Serum Leptin, Resistin, and Lipid Levels in Patients with End Stage Renal Failure with Regard to Dialysis Modality

Abstract

Little information is available on the relationship between serum resistin levels and other adipokines with serum lipid levels and insulin resistance in uremic patients under different dialysis modalities. Methods. This study investigated the effects of dialysis modality on serum leptin, adiponectin, resistin, interleukin 6 (IL-6), and tumor necrosis factor (TNF) α levels in age, sex, and total adipose tissue mass (TATM); matched 30 hemodialysis (HD) patients, 30 continuous peritoneal dialysis (CAPD) patients, and 30 healthy controls; and evaluated the relationship between these adipokines and dyslipidemia and insulin resistance. Results. Serum resistin, adiponectin, IL-6, TNF-α, and high sensitive C reactive protein (hsCRP) levels were significantly increased in dialysis patients compared to controls (p < 0.05). In CAPD patients, serum leptin, resistin, triglycerides, and total cholesterol levels were higher than those in HD patients (p < 0.05). Leptin levels were positively correlated with TATM, serum triglycerides, total cholesterol, and low density lipoprotein (LDLc) levels in both dialysis groups. Resistin levels were found to positively correlate with TATM and triglycerides in CAPD patients. No relationship was found between the homeostasis model assessment-insulin resistance index (HOMA-IR) and adipokines studied. Conclusion. Serum leptin, resistin, triglycerides, and total cholesterol levels were higher in CAPD patients. Leptin levels were positively correlated with TATM, serum triglycerides, total cholesterol, and LDLc levels in dialysis patients. Resistin levels were positively correlated with TATM and triglycerides in CAPD patients. Glucose load during CAPD may be an important factor in increased in leptin, resistin, triglycerides, and total cholesterol levels in CAPD patients. These results highlight the importance of leptin and resistin as determinants of dyslipidemia, especially in CAPD patients.

Keywords:

• resistin
• adiponectin
• leptin
• interleukin-6
• dialysis
• lipids
• insulin resistance

INTRODUCTION

Adipocytes produce and secrete several proteins that act as veritable hormones, collectively called adipokines, that play important roles in the inflammatory and atherosclerotic processes. These include tumor necrosis factor (TNF) α, leptin, interleukin (IL)-6, adiponectin, resistin, and plasminogen activator inhibitor-1.[1] These factors play a central role in whole body homeostasis by influencing a variety of biological and physiological processes, including food intake, insulin action, lipid and glucose metabolism, regulation of energy balance, coagulation, angiogenesis, and vascular remodeling. Recent studies have shown that an alteration in these hormone/cytokine levels has been implicated in the pathogenesis of insulin resistance and dyslipidemia.[2],[3] Insulin resistance and dyslipidemia in uremic patients has been recognized for many years.[4]

Blood levels of hsCRP, interleukin (IL)-10, IL-6, IL 8, TNF-α, and leptin have all been reported to be elevated in dialysis patients,[5],[6] but little information is available on serum adiponectin and resistin levels in patients under different modalities of dialysis therapy.

The aims of this study were to compare serum adiponectin, resistin, leptin, TNF, IL-6 levels, and high sensitive C-reactive protein (hsCRP) in hemodialysis (HD) and continuos peritoneal dialysis (CAPD) patients and examine the relationship between them and total adipose tissue (TATM), serum triglycerides, total and low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc) levels, and insulin resistance estimated using homeostasis model assessment (HOMA–IR).

PATIENTS AND METHODS

Thirty HD patients (17 men, 13 women; mean age, 48.2 ± 15.5 years) and 30 CAPD patients (18 men, 12 women; mean age, 43.2 ± 10.8 years) on dialysis for longer than two months were studied. Thirty healthy persons (17 men, 13 women, mean age 40.9 ± 10.0 years) acted as controls.

Diabetic, clinically unstable patients, those with tumors or inflammatory diseases (such as chronic pulmonary disease, and malnutrition), and those treated with immunosuppressives were excluded. No patient showed signs of inflammation or infection during the study period. No participant had morbid obesity (BMI >40 kg/m²), infections, or any other serious medical problems. The local ethical committee approved the study protocol.

Information on patient's demographics and clinical parameters, including type and prescription of peritoneal dialysis, adequacy of dialysis parameters, and list of current and prior use of medications, were obtained from the medical recordings and from patients themselves using a case report form.

All HD patients received regular dialysis using a hemophage hollow-fiber dialyzer three times per week in sessions lasting four hours each. The dialysate was bicarbonate buffered and contained 140 mmol/L sodium. The dialysate flow rate was 500 mL/min, and blood flow ranged from 300 to 350 mL/min. All patients on CAPD received four exchanges per day using standard dialysis bags (8 L/day).

Body mass index (BMI) was calculated by dividing the dry weight in kilograms by the square of the height in meters. Total adipose tissue mass (TATM) was assessed by means of bioelectrical impedance analysis using a commercially available bioimpedance analyzer (Body Composition analyzer Type TBF-300 m).

From the serum fasting levels of glucose and insulin, the homeostasis assessment ratio (HOMA-IR) was calculated according to the following formula[7]:

Fasting samples of venous blood were obtained from an antecubital vein between 08:30 and 09:00 h in all subjects for the estimation of hormonal and general analytical data. In HD patients, blood samples were obtained immediately before the first dialysis session of the week (i.e., 72 h after the last dialysis session). Blood samples were centrifuged immediately, and the serum samples were stored at −20°C until analysis of the data.

Serum C-reactive protein (CRP) was estimated with a high-sensitive (hs) enzyme-linked immunosorbent assay kit (BioCheck, Foster City, California, USA). Serum adiponectin and resistin levels were determined using enzyme-linked immunosorbent assay kits (Linco Research, St. Charles, Missouri, USA). Serum leptin was measured using enzyme-linked immunosorbent assay kit (DRG instruments GmbH, Marburg, Germany). TNF and IL-6 were measured in serum by using an enzyme-linked immunosorbent assay kit (Beckman Coulter, Marseille, France). The insulin levels were determined by chemiluminescent based Immulite 2000 system (DPC, Los Angeles, California, USA).

Serum total cholesterol, HDLc, LDLc, triglycerides, glucose levels (hexokinase method), and urine and serum creatinine were analyzed on a fully automated Olympus AU 2700 (Olympus Diagnostica GmbH, Hamburg, Germany). To evaluate residual creatinine clearance (Ccr), urine creatinine was measured in 24 hour collection.

Statistical Analyses

Adjustment to normal distribution was tested by the Kolmogorov-Smirnov test. For comparisons of means between two groups of subjects the Mann-Whitney U-test was used. The Kruskal-Wallis analysis of variance by ranks was used to detect differences if data were nonparametric. For ratio comparisons, the χ²-test or Fisher exact test was used. Correlation analyses were examined by using Pearson test or Spearman rank test. Differences were considered significant when p < 0.05. All results are expressed as mean ± SD.

RESULTS

Causes of renal failure in HD patients included chronic glomerulonephritis (n = 5), hypertensive nephrosclerosis (n = 5), polycystic kidney disease (n = 2), others (n = 8), and unknown (n = 10). Causes of renal failure in CAPD patients were chronic glomerulonephritis (n = 6), hypertensive nephrosclerosis (n = 5), polycystic kidney disease (n = 1), others (n = 8), and unknown (n = 10).

The dialysis groups were matched with regard to sex distribution, age, TATM, and dialysis duration. The control group had similar mean age, TATM, and sex distribution as the dialysis groups (p ( 0.05). Demographic and laboratory characteristics of all patients and controls were shown at Table 1.

Table 1 Demographic and laboratory findings of three groups

	HD patients	CAPD patients	Controls	p
Sex (Female/male)	17/13	18/12	17/13	NS
Age (years)	48.2 ± 15.5	43.3 ± 10.8	40.9 ± 10.0	NS
Weight (kilogram)	61.3 ± 14.8	66.4 ± 14.0	64.4 ± 11.3	NS
BMI (kg/m^2)	23.3 ± 4.7	25.4 ± 5.9	25.1 ± 3.9	NS
Glucose (mg/dL)	93.1 ± 23.3	98.9 ± 20.1	83.4 ± 7.4	NS
HsCRP (mg/L)	8.7 ± 8.9	8.6 ± 11.9	2.9 ± 1.1	p < 0.05^†^‡
Leptin (ng/mL)	11.5 ± 22.3	59.8 ± 61.5	7.9 ± 7.0	p < 0.05^‡
Adiponectin (ng/mL)	21.6 ± 10.1	37.7 ± 36.5	11.6 ± 5.1	p < 0.05^†^‡
Resistin (ng/mL)	41.9 ± 10.3	65.3 ± 28.5	17.9 ± 8.0	p < 0.05^†^‡
Interleukin-6 (pg/mL)	14.4 ± 14.9	15.9 ± 24.0	6.0 ± 5.4	p < 0.05^†^‡
TNF-α (pg/mL)	25.1 ± 7.3	30.3 ± 13.2	8.1 ± 3.1	p < 0.05^†^‡
Insulin (uIU/mL)	17.6 ± 11.6	27.9 ± 20.8	15.1 ± 14.0	p < 0.05^‡
HOMA-IR index	3.8 ± 2.8	5.6 ± 3.5	2.9 ± 2.4	p < 0.05^‡

Abbreviations: BMI = body mass index, TNF-α = tumor necrosis factor, HsCRP = high sensitive C reactive protein, IL-6 = interleukin 6.

*HD-CAPD significant, ^†HD-controls significant, ^‡CAPD-controls significant.

However, the only purpose of including healthy controls was to confirm that levels of hsCRP, leptin, adiponectin, resistin, IL-6, and TNF-α levels were increased in dialysis patients, and therefore no further statistical analysis was performed in this group.

In patients on both HD and CAPD, hsCRP, IL-6, TNF-α, adiponectin, and resistin levels were significantly higher than those in controls (p < 0.05). Serum leptin levels were higher in CAPD patients compared with in HD patients and controls, but not in HD patients compared with controls. Serum insulin levels and HOMA-IR were significantly and statistically higher in CAPD patients than those of controls, but no difference in serum insulin levels and HOMA-IR was detected between HD patients and controls (see Table 1).

Serum resistin levels were markedly greater in CAPD patients than those of HD patients (p < 0.05). Serum adiponectin, TNFα, IL-6, insulin levels, HOMA-IR in CAPD patients were not significantly different from in HD patients (p > 0.05).There were no significant differences between HD and CAPD patients with regards to LDLc levels (p > 0.05). Serum triglycerides, total cholesterol, HDLc, and glucose levels were significantly and statistically higher in CAPD patients than those of HD patients (p < 0.05). Urinary Ccr was significantly higher in CAPD compared to those of HD patients (p < 0.05; see Table 2).

Table 2 Demographic and laboratory findings of dialysis patients

	Hemodialysis 30 patients	Peritoneal dialysis 30 patients	p
Duration of dialysis (months)	35.1 ± 32.4	38.7 ± 32.6	NS
Urinary Ccr (mL/min/)	2.6 ± 2.8	4.3 ± 3.1	p < 0.05
Glucose (mg/dL)	93.1 ± 23.3	98.9 ± 20.1	NS
Triglycerides (mg/dL)	152.0 ± 53.4	192.0 ± 74.9	p < 0.05
Total cholesterol (mg/dL)	149.2 ± 34.8	186.8 ± 33.7	p < 0.05
HDLc (mg/dL)	35.0 ± 8.7	47.7 ± 13.2	p < 0.05
LDLc (mg/dL)	81.0 ± 28.3	93.7 ± 37.8	NS

Abbreviations: Ccr = Creatinine clearance, HDLc = high-density lipoprotein cholesterol, LDLc = low-density lipoprotein cholesterol, BUN = blood urea nitrogen, NS = non-significant.

Results of Correlation Analyses in HD Patients

Leptin was positively related to TATM, serum triglycerides, total cholesterol, and serum LDLc (p < 0.05). A negative association was found between adiponectin and weight, and a positive association was found between serum adiponectin and HDLc (p < 0.05). Resistin was negatively related to weight (p < 0.05). A positive association was shown between IL-6 and age (p < 0.05). HsCRP was positively related to age and weight and negatively to adiponectin (p < 0.05; see Table 3).

Table 3 Results of correlation analyses in HD patients

	HsCRP r	Leptin r	Adiponectin r	Resistin r	IL-6 r	TNF-α r
Age (years)	0.648	NS	NS	NS	0.436	NS
Duration of dialysis (months)	NS	NS	NS	NS	NS	NS
Weight (kilogram)	0.394	NS	−0.446	−0.390	NS	NS
BMI (kg/m^2)	NS	0.466	−0.408	NS	NS	NS
Total adipose tissue mass	NS	0.585	NS	NS	NS	NS
Total cholesterol (mg/dL)	NS	0.391	NS	NS	NS	NS
LDLc (mg/dL)	NS	0.372	NS	NS	NS	NS
HDLc (mg/dL)	NS	NS	0.491	NS	NS	NS
Triglycerides (mg/dL)	NS	0.381	NS	NS	NS	NS
HsCRP (mg/L)	NS	NS	−0.393	NS	NS	NS
IL-6 (pg/mL)	NS	NS	NS	NS	NS	NS
TNF-α (pg/mL)	NS	NS	NS	NS	NS	NS
Leptin (ng/mL)	NS	NS	NS	NS	NS	NS
Adiponectin (ng/mL)	−0.393	NS	NS	NS	NS	NS
Resistin (ng/mL)	NS	NS	NS	NS	NS	NS
Insulin (uIU/mL)	NS	NS	NS	NS	NS	NS
Insulin resistance	NS	NS	NS	NS	NS	NS
Urinary Ccr(mL/min)	NS	NS	NS	NS	NS	NS

Abbreviations: NS = non-significant, BMI = body mass index, HDLc = high-density lipoprotein cholesterol, LDLc = low-density lipoprotein cholesterol, TNF-α: tumor necrosis factor, HsCRP = high sensitive C reactive protein, IL-6 = interleukin 6, Ccr = creatinine clearance.

*p < 0.05.

Results of Correlation Analyses in CAPD Patients

In CAPD patients, leptin showed a negative correlation with adiponectin but a positive correlation with TATM, triglycerides, total cholesterol, and LDLc (p < 0.05). Serum adiponectin was positively related to duration of dialysis and LDLc but negatively to triglycerides (p < 0.05). Resistin was found to correlate positively and significantly with weight, BMI, TATM, and triglycerides (p < 0.05). Serum IL-6 was positively related to age (p < 0.05). HsCRP was positively related to TATM, HOMA-IR, and TNF-α (p < 0.05; see Table 4).

Table 4 Results of correlation analyses in CAPD patients

	HsCRP r	Leptin r	Adiponectin r	Resistin r	IL6 r	TNFr
Age (years)	NS	NS	NS	NS	0.436	NS
Weight (kilogram)	NS	NS	NS	0.694	NS	NS
Duration of dialysis (months)	NS	NS	0.643	NS	NS	NS
BMI (kg/m^2)	NS	NS	NS	0.839	NS	NS
Total cholesterol (mg/dL)	NS	0.391	NS	NS	NS	NS
Total adipose tissue mass (kg)	0.622	0.585	NS	0.862	NS	NS
LDLc (mg/dL)	NS	0.372	0.606	NS	NS	NS
HDLc (mg/dL)	NS	NS	NS	NS	NS	NS
Triglycerides (mg/dL)	NS	0.381	−0.515	0.751	NS	NS
HsCRP (mg/L)	NS	NS	NS	NS	NS	0.588
IL-6 (pg/mL)	NS	NS	NS	NS	NS	NS
Leptin (ng/mL)	NS	NS	NS	NS	NS	NS
Adiponectin (ng/mL)	NS	−0.393	NS	NS	NS	NS
Resistin (ng/mL)	NS	NS	NS	NS	NS	NS
TNF-α (pg/mL)	0.588	NS	NS	NS	NS	NS
Insulin (uIU/mL)	NS	NS	NS	NS	NS	NS
HOMA-IR index	0.700	NS	NS	NS	NS	NS
Urinary Ccr(mL/min)	NS	NS	NS	NS	NS	NS

Abbreviations: NS = non-significant, BMI = body mass index, BUN = blood urea nitrogen, HDLc = high-density lipoprotein cholesterol, LDLc = low-density lipoprotein cholesterol, TNF-α = tumor necrosis factor, HsCRP = high sensitive C reactive protein, IL-6 = interleukin 6, Ccr = Creatinine clearance.

*p < 0.05.

No relationship was found between creatinine clearance and hsCRP, HOMA-IR, insulin, IL-6, TNF-α, leptin, adiponectin, and resistin levels in neither HD nor CAPD patients. No correlation was found between TNF-α levels and studied parameters in both dialysis groups.

DISCUSSION

Clinical studies have shown that circulating levels of adipokines, such as leptin, adiponectin, TNF-α, and IL-6, are increased in patients with end stage renal failure (ESRD). Although decreased elimination may be a major cause of elevated adipokines in patients with ESRD, increased generation also may play a role.[6],[8–11]

It was found that the hsCRP, resistin, adiponectin, IL-6, and TNF-α levels were significantly increased in both HD and CAPD patients compared to those of healthy controls. Serum leptin levels were higher in CAPD patients compared with in HD patients and controls, but not in HD patients compared with controls. Resistin levels were higher in CAPD patients than in HD patients. Similar levels of adiponectin, TNF-α, and IL-6 in both dialysis groups were detected.

Diez et al.[12] reported differences in serum adipocytokines according to the modality of dialysis therapy. These authors found that CAPD patients had higher serum leptin levels than those of patients on HD or conservative management, that adiponectin concentrations were similar in the three groups of patients, and that patients treated by both CAPD and HD exhibited resistin concentrations significantly higher than those found in controls.

Leptin is a circulating factor and acts both centrally and peripherally to regulate several metabolic and inflammation-related functions and control appetite and energy expenditure through hypothalamic pathways.[13] The CAPD patients have higher leptin levels than those of HD patients and controls, as previously reported by others.[8],[9],[12] However, because BMI and body fat mass were not different in CAPD patients compared to HD patients, it is probable that factors other than increased body fat content also contribute to the elevated leptin levels in CAPD patients. In CAPD, it is well documented that the continuous glucose load will result in chronic hyperinsulinemia, which in this context is of interest, as insulin has been shown to regulate leptin gene expression. Data from experiments in isolated adipocytes[14] and clinical studies in human subjects[15] support the idea that insulin increases leptin production indirectly via its effects to increase glucose utilization and oxidative glucose metabolism in adipocytes[16] at the transcriptional level.[17] However, no relationship between insulin and leptin levels in both dialysis groups, though in CAPD patients serum insulin levels were significantly higher than those of HD patients. It is possible that continuous glucose load during CAPD led to an increase in leptin levels. Many aspects of glucose sensing have been shown to occur as a result of the metabolic conversion of glucose to hexosamines.[18–20] Increased tissue concentrations of the end product of the hexosamine biosynthetic pathway, UDP-N-acetylglucosamine (UDP-GlcNAc), result in rapid and marked increases in leptin messenger RNA and protein levels, although these levels were much lower than those in fat. Plasma leptin levels and leptin mRNA and protein levels in adipose tissue also increase. Most important, the stimulation of leptin synthesis is reproduced by either hyperglycemia or hyperlipidemia, which also increases tissue levels of UDP-N-acetylglucosamine in conscious rodents. Incubation of 3T3-L1 pre-adipocytes and L6 myocytes with glucosamine rapidly induces ob leptin gene expression.[21]

In this study, serum leptin levels were positively related to adipose tissue mass, serum triglycerides, total cholesterol, and LDLc in both dialysis groups. Serum triglycerides and total cholesterol levels were significantly higher in CAPD patients than HD patients (p < 0.05). Nakozono et al. reported that the serum leptin concentration is a valuable clinical marker of the body fat content and may also contribute to the evaluation of hyperlipidemia in HD patients.[22]

Adiponectin is a multifunctional protein with protective roles against the development of insulin resistance, dyslipidemia, nonalcoholic fatty liver disease, atherosclerosis, cardiac hypertrophy, and ischemic injury.[23],[24] Obesity is associated with decreased adiponectin levels.[24] Hypertriglyceridemia, low HDLc, and decreased LDL particle size were shown in humans to be correlated with low plasma adiponectin levels independent of the degree of intra-abdominal fat and insulin resistance.[24],[25]

In accordance with a recent report, adiponectin levels have been found to be similar in HD and CAPD patients and patients on conservative management.[12] In this study, adiponectin was negatively correlated with weight and positively correlated with HDLc in HD patients, while serum adiponectin was positively related to duration of dialysis and negatively to triglycerides in CAPD patients. As reported previously in healthy persons,[23–25] an inverse relationship was found between adiponectin and leptin in CAPD patients.

Resistin is a member of the newly discovered family of cysteine-rich secretory proteins called resistin-like molecules.[26] The role of resistin in the pathogenesis of insulin resistance remains questionable, with conflicting data in animal models and negative findings in clinical observations.[27] Two cross-sectional studies in human beings have failed to show an association between resistin and indices of obesity or insulin resistance.[28],[29]

Resistin levels have been shown to be increased in patients with moderate renal failure, and this increase has been inversely related to glomerular filtration rate. Filippidis et al.[30] reported that resistin serum levels are significantly elevated in non-diabetic patients on HD. These authors found that increased serum resistin levels in HD patients are not related to reduced insulin sensitivity encountered in uremia. While several studies investigated the effect of dialysis modality on serum leptin levels, only one study evaluated the effect of dialysis modality on resistin levels in patients with end stage renal failure, as Diez et al.[12] reported similar resistin levels in HD and CAPD patients. The present study shows that resistin levels are higher in CAPD patients than in HD patients and that resistin levels were positively related to TATM and serum triglyceride levels in CAPD patients. No correlation was found between resistin and insulin resistance in both dialysis groups.

IL-6, a pleiotropic circulating cytokine, is reported to have multiple effects, ranging from inflammation to host defense and tissue injury. It is secreted by many cell types, including immune cells, fibroblasts, endothelial cells, skeletal muscle, and adipose tissue. Plasma IL-6 concentrations correlate positively with human obesity and insulin resistance.[31] However, only about 10–30% of the total IL-6 appear to be produced exclusively by fat cells.[32] No relationship was found between IL-6 and TATM nor insulin resistance and dyslipidemia in both dialysis groups. Serum IL-6 levels were highly correlated with age in both dialysis groups.

Espinazo et al. reported that peritoneal dialysis patients with hypertriglyceridemia or taking lipid-lowering agents showed a positive linear correlation between TNF-α and triglycerides.[33] However, no relationship was found between TNF-α and dyslipidemia and insulin resistance in dialysis patients.

It has been suggested that CRP is involved in the metabolic syndrome and that higher body weight is associated with increased levels of CRP.[34] The mechanism linking increased fat mass with high CRP levels has not been exhaustively explained. It has been suggested that adipose tissue-produced cytokines, including IL-6, TNF-α, and IL-1β, represent the causal link between increased body fat and high CRP levels. However, Fujino et al.[35] reported that in hemodialysis patients, adipose tissue mass change appears to be a parameter indicative of nutritional changes. Chronic inflammation, represented by higher CRP levels, is a significant factor affecting a decrease in adipose tissue mass and is related to poorer nutritional status. However, because this study excluded clinically unstable patients, no negative association was found between CRP and adipose tissue mass. In the present study, hsCRP was positively related to age and weight and negatively related to adiponectin levels in HD patients, while hsCRP was positively related to TATM, HOMA-IR, serum glucose, and TNF-α levels in CAPD patients.

In conclusion, these results confirm that serum resistin, leptin, adiponectin, IL-6, hsCRP, and TNF-α levels are significantly increased in patients on dialysis compared to healthy controls. CAPD patients showed significantly higher serum leptin, resistin, triglycerides, and total cholesterol levels than those of HD patients. Leptin levels were positively correlated with TATM, serum triglycerides, total cholesterol, and LDLc levels in both patient groups. Resistin levels were positively correlated with TATM and triglycerides in CAPD patients. Glucose load during CAPD may be important factor in increased in leptin, resistin, triglycerides, and total cholesterol levels in CAPD patients. These results highlight the importance of leptin and resistin as a determinant of dyslipidemia, especially in CAPD patients.

ACKNOWLEDGMENT

This study was supported by Inonu University Research Center.