Differential impact of glucose levels and advanced glycation end-products on tubular cell viability and pro-inflammatory/ profibrotic functions Benoit Franko a,b , Julie Brault b,c , Thomas Jouve a , Sylvain Beaumel b,c , Pierre-Yves Benhamou d , Philippe Zaoui a , Marie José Stasia b,c,⇑ a Nephrology Clinic, Grenoble University Hospital, Grenoble F-38043, France b TIMC/Therex Laboratory, UMR 5525 (CNRS-UJF) Faculty of Medicine, Univ. Grenoble Alpes, Grenoble F-38041, France c Chronic Granulomatous Disease Diagnosis and Research Centre (CDiReC), Pôle Biologie, CHU de Grenoble, Grenoble F-38043, France d Department of Endocrinology, Grenoble University Hospital, Grenoble F-38043, France article info Article history: Received 4 August 2014 Available online xxxx Keywords: Diabetic nephropathy Tubular cells Glucose AGE Inflammation ROS abstract High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally used to mimic diabetes in cellular models. Both models have shown an increase of apoptosis, oxidative stress and pro-inflammatory cytokine production in tubular cells. However, the impact of the two conditions com- bined has rarely been studied. In addition, the impact of glucose level variation due to cellular consump- tion is not clearly characterized in such experiments. Therefore, the aim of this study was to compare the effect of HG and AGE separately and of both on tubular cell phenotype changes in the HK2 cell line. More- over, glucose consumption was monitored every hour to maintain the glucose level by supplementation throughout the experiments. We thus observed a significant decrease of apoptosis and H 2 O 2 production in the HK2 cell. HG or AGE treatment induced an increase of total and mitochondrial apoptosis as well as TGF-b release compared to control conditions; however, AGE or HG led to apoptosis preferentially involv- ing the mitochondria pathway. No cumulative effect of HG and AGE treatment was observed on apopto- sis. However, a pretreatment with RAGE antibodies partially abolished the apoptotic effect of HG and completely abolished the apoptotic effect of AGE. In conclusion, tubular cells are sensitive to the lack of glucose as well as to the HG and AGE treatments, the AGE effect being more deleterious than the HG effect. Absence of a potential synergistic effect of HG and AGE could indicate that they act through a common pathway, possibly via the activation of the RAGE receptors. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Diabetic nephropathy (DN) remains the main cause of end- stage renal disease and is still a challenge [1], affecting vascular, glomerular and tubular components. Several pathways are studied in glomerular cells involving polyol and hexosamine pathways [2], such as oxidative stress [3]. Nonenzymatic linkage of glucose with amino groups leads to advanced glycation end-products (AGE), whose levels increase in diabetes [4] and chronic kidney diseases [5]. They are responsible for oxidative stress by inducing reactive oxygen species (ROS) production. ROS production is triggered by PKC, NADPH oxydase (NOX) or mitochondrial activation [6]. Fur- thermore, AGE and HG induce TGF-b1 activation and ROS, leading to increased connective tissue growth factor [7], followed by extra- cellular matrix expansion, epithelial–mesenchymal transition and tubular atrophy [8]. Tubular injuries in DN have only recently been investigated and seem to be preferentially associated with the loss of kidney function rather than glomerular lesions [9]. Two in vitro conditions are currently available to mimic diabe- tes: high glucose (HG) and AGE exposure. However, only few stud- ies have analyzed the effect of HG and AGE together. The effects of http://dx.doi.org/10.1016/j.bbrc.2014.08.042 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. Abbreviations: AGE, advanced glycation end-products; AGE-BSA, glycated bovine serum albumin; BSA, bovine serum albumin; DHR, Dihydrorhodamine; DIOC6, 3,3 0 -dihexyloxacarbocyanine iodide; DMEM, Dulbecco’s minimum essential medium; DN, diabetic nephropathy; GLUT, glucose transporter; HG, high glucose condition; KSFM, keratinocyte-serum free medium; LG, low-glucose condition; M, Mannitol condition; NOX, NADPH oxidase; RAGE, receptor for advanced glycation end-product; ROS, reactive oxygen species; SGLT2, sodium/glucose co-transporter 2; TGF b, transforming growth factor beta. ⇑ Corresponding author at: CDiReC, Institut de Biologie et Pathologie, CHU de Grenoble, BP 217, 38043 Grenoble Cedex 9, France. Fax: +33 476765608. E-mail address: mjstasia@chu-grenoble.fr (M.J. Stasia). Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Please cite this article in press as: B. Franko et al., Differential impact of glucose levels and advanced glycation end-products on tubular cell viability and pro-inflammatory/ profibrotic functions, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.08.042