Association between Deletion Polymorphism of Angiotensin Converting Enzyme Gene and Proteinuria in Japanese Overweight Men

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1 J Occup Health 2001; 43: Journal of Occupational Health Association between Deletion Polymorphism of Angiotensin Converting Enzyme Gene and Proteinuria in Japanese Overweight Men Yoshiaki HASHIMOTO 1 *, Azusa FUTAMURA 1, Hideo NAKARAI 1, Hiromitsu YOKOTA 1, Miho OMURA 1, Kazuhisa TSUKAMOTO 2, Masako TOGO 2, Hiroaki SATO 2, Masumi HARA 2, Naoyuki ISOO 2, Jun-ichi TAGUCHI 2 and Kazuhiko NAKAHARA 1 1 Departments of Clinical Laboratory Medicine and 2 Internal Medicine, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo , Japan Abstract: Association between Deletion Polymorphism of Angiotensin Converting Enzyme Gene and Proteinuria in Japanese Overweight Men: Yoshiaki HASHIMOTO, et al. Departments of Clinical Laboratory Medicine, The University of Tokyo Hospital A cross-sectional study was performed in 237 overweight men (BMI: kg/m 2 ) to investigate the relationship between an insertion (I)/ deletion (D) polymorphism of intron 16 of the angiotensin converting enzyme (ACE) gene and proteinuria. Proteinuria (± or greater) was determined with a reagent strip. The prevalences of proteinuria were 9.2, 13.3 and 24.3% in the subjects with the ACE II, ID and DD genotype, respectively. There was a significant difference between the II and the DD genotypes in the prevalence of proteinuria. No difference among the three genotypes was observed in blood pressure, total cholesterol, HDL-cholesterol, triglycerides and HbA 1C. When the subjects were divided into two groups according to the result of urinalysis, the prevalence of the DD genotype was significantly higher in the group with proteinuria (28.1%) than in the group without it (13.7%). Multiple logistic regression analysis showed that the ACE DD genotype was independently related to proteinuria. The results suggest that the ACE DD genotype may be an independent risk factor of proteinuria in Japanese overweight men. It is specially important for subjects with the ACE DD genotype to prevent the other risk factors of proteinuria such as diabetes, hypertension and obesity by keeping healthy life-style habits. (J Occup Health 2001; 43: 80 84) Received July 7, 2000; Accepted Dec 27, 2000 Correspondence to: Y. Hashimoto, Department of Clinical Laboratory Medicine, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo , Japan Key words: Angiotensin converting enzyme gene polymorphism, Proteinuria, Hypertension, HbA 1C, Smoking, Obesity In Japan, many adults undergo an annual health check up which includes a qualitative urine examination. Proteinuria (+ or greater, as determined using a reagent strip) is detected in % of Japanese adults 1, 2). In most cases, the etiology of proteinuria detected during a health check up remains unknown, because renal biopsy is rarely performed in the case of intermittent, orthostatic and mild to moderate persistent proteinuria. Fewer than 1.5% of asymptomatic patients with proteinuria may have serious urinary tract disorders 3), but several studies have suggested that microalbuminuria 4 7) in addition to proteinuria 8, 9) was a predictor of increased mortality or vascular diseases. It is therefore important to investigate the factors associated with proteinuria and microalbuminuria. Cross-sectional population studies have suggested that proteinuria is associated with various factors such as blood pressure, the plasma glucose level, body-mass index and smoking 2, 10 12). 13, 14) Recent studies suggested that albuminuria in type 1 and type ) diabetes, and hypertension 19) might be associated with angiotensin converting enzyme (ACE) gene polymorphism, which is characterized by an insertion/ deletion (I/D) of intron 16. The ACE gene polymorphism is related to the serum and cellular levels of ACE, and accounts for about half of individual variation in the serum ACE level 20). The highest and lowest ACE levels are found in DD and II homozygotes, respectively 20). ACE converts angiotensin I into the vasoactive angiotensin II and inactivates bradykinin, which results in increased intraglomerular pressure due to increased efferent arteriolar resistance 21). Inhibitors of ACE have an antiproteinuric

2 Yoshiaki HASHIMOTO, et al.: ACE Gene Polymorphism and Proteinuria 81 effect in patients with diabetic and nondiabetic nephropathy, which cannot be explained only by a decrease in systemic blood pressure 22 24). Based on these findings, it is conceivable that proteinuria or microalbuminuria in the general population may also be associated with the ACE gene polymorphism. In this study, we examined the relationship between proteinuria (± or greater) determined with a reagent strip and the ACE genotypes in overweight men, who had a higher prevalence of proteinuria. Subjects and Methods Subjects The subjects for this study were 237 men aged yr with a body-mass index of They visited the obesity clinic of a company after an annual health check up, seeking advice on how to change their lifestyles and lose weight. Informed consent was obtained from them. This study was approved by the Ethics Committee of the workers company. Methods We utilized the data from an annual health check up. Body weight was measured with light clothes on, but without shoes. Blood and urine samples were taken after a 12-h or longer fast in the morning. Urine was qualitatively examined with a reagent strip. Proteinuria was defined when the reagent strip indicated ± and hematuria when it indicated 1+. Serum triglycerides, total cholesterol and HDL-cholesterol were measured by commercially available enzymatic methods. HbA 1C levels were measured by high-performance liquid chromatography. The reference range of HbA 1C was %. ACE genotype DNA was extracted from peripheral blood leucocytes with a commercially available DNA extraction kit, Dr. GenTLE (Takara Biomedicals, Otsu, Japan). The ACE genotype was determined by polymerase chain reaction with primers flanking the polymorphic region of intron 16 25). To avoid mistyping heterozygotes as DD, DNA from subjects with the DD genotype was amplified again according to the method of Lindpaintner et al. 26) The PCR products were separated on a 2% agarose gel containing ethidium bromide. Statistical analysis Data was analyzed by the Statistical Analysis System (SAS Institute, Cary, NC). The two-tailed Fisher s exact test and the Dunnett s test were used to compare group proportions and group means, respectively. The multiple logistic regression model procedure was used to examine the variables associated with proteinuria. Differences with a p value of <0.05 were considered statistically significant. Results Among the 237 overweight men, the prevalences of the II, ID and DD genotypes were 36.7, 47.7 and 15.6%, respectively (Table 1), which were similar to those reported previously in healthy Japanese 27 29). The prevalences of proteinuria were 9.2, 13.3 and 24.3% in the subjects with the II, ID and DD genotype, respectively. There was a significant difference in the prevalence of proteinuria between the subjects with the II genotype and those with the DD genotype. Among the 32 subjects with proteinuria, only 3 showed signs of both proteinuria and hematuria. Seven subjects with proteinuria had a HbA 1C of 6% or higher, and/or were taking medication for diabetes. No differences were observed in blood pressure, total cholesterol, HDL-cholesterol, triglycerides and HbA 1C among those three ACE genotype groups. When the subjects were divided into two groups according to the result of urinalysis, the prevalence of the DD genotype was significantly higher in the group with proteinuria (28.1%) than in the group without it (13.7%) (Table 2). There was no significant difference between the two groups in the prevalences of the II and the ID genotypes. Multiple logistic regression analysis showed that the DD genotype was the most important factor with an odds ratio of 4.1 (Table 3). Neither obesity (p=0.078), age, blood pressure, HbA 1C (p=0.052) nor smoking was associated with proteinuria. Discussion The present study showed that the ACE DD genotype might be associated with proteinuria in Japanese overweight men. The relationship between the ACE D allele and proteinuria has been studied mostly in type 1 13, 14, 30 33) and type 2 diabetes 15 18, 31, 34, 35). Some studies showed an association betweeen the two 13-18), but others failed to confirm those findings 30 35). A meta-analysis did not suggest that this factor played a role in the initiation of diabetic nephropathy 36), but studies seem to support an association of the ACE D allele with progression of renal disease in type 2 diabetes 28). There are also conflicting data on the association between this ACE genotype and the severity of primary renal diseases such as immunoglobulin A nephropathy and polycystic kidney 37). In the present study, the etiology of proteinuria was not identified. Most of the subjects with proteinuria were not considered as diabetics judging from their HbA 1C levels and prevalence of the subjects who were taking medication for diabetes. In the urine screening of school children with a reagent strip, only 10 20% of those who had proteinuria ( +) in the first urinalysis were reported to have proteinuria in the subsequent examination 38, 39). This suggests that proteinuria in a single urinalysis may correspond to benign isolated proteinuria such as idiopathic, functional and postural proteinurias in most

3 82 J Occup Health, Vol. 43, 2001 Table 1. Clinical characteristics of the subjects distributed according to their angiotensin converting enzyme genotype Genotype II ID DD n Age (yr) 44.2 (7.4) 45.8 (7.4) 44.6 (7.6) Body-mass index (kg/m 2 ) 27.3 (2.0) 27.3 (1.6) 27.3 (2.0) Exercise (times/month) 3.0 (3.5) 2.3 (3.3) 1.7 (2.9)* Alcohol (ml/week) 7.4 (5.8) 8.2 (7.7) 6.2 (5.7) Present and past smoker (%) Systolic BP (mmhg) 128 (13) 130 (13) 125 (15) Diastolic BP (mmhg) 83 (11) 84 (10) 80 (9) Total cholesterol (mg/dl) 213 (33) 208 (36) 204 (31) HDL-cholesterol (mg/dl) 53 (12) 52 (11) 49 (12) Triglycerides (mg/dl) 161 (85) 162 (82) 163 (71) HbA 1C (%) 5.4 (0.7) 5.3 (0.6) 5.4 (0.8) proteinuria (%) ± ± * Hematuria (%) Medication for Hypertension (%) Hyperlipidemia (%) Diabetes (%) Values are means (SD). BP: blood pressure. *: P<0.05 between II and DD. Table 2. Prevalences of the ACE genotypes in subjects with and without proteinuria Proteinuria + n ACE genotype % II ID DD * *p<0.05. ACE: angiotensin converting enzyme. cases. Because in our study even those with a ± were included among the subjects with proteinuria and proteinuria was judged based on the results of one urinalysis, most of our proteinuric patients may not have presented histologically abnormal changes in the kidney. Thus benign isolated proteinuria may be associated with the ACE genotype. The subjects of the present study were overweight men. The correlation between massive obesity and proteinuria has been previously reported 40 42). In recent studies, we have shown that moderate obesity (mean BMI: 26.9) may also be a risk factor of proteinuria independently of blood pressure and HbA 1C levels 2, 43). Although the mechanism of this association between obesity and proteinuria remains unclear, glomerular hyperfiltration has been considered to play an important role 44, 45). Increased blood flow volume, renal blood flow 46) and glomerular filtration rate 47), and nutritional factors such as protein intake 48) may contribute to this glomerular hyperfiltration in obese patients. An increased ACE level in DD homozygotes may further accelerate glomerular filtration. There were some limitations to our study. Proteinuria was determined based on the results of a single qualitative urinalysis and included those with ± proteinuria. In addition, the etiology of proteinuria was not identified. Nonetheless, the present study suggests that Japanese overweight men with the ACE DD genotype may have a higher prevalence of proteinuria. Because proteinuria may be associated with increased mortality and vascular diseases 4 9), overweight men, in particular those with the ACE DD genotype, should try to maintain healthy lifestyle habits and lose weight to prevent the other risk factors of proteinuriat. Further cross-sectional and prospective studies are needed to draw a definite conclusion.

4 Yoshiaki HASHIMOTO, et al.: ACE Gene Polymorphism and Proteinuria 83 Table 3. Multiple logistic regression analysis of variables associated with proteinuria Variables Odds ratio p ACE genotype ID vs II 1.5 (0.6~4.0) DD vs II 4.1 (1.3~12.7) Age (10-yr increase) 1.4 (0.8~2.5) Body-mass index (1 kg/m 2 increase) 1.2 (1.0~1.5) Systolic BP (10 mmhg increase) 1.2 (0.9~1.5) HbA 1C (1% increase) 1.6 (1.0~2.4) Smoking ((past and current) vs no) 1.6 (0.7~3.7) Values are odds ratios (ninety-five percent confidence intervals). ACE: angiotensin converting enzyme, BP: blood pressure. Acknowledgments: The present study was supported in part by a grant from the Clinical Pathology Research Foundation of Japan. References 1) Yamagata K, Yamagata Y, Kobayashi M, Koyama A. A long-term folloe-up study of asymtomatic hematuria/ or proteinuria in adults. Clin Nephrol 1996; 45: ) Hashimoto Y, Futamura A, Watanabe N, et al. Relationship between glycosylated hemoglobin and the prevalence of proteinuria in Japanese men. Intern Med 1999; 38: ) Woolhandler S, Pels RJ, Bor DH, Himmelstein DU, Lawrence RS. Dipstick urinalysis screening of asymptomatic adults for urinary tract disorders. J Am Med Assoc 1989; 262: ) Mogensen CE. Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes. 1984; 310: ) Yudkin JS, Forrest RD, Jackson CA. Microalbuminuria as predictor of vascular disease in non-diabetic subjects: Islington Diabetes Survey. Lancet 1988: ) Damsgaard EM, Frøland A, Jørgensen OD, Mogensen CE. Microalbuminuria as predictor of increased mortality in elderly people. Br Med J 1990; 300: ) Mattock MB, Morrish NJ, Viberti G, et al. Prospective study of microalbuminuria as predictor of mortality in NIDDM. Diabetes 1992; 41: ) Fuller JH, Head J, the WHO Multinational Study Group. Blood pressure, proteinuria and their relationship with circulatory mortality: the WHO Multinational Study of Vascular Disease in Diabetics. Diabete Metab 1989; 15: ) Morrish NJ, Stevens LK, Head J, Fuller JH, Jarrett RJ, Keen H. A prospective study of mortality among middle-aged diabetic patients (the London cohort of the WHO Multinational Study of Vascular Disease in Diabetics) II: associated risk factors. Diabetologia 1990; 33: ) Collins VR, Dowse GK, Finch CF, Zimmet PZ, Linnane AW. Prevalence and risk factors for microand macroalbuminuria in diabetic subjects and entire population of Nauru. Diabetes 1989; 38: ) Metcalf P, Baker J, Scott A, Wild C, Scragg R, Dryson E. Albuminuria in people at least 40 years old: effect of obesity, hypertension, and hyperlipidemia. Clin Chem 1992; 38: ) Mogensen CE, Vestbo E, Poulsen PL, et al. Microalbuminuria and potential confounders. Diabetes Care 1995; 18: ) Marre M, Bernadet P, Gallois Y, et al. Relationship between angiotensin I, converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 1994; 43: ) Barnas U, Schmidt A, Illievich A, et al. Evaluation of risk factors for the development of nephropathy in patients with IDDM: insetion/deletion angiotensin converting enzyme gene polymorphism, hypertension and metabolic control. Diabetologia 1997; 40: ) Doi Y, Yoshizumi H, Yoshinari M, et al. Association between a polymorphism in the angiotensin converting enzyme gene and microvascular complications in Japanese patients with NIDDM. Diabetologia 1996; 39: ) Mizuiri S, Hemmi H, Inoue A, et al. Angiotensin converting enzyme polymorphism and development of diabetic nephropathy in non-insulin-dependent diabetes mellitus. Nephron 1995; 70: ) Ohno T, Kawazu S, Tomono S. Association analyses of the polymorphisms of the angiotensin-converting enzyme and angiotensinogen genes with diabetic nephropathy in Japanese non-insulin-dependent diabetics. Metabolism 1996; 45: ) Dudley CRK, Keavney B, Stratton IM, Turner RC, Ratcliffe PJ. U.K. Prospective Diabetes Study XV: relationship of reninangiotensin system gene polymorphisms with microalbuminuria in NIDDM. Kidney Int 1995; 48:

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Paper. Abdolrahim Nikzamir, * Alireza Esteghamati, Mostafa Feghhi, # Manouchehr Nakhjavani, Armin Rashidi, Javad Zavar Reza^*

Paper. Abdolrahim Nikzamir, * Alireza Esteghamati, Mostafa Feghhi, # Manouchehr Nakhjavani, Armin Rashidi, Javad Zavar Reza^* The insertion/deletion polymorphism of the angiotensin-converting enzyme gene is associated with progression, but not development, of albuminuria in Iranian patients with type 2 diabetes Abdolrahim Nikzamir,