Pentoxifylline suppresses renal tumour necrosis factor-{alpha} and ameliorates experimental crescentic glomerulonephritis in rats

Yung-Ming Chen1, Yee-Yung Ng2, Shuei-Liong Lin2, Wen-Chih Chiang2, Hui Y. Lan3 and Tun-Jun Tsai1

1Department of Medicine, National Taiwan University Hospital and College of Medicine National Taiwan University, 2Veterans General Hospital, Yang-Ming University, Taipei, 10016, Taiwan and 3Department of Medicine-Nephrology, Baylor College of Medicine, Houston, USA

Correspondence and offprint requests to: Dr Tun-Jun Tsai, Department of Medicine, National Taiwan University Hospital, College of Medicine National Taiwan University. No. 7, Chung-Shan South Road, Taipei, 10016, Taiwan. Email: paul{at}ha.mc.ntu.edu.tw



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Crescentic glomerulonephritis is a rapidly progressive form of glomerulonephritis, but treatment remains non-specific. The methylxanthine derivative pentoxifylline (PTX) is a clinically available phosphodiesterase inhibitor with anti-inflammatory and immunoregulatory activities. This study examined whether PTX has beneficial effects in a rat model of anti-glomerular basement membrane (GBM) crescentic glomerulonephritis.

Methods. Experimental crescentic glomerulonephritis was induced in Wistar rats by intravenous injection of rabbit anti-rat GBM serum and treated with either vehicle (phosphate-buffered saline) or PTX (0.1 g/kg/day) intravenously on a daily basis. Groups of six animals were euthanized at days 3, 7, 14 or 28 after induction of disease. Effects of PTX on renal function, histology and expression of cytokines, chemokines and adhesion molecules were determined.

Results. Compared with the vehicle-treated nephritic rats, PTX treatment beginning at the start of the nephritis significantly suppressed mRNA expression of tumour necrosis factor (TNF)-{alpha}, but not interleukin-1ß, throughout the course of nephritis. Moreover, PTX decreased renal mRNAs for intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted (RANTES) and osteopontin (OPN) at all time points examined. These effects were associated with a significant inhibition of macrophage and T-cell infiltration, a reduction of 24-h urinary protein excretion (50–75%, P<0.05), an improvement of histological damage including glomerular crescent formation (60–70%, P<0.01) and a decrease of cortical mRNAs for type I ({alpha}1) collagen and fibronectin. The efficacy of PTX could also be seen, though to a lesser extent, in rats with established nephritis.

Conclusions. PTX is an effective anti-inflammatory and immunomodulatory agent capable of suppressing rat crescentic glomerulonephritis. Inhibition of renal TNF-{alpha}, ICAM-1, RANTES, MCP-1 and OPN expression may be a mechanism whereby PTX suppresses progressive renal injury in rat crescentic glomerulonephritis.

Keywords: adhesion molecules and chemokines; crescents; glomerulonephritis; pentoxifylline; TNF-{alpha}



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Crescentic glomerulonephritis, characterized by glomerular crescent formation, is the most severe form of glomerulonephritis. It is well established that cell-mediated mechanisms play a pivotal role in the pathogenesis of crescentic glomerulonephritis [1]. Indeed, macrophages and T cells are the major effector cells in both human and experimental crescentic glomerulonephritis [2]. These inflammatory cells may be recruited into the glomeruli and interstitium by upregulation of adhesion molecules [e.g. intercellular adhesion molecule-1 (ICAM-1)], chemokines [e.g. monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted (RANTES)] and other adhesion and chemotactic proteins [e.g. osteopontin (OPN)] [35]. Inhibition of chemotactic/adhesion molecule expression and macrophage/T-cell accumulation by blocking interleukin-1 (IL-1) and/or tumour necrosis factor-{alpha} (TNF-{alpha}) in experimental crescentic glomerulonephritis exemplifies the importance of proinflammatory cytokines in the pathogenetic mechanisms of crescentic glomerulonephritis [6,7].

Pentoxifylline (PTX) is a methylxanthine phosphodiesterase inhibitor that has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with peripheral vascular diseases and cerebrovascular disorders [8]. In addition to its well-known haemorheological activity, several lines of evidence suggest that PTX also possesses anti-inflammatory and/or immunoregulatory activities. It is of note that PTX suppresses TNF-{alpha} production by monocytes/macrophages [9], reduces ICAM-1 or MCP-1 expression by mononuclear cells and proximal renal tubular cells [1012], and interferes with chemotaxis of leukocytes [13]. In vivo, PTX has shown its efficacy in different models of renal diseases where TNF-{alpha}, MCP-1 or ICAM-1 is involved. These include rat anti-Thy1 nephritis [14], murine lupus nephritis [15], rabbit ischaemia/reperfusion renal injury [16], mouse cisplatin nephrotoxicity [17] and rat remnant kidney model [12]. Parallel to these findings, clinical trials in patients with diabetic nephropathy [18] and membranous glomerulonephritis [19] have also shown that PTX can lower endogenous TNF-{alpha} and attenuate proteinuria. Taken together, these data raise the possibility that PTX may have promise as an anti-inflammatory agent via its ability to antagonize TNF-{alpha} and multiple inflammatory mediators. In this study, we investigated this potential usefulness of PTX in a rat model of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Induction of rat crescentic glomerulonephritis and experimental design
Crescentic glomerulonephritis was induced as previously described [20]. Briefly, male Wistar rats obtained from the animal centre of National Taiwan University Hospital, weighing 190–200 g, were immunized by a subcutaneous injection of 5 mg of normal rabbit immunoglobulin G (IgG) in Freund's complete adjuvant. Five days later, groups of six animals were injected intravenously (i.v.) with 0.5 ml of rabbit anti-rat GBM serum (2.5 ml/kg body weight, 12.5 mg IgG/ml), followed immediately by either PTX (0.1 g/kg/day, Hoechst, Frankfurt, Germany) or vehicle [1x phosphate-buffered saline (PBS)] via i.v. infusion over 1 h on a daily basis until being killed on days 3, 7, 14 or 28. A separate set of experiments was performed in groups of six rats in which PTX or PBS was administered 7 days after the induction of nephritis, i.e. after the nephritis was established. These experiments were approved by the Animal Experimentation Committee and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Examination of renal function and proteinuria
Blood samples and 24-h urine collections were taken on days 0, 3, 7, 14, 21 and 28. Creatinine levels were measured using a standard colorimetric method (Jaffe rate reaction), and urine protein concentrations were quantitated by Biorad's method. All analyses were performed by the Department of Laboratory Medicine, National Taiwan University Hospital.

Measurement of TNF-{alpha} by enzyme-linked immunosorbent assay (ELISA)
Blood and urine samples were collected in endotoxin-free tubes at different time points and TNF-{alpha} concentrations (pg/ml) were measured using a commercially available ELISA kit specific for rat TNF-{alpha} (Endogen, Woburn, MA).

Renal histopathology
Kidney tissues for histological examination were fixed in 10% formalin, and 4 µm paraffin sections were stained with haematoxylin and eosin (H&E) or periodic acid–Shiff reagent (PAS). The severity of glomerular and interstitial injury was examined in H&E-stained sections, and the percentage of glomeruli exhibiting crescentic formation was assessed by examination of at least 50 glomerular cross-sections (GCS) per animal in PAS-stained sections as previously described [20]. All analyses were performed blind on coded slides.

Immunohistochemistry
Immunohistochemical staining using monoclonal antibodies (mAbs) to monocytes/macrophages (ED1; Chemicon, Temecula, CA) and CD8 T cells (MRC OX-8, Oxford, UK) was performed on cryostat sections of tissues fixed in 2% paraformaldehyde–lysine–periodate, as previously described [20]. Briefly, sections were pre-incubated with 10% fetal calf serum and 10% normal goat serum in 1x PBS for 20 min, drained, and labelled with mouse mAb overnight at 4°C, washed three times in 1x PBS and endogenous peroxidase inactivated by incubation in 0.5% H2O2 in methanol. Sections then were washed in 1x PBS, and incubated with biotin-conjugated goat anti-mouse IgG for 60 min at room temperature. After washing, sections were incubated with the avidin–biotin–peroxidase reagent (Dakopatts, Glostrup, Denmark) for 60 min and developed with 3,3-diaminobenzidine (DAB) to produce a brown product. Normal mouse IgG was used as a negative control, and no staining was seen in these sections. Sections were counterstained with haematoxylin and the number of positive cells was counted in 50 GCS per animal, or in 25 consecutive high-power fields (x400) in the cortical interstitium for each section. These fields progressed from the outer to inner cortex, avoiding only large vessels and glomeruli. No adjustment of the interstitial cell count was made for tubules or the luminal space. All counting was performed on blinded slides.

A separate experiment was performed for the analysis of possible effects of PTX on glomerular binding of anti-GBM antibodies. Nephrectomy was performed in vehicle- and PTX-treated nephritic rats 3 days after initiation of nephritis. Renal sections were stained with a biotin-conjugated anti-rabbit IgG (1:250; Dakopatts) for 1 h at room temperature and were detected by the avidin–biotin–peroxidase method using DAB as substrate as described above.

Measurement of mRNA expression by reverse transcription–polymerase chain reaction (RT–PCR) and northern blots
Total RNA from the renal cortex was isolated using the guanidinium thiocyanate/acid phenol method [14]. Because of relatively low expression of TNF-{alpha} and IL-1ß genes, the present study used RT–PCR to measure their mRNA levels, using the specific primers outlined in Table 1. The DNA amplifications were performed for 26 cycles in an air thermal cycler, as described previously [14]. For synthesis of type I ({alpha}1) collagen and fibronectin riboprobes, cDNAs for human type I ({alpha}1) collagen and human fibronectin-1 were purchased from American Type Culture Collection (Rockville, MD). A 1.5 kb EcoRI fragment of type I ({alpha}1) collagen and a 1.2 kb EcoRI fragment of fibronectin cDNAs were subcloned, respectively, into the pBSII/SK- vector (Stratagene, LA Jolla, CA). For synthesis of rat ICAM-1, MCP-1, RANTES and OPN riboprobes, cDNA fragments were first amplified by RT–PCR from renal cortical total RNA of vehicle-treated nephritic rats, using specific upstream and downstream primers (Table 1). The products subsequently were subcloned into the pGEM-dT vector (Promega, Madison, WI). The cloned cDNAs were then linearized and used as templates for in vitro transcription of antisense digoxigenin-conjugated riboprobes according to the supplier's instructions (Roche Molecular Biochemicals, Mannheim, Germany).


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Table 1. Primers for RT–PCR amplification of cDNA fragments

 
A 10 µg aliquot of total RNA isolated from the renal cortex was electrophoresed on formaldehyde-denatured 1% agarose gels and subsequently transferred to nylon membranes according to standard protocols. Membranes were hybridized overnight at 65°C with a specific digoxigenin-labelled cRNA probe in 5 x SSPE, 50% formamide, 0.2 mg/ml herring sperm DNA, 0.2 mg/ml yeast RNA, 0.5% SDS and 5 x Denhardt's solution. After hybridization, the northern blots were developed using CSPD® (Roche Molecular Biochemicals) as the substrate for alkaline phosphatase. The intensity of RT–PCR products and northern blot signals was quantified with computerized densitometry and normalized against the signal of glyceraldehyde-3-phosphate dehydrogenase messages.

Statistical analysis
Data are expressed as mean±SEM. All comparisons were done by analysis of variance followed by Dunnett's t-test using the Stat-View® package for the Macintosh computer (Abacus Concepts, Berkeley, CA). A P-value of <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Effect of PTX treatment on urinary protein excretion and renal function
During the course of the experiment, animals which received PTX treatment showed no difference in body weight, fluid intake or urinary volume compared with animals which received the vehicle control (data not shown). However, vehicle-treated anti-GBM nephritic rats developed frank proteinuria during days 3 and 28, and PTX treatment significantly reduced urinary protein excretion throughout the course (P<0.05 vs vehicle-treated rats, Figure 1A). In addition, compared with vehicle-treated rats, PTX treatment also partially improved renal function on day 3 as reflected by creatinine clearance (P<0.05, Figure 1B). In nephritic rats whose treatment began 7 days after the induction of nephritis, i.e. after the nephritis was established, the urinary protein excretion was reduced at day 28 (P<0.05), but not at day 14 or 21 (Figure 1C).



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Fig. 1. Effects of PTX on 24-h proteinuria and renal function. (A) Proteinuria (mg/day) and (B) creatinine clearance (ml/min) in rats with anti-GBM nephritis that were treated with PBS (vehicle, filled bars) or PTX (open bars) immediately after the induction of nephritis. (C) Proteinuria (mg/day) in rats with anti-GBM nephritis that were treated with PBS (vehicle, filled squares) or PTX (open squares) 7 days after the induction of nephritis, i.e. after the nephritis was established. Each bar represents the mean±SEM for a group of six rats. *P<0.05 compared with vehicle-treated nephritic rats.

 
Effect of PTX treatment on renal histopathology and matrix gene expression
To determine whether treatment with PTX could cause a reduction in the amount of rabbit Ig deposited in glomeruli, kidney sections from both vehicle-treated and PTX-treated anti-GBM nephritic rats on day 3 were stained to reveal the presence of rabbit IgG. The results showed similar intensity of DAB staining along glomerular capillary walls between the two groups, indicating no inhibition by PTX therapy of the glomerular binding of rabbit anti-GBM antibodies (Figure 2).



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Fig. 2. Effect of PTX on binding of rabbit anti-GBM antibodies. Immunostaining micrograph of glomeruli stained for rabbit IgG in kidney sections from vehicle-treated nephritic rats at day 3 that demonstrated similar intensity of DAB staining along glomerular capillary walls (A), which can be compared with sections from nephritic rats treated with PTX (B) (original magnification x120).

 
In vehicle-treated anti-GBM nephritic rats, there were severe proliferative and inflammatory changes in both glomeruli and tubulointerstitium, including glomerular hypercellularity, matrix accumulation and crescent formation, as well as interstitial mononuclear cell infiltrates, tubular atrophy and interstitial fibrosis (Figure 3A and B). Strikingly, PTX treatment beginning at the start of the nephritis significantly attenuated all parameters of glomerular and interstitial injury, particularly glomerular crescentic formation (Figures 3C and D, and 4). These findings were associated with a significant reduction of the augmented renal cortical mRNA levels of type I ({alpha}1) collagen and fibronectin throughout the course of the nephritis (Figure 5A). PTX treatment beginning after the establishment of nephritis also resulted in a reduction of glomerular crescent formation at day 28 (vehicle-treated nephritic rats: 9±2% vs PTX-treated nephritic rats: 2±1%, P<0.05, n = 6 for each group). Additionally, the suppressive effect of PTX on renal cortical mRNAs for type I ({alpha}1) collagen and fibronectin was also evident in rats with established nephritis (Figure 5B).



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Fig. 3. Effects of PTX on renal histopathology. (A and B) Cortical sections of kidney at day 28 from the vehicle-treated nephritic rats show proliferative and inflammatory glomerular and interstitial changes, including glomerular hypercellularity, matrix deposition and crescent formation, as well as periglomerular and peritubular interstitial mononuclear cell infiltrates, tubular atrophy and interstitial fibrosis. (C and D) Cortical sections at day 28 from the PTX-treated nephritic rats show a significant reduction in both glomerular and tubulointerstitial damage including glomerular crescentic formation (H&E-stained paraffin sections; original magnification: A and C, x120; B and D, x380). Shown are representative sections for six animals per group.

 


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Fig. 4. Quantitation of glomerular crescentic formation. Each bar represents the mean±SEM for a group of six rats with anti-GBM nephritis that were treated with PBS (vehicle, filled bars) or PTX (open bars) immediately after the induction of nephritis. **P<0.01; *P<0.05 compared with vehicle-treated nephritic rats.

 


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Fig. 5. Effects of PTX on matrix gene expression. (A) Representative northern blots demonstrating the effects of PTX on renal cortical mRNA levels of type I ({alpha}1) collagen and fibronectin. Messages for GAPDH are shown in the lower graph at each time point. Right panels show the corresponding densitometer readings corrected for GAPDH and relative to that of control. Values are the mean±SEM of three experiments. *P < 0.05 PTX-treated vs vehicle-treated nephritic rats; +P < 0.05 vehicle-treated nephritic rats vs normal control rats. Open circles, normal control rats; filled squares, vehicle-treated nephritic rats; open squares, PTX-treated nephritic rats. (B) Representative northern blots at day 28 of the nephritis showing effects of PTX administered after the nephritis was established on renal cortical mRNAs for fibronectin and type 1 collagen ({alpha}1). The lower panel shows corresponding densitometer readings corrected for 28S and relative to that of control. Values are the mean±SEM of three experiments. *P<0.05 PTX-treated vs vehicle-treated nephritic rats; +P<0.05 vehicle-treated nephritic rats vs normal control rats.

 
Effect of PTX treatment on accumulation of macrophages and T cells
After induction of anti-GBM nephritis, vehicle-treated rats developed marked infiltration of ED1-positive macrophages and CD8-positive T cells in the glomeruli and the interstitium of the kidney. PTX-treated nephritic rats, in contrast, had significantly less glomerular and interstitial macrophage and T-cell accumulation than the vehicle-treated group (P<0.05, Figure 6A and B). These beneficial effects of PTX could also be seen in rats with the established form of nephritis (Figure 6C).



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Fig. 6. Effects of PTX on macrophage and T-cell infiltration. (A) Left panels: quantitation of ED1-positive (upper) and CD8-positive (lower) cells in the glomeruli. Right panels: quantitation of ED1-positive (upper) and CD8-positive (lower) cells in the interstitium from rats with anti-GBM nephritis that were treated with PBS (vehicle, filled bars) or PTX (open bars) immediately after the induction of nephritis. (B) Immunohistochemistry showing ED1-positive or CD8-positive cells in the glomeruli and interstitium of nephritic kidneys at day 28 with or without PTX treatment (original magnification x 100). (C) Effects of PTX administered after the nephritis was established on glomerular and interstitial ED1- or CD8-positive cells at day 28. Each bar represents the mean±SEM for a group of six rats. *P<0.05 compared with vehicle-treated nephritic rats. GCS = glomerular cross-section.

 
Effect of PTX treatment on renal TNF-{alpha}, IL-1ß, ICAM-1, MCP-1, RANTES and OPN mRNA expression
We next investigated the potential mechanisms whereby PTX suppressed rat crescentic glomerulonephritis. TNF-{alpha} and IL-1ß, both key proinflammatory cytokines in anti-GBM glomerulonephritis, and a group of chemotactic and adhesion molecules including ICAM-1, MCP-1, RANTES and OPN, were examined. In vehicle-treated nephritic rats, there was a substantial increase in renal mRNA expression of TNF-{alpha}, IL-1ß, ICAM-1, MCP-1, RANTES and OPN. Treatment with PTX significantly reduced upregulation of all inflammatory genes examined (P<0.05) except IL-1ß (Figure 7A and B). Furthermore, PTX was capable of attenuating renal cortical mRNAs for ICAM-1, MCP-1 and OPN even when administered after the establishment of nephritis (Figure 7C).



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Fig. 7. Effects of PTX on inflammatory gene expression. (A) Representative RT–PCR showing the effects of PTX on renal cortical mRNA levels of TNF-{alpha} and IL-1ß. Messages for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) are shown in the lower graph at each time point. (B) Representative northern blots demonstrating the effects of PTX on renal cortical mRNA levels of ICAM-1, MCP-1, RANTES and OPN. Messages for GAPDH are shown in the lower graph at each time point. Right panels show the corresponding densitometer readings corrected for GAPDH and relative to that of control. Open circles, normal control rats; filled squares, vehicle-treated nephritic rats; open squares, PTX-treated nephritic rats. (C) Representative northern blots at day 28 of the nephritis showing the effects of PTX administered after the nephritis was established on renal cortical mRNAs for ICAM-1, MCP-1 and OPN. The lower panel shows the corresponding densitometer readings corrected for 18S and relative to that of control. Values are the mean±SEM of three experiments. *P<0.05 PTX-treated vs vehicle-treated nephritic rats; +P<0.05 vehicle-treated nephritic rats vs normal control rats.

 
To demonstrate further the anti-inflammatory effect of PTX on rat crescentic glomerulonephritis, we determined the TNF-{alpha} levels within the urine and serum by ELISA. Results demonstrated that compared with vehicle, PTX treatment significantly reduced urinary TNF-{alpha} levels, corrected by creatinine clearance rates, throughout the entire disease course (P<0.05, Figure 8). Interestingly, the serum levels of TNF-{alpha} were not different between the two groups, indicating that PTX treatment may primarily suppress the local immune and inflammatory response within the diseased kidney.



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Fig. 8. Effects of PTX on urinary and serum TNF-{alpha} levels. (A) Urinary TNF-{alpha} levels corrected by creatinine clearance rate (pg/ml/ml/min), and (B) serum TNF-{alpha} levels (pg/ml) in rats with anti-GBM nephritis that were treated with PBS (vehicle, filled bars) or PTX (open bars) immediately after the induction of nephritis. Each bar represents the mean±SEM for a group of six rats. *P<0.05 compared with vehicle-treated nephritic rats.

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study demonstrates that PTX, when administered at the start of the disease, is an effective anti-inflammatory and immunomodulatory agent capable of reducing proteinuria, suppressing macrophage and T-cell infiltration, and ameliorating histological damage including glomerular crescent formation and cortical matrix gene expression in a rat model of experimental crescentic glomerulonephritis. These beneficial effects of PTX were not due to non-specific blockade of glomerular binding of anti-GBM antibodies, and were still evident, though to a lesser extent, in rats that were treated after the nephritis was established. Our results indicate that inhibition of the upregulated proinflammatory cytokine TNF-{alpha}, and a variety of chemokines and adhesion molecules such as MCP-1, RANTES, ICAM-1 and OPN, may be a key mechanism whereby PTX suppresses rat crescentic glomerulonephritis.

Crescentic glomerulonephritis in the early phase is characterized by activation of an array of cytokines, chemokines and adhesion molecules, in which TNF-{alpha} is thought to play a crucial role [1,2,7]. PTX is a methylxanthine phosphodiesterase inhibitor that may elevate intracellular cAMP [8]. It has been reported that cAMP is capable of inhibiting TNF-{alpha} production in different cell types [21]. Consistent with this finding, studies with cultured monocytes reveal that PTX and a selective type IV phosphodiesterase inhibitor, rolipram, also inhibit lipopolysaccharide-induced increases in TNF-{alpha} [9,22]. In addition, PTX treatment has been shown to reduce the production of TNF-{alpha} in several animal models including murine lupus nephritis [15], rabbit ischaemia/reperfusion renal injury [16] and mouse cisplatin nephrotoxicity [17]. In the present study, we further demonstrated that inhibition of intrarenal TNF-{alpha} production could be a central mechanism for the suppressive effect of PTX on rat crescentic glomerulonephritis. This finding is consistent with previous reports that PTX lowers urinary excretion of TNF-{alpha} and reduce proteinuria in human diabetic nephropathy and membranous glomerulonephritis [18,19].

TNF-{alpha} may contribute to renal damage by inciting an inflammatory response within the kidney via induction of a variety of chemokines and adhesion molecules [1,2]. The present study shows that PTX is capable of suppressing upregulated mRNAs for ICAM-1, MCP-1, RANTES and OPN in nephritic kidneys. Parallel to these findings, we have previously reported that PTX suppresses mRNA expression for ICAM-1 and MCP-1 in rat anti-Thy1 disease and remnant kidney model [12,14]. In vitro, there are reports showing that PTX inhibits ICAM-1 expression in monocytes [10], and downregulates MCP-1 induction in peripheral blood mononuclear cells and renal proximal tubular cells [11,12]. Because the pathogenic role of these chemotactic/adhesion molecules in crescentic glomerulonephritis is well documented [35], we surmise that downregulation of their gene expression by PTX may account for the significant inhibition of macrophage and T-cell infiltration within the nephritic kidneys, and thereby explain at least partially the beneficial effects of PTX in this disease.

It should be pointed out that PTX treatment could not completely inhibit the development of rat crescentic glomerulonephritis. This may be attributed, at least in part, to the presence of TNF-{alpha}-independent pathways in the pathogenesis of crescentic glomerulonephritis. Indeed, the present study demonstrated that mRNA for IL-1ß, another potent proinflammatory cytokine causally associated with crescentic glomerulonephritis [6], is elevated in nephritic kidneys. However, PTX treatment was unable to inhibit the upregulated IL-1ß gene expression, although it substantially inhibited TNF-{alpha} mRNA expression. Consistent with this finding, several in vitro and in vivo reports have also shown that cAMP-elevating agents, including PTX and other more selective phosphodiesterase inhibitors, do not affect IL-1ß mRNA or protein expression despite their ability to suppress TNF-{alpha} production [23,24]. The mechanism whereby PTX differentially inhibits TNF-{alpha} and IL-1ß awaits further studies.

In summary, this study demonstrates that PTX suppresses intrarenal TNF-{alpha} production and attenuates glomerular and tubulointerstitial inflammation in a rat model of crescentic glomerulonephritis. Given the complexity and redundancy of the inflammatory mediators involved in this disease, a clinically available anti-TNF-{alpha} agent such as PTX that inhibits the activation of a variety of chemotactic and adhesion molecules within the kidney may have therapeutic implications for rapidly progressive glomerulonephritis in humans.



   Acknowledgments
 
This work was supported by grants from the National Science Council, 89-2314-B-002-511 (to Y.-M.C.), the Ta-Tung Kidney Foundation and the Mrs Hsiu-Chin Lee Kidney Research Fund, Taipei, Taiwan.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
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Received for publication: 8. 5.03
Accepted in revised form: 17.12.03