NADPH Oxidase Nox5 Accelerates Renal Injury in Diabetic Nephropathy Jay C. Jha, 1,2 Claudine Banal, 1 Jun Okabe, 2,3 Stephen P. Gray, 1 Thushan Hettige, 1 Bryna S.M. Chow, 1,2 Vicki Thallas-Bonke, 1 Lisanne De Vos, 1 Chet E. Holterman, 4 Melinda T. Coughlan, 1,2 David A. Power, 5 Alison Skene, 6 Elif I. Ekinci, 7 Mark E. Cooper, 1,2 Rhian M. Touyz, 8 Chris R. Kennedy, 4 and Karin Jandeleit-Dahm 1,2 Diabetes 2017;66:2691–2703 | https://doi.org/10.2337/db16-1585 NADPH oxidase–derived excessive production of reac- tive oxygen species (ROS) in the kidney plays a key role in mediating renal injury in diabetes. Pathological changes in diabetes include mesangial expansion and accumula- tion of extracellular matrix (ECM) leading to glomerulo- sclerosis. There is a paucity of data about the role of the Nox5 isoform of NADPH oxidase in animal models of dia- betic nephropathy since Nox5 is absent in the mouse ge- nome. Thus, we examined the role of Nox5 in human diabetic nephropathy in human mesangial cells and in an inducible human Nox5 transgenic mouse exposed to streptozotocin- induced diabetes. In human kidney biopsies, Nox5 was iden- ti fied to be expressed in glomeruli, which appeared to be increased in diabetes. Colocalization demonstrated Nox5 expression in mesangial cells. In vitro, silencing of Nox5 in human mesangial cells was associated with attenuation of the hyperglycemia and TGF-b1–induced enhanced ROS production, increased expression of profibrotic and proin- flammatory mediators, and increased TRPC6, PKC-a, and PKC-b expression. In vivo, vascular smooth muscle cell/ mesangial cell –specific overexpression of Nox5 in a mouse model of diabetic nephropathy showed enhanced glomer- ular ROS production, accelerated glomerulosclerosis, mesangial expansion, and ECM protein (collagen IV and fibronectin) accumulation as well as increased macro- phage infiltration and expression of the proinflammatory chemokine MCP-1. Collectively, this study provides evi- dence of a role for Nox5 and its derived ROS in promoting progression of diabetic nephropathy. Diabetic nephropathy (DN), a leading cause of end-stage renal disease, is characterized by the progressive expansion of the mesangium with accumulation of extracellular matrix (ECM) components in both the glomerular mesangium and basement membrane, ultimately leading to glomeruloscle- rosis (1). Multiple factors in diabetes contribute to the de- velopment of increased accumulation of mesangial matrix (2). The activation of profibrotic growth factors and cyto- kines by hyperglycemia leads to an increase in the synthesis of most collagens and fibronectin (3). Several studies support the finding that renal reactive oxygen species (ROS) play a central role in mediating renal injury in diabetes (4–6). A range of enzymes are capable of generating ROS, but the prooxidant enzyme family of NADPH oxidases, Nox, are the only enzymes known to be solely dedicated to ROS generation in the kidney (7–10). Indeed, we previously showed that the Nox4 isoform con- tributes to renal injury in a mouse model of DN and that targeting of Nox4 using global or podocyte-specific genetic deletion or pharmacological inhibition resulted in attenuation 1 JDRF Danielle Alberti Memorial Centre for Diabetic Complications, Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia 2 Department of Diabetes, Central Clinical School, Monash University, Mel- bourne, Australia 3 Human Epigenetics Laboratory, Baker IDI Heart and Diabetes Institute, Mel- bourne, Australia 4 Kidney Research Centre, Department of Medicine, Ottawa Hospital Research Institute, Ottawa, Canada 5 Department of Nephrology and Institute of Breathing and Sleep, Austin Health, Heidelberg, Australia 6 Department of Anatomical Pathology, Austin Health, Heidelberg, Australia 7 Endocrine Centre, Austin Health, Repatriation Campus, Heidelberg, Australia 8 Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glas- gow, U.K. Corresponding author: Karin Jandeleit-Dahm, karin.jandeleit-dahm@monash .edu. Received 4 January 2017 and accepted 18 July 2017. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1585/-/DC1. © 2017 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Diabetes Volume 66, October 2017 2691 COMPLICATIONS Downloaded from http://diabetesjournals.org/diabetes/article-pdf/66/10/2691/534390/db161585.pdf by guest on 09 November 2022