 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pathophysiology of Renal Disease and Progression |
Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Address correspondence to: Dr. Kenichi Shikata, Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, Japan 700-8558. Phone: +81-86-235-7235; Fax: +81-86-222-5214; shikata{at}md.okayama-u.ac.jp
Received for publication November 28, 2004. Accepted for publication August 1, 2005.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and type IV collagen, and intercellular adhesion molecule-1 in glomeruli. MTX also reduced the high glucose-induced NF-
B activation in vitro and in vivo. The results indicate that intermittent administration of MTX prevented renal injuries without changes in blood glucose level and BP in experimental diabetic rats. The protective effects of MTX are suggested to be mediated by its anti-inflammatory actions through inhibition of NF-B activation and consequent reduction of intercellular adhesion molecule-1 expression and macrophage infiltration. The results suggest that anti-inflammatory agents might be beneficial for the treatment of diabetic nephropathy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and overproduction of ECM by mesangial cells (2); acceleration of the polyol pathway; oxidative stress; and hemodynamic changes. In addition to these factors, the emerging roles of inflammatory mechanisms, such as overexpression of cell adhesion molecules and chemokines that induce leukocyte infiltration, are recognized in diabetic nephropathy.
Infiltration of macrophages in the glomeruli and interstitium is one of the characteristic features of diabetic nephropathy, in addition to expansion of glomerular ECM and interstitial fibrosis (3,4). Leukocyte influx begins with leukocyte rolling and firm attachment to the endothelium, followed by transmigration across the endothelial surface into inflammatory or atherosclerotic lesions. Intercellular adhesion molecule-1 (ICAM-1), a cell-surface protein with five Ig-like domains, is one of the major molecules involved in the process of leukocyte firm attachment on vascular endothelium by the binding to 2 leukocyte integrins (5). We previously described overexpression of leukocyte adhesion molecules, including ICAM-1, and infiltration of macrophages in both the glomeruli and interstitium of patients with diabetic nephropathy (6). We further demonstrated that upregulated expression of ICAM-1 on glomerular endothelial cells plays a critical role in the infiltration of macrophages into glomeruli of streptozotocin (STZ)-induced diabetic rats at an early stage (1 to 2 wk) (7). Moreover, we showed recently that ICAM-1–deficient mice are protected from renal injuries after induction of diabetes, indicating that ICAM-1 plays a critical role in the progression of diabetic nephropathy (8).
Methotrexate (MTX) is a folic acid antagonist on the basis of its competitive binding to dihydrofolate reductase, an enzyme involved in de novo synthetic pathways for purine and pyrimidine precursors of DNA and RNA required for cell proliferation (9). Therefore, MTX has been used extensively for treatment of neoplastic diseases in doses of 20 to 250 mg/kg. Low-dose MTX, which is administered in a weekly dose of 0.1 to 0.3 mg/kg, is among the most widely used treatments for graft-versus-host diseases (10), inflammatory diseases such as rheumatoid arthritis (11), psoriasis (12), bullous pemphigoid (13), and Crohn’s disease (14). Recently, it was reported that treatment with low-dose MTX reduces the cardiovascular mortality in patients with rheumatoid arthritis (15), suggesting that MTX exerts anti-atherosclerotic effects via its anti-inflammatory actions.
In this study, we evaluated the effects of MTX on experimental diabetic nephropathy to clarify the role of inflammatory mechanisms in the development of diabetic nephropathy. We further investigated the mechanisms of anti-inflammatory effects of MTX using human umbilical vein endothelial cells (HUVEC).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental Design
Sprague-Dawley rats were randomly divided into five groups of 12 rats each: (1) Nondiabetic control rats, (2) STZ-induced diabetic rats that received vehicle alone (DM), (3) STZ-induced diabetic rats that were treated with prophylactic MTX at a dose of 1.0 mg/kg per wk starting the day before the STZ injection (DM+pre1.0MTX), (4) STZ-induced diabetic rats that were treated with MTX at a dose of 0.5 mg/kg per wk commencing 1 wk after STZ injection (DM+0.5MTX), and (5) STZ-induced diabetic rats that were treated with MTX at a dose of 1.0 mg/kg per wk starting 1 wk after STZ injection (DM+1.0MTX). The effective concentration of MTX was decided on the basis of previously published data (16,17). Diabetes was induced by a single intravenous injection of 65 mg/kg STZ (Sigma, St. Louis, MO) in citrate buffer (pH 4.5) at the age of 5 wk, as described previously (18). Nondiabetic control rats received an injection of citrate buffer alone. MTX (Sigma) was dissolved in PBS, and animals in the MTX treatment groups were treated weekly with MTX intraperitoneal injections at a dose of 0.5 or 1.0 mg/kg. Other groups received PBS alone in the same schedule as the MTX treatment groups. At 8 wk after induction of diabetes, the rats were killed under ether anesthesia, and both kidneys were harvested and weighed. All procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (No. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6).
Metabolic Data
At 1, 4, and 8 wk after induction of diabetes, systolic BP (SBP) was measured by tail-cuff plethysmography (BP-98A; Softron, Tokyo, Japan) in conscious prewarmed rats, and then blood samples were obtained from the tail vein, and each rat was placed in an individual metabolic cage to collect urine over 24 h. Hemoglobin A1c (HbA1c) was measured by HPLC; serum and urine creatinine (Cr) and glutamic pyruvic transaminase (GPT) were measured using enzymatic methods. The urinary albumin concentration in a 24-h urine collection was measured by nephelometry using anti-rat albumin antibody (ICN Pharmaceuticals, Aurora, OH). Creatinine clearance (Ccr; µl/min per 100 g body wt) was calculated as urinary Cr x urine volume/serum Cr/body weight. We measured the plasma concentration and urinary excretion of MTX at 1, 6, 12, and 24 h after intraperitoneal injection of MTX at a dose of 1.0 mg/kg in nondiabetic Sprague-Dawley rats (n = 6).
Kidney Morphology
The renal tissue was fixed in 10% formalin and embedded in paraffin. Paraffin sections (4 µm thick) then were stained with periodic acid-methenamine. For evaluating glomerular size and mesangial matrix area, 30 glomeruli per animal were examined. The glomerular tuft area was measured by manually tracing the glomerular tuft using Photoshop software version 6 (Adobe systems, San Jose, CA) and Scion Image analysis software (Scion Corp., Frederick, MD). The mesangial matrix area was defined as the periodic acid-methenamine–positive area within the tuft area. The mesangial matrix index represented the ratio of mesangial matrix area divided by the tuft area, as described previously (8).
Immunoperoxidase Staining for Macrophages
Infiltration of macrophages was evaluated by immunoperoxidase staining using the ABC kit (Vector Laboratories, Burlingame, CA) as described previously (7). In brief, the frozen sections (4 µm thick) were fixed with cold acetone for 3 min, and nonspecific protein binding was blocked by incubation with normal goat serum for 30 min. The sections first were incubated with a mouse mAb against rat monocyte/macrophage (ED1; Serotec, Oxford, UK) for 12 h at 4°C. Sections then were incubated with biotin-labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 min at room temperature. Endogenous peroxidase activity was blocked by incubating the sections in methanol that contained 0.3% H2O2 for 30 min. The sections then were incubated with ABC reagent that contained avidin and biotinylated horseradish peroxidase for 30 min at room temperature. Peroxidase activity was developed in 3,3-diaminobenzidine and H2O2. The sections then were counterstained with Mayer’s hematoxylin. The average number of ED1-positive cells per glomerulus was calculated by counting the cells in 30 glomeruli per animal.
Immunofluorescence Staining for ICAM-1
ICAM-1 expression was detected by indirect immunofluorescence as described previously (7). In brief, the frozen sections (4 µm thick) were fixed with cold acetone for 3 min and incubated with mouse anti-rat ICAM-1 (CD54) mAb (Seikagaku Co., Tokyo, Japan) for 24 h at 4°C. For identifying the ICAM-1–positive cells in the glomeruli, serial sections were prepared and incubated with mouse anti-rat RECA-1 mAb (Serotec) for endothelial cell maker. The sections then were incubated with FITC-conjugated goat anti-mouse IgG Ab (Jackson Immunoresearch Laboratories) for 30 min at room temperature and observed using a laser-scanning confocal microscope (LSM; LSM-510, Carl Zeiss, Jena, Germany), and digitized images were obtained by LSM software version 5.0. Quantification of ICAM-1 immunofluorescence intensity was calculated by the modified method as described previously (19). In brief, color images were obtained as TIFF target image files by LSM-510. The brightness of each image file was uniformly enhanced by Photoshop software version 6, followed by analysis using Scion Image analysis software. TIFF target image files were inverted and opened in gray scale mode. The ICAM-1 index was calculated using the formula, [positive area mean density x positive area (µm2)/glomerular total aria (µm2)], where the staining density is indicated by a number from 0 to 256 in gray scale. Thirty glomeruli were evaluated per animal.
Quantitative Real-Time PCR
Total RNA was extracted from the renal cortex using an RNeasy Midi kit (Qiagen, Valencia, CA). Single-strand complementary DNA (cDNA) was synthesized from the extracted RNA using a reverse transcription–PCR (RT-PCR) kit (Perkin Elmer, Foster City, CA) according to the instructions provided by the manufacturer. To evaluate mRNA expression of TGF-1, collagen IV
1, and -actin in renal cortex, we performed quantitative real-time RT-PCR using a Light Cycler and LightCycler-FastStart SYBR Green 1 (Roche Diagnostics, Tokyo, Japan). After the addition of specific forward and reverse primers (0.5 µM), MgCl2 (3 mM), and template DNA to the master mix, 40 cycles of denaturation (95°C for 10 s), annealing (60°C for 15 s), and extension (72°C for 10 s) were performed. To determine the specificity of each primer set, we performed melting curve analysis after the completion of PCR amplification. Accumulated levels of fluorescence were analyzed by the fit-point method after the melting curve analysis. TGF-1 and collagen IV1 mRNA levels were normalized with a housekeeping gene (-actin) in each sample and calculated as relative expression ratio. The following oligonucleotide primers specific for rat TGF-1 (GenBank accession no. X52498), rat collagen IV1, and rat -actin (GenBank accession no. NM_031144) were used: TGF-1 5'-GCAACAACGCAATCTATGAC-3' (forward) and 5'-CCTGTATTCCGTCTCCTT-3' (reverse), collagen IV1 5'-TCCGGGAGAGATTGGTTT-3' (forward) and 5'-CACCTTTGAGTCGCATGT-3' (reverse), and -actin 5'-CCTGTATGCCTCTGGTCGTA-3' (forward) and 5'-CCATCTCTTGCTCGAAGTCT-3' (reverse). Each experiment was performed twice.
Isolation of Glomeruli for Western Blot Analysis
Glomeruli were isolated by a fractional sieving method as described previously (20). The yield and purity of glomeruli were comparable (purity >90%). Glomeruli were isolated and centrifuged in PBS. The pellet was homogenated and resuspended in 1.5 ml of ice-cold cell lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.2 mM Vanadate, 1 mM PMSF, 1% Triton X-100, and 0.5% Nonidet P-40) for 60 min. After centrifugation, the protein concentration was determined with a DC protein assay kit (Bio-Rad, Hercules, CA). Immunoblotting was fundamentally performed as described previously (21). Briefly, samples that contained 10 µg of protein were boiled for 3 min and then subjected to 12.5% SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride membranes by electrotransfer. The transferred membranes were subsequently blocked with 5% BSA in Tris-buffered saline (TBS) that contained 0.1% Tween-20 at room temperature for 60 min. The membranes were incubated with mouse anti-rat ICAM-1 mAb (R&D Systems, Minneapolis, MN) or rabbit anti-human TGF-1,2,3 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-human type IV collagen polyclonal Ab (Santa Cruz, CA) at 4°C overnight. After washing three times for 10 min with TBS that contained 0.1% Tween-20, the membranes were incubated with horseradish peroxidase–linked anti-mouse or anti-rabbit IgG secondary antibodies (Amersham Biosciences, Piscataway, NJ) at room temperature for 1 h. The blots then were visualized with the enhanced chemiluminescence Western blot detection system (Amersham Biosciences). The amount of ICAM-1, TGF-1, and type IV collagen were analyzed using Image QuaNT analysis software version 4.2-J (Molecular Dymamics, Sunnyvale, CA).
Preparation of Nuclear Extracts of Renal Cortex
A nuclear extract was prepared from renal cortex as described previously (19). In brief, frozen renal cortex was minced and suspended in 1 ml of ice-cold TBS buffer (25 mM Tris-HCl [pH 7.4], 130 mM NaCl, and 5 mM KCl) and homogenized. The homogenate was centrifuged at 7000 x g for 2 min at 4°C, the pellet was lysed in 1 ml of ice-cold buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA [pH 7.5], 0.1 mM EGTA [pH 7.5], 1 mM PMSF, and 1 mM dithiothreitol [DTT]) and incubated on ice for 20 min. Next, 100 µl of 10% Nonidet P-40 was added and vigorously vortexed, and the extract was centrifuged at 12,000 x g for 7 min at 4°C. The nuclei then were extracted with 100 µl of ice-cold buffer C (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA [pH 7.5], 1 mM EGTA [pH 7.5], 1 mM PMSF, and 1 mM DTT), incubated on ice for 2 h, and centrifuged at 12,000 x g for 7 min at 4°C. The supernatant fraction was stored at –80°C.
Electrophoretic Mobility Shift Assay
Nuclear extract was prepared from renal cortex as described above. For electrophoretic mobility shift assay, the following oligonucleotide with the NF-B consensus binding sequence was used: 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Santa Cruz Biotechnology). A mutant motif with G to C substitution (5'-AGTTGAGGCGACTTTCCCAGGC-3'; Santa Cruz Biotechnology) served as a control. The consensus oligonucleotide was labeled with [
-32P]ATP (Amersham Biosciences) using T4 poly-nucleotide kinase (Promega Corp., Madison, WI). The binding reaction was performed with 45 µg of nuclear extract in a binding buffer that contained 20 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl2, 4% glycerol, and 2 µg of poly(dl-dc) for 20 min at room temperature, and 1 ng of 32P-labeled probe was added in a final volume of 30 µl. Reaction mixture was incubated for 20 min at room temperature. Individual samples were loaded on a 4% polyacrylamide gel and were electrophoresed for 90 min at 150 V. After electrophoresis, the gel was dried and exposed to Fuji Imaging Plate (Fujifilm, Tokyo, Japan) for 60 min, and the autoradiogram was read using FUJIX BAS-2000 (Fujifilm) and amount of NF-B was analyzed using Image QuaNT analysis software version 4.2-J. For the competition assay, 100-fold excess unlabeled NF-B oligonucleotide was incubated with reaction mixture for 20 min before the addition of radiolabeled NF-B oligonucleotide. Each experiment was performed three times.
Cell Culture
HUVEC were purchased from Clonetics (San Diego, CA) and cultured in EBM-2 medium that contained 5.5 mM d-glucose and 5% FBS with 1 x 10–2 ng/ml hEGF and 1 µg/ml hydrocortisone to gelatin precoated T-75 flasks. This is referred to as HUVEC medium. The cultures were maintained at 37°C in 5% CO2 and 95% air. For experiments, they were trypsinized and passaged to gelatin-precoated 100-mm dishes. All HUVEC were used throughout passages 2 to 4.
Effect of MTX and Lactacystin for HG-Induced ICAM-1 Expression on HUVEC
To clarify the relationship with ICAM-1 expression and NF-B activation, we used a transcription inhibitor, the proteasome inhibitor lactacystin (Calbiochem, San Diego, CA). To evaluate the hyperglycemic effects, we used the agents of d-glucose (Sigma, St. Louis, MO). Confluent cultures of HUVEC in 100-mm dishes were preincubated with or without 10 mM MTX for 2 h or 10 mM lactacystin for 1 h. The effective concentrations of MTX and lactacystin were decided on the basis of published data (22–24) and our own preliminary study. After preincubation of MTX or lactacystin, they were added to 24.5 mM d-glucose (high glucose [HG]) and incubated for 24 h. After incubation, cells were collected using a cell scraper and were lysed in TNE Buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, and 20 mM EDTA) for 30 min on ice. Western immunoblotting was performed as described above. Briefly, the transferred membranes were incubated with mouse anti-human ICAM-1 mAb (Santa Cruz Biotechnology) at 4°C overnight. After washing three times for 10 min with TBS that contained 0.1% Tween-20, the membranes were incubated with horseradish peroxidase–linked anti-mouse IgG secondary antibodies at room temperature for 1 h. The blots then were visualized and analyzed as described above.
Effect of MTX for HG-Induced NF-B Activation on HUVEC
Confluent cultures of HUVEC in 100-mm dishes were preincubated with or without 10 mM MTX for 2 h and added to 24.5 mM d-glucose (HG). After incubation for 24 h, nuclear and cytosolic extracts were prepared as described previously (25). Briefly, cells were collected using a cell scraper and were lysed in 300 µl of buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, and 19 mM KCl) and 0.1% Nonidet P-40 on ice for 10 min and then centrifuged at 600 x g for 3 min. The supernatant was saved as cytosolic extract. The nuclear pellet then was washed in 1 ml of buffer A at 4200 x g for 3 min, resuspended in 30 µl of buffer C (20 mM HEPES ([pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA), incubated on ice for 30 min, and centrifuged at 14,300 x g for 20 min at 4°C. The supernatant was used as nuclear extract. The cytosolic and nuclear extracts then were analyzed for protein concentration using a DC protein assay kit. Western immunoblotting was performed as described above. Briefly, the transferred membranes were incubated with mouse anti-human NF-B p65 mAb (Santa Cruz Biotechnology) at 4°C overnight. After washing three times for 10 min with TBS that contained 0.1% Tween-20, the membranes were incubated with horseradish peroxidase–linked anti-mouse IgG secondary antibody at room temperature for 1 h. The blots then were visualized and analyzed as described above.
Statistical Analyses
All values are expressed as mean ± SEM. Differences between groups were examined for statistical significance using one-way ANOVA followed by Scheffe test. P < 0.05 was considered a statistically significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Urinary albumin excretion levels at each time point in each group are shown in Figure 1. In the DM group, albuminuria gradually increased during the study. There were no statistically significant changes in albuminuria in the control group at any point. At 8 wk after induction of diabetes, urinary albumin excretion was significantly increased in the DM group compared with control group and was significantly reduced in the MTX treatment groups in a dose-dependent manner compared with the DM group. Urinary albumin excretion at 8 wk after induction of diabetes were as follows: Control 221.4 ± 55.0, DM 1123.7 ± 201.6, DM+0.5MTX 629.4 ± 98.0, DM+1.0MTX 386.2 ± 59.9, DM+pre1.0MTX 377.6 ± 39.1 µg/d.
|
Kidney Morphology
Mesangial matrix index was significantly increased in the DM group (Figure 2B) compared with the control group (Figure 2A), and significantly reduced in MTX treatment group (Figure 2, C through F), Mesangial matrix indexes were as follows: Control 8.3 ± 0.10, DM 12.9 ± 0.17, DM+0.5MTX 12.1 ± 0.20, DM+1.0MTX 10.1 ± 0.15, DM+pre1.0MTX 10.5 ± 0.15%. Mean value of glomerular surface area was higher in the DM group than in the control group, although there was no statistically significant difference in all groups (control 9156.7 ± 109.1, DM 9312.9 ± 115.5, DM+0.5MTX 8940.7 ± 155.4, DM+1.0MTX 9347.5 ± 152.7, DM+pre1.0MTX 9002.8 ± 98.0 µm2).
|
|
|
|
and Type IV Collagen mRNA in Renal Cortex1 and collagen IV1 mRNA levels in renal cortex. TGF-1 mRNA expression was significantly increased in the DM group compared with the control group and reduced in the DM+pre1.0MTX group (Figure 6A). The relative expression levels of TGF-1 mRNA were as follows: Control 100.0 ± 23.7, DM 391.2 ± 47.4, DM+pre1.0MTX 238.5 ± 45.9% of control. Collagen IV1 mRNA expression was significantly increased in the DM group compared with the control group and reduced in the DM+pre1.0MTX group (Figure 6B). The relative expression levels of collagen IV1 mRNA were as follows: Control 100.0 ± 29.0, DM 245.8 ± 32.5, DM+pre1.0MTX 105.6 ± 16.0% of control.
|
, and Type IV Collagen in Glomeruli, and type IV collagen in glomeruli, we used Western blot analysis. The protein levels were significantly increased in the DM group compared with the control group and reduced in the DM+pre1.0MTX group (Figure 7). The amounts of these proteins were as follows: ICAM-1: control 100.0, DM 288.9 ± 34.7, DM+pre1.0MTX 146.6 ± 7.5% of control; TGF-: control 100.0, DM 167.0 ± 17.0, DM+pre1.0MTX 111.6 ± 6.1% of control; type IV collagen: control 100.0, DM 221.5 ± 11.5, DM+pre1.0MTX 139.9 ± 12.5% of control.
|
B Activation in Renal CortexB DNA binding activity in the renal cortex by electrophoretic mobility shift assay. As presented in Figure 6C, NF-B activation of the renal cortex was increased in the DM group (Figure 6C, lane 2) compared with the control group (Figure 6C, lane 1) and diminished in the DM+pre1.0MTX group (Figure 6C, lane 3). To ensure that the mobility shift bands under these experimental conditions were the NF-B/oligonucleotide complex, we used an NF-B mutant oligonucleotide with G to C substitution in the NF-B binding motif as a control. Addition of this unlabeled NF-B mutant oligonucleotide (100-fold excess) did not affect the bands (Figure 6C, lane 5) that were associated with the labeled NF-B oligonucleotide probe, whereas the unlabeled consensus NF-B oligonucleotide (100-fold excess) completely abolished these bands (Figure 6C, lane 4). The NF-B level of nuclear extracts in renal cortex are shown in Figure 6D and were as follows: Control 100, DM 213.1 ± 22.6, DM+pre1.0MTX 147.2 ± 8.1% of control.
Effect of MTX and Lactacystin for HG-Induced ICAM-1 Expression on HUVEC
We evaluated the effects of MTX and lactacystin for HG-induced ICAM-1 expression on HUVEC by Western blot analysis. ICAM-1 expression was significantly increased in HG compared with 5.5 mM normal glucose (Control). HG-induced ICAM-1 expression was significantly inhibited by MTX and lactacystin (Figure 8, A and B). The amount of ICAM-1 were as follow: Control 100, HG 356.4 ± 43.0, HG+MTX 138.1 ± 18.9, HG+lactacystin 130.9 ± 11.5% of control.
|
B Activation on HUVECB. To this purpose, we performed a Western blot analysis for NF-B p65 protein in the cytosolic and nuclear fractions of HUVEC. The results show that p65 protein of cytosolic extracts was significantly decreased in HG culture compared with normal glucose. In contrast, p65 protein of nuclear extracts was significantly increased in HG culture compared with normal glucose. The effect of HG on the translocation of NF-B was significantly inhibited by treatment of the cells with MTX (Figure 8, C through F). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is thought to play a central role in the enhancement of glomerular ECM production in diabetic nephropathy (27,28). In this study, gene expression and protein level of TGF- and type IV collagen were reduced by MTX treatment on renal cortex and glomeruli of diabetic rats.
Recently, several studies described the involvement of macrophages in the pathogenesis of diabetic nephropathy. Sassy-Prigent et al. (29) reported that the depletion of leukocytes by irradiation decreased the gene expression of TGF- and type IV collagen in the glomeruli of diabetic rats at 4 wk after induction of diabetes. We have demonstrated that renal infiltration of macrophages is prevented in ICAM-1–deficient mice after induction of diabetes, resulting in decrease in albuminuria, glomerular hypertrophy, mesangial expansion, and glomerular expression of TGF- and type IV collagen (8). Mesangial matrix proteins, including type IV collagen, are produced by mesangial cells. With respect to the interaction between macrophages and mesangial cells, it was reported that culture supernatant of macrophages stimulated mesangial cells to produce fibronectin in vitro (30). In line with these notions described above, we can speculate that infiltrated macrophages stimulate mesangial cells to secrete TGF-, which induces production of ECM proteins in an autocrine or paracrine manner. However, macrophages can secrete TGF-, which can stimulate mesangial cells to produce ECM proteins (31). Thus, macrophages could stimulate mesangial cells to produce mesangial matrix directly and/or indirectly via TGF- in the development of diabetic nephropathy.
Low-dose MTX treatment is widely used for inflammatory diseases such as rheumatoid arthritis, psoriasis, bullous pemphigoid, and Crohn’s disease. However, the adverse effects of MTX, including hepatotoxicity and nephrotoxicity, must be considered. In this study, MTX treatment did not induce hepatotoxicity and nephrotoxicity. We measured plasma concentrations and urinary excretion of MTX after intraperitoneal injection to Sprague-Dawley rats. The plasma concentration of MTX increased within 1 h after intraperitoneal injection and rapidly decreased. Approximately 55% of MTX was excreted into urine within 24 h. These data of pharmacokinetics of MTX were similar with those of low-dose MTX treatment in human (32). The adverse effect of MTX was not detected in this study; however, we need to consider them because several adverse effects, including liver injury, were reported in chronic use of MTX in human.
It was reported that long-term low-dose MTX treatment may increase concentrations of homocysteine (33), which may promote atherosclerosis and thrombosis (34). However, Choi et al. (15) reported that treatment with low-dose MTX reduces cardiovascular mortality in the patients with rheumatoid arthritis. Several similarities have emerged between the inflammatory mechanisms in the pathogenesis of atherosclerosis (35) and diabetic nephropathy. As we described previously, ICAM-1–dependent infiltration of macrophages plays a critical role in the development of diabetic nephropathy (8).
Several studies have reported the inhibitory effect on ICAM-1 expression by MTX (13,24,36,37), although the mechanisms of the inhibition of ICAM-1 by MTX are unclear. Expression of ICAM-1 can be induced by a variety of factors, including inflammatory cytokines, reactive oxygen species, and shear stress (7,38). In addition, NF-B is one of the most important transcription factors regulating ICAM-1 expression (39). NF-B is composed of proteins with molecular masses of 50 kD (p50) and 65 kD (p65) and is retained in the cytoplasm by an inhibitory subunit, IB. In its unstimulated form, NF-B is activated by a wide variety of inflammatory stimuli and is induced by the phosphorylation-dependent degradation of IB proteins via the proteasome-dependent pathway, allowing active NF-B to translocate into the nucleus and promote the transcriptional activation of target genes. The signal is eventually terminated by the new synthesis of IB (28). Recently, it was shown that MTX suppresses NF-B activation through inhibition of IB phosphorylation and degradation (40).
Expression of ICAM-1 on endothelial cells is upregulated by several stimuli, such as IL-1, TNF- (24,41), and lysophospholipids (42). In addition, recent studies showed that ICAM-1 expression on HUVEC is increased by HG and high osmotic condition (43,44). It was also shown that increased ICAM-1 expression can be caused by plasma hyperosmolarity (43) and hyperglycemia (45) in the endothelial cells of diabetic rat glomeruli. MTX has been reported to reduce TNF-–induced ICAM-1 expression on HUVEC (24); however, the effect of MTX for HG-induced ICAM-1 expression on HUVEC has remained unclear.
In this study, we demonstrated the inhibitory effects of MTX against HG-induced ICAM-1 expression on HUVEC. Moreover, to clarify the mechanism of inhibitory effects of MTX, we performed Western blot analysis for NF-B p65 protein in the cytosolic and nuclear fractions of HUVEC. As a result, MTX inhibited the HG-induced translocation of NF-B p65 into nucleus in HUVEC. Lactacystin is a proteasome inhibitor that inactivates NF-B and reduces TNF-–induced ICAM-1 expression on HUVEC (22,23). In this study, lactacystin also reduced HG-induced ICAM-1 expression on HUVEC. These results suggest that MTX inhibits the expression of ICAM-1 via inactivation of NF-B in endothelial cells under HG condition. In this study, renoprotective effects of MTX may be mediated by an anti-inflammatory action via inhibition of NF-B activation, ICAM-1 expression, and macrophage infiltration, although the antiproliferative effects of MTX on macrophages and mesangial cells may also contribute to the protective effects.
Recent studies have provided evidence that treatment with mycophenolate mofetil, an antilymphocyte drug with immunosuppressive and anti-inflammatory effects aimed at limiting renal inflammation, can largely attenuate renal injury in experimental models of progressive renal failure (46) and diabetic nephropathy (47). We previously reported that a HMG-CoA reductase inhibitor prevents glomerular injury in diabetic rats via pleiotropic effects, including an anti-inflammatory action (19). In this way, there are some reports that anti-inflammatory agents are effective for diabetic nephropathy.
In summary, MTX treatment prevented glomerular injury without altering blood glucose levels or BP in experimental diabetic rats. Furthermore, MTX inhibited HG-induced ICAM-1 expression and NF-B activation in endothelial cells. The protective effects of MTX may be mediated by an anti-inflammatory action via inhibition of NF-B activation, ICAM-1 expression, and macrophage infiltration. These results suggest that anti-inflammatory agents might be beneficial for diabetic nephropathy.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  Y. Qian, E. Feldman, S. Pennathur, M. Kretzler, and F. C. Brosius III From Fibrosis to Sclerosis: Mechanisms of Glomerulosclerosis in Diabetic Nephropathy Diabetes, June 1, 2008; 57(6): 1439 - 1445. [Full Text] [PDF]
|
|
|
|
 |
|
 X. Y. Shi, F. F. Hou, H. X. Niu, G. B. Wang, D. Xie, Z. J. Guo, Z. M. Zhou, F. Yang, J. W. Tian, and X. Zhang Advanced Oxidation Protein Products Promote Inflammation in Diabetic Kidney through Activation of Renal Nicotinamide Adenine Dinucleotide Phosphate Oxidase Endocrinology, April 1, 2008; 149(4): 1829 - 1839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Ninichuk, S. Clauss, O. Kulkarni, H. Schmid, S. Segerer, E. Radomska, D. Eulberg, K. Buchner, N. Selve, S. Klussmann, et al. Late Onset of Ccl2 Blockade with the Spiegelmer mNOX-E36-3'PEG Prevents Glomerulosclerosis and Improves Glomerular Filtration Rate in db/db Mice Am. J. Pathol., March 1, 2008; 172(3): 628 - 637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Ninichuk, A. G. Khandoga, S. Segerer, P. Loetscher, A. Schlapbach, L. Revesz, R. Feifel, A. Khandoga, F. Krombach, P. J. Nelson, et al. The Role of Interstitial Macrophages in Nephropathy of Type 2 Diabetic db/db Mice Am. J. Pathol., April 1, 2007; 170(4): 1267 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ohga, K. Shikata, K. Yozai, S. Okada, D. Ogawa, H. Usui, J. Wada, Y. Shikata, and H. Makino Thiazolidinedione ameliorates renal injury in experimental diabetic rats through anti-inflammatory effects mediated by inhibition of NF-{kappa}B activation Am J Physiol Renal Physiol, April 1, 2007; 292(4): F1141 - F1150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lee, W. Kim, S.-O. Moon, M. J. Sung, D. H. Kim, K. P. Kang, K. Y. Jang, S. Y. Lee, B. H. Park, G. Y. Koh, et al. Renoprotective effect of COMP-angiopoietin-1 in db/db mice with type 2 diabetes Nephrol. Dial. Transplant., February 1, 2007; 22(2): 396 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. K. Usui, K. Shikata, M. Sasaki, S. Okada, M. Matsuda, Y. Shikata, D. Ogawa, Y. Kido, R. Nagase, K. Yozai, et al. Macrophage Scavenger Receptor-A-Deficient Mice Are Resistant Against Diabetic Nephropathy Through Amelioration of Microinflammation Diabetes, February 1, 2007; 56(2): 363 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ozawa, H. Kobori, Y. Suzaki, and L. G. Navar Sustained renal interstitial macrophage infiltration following chronic angiotensin II infusions Am J Physiol Renal Physiol, January 1, 2007; 292(1): F330 - F339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Schmid, A. Boucherot, Y. Yasuda, A. Henger, B. Brunner, F. Eichinger, A. Nitsche, E. Kiss, M. Bleich, H.-J. Grone, et al. Modular Activation of Nuclear Factor-{kappa}B Transcriptional Programs in Human Diabetic Nephropathy Diabetes, November 1, 2006; 55(11): 2993 - 3003. [Abstract] [Full Text] [PDF]
|
|
|
