Clinical Science (2013) 124, 191–202 (Printed in Great Britain) doi: 10.1042/CS20120330 Renoprotective effects of a novel Nox1/4 inhibitor in a mouse model of Type 2 diabetes Mona SEDEEK ∗ , Alex GUTSOL ∗ , Augusto C. MONTEZANO ∗ , Dylan BURGER ∗ , Aurelie NGUYEN DINH CAT ∗ , Chris R. J. KENNEDY ∗ , Kevin D. BURNS ∗ , Mark E. COOPER†, Karin JANDELEIT-DAHM†, Patrick PAGE‡, Cedric SZYNDRALEWIEZ‡, Freddy HEITZ‡, Richard L. HEBERT ∗ and Rhian M. TOUYZ ∗ § ∗ Kidney Research Center, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada †Diabetic Complications Division, Baker IDI Heart and Diabetes Research Institute, Melbourne, Australia ‡GenKyoTex S.A., Geneva, Switzerland §Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, U.K. Abstract Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFβ (transforming growth factor β ) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process. Key words: db/db mouse, diabetes, kidney, NADPH oxidase, NOX inhibitor, oxidative stress INTRODUCTION Diabetes mellitus is the leading cause of ESRD (end-stage renal disease), with nephropathy accounting for >35 % of incident cases of ESRD in developed countries [1,2]. Clinical features of diabetic nephropathy include albuminuria and reduced GFR (glomerular filtration rate), which occur in association with in- creased BP (blood pressure), ultimately leading to ESRD [3]. These changes develop as a consequence of glomerular, tubular, Abbreviations: AGE, advanced glycation end-product; BP , blood pressure; CCSA, capillary cross-sectional area; CKD, chronic kidney disease; ERK, extracellular-signal-regulated kinase; ESRD, end-stage renal disease; GCSA, glomerular cross-sectional area; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MCSA, mesangial cross-sectional area; NMC, number of mesangial cells; Nox, NADPH oxidase; ROS, reactive oxygen species; SOD-1, superoxide dismutase 1; TBARS, thiobarbituric acid-reacting substances; TGFβ, transforming growth factor β; VCAM-1, vascular cell adhesion molecule 1. Correspondence: Professor Rhian M. Touyz (email rhian.touyz@glasgow.ac.uk or rtouyz@uottawa.ca). vascular and interstitial damage, characterized by thickening of the glomerular basement membrane, glomerulosclerosis and tu- bulointerstitial fibrosis. Associated with these structural changes is monocyte–macrophage invasion, mesangial cell hyperplasia, matrix expansion and podocytopenia [4,5]. Molecular mechanisms underlying diabetic nephropathy remain elusive, but hyperglycaemia and activation of the RAS (renin–angiotensin system) are important [6,7]. Com- mon to these processes is oxidative stress, defined as damage www.clinsci.org 191