Download Hepatocyte-Cfh -/- mice have reduced plasma FH and C3

Partial complement factor H deficiency is associated with both C3
glomerulopathy and thrombotic microangiopathy
Katherine A. Vernon1, Marieta M. Ruseva1; H. Terence Cook1, Marina Botto1, Talat
H. Malik1, Matthew C. Pickering1
1
Centre for Complement and Inflammation Research, Imperial College, London
Running title: Partial FH deficiency and renal disease
Abstract:
199
words
Text:
2376
words
CORRESPONDING AUTHOR
Matthew Pickering
[email protected]
Centre for Complement and Inflammation Research
Division of Immunology and Inflammation
Imperial College London
Hammersmith Campus
Du Cane Road
London W12 0NN
Tel: +44 (0) 20 8383 2398
Fax: +44 (0) 20 8383 2379
ABSTRACT
The complement-mediated renal diseases, C3 glomerulopathy (C3G) and atypical
haemolytic uraemic syndrome (aHUS), are strongly associated with inherited and
acquired abnormalities in the regulation of the complement alternative pathway (AP).
The major negative AP regulator is the plasma protein complement factor H (FH).
Abnormalities in FH result in uncontrolled activation of complement C3 through the
AP and are associated with susceptibility to both C3G and aHUS. FH is
predominantly synthesized in the liver. We generated mice with hepatocyte-specific
FH deficiency and demonstrated that these animals have reduced plasma FH levels
with secondary reduction in plasma C3. Unlike mice with complete FH deficiency,
hepatocyte-specific FH-deficient animals did not develop either plasma C5 depletion
or accumulation of C3 along the glomerular basement membrane. In contrast, the
sub-total FH deficiency was associated with mesangial C3 accumulation consistent
with C3G. Although, there was no evidence of
spontaneous thrombotic
microangiopathy (TMA), the hepatocyte-specific FH-deficient animals developed
severe C5-dependent TMA following induction of complement activation within the
kidney using accelerated serum nephrotoxic nephritis. Taken together, our data
indicate that sub-total FH deficiency can give rise to either spontaneous C3G or
aHUS following a complement-activating trigger within the kidney and that the latter
is C5-dependent.
INTRODUCTION
Both atypical haemolytic uraemic syndrome (aHUS) and C3 glomerulopathy (C3G)
can develop in the setting of abnormal complement regulation
1-3
. C3G includes the
entities dense deposit disease and C3 glomerulonephritis, and is characterized by
the abnormal accumulation of C3 within the glomerulus which may or may not be
associated with inflammation, such as membranoproliferative glomerulonephritis 4. In
practice, it is considered when there is C3-dominant glomerulonephritis, with
dominance defined as C3 reactivity at least two orders of magnitude more intense
than any other immune reactant 5. In aHUS, there is complement-mediated damage
to
the
renal
endothelium
with
consequent
development
of
thrombotic
microangiopathy (TMA) 2.
C3G and aHUS are typically associated with genetic and acquired abnormalities that
result in uncontrolled activation of the complement alternative pathway (AP).
Complement factor H (FH) is the major negative regulator of the AP and
abnormalities in FH have been associated with aHUS and C3G 3. Genetic changes in
complement components in aHUS include loss of function in regulators (FH,
complement factor I [FI], CD46) and gain of function in activation proteins
(complement factor B and C3) 2. In C3G genetic abnormalities are less frequent and
include loss of function changes in FH, gain of function change in C3 and structural
changes within the factor H-like gene family
1
. Acquired factors include
autoantibodies that enhance AP activation, most commonly C3 nephritic factors in
the case of C3G and anti-FH autoantibodies in aHUS 1,2.
Intriguingly, case reports have shown that complete FH deficiency can manifest as
aHUS or C3G 3. The factors that determine which pathology develops in the setting
of FH deficiency are not fully understood but mechanistic insights into C3G and
aHUS have derived from functional characterization of the genetic defects together
with the study of animal models that mimic some of these defects
3,6
. Pigs
7
and
mice 8 with complete FH deficiency develop spontaneous C3G but not aHUS. In both
species, the deficiency results in uncontrolled AP activation and secondary
consumption of C3
3
and C5
7,9
, which results in absence of plasma complement
haemolytic activity. In FH-deficient mice (Cfh-/-) spontaneous C3G depended on AP
activation 8 but not on C5 10 and developed in wild-type kidneys transplanted into Cfh/-
recipients
11
. Notably, spontaneous C3G did not develop in the heterozygous FH-
deficient pig or mouse strain
7,8
. FH consists of 20 short consensus repeat (SCR)
domains with SCR1-4 essential for C3 regulation and SCR19-20 important in
interacting with surface polyanions and C3b. FH mutations in aHUS predominantly
affect the surface recognition sites within SCR19-20 6. C5-dependent spontaneous
aHUS did occur in Cfh-/- mice transgenic for a FH protein (FHΔ16-20) that lacked
surface recognition domains
9,12
. This led to the paradigm that aHUS occurs when
there is a combination of defective regulation of complement activation along the
glomerular endothelium (e.g. in the presence of FH mutations that impair surface
recognition) and sufficient circulating intact C3 and C5 to enable complementmediated endothelial injury to occur 9,12.
Here we report the phenotype of hepatocyte-specific FH-deficient mice (hepatocyteCfh-/-) and show that this strain has plasma FH levels of approximately 20% of
normal. This sub-total FH deficiency resulted in plasma C3 dysregulation and
accumulation of C3 within mesangial areas. However, unlike mice with total FH
deficiency, hepatocyte-Cfh-/- mice did not develop plasma C5 deficiency or
glomerular basement membrane (GBM) C3 deposition. The hepatocyte-Cfh-/animals did not develop spontaneous aHUS but, following experimentally triggered
renal injury using accelerated serum nephrotoxic nephritis (NTN), they developed a
severe C5-dependent TMA.
RESULTS
Hepatocyte-Cfh-/- mice have reduced plasma FH and C3 but normal plasma C5
The conditional FH-deficient (CfhloxP/loxP) strain was developed using recombineering
techniques
13
to introduce loxP sites either side of exons 2 and 3 of the Cfh gene in
embryonic stem cells (Figure 1A). CfhloxP/loxP mice were viable and back-crossed onto
the C57BL/6 genetic background. To generate hepatocyte-specific FH-deficient mice
(hepatocyte-Cfh-/-), the CfhloxP/loxP strain was inter-crossed with mice selectively
expressing Cre recombinase in hepatocytes using the albumin promoter/enhancer
sequence (Alb-Cre)14. Hepatocyte Cre recombinase-mediated excision of exons 2
and 3 was confirmed by analysis of hepatic RNA (supplemental Figure 1). Consistent
with hepatocytes being the predominant source of plasma FH synthesis, circulating
FH levels were significantly reduced in hepatocyte-Cfh-/- mice (Figure 1B). This subtotal FH deficiency was associated with marked reduction in plasma C3 levels
although not as low as that seen in mice with complete FH deficiency (Figure 2A).
However, unlike Cfh-/- mice, plasma C5 levels appeared normal when assessed
semi-quantitatively by western blotting (Figure 2B). Unexpectedly CfhloxP/loxP animals
had significantly lower plasma FH and C3 compared to Alb-Cre mice (Figure 1B and
2A). This indicated that the production of FH from the targeted cfh allele was
impaired.
Hepatocyte-Cfh-/- mice develop spontaneous mesangial but not GBM C3
deposition
Cfh-/- mice spontaneously develop C3 accumulation along the GBM 8. To determine if
sub-total FH deficiency was associated with the same renal phenotype we examined
renal histology in 9 month old hepatocyte-Cfh-/- and Cfh-/- mice. We included
CfhloxP/loxP and Alb-Cre strains as control groups (supplemental table 1). We found
that serum urea levels were <20mmol/l with the exception of one animal in the
hepatocyte-Cfh-/- cohort (26.5mmol/l) and two animals in the Cfh-/- cohort (20.5 and
21.7mmol/l). Albuminuria was only detectable in one of the hepatocyte-Cfh-/(2.1mg/ml) mice. Haematocrits and peripheral blood smears were normal
(supplemental Figure 2). Renal light microscopy showed minimal glomerular
hypercellularity in hepatocyte-Cfh-/- and Cfh-/- mice and no evidence of glomerular
thrombosis (Figure 2C and D). As expected all Cfh-/- mice had linear glomerular
capillary wall C3 staining8. In contrast, glomerular C3 deposition was granular and
mesangial in distribution in age-matched hepatocyte-Cfh-/- mice (figure 2E). The
normal tubulo-interstitial C3 staining pattern (seen in the control strains, Figure 2E)
was reduced in intensity in the hepatocyte-Cfh-/- strain and, as previously shown 8,
absent in Cfh-/- animals. These data indicated that sub-total FH deficiency was
sufficient to prevent C3 accumulation along the GBM.
Hepatocyte-Cfh-/- and CfhloxP/loxP mice develop TMA during NTN
To test the hypothesis that the impaired complement regulation associated with subtotal FH deficiency predisposed to enhanced renal damage during complement
activation within the glomerulus, we examined the response of hepatocyte-Cfh-/- mice
to NTN. Hepatocyte-Cfh-/- mice rapidly developed renal failure and animals were
humanely culled on day 3 after administration of nephrotoxic serum (NTS, Figure 3).
In a representative experiment, 2 out of 14 hepatocyte-Cfh-/- mice were culled 3 days
after receiving NTS, whereas there were no deaths in the wild-type (n=9) and
CfhloxP/loxP (n=12) groups. Maximal haematuria developed in all hepatocyte-Cfh-/- mice
1 day post-NTS (supplemental Figure 3). In comparison to wild-type mice, at day 3
post-NTS hepatocyte-Cfh-/- animals had greater haematuria, higher urea levels, lower
haematocrits, marked red cell fragmentation and glomerular thrombosis (Figure 3
and 4). Glomerular C3 was highest in the hepatocyte-Cfh-/- mice (Figure 3F).
Differences in glomerular IgG and sheep IgG were minimal and the immune
response to sheep IgG in adjuvant was equivalent between the experimental groups
(supplemental Figures 4-6). CfhloxP/loxP mice, in which we had demonstrated slight
reduction in plasma FH levels (Figure 1B), developed a phenotype intermediate in
severity between wild-type and hepatocyte-Cfh-/- mice. Together these data indicated
that sub-total FH deficiency was associated with a hypersensitive response during
NTN and that the severity of TMA was related to the degree of FH deficiency.
The hypersensitive response of hepatocyte-Cfh-/- mice during NTN is
dependent on C5.
Since hepatocyte-Cfh-/- animals had normal C5 levels we hypothesized that the
enhanced renal damage during nephrotoxic nephritis could be influenced by C5
activation. To test this hypothesis we administered either a monoclonal anti-mouse
C5 antibody (BB5.1) or an isotype-matched control antibody to hepatocyte-Cfh-/mice. Administration of BB5.1 ameliorated the renal damage with reduced
haematuria, urea, red cell fragmentation and glomerular thrombosis and neutrophil
scores (Figure 5). Notably the thrombosis was lower in the isotype-matched antibody
treated group than that typically seen in untreated hepatocyte-Cfh-/- mice during NTN
(Figure 3E). These data indicated that the hypersensitive response of hepatocyteCfh-/- mice during NTN was dependent on C5.
DISCUSSION
Mice with hepatocyte-specific FH deficiency were established by inter-crossing
conditional FH-deficient mice with mice expressing Cre recombinase under the
control of a hepatocyte-specific promoter. The hepatocyte-Cfh-/- mice had low plasma
FH and C3 levels and spontaneous development of mesangial C3 deposition. This
15
type of lesion has been reported as one morphological type of human C3G
.
Heterozygous FH-deficient animals have normal glomerular C3 staining pattern with
slight but significantly lower plasma C3 levels than wild-type mice 8. The FH levels in
the hepatocyte-Cfh-/- mice (~20% of normal) were lower than those in heterozygous
FH-deficient mice
12
, but still sufficient to prevent glomerular C3 accumulation along
the GBM, a characteristic feature of mice with complete FH-deficiency 8. However,
the sub-total FH level in the hepatocyte-Cfh-/- mice resulted in spontaneous
mesangial C3 deposition. Paramesangial deposits composed solely of C3c have
been reported in DDD specifically during episodes of hypocomplementaemia
16
. It
was hypothesized that these derived from plasma C3c that had been released
following the FI-mediated cleavage of iC3b to C3dg. This mechanism could be
operating in the hepatocyte-Cfh-/- mice continuously due to the low FH levels. The
normal tubulo-interstitial C3 staining seen in wild-type mice was reduced in intensity
in hepatocyte-Cfh-/- mice. This is most likely due to the reduced plasma C3 levels
since tubulo-interstitial C3 staining in mice is dependent on circulating C3 11,17,18.
Renal disease in C3G may become clinically evident or be exacerbated by an
environmental trigger, such as infection. For example, upper respiratory tract
infection is an exacerbating factor in IgA nephropathy (synpharyngitic macroscopic
haematuria) and C3G
19,20
. The hypothesis is that the inability to regulate the AP not
only increases susceptibility to spontaneous C3G but also increases the likelihood of
renal injury during an inter-current event that triggers complement activation within
the kidney. To model this we examined the response of hepatocyte-Cfh-/- animals to
NTN. Our observations showed that partial FH deficiency enhanced renal injury
during experimental NTN and that this was C5-dependent. Hepatocyte-Cfh-/- mice
were extremely sensitive to renal injury in this model and developed a renal
phenotype consistent with aHUS. Our interpretation is that hepatocyte-Cfh-/- mice
were susceptible during NTN due to a combination of the inability of the partial FH
deficiency to adequately regulate complement within the kidney and the ability of the
partial FH deficiency to preserve sufficient C5 in plasma to mediate injury. The
hepatocyte-Cfh-/- mice were housed in specific pathogen-free conditions so we do not
know how the renal phenotype might be influenced by infection. The C5-dependence
of the TMA in our experimental model suggests that the therapeutic utility of C5
inhibition in C3G may be particularly useful in patients with C3G and defective
complement regulation who present with features of intercurrent TMA.
The spontaneous phenotype (mesangial C3 glomerulopathy with no evidence of
TMA) and the phenotype observed during NTN (acute severe TMA) were distinct.
There is clear evidence of phenotypic variation within these diseases among
individuals with the same complement mutation. For example, in CFHR5
nephropathy the spectrum of renal disease varies from microscopic haematuria to
end-stage renal failure
21
. Similarly, in aHUS incomplete penetrance in the setting of
complement mutations is common and clinical onset is typically preceded by an
environmental change e.g. infection, drugs and pregnancy
22
. However, variation
between C3G and aHUS phenotypes within the same individual is less well
understood. Case reports of C3G as the presenting feature with aHUS occurring later
in the clinical course include: an adult male with complete FH deficiency who
presented with biopsy-proven C3G (membranoproliferative glomerulonephritis,
[MPGN]) but approximately one year later developed aHUS 23; an individual with antiFH autoantibodies who presented with C3G (MPGN) and developed recurrent C3G
in the first renal transplant and TMA in the second transplant
24
; a young male with
mutations in both C3 and CD46 and a family history of aHUS who presented initially
with C3G (MPGN) before developing clinical features of aHUS
25
; a young male with
heterozygous FH mutation who presented with C3G (MPGN) and later developed
aHUS
26,27
; an adult female with C3G (MPGN) with no complement mutations who
later developed aHUS
27
and an adult male with C3G (mesangial C3 deposits) with
no complement mutations who later developed concurrent aHUS
27
. Case reports of
aHUS as the presenting feature with C3G occurring later in the clinical course appear
less numerous but include: a young male with combined heterozygous FH and FI
mutations who presented with aHUS and subsequently developed glomerular C3
deposits in the transplant kidney
28
and a young male with homozygous FH
deficiency who presented with aHUS and then developed isolated glomerular C3
deposits in the absence of TMA within the transplant kidney
28
. In this context the
hepatocyte-Cfh-/- mice recapitulate the clinical observations and provide definitive
experimental evidence that the same genetic defect can predispose to C3G and
aHUS.
The mice carrying the targeted Cfh alleles had significantly reduced basal plasma FH
and C3 levels indicating that expression of FH by the targeted locus was impaired.
These animals were also hypersensitive to renal injury during NTN which, based on
the hepatocyte-Cfh-/- NTN phenotype, was likely a consequence of the reduced FH
levels. Impaired expression of targeted genes due to the introduction of loxP sites
and/or selectable markers is well recognized and may be rectified by removal of the
selectable marker
29
. In our construct FRT sites were placed either side of the
neomycin resistance gene to enable the neomycin resistance gene to be excised by
Flp recombinase and this is currently in progress. With our present dataset, the
impaired FH expression from the targeted allele prevented us from deriving any firm
conclusions with respect to the contribution of extra-hepatocyte FH to the total FH
pool.
In summary, our experimental data demonstrated that two distinct renal phenotypes,
C3G and aHUS, can develop in the setting of sub-total FH deficiency. Like
spontaneous aHUS in a murine model of FH-associated aHUS 9, experimentally
triggered aHUS in hepatocyte-Cfh-/- animals was C5-dependent, further supporting
the key role of C5 in complement-mediated glomerular TMA. Our data illustrate the
complex relationship between FH insufficiency and complement-mediated renal
injury and that the therapeutic utility of C5 inhibition in this setting depends on the
renal phenotype.
CONCISE METHODS
Mice
Mice were housed in specific pathogen-free conditions and all animal procedures
were performed in accordance with institutional guidelines and approved by the UK
government. Recombineering reagents, EL350 cells, PL451 plasmid and PL452
plasmid were obtained from NCI at Frederick (www.ncifrederick.cancer.gov). Mice
used in models of experimental renal disease were matched for age and sex, and
aged between 10 to 12 weeks at the time of the experiment. Mice expressing Cre
recombinase under the control of the murine albumin promoter (Alb-Cre) were
purchased from the Jackson laboratories (B6.Cg-Tg[Alb-cre]21Mgn/J, strain number
003574, http://jaxmice.jax.org/strain/003574.html). Genotyping was performed by
PCR
on
digested
tissue
using
primers
pairs.
Alb-Cre
primer
pairs:
5’-
GCGGTCTGGCAGTAAAAACTATC-3’ and 5’-GTGAAACAGCATTGCTGTCACTT-3’
generated a 100bp product in presence of Alb-Cre allele. Targeted Cfh allele primer
pairs: 5’-AGGGGGAAAGAGAGAGATG-3 and 5’-TCCTAGTAGCAAAGCTAAACAG3’ generated a 450bp or 527bp amplicon in wild-type and targeted Cfh alleles
respectively. Floxed Cfh allele primer pairs: 5’-CTGTCAGCAAGAATTATTTGGC-3’
and 5’-ACACATCGTGGCTTTTCATTGC-3’ generated a 610bp or 318bp amplicon in
wild-type and floxed Cfh alleles respectively. The 9 month old cohorts comprised:
hepatocyte-Cfh-/- (n=25; 12M and 13F), Cfh-/- (n=23; 8M and 15F), CfhloxP/loxP (n=9;
4M and 5F) and Alb-Cre (n=8; 2M and 6F) mice. Two unexplained deaths occurred:
one hepatocyte-Cfh-/- mouse aged 5 weeks and one Alb-Cre mouse aged 17 weeks.
In both cases renal histology was normal by light microscopy.
Measurement and detection of mouse FH, C3 and C5
Mouse blood samples were collected directly into EDTA-containing Eppendorf tubes
and kept on ice until centrifugation and aliquots stored at -80°C. Repeated
freeze/thaw cycles were avoided. Plasma FH levels were measured by enzymelinked immunosorbent assay (ELISA) using a polyclonal sheep anti-human FH
antibody (antibodies-online.com) and a biotinylated goat anti-human FH antibody
(Quidel, San Diego, USA). FH levels in the samples were quantified relative to
normal wild-type mouse plasma used in the standard curve. Measurement of plasma
C3 was performed by ELISA as previously described
30
using a unconjugated and
horse radish peroxidase-conjugated polyclonal goat anti-mouse C3 antibody (MP
Biomedical, Cambridge, UK) with a standard curve generated using acute phase
serum containing a known quantity of C3 (Calbiochem, Hertfordshire, UK). Western
blotting for mouse C5 was performed as previously described 31.
Assessment of renal and haematological function
Haematuria was detected using Hema-Combistix urine reagent strips (Siemens,
Frimley, UK) using the following scale: negative, trace, 1+, 2+ and 3+. Plasma urea
was measured using an ultraviolet method according to the manufacturer’s
instructions (R-Biopharm, Glasgow, UK). Plasma samples were diluted 1:40 in PBS
and a standard preparation of urea of known concentration was included as an
internal control. Albuminuria was measured using radial immunodiffusion as
previously described
10
with a rabbit anti-mouse albumin antibody (AbD Serotec,
Oxford, UK) and purified mouse albumin standard (Sigma-Aldrich, Dorset, UK).
Haematocrit was calculated after centrifuging EDTA-treated blood in ammoniumheparin capillary tubes (VWR International Ltd, Lutterworth, UK) at 13,000rpm for 5
minutes and measuring the packed cell height to total height ratio. Peripheral blood
smears were prepared on glass slides and stained using the Diff Quick staining kit
(Polysciences, Warrington, USA). Red cell fragments were quantified as the number
per 200 red blood cells counted at high power (x40).
Assessment of renal histology
Kidneys were fixed in Bouin’s solution (Sigma-Aldrich, Dorset, UK) for 2-4 hours and
then transferred to 70% ethanol. Sections were embedded in paraffin and stained
with periodic acid-Schiff (PAS) reagent and examined by light microscopy. Sections
were scored for glomerular thrombosis and glomerular neutrophil infiltration.
Analyses were all done blinded to the sample identity with 20 glomeruli scored per
section. Glomerular thrombosis was scored according to the amount of PAS-positive
material per glomerular cross-section as follows: grade 0, no PAS-positive material;
grade 1, 0-25%; grade 2, 25-50%; grade 3, 50-75%; grade 4, 75-100%. Glomerular
neutrophils were scored according to the number of neutrophils per glomerular crosssection. Glomerular histology was graded as follows: grade 0, normal; grade 1,
segmental hypercellularity in 10–25% of the glomeruli; grade 2, hypercellularity
involving >50% of the glomerular tuft in 25–50% of glomeruli; grade 3,
hypercellularity involving >50% of the glomerular tuft in 50–75% of glomeruli; grade
4, glomerular hypercellularity in >75% or crescents in >25% of glomeruli.
Immunostaining for C3, IgG and sheep IgG was performed on acetone-dried 5µm
frozen sections as previously described
32
. Antibodies used were: fluorescein-
isothiocyanate (FITC)-labeled polyclonal goat anti-mouse C3 (MP Biomedical,
Cambridge, UK), FITC-conjugated polyclonal goat anti-mouse IgG Fc γ-chain
specific antibody (Sigma-Aldrich, Dorset, UK) and a FITC-conjugated monoclonal
mouse
anti-goat/sheep
IgG
(Sigma-Aldrich,
Dorset,
UK)
Quantitative
immunofluorescence analysis was performed using an Olympus fluorescent
microscope with digital camera (Olympus, Southend, UK), Image-Pro Plus software
(Media Cybernetics, Silver Spring, USA) and a minimum of 10
examined per section as previously described
glomeruli were
32
. Mean fluorescence intensity was
expressed in arbitrary fluorescence units (AFU). Glomerular C3 from NTN was
scored according to the number of glomerular cross-section quadrants that stained
for C3. Grade 0, 1, 2, 3 and 4 representing staining of 0, 1, 1-2, 2-3, or all 4
quadrants respectively.
Induction of serum nephrotoxic nephritis
Nephrotoxic nephritis (NTN) was induced by the intravenous injection of 200μl of
sheep nephrotoxic serum (NTS; a sheep IgG serum fraction containing anti-mouse
GBM antibodies), in to mice that had been pre-immunised with an intra-peritoneal
injection of 200µg of sheep IgG (Sigma-Aldrich, Dorset, UK) in complete Freund’s
adjuvant (CFA) (Sigma-Aldrich, Dorset, UK) as previously described
32
. For anti-C5
antibody treatment mice received either 2mg of BB5.1 or isotype-matched antibody
subcutaneously 24 hours before NTS. The animals received two more doses of
either BB5.1 or isotype-matched antibody on day 1 and 3 post-NTS administration.
To assess the immune response to sheep IgG hepatocyte-Cfh-/- (n=8), CfhloxP/loxP
(n=7), and wild-type (n=10) mice were injected with 200 µg of sheep IgG in CFA.
Plasma samples were collected at day 2, 4 and 9 post-immunization. IgG response
to sheep IgG was measured by ELISA on microtitre plates coated with 3.5 µg/ml of
sheep IgG. The plates were incubated with mouse plasma serially diluted in PBS
containing 1% BSA. Bound mouse IgG was detected with a peroxidase-conjugated
sheep anti-mouse IgG antibody (product number: 515-035-062; Jackson Immune
Research, US). The antibody titre was expressed as the inverse of the greatest
serum dilution that gave two-fold the background reading.
Statistical analysis
Non-parametric data was represented as the median with the range of values given
in parentheses. Two groups were analysed using the Mann-Whitney test whilst oneway analysis of variance with Bonferroni multiple comparison test was used to
analyse three or more groups. Data were analyzed using GraphPad Prism version
3.0 for Windows (GraphPad Software, San Diego, USA).
ACKNOWLEDGEMENTS
KAV was a funded by Kidney Research UK Clinical Fellowship (TF8/2009). MCP is a
Wellcome Trust Senior Fellow in Clinical Science (WT082291MA) and MMR is
funded by this fellowship.
STATEMENT OF COMPETING FINANCIAL INTERESTS
MCP has received fees from Alexion Pharmaceuticals for invited lectures and for preclinical testing of complement therapeutics and consultancy fee from Achillion
Pharmaceuticals.
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FIGURE LEGENDS
Figure 1. Generation of conditional FH-deficient mouse. (A) The targeting vector
contained loxP sites (blue arrows) located either side of exons 2 and 3 of the Cfh
gene. The targeted locus contained a neomycin-resistant (Neo) positive selectable
marker flanked by flippase (Flp) recombinase recognition target (FRT) sites (black
arrows). Cre recombinase-mediated recombination between the loxP sites results in
excision of exons 2 and 3 and the positive selectable marker (floxed allele). This
causes a reading frameshift and generation of a premature stop codon in exon 4 and
therefore a null allele. Vertical numbered boxes denote exons; NcoI – Nco1
restriction enzyme; black box denotes probe location used to identify correctly
targeted cells. (B) Plasma FH levels in 12-week old Alb-Cre (median 93% of wildtype, range 71-137%, n=6), CfhloxP/loxP (median 59.5% of wild-type levels, range 37103%, n=8) and hepatocyte-Cfh-/- (median 19% of wild-type levels, range 14-29%,
n=7) mice. Horizontal bars denote median values. *P=0.032, **P=0.002, P values
derived from Bonferroni multiple comparison test.
Figure 2. Plasma complement and spontaneous renal phenotype in conditional
and hepatocyte-specific FH-deficient mice. (A) Plasma C3 levels in 9 month old
Alb-Cre
(median
171.5µg/ml,
range
158.8-273.1,
n=8),
-/-
131.1µg/ml, range 118.4-272.8, n=9), hepatocyte-Cfh
CfhloxP/loxP
(median
(median 41.5µg/ml, range
28.6-82.2, n=25) and Cfh-/- (median 15.8µg/ml, range 7.9-26.6, n=23) mice.
Horizontal bars denote median values. * P<0.05, **P<0.001, ***P<0.0001, P values
derived from Bonferroni multiple comparison test. (B) Assessment of plasma C5 in
hepatocyte-Cfh-/- mice by western blotting. The intensity of the C5 protein band was
comparable between hepatocyte-Cfh-/- (lanes 1-4) and wild-type (lanes 5-7) mice. No
C5 is seen in Cfh-/- plasma (lane 8) and lane 9 contained 100ng of purified human C5
as reference. (C) Glomerular cellularity scores and (D) representative PAS-stained
glomerular images in 9-month old Alb-Cre, CfhloxP/loxP, hepatocyte-Cfh-/- and Cfh-/mice. Horizontal bars denote median values. Original magnification x40. (E)
Representative immunostaining images for glomerular C3 and IgG in 9-month old
Alb-Cre, CfhloxP/loxP, hepatocyte-Cfh-/- and Cfh-/- mice. The characteristic linear
capillary wall C3 deposition seen in Cfh-/- mice was replaced by a mesangial staining
pattern in hepatocyte-Cfh-/- mice. Tubulo-interstitial C3 deposition was absent in Cfh-/mice and normal in CfhloxP/loxP and Alb-Cre animals. It was detectable but reduced in
the hepatocyte-Cfh-/- mice. Mesangial IgG is commonly seen in older mice and was
detectable in small amounts in all of the cohorts.
Figure 3. Response of conditional and hepatocyte-specific FH-deficient mice to
accelerated serum nephrotoxic nephritis. (A) Hematuria, (B) serum urea, (C)
hematocrit, (D) red cell fragments, (E) glomerular thrombosis score and (F)
glomerular C3 deposition at day 3 following administration of nephrotoxic serum to
pre-immunized wild-type, CfhloxP/loxP and hepatocyte-Cfh-/- mice. Horizontal bars
denote median values. * P<0.001, ** P<0.01, P values derived from Bonferroni
multiple comparison test. Data representative of three independent experiments.
Figure 4. Response of conditional and hepatocyte-specific FH-deficient mice to
accelerated serum nephrotoxic nephritis. Representative images of peripheral
blood smears (upper panel), renal sections (middle panel) and glomerular C3
immunostaining (lower panel) in wild-type, CfhloxP/loxP and hepatocyte-Cfh-/- mice.
Figure 5. Antibody-mediated C5 inhibition in hepatocyte-specific FH-deficient
mice during accelerated serum nephrotoxic nephritis. (A) Hematuria, (B) serum
urea, (C) red cell fragments, (D) glomerular thrombosis score and (E) glomerular
neutrophil numbers at day 4 following administration of nephrotoxic serum to preimmunized hepatocyte-Cfh-/- mice treated with either BB5.1 (n=11) or isotypematched antibody (n=10). Horizontal bars denote median values. P values derived
from Mann Whitney Test.