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Kidney International, Vol. 63 (2003), pp. 2230–2235

Microsatellite DNA of human adrenomedullin in type 2 diabetic patients with renal failure

TOSHIHIKO ISHIMITSU,KOHJU TSUKADA,JUNICHI MINAMI,HIDEHIKO ONO,MASAMI OHRUI, JUN HINO,KENJI KANGAWA, and HIROAKI MATSUOKA

Department of and Cardiorenal Medicine, and Department of Health Care, Dokkyo University School of Medicine, Mibu, Tochigi, Japan; Department of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka, Japan

Microsatellite DNA polymorphism of human adrenomedullin Adrenomedullin (AM) is a hypotensive peptide pro- gene in type 2 diabetic patients with renal failure. duced in cardiovascular tissues such as heart, lung, kid- Background. Adrenomedullin (AM) is a hypotensive pep- ney, and vascular wall [1, 2]. Besides the potent vasodila- tide widely produced in the cardiovascular organs and tissues such as the heart, kidney, and the vascular cells. We have pre- tor action, AM has also been shown to cause natriuresis viously cloned and sequenced the genomic DNA encoding hu- in the kidney [3]. A significant level of AM has been also man AM gene, and determined that the gene is located in the identified in human plasma [1, 4] and we have previously short arm of chromosome 11. The 3Ј-end of the gene is flanked reported that plasma AM levels are increased in patients by the microsatellite marker of cytosine adenine (CA) repeats. with renal failure and hypertension [5]. Moreover, it has In this study, we investigated the association between DNA variations in AM gene and the predisposition to develop ne- been recently reported that gene delivery or chronic phropathy in type 2 diabetes mellitus. administration of AM alleviates renal injuries in hyper- Methods. Genomic DNA was obtained from the peripheral tensive rats [6–10]. These findings suggest that AM has leukocytes of 233 normal healthy subjects (NH), 139 type 2 implications in pathophysiology of the renal diseases. diabetic patients on hemodialysis (DM-HD), 106 control pa- We have cloned and sequenced the genomic DNA tients with type 2 diabetes without nephropathy (DM-C) and 318 hemodialysis patients due to chronic glomerulonephritis encoding human AM gene, and determined that the gene (CGN-HD). The genomic DNA was subject to polymerase chain is located in the short arm of chromosome 11 [11]. The reaction (PCR) using a fluorescence-labeled primer, and the gene consists of four exons and whole nucleotide se- number of CA repeats were determined by polyacrylamide gel quence corresponding to the 52 amino acid residues of electrophoresis (PAGE). mature AM is included in the fourth exon. Nucleotide Results. In our Japanese subjects, there existed four types sequencing of genomic DNA adjacent to the AM gene of alleles with different CA-repeat number; 11, 13, 14, and 19. Ј The frequencies of these alleles were 11: 27.7%, 13: 32.8%, 14: revealed that the 3 -end of the gene is flanked by the 35.6%, and 19: 3.9% in NH. These allele frequencies were microsatellite marker with a variable number of cytosine not significantly different in DM-C and CGN-HD. However, adenine (CA) repeat [12]. This microsatellite marker is DM-HD showed significantly different distribution of allele located approximately 4 kb downstream of the AM gene. ␹2 ϭ ϭ frequency from other groups ( 18.9, P 0.026). Namely, Considering the possible implications of AM in the car- the frequency of 19-repeat allele in DM-HD was higher (9.0%) than NH, DM-C, and CGN-HD (P ϭ 0.005, 0.041, and 0.004, diovascular system, it seems of interest to elucidate respectively). whether this gene variation has any relation to the etiol- Conclusion. The microsatellite DNA polymorphism of AM ogy of renal diseases. gene may be associated with the genetic predisposition to de- With regard to the genetic background of renal dis- velop nephropathy in Japanese patients with type 2 diabetes eases, it has been demonstrated that diabetic nephropa- mellitus. thy, not only type 1 but also type 2, occurs in familial clusters, suggesting that genetic factors may be involved in the pathogenesis [13, 14]. In this aspect, numbers of studies have been performed as to the association of Key words: adrenomedullin, gene polymorphism, diabetes mellitus, various gene polymorphisms with genetic susceptibility diabetic nephropathy, microsatellite repeats, genetic markers. to suffer diabetic nephropathy. In the present study, we Received for publication September 26, 2002 investigated the relationship between the microsatellite and in revised form November 30, 2002 DNA polymorphism adjacent to the AM gene and ge- Accepted for publication January 28, 2003 netic predisposition to develop nephropathy in Japanese  2003 by the International Society of Nephrology type 2 diabetic patients.

2230 Ishimitsu et al: Adrenomedullin gene polymorphism in diabetic nephropathy 2231

METHODS was performed with 0.5 ␮g of genomic DNA and 25 pmol Ј Study subjects of each primer (sense 5 -AAGAGGCTGAGTCAGAA GGATTGG-3Ј and antisense 5Ј-GCAACATCATTTT A group of 139 hemodialysis patients with type 2 dia- AATATCCTGCACAG-3Ј) in a final volume of 50 ␮L betes (DM-HD) were selected from the hemodialysis containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L unit of Dokkyo University School of Medicine Hospital KCl, 1.5 mmol/L MgCl ,10␮g/mL gelatin, 0.2 mmol/L and eight nearby hemodialysis hospitals and clinics. 2 of each deoxynucleoside triphosphate (dNTP), 1 unit of Type 2 diabetes was diagnosed when diabetes was de- Taq DNA polymerase (Perkin-Elmer, Branchburg, New tected at 30 years old or older and insulin therapy was Jersey). The 5Ј end of sense primer was labeled with the not given during 2 years after the onset [15]. From the blue fluorescent dye 6-FAM. The DNA was amplified same hospitals and clinics, 318 hemodialysis patients due for 30 cycles with denaturation at 94ЊC for 45 seconds, to chronic glomerulonephritis (CGN-HD) were involved annealing at 60ЊC for 1 minute, and extension at 72ЊC in this study. Glomerulonephritis was diagnosed by pre- ϩ for 2 minutes in a thermal cycler. Then, an aliquot of the vious renal biopsy or the presence of 2 or more protein- PCR product was mixed with the internal DNA size uria and hematuria and exclusion of other renal diseases standard labeled with the red fluorescent dye ROX. The such as diabetic nephropathy, hypertensive nephrosclero- mixture was electrophoresed on a 6% urea-polyacryl- sis, lupus nephritis, polycystic kidney, and pyelonephritis. amide gel using ABI 373 DNA sequencer (Perkin-Elmer). A hundred and six control type 2 diabetes patients without The length of PCR product was calculated from the cali- nephropathy (DM-C) were selected from the outpatient bration curve of internal standard using GENESCAN clinic of Dokkyo University School of Medicine Hospi- 672 software (Perkin-Elmer). tal. In addition to the criteria for the diagnosis of type 2 diabetes, three more criteria were applied to select DM-C Biochemical measurement Ͻ subjects: (1) normal serum creatinine (men 1.5 mg/dL Peripheral blood sample was obtained in the morning Ͻ and women 1.3 mg/dL), (2) absence of microalbumin- after overnight fast in NH and DM-C. In DM-HD and uria, and (3) 5 or more years of known history of diabetes. CGN-HD, blood sample was taken before hemodialysis. Microalbuminuria was denied when the albumin in spot Serum creatinine was measured by automatic analyzer urine was less than 30 mg/g creatinine on multiple occa- (HITACHI 736-60; Hitachi, Tokyo, Japan) and blood sions [16]. The normal healthy group (NH) included 233 hemoglobin A1c was determined by high-performance healthy subjects age 50 years old or older. They were re- liquid chromatography (HPLC) using ADAMS hemo- cruited from participants of the health check program globin A1c HA-8160 (ARKRAY, Inc., Kyoto, Japan). of Dokkyo University School of Medicine Hospital. Dis- eases were ruled out through comprehensive checkup, in- Statistical analysis cluding a full medical history taking and physical exami- Data are presented as means Ϯ SD. Statistical analyses nations, urinalysis, a stool examination, blood cell counts, were performed using Stat View software (version 5.0; blood chemistry, a glucose tolerance test, serologic tests SAS Institute Inc., Cary, NC, USA). Clinical characteris- for hepatitis viruses, a chest x-ray film, an electrocardio- tics between the case and control groups were compared gram, a respiratory function, an alimentary examination by one-way analysis of variance (ANOVA) for paramet- of the upper gastrointestinal tract, and abdominal ultra- ric data and by chi-square test for categoric data. If the sonography. The physical examination was performed by ANOVA showed statistical significance, Scheffe’s test an internist, a surgeon, an ophthalmologist, an otorhino- was applied for the post-hoc comparison between groups. laryngologist, and a gynecologist for women. All subjects The frequencies of alleles and genotypes in the four were Japanese and unrelated each other. groups were compared by chi-square test and Fisher’s The study protocol was approved by the Institutional exact test. Differences of parametric data in two groups Ethical Committee, and informed consent was obtained were analyzed by unpaired Student t test. A P value less from each subject. than 0.05 was considered to indicate statistical signifi- cance. Genotype analysis The number of CA repeats adjacent to AM gene was determined by fluorescence-labeled polymerase chain RESULTS reaction and polyacrylamide gel electrophoresis (PCR- Clinical characteristics of study subjects PAGE) as described previously [12, 17]. Genomic DNA Table 1 lists the physical and laboratory findings of was extracted from peripheral leukocytes using Easy the study subjects. Average age and the gender ratio DNA kit (Invitrogen, Carlsbad, CA, USA). The micro- were not significantly different among the four groups. satellite region containing the CA repeats 4 kb down- Body mass index was greater in DM-C than in NH (P ϭ stream of the AM gene was amplified by PCR. The PCR 0.026). However, the body mass index of DM-HD was 2232 Ishimitsu et al: Adrenomedullin gene polymorphism in diabetic nephropathy

Table 1. Clinical characteristics of the study subjects NH DM-C DM-HD CGN-HD Parameter N ϭ 233 N ϭ 106 N ϭ 139 N ϭ 318 Age years 57Ϯ659Ϯ16 60Ϯ10 57Ϯ12 Sex male/female 154/79 64/42 93/46 184/134 Body mass index kg/m2 23.7Ϯ2.8 24.9Ϯ4.2a 21.1Ϯ2.8b,d 20.7Ϯ2.7b Systolic blood pressure mm Hg 117Ϯ11 146Ϯ25b 164Ϯ15b,d,e 153Ϯ19b Diastolic blood pressure mm Hg 73Ϯ885Ϯ15b 81Ϯ8b,c 81Ϯ9b Duration of diabetes years 14Ϯ615Ϯ7 (until nephropathy was revealed) (8Ϯ6) Duration of dialysis years 5Ϯ3e 9Ϯ6 Serum creatinine mg/dL 0.9Ϯ0.1 1.0Ϯ0.2 10.3Ϯ2.9b,d,e 11.9Ϯ2.7b d Blood hemoglobin A1c 9.5Ϯ2.3 6.7Ϯ1.6 Abbreviations are: NH, normal healthy subjects; DM-C, control diabetic patients without nephropathy; DM-HD, diabetic patients on hemodialysis; CGN-HD, hemodialysis patients due to chronic glomerulonephritis. Data are mean Ϯ SD. F values of ANOVA for body mass index, systolic blood pressure, diastolic blood pressure, and serum creatinine are 72.0 (P Ͻ 0.001), 250.4 (P Ͻ 0.001), 48.7 (P Ͻ 0.001), and 1422.8 (P Ͻ 0.001), respectively. a P Ͻ 0.01; b P Ͻ 0.001 vs. NH; c P Ͻ 0.01; d P Ͻ 0.001 vs. DM-C; and e P Ͻ 0.001 vs. CGN-HD

Table 2. Genotype frequencies of microsatellite DNA Table 3. Allele frequencies of microsatellite DNA polymorphism polymorphism adjacent to the human adrenomedullin adjacent to the human adrenomedullin gene in the study subjects gene in the study subjects NH DM-C DM-HD CGN-HD Genotype NH DM-C DM-HD CGN-HD Allele N ϭ 233 N ϭ 106 N ϭ 139 N ϭ 318 N (%) N ϭ 233 N ϭ 106 N ϭ 139 N ϭ 318 11 CA repeats 27.7% 28.8% 31.6% 27.8% 11/11 22 (9.4%) 8 (7.6%) 14 (10.0%) 18 (5.7%) 13 CA repeats 32.8% 37.3% 27.8% 33.7% 11/13 44 (18.9%) 19 (17.9%) 30 (21.6%) 54 (17.0%) 14 CA repeats 35.6% 29.7% 31.6% 34.6% 11/14 37 (15.9%) 23 (21.7%) 24 (17.3%) 76 (23.9%) 19 CA repeats 3.9% 4.2% 9.0%a 3.9% 11/19 4 (1.7%) 3 (2.8%) 6 (4.3%) 11 (3.4%) Abbreviations are: NH, normal healthy subjects; DM-C, control diabetic pa- 13/13 23 (9.9%) 20 (18.9%) 8 (5.8%) 36 (11.3%) tients without nephropathy; DM-HD, diabetic patients on hemodialysis; CGN- 13/14 54 (23.2%) 17 (16.1%) 22 (15.8%) 81 (25.5%) HD, hemodialysis patients due to chronic glomerulonephritis. 13/19 9 (3.9%) 3 (2.8%) 9 (6.5%) 7 (2.2%) a ␹2 ϭ 18.9, P ϭ 0.026 14/14 35 (15.0%) 10 (9.4%) 16 (11.5%) 28 (8.8%) 14/19 5 (2.1%) 3 (2.8%) 10 (7.2%) 7 (2.2%) 19/19 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 11, 13, 14, and 19. The alleles with 11, 13, 14, and 19 CA Abbreviations are: NH, normal healthy subjects; DM-C, control diabetic pa- tients without nephropathy; DM-HD, diabetic patients on hemodialysis; CGN- repeat yield DNA fragment length of 248, 252, 254, and HD, hemodialysis patients due to chronic glomerulonephritis. 264 bp, respectively. Combination of the four alleles yields ten possible genotypes: 11/11, 11/13, 11/14, 11/19, smaller than that of NH and comparable to that of 13/13, 13/14, 13/19, 14/14, 14/19, and 19/19. Table 2 shows CGN-HD. Systolic and diastolic blood pressures were observed frequencies of these ten genotypes of this mi- obviously higher in DM-C, DM-HD, and CGN-HD than crosatellite gene polymorphism in the study subjects, and in NH (P Ͻ 0.001, respectively). Especially, DM-HD Table 3 lists the frequency of each allele calculated from had even higher systolic blood pressure than DM-C and the genotype frequencies. The genotype frequencies were CGN-HD (P Ͻ 0.001, respectively), and DM-C had not significantly deviated from those expected from the higher diastolic pressure than DM-HD (P ϭ 0.007) and Hardy-Weinberg’s equilibrium in each group. The fre- CGN-HD (P ϭ 0.008). Duration of diabetes was compa- quencies of 11, 13, and 14, repeat alleles were approxi- rable between DM-C and DM-HD, however, the dura- mately 30%, while the frequency of 19 repeat allele was tion of diabetes in DM-C was longer than the duration less than 10% in each group. There were no homozygotes of diabetes until nephropathy was revealed in DM-HD, of the least frequent 19 repeat allele in the examined P Ͻ 0.001. In hemodialysis patients, DM-HD had shorter subjects. Distributions of the genotypes were not signifi- duration of dialysis than CGN-HD, P Ͻ 0.001. Serum cantly different among the four groups. On the other hand, creatinine did not significantly differ between NH and the allele frequencies were significantly different between DM-C. Of course, DM-HD and CGN-HD had much the groups (␹2 ϭ 18.9, P ϭ 0.026). Figure 1 depicts the dif- higher serum creatinine than NH and DM-C (P Ͻ 0.001, ference in allele frequency more clearly. The frequency respectively). In hemodialysis patients, predialysis serum of 19 repeat allele was higher in DM-HD than in NH, creatinine was lower in DM-HD than in CGN-HD, P Ͻ DM-C, or CGN-HD. The frequencies of 11, 13, and 14 0.001. Blood hemoglobin A1c was lower in DM-HD than repeat alleles did not show significant group differences. in DM-C, P Ͻ 0.001.

Genotype and allele frequencies DISCUSSION As we have reported previously [12, 17], there existed Nowadays, diabetic nephropathy is the leading cause four types of alleles with different CA repeat number; of end-stage renal failure in Japan as well as in United Ishimitsu et al: Adrenomedullin gene polymorphism in diabetic nephropathy 2233

DNA polymorphism adjacent to the AM gene with the genetic predisposition to suffer diabetic nephropathy. Namely, the existence of 19 CA repeat allele is supposed to be associated with the risk of developing nephropathy in Japanese type 2 diabetic patients. However, because the frequency of this 19 repeat allele was small, this gene variation alone is not thought to reflect the large part of genetic predisposition to diabetic nephropathy. Any single gene polymorphism, which has been previously shown to be associated with diabetic nephropathy, does not represent the large part of genetic risk for diabetic nephropathy. It is supposed that multiple contrib- ute to the development of diabetic nephropathy. In the future direction, information as to the relation between gene polymorphisms and the risk of diabetic nephropa- Fig. 1. Frequencies of 11, 13, 14, and 19 repeat alleles in normal healthy thy should be gathered and analyzed comprehensively subjects (NH), control diabetic subjects without nephropathy (DM-C), to construct a way to evaluate the genetic risk of diabetic diabetic patients on hemodialysis (DM-HD), and hemodialysis patients nephropathy precisely. In this sense, The DNA polymor- due to chronic glomerulonephritis (CGN-HD).*P Ͻ 0.05; **P Ͻ 0.01. phism examined in this study seems to add information to the prediction of the development of diabetic ne- phropathy. States and European countries. Because type 2 diabetes Some gene polymorphisms have been shown to affect is much more frequent than type 1 diabetes in Japan, expression of the genes or activity of the gene products. prevention of nephropathy in type 2 diabetic patients is For instance, the serum ACE activity is known to be of primary importance. In order to reduce the incidence increased in individuals carrying the deletion allele of of diabetic nephropathy effectively, it is necessary to the gene [28], and it has been reported that the methio- identify the patients at high risk. It is known that the nine to threonine substitution at amino acid residue 235 influence of a genetic factor on the onset of diabetes is (M235T) of angiotensinogen is associated with an in- greater in type 2 diabetes than in type 1 diabetes. Al- crease in the gene expression [29]. Because the micro- though the familial clustering of diabetic nephropathy satellite polymorphism examined in this study is located has first been reported in type 1 diabetes [13], later evi- 3Ј downstream of the AM gene, it is unlikely that the dence has also indicated inherited susceptibility to ne- gene transcription is affected by this polymorphism. In- phropathy in type 2 diabetes [14, 18, 19]. In order to deed, the plasma AM levels were not significantly differ- delineate the genetic background of type 2 diabetic ne- ent among the genotypes of this polymorphism in normal phropathy, numbers of studies have investigated the as- subjects [12]. Namely, the subjects carrying 19 repeat sociation of this complication with various gene polymor- allele and the homogygotes of 11, 13, and 14 repeat allele phisms. So far, several gene polymorphisms have been showed comparable plasma AM levels. Therefore, the indicated to have relation with the predisposition to de- association of 19 CA repeat allele with diabetic nephrop- velop nephropathy in type 2 diabetic patients. The gene athy is not likely mediated by the gene expression and polymorphisms include genes of renin-angiotensin sys- the action of AM itself. It may be possible that this tem such as angiotensin-converting enzyme (ACE) and microsatellite polymorphism is associated with the ex- angiotensinogen, and genes of endothelial nitric oxide pression of other genes. Near the location of AM gene synthase, glucose transporter, apolipoprotein E, methyl- in the short arm of chromosome 11, there exist such enetetrahydrofolate reductase and G-protein ␤3 subunit genes as sphyngomyelinase, parathyroid hormone, and [20–27]. lactate dehydrogenase [30–32]. Among these, so far the In the present study, we examined the microsatellite literature does not suggest relation of sphyngomyelinase DNA polymorphism lying 3Ј downstream of the AM and lactate dehydrogenase genes to diabetic nephropa- gene in diabetic patients and patients with end-stage thy. With regard the parathyroid hormone gene, it has renal disease. Among the four types of alleles with CA been shown that the secretion of parathyroid hormone repeat number of 11, 13, 14, and 19, the frequency of 19 is impaired in patients with diabetic nephropathy [33, 34], repeat allele was higher in DM-HD than in NH, DM-C, which suggests relation between the development of dia- and CGN-HD. The frequency of 19 repeat allele in betic nephropathy and the expression of parathyroid hor- DM-HD was more than twice as much as other groups. mone gene, although this may be caused by the long du- This result suggests the association of microsatellite ration of poor glycemic control as well. Besides these, 2234 Ishimitsu et al: Adrenomedullin gene polymorphism in diabetic nephropathy the microsatellite polymorphism downstream of AM gene 24–26, 42, 44–46]. In this respect, the subjects of this study may be linked to other yet unidentified gene variations. are all Japanese and ethnically unanimous. However, Microsatellite markers, like the one examined in this inharmonious results have been sometimes obtained study, consist of variable number of repeats of short even in the same or the closely related ethnic groups nucleotides, and more than hundreds of such repeat [23, 27, 43, 47]. In some studies, microalbuminuria was markers are scattered throughout the genomic DNA. assumed to indicate the existence of diabetic nephropa- These microsatellite markers can be utilized to locate thy. However, it has been reported that type 2 diabetic the genomic region responsible for hereditary diseases patients with microalbuminuria have heterogenous renal or traits. Until now, several diseases have been shown lesions [48]. Therefore, the relations between gene poly- to be associated with such microsatellite DNA polymor- morphisms and type 2 diabetic nephropathy may be af- phism. For instance, it has been reported that the CA fected by the criteria used for the diagnosis of diabetic repeat polymorphism lying upstream of aldose reductase nephropathy. gene affects the development of nephropathy and reti- The results of this study revealed the association of nopathy in type 1 diabetes mellitus [35, 36]. However, the microsatellite CA repeat polymorphism adjacent to inconsistent results have been reported as to the associa- the AM gene with genetic predisposition to type 2 dia- tion of this microsatellite polymorphism with type 2 dia- betic nephropathy in Japanese population. The frequency betic nephropathy [37–39]. On the other hand, genetic of 19 repeat allele was significantly increased in patients predisposition to essential hypertension has been shown with end-stage renal disease due to type 2 diabetes. Al- to have significant linkage to a certain number of TCAT though this DNA polymorphism is not likely to affect repeat in the intron 1 of tyrosine hydroxylase gene and the AM gene transcription, the existence of 19 CA repeat the GT repeat in the intron 2 of type B natriuretic peptide allele is supposed to reflect a certain part of the genetic receptor gene [40, 41]. We have also reported that the predisposition to develop nephropathy in Japanese type 19 CA repeat allele examined in this study is increased 2 diabetic patients. in patients with essential hypertension [17]. The relation of these microsatellite polymorphisms to diabetic ne- ACKNOWLEDGMENTS phropathy has not yet been examined. However, it seems The authors thank Ms. Masako Minato, Ms. Machiko Sakata, Ms. of interest because hypertension is a major risk factor Mika Nomura, Ms. Yasuko Mamada, Ms. Jun Chou, and Dr. Kazumi for the deterioration of renal function in diabetic pa- Akimoto for technological assistance. We also thank Dr. Hisamoto Kuroda, Dr. Tsutomu Nakamura, and Dr. Yoshiyuki Hattori of Depart- tients. As to the frequency of hypertension in our study ment of Endocrinology and Metabolism, Dokkyo University School subjects, 72 out of 106 DM-C patients and 111 out of of Medicine for recruiting diabetic patients. This study was supported DM-HD patients showed blood pressure exceeding 140/ in part by the grant-in-aid for scientific research (10218209) from the Ministry of Education, Science and Culture of Japan. 90 mm Hg or were given antihypertensive medication. However, the duration of hypertension, especially before Reprint requests to Toshihiko Ishimitsu, M.D., Department of Hyper- developing nephropathy, was inevitably inaccurate and tension and Cardiorenal Medicine, Dokkyo University School of Medi- cine, Mibu, Tochigi 321-0293, Japan. we could not collect sufficient information. 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