Free serum leptin but not bound leptin concentrations are elevated in patients with end-stage renal disease
Adji Widjaja1,,
Jan T. Kielstein2,
Rüdiger Horn1,
Alexander von zur Mühlen1,
Volker Kliem2 and
Georg Brabant1
1 Departments of Endocrinology and
2 Nephrology, Hannover Medical School, Hannover, Germany
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Abstract
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Background. Leptin is a 16-kDa protein that is thought to be a regulator of food intake and body weight. Although total serum leptin levels have been reported to be elevated in obese and normal weight patients with end-stage renal disease (ESRD), it is not known whether serum-free leptin concentrations are also increased in patients with ESRD with no apparent nutritional problems. Furthermore, there are no data on how different dialysis modes (high-flux haemodiafiltration and low-flux dialysis) influence serum leptin subfractions.
Methods. We measured fasting serum free and bound leptin levels in three groups of male subjects: patients on haemodiafiltration with high flux dialysers (n=11), patients on haemodialysis with low-flux dialysers (n=17) and healthy age (61±8 years) and BMI (23.8±3.1 kg/m2) matched control subjects (n=28). Both leptin components were determined before and after a single dialysis session.
Results. Body mass indices were correlated with serum free leptin levels in both patients (r=0.69, P<0.001) and controls (r=0.77, P<0.001). Mean (SD) serum free leptin levels were significantly higher in ESRD patients than in control subjects (91±33 vs 41± 21 pmol/l; P<0.01). Bound leptin levels did not differ in both groups (0.67±0.12 vs 0.56±0.11 nmol/l, NS). Elevated serum-free leptin levels in ESRD patients could be reduced by haemodiafiltration with high-flux membranes, but not with low-flux haemodialysis membranes.The former led to a reduction of initial serum free leptin values to 76±17% (P<0.01), whereas bound leptin remained unaffected.
Conclusion. Serum-free leptin levels are elevated in ESRD without any apparent effect on body weight. In contrast, serum bound leptin levels remain stable, thus central feedback regulation via the bound form of the hormone may serve as an alternative explanation in the regulation of food intake and energy expenditure in chronic patients on haemodialysis with no apparent nutritional problems.
Keywords: bound leptin; free leptin; haemodialysis; nutritional status; post-dialysis; pre-dialysis
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Introduction
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Malnutrition due to poor food intake is a common clinical problem in patients with end-stage renal disease (ESRD) [12] and is associated with an increase in morbidity and mortality in patients on haemodialysis treatment [3]. The role of leptin in controlling food intake in humans is not clear, however, in rodents it has been shown that leptin injection can decrease body weight and increase energy expenditure [4]. In animals and humans, circulating total leptin levels are positively correlated with body fat [5,6]. Moreover, it has been reported that patients with ESRD with [7,8] and without ongoing dialysis treatment [9,10] have markedly elevated total serum leptin levels without an increase in body fat mass. Despite these elevated serum levels, it has been shown that patients with ESRD have a significant lower expression of the leptin gene compared with body mass index (BMI) matched controls, possibly through a negative feed back mechanism due to a decreased plasma clearance [11]. In addition, it has been reported that high-flux but not low-flux dialysers can decrease total leptin levels [1214]. However, serum leptin has been noted to exist in a bound and free form [15]. Thus, we hypothesized, that elevated total leptin levels in ESRD are due to elevated free leptin levels which do not necessarily contribute to the anorexia found in uraemia.
We measured free and bound leptin levels with specific radioimmunoassays in male patients undergoing regular dialysis with no apparent nutritional problems and compared their levels with a BMI and age matched control group. Both components of the leptin system were also determined before and after different modes of haemodialysis treatment.
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Subjects and methods
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Patients and study design
All subjects who were included in this study had given informed consent for participation and ethics committee approval was obtained. Fasting serum free leptin and bound leptin levels were measured in 28 male anuric patients (mean age 61±11 years) who had been treated with dialysis for a median of 48 months (range 6221 months) and in 28 healthy age (61±8 years) and BMI (23.8±3.1 kg/m2) matched males who served as controls.
All pre-dialysis blood samples from fasting subjects were drawn at morning sessions starting at 07:00 hours after a long interval. The mean (SD) BMI (23.8±3.2 kg/m2), serum albumin concentration (38.4±2.9 g/l) and mean haemoglobin levels (10.97±0.99 g/l) were stable for at least 6 months. The principal causes of ESRD were chronic glomerulonephritis (9), hypertension (7), adult polycystic kidney disease (5), insulin-dependent diabetes mellitus (3), interstitial nephritis (2), and Alport syndrome (1). Patients underwent standard bicarbonate haemodialysis or haemodiafiltration three times a week. The average dialysis time was 289±26 min, blood flow was 236±26 ml/min, and dialysate flow was 500 ml/min. The delivered dialysis dose measured by Kt/Vurea was 1.33±0.18.
For a further step analysis, the 28 patients were divided into two groups dialysed with different modes of haemodialysis. Group A, 17 patients with conventional haemodialysis with a low flux polysulphone membrane (F6 HPS, Fresenius, Germany; cut-off point: molecular mass of approximately 8 kDa); group B, 11 patients with haemodiafiltration with a high-flux polyamide membrane (F60 S, Fresenius, Germany; cut-off point: molecular mass of approximately 50 kDa). In these two groups, pre- and post-dialysis serum leptin levels were measured. The serum leptin concentrations after haemodialysis were adjusted to the change in haematocrit in order to decrease the influence of haemoconcentration caused by ultrafiltration during haemodialysis. Mean (SD) BMI of patients on low flux haemodialysis was 22.3±2.6 kg/m2 and did not differ from patients on haemodiafiltration 23.7±3.7 kg/m2 (NS). Haemoglobin and serum albumin concentrations as well as duration of dialysis sessions were not different in the two groups.
Laboratory measurements
Our assays for the determination of free leptin [16] and bound leptin in serum [17,18] have earlier been described in detail. In brief, polyclonal antibodies to a carboxy-terminal (amino acids 126140) and an amino-terminal fragment (amino acids 2539; 25-Tyr), both coupled to haemocyanin by the carbodimide method, were generated in rabbits. Labelled carboxy- and amino-terminal fragments served as tracer. The specificity of the fragments assays was characterized by Sephadex G-200 gel chromatography.
Statistical analyses
Results are expressed as mean±SD. Data were analysed using Student's two-tailed t-test or the signed rank test for non-normally distributed data. Pearson's correlation was used to assess relationships between variables. Statistical significance was accepted as P<0.05.
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Results
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There was a strong positive correlation between log serum free leptin levels and BMI in both controls and patients (n=28; Figure 1
). No correlation was found between BMI and bound leptin levels in both groups (r=0.05 and r=-0.41; NS for both).

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Fig. 1. Positive correlation between BMI and serum-free leptin concentrations in controls and patients with ESRD.
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Higher serum free leptin levels were determined in patients (n=28) before dialysis treatment compared to controls (Figure 2a
). Serum-free leptin levels (pre-dialysis) remain elevated in both patients on high- flux haemodiafiltration (85±30 pmol/l) and low-flux dialysis (94±35 pmol/l) vs controls (41±21 pmol/l; P<0.01 for both). However, pre-dialysis serum free leptin levels were not different between the treatment groups (NS).

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Fig. 2. Boxplot of serum-free (a) and bound leptin levels (b) in controls and patients with ESRD. Serum-free leptin levels were significantly elevated in patients with ESRD compared with controls.
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Mean serum-free leptin levels in patients treated with high-flux haemodiafiltration decreased during the procedure from 85±30 to 65±27 pmol/l (P<0.01) and the mean percentage after haemodiafiltration was 76±17% of the initial value (Figure 3
). In contrast, no significant change of mean serum-free leptin levels on low-flux haemodialysis (pre, 94±35 vs post, 105±40 pmol/l; NS) or mean percentage after low-flux dialysis were seen (Figure 3
).

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Fig. 3. Boxplot of reduction rates (expressed as percentage of initial value and corrected for haemoconcentration during treatment) of serum-free and bound leptin levels after different modes of treatment. Only serum-free leptin levels were significantly reduced after haemodiafiltration with high-flux membranes.
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Pre-dialysis bound leptin levels did not differ between patients (670±120 pmol/l; n=28) and controls (560±110 pmol/l, NS; Figure 2b
). No difference was found even when patients were separated in high-flux haemodiafiltration (630±100 pmol/l) and low-flux dialysis (690±140 pmol/l; NS) and compared with age and BMI matched controls (540±140 and 590± 110 pmol/l; NS for both).
Mean serum bound leptin levels were not altered by high-flux haemodiafiltration (pre, 630±100 vs post, 660±130 pmol/l; NS) or low-flux haemodialysis (pre, 690±140 vs post, 81±190 pmol/l; NS). The mean percentage of the initial value did not differ during both procedures (Figure 3
).
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Discussion
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Our data show that serum-free but not bound leptin levels are elevated in patients with ESRD who do not have any apparent nutritional problems. In addition, we found that high-flux haemodiafiltration but not low-flux haemodialysis was able to reduce serum free leptin concentrations.
Both in humans and in rodents leptin is known to circulate in free and bound forms [15,18,19] and obese subjects have elevated levels of free leptin, probably because of a saturation of leptin binding proteins [19]. Our data show in haemodialysis patients as well as in control subjects a positive relationship between serum-free leptin levels and the degree of adiposity as determined by BMI. This result is consistent with previous reports in normal weight and obese subjects with type 2 diabetes mellitus [6]. Moreover, it is known that the rate of increase of total leptin concentrations with BMI is significantly greater in ESRD patients compared with normal controls [7]. We could confirm that serum-free leptin levels are higher in ESRD patients independent from the mode of dialysis treatment. However, the exact mechanism that causes inappropriately high plasma leptin levels in uraemia is not clear. It could be either as a result of an increased production, decreased metabolism, or elimination.
Leptin production is known to be stimulated by i.e. insulin [20] or TNF-
[21], which appear to be elevated in patients on haemodialysis [22,23]. However, a low leptin gene expression was determined in patients with ESRD, which suggest that elevated leptin levels, possibly due to a decreased plasma clearance, down-regulate the production of leptin [11]. Our data on serum bound leptin levels would fit in this context, since its concentrations did not differ between patients and controls. Sharma et al. [24] have also reported that the elevation of leptin in dialysis patients was due to an increased free but not bound leptin fraction. However, they only calculated the different fractions from total leptin, whereas we used specific radioimmunoassays to determine free and bound leptin in sera. Furthermore, we have studied the biovariability of serum free leptin over a 12-day period in 12 normal and body-weight-stable subjects, and showed that the within-subject CV in this group of healthy subjects was small compared with the variation between subjects, implying relative stability over a 12-day period with no association to food intake [25]. In addition, subcutaneous recombinant free leptin injections appeared to induce moderate weight loss only in some obese subjects with elevated endogenous serum leptin concentrations [26]. Thus, these data support the concept that free leptin does not significantly contribute to the homeostatic regulation of body weight. If leptin is able to regulate food intake and energy expenditure in humans, normal bound leptin levels in ESRD patients may be one explanation why our patients did not suffer from any apparent nutritional problems.
Alternatively, it seems reasonable to speculate that serum leptin also accumulates in renal failure due to reduced renal clearance. However, many individuals without renal function (including surgically anephric subjects) have leptin concentrations appropriate for their degree of adiposity, and leptin concentrations do not correlate strongly with measures of residual renal function such as urinary clearance [710,24]. Thus, the idea that leptin simply accumulates as renal elimination decreases is untenable, but is probably due to other unknown factors [7,24].
In addition, our results demonstrated that serum-free leptin levels can be decreased by 24% in patients on high-flux haemodiafiltration, whereas no change was found after low-flux dialysis. This result is in agreement with others who have described a 2430% decrease in total leptin levels in patients treated by haemodiafiltration [12,14]. The reduction rate of total and free leptin levels can be explained by the pore size, which is larger in high-flux than in low-flux membranes dialysers, and which permit the removal of solutes up to 50 kDa. Thus, free leptin, which has a molecular weight of 16 kDa is able to be filtrated by high-flux membranes, whereas bound leptin with a much higher molecular weight of 200 kDa is not [15,18].
In conclusion, serum-free leptin levels are elevated in ESRD but do not appear to cause decreased weight. In contrast, serum bound leptin levels remain stable, thus central feedback regulation via the bound form of the hormone may serve as an alternative explanation in the regulation of food intake and energy expenditure in healthy patients on haemodialysis with no apparent nutritional problems.
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Notes
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Correspondence and offprint requests to: Dr A. Widjaja, Department of Endocrinology, Hannover Medical School, Carl-Neubergstr. 1, D-30625 Hannover, Germany. 
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Received for publication: 18. 8.99
Revision received 15.12.99.