1 Department of Internal Medicine, School of Medicine, University of Ioannina, Ioannina, Greece and 2 Molecular Genetics Department B-DNA Identification Laboratory, the Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
Correspondence and offprint requests to: Moses S. Elisaf, MD, FACA, FRSH, Department of Internal Medicine, University of Ioannina, 451 10 Ioannina, Greece. Email: egepi{at}cc.uoi.gr
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Abstract |
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Methods. 290 apparently healthy individuals were studied. ApoE genotyping was performed by the polymerase chain reaction; the Modification of Diet in Renal Disease equation (MDRD) predicted the GFR.
Results. ApoE2 was associated with lower levels of total cholesterol, low-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol, as well as with higher levels of triglycerides in our population. Furthermore, the ApoE2 allele was associated with increased serum creatinine levels compared with both the E3 and E4 alleles (1.04±0.13 vs 0.92±0.13 vs 0.88± 0.11 mg/dl, respectively, P = 0.0077), while the MDRD-predicted GFR was decreased in ApoE2 carriers compared with both E3 and E4 carriers (80.3±10.2 vs 88.1±9.6 vs 89.3±9.7 ml/min/1.73 m2, respectively, P = 0.031). These observations remained significant statistically even if the effect of ApoE polymorphisms on age- and body-mass index-adjusted serum creatinine and MDRD-predicted GFR was separately analysed in both men and women. Although, ApoE4 carriers tended to exhibit lower levels of serum creatinine and higher values of predicted GFR compared with the E3 carries, these differences did not reach statistical significance.
Conclusions. ApoE2 allele seems to be associated with increased serum creatinine levels and decreased MDRD-predicted GFR in healthy subjects.
Keywords: apolipoprotein E; creatinine; GFR; lipids; lipoproteins; renal function
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Introduction |
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Beyond the known influence of ApoE polymorphisms on serum lipid profile, on the pathogenesis of atherosclerosis and the development of neurodegenerative disorders, ApoE also exerts a major role in the pathogenesis and the progression of a variety of renal diseases, as well as in the atherosclerotic complications associated with them (recently reviewed in [3]). However, there is much discrepancy in the literature concerning the possible ApoE polymorphism-mediated predisposition to the deterioration of renal function and the development of end-stage renal disease in both diabetic and non-diabetic, as well as in paediatric and adult patients [3]. Some studies have provided evidence that ApoE2 allele carries such a genetic risk [410], whereas others have shown that the ApoE4 allele is a risk factor for the progression of renal failure [1113]. On the other hand, some studies have provided evidence that the ApoE4 allele protects from the progression of diabetic nephropathy in type 2 diabetics [9,14]. Furthermore, the ApoE4 allele has been associated with reduced postoperative increase in serum creatinine levels after cardiac coronary bypass surgery, whereas there was no difference in the preoperative serum creatinine concentrations among the different ApoE allele groups [15]. The ApoE4 allele has been associated with better creatinine clearance in kidney transplanted patients [16], whereas the ApoE2 allele has been reported to be a negative predictor of creatinine clearance in type 1, but not in type 2 diabetic patients [6]. In fact, there are experimental data for a protective role of kidney ApoE, with the ApoE2 isoform being less protective against mesangial cell proliferation than the ApoE3 and ApoE4 isoforms [17].
However, there are no studies concerning the possible effect of ApoE polymorphisms on the levels of serum creatinine and glomerular filtration rate (GFR) in healthy individuals without renal disease. Thus, we undertook the present study to test the hypothesis that ApoE polymorphisms could influence serum creatinine levels and predicted GFR values in healthy subjects.
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Subjects and methods |
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Laboratory measurements
In all participants blood samples were obtained after a 14-h overnight fast for gene genotype detection, as well as for the determination of serum laboratory parameters. Blood samples were centrifuged for 30 min (3600 g) and then the serum was separated and stored at 80°C for analysis of laboratory parameters.
Concentrations of total cholesterol and triglycerides were determined enzymatically on the Olympus AU600 clinical chemistry analyser (Olympus Diagnostica, Hamburg, Germany). HDL-cholesterol (HDL-C) was determined in the supernatant, after precipitation of the ApoB-containing lipoproteins with dextran sulphate-Mg2+ (Sigma Diagnostics, St Louis, MO, USA). LDL-cholesterol (LDL-C) was calculated using the Friedewald formula, while the non-HDL-C was calculated by the equation: non HDL-C = total cholesterol HDL-C.
Finally, serum creatinine levels were determined using the Jaffé method [kinetic alkaline picrate reaction, normal range 0.71.4 mg/dl (62124 µmol/l)], while serum albumin and serum urea nitrogen were measured according to the bromcresol green and enzymatic urease methods, respectively.
ApoE genotyping
DNA was extracted from the whole blood specimens according to standard procedures. ApoE genotyping was performed as described by Hixson and Vernier. Polymerase chain reaction (PCR) was used to amplify a 244-bp sequence of the ApoE gene, including the two polymorphic sites. The PCR products were then digested with the restriction enzyme HhaI and the different genotypes were detected after electrophoresis on 8% non-denaturing polyacrylamide gels, treated with ethidium bromide and visualized in ultra-violet radiation. Subjects were classified as ApoE2 carriers if they had the ApoE2/3 or the ApoE2/2 genotype, ApoE3 carriers if they had the ApoE3/3 genotype, and ApoE4 carriers if they had the ApoE4/3 or the ApoE4/4 genotype. Subjects with the ApoE4/2 (n = 3) were excluded from the study due to their small number and the difficulties in their classification. Thus, 287 subjects were finally included in the analysis.
Estimation of GFR
GFR was predicted by a recently described formula, which was developed based on the data derived from the Modification of Diet in Renal Disease (MDRD) study in patients with renal dysfunction [18]:
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Statistical analysis
Statistical analysis was performed with STATISTICA 6.0 statistical software. The effect of the ApoE gene polymorphisms on laboratory parameters was tested using the one-way analysis of variance (one-way ANOVA) followed by the LSD test (in case of significant effects) for multiple pairwise comparisons, except for serum triglycerides, where the KruskalWallis ANOVA median test was used followed by the MannWhitney U test for pairwise comparisons because of their skewed distribution. Furthermore, the effect of ApoE polymorphisms on the serum creatinine and MDRD-predicted GFR values was adjusted for age and body-mass index (BMI) by the analysis of covariance (ANCOVA) in both men and women. Finally, multiple linear regression analysis was performed to test the effect of ApoE polymorphisms and other factors (sex, age and BMI) on serum creatinine levels.
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Results |
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Finally, Table 3 shows the age- and BMI-adjusted effect of ApoE polymorphisms on serum creatinine and MDRD-predicted GFR levels separately in men and women. The above-described association of ApoE2 allele with higher serum creatinine concentrations and lower MDRD-predicted GFR values remained significant statistically and to the same extent in both male and female subgroups, and this association persisted even after correction for age and BMI.
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Discussion |
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There is only one report in the literature concerning the effect of ApoE polymorphisms on serum creatinine levels. This study did not reveal any difference in serum baseline pre-operative creatinine among the different ApoE allele groups in surgical coronary bypass patients [15]. However, the study population consisted of patients with coronary artery disease, including diabetics, while there was no report on the prevalence of hypertension or dyslipidaemia in these patients. Additionally, ApoE2 has been associated with decreased creatinine clearance in type 1, but not in type 2 diabetics [6], while ApoE4 was linked with better creatinine clearance in kidney transplanted patients [16].
Our study suggests that ApoE2 allele may have an unfavourable effect on renal function, and the serum creatinine levels and the MDRD-predicted GFR in healthy individuals reflect this. This observation is in good agreement with the above mentioned study in diabetic patients [6], and with studies that have shown that the ApoE2 allele is a genetic risk factor for the progression of renal disease in type 1 diabetics [5,6], in type 2 diabetics [79], in patients with IgA nephropathy [10] and in patients with different renal diseases [4]. However, other studies have failed to show any association between ApoE polymorphisms and diabetic nephropathy in type 1 diabetics [20]. The reasons for these conflicting results are poorly understood, but may be related to ethnic differences, different study protocols, sampling errors, different prevalence of ApoE alleles among the studied populations, or to interactions with other genetic or environmental factors [3].
ApoE4 carriers in our healthy population tended to have the lowest serum creatinine levels and the highest MDRD-predicted GFR, although there were not significant differences from E3 carriers. However, this is in keeping with the studies that have shown a protective effect for the ApoE4 allele from the deterioration of renal function in type 2 diabetics [9,14], and from acute renal impairment in post-bypass surgical patients [15], as well as with the study that showed a better renal function in ApoE4 kidney transplanted patients [16], and with our findings showing a significantly lower prevalence of the ApoE4 allele in patients with end-stage renal disease compared with the healthy population (7.3 vs 13.0%, respectively, P<0.01). However, other studies have related the ApoE4 allele with the progression of renal disease in diabetic patients [12,13] and in renal transplant recipients [11].
As it was anticipated, ApoE polymorphisms significantly affected serum lipid levels in our participants; ApoE2 had a cholesterol-lowering effect, while ApoE4 had a cholesterol-raising effect compared with the ApoE3 allele. This cholesterol-lowering effect of 2 allele can be seen in almost all studies involving healthy subjects [2]. Because of the failure of ApoE2 isoform to bind effectively to the LDL and the ApoE receptors, less cholesterol from the chylomicrons, the VLDL and their remnants enters the hepatocytes, resulting in an up-regulation of the LDL receptors, which in turn lowers serum ApoB-containing lipoproteins [2]. Moreover, LDL clearance is probably enhanced due to the higher affinity of the LDL particles for the LDL receptor compared with the remnant lipoproteins carrying the defective ApoE2 [2]. On the other hand, the ApoE4-induced increase of total and LDL cholesterol levels is attributed to an increase in the intestinal absorption of dietary cholesterol, and to a down-regulation of LDL receptors in the surface of hepatic cells. The latter results from an increased delivery of cholesterol to the hepatic cells owing to the enhanced interaction of ApoE4-containing remnants and the ApoE receptors. Furthermore, the ApoE genotype can influence the location of ApoE in lipoproteins, with the E4 isoform being preferentially located in the VLDL and with the E3 and E2 isoforms in HDL. Finally, ApoE2 was associated with higher serum triglycerides in our healthy individuals, probably due to the delayed receptor-mediated clearance of the triglyceride-rich lipoproteins.
The underlying mechanisms for the observed association between E2 allele and higher creatinine concentrations, as well as lower predicted GFR values are not well understood. First, ApoE2 is associated with higher levels of triglycerides, and thus higher concentrations of triglyceride-rich lipoproteins in our healthy population. An increase of triglyceride-rich lipoproteins could stimulate the accumulation of cholesteryl esters by human mesangial cells (HMCs) leading to a change in the properties of the mesangial matrix and glomerulosclerosis, thus impairing renal function. In fact, triglyceride-rich lipoproteins from ApoE2 diabetic patients enhanced cholesteryl ester accumulation by HMCs significantly more than those from ApoE3 patients [9]. Furthermore, ApoE2/2-induced type III hyperlipoproteinaemia is closely associated with glomerular lipidosis or a lipoprotein glomerulopathy-like disease [3]. On the other hand, the ApoE4 isoform has been reported to be more effective in modulating the direct uptake and conversion of remnant lipoproteins to LDL, leading to decreased levels of triglyceride-rich lipoproteins, thus preserving renal function [14]. Secondly, ApoE has been shown to inhibit mesangial cell proliferation induced by cytokines or LDL in experimental models [17]. In these experiments, ApoE2 was found to be the least effective isoform independent of the presence of hyperlipidaemia. Moreover, the ApoE2 isoform has also been shown to have no significant antiproliferative effect on smooth muscle cells [17]. Therefore, the inability of ApoE2 to regulate mesangial expansion due to different stimuli may result in restriction of glomerular capillary luminal volume, which in turn diminishes filtration surface [6]. This mechanism may partially explain the observed unfavourable effect of ApoE2 allele on renal function. Thirdly, ApoE2 may not be the truly unfavourable gene, but it may be in linkage disequilibrium with another renal harmful gene. This hypothesis, however, was rejected in another study, which examined the effect of ApoE polymorphisms on the development of diabetic nephropathy in type 1 diabetics [5].
On the other hand, the observed association between the ApoE polymorphism and the levels of serum creatinine and the creatinine-based MDRD-predicted GFR could be the consequence of a mechanism unrelated to the renal function, for instance creatinine metabolism. Yet, there are no literature data to support this hypothesis.
One limitation of our study is that the determination of serum creatinine was performed using the Jaffé kinetic method, which is less accurate compared with the enzymatic methods for creatinine measurement due to the presence of non-creatinine chromogens. However, since the Jaffé method was also used in the MDRD study [18], measurement of creatinine with another laboratory method could have resulted in inaccurate results regarding the use of the MDRD-derived formula. Moreover, no medical condition known to interfere with creatinine assay in the Jaffé method (i.e. diabetic ketoacidosis, administration of certain cephalosporins, lipaemia or jaundice) was present in any of our study subjects. Finally, the Jaffé method used in our laboratory has been compared with an enzymatic method for creatinine determination (creatinine deiminase, Randox, Mauguio, France). We found a high correlation between the two methods (r = 0.99), when considering creatinine concentrations ranging from 0.70 to 11.3 mg/dl (621.000 µmol/l). In fact, all creatinine values in our study were within this range.
Additionally, our study participants consisted of apparently healthy individuals visiting our outpatient clinic for a regular medical check-up. However, this population may not be representative of a general healthy population, since their mean age was 60 years old, and their mean BMI was in the overweight range. Therefore, the results obtained from our study should be carefully extrapolated in the general population.
In conclusion, we provide for the first time data on the possible effect of ApoE polymorphisms on serum creatinine levels and predicted GFR values in apparently healthy subjects; the ApoE2 allele seems to be associated with the highest creatinine concentrations and the lowest predicted GFR values in these healthy individuals. Further larger prospective studies using more accurate measurements of renal function are necessary to better clarify the association between ApoE polymorphisms and renal disease.
Conflict of interest statement. None declared.
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References |
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