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,1
* 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
1Correspondence: 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{at}ncl.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: proteinuria • renal failure • innate immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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 function (3)
. These observations have led to the widely accepted hypothesis that proteinuria mediates TI injury, typically interstitial fibrosis and tubular atrophy, 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. Alternatively, specific proteins in the glomerular filtrate may be toxic to tubules, for example, transferrin or fatty acid-conjugated albumin (4)
. Another possible mediator 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 activation 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 proteinuric 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 proteinuria in animals complement deficiency or inhibition reduces the level of histological injury and the loss of renal function (9
10
11
12)
.
An additional dimension regarding the action of complement is the ability of the kidney to synthesize complement proteins. The kidney contributes measurably to the circulating pool of C3 (13)
, the pivotal complement protein. In addition, increased C3 gene 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. However, 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 confirm the importance of C3 activation in the development 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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 FACSlyse (BD Biosciences, Oxford, UK). Age- and sex-matched 6- to 8-wk-old female BALB/b mice were purchased from Harlan, 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 (Amersham 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 performed as described previously (20)
. One native nephrectomy 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 immunodiffusion through 1.2% agarose gel containing 15 µl/ml rabbit anti-mouse albumin globulin fraction (Biogenesis, Poole, 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 (Clindia, Leusden, Netherlands) following manufacturer’s instructions.
Histological assessment
Formalin-fixed, paraffin-embedded sections (2 µm) were cut and stained with Periodic Acid Schiff. Ten random high-power (x400) 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 = 
25%, 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-µm frozen sections fixed in acetone at 4°C for 5 min and blocked with 20% goat serum. For C3, sections were sequentially 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 collagen 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 myofibroblasts, 5-µm 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 temperature. A tertiary layer of streptavidin-alkaline phosphatase (Dako) diluted to 1:50 was used for 35 min at room temperature and developed in Vector Red substrate (Vector Laboratories, Peterborough, UK).
To quantify C3 deposition, 10 random cortical fields (x400 magnification) were photographed, and the average fluorescence 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 x 9 grid was superimposed over 10 randomly chosen x400 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 described 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-labeled oligonucleotide probe in prehybridization buffer at 37°C overnight. After washing with 2x standard saline citrate, the DIG-labeled probe was visualized using HRP-conjugated sheep polyclonal anti-DIG antibody (Boehringer Mannheim) 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 µg/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), streptavidin-horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine (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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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). Albuminuria was evident 1 wk after injection of adriamycin in both wild-type C3+/+ and C3–/– mice and persisted up to week 6. The albumin excretion rate, measured at weekly intervals in surviving mice, was equivalent in the two groups of mice (Fig. 1
A). 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 associated 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.
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C3 deposition and synthesis in the kidneys of mice with adriamycin nephropathy
C3 staining and thus evidence of complement activation was evident in the kidneys of control saline-injected mice in a peritubular distribution (Fig. 2
A). This is consistent with previous reports that have suggested 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 tubular 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 epithelial cells of mice with nephropathy, particularly within damaged tubules (Fig. 2D
).
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Histological assessment of injury
Consistent with our functional data, damage to the TI compartment, as assessed by semiquantitative scoring, was greater in the wild-type C3+/+ mice (Fig. 3
A, C) than C3–/–mice (Fig. 3B, C
). When tissue was available 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.
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The injury that occurs during proteinuria includes the deposition of collagen type IV. Immunostaining for collagen IV clearly demonstrated increased deposition within the renal interstitium in wild-type C3+/+ mice (Fig. 4
A, C) compared to C3–/– mice (Fig. 4B, C
; P<0.002). In addition, there was increased accumulation of -smooth muscle actin expressing myofibroblasts 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.
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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. Adriamycin 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. 5
A). 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 (Fig. 5C
) in recipients of wild-type C3+/+ kidneys. These data on albuminuria and serum urea only presented to 3 wk because of reduced numbers of recipients of a wild-type kidney due to the high mortality.
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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. 6
A, 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 difference between these two groups of mice prior to induction 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
10
11
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 reports of complement activity influencing the progression 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 inflammatory renal injury in both native (14)
and transplant kidney disease (25)
. Animal studies have provided confirmatory evidence that local complement production increases during disease development, with a temporal association between the development of injury and increased complement gene expression (26)
. The amount of complement proteins produced by the kidney is significant and may account for 9% of the circulating pool of C3 (13)
. Although studies of transplant-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 evidence 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 synthesis. 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 progression in several ways. There may be a concentration effect. The alternative pathway is important in this disease model (24)
, and its activation is critically dependent on the concentration of complement proteins available. Local C3 synthesis may generate a concentration 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 expression increases during disease (27)
. However, their function has not been assessed in this series of experiments.
Once complement is activated, generation of sublytic concentrations of C5b-9 can alter epithelial cell function, inducing morphological changes, up-regulation of collagen gene expression (28)
, and production of inflammatory cytokines (29)
. These cytokines may induce cellular infiltration, which may be further enhanced 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 accumulation (33)
at least in part due to the generation of C5b-9.
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 effacement and proteinuria (35)
. Susceptibility in the mouse maps to a locus at chromosome 16A1-B1 (23)
, DOXNPH. The susceptible genotype at the DOXNPH locus strongly predicts a low level of the arginine methyltransferase, Prmt7 (chromosome 8). Methylation of adriamycin 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 complement 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 adriamycin 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 association with proteinuria, or activation could be occurring at this site. In the native kidney, the glomerular injury was worse (although not reaching statistical significance) in the wild-type mice; following transplant, 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 explanation is that the glomerular changes are secondary to tubular damage, and the primary site affected by complement is the tubulointerstitium. In a model of antibody-mediated glomerular injury, there was no evidence that the local production of C3 within the glomerulus altered disease development (17)
. The glomerular histology may, therefore, reflect changes further 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 inflammatory tissue injury at other sites.
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ACKNOWLEDGMENTS |
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Received for publication July 3, 2007. Accepted for publication October 11, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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