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NDT Advance Access originally published online on August 5, 2006
Nephrology Dialysis Transplantation 2006 21(10):2736-2744; doi:10.1093/ndt/gfl431
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

TAK-603, an anti-inflammatory compound, reduces crescentic glomerulonephritis and preserves renal function in WKY rats

Junya Yamahana1, Takashi Wada1,, Kengo Furuichi1, Norihiko Sakai1, Hitoshi Yokoyama2 and Shuichi Kaneko1

1Department of Gastroenterology and Nephrology, Graduate School of Medical Science, Kanazawa University, Kanazawa and 2Division of Nephrology, Kanazawa Medical University, Kahoku, Japan

Correspondence and offprint requests to: Dr Takashi Wada, Division of Blood Purification, Department of Gastroenterology and Nephrology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-Machi, Kanazawa 920-8641. Japan. Email: twada{at}medf.m.kanazawa-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. The therapeutic efficacy of the regulation of T helper (Th)-1-predominant immune responses remains to be investigated. Therefore, the effects of the anti-inflammatory compound TAK-603 were investigated in a model of crescentic glomerulonephritis induced by a small dose of nephrotoxic serum in Wistar–Kyoto rats.

Methods. TAK-603 (50 mg/kg body weight) was administered orally, starting at the time of induction of glomerulonephritis. In group 1, the drug was administered daily for the initial 6 days. TAK-603 was administered on day 0 only in group 2, and from day 3 to 5 in group 3. In each group, nephritic rats were killed on days 6 and 56.

Results. In group 1 consisting of rats treated with TAK-603 daily from day 0 to 5, glomerular damage, including crescent formation, was improved on day 6, with reductions in the numbers of CD4, CD8 and ED-1 positive cells, as well as in urinary protein excretion. Protein and transcript levels of Th1 cytokines in the diseased kidneys were markedly decreased by TAK-603 treatment. Renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. Elevated levels of serum creatinine showed concomitant improvement. In group 3, in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. Treatment only on the first day in group 2, partially rescued renal dysfunction.

Conclusions. These results suggest the possible therapeutic application of inhibition of Th1-predominant immune responses in progressive crescentic glomerulosclerosis.

Keywords: crescentic glomerulonephritis; Th1/Th2; WKY rats



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Crescentic glomerulonephritis is characterized clinically by rapid deterioration of renal function, and histologically by mononuclear cell infiltration of glomeruli, glomerular cell proliferation, crescent formation and eventual glomerulosclerosis. A number of studies have attempted to regulate cellular pathways that contribute to immune responses and subsequent inflammatory cascade [1]. However, the mechanisms involved in the progression of crescentic glomerulonephritis remain poorly understood. Although an imbalance of the T helper 1 (Th1)/Th2 ratio is thought to play a crucial role in the pathogenesis of crescentic glomerulonephritis [1,2], the impact of the inhibition of Th1-predominant responses remains to be investigated.

In Wistar–Kyoto (WKY) rats, a very small dose of nephrotoxic serum induces severe proliferative and necrotizing glomerulonephritis with crescentic formation, resembling human crescentic glomerulonephritis. Renal damage leads eventually to glomerulosclerosis and interstitial fibrosis in this model [3]. It is notable that WKY rats are prone to Th1 immune responses, and serve as a useful model in which to analyse the mechanisms of crescentic glomerulonephritis in addition to examining the impact of anti-inflammatory agents on progressive glomerular and interstitial lesions.

The anti-inflammatory drug TAK-603, (ethyl 4-(3, 4-dimethoxyphenyl)-6, 7-dimethoxy-2-(1, 2, 4-triazol-1-ylmethyl)-quinoline-3-carboxylate; Takeda Ltd., Co, Tokyo, Japan), reduces Th1 cytokines, including {gamma}-interferon ({gamma}-IFN), interleukin (IL)-2, IL-12, although the detailed molecular mechanisms of its actions remain unclear [4]. This drug ameliorates the progression of synovial injury and arthritis in an adjuvant arthritis model in rats [5], and has a therapeutic effect on graft vs host disease [4], as well as on rat skin allograft survival [6].

Therefore, we hypothesized that the initial inhibition of Th1 immune responses may provide a key to the long-term therapeutic impact on crescentic glomerulonephritis in this study. We reported here that the suppression of Th1 immune responses via TAK-603 treatment represents a beneficial therapeutic approach for reducing glomerulosclerosis and interstitial fibrosis and preserving renal function in crescentic glomerulonephritis in WKY rats.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Inbred male WKY rats, aged 8–10 weeks, were purchased from Charles River Japan Inc. (Yokohama, Japan). All procedures used in animal experiments complied with standards in the Guidelines for the Care and Use of Laboratory Animals in Takara-machi Campus of Kanazawa University.

Preparation of anti-rat glomerular basement membrane antibodies
Rat glomerular basement membrane (GBM) was prepared according to the method of Krakower and Greenspon [7]. Preparation of anti-rat GBM antibodies was described previously [3]. Specificity was confirmed by in vitro indirect immunofluorescence using fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG; no.38236; Organon Teknika Corp., Durham, NC, USA) on frozen sections of normal Wistar rat kidneys. Sharp linear immunofluorescence was observed only along the GBM.

Experimental design
The experimental design is described in Figure 1. A total of 48 male WKY rats were injected intravenously with 0.1 ml of nephrotoxic serum on day 0. TAK-603 (50 mg/kg body weight), dissolved in methylcellulose, was administered, starting at the time of induction of glomerulonephritis. Nephrotoxic rats were divided into three groups as follows:


Figure 1
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  		Fig. 1. Experimental design. TAK-603 was orally administrated in WKY rats (group 1, from day 0 to 5; group 2, at day 0; group 3, from day 3 to 5). Rats were sacrificed on day 6 or 56.

 
Group 1: 12 of 48 rats were administered TAK-603 daily for the initial 6 days.

Group 2: 12 rats received a single administration of TAK-603 on day 0.

Group 3: 12 rats were administered TAK-603 from day 3 to 5.

In all the three groups, six nephritic rats were killed on day 6, and the remaining six rats were killed on day 56.

Histopathological studies
A portion of renal tissue was fixed in 10% buffered formalin, followed by embedding in paraffin and staining with haematoxylin and eosin, as well as periodic acid-Schiff reagent. To avoid variation in the shape of glomeruli, glomeruli of the same diameter from a vascula pole were chosen. Crescentic formation was counted from more than 100 glomeruli for each rat and expressed as a percentage of positive glomeruli to the total number. Cell populations infiltrating glomeruli were counted in 100 glomeruli, and the results are expressed as total cell number per glomerulus. The extent of glomerular sclerosis was expressed as a percentage of the periodic acid-methenamine-silver strain (PAM)-positive area per whole glomerular area. Each area was measured by computer-aided manipulation using Mac Scope version 6.02 (Mitani Shoji Co., Ltd., Fukui, Japan). The whole glomerular area was measured by tracking out the glomerular tuft. The extent of interstitial fibrosis, stained blue by Mallory-Azan staining, was evaluated from the whole area of the cortex in the individual kidney sections and expressed as a percentage of the field using Mac Scope version 6.02.

Immunohistology
To determine whether glomerulonephritis was induced equally in both vehicle-treated and TAK-603-treated rats, the depositions of rat IgG and C3 were examined. FITC-conjugated anti-rat C3 and FITC-conjugated anti-rat-IgG (Organon Teknika Corp., Durham, NC, USA) antibodies were used for direct immunofluorescence. To analyse cell populations infiltrating glomeruli, cryostat sections were stained with either a mouse monoclonal antibody against ED-1 (BMA Biomedicals Ltd., Augst, Switzerland) for monocytes and macrophages, or with anti-CD4 (BMA Biomedicals Ltd., Switzerland) and anti-CD8 (Oxford Biotechnology Ltd., UK) antibodies for CD4 and CD8 positive cells lymphocytes, followed by FITC-conjugated rabbit anti-mouse IgG (Cappel, West Chester, PA, USA). Positive cells were counted in at least 100 randomly chosen glomeruli and the results are expressed as number of positive cells per glomerular cross section.

Electron microscopy
One portion of renal tissue was fixed in 2.5% glutaraldehyde and 4% osmic acid, followed by embedding in Epon812 (Oken, Tokyo, Japan) and staining with uranyl acetate. These specimens were observed by electron microscopy (Hitachi, Tokyo, Japan).

Determination of urinary protein concentration and creatinine level
Urinary protein concentrations were determined before immunization on days 0, 6 and 56. Urinary protein excretion was expressed as the ratio of urinary protein to urinary creatinine. Creatinine levels in urine and serum were measured using an automated analyser (Hitachi, Tokyo, Japan) according to the manufacturer's instructions.

Determination of serum and urinary cytokines
To investigate the effects of TAK-603 administration, Th1/Th2 cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Rat ELISA kits for IL-10, {gamma}-IFN, and IL-4 were purchased from R&D systems (Minneapolis, MN, USA), and a rat IL-12 kit was from Biosource Int. Inc. (Camarillo, CA, USA). Urinary levels of these cytokines were corrected by urinary creatinine levels.

RNA isolation and reverse transcriptase polymerase chain reaction
Total RNA was isolated from the renal cortices, and analyses were performed using reverse transcriptase polymerase chain reaction (RT–PCR), as described previously [8]. Reverse transcription of aliquots of 6 µg of total RNA from six rats in each group (1 µg RNA per rat) was performed using an RT–PCR kit (Perkin Elmer, Foster City, CA, USA). The complementary DNA product (1 µg) was amplified by PCR with the following primers: IL-2, GCG CAC CCA CTT CAA GCC CT (sense) and CCA CCA CAG TTG CTG GCT CA (antisense) [9]; {gamma}-IFN, CCC TCT CTG GCT GTT ACT GC (sense) and CTC CTT TTC CGC TTC CTT AG (antisense) [9]; IL-4, CAG AAA AAG GGA CTC CAT GCA (sense) and GCT CGT TCT CCG TGC TGT TC (antisense) [10]. Aliquots of 10 µl of PCR products were analysed by 2% agarose gel electrophoresis and stained with ethidium bromide. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for PCR.

Statistical analyses
The mean values and SE were calculated for all parameters determined in this study. Statistical analyses were performed by ANOVA and P-values of <0.05 were accepted as statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
TAK-603 reduced renal pathology in WKY rats
Immunofluorescence analysis revealed no deposition of rabbit IgG in glomeruli from normal WKY rats (data not shown). Rabbit IgG was detected in an intense linear pattern along the glomerular capillaries in specimens from nephrotoxic serum-injected rats treated with TAK-603 or vehicle alone. Rat IgG (data not shown) and C3 (Figure 2) were also detected, but the intensities of staining were faint. Semiquantitative evaluation of deposition revealed no significant differences in the deposition of rabbit IgG, rat IgG and rat C3 between glomeruli from rats treated with TAK-603 and those from rats treated with vehicle only (data not shown).


Figure 2
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  		Fig. 2. Immunofluorescence study of rat C3. Immunofluorescence analysis showed faint labelling of rat C3. There were no significant differences between glomeruli from rats treated with vehicle (A) and from those treated with TAK-603 (B).

 
On day 6, glomeruli exhibited endocapillary proliferation, severe necrotizing lesions and crescentic lesion formation (Figure 3A). Conversely, administration of TAK-603 markedly reduced severe crescentic and necrotizing lesions in group 1 (Figure 3B). TAK-603 treatment decreased the development of crescents per 100 glomeruli in a section (Figure 3C). The total number of glomerular cells was also reduced in rats treated with TAK-603 (Figure 3D).


Figure 3
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  		Fig. 3. Renal pathology was reduced in TAK-603-treated WKY rats. On day 6, WKY rats developed severe proliferative glomerulonephritis (A). Renal pathology was decreased in TAK-603-treated WKY rats (B). Reduction of the number of crescents (C) and glomerular cells was observed (D).

 
To identify the cell phenotypes of leukocytes affected by this treatment, CD4, CD8 and ED-1-positive cells in glomeruli were examined. With the administration of TAK-603, the numbers of CD4 and ED-1 positive cells (Figure 4A and B, respectively) were reduced markedly in all the three groups on day 6. The number of infiltrating CD8 positive cells tended to be reduced by TAK-603 treatment (Figure 4C).


Figure 4
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  		Fig. 4. Reduced cell infiltration in glomeruli in TAK-603-treated WKY rats. There were significant reductions in the numbers of CD4, and ED-1 positive cells (A and B, respectively). The number of CD8 positive cells tended to be reduced (C). *P < 0.05 compared with untreated rats.

 
On day 56, interstitial fibrosis and glomerular sclerosis were observed in rats treated with vehicle only (Figure 5A). Interstitial fibrosis was significantly decreased in groups 1 and 3 (Figure 5B and C). However, the reduction of glomerular sclerosis with the administration of TAK-603 in group 2 was not significant (Figure 5D).


Figure 5
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  		Fig. 5. TAK-603 reduced interstitial fibrosis and glomerular sclerosis. On day 56, WKY rats exhibited severe interstitial fibrosis and glomerular sclerosis (A). In contrast, administration of TAK-603 reduced renal pathology in group 1 (B). The mean interstitial fibrosis (C) and glomerular sclerosis (D) expressed as percentage involvement of the field were reduced in TAK-603-treated rats. *P < 0.05 compared with untreated rats. Magnification 200x.

 
Effect of TAK-603 on urinary protein excretion
Normal rats showed excretion of minute amounts of protein in the urine on day 6. In contrast, vehicle-treated nephritic rats showed excretion of markedly elevated amounts of protein in the urine on day 6. Notably, proteinuria was markedly decreased to almost normal levels by administration of TAK-603 in group 1. In group 3, administration of TAK-603, even after the induction of glomerulonephritis, had a mild protective effect on urinary excretion of protein. However, a single administration of TAK-603 on day 0 in group 2 showed little effect on urinary excretion of protein. In contrast to the urinary excretion of protein on day 6, proteinuria was decreased in all three treated groups on day 56, especially in rats treated with TAK-603 for the initial 6 days (Figure 6A). To further evaluate the mechanisms involved in urinary protein excretion, foot processes were examined by electron microscopy. Extensive fusion of the foot processes around the infiltrating leucocytes was observed in untreated rats (Figure 6B), whereas mild fusion was detected in the tufts even with infiltrated leucocytes in TAK-603-treated rats (Figure 6C).


Figure 6
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  		Fig. 6. TAK-603 reduced urinary protein excretion. Vehicle-treated rats showed elevated levels of urinary protein excretion on day 6. Administration of TAK-603 for the initial 6 days reduced proteinuria. Proteinuria was decreased in all the treated groups on day 56 (A). Under an electron microscopic evaluation, an extensive fusion of the foot processes around the infiltrated leucocytes was observed in untreated rats (B), whereas a mild fusion was detected in the tufts even with infiltrated leucocytes (C). *P < 0.05 compared with untreated rats. #P < 0.05 compared with TAK.

 
Effect of TAK-603 on renal function
Serum creatinine concentrations on day 6 remained low in all groups. In contrast, on day 56, serum creatinine levels were markedly elevated in vehicle-treated nephritic rats. Notably, serum creatinine levels were decreased by administration of TAK-603 in both groups 1 and 3. These results together with the histological improvement suggest beneficial effects of TAK-603 on renal protection from progressive renal insults (Figure 7).


Figure 7
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  		Fig. 7. TAK-603 treatment preserved renal function. Serum creatinine concentrations on day 6 remained low in all groups. On day 56, serum creatinine levels were markedly elevated in vehicle-treated nephritic rats. In groups 1 and 3, serum creatinine levels were decreased by the administration of TAK-603. *P < 0.05 compared with untreated rats.

 
TAK-603 treatment on Th1/Th2 cytokines
To investigate the effects of TAK-603 administration on Th1/Th2 cytokines, mRNAs of {gamma}-IFN, IL-2 and IL-4 were evaluated in the renal cortices. In untreated rats, levels of {gamma}-IFN and IL-2 mRNA were elevated, but IL-4 mRNA was not detected. TAK-603 treatment down-regulated expression of {gamma}-IFN and IL-2 mRNA in group 1 (Figure 8), and had a mild impact in groups 2 and 3 (data not shown).


Figure 8
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  		Fig. 8. Effects of TAK-603 treatment on {gamma}-IFN and IL-2 transcript levels. Both of {gamma}-IFN (A) and IL-2 (B) transcripts in diseased kidneys were up-regulated in vehicle-treated rats on day 6, compared with the faint expression in normal rats. Transcript levels were markedly decreased in TAK-603-treated rats in group 1 compared with vehicle treated rats on day 6. Transcript level were not significantly different between TAK-603 trated rats in group 1 and vehicle treated rats on day 56. *P < 0.05 compared with untreated rats.

 
To further evaluate the effects of TAK-603 treatment on Th1/Th2 cytokines, the concentrations of {gamma}-IFN and IL-12 as Th1 cytokines and IL-4 and IL-10 as Th2 cytokines were measured by ELISA. In vehicle-treated rats, the levels of IL-12 in serum were higher than those of normal rats. Administration of TAK-603 significantly decreased these levels (Figure 9). In contrast, urinary IL-12 levels were undetectable in this model, and were not altered by TAK-603 treatment (data not shown). Further, IL-4 and IL-10 were not detected in serum or urine (data not shown).


Figure 9
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  		Fig. 9. Effects of TAK-603 treatment on serum IL-12 levels. Compared with normal rats, serum IL-12 levels increased in vehicle-treated rats on day 6. TAK-603-treated rats in all the three groups showed decreased levels of serum IL-12. *P < 0.01 compared with untreated rats.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
This study demonstrated that the administration of an anti-inflammatory drug, TAK-603, markedly reduced crescentic glomerulonephritis with the marked decreases in infiltrates as well as in urinary protein excretion. In addition, TAK-603 treatment reduced glomerulosclerosis and interstitial fibrosis, concomitantly with preservation of renal function. Semiquantitative evaluation revealed no significant differences in the deposition of rabbit IgG, rat IgG, or rat C3 between glomeruli from rats treated with TAK-603 and those from rats treated with vehicle only. These results suggest that the induction of glomerulonephritis was achieved equally. The results of this study, taken together, suggest that the initial inhibition of Th1 immune responses by TAK-603 may be effective in the treatment of progressive crescentic glomerulonephritis in WKY rats.

Administration of TAK-603 improved crescentic glomerulonephritis concomitantly with a decrease in urinary protein excretion. Of interest, WKY rats are prone to be Th1-dominant. There is accumulating evidence that up-regulated Th1 cytokines govern the progression of crescentic glomerulonephritis in WKY rats, whereas the administration of Th2 cytokines ameliorates renal pathology [11,12]. It is notable that TAK-603 inhibits the expression of Th1 cytokines including {gamma}-IFN, IL-2 [4], although the detailed molecular mechanisms involved in these effects remain to be determined. Crescentic formation is related to renal dysfunction in rats [3,13]. Furthermore, the presence of crescentic formation is important because of the poor prognosis of renal function [14,15]. Thus, the initial inhibition of Th1 immune responses, e.g. by the administration of TAK-603, may be therapeutically effective in crescentic glomerulonephritis.

The balance of Th1 and Th2 immune responses has been reported to play a role in various immune disorders [16]. Th1 inhibition may be associated with up-regulation of Th2 responses. In addition, up-regulation of Th2 immune responses may contribute to organ fibrosis [17,18]. Therefore, in this study, to determine whether TAK-603 treatment up-regulates Th2 immune responses, mRNA and protein levels of Th1/Th2 cytokines were evaluated. Th1 cytokines were suppressed by TAK-603 treatment. On the other hand, TAK-603 treatment had little effect on the levels of Th2 cytokines. Supporting this notion, TAK-603 suppresses {gamma}-IFN but hardly affects IL-4 expression in established Th1- and Th2-type T-cell lines [5]. The {gamma}-IFN expression in diseased kidneys, which was inhibited by TAK-603 treatment in the present study, induces mesangial cells to produce monocyte chemoattractant protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-1{alpha} (MIP-1{alpha})/CCL3, and regulated on activation normal T-cells expressed and secreted (RANTES)/CCL5 [19,20]. In addition, MIP-1{alpha} and RANTES through their receptors, CCR1 and CCR5, may be involved in human crescentic glomerulonephritis [21,22]. These chemokines have the capacity to be chemotactic to macrophages and T cells. In fact, in this study, the numbers of CD4, CD8 and ED-1 positive cells were decreased. Interestingly, MCP-1 and its cognate receptor CCR2 have been reported to be involved in crescentic glomerulonephritis, leading to glomerulosclerosis and interstitial fibrosis [3,23]. Although, the impact of TAK-603 on these chemokines remains to be investigated, TAK-603 treatment may have reduced glomerulosclerosis and interstitial fibrosis via {gamma}-IFN-induced chemokine expression without IL-4 up-regulation.

The administration of TAK-603 from day 0 to 6 returned proteinuria to almost normal, and reduced glomerulosclerosis and interstitial fibrosis. Previously, we reported that the initial inhibition of p38 mitogen-activated protein kinase prevented proteinuria, glomerulosclerosis and interstitial fibrosis, suggesting that the initial inflammatory events may be pivotal for long-term renal pathology [12]. Similar to the previous report, TAK-603 treatment for 6 days completely reduced proteinuria. Supporting this notion, TAK-603 treatment prevented the fusion of epithelial foot processes. In crescentic glomerulonephritis, a Th1 response induces a severe crescentic pattern of glomerulonephritis. Treatment with monoclonal anti-{gamma}-IFN antibody significantly reduced glomerular injury and crescent formation [24]. In addition, proteinuria is thought to be a result of glomerular injury and to cause long-term interstitial injury, resulting in interstitial fibrosis [25]. Therefore, these findings suggest that treatment of TAK-603 for 6 days markedly ameliorated crescentic glomerulonephritis at the initial phase in group 1, thereby preventing long-term renal progressive insults.

In this study, a single injection of TAK-603 in group 2 had less effect on glomerulosclerosis and interstitial fibrosis as compared with those in group 1. After oral administration of TAK-603, a period of at least 2 h is required before the systemic concentration of TAK-603 reaches therapeutic levels; the half-life of TAK-603 is therefore almost 4 h [26]. Tumour necrosis factor-{alpha} and MCP-1/CCL2, continue to be up-regulated in crescentic glomerulonephritis, up to several days after the injection of nephrotoxic serum [3,27]. Therefore, these findings in group 2 may be derived from the short-acting effect with a single injection of TAK-603. To further determine the therapeutic impacts of TAK-603 on the already-established crescentic glomerulonephritis, TAK-603 was administered from day 3 to 5. Partial but significant reductions in renal pathology and urinary protein excretion were observed in group 3, suggesting that TAK-603 treatment for a sufficient duration may be effective even if crescentic glomerulonephritis has already begun. These results are attractive because the regulation of Th1 cytokines in the early period of crescent formation may protect against renal function in the long-term. Taken together, these observations suggest that Th1 inhibition may be useful for the therapeutic application in human crescentic glomerulonephritis.

In summary, we established that TAK-603 treatment markedly ameliorates crescentic glomerulonephritis in a WKY rat model, resulting in preserved renal function. These results suggest that the initial inhibition of Th1 immune response has an appealing therapeutic potential for crescentic glomerulonephritis.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
T.W. is a recipient of a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture in Japan. This work is supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 3. 2.06
Accepted in revised form: 22. 6.06


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