PMC9186056_01
(A) Course of S-Creatinine values previous to treatment. (B) Representative microphotographs of periodic acid Schiff (PAS) and Masson-Goldner-Elastica (MGE) stained kidney sections show a mesangioproliferative pattern (PAS) and signs of chronic endothelial damage in an arteriole (MGE). Immunohistochemical staining reveal strong positivity for C3, but no deposition of Immunoglobulin G (IgG). (C) Genetic analysis revealed introduction of a stop codon in domain 7 with a (D) consecutive a lack of Complement Factor H (Factor H) and Factor H like Protein1 (FHL1) in serum of the patient.
fimmu-13-826513-g001.jpg
PMC9186056_01_fimmu-13-826513-g001.jpg
f1
Due to ongoing rise of serum creatinine the patient was sent to our hospital ( Figure 1A ). In contrast, there was dominant mesangial positivity for C3 ( Figure 1B ), leading to the diagnosis of a mesangioproliferative glomerulonephritis with dominant C3 deposition. SCR7 ( Figure 1C ). Such heterozygous Factor H mutations in domain 7 are not described so far for C3G and to our knowledge most mutations in the Factor H gene associated with C3G or MPGN type II present as homozygous or compound heterozygous settings. Indeed, plasma protein levels of both Factor H and FHL1 were low when compared to healthy subjects ( Figure 1D ). Indeed, the initial biopsy showed some intimal sclerosis without elastosis ( Figure 1B ), which could be regarded as a former or chronic thrombotic microangiopathy, indicating an ongoing damage of endothelial cells.
(A) Measurement of the complement protein 3 (C3), the cleavage product C3a, Factor Ba, fragment, complement anaphylatoxin C5a, and the soluble form of the membrane attack complex (sC5b-9) in sera of the patient, a healthy subject (NHS) and a patient suffering from hemolytic uremic syndrome induced by deficiency of complement Factor H (positive control) by ELISA. By following the levels of C3, the central complement components indicative of continuous ongoing complement activation at the early levels. The consumption of C3 combined with elevated levels of C3a, Ba, C5a and sC5b-9 shows that complement is activated and that activation proceeds to the C3 convertase, as well as C5 convertase level and processes to the terminal pathway. Such strong fluid phase activation is in agreement with reduced Factor H and FHL1 plasma levels. (B) Representative immunohistochemical staining for C3 and the membrane attack complex (C5b-9). (C)
In vitro effect of Factor H, CPV-101, FHL1, sCR1 Eculizumab and Compstatin in in vitro hemolysis assays using patient serum (white circles) NHS (black circles), H2O (grey circles) or buffer (dark grey circles).
fimmu-13-826513-g002.jpg
PMC9186056_01_fimmu-13-826513-g002.jpg
f2
Using ELISA, a series of complement markers and activation product were analyzed in plasma to follow the complete complement pathways and compared their pattern with a patient with atypical hemolytic uremic syndrome as with other patients and also healthy subjects ( Figure 2A ). Indeed, we detected the terminal complement cascade products to be elevated (C5a and C5b-9) ( Figure 2A ). Analyzing the kidney biopsy, prominent immunohistochemical staining for C3 and, even more impressive, C5b-9 we observed ( Figure 2B ) arguing for activation of the terminal complement pathway also on the surface of the target organ. Moreover, Eculizumab, at that time the only clinically approved inhibitor, also inhibited lyses ( Figure 2C ).
(A) Representative immunohistochemical staining for complement C3 with a mild and the membrane attack complex (C5b-9) with a significant reduction after treatment with Eculizumab. (B) Analyzing density of mesangial and endocapillary mononuclear cells shows a reduction of both cell types. (C) Quantification of the signal density for the classical and the alternative C3 convertase revealed no major changes, especially no reduction of the alternative convertase. (D) Complement marker profile before and after Eculizumab treatment. C3 plasma levels increased upon Eculizumab therapy. The proximal activation markers C3a, and Ba did not change. Distal activation markers, e.g. the anaphylatoxin C5a and the soluble membrane attack complex (sC5b-9) showed reduction after treatment with Eculizumab.
fimmu-13-826513-g003.jpg
PMC9186056_01_fimmu-13-826513-g003.jpg
f3
Upon treatment we observed some reduction of C3 and considerable reduction of C5b-9 deposition in the glomeruli ( Figure 3A ). Using a computer-based deep-learning assisted detection of cell nuclei, we quantified mesangial and endocapillary mononuclear cells ( Figure 3B ). As previously described, we are also able to visualize the alternative and classical C3 convertase using proximity ligation assay (PLA) ( Figure 3C ). Ba, were not affected ( Figure 3D ). By including these PLA methods, theoretically it is also possible to distinguish whether the reduction of C5b-9 deposition is a therapy effect or reflects a decreased natural disease activity.