That MPGN2 and AMD, but not aHUS, have pathological similarities was recapitulated in the at-risk single nucleotide polymorphism (SNP; Table S1, available at http://www.jem.org/cgi/content/full/jem.20070301/DC1
) and haplotype () association data derived from a comparative genetic analysis, using a minimal set of informative CFH
SNPs, in Spanish subjects with aHUS, AMD, and MPGN2. No overlapping between CFH
aHUS-associated at-risk alleles or at-risk haplotypes was seen with the other conditions, consistent with previous data (19
haplotype H1 (-332C, c.184G, c.1204C, c.2016A, and c.2808G) was significantly increased in AMD and MPGN2 versus controls but not in aHUS patients. Conversely, haplotype H3 (-332T, c.184G, c.1204T, c.2016G, and c.2808T) was significantly increased in aHUS patients but not in either AMD or MPGN2 patients. Notably, haplotype H2 (-332C, c.184A, c.1204T, c.2016A, and c.2808G), previously shown to protect from AMD (19
), was markedly decreased in all three conditions, suggesting that this haplotype may be associated with increased FH regulatory activity and reduced AP activation. A strong correlation between the CFH
genotypes and the pathological outcome is further supported by the observation that the carboxy-terminal CFH
mutations, frequently found in aHUS patients (2
), were not detected in either healthy controls or subjects with MPGN2 or AMD (Table S2). These genetic data support the hypothesis that distinct functional alterations in FH are critical in the pathogenesis of aHUS and AMD/MPGN2.
Figure 1. Association analysis of CFH haplotypes with aHUS, AMD, and MPGN2 within a single population. Schematic illustration of the CFH exon structure demonstrating the location of the five SNPs included in these studies. These SNPs represent a minimal informative (more ...)
To test this hypothesis and to establish that a combination of effective plasma C3 regulation and defective regulation on renal endothelium is required for aHUS to develop, we generated transgenic mice expressing a mouse FH protein (FHΔ16-20) that lacked the terminal five SCR domains (), the equivalent mouse location of the majority of aHUS-associated FH human mutations (13
). These animals were intercrossed with FH-deficient (Cfh−/−
) mice to generate mice expressing either the mutant protein alone (Cfh−/−
.FHΔ16-20) or in combination with the full-length mouse protein (Cfh+/−
.FHΔ16-20 mice were viable, and FHΔ16-20 was detectable in plasma () at levels comparable to FH in Cfh+/−
mice (). Analogous to aHUS-associated FH human mutants, FHΔ16-20 retained complement regulatory activity but showed impaired binding to heparin and human umbilical vein endothelial cells (HUVECs) in vitro ().
Figure 2. The development of Cfh−/−.FHΔ16-20 mice. (A) Schematic representation of the mouse FH protein and the mutant mouse FHΔ16-20 protein. SCR domains are numbered incrementally from the amino terminus. Complement regulatory (more ...)
Figure 3. Functional characterization of FHΔ16-20. (A and B) Heparin binding assay. Cfh−/+.FHΔ16-20 mouse plasma was applied to a heparin–sepharose column, and the proteins bound to the column were eluted with a NaCl linear (more ...) Cfh−/−
mice have secondary plasma C3 depletion (12
), enabling us to assess the ability of FHΔ16-20 to regulate AP activation in vivo by measuring C3 levels in the Cfh−/−
.FHΔ16-20 mice. C3 levels were significantly higher in the Cfh−/−
.FHΔ16-20 mice compared with Cfh−/−
littermate controls (). C3 levels in Cfh+/−
.FHΔ16-20 mice were also significantly higher compared with age-matched Cfh+/−
mice, reaching wild-type C3 levels (). Thus, FHΔ16-20 retained the ability to regulate plasma C3 activation in vivo. Spontaneous GBM C3 deposition is seen in Cfh−/−
). To assess the ability of FHΔ16-20 to regulate C3 activation within the kidney, we compared glomerular C3 staining in 3-wk-old Cfh−/−
.FHΔ16-20, and Cfh−/−
mice (). In striking contrast to the linear GBM C3 staining pattern evident in the Cfh−/−
mice, only a granular mesangial C3 staining pattern was detected in Cfh−/−
.FHΔ16-20 mice. Thus, FHΔ16-20 also efficiently prevented accumulation of C3 along the GBM.
Figure 4. Plasma and glomerular C3 regulation in Cfh−/−.FHΔ16-20 mice. (A) Plasma C3 levels in Cfh−/−.FHΔ16-20 mice. Median plasma C3 levels in the Cfh−/−.FHΔ16-20 mice were 79.5 mg/liter (range (more ...)
During our initial observations, all Cfh−/−
.FHΔ16-20 mice developed hematuria and anasarca or died before 12 wk of age. Hence, we monitored cohorts of Cfh−/−
= 15) and Cfh+/−
= 11) mice over an 8-wk period. At 8 wk, 9 out of the 15 Cfh−/−
.FHΔ16-20 mice (60%) had developed hematuria and anasarca, necessitating death, whereas all Cfh+/−
.FHΔ16-20 animals remained well. Renal histology in the Cfh−/−
.FHΔ16-20 mice with hematuria demonstrated thrombotic microangiopathy (). Endothelial damage characteristic of thrombotic microangiopathy was evident on ultrastructural examination of these animals (). Importantly, electron-dense GBM deposits, an ultrastructural feature of MPGN2 that we have previously shown to be present at this age in Cfh−/−
), were absent. No renal histological abnormalities were seen in the 8-wk-old Cfh+/−
.FHΔ16-20 mice, and in a separate cohort of Cfh+/−
.FHΔ16-20 mice (n
= 4), renal histology remained normal at 6 mo (unpublished data).
Figure 5. HUS in Cfh−/−.FHΔ16-20 mice. (A) Renal histology in Cfh−/−.FHΔ16-20 mice. Normal glomerulus from a 2-mo-old Cfh+/−.FHΔ16-20 mouse (i), and light microscopic features of thrombotic (more ...)
In all of the Cfh−/−
.FHΔ16-20 mice with hematuria, there was significant elevation of blood urea (median = 31.8 mmol/liter, range = 26.3–42.8 mmol/liter; n
= 8) compared with normal values in the age-matched Cfh+/−
.FHΔ16-20 mice (median = 10 mmol/liter, range = 4.8–16.5 mmol/liter; n
= 11; P = 0.0003; Table S3, available at http://www.jem.org/cgi/content/full/jem.20070301/DC1
). Red cell fragmentation was evident on the peripheral blood films in all of the Cfh−/−
.FHΔ16-20 mice with hematuria (, arrows). Furthermore, these mice had significantly reduced platelet counts (median = 64 × 109
platelets/liter, range = 28–291 platelets/liter; n
= 7) compared with normal values in the Cfh+/−
.FHΔ16-20 mice (median = 517 × 109
platelets/liter, range = 445–584 platelets/liter; n
= 4; P = 0.0061). Thus, renal thrombotic microangiopathy in Cfh−/−
.FHΔ16-20 mice was associated with renal failure, red cell fragmentation, and thrombocytopenia, all cardinal features of aHUS. Immunofluorescence studies in the Cfh−/−
.FHΔ16-20 mice with hematuria showed C3 deposition along the endothelium and within the smooth muscle of renal arteries (), in addition to abnormal deposition within the glomerular mesangium and capillary walls (). In contrast, no abnormal C3 staining was seen in age-matched Cfh+/−
.FHΔ16-20 mice (). Thus, consistent with the in vitro data, FHΔ16-20 failed to regulate C3 activation on renal endothelium.
That a degree of plasma C3 regulation is required to enable thrombotic microangiopathy to develop derived from our observations in a second transgenic line (Cfh−/−.FHΔ16-20low) with a median plasma FHΔ16-20 level of only 2% of normal wild-type FH levels. Median plasma C3 levels were 34.8 mg/liter (range = 20.7–50.1 mg/liter; n = 6), significantly less than the median value measured in the Cfh−/−.FHΔ16-20 mice (79.5 mg/liter; P < 0.001) but greater than median C3 levels in Cfh−/− animals (14.3 mg/liter; P < 0.01). At 8 mo of age, renal histology in the Cfh−/−.FHΔ16-20low mice (n = 6) demonstrated only mild mesangial expansion with no evidence of thrombotic microangiopathy. Furthermore, these mice did not develop hematuria or red cell fragmentation, and serum urea levels remained normal at the time of death (median = 10.6 mmol/liter, range = 8.9–11.5 mmol/liter). Capillary wall C3 staining was reduced in comparison to age-matched Cfh−/− mice, and subendothelial electron-dense GBM deposits were infrequent. Hence, the plasma C3 regulation in the Cfh−/−.FHΔ16-20low mice was insufficient for aHUS to develop but did prevent the development of MPGN2 up to the time point examined. The data from both transgenic lines, together with the observation that aHUS did not develop in Cfh−/− mice that have secondary C3 depletion, demonstrated that C3 activation is a key effector mechanism in aHUS.
There is now overwhelming evidence that aHUS is associated with defective regulation of the AP of complement activation. Mutations affecting the cofactors for the factor I–mediated proteolytic inactivation of activated C3 in plasma (FH; references 2
) and on cell surfaces (membrane cofactor protein; references 15
), in addition to mutations affecting the serine protease factor I itself (23
), predispose to the development of aHUS. Similarly, gain-of-function mutations in the complement activator factor B also predispose to aHUS, further supporting the critical role of C3 activation in the pathogenesis of aHUS (24
). The spontaneous pathology in the Cfh−/−
.FHΔ16-20 mice, like that of humans with functionally similar FH mutations, targeted the renal vasculature, suggesting that there are unique anatomical and/or physiological properties of this endothelial bed that render it particularly sensitive to complement-mediated damage.
Interestingly, aHUS-associated mutations in complement genes are normally found in heterozygosis in aHUS patients and are frequently associated with incomplete penetrance. In this respect, it is notable that Cfh+/−
.FHΔ16-20 mice did not spontaneously develop aHUS, suggesting that, like in some human patients, multiple genetic defects affecting complement regulators are required for aHUS to develop in mice (25
). Furthermore, infection, immunosuppressive drugs, cancer therapies, oral contraceptive agents, pregnancy, or postpartum period are all factors that may trigger aHUS in individuals carrying CFH
mutations in heterozygosis, and the syndrome has developed in the native kidney of live-related kidney donors who had previously unidentified FH mutations (27
). Thus, Cfh+/−
.FHΔ16-20 mice may only develop aHUS after an additional insult, either genetic or environmental (or both), although interspecies differences in the regulation of C3 on cell surfaces by FH and other complement regulators may also be relevant to the apparent resistance of Cfh+/−
.FHΔ16-20 mice to aHUS.
Treatment of aHUS associated with FH mutations is difficult. Renal transplantation is associated with a high incidence of disease recurrence (28
). Plasma infusions as a source of FH have been beneficial (29
) but can result in hyperproteinemia, requiring plasma exchange (30
). The principle source of FH is hepatic, and hence, the expected definitive treatment would be combined liver and renal transplantation, which has produced mixed results (31
). Our data define an important aspect of therapy. Agents that attempt to restore C3 regulation must critically achieve this on cell surfaces. Indeed, our observations suggest that restoration of fluid-phase regulation alone may, by increasing the circulating plasma C3 levels, be deleterious.
In conclusion, the similarities between the surface recognition domains of mouse and human FH (34
) enabled us to mutate mouse FH to functionally mimic aHUS-associated human FH mutations. Cfh−/−
mice expressing this mutant FH protein spontaneously developed aHUS, not MPGN2. Our data provide the first in vivo proof of principle evidence that FH mutations specifically impairing surface recognition can result in spontaneous aHUS and define the molecular pathogenesis of aHUS-associated FH mutations.