Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells

RESEARCH ARTICLE Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells Soon-Youn Choi1,2,3, Hye-Myung Ryu1,3, Eun-Joo Oh1, Ji-Young Choi1, Jang-Hee Cho1, Chan-Duck Kim1,3, Yong-Lim Kim1,2,3, Sun-Hee Park1,3* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Division of Nephrology and Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Korea, 2 BK21 Plus Biomedical Convergence Program, Department of Biomedical Science, Kyungpook National University, Daegu, Korea, 3 Cell and Matrix Research Institute, Kyungpook National University, Daegu, Korea * Abstract OPEN ACCESS Citation: Choi S-Y, Ryu H-M, Oh E-J, Choi J-Y, Cho J-H, Kim C-D, et al. (2017) Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells. PLoS ONE 12(7): e0180393. https://doi.org/10.1371/ journal.pone.0180393 Editor: Xing-Ming Shi, Augusta University, UNITED STATES Received: March 15, 2017 Accepted: June 14, 2017 Published: July 7, 2017 Copyright: © 2017 Choi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are included within the paper. Funding: This study was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (HI15C0001; HI13C1232), funded by the Ministry of Health & Welfare, Republic of Korea (www.khidi.or.kr) (YLK, CDK). The funders had no role in study design, data collection and Although dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, have various pleiotropic effects, it remains undetermined whether gemigliptin has a beneficial effect on vascular calcification. Therefore, this study was performed to evaluate the effect of gemigliptin on vascular calcification in a rat model of adenine-induced chronic kidney disease and in cultured vascular smooth muscle cells. Gemigliptin attenuated calcification of abdominal aorta and expression of RUNX2 in adenine-induced chronic kidney disease rats. In cultured vascular smooth muscle cells, phosphate-induced increase in calcium content was reduced by gemigliptin. Gemigliptin reduced phosphate-induced PiT-1 mRNA expression, reactive oxygen species generation, and NADPH oxidase mRNA expression (p22phox and NOX4). The reduction of oxidative stress by gemigliptin was associated with the downregulation of phospho-PI3K/AKT expression. High phosphate increased the expression of frizzled-3 (FDZ3) and decreased the expression of dickkopf-related protein-1 (DKK-1) in the Wnt pathway. These changes were attenuated by gemigliptin treatment. Gemigliptin restored the decreased expression of vascular smooth muscle cells markers (α-SMA and SM22α) and increased expression of osteogenic makers (CBFA1, OSX, E11, and SOST) induced by phosphate. In conclusion, gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps including downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway. Introduction Vascular calcification (VC) occurs more frequently in patients with chronic kidney disease (CKD) and diabetes mellitus (DM), and usually affects blood vessels including the aorta as well PLOS ONE | https://doi.org/10.1371/journal.pone.0180393 July 7, 2017 1 / 18 DPP-4 inhibitor gemigliptin protects against vascular calcification analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. as medium- and small-sized vessels such as coronary arteries [1]. It is characterized by accelerated mineral deposition within the medial layer of arteries. VC increases the stiffness of the arterial wall and negatively influences heart function by increasing cardiac afterload and left ventricular hypertrophy, and decreasing coronary blood flow. Clearly, VC has an impact on cardiovascular events and mortality in CKD patients as well as patients with DM [2–4]. Hyperphosphatemia, one of the major abnormalities in CKD-mineral bone disorder (MBD), is primarily associated with VC in patients with kidney disease. Previously, high phosphate-induced VC was reported to indicate passive calcium-phosphate deposition [5]. However, recently VC has been recognized as a highly active process. It is associated with a multifactorial mechanism, which includes calcium/phosphate dysregulation, calciprotein particles, impaired anti-calcific mechanism such as dysfunction of inhibitors, and trans-differentiation of vascular smooth muscle cell (VSMC) phenotype. VSMCs trans-differentiation is characterized by loss of VSMC marker proteins [smooth muscle (SM) α-actin and SM22α] and gain of osteoblast marker proteins [runt-related transcription factor-2 (RUNX2; also called CBFA1), osterix (OSX), osteocalcin (OC), DMP-1, sclerostin (SOST), and E11]. This is a process similar to physiological bone formation [6]. In addition, Wnt signaling has been reported as a main master regulator for activating the expression of osteoblast trans-differentiation markers to induce VC [7]. Wnt proteins bind to the plasma membrane frizzled (FDZ) receptors and low-density lipoprotein receptor-related protein-5/6 (LRP5/6) co-receptor, and regulate downstream signaling by dephosphorylation of β-catenin. Activation of Wnt signaling regulates trans-differentiation of the osteogenic phenotype through the expression of several bone-related proteins such as osterix (OSX), osteocalcin (OC), and sclerostin (SOST) [8]. Dipeptidyl peptidase-4 (DPP-4) inhibitors, novel antidiabetic drugs, have the ability to control blood glucose by inhibiting the degradation of incretin hormones such as type I glucagonlike peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) without the risk of hypoglycemia or body weight gain [9, 10]. Besides, recent studies have suggested that DPP-4 inhibitors have cardio-protective effects in addition to their glucose-lowering effect in experimental studies [11–13]. The DPP-4 inhibitor des-fluoro-sitagliptin reduces restenosis in the carotid artery following balloon injury in type 2 DM rats [14]. Sitagliptin treatment in ApoE KO mice reduced plaque inflammation through inhibition of monocyte migration and macrophage MMP-9 release [15]. Linagliptin significantly reduced neointima formation in a vascular injury model of non-diabetic mice [13], and in obstructed aortic and endothelial stiffness induced by a western diet in female mice [16]. Cardiovascular pleiotropic actions of DPP-4 inhibitors in (...truncated)