Haemodialysis with the biocompatible high permeability AN-69 membrane does not alter plasma insulin-like growth factor-I and insulin-like growth factor binding protein-3

Julien Bohé1,, Marie-Odile Joly2, Walid Arkouche3, Maurice Laville3 and Denis Fouque3

1 Medical Intensive Care Unit, Lyon-Sud Hospital, Pierre-Bénite, 2 Department of Biochemistry, Debrousse Hospital, Lyon 3 Department of Nephrology and Hypertension, E Herriot Hospital, Lyon, France



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Insulin-like growth factor-I (IGF-I) bioactivity has been reported to be decreased in maintenance haemodialysis patients and this may affect their nutritional status. Clearances of IGF-I and its binding proteins (IGFBPs) during haemodialysis sessions using a high permeability biocompatible membrane are unknown.

Methods. Five well nourished, non-diabetic adult patients were studied during one 4-h morning haemodialysis treatment using the high permeability biocompatible AN-69 dialyser. Blood was collected at the arterial and venous ports of the dialyser at 0, 1, 2 and 4 h of dialysis for haematocrit, plasma IGF-I, IGFBP-3 and insulin measurements. IGF-I, IGFBP-3 and insulin concentrations were adjusted for haemoconcentration before comparisons were made.

Results. At the beginning of the dialysis session, plasma IGF-I, IGFBP-3 and insulin levels were within the normal range (297±47 ng/ml (mean±SEM), 4.3±0.6 µg/ml and 11.8±3.4 µIU/ml, respectively). During the session, insulin tended to be cleared through the dialyser, whereas plasma IGF-I and IGFBP-3 values did not vary significantly.

Conclusion. Dialysis with the high permeability AN69 membrane did not alter the main blood compounds of the IGF system in well nourished chronic haemodialysis patients, and it is unlikely that the malnutrition frequently observed in such patients would result from alterations of the IGF system during haemodialysis.

Keywords: haemodialysis; high permeability dialyser; insulin-like growth factor binding protein-3; insulin-like growth factor-I; nutrition



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Malnutrition and protein wasting occur in up to 10–40% of maintenance haemodialysis (MHD) patients and are associated with increased morbidity and mortality [1]. Nutritional status of MHD patients has been reported to improve in some studies using biocompatible membranes [2], but malnutrition still occurs in this population.

Insulin-like growth factor I (IGF-I) is a potent anabolic hormone which affects growth in children [3] and lean body mass in adults. More than 95–98% of plasma IGF peptides circulate bound to specific IGF binding proteins (IGFBPs) that regulate IGF-I bioavailability [4]. Among these binding proteins, IGFBP-3 is the quantitatively most important carrier in human plasma [4].

The normal kidney is an important site that regulates the IGF system and alterations of plasma IGF [512], and IGFBP levels have been reported in patients with end stage renal disease (ESRD) [5,6,8,9,12]. Interactions between the dialysis membrane and the blood further alters the IGF-I system in ESRD patients undergoing MHD [9,1315]. The causes for these alterations and their implication in malnutrition are not fully understood. However, low plasma IGF-I levels have been reported in MHD patients and correlated positively with other markers of malnutrition [7].

Since bioincompatible dialysers accelerate net protein breakdown [16], it has also been postulated that IGF with a low molecular weight (7.5 kDa) and small size IGFBPs could be cleared through high permeability biocompatible dialysers, and therefore contribute to a reduced IGF-I bioactivity and malnutrition in these patients. To investigate this question, we examined the kinetics of both plasma IGF-I and IGFBP-3 during a unique MHD session using the biocompatible high permeability polyacrylonitrile AN-69 membrane (Biospan, Hospal, Lyon, France).



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
We studied five aneuric MHD patients (three females, two males) in one single outpatient facility after informed consent was obtained. Patients were 64±5.6 (mean±SEM) years old, had a body weight of 59.2±4.4 kg and none of them were diabetic or had suffered from malignancy or inflammatory diseases. Causes for ESRD were polycystic kidney disease (one patient), interstitial nephropathy (two patients), hypertensive nephrosclerosis (one patient) and chronic glomerulonephritis (one patient). All five patients have been receiving regular MHD treatment, three times a week, for 27±9 months (from 5 to 54 months). A high permeability AN-69 dialyser membrane was used in every patient for >1 month. Kt/V urea was 1.2±0.1 and haemoglobin 11.7±0.3 g/dl. Patients were clinically stable when enrolled. A specifically trained dietitian interviewed each patient and estimation for daily nutrient intakes was made from a 3-day dietary report. Patients were well nourished according to their energy and protein intakes (30±1.7 kcal/kg bodyweight and 1.3±0.1 g/kg bodyweight, respectively), and plasma albumin was within the normal range (4.2±0.4 g/dl).

Haemodialysis
Patients were fasted overnight and underwent a mid-week, 4-h morning MHD session. MHD was processed using standard bicarbonate 5.5 mM glucose containing dialysate and a high permeability AN-69 dialyser (Crystal 2800 or 3400, Hospal, Lyon, France). Blood and dialysate flows were set to a constant 200 and 500 ml/min, respectively. Ultrafiltration flow rate was constant throughout the session and the total ultrafiltrated volume was <3.5 l for each subject. The recirculation flow rate was <5% for each patient. Patients were allowed to have breakfast 1 h after the beginning of the session.

Blood sampling
Blood samples were collected at the arterial and venous ports of the dialyser at time 0 (just after the beginning of the MHD session), 1, 2 and 4 h (just before blood restitution) (H0, H1, H2 and H4) and immediately chilled on ice. An aliquot of each sample was microcentrifuged for 10 min and haematocrit was measured. Blood samples were centrifuged at 3000 g for 20 min before plasma was collected and stored at -20°C until further processing.

Plasma values of the tested compounds
Insulin and IGFBP-3 were measured by the Bi Insulin IRMA kit (Institut Pasteur, Paris, France) and the ‘active IGFBP-3’ kit (Diagnostic System Laboratories, Webster, TX, USA), respectively. IGF-I was measured by radioimmunoassay (RIA) after acid/ethanol extraction using recombinant human IGF-I calibrated against the international standard, IRR IGF-I #87/518. The mean IGF-I value for a normal adult was 283 ng/ml (range 130–486 ng/ml).

Standardization for comparison
In order to compensate non-specific variations of the three tested compounds due to intra-individual variation of the haematocrit, original values obtained from each patient were corrected using the formula:


(|<|(|>|1|<|)|>|)
where corX represents the corrected value for the tested compound, X the observed value for the tested compound, Htx the haematocrit value at the time of the sampling, and HtA0 the haematocrit value measured at the arterial port of the dialyser at H0.

Statistical analysis
All data are expressed as mean±SEM. For serial measurement of each compound throughout the dialysis session, analysis of variance for repeated measures was performed. If a global statistical difference was observed, the presence of a statistically different significance between any two time points was determined by the post-hoc Scheffe test. Concentrations obtained at the same time point from the arterial and the venous port of the dialyser were also compared with analysis of variance. P<0.05 was considered significant.



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
The results are presented in Figure 1Go. At the beginning of the MHD session, plasma IGF-I, IGFBP-3 and insulin levels measured at the arterial port of the dialyser were within the normal range, i.e. 297±47 ng/ml, 4.3±0.6 µg/ml and 11.8±3.4 µIU/ml, respectively. For each time point, haematocrit was significantly higher at the venous side. Irrespective of the sampling site, H4 haematocrit values were also significantly higher than H0 haematocrit values.



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Fig. 1. Time course of haematocrit (a), plasma IGF-I (b), IGFBP-3 (c) and insulin (d) during a single haemodialysis treatment with a high permeability AN-69 membrane dialyser in five well nourished ESRD patients. Values were measured at the arterial port ({blacksquare}) and at the venous port ({circ}) of the dialyser at the beginning (H0), and at 1 (H1), 2 (H2) and 4 h (H4) of the dialysis session. For each subject and for each time point, plasma values for IGF-I, IGFBP-3 and insulin were standardized according to the arterial haematocrit level measured at H0 (see Subjects and methods). A significant but transient increase in plasma insulin occurred at H2 (36.5±7.1 µIU/ml at the arterial port and 30±5.2 µIU/ml at the venous port), which mainly reflected the breakfast intake. Since this alteration was not related to the effect of the dialyser, plasma insulin levels for H2 were not plotted. a and b, P<0.05 and P<0.01 vs time corresponding arterial port value. c, P<0.01 vs H0 value. Values are represented as the mean±SEM.

 
Plasma IGF-I and IGFBP-3 values, corrected for haemoconcentration, did not vary throughout the MHD session, whatever the site or the time of collection. In contrast, plasma insulin values were lower in samples collected at the venous side, although the differences between arterial and venous samples were significant only for H1 and H2 values. In addition, insulin concentration progressively decreased throughout the session despite a marked peak at H2 in response to breakfast intake (36.5±7.1 µIU/ml at the arterial port and 30±5.2 µIU/ml at the venous port).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The IGF/IGFBP system plays an important role in growth in children [3] and in maintenance of lean body mass in adults. The normal kidney is an important site for regulating the IGF/IGFBP system as it synthesizes and degrades IGF-I and most IGFBPs, and clears the bloodstream of damaged compounds of the system [17]. Alterations in plasma IGF [512] and IGFBP levels [5,6,8,9,12] have been reported in patients with ESRD. Therefore, the IGF/IGFBP system of ESRD patients undergoing MHD may be affected by two independent mechanisms. First, ESRD patients lack the normal renal function necessary to regulate the system. Secondly, each dialysis session can independently affect some compounds of the system (IGF, IGFBPs, damaged IGFBP fractions and circulating IGF inhibitors) by filtering, fragmenting or adsorbing on the dialyser [9,15]. The characteristics of the dialyser membrane represents an important factor [2,15]. High permeability dialysers are better at removing middle-size molecules, e.g. beta-2 microglobulin (11.8 kDa), than low permeability dialysers. Free IGF-I (7.5 kDa), small size IGFBPs and fragments of these proteins, which are normally present in human urine, are present in this class of molecules. These compounds are therefore likely to be removed during MHD with high permeability dialysers. Altogether these modifications would alter IGF-I bioavailability and subsequently the long-term clinical outcome of MHD patients.

Pre-dialysis levels for plasma IGF-I and IGFBP-3 in our five well nourished MHD patients were within the normal range that has been reported previously [9,12]. With measured plasma levels corrected for haematocrit variation during the dialysis, we found that neither plasma IGF-I nor IGFBP-3 levels were modified during the time course of the MHD session. To date, only a few studies have addressed the removal of IGF/ IGFBP system compounds during MHD [9,1315] and only one was conducted with a high permeability biocompatible membrane [15]. The absence of IGF-I [9,13,14] and IGFBP-3 [9] level modifications during an MHD session was reported previously, but measured values were not corrected relative to haemoconcentration.

Despite the fact that chronic metabolic acidosis was shown to depress serum IGF-I in normal humans [18], no reduced IGF-I level has been reported in MHD patients. In 7123 MHD patients, Chauveau et al. have reported pre-dialysis serum bicarbonate levels of 22.8±3.5 mM [19]. This moderate metabolic acidosis is probably not sufficient to compromise liver IGF-I production.

Indeed, the IGF/IGFBP axis has been showed to be altered during chronic renal failure (CRF). We and others have previously shown that low molecular weight IGFBPs are increased during CRF [6]. These findings have been confirmed recently by Frystyk et al. [5]. With respect to IGFBP-3, RIA and western ligand blotting studies usually found normal range values in CRF adults. Since IGFBP-3 serum proteolytic activity has been evoked in pre-dialysis CRF patients [5], it is possible that MHD patients present such an IGFBP-3 proteolytic activity. By western immunoblotting, these altered IGFBP-3 fragments can be identified, they are unable to bind IGF-I and are therefore considered inactive. These inactive fragments may artificially increase the IGFBP-3 serum levels. Whether the dialysis procedure itself can increase IGFBP-3 proteolysis and the subsequent free IGF-I fraction—a determination method [5] not available when we performed this study—is an important question that should be addressed using a specifically designed study.

Considering the removal of IGF-I and IGFBP-3 during the MHD session, it seems unlikely that the membrane handles these compounds differently in malnourished subjects, and we propose that malnutrition in MHD patients treated with the AN-69 membrane does not result from main IGF system compound alterations during MHD. Further studies are needed to determine whether other high permeability biocompatible membranes present the same properties.

Interestingly, insulin concentration fell during the dialysis session. The transient increase in insulin level at the H2 time point is explained by the physiological response to the breakfast intake. Therefore, this point may be considered as an artifact when analysis of insulin level is performed throughout the session. The fall in plasma insulin level would probably have been more pronounced if patients did not eat 1 h after the session was started. We therefore showed that the AN-69 dialyser could remove insulin, whose molecular weight is close to that of free IGF-I. Other authors found a similar result during MHD with a high permeability biocompatible polysulfone dialyser [20]. Given their low molecular weights, IGF-I and insulin could theoretically be filtered across high permeability dialysers. Nevertheless, the differential removal of insulin and IGF-I probably results from the presence of IGFBPs in plasma. Indeed, >95–98% of IGF-I in the circulation is bound to IGFBPs (especially to IGFBP-3-containing complex), whereas insulin is present only in the free form. This greatly exposes insulin to interactions with the dialyser membrane. Of note, Wizemann et al. did not find any trace of insulin in the ultrafiltrate and proposed that insulin was adsorbed onto the high permeability synthetic membrane [20].

In summary, whereas plasma insulin was partly cleared by the dialyser, we found that plasma IGF-I and IGFBP-3 levels remained within the normal range in well nourished MHD patients and that none of these compounds was significantly removed during MHD treatment with the high permeability AN-69 dialyser. Therefore, with respect to the IGF-I/IGFBP status, the AN-69 membrane does not induce a particular anti-anabolic response in MHD patients.



   Acknowledgments
 
We are indebted to the renal dieticians who performed the diet records: M. Thevenet, C. Monnet and C. Cléaud. We thank Dr R. N. Charrel for his critical review of this manuscript. This work was supported by a grant from Hospal, Lyon, France.



   Notes
 
Correspondence and offprint requests to: Julien Bohé, Service de réanimation médicale, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 5.00
Revision received 4.10.00.