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Mechanical Stretch Induces Monocyte Chemoattractant Activity via an NF-B-Dependent Monocyte Chemoattractant Protein-1-Mediated Pathway in Human Mesangial Cells: Inhibition by Rosiglitazone

Gabriella Gruden^*, Giorgia Setti^*, Anthea Hayward^*, David Sugden, Sara Duggan^*, Davina Burt^*, Robin E. Buckingham^*, Luigi Gnudi^* and Giancarlo Viberti^*

^* Department of Diabetes and Endocrinology, Cardiovascular Division, and Endocrinology and Reproduction Research Group, King’s College, London, United Kingdom

Address correspondence to: Dr. Gabriella Gruden, Department of Internal Medicine, C/so AM Dogliotti 14, Turin, Italy. Phone: 39-011-6336448; Fax: 39-011-6634751; E-mail: ggruden{at}hotmail.com

	Abstract

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Hemodynamic abnormalities are important in the pathogenesis of the glomerular damage in diabetes. Glomerular macrophage infiltration driven by the chemokine monocyte chemoattractant protein-1 (MCP-1) is an early event in diabetic nephropathy. The thiazolidinedione rosiglitazone ameliorates albumin excretion rate in diabetic patients with microalbuminuria and has anti-inflammatory properties, raising the possibility of a relationship between its renoprotective and anti-inflammatory activity. Investigated was whether mesangial cell stretching, mimicking in vitro glomerular capillary hypertension, enhances MCP-1 expression and monocyte chemoattractant activity. The effect of the combination of stretch with high glucose on MCP-1 production was studied and, finally, the effect of rosiglitazone on these processes was assessed. Stretching of human mesangial cells significantly enhanced their monocyte chemoattractant activity. This effect was mediated by MCP-1 as it was paralleled by a significant rise in both MCP-1 mRNA and protein levels and was completely abolished by MCP-1 blockade. Combined exposure to both stretch and high glucose further increased MCP-1 production. Stretch activated the IB-NF-B pathway, and NF-B inhibition, with the use of the specific inhibitor SN50, completely abolished stretch-induced MCP-1, indicating that stretch-induced MCP-1 was NF-B dependent. The addition of rosiglitazone significantly diminished stretch-induced NF-B activation, MCP-1 production, and monocyte chemotaxis. In conclusion, stretching of mesangial cells stimulates their monocyte chemoattractant activity via an NF-B-mediated, MCP-1-dependent pathway, and this effect is prevented by rosiglitazone.

	Introduction

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Glomerular hypertension is a cause of injury in diabetic and other progressive glomerulopathies (1). The molecular mechanisms that translate this mechanical insult into tissue damage are currently the object of active investigation. Increased intracapillary pressure, by expanding glomerular structures, induces mesangial cell stretching, leading to overproduction of extracellular matrix molecules and cytokines (2, 3)

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine produced by mesangial cells. In both experimental diabetes and the remnant kidney model, MCP-1 is overexpressed in the glomeruli, an event that precedes and closely correlates with macrophage infiltration (4–7). Intervention strategies that suppress macrophages recruitment have antiproteinuric and renoprotective effects, suggesting a contribution of MCP-1-driven macrophage infiltration in the pathogenesis of the glomerular damage (8, 9). In vitro, in mesangial cells, high glucose stimulates MCP-1 expression through activation of NF-B (10), but the effect of cyclic stretching on MCP-1 remains unknown

Thiazolidinediones (TZD) are insulin-sensitizing agents that ameliorate microalbuminuria in patients with type 2 diabetes (11) and have renoprotective effects in animal models of diabetes (12–15). This seems, at least in part, independent of their metabolic action as beneficial effects are also seen in nondiabetic models of glomerulosclerosis such as the spontaneous hypertensive rat and the remnant kidney model (16, 17). Of interest, TZD are also potent monocyte inhibitors (18) and prevent MCP-1 overexpression in a rat model of myocardial infarction (19), raising the possibility that their renoprotective properties are mediated by their anti-inflammatory action. TZD are agonists of the nuclear receptor peroxisome proliferator-activated receptor- (PPAR-), which is both expressed and functionally active in mesangial cells (20). Furthermore, these compounds can downregulate inflammatory responses independent of PPAR- in embryonic stem cell-derived macrophages (21).

The effect of TZD on mesangial cell MCP-1 expression and monocyte chemoattractant activity has not been investigated. Therefore, in this study, we tested in human mesangial cells whether stretch induces MCP-1 expression and whether this response is modulated by different ambient glucose concentrations. Furthermore, we investigated the signal transduction pathways involved and examined whether rosiglitazone, a potent TZD, interfered in this process

	Materials and Methods

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All materials were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. FCS was obtained from Life Technologies-BRL (Paisley, UK), and Flex I and Flex II plates were obtained from Flexcell International (McKeensport, PA). MCP-1 ELISA and anti-MCP-1 neutralizing antibody were obtained from R&D Systems (Minneapolis, MN). SN50 and SN50M were from Affiniti (Exeter, UK). The monocyte negative isolation kit and the microchemotaxis chamber were obtained from Dynal Bioteck UK (Bromborough, UK) and Neuroprobe (Oxon, UK), respectively. The reverse transcription system was purchased from Promega (Southampton, UK), and QuantiTect SYBR Green PCR was purchased from Qiagen Ldt (West-Sussex, UK). The anti-phospho-IkB- antibody was from New England Biolabs Ldt (Hertfordshire, UK), and calphostin C and herbimycin A were from Calbiochem (Nottingham, UK). BRL-49653 (rosiglitazone) was provided by GlaxoSmithKline (Worthing, UK)

Cell Culture
Human mesangial cells were isolated as described previously (22, 23). Briefly, normal renal cortex was obtained from three donor nephrectomies that were found to be unsuitable for transplantation on the basis of an abnormal vascular supply. Intact glomeruli, collected from cortical homogenates by serial sieving, were digested with type IV collagenase and then seeded in culture flasks. After the outgrowth of mesangial cells, the glomeruli were removed and the cells were cultured in RPMI-1640, supplemented with insulin-transferrin-selenium and -glutamine, and contained 20% heat-inactivated FCS, 7 mM glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO₂ incubator at 37°C. The cells were stellate or fusiform in appearance, grew in multilayers, formed hillocks in long-term culture, and stained for -smooth muscle actin by direct immunofluorescence. Cells did not stain for cytokeratin, factor VIII, common leukocyte antigen, and Thy-1 (DAKO, High Wycombe, UK), excluding contamination of other glomerular cell types. Studies were performed between passages 4 and 7, while the cells retained the characteristic morphologic features described above

Mesangial Cell Exposure to Mechanical Stretch under Various Glucose Concentrations
Mesangial cells were seeded in equal number into six-well type I collagen-coated silicon elastomer-base culture plates (Flex I plates) and control plates (Flex II plates). After insulin and serum deprivation for 24 h, cells were subjected to repeated stretch/relaxation cycles by mechanical deformation using a Flexercell Strain Unit (FX3000; Flexcell Int.). The stress unit is a modification of the unit initially described by Banes et al. (24) and consists of a vacuum unit and a base plate. A vacuum was cyclically applied (60 cycles/min) to the rubber base plates via the base plate, which was placed in a humidified incubator with 5% CO₂ at 37°C. Cells were exposed to an average 10% uniaxial elongation, which mimics that present in vivo in glomeruli exposed to supernormal pressure levels (3, 23). Control cells were grown in nondeformable but otherwise identical plates (Flex II plates). We applied a cyclical mechanical stretch on the evidence that, in the normal glomerulus, capillary pressure is pulsatile and that, in situations such as diabetes, this pulsatility may be enhanced because of defective autoregulation (25, 26). A rate of 60 cycles/min, which approximates the pulse frequency, has been used in previous studies on mesangial cells exposed to stretch (23)

A subset of experiments were performed on human mesangial cells that were exposed to high glucose concentrations for 48 h. Media that contained normal (5.5 mM) and high (25 mM) glucose concentrations were made iso-osmolar with the addition of mannitol. Stretch was applied during the last 4 h of the glucose incubation period

mRNA Analysis
Total RNA was extracted using RNeasy Minispin columns (Qiagen) and reverse transcribed (2 µg) according to standard protocols using avian myeloblastosis virus reverse transcriptase and oligod(T). Real-time PCR was carried out with the Light Cycler Instrument (Roche Diagnostics Ldt, Lewes, UK) using the DNA binding dye SYBR Green I for the detection of PCR products. PCR was set up using 8 µl of PCR master mix that contained QuantiTect SYBR Green PCR supplemented with 0.5 µM primers and 2 µl of either external standards or a cDNA equivalent to 20 ng of total RNA to give a final reaction volume of 10 µl. Prime sequences were sense 5`-GATCTCAGTGCAGAGGCTCG-3` and antisense 5`-TGCTTGTCCAGGTGGTCCAT-3`. The settings for the thermal profile were as follows: Initial denaturation (15 min at 95°C), followed by 40 amplification cycles (15 s at 94°C; 20 s at 65°C; 8 s at 72°C). Fluorescence was measured after extension of each cycle. Melting curve analysis was used to confirm generation of a single product. Expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was determined in parallel and used as reference gene. Primers sequences were sense 5`-CCCATCACCATCTTCCAGGAGC-3` and antisense 5`-CCAGTGAGCTTCCCG TTCAGC-3`. The light cycler conditions were as follows: Initial denaturation (15 min at 95°C), followed by 40 amplification cycles (15 s at 95°C; 20 s at 55°C; 24 s at 72°C). External standards were PCR products purified after gel electrophoresis (Qiaquick purification kit; Qiagen) and quantified by densitometry against a known amount of a molecular weight standard. Standards were serially diluted and stored in small aliquots at –80°C before use. Experiments were analyzed by real-time PCR in separate runs, and to account for interassay variability, data from individual experiments were transformed in percentage change over control before pooling

MCP-1 Protein Measurement
Culture supernatants from all experimental conditions were collected, centrifuged to remove cell debris, and stored at –70°C for analysis. MCP-1 protein concentration was measured by a quantitative sandwich ELISA using a mouse monoclonal and a rabbit polyclonal anti-human MCP-1 (range, 5 to 2000 pg/ml; intra- and interassay coefficients of variation, 4.9 and 4.8%, respectively). Results were corrected for cell numbers

Isolation of Human Monocytes
Total blood was collected from healthy volunteers, and peripheral blood mononuclear cells were separated using the Lymphoprep density gradient centrifugation method. Monocytes were negatively isolated from peripheral blood mononuclear cells using antibody-coated magnetic beads that selectively remove contaminant cells. Cell viability and purity were 98 and 95%, respectively, as assessed by Trypan blue exclusion test and nonspecific esterase staining

Determination of Chemotactic Activity
Chemotaxis assay was performed on freshly isolated human blood monocytes in a 48-well Micro-Chemotaxis Chamber with 5-µm pore size polycarbonate filter. Aliquots of monocyte suspension (2 x 10⁵ cells/ml) and conditioned media from both stretched and nonstretched mesangial cells were placed respectively in the upper and lower compartments of the Micro-Chemotaxis Chamber. After 2 h of incubation at 37°C, the filter was removed, and the migrated cells were fixed, stained with Diff-Quick, and counted under light microscopy. Formyl-methionyl-leucyl-phenylalanine (f-MLP 10^–7 M) and serum-free media were used in the assay as positive and negative control, respectively

Electrophoretic Mobility Shift Assay
NF-B DNA binding activity was determined by electrophoretic mobility shift assay. Nuclear proteins were extracted using the NE-PER nuclear extraction kit (Perbio Science UK Ltd., Tattenhall, UK), and protein concentration was determined using the DC Protein Assay (Bio-Rad, Hertfordshire, UK). The following double-stranded oligonucleotides that contained the consensus NF-B motif were used as probes: 5`-AGTTGAGGGGACTTTCCCAGG C-3` and 3`-TCAACTCCCCTGAAAGGGTCCG-5` (Promega, Southampton, UK). Probes were prepared by end labeling with [-³²P]ATP using T4 polynucleotide Kinase (Roche, Herts, UK) and purified on G-25 MicroSpin columns (Amersham Pharmacia Biotech, Little Chalfont, UK). Nuclear proteins (5 µg) were incubated with the labeled probes for 45 min on ice and separated through a 4.5% nondenaturing polyacrylamide gel in 0.25x Tris-borate-EDTA buffer. Gels were dried, and protein-DNA complexes were visualized by autoradiography. Specificity of the binding reaction was established through competition with a 100-fold excess of unlabeled probe and a lack of competition with an unlabeled and unrelated oligonucleotide (Oct-1). Bound proteins were identified by supershift after 30 min of preincubation of the nuclear protein with antibodies to p50 and p65 (Santa Cruz Biotechnologies, Santa Cruz, CA)

Measurement of Phospho-IB Levels
Cells were harvested in a Tris (40 mM, pH 7.6) lysis buffer that contained 150 mmol/L NaCl, 0.25% deoxycholate, 1% NP40, 10 mmol/L NaF, 2 mmol/L EGTA, 1 mmol/L PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. For determining phospho-IB levels, total protein extracts were separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. The membranes, blocked in 5% skim milk, were incubated with a rabbit anti-phospho-IB antibody, which binds to IB exclusively when phosphorylated in Ser32, and then with a horseradish peroxidase-linked secondary antibody. Detection was performed by enhanced chemiluminescence, and the band intensity was quantified by densitometry. Equal protein loading of each lane was verified with Ponceau S staining of total proteins on the nitrocellulose membranes

Inhibition Experiments
Cells were exposed to cyclical stretch in the presence and in the absence of (1) SN50 or SN50M (18 µM); (2) rosiglitazone (1 and 10 µM); (3) herbimycin A (3.4 µM); and (4) calphostin C (1µM), added to the culture media 1 to 4 h before the experiment. SN50 contains the nuclear localization sequence (NLS residues 360 to 369) of the transcription factor NF-B p50 linked to a peptide cell-permeabilization sequence, the hydrophobic region (h-region) of the signal peptide of Kaposi fibroblast growth factor. This peptide inhibits translocation of the NF-B active complex into the nucleus, and it is the most specific NF-B inhibitor available. The control peptide SN50M is identical to SN50 except for two mutations, which make it inactive (27)

Statistical Analyses
The number of experiments, which were performed either in duplicate or in triplicate, is reported in the legends. Data are expressed as fold change over control. This normalization was conducted to allow for pooling of data from separate experiments without increasing the overall variability of final results. All data are presented as mean ± SEM. A t test was used for the comparison between two groups. When more than two groups were studied, data were analyzed by ANOVA, and, if significant, the Newman-Keuls procedure was used for post hoc comparisons. Values for P < 0.05 were considered significant

	Results

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Stretch Induces MCP-1 Gene Expression and Protein Secretion in Human Mesangial Cells
Application of mechanical stretch (10% elongation) resulted in a significant increase in MCP-1 protein levels at 4 h (2.16 ± 0.27-fold increase; P < 0.001) with a return to the baseline by 33 h (Figure 1A). The rise in MCP-1 protein levels was preceded by a significant and transient increase in MCP-1 mRNA (1.70 ± 0.29-fold increase over control at 3 h; P < 0.05; Figure 1B). Light cycler gave one single product, and amplification of total RNA gave no product, excluding contamination with genomic DNA

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of mechanical stretch on monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels. Serum- and insulin-deprived human mesangial cells were exposed to mechanical stretch for the indicated time periods. MCP-1 (A) protein (n = 5; *P < 0.001 stretch at 4 h versus others) and (B) mRNA (n = 5; #P < 0.05 stretch at 3 h versus others) levels are expressed as fold increase versus control nonstretched cells (dotted line).

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effect of combined exposure to stretch and high glucose on MCP-1 production. Serum- and insulin-deprived human mesangial cells were exposed to high glucose (25 mM) for 48 h and/or mechanical stretch for 4 h. MCP-1 protein levels were measured in the supernatants as described in the Materials and Methods section and expressed as fold increase over control (n = 4); *P < 0.05 stretch + high glucose, stretch, and high glucose versus control.

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effect of mesangial cell stretching on monocyte chemoattractant activity. Serum- and insulin-deprived human mesangial cells were exposed to stretch for 4 h; conditioned media were collected and tested in a chemotaxis assay as described in the Materials and Methods section. (A) Number of migrated monocytes in the presence of conditioned media from nonstretched and stretched mesangial cells, serum-free media (Sfm), and formyl-methionyl-leucyl-phenylalanine (f-MLP; 10–7 M; n = 4). *P < 0.05 stretched versus control nonstretched cells and Sfm; **P < 0.01 f-MLP versus others. (B) Number of migrated monocytes in nonstretched and stretched mesangial cells (n = 3) in the presence and in the absence of an anti-MCP-1 neutralizing antibody. #P < 0.05 stretch versus others.

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effect of rosiglitazone on both stretch-induced MCP-1 and monocyte chemoattractant activity. Serum- and insulin-deprived human mesangial cells were exposed to stretch in the presence and in the absence of various concentrations of rosiglitazone (RSG) for 4 h. (A) MCP-1 protein levels in the supernatants is expressed as fold increase over controls (n = 3). *P < 0.01 stretch versus nonstretch, nonstretch + RSG 10 µM, stretch + RSG (1 µM), stretch + RSG (10 µM). (B) Monocyte chemoattractant activity of conditioned media tested in a chemotaxis assay and expressed as fold increase over control (n = 3). *P < 0.05 stretch versus nonstretch, nonstretch + RSG 10 µM, stretch + RSG (1 µM), stretch + RSG (10 µM).

NF-B Mediates Stretch-Induced MCP-1 Production
NF-B, a transcription factor that is important in the transcriptional regulation of inflammatory genes, controls MCP-1 expression in various cell types, including mesangial cells (10, 28, 29). To establish whether NF-B plays a role in stretch-induced MCP-1 expression, we tested the effect of stretch on NF-B-DNA-binding activity. Mesangial cell exposure to stretch for 60 min induced a significant increase in the binding of NF-B to DNA (Figure 5A, lines 1 to 2). Supershift experiments revealed that the p65-p50 heterodimer was the major NF-B activated form (Figure 5A, lines 3 to 4). The band was specific as it was abolished by an excess of unlabeled probe, whereas it was not affected by an excess of unlabeled unrelated probe (Oct-1; Figure 5A, lines 5 to 6). for investigating whether stretch-induced NF-B activation plays a role in MCP-1 overexpression, mesangial cells were pre-exposed to either SN50, a specific NF-B inhibitor, or the control peptide SN50M for 1 h and then exposed to stretch for 4 h. The addition of SN50 completely abolished MCP-1 response to stretch, whereas the control peptide SN50M was ineffective (Figure 5B). Preincubation with either calphostin C, a protein kinase C (PKC) inhibitor, or herbimycin A, a protein tyrosine kinase inhibitor, did not affect stretch-induced MCP-1 production (calphostin C, 5%; herbimycin A, 7% percentage reduction; NS)

View larger version (39K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Role of NF-B in stretch-induced MCP-1 expression in human mesangial cells. (A) Serum- and insulin-deprived human mesangial cells were exposed to mechanical stretch for 1 h. Nuclear proteins were prepared, and NF-B activity was assessed by electrophoretic mobility shift assay (EMSA) as described in the Materials and Methods section. Control nonstretched cells are shown in line 1, and stretched mesangial cells are shown in line 2. Bound proteins were identified by supershift after 30 min of preincubation of the nuclear proteins with antibodies to p50 (line 3) and p65 (line 4). Specificity of the binding reaction was established through competition with a 100-fold excess of unlabeled probe (line 5) and a lack of competition with unlabeled unrelated Oct-1 oligonucleotide (line 6). The immunoblot is representative of three EMSA performed on different nuclear protein extracts. (B) Serum- and insulin-deprived human mesangial cells were exposed to mechanical stretch for 4 h in the presence and in the absence of SN50 (18 µM), a specific NF-B inhibitor, and SM50, the inactive peptide. MCP-1 protein levels were measured as described in the Materials and Methods section and expressed as fold increase over control nonstretched cells (n = 4). *P < 0.05 stretch and stretch + SM50 versus nonstretch, nonstretch + SN50, stretch + SN50.

Stretch-Induced NF-B Activation Is IB Dependent

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of stretch and RSG on IB phosphorylation and NF-B activation. Serum- and insulin-deprived human mesangial cells were pretreated with RSG (10 µM) or vehicle for 1 h, then exposed to stretch. Total proteins were extracted, and phospho-IB was assessed by immunoblotting. Nuclear proteins were prepared, and NF-B activity was assessed by EMSA. (A) Representative immunoblots. (B) Densitometric analysis of three EMSA performed on different nuclear protein extracts. *P < 0.05 stretch versus nonstretch, nonstretch + RSG, and stretch + RSG.

Rosiglitazone Diminished Stretch-Induced MCP-1 Production by Inhibiting NF-B

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have demonstrated that in human mesangial cells, mechanical stretch induces monocyte chemotaxis via an NF-B-MCP-1-dependent pathway and that rosiglitazone prevents this effect

Mesangial cells that were subjected to cyclic stretching, to simulate an in vivo hemodynamic insult, producing a 10% cell elongation, showed a significant twofold increase in MCP-1 protein levels. This was preceded by a 1.7-fold rise in MCP-1 mRNA expression and paralleled by a 2-fold enhancement in monocyte chemoattractant activity. This is the first demonstration of stretch-induced MCP-1 in mesangial cells. These results, together with the recent report showing that mesangial cell compression augments MCP-1 expression (32), underlie the importance of physical forces in modulating mesangial cell MCP-1 production

The magnitude of the MCP-1 response to stretch was comparable to that reported in endothelial and vascular smooth muscle cells that were exposed to stretch (33, 34) or in mesangial cells that were exposed to high glucose (10). Inflammatory cytokines, such as TNF-, IL-1, and IFN-, are more potent MCP-1 inducers (35, 36). Consistently, in diabetic and hypertensive glomerulopathies, the degree of macrophage infiltration is much lower than that observed in inflammatory glomerular diseases (5–7)

Stretch-induced MCP-1 protein rose at 2 h, peaked at 4 h, and declined thereafter. We showed previously that in mesangial cells, vascular endothelial growth factor, another cytokine with chemoattractive properties, is induced by stretch with a similar time course (23). Furthermore, the time course of stretch-induced MCP-1 is similar to that observed in response to PDGF, a potent MCP-1 inducer (37), suggesting that MCP-1 induction is tightly controlled over time to prevent exaggerated activation of the inflammatory cascade. Although transient in time, stretch-induced MCP-1 can trigger a cascade of inflammatory processes and, thus, have prolonged downstream effects. In addition, the response to MCP-1 is likely to be sustained longer than the rise in MCP-1 because monocytes are known to continue to migrate toward the source of MCP-1 even when MCP-1 is no longer present (38)

Stretch-induced MCP-1 production was paralleled by a significant increase in monocyte chemoattractant activity, which was abolished by the addition of a specific MCP-1 neutralizing antibody. This indicates that MCP-1 released in response to stretch is functionally active and mediates stretch-induced monocyte recruitment

Our data provide evidence that the combined exposure to high glucose and stretch enhances MCP-1 production even further in this cell type. This is consistent with previous reports showing that high glucose upregulates MCP-1 in mesangial cells (10, 39) and suggests that both metabolic and hemodynamic abnormalities, to which mesangial cells are exposed in vivo in the diabetic glomerulus, may contribute to the local increase in MCP-1

Stretch-induced MCP-1 expression was mediated by the transcription factor NF-B as stretch induced a twofold increase in NF-B activity and SN50, a highly specific NF-B inhibitor, completely abolished stretch-induced MCP-1. This finding has particular relevance for diabetes in light of recent studies suggesting that multiple mechanisms of glucose-induced cell damage result in NF-B activation (40). Furthermore, both high glucose and angiotensin II have been shown to induce MCP-1 in mesangial cells via an NF-B-dependent pathway (10, 29). Although previous studies have shown that stretch activates NF-B in cardiomyocytes and human fibroblasts (41, 42), this is the first evidence of stretch-induced NF-B activation in mesangial cells

IB phosphorylation at serine 32/36 by IB kinase leads to IB ubiquitination/degradation, followed by the unmasking of the nuclear localization signal of NF-B and its translocation to the nucleus. Mesangial cell stretching activated NF-B via this classical pathway as we observed a 2.8-fold increase in phospho-Ser32/36-IB levels. In mesangial cells, both PKC and protein tyrosine kinases (PTK) are intracellular mediators of stretch, and high glucose-induced MCP-1 expression is PKC dependent (10, 23, 39). However, in this work, neither calphostin C nor herbimycin A altered stretch-induced MCP-1 production, indicating that this effect was PKC- and PTK-independent

Rosiglitazone significantly reduced stretch-induced monocyte chemotaxis by inhibiting NF-B activation and hence MCP-1 expression. This effect was observed with rosiglitazone concentrations within the pharmacologic range in humans (43) and comparable to those used in previous studies (44–46). Of interest, the inhibitory effect of rosiglitazone on stretch-induced MCP-1 production was also observed in mesangial cells that were cultured in high glucose ambient concentration, indicating that the diabetic milieu does not interfere with the anti-inflammatory activity of this compound. There was no direct rosiglitazone effect on monocyte chemotaxis, in agreement with previous studies showing that monocytes express PPAR- only when they differentiate into macrophages (47). The mechanism of NF-B inhibition by rosiglitazone was not investigated; however, mesangial cells express a functionally active PPAR- in the nucleus (20), and rosiglitazone, which binds and potently activates PPAR-, may interfere with NF-B activity within the nucleus via PPAR-. Accordingly, in our study, rosiglitazone did not affect stretch-induced IB phosphorylation, which occurs in the cytosol, but reduced nuclear NF-B DNA binding activity

These in vitro findings may have important implications for in vivo pathophysiologic conditions in renal disease. Rises in intraglomerular pressure as measured in experimental diabetes and remnant kidney models can be calculated to result in an expansion in glomerular volume, which entails approximately a 10% radial elongation (48, 49). Application of this degree of elongation to human mesangial cells in vitro resulted in a significant enhancement in MCP-1-driven monocyte recruitment. In an advanced stage of the disease, sclerosis reduces glomerular compliance, and glomerular capillary hypertension may lead to mesangial cell compression. Of interest, mesangial cell exposure to high pressure, mimicking cell compression, has also been reported to induce mesangial cell MCP-1 expression, although through a different intracellular signaling pathway (32)

In rat models of glomerular capillary hypertension, such as experimental diabetes and the remnant kidney models, there is an early increase in glomerular MCP-1 expression, followed by a low-grade macrophage infiltration (5–7). Our study suggests that hemodynamic and metabolic perturbations may directly participate in the monocyte recruitment through production of biologically active MCP-1 by resident mesangial cells. Accordingly, in experimental diabetes, glomerular capillary pressure-lowering agents, such as angiotensin-converting enzyme inhibitors, prevent both glomerular MCP-1 overexpression and macrophage infiltration (6)

Blockade of glomerular macrophage infiltration by treatment with the immunosuppressive agent mycophenolate mofetil or by knockout of the intercellular adhesion molecule ameliorates proteinuria and prevents glomerulosclerosis in experimental diabetes (8, 9). In vitro inflammatory cytokines released by activated macrophages magnify the mesangial cell response to the prosclerotic cytokine TGF-1, providing a molecular mechanism whereby locally recruited macrophages may contribute to glomerulosclerosis (50). This raises the possibility that the deleterious effect of both glomerular hypertension and hyperglycemia may result not only from a direct mechanical insult but also from MCP-1-driven macrophage infiltration

Our observation that rosiglitazone can prevent glomerular monocyte recruitment by inhibiting NF-B, the most important transcriptional regulator of inflammatory genes, suggests a new mechanism of rosiglitazone renoprotective action that is independent of amelioration of blood glucose and BP control. This may help explain the beneficial effects of TZD in animal models of glomerulosclerosis that are not associated with insulin resistance (16, 17) and extend the potential relevance of these compounds to glomerulopathies that are mediated by inflammatory mechanisms. Accordingly, a recent study showed that TZD renoprotective activity in crescentic glomerulonephritis is paralleled by inhibition of glomerular monocyte infiltration (51). However, in vivo data are still conflicting as TZD inhibit monocyte infiltration in crescentic glomerulonephritis (51), whereas they enhance it in another experimental model of glomerulonephritis (52). Furthermore, we cannot exclude the possibility that rosiglitazone antiproteinuric activity is due to direct effects on podocytes, a glomerular cell type expressing PPAR- and having a key role in maintaining the integrity of the glomerular filtration barrier. According to this hypothesis, it was reported recently that TZD reduce podocyturia in human diabetes (53)

TZD are currently in use in patients with type 2 diabetes as insulin-sensitizing agents, and they seem to ameliorate microalbuminuria in these patients, independent of glucose control (11). The potential direct renoprotective effects of these compounds in the treatment of patients with type 2 diabetes with incipient nephropathy will have to be addressed by purpose-designed long-term trials

	Acknowledgments

 
This work was supported by a project grant from GlaxoSmithKline. An abstract from this work was presented at the European Association for the Study of Diabetes Meeting, August 24–29, 2003

	References

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