Original Paper Received: February 20, 2009 Accepted: May 18, 2009 Published online: August 15, 2009
Nephron Clin Pract 2009;113:c258–c261 DOI: 10.1159/000235250
Plasma Cell-Free DNA Levels in Children on Peritoneal Dialysis O. Ozkaya a K. Bek a A. Bedir b Y. Açıkgöz a T. Özdemir b Departments of a Pediatric Nephrology, and b Biochemistry, Ondokuz Mayıs University, School of Medicine, Samsun, Turkey
Key Words Cell-free DNA ⴢ Children ⴢ Peritoneal dialysis
Abstract Background/Aims: Plasma levels of cell-free DNA (cfDNA) are elevated in various clinical conditions including cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. Previously, increased cfDNA levels were reported during hemodialysis. However, there is limited data regarding cfDNA levels in peritoneal dialysis (PD) patients. The aim of this study was to investigate the levels of cfDNA in children on PD. Methods: Twenty-one children on PD (median age: 12; range: 4–18 years) and 21 healthy children (median age: 10; range: 6–16 years) were enrolled into the study. Plasma cfDNA was measured using a real-time quantitative PCR for the beta-globin gene. Results: The median concentrations of cfDNA in the plasma of PD patients and healthy controls were 2,205 genome-equivalents/ml of plasma (range: 39–5,845) and 1,033 genome-equivalents/ml of plasma (range: 254–5,116), respectively (p = 0.026). A significant positive correlation was observed between C-reactive protein levels and plasma cfDNA levels (r: 0.52, p ! 0.0001). Conclusion: Our data have demonstrated for the first time that cfDNA is increased in children on PD treatment. However, the mechanism by
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which the levels of cfDNA is increased and the clinical significance of this finding in PD patients is unclear. Further studies are warranted to clarify the precise mechanism and clinical significance of elevated cfDNA in children on PD. Copyright © 2009 S. Karger AG, Basel
Introduction
Cell-free plasma DNA (cfDNA) has been detected in small amounts in healthy individuals. In a healthy person, DNA probably enters the circulation via apoptosis of lymphocytes and other nucleated cells [1, 2]. Increased levels of cfDNA have been found in various clinical conditions including cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications [2–9]. This finding has led to the idea that circulating nucleic acid measurements can be used as a relatively less invasive, rapid, sensitive and accurate diagnostic tool in several diseases [2]. Previously, increased cfDNA levels were reported during hemodialysis (HD) [10–12]. However, there is limited data regarding cfDNA levels in peritoneal dialysis (PD) patients. The aim of this study was to investigate the levels of cfDNA in children on PD.
Ozan Ozkaya, Assoc. Prof. of Pediatrics Ondokuz Mayıs University School of Medicine Department of Pediatric Nephrology TR–55139 Samsun (Turkey) Tel. +90 362 312 1919, Fax +90 362 457 6041, E-Mail
[email protected]
Material and Methods Patients Twenty-one children on PD (F/M: 13/8; median age: 12; range 4–18 years) and 21 healthy children (F/M: 12/9; median age: 10; range 6–16 years) were enrolled in the study. The criteria for recruitment were as follows: (a) between 4 and 18 years, (b) at least 3 months of dialysis therapy. Patients were excluded from the study if they had one of the following conditions: (a) presence of any severe infection such as peritonitis, sepsis, exit-site infection for PD patients, or (b) presence of any disease which may affect liver function. The underlying renal diseases of the patients were as follows: reflux nephropathy (n = 8), posterior urethral valve (n = 2), hypoplastic kidneys (n = 1), nephrolithiasis (n = 1), Alport syndrome (n = 1), renal vein thrombosis (n = 1), unknown (n = 7). Patients were dialyzed using either Baxter Twin Bag (Baxter Healthcare SA, Castlebar, Ireland) or Fresenius ANDY Plus (Fresenius Medical Care GmbH, Bad Homburg, Germany) systems. The glucose concentration in dialysis solutions ranged from 1.36– 1.5 to 2.27–1.5%. Extraneal (7.5% icodextrin) and Nutrineal (1.1% amino-acid-containing solution) solutions were prescribed as needed. All of the patients were prescribed vitamin D analogues, phosphate binders and erythropoietin, according to suggested doses and the individual assessment of each patient. The present study was conducted after obtaining written informed consent and was approved by the Ethical Committee. Blood Sampling, Measurements All the samples in the patients and the control group were taken in the morning at 8.00 a.m. after an overnight fast and stored at –70 ° C until analyzed. Our current protocol, based on the study of Chiu et al. [13], involves initial centrifugation of blood samples at 1,600 g followed by a second centrifugation at 16,000 g. Peripheral blood was collected from each participant into EDTA tubes. Blood samples were centrifuged at 1,600 g for 10 min at 4 ° C, and plasma was carefully transferred into new tubes, followed by further centrifugation at 16,000 g at 4 ° C. The plasma samples were stored at –80 ° C until analysis. DNA was extracted from 600 l of plasma according to the protocol of the High Pure viral nucleic acid kit (Roche Diagnostics GmbH, Penzberg, Germany), and eluted in 100 l of H2O. We then subjected 10 l of the DNA to real-time quantitative PCR for the -globin gene as described in the protocol of LightCycler Control Kit DNA using LightCycler FastStart DNA Master HybProbe on LightCycler version 1.5 to amplify the 110-bp fragments. Amplification of target DNA was monitored using Hybridization Probes that hybridize to an internal sequence of the amplified fragment. Results were expressed as genomeequivalents/ml of plasma by use of the conversion factor of 3.3 pg of DNA. Statistical Methods Data are given as median (range). Statistical comparisons between the 2 groups were made with Mann-Whitney U tests. Correlations were calculated by using Spearman correlation. A p value ! 0.05 was considered significant.
Cell-Free DNA Levels in Peritoneal Dialysis
Results
The median age was 12 years (range: 4–18) and 10 years (range: 6–16) in the PD patients and control group, respectively (p 1 0.05). The median duration of dialysis was 26 months (range: 7–79). The median KT/V was 2.2 (range: 1.4–3.2). The median concentration of cfDNA in the plasma was 2,205 genome-equivalents/ml of plasma (range: 39–5,845) and 1,033 genome-equivalents/ml of plasma (range: 254–5,116) in PD patients and healthy control patients, respectively. There was a significant difference in the levels of cfDNA concentration between PD patients and healthy controls (p = 0.026) (fig. 1). C-reactive protein (CRP) levels were higher in PD patients (median: 3.5; range: 3–16.4 mg/l) than in controls (median: 2; range: 3–5 mg/l) (p ! 0.0001). In addition, a significant correlation was observed between CRP levels and cfDNA levels (r: 0.52, p ! 0.0001) (fig. 2). No correlation was found between cfDNA levels and KT/V. There was no significant correlation between duration of dialysis treatment and cfDNA levels. The patients were divided into groups according to the peritoneal equilibrium test classification: high transport rate group (n = 6), high-average transport rate group (n = 9), low-average transport rate group (n = 3), and low transport rate group (n = 3). No significant difference was found in the cfDNA levels among groups (p = 0.93).
Discussion
The mechanism by which RNA is released into the circulation is still not clear. Possible mechanisms suggested are an active process and a nonspecific release from apoptotic or necrotic cells [1, 2]. Previously, several studies reported increased plasma cfDNA levels during HD [10– 12]. Atamaniuk et al. [10] reported increases in cfDNA levels, anexin V expression, and 7-AAD uptake in leukocytes during HD. The authors concluded that the predominant portion of cfDNA in HD patients originates from apoptotic leukocytes. Our study demonstrated that cfDNA levels were significantly higher in patients on PD than in healthy controls. We evaluated, for the first time, the level of cfDNA in children on PD; therefore, we cannot compare our results with the studies carried out in adults. Also, there is limited data regarding the cfDNA levels in adult PD patients. Korabecna et al. [14] showed that plasma cfDNA levels of adult patients on HD and PD treatment taken prior to the dialysis procedures did not significantly differ from those of healthy volunteers. In Nephron Clin Pract 2009;113:c258–c261
c259
6,000.00
p = 0.026
* *
4,000.00
2,000.00
cfDNA concentration (genome-equivalents/ml of plasma)
cfDNA concentration (genome-equivalents/ml of plasma)
6,000.00
4,000.00
2,000.00
r: 0.52 p < 0.0001
0 0
n = 21 PD
n = 21
4.00
Control
Fig. 1. cfDNA levels in the PD and control groups.
8.00 12.00 CRP (mg/l)
16.00
Fig. 2. CRP levels and cfDNA levels in the PD and control
groups.
the same study, PD patients were divided into 2 groups according to the ratio of dialysate cfDNA to plasma cfDNA (D/P ratio). A subgroup of patients with a D/P ratio 11 had been on PD treatment for a shorter period of time than a subgroup with a D/P ratio !1. The authors concluded that the level of cfDNA in overnight effluent correlated inversely with the duration of PD treatment and these differences may include changes in the quality and quantity of the cell population of peritoneum [14]. One limitation of our study is that we did not study cfDNA levels of peritoneal effluent. Dynamics of interactions between cfDNA levels in peritoneal effluent and plasma during PD are not clear, necessitating further studies. In the literature, the size of genomic DNA sequences was reported to be different depending on the necrotic or apoptotic origin. While large DNA genomic sequences of 11,000 base pairs represent a necrotic origin, those within the range of 80–200 base pairs represent mainly an apoptotic origin [15, 16]. In our study, we analyzed 110-bp fragments, which are considered to be of apoptotic origin. Although the lack of analysis of other apoptotic markers is another limitation of our study, it can be speculated that the source of the cfDNA in our study might be apoptosis because of the size of genomic DNA sequences studied. Recent studies suggest that the state of uremia is associated with accelerated apoptosis of lymphocytes, monocytes and neutrophils [17–20]. Furthermore, casc260
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pase-dependent apoptosis of mononuclear cells, neutrophils and mesothelial cells induced by conventional glucose-containing PD solutions with a high glucose degradation product content have been reported [21–24]. Elevated cfDNA levels in our patients may be explained by the accelerated apoptosis previously reported in PD patients. Our data have demonstrated for the first time that cfDNA levels positively correlated with CRP. Since this is an observational study, and correlational data can not prove causality, it is difficult to clearly define the relationship between cfDNA and CRP based on these results. Uremia is a state of microinflammation, with elevated cytokine and positive acute-phase protein levels [25]. Our findings raise the possibility that the amount of free DNA released might depend on the extent of inflammation. Further in vivo and in vitro studies investigating the association of cfDNA with inflammation may contribute to a better understanding of this relationship. One can speculate that decreased clearance of DNA due to end-stage renal disease might have led to enhanced levels of cfDNA in our patients. The clearance mechanisms for circulating DNA are poorly understood. Botezatu et al. [26] show that ⬃0.5–2% of the free DNA is excreted in the urine. Therefore, it is not possible to explain increased cfDNA levels in our patients only with impaired renal clearance. Our data have demonstrated for the first time that cfDNA is increased in children on PD treatment. HowOzkaya /Bek /Bedir /Açıkgöz /Özdemir
ever, the mechanism by which the levels of cfDNA are increased and the clinical significance of this finding in PD patients is unclear. Further studies are warranted to clarify the precise mechanism and clinical significance of elevated cfDNA in children on PD.
Acknowledgement This study was supported by the Research Fund of Ondokuz Mayıs University (Grant Number: T-411).
References 1 Gahan PB, Swaminathan R: Circulating nucleic acids in plasma and serum. Recent developments. Ann N Y Acad Sci 2008; 1137: 1–6. 2 Swarup V, Rajeswari MR: Circulating (cellfree) nucleic acids – a promising, non-invasive tool for early detection of several human diseases. FEBS Lett 2007;581:795–799. 3 Shao ZM, Wu J, Shen ZZ, Nguyen M: p53 mutation in plasma DNA and its prognostic value in breast cancer patients. Clin Cancer Res 2001;8:3027. 4 Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, Lederrey C, Anker P: Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996;2:1033–1035. 5 Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA: Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000; 46:319–323. 6 Rainer TH, Wong LK, Lam W, Yuen E, Lam NY, Metreweli C, Lo YM: Prognostic use of circulating plasma nucleic acid concentrations in patients with acute stroke. Clin Chem 2003;49:562–569. 7 Chang CP, Chia RH, Wu TL, Tsao KC, Sun CF, Wu JT: Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin Chim Acta 2003;327:95–101. 8 Koffler D, Agnello V, Winchester R, Kunkel HG: The occurrence of single-stranded DNA in the serum of patients with systemic lupus erythematosus and other diseases. J Clin Invest 1973;52:198–204. 9 Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS: Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–487. 10 Atamaniuk J, Ruzicka K, Stuhlmeier KM, Karimi A, Eigner M, Mueller MM: Cell-free plasma DNA: a marker for apoptosis during hemodialysis. Clin Chem 2006;52:523–526.
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11 Steinman CR, Ackad A: Appearance of circulating DNA during hemodialysis. Am J Med 1977;62:693–697. 12 García Moreira V, de la Cera Martínez T, Gago González E, Prieto García B, Alvarez Menéndez FV: Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR. Clin Chem Lab Med 2006;44:1410–1415. 13 Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM: Effects of blood processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;47:1607–1613. 14 Korabecna M, Opatrna S, Wirth J, Rulcova K, Eiselt J, Sefrna F, Horinek A: Cell-free plasma DNA during peritoneal dialysis and hemodialysis and in patients with chronic kidney disease. Ann N Y Acad Sci 2008;1137: 296–301. 15 Rainer TH, Lam NY: Circulating nucleic acids and critical illness. Ann N Y Acad Sci 2006;1075:271–277. 16 Wang BG, Huang HY, Chen YC, Bristow RE, Kassauei K, Cheng CC, Roden R, Sokoll LJ, Chan DW, Shih IeM: Increased plasma DNA integrity in cancer patients. Cancer Res 2003;63:3966–3968. 17 Matsumoto Y, Shinzato T, Amano I, Takai I, Kimura Y, Morita H, Miwa M, Nakane K, Yoshikai Y, Maeda K: Relationship between susceptibility to apoptosis and Fas expression in peripheral blood T cells from uremic patients: a possible mechanism for lymphopenia in chronic renal failure. Biochem Biophys Res Commun 1995;215:98–105. 18 Heidenreich S, Schmidt M, Bachmann J, Harrach B: Apoptosis of monocytes cultured from long-term hemodialysis patients. Kidney Int 1996;49:792–799.
19 Cendoroglo M, Jaber BL, Balakrishnan VS, Perianayagam M, King AJ, Pereira BJ: Neutrophil apoptosis and dysfunction in uremia. J Am Soc Nephrol 1999; 10:93–100. 20 Jaber BL, Cendoroglo M, Balakrishnan VS, Perianayagam MC, King AJ, Pereira BJ: Apoptosis of leukocytes: basic concepts and implications in uremia. Kidney Int 2001; 78(suppl):197–205. 21 Catalán MP, Reyero A, Egido J, Ortiz A: Acceleration of neutrophil apoptosis by glucose-containing peritoneal dialysis solutions: role of caspases. J Am Soc Nephrol 2001;12:2442–2449. 22 Catalán M, Santamaría B, Reyero A, Ortiz A, Egido J, Ortiz A: 3,4-di-deoxyglucosone-3ene promotes leukocyte apoptosis. Kidney Int 2005;68:1303–1311. 23 Pulm J, Lordnejad MR, Grabensee B: Effect of alternative peritoneal dialysis solutions on cell viability, apoptosis/necrosis and cytokine expression in human monocytes. Kidney Int 1998;54:224–235. 24 Cendoroglo M, Sundaram S, Groves C, Ucci AA, Jaber BL, Pereira BJ: Necrosis and apoptosis of polymorphonuclear cells exposed to peritoneal dialysis fluids in vitro. Kidney Int 1997;52:1626–1634. 25 Herbelin A, Nguyen AT, Zingraff J, Urena P, Descamps-Latscha B: Influence of uremia and hemodialysis on circulating interleukin1 and tumor necrosis factor alpha. Kidney Int 1990;37:116–125. 26 Botezatu I, Serdyuk O, Potapova G, Shelepov V, Alechina R, Molyaka Y, Ananev V, Bazin I, Garin A, Narimanov M, Knysh V, Melkonyan H, Umansky S, Lichtenstein A: Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin Chem 2000;46:1078–1084.
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