Functional Polymorphism of the Myeloperoxidase Gene in Hypertensive Nephrosclerosis Dialysis Patients

1193 Hypertens Res Vol.30 (2007) No.12 p.1193-1198 Original Article Functional Polymorphism of the Myeloperoxidase Gene in Hypertensive Nephrosclerosis Dialysis Patients Kent DOI1),2), Eisei NOIRI1),3), Rui MAEDA1),3), Akihide NAKAO1), Shuzo KOBAYASHI4), Katsushi TOKUNAGA2), and Toshiro FUJITA1) Myeloperoxidase (MPO) may play an important role not only in host defense reactions but also in local inflammations, especially in atherosclerotic diseases such as hypertensive nephrosclerosis (HN). Paradoxically, MPO-deficient mice have been reported to show increased atherosclerosis compared with wild mice, although higher MPO levels are thought to exacerbate atherosclerotic disease. To clarify the genetic role of MPO in HN, we examined the function and distribution of the – 463G/A polymorphism located in the promoter region of the MPO gene with ex vivo flow cytometry analysis and a study in end-stage renal disease patients, respectively. This polymorphism has been reported to have a functional significance in vitro, with the A allele being associated with lower MPO expression. In the present study, we also found significantly higher reactive oxygen species (ROS) production with peripheral neutrophils isolated from subjects with the GG genotype compared with those from subjects with other genotypes by flow cytometry assay with 2-[6(4′-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), which shows higher sensitivity with hypochlorite (OCl –). Genotyping the – 463G/A polymorphism in HN, chronic glomerulonephritis (CGN) and diabetic nephropathy (DM) patients who were under hemodialysis treatment demonstrated that the GG genotype was more frequent in the HN group than in the CGN and DM groups. However, the distribution of the GG genotype in the HN group was similar to that in healthy individuals. Although the – 463G/A polymorphism is associated with ROS production, careful interpretation may be required to conclude that the – 463G/A polymorphism can serve as a useful marker of atherosclerosis and cardiovascular events in dialysis patients. (Hypertens Res 2007; 30: 1193–1198) Key Words: hypertensive nephrosclerosis, dialysis, myeloperoxidase, polymorphism, flow cytometry Introduction Myeloperoxidase (MPO) is a heme protein derived from leukocytes and generates specific oxidants such as hypochlorous acid. It may play an important role not only in host defense reactions but also in local inflammations, especially in atherosclerotic lesions (1). Zhang et al. (2) demonstrated that elevated MPO levels were associated with the presence of coronary artery disease. Given the presence of MPO in ath- From the 1)Department of Nephrology and Endocrinology, 2)Department of Human Genetics, and 3)Center for Nanobiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and 4)Department of Nephrology and Kidney and Dialysis Center, Shonan Kamakura General Hospital, Kamakura, Japan. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) “Medical Genome Science” (to K.T.), a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (14370315 to E.N.), the BioBank Japan Project on the Implementation of Personalized Medicine, MEXT, Japan (3023168 to E.N.), and a grant from the Cell Science Research Foundation (to E.N.). Address for Reprints: Eisei Noiri, M.D, Ph.D., Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, 7–3– 1 Hongo, Bunkyo-ku, Tokyo 113–8655, Japan. E-mail: Received March 13, 2007; Accepted in revised form July 11, 2007. 1194 Hypertens Res Vol. 30, No. 12 (2007) b Cell count 100 80 GA GG AA P = 0.002 Relative fluorescence intensity a 60 40 20 4 3 2 1 0 100 101 102 Fluorescence intensity 103 GG n=6 GA+AA n=4 Fig. 1. Association of the − 463G/A polymorphism with ROS production in neutrophils. A representative histogram of the fluorescence intensity measured by APF (a) and results of the quantitative analysis of ROS production (b) are shown. ROS, reactive oxygen species; APF, 2-[6-(4′-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid. erosclerotic plaques, higher MPO levels may exacerbate atherosclerotic disease. However, the role of MPO in atherosclerosis is still unclear because MPO-deficient mice unexpectedly show increased atherosclerosis (3) and increased cerebral infarction volume (4) compared with wild mice. The −463G/A polymorphism in the promoter region of the MPO gene has been detected in patients with leukemia (5). This polymorphism is in an Alu element in the promoter region, and the transition from G to A results in the loss of an SP1-binding site. SP1 is a strong transcription factor, and therefore the A allele is associated with lower MPO expression in vitro (6). In the present study, we investigated the association of the −463G/A polymorphism with ex vivo reactive oxygen species (ROS) production in peripheral neutrophils. The −463G/A polymorphism has also been associated with diseases such as acute leukemia (7), Alzheimer’s disease (8) and lung cancer (9). Recently, Asselbergs et al. (10) showed that the GG genotype, which might be associated with higher MPO levels, was related to an increased risk of cardiovascular events. Pecoits-Filho et al. (11) reported that the GG genotype is associated with higher prevalence of cardiovascular complications in predialysis end-stage renal disease (ESRD) patients. Hypertensive nephrosclerosis (HN) has been recognized as a clinical syndrome characterized by long-term essential hypertension, hypertensive retinopathy, left ventricular hypertrophy, proteinuria, and progressive renal insufficiency. Long-term severe hypertension causes atherosclerotic changes in the renal vessels and subsequent renal hypoxia, which plays an important role in the progression of kidney disease (12). Mesangial α-smooth muscle actin and collagen type III expression have also been reported in renal biopsy specimens of HN patients (13). HN is the third most frequent diagnosis in patients starting dialysis and its incidence is increasing in Japan (Annual Report of the Japanese Society of Dialysis Therapy 2005). In the present study, we hypothesized that the −463G/A polymorphism might be associated with HN and examined the association of the −463G/A polymorphism with HN and non-HN dialysis patients and healthy controls in order to clarify the effects of MPO genetic polymorphism on ESRD caused by HN. Methods Measurement of ROS Production of Peripheral Neutrophils We examined ROS production of neutrophils by flow cytometry in 10 healthy individuals (−463GG: n= 6; −463GA: n= 3; −463AA: n= 1). Peripheral polymorphemic neutrophils (PMNs) were isolated using Polymorphprep (Axis-Shield PoC AS, Oslo, Norway). After incubation with 2-[6-(4′amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF; Dai-ichi Chem, Tokyo, Japan) at a concentration of 10 μmol/ L for 30 min at room temperature, dye-loaded PMNs were stimula (...truncated)