The monocyte chemotactic protein synthesis inhibitor bindarit prevents mesangial cell proliferation and extracellular matrix remodeling

Pharmacological Research 66 (2012) 526–535

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The monocyte chemotactic protein synthesis inhibitor bindarit prevents mesangial cell proliferation and extracellular matrix remodeling Sara Paccosi a , Claudia Musilli a , Giorgina Mangano c , Angelo Guglielmotti c , Astrid Parenti a,b,∗ a b c

Department of Pharmacology, University of Florence, Florence, Italy CIMMBA, University of Florence, Florence, Italy Angelini Research Center, S. Palomba-Pomezia, Rome, Italy

a r t i c l e

i n f o

Article history: Received 31 July 2012 Received in revised form 6 September 2012 Accepted 6 September 2012 Keywords: Bindarit Mesangial cell proliferation Chemotactic protein-1/chemokine (C–C motif) ligand-2 Extracellular matrix remodeling Collagen

a b s t r a c t Glomerular expression of chemotactic protein-1/chemokine (C–C motif) ligand-2 (MCP-1/CCL2) correlates with the degree of renal damage, suggesting a role of this chemokine in the pathogenesis of renal diseases. Bindarit is an original indazolic derivative able to inhibit MCPs synthesis and to significantly decrease MCP-1/CCL2 urinary excretion in patients with Lupus Nephritis, in correlation with reduction in albuminuria. Aim of the present work was to elucidate the effect of MCP-1/CCL2 synthesis inhibition on in vitro models of mesangial cell dysfunction. ET1 (10 nM) and AngII (10 nM) significantly stimulated MCP-1/CCL2 release by human renal mesangial cells (HRMCs) after 3–12 h stimulation. Bindarit (10–300 ␮M) significantly inhibited MCP-1/CCL2 release in response to both stimuli within 12 h. Bindarit also inhibited mRNA MCP-1/CCL2 expression, confirming an effect of the drug at transcriptional level. Bindarit significantly and concentration-dependently inhibited HRMC proliferation, measured as either cell duplication or total DNA/well, and impaired mRNA collagen IV expression, collagen deposition and fibronectin expression induced by AngII and ET1. Exposure of HRMCs to bindarit also impaired MMP2 activation in response to both stimuli, measured by means of gelatin zymography. These data confirm the important role of MCP-1/CCL2 synthesis in mesangial cell dysfunction and support the potential of therapeutic intervention targeting this chemokine in kidney disease. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Mesangial cells are specialized smooth muscle cells important in maintaining the normal structure and function of the glomerulus, producing various cytokines and growth factors and regulating the turnover of the extracellular matrix (ECM) in the structure [1]. Glomerulosclerosis, the final common pathway in a variety of renal diseases, is characterized by phenotype transition of mesangial cells and increased extracellular matrix formation. Deposition and accumulation of ECM proteins such as fibronectin, laminin, collagen and the expression and activity of gelatinases (MMP-2, MMP-9) are prominent crucial features of ECM remodeling with likely implications for glomerular function. It has been demonstrated that the degree of ECM accumulation correlates with the extent of renal

Abbreviations: AngII, angiotensin II; ECM, extracellular matrix; ET1, endothelin 1; FCS, fetal calf serum; GMCs, glomerular mesangial cells; HRMCs, human renal mesangial cells; MCP-1/CCL2, monocyte chemotactic protein-1/chemokine (C–C motif) ligand-2; MMP-2-9, metalloproteinase-2-9; PBS, phosphate buffered saline. ∗ Corresponding author at: Vascular Pharmacology Unit, Department of Preclinical and Clinical Pharmacology, University of Florence, V.le G. Pieraccini, 6, 50139 Florence, Italy. Tel.: +39 0554271330; fax: +39 0554271280. E-mail address: astrid.parenti@unifi.it (A. Parenti). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.09.006

insufficiency and proteinuria. Glomerular cell proliferation and hypertrophy are other important hallmarks of progressive renal diseases [1,2]. A number of growth factors and cytokines, both released by glomerular cells and infiltrating leukocytes, appear well suited to mesangial cell changes. Among them, MCP-1/CCL2 has been indicated to play a major role in the pathogenesis of renal diseases [3]. There is evidence that glomerular expression of MCP-1/CCL2 correlates with the degree of renal damage. Among molecules able to increase MCP-1/CCL2 production, endothelin 1 (ET1) and angiotensin II (AngII) seem to play an important role in the pathogenesis of renal damage. ET1 has been shown to be produced by intrinsic glomerular cells and tissue levels of AngII are increased under renal pathologic conditions upon the local activation of the renin–angiotensin system. AngII and ET1 are able to increase mesangial cell contraction (and/or proliferation), to reduce glomerular filtration, leading to decrease of renal blood flow and microalbuminuria. Most of ET1 and AngII effects reported above are mediated by the release of cytokines and chemokines such as MCP1/CCL2 [4,5]. In diabetic nephropathy, MCP-1/CCL2 expression is increased in animal models and humans, and urinary excretion of MCP-1/CCL2 correlates not only with renal interstitial disturbance, but also with glomerular damage and albuminuria [6,7]. Moreover, the block of the CCR2 receptor in type 1 diabetic mice reduces

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glomerular deposition of TGF-␤1 and collagen IV and of the mesangial matrix fraction [8]. It has been proposed that urinary levels of MCP-1/CCL2 can be used to assess the efficacy of treatments in reducing renal inflammation. Treatment of patients with the ACEinhibitor lisinopril reduced urine MCP-1/CCL2 in type 2 diabetic nephropathy and this correlates with the decline of proteinuria [4]. Bindarit, 2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid, is an original indazolic derivative with anti-inflammatory activity in a number of experimental diseases [9–14]. These pharmacological activities have been associated with the ability of bindarit to interfere with monocyte recruitment, which has been ascribed to a selective inhibitory effect on the synthesis of the monocyte chemotactic protein (MCP) subfamily of CC inflammatory chemokines, including MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8 [15]. Phase II trials in patients with type 2 diabetes and Lupus Nephritis have shown that bindarit was well tolerated and it significantly reduced urinary MCP-1/CCL2 and albumin excretion in kidney disease [16,17]. However the mechanisms by which bindarit decreases proteinuria have not been clarified. The aim of this paper is to analyze the effects of bindarit on in vitro models of mesangial cell dysfunction. We evaluated the effect of bindarit on human renal mesangial cell (HRMCs) proliferation and ECM remodeling induced by ET1 and AngII. 2. Materials and methods 2.1. Materials 2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3Bindarit, yl]methoxy]propanoic acid (MW 324.38), was synthesized by Angelini (Angelini Research Center – ACRAF, Italy). A 100 ␮M bindarit stock solution was prepared by adding NaOH 1N, then distilled water and finally, to obtain the final starting product solution, by adding a neutralizing buffer at pH 7.4 (without calcium) up to the right volume. The concentrations of bindarit used in our experiments have been chosen based on previous in vitro and in vivo studies and are related to bindarit plasma levels obtained following oral administration either in preclinical or clinical experiments, which are reported to be in the range of 150–450 ␮M (Product Data Sheet, Angelini Research Center). AngII and ET-1 were purchased from Sigma (St. Louis, MO, USA) and were dissolved in phosphate buffered saline (PBS) with 0.1% bovine serum albumin at a concentration of 10 mM. 2.2. Experimental design Following starving conditions (0.1% FCS) HRMCs were pretreated or not pretreated (control group) for 1 h with bindarit and

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then stimulated with 10 nM AngII or 10 nM ET1 for different periods of time, as reported in Section 2. 2.3. Cell culture Human renal mesangial cells (HRMCs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and were grown in Mesangial cell medium, supplemented with growth factors 100 U/ml penicillin, 100 ␮g/ml streptomycin and 5% fetal calf serum (FCS). 2.4. MCP-1/CCL2 production HRMCs were seeded onto 48-well plastic culture plates at a density of 1 × 104 cells/well. After 24 h starvation in 0.1% FCS, cells were pretreated or not pretreated (control group) for 1 h with bindarit (10–300 ␮M, MW 324.38) in 1% FCS medium and then stimulated with 10 nM AngII or ET1. After 3, 6, 12 and 24 h media were collected, centrifuged at 2000 × g for 10 min at 4 ◦ C and supernatants were immediately frozen at −80 ◦ C until used for ELISA (OptEIA, BD, San Diego, USA). 2.5. RT-PCR HRMCs were seeded at 80% confluence onto 6-cm-diameter Petri dishes in 10% FCS medium. Cells were starved for 24 h (0.1% FCS medium), pretreated for 1 h with bindarit (300 ␮M) in 1% FCS medium and then stimulated with AngII or ET1 (10 nM) for 3, 6, 12 and 24 h. Total RNA was isolated according to the manufacturer’s protocol (Nucleospin RNAII, Nacherey-Nagel) and was reverse transcribed by using random primers (Omniscript-Qiagen). cDNA was amplified (HotStarTaq; Qiagen) by specific primer sequences (Table 1). Amplificates were electrophoresed in 1% agarose gel and quantified as the ratio between optical density of the target genes and GAPDH housekeeping gene amplification products. 2.6. Quantitative assessment of collagen accumulation in the extracellular matrix The colorimetric Sircol collagen assay uses a dye that specifically binds to the native triple helix structure of collagen released from cells (Biocolor, Northern Ireland, UK). HRMCs were cultured for 24–48 h with AngII or ET1 (10 nM) in presence or absence of 300 ␮M bindarit. Then cells and matrix were recovered in 0.5 M acetic acid. According to manufacturer’s instructions, samples were concentrated (1:10), mixed with the dye and centrifuged (10,000 × g for 10 min). The pellet of collagen with bound dye was then dissolved in an acidic solution. Absorbance of each sample at 540 nm was measured (after blanking) and averaged using a multilabel plate counter (VICTOR2 – Wallac).

Table 1 Primers and PCR conditions. Gene

Primers

Annealing temp.

Cycles

GAPDH

F 5 -CCC ATC ACC ATC TTC CAG-3 R 5 -AGT CCT TCC ACG ATA CCA-3 F 5 -GAA ACA TCC AAT TCT CAA AC-3 R 5 -AGT GTT CAA GTC TTC GGA GT-3 F 5 -ATG TTC AGC TTT GTG GAC CT-3 R 5 -TCG TCA CAG ATC ACG TCA TC-3 F 5 -ATG CTC AGC TTT GTG GAT AC-3 R 5 -TGG TCC AAC AAC TCC TCT CT-3 F 5 -TGC TGG CTC TGG CTG TGG CAA A-3 R 5 -TCA TCC CTG GTA AGC CTG GTG GTC C-3 F 5 -TTC CCC TTC TTG TTC AAT GG-3 R 5 -ATT TGT TGC CCA GGA AAG TG-3

56 ◦ C

28

56 ◦ C

35

56 ◦ C

28

MCP-1/CCL2 Collagen I-␣1 Collagen I-␣2 Collagen IV MMP-2

◦

56 C

28

65 ◦ C

28

56 ◦ C

28

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2.7. Gelatin zymography Cells were cultured in 48-well culture plates in 10% FCS medium until 90% confluence was achieved. Starved cells were stimulated with ET1 or AngII (10 nM) in the presence or absence of bindarit (300 ␮M). After 3, 6, 12 and 24 h the media were collected, clarified by centrifugation and subjected to electrophoresis onto 8% SDSPAGE containing 1 mg/ml gelatin under non-denaturing conditions. Following electrophoresis the gels were washed with 2.5% Triton X-100 to remove SDS and incubated for 24 h at 37 ◦ C in 50 mM Tris buffer containing 200 mM NaCl and 20 mM CaCl2, pH 7.4. Gels were then stained with 0.5% Coomassie brilliant blue R-250 in 10% acetic acid and 45% methanol and destained with 10% acetic acid and 45% methanol. Bands of gelatinase activity appeared as transparent areas against a blue background. Gelatinase activity was then evaluated by quantitative densitometry. 2.8. Cell proliferation HRMC proliferation was quantified by the total cell number as well as the total DNA/well via a fluorescent dye (Cell proliferation kit, Invitrogen). Cells (1.5 × 103 /100 ␮l) suspended in 10% FCS medium were plated on flat-bottom 96-multiwell plates and allowed to adhere overnight. Cells were kept in starving conditions (0.1% FCS) for 24 h, then media were removed and replaced with 1% FCS medium containing test stimuli. In order to assess bindarit effects, the drug was added 1 h prior to the addition of growth factors. After 48 h, cells were fixed with methanol and stained with Diff-Quik. Cell duplication was assessed by counting the total cell number in 10 random fields of each well at 200× with the aid of a 21-mm2 grid. Moreover, proliferation was assessed by total DNA/well. After 48 h, 100 ␮l of dye binding solution were added to each microplate well and incubated at 37 ◦ C for 30 min. This incubation period is required for equilibration of dye-DNA binding, resulting in a stable fluorescence endpoint. The fluorescence

intensity was read using a fluorescence microplate reader with excitation at ∼485 nm and emission detection at ∼530 nm. 2.9. Western blot analysis HRMCs were lysed in buffer followed by centrifugation at 14,000 × g for 10 min at 4 ◦ C. The cell lysate (15 ␮g) was run on 7% SDS-polyacrylamide gel electrophoresis, blotted onto PVDF membrane (Millipore) and immunostained with mouse monoclonal anti-fibronectin antibody (1:10,000, BD) and with anti ␤-tubulin monoclonal antibody (1:1000, Sigma–Aldrich). The antigen–antibody complexes were visualized using appropriate secondary antibodies and the ECL detection system, as recommended by the manufacturer (Amersham Corp.). 2.10. Statistical analysis Results are expressed as the mean ± SEM of multiple experiments. Statistical analysis was performed using the ANOVA test (Tukey’s Multiple Comparison Test); Student’s t test for unpaired data was used where appropriate. A P < 0.05 was considered significant. 3. Results 3.1. Effect of bindarit on MCP-1/CCL2 release HRMCs were stimulated with 10 nM AngII or 10 nM ET1, in order to assess MCP-1/CCL2 release by means of an ELISA assay. Both AngII and ET1 induced a time-dependent release of MCP1/CCL2 which resulted faster in response to AngII, compared to ET1. MCP-1/CCL2 release was maximal following 6 h incubation with either AngII or ET1, exerting a 63 ± 8% and 64.8 ± 7% increase of MCP-1/CCL2 over control, respectively. The effects of AngII and ET1 decreased at 12 h and disappeared within 24 h. Bindarit

Fig. 1. MCP-1/CCL2 release by HRMCs. Time-dependent release of MCP-1/CCL2 in response to AngII and ET1 (10 nM). Cells were pretreated or not pretreated (control, ctr) with increasing concentrations of bindarit and then stimulated with AngII or ET1. Results are expressed as mean ± SEM of ng/ml MCP-1/CCL2 (7 experiments). ˆP < 0.05 vs. unstimulated cells (0); *P < 0.05 vs. ET1 or AngII alone.

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Fig. 2. MCP-1/CCL2 mRNA expression by HRMCs. RT-PCR semi-quantitative analysis of MCP-1/CCL2 mRNA expression in cultured mesangial cells following ET1 (a) or AngII (b) stimulation with or without (control, ctr) bindarit. Data are expressed as densitometric (OD) ratio between MCP-1/CCL2 and housekeeping gene. Mean ± SEM of 4 experiments. ˆP < 0.05 vs. unstimulated cells (0); *P < 0.05 vs. AngII or ET1 alone.

(10–300 ␮M), concentration-dependently inhibited MCP-1/CCL2 production in response to both stimuli (Fig. 1). The inhibition of MCP-1/CCL2 release from HRMCs by bindarit appeared related to the inhibition of specific mRNA expression, as shown in Fig. 2. AngII and ET1 (10 nM) induced an increase of MCP-1/CCL2 mRNA which was significantly inhibited by 300 ␮M bindarit. 3.2. Effect of bindarit on HRMCs proliferation Since mesangial cells proliferation is a key event in glomerular dysfunction, the effect of bindarit on AngII- and ET1-induced HRMCs proliferation was assessed. AngII (10 nM) and ET1 (10 nM) significantly stimulated HRMC proliferation, measured as either total DNA/well (Fig. 3a) or total cells/well (Fig. 3b). The addition of bindarit (300 ␮M), significantly impaired cell proliferation induced by both stimuli. The inhibition of cell proliferation induced by bindarit was concentration-dependent as shown in Fig. 3c, whose maximal effect was observed at 600 ␮M, concentration which inhibited cell growth induced by ET1 and AngII by 71% and 73%, respectively (Fig. 3c). At the highest concentration (600 ␮M) bindarit did not affect basal proliferation and it was devoid of any toxic effect, as shown by trypan blue exclusion observations (data not shown). To confirm that the production of MCP-1/CCL2 is responsible of the autocrine and paracrine stimulation of mesangial cell proliferation, recombinant human MCP-1/CCL2 was added to HRMCs to assess cell growth. Fig. 3d shows that 0.1–10 ng/ml MCP-1/CCL2, concentrations corresponding to those measured following ET1 and AngII stimulation (Fig. 1), significantly increased HRMCs proliferation, with maximal effect obtained in response to 1 ng/ml (30% increase over unstimulated cells, ˆˆP < 0.01). 3.3. Effect of bindarit on collagen expression and deposition Since collagen expression and deposition is a hallmark for mesangial cell dysfunction, we assessed the effect of bindarit on collagen IV and I mRNA expression in HRMCs stimulated with AngII

and ET1. ET1 (Fig. 4a) and AngII (Fig. 4b) significantly stimulated collagen IV mRNA expression. Maximal effects were obtained for both vasoactive peptides at 12 h stimulation. Bindarit (300 ␮M) significantly inhibited collagen IV mRNA expression in response to either AngII or ET1, with maximal effects following 12 h stimulation (Fig. 4a and b). Conversely, in the experimental conditions used, CollagenI ␣1 and ␣2 mRNA was not modulated at any time point studied (data not shown). We then assessed the effect of bindarit on AngII- and ET1-induced total collagen deposition by mesangial cells. Following cell starvation, HRMCs were stimulated with AngII or ET1 for 12, 24 and 48 h. Then cells and matrix were collected in order to measure total collagen deposition. After 48 h stimulation with AngII (10 nM) total collagen accumulation increased by 35 ± 3.7% compared with untreated cells (0, Fig. 5). AngII-induced collagen accumulation was prevented by the addition of 300 ␮M bindarit. Conversely, no significant modulation of collagen deposition was measured in response to ET1. 3.4. Effect of bindarit on fibronectin expression Fibronectin expression and deposition is also involved in mesangial extracellular matrix remodeling. The expression of fibronectin was then measured by Western blot analysis. AngII and with a lesser extent ET1, induced fibronectin expression by mesangial cells at the time points measured. Bindarit significantly inhibited fibronectin expression in response to both stimuli, being more active on AngIIinduced expression (Fig. 6). 3.5. Effect of bindarit on MMP-2 and 9 activity MMP-2 and, with a lesser extent, MMP-9 are involved in matrix remodeling and in mesangial cell proliferation. The effect of bindarit was then assessed on AngII and ET1-induced MMP2 and -9 activation. Gelatin zymography of control supernatants showed constitutive release of the latent forms of MMP-2, visualized as a band at 72 kDa (Fig. 7b and d). ET1 and AngII stimulated the release of MMP-2 within 3 h and induced its activation as revealed

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Fig. 3. Effect of bindarit on HRMC proliferation. Cells were pretreated or not pretreated (control, ctr) for 1 h with bindarit and then stimulated with ET1 or AngII. Proliferation was evaluated after 48 h incubation as total DNA/well (a) by means of a fluorescent dye, or as total cells/well (b); Mean ± SE n = 7, ˆP < 0.05, ˆˆP < 0.01 vs. unstimulated cells (0). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AngII or ET1 alone (Student’s t test). (c) Concentration-dependent effect of bindarit (10–600 ␮M). Results are reported as percent of AngII or ET1 effect (control). Mean ± SE n = 8; *P < 0.05, **P < 0.01 vs. AngII or ET1 alone; (d) HRMC proliferation in response to increasing concentrations of human recombinant MCP-1/CCL2 (0.1–100 ng/ml). Proliferation was evaluated after 48 h as total DNA/well. Mean ± SE n = 6, ˆP < 0.05, ˆˆP < 0.01 vs. unstimulated cells (0).

by the appearance of the 62 kDa form (Fig. 7b and d). Bindarit significantly inhibited only the activated MMP-2 form within few hours in response to ET1 (Fig. 7a and b) or AngII (Fig. 7c and d). Consistently, neither AngII nor ET1 significantly modulated MMP-2 at transcriptional level, as demonstrated by MMP-2 mRNA expression (Fig. 7e). However, no activation or release of MMP-9 was observed in response to ET1 and AngII (data not shown). 4. Discussion Bindarit is an original indazolic derivative devoid of immunosuppressive effects and with no activity on arachidonic acid metabolism that was shown to have anti-inflammatory activity in a number of experimental diseases including nephritis, arthritis, pancreatitis, and colitis [9,10,14,18,19]. These pharmacological activities have been associated with bindarit ability to interfere with monocyte recruitment, which has been ascribed to a selective inhibitory effect on the synthesis of monocyte chemotactic protein (MCP) subfamily of CC inflammatory chemokines, including MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8 [15]. In in vivo studies bindarit has been demonstrated to reduce renal dysfunction and microvascular dysfunction in a swine renovascular hypertension model [11,20]. In lupus subjects with active nephritis, bindarit significantly reduced albuminuria and urinary MCP-1/CCL2 levels [17]. Benefits of bindarit therapy have also been demonstrated in a prospective, randomized double-blind and placebo-controlled trial, in which bindarit significantly and safely reduced albuminuria

in type 2 diabetic patients [16], thus confirming MCP-1/CCL2 as a potential target for renal disease. Since the cellular mechanisms underlying these renal protective effects of bindarit have not been investigated till now, we proposed an in vitro experimental model of mesangial cell dysfunction, in which bindarit effect was assessed. The results provided in this study show that bindarit significantly prevented AngII- and ET1-induced human mesangial cells proliferation and matrix remodeling. All these effects were correlated with the impairment of MCP-1/CCL2 synthesis exerted by bindarit. Glomerular mesangial cells (GMCs) play important roles in kidney physiological and pathological processes. GMC dysfunction has been postulated to be a key contributor to glomerulosclerotic lesions in diabetic patients [21]. GMC proliferation and hypertrophy, ECM accumulation, and consequent renal fibrosis have been recognized as major pathogenic events in the progression of renal failure in diabetic nephropathy [22]. Most of the human and experimental glomerular diseases associated with sclerosis are characterized by the infiltration of macrophages into the glomerulus in the early stages of the disease, before the development of extracellular matrix expansion and glomerulosclerosis [2,3]. Monocyte chemoattractant protein-1 (MCP-1/CCL2), a specific chemoattractant for monocytes, is implicated in recruiting and activating monocytes/macrophages to the glomerulus in proliferative glomerular diseases. Biologically active MCP-1/CCL2 is present in the urine of patients with various glomerular diseases and the urinary levels of MCP-1/CCL2 correlate with both the extent of proteinuria and the number of glomerular macrophages [4,7,23]. It

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Fig. 4. Collagen IV mRNA expression. RT-PCR semi-quantitative analysis of Collagen IV mRNA expression in cells treated or not treated (control, ctr) with bindarit. HRMCs were stimulated with 10 nM ET1 (a) or AngII (b) with or without bindarit (300 ␮M). Data are expressed as densitometric (OD) ratio. Mean ± SEM of 3 experiments. ˆP < 0.05; ˆˆP < 0.01 vs. unstimulated cells (0); *P < 0.05 vs. ET1 or AngII alone (Student’s t test).

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has been demonstrated that administration of antibodies to MCP1/CCL2 selectively reduces glomerular monocytes/macrophage recruitment [24], decreases the extent of proteinuria, reduces glomerulosclerosis, and improves renal dysfunction in experimental crescent glomerulo-nephritis [25,26]. Consistently, in patients with Lupus Nephritis, bindarit significantly decreased MCP-1/CCL2 urinary excretion which correlated with improvement in albuminuria [17]. However, the mechanisms through which bindarit exerts its antiproteinuric activity have been not yet clarified. It has been previously demonstrated that the anti-inflammatory effect of bindarit is the modulation of MCP-1/CCL2 axis by inhibition of the NF-␬B canonical pathway [14]. This mechanism of action is specific, since bindarit does not affect other promoters. This paper provides cellular mechanistic information which might explain the protective effects of bindarit observed in vivo model of renal dysfunction and in patients with Lupus Nephritis. We demonstrated that AngII and ET1, powerful and proven mediators of mesangial dysfunction, induced a fast and time-dependent release of MCP-1/CCL2, which was maximal after 6 h incubation and decreased within 24 h. This fast and short-lasting effect of AngII on MCP-1/CCL2 release has been previously demonstrated in mesangial cells [27] as well as in proximal tubular cells [28]. It is likely that this short-lasting effect of AngII and ET1 results from their rapid degradation, since they were added to the cells once at the beginning of the experimental procedure (time 0). Bindarit concentration-dependently inhibited MCP-1/CCL2 release in response to both stimuli. We also demonstrated that proliferation of human renal mesangial cells (HRMCs) induced by ET1 and AngII, was significantly impaired by bindarit. The proliferative effect of both vasoactive peptides was associated to MCP-1/CCL2 synthesis and release by cells, condition confirmed by bindarit inhibition of MCP-1/CCL2 release and cell proliferation, in response to both stimuli. The addition of an amount of recombinant human MCP-1/CCL2, comparable to that measured in the conditioned media of HRMCs cells stimulated with AngII and ET1, promoted their proliferation. Thus, obtained results confirm that the hyperplastic effect of AngII and ET1 on mesangial cells is partially mediated by this chemokine. Evidences suggest that proliferation of mesangial cells can occur in the very early stages of glomerular dysfunction [29], but not in later stages, in which glomerular lesions are characterized by

Fig. 5. Total collagen deposition by HRMCs. Collagen deposition by cells was measured after 48 h from stimuli addition by the Sircol assay. Cells were pretreated or not pretreated (control, ctr) with 300 ␮M bindarit and then stimulated with 10 nM AngII or ET1. Data are expressed as ␮g/ml collagen. Mean ± SEM of 3 experiments. ˆP < 0.05 vs. unstimulated cells (0); *P < 0.05 vs. AngII alone.

expansion of the mesangial matrix, leading to encroachment of cellular elements, loss of filtration area and thickening of the peripheral capillary basement membrane [30]. Up-regulation of mesangial matrix results from increased deposition of matrix, reduced catabolism, or possibly the combination of both [31]. A number of growth factors and cytokines, both released by glomerular cells and infiltrating leukocytes, appear well suited to stimulate matrix accumulation. Among them, AngII and ET1 are involved. Over-expression of ET1 in transgenic mice causes mesangial remodeling, glomerulosclerosis with collagen deposition, fibrosis, and loss of organ function in the kidney, heart, and lung [32–34]. These ET1-directed changes in mesangial phenotype lead to progressive renal injury and a decline in glomerular filtration rate. It has been recently demonstrated that 100 nM ET1 increases collagen deposition in the extracellular matrix in human mesangial cells in vitro, by inducing an autocrine signaling loop involving secretion of MCP-1/CCL2 and IL-6 [35]. Our present data demonstrate that 10 nM ET1 significantly increases collagen IV mRNA expression, which is prevented by bindarit. Bindarit also

Fig. 6. Fibronectin expression by HRMCs. Cells pretreated or not pretreated (control, ctr) with bindarit were stimulated for 24 and 48 h with 10 nM AngII or ET1. Fibronectin expression was assessed by means of Western blot analysis. (a) Mean ± SEM of 3 experiments. ˆP < 0.05 vs. unstimulated cells (0); *P < 0.05, **P < 0.01 vs. AngII or ET1 alone (Student’s t test). (b) Representative experiment; AII = 10 nM Ang II, ET = 10 nM ET1, b = 300 ␮M bindarit.

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Fig. 7. Gelatin zymography for MMP-2 activity. Changes of gelatinolytic activity in cells stimulated for 3–24 h with 10 nM AngII or ET1 in the presence or absence of bindarit. A lysis zone was observed in unstimulated cells (∼72 kDa, latent MMP2), and a ∼67 kDa band of activated MMP2 appeared in response to either ET1 (b) or AngII (d). (a and c) Densitometric analysis showing the effect of bindarit (300 ␮M) on both latent and activated MMP-2 in response to ET1 (a) or AngII (c). Mean ± SEM of 3 experiments. ˆP < 0.05, vs. unstimulated cells (0); *P < 0.05 vs. ET1 or AngII alone, Student’s t test. (b and d) Representative zymogram for MMP-2 following 3 and 6 h stimulation with ET1 (b) or AngII (d) in the presence or absence of 300 ␮M bindarit (bin). (e) MMP-2 mRNA expression in HRMCs stimulated for 3–12 h with AngII (AG) or ET1 (ET) in the presence or absence of 300 ␮M bindarit (bin). Experiment representative of 3 replicates.

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impaired the expression of fibronectin stimulated by ET1. Our present data also show that bindarit prevents AngII-induced ECM remodeling. We demonstrated that AngII significantly stimulated human mesangial extracellular matrix remodeling, by increasing expression and deposition of collagen and fibronectin expression. All these effects were significantly impaired by bindarit, thus confirming a pivotal role of MCP-1/CCL2 in the hyperplastic and pro-fibrotic effects of AngII. Most of the AngII-effects in the kidney are mediated by the release of several fibrogenic chemokines, namely MCP-1/CCL2 and TGF-␤ [4]. Because MCP-1/CCL2 induces monocyte migration and differentiation to macrophages, which augment extracellular matrix production and tubulointerstitial fibrosis, pharmacological reduction of AngII effects by means of ACEIs may also exert its beneficial effects in diabetic nephropathy by downregulating renal MCP-1/CCL2, even if a direct effect of MCP-1/CCL2 on mesangial cells cannot be excluded. Our data also show that bindarit impaired matrix metalloproteinase-2 (MMP-2) activation in HRMCs, in response to AngII and ET1 stimulation. The effect was evident only on the active form of the gelatinase and within few hour of stimulation (3–12 h) which correlates with the time-dependent release of MCP-1/CCL2. No effect was observed on MMP-9 activity. It is well documented that human mesangial cells in culture express only the latent MMP-2 form [36], which may be stimulated or inhibited by pro-fibrotic and vasoactive stimuli, depending on the in vitro experimental models. ET1 or AngII were demonstrated to inhibit MMP-2 expression and/or activity in in vitro or in vivo models of glomerulonephritis. However, changes in the status and composition of the ECM have been shown to affect the function of a variety of different cells and to modulate the synthesis and release of MMP in other systems [37,38]. Moreover, ET1 has been demonstrated to stimulate activity of MMP-2 in rat mesangial cell-induced collagen gel contraction [39]. In addition to its matrix remodeling, it has been demonstrated that active MMP-2 may play a role in the proliferation and in the inflammatory phenotype of rat mesangial cells [40]. In conclusion this study demonstrates that bindarit is effective in preventing human mesangial cell proliferation and matrix remodeling in in vitro models of mesangial cell dysfunction, suggesting a potential therapeutic role of this compounds in several renal diseases in which mesangial cell dysfunction is well documented.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

Acknowledgements This work was supported by the Angelini (ACRAF, Italy; 004FA09442; 004FA11089) awarded to A.P.

[24]

[25]

References [1] Schlöndorff D, Banas B. The mesangial cell revisited: no cell is an island. Journal of the American Society of Nephrology 2009;20(6):1179–87. [2] Dussaule JC, Guerrot D, Huby AC, Chadjichristos C, Shweke N, Boffa JJ, et al. The role of cell plasticity in progression and reversal of renal fibrosis. International Journal of Experimental Pathology 2011;92(3):151–7. [3] Eardley KS, Zehnder D, Quinkler M, Lepenies J, Bates RL, Savage CO, et al. The relationship between albuminuria MCP-1/CCL2, and interstitial macrophages in chronic kidney disease. Kidney International 2006;69(7):1189–97. [4] Amann B, Tinzmann R, Angelkort B. ACE inhibitors improve diabetic nephropathy through suppression of renal MCP-1. Diabetes Care 2003;26(8):2421–5. [5] Saleh MA, Pollock JS, Pollock DM. Distinct actions of endothelin A-selective versus combined endothelin A/B receptor antagonists in early diabetic kidney disease. Journal of Pharmacology and Experimental Therapeutics 2011;338(1):263–70. [6] Banba N, Nakamura T, Matsumura M, Kuroda H, Hattori Y, Kasai K. Possible relationship of monocyte chemoattractant protein-1 with diabetic nephropathy. Kidney International 2000;58(2):684–90. [7] Tesch GH. MCP-1/CCL2: a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. American Journal of Physiology Renal Physiology 2008;294(4):F697–701. [8] Kanamori H, Matsubara T, Mima A, Sumi E, Nagai K, Takahashi T, et al. Inhibition of MCP-1/CCR2 pathway ameliorates the development of

[26]

[27]

[28]

[29]

[30]

[31]

diabetic nephropathy. Biochemical and Biophysical Research Communications 2007;360(4):772–7. Zoja C, Corna G, Morigi M, Donarelli R, Guglielmotti A, Pinza M, et al. Bindarit retards renal disease and prolongs survival in murine lupus autoimmune disease. Kidney International 1998;53(3):726–34. Bhatia M, Ramnath RD, Chevali L, Guglielmotti A. Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis. American Journal of Physiology Gastrointestinal and Liver Physiology 2005;288(6):G1259–65. Zhu XY, Chade AR, Krier JD, Daghini E, Lavi R, Guglielmotti A, et al. The chemokine monocyte chemoattractant protein-1 contributes to renal dysfunction in swine renovascular hypertension. Journal of Hypertension 2009;27(10):2063–73. Ialenti A, Grassia G, Gordon P, Maddaluno M, Di Lauro MV, Baker AH, et al. Inhibition of in-stent stenosis by oral administration of bindarit in porcine coronary arteries. Arteriosclerosis, Thrombosis, and Vascular Biology 2011;31(11):2448–54. Rulli NE, Rolph MS, Srikiatkhachorn A, Anantapreecha S, Guglielmotti A, Mahalingam S. Protection from arthritis and myositis in a mouse model of acute chikungunya virus disease by bindarit, an inhibitor of monocyte chemotactic protein-1 synthesis. Journal of Infectious Diseases 2011;204(7):1026–30. Mora E, Guglielmotti A, Biondi G, Sassone-Corsi P. Bindarit: an antiinflammatory small molecule that modulates the NF␬B pathway. Cell Cycle 2012;1(1):159–69. Mirolo M, Fabbri M, Sironi M, Vecchi A, Guglielmotti A, Mangano G, et al. Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins. European Cytokine Network 2008;9(3):119–22. Ruggenenti P. Effects of MCP-1 inhibition by bindarit therapy in type 2 diabetes subjects with micro- or macro-albuminuria. Journal of the American Society of Nephrology 2010;21:44A [Abstract]. Ble A, Mosca M, Di Loreto G, Guglielmotti A, Biondi G, Bombardieri S, et al. Antiproteinuric effect of chemokine C–C motif ligand 2 inhibition in subjects with acute proliferative lupus nephritis. American Journal of Nephrology 2011;34(4):367–72. Guglielmotti A, D’Onofrio E, Coletta I, Aquilini L, Milanese C, Pinza M. Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an inhibitor of MCP-1 and TNF-alpha production. Inflammation Research 2002;51(5):252–8. Bhatia M, Landolfi C, Basta F, Bovi G, Ramnath RD, de Joannon AC, et al. Treatment with bindarit, an inhibitor of MCP-1 synthesis, protects mice against trinitrobenzene sulfonic acid-induced colitis. Inflammation Research 2008;57(10):467–71. Lin J, Zhu X, Chade AR, Jordan KL, Lavi R, Daghini E, et al. Monocyte chemoattractant proteins mediate myocardial microvascular dysfunction in swine renovascular hypertension. Arteriosclerosis, Thrombosis, and Vascular Biology 2009;29(11):1810–6. Molitch ME, Steffes M, Sun W, Rutledge B, Cleary P, de Boer IH, et al. Development and progression of renal insufficiency with and without albuminuria in adults with type 1 diabetes in the diabetes control and complications trial and the epidemiology of diabetes interventions and complications study. Diabetes Care 2010;33(7):1536–43. Raptis AE, Viberti G. Pathogenesis of diabetic nephropathy. Experimental and Clinical Endocrinology and Diabetes 2001;109(Suppl. 2):S424–37. Rovin BH, Doe N, Tan LC. Monocyte chemoattractant protein-1 levels in patients with glomerular disease. American Journal of Kidney Diseases 1996;27(5):640–6. Panzer U, Thaiss F, Zahner G, Barth P, Reszka M, Reinking RR, et al. Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocytes recruitment in experimental glomerulonephritis. Kidney International 2001;59(5):1762–9. Wada T, Yokoyama H, Furiichi K, Kobayashi KI, Harada K, Naruto M, et al. Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1). FASEB Journal 1996;10(12):1418–25. Lee EY, Chung CH, Khoury CC, Yeo TK, Pyagay PE, Wang A, et al. The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin permeability. American Journal of Physiology Renal Physiology 2009;297(1):F85–94. Ruiz-Ortega M, Ruperez M, Lorenzo O, Esteban V, Blanco J, Mezzano S, et al. Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney. Kidney International Supplement 2002;82: S12–22. Tanifuji C, Suzuki Y, Geot WM, Horikoshi S, Sugaya T, Ruiz-Ortega M, et al. Reactive oxygen species-mediated signaling pathways in angiotensin II-induced MCP-1 expression of proximal tubular cells. Antioxidants and Redox Signalling 2005;7(9–10):1261–8. Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, et al. Cellular events in the evolution of experimental diabetic nephropathy. Kidney International 1995;47(3):935–44. Osterby R, Parving HH, Hommel E, Jorgensen HE, Lokkegaard H. Glomerular structure and function in diabetic nephropathy. Early to advanced stages. Diabetes 1990;39(9):1057–63. Couchman JR, Beavan LA, McCarthy KJ. Glomerular matrix: synthesis, turnover and role in mesangial expansion. Kidney International 1994;45(2): 328–35.

S. Paccosi et al. / Pharmacological Research 66 (2012) 526–535 [32] Shindo T, Kurihara H, Maemura K, Kurihara Y, Ueda O, Suzuki H, et al. Renal damage and salt-dependent hypertension in aged transgenic mice overexpressing endothelin-1. Journal of Molecular Medicine (Berlin) 2002;80(2):105–16. [33] Mishra R, Leahy P, Simonson MS. Gene expression profile of endothelin-1induced growth in glomerular mesangial cells. American Journal of Physiology Cell Physiology 2003;285(5):C1109–15. [34] Yang LL, Gros R, Kabir MG, Sadi A, Gotlieb AI, Husain M, et al. Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation 2004;109(2):255–61. [35] Simonson MS, Ismail-Beigi F. Endothelin-1 increases collagen accumulation in renal mesangial cells by stimulating a chemokine and cytokine autocrine signaling loop. Journal of Biological Chemistry 2011;286(13): 11003–8. [36] Kazes I, Delarue F, Hagège J, Bouzhir-Sima L, Rondeau E, Sraer JD, et al. Soluble latent membrane-type 1 matrix metalloprotease secreted by human

[37]

[38]

[39]

[40]

535

mesangial cells is activated by urokinase. Kidney International 1998;54(6): 1976–84. Werb Z, Tremble PM, Behrendtsen O, Crowley E, Damsky CH. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. Journal of Cell Biology 1989;109(2):877–89. Martin J, Eynstone L, Davies M, Steadman R. Induction of metalloproteinases by glomerular mesangial cells stimulated by proteins of the extracellular matrix. Journal of the American Society of Nephrology 2001;12(1):88–96. Kitamura A, Kagami S, Urushihara M, Kondo S, Yoshizumi M, Tamaki T, et al. Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells. Biochemical and Biophysical Research Communications 2002;299(4):555–61. Turck J, Pollock AS, Lee LK, Marti HP, Lovett DH. Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation. Journal of Biological Chemistry 1996;271(25):15074–83.