Plasma and dialysate IL-6 and VEGF concentrations are associated with high peritoneal solute transport rate

Roberto Pecoits-Filho1,2,, Maria Regina T. Araújo1, Bengt Lindholm2, Peter Stenvinkel2, Hugo Abensur1, João Egídio Romão, Jr1, Marcelo Marcondes1, André H. Freiria de Oliveira1 and Irene L. Noronha1

1 Nephrology Division, University of São Paulo School of Medicine, São Paulo, Brazil and 2 Divisions of Baxter Novum and Renal Medicine, Karolinska Institutet, Stockholm, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. It has been speculated that increased levels of circulating or intraperitoneal pro-inflammatory cytokines such as interleukin 6, and pro-angiogenic vascular endothelial growth factor (VEGF) may contribute to high peritoneal small-solute transport rate (PSTR) in continuous ambulatory peritoneal dialysis (CAPD) patients. In this study we evaluated possible relationships between plasma and dialysate IL-6 and VEGF levels and PSTR.

Methods. Forty CAPD patients (mean age±SD of 58±14 years) with no apparent inflammation process or disease, who had been on CAPD for 19±15 months (range 3–56 months) were included in the study. Peritoneal equilibration test (PET) was used to evaluate PSTR. Patients were divided into two groups: high-average and high transporters (H/A; D/Pcreat>=0.65) and low-average and low transporters (L/A; D/Pcreat<0.64). Albumin and IgG clearances were used in the evaluation of permeability to larger solutes. Plasma and overnight dialysate levels of IL-6 and VEGF were measured.

Results. Plasma IL-6 (7.6 vs 4.3 pg/ml) and VEGF (342 vs 163 pg/ml) as well as dialysate IL-6 (174 vs 80 pg/ml) and VEGF (96 vs 69 pg/ml) levels were significantly higher in the H/A than in the L/A group. The dialysate appearance of IL-6 and VEGF correlated with D/Pcreat, as well as with albumin and IgG clearances. Moreover, significant correlations were noted between dialysate IL-6 and dialysate VEGF levels.

Conclusions. The findings of (i) increased plasma and dialysate levels of IL-6 and VEGF in the H/A group compared to the L/A group, (ii) an association between PSTR and both plasma and dialysate IL-6 and VEGF levels, and (iii) a significant correlation between dialysate IL-6 and VEGF concentrations suggest that inflammation, angiogenesis, and peritoneal transport may be interrelated and involved in the pathophysiology of high PSTR in CAPD patients. However, due to the cross-sectional design of this study, the cause and effect relationships between plasma and dialysate IL-6 and VEGF concentrations and high PSRT remain unclear.

Keywords: angiogenesis; IL-6; inflammation; peritoneal dialysis; peritoneal transport; VEGF



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
During long-term continuous ambulatory peritoneal dialysis (CAPD) treatment, the peritoneal membrane undergoes functional as well as structural alterations, which may be the consequence of many factors such as peritonitis and continuous exposure to dialysis solutions with high concentrations of glucose and glucose degradation products, low pH, and high osmolality. The bioincompatibility of dialysis solutions induces chronic generation of inflammatory mediators in the peritoneal cavity. Concurrently, a persistent systemic inflammatory state has been described in end-stage renal disease (ESRD) patients, both before and after initiation of dialytic treatment [1]. Libetta et al. [2] showed higher IL-6 production from cultured peripheral blood mononuclear cells (PBMC), in peritonitis-free CAPD patients compared to healthy individuals; however, the systemic inflammatory effects of CAPD were comparable to those observed in HD.

The most common functional alteration during long-term CAPD is increased peritoneal small-solute transport rate (PSTR) [3], resulting in impaired ultrafiltration and decreased dialysis efficiency [4]. In addition, for reasons yet to be clarified, high PSTR represents an independent risk factor for increased mortality [5]. In parallel with these alterations, the peritoneum undergoes typical structural modifications including denudation of the mesothelial cell layer, thickening of the sub-mesothelial space, neoangiogenesis, and thickening of the vascular wall by type IV collagen [6].

Interleukin 6 (IL-6), a cytokine involved in the acute-phase inflammatory reaction, increases the permeability of the endothelium in vitro [7]. Plasma IL-6 is a reliable marker of systemic inflammation and furthermore predicts outcome in dialysis patients [8]. IL-6 can be detected in the dialysate of CAPD patients with no apparent sign of inflammatory process or disease [9]. Its levels are much higher in the dialysate than in plasma, showing a further increase during peritonitis [10]. The local intraperitoneal production of IL-6 has been demonstrated and may reflect an intraperitoneal inflammatory state. Vascular endothelial growth factor (VEGF) is a pro-angiogenic cytokine involved in neovascularization and vascular permeability [11], and increased plasma levels of VEGF have been described in active inflammatory disorders [12]. VEGF is also produced in the peritoneal cavity of stable CAPD patients [13].

The aim of the present study was to analyse the association between systemic and intraperitoneal IL-6 and VEGF concentrations, and their possible correlation to peritoneal solute transport rate in stable CAPD patients.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patient population
During the period from September 1999 to September 2000, 40 patients on CAPD in the Dialysis Unit of the University of São Paulo Hospital das Clínicas were included in the study, representing all of the clinically stable patients treated in the centre. This population consisted of 18 male and 22 female patients with a mean age of 58±14 years (range 26–82 years), who had been treated with CAPD for 19±15 months (3–56 months). The causes of ESRD were diabetes mellitus (DM) in 19 (47.5%) patients, nephrosclerosis in 10 (25%) patients, chronic glomerulonephritis in seven patients (17.5%), and other causes in four (10%) patients. The overall peritonitis rate was one episode/35 patient-months, and the total Kt/V (peritoneal+renal) was 1.96±0.23. All patients had been peritonitis-free for at least 3 months before and 1 month after the study. The ethics committee of the University of São Paulo Hospital das Clínicas approved the protocol. All subjects gave their informed consent to participate.

Protocol
On the night prior to the study, patients were asked to perform a dialysis exchange using a 2.5% glucose solution (Dianeal®, Deerfield, IL, USA) and the exact time of the exchange was recorded. The patients arrived at the dialysis centre the next morning and the dialysis fluid was drained. A 10-ml sample of dialysate was collected from the drained volume and the exact time of the collection was recorded. The material was immediately stored at -70°C. At this time a standard fast peritoneal equilibrium test (PET) was initiated [14]. Dialysate samples (D) for glucose and creatinine were collected at time 0 and 240 min. A blood sample with anticoagulant was collected at 120 min, centrifuged, and the plasma (P) was immediately stored at -70°C.

Evaluation of peritoneal transport rate of solutes
For the evaluation of PSTR we used the dialysate to plasma concentration ratio of creatinine (D/Pcreat). The patients were divided according to the PET classification into high (D/Pcreat>=0.81; n=6), high average (D/Pcreat 0.65–0.80; n=15), low average (D/Pcreat 0.51–0.64; n=14), and low PSTR (D/Pcreat<=0.50; n=5).

The peritoneal membrane transport rate of larger molecules was assessed by the clearance of albumin (alb) and immunoglobulin G (IgG) according to the formula: clearance=CdxVd/Cpxt, where Cd is the concentration in the dialysate in µg/dl, Vd is the drained volume in ml, Cp is the plasma concentration in mg/dl, and t is the dwell time in min. Nephelometry was used to measure levels of alb and IgG in plasma and dialysate (Behring Nephelometer, Analyse II, Dade Behring, Marburg, Germany).

Biochemistry
Creatinine was determined by the Jaffé method using the Cobas Integra® (Roche Diagnostic System, Somerville, NJ, USA). Alb and IgG were determined by nephelometry (Behring Nephelometer).

Determination of cytokines
The concentrations of cytokines were determined in the plasma and dialysis effluent after the night dwell using the ELISA technique. All samples were run simultaneously and in duplicate for each mediator to avoid intra- and inter-assay variations. IL-6 and VEGF levels were determined using Quantikine® kits (R&D Systems Inc, Minneapolis, MN, USA). For plasma IL-6, a high-sensitivity kit was used. The intra- and inter-assay variations were 2.3 and 4.2% respectively for IL-6, and 5.2 and 7.1% respectively for VEGF. The sensitivity was 0.70 pg/ml for IL-6 (0.09 pg/ml for the high sensitivity test) and 5 pg/ml for VEGF. Both of the assays are considered highly specific for the cytokines, and no significant cross-reactivity was observed. The plates were read using the ELISA VERSAmax reader (Molecular Devices Corporation, Sunnyvale, CA, USA) and the data were analysed with the SoftmaxPRO® software (Molecular Devices Corporation). For correlation and comparisons, dialysate appearance rate of IL-6 and VEGF was calculated as dialysate concentration times the drained volume divided by the dwell time and expressed as pg/ml/min.

Statistical analysis
Results are expressed as mean values ±standard deviation (SD), whereas median and range were used for non-normally distributed parameters such as IL-6 and VEGF. To compare differences between two transport groups, the unpaired Student t-test was used. When normal distribution was not present, non-parametric analysis (Mann–Whitney test) was applied. To analyse individual clearances or D/Pcreat values in comparison to their respective cytokine levels, we used linear regression analysis. Log-transfer was applied to non-normally distributed values before entering regression analysis. The statistical tests were performed using GraphPad Prism© version 3.00 for Windows (GraphPad Software, San Diego, CA, USA).



   Results
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 Subjects and methods
 Results
 Discussion
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Peritoneal membrane transport rate of solutes
D/Pcreat was 0.64±0.15 for the entire group. The drained volume after 4 h was 2394±178 ml. Alb and IgG clearances were 89.6±56.1 and 56.6±38.9 µl/min respectively.

The patients were subdivided into two groups according to the peritoneal transport characteristics to allow statistical evaluation: low and low average (D/Pcreat<0.64; L/A), and the high and high average (D/Pcreat>0.65; H/A) PSTR. The characteristics of the two transport groups are described in Table 1Go. There was a significant difference between the groups regarding prevalence of diabetes (P<0.05) and peritonitis rate (P<0.01). Furthermore, alb levels were significantly lower in the H/A group (P<0.05 vs L/A). Significantly higher clearances of alb (P<0.05) and IgG (P<0.05) were found in H/A compared to the L/A group.


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Table 1.  Clinical and biochemical characteristics in low and low average (L/A), and high and high average (H/A) peritoneal transport rate groups (means±SD or median and range)

 

Concentration of cytokines in the plasma and in the dialysate
The plasma and dialysate concentrations of IL-6 and VEGF are given in Figure 1Go. VEGF concentration was lower in dialysate compared to plasma (D/P of VEGF, median 0.2 (range 0.06–3.6)); dialysate and plasma concentrations did not correlate. In contrast, IL-6 levels were on average 26 times greater in dialysate compared to plasma (D/P for IL-6, median 14.6 (range 1.4–138)); dialysate and plasma concentrations did not correlate.



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Fig. 1.  Plasma and dialysate concentrations of (A) IL-6 and (B) VEGF.

 

Plasma cytokines and peritoneal transport rate of solutes
In plasma, the concentrations of IL-6 (P<0.05) and VEGF (P<0.01) were significantly higher in the H/A group, when compared to the L/A group (Figure 2Go). Plasma VEGF correlated significantly with D/Pcreat (r=0.36, P<0.05).



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Fig. 2.  Plasma concentration and dialysate appearance rate of IL-6 and VEGF in H/A and L/A groups (P<0.01 vs L/A; P<0.05 vs L/A). Box plot representation: 75% percentile, 25% percentile, median, and maximum and minimum values.

 

Dialysate IL-6 and VEGF and peritoneal transport rate of solutes
The dialysate appearance rate of IL-6 correlated with all investigated transport parameters. Thus, the IL-6 dialysate appearance rate was significantly higher in the H/A group than in the L/A group (Figure 2Go). A positive and significant correlation was found between D/Pcreat and IL-6 dialysate appearance rate (r=0.54; P<0.0005). Furthermore, IL-6 dialysate appearance rate correlated with alb and IgG clearances (Figure 3Go).



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Fig. 3.  Relationship between log IL-6 and log VEGF dialysate appearance rates and clearances of alb and IgG.

 
The dialysate appearance rate of VEGF was significantly higher in the H/A than in the L/A group (Figure 2Go). Finally, the dialysate appearance rate of VEGF correlated with D/Pcreat (r=0.36; P<0.05), as well as with the clearance of alb and clearance of IgG (Figure 3Go).

Correlation between dialysate IL-6 and VEGF
A significant correlation was noted between the dialysate appearance rates of IL-6 and VEGF (r=0.69, P<0.0001) (Figure 4Go).



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Fig. 4.  Relationship between VEGF and IL-6 dialysate appearance rates.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The results of the present study indicate that increased plasma and dialysate concentrations of IL-6 and VEGF were associated with increased peritoneal transport rate of solutes, suggesting a link between inflammation, angiogenesis, and small-solute peritoneal transport rate. Perhaps unexpectedly, the associations between plasma and dialysate concentrations of these cytokines were weak.

Since cytokines may interfere with the permeability of membranes [15], it is likely that they also have the same influence on the peritoneum. However, until now a significant relationship between plasma concentration of cytokines and peritoneal transport characteristics has not been reported. High PSTR represents an independent risk factor for mortality in CAPD [5]. Plasma IL-6 has been related to poor outcome in haemodialysis patients [8] and CAPD patients [16], but it is not known if plasma VEGF is related to clinical outcome in dialysis patients. In the present study, we observed significantly higher levels of plasma IL-6 and plasma VEGF in the group with higher PSRT. These findings support the hypothesis that the poor outcome of high transporters may be related at least in part to the presence of systemic inflammation. Beyond the active inflammatory response as such, higher prevalence of co-morbidities in high transporters, as described by Chung et al. [17], may also contribute to poor outcome in high transporters. In our study, high transporters were characterized by an increased incidence of diabetes mellitus and higher peritonitis rates when compared to low transporters.

On the local side, various factors are involved in the regulation of peritoneal permeability. Permeability changes due to several local factors have been demonstrated with the administration of vasoactive drugs such as nitroprusside [18], or in severe inflammation such as peritonitis [10]. The intraperitoneal levels of IL-6 were much higher than the plasma concentrations (Figure 1Go), indicating local production of IL-6. Interestingly, IL-6 appearance in the dialysate correlated to all investigated transport parameters. In another study, intraperitoneal IL-6 concentration was correlated to increased transport rate of large solutes, but not to smaller molecules in stable CAPD patients [9]. Intraperitoneal production of IL-6 can probably be attributed to mesothelial cells [19], macrophages or endothelial cells [20]. It appears from recent studies that IL-6 is a central mediator of the inflammatory response in the peritoneal cavity. Hurst et al. [21], combining clinical and experimental evidence, recently showed that IL-6 controls the pattern of leukocyte recruitment during inflammation. Additionally, these authors showed that the soluble IL-6 receptor represents a key factor in the transition between early (neutrophil-mediated) and late (mononuclear leukocyte-mediated) phase of the intraperitoneal inflammatory response. In vitro studies demonstrated higher IL-6 production in response to the exposure to more biocompatible dialysis solutions, in which case IL-6 is presumably a marker of cell vitality [22,23]. In contrast, ex vivo studies showed lower cytokine production when more biocompatible solutions were used [24]. In fact, recent in vivo clinical studies using a bicarbonate/lactate solution have shown that the intraperitoneal cellular response to the more biocompatible solution leads to lower production of IL-6 in comparison to standard solutions, which points to reduced inflammatory response [25]. Also, in a clinical evaluation, Fujimori et al. [26] showed that higher glucose concentration induced higher production of intraperitoneal IL-6. Therefore, in accordance with Cooker et al. [25], we propose that intraperitoneal IL-6 concentration may be regarded a marker of ongoing intraperitoneal inflammation.

In our study, the dialysate appearance of VEGF in the peritoneal cavity was associated with increased peritoneal transport rate for both small and large solutes. VEGF is strongly involved in neoangiogenesis and altered permeability, both events potentially related to high PSTR [11]. An increased peritoneal vascular surface area has been proposed as a major determinant of high PSTR [6], but conclusive studies are lacking. A recent study demonstrated that the use of glucose-based solutions was associated with higher levels of VEGF in comparison to treatment with non-glucose solutions, suggesting a link between either glucose exposure or bioincompatibility or both, and intraperitoneal VEGF generation [27]. Interestingly, in a peritoneal dialysis animal model, VEGF induced increased peritoneal permeability, and the effect was reversed by anti-VEGF treatment [28].

In support of a possible link between dialysate bioincompatibility and cytokine levels, a recent clinical study showed that dialysate levels of both IL-6 and VEGF were lower in the group treated with more biocompatible solutions, when compared to standard glucose based solutions [25]. However, due to the cross-sectional design of the present study, the cause-and-effect relationship of IL-6 and VEGF cannot be addressed appropriately.

Finally, a highly significant correlation was found between the intraperitoneal appearance of VEGF and IL-6. This relationship may suggest that a common factor stimulates the generation of both VEGF and IL-6, and that VEGF subsequently mediates morphological and functional alterations of the peritoneal membrane such as angiogenesis, in response to this stimulating factor (inflammation?). It should be noted that IL-6 induces the proliferation of brain microvascular endothelial cells in vitro, coinciding with the expression of VEGF mRNA in a brain-injury model [29]. In addition, both VEGF and IL-6 have well documented potent effects on microvascular permeability [7,11], which may result in increased peritoneal solute transport rate of small (creatinine) and larger (alb and IgG) solutes. In fact, Combet et al. [30] recently demonstrated that angiogenesis and increased endothelial area (simultaneously with enhanced NOS activity and endothelial NOS upregulation) are involved in the permeability changes observed in long-term PD. The authors also present evidence for the participation of local advanced glycation end-product deposits and VEGF peritoneal tissue expression in that process.

In conclusion, high peritoneal solute transport rate was associated with higher dialysate and plasma concentrations of IL-6 and VEGF in a cross-sectional analysis of CAPD patients with no apparent inflammation process or disease. Our findings suggest a link between inflammation, angiogenesis, and peritoneal solute transport rate. Relationships between mortality, morbidity, and inflammation, as well as between inflammation and peritoneal fibrotic changes have been proposed in peritoneal dialysis patients. Therefore, further analysis of systemic and intraperitoneal inflammation and its relationship to transport parameters is essential to clarify whether or not reducing systemic and intraperitoneal inflammation can improve patient and technique survival in CAPD.



   Acknowledgments
 
This work was supported by grants from the São Paulo Foundation for Research Support (FAPESP; process number 99–08096–8). During these studies, Roberto F. S. Pecoits-Filho was a recipient of a Research Award number 98/13385–6 from the São Paulo Foundation for Research Support (FAPESP). We are thankful to Wagner Vasques Dominguez for his expert technical assistance and Martha Garrett Pecoits for her valuable language review of the manuscript.



   Notes
 
Correspondence and offprint requests to: Roberto Pecoits-Filho, Renal Physiopathology Laboratory (LIM-16), University of São Paulo School of Medicine, Av. Dr Arnaldo, 455 – Sala 3342, São Paulo – SP, Brazil CEP – 01246–903. Email: pecoits{at}usp.br Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 27. 7.01
Accepted in revised form: 20. 2.02