Defective Complement Regulation Research Articles

Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice

Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin-producing enteropathic bacteria (Stx-associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx-associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx-2. Stx-2 was administered together with lipopolysaccharide to wild-type and Cfh(+/-) C57BL/6 animals. Forty-eight hours after administration of the first Stx-2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild-type or Cfh(+/-) Stx-2-treated mice. Uraemia and renal tubular apoptosis did not develop in wild-type or Cfh(+/-) animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx-2 treated animals. In summary, Stx-2 administration did not result in TMA in either Cfh(+/-) or wild-type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx-2-induced renal tubular injury.

Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency

AbstractAtypical hemolytic uremic syndrome (aHUS) is a severe renal disease that is associated with defective complement regulation caused by multiple factors. We previously described the deficiency of factor H–related proteins CFHR1 and CFHR3 as predisposing factor for aHUS. Here we identify in an extended cohort of 147 aHUS patients that 16 juvenile individuals (ie, 11%) who either lacked the CFHR1/CFHR3 completely (n = 14) or showed extremely low CFHR1/CFHR3 plasma levels (n = 2) are positive for factor H (CFH) autoantibodies. The binding epitopes of all 16 analyzed autoantibodies were localized to the C-terminal recognition region of factor H, which represents a hot spot for aHUS mutations. Thus we define a novel subgroup of aHUS, termed DEAP HUS (deficiency of CFHR proteins and CFH autoantibody positive) that is characterized by a combination of genetic and acquired factors. Screening for both factors is obviously relevant for HUS patients as reduction of CFH autoantibody levels represents a therapeutic option.

Membrane cofactor protein mutations in atypical hemolytic uremic syndrome (aHUS), fatal Stx-HUS, C3 glomerulonephritis, and the HELLP syndrome

The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in complement control protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective complement regulation. Permanent cell lines expressing the mutated proteins were complement "challenged," and membrane control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to control the alternative pathway of complement activation on a cell surface, illustrating the importance of modeling transmembrane proteins in situ.

Anti–factor H autoantibodies block C-terminal recognition function of factor H in hemolytic uremic syndrome

AbstractThe atypical form of the kidney disease hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. In addition to mutations in complement regulators, factor H (FH)–specific autoantibodies have been reported for aHUS patients. The aim of the present study was to understand the role of these autoantibodies in aHUS. First, the binding sites of FH autoantibodies from 5 unrelated aHUS patients were mapped using recombinant FH fragments and competitor antibodies. For all 5 autoantibodies, the binding site was localized to the FH C-terminus. In a functional assay, isolated patient IgG inhibited FH binding to C3b. In addition, autoantibody-positive patients' plasma caused enhanced hemolysis of sheep erythrocytes, which was reversed by adding FH in excess. These results suggest that aHUS-associated FH autoantibodies mimic the effect of C-terminal FH mutations, as they inhibit the regulatory function of FH at cell surfaces by blocking its C-terminal recognition region.

Deletion of Complement Factor H–Related Genes CFHR1 and CFHR3 Is Associated with Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H–related 1 (CFHR1) and complement factor H–related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of ∼84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.

Unrestricted C3 Activation Occurs in Crry-Deficient Kidneys and Rapidly Leads to Chronic Renal Failure

Deficiency of the C3 convertase regulator Crry is embryonic lethal in mice unless C3 also is absent. For evaluation of the effect of local kidney Crry deficiency in the setting of an intact complement system, Crry(-/-)C3(-/-) mouse kidneys were transplanted into syngeneic C57BL/6 wild-type mice. These Crry-deficient kidneys developed marked inflammatory cell infiltration, tubular damage, and interstitial fibrosis, whereas similar changes were absent in control transplanted kidneys. Strong C3 deposition in the vessels and tubules that correlated significantly with measures of disease supported that complement activation was pathogenic in this model. Microarray studies showed upregulation of a number of chemokine and extracellular matrix genes, which were validated for CCL2 and CXCL10 mRNA and collagen III protein. The functional significance of these pathophysiologic findings was evaluated by removing both native kidneys, so the transplanted kidney alone provided renal function. Within 21 d of transplantation, seven of eight Crry-deficient kidneys in complement-sufficient wild-type hosts failed, compared with two of 13 controls (P = 0.001), with final blood urea nitrogen levels of 133.9 +/- 33.0 and 55.6 +/- 8.3 mg/dl, respectively (P = 0.015). These data show that mouse Crry is a critical complement regulator in the kidney. When absent, unrestricted complement activation occurs and quickly leads to marked inflammation and progressive renal failure, with features relevant to human diseases with underlying defects in complement regulation, such as hemolytic uremic syndrome.

Hemolytic Uremic Syndrome

Defective complement regulation results in hemolytic uremic syndrome (HUS), a disease that is characterized by microangiopathy, thrombocytopenia, and acute renal failure and that causes endothelial cell damage. For characterization of how defective complement regulation relates to the pathophysiology, the role of the complement regulator factor H and also of a mutant factor H protein was studied on the surface of human umbilical vein endothelial cells. The mutant 145-kD factor H protein was purified to homogeneity, from plasma of a patient with HUS, who is heterozygous for a factor H gene mutation G3587T, which introduces a stop codon at position 1172. Functional analyses show that the lack of the most C-terminal domain short consensus repeats 20 severely affected recognition functions (i.e., binding to heparin, C3b, C3d, and the surface of endothelial cells). Wild-type factor H as well as the mutant protein formed dimers in solution as shown by cross-linking studies and mass spectroscopy. When assayed in fluid phase, the complement regulatory activity of the mutant protein was normal and comparable to wild-type factor H. However, on the surface of endothelial cells, the mutant factor H protein showed severely reduced regulatory activities and lacked protective functions. Similarly, with the use of sheep erythrocytes, the mutant protein lacked the protective activity and caused increased hemolysis when it was added to factor H-depleted plasma. This study shows how a mutation that affects the C-terminal region of the factor H protein leads to defective complement control on cell surfaces and damage to endothelial cells in patients with HUS. These effects explain how mutant factor H causes defective complement control and in HUS-particularly under condition of inflammation and complement activation-causes endothelial cell damage.

MPGN II – genetically determined by defective complement regulation?

MPGN II is a rare disease which is characterized by complement containing deposits within the GBM. The disease is characterized by functional impairment of the GBM causing progressive loss of renal function eventually resulting in end stage renal disease. It now becomes evident that in addition to C3NeF, which inhibits the inactivation of the alternative C3 convertase C3bBb, different genetically determined factors are also involved in the pathogenesis of MPGN II. These factors though different from C3NeF also result in defective complement regulation acting either through separate pathways or synergistically with C3NeF. Following the finding of MPGN II in Factor H deficient animals, patients with MPGN II were identified presenting with an activated complement system caused by Factor H deficiency. Factor H gene mutations result in a lack of plasma Factor H or in a functional defect of Factor H protein. Loss of Factor H function can also be caused by inactivating Factor H autoantibodies, C3 mutations preventing interaction between C3 and Factor H, or autoantibodies against C3. Identification of patients with MPGN II caused by defective complement control may allow treatment by replacement of the missing factor via plasma infusion, thus possibly preventing or at least delaying disease progress.

Implications of the initial mutations in membrane cofactor protein (MCP; CD46) leading to atypical hemolytic uremic syndrome

The hemolytic uremic syndrome is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. There are two general types. One occurs in epidemic form and is diarrheal associated (D + HUS). It has a good prognosis. The second is a rare form known as atypical (aHUS), which may be familial or sporadic, and has a poor prognosis. aHUS is increasingly recognized to be a disease of defective complement regulation, particularly cofactor activity. Mutations in membrane cofactor protein (MCP; CD46) that predispose to the development of aHUS were first identified in 2003. MCP is a membrane-bound complement regulator that acts as a cofactor for the factor I-mediated cleavage of C3b and C4b deposited on host cells. More than 20 different mutations in MCP have now been identified in patients with aHUS. Many of these mutants have been functionally characterized and have helped to define the pathogenic mechanisms leading to aHUS development. Over 75% of the reported mutations cause a reduction in MCP expression, due to homozygous, compound heterozygous or heterozygous mutations. This deficiency of MCP leads to inadequate control of complement activation on endothelial cells after an initiating injury. The remaining MCP mutants are expressed, but demonstrate reduced ligand (C3b/C4b) binding capacity and cofactor activity of MCP. MCP mutations in aHUS demonstrate incomplete penetrance, indicating that additional genetic and environmental factors are required to manifest disease. MCP mutants as a cause of aHUS have a favorable clinical outcome in comparison to patients with factor H ( CFH) or factor I ( IF) mutations. In 90% of the renal transplants performed in patients with MCP-HUS, there has been no recurrence of the primary disease, whilst >50% of factor I or factor H deficient patients have had a prompt recurrence. This highlights the importance of defining and characterizing the underlying genetic defects in patients with aHUS.

Is complement a target for therapy in renal disease?

Complement deposition in the injured kidney is common, especially in glomerulonephritis. The precise role of the complement system in the mediation of tissue injury in the kidney has been defined in recent years, and this has assumed extra importance with the recent development of specific forms of therapy directed at the complement pathway. As well as the induction of cell lysis, complement has many subtle effects on cell biology, particularly on endothelial cells. Complement components are produced locally in the kidney. Detailed studies of certain rare forms of nephritis have provided evidence that complement activation can directly cause tissue injury. Appreciation of the importance of complement in hyperacute rejection of xenotransplants has given new impetus to the development of complement inhibitors. A narrative review is provided, with a brief overview of the complement pathway and its regulatory mechanisms, mechanisms of complement-induced tissue injury, local complement production, and the renal consequences of complement dysregulation. Currently available forms of therapy aimed at the complement system are reviewed, and possible future therapeutic strategies are suggested. The complement system plays a direct causal role in tissue injury in certain forms of renal disease. Specific forms of therapy are becoming available that can selectively interrupt complement activation or promote its regulation. Much of the drive for the development of these therapies comes from the field of xenotransplantation, but these forms of therapy should also be tested in various primary renal diseases.

Defective Regulation of Complement by the Sickle Erythrocyte: Evidence for a Defect in Control of Membrane Attack Complex Formation

A prominent clinical manifestation of sickle cell disease (SCD) is hemolytic anemia. Although complement activation can lead to intravascular hemolysis, its role in the hemolysis of SCD is not known. Because normal red blood cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement and because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte; therefore, defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. These studies show a defect in the regulation of membrane attack complex (C5b-9) formation in sickle erythrocytes, particularly in the most dense cells. The defect is characterized by increased binding of C5b-7 and of C9 to denser sickle cells and results in increased susceptibility of sickle cells to C5b-9-mediated (reactive) lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells are especially sensitive to reactive lysis. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement-mediated hemolysis could play a role in the anemia of SCD.

Defective regulation of complement by the sickle erythrocyte: evidence for a defect in control of membrane attack complex formation

A prominent clinical manifestation of sickle cell disease (SCD) is hemolytic anemia. Although complement activation can lead to intravascular hemolysis, its role in the hemolysis of SCD is not known. Because normal red blood cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement and because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte; therefore, defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. These studies show a defect in the regulation of membrane attack complex (C5b-9) formation in sickle erythrocytes, particularly in the most dense cells. The defect is characterized by increased binding of C5b-7 and of C9 to denser sickle cells and results in increased susceptibility of sickle cells to C5b-9-mediated (reactive) lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells are especially sensitive to reactive lysis. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement-mediated hemolysis could play a role in the anemia of SCD.