measures total hemolytic activity of the classical and terminal pathways using sensitized sheep erythrocytes. CH50 can be low if complement components in the classical pathway (C1, C4, C2, C3) or terminal pathway (C5 through C9) are reduced or absent. Since most patients with C3G (especially DDD) have exceedingly low plasma C3 levels, their CH50s are also typically low.

The Alternative Pathway Functional Assay (APFA) measures activity of the alternative pathway (AP) of complement. Specific initiators are used to stimulate the AP on patient-derived sera; neoantigens of C9 produced as a result of terminal complement complex (C5b-9) activation are measured. Consumption or depleted of AP complement proteins will result in a low (abnormal) APFA.

Dense Deposit Disease, C3 Glomerulonephritis and atypical Hemolytic Uremic Syndrome Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two ultra-rare renal diseases. Both diseases are characterized by fluid-phase dysregulation of the AP that often leads to partial or complete consumption of circulating complement components, including complement C3, factor B, properdin and C5 (see Zhang et al. Defining the complement biomarker profile of C3 glomerulopathy, CJASN 2014). As a consequence, APFA can be low.

The C3b deposition assay (C3bDA) quantifies complement-mediated C3b deposition on cell surfaces following activation of the alternative pathway (AP). In brief, patient serum is diluted in a buffer conducive to AP activation and applied to cultured MES-13 cells, which are then incubated at 37°C for 20 minutes. C3b deposition on cell surfaces is visualized using Alexa-488 labeled anti-C3 antibody.

Immunofixation electrophoresis (IFE) integrates electrophoresis with immunofixation to identify C3 degradation products, providing an indirect assessment of dysregulation of C3 convertase. The quantification of the conversion of C3 to C3-activation products is performed.

Dense Deposit Disease (DDD, aka Membranoproliferative Glomerulonephritis Type II, MPGNII) Factor H autoantibodies have been associated with DDD (Meri, et al., 1992). In patients with DDD, these autoantibodies bind to and block the N-terminal region of the Factor H protein, which compromises its fluid-phase regulatory function.

Atypical Hemolytic-Uremic Syndrome
Factor H autoantibodies are identified in ~10% patients with aHUS (Dragon-Durey, et al., 2005, Moore, et al., 2010). Most but not all patients with aHUS who develop Factor H autoantibodies are homozygous for a known polymorphism, del(CFHR3-CFHR1). Homozygosity for this deletion is seen in 15% of patients with aHUS as compared to 5% of controls of northern European ancestry (Zipfel, et al., 2007, Skerka, et al., 2009). The Factor H autoantibodies in aHUS patients bind to and block the C-terminal region of the Factor H protein, which interferes with its surface regulatory function (Józsi, et al., 2007).

Dense Deposit Disease (DDD, aka Membranoproliferativee Glomerulonephritis Type II, MPGNII)
C3 Glomerulonephritis (C3GN)
Atypical Hemolytic Uremic Syndrome (aHUS)
Factor B autoantibodies (FBAAs) have been associated with DDD (Strobel, et al. 2010; Chen, et al. 2011). FBAA binds to factor B and/or the individual component Bb part of C3 convertase. More importantly, FBAA enhances C3 convertase activity, which often leads to increased complement breakdown products. FBAA represents an additional acquired driver of disease in DDD, C3GN and aHUS. Although rare, FBAAs should be considered in the comprehensive evaluation of patients with DDD, C3GN and aHUS.

Dense Deposit Disease (DDD, aka Membranoproliferative Glomerulonephritis Type II, MPGNII) C3 nephritic factors (C3Nefs), C5 nephritic factors (C5Nefs) and nephritic factor activity (nef activity) are defined as IgG autoantibodies to C3 convertase (C3bBb) that were first described by Spitzer and colleagues in 1969 as a substance in patient serum that constantly activated the alternative pathway cascade (Spitzer, 1969). They can be detected in ~80% of DDD patients and interfere with innate mechanisms that would otherwise control C3 convertase activity. Nephritic factors can also be detected in patients with partial lipodystrophy, meningococcal meningitis and post-streptococcal acute glomerulonephritis (Savage et al., 2009; Fremeaux-Bacchi, et al., 1994; Hulton, et al., 1992).

Dense Deposit Disease and C3 Glomerulonephritis
Testing is appropriate for patients with C3 glomerulopathy (DDD and C3GN). C4 nephritic factor (C4Nef) is an autoantibody to the classical pathway C3 convertase (C4b2a). By stabilizing C4b2a, it prolongs the half-life of this convertase and protects C4b2a against decay dissociation by C4 binding protein (C4BP). C4Nef has been associated with several diseases including post-infectious glomerulonephritis (Halbwachs et al, 1980), systemic lupus erythematosus (Daha and van Es, 1980), membranoproliferative glomerulonephritis (Tanuma, et al. 1989) and meningococcal disease (Miller, et al. 2012).