Modeling C3 glomerulopathies: C3 convertase regulation on an extracellular matrix surface

Abstract

Introduction: C3 glomerulopathies (C3G) are ultra-rare complement-mediated diseases that lead to end-stage renal disease (ESRD) within 10 years of diagnosis in ~50% of patients. Overactivation of the alternative pathway (AP) of complement in the fluid phase and on the surface of the glomerular endothelial glycomatrix is the underlying cause of C3G. Although there are animal models for C3G that focus on genetic drivers of disease, in vivo studies of the impact of acquired drivers are not yet possible.

Methods: Here we present an in vitro model of AP activation and regulation on a glycomatrix surface. We use an extracellular matrix substitute (MaxGel) as a base upon which we reconstitute AP C3 convertase. We validated this method using properdin and Factor H (FH) and then assessed the effects of genetic and acquired drivers of C3G on C3 convertase.

Results: We show that C3 convertase readily forms on MaxGel and that this formation was positively regulated by properdin and negatively regulated by FH. Additionally, Factor B (FB) and FH mutants impaired complement regulation when compared to wild type counterparts. We also show the effects of C3 nephritic factors (C3Nefs) on convertase stability over time and provide evidence for a novel mechanism of C3Nef-mediated C3G pathogenesis.

Discussion: We conclude that this ECM-based model of C3G offers a replicable method by which to evaluate the variable activity of the complement system in C3G, thereby offering an improved understanding of the different factors driving this disease process.

Keywords: C3 convertase; C3 glomerulopathies; C3 nephritic factor (C3Nef); complement regulation; extracellar matrix; factor B (FB); factor H (FH).

Figure 1

Complement control in the glomerular microenvironment. The glomerulus is the filtration unit of the kidney. Its filtration barrier is composed of the glycocalyx, glomerular endothelial cells (GEnC), glomerular basement membrane (GBM) and podocytes. The glycocalyx, a network of proteoglycans and glycoproteins, overlays highly fenestrated GEnCs. GEnC pores comprise ~20-50% of total cell surface area (–16), allowing filtration of waste products through the glycomatrix (glycocalyx and the GBM), underlying podocytes and further into the Bowman’s capsule to be excreted as urine. Complement activity on GEnCs is controlled by both cell-bound (MCP and DAF depicted) and fluid-phase complement regulators (Factor H (FH) and Factor I (FI) depicted), while complement control over the GEnC pores relies on fluid-phase regulators alone. A healthy glomerular microenvironment exhibits adequate cell-surface and fluid-phase complement control thereby preventing injury to the GEnCs and complement deposition in the glycomatrix. In contrast, in the C3G glomerular microenvironment, fluid-phase complement dysregulation occurs (an example of C3G driven by FH deficiency depicted) in presence of adequate cell-surface complement control. Here, GEnCs are still protected by the cell-bound regulators, but the lack of fluid-phase complement control over the GEnC pores allows complement amplification and complement deposition to occur as reflected by changes in the lamina densa of the GBM.

Figure 2

C3 convertase assays on MaxGel surface. (A) Preparation for all assays involves coating microtiter plates with MaxGel (ECM) followed by coating with C3b. (B) Formation assay: Factor B (FB) and Factor D (FD) are added to C3b-coated MaxGel, subsequently generating C3bBb over the course of 10 min in the absence (i) or presence (ii) of Properdin. Decay assay: C3bBb is generated as described above in absence (iii, v) or presence of Properdin (iv), and then allowed to decay over time naturally (iii, iv) or in presence of Factor H (v). (C) The amount of C3 convertase present on MaxGel surface at the time of elution is quantitated by western blot and observing the Bb complement fragment.

Figure 3

C3 convertase assembles, decays on ECM surface and is regulated by RCA proteins. (A) Western blot analysis of C3 convertase formation on ECM surface. MaxGel does not contain complement proteins necessary to form C3 convertase (lanes 1-5). C3 convertase alone (CA) forms in presence of C3b, FB and FD (lane 6), and is regulated by inhibitory FH (lane 7), stabilizing FP (lane 8) or a mix of FH+FP (lane 9). Results were normalized to CA and presented as relative protein expression. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of four independent experiments. Significance calculated for CA. (B) Western blot analysis of timed decay (1-15 min) of CA formed alone or in presence of Properdin. FP increases C3bBb half-life more than 2-fold (CA t_{1/2 =} 3.1min; FP t_{1/2 =} 7.4min). (C) Western blot analysis of timed decay (1-15 min) of CA formed alone or in presence of properdin (C3bBb(P)), followed by FH-mediated decay of C3bBb(P). FH increases decay of C3bBb(P) (CA t_{1/2 =} 3.2min; C3bBb(P)+FH t_1/2 <1min). Results were normalized to CA at 1 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Mean ± SD of four independent experiments. Significance calculated for CA at respective timepoints. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 4

FH inhibits C3 convertase formation in dose dependent manner. (A) Western blot analysis of C3 convertase formed in presence of varying amounts of FH. Decreasing the concentration of FH (lanes 3-5) leads to decreased inhibition of C3 convertase formation in dose-depended manner, as compared to the normal physiological concentration of FH (lane 2). Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of four independent experiments. (B) Western blot analysis of C3 convertase formed in presence of varying amounts of properdin. Increasing the concentration of FP (lanes 3-5) leads to increased formation of C3 convertase in dose-depended manner, as compared to convertase alone (CA, lane 1). (C) Western blot analysis of C3 convertase formed in presence of decreasing FH : FP ratio. Decreasing the ratio of FH : FP (lanes 5-9) leads to decreased FH inhibition of complement formation in dose-dependent manner. Low FH : FP ratios allow for a significant increase (lanes 7-9) in convertase formation as compared to the physiological FH : FP ratio (lane 4). FH and FP biomarkers of two C3G patients with elevated FP levels from MORL C3G cohort are shown in lanes 10 (FH: 287ug/mL, FP: 43.2 ug/mL) and 11 (FH: 363 ug/mL, FP: 38.4 ug/mL). Results were normalized to CA and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of three independent experiments. Significance calculated for CA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 5

Variants in CFB can affect its affinity for C3b binding and rate of C3 convertase decay in presence/absence of FH. (A) Schematic of recombinant FB proteins, blue arrows denoting the positions of specific mutants. (B) Western blot analysis of C3 convertase formation. Recombinant WT FB (FB his) shows decreased ability to form C3 convertase (lanes 3) as compared to WT FB (CA, lanes 1). There is no formation in presence of FH (lanes 2 and 4). Both p.Arg138Trp (lanes 5-6) and p.Phe286Leu (lanes 9-10) fail to form C3bBb in absence/presence of FH. p.Asp279Gly increases C3 convertase formation ~1.5-fold (lane 7) as compared to CA (lane 1) and is able to form C3bBb even in presence of FH (lane 8). Results were normalized to CA and presented as relative protein expression. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of three independent experiments. Significance calculated for WT FB. (C) Western blot analysis of CA formed using WT FB or FB his, then decayed in presence/absence of FH. Decrease in C3 convertase at 0 and 2 min is observed as compared to WT FB. (D) Western blot analysis of CA formed using WT or p.Arg138Trp, then decayed in absence/presence of FH. No mutant convertase activity is observed at 0 min as compared to WT FB. (E) Western blot analysis of CA formed using WT or p.Asp279Gly, then decayed in absence/presence of FH. Decrease in natural and FH-mediated decay of p.Asp279Gly C3bBb is observed as compared to WT FB at all timepoints. (F) Western blot analysis of CA formed using WT or p.Phe286Leu, then decayed in absence/presence of FH. No mutant convertase activity is observed at 0 min as compared to WT FB. For all decay experiments, results were normalized to CA at 0 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Statistical significance shown for comparisons between CA/recombinant protein and CA+FH/recombinant protein+FH respectively at the same timepoint. Mean ± SD of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 6

Variants in CFH can affect its ability to inhibit C3 convertase formation and promote Decay Acceleration Activity. (A) Schematic of recombinant FH proteins, blue arrows denoting the positions of specific mutants. (B) Western blot analysis of C3 convertase formed alone (CA), or in presence of WT FH or recombinant WT FH (FH his). Recombinant WT FH inhibits C3bBb formation to the same degree as WT FH. (C) Western blot analysis of CA formed alone, or in presence of recombinant FH variants or WT FH. p.Arg53Cys inhibits C3bBb formation to the same degree as WT FH, while p.Trp134Arg and p.Arg175Pro shows significant impairment in their ability to prevent C3 convertase formation. Results were normalized to CA and presented as relative protein expression. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of three independent experiments. Significance calculated for WT FH. (D) Western blot analysis of CA formed alone, then decayed in buffer, or with WT FH or recombinant WT FH. There is no difference between the two FH conditions. (E) Western blot analysis of CA formed alone, then decayed in buffer, or with p.Arg53Cys or WT FH. p.Arg53Cys almost reached significance at 1 min as compared to WT FH (p=0.055). (F) Western blot analysis of CA formed alone, then decayed in buffer, or with p.Trp134Arg or WT FH. p.Trp134Arg (t_{1/2 =} 2  _min , t_{1/2 =} 2.5  _min ) shows very poor DAA, providing only 20% decrease in C3bBb half-life as compared to CA. (G) Western blot analysis of CA formed alone, then decayed in buffer, or with p.Arg175Pro or WT FH. p.Arg175Pro (t_1/2 ~1.5 min, CA t_{1/2 =} 3.1  _min ) decreases DAA as compared to WT FH. For all decay experiments, results were normalized to CA at 0 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Mean ± SD of three independent experiments. Statistical significance shown for comparisons between WT FH/recombinant FH at the same timepoint. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 7

IgG from C3Nef+ C3G patients stabilize C3 convertase and decrease its decay rate. (A) Western blot analysis of CA formed alone, or in presence of NHS, P1, P2 or P3 IgG. P3 IgG significantly increases C3bBb formation. Results were normalized to CA and presented as relative protein expression. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of six independent experiments. Significance calculated for NHS IgG. (B) Western blot analysis showing C3 convertase formed alone (CA) or in presence of NHS IgG, then decayed for up to 30 min. NHS IgG do not stabilize C3 convertase (NHS IgG t_{1/2 =} 3 _min, CA t_{1/2 =} 3.36 _min). (C–E) Western blot analyses showing C3 convertase formed alone (CA) or in presence of IgG derived from C3Nef+ patients, then decayed for up to 30 min. (C) P1 IgG stabilizes C3bBb 2-fold (P1 t_{1/2 =} 8.9 _min, CA t_{1/2 =} 4.3min). (D) P2 IgG stabilizes C3bBb 1.9-fold (P2 t_{1/2 =} 9.4 _min, CA t_{1/2 =} 4.9 _min). (E) P3 IgG stabilizes C3bBb 1.7-fold (P3 t_{1/2 =} 8.7 _min CA t_{1/2 =} 5 _min). Mean ± SD of three independent experiments. Statistical significance shown for comparisons between CA/Patient IgG at the same timepoint. (F) Composite box plots showing C3 convertase stabilization by IgG from C3Nef+ C3G patients after 10 min of decay in buffer. For all decay experiments, results were normalized to CA at 0 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Box plots show median, and the individual data points of three independent experiments. Significance calculated for NHS IgG at 10 min. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 8

IgG from C3Nef- C3G patients do not promote C3 convertase formation but can increase its stability. (A) Western blot analysis of CA formed alone, or in presence of NHS, P4, P5 or P6 IgG. IgG from C3Nef-negative C3G patients does not affect C3bBb formation. Results were normalized to CA and presented as relative protein expression. Box plots show median, 1^st and 3^rd quartile ranges, and the individual data points of six independent experiments. Significance calculated for NHS IgG. (B) Western blot analysis showing C3 convertase formed alone (CA) or in presence of NHS IgG, then decayed for up to 30 min. NHS IgG do not stabilize C3 convertase (NHS IgG t_{1/2 =} 3 _min, CA t_{1/2 =} 3.36 _min). (C–E) Western blot analyses shows C3 convertase formed alone (CA) or in presence of IgG derived from C3Nef- patients, then decayed for up to 30 min. (C) P4 IgG increases C3bBb half-life 1.3-fold (P4 t_{1/2 =} 7 _min, CA t_{1/2 =} 5.5 _min). (D) P5 IgG increases C3bBb half-life 1.7-fold (P5 t_{1/2 =} 5.5 _min, CA t_{1/2 =} 3.3 _min). (E) P6 IgG increases C3bBb half-life 1.2-fold (P6 t_{1/2 =} 5.6 _min CA t_{1/2 =} 4.6 _min). Mean ± SD of three independent experiments. Statistical significance shown for comparisons between CA/Patient IgG at the same timepoint. (F) Composite box plots showing C3 convertase stabilization by IgG from C3Nef-negative patients (P4, P5) after 10 min of decay in buffer. For all decay experiments, results were normalized to CA at 0 min and presented as relative protein expression. Ponceau S Staining was used as a measure of total protein load. Box plots show median, and the individual data points of three independent experiments. Significance calculated for NHS IgG at 10 min. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.