Abstract
To compare the interleukin-6 (IL-6) gene expression in the peripheral blood mononuclear cells (PBMCs) and plasma IL-6 levels in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) with those in patients undergoing hemodialysis.
Eleven hemodialysis patients, 10 CAPD patients, 15 non-dialyzed patients with end-stage kidney disease (ESKD), and 7 healthy controls were included in this study. PBMCs were collected by differential centrifugation. Plasma IL-6 concentration was measured by enzyme immunoassay.
Plasma IL-6 levels were significantly increased in the hemodialysis and CAPD patients as compared with non-dialyzed ESKD patients and normal subjects (p < 0.01). Following hemodialysis, plasma IL-6 levels exceeded those before hemodialysis. No significant difference was found in plasma IL-6 levels in CAPD patients and in hemodialysis patients when blood was drawn before hemodialysis. Low but steady-state levels of IL-6 mRNA expression were observed in the non-dialyzed ESKD patients. The expression of IL-6 mRNA in PBMCs was significantly increased in the patients undergoing hemodialysis or CAPD as compared with the non-dialyzed ESKD patients. The PBMC IL-6 mRNA was significantly lower in CAPD patients than in hemodialysis patients (p < 0.01). A significant correlation was found between the plasma concentration of IL-6 and the expression of IL-6 mRNA in PBMCs from patients undergoing hemodialysis or CAPD (p < 0.01).
The hemodialysis or CAPD procedure contributed to the increase in PBMC IL-6 mRNA expression and plasma IL-6 concentration. CAPD treatment stimulated the production of IL-6 to a lesser extent than hemodialysis treatment.
INTRODUCTION
Interleukin(IL)-6 is a multifunctional cytokine that activates T cells and B cells, immunoglobulin secretion, and acute-phase protein production in a variety of cell types Citation[[1]]. IL-6 is known to play a key role in the cytokine network as it exerts diverse effects on the immune system. Inflammatory cytokines, including IL-6 are reportedly responsible for such acute complications in patients with end-stage kidney disease as fever, headache, and hypotension Citation[2-4] and for such chronic complications as immunological disorders Citation[[5]], muscle wasting, and amyloidosis Citation[[6]]. These dialysis-related complications are considered to be mediated, at least in part, by the pro-inflammatory cytokines including IL-6 derived from the monocyte/ macrophage Citation[[7]]. Recent controversy about the biocompatibility of dialysis membrane in hemodialysis has focused on the roles of TNF-α, IL-1β, and IL-6 Citation[8-9]. The contact of lymphocytes or monocytes with the dialysis membranes reportedly stimulates the production of IL-6 by these cells and affects plasma IL-6 levels in the hemodialysis patients Citation[10-11]. However, it is not known how IL-6 production is regulated in patients with end-stage kidney disease.
We previously reported that patients with IgA nephropathy exhibited significantly higher urinary IL-6 levels than in healthy controls Citation[[12]], and that abnormally regulated IL-6 mRNA in peripheral blood T cells may be associated with disease activity in patients with IgA nephropathy Citation[[13]]. In the present study, we measured the circulating plasma level of IL-6 and the expression of IL-6 mRNA in peripheral blood mononuclear cells (PBMCs) to assess the regulation of IL-6 production in patients undergoing chronic hemodialysis or continuous ambulatory peritoneal dialysis (CAPD).
PATIENTS AND METHODS
Patients
We evaluated 21 patients who were undergoing maintenance dialysis for end-stage kidney disease including 11 undergoing hemodialysis (7 males and 4 females) and 10 undergoing CAPD (6 males and 4 females). In addition, we evaluated 15 non-dialyzed patients with end-stage kidney disease (ESKD) (9 males and 6 females). Seven healthy volunteers served as control subjects. The etiology of chronic renal failure was chronic glomerulonephritis in 29 patients, diabetic nephropathy in 6, and polycystic kidney disease in 1. The mean age was 52.0 ± 17.5 years for hemodialysis patients, 43.8 ± 9.4 years for CAPD patients, 61.3 ± 12.4 years for non-dialyzed ESKD patients, and 32.4 ± 4.9 years for controls. Serum creatinine levels of the non-dialyzed ESKD patients were 4.0 ± 1.0 mg/100 mL in group 1 and 9.4 ± 2.4 mg/100 mL in group 2. The mean duration of maintenance hemodialysis and CAPD was 25.7 months (range, 1 to 85 months), and 10.8 months (range, 1 to 50 months), respectively (Table). In hemodialysis, a hollow-fiber dialyzer with triacetate cellulose membrane was used for 3.5 or 4.0 hours, three times a week. In CAPD, a PD-2 Dianeal was used (Baxter Healthcare, Tokyo, Japan), with the CAPD fluid exchanged four times daily. No hemodialysis or CAPD patient showed evidence of C-reactive protein or had a recent history of injury or surgery. Endotoxin was not detected in the dialysate of any hemodialysis procedure. Informed consent was obtained from all participants.
Table. Patient characteristics
ISOLATION OF MONONUCLEAR CELLS
Blood was collected from the arterial side of the arteriovenous fistula of each hemodialysis patient at the beginning and the end of each dialysis session. In the CAPD patients, blood was collected from a peripheral vein after the first chance of CAPD fluid change in the morning. Blood was also collected from a peripheral vein of the ESKD patients in the morning. Serum levels of urea, creatinine, α2-macroglobulin, and hemoglobin were measured by standard chemical methods.
PBMCs were collected by differential centrifugation as previously described Citation[[14]]. Briefly, heparinized blood was layered on Ficoll-Hypaque (Pharmacia, Milwaukee,WI,USA), centrifuged at 400xg for 20 minutes and the PBMC layer was collected. After being washed with phosphate-buffered saline (PBS), PBMCs were counted by hematocytometry or Coulter counter (Coulter Electronics, Mijdrecht, The Netherlands), then stored at −70°C until RNA was prepared. We standardized the number of mononuclear cells at 106 cells in each study group. We did not differentiate between lymphocytes and monocytes. The mean circulating levels of lymphocytes and monocytes did not differ among the three groups.
Plasma IL-6 Concentration
Blood was collected into chilled tubes containing ethylene diaminetetraacetate (EDTA) (2mg/mL of blood) and centrifuged immediately at 2000×g for 10 minutes at 4°C. The plasma concentration of IL-6 was measured by enzyme immunoassay (IL-6 assay kit, Otsuka, Tokyo, Japan), with a detection limit of 10.0 pg/mL.
RNA Preparation and cDNA Probes
Total cellular RNA was extracted with vanadylribonucleoside complexes (Sigma, St. Louis, MO, USA) Citation[[15]]. The total concentration of RNA was determined spectrophotometrically by optical absorbance at 260 nm. The purity of the RNA preparations was assessed by measuring the optical density ratio of 260 nm to 280 nm. For Northern blot analysis, RNA samples (5 μg) were denatured at 65°C, electrophoresed through 0.7% agarose gels containing 2.2M formaldehyde, and transferred to nylon filters (New England Nuclear, Boston, MA, USA). Before transferring RNA onto the nylon filters, the ethidium-stained gels were visualized under ultraviolet illumination to determine the position of 28S and 18S ribosomal RNA bands, to assess the integrity of RNA, and to verify that equal amounts of RNA were loaded. Serial twofold dilutions of RNA were applied to the nylon filters with a 96-well vacuum manifold (Schleicher & Schuell, Keene, NH, USA) to quantitate steady-state mRNA levels. cDNAs for human IL-6 and human glyceraldehyde -3-phosphate dehydrogenase (GAPDH) were obtained from the American Type Culture Collection (Rockville, MD, USA). These cDNA probes were labeled with 32P by the random primed method (Boehringer Mannheim, Mannheim, Germany) Citation[[16]]. The specific activity of the cDNA was 109 cpm. The GAPDH cDNA was used to confirm that similar amounts of RNA were applied to each well. The IL-6 mRNA levels were quantified by initial standardization to the GAPDH mRNA levels. Hybridization was carried out in 1% bovine serum albumin (BSA), 7% sodium dodecylsulfate (SDS), 0.5M NaH2PO4, 1 mM EDTA and 100 μg/mL sonicated salmon sperm DNA for 20 hours at 65°C Citation[17-18]. After hybridization, the filters were washed three times in 0.5% BSA, 5% SDS, 40 mM NaH2PO4 and 1 mM EDTA at 65°C. The filters were dried, and exposed to Kodak X-OMAT AR films (Eastman Kodak, Rochester, NY, USA) at −70°C, and scanned with a Shimadzu densitometer (Shimadzu, Kyoto, Japan).
Statistical Analysis
The results are presented as means ± SD. The Wilcoxon signed ranks test and the Mann-Whitney U test were used to assess the statistical significance. Significance was assigned at the p < 0.05 level.
RESULTS
Plasma IL-6 Concentration
Plasma IL-6 levels in healthy subjects and non-dialyzed ESKD patients (groups 1 and 2) were less than 10 pg/mL. In contrast, plasma IL-6 concentration was elevated in patients on hemodialysis (predialysis, 27.8 ± 18.6 pg/mL; postdialysis, 54.9 ± 21.9 pg/mL) and in patients on CAPD (23.9 ± 26.1 pg/mL) (). A significant difference in plasma IL-6 levels was found between before and after hemodialysis. In CAPD patients, however, plasma IL-6 levels were two-fold lower than the post-dialysis levels in the hemodialysis patients (p < 0.05). There was no significant difference in plasma IL-6 levels between predialysis hemodialysis patients and CAPD patients.
Figure 1. Plasma IL-6 concentrations in hemodialysis patients (HD) (n = 11; predialysis and post-dialysis), CAPD patients (n = 10), non-dialysis ESKD patients (n = 15; group 1, serum creatinine 4.0 ± 1.0 mg/dL; group 2, serum creatinine 9.4 ± 2.4 mg/dL) and healthy controls (n = 7). Data are presented as mean ± SD. *p < 0.05.
IL-6 mRNA Expression in Peripheral Blood Mononuclear Cells
The expression of IL-6 mRNA was hardly detectable in PBMCs from the healthy subjects. IL-6 mRNA expression was elevated in CAPD patients and hemodialysis patients. Low but significantly increased levels of IL-6 mRNA expression were observed in the non-dialyzed ESKD patients compared to that in healthy controls, however, no significant difference was found between the non-dialyzed ESKD patients with high serum creatinine levels (group 2) and those with low serum creatinine levels (group 1). The IL-6 mRNA expression in PBMC from the non-dialyzed ESKD patients was significantly lower than that from patients on CAPD or hemodialysis. The IL-6 mRNA levels in PBMCs from CAPD patients were significantly lower than predialysis or post-dialysis levels in PBMCs from hemodialysis patients (p < 0.01). In hemodialysis patients, we observed a significantly higher PBMC L-6 mRNA expression after dialysis than those before dialysis (p < 0.05) (). In CAPD patients, no significant changes were found in IL-6 mRNA levels between PBMC obtained at the time of bag exchange and that obtained 4 hours later (data not shown). Dot blot analyses of IL-6 mRNA expression in PBMCs from the dialysis patients and the control subjects are shown in .
Figure 2. The IL-6 mRNA levels in peripheral blood mononuclear cells in hemodialysis patients (HD) (n = 11); predialysis and postdialysis), CAPD patients (n = 10), non-dialyzed ESKD patients (n15;group 1, serum creatinine 4.0 ± 1.0 mg/dL;group 2, serum creatinine 9.4 ± 2.4 mg/dL) and healthy controls (n = 7). Data are presented as mean ± SD. *p < 0.05, **p < 0.01.
Figure 3. Hybridization of the IL-6 cDNA probe to the total RNA extracted from PBMC obtained from hemodialysis patients. CAPD patients and healthy controls. Total RNA was spotted onto a nylon filter. The amount of RNA in each dot is shown along the left. The filter was hybridized with a random primer-labeled specific IL-6 cDNA probe. Each number designates a different subject (A, pre-dialysis: B, post-dialysis).
Correlation of Plasma IL-6 Concentration and PBMC IL-6 mRNA Expression with Other Clinical Parameters
A significant correlation was found between plasma IL-6 concentration and the PBMC IL-6 mRNA expression in patients undergoing hemodialysis or CAPD (p < 0.01). However, IL-6 mRNA expression in PBMCs did not correlate with age, C-reactive protein levels, serum urea or creatinine concentration, dose of erythropoietin and duration of dialysis.
DISCUSSION
In the present study, we have shown that the plasma IL-6 concentration and IL-6 mRNA expression in PBMCs increased significantly in hemodialysis and CAPD patients when compared with non-dialyzed ESKD patients and normal subjects. Some investigators have reported elevated plasma IL-6 levels in patients on hemodialysis Citation[[11]], Citation[[13]], Citation[[19]]. In contrast, Anderson et al. Citation[[20]] did not detect plasma IL-6 in any hemodialysis patients. In CAPD patients without peritonitis plasma IL-6 has been reported to be undetectable Citation[20-22]. Differences in the IL-6 assay methods, including sensitivity of ELISA or bioassay, may explain partly this discrepancy. In the present study, we could find the low levels of plasma IL-6 (from 4.2 to 8.9 pg/mL) by different sensitive ELISA assay in some patients, however, other patients showed less than detection limit of plasma IL-6 concentrations. Serum α2-macroglobulin, known to combine with IL-6 without modifying its activity, may inhibit the ELISA assay Citation[[23]], although we could not find any correlation between plasma IL-6 levels and serum α2-macroglobulin concentration (unpublished data). It is likely that the short half-life of IL-6 Citation[[24]] and the existence of anti IL-6 auto-antibodyCitation[[25]] may also influence plasma IL-6 levels. Thus, the plasma IL-6 concentration reflects the net result of the balance between cytokine release and its binding to soluble receptors or inhibitors Citation[26-28].
Expression of the IL-6 gene in PBMCs had not been studied in hemodialysis patients or CAPD patients. In the present study, we have demonstrated that low but steady-state levels of IL-6 mRNA expression in non-dialyzed ESKD patients and that higher IL-6 mRNA expression in PBMCs was observed in hemodialysis patients than that in CAPD patients. These data suggest that uremia itself is the origin of elevated IL-6 mRNA expression, and that abnormal stimulation also may contribute to an increase in the IL-6 mRNA as a result of the dialysis. Higher mRNA expression levels seen in hemodialysis patients suggests that the stimulation of monocyte/macrophages by an artificial membrane may be stronger than that caused by physiologic membrane in CAPD. In addition, the fact that the plasma IL-6 concentration and PBMC IL-6 mRNA expression are higher after dialysis than before dialysis suggests that the hemodialysis procedure itself stimulates IL-6 production at transcriptional levels. Significant correlation was observed between the plasma IL-6 concentration and IL-6 mRNA expression in patients on hemodialysis or CAPD, but not in non-dialyzed ESKD patients. These data indicate that the measurement of cytokine mRNA expression in PBMCs may contribute to assess the intensity of stimulation for cytokine production in dialysis patients. In addition, there was no correlation between the C-reactive protein level and plasma IL-6 concentration or of PBMC IL-6 mRNA expression, suggesting that increased levels of plasma IL-6 concentration and PBMC IL-6 mRNA may not always reflect directly the acute phase inflammatory response, but chronic stimulation in the host for complications in hemodialysis and CAPD patients.
In summary, we demonstrated a significant increase in plasma IL-6 and steady-state IL-6 mRNA expression in PBMCs in hemodialysis, CAPD, and non-dialyzed ESKD patients. In CAPD patients, the plasma IL-6 level and PBMC IL-6 mRNA expression were significantly lower than that in hemodialysis patients after dialysis. These results suggest that the biomembrane in CAPD therapy may be less stimulant to IL-6 production and secretion than the artificial membrane in hemodialysis.
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