Associations between apolipoprotein CIII concentrations and microalbuminuria in type 2 diabetes

Microalbuminuria (MAU) is a strong predictor of diabetic nephropathy (DN), which is the main cause of morbidity and mortality in patients with diabetes mellitus (DM). Dyslipidemia exists in the majority of patients with DM and contributes to micro- and macrovascular complications associated with DM. Apolipoprotein CIII (apoCIII) is an inhibitor of the activity of lipoprotein lipase, which metabolizes triglyceride (TG) in very low-density lipoprotein (VLDL) and facilitates its clearance from plasma. The aim of the present study was to investigate the associations between apoCIII and MAU and the effects of atorvastatin in type 2 diabetes. In total, 120 subjects were divided into type 2 diabetes and type 2 DN groups, while 60 healthy subjects were selected as controls. The patients with DN were administered 20 mg atorvastatin daily for 16 weeks. Blood pressure, body mass index (BMI) and levels of HbA1c, FBG, TG, VLDL-cholesterol (VLDL-C), apoCIII and MAU were markedly elevated in the type 2 diabetes and type 2 DN groups compared with those in the control group (P<0.01), while high-density lipoprotein-cholesterol (HDL-C) levels were decreased significantly (P<0.01). All patients with type 2 DN showed significantly elevated blood pressure, apoCIII levels, MAU, course of the disease and rate of stroke and retinopathy compared with the patients with type 2 diabetes (P<0.01). MAU was significantly positively correlated with the course of the disease, systolic blood pressure, diastolic blood pressure, BMI and HbA1c, FBG, TG, total cholesterol, low-density lipoprotein-cholesterol, VLDL-C and apoCIII levels (P<0.05), whereas negatively correlated with HDL-C levels (r=−0.194, P=0.020). Logistic regression analysis showed that apoCIII levels were independently associated with MAU (odds ratio, 1.100; 95% confidence interval, 1.037–1.153; P<0.001). Atorvastatin improved the lipid profile and MAU in patients with type 2 DN (P<0.01). Therefore, the present study demonstrated that an independent positive correlation exists between the levels of apoCIII and MAU in patients with type 2 diabetes. Furthermore, atorvastatin may be used to improve the lipid profile and MAU in type 2 DN.


Introduction
Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD). One of the early markers of DN and vascular disease in patients with diabetes is the presence of microalbuminuria (MAU). Without specific intervention, 20-40% of type 2 diabetic patients with MAU progress to overt nephropathy (1). Therefore, screening for MAU and timely therapeutic intervention has become the standard in care worldwide.
Plasma triglyceride (TG) levels are increased in patients with DN. The abnormal synthesis and clearance of TG and the lipoprotein particles associated with TG may be involved in the development of DN (2). There is evidence suggesting that TG-rich lipoprotein (TRL) particles predominantly containing apolipoproteins (apos) E, C and B may be major promoters of DN (2). In patients with DN, plasma TG levels may increase, partly due to the reduced activity of hepatic lipase (HL) and lipoprotein lipase (LPL), which hydrolyze TG (3).
ApoCIII is a protein composed of 79 amino acid residues that is predominantly synthesized in the liver, although synthesis also occurs in the intestine. ApoCIII is an exchangeable protein moiety between chylomicron remnants, very low-density lipoproteins (VLDLs) and high-density lipoproteins (HDLs) (4,5). In the blood circulation, ApoCIII is associated mainly with TRLs, HDLs and, to a lesser extent, low-density lipoproteins (LDLs) (6)(7)(8). Total plasma apoCIII levels have been found to be key determining factors of serum TG levels. Studies involving gene variation and circulating protein levels have implicated apoCIII as a risk factor for cardiovascular disease (9)(10)(11)(12). ApoCIII is an inhibitor of the activity of LPL (13), which metabolizes TG in VLDL and facilitates its clearance from plasma. In addition, ApoCIII reduces the plasma clearance of VLDL and LDL by inhibiting their interaction with hepatic lipoprotein receptors (14,15).
There is considerable information regarding the effects of apoCIII on lipoprotein metabolism (10,16,17). Statins have been suggested to lower plasma apoCIII levels. In 27 patients with primary hypertriglyceridemia, administration of 20-40 mg atorvastatin for four weeks reduced plasma apoCIII levels by 18-30% (18). However, little information is available regarding the association between microalbuminuria (MAU) and circulating apoCIII levels in DN. To enhance the understanding of this issue, the present study aimed to assess serum apoCIII levels and the association between apoCIII levels and MAU, and to evaluate the effectiveness of statins in lowering apoCIII levels and therefore decreasing MAU in type 2 diabetes.

Materials and methods
Patients. Subjects with type 2 diabetes (n=120) aged 48-66 years were recruited from Hebei General Hospital (Shijiazhuang, China). A group of 60 healthy subjects aged 47-65 years (30 male and 30 female) was selected as control subjects. The study was approved by the Institutional Ethic Committee of Hebei General Hospital and written informed consent was obtained from all subjects. Written informed consent was obtained from all subjects. The baseline characteristics of the type 2 diabetes and type 2 DN groups are shown in Table I. The patients with DN were administered atorvastatin (20 mg) daily. Patients were interviewed every four weeks during treatment to assess drug adherence up to a total of 16 weeks.

Samples.
A blood sample was collected following an overnight fast of ≥8 h and prior to insulin administration from the 120 patients with type 2 diabetes and the 60 healthy subjects. Fasting blood samples were taken for the measurement of serum glucose, glycosylated hemoglobin (HbA1c) and the lipid profile. Timed urine samples (24 h) were collected from all patients for the determination of MAU. Fasting blood glucose (FBG) was determined on a Beckman CX9 automatic analyzer (Beckman Coulter, Miami, FL, USA) using a glucose oxidase method. HbA1c levels were determined by high-performance liquid chromatography, using the BioRad Variant Hemoglobin Analyzer (Bio-Rad, Hercules, CA, USA).
For the lipoprotein studies, blood was placed on ice in polypropylene tubes containing a solution of lipoprotein preservatives comprising 2.8 mmol/l EDTA, 62 µmol/l chloramphenicol, 50 µg/ml gentamycin sulfate, 10 mmol/l ε-aminocaproic acid and 100 mmol/l 5,5'-dithiobis-(2-nitrobenzoic acid) (final concentrations). Samples were immediately centrifuged at 1,008 x g for 25 min to sediment blood cells and were subsequently stored at -80˚C until analysis. Total cholesterol (TC) and TG levels were determined using the enzymatic colorimetric method on the Beckman CX9 automatic analyzer and HDL-cholesterol (HDL-C) was measured using a direct enzymatic HDL-C method based on polyethylene glycol-modified enzymes on the Beckman CX9 automatic analyzer. VLDL-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) levels were estimated using the Friedewald formula (19). ApoCIII concentration in the serum was determined using a competitive ELISA method (Bioworld Technology, Shanghai, China) (18). The standard curve was fitted to a four-parameter sigmoidal curve. The inter-and intra-assay variability for the assay was <7%.
Body mass index (BMI) and blood pressure (BP) were measured for all the subjects. Obesity was defined as BMI ≥25 kg/m 2 . Hypertension was defined as BP >140/90 mmHg or the administration of anti-hypertensive drug treatment. Fasting blood samples were measured for biochemistry and metabolic profile analysis. Dyslipidemia was defined as TG ≥1.7 mmol/l and/or HDL-C <1.1 mmol/l in males and <1.3 mmol/l in females and/or LDL-C > 2.6 mmol/l or treatment with lipid-lowering drugs. DN was defined as MAU ≥30 mg/24 h in two collections of timed urine samples (24 h) and serum creatinine (Cr) ≤132 µmol/l. Patients with urinary tract infection, hematuria shown by urine microscopy or obstructive uropathy shown by kidney ultrasound suggestive of non-diabetes-related causes were excluded. Type 2 diabetic patients without DN had serum Cr ≤132 µmol/l and MAU <30 mg/24 h in two collections of timed urine samples. Control subjects without diabetes had no known history of diabetes, exhibited normal glucose tolerance at the 75 g oral glucose tolerance test (1998 World Health Organization criteria) (20) and had normal blood biochemistry. The blood and urine samples of patients with type 2 DN were collected after 16 weeks of therapy.
Statistical analysis. Analyses were performed using SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data are expressed as percentages or as the mean ± standard deviation. The mean differences of continuous variables between the type 2 diabetes and type 2 DN groups were analyzed using the Student's t-test. The mean differences of categorical variables between these two groups were analyzed using the χ 2 test. The mean differences among groups were analyzed using analysis of variance, and the Newman-Keuls post hoc test was used in the event of a significant F-ratio. Pearson correlation was performed between MAU and the other variables. Logistic regression analysis was used to assess the association between MAU and other clinically relevant variables, where the clinical variables were considered as independent and MAU as dependent. Estimated odds ratios (ORs) are expressed with their 95% confidence intervals (95% CIs). All significant tests were two-sided and were considered statistically significant at P<0.05.

Results
Comparisons of basic clinical data. Compared with the type 2 diabetes group, the type 2 DN group showed a longer course of disease (P<0.01). The stroke and retinopathy rates in the patients with type 2 DN were significantly higher than those in patients without DN (P<0.05). However the occurrence rate of coronary heart disease exhibited no statistical difference between the two groups (P>0.05) ( Table I).
Comparisons of BP, BMI, HbA1c, FBG, lipid profile, apoCIII and MAU. Compared with the control group, systolic BP (SBP), diastolic BP (DBP), BMI and levels of HbA1c, FBG, TG, VLDL-C, apoCIII and MAU were markedly elevated in the type 2 diabetes and type 2 DN groups (P<0.01), while HDL-C levels were decreased significantly (P<0.01). All patients with type 2 DN showed significantly elevated TC and LDL-C levels (P<0.05) (Table II). Although the BMI and levels of HbA1c, FBG, TG, TC, LDL-C and VLDL-C were elevated in the type 2 DN group compared with those in the type 2 diabetes group, the differences were not significant (P>0.05). However, the levels of SBP, DBP, apoCIII and MAU in this group were more notably increased (P<0.01) (Table II).  (Table IV).
Atorvastatin improves the lipid profile and MAU levels in patients with type 2 DN. After 16 weeks of atorvastatin administration in patients with type 2 DN, levels of TG, TC, LDL-C, VLDL-C and apoCIII were significantly decreased compared with those prior to treatment, while HDL-C levels were found to have increased markedly (P<0.05).  MAU levels were observed to have decreased significantly (P<0.01) ( Table V).

Discussion
There is currently a global epidemic of type 2 diabetes mellitus (DM), which accounts for 40-50% of all new cases of ESRD. Among the clinical signs of nephropathy, the appearance of low but abnormal levels (>30 mg/day) of albumin in the urine, known as MAU, is first to occur. MAU is a leading cause of DM-related morbidity and mortality (21). In type 2 diabetes, lipotoxicity and glucotoxicity increase the risk of diabetic micro-and macrovascular complications (3,22). In the majority of type 2 diabetic patients, insulin resistance is a key pathophysiological feature. Lipases are insulin-sensitive enzymes that hydrolyze TG in TRL particles. Diabetic dyslipidemia is typically characterized by high TG and low HDL-C levels. The high TG levels in turn can alter the composition of LDL-C and HDL-C, making these lipid particles more atherogenic (23). Nephritis can be exacerbated through increased TG levels and oxidized remnant lipoprotein particles, which induce mesangial cells to proliferate and secrete cytokines (24). Following the initiation of proteinuria, the loss of lipoprotein particles in the urine can enhance their synthesis in the liver. Furthermore, the lipoprotein particles can cause renal damage by binding to glomerular basement membranes and renal tubular cells. This can initiate a vicious cycle of proteinuria and dyslipidemia (22). Patients with renal disease, whether associated with diabetes or not, have increased TG and remnant lipoprotein levels and decreased LPL and Values are presented as the mean ± standard deviation (n=60). TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; VLDL-C, very low-density lipoprotein-cholesterol; apoCIII, apolipoprotein C-III; MAU, microalbuminuria. HL activity (3). In addition to the effects of lipotoxicity on the kidney, increased von Willebrand factor and decreased heparin-releasable LPL levels lead to vascular endothelial damage. Reduced lipase binding to the damaged endothelium may further increase TG levels in DN (3).
The present study showed that BP, BMI and levels of HbA1c, FBG, TG, VLDL-C, apoCIII and MAU were markedly elevated in the type 2 diabetes and type 2 DN groups compared with those in the control group (P<0.01), while HDL-C levels were decreased significantly (P<0.01). These findings were consistent with those in other studies (3,23). All patients with type 2 DN showed significantly elevated BP, apoCIII, MAU, course of the disease and stroke and retinopathy rates compared with the patients with type 2 diabetes (P<0.01).
ApoCIII is an effective inhibitor of LPL activity and is present in TRLs and HDLs (13). Sections of apoCIII can be exchanged between these lipoproteins (24). Upon an elevation in TRL concentration, ApoCIII is transferred from HDLs to TRLs, which causes a decrease in HDL apoCIII concentration. Following the degradation of TG in TRLs by LPLs, apoCIII transfers back to the HDLs, such that clearance of the TRL particles may proceed uninhibited (25). In a study by Klein et al (26), an independent positive association between apoCIII levels and type 1 diabetes-related microvascular complications was found in the cohort of patients.
The present study revealed that MAU was significantly positively correlated with the course of the disease, SBP, DBP, BMI and HbA1c, FBG, TG, TC, LDL-C VLDL-C and apoCIII levels, whereas negatively correlated with HDL-C levels (P<0.05). MAU exhibited no association with age and gender (P>0.05). Logistic regression analysis revealed that only apoCIII was independently associated with MAU (OR, 1.100; 95% CI, 1.037-1.153; P<0.001). Other factors were not independent predisposing factors for MAU in type 2 diabetes.
Atorvastatin is a potent 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor that has been shown to effectively reduce total plasma TC and TG levels. Statins have become the preferred agents as lipid-lowering drugs in diabetic patients (27). Statins have been reported to exert cardioprotective and antioxidant effects, regardless of their effect on LDL-C reduction (28). In a study of 305 patients with primary hypercholesterolemia, atorvastatin and pravastatin reduced levels of lipoprotein particles containing apoCIII and apoB by 30% (29). The present study indicated that, by the end of 16 weeks, atorvastatin decreased TG, TC, LDL-C, VLDL-C and apoCIII levels significantly (P<0.05), while increasing HDL-C markedly (P<0.05) in patients with type 2 DN. A significant decrease in MAU was also observed (P<0.01). Rutter et al (30) compared the renal effects of treatment with low-versus high-dose atorvastatin in patients with type 2 DM and optimally managed early renal disease. No statistical difference in renal function was identified between the high-and low-dose atorvastatin treatment groups over two years. The beneficial effect of a glomerular filtration rate of <1.6 ml/min/1.73 m 2 /year, as estimated using the Modification of Diet in Renal Disease equation, was observed. However, such an effect may have been due to blood pressure management and/or a renin-angiotensin system blocker use (30,31). With regard to the present findings, the decrease in MAU in patients with type 2 DN may have been due to comprehensive treatment. In conclusion, in type 2 diabetes, circulating apoCIII levels were independently correlated with MAU. Atorvastatin improved the lipid profile in patients and may contribute to decreasing MAU levels in type 2 DN.