The association of serum adiponectin with abdominal aortic calcification in Japanese male hemodialysis patients: a cross-sectional observational study

The negative relation of serum adiponectin to atherosclerosis becomes a positive association in patients with chronic kidney disease (CKD). We conducted a small-scale cross-sectional observational study, in 101 Japanese male hemodialysis patients, to examine the relationship of serum adiponectin and leptin to abdominal aortic calcification (AAC). The presence of AAC was evaluated from simple X-ray radiographs of the left lateral abdomen. Serum adiponectin was significantly higher in AAC-positive patients [18.8 (13.0–28.1) μg/mL] than in AAC-negative patients [15.4 (8.9–22.8) μg/mL] (p = 0.03), whereas serum leptin did not differ significantly between the two groups. Multiple logistic regression analysis showed that log adiponectin, but not log leptin, was independently and significantly associated in a positive manner with AAC (odds ratio: 16.31, 95% confidence interval: 1.70–156.41, p = 0.02), after adjustment for age, body weight, percentage body fat, hemodialysis duration, prevalence of diabetes mellitus, and other risk factors. In conclusion, we found a positive and independent association of serum adiponectin with AAC in male hemodialysis patients, indicating that the reversed association between serum adiponectin and atherosclerosis in patients with CKD dose not result from increased serum adiponectin due to the impaired urinary secretion.


Results
Clinical characteristics of patients, and comparisons between hemodialysis patients with and without AAC. Table 1 showed the characteristics of 101 hemodialysis patients, among whom 55 (54%) were positive for AAC on left abdominal X-ray radiographs. The serum level of adiponectin was 17.0 (11.3-24.9) µg/ mL, which was approximately 3-fold higher than the reported value of 5.4 ± 2.3 µg/mL in male subjects without CKD, whereas the serum levels of leptin did not differ from those in subjects without CKD 8 . The serum adiponectin and leptin levels were significantly correlated in a negative and positive manner, respectively, with percentage body fat (adiponectin: ρ = −0.40, p < 0.001; leptin: ρ = 0.81, p < 0.001) in hemodialysis patients, which were consistent with the significant correlations reported previously in the general population 9,10 .
Various clinical parameters in hemodialysis patients were compared between those with and without AAC. AAC-positive hemodialysis patients were significantly older and had significantly higher prevalence of DM (p < 0.001) than their AAC-negative counterparts. Furthermore, AAC-positive patients showed a tendency towards having a higher serum high-sensitivity C-reactive protein (hs-CRP) (p = 0.07) than AAC-negative patients; however, the difference was not significant.

Multiple logistic regression analysis of the association between AAC and other factors.
Multiple logistic regression analysis was employed to examine whether serum adiponectin was independently and significantly associated with the presence of AAC (Table 2). Model 1, which included log adiponectin, in addition to age, body weight, percentage body fat, hemodialysis duration, presence/absence of DM, serum calcium and

Discussion
The present cross-sectional observational study demonstrated that higher serum adiponectin, but not leptin, was significantly and positively associated with the prevalence of AAC in male hemodialysis patients, suggesting higher serum adiponectin as a definite risk for AAC, independent of other risk factors for AAC, such as age, percentage body fat, hemodialysis duration, presence/absence of DM, and serum levels of calcium, phosphate, intact PTH, and hs-CRP. The presence of DM was significantly and positively associated with the prevalence of AAC in male hemodialysis patients, as we reported previously 11 . Furthermore, the present study clearly demonstrated that the positive association of increased serum adiponectin with the presence of AAC thus far reported in pre-dialysis CKD patients could not be explained by the apparent increase of serum adiponectin due to the impaired renal excretion of adiponectin into urine, as hemodialysis patients without any appreciable residual renal function exhibited a significant, independent, and positive association between serum adiponectin and AAC. Previous studies showed that patients with CKD exhibited higher serum adiponectin due to impaired urinary adiponectin excretion, while they had a higher incidence of vascular calcification due to higher serum calcium × phosphate products, making the relationship between serum adiponectin and AAC significant and positive. Therefore, it remains difficult to distinguish whether increased serum adiponectin by itself might be a true risk factor that is intimately involved in the development of AAC, or simply a surrogate marker of renal dysfunction. However, the present study first determined that higher serum adiponectin could be independently and positively associated with the development of AAC in patients with CKD stage 5D without appreciable residual renal function. Because the enrolled hemodialysis patients had lost almost all residual renal function and thus could not produce a significant difference in the apparent increase of serum adiponectin due to impaired renal function, the present study clearly shows that increased serum adiponectin is by itself a positive factor associated with the prevalence of AAC and thus a definite risk for CVD in patients with CKD.
Epidemiologic studies, thus far performed mainly in patients with metabolic syndrome, have established the notion that serum adiponectin is negatively and independently associated with predictors for CVD risks and CVD events, by using various clinically useful markers for CVD risk, such as carotid intima-media thickness 12 , endothelial dysfunction of the coronary arteries, acute coronary syndrome, and multi-vessel coronary atherosclerosis 13 . Moreover, in a case-control study involving men without pre-existing CVD, higher adiponectin was found to be associated with a lower risk of myocardial infarction 2 . In basic studies, adiponectin-deficient mice developed arterial calcification in vivo, while adiponectin inhibited the differentiation of vascular smooth muscle cells into osteoblasts in vitro 14 . However, contrary to the accepted hypothesis that reduced serum adiponectin is associated with an increased CVD risk or CVD mortality in men, such a relationship has been reported to be reversed, particularly in elderly subjects, in patients with pre-existing CVD 15 , and in pre-dialysis CKD patients 16 .
Among the various studies investigating the relationship between serum adiponectin and atherosclerosis in human subjects, only a few have investigated the relationship of serum adiponectin with vascular calcification. Serum adiponectin was not associated with calcified atherosclerotic plaques in patients with diabetes, with coronary artery calcification (in Caucasians) 17 , or with calcified aortic stenosis 18 . There are limited data suggesting that higher adiponectin levels are directly associated with vascular calcification in patients with CKD. The present study demonstrated that higher serum adiponectin levels were associated with a higher prevalence of AAC in hemodialysis patients. It is known that as renal dysfunction progresses to end-stage renal disease, the prevalence of vascular calcification becomes higher, and the presence and degree of AAC are independent predictors of future CVD events and mortality, as we previously reported 7 . Combining the data from our present study with the data from previous study, indicating the increase of serum adiponectin along with a decline in the estimated glomerular filtration rate in pre-dialysis CKD patients, it seems likely that the increase of serum adiponectin in pre-dialysis CKD patients might be involved in the development of AAC, leading to increased mortality. However, adiponectin has beneficial effects on cardiovascular cells through its antidiabetic, anti-inflammatory, and antiatherogenic action in vitro 19 . The mechanism of why adiponectin positively induces AAC in hemodialysis patients might be explained by its effect on the nutritional and inflammatory status, which is a very common condition associated with the development of atherosclerosis including AAC. This condition, known as MIA (malnutrition, inflammation, atherosclerosis) -syndrome, is composed of malnutrition including decreases in body fat, which might be accompanied by an increase in serum adiponectin, and atherosclerosis including vascular calcification. Therefore, it is possible that the frequent occurrence of malnutrition in hemodialysis patients will reverse the association between serum adiponectin and AAC. However, in this study, the association between adiponectin and increased vascular calcification risk was not modified by body weight or percentage body fat. This suggests that decreases in body fat cannot fully account for the positive association between serum adiponectin and AAC. Alternatively, increased serum adiponectin might protect against the development of AAC in situations that stimulate AAC; however, its protective effect is insufficient 20 . It has been suggested that such counter-regulatory vascular protective mechanisms lead to increases in adiponectin levels. Finally, it was reported that adiponectin resistance may also induce these paradoxical responses to increased serum adiponectin in patients with AAC 21 .
Several studies have reported that increased serum leptin is associated with vascular calcification. For example, serum leptin was positively associated with higher coronary artery calcification in asymptomatic participants without DM 5 . Moreover, in Caucasians, serum leptin has been independently associated with coronary artery calcification after adjustment for age, sex, family history of CVD, DM status, exercise, and major cardiometabolic medications 17 . Furthermore, in basic research, leptin has been shown to promote osteoblast differentiation and mineralization in primary cultures of vascular smooth muscle cells 22 . However, in the present study, serum leptin was not associated with AAC.
This study has several limitations. First, because we determined the presence of AAC based on plain radiographs, which could not differentiate between intimal calcification (atherosclerosis) and medial calcification (arteriosclerosis), the presence of AAC in the present study did not equal to atherosclerosis. However, as not only intimal but also medial calcification is associated independently with mortality 23 , it provided a clinically relevant measure for CVD risk in hemodialysis patients, as we reported previously 7 . Second, all patients were Japanese men and the sample size was relatively small. Furthermore, because this was a cross-sectional observational study, we could not determine causality in the relationships between the studied variables. Lastly, recent studies have reported that there exist several adiponectin isoforms with different metabolic activities; we did not address this point in the present study.
In conclusion, high serum adiponectin is associated with AAC in hemodialysis patients. This association remains significant even after adjustment for age, percentage body fat, hemodialysis duration, DM, and serum levels of calcium, phosphate, intact PTH, hs-CRP, and leptin. The present study indicated that adiponectin is an independent factor that is positively associated with the prevalence of AAC in hemodialysis patients.

Methods
Subjects and study design. A cross-sectional observational study was conducted in 101 male Japanese hemodialysis patients in Shirasagi Hospital, Osaka, Japan, in 2003. Each patient provided written informed consent before being enrolled in the study. This study was approved by the Ethics Review Committee of Shirasagi Hospital 24 , and conducted in accordance with the principles of the Declaration of Helsinki. The enrolled patients were restricted to male to avoid the sex differences of serum adiponectin and/or leptin due to the sex differences in adiposity. Those who had acute illness, infection, or malignancy were excluded from the present study. The underlying kidney diseases of the enrolled patients were as follows: diabetic nephropathy (n = 42), chronic glomerulonephritis (n = 35), nephrosclerosis (n = 11), polycystic kidney disease (n = 2), other disease (n = 7), and unknown disease (n = 4). None of the patients had undergone parathyroidectomy or renal transplantation. All patients underwent three sessions of hemodialysis per week, each lasting for 4-5 hours, by using a hollow-fiber dialyzer, and a bicarbonate dialysate containing 3.0 mEq/L calcium. DM was diagnosed when the patient had a history of the disease or on the basis of the criteria of the American Diabetes Association 25 .