Search

Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence.

Background: Complement deficiency predisposes to autoimmune renal disease. Since complement deficient mice are increasingly used to study the immunopathogenesis of renal disease we have determined whether mice deficient in C3 or C4 are susceptible to spontaneous immune-mediated renal injury., Methods: C3-deficient, C4-deficient and complement-sufficient, wild-type mice were maintained in standard conditions for 1 year at which stage renal function, renal histology, circulating antibody and autoantibody levels were assessed., Results: No significant decline in renal function was demonstrated in the complement-deficient mice. However, there was histological evidence of glomerular injury in both the C3- and C4-deficient mice, but of insufficient severity to alter function. Serum IgG2a concentration was significantly lower in C3- and C4-deficient mice. In contrast C3-deficient mice had higher concentrations of serum IgG2b. There was a tendency for mice from all groups, including the complement-sufficient mice, to develop autoantibodies. C4-deficient mice had higher titres of anti-dsDNA IgG but otherwise deficient mice had similar autoantibody titres to controls., Conclusion: We conclude that C4-deficient mice demonstrate a small increase in autoantibody production at 1 year of age compared to C3-deficient and wild-type mice. Furthermore, although complement-deficient mice exhibit glomerular changes, they are of minor functional significance, and are unlikely to affect the study of experimentally induced renal disease in these mice., (Copyright 2004 S. Karger AG, Basel)

Background: The mechanisms controlling the production of antibodies against histocompatibility antigens are of prime importance in organ transplantation., Methods: We investigated the role of complement in the response to allogeneic stimulation, using mice deficient in C3, C4, or C5 to dissect the role of the alternative, classical, and terminal complement pathways., Results: After fully major histocompatibility complex disparate skin grafts, the allospecific immunoglobulin (Ig)G response was markedly impaired in C3- and C4-, but not in C5-deficient mice. This defect was most pronounced for second set responses. C3-deficient mice also demonstrated a decreased range of IgG isotypes. In contrast, there was no impairment of the allospecific IgM response. In functional T cell assays, the proliferative response and interferon-gamma secretion of recipient lymphocytes restimulated in vitro with donor antigen was decreased two- to threefold in C3-deficient mice., Conclusions: These data show impairment of allogeneic T cell and B cell function in mice with defective complement activation and suggest a predominant role for the classical pathway in stimulating alloimmunity. The terminal pathway seems unimportant in this regard. This extends the results reported for soluble protein antigens and demonstrates a surprisingly marked effect on the alloresponse despite the presence of a stringent antigenic stimulus. These results have implications for the prevention of sensitization in naïve transplant recipients.

To assess the role of complement in renal infection, we studied a model of Escherichia coli-induced pyelonephritis in mice deficient in complement components C3 and C4. Renal infection occurred less frequently in C3- and C4-deficient mice compared with wild-type mice. In vitro, renal epithelial cells internalized fewer bacteria in the absence of C3 or in the presence of blockade of C3 bound to the bacteria. Moreover, upregulation of epithelial C3 production by stimulation with lipopolysaccharide enhanced bacterial internalization. Here we provide evidence that uropathogenic E. coli might use host C3 to invade the renal epithelium and that local complement production is sufficient for the bacteria to achieve this effect.

The role of complement in autoimmune glomerulonephritis (as in other autoimmune diseases) is paradoxical, in that complement activation mediates acute inflammatory injury, yet inherited deficiency of complement may predispose to immune complex disease in particular immune complex glomerulonephritis. We have investigated the role of complement in experimentally induced glomerulonephritis in C3-deficient mice, using antibodies against the mouse glomerular basement membrane (GBM). In the acute phase of the disease, which is initiated by binding of heterologous antibody to the GBM, we confirmed that the inflammatory injury was positively complement dependent, with C3-deficient mice developing less severe injury. In contrast, in the autologous phase of the disease, mediated by the immune response against the heterologous antibody fixed in the GBM, the disease was negatively complement dependent. That is, by 14 days after disease induction the C3-deficient mice had heavier proteinuria and more severe uremia (p < 0.001) compared to the complement sufficient mice. The C3-deficient mice also showed a greater accumulation of electron-dense deposits in the GBM. These findings were reproduced in an accelerated model of this disease in which C3-deficient mice also develop more severe functional disturbance and demonstrate a higher rate of immune complex deposition. These data illustrate the potential for the net effect of complement to switch from a detrimental to a protective mode at different stages of autoimmune injury.

Complement is increasingly implicated in the pathogenesis of progressive renal disease resulting from persistent proteinuria. We have previously shown that apical serum proteins stimulate C3 in cultured human proximal tubular epithelial cells (PTECs), and that the stimulant is a nonalbumin compound of 30 to 100 kd. We postulated in this study that transferrin and apotransferrin, also important components of proteinuric urine in this molecular-weight range, might be the culprit. Human PTECs were obtained by differential sieving of renal cortical tissue from the normal pole of tumor nephrectomy specimens and characterized to be predominantly of proximal tubular origin. Complement C3 messenger RNA (mRNA) expression was analyzed in confluent growth-arrested PTEC monolayers in media containing different concentrations (2.5 to 20 mg/mL) of transferrin by reverse transcription and polymerase chain reaction. Pure human albumin was used as a control protein. C3 protein secretion was detected and quantified by a sandwich enzyme-linked immunosorbent assay on cell culture supernatants after distinct time points. Transferrin enhanced the rate of C3 secretion in a dose-dependent manner, reaching maximal stimulation at doses of 10 mg/mL. Selected experiments using the Transwell technique showed that C3 release was predominantly apical in the resting state. The addition of 10 mg/mL of transferrin apically but not basolaterally stimulated both apical and basolateral C3 secretion and increased the basolateral-apical ratio of C3 secretion from 0.45 +/- 0.16 to 0.93 +/- 0.24 (P: < 0.02). Constitutive C3 mRNA expression was upregulated by transferrin in a time- and dose-dependent fashion, reaching a peak after 24 hours. A similar degree of C3 upregulation was reproduced when iron-poor transferrin, apotransferrin, was used instead. These results indicate that C3 synthesis in PTECs is upregulated by transferrin, for which protein rather than iron moiety may account for the observed effects. These findings provide evidence linking proteinuria with overexpression of tubular complement.

Recent evidence has implicated complement in renal transplant injury and identified the kidney as a source of complement components. We therefore investigated the local gene expression of complement component C3, pivotal to complement activation pathways and a mediator of inflammatory injury, in a rat renal transplant model. By reverse transcriptase-polymerase chain reaction, the expression of C3 mRNA increased in two phases. The first phase coincided with post-ischemic injury over 2 days post-transplantation and was localized by in situ hybridization to vessels and glomerular mesangial cells in allogeneic and syngeneic (control) kidney transplants. In allografts only, a second phase was found in tubular epithelial cells, glomerular parietal cells, vessel walls and some infiltrating cells, which peaked on day 4 together with rapid influx of leukocytes, tubule cell damage, the induction of interleukin-2 and interferon-gamma mRNA, and the up-regulation of tumor necrosis factor-alpha and interleukin-1beta mRNA in the graft. In vitro studies showed that interleukin-2 and interferon-gamma up-regulate C3 production in renal tubule cells. We conclude that post-ischemic injury led to transient up-regulation of glomerular expression of C3 mRNA. Subsequent cellular rejection was associated with tubulointerstitial/glomerular parietal cell expression of C3 mRNA. This differential expression of local C3, immediately post-transplant or associated with acute rejection, may have implications for putative therapeutic complement inhibition in clinical transplantation.

We have studied the role of complement in a model of glomerular inflammation induced by the in situ formation of immune complexes along the glomerular basement membrane. In C3-deficient mice, produced by homologous recombination, immune complex formation occurs initially in the subendothelial site and progresses slowly to the subepithelial position, whereas wild-type mice do not develop subendothelial deposits. In addition, the accumulation of electron-dense deposits is greater in the complement-deficient mice. Complement therefore influences glomerular handling of immune complexes, possibly because of changes in the physiochemical characteristics of the immune complexes. However, despite evidence of complement activation in the wild-type mice, as demonstrated by immunohistochemical detection of C3, C4 and C9, the degree of proteinuria was similar in C3-deficient mice. We conclude that, although complement is required for the normal glomerular metabolism of immune complexes, other, complement-independent, factors are involved in the generation of glomerular injury in this model.

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.

There is increasing evidence to suggest that the renal tubular epithelium is important in the pathogenesis of progressive renal failure resulting from persistent proteinuria. The role of complement in the progression of chronic renal failure is not well defined. The purpose of this study was to characterize the production of complement by human proximal tubular epithelial cells exposed to serum proteins at the apical surface. Complement C3 gene expression was analyzed by reverse transcription and PCR. C3 protein biosynthesis was confirmed by metabolic labeling followed by immunoprecipitation and quantified by enzyme-linked immunosorbent assay. In the quiescent state, proximal tubular epithelial cells grown on permeable membrane supports secreted C3 predominantly into the apical medium. The addition of 5 mg/ml serum proteins led to an 8.9-fold increase in basolateral C3 secretion and a 2.1-fold increase in apical C3 secretion, altering the ratio of basolateral: apical C3 secretion from 0.44 +/- 0.16 to 1.87 +/- 0.52. C3 mRNA expression was also upregulated in a time- and dose-dependent manner. Serum fractionation demonstrated that the stimulant responsible for these effects was in the molecular weight range 30 to 100 kD. The observed phenomenon was not reproduced when purified human albumin alone was used as the stimulant. These findings could provide a possible mechanism for the link between proteinuria and interstitial fibrosis. This may have potential implications for strategies directed against complement in retarding the progression of chronic renal failure.

Mice rendered completely deficient of the complement components C3 or C4 were used to determine the influence of complement activation in the heterologous phase of the anti-GBM disease model. In wild-type animals the disease is characterized by a neutrophil infiltrate, capillary thrombosis, proteinuria and C3 and C4 deposited within the glomerulus. The early infiltration of neutrophils into the glomeruli is greater in wild-type mice (2.8 +/- 0.3) compared with C3-deficient (1.4 +/- 0.2) and C4-deficient (1.2 +/- 0.003) mice. Deficiency also protects against the subsequent development of proteinuria (2.99 +/- 1.11 mg/24h, 0.059 mg/24h and 0.327 +/- 0.14 mg/24h in wild-type, C3-deficient and C4-deficient mice, respectively) and decreases glomerular capillary thrombosis in both C3- and C4-deficient mice. The degree of protection is greater in the C3-deficient than the C4-deficient animals, suggesting both classical and alternative pathway involvement. These studies support a critical role for complement in the development of anti-GBM disease. However, the protective effect of complement deficiency can be broken if the dose of nephritogenic antibody is increased.

Passive Heymann nephritis (PHN), a model of human membranous nephropathy, is an immune-complex-mediated glomerulonephritis characterized by the presence of complement-dependent tissue injury. Recent studies have confirmed the synthesis of C3, involved in both the classical and alternative pathways of complement, in injured human and animal renal tissues. However, there is little clear information on the role of local C3 synthesis in the pathogenesis of nephritides such as PHN. In the present study, using nonradioactive in situ hybridization and semiquantitative reverse transcriptase polymerase chain reaction, we examined C3 synthesis in the kidney and its contribution to tissue injury in a rat model of PHN induced by the injection of polyclonal anti-gp330 antibody. C3 mRNA was localized in mesangial cells, glomerular epithelial cells, and cells of Bowman's capsule. During the early stages of PHN, C3 mRNA expression was detected in mesangial cells and glomerular epithelial cells, whereas such expression was limited to mesangial cells during the late stages of the disease. Focal, weak C3 mRNA expression was detected in tubular epithelial cells and occasionally in the interstitium. Semiquantitative polymerase chain reaction demonstrated that the level of C3 mRNA expression correlated with that of proteinuria. Our results suggest that renal cells synthesize C3 mRNA in PHN in a site-specific manner and that locally produced C3 is associated with the development of proteinuria in this model.

Glomerular endothelial cells are an important site of interaction with the cellular and soluble components of inflammation. To investigate the capacity of these cells to synthesize complement they were cloned from isolated rat glomeruli. Messenger RNA (mRNA) was extracted from the cells, reverse transcribed and used as the template to identify specific gene transcripts with the polymerase chain reaction (PCR). mRNA coding for the third component of the complement cascade (C3) was detected in unstimulated endothelial cells, whereas no message for the fourth component (C4) could be demonstrated. Using a semiquantitative method of PCR, we found that the expression of C3 is up-regulated by the cytokine tumor necrosis factor-alpha (TNF-alpha), but not by the cytokines interferon-gamma (IFN-gamma) and interleukin 1 alpha (IL-1 alpha). The increase in levels of C3 mRNA occurred in a time and dose dependent manner. This increase was dependent on new protein synthesis. Production of the C3 protein was demonstrated by radiolabeling and immunoprecipitation, and this also was stimulated by TNF-alpha. In conclusion, we demonstrate the production of C3 by microvascular endothelium of glomerular origin and its stimulation by TNF-alpha. We believe that this local synthesis could have a role in the pathogenesis of disease, however, the nature of this role at present remains unclear.

On pages 2428 to 2433 of this issue, Miyazaki et al. provide evidence that complement component C3 is synthesized within the glomeruli of patients with mesangial glomerulonephritis. Although it has been known for several years that cells isolated from the human kidney can synthesize a range of complement components, and that immune complex disease can increase the expression of these genes, it is only now that the link between intraglomerular synthesis of complement and human glomerulonephritis can be made. In view of the long interest in the role of complement in the pathogenesis of glomerulonephritis, this begs the question to what extent local complement synthesis may be involved in the generation of tissue injury. Given also the double-sided nature of complement-proinflammatory and protective-It remains to be seen if the roles of local and systemic complement differ in these respects. To weigh these questions, we examine the current evidence that connects local complement synthesis with renal injury, and ask what studies are needed to provide more definitive answers.

The third component of complement (C3) exists in two main allotypic forms, C3S and C3F, which can be distinguished at the molecular level using a variation of the polymerase chain reaction. An increased frequency of the C3F allele has been noted in a number of autoimmune and inflammatory conditions affecting the kidney, including systemic vasculitis, IgA nephropathy, and type II mesangiocapillary nephritis. Recently, in an unrelated study, we found (with small numbers) an increased incidence of graft loss associated with the presence of the C3F allele. To further assess this, we analyzed the S/F polymorphism in 183 donor-recipient pairs of patients undergoing renal transplantation. Forty-one of 183 grafts were lost, but graft loss was not associated with the C3F allele over 14-month follow-up. However, the presence of the C3F allele predicted an increased risk of graft dysfunction (defined as serum creatinine > 150 mumol/L): 61/105 versus 36/78, with a relative risk of 1.4 (P < 0.05). The C3F allele predisposed toward graft dysfunction when present in either donor or recipient. The presence of two C3F alleles gave a relative risk for graft dysfunction of 1.8, suggesting a dose-dependent effect, although numbers were small. The presence of the C3F allele was not significantly correlated with the number of rejection episodes, serum creatinine, or duration of primary nonfunction. These findings suggest that C3F may be a susceptibility allele for allograft injury. Possible mechanisms for this association are discussed.

Recent evidence suggests that the third component of complement, C3, is synthesized in renal tissue, and that increased C3 synthesis occurs in allograft rejection and immune complex-mediated nephritis. However, it is unclear whether intrinsic renal cells or migratory cells in the inflammatory infiltrate, possibly of recipient bone marrow origin, are the source of the C3 detected. This was investigated by determining the C3 allotypes of mRNA and protein produced by transplanted human kidney. Twenty donor-recipient pairs were examined, of which nine pairs had C3 allotypes that were informatively mismatched at the C3 F/S locus. Reverse transcriptase polymerase chain reaction (RT-PCR) followed by amplification refractory mutation system analysis showed intracellular donor-specific mRNA expression in six of these nine cases, at up to 61 days post-transplantation. Nested PCR reactions and the size of PCR products excluded contamination by genomic DNA. Allotype-specific staining of frozen sections of renal cortex demonstrated donor-derived C3 protein in both glomeruli and tubules of all biopsies examined, in a predominantly tubular distribution. These results imply that at least some of the pro-inflammatory effects of complement arise from intrinsic tissue synthesis of donor C3, and that this may represent a previously unrecognized source of tissue injury. The occurrence of local synthesis of C3 of donor allotype may have functional implications related to C3 allotype, and may also be relevant to strategies to inhibit intrarenal complement-mediated injury.

Extra-hepatic synthesis of complement is thought to mediate local tissue inflammatory injury. To investigate this phenomenon in the glomerular epithelial cell (GEC), we examined the biosynthesis and regulation of gene expression of the third component of complement in isolated human GEC derived from normal tissue. Metabolic labelling and immunoprecipitation studies demonstrated that C3 protein was synthesized, processed and secreted by GEC under basal conditions. The secreted C3 alpha and beta polypeptide chains had identical electrophoretic mobilities with those of hepatic C3. Examination of cellular RNA using semi-quantitative polymerase chain reaction (PCR) showed that C3 gene expression was present in unstimulated GEC and was increased by stimulation with interferon-gamma (IFN-gamma) in a time- and dose-dependent manner. Tumour necrosis factor-alpha (TNF-alpha), while mediating an increase in monocyte U937 C3 expression, revealed no evidence of regulation of GEC C3 gene expression. These results indicate that human GEC spontaneously express the C3 gene and that increased gene expression is regulated by IFN-gamma. These observations may reflect part of a wider mechanism of protection against or mediation of local, immune-mediated tissue injury.