- Email: [email protected]
Acute and chronic inflammation in pediatric patients receiving hemodialysis
ACUTE AND CHRONIC INFLAMMATION IN PEDIATRIC PATIENTS RECEIVING HEMODIALYSIS STUART L. GOLDSTEIN, MD, HELEN CURRIER, BSN, RN, CNN, LYNNE WATTERS, BS, RN, JAMES M. HEMPE, PHD, RITA D. SHETH, MD, AND DOUGLAS SILVERSTEIN, MD
Objectives
To assess chronic and acute inflammation in children receiving maintenance hemodialysis.
Study design To assess markers of acute inflammation, serum levels (ELISA) of the cytokines tumor necrosis factor (TNF)a, interleukin (IL)-1b, IL-10, and IL-6, 3 to 5 mL of serum was obtained from 13 pediatric patients (mean patient weight, 37.0 ± 15.2 kg; mean age, 14.6 ± 4.6 years) before and 30 minutes and 24 hours after a routine midweek hemodialysis treatment session. Chronic inflammation was assessed by serum C-reactive protein (CRP) levels. Results Early-response cytokines TNF-a at 30 minutes (5.84 ± 0.94 to 9.67 ± 0.92 pg/mL; P = .002) and 24 hours (5.84 ± 0.94 to 9.54 ± 1.05 pg/mL; P = .008) and IL-1b at 30 minutes (17.19 ± 2.00 to 26.17 ± 1.12 pg/mL; P = .001) and 24 hours (17.19 ± 2.00 to 23.01 ± 1.13 pg/mL; P = .02) increased significantly after hemodialysis. Later-response cytokines IL-10 and IL-6 activation was not significant. CRP levels were elevated in 10 of 13 patients (mean, 14.7 ± 9.5mg/L; range, 7.2-38.8 mg/ L) and correlated with dialysis vintage. Baseline IL-6 and IL-10 levels correlated with dialysis vintage and correlated negatively with eqKt/V. Conclusions We observed a chronic inflammatory state in pediatric hemodialysis patients not related to the hemodialysis treatment but rather dialysis vintage and hemodialysis adequacy. We suggest that either more frequent dialysis or enhanced cytokine clearance may ameliorate the chronic inflammatory state observed in pediatric patients receiving hemodialysis. (J Pediatr 2003;143:653-7)
herapeutic and technical advances over the past decade have reduced the mortality rates of children and young adults with end-stage renal disease (ESRD) receiving hemodialysis.1 Young adults with ESRD have a 1000-fold increased risk of cardiovascular death when compared with the general population,2 which may be caused, in part, by cardiovascular calcifications. A recent study of adult patients with ESRD has shown that inflammation may promote cardiovascular calcification development.3 Substantial research has examined the association between various inflammatory diseases and proinflammatory cytokine generation in adult patients.4-7 The association between inflammation and cytokine generation has also been established in adult patients receiving hemodialysis.8-12 Early results have shown that dialyzer membranes composed of biocompatible materials led to less proinflammatory cytokine generation.8,9 To date, there are minimal data addressing the inflammatory state of children with ESRD receiving hemodialysis, and the results are often conflicting.13-16 One study demonstrated elevated serum concentrations of interleukin (IL)-2 and tumor necrosis factor (TNF)-a in children with chronic renal failure and ESRD, although dialysis treatment had no effect on serum cytokine levels.13 In another study,14 IL-1b and TNF-a mRNA and protein levels were not elevated in peripheral blood mononuclear cells derived from hemodialysis patients compared with age-matched control subjects; in another study, it was reported that serum IL-1b was undetectable in all patients, whereas TNF-a levels
T
CRP ESRD IL
C-reactive protein End-stage renal disease Interleukin
TNF RT
Tumor necrosis factor Room temperature
From Baylor College of Medicine, Renal Dialysis Unit, Texas Children’s Hospital, Houston, Texas; and Louisiana State University Health Sciences Center, New Orleans, Louisiana. Supported by a grant from Toray, Incorporated. Submitted for publication Dec 26, 2002; revision received June 13, 2003; accepted Aug 22, 2003. Reprint requests: Stuart L. Goldstein, MD, Texas Children’s Hospital, 6621 Fannin St, MC 3-2482, Houston, TX 77030. E-mail: [email protected]. Copyright ª 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0 10.1067/S0022-3476(03)00534-1
653
were higher in predialysis and dialysis patients.15 Finally, TNF-a but not IL-6 levels were significantly higher in patients with either chronic renal failure or ESRD, although intradialytic and interdialytic changes were not measured.16 No study has aimed to assess simultaneously various markers of inflammation in children receiving hemodialysis. Nevertheless, such information is critical, since if inflammation contributes to the high risk for cardiac morbidity seen in young adults, children probably have inflammation as they progress to ESRD in childhood or adolescence. Therefore, we assessed the potential effect of hemodialysis on cytokine generation and the association between chronic inflammation and hemodialysis adequacy.
METHODS Patient Population All patients had been receiving maintenance hemodialysis for at least 6 months at the Texas Children’s Hospital Renal Dialysis Unit, Houston, Texas. Patients with underlying systemic chronic inflammatory disease (eg, systemic lupus erythematosus) or active acute systemic inflammatory disease or infection were excluded from participation. The institutional review boards of the Baylor College of Medicine and the Louisiana State University Health Sciences Center approved the study. Patients were entered into study after obtaining informed consent. The diseases leading to ESRD in the study population were obstructive uropathy (n = 2), dysplasia (n = 3), focal segmental glomerulosclerosis (n = 2), rapidly progressive glomerulonephritis (n = 1), Jeune syndrome (n = 1), chronic idiopathic glomerulonephritis (n = 1), branchio-oto-renal syndrome (n = 1), membranoproliferative glomerulonephritis type II (n = 1), and unknown (n = 1).
Cytokine Assays Three to five milliliters of serum was obtained from each patient before a routine midweek hemodialysis treatment session and 30 minutes and then 24 hours after dialysis session termination. The samples were immediately centrifuged and the serum decanted, aliquoted, and stored at 808C until analysis. Serum levels from each time point were analyzed for the cytokines TNF-a, IL-1b, IL-10, and IL-6.
Measurement of Serum Cytokine Levels OptEIA assay kits to measure all cytokines were all purchased from BD PharMingen (San Diego, Calif ). The protocol for the analysis of each cytokine was similar. Briefly, all patient serum samples and reagents were brought to room temperature (RT). First, standards were prepared from a 500pg/mL stock. The appropriate dilutions were made to establish a standard curve. Fifty microliters of ELISA diluent was pipetted into each well of the 96-well plates. Then, 100 lL of the standards and samples (all done in duplicate) were pipetted into wells and mixed with the diluent. The plate was gently shaken and covered with a plate sealer, and the samples were incubated for 2 hours at RT. After incubation, the 654
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standards and samples were aspirated from the wells and washed with buffer; 100 lL of a solution containing a detection antibody (avidin-horseradish peroxidase conjugate) preserved in 0.15% Pro-Clin-150 was then added. This was incubated for 1 hour at RT. The wells were then washed with buffer and 100 lL 3,39,5,59-tetramethylbenzidine (TMB) was subsequently added. The plate was then incubated in the dark at RT for 30 minutes. Afterward, a 50-lL solution containing 1 mol/L phosphoric acid was added. Absorbance was read at 450 nm. Results were initially calculated by optical density and converted to picograms per milliliter by log-log computation with application according to the standard curve. For each time point studied, all samples were run in duplicate to ensure reproducibility of the ELISA assay itself. Both samples from each patient for each cytokine measured were run on the same day on one ELISA plate, with two sets of internal standards, also ensuring consistency and reliability.
Hemodialysis Adequacy Midweek hemodialysis adequacy assessment occurred during the same midweek hemodialysis when cytokine levels were obtained. Single-pool Kt/V was derived through the use of formal urea kinetic modeling. Equilibrated double-pool Kt/ V was estimated by the logarithmic extrapolation method of Goldstein.17 Since 1998, patients in our program have been dialyzed exclusively with membranes composed of polymethylmethacrylate (PMMA; Toray, Inc, Tokyo, Japan), which are sterilized in the production plant by gamma irradiation. We do not use dialyzer reuse in the Texas Children’s Hospital dialysis program and therefore do not run the risk of chemical dialyzer sterilization affecting patient inflammatory response. Dialyzer surface area was prescribed to limit the dialysis circuit volume to <10% of patient blood volume. To ensure eqKt/V values obtained on the day of analysis represented the same delivered dialysis dose for an entire month, no patient dialysis prescription was changed, and all patients received the prescribed liters of blood processed for the month before the date of cytokine and adequacy assessment.
Statistical Analysis The effect of the hemodialysis treatment on cytokine production was evaluated by 1-way analysis of variance with Bonferroni multiple comparison test. Potential associations between eqKt/V and baseline pretreatment cytokine levels were assessed by linear regression analysis. Potential associations between dialysis-induced cytokine level changes and chronic inflammation manifested by serum C-reactive protein (CRP) levels were assessed by independent t test. The CRP assay used in the current study reported levels >6.9 mg/L; levels <6.9 g/L were considered to be within normal limits (Spectra Laboratories, Fremont, Calif). Potential associations between spKt/V, eqKt/V, and CRP levels were assessed by independent t test. The potential association between dialysis vintage (ie, the number of months a patient has been receiving dialysis) and CRP and baseline cytokine levels was assessed by The Journal of Pediatrics November 2003
linear regression analysis. A P value of <.05 was considered significant.
detectable CRP levels. Mean spKt/V and eqKt/V were not significantly different between patients with elevated versus nondetectable CRP levels.
RESULTS Patient Demographics
DISCUSSION
The mean spKt/V was 1.51 ± 0.20 (range, 1.11-1.94) and the mean equilibrated Kt/V (eqKt/V) was 1.29 ± 0.24 (range, 0.76-1.70). We observed significant negative correlation between prehemodialysis treatment levels of the lateresponse cytokine IL-6 and spKt/V (r = 0.72, P < .01) and between prehemodialysis treatment levels of late-response cytokines IL-10 (r = 0.77, P = .003) and IL-6 (r = 0.73, P = .010) and eqKt/V. No correlation was observed between prehemodialysis treatment levels of the early-response cytokines TNF-a or IL-1b and either spKt/V or eqKt/V. Ten of 13 patients had elevated CRP levels (mean, 14.7 ± 7.5 mg/L; range, 7.2-38.8 mg/L). Neither the mean pre-HD nor the percent change in serum cytokine levels was significantly different for patients with elevated versus non-
Our study was primarily designed to assess inflammation, a major risk factor for cardiovascular disease in pediatric patients receiving maintenance hemodialysis. To this end, we measured various serum cytokines. Our results show that after 30 minutes of hemodialysis, serum levels of the early-response serum cytokines TNF-a and IL-1b were significantly elevated compared with pre-HD levels. These changes persisted for at least 24 hours after the dialysis session. In contrast, the serum levels of two late-response cytokines, IL-10 and IL-6, did not change significantly after dialysis treatment. We did not measure cytokine levels in age-matched control subjects. However, our pediatric hemodialysis patients had elevated levels of multiple serum cytokines when compared with published age-matched control subjects. Serum levels of TNF-a in pediatric control subjects (2.2 ± 0.5 pg/mL), as shown in a study by Sylvester et al,18 were distinctly lower than those obtained in our dialysis patients: 5.8 ± 0.9 pg/mL (preHD); 9.7 ± 0.9 pg/mL (30 minutes post-HD); 9.5 ± 1.1 pg/ mL (24 hours post-HD). Similarly, serum levels of IL-1b were low (<10 pg/mL) in their control subjects compared with the values at all time points in our dialysis patients: 17.2 ± 2.0 pg/ mL (pre-HD); 25.8 ± 1.0 pg/mL (30 minutes post-HD); 23.0 ± 1.1 pg/mL (24 hours post-HD). Serum levels of IL-6 in their pediatric control patients (0.7 ± 0.2 pg/mL) were markedly lower than the values we measured at all time points (10.5 ± 1.9 pg/mL [pre-HD]; 13.5 ± 2.7 pg/mL [30 minutes post-HD]; 9.3 ± 0.6 pg/mL [24 hours post-HD]) in our dialysis population. Serum IL-10 levels in our patients were higher (13.2 ± 8.0 pg/mL [pre-HD]; 21.3 ± 10.4 pg/mL [30 minutes post-HD]; 15.5 ± 9.3 pg/mL [24 hours post-HD]) compared with the control pediatric patients (4.4 ± 1.5 pg/mL) reported by Katayama et al.19 Since all studied serum cytokines were elevated at all time points, we suggest that a chronic inflammatory state exists in pediatric patients with maintenance hemodialysis. Uremia must, in part, explain the elevated serum cytokine levels, since we observed a positive correlation between dialysis vintage and CRP levels. In addition, adult patients with chronic renal failure had elevated cytokine levels before starting dialysis therapy,20,21 which supports the role of uremia in chronic inflammation. Our data are consistent with that obtained from adult hemodialysis patients.8-12 The fact that we observed significant changes during the hemodialysis treatment for only TNF-a and IL-1b may be interpreted in a variety of ways, but the changes in these early-response cytokines is most likely induced by the treatment itself, yet the lack of IL-10 and IL-6 stimulation suggests that the hemodialysis treatment does not contribute to the chromic inflammatory state seen in these patients. Although serum levels of the late-response cytokines IL-10 and IL-6 levels did not change significantly after
Acute and Chronic Inflammation in Pediatric Patients Receiving Hemodialysis
655
Thirteen children receiving maintenance hemodialysis were enrolled. Mean patient weight was 37.0 ± 15.2 kg (range, 15.6-63.9 kg; 6 patients <40 kg). Mean patient age was 14.6 ± 4.6 years (range, 5.9-20.7 years).
Hemodialysis and Cytokine Generation We measured serum cytokines in all patients before a routine midweek hemodialysis treatment (pre-HD), 30 minutes after treatment (30 minutes post-HD) termination, and 24 hours after the treatment (24 hours post-HD) termination. Our results show that serum levels of the earlyresponse cytokine TNF-a rose significantly 30 minutes (5.8 ± 0.9 [pre-HD] to 9.7 ± 0.9 [30 minutes post-HD] pg/ mL; P = .0008) and 24 hours (5.8 ± 0.9 [pre-HD] to 9.5 ± 1.1 [24 hours post-HD] pg/mL; P = .02) after a routine the dialysis session. In addition, there was a significant increase in the serum levels of the early-response cytokine IL-1b 30 minutes (17.2 ± 2.0 [pre-HD] to 25.8 ± 1.0 [30 minutes postHD] pg/mL; P = .008) and 24 hours (17.2 ± 2.0 [pre-HD] to 23.0 ± 1.1 [24 hours post-HD] pg/mL; P = .01) after a routine dialysis session. In contrast, although the serum levels of the later-response cytokines IL-10 and IL-6 increased after dialysis, this rise was not statistically significant, and this slight rise of serum IL-10 and IL-6 levels was not sustained 24 hours after the dialysis treatment (Figure).
Inflammation and Dialysis Vintage Mean dialysis vintage was 33.7 ± 30.4 months (range, 7.6-122.6 months). Median dialysis vintage was 27.2 months. A significant positive correlation was observed between dialysis vintage and CRP (r = 0.89, P < .001), IL-6 levels (r = 0.89, P < .001) and IL-10 levels (r = 0.66, P = .03). No correlation was noted between dialysis vintage and levels of earlyresponse cytokines TNF-a or IL-b.
Inflammation and Hemodialysis Adequacy
Figure. Serum cytokine levels in hemodialysis patients. Serum cytokine levels were measured immediately before (open bars) and 30 minutes (solid bars) and 24 hours (hatched bars) after routine dialysis treatment. There was a significant (P < .05) increase in serum levels of (A) TNF-a and (B) IL-1b *30 minutes and #24 hours after a dialysis session as compared with predialysis levels. Data are portrayed as mean ± SEM.
a dialysis treatment, their pre-HD levels correlated strongly with patient dialysis vintage. In addition, the levels of lateresponse cytokines IL-10 and IL-6 were inversely related to delivered eqKt/V, suggesting that higher delivered doses of dialysis may reduce baseline chronic IL-10 and IL-6 levels. Although dialyzer membrane composition is known to affect the inflammatory response8,9 and the PMMA membranes lead to higher b-2 microglobulin clearance and less cytokine activation than previous generation cuprophane or cellulose acetate membranes, it was not the purpose of this study to evaluate the relative biocompatibility of PMMA membranes. Such analysis should be the subject of future study to assess the impact of various different biocompatible membranes on chronic inflammation in children receiving hemodialysis. Our study suggests that the hemodialysis treatment causes a transient increase in only an acute inflammatory 656
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response. The chronic inflammatory state observed in our patients probably is due to chronic uremia. The extremely elevated CRP levels further demonstrate a chronic inflammatory state in a majority of patients. Interpretation of our results is limited by the fact that we measured cytokine levels around only a single hemodialysis treatment. It is possible that patient cytokine levels might vary from treatment to treatment, but since none of the patients had fever or other evidence of active infection and since we observed chronic inflammation in the majority of patients, our data may suggest that factors other than the acute inflammatory response caused by the hemodialysis treatment lead to the chronic inflammation observed. Further study is necessary to assess the relations between inflammation and cardiovascular disease and to evaluate the effects of potential therapeutic options, which include antiinflammatory agents, daily hemodialysis, and chronic The Journal of Pediatrics November 2003
1. US renal data System. Excerpts for the USRDS 2001 Annual Data Report. Am J Kidney Dis 2001;38(Suppl):S1-248. 2. Sarnak MJ, Levey AS. Cardiovascular disease and maintenance renal disease: a new paradigm. Am J Kidney Dis 2000;35(Suppl 1):S117-31. 3. Wang AY, Woo J, Wang M, Sea MM, Ip R, Li PK, et al. Association of inflammation and malnutrition with cardiac valve calcification in continuous ambulatory peritoneal dialysis patients. J Am Soc Nephrol 2001;12:1927-36. 4. Kim PK, Deutschman CS. Inflammatory responses and mediators. Surg Clin North Am 2000;80:885-94. 5. Dinarello CA. Pro-inflammatory cytokines. Chest 2000;118:503-8. 6. Brivet FG, Emilie D, Galanaud P. Pro- and anti-inflammatory cytokines during acute severe pancreatitis: an early and sustained response, although unpredictable of death. Crit Care Med 1999;27:749-55. 7. Gloor B, Uhl W, Tcholakov O, Roggo A, Muller CA, Worni M, et al. Hydrocortisone treatment of early SIRS in acute experimental pancreatitis. Dig Dis Sci 2001;456:2154-61. 8. Horl WH. Hemodialysis membranes: Interleukins, biocompatibility, and middle molecules. J Am Soc Nephrol 2002;13(Suppl 1):S62-71. 9. Herbelin A, Nguyen AT, Zingraff J, Urena P, Descamps-Latscha B. Influence of uremia and hemodialysis on circulating interleukin-1 and tumor necrosis factor alpha. Kidney Int 1990;37:116-25. 10. Girndt M, Kaul H, Sester U, Ulrich C, Sester M, Georg T, et al. Antiinflammatory interleukin-10 genotype protects dialysis patients from cardiovascular events. Kidney Int 2002;62:949-55. 11. Nitta K, Akiba T, Kawashima A, Kimata N, Miwa N, Nishida E, et al. Characterization of TH1/TH2 profile in uremic patients. Nephron 2002; 91:492-5.
12. Herbelin A, Urena P, Nguyen AT, Zingraff J, Descamps-Latscha B. Elevated circulating levels of interleiukin-6 in patients with chronic renal failure. Kidney Int 1991;39:954-60. 13. Zwolinska D, Medynska A, Szprynger K, Szczepanska M. Serum concentration of IL-2, IL-6, TNF-a and their soluble receptors in children on chronic hemodialysis. Nephron 2000;86:441-6. 14. Neu AM, Fivush BA, Lederman HM. Cytokine production by peripheral blood mononuclear cells from pediatric chronic peritoneal dialysis patients. Pediatr Nephrol 1995;9:333-6. 15. Besbas N, Ozdemir S, Saatci U, Coskun T, Ozen S, Topaloglu R, et al. Nutritional assessment of children on haemodialysis: value of IGF-I, TNFalpha and IL-1beta. Nephrol Dial Transplant 1994;13:1484-8. 16. Sebekova K, Podracka L, Heidland A, Schinzel R. Enhanced plasma levels of advanced glycation end products (AGE) and pro-inflammatory cytokines in children/adolescents with chronic renal insufficiency and after renal replacement therapy by dialysis and transplantation: are they interrelated? Clin Nephrol 2001;56:S21-26. 17. Goldstein SL, Brewer ED. Logarithmic extrapolation of a 15-minute postdialysis BUN to predict equilibrated BUN and calculate double-pool Kt/ V in the pediatric hemodialysis population. Am J Kidney Dis 2000;36: 98-104. 18. Sylvester FA, Wyzga N, Hyams JS, Gronowicz GA. Effect of Crohn’s disease on bone metabolism in vitro: a role for Interleukin-6. J Bone Miner Res 2002;17:695-702. 19. Katayama K, Matsubara T, Fujiwara M, Koga M, Furukawa S. CD14+ CD16+ monocyte subpopulation in Kawasaki disease. Clin Exp Immunol 2000;121:566-70. 20. Panichi V, Migliori M, De Pietro S, Taccola D, Bianchi AM, Norpoth M, et al. C reactive protein in patients with chronic renal diseases. Ren Fail 2001;23:551-62. 21. Bolton CH, Downs LG, Victory JG, Dwight JF, Tomson CR, Mackness MI, et al. Endothelial dysfunction in chronic renal failure: roles of lipoprotein oxidation and pro-inflammatory cytokines. Nephrol Dial Transplant 2001;6:1189-97.
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hemodiafiltration on chronic inflammation in children with ESRD. We thank David Marsh for his support of the project.
REFERENCES
Objectives
To assess chronic and acute inflammation in children receiving maintenance hemodialysis.
Study design To assess markers of acute inflammation, serum levels (ELISA) of the cytokines tumor necrosis factor (TNF)a, interleukin (IL)-1b, IL-10, and IL-6, 3 to 5 mL of serum was obtained from 13 pediatric patients (mean patient weight, 37.0 ± 15.2 kg; mean age, 14.6 ± 4.6 years) before and 30 minutes and 24 hours after a routine midweek hemodialysis treatment session. Chronic inflammation was assessed by serum C-reactive protein (CRP) levels. Results Early-response cytokines TNF-a at 30 minutes (5.84 ± 0.94 to 9.67 ± 0.92 pg/mL; P = .002) and 24 hours (5.84 ± 0.94 to 9.54 ± 1.05 pg/mL; P = .008) and IL-1b at 30 minutes (17.19 ± 2.00 to 26.17 ± 1.12 pg/mL; P = .001) and 24 hours (17.19 ± 2.00 to 23.01 ± 1.13 pg/mL; P = .02) increased significantly after hemodialysis. Later-response cytokines IL-10 and IL-6 activation was not significant. CRP levels were elevated in 10 of 13 patients (mean, 14.7 ± 9.5mg/L; range, 7.2-38.8 mg/ L) and correlated with dialysis vintage. Baseline IL-6 and IL-10 levels correlated with dialysis vintage and correlated negatively with eqKt/V. Conclusions We observed a chronic inflammatory state in pediatric hemodialysis patients not related to the hemodialysis treatment but rather dialysis vintage and hemodialysis adequacy. We suggest that either more frequent dialysis or enhanced cytokine clearance may ameliorate the chronic inflammatory state observed in pediatric patients receiving hemodialysis. (J Pediatr 2003;143:653-7)
herapeutic and technical advances over the past decade have reduced the mortality rates of children and young adults with end-stage renal disease (ESRD) receiving hemodialysis.1 Young adults with ESRD have a 1000-fold increased risk of cardiovascular death when compared with the general population,2 which may be caused, in part, by cardiovascular calcifications. A recent study of adult patients with ESRD has shown that inflammation may promote cardiovascular calcification development.3 Substantial research has examined the association between various inflammatory diseases and proinflammatory cytokine generation in adult patients.4-7 The association between inflammation and cytokine generation has also been established in adult patients receiving hemodialysis.8-12 Early results have shown that dialyzer membranes composed of biocompatible materials led to less proinflammatory cytokine generation.8,9 To date, there are minimal data addressing the inflammatory state of children with ESRD receiving hemodialysis, and the results are often conflicting.13-16 One study demonstrated elevated serum concentrations of interleukin (IL)-2 and tumor necrosis factor (TNF)-a in children with chronic renal failure and ESRD, although dialysis treatment had no effect on serum cytokine levels.13 In another study,14 IL-1b and TNF-a mRNA and protein levels were not elevated in peripheral blood mononuclear cells derived from hemodialysis patients compared with age-matched control subjects; in another study, it was reported that serum IL-1b was undetectable in all patients, whereas TNF-a levels
T
CRP ESRD IL
C-reactive protein End-stage renal disease Interleukin
TNF RT
Tumor necrosis factor Room temperature
From Baylor College of Medicine, Renal Dialysis Unit, Texas Children’s Hospital, Houston, Texas; and Louisiana State University Health Sciences Center, New Orleans, Louisiana. Supported by a grant from Toray, Incorporated. Submitted for publication Dec 26, 2002; revision received June 13, 2003; accepted Aug 22, 2003. Reprint requests: Stuart L. Goldstein, MD, Texas Children’s Hospital, 6621 Fannin St, MC 3-2482, Houston, TX 77030. E-mail: [email protected]. Copyright ª 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0 10.1067/S0022-3476(03)00534-1
653
were higher in predialysis and dialysis patients.15 Finally, TNF-a but not IL-6 levels were significantly higher in patients with either chronic renal failure or ESRD, although intradialytic and interdialytic changes were not measured.16 No study has aimed to assess simultaneously various markers of inflammation in children receiving hemodialysis. Nevertheless, such information is critical, since if inflammation contributes to the high risk for cardiac morbidity seen in young adults, children probably have inflammation as they progress to ESRD in childhood or adolescence. Therefore, we assessed the potential effect of hemodialysis on cytokine generation and the association between chronic inflammation and hemodialysis adequacy.
METHODS Patient Population All patients had been receiving maintenance hemodialysis for at least 6 months at the Texas Children’s Hospital Renal Dialysis Unit, Houston, Texas. Patients with underlying systemic chronic inflammatory disease (eg, systemic lupus erythematosus) or active acute systemic inflammatory disease or infection were excluded from participation. The institutional review boards of the Baylor College of Medicine and the Louisiana State University Health Sciences Center approved the study. Patients were entered into study after obtaining informed consent. The diseases leading to ESRD in the study population were obstructive uropathy (n = 2), dysplasia (n = 3), focal segmental glomerulosclerosis (n = 2), rapidly progressive glomerulonephritis (n = 1), Jeune syndrome (n = 1), chronic idiopathic glomerulonephritis (n = 1), branchio-oto-renal syndrome (n = 1), membranoproliferative glomerulonephritis type II (n = 1), and unknown (n = 1).
Cytokine Assays Three to five milliliters of serum was obtained from each patient before a routine midweek hemodialysis treatment session and 30 minutes and then 24 hours after dialysis session termination. The samples were immediately centrifuged and the serum decanted, aliquoted, and stored at 808C until analysis. Serum levels from each time point were analyzed for the cytokines TNF-a, IL-1b, IL-10, and IL-6.
Measurement of Serum Cytokine Levels OptEIA assay kits to measure all cytokines were all purchased from BD PharMingen (San Diego, Calif ). The protocol for the analysis of each cytokine was similar. Briefly, all patient serum samples and reagents were brought to room temperature (RT). First, standards were prepared from a 500pg/mL stock. The appropriate dilutions were made to establish a standard curve. Fifty microliters of ELISA diluent was pipetted into each well of the 96-well plates. Then, 100 lL of the standards and samples (all done in duplicate) were pipetted into wells and mixed with the diluent. The plate was gently shaken and covered with a plate sealer, and the samples were incubated for 2 hours at RT. After incubation, the 654
Goldstein et al
standards and samples were aspirated from the wells and washed with buffer; 100 lL of a solution containing a detection antibody (avidin-horseradish peroxidase conjugate) preserved in 0.15% Pro-Clin-150 was then added. This was incubated for 1 hour at RT. The wells were then washed with buffer and 100 lL 3,39,5,59-tetramethylbenzidine (TMB) was subsequently added. The plate was then incubated in the dark at RT for 30 minutes. Afterward, a 50-lL solution containing 1 mol/L phosphoric acid was added. Absorbance was read at 450 nm. Results were initially calculated by optical density and converted to picograms per milliliter by log-log computation with application according to the standard curve. For each time point studied, all samples were run in duplicate to ensure reproducibility of the ELISA assay itself. Both samples from each patient for each cytokine measured were run on the same day on one ELISA plate, with two sets of internal standards, also ensuring consistency and reliability.
Hemodialysis Adequacy Midweek hemodialysis adequacy assessment occurred during the same midweek hemodialysis when cytokine levels were obtained. Single-pool Kt/V was derived through the use of formal urea kinetic modeling. Equilibrated double-pool Kt/ V was estimated by the logarithmic extrapolation method of Goldstein.17 Since 1998, patients in our program have been dialyzed exclusively with membranes composed of polymethylmethacrylate (PMMA; Toray, Inc, Tokyo, Japan), which are sterilized in the production plant by gamma irradiation. We do not use dialyzer reuse in the Texas Children’s Hospital dialysis program and therefore do not run the risk of chemical dialyzer sterilization affecting patient inflammatory response. Dialyzer surface area was prescribed to limit the dialysis circuit volume to <10% of patient blood volume. To ensure eqKt/V values obtained on the day of analysis represented the same delivered dialysis dose for an entire month, no patient dialysis prescription was changed, and all patients received the prescribed liters of blood processed for the month before the date of cytokine and adequacy assessment.
Statistical Analysis The effect of the hemodialysis treatment on cytokine production was evaluated by 1-way analysis of variance with Bonferroni multiple comparison test. Potential associations between eqKt/V and baseline pretreatment cytokine levels were assessed by linear regression analysis. Potential associations between dialysis-induced cytokine level changes and chronic inflammation manifested by serum C-reactive protein (CRP) levels were assessed by independent t test. The CRP assay used in the current study reported levels >6.9 mg/L; levels <6.9 g/L were considered to be within normal limits (Spectra Laboratories, Fremont, Calif). Potential associations between spKt/V, eqKt/V, and CRP levels were assessed by independent t test. The potential association between dialysis vintage (ie, the number of months a patient has been receiving dialysis) and CRP and baseline cytokine levels was assessed by The Journal of Pediatrics November 2003
linear regression analysis. A P value of <.05 was considered significant.
detectable CRP levels. Mean spKt/V and eqKt/V were not significantly different between patients with elevated versus nondetectable CRP levels.
RESULTS Patient Demographics
DISCUSSION
The mean spKt/V was 1.51 ± 0.20 (range, 1.11-1.94) and the mean equilibrated Kt/V (eqKt/V) was 1.29 ± 0.24 (range, 0.76-1.70). We observed significant negative correlation between prehemodialysis treatment levels of the lateresponse cytokine IL-6 and spKt/V (r = 0.72, P < .01) and between prehemodialysis treatment levels of late-response cytokines IL-10 (r = 0.77, P = .003) and IL-6 (r = 0.73, P = .010) and eqKt/V. No correlation was observed between prehemodialysis treatment levels of the early-response cytokines TNF-a or IL-1b and either spKt/V or eqKt/V. Ten of 13 patients had elevated CRP levels (mean, 14.7 ± 7.5 mg/L; range, 7.2-38.8 mg/L). Neither the mean pre-HD nor the percent change in serum cytokine levels was significantly different for patients with elevated versus non-
Our study was primarily designed to assess inflammation, a major risk factor for cardiovascular disease in pediatric patients receiving maintenance hemodialysis. To this end, we measured various serum cytokines. Our results show that after 30 minutes of hemodialysis, serum levels of the early-response serum cytokines TNF-a and IL-1b were significantly elevated compared with pre-HD levels. These changes persisted for at least 24 hours after the dialysis session. In contrast, the serum levels of two late-response cytokines, IL-10 and IL-6, did not change significantly after dialysis treatment. We did not measure cytokine levels in age-matched control subjects. However, our pediatric hemodialysis patients had elevated levels of multiple serum cytokines when compared with published age-matched control subjects. Serum levels of TNF-a in pediatric control subjects (2.2 ± 0.5 pg/mL), as shown in a study by Sylvester et al,18 were distinctly lower than those obtained in our dialysis patients: 5.8 ± 0.9 pg/mL (preHD); 9.7 ± 0.9 pg/mL (30 minutes post-HD); 9.5 ± 1.1 pg/ mL (24 hours post-HD). Similarly, serum levels of IL-1b were low (<10 pg/mL) in their control subjects compared with the values at all time points in our dialysis patients: 17.2 ± 2.0 pg/ mL (pre-HD); 25.8 ± 1.0 pg/mL (30 minutes post-HD); 23.0 ± 1.1 pg/mL (24 hours post-HD). Serum levels of IL-6 in their pediatric control patients (0.7 ± 0.2 pg/mL) were markedly lower than the values we measured at all time points (10.5 ± 1.9 pg/mL [pre-HD]; 13.5 ± 2.7 pg/mL [30 minutes post-HD]; 9.3 ± 0.6 pg/mL [24 hours post-HD]) in our dialysis population. Serum IL-10 levels in our patients were higher (13.2 ± 8.0 pg/mL [pre-HD]; 21.3 ± 10.4 pg/mL [30 minutes post-HD]; 15.5 ± 9.3 pg/mL [24 hours post-HD]) compared with the control pediatric patients (4.4 ± 1.5 pg/mL) reported by Katayama et al.19 Since all studied serum cytokines were elevated at all time points, we suggest that a chronic inflammatory state exists in pediatric patients with maintenance hemodialysis. Uremia must, in part, explain the elevated serum cytokine levels, since we observed a positive correlation between dialysis vintage and CRP levels. In addition, adult patients with chronic renal failure had elevated cytokine levels before starting dialysis therapy,20,21 which supports the role of uremia in chronic inflammation. Our data are consistent with that obtained from adult hemodialysis patients.8-12 The fact that we observed significant changes during the hemodialysis treatment for only TNF-a and IL-1b may be interpreted in a variety of ways, but the changes in these early-response cytokines is most likely induced by the treatment itself, yet the lack of IL-10 and IL-6 stimulation suggests that the hemodialysis treatment does not contribute to the chromic inflammatory state seen in these patients. Although serum levels of the late-response cytokines IL-10 and IL-6 levels did not change significantly after
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Thirteen children receiving maintenance hemodialysis were enrolled. Mean patient weight was 37.0 ± 15.2 kg (range, 15.6-63.9 kg; 6 patients <40 kg). Mean patient age was 14.6 ± 4.6 years (range, 5.9-20.7 years).
Hemodialysis and Cytokine Generation We measured serum cytokines in all patients before a routine midweek hemodialysis treatment (pre-HD), 30 minutes after treatment (30 minutes post-HD) termination, and 24 hours after the treatment (24 hours post-HD) termination. Our results show that serum levels of the earlyresponse cytokine TNF-a rose significantly 30 minutes (5.8 ± 0.9 [pre-HD] to 9.7 ± 0.9 [30 minutes post-HD] pg/ mL; P = .0008) and 24 hours (5.8 ± 0.9 [pre-HD] to 9.5 ± 1.1 [24 hours post-HD] pg/mL; P = .02) after a routine the dialysis session. In addition, there was a significant increase in the serum levels of the early-response cytokine IL-1b 30 minutes (17.2 ± 2.0 [pre-HD] to 25.8 ± 1.0 [30 minutes postHD] pg/mL; P = .008) and 24 hours (17.2 ± 2.0 [pre-HD] to 23.0 ± 1.1 [24 hours post-HD] pg/mL; P = .01) after a routine dialysis session. In contrast, although the serum levels of the later-response cytokines IL-10 and IL-6 increased after dialysis, this rise was not statistically significant, and this slight rise of serum IL-10 and IL-6 levels was not sustained 24 hours after the dialysis treatment (Figure).
Inflammation and Dialysis Vintage Mean dialysis vintage was 33.7 ± 30.4 months (range, 7.6-122.6 months). Median dialysis vintage was 27.2 months. A significant positive correlation was observed between dialysis vintage and CRP (r = 0.89, P < .001), IL-6 levels (r = 0.89, P < .001) and IL-10 levels (r = 0.66, P = .03). No correlation was noted between dialysis vintage and levels of earlyresponse cytokines TNF-a or IL-b.
Inflammation and Hemodialysis Adequacy
Figure. Serum cytokine levels in hemodialysis patients. Serum cytokine levels were measured immediately before (open bars) and 30 minutes (solid bars) and 24 hours (hatched bars) after routine dialysis treatment. There was a significant (P < .05) increase in serum levels of (A) TNF-a and (B) IL-1b *30 minutes and #24 hours after a dialysis session as compared with predialysis levels. Data are portrayed as mean ± SEM.
a dialysis treatment, their pre-HD levels correlated strongly with patient dialysis vintage. In addition, the levels of lateresponse cytokines IL-10 and IL-6 were inversely related to delivered eqKt/V, suggesting that higher delivered doses of dialysis may reduce baseline chronic IL-10 and IL-6 levels. Although dialyzer membrane composition is known to affect the inflammatory response8,9 and the PMMA membranes lead to higher b-2 microglobulin clearance and less cytokine activation than previous generation cuprophane or cellulose acetate membranes, it was not the purpose of this study to evaluate the relative biocompatibility of PMMA membranes. Such analysis should be the subject of future study to assess the impact of various different biocompatible membranes on chronic inflammation in children receiving hemodialysis. Our study suggests that the hemodialysis treatment causes a transient increase in only an acute inflammatory 656
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response. The chronic inflammatory state observed in our patients probably is due to chronic uremia. The extremely elevated CRP levels further demonstrate a chronic inflammatory state in a majority of patients. Interpretation of our results is limited by the fact that we measured cytokine levels around only a single hemodialysis treatment. It is possible that patient cytokine levels might vary from treatment to treatment, but since none of the patients had fever or other evidence of active infection and since we observed chronic inflammation in the majority of patients, our data may suggest that factors other than the acute inflammatory response caused by the hemodialysis treatment lead to the chronic inflammation observed. Further study is necessary to assess the relations between inflammation and cardiovascular disease and to evaluate the effects of potential therapeutic options, which include antiinflammatory agents, daily hemodialysis, and chronic The Journal of Pediatrics November 2003
1. US renal data System. Excerpts for the USRDS 2001 Annual Data Report. Am J Kidney Dis 2001;38(Suppl):S1-248. 2. Sarnak MJ, Levey AS. Cardiovascular disease and maintenance renal disease: a new paradigm. Am J Kidney Dis 2000;35(Suppl 1):S117-31. 3. Wang AY, Woo J, Wang M, Sea MM, Ip R, Li PK, et al. Association of inflammation and malnutrition with cardiac valve calcification in continuous ambulatory peritoneal dialysis patients. J Am Soc Nephrol 2001;12:1927-36. 4. Kim PK, Deutschman CS. Inflammatory responses and mediators. Surg Clin North Am 2000;80:885-94. 5. Dinarello CA. Pro-inflammatory cytokines. Chest 2000;118:503-8. 6. Brivet FG, Emilie D, Galanaud P. Pro- and anti-inflammatory cytokines during acute severe pancreatitis: an early and sustained response, although unpredictable of death. Crit Care Med 1999;27:749-55. 7. Gloor B, Uhl W, Tcholakov O, Roggo A, Muller CA, Worni M, et al. Hydrocortisone treatment of early SIRS in acute experimental pancreatitis. Dig Dis Sci 2001;456:2154-61. 8. Horl WH. Hemodialysis membranes: Interleukins, biocompatibility, and middle molecules. J Am Soc Nephrol 2002;13(Suppl 1):S62-71. 9. Herbelin A, Nguyen AT, Zingraff J, Urena P, Descamps-Latscha B. Influence of uremia and hemodialysis on circulating interleukin-1 and tumor necrosis factor alpha. Kidney Int 1990;37:116-25. 10. Girndt M, Kaul H, Sester U, Ulrich C, Sester M, Georg T, et al. Antiinflammatory interleukin-10 genotype protects dialysis patients from cardiovascular events. Kidney Int 2002;62:949-55. 11. Nitta K, Akiba T, Kawashima A, Kimata N, Miwa N, Nishida E, et al. Characterization of TH1/TH2 profile in uremic patients. Nephron 2002; 91:492-5.
12. Herbelin A, Urena P, Nguyen AT, Zingraff J, Descamps-Latscha B. Elevated circulating levels of interleiukin-6 in patients with chronic renal failure. Kidney Int 1991;39:954-60. 13. Zwolinska D, Medynska A, Szprynger K, Szczepanska M. Serum concentration of IL-2, IL-6, TNF-a and their soluble receptors in children on chronic hemodialysis. Nephron 2000;86:441-6. 14. Neu AM, Fivush BA, Lederman HM. Cytokine production by peripheral blood mononuclear cells from pediatric chronic peritoneal dialysis patients. Pediatr Nephrol 1995;9:333-6. 15. Besbas N, Ozdemir S, Saatci U, Coskun T, Ozen S, Topaloglu R, et al. Nutritional assessment of children on haemodialysis: value of IGF-I, TNFalpha and IL-1beta. Nephrol Dial Transplant 1994;13:1484-8. 16. Sebekova K, Podracka L, Heidland A, Schinzel R. Enhanced plasma levels of advanced glycation end products (AGE) and pro-inflammatory cytokines in children/adolescents with chronic renal insufficiency and after renal replacement therapy by dialysis and transplantation: are they interrelated? Clin Nephrol 2001;56:S21-26. 17. Goldstein SL, Brewer ED. Logarithmic extrapolation of a 15-minute postdialysis BUN to predict equilibrated BUN and calculate double-pool Kt/ V in the pediatric hemodialysis population. Am J Kidney Dis 2000;36: 98-104. 18. Sylvester FA, Wyzga N, Hyams JS, Gronowicz GA. Effect of Crohn’s disease on bone metabolism in vitro: a role for Interleukin-6. J Bone Miner Res 2002;17:695-702. 19. Katayama K, Matsubara T, Fujiwara M, Koga M, Furukawa S. CD14+ CD16+ monocyte subpopulation in Kawasaki disease. Clin Exp Immunol 2000;121:566-70. 20. Panichi V, Migliori M, De Pietro S, Taccola D, Bianchi AM, Norpoth M, et al. C reactive protein in patients with chronic renal diseases. Ren Fail 2001;23:551-62. 21. Bolton CH, Downs LG, Victory JG, Dwight JF, Tomson CR, Mackness MI, et al. Endothelial dysfunction in chronic renal failure: roles of lipoprotein oxidation and pro-inflammatory cytokines. Nephrol Dial Transplant 2001;6:1189-97.
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hemodiafiltration on chronic inflammation in children with ESRD. We thank David Marsh for his support of the project.
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