Antibodies against mesangial cells in a rat model of chronic renal allograft rejection

Simone A. Joosten, Vanessa van Ham, Maria C. Borrias, Cees van Kooten and Leendert C. Paul

Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands

Correspondence and offprint requests to: Dr Cees van Kooten, Department of Nephrology, C3p, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Email: kooten{at}lumc.nl



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Chronic renal allograft rejection (CR) is the leading cause of late renal transplant failure. The histological lesions of CR may comprise glomerular basement membrane (GBM) duplications and mesangiolysis. Its pathogenesis is not yet completely understood, although lately humoral responses have been suggested to be important. Recently, we identified antibody responses directed against GBM antigens in the Fischer (F344) to Lewis (LEW) renal transplantation model. Immunofluorescent studies in this model also suggested deposition of antibodies on mesangial cells. Therefore, we hypothesized that antibodies were not only directed at GBM antigens but also to mesangial cell antigens.

Methods. F344 to LEW renal transplantations were performed and sera were collected. Pre- and post-transplantation sera were tested for antibody binding to donor rat mesangial cells (RMCs) cultured from F344 kidneys. Anti-mesangial cell antibodies were compared with anti-GBM antibodies measured in the same sera.

Results. Post-transplant sera of F344 to LEW renal transplantations, but not LEW to F344, bound to F344 RMC in a dose-dependent manner. Whereas antibodies reactive with RMCs were not present before transplantation, all rats with CR developed antibodies. The antibodies were predominantly of the IgG1 isotype. Antibody binding to RMCs correlated with binding to F344 GBM. Pre-incubation with RMCs partially inhibited GBM binding, and RMC binding was inhibited by GBM. Antibody binding to RMCs did not result in complement activation or cell lysis.

Conclusion. LEW recipients of F344 grafts produce antibodies reactive with F344 RMCs. The antigens involved are similar to or at least share antigenic epitopes with antigens recognized in the GBM.

Keywords: antibodies; chronic rejection; humoral rejection; kidney transplantation; mesangial cells; mesangiolysis; transplant glomerulopathy



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chronic rejection (CR) is the most prevalent cause of renal transplant failure after the first few months post-transplantation (Tx). Histologically, CR is characterized by vasculopathy, glomerulopathy, tubular atrophy and interstitial fibrosis. In the glomeruli, glomerular basement membrane (GBM) abnormalities can be found as well as mesangiolysis [1,2]. Mesangiolysis is defined as the dissolution of mesangial matrix and degeneration of mesangial cells [3]. Mesangiolysis is found in a variety of renal diseases, and might be the result of direct antibody-mediated mesangial damage [3]. For example, in Thy-1.1 nephritis, injected antibodies bind rapidly to the mesangium, resulting in mesangiolysis [4,5]. In Thy-1.1 nephritis, anti-Thy-1.1 antibodies are injected and bind the Thy-1.1 antigen on mesangial cells. Antibody binding results in activation of the complement system, resulting in formation of the membrane attack complex (C5b-9) and inflammation [6–8]. In vitro analyis of the interaction of anti-Thy-1.1 antibodies and mesangial cells demonstrates complement-mediated lysis or apoptosis of the mesangials cells depending on the dose of antibodies [9]. In addition, mesangial cells that bound antibodies on their surface can be recognized by CD16 (Fc-{gamma}-RIII) on macrophages resulting in Fas-ligand (Fas-L)-mediated apoptosis [10].

The pathogenesis of CR is complex and involves multiple antigen-dependent and antigen-independent factors. These may include both cellular and humoral responses directed against major histocompatibility complex (MHC) antigens and tissue-specific antigens [11]. Previously, we identified a humoral response against GBM antigens in a rat model of chronic rejection [1,12]. In the Fischer (F344) to Lewis (LEW) rat model, transplantation of a F344 kidney into a LEW recipient results in acute rejection and subsequent CR. The reverse combination, transplantation of a LEW kidney into a F344 recipient, results in acute rejection, but no CR develops and the kidneys show long-term function. In this model, we found antibodies, only in LEW recipients of a F344 graft, that were reactive with donor, but not recipient type GBM [1]. The antigens recognized by these antibodies were at least partially identified and include the heparan sulfate proteoglycan (HSPG) perlecan and the {alpha}1 chain of collagen type VI in association with the {alpha}5 chain of collagen type IV [1].

Recently, we also studied humoral responses in patients with transplant glomerulopathy. In these patients, we identified antibodies reactive with GBM antigens and identified another important HSPG molecule, agrin, as the antigen recognized by these patients [13]. The finding of antibodies against GBM antigens both in an experimental model and in patients with transplant glomerulopathy suggests a role in the pathogenesis of the glomerular abnormalities.

Immunofluorescent studies in the F344 to LEW model also suggested deposition of antibodies on mesangial cells [1]. Furthermore, the antigens identified in the GBM, perlecan and collagens, can be expressed in the mesangial matrix [14,15]. Hence, we hypothesized that in the F344 to LEW model, antibodies against rat mesangial cells (RMCs) might be present and that these antibodies are involved in mesangiolysis.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and kidney transplantation
Male Fischer (F344, RT1lv1) and Lewis (LEW, RT1l) rats were purchased from Harlan (Horst, The Netherlands). Animal care and experimentation were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animals had free access to water and standard rat chow.

Kidney transplantations were performed as previously described [1]. Under halothane anaesthesia, the left kidney of the recipient is removed and replaced by a donor kidney in the orthotopic position. The remaining native kidney was removed 7 days after transplantation. Animals received 1 mg/kg body weight of Temgesic (Buprenorphine-hydrochloride; Schering-Plough B.V., Amstelveen, The Netherlands) on the first post-operative day for pain relief. All transplantations were performed in the absence of immunosuppression. Blood samples were collected weekly by tail vein puncture and sera were stored at –80°C.

F344 to LEW (n = 19) and LEW to F344 (n = 9) transplantations were performed and rats were sacrificed at day 60 post-Tx.

Rat mesangial cell isolation and culture
Normal F344 rat kidneys (rats 6 weeks of age) were dissected and the renal cortex was homogenized. Subsequently, glomeruli were harvested by pressing through a series of sieves of 80, 150 and 200 mesh [16]. Glomeruli were digested with type Ia collagenase for 10 min at 37°C (Sigma Chemical Co, St Louis, MO). Isolated glomeruli were plated in T75 culture flasks (Greiner, Frickenhausen, Germany) in RPMI 1640 (Gibco/Life Technologies, Breda, The Netherlands) supplemented with 20% heat-inactivated fetal calf serum (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), and allowed to attach for 10 days. RMC outgrowth was confirmed by morphological and immunohistochemical criteria as described [16].

Flow cytometric analysis
Antibody binding to RMCs was determined using flow cytometry. RMCs were harvested and washed in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA, Sigma) and 0.02% NaN3. Pre- and post-Tx sera were diluted in PBS/1% BSA and incubated for 30 min on ice. Subsequently, the cells were incubated with R-phycoerythrin (R-PE)-conjugated goat-anti-rat Ig (BD Biosciences, Alphen aan den Rijn, The Netherlands). The cells were assessed for fluorescence intensity by fluorescence-activated cell sorting (FACS) on a FACS-Calibur with Cell Quest software (Becton Dickinson, Mountain View, CA).

Isotype-specific responses were detected by incubation of the RMCs with post-Tx sera and detection with rat isotype-specific monoclonal antibodies. FITC (fluorescein isothiocyanate)-conjugated monoclonal antibodies specific for rat IgA, IgM, IgG1, IgG2a, IgG2b and IgG2c were used (Professor H. Bazin, Leuven University, Leuven, Belgium).

GBM-ELISA
GBM was isolated from F344 and LEW kidneys as previously described [1,12], and a GBM-enzyme-linked immunosorbent assay (ELISA) was performed as previously described [1,12]. Briefly, glomeruli were isolated by differential sieving and collected on a 75 nm sieve. After washing, glomeruli were sonicated and washed in ultrapure H2O. Finally membrane fragments were digested using type Ia collagenase (Sigma) overnight at 37°C in 100 mM Tris–HCl (pH 7.4) and 10 mM CaCl2. After centrifugation, GBM is present in the supernatant. The purity of this preparation was tested using monoclonal antibodies against MHC class I (OX-18), mesangial cells (Thy1.1) and a polyclonal serum against podocytes. No reactivity of these antibodies was detected in our GBM preparations and the GBM was thus considered free of cell membranes. GBM proteins were used as coating in a 96-well ELISA plate (Greiner, Alphen aan den Rijn, The Netherlands); 0.3 µg of total protein per well in a carbonate buffer at pH 9.6. After blocking with PBS/1% BSA, sera were incubated in a 1:25 dilution (in PBS/0.05% Tween-20/1% BSA). Subsequently, a digoxigenin (DIG, Roche Diagnostics, Almere, The Netherlands)-conjugated mouse monoclonal-anti-rat {kappa} light chains (His 8; Professor P. Nieuwenhuis, University of Groningen, The Netherlands) was applied to the wells, followed by horseradish peroxidase (HRP)-conjugated sheep F(ab') fragments anti-DIG (Roche Diagnostics) and the substrate ABTS (2,2'-amino-bis-3-ethylbenzthiazoline-6-sulfonic acid; Sigma).

Inhibition studies
Post-Tx sera to be used in the GBM-ELISA were pre-incubated with F344 RMCs. Sera diluted 1:10 were pre-incubated (30 min on ice) on 1 x106 cells serially four times before further dilution to 1:40 and incubation on F344 GBM-coated wells. The GBM-ELISA was performed as described above.

Alternatively, sera were pre-incubated with GBM before staining of mesangial cells was performed. Sera (1:10 diluted) were incubated for 1 h on F344 or LEW GBM-coated ELISA plates; subsequently the sera were diluted two, four or eight times and incubated on F344 RMCs. FACS analysis was performed as described above.

C3 deposition
To study complement activation upon antibody binding, C3 deposition was studied in a modified GBM-ELISA. Ninety-six well plates were coated with 300 ng/well F344 GBM or (6 ng/ well) rabbit IgG as a positive control. Wells were incubated with heat-inactivated, 1:20 diluted post-Tx sera in PBS/Tween/1% BSA for 1 h at 37°C. After washing the wells with PBS/Tween, normal rat serum (1:15, with active complement) diluted in 0.5 mM Mg2+ and 2 mM Ca2+ buffer was added and incubated for 60 min at 37°C. Subsequently, wells were stained with biotinylated rabbit anti-rat C3 polyclonal antibodies (our own laboratory) and streptavidin–HRP. Finally wells were stained with ABTS.

Apoptosis detection by nuclear morphology
To investigate the effect of antibody binding on cell survival, RMCs were incubated with post-Tx sera and nuclear fragmentation was assessed. RMCs were plated in 24-well plates (Costar, Corning, NY), 1–5 x 104 cells per well, and rested for 24 h. Cells were incubated with 10% heat-inactivated post-Tx or normal rat sera diluted in medium for 1 h at 37°C. Subsequently, 1:5 diluted normal rat serum (with active complement) was added to the wells and incubated for 24 h at 37°C. Staurosporin (0.2 µg/ml; Sigma) was used as a positive control. After 24 h, cells were trypsinized, fixed with 1% paraformaldehyde and cytospins were prepared. Cells were stained with 1 µg/ml Hoechst-33258 (Molecular Probes, Leiden, The Netherlands) and the number of fragmented nuclei was counted using fluorescence microscopy. Alternatively, cells were harvested using trypsin and counted using 0.4% trypan blue solution (Sigma).

Statistical analysis
Statistical analysis was performed to test the differences between F344 to LEW and LEW to F344 transplantation using an unpaired t-test. To compare pre- and post-Tx samples of the same rats, a paired t-test was performed, and to correlate different types of experiments using the same sera the Pearson correlation was used. In all tests, P<0.05 was considered significant.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transplantation of a F344 kidney into a LEW recipient resulted in acute rejection followed by CR in all animals. Transplantation of a LEW kidney into a F344 recipient resulted in acute rejection but without subsequent development of CR. Mesangiolysis was found in all 19 F344 kidneys with CR, being generalized in 17 out of 19 and focal in two cases. Mesangiolysis was not present in LEW to F344 transplants.

Post-Tx sera bind F344 rat mesangial cells
RMCs were cultured from F344 kidneys to investigate the possible development of a humoral response against components of mesangial cells. We found that F344 to LEW post-Tx sera had a specific reactivity with F344 RMC (Figure 1A). Binding of post-Tx antibodies to F344 RMC was dose dependent (Figure 1B). Binding to F344 RMCs was observed in F344 to LEW post-Tx sera but not in LEW to F344 post-Tx sera (P = 0.0014) (Figure 1C).



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Fig. 1. Post-transplantation antibodies bind to rat mesangial cells. (A) Representative example of post-Tx antibodies of a F344 to LEW and a LEW to F344 renal Tx (day 60). F344 RMCs were incubated with a 1:5 diluted serum and stained for total immunoglobulin binding. In grey is the post-Tx serum and in white is a normal rat serum. (B) Serial dilutions of two different F344 to LEW sera were used in FACS analysis on F344 RMCs; a normal rat serum was used as a control (dashed line). (C) Binding to F344 RMCs of F344 to LEW (n = 19) and LEW to F344 (n = 9) sera at day 60 post-Tx in a 1:5 dilution; indicated are individual rat sera and the mean±SD of all, P = 0.0014 (unpaired t-test).

 
Antibodies against RMCs are produced post-Tx
Post-Tx sera (day 60), but not pre-Tx sera, of F344 to LEW transplants bound to RMCs (Figure 2A). All but one F344 to LEW post-Tx sera showed significantly higher binding to RMCs compared with the respective pre-Tx sera (P<0.0001) (Figure 2B). More detailed kinetic analysis showed antibody binding to RMCs from ~3 weeks post-Tx that increased with time (Figure 2C). The antibodies were predominantly of the IgM and IgG1 isotype, whereas no IgA, IgG2a, IgG2b or IgG2c responses were detected (Figure 3).



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Fig. 2. Antibodies are produced post-Tx. (A) Representative example of pre- and post-Tx (day 60) (1:5 diluted) sera of a F344 to LEW renal Tx binding to F344 RMCs. In grey are the pre- and post-Tx sera and in white is a normal rat serum. (B) All F344 to LEW pre- and post-Tx samples (day 60) binding to F344 RMCs show that the antibodies are produced after renal Tx; P<0.0001 (paired t-test). (C) Follow-up in time post-Tx in three different F344 to LEW rat sera binding to F344 RMCs. Note that (B) and (C) are derived from independent experiments using RMCs from different passages; therefore, the maximum binding is not similar.

 


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Fig. 3. Anti-RMC antibodies are predominantly of the IgG1 isotype. Binding of post-Tx sera (1:5 diluted) was detected using monoclonal antibodies specific for rat immunoglobulin isotypes. Representative example of seven post-Tx sera were analysed. In grey are the post-Tx sera and in white is the control without monoclonal antibodies.

 
Antibody responses against RMCs and GBM are related
Since antigens present in the GBM can be produced by mesangial cells, similarities between GBM and RMC antigens were studied. In agreement with our previous findings, anti-F344 GBM antibodies were only present in F344 to LEW post-Tx sera and not in LEW to F344 post-Tx sera (P = 0.0024) (Figure 4A). The antibody binding of the F344 to LEW post-Tx sera is specific for the donor-type GBM [1]. The binding of F344 to LEW sera to F344 RMCs correlated with binding of the same sera to F344 GBM isolates (Pearson correlation R = 0.9010; P<0.0001) (Figure 4B). The binding of F344 to LEW post-Tx antibodies to RMCs was not dependent on the concentration of total serum immunoglobulins (Pearson correlation R = 0.038; P = 0.8752) (data not shown).



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Fig. 4. Binding of post-Tx antibodies is correlated with binding to GBM. (A) All F344 to LEW (n = 19) and LEW to F344 (n = 9) sera at day 60 post-Tx were measured in a F344 GBM-ELISA (diluted 1:25); indicated are individual rat sera and the mean±SD of all, P = 0.0024 (unpaired t-test). (B) The binding of F344 to LEW sera to F344 RMCs and F344 GBM is correlated; Pearson R = 0.9010, P<0.0001. (C) Inhibition of binding of F344 to LEW post-Tx serum to F344 GBM isolates using a pre-incubation with F344 RMCs (representative example of three experiments).

 
Since these data suggested a resemblance between mesangial cell and GBM antigens recognized by post-Tx sera, we performed direct inhibition experiments. Pre-incubation of post-Tx sera with F344 RMCs resulted in decreased binding to GBM compared with non-pre-incubated sera (Figure 4C). The inhibition obtained varied between 32 and 48%. In a reciprocal experiment, binding to RMCs was inhibited (ranging from 25 to 40%) by pre-incubation of the sera with F344 GBM preparations (data not shown). No inhibition was observed when sera were pre-incubated with LEW GBM.

Antibody binding to RMCs does not induce cell death
C3 deposition as a marker of complement activation was studied in a GBM-ELISA. Rabbit IgG-coated plates demonstrated that our serum contained active complement as detected by C3 deposition (Figure 5A). Incubation of GBM-coated plates with post-Tx serum did not result in detectable C3 depositions (Figure 5A); similarly, no C3 deposition was detected in FACS analysis (data not shown). To study directly the effect of antibody binding on RMC survival, F344 RMCs were incubated with post-Tx sera in the presence of serum containing active complement, and nuclear fragmentation was studied [9]. Incubation of RMCs with up to 10% post-Tx serum did not induce nuclear fragmentation as a marker of apoptosis (Figure 5B). In addition, counting the viable cells in the wells did not show decreased survival (Figure 5C).



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Fig. 5. Post-Tx antibodies do not induce apoptosis or complement activation. (A) Complement activation of post-Tx sera was assessed by ELISA. Sera (1:20) were incubated on GBM-coated plates, incubated with 7% normal rat serum (with active complement) and C3 deposition was measured. Rabbit IgG (Rb IgG) was used as a positive control to demonstrate the presence of active complement components in the normal rat serum. Data are expressed as the ratio of the 1:20 diluted sample to the blank (mean of two experiments±SD). (B) F344 mesangial cells were incubated with F344 to LEW post-Tx sera (1:10) for 1 h; subsequently, 20% normal rat serum (with active complement) was added and cells were incubated for 24 h. Cells were harvested, cytospins were prepared and nuclear fragmentation was assessed after Hoechst staining. The percentage nuclear condensation is expressed after counting 200 nuclei per sample. A representative experiment out of two experiments is shown (mean±SD). (C) F344 mesangial cells were incubated with F344 to LEW post-Tx sera (1:10) for 1 h; subsequently, 20% normal rat serum (with active complement) was added and cells were incubated for 24 h. Cells were harvested and the number of viable cells was counted using trypan blue exclusion. A representative experiment out of two experiments is shown (mean±SD).

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mesangiolysis was observed in all F344 kidneys transplanted in LEW recipients at 60 days post-Tx. These LEW recipients of F344 allografts produced antibodies upon renal Tx reactive with F344 RMCs. In F344 to LEW post-Tx sera, antibody binding to RMCs correlated with the anti-GBM antibody response. Furthermore, antibody binding to GBM and RMCs could be inhibited by pre-incubation with RMCs and GBM, respectively. These results suggest that antigens recognized by post-Tx sera are at least partially comparable between RMCs and GBM.

Antibody binding to mesangial cells could be the recognition of MHC or non-MHC antigens on the cell surface. To test for the presence of antibodies directed against MHC antigens, peripheral blood mononuclear cells of donor and recipient strains were incubated with post-Tx sera. No antibody binding was detected (data not shown), suggesting that the antibodies were directed against non-MHC antigens on the surface of the mesangial cells. However, we cannot exclude a contribution of cellular or humoral anti-MHC responses in the complex pathogenesis of CR.

Previously, it has been shown that F344 to LEW post-Tx sera do not bind to LEW mesangial cells [17]. In the present study, only F344 RMCs were used, because we were not able to culture RMCs from LEW origin. Since the responses against RMCs parallel the anti-GBM responses, which were donor specific in all cases, similar donor specificity could be hypothesized for the anti-RMC antibodies. Furthermore, F344 recipients of LEW grafts did not produce antibodies reactive with F344 RMCs comparable with the absence of anti-GBM antibodies in these rats. Together, these data suggest that differences in glomerular antigens between F344 and LEW rats exist, resulting in antibody production by LEW recipients of F344 grafts, but not by F344 recipients of LEW grafts. More detailed analysis of rat perlecan and collagen types IV and VI is necessary to find differences between these strains.

Another argument for a parallel in the antigens between GBM and RMCs is derived from the isotype of the antibody responses. As previously shown for anti-GBM antibodies, the antibodies binding to F344 RMCs are predominantly of the IgG1 isotype. In normal rat sera, the levels of IgG1 are comparable with the levels of IgG2a [18], as was found in the post-Tx sera (data not shown). This suggests that in LEW recipients of F344 kidney grafts, there is a bias towards IgG1 production.

Antibody binding to RMCs might be detrimental, as has been shown in Thy1.1-nephritis [4,5]. At present, the effects of binding of post-Tx antibodies to RMCs are unknown. In the F344 to LEW model, antibodies were only found in rats that had mesangial cell damage (all F344 to LEW transplants) and not in rats that remained free of these lesions (LEW to F344 transplants). However, this remains indirect evidence that the antibodies are involved in mesangial cell damage. Antibody binding to cells might either result in direct cytotoxicity via complement-mediated lysis or can result in increased Fc receptor activation and thereby to increased inflammation. To test the first hypothesis, F344 RMCs were incubated with post-Tx serum (up to 20%) in the presence of active normal serum as a complement source and cell death was assessed after 24 h. No apoptosis or necrosis was detected and the cells appeared normal. Despite active complement in the normal rat serum, no C3 deposition was found after incubation of post-Tx sera on F344 GBM or RMCs (data not shown). Together, these results suggest that the post-Tx antibodies had no complement-activating capacity and had no direct effect on the growth and survival of F344 RMCs in vitro. One explanation might be that antibodies that bound to RMCs were predominantly of the IgG1 isotype as found for anti-GBM antibodies (Figure 3) [1]. Rat IgG1 antibodies might not be the best complement-activating antibodies and have a lower efficiency to induce killing of the target cell [18].

In vivo, Fc receptor-mediated interactions with inflammatory cells might also result in killing. Recently, it has been shown that in vitro, human macrophages recognize antibodies on mesangial cells via CD16 (Fc-{gamma}-RIII), resulting in Fas-L-mediated apoptosis [10]. Although, it is known that during CR many mononuclear cells infiltrate the glomeruli, it remains speculative to link this to mesangiolysis. In vivo experiments will be necessary to demonstrate whether the antibodies play a role in the pathogenesis of CR and if so whether Fc receptors are involved in the development of mesangiolysis upon Tx. Demonstration of a pathogenetic role for these antibodies in both the induction of mesangiolysis and CR was outside the scope of the present study.

In conclusion, LEW recipients of F344 renal grafts produce antibodies reactive with F344 RMCs upon Tx. The antigens seem to be similar to GBM antigens recognized by the same sera. Additional experiments will have to be performed to demonstrate a role for these and other antibodies in the pathogenesis of CR and identify the exact antigens involved.



   Acknowledgments
 
We are grateful to Harmen Draisma for his contributions to the initial experiments. This work was supported by a grant from the Dutch Kidney Foundation (grant number C98.1783).

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 15.10.04
Accepted in revised form: 5. 1.05





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