The chemokine (C-C motif) ligand protein synthesis inhibitor bindarit prevents cytoskeletal rearrangement and contraction of human mesangial cells Sara Paccosi a , Matelda Giachi a , Paola Di Gennaro c , Angelo Guglielmotti b , Astrid Parenti a,⇑ a Department of Health Sciences, Clinical Pharmacology and Oncology Section, University of Florence, Florence, Italy b Angelini Research Center, S. Palomba-Pomezia, Rome, Italy c Unit of Plastic and Reconstructive Surgery – Regional Melanoma Referral Center, Tuscan Tumor Institute (ITT), Santa Maria Annunziata Hospital, Florence, Italy article info Article history: Received 19 January 2016 Received in revised form 6 June 2016 Accepted 7 June 2016 Keywords: CCL2 Bindarit Mesangial cell contraction Cytoskeletal rearrangement a-smooth muscle actin Vinculin abstract Intraglomerular mesangial cells (MCs) maintain structural and functional integrity of renal glomerular microcirculation and homeostasis of mesangial matrix. Following different types of injury, MCs change their phenotype upregulating the expression of a-smooth muscle actin (a-SMA), changing contractile abilities and increasing the production of matrix proteins, chemokines and cytokines. CCL2 is a chemo- kine known to be involved in the pathogenesis of renal diseases. Its glomerular upregulation correlates with the extent of renal damage. Bindarit is an indazolic derivative endowed with anti-inflammatory activity when tested in experimental diseases. It selectively inhibits the synthesis of inflammatory C-C chemokines including CCL2, CCL7 and CCL8. This work aims to analyse bindarit effects on ET1-, AngII- and TGFb-induced mesangial cell dysfunction. Bindarit significantly reduced AngII-, ET1- and TGFb-induced a-SMA upregulation. In a collagen contraction assay, bindarit reduced AngII-, ET1- and TGFb-induced HRMC contraction. Within 3–6 h stimulation, vinculin organization and phosphorylation was significantly impaired by bindarit in AngII-, ET1- and TGFb-stimulated cells without any effect on F-actin distribution. Conversely, p38 phosphorylation was not significantly inhibited by bindarit. Our data strengthen the importance of CCL2 on ET-1, AngII- and TGFb-induced mesangial cell dysfunction, adding new insights into the cellular mechanisms responsible of bindarit protective effects in human MC dysfunction. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction Intraglomerular mesangial cells (MCs) are secretory and contractile glomerular cells located among the glomerular capillaries within kidney’s renal corpuscles. They offer structural support to glomerular vascular architecture, regulate the turnover of glomerular extracellular matrix and concur to the regulation of glomerular filtration [1]. Many injurious stimuli can modulate MC’s phenotype: they can acquire characteristics of smooth muscle cells (SMC) or fibroblasts, like a-SMA expression [2] or interstitial collagens, fibronectin, laminin production, thus also changing their contractile capacity [1]. Angiotensin II (AngII), arginine, vaso- pressin, endothelin-1 (ET1), TGFb, histamine and thrombin are among agonists involved in mesangial dysfunction [3–5]. The role of AngII in the pathogenesis of renal damage is sup- ported by beneficial effect of ACE inhibitors on microalbuminuria and reduced CCL2 excretion in type 2 diabetic patients [6]. ET1 over-expression has been reported to cause mesangial cell remod- eling in vitro and glomerulosclerosis and collagen deposition in in vivo animal model [7]. TGFb plays a significant role in progres- sive renal disease, since it stimulates collagen deposition from mesangial cells [8] and mediates AngII effects on renal fibrosis [9]. CCL2 has also been suggested playing a key role, since its tubular up-regulation has been demonstrated in animal models of diabetic nephropathy [10,11]. In human it has been shown correlation in progressive renal diseases [12,13] with urinary CCL2 [14], albumin http://dx.doi.org/10.1016/j.cyto.2016.06.012 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved. Abbreviations: AngII, angiotensin II; CCL2, Chemokine (C-C motif) ligand-2; ECM, extracellular matrix; ET1, endothelin 1; FCS, fetal calf serum; GMCs, glomerular mesangial cells; HRMCs, human renal mesangial cells; MAPK, mitogen-activated protein kinases; MCs, mesangial cells; PBS, phosphate buffered saline; SMCs, smooth muscle cells; a-SMA, a-smooth muscle actin; TGFb, trans- forming growth factor-b. ⇑ Corresponding author at: Department of Health Sciences, Clinical Pharmacology and Oncology Section, University of Florence, V.le G. Pieraccini, 6, 50139 Florence, Italy. E-mail address: astrid.parenti@unifi.it (A. Parenti). Cytokine 85 (2016) 92–100 Contents lists available at ScienceDirect Cytokine journal homepage: www.journals.elsevier.com/cytokine