Association between apolipoprotein E polymorphism and macroalbuminuria in patients with non-insulin dependent diabetes mellitus

Sung-Kyu Ha, Hong Su Park, Kyung Wook Kim, Seung Jung Kim1, Do-Hun Kim1, Jung Ho Kim2, Ho Yung Lee and Dae Suk Han

Departments of Internal Medicine and 2 Clinical Pathology, Yonsei University College of Medicine, Seoul, Korea and 1 Department of Internal Medicine, Ajou University College of Medicine, Suwon, Korea

Correspondence and offprint requests to: Sung-Kyu Ha MD, Department of Internal Medicine, Yongdong Severance Hospital, Yonsei University College of Medicine, Young Dong, PO Box 1217, Seoul, Korea.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Objectives. Apolipoprotein E (apo E) is known to play an important role in lipoprotein metabolism through its ability to bind to the receptors as a ligand. Three different apo E alleles ({varepsilon}2, {varepsilon}3 and {varepsilon}4) produce six apo E genotypes ({varepsilon}2/2, {varepsilon}2/3, {varepsilon}2/4, {varepsilon}3/3, {varepsilon}3/4 and {varepsilon}4/4). The objective of this study was to investigate an association between apo E gene polymorphism and macroalbuminuria in 167 Korean patients with non-insulin dependent diabetes mellitus (NIDDM).

Methods. The patients in the macroalbuminuria group (n=74) represent those in whom 24 h urinary albumin excretion was above 300 mg. The patients in the normoalbuminuria group (n=93) represent those in whom 24 h urinary albumin excretion was below 30 mg and serum creatinine levels were less than 1.2 mg/dl. The duration of diabetes in all patients was at least 8 years.

Results. There were no significant differences in terms of age, sex, body mass index, HbA1c, total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol between the two groups. In the macroalbuminuria group, the distribution of apo E genotypes revealed {varepsilon}2/2 2 (2.7%), {varepsilon}2/3 14 (18.9%), {varepsilon}2/4 0 (0%), {varepsilon}3/3 47 (63.5%), {varepsilon}3/4 11 (14.9%) and {varepsilon}4/4 0 (0%). In the normoalbuminuria group, the distribution of apo E genotypes revealed {varepsilon}2/2 0 (0%), {varepsilon}2/3 7 (7.5%), {varepsilon}2/4 1 (1.1%), {varepsilon}3/3 72 (77.4%), {varepsilon}3/4 12 (12.9%) and {varepsilon}4/4 1 (1.1%). There was no significant difference in the distribution of apo E genotypes between the two groups. However, there was a significant difference in the allele frequencies, {varepsilon}2 frequency was significantly higher in macroalbuminuria group compared to normoalbuminuria group (12.2% vs 4.3%, P<0.05). Also, we compared apo E carrier frequencies between the two groups. {varepsilon}2 carrier frequency was significantly higher in macroalbuminuria group compared to normoalbuminuria group (21.6% vs 7.6%, P<0.05). In each group, there was no significant difference in the degree of lipid abnormalities between apo {varepsilon}2 carrier ({varepsilon}2/2, {varepsilon}2/3 genotypes), {varepsilon}3 carrier ({varepsilon}3/3 genotype) and {varepsilon}4 carrier ({varepsilon}3/4, {varepsilon}4/4 genotype).

Conclusion. Apo {varepsilon}2 allele and {varepsilon}2 carrier frequencies were significantly higher in macroalbuminuria group. These results suggest that {varepsilon}2 allele may be associated with the development of clinical albuminuria in Korean patients with NIDDM.

Keywords: apolipoprotein E; polymorphism; macroalbuminuria; normoalbuminuria



   Introduction
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Diabetic nephropathy is a major cause of morbidity and premature mortality in patients with non-insulin dependent diabetes mellitus (NIDDM). The pathophysiological mechanism for the development and progression of diabetic nephropathy is not well known, but several factors have been linked to this. Among these factors, it has been proposed that lipid abnormalities possibly contribute to the development and progression of the kidney diseases including diabetic nephropathy [16]. The human apolipoprotein E (apo E) is composed of 299 amino acids and is a polymorphic glycoprotein [7]. Recently, apo E alleles have been reported to be important genetic markers for dyslipidaemia associated with coronary artery disease [8], and there appear to be similar pathophysiological mechanisms in the development of glomerulosclerosis and atherosclerosis [4,9]. It is possible that the development of diabetic nephropathy in patients with NIDDM is associated with apo E polymorphism. Apo E is not only a major protein of very low density lipoprotein (VLDL), but also present in almost all kinds of lipoproteins including chylomicron, chylomicron remnants, intermediate density lipoproteins (IDL), and the cholesterol-rich subclass of high density lipoproteins [10]. Apo E is known to play an important role as a ligand of lipoprotein receptors, namely low density lipoprotein (LDL) receptor or apo E receptor (or LDL receptor-related protein: LRP) [10,11].

Apo E gene is located on chromosome 19 and its genetic polymorphism is well known [12]. It has three common alleles coding for three isoforms of apo E protein, {varepsilon}2, {varepsilon}3 and {varepsilon}4 at a single genetic locus producing the apo E isoproteins, apo E2, E3 and E4. Apo E3, the native form (wild type), is the most common apo E isoproteins. Apo E2 is commonly caused by a change of residue 158 from Arg to Cys (Arg 158->Cys). In apo E4, Cys is substituted by Arg at 112th amino acid (Cys 112->Arg). According to the genetic models of the three alleles, six apo E genotypes ({varepsilon}2/2,{varepsilon}2/3,{varepsilon}3/3, {varepsilon}2/4,{varepsilon}3/4, {varepsilon}4/4) and six phenotypes (E2/2, E2/3, E3/3, E2/4, E3/4, E4/4) are recognized by isoelectric focusing [10]. This polymorphism of apo E gene is known to influence the lipid metabolism because the product of {varepsilon}2 and {varepsilon}4 alleles has different characteristics from that of {varepsilon}3 allele. Apo {varepsilon}2 exhibits reduced binding to the receptors, and contributes to the accumulation of remnant particles in plasma derived from the partial catabolism of triglyceride-rich lipoproteins. On the other hand, apo {varepsilon}4 has increased binding to the receptors leading to increased metabolism of triglyceride-rich lipoprotein and reduced triglyceride level and increased LDL-cholesterol in plasma [10]. Recent studies of the association of apo E polymorphism on the risk of diabetic nephropathy have yielded conflicting results [1318]. There are ethnic differences in the relative frequencies of the {varepsilon} alleles and these frequency differences can influence the study results [19].

The purpose of this study was to investigate the association between apo E gene polymorphism and macroalbuminuria in 167 Korean patients with NIDDM. We compared the apo E allele frequencies in Korean NIDDM patients with normoalbuminuria and macroalbuminuria.



   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Subjects
One hundred and sixty seven NIDDM patients were recruited from renal and endocrine units of the Department of Internal Medicine, Yongdong Severance Hospital, Yonsei University, from March 1994 to July 1997. Patients were composed of 74 diabetics with macroalbuminuria and 93 normoalbuminuric patients. They were all Koreans. The macroalbuminuric diabetic group (n=74) represents those in whom 24 h urinary albumin excretion was above 300 mg. The normoalbuminuria group (n=93) represents those in whom 24 h urinary albumin excretion was below 30 mg and normal renal function (serum creatinine <=1.2 mg/dl). The duration of diabetes mellitus in all patients was at least 8 years.

We excluded patients with microalbuminuria in this study because in NIDDM, microalbuminuria may be present more frequently for reasons other than diabetic nephropathy compared with macroalbuminuria. Familial hyperlipidaemia, liver disease, thyroid disease and non-diabetic renal disease were excluded.

A diagnosis of hypertension was made if the patient had two consecutive blood pressure readings >140/90 mmHg or mean arterial pressure >105 mmHg, or was on antihypertensive therapy at the time of study. Antihypertensive regimens included diuretics, ACE inhibitors, calcium channel blockers, beta-blockers, alpha-blockers and combinations of the above drugs. Forty-eight out of 74 patients in macroalbuminuria group and 41 out of 93 patients in normoalbuminuria group were on antihypertensive therapy. In macroalbuminuria group, 21 out of 48 (43.8%) patients used ACE inhibitors. In normoalbuminuria group, 9 out of 41 (22.0%) patients used ACE inhibitors which was low compared to macroalbuminuria group. We also used lovastatin and gemfibrosil for the purposes of lowering serum cholesterol and triglyceride. In macroalbuminuria group, 22 out of 74 (29.7%) patients used gemfibrosil and 7 out of 74 (9.5%) patients used lovastatin. In normoalbuminuria group, 10 out of 93(10.8%) and 3 out of 93 (3.2%) patients used gemfibrosil and lovastatin, respectively.

Methods
Specimen collection.
After 12 h of overnight fast, 5 ml of EDTA-anti coagulated blood samples were drawn from the patients, and were centrifuged within 4 h. Buffy coat layers were refrigerated for DNA extraction at -20°C. The total cholesterol, triglyceride and HDL-cholesterol were measured from the separated plasma. Urinary albumin excretion and creatinine clearance were measured by 24 h urine collection.

Measurement of plasma lipids, lipoproteins and other parameters.
Total cholesterol was determined with cholesterol oxidase method. Triglyceride was determined with glycerol 3-phosphate oxidase-peroxidase (without glycerol blanks) on the Synchron CX5 (Beckman Instruments, Brea, CA, USA or Hitachi Co., Hitachi, Japan). LDL-cholesterol was calculated by Friedewald and Delong's equation. HbA1c was determined with ion capture assay, and 24 h urinary albumin was measured with nephelometric method (Behring Nephelometer 100, Marburg, Germany). To separate the group according to albumin excretion rate, we performed 24 h urine collection every month for 3 consecutive months.

Normoalbuminuria group was defined as those in whom three collections done in 3 month period showed less than 30 mg and normal renal function (serum creatinine <=1.2 mg/dl). Macroalbuminuria group was defined as those in whom at least two of the three collections showed elevated levels (>300 mg/24 h). We took the mean of the last two measurements of urinary albumin excretion. Because of potential antiproteinuric effect of antihypertensive therapy, we temporarily discontinued antihypertensive drugs 2 days before urine samples were collected.

Determination of apolipoprotein E genotypes.
ApoE genotyping was performed with PCR-restriction fragment length polymorphism as Hixon and Vernier [20] with slight modification as follows. The oligonucleotide sequences were:

F4: 5'-ACA GAA TTC GCC CCG GCC TGG TAC AC-3' F6: 5'-TAA GCT TGG CAC GGC TGT CCA AGG A-3' to amplify the specific fragment apoE genomic DNA in preparation for subsequent digestion with restriction endonuclease. Final concentration of each constituent in 50 µl reaction was as follows: dNTP (Pharmacia Biotech), 0.2 mM; each upstream (F4) and downstream (F6) oligonucleotide primer, 0.2 µM; genomic DNA, 100 ng (500–200 ng); MgCl2, 2 mM; KCl, 50 mM; Tris-HCl, pH 8.3, 10 mM. We denatured DNA first at 95°C for 5 min and added 1.5 Unit of Taq polymerase (Perkin Elmer) to each sample at 72°C. We amplified DNA as follows: 95°C for 40 s, 60°C for 30 s, 72°C for 30 s for 35 cycles, and final extension time 10 min at 72°C. We checked the 244 bp PCR product and digested with HhaI (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) with concentration of 0.5 U/µl for 3 h at 37°C. We separated the digested PCR product at 4% MetaPhor TM agarose (FMC BioProduct, ME, USA) and 0.5 µg/l ethidium bromide gel and determined the apoE genotyping.

Statistical analysis.
The distribution of Apo-E allele was in the Hardy-Weinberg equilibrium. Variables with normal distribution were tested with t-test. Variables with skewed distribution (e.g. cholesterol, triglyceride, urinary albumin excretion, BUN, Cr) were logarithmically transformed and then tested with t-test. Concomitantly the non-parametric Mann-Whiney U tests were used to analyse variable with skewed distribution. The test results of logarithmically transformed data and non-parametric test were the same as the results of parametric test. Differences of ApoE allele frequency between the two groups were analysed with Chi-square test. ApoE allele frequency between the two groups was significantly different and after Bonnferroni's correction (for multiple comparison), apo E2 allele and carrier frequencies between the two groups were significantly different but apo E3 and apo E4 were not. To determine factors predicting development of macroalbuminuria, we also calculated odds ratio by failure time anlaysis using cox-proportional hazard model. The level of significance was defined as P<0.05.



   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Clinical characteristics of the patients (Table 1Go)
The mean age of the 74 NIDDM with macroalbuminuric patients was 59.9±10.0 years, and that of 93 normoalbuminuric patients was 58.2±11.2 years. There were no significant differences in age, sex, body mass index, duration of diabetes, total cholesterol, HDL-cholesterol, LDL-cholesterol, and HbA1C between the two groups. In the macroalbuminuric diabetic group, the level of blood urea nitrogen was 45.8±23.7 mg/dl, and in the normoalbuminuric diabetic group 17.3±7.7 mg/dl (P<0.05). The level of serum creatinine was 4.7±3.6 mg/dl in the macroalbuminuric diabetic group, and 1.0±0.2 mg/dl in the normoalbuminuric group (P<0.05). Creatinine clearances revealed 24.4±22.3 and 77.8±26.1 ml/min and 24 h urine albumin excretions were 2556.2±2069.7 and 17.8±18.9 mg in each group, respectively (P<0.05). Hypertension was accompanied by 48 out of 74 (64.9%) in the macroalbuminuric diabetic group, and by 52 out of 93 (55.9%) in the normoalbuminuric diabetic group (P<0.05). In the macroalbuminuric diabetic group, dialysis was performed in 22 out of 74 (29.7%) patients. We carried out ophthalomoscopic examinations on 141 out of 167 patients (macroalbuminuria, 72 patients; normoalbuminuria, 69 patients). In the macroalbuminuric group, 35 (48.6%) patients had proliferative diabetic retinopathy, 36 (50.0%) patients had background retinopathy and one (1.4%) patient had no retinopathy. In the normoalbuminuric group, 2 (2.9%) patients had proliferative diabetic retinopathy, 19 (27.5%) patients had background retinopathy and 48 (69.6%) patients had no retinopathy. Percentage of proliferative and background retinopathy in macroalbuminuric group was significantly higher than normoalbuminuric group (98.6% vs 30.4%, P<0.05).


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Table 1. Clinical characteristics of NIDDM patients with macroalbuminuria and normoalbuminuria.
 
Apo E genotype and allele frequencies and apo E carrier frequencies (Tables 2 and 3GoGo, Figure 1Go)
In the macroalbuminuric diabetic group, apo E genotypes revealed 2 (2.7%) patients with {varepsilon}2/2, 14 (18.9%) with {varepsilon}2/3, 0 (0%) with {varepsilon}2/4, 47 (63.5%) with {varepsilon}3/3, 11 (14.9%) with {varepsilon}3/4 and 0 (0%) with {varepsilon}4/4. In the normoalbuminuric diabetic group, apo E genotypes revealed 0 (0%) patients with {varepsilon}2/2, 7 (7.5%) with {varepsilon}2/3, 1 (1.1%) with {varepsilon}2/4, 72 (77.4%) with {varepsilon}3/3, 12 (12.9%) with {varepsilon}3/4 and 1 (1.1%) with {varepsilon}4/4. There was no significant difference between the two groups in the distribution of apo E genotypes. However, the frequency of apo {varepsilon}2 allele was significantly higher in the macroalbuminuric diabetic group compared to the normoalbuminuric group (12.2% vs 4.3%, P<0.05). Also, patients were grouped according to their apo {varepsilon} allele carrier status as one of the following: {varepsilon}2 carrier ({varepsilon}2/2, {varepsilon}2/3 genotypes); {varepsilon}3 carrier ({varepsilon}3/3 genotype) and {varepsilon}4 carrier ({varepsilon}3/4, {varepsilon}4/4 genotype). The single patient carrying {varepsilon}2/4 genotype in normoalbuminuric group was excluded. As shown in Table 2Go and Figure 1Go, {varepsilon}2 carrier frequency was significantly higher in the macroalbuminuric diabetic group than in the normoalbuminuric group (21.6% vs 7.6%, P<0.05). To determine predictors of the development of macroalbuminuria, odds ratio was calculated by failure time analysis using cox-proportional harzard model. As shown in Table 3Go, odds ratio for the carriage of apo {varepsilon}3 allele and development of macroalbuminuria was presumed to 1, odds ratios of apo {varepsilon}2 and {varepsilon}4 alleles were 3.466 (95% CI 1.120–10.727, P=0.0310) and 1.108 (95% CI 0.330–3.719, P=0.8682), respectively.


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Table 2. The frequencies of apo E genotypes, alleles, and carrier in NIDDM
 

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Table 3. Factors affecting development of macroalbuminuria in NIDDM patients
 


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Fig. 1. Distribution of apo E allele and carrier frequencies between the two groups. (*P<0.05).

 
Other factors such as sex, presence or absence of hypertension, age, body mass index, HbA1c, serum triglyceride, LDL-cholesterol, and HDL-cholesterol were not significant predictors for the development of macroalbuminuria.

Comparison of serum lipid profiles between apo {varepsilon}2 carrier, {varepsilon}3 carrier and {varepsilon}4 carrier in each group (Table 4Go)
In the macroalbuminuric diabetic group, the mean levels of total cholesterol were 213.1±48.1 mg/dl in apo {varepsilon}2 carrier, 199.9±72.6 mg/dl in apo {varepsilon}3 carrier, and 210.4±89.8 mg/dl in apo {varepsilon}4 carrier (P>0.05). In the normoalbuminuric group, the mean levels of total cholesterol were 194.6±71.5 mg/dl in apo {varepsilon}2 carrier, 195.1±41.3 mg/dl in apo {varepsilon}3 carrier, and 225.8±56.3 mg/dl in apo {varepsilon}4 carrier (P>0.05). The mean levels of triglyceride, HDL-cholesterol, and LDL-cholesterol are shown in Table 4Go. There were no significant differences between apo {varepsilon}2 carrier ({varepsilon}2/2, {varepsilon}2/3 genotypes), {varepsilon}3 carrier ({varepsilon}3/3 genotype) and {varepsilon}4 carrier ({varepsilon}3/4, {varepsilon}4/4 genotypes) in each group in the levels of cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol (P>0.05).


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Table 4. The lipid values in Apo {varepsilon}2, {varepsilon}3, and {varepsilon}4 carrier groups
 


   Discussion
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
In this study, serum lipid profiles were not significantly different between macroalbuminuric diabetic and normoalbuminuric groups. Onuma et al. [16] reported that serum cholesterol and triglyceride levels were higher in patients with proteinuria than in those with normoalbuminuria. Mulec et al. [1] also reported that in type I diabetic patients with nephropathy, patients with low serum cholesterol concentration exhibited considerably slower decline in kidney function than those with high serum cholesterol concentration, suggesting that hypercholesterolaemia could contribute to more rapid progression of renal disease in patients with diabetic nephropathy. Recently, the significance of hypercholesterolaemia for progressive renal damage has been established in experimental models of kidney disease. Specially, hypercholesterolaemia can markedly aggravate nephron injury in the presence of other glomerular diseases [21,22]. Lipid-lowering therapies can prevent or attenuate this injury. However, very little is known about the long-term effects of elevations of serum cholesterol or other lipids on kidney disease in man. As previously mentioned, total cholesterol and triglyceirde levels are higher in patients with NIDDM and nephropathy but our study revealed no significant differences. This is probably due to the effect of treatment at the time of study (we used lovastatin and gemfibrosil for the purposes of lowering serum cholesterol and triglyceride, respectively).

In this study, the distribution of apo E genotypes in patients with macroalbuminuria was similar to patients with normoalbuminuria but {varepsilon}2 allele frequency was significantly higher in diabetic patients with macroalbuminuria. Distribution of apo E allele frequency of our normoalbuminuric diabetic group was similar to Japanese population. Recent studies have reported inconsistent associations between apo E polymorphism and diabetic nephropathy in various populations [1318]. Ukkola et al. [13] showed that apo E2 phenotype including apo E2/3 and E2/4 protected from macro- and micro-vascular complications in NIDDM. In contrast, apo E4/4 and E4/3 tended to increase the risk for macroangiopathy. The lower prevalence of macroangiopathy in patients with apo E2 phenotype was associated with lower plasma total and LDL cholesterol levels. They concluded that apo E phenotype modulated the risk for diabetic complications in patients with NIDDM. Boize et al. [14] in Europe also reported that the prevalence of nephropathy in NIDDM was significantly reduced in {varepsilon}2 allele carrier. They supported evidence for the role of LDL in the development of diabetic nephropathy as in atherosclerosis. Diabetics and non-diabetic subjects carrying the E2 allele had lower total and LDL cholesterol concentrations. In subsequent years, Horita et al. [15] and Eto et al. [16] from Japan reported that the {varepsilon}2 allele frequency was significantly higher in diabetic patients with nephropathy and renal failure, which suggested that apo {varepsilon}2 would be associated with nephropathy and renal insufficiency in NIDDM. They suggested that increased plasma levels of remnants and triglyceride-rich lipoproteins which were associated with apo E2 in NIDDM contributed to the renal damage in NIDDM, although there was no evidence to support a direct link between remnants and renal damage. There were several case reports of so called lipoprotein glomerulopathy in recent years [23,24]. This entity is characterized by the presence of apo E2, type III hyperlipoproteinaemia-like lipoprotein profiles and proteinuria. These reports supported the possibility that increased remnants in plasma associated with apo E2 may contribute to renal damage but a direct link between apo E2 and diabetic nephropathy remains speculative. Our study showed the same finding as Horita et al. and Eto et al. as far as the relationship between apo E2 and macroalbuminuria. Onuma et al. [17] investigated the relationship between apo E polymorphism and diabetic nephropathy in Caucacian patients with IDDM. They did not see any difference in the distribution of apolipoprotein E genotypes and allele frequencies in the proteinuric, microalbuminuric, or normoalbuminuric groups. And they also found no significant association between the lipid levels and the apo E genotypes in the IDDM patients. Recently, Kimura et al. [18] showed different results compared with ours. They showed that the presence of apo E4 allele was protective against the progression of diabetic nephropathy in Japanese population. We did not see any protective effect of the E4 allele in our subjects. Also our report did not support E3 allele being a risk factor. Kimura's report and ours were the same with respect to the E2 allele.

Some argue that duration of diabetes of at least 8 years is too short in the normoalbuminuria group which may develop microalbuminuria later and then progress to macroalbuminuria. In type 2 diabetes, diabetes is actually present for many years before the diagnosis is made [25]. Mean duration of diabetes in our study population (around 13 years) were not different. Also, we calculated odds ratio by failure time anlaysis using cox-proportional hazard model and revealed that the carriage of apo {varepsilon}2 allele has a 3.466 times higher chance to macroalbuminuria in the progression of diabetic nephropathy compared with apo {varepsilon}3 allele. What are the reasons for these different results? First, the distribution of apo E frequencies is different between various ethnic groups. Secondly, there are differencies in how diabetic nephropathy was defined. Thirdly, sampling errors influence the study results as in ACE I/D polymorphism in diabetes mellitus.

In summary, although our study was somewhat limited due to the size of sample, we found a significant association between both apo {varepsilon}2 allele and {varepsilon}2 carrier frequencies and macroalbuminuria in Korean NIDDM patients. The frequency of apo {varepsilon}3 and {varepsilon}4 alleles were slightly lower in patients with macroalbuminuria when compared with normoalbuminuric patients, but these differences were not statistically significant. Future stuies with a prospective longitudinal approach and a larger sample size will provide more valuable information on the association of apo E polymorphism and genetic susceptibility for diabetic nephropathy.



   Acknowledgments
 
This work was supported by Professor Research Grant of Yonsei University College of Medicine (1997–04).



   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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