Ezetimibe Reduces Urinary Albumin Excretion in Hypercholesterolaemic Type 2 Diabetes Patients with Microalbuminuria

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1 The Journal of International Medical Research 2012; 40: Ezetimibe Reduces Urinary Albumin Excretion in Hypercholesterolaemic Type 2 Diabetes Patients with Microalbuminuria T NAKAMURA 1, E SATO 1, M AMAHA 1, Y KAWAGOE 1, S MAEDA 2, H INOUE 3 AND SI YAMAGISHI 2 1 Division of Nephrology, Department of Internal Medicine, Shinmatsudo Central General Hospital, Chiba, Japan; 2 Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan; 3 Department of Chemistry, Keio University School of Medicine, Yokohama, Japan OBJECTIVE: This study investigated the effects of ezetimibe, an inhibitor of intestinal cholesterol absorption, on early phase diabetic nephropathy. METHODS: A total of 32 hypercholesterolaemic type 2 diabetes patients with microalbuminuria, defined as a urinary albumin excretion (UAE) 30 but < 300 mg/g creatinine, were enrolled. Various clinical and laboratory parameters were determined at baseline and after 6 months of treatment with 10 mg/day ezetimibe. RESULTS: Ezetimibe treatment significantly decreased glycated haemoglobin (HbA 1c ), low-density lipoprotein-cholesterol (LDL-C), triglycerides and UAE, and significantly increased high-density lipoproteincholesterol and albumin. It also decreased the serum level of monocyte chemoattractant protein-1 (MCP-1), but this difference was not statistically significant. Univariate analyses showed a correlation between UAE and body mass index, systolic and diastolic blood pressures, HbA 1c, LDL-C, estimated glomerular filtration rate (inverse), creatinine and MCP-1. Since these parameters may be closely correlated with each other, multiple stepwise regression analysis was performed and demonstrated that HbA 1c and MCP-1 were independent determinants of UAE. CONCLUSIONS: Ezetimibe may be a promising therapeutic strategy for improving albumin excretion, partly through its anti-inflammatory properties, and for reducing LDL-C in hypercholesterolaemic type 2 diabetes patients with microalbuminuria. KEY WORDS: EZETIMIBE; DIABETIC NEPHROPATHY; ALBUMINURIA; HYPERCHOLESTEROLAEMIA; MONOCYTE CHEMOATTRACTANT PROTEIN-1 Introduction Microalbuminuria in type 2 diabetes is not only a marker for diabetic nephropathy, but is also a predictor for future cardiovascular events. 1 Hypercholesterolaemia, a wellknown risk factor for cardiovascular disease (CVD), has been reported to contribute to the progression of chronic kidney disease 798

2 (CKD). 2,3 Since statins have been shown to offer significant reductions in cardiovascular and/or renal morbidity in type 2 diabetes with albuminuria, 2,3 control of hypercholesterolaemia may be an appropriate therapeutic target for preventing the onset of CVD and slowing the progression of CKD in these patients. There is, however, still controversy as to whether control of hypercholesterolaemia by statins is effective in reducing urinary albumin excretion (UAE) in type 2 diabetes patients with microalbuminuria. 3,4 Ezetimibe is a lipid-lowering agent that selectively inhibits intestinal cholesterol absorption. 5 To our knowledge, little has been published on the effects of ezetimibe on microalbuminuria in patients with type 2 diabetes. The present study investigated the effect of ezetimibe treatment on UAE in type 2 diabetes patients with microalbuminuria. Patients and methods STUDY POPULATION Consecutive type 2 diabetes patients with hypercholesterolaemia, defined as a lowdensity lipoprotein-cholesterol (LDL-C) serum concentration 140 mg/dl, and micro - albuminuria, defined as a UAE 30 mg/g creatinine but < 300 mg/g creatinine, from Shinmatsudo Central General Hospital, Chiba, Japan, were enrolled in the study between January and December Patients with chronic pulmonary disease, collagen disease, liver disease, neoplastic disorders, those who had recent (< 6 months) acute coronary syndrome, stroke or any acute infections, and those with a serum creatinine level > 1.2 mg/dl were excluded from the study. All patients received 10 mg ezetimibe once daily, with no statins or other antihypercholesterolaemic agents, for 6 months. During the study period, subjects were instructed not to change their lifestyles and to continue taking the same dose of any concomitant drugs. Written informed consent was obtained from all the study participants. The study protocol was approved by the Ethical Committee of Shinmatsudo Central General Hospital and the study complied with the principles of the Helsinki Declaration. CLINICAL AND LABORATORY ASSESSMENTS Patients were assessed at baseline and following 6 months of treatment with ezetimibe. Height and weight were measured, and body mass index (BMI) was calculated. Measurements of systolic and diastolic blood pressure were made in the sitting position using an upright standard sphygmomanometer. Blood was collected into Vacutainer tubes (BD, Franklin Lakes, NJ, USA) without ethylenediaminetetraacetic acid or heparin after a 12-h fast. Serum was then separated by centrifugation (3000 rpm, 15 min) and stored at 80 C until use for determination of serum glycated haemoglobin (HbA 1c ), serum creatinine, blood urea nitrogen, serum albumin, serum monocyte chemoattractant protein-1 (MCP- 1) and serum lipid profiles, including LDL-C, triglycerides (TG) and HDL-cholesterol (HDL- C. Standard enzymatic methods or enzymelinked immunosorbent assays were used as described previously. 5,6 The LDL-C level was calculated using Friedewald s formula. 7 A 24-h urine sample was collected from each patient and stored at 80 C until use. Urinary albumin excretion was measured using an immunoturbidimetry assay in a Hitachi 7070 automated analyser (Hitachi, Tokyo, Japan). Estimated glomerular filtration rate (egfr) was calculated according to the Modification of Diet in Renal Disease equation modified for the Japanese population

3 STATISTICAL ANALYSES All statistical analyses were performed using SPSS statistical software, version 18 (SPSS Inc., Chicago, IL, USA) for Windows. The mean ± SD for data were calculated as appropriate. For statistical comparisons of values at baseline and after ezetimibe treatment, a paired, two-tailed dependent t- test was used. Correlations between clinical and laboratory parameters and UAE were calculated using univariate analyses, and independent determinants of UAE were determined using multiple stepwise regression analysis. A P-value < 0.05 was considered to be statistically significant. Results A total of 32 type 2 diabetes patients with hypercholesterolaemia and microalbumin - uria were enrolled in the study. Their demographic and clinical characteristics are given in Table 1. Clinical and laboratory variables at baseline and after 6 months of ezetimibe treatment are shown in Table 2. Ezetimibe treatment was associated with significant decreases in serum HbA 1c, LDL-C, TG and UAE, and significant increases in serum HDL-C and albumin. The mean concentration of serum MCP-1 decreased after ezetimibe treatment, but the difference between the two groups was not statistically significant. Univariate analysis showed a significant correlation between UAE and BMI, systolic and diastolic blood pressures, HbA 1c, LDL-C, egfr (inverse), creatinine and MCP-1 (P < 0.05 for all comparisons; Table 3). Since these parameters may be closely correlated with each other, multiple stepwise regression analysis was performed and demonstrated that HbA 1c and MCP-1 were independent determinants of UAE (coefficient of determination R 2 = 0.280). Discussion The present study demonstrated that ezetimibe treatment for 6 months significantly reduced UAE in type 2 diabetes patients with hypercholesterolaemia and microalbumin - uria. Although ezetimibe treatment only modestly and nonsignificantly decreased serum MCP-1 protein levels, multiple regression analysis showed that serum MCP-1 and serum HbA 1c, but not lipid parameters, were independent determinants of UAE. The level of circulating MCP-1 has been shown to be significantly higher in type 1 diabetes patients with microalbuminuria and poorer glycaemic control compared with normoalbuminuric patients and healthy controls. 9 Furthermore, type 2 diabetes patients with macroalbuminuria have a significantly higher circulating MCP-1 concentration than those with normo- or microalbuminuria. 10 Given the pathological role of MCP-1 in the early phase of diabetic TABLE 1: Demographic and clinical characteristics of type 2 diabetes patients with hypercholesterolaemia and microalbuminuria (n = 32) Age, years 61.5 ± 8.6 Gender Males 11 Females 21 Diabetic retinopathy Simple 18 Proliferative 10 Cardiovascular disease 10 Diabetes medication Sulphonylureas 8 Biguanides 2 α-glucosidase inhibitors 11 Pioglitazone 6 Insulin 9 Hypertension medication Angiotensin II type 1 receptor blockers 16 Calcium channel blockers 11 Data presented as mean ± SD or n of patients. 800

4 TABLE 2: Clinical and laboratory parameters in type 2 diabetes patients with hypercholesterolaemia and microalbuminuria (n = 32) at baseline and after 6 months of treatment with ezetimibe After ezetimibe Statistical Parameter Baseline treatment significance Body mass index, kg/m ± ± 1.3 NS Systolic blood pressure, mmhg ± ± 3.6 NS Diastolic blood pressure, mmhg 77.8 ± ± 3.5 NS Glycated haemoglobin, % 7.2 ± ± 0.9 P < 0.01 Low-density lipoprotein-cholesterol, mg/dl ± ± 23.0 P < 0.01 High-density lipoprotein-cholesterol, mg/dl 59.5 ± ± 15.0 P < 0.05 Triglycerides, mg/dl ± ± 57.0 P < 0.01 Blood urea nitrogen, mg/dl 17.7 ± ± 3.7 NS Estimated glomerular filtration rate, ml/min 65.3 ± ± 12.8 NS Creatinine, mg/dl 0.8 ± ± 0.2 NS Albumin, g/dl 4.1 ± ± 0.3 P < 0.05 Urinary albumin excretion, mg/g creatinine 89.2 ± ± 54.9 P < 0.05 Monocyte chemoattractant protein-1, pg/ml ± ± 79.8 NS Data presented as mean ± SD. Statistical significance was calculated using a two-tailed dependent t-test. NS, no statistically significant difference (P 0.05). nephropathy and that MCP-1 is regulated by oxidative stress, 11,12 the mechanism of the effects of ezetimibe on UAE observed in the present study may be partly due to its antioxidative properties, which may be linked to blockade of the intestinal TABLE 3: Univariate and multiple stepwise regression analyses of the association between urinary albumin excretion and clinical and laboratory parameters in type 2 diabetes patients (n = 32) with hypercholesterolaemia and microalbuminuria Multiple stepwise Univariate analysis analysis Regression Statistical Regression coefficient Parameter coefficient significance (statistical significance) Body mass index P < 0.01 NS Systolic blood pressure P = NS Diastolic blood pressure P = NS Glycated haemoglobin P < (P < 0.01) Low-density lipoprotein-cholesterol P = NS High-density lipoprotein-cholesterol NS NS Triglycerides NS NS Blood urea nitrogen NS NS Estimated glomerular filtration rate P = NS Creatinine P = NS Albumin NS NS Monocyte chemoattractant protein P = (P < 0.01) NS, no statistically significant difference (P 0.05). 801

5 absorption of oxidized cholesterol. This is consistent with a previous report that ezetimibe decreased proteinuria and urinary excretion of 8-hydroxydeoxyguanosine (8- OHdG), an oxidative stress marker, in nondiabetic patients with CKD. 5 In addition, it has recently been shown that ezetimibe reduced the oxidized LDL-C level in patients with hypercholesterolaemia. 13 Although the decrease in HbA 1c after ezetimibe treatment was slight and may or may not have been related to ezetimibe therapy, it was statistically significant. Furthermore, HbA 1c was found to be an independent determinant of UAE. Yagi et al. 14 reported that ezetimibe reduced UAE, urinary 8-OHdG and circulating levels of inflammatory biomarkers, such as tumour necrosis factor-α and C-reactive protein, and ameliorated insulin resistance in hyper - cholesterol aemic patients with micro - albumin uria. Thus, ezetimibe may reduce UAE partly by improving insulin sensitivity and glucose metabolism. Ezetimibe has also been shown to improve atherogenic lipoprotein profiles, but its effect on albuminuria was modest and not significant in type 2 diabetes patients already receiving simvastatin. 15 The differences in duration of ezetimibe therapy (6 months versus 2 months), degree of albuminuria (UAE of approximately 90 mg/g creatinine versus 5 µg/min creatinine, equivalent to approximately 7 mg/g creatinine), and statin use could explain the discrepancy between the results of Ruggenenti et al. 15 and the present study. In conclusion, the present study suggests that ezetimibe may ameliorate albuminuria partly via its anti-inflammatory properties in hypercholesterolaemic type 2 diabetes patients with microalbuminuria. Reduction of LDL-C by ezetimibe may be a promising strategy for halting the progression of CKD and CVD in these high-risk subjects. Acknowledgement This work was supported in part by the Grants of Collaboration with Venture Companies Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (SI Yamagishi). Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 28 November 2011 Accepted subject to revision 30 November 2011 Revised accepted 22 February 2012 Copyright 2012 Field House Publishing LLP References 1 Adler AI, Stevens RJ, Manley SE, et al: Development and progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int 2003; 63: Yamagishi S, Matsui T, Nakamura K: Atorvastatin and diabetic vascular complications. Curr Pharm Des 2006; 12: Colhoun HM, Betteridge DJ, Durrington PN, et al. for CARDS Investigators: Effects of atorvastatin on kidney outcomes and cardiovascular disease in patients with diabetes: an analysis from the Collaborative Atorvastatin Diabetes Study (CARDS). Am J Kidney Dis 2009; 54: Nakamura T, Sugaya T, Kawagoe Y, et al: Effect of pitavastatin on urinary liver-type fatty acidbinding protein levels in patients with early diabetic nephropathy. Diabetes Care 2005; 28: Nakamura T, Sato E, Fujiwara N, et al: Ezetimibe decreases serum levels of asymmetric dimethylarginine (ADMA) and ameliorates renal injury in non-diabetic chronic kidney disease patients in a cholesterol-independent manner. Pharmacol Res 2009; 60: Nakamura K, Yamagishi S, Adachi H, et al: Circulating advanced glycation end products 802

6 (AGEs) and soluble form of receptor for AGEs (srage) are independent determinants of serum monocyte chemoattractant protein-1 (MCP-1) levels in patients with type 2 diabetes. Diabetes Metab Res Rev 2008; 24: Laker MF: Laboratory testing and biochemical analysis of hyperlipidaemias. Postgrad Med J 1993; 69(suppl 1): S12 S Imai E, Horio M, Nitta K, et al: Estimation of glomerular filtration rate by MDRD study equation modified for Japanese patients with chronic kidney disease. Clin Exp Nephrol 2007; 11: Chiarelli F, Cipollone F, Mohn A, et al: Circulating monocyte chemoattractant protein- 1 and early development of nephropathy in type 1 diabetes. Diabetes Care 2002; 25: Takebayashi K, Matsumoto S, Aso Y, et al: Association between circulating monocyte chemoattractant protein-1 and urinary albumin excretion in nonobese type 2 diabetic patients. J Diabetes Complications 2006; 20: Fornoni A, Ijaz A, Tejada T, et al: Role of inflammation in diabetic nephropathy. Curr Diabetes Rev 2008; 4: Matsui T, Yamagishi S, Takeuchi M, et al: Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation. Biochem Biophys Res Commun 2009; 385: Ueda S, Miyake I, Takata K, et al: Ezetimibe, an inhibitor of intestinal cholesterol absorption, decreases serum level of malondialdehydemodified low-density lipoprotein in patients with hypercholesterolemia. Int J Cardiol 2011; 146: Yagi S, Akaike M, Aihara K, et al: Ezetimibe ameliorates metabolic disorders and microalbuminuria in patients with hypercholesterolemia. J Atheroscler Thromb 2010; 17: Ruggenenti P, Cattaneo D, Rota S, et al: for the Ezetimibe and Simvastatin in Dyslipidemia of Diabetes (ESD) Study Group: Effects of combined ezetimibe and simvastatin therapy as compared with simvastatin alone in patients with type 2 diabetes: a prospective randomized double-blind clinical trial. Diabetes Care 2010; 33: Author s address for correspondence Dr Sho-ichi Yamagishi Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume , Kurume, Japan. shoichi@med.kurume-u.ac.jp 803