The FASEB Journal • Research Communication
Synthesis of complement protein C3 in the kidney is an
important mediator of local tissue injury
Neil S. Sheerin,*,†,1 Paul Risley,* Katsu Abe,‡ Ziyong Tang,*,§ Wilson Wong,*
Tao Lin,* and Steven H. Sacks*
*King’s College London, Department of Nephrology and Transplantation, Guy’s Hospital, London,
UK; †School of Clinical Medical Sciences, Newcastle University, Newcastle upon Tyne, UK; ‡Division
of Nephrology, Second Department of Internal Medicine, Nagasaki University School of Medicine,
Nagasaki, Japan; and §Department of Nephrology, Peking University Third Hospital, Beijing, China
ABSTRACT Increased exposure of the tubular epi-
thelium to filtered protein is a proposed mechanism of
progressive renal failure associated with glomerular
disease, but how this protein overload translates into
tubular damage remains unclear. We have examined a
model of adriamycin-induced proteinuria to determine
the effect of locally synthesized C3, the central proin-
flammatory protein of the complement cascade.
C3ⴚ/ⴚ kidney isografts placed in wild-type C3ⴙ/ⴙ
mice were protected from proteinuria-associated com-
plement activation, tubular damage, and progressive
renal failure despite the presence of abundant circulat-
ing C3. The quantity of urinary protein was unaffected
by the absence of C3, and thus the influence of C3 was
not explained by alteration in the filtered protein load.
These results suggest that local synthesis of comple-
ment from renal epithelial cells is a critical mediator of
tubular damage in proteinuria-associated renal disease.
Our results concur with previous findings of increased
synthesis of C3 in human tubular epithelium exposed
to high concentrations of protein in vitro. Because
progressive renal damage in humans associates with
proteinuria regardless of cause, our findings have im-
plications for the pathogenesis and treatment of renal
failure from many common causes, immunological and
nonimmunological.—Sheerin, N. S., Risley, P., Abe, K.,
Tang, Z., Wong, W., Lin, T., Sacks, S. H. Synthesis of
complement protein C3 in the kidney is an important
mediator of local tissue injury. FASEB J. 22, 1065–1072
(2008)
Key Words: proteinuria 䡠 renal failure 䡠 innate immunity
There is a well-established association between
proteinuria, injury to the tubulointerstitial (TI) com-
partment of the kidney, and progressive loss of renal
function (1). In addition, the amount of proteinuria is
predictive of the rate of loss of renal function (2), and
strategies that reduce proteinuria, e.g., inhibition of the
renin-angiotensin system, slow the loss of renal func-
tion (3). These observations have led to the widely
accepted hypothesis that proteinuria mediates TI in-
jury, typically interstitial fibrosis and tubular atrophy,
0892-6638/08/0022-1065 © FASEB
and therefore progressive loss of function. However, it
is not known how proteinuria causes TI injury.
It might be that excessive glomerular protein leak
exceeds the safe resorptive capacity of the tubules
placing an injurious metabolic stress on the tubular
epithelium, worsened by relative tubular ischemia. Al-
ternatively, specific proteins in the glomerular filtrate
may be toxic to tubules, for example, transferrin or fatty
acid-conjugated albumin (4). Another possible media-
tor of damage is the complement system.
The complement system is a complex set of soluble
and membrane-bound proteins and constitutes a major
element of the innate immune system. The generation
of anaphylotoxins, opsonization, and the formation of
the cytopathic membrane attack complex, C5b-9, all
contribute to pathogen removal and augmentation of
the adaptive immune response. Despite a series of
inhibitory proteins, excessive or inappropriate activa-
tion of complement can cause tissue injury. Evidence
that this may be the case during proteinuria comes
from both clinical and animal studies. Complement
activation products are found in the urine of protein-
uric patients even when the glomerular lesion has a
nonimmune basis, suggesting activation of complement
within tubules (5). Proximal tubular epithelial cells can
activate complement (6) due to intrinsic convertase
activity or a paucity of inhibitors (7). Histological
examination of damaged kidney demonstrates a spatial
relationship between tissue injury and complement
protein staining (8). Furthermore, in studies of pro-
teinuria in animals complement deficiency or inhibi-
tion reduces the level of histological injury and the loss
of renal function (9 –12).
An additional dimension regarding the action of
complement is the ability of the kidney to synthesize
complement proteins. The kidney contributes measur-
ably to the circulating pool of C3 (13), the pivotal
complement protein. In addition, increased C3 gene
1
Correspondence: School of Clinical Medical Sciences, 4th
Fl., William Leech Bldg., The Medical School, Framlington
Pl., Newcastle University, Newcastle upon Tyne, NE2 4HH,
UK. E-mail: neil.sheerin@ncl.ac.uk
doi: 10.1096/fj.07-8719com
1065
expression occurs during renal inflammation (14), in
proteinuric diseases (15) and can be stimulated in vitro
by exposure of tubular epithelial cells to serum proteins
(16). The main source of renal-derived C3 is the
tubular compartment although glomerular cells have
the capacity to synthesize complement proteins. How-
ever, no effect of local renal production of C3 was seen
in a mouse model of antibody-mediated glomerular
disease (17). However, in renal transplant injury, where
the tubular compartment is the main target of injury,
local complement synthesis is a critical mediator of
both ischemia reperfusion injury (18) and transplant
rejection (19, 20).
This study was performed to understand the role of
locally synthesized C3 in the development of TI injury.
We have used C3-deficient mice (C3⫺/⫺) rendered
proteinuric by the glomerulotoxin, adriamycin, to con-
firm the importance of C3 activation in the develop-
ment of TI injury. Using a kidney transplantation
strategy between wild-type C3⫹/⫹ and C3⫺/⫺ mice,
we have then specifically addressed the role of locally
synthesized C3.
MATERIALS AND METHODS
Animals
C3⫺/⫺ mice were generated by homologous recombination
as described previously (21). Mice with C3⫺/⫺ on a
C57BL/6 background (H-2b) were backcrossed with BALB/c
(H-2d) mice for 7 generations. C3 was not detected in the
plasma of C3⫺/⫺ by ELISA (sensitivity 10 ng/ml). In mice
the gene for C3 on chromosome 17 is ⬃15 cM from the H2
region, with H-2d closest to the C3 locus. The H-2d allotype of
mice was determined by FACS on heparinized peripheral
blood by sequential incubation with biotinylated mAb against
H-2Db or H-2Dd, streptavidin-phycoerythrin-Cy5 and FACS-
lyse (BD Biosciences, Oxford, UK). Age- and sex-matched 6-
to 8-wk-old female BALB/b mice were purchased from Har-
lan, UK. All procedures were performed in accordance with
UK Home Office regulations.
Induction of adriamycin nephropathy
A single intravenous injection of 10 mg/kg adriamycin (Am-
ersham Pharmacia, Little Chalfont, Buckinghamshire, UK)
was administered. Mice were housed in metabolic cages for
24 h prior to injection and at weekly intervals for 6 wk. Serum
was taken at baseline and at 3 and 6 wk. Mice were killed at 6
wk, and kidneys were harvested for analysis. To assess injury in
a transplanted kidney, heterotopic transplants were per-
formed as described previously (20). One native nephrec-
tomy was performed at the time of transplantation and a
second 2 wk later. Adriamycin nephropathy was induced after
a further 2 wk as above. To generate control tissue, BALB/b
mice (n⫽8) were injected with normal saline.
Measurement of proteinuria and renal function
Urine albumin output was measured by radial immunodiffu-
sion through 1.2% agarose gel containing 15 ␮l/ml rabbit
anti-mouse albumin globulin fraction (Biogenesis, Poole,
1066 Vol. 22 April 2008 The FASEB Journal
UK) against mouse albumin standards (Sigma, Dorset, UK).
Gels were blotted onto Gelbond (Cambrex, Nottingham, UK)
and stained with Coomassie blue. Blood urea nitrogen was
assayed using the Urea Liquid Stable Reagent System (Clin-
dia, Leusden, Netherlands) following manufacturer’s instruc-
tions.
Histological assessment
Formalin-fixed, paraffin-embedded sections (2 ␮m) were cut
and stained with Periodic Acid Schiff. Ten random high-
power (⫻400) cortical fields per section were scored by an
observer in a blinded procedure. Tubulointerstitial injury
(tubular dilatation, cast formation, loss of brush border, and
epithelial cell flattening) was assessed on a 5-point scale: 0 ⫽
no damage, 1 ⫽ ⱕ25%, 2 ⫽ 26 –50%, 3 ⫽ 51–75%, 4 ⫽
76 –100% of the tubulointerstitium injured, as described
previously (10). The mean score of the 10 fields was used. The
same semiquantitative scale was used to assess glomerular
sclerosis, with a minimum of 25 glomeruli scored for each
mouse.
Immunochemistry for C3 and collagen IV was performed
on 5-␮m frozen sections fixed in acetone at 4oC for 5 min and
blocked with 20% goat serum. For C3, sections were sequen-
tially incubated with 1:200 rabbit anti-human C3d (Dako,
Cambridgeshire, UK) and 1:100 fluorescein-conjugated goat
anti-rabbit IgG (Stratech Scientific, Suffolk, UK). For colla-
gen IV, sections were incubated with 1:200 rabbit anti-
collagen IV (Abcam, Cambridge, UK), 1:200 HRP-conjugated
goat anti-rabbit Ig (Dako), and color was developed with
diaminobenzidine (DAB). To identify activated myofibro-
blasts, 5-␮m sections were dewaxed and taken through graded
alcohols to PBS and microwaved for 5 to 10 min in sodium
citrate (2.94 g/L). Slides were preincubated in 4% normal
rabbit serum for 10 min and then incubated in 1:4000 mouse
anti-␣-SMA (Clone 1A4, A-2547, Sigma) for 35 min at room
temperature. The secondary antibody was a biotinylated
rabbit anti-mouse (Dako), applied for 35 min at room tem-
perature. A tertiary layer of streptavidin-alkaline phosphatase
(Dako) diluted to 1:50 was used for 35 min at room temper-
ature and developed in Vector Red substrate (Vector Labo-
ratories, Peterborough, UK).
To quantify C3 deposition, 10 random cortical fields (⫻400
magnification) were photographed, and the average fluores-
cence intensity across the field was measured using Lucia
software (Nikon, Surrey, UK). A method previously described
by Anders et al. (22) was used to quantify collagen IV and
␣SMA staining. A 7 ⫻ 9 grid was superimposed over 10
randomly chosen ⫻400 cortical sections, and the number of
intersections overlying positive staining was counted. This
number was expressed as a percentage of total points.
In situ hybridization
In situ hybridization for C3 mRNA was performed as de-
scribed previously (17). Briefly, the probe was a 30-base
antisense oligonucleotide probe, corresponding to base 167
to 196 of mouse C3 cDNA. The probe was labeled using
digoxigenin (DIG) oligonucleotide tailing kit, according to
the manufacturer’s instructions (Boehringer Mannheim,
Lewes, UK). Four-micrometer frozen sections were fixed with
4% paraformaldehyde in phosphate-buffered saline. The
sections were deproteinized using HCl and proteinase K
(Sigma), prehybridized, and then hybridized with DIG-la-
beled oligonucleotide probe in prehybridization buffer at
37°C overnight. After washing with 2⫻ standard saline citrate,
the DIG-labeled probe was visualized using HRP-conjugated
sheep polyclonal anti-DIG antibody (Boehringer Mannheim)
SHEERIN ET AL.
and DAB. Control studies with a sense probe and competitive
binding studies with sense and antisense probe confirmed
specificity.
C3a ELISA
ELISA plates were coated with 1 ␮g/ml rat anti-mouse C3a
(Clone 187-1162, BD Biosciences, Oxford, UK) in phosphate
buffer, pH 6.5. Test or control samples (purified mouse C3a,
BD Biosciences) were applied to the plate for 1 h at room
temperature. Bound C3a was detected with biotinylated rat
anti-mouse C3a (Clone 187– 419, BD Biosciences), streptavi-
din-horseradish peroxidase, and 3,3⬘,5,5⬘-tetramethylbenzi-
dine (TMB).
Statistical analysis
Mann-Whitney U tests or two-way ANOVAs were used for data
analysis. Values of P ⬍ 0.05 were regarded as significant.
RESULTS
Adriamycin nephropathy in mice
The development of proteinuria in mice after adriamy-
cin injection is strain restricted and is best described in
mice on a BALB genetic background. In contrast,
C57BL/6 mice, including many knockout strains, are
resistant to the nephropathic effect of adriamycin.
Therefore the disrupted C3 allele was backcrossed onto
a BALB background. After backcrossing for seven gen-
erations, the disrupted C3 gene remained in linkage
disequilibrium with the H-2 region. Over 90% of mice
screened were C3⫺/⫺b/b, with only a small number of
C3⫺/⫺ mice heterozygotic at the H-2D locus. Therefore
BALB/b mice (a BALB/c congenic strain expressing
H-2b) were used as the wild-type strain for C3⫺/⫺H-
2b/b mice. This would overcome any potential effect of
genes expressed in the H2 region on the progression of
kidney injury. Skin grafts between BALB/b and back-
crossed C3⫺/⫺ mice were accepted indefinitely (data
not shown). Susceptibility to adriamycin nephropathy is
thought to be an inherited trait in mice through a yet
unidentified gene on chromosome 16 (23); therefore,
it should be inherited independently of either the C3
locus or the H2 region.
The effect of adriamycin on renal function is
dependent on C3
Adriamycin was injected into C3⫺/⫺ (n⫽9) and age-
and sex-matched BALB/b C3⫹/⫹ mice (n⫽12). Albu-
minuria was evident 1 wk after injection of adriamycin
in both wild-type C3⫹/⫹ and C3⫺/⫺ mice and per-
sisted up to week 6. The albumin excretion rate,
measured at weekly intervals in surviving mice, was
equivalent in the two groups of mice (Fig. 1A). The
urinary albumin loss resulted in a fall in serum albumin
that was again similar in the two groups of mice (Fig.
1B). In contrast to the development of albuminuria,
activation of the complement system through C3 had a
profound effect on renal function. The serum urea in
the wild-type C3 ⫹/⫹ mice was significantly elevated by
3 wk after adriamycin injection (P⬍0.001). However,
there was no significant rise in the serum urea in the
C3⫺/⫺ mice, suggesting preservation of renal function
in this group. Comparing the serum urea in the two
groups, there was a significantly higher serum urea
concentration in the wild-type C3⫹/⫹ compared to
C3⫺/⫺ mice (P⬍0.001, Fig. 1C). The worse renal
function in the wild-type C3⫹/⫹ mice was also associ-
ated with a significantly higher mortality (Fig. 1D), with
7 of this group dying before the end of the 6-wk
protocol period. Mice that died in this group invariably
had a high serum urea level prior to death.

Figure 1. A) Twenty-four-hour urine albumin excretion was measured in

wild-type C3⫹/⫹ (n⫽12) and C3⫺/⫺ (n⫽9) mice at weekly intervals after
adriamycin (10 mg/kg) injection. B, C) Serum albumin (B) and urea (C) were
measured immediately prior to and 3 and 6 wk after adriamycin injection. D)
Kaplan-Meier survival curve of wild-type mice and C3⫺/⫺ mice injected with
adriamycin.

RENAL C3 SYNTHESIS CAUSES INJURY IN PROTEINURIA 1067

 Figure 2. A, B) Complement C3 staining in the kidneys of control, saline-injected
wild-type mice (A) and wild-type mice C3⫹/⫹ 6 wk after adriamycin injection (B).
Staining is predominantly along the tubular basement membranes, but some
apical staining is seen (white arrow). Glomerular (G) staining is also seen in the
adriamycin-treated mice. C) The intensity of fluorescence staining was measured.
D) In situ hybridization for C3 message demonstrated staining in the epithelial
cells of damaged, dilated tubules (arrowheads). Staining with the sense probe was
negative (not shown). Original images ⫻400.

C3 deposition and synthesis in the kidneys of mice Histological assessment of injury

with adriamycin nephropathy
C3 staining and thus evidence of complement activa-
tion was evident in the kidneys of control saline-
injected mice in a peritubular distribution (Fig. 2A).
This is consistent with previous reports that have sug-
gested that the C3 detected may be derived from the
tubular epithelium (18). Significantly greater staining
for C3 was demonstrated in kidneys from mice with
adriamycin nephropathy (Fig. 2B, C) when compared
to saline-injected control mice (Fig. 2A, C). Again, the
predominant site for C3 staining was around the tubu-
lar basement membranes. Minor staining was present
on apical tubular membranes and within the glomeruli.
No staining for C3 was seen in the kidneys of C3⫺/⫺
mice (not shown). In situ hybridization demonstrated
the presence of C3 messenger RNA in tubular epithe-
lial cells of mice with nephropathy, particularly within
damaged tubules (Fig. 2D).
Consistent with our functional data, damage to the TI
compartment, as assessed by semiquantitative scoring,
was greater in the wild-type C3⫹/⫹ mice (Fig. 3A, C)
than C3⫺/⫺mice (Fig. 3B, C). When tissue was avail-
able from mice dying prior to the end of the 6-wk
protocol histology was performed and included in the
data analysis. In the wild-type mice, there was evidence
of tubular dilatation with proteinaceous cast formation,
epithelial cell flattening, and interstitial expansion.
These changes were less evident in the C3⫺/⫺ mice.
The severity of glomerular injury in wild-type C3⫹/⫹
mice (2.12⫾0.37, mean score⫾se) was not significantly
worse than for C3⫺/⫺ mice (1.11⫾0.34; P⫽0.073).
This is consistent with the similar level of albuminuria,
a surrogate marker of the severity of glomerular injury,
in both groups.
The injury that occurs during proteinuria includes
the deposition of collagen type IV. Immunostaining for
collagen IV clearly demonstrated increased deposition

Figure 3. Representative PAS-stained sections from wild-type C3⫹/⫹ (A) and C3⫺/⫺ (B) mice 6 wk after adriamycin injection
(original images ⫻400). TI injury was quantified in 10 random cortical fields in the two groups of mice; the mean score for each
animal is presented (C).

1068 Vol. 22 April 2008 The FASEB Journal SHEERIN ET AL.

within the renal interstitium in wild-type C3⫹/⫹ mice
(Fig. 4A, C) compared to C3⫺/⫺ mice (Fig. 4B, C;
P⬍0.002). In addition, there was increased accumu-
lation of ␣-smooth muscle actin expressing myofibro-
blasts in the interstitium of wild-type C3⫹/⫹ mice
compared to C3⫺/⫺ mice (Fig. 4D–F; P⬍0.05).
These cells may represent the source of the increased
collagen deposited in the interstitium and their
presence in clinical biopsies correlates with a poor
renal prognosis.
Renal functional injury is dependent on local C3
synthesis
Kidneys from C3⫺/⫺ (n⫽5) or wild-type C3⫹/⫹
(n⫽7) donors were transplanted into wild-type C3⫹/⫹
recipients. Both native kidneys were removed so the
mice were reliant on the transplant for function. Adria-
mycin nephropathy was induced in these mice. As with
untransplanted mice, the level of albuminuria that
developed was similar irrespective of the complement
status of the donor (Fig. 5A). However, the loss of renal
function in mice receiving a kidney from a wild type
C3⫹/⫹ donor was significantly greater than if the
donor was C3⫺/⫺ (Fig. 5B). The serum urea of
recipients of a C3⫺/⫺ kidney did not rise significantly
above predisease levels. In contrast, the serum urea of
recipients of a wild-type C3⫹/⫹ kidney had risen 4-fold
compared to baseline by 2 wk after disease induction
(P⬍0.02) and was significantly higher than that in
recipients of a C3⫺/⫺ kidney (P⬍0.001). The worse
renal function was also reflected in a higher mortality
Figure 4. Collagen IV and ␣SMA staining from wild-type C3⫹/⫹ (A, D) and C3⫺/⫺ (B, E) mice 6 wk after adriamycin injection
(original images ⫻400 for collagen IV, ⫻1000 for ␣SMA). The area of positive staining was assessed for both collagen IV (C)
and ␣SMA (F), as described in Materials and Methods.
RENAL C3 SYNTHESIS CAUSES INJURY IN PROTEINURIA
(Fig. 5C) in recipients of wild-type C3⫹/⫹ kidneys.
These data on albuminuria and serum urea only pre-
sented to 3 wk because of reduced numbers of recipi-
ents of a wild-type kidney due to the high mortality.
There was evidence of complement activation in the
urinary space of the proteinuric transplant recipients.
The concentration in the urine of C3a, a peptide
generated on C3 activation, was low prior to disease
induction (1.7⫾0.15 ng/ml) but rose significantly 1 wk
after disease induction in recipients of a C3⫹/⫹ kidney
(72,425⫾24,544 ng/ml; P⬍0.001). There was a smaller
rise in urinary C3a concentration in the recipient of a
C3⫺/⫺ kidney, but this was not significantly different
from than seen in recipients of a wild-type C3⫹/⫹
kidney (Fig. 5D).
Histological analysis of transplanted kidneys with
adriamycin nephropathy
The mice receiving a kidney from a wild-type C3⫹/⫹
donor had significantly greater TI injury (Fig. 6A, C)
than mice receiving a kidney from a C3⫺/⫺ donor
(Fig. 6B, C). These changes were similar to those seen
in native kidneys, with tubular dilatation, epithelial cell
flattening, and interstitial infiltrates. The only differ-
ence between these two groups of mice prior to induc-
tion of nephropathy was their ability to synthesize C3 in
the transplanted kidney. In addition, glomeruli from
wild-type C3⫹/⫹ donor kidneys had more severe injury
than those from C3⫺/⫺ mice (3.21⫾0.38 vs.
1.14⫾0.55, respectively; P⬍0.05).
1069
 Figure 5. A, B)Twenty-four-hour urinary albumin excretion (A) and serum urea
(B) were measured at weekly intervals after the induction of adriamycin
nephropathy in wild-type C3⫹/⫹ recipients of either a wild-type C3⫹/⫹ or
C3⫺/⫺ kidney transplant. P values represent a comparison of the two groups.
C) Kaplan-Meier survival curve of wild-type recipients of a wild-type C3⫹/⫹ or
C3⫺/⫺ kidney after the induction of adriamycin nephropathy. D) Urinary C3a
concentration in recipients of a wild-type C3⫹/⫹ or C3⫺/⫺ kidney after the
induction of adriamycin nephropathy.

DISCUSSION reports of complement activity influencing the progres-

This study clearly demonstrates that the activation of
the complement system is involved in the development
of renal injury associated with proteinuria. Despite
equivalent levels of albuminuria, complement activa-
tion through C3 in wild-type C3⫹/⫹ mice results in
greater tubular damage and functional disturbance
than is seen in C3⫺/⫺ mice during the development of
adriamycin nephropathy. In addition, the results pre-
sented demonstrate that C3 synthesized locally within
the kidney is, at least in part, responsible for comple-
ment-mediated injury.
There is an increasing body of evidence supporting
the hypothesis that complement activation increases
tubulointerstitial injury and reduces renal function
during proteinuria. This has been shown previously in
animal studies (9 –12, 24) and is supported by clinical
observation (8). Complement proteins may access the
tubular compartment via glomerular filtration or may
be synthesized locally by native renal cells. The previous
sion of proteinuria-associated interstitial disease have
not distinguished between these two sources.
For more than 15 years, there have been reports of
the ability of native kidney cells, including endothelial
and epithelial cells of both glomerular and tubular
origin, to produce many, if not all, of the proteins of
the complement activation cascade. In vitro studies have
demonstrated that production of complement proteins
is under the control of proinflammatory cytokines,
suggesting that local factors within the kidney may
control local levels of complement protein expression.
This is supported by the observation that intrarenal
complement gene expression is increased during in-
flammatory renal injury in both native (14) and trans-
plant kidney disease (25). Animal studies have provided
confirmatory evidence that local complement produc-
tion increases during disease development, with a tem-
poral association between the development of injury
and increased complement gene expression (26). The

Figure 6. Representative PAS-stained sections from wild-type recipients of wild-type C3⫹/⫹ (A) or C3⫺/⫺ (B) kidneys 6 wk after
adriamycin injection (original images ⫻160). TI injury was quantified in 10 random cortical fields in the two groups of mice;
the mean score for each animal is presented (C).

1070 Vol. 22 April 2008 The FASEB Journal SHEERIN ET AL.

amount of complement proteins produced by the kid-
ney is significant and may account for 9% of the
circulating pool of C3 (13). Although studies of trans-
plant-related injury have shown that local production of
C3 is important in the development of kidney injury
(18, 20), this is the first study to show this in native
disease.
In the transplant study, both groups of mice had
intact systemic circulating C3 primarily derived from
the liver. The groups only differed by the capacity of
the transplanted kidney to produce C3. Despite evi-
dence of complement activation in the recipients of
both C3⫹/⫹ and C3⫺/⫺ kidneys, the degree of injury
was significantly influenced by this difference, being
markedly attenuated by the absence of local C3 synthe-
sis. This provides compelling evidence that, not only is
the production of C3 from within the kidney a key
mediator of the interstitial renal injury in adriamycin
nephropathy but that the site of production, and
therefore activation, is equally critical.
Locally synthesized C3 could influence disease pro-
gression in several ways. There may be a concentration
effect. The alternative pathway is important in this
disease model (24), and its activation is critically depen-
dent on the concentration of complement proteins
available. Local C3 synthesis may generate a concentra-
tion of C3 permissive for triggering of this pathway.
Another possibility is that local C3 is produced from the
basolateral side of cells into a site that is inaccessible to
filtered proteins. Therefore, only locally synthesized C3
will be activated at the basement membrane and within
the interstitium. The available data are consistent with
the latter. C3a is formed in the urine, irrespective of the
C3 status of the transplanted kidney, suggesting that
the absence of local C3 is not limiting. We show that C3
deposition in the adriamycin-treated wild-type C3⫹/⫹
mice is predominantly basolateral and interstitial, and
it has previously been shown that most of the C3
deposited at this site is locally produced (18). Other
proteins required to activate C3 could also be produced
at this site. Gene expression of alternative pathway
proteins can be seen in the tubular cells, and expres-
sion increases during disease (27). However, their
function has not been assessed in this series of experi-
ments.
Once complement is activated, generation of sublytic
concentrations of C5b-9 can alter epithelial cell func-
tion, inducing morphological changes, up-regulation
of collagen gene expression (28), and production of
inflammatory cytokines (29). These cytokines may in-
duce cellular infiltration, which may be further en-
hanced by the generation of the anaphylotoxins C3a
and C5a. These anaphylotoxins may also have direct
effect on tubular epithelial cells, increasing collagen
gene expression (30). The TI injury during proteinuric
disease is known to depend on both cellular infiltration
(31) and the generation of C5a (32). Complement
activation also increases interstitial myofibroblast accu-
mulation (33) at least in part due to the generation of
C5b-9.
RENAL C3 SYNTHESIS CAUSES INJURY IN PROTEINURIA
Adriamycin is thought to be toxic to the glomerular
epithelial cell, direct exposure of the kidney to the drug
(34) resulting in early epithelial cell foot process efface-
ment and proteinuria (35). Susceptibility in the mouse
maps to a locus at chromosome 16A1-B1 (23), DOX-
NPH. The susceptible genotype at the DOXNPH locus
strongly predicts a low level of the arginine methyltrans-
ferase, Prmt7 (chromosome 8). Methylation of adria-
mycin may reduce its toxicity and, although it has not
been proven that Prmt7 methylates adriamycin, low
levels of Prmt7 could explain why some mouse strains
are susceptible to nephropathy. Our understanding of
the mechanism of adriamycin-induced proteinuria
would not predict a major role for the complement
system in the development of glomerular injury. To
support this, albuminuria was independent of the com-
plement status of the mice. Adriamycin is toxic to the
glomerular podocyte, causing foot process effacement
and loss of the podocyte contribution to glomerular
permselectivity. However, there is some evidence that T
cell function, particularly that of CD8 cells (31), is
involved in the development of injury, suggesting that
some immunological factors may be involved in adria-
mycin nephropathy. In addition, C3 immunostaining
was evident in the glomeruli of wild-type C3⫹/⫹ mice
with adriamycin nephropathy, as has been reported
previously (24). Complement proteins may be trapped
nonspecifically within the damaged glomerulus in asso-
ciation with proteinuria, or activation could be occur-
ring at this site. In the native kidney, the glomerular
injury was worse (although not reaching statistical
significance) in the wild-type mice; following trans-
plant, glomerular injury was worse in the recipients of a
wild-type kidney. This does suggest that complement,
including locally produced protein, is contributing to
glomerular injury in this model. An alternative expla-
nation is that the glomerular changes are secondary to
tubular damage, and the primary site affected by com-
plement is the tubulointerstitium. In a model of anti-
body-mediated glomerular injury, there was no evi-
dence that the local production of C3 within the
glomerulus altered disease development (17). The glo-
merular histology may, therefore, reflect changes fur-
ther along the nephron.
Overall, this study clearly demonstrates that renal
injury in adriamycin nephropathy is dependent on the
activation of complement through C3. However, we
have additionally shown that the local production of C3
within the kidney is a critical mediator of tissue injury.
Although the capacity of the kidney to produce C3 is
well recognized, this is the first report to show that this
local production of C3 is important in the development
of nontransplant kidney disease. It also raises the
possibility that local tissue production of complement
proteins may be involved in the development of inflam-
matory tissue injury at other sites.
This work was supported by an unconditional grant from
the Wellcome Trust.
1071
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Received for publication July 3, 2007.
Accepted for publication October 11, 2007.
SHEERIN ET AL.