Concentrations of representative uraemic toxins in a healthy versus non-dialysis chronic kidney disease paediatric population

Concentrations of representative uraemic toxins in a healthy versus non-dialysis chronic kidney... ABSTRACT Background Chronic kidney disease (CKD) in childhood is poorly explained by routine markers (e.g. urea and creatinine) and is better depicted in adults by other uraemic toxins. This study describes concentrations of representative uraemic toxins in non-dialysis CKD versus healthy children. Methods In 50 healthy children and 57 children with CKD Stages 1–5 [median estimated glomerular filtration rate 48 (25th-75th percentile 24–71) mL/min/1.73 m2; none on dialysis], serum concentrations of small solutes [symmetric and asymmetric dimethyl-arginine (SDMA and ADMA, respectively)], middle molecules [β2-microglobuline (β2M), complement factor D (CfD)] and protein-bound solutes [p-cresylglucuronide (pCG), hippuric acid (HA), indole-acetic acid (IAA), indoxyl sulphate (IxS), p-cresyl sulphate (pCS) and 3-carboxy-4-methyl-5-propyl-furanpropionic acid (CMPF)] were measured. Concentrations in the CKD group were expressed as z-score relative to controls and matched for age and gender. Results SDMA, CfD, β2M, IxS, pCS, IAA, CMPF and HA concentrations were higher in the overall CKD group compared with controls, ranging from 1.7 standard deviations (SD) for IAA and HA to 11.1 SD for SDMA. SDMA, CfD, β2M, IxS and CMPF in CKD Stages 1–2 with concentrations 4.8, 2.8, 4.5, 1.9 and 1.6 SD higher, respectively. In contrast, pCS, pCG and IAA concentrations were only higher than controls from CKD Stages 3–4 onwards, but only in CKD Stage 5 for ADMA and HA (z-score 2.6 and 20.2, respectively). Conclusions This is the first study to establish reference values for a wide range of uraemic toxins in non-dialysis CKD and healthy children. We observed an accumulation of multiple uraemic toxins, each with a particular retention profile according to the different CKD stages. child, chronic kidney disease, reference values, uraemic toxins INTRODUCTION Chronic kidney disease (CKD) affects 56–75 children per million of age-related population (pmarp) in Europe [1]. It is a complex multisystem disease that negatively impacts the quality of life (QoL) and lifespan of children [2–4]. As the kidney function of these children deteriorates, several uraemic toxins accumulate in their bodies. These uraemic toxins can be subdivided into three major classes according to their removal pattern during dialysis: (i) small water-soluble compounds (≤500 Da), which can be removed during dialysis by passive diffusion; (ii) middle molecules (>500 Da), which can be removed using high-flux dialyser membranes and/or by convective transport; and (iii) protein-bound compounds, which are poorly removed by dialysis as they are bound to protein [5, 6]. In the last decades, uraemic toxins were recognized to play a predominant role in the pathophysiology of CKD in adults. For example, cardiovascular toxicity was attributed to elevated concentrations of asymmetric dimethyl-arginine (ADMA), symmetric dimethyl-arginine (SDMA), p-cresyl sulphate (pCS) and indoxyl sulphate (IxS), both in vitro and in vivo (e.g. affecting leucocyte, endothelial and vascular smooth muscle cell function) [7–13]. Furthermore, in adults, concentrations of IxS and pCS have been associated with infection, faster progression of kidney dysfunction and overall mortality [13–15]. While our understanding of uraemic toxicity in adults improved substantially over the last decades, the impact of uraemic toxicity in children remains to be determined. Adult pathophysiological mechanisms cannot be extrapolated to children, as there are many differences in the aetiology of primary kidney disease and comorbidities (e.g. growth failure) [3, 16]. Furthermore, unique for the paediatric population is the contribution of maturation and development of all organ systems, e.g., neurocognitive function [3, 16]. Paediatric expert groups favour alternative dialysis regimens to target more efficient removal of these uraemic toxins, despite the lack of evidence on the impact of uraemic toxicity in children [17–20]. As a first essential step, efforts have to be made to obtain reference values for concentrations of representative uraemic toxins in children. Therefore we aimed to describe reference values of a set of representative uraemic toxins in a group of children with CKD Stages 1–5 (not on dialysis) and in a control group of healthy children. MATERIALS AND METHODS Patients, sampling and analysis Two groups of children (between 0 and 18 years of age) were prospectively included in this cross-sectional, multicentric study: (i) the healthy control group and (ii) the CKD Stages 1–5 group. Healthy children (n = 50) were recruited at the ambulatory surgical centre of the Ghent University Hospital (Ghent, Belgium) between November 2014 and July 2016. They were admitted for minor surgery such as restorative dental treatment (n = 14), tympanostomy tube insertion (n = 10), adenotonsillectomy and other otorhinolaryngology surgery (n = 6), phimosis correction (n = 6), plastic surgery (n = 3) and other (n = 11). Children with CKD Stages 1–5 (n = 57) were recruited from two paediatric nephrology departments in Belgium (Ghent University Hospital and University Hospital Antwerp) between August 2014 and June 2016. CKD was defined according to the Kidney Disease: Improving Global Outcomes guidelines as an abnormality of the kidney structure or function present for ≥3 months and with implications on health. They were classified in different stages (1–5) according to their estimated glomerular filtration rate (eGFR), determined by the updated Schwartz equation [21]. None of the children were treated with any type of dialysis. All samples were drawn during a stable disease status; children with active inflammatory disease or malignancy were excluded. The study protocol was approved by the Ethical Committee and written informed consent was obtained from each parent or caregiver and from all patients who were ≥12 years of age (B670201524922, B670201422206). One blood sample from every healthy child was withdrawn immediately after induction of anaesthesia (inhalation of sevoflurane) during intravenous line placement in young children and preoperatively during intravenous line placement prior to intravenous anaesthesia induction in adolescents. One blood sample from every child with CKD Stages 1–5 was withdrawn during routine ambulatory visits. Consequently, blood samples were centrifuged [2095g, 10 min, 4°C), aliquoted and stored at −80°C until batch analysis. Concentration quantification was performed for small water-soluble solutes (creatinine, SDMA and ADMA), middle molecules [β2-microglobuline (β2M), complement factor D (CfD)] and protein-bound solutes [p-cresylglucuronide (pCG), hippuric acid (HA), indole acetic acid (IAA), IxS, pCS, and 3-carboxy-4-methyl-5-propyl-furanpropionic acid (CMPF)]. Urinary protein, urinary α1-microglobuline, serum creatinine (enzymatic analysis) and serum protein concentrations were analysed at the Clinical Laboratory at Ghent University Hospital, using standard laboratory methods. The protein-bound solutes were determined by reverse-phase high-performance liquid chromatography (HPLC) using an Alliance 2695 device (Waters, Zellik, Belgium). HA and CMPF were detected with a Waters 996 photodiode array detector at 245 nm and 254 nm, respectively. IxS (λex: 272 nm, λem: 374 nm), pCS and pCG (λex: 264 nm, λem: 290 nm), IAA (λex: 272 nm, λem: 340 nm) and the internal standard fluorescein (λex: 443 nm, λem: 512 nm) were detected by a Waters 2475 fluorescence detector. Plasma concentrations of the following solutes were determined by enzyme-linked immunosorbent assays (ELISAs): SDMA and ADMA competitive ELISA (DLD Diagnostika, Hamburg, Germany), CfD sandwich ELISA (R&D Systems, Abingdon, UK) and β2M sandwich ELISA (ORGENTEC Diagnostika, Mainz, Germany). ELISAs were used according to the manufacturer’s guidelines. ELISAs were analysed using the EL808 Ultra Microplate Reader from Bio-Tek Instruments (Winooski, VT, USA) using KC4 version 3.0 analysis software (Bio-Tek Instruments). Statistical analysis To determine the appropriate number of cases per age category, a power calculation was performed prior to the study. Sample size was estimated using the means and SDs of uraemic concentrations in CKD and healthy adults as published by Duranton et al. (2012) [22]. Performing the Mann–Whitney U-test on assumed normal distributions in the healthy and CKD groups with a significance level of α = 0.05, a minimum sample size of 6–12 in each age category was necessary to reach a power of 80% (SAS Power and Sample Size software, SAS Institute, Cary, NC, USA). Continuous variables were summarized as mean ± SD if normally distributed, otherwise median value with interquartile range (IQR) was reported. Categorical variables were expressed as frequencies and percentages. Percentages of protein binding were calculated using the total and free determined fraction of protein-bound toxin concentrations. The influence of sex on uraemic toxin concentrations was evaluated by a Mann–Whitney U-test. Pearson (r) or Spearman’s rho (rs) correlation coefficients, as appropriate, were calculated to correlate uraemic toxins with age. Uraemic toxin concentrations of the CKD Stages 1–5 group were expressed as a z-score (for child i, zi  =  (xi – xc-)/sdxc, where xi is the concentration of toxin in child i, xc- is the average toxin level in the control group and sdxc is the SD of uraemic toxin in the control group). The z-scores are always relative to the healthy control group and can be interpreted in terms of SD away from the average toxin level in the control group. More concretely, the z-score of a child in CKD Stages 1–5 denotes how the concentrations are related to the control group. For the uraemic toxins that were found to vary with age, z-scores were calculated for three age categories: 0–6 years, 6–12 years and 12–18 years. Mann–Whitney U-test or independent sample t-test, as appropriate, was performed to compare the uraemic toxin concentrations (z-scores) between the non-dialysis CKD and healthy groups and between the different CKD stages. Bonferroni correction was applied to deal with multiple testing. A P-value < 0.05 was considered statistically significant. All statistical analyses were performed using R version 3.1.1 (R Project for Statistical Computing, Vienna, Austria) [23]. RESULTS Study population The characteristics of 50 healthy children and 57 CKD Stages 1–5 children are presented in Table 1. There were slightly more males in the CKD Stages 1–5 group compared with the healthy control group. The median eGFR in the CKD Stages 1–5 group was 48 (25th–75th percentile 24–71) mL/min/1.73 m2 compared with 137 (119–159) in the healthy control group. None of the children were treated with dialysis. Of the children with CKD Stage 1–5, 33.3% were diagnosed with CKD Stages 1–2, 36.8% with Stage 3, 15.8% with Stage 4 and 14.0% with Stage 5. The majority of cases involved congenital anomalies of the kidney and urinary tract (CAKUT) (54.4%). Table 1 Patient characteristics of the healthy and CKD Stages 1–5 children (overall and per CKD stage) [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  Data are median (25th; 75th percentile), or number (percentage) as appropriate. eGFR according to Schwartz et al. [21]. CAKUT, congenital anomalies of the kidney and urinary tract. Table 1 Patient characteristics of the healthy and CKD Stages 1–5 children (overall and per CKD stage) [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  Data are median (25th; 75th percentile), or number (percentage) as appropriate. eGFR according to Schwartz et al. [21]. CAKUT, congenital anomalies of the kidney and urinary tract. Reference values of uraemic toxins in healthy children, stratified by age and sex Table 2 summarizes the mean concentration of each evaluated uraemic toxin in the control group. Only the total fraction of the protein-bound toxins, including the percentage of protein binding, is presented in Table 2. The protein binding varied from 17% for pCG to 100% for CMPF. For none of the studied uraemic toxins was a difference in concentration observed between males and females (Table 2). Age was found to correlate with CfD (rs = 0.532, P < 0.001) and HA (rs = −0.434, P = 0.002). Table 2 Reference values, stratified by age and sex, of uraemic toxin concentrations in the healthy children group and compared with previously published studies in adults and paediatrics [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  Data are median (25th–75th percentile) or mean ± SD as appropriate. Additionally, previously published adult and paediatric reference values and the degree of protein binding (%) are shown as Spearman (rs) or Pearson (r) correlation coefficients, as appropriate, and are only displayed there was if significant correlation between age/sex and uraemic toxins. MW, molecular weight; ND, no data; NS, not significant. a Only mean concentrations from previously published studies are displayed. Table 2 Reference values, stratified by age and sex, of uraemic toxin concentrations in the healthy children group and compared with previously published studies in adults and paediatrics [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  Data are median (25th–75th percentile) or mean ± SD as appropriate. Additionally, previously published adult and paediatric reference values and the degree of protein binding (%) are shown as Spearman (rs) or Pearson (r) correlation coefficients, as appropriate, and are only displayed there was if significant correlation between age/sex and uraemic toxins. MW, molecular weight; ND, no data; NS, not significant. a Only mean concentrations from previously published studies are displayed. Uraemic toxin concentrations in children with CKD Stages 1–5 The concentrations of the studied uraemic toxins are outlined in Table 3 and illustrated per uraemic toxin class in Figures 1–3. SDMA, CfD, β2M, IxS, pCS, IAA, CMPF and HA concentrations were higher in the overall CKD Stages 1–5 group compared with the control group, with concentrations from 1.7 SD higher for IAA (25th–75th percentile 0.3–3.1) and HA (−0.1–5.3) to 11.1 SD (5.2–20.7) higher for SDMA (all P < 0.05; see Table 3). Table 3 Creatinine and studied uraemic toxin concentrations (in z-score) in the overall paediatric CKD Stages 1–5 group and according to the different CKD stages [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  The uraemic toxin concentrations were expressed for each participant as a z-score relative to our control population, using age categories as appropriate. Data are median (25th–75th percentiles) or number (percentage). Bonferroni correction was applied to address multiple testing. * P < 0.05 versus Stages 1–2. + P < 0.05 versus stage 3. • P < 0.05 versus healthy children. Table 3 Creatinine and studied uraemic toxin concentrations (in z-score) in the overall paediatric CKD Stages 1–5 group and according to the different CKD stages [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  The uraemic toxin concentrations were expressed for each participant as a z-score relative to our control population, using age categories as appropriate. Data are median (25th–75th percentiles) or number (percentage). Bonferroni correction was applied to address multiple testing. * P < 0.05 versus Stages 1–2. + P < 0.05 versus stage 3. • P < 0.05 versus healthy children. FIGURE 1 View largeDownload slide Creatinine and water-soluble uraemic toxin concentrations in a non-dialysis paediatric CKD population according to CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). Significantly elevated ADMA concentrations were only found for CKD Stage 5 (P = 0.04). All box plots of SDMA were significantly elevated compared with the healthy controls (all P < 0.001). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 1 View largeDownload slide Creatinine and water-soluble uraemic toxin concentrations in a non-dialysis paediatric CKD population according to CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). Significantly elevated ADMA concentrations were only found for CKD Stage 5 (P = 0.04). All box plots of SDMA were significantly elevated compared with the healthy controls (all P < 0.001). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 2 View largeDownload slide Middle molecule concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group. *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 2 View largeDownload slide Middle molecule concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group. *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 3 View largeDownload slide Protein-bound uraemic toxin concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 3 View largeDownload slide Protein-bound uraemic toxin concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. The degree of accumulation of the evaluated uraemic toxins according to CKD stages was very different for each solute. SDMA, CfD, β2M, IxS and CMPF accumulated in the very early stages of CKD: median concentrations in children with CKD Stages 1–2 were 4.8 (25th–75th percentile 3.5–7.0), 2.8 (1.8–6.2), 4.5 (2.4–5.9), 1.9 (0.0–4.6), and 1.6 (0.0–3.7) SD higher than in healthy children, respectively. The median of the normalized SDMA [z = 11.1 (5.2–20.7)] and CfD [z = 9.5 (3.9–21.2)] concentrations in children with CKD Stages 1–2 were even higher than those of creatinine [z = 8.9 (25th–75th percentile 4.1–21.7)]. In the cohort with different CKD stages, the median concentrations of SDMA, CfD, β2M and IxS further increased to 33.2 (25th–75th percentile 23.4–50.1), 36.1 (28.3–42.0), 48.1 (32.3–65.1) and 32.2 (21.1–52.9) SD relative to the controls, respectively. In contrast, CMPF concentrations were equally elevated in the different stages of CKD (P > 0.05 when comparing concentrations with Stages 1–2 and 3). In contrast, pCS, pCG and IAA concentrations were only higher than the control group in CKD Stages 3, 4 and 5. Additionally, ADMA and HA concentrations were only increased in the cohort with end-stage renal disease (CKD Stage 5; z=2.6 (25th–75th percentile −0.1–3.7) and 20.2 (14.3–21.6), respectively. DISCUSSION The main contribution of the present study is the reporting of reference values of a representative set of uraemic toxins in a healthy paediatric population stratified by age and sex. In addition to these reference values, we report concentrations of uraemic toxins in a cohort of paediatric patients with non-dialysis CKD Stages 1–5. First, our report presents reference values for uraemic toxins in a large group of healthy children covering all age categories. In the paediatric setting, it is of utmost importance to include a sufficient subset of children of all age categories, because there might be an association between normal values and age, as is the case, for example, for creatinine. Our results indicate that such an association is present for CfD and HA but not for the other evaluated uraemic toxins. The age dependency of HA may reflect changes in fruits and vegetables consumption with age, as HA is produced by the liver from aromatic compounds (phenolic acids and hydroxycinnates) derived from dietary sources of polyphenol [41, 42]. The age independency of ADMA diverges from the observations by Lücke et al. [25], who included both children and adults and found a decrease of ADMA from 1.0 µmol/L in infancy to ∼0.4 µmol/L in adolescence. The results of Lücke et al., correspond with those of other reports in young healthy adolescents and young healthy adults [26, 27]. We hypothesize that the lack of association between age and ADMA concentrations is because of the predominance of younger children and the absence of adolescents >18 years of age in our healthy paediatric population. Compared with healthy adults, we observed similar concentrations for SDMA, IxS, pCS, IAA and β2M in our control group [22]. In contrast, lower concentrations compared with adults were found for CfD, CMPF, HA and pCG [22]. For CfD, this correlates with the increase in CfD concentrations according to age, as we observed in the current study, and the lower concentrations demonstrated in previous paediatric studies [22, 28, 29]. As mentioned earlier, the lower concentrations of CMPF and HA may be attributable to differences in dietary intake in different age groups, as, for example, strong correlations of CMPF with intake of fatty fish were described [43]. Our study is the first to present reference values for pCG, IAA, CMPF and HA in healthy children. Therefore, no comparison for these substances to other reports can be made. Although the number of publications for the paediatric population are scant, the concentrations of ADMA, SDMA and β2M concentrations observed in our control group are consistent with previously published mean/median concentrations in healthy children [25–27, 30–39]. In contrast, we observed higher CfD concentrations than two previous studies in a paediatric population [28, 29]. The higher CfD concentrations in the current study may reflect a difference in age distributions among studies, as especially younger children (all <5–10 years) were included in the previously reported studies, which fits with the increased CfD concentrations according to age demonstrated in the current study [28, 29]. We also found higher concentrations of IxS and lower concentrations of pCS compared with one small study evaluating 16 healthy children [40]. Second, our report describes concentrations of uraemic toxins in children with mild to severe CKD. To the best of our knowledge, this is the first study determining pCG, HA, IAA, IxS, pCS and CMPF concentrations in children with CKD Stages 1–5. We demonstrate that serum concentrations of IxS and CMPF were increased already in patients with early stages of CKD, and this is in line with observations in adults [13, 22, 44]. CMPF concentrations were equally elevated in CKD Stages 1–4, with a sharp further increase in the CKD Stage 5 cohort. In contrast, pCS, pCG, IAA and HA concentrations appeared to be elevated only in patients with more advanced CKD. This pattern was comparable to previously published adult studies for IAA, but we found that pCS and pCG were increased already in children with more limited CKD, whereas in adults, pCS concentrations were only elevated in patients with CKD Stage 5–5D and pCG concentrations even only in haemodialysis patients [14, 22, 45]. The increase of pCS and pCG in patients with earlier stages of CKD in this paediatric population may be attributable to differences in diet, microbiome or metabolism. Additional studies will be necessary to explore the underlying mechanisms. In contrast, few paediatric studies have been published for ADMA, SDMA, CfD and β2M. For ADMA, we did not observe elevated concentrations in the overall CKD group compared with the healthy group, which is in accordance with other paediatric studies but in contrast with two recent studies reporting elevated concentrations [26, 32, 34, 39]. A similar discrepancy in ADMA concentrations between studies is present in reports of adult CKD populations; these differences were explained by the participants, e.g. kidney function and underlying primary kidney disease [46–49]. We observed an elevation in ADMA concentrations in more advanced CKD stages, in line with other observations and several studies in adults [32, 39, 50]. Moreover, differences in ADMA accumulation patterns according to underlying kidney disease are plausible, since impaired ADMA degradation by the renal dimethylarginine dimethylamino hydrolase (DDAH) enzyme (abundant in endothelial and renal tubular cells) rather than reduced renal filtration plays a central role in the accumulation of ADMA in CKD [46–49]. For SDMA, β2M and CfD, we found an increase in concentrations in patients with minor renal insufficiency, with a further exponential increase according to CKD stage, which dovetails nicely with previously published studies in adults and children [27, 30, 51–57]. It is worthwhile noting that the normalized difference in SDMA and CfD concentrations between healthy children and those with CKD Stages 1–2 was even greater than that for creatinine. These molecules might thus be more sensitive predictors of an early decrease in GFR rather than creatinine [27, 52]. Without doubt, clinical uraemic toxicity is even more complex, as only a small selection of uraemic toxins are presented here and a host of retained compounds remain unidentified [5]. Nevertheless, the uraemic toxins described in this article are the state of the art to represent uraemic toxicity and can be used to model their removal during dialysis. However, further studies are needed to correlate serum concentrations of these toxins with hard clinical outcomes to determine which ones do really contribute to the toxicity of the uraemic syndrome. The paediatric setting is ideally suited for this goal, as in these patients several hard outcomes related to uraemic toxicity, such as growth and cognitive functioning, can be easily followed over time. In conclusion, this study reported reference values for a set of representative uraemic toxins of different classes in a healthy population. The complexity of uraemic retention in childhood is clearly illustrated by the variability in the accumulation patterns of the different uraemic toxins in this study [5]. Understanding the complexity of uraemic retention, especially in the paediatric population, is necessary since childhood CKD is known to differ from adult CKD in terms of aetiology, comorbidities and organ maturation [3]. Consequently, this article is a first step towards constructing a paediatric reference frame of uraemic toxins in healthy and CKD children to ensure the use of biologically relevant uraemic toxin concentrations in experimental settings in the future. ACKNOWLEDGEMENTS The authors are indebted to our laboratory staff—Sophie Lobbestael, Tom Mertens and Maria Van Landschoot—for their technical support. FUNDING This study was funded by the Agency for Innovation by Science and Technology (IWT) from the ‘Applied Biomedical Research with a Primary Societal Goal’ (TBM) programme in Flanders (Belgium) (UToPaed project, grant number IWT-TBM 150195). AUTHORS’ CONTRIBUTIONS E.S. provided substantial contributions to the conception and design; the acquisition, analysis and interpretation of data; drafted the work and approved the final version and agreed to be accountable for all aspects of the work. W.V.B. provided substantial contributions to the conception and design; interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. A.R., J.V.W and S.E. provided substantial contributions to the conception and design, interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. G.G. provided substantial contributions to the conception and design; analysis and interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. V.V.B., K.V.H. and M.C. provided substantial contributions to the acquisition of data; critically revised the intellectual content of the work and approved the final version and agreed to be accountable for all aspects of the work. S.R. provided substantial contributions to the analysis and interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. CONFLICT OF INTEREST STATEMENT W.V.B. received lecture fees, travel and grant support from Fresenius Medical Care, Baxter Gambro, Leo Pharma and Astellas. A.R. received lecture fees and travel support from Ferring Pharmaceuticals. G.G. received lecture fees from Fresenius Medical Care and Baxter and travel support from Baxter. J.V.W. served on paid advisory boards for Alexion, Astellas and Ferring Pharmaceuticals for the last 2 years and received lecture fees from Alexion, Astellas and Ferring Pharmaceuticals. S.E. received lecture fees and travel support from Fresenius Medical Care. The other authors have no conflicts of interest to declare. REFERENCES 1 Harambat J, van Stralen KJ, Kim JJ et al.  . Epidemiology of chronic kidney disease in children. Pediatr Nephrol  2012; 27: 363– 373 Google Scholar CrossRef Search ADS PubMed  2 McDonald SP, Craig JC, Australian et al.  . Long-term survival of children with end-stage renal disease. N Engl J Med  2004; 350: 2654– 2662 Google Scholar CrossRef Search ADS PubMed  3 Kaspar CD, Bholah R, Bunchman TE. A review of pediatric chronic kidney disease. Blood Purif  2016; 41: 211– 217 Google Scholar CrossRef Search ADS PubMed  4 Shroff R, Ledermann S. 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Serum level of complement factor D in systemic lupus erythematosus—an indicator of glomerular filtration rate. Acta Med Scand  1984; 216: 171– 177 Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

Concentrations of representative uraemic toxins in a healthy versus non-dialysis chronic kidney disease paediatric population

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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0931-0509
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1460-2385
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10.1093/ndt/gfx224
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Abstract

ABSTRACT Background Chronic kidney disease (CKD) in childhood is poorly explained by routine markers (e.g. urea and creatinine) and is better depicted in adults by other uraemic toxins. This study describes concentrations of representative uraemic toxins in non-dialysis CKD versus healthy children. Methods In 50 healthy children and 57 children with CKD Stages 1–5 [median estimated glomerular filtration rate 48 (25th-75th percentile 24–71) mL/min/1.73 m2; none on dialysis], serum concentrations of small solutes [symmetric and asymmetric dimethyl-arginine (SDMA and ADMA, respectively)], middle molecules [β2-microglobuline (β2M), complement factor D (CfD)] and protein-bound solutes [p-cresylglucuronide (pCG), hippuric acid (HA), indole-acetic acid (IAA), indoxyl sulphate (IxS), p-cresyl sulphate (pCS) and 3-carboxy-4-methyl-5-propyl-furanpropionic acid (CMPF)] were measured. Concentrations in the CKD group were expressed as z-score relative to controls and matched for age and gender. Results SDMA, CfD, β2M, IxS, pCS, IAA, CMPF and HA concentrations were higher in the overall CKD group compared with controls, ranging from 1.7 standard deviations (SD) for IAA and HA to 11.1 SD for SDMA. SDMA, CfD, β2M, IxS and CMPF in CKD Stages 1–2 with concentrations 4.8, 2.8, 4.5, 1.9 and 1.6 SD higher, respectively. In contrast, pCS, pCG and IAA concentrations were only higher than controls from CKD Stages 3–4 onwards, but only in CKD Stage 5 for ADMA and HA (z-score 2.6 and 20.2, respectively). Conclusions This is the first study to establish reference values for a wide range of uraemic toxins in non-dialysis CKD and healthy children. We observed an accumulation of multiple uraemic toxins, each with a particular retention profile according to the different CKD stages. child, chronic kidney disease, reference values, uraemic toxins INTRODUCTION Chronic kidney disease (CKD) affects 56–75 children per million of age-related population (pmarp) in Europe [1]. It is a complex multisystem disease that negatively impacts the quality of life (QoL) and lifespan of children [2–4]. As the kidney function of these children deteriorates, several uraemic toxins accumulate in their bodies. These uraemic toxins can be subdivided into three major classes according to their removal pattern during dialysis: (i) small water-soluble compounds (≤500 Da), which can be removed during dialysis by passive diffusion; (ii) middle molecules (>500 Da), which can be removed using high-flux dialyser membranes and/or by convective transport; and (iii) protein-bound compounds, which are poorly removed by dialysis as they are bound to protein [5, 6]. In the last decades, uraemic toxins were recognized to play a predominant role in the pathophysiology of CKD in adults. For example, cardiovascular toxicity was attributed to elevated concentrations of asymmetric dimethyl-arginine (ADMA), symmetric dimethyl-arginine (SDMA), p-cresyl sulphate (pCS) and indoxyl sulphate (IxS), both in vitro and in vivo (e.g. affecting leucocyte, endothelial and vascular smooth muscle cell function) [7–13]. Furthermore, in adults, concentrations of IxS and pCS have been associated with infection, faster progression of kidney dysfunction and overall mortality [13–15]. While our understanding of uraemic toxicity in adults improved substantially over the last decades, the impact of uraemic toxicity in children remains to be determined. Adult pathophysiological mechanisms cannot be extrapolated to children, as there are many differences in the aetiology of primary kidney disease and comorbidities (e.g. growth failure) [3, 16]. Furthermore, unique for the paediatric population is the contribution of maturation and development of all organ systems, e.g., neurocognitive function [3, 16]. Paediatric expert groups favour alternative dialysis regimens to target more efficient removal of these uraemic toxins, despite the lack of evidence on the impact of uraemic toxicity in children [17–20]. As a first essential step, efforts have to be made to obtain reference values for concentrations of representative uraemic toxins in children. Therefore we aimed to describe reference values of a set of representative uraemic toxins in a group of children with CKD Stages 1–5 (not on dialysis) and in a control group of healthy children. MATERIALS AND METHODS Patients, sampling and analysis Two groups of children (between 0 and 18 years of age) were prospectively included in this cross-sectional, multicentric study: (i) the healthy control group and (ii) the CKD Stages 1–5 group. Healthy children (n = 50) were recruited at the ambulatory surgical centre of the Ghent University Hospital (Ghent, Belgium) between November 2014 and July 2016. They were admitted for minor surgery such as restorative dental treatment (n = 14), tympanostomy tube insertion (n = 10), adenotonsillectomy and other otorhinolaryngology surgery (n = 6), phimosis correction (n = 6), plastic surgery (n = 3) and other (n = 11). Children with CKD Stages 1–5 (n = 57) were recruited from two paediatric nephrology departments in Belgium (Ghent University Hospital and University Hospital Antwerp) between August 2014 and June 2016. CKD was defined according to the Kidney Disease: Improving Global Outcomes guidelines as an abnormality of the kidney structure or function present for ≥3 months and with implications on health. They were classified in different stages (1–5) according to their estimated glomerular filtration rate (eGFR), determined by the updated Schwartz equation [21]. None of the children were treated with any type of dialysis. All samples were drawn during a stable disease status; children with active inflammatory disease or malignancy were excluded. The study protocol was approved by the Ethical Committee and written informed consent was obtained from each parent or caregiver and from all patients who were ≥12 years of age (B670201524922, B670201422206). One blood sample from every healthy child was withdrawn immediately after induction of anaesthesia (inhalation of sevoflurane) during intravenous line placement in young children and preoperatively during intravenous line placement prior to intravenous anaesthesia induction in adolescents. One blood sample from every child with CKD Stages 1–5 was withdrawn during routine ambulatory visits. Consequently, blood samples were centrifuged [2095g, 10 min, 4°C), aliquoted and stored at −80°C until batch analysis. Concentration quantification was performed for small water-soluble solutes (creatinine, SDMA and ADMA), middle molecules [β2-microglobuline (β2M), complement factor D (CfD)] and protein-bound solutes [p-cresylglucuronide (pCG), hippuric acid (HA), indole acetic acid (IAA), IxS, pCS, and 3-carboxy-4-methyl-5-propyl-furanpropionic acid (CMPF)]. Urinary protein, urinary α1-microglobuline, serum creatinine (enzymatic analysis) and serum protein concentrations were analysed at the Clinical Laboratory at Ghent University Hospital, using standard laboratory methods. The protein-bound solutes were determined by reverse-phase high-performance liquid chromatography (HPLC) using an Alliance 2695 device (Waters, Zellik, Belgium). HA and CMPF were detected with a Waters 996 photodiode array detector at 245 nm and 254 nm, respectively. IxS (λex: 272 nm, λem: 374 nm), pCS and pCG (λex: 264 nm, λem: 290 nm), IAA (λex: 272 nm, λem: 340 nm) and the internal standard fluorescein (λex: 443 nm, λem: 512 nm) were detected by a Waters 2475 fluorescence detector. Plasma concentrations of the following solutes were determined by enzyme-linked immunosorbent assays (ELISAs): SDMA and ADMA competitive ELISA (DLD Diagnostika, Hamburg, Germany), CfD sandwich ELISA (R&D Systems, Abingdon, UK) and β2M sandwich ELISA (ORGENTEC Diagnostika, Mainz, Germany). ELISAs were used according to the manufacturer’s guidelines. ELISAs were analysed using the EL808 Ultra Microplate Reader from Bio-Tek Instruments (Winooski, VT, USA) using KC4 version 3.0 analysis software (Bio-Tek Instruments). Statistical analysis To determine the appropriate number of cases per age category, a power calculation was performed prior to the study. Sample size was estimated using the means and SDs of uraemic concentrations in CKD and healthy adults as published by Duranton et al. (2012) [22]. Performing the Mann–Whitney U-test on assumed normal distributions in the healthy and CKD groups with a significance level of α = 0.05, a minimum sample size of 6–12 in each age category was necessary to reach a power of 80% (SAS Power and Sample Size software, SAS Institute, Cary, NC, USA). Continuous variables were summarized as mean ± SD if normally distributed, otherwise median value with interquartile range (IQR) was reported. Categorical variables were expressed as frequencies and percentages. Percentages of protein binding were calculated using the total and free determined fraction of protein-bound toxin concentrations. The influence of sex on uraemic toxin concentrations was evaluated by a Mann–Whitney U-test. Pearson (r) or Spearman’s rho (rs) correlation coefficients, as appropriate, were calculated to correlate uraemic toxins with age. Uraemic toxin concentrations of the CKD Stages 1–5 group were expressed as a z-score (for child i, zi  =  (xi – xc-)/sdxc, where xi is the concentration of toxin in child i, xc- is the average toxin level in the control group and sdxc is the SD of uraemic toxin in the control group). The z-scores are always relative to the healthy control group and can be interpreted in terms of SD away from the average toxin level in the control group. More concretely, the z-score of a child in CKD Stages 1–5 denotes how the concentrations are related to the control group. For the uraemic toxins that were found to vary with age, z-scores were calculated for three age categories: 0–6 years, 6–12 years and 12–18 years. Mann–Whitney U-test or independent sample t-test, as appropriate, was performed to compare the uraemic toxin concentrations (z-scores) between the non-dialysis CKD and healthy groups and between the different CKD stages. Bonferroni correction was applied to deal with multiple testing. A P-value < 0.05 was considered statistically significant. All statistical analyses were performed using R version 3.1.1 (R Project for Statistical Computing, Vienna, Austria) [23]. RESULTS Study population The characteristics of 50 healthy children and 57 CKD Stages 1–5 children are presented in Table 1. There were slightly more males in the CKD Stages 1–5 group compared with the healthy control group. The median eGFR in the CKD Stages 1–5 group was 48 (25th–75th percentile 24–71) mL/min/1.73 m2 compared with 137 (119–159) in the healthy control group. None of the children were treated with dialysis. Of the children with CKD Stage 1–5, 33.3% were diagnosed with CKD Stages 1–2, 36.8% with Stage 3, 15.8% with Stage 4 and 14.0% with Stage 5. The majority of cases involved congenital anomalies of the kidney and urinary tract (CAKUT) (54.4%). Table 1 Patient characteristics of the healthy and CKD Stages 1–5 children (overall and per CKD stage) [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  Data are median (25th; 75th percentile), or number (percentage) as appropriate. eGFR according to Schwartz et al. [21]. CAKUT, congenital anomalies of the kidney and urinary tract. Table 1 Patient characteristics of the healthy and CKD Stages 1–5 children (overall and per CKD stage) [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  [AuthorQuery id="AQ9" rid="9"]?>Patient Characteristics  Healthy  Overall CKD Stages 1–5  Stages 1–2 (≥60 mL/min/ 1.73 m2)  Stage 3  (30–59 mL/min/ 1.73m2)  Stage 4 (15–29mL/min/ 1.73 m2)  Stage 5 (<15 mL/min/ 1.73 m2)  Number (%)  50  57  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Age (years)  6.7 (4.2–9.8)  8.8 (5.1–14.7)  6.0 (4.9–11.0)  14.4 (9.8–16.0)  7.5 (3.8–11.4)  7.6 (1.3–14.5)  Age, categories (%)               < 6 years  20 (40)  19 (33)  9 (47)  4 (20)  3 (33)  3 (38)   6–12 years  23 (46)  17 (30)  6 (32)  4 (20)  4 (44)  3 (38)   >12 years  7 (14)  21 (37)  4 (21)  12 (60)  2 (22)  2 (25)  Boys (%)  29 (58)  44 (77)  13 (68)  18 (86)  7 (78)  6 (75)  eGFR (mL/min/1.73 m2)  137 (119–159)  48 (24–71)  74 (67–103)  47 (35–55)  20 (18–26)  11 (9–13)  Primary CKD diagnosis  /             Glomerular    7 (12.3)  3 (15.8)  2 (9.5)  1 (11.1)  1 (12.5)   CAKUT    31 (54.4)  7 (36.9)  12 (57.1)  6 (66.7)  6 (75.0)   Cystic disease    4 (7.0)  1 (5.3)  2 (9.5)  1 (11.1)  0 (0.0)   Other non-glomerular    15 (26.3)  8 (42.1)  5 (23.8)  1 (11.1)  1 (12.5)  Serum total protein (g/L)  66 (63–70)  70 (65–73)  68 (65–71)  72 (67–77)  72 (68–74)  65 (60–70)  Glomerular proteinuria  /  26 (45.6)  5 (26.3)  10 (45.5)  5 (55.6)  8 (100)  Tubular proteinuria  /  31 (54.4)  5 (26.3)  10 (47.6)  9 (100)  9 (100)  Medication use  /             Phosphate binder    12 (21.1)  0 (0.0)  4 (19.0)  4 (44.4)  4 (50.0)   Cholecalciferol    28 (49.1)  11 (57.9)  8 (38.1)  4 (44.4)  5 (62.5)   Alfacalcidol    29 (50.9)  3 (15.8)  11 (52.4)  8 (88.9)  7 (87.5)   Antihypertensive agent    29 (50.9)  8 (42.1)  10 (47.6)  6 (66.7)  5 (62.5)   Immune suppression    5 (8.8)  2 (10.5)  3 (14.3)  0 (0.0)  0 (0.0)   Erythropoietin    11 (19.3)  0 (0.0)  3 (14.3)  3 (33.3)  5 (75.0)   Growth hormone    7 (12.3)  0 (0.0)  2 (9.5)  3 (33.3)  2 (25.0)   Steroids    2 (3.5)  0 (0.0)  2 (9.5)  0 (0.0)  0 (0.0)  Data are median (25th; 75th percentile), or number (percentage) as appropriate. eGFR according to Schwartz et al. [21]. CAKUT, congenital anomalies of the kidney and urinary tract. Reference values of uraemic toxins in healthy children, stratified by age and sex Table 2 summarizes the mean concentration of each evaluated uraemic toxin in the control group. Only the total fraction of the protein-bound toxins, including the percentage of protein binding, is presented in Table 2. The protein binding varied from 17% for pCG to 100% for CMPF. For none of the studied uraemic toxins was a difference in concentration observed between males and females (Table 2). Age was found to correlate with CfD (rs = 0.532, P < 0.001) and HA (rs = −0.434, P = 0.002). Table 2 Reference values, stratified by age and sex, of uraemic toxin concentrations in the healthy children group and compared with previously published studies in adults and paediatrics [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  Data are median (25th–75th percentile) or mean ± SD as appropriate. Additionally, previously published adult and paediatric reference values and the degree of protein binding (%) are shown as Spearman (rs) or Pearson (r) correlation coefficients, as appropriate, and are only displayed there was if significant correlation between age/sex and uraemic toxins. MW, molecular weight; ND, no data; NS, not significant. a Only mean concentrations from previously published studies are displayed. Table 2 Reference values, stratified by age and sex, of uraemic toxin concentrations in the healthy children group and compared with previously published studies in adults and paediatrics [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  [AuthorQuery id="AQ9" rid="9"]?>    Current study   Previously published studies   Uraemic toxins  MW (Da)  Protein binding (%)  Concentration  Age (r or rs)  Sex  Adults [22, 24]  Paediatrics [25–29, 30–40]  Water-soluble toxins                 Creatinine, mg/dL  113  –  0.39 ± 0.16  0.867  NS  –  –   SDMA, µmol/L  202  –  0.64 ± 0.08  NS  NS  0.38–1.10a  0.37–1.18a   ADMA, µmol/L  202  –  0.67 ± 0.11  NS  NS  0.43 ± 0.7  0.57–0.78a  Middle molecules                 CfD, µg/mL  23 750  –  1.71 ± 0.43  0.532  NS  1.90 ± 0.50  0.74–1.17a   β2M, µg/mL  11 818  –  1.74 ± 0.34  NS  NS  1.90 ± 1.60  1.45–1.78a  Protein-bound toxins                 pCG total, mg/dL  284  17 (0–31)  0.006 ± 0.005  NS  NS  0.035 ± 0.003  ND   HA total, mg/dL  179  64 (53–70)  0.044 ± 0.037  −0.434  NS  0.300 ± 0.200  ND   IAA total, mg/dL  175  90 (88–94)  0.023 ± 0.010  NS  NS  0.050 ± 0.030  ND   IxS total, mg/dL  213  94 (89–99)  0.056 ± 0.025  NS  NS  0.053 ± 0.029  0.174 ± 0.140   pCS total, mg/dL  188  95 (91–98)  0.244 ± 0.179  NS  NS  0.190 ± 0.130  0.060 ± 0.027   CMPF total, mg/dL  240  ∼100  0.010 ± 0.012  NS  NS  0.360 ± 0.020  ND  Data are median (25th–75th percentile) or mean ± SD as appropriate. Additionally, previously published adult and paediatric reference values and the degree of protein binding (%) are shown as Spearman (rs) or Pearson (r) correlation coefficients, as appropriate, and are only displayed there was if significant correlation between age/sex and uraemic toxins. MW, molecular weight; ND, no data; NS, not significant. a Only mean concentrations from previously published studies are displayed. Uraemic toxin concentrations in children with CKD Stages 1–5 The concentrations of the studied uraemic toxins are outlined in Table 3 and illustrated per uraemic toxin class in Figures 1–3. SDMA, CfD, β2M, IxS, pCS, IAA, CMPF and HA concentrations were higher in the overall CKD Stages 1–5 group compared with the control group, with concentrations from 1.7 SD higher for IAA (25th–75th percentile 0.3–3.1) and HA (−0.1–5.3) to 11.1 SD (5.2–20.7) higher for SDMA (all P < 0.05; see Table 3). Table 3 Creatinine and studied uraemic toxin concentrations (in z-score) in the overall paediatric CKD Stages 1–5 group and according to the different CKD stages [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  The uraemic toxin concentrations were expressed for each participant as a z-score relative to our control population, using age categories as appropriate. Data are median (25th–75th percentiles) or number (percentage). Bonferroni correction was applied to address multiple testing. * P < 0.05 versus Stages 1–2. + P < 0.05 versus stage 3. • P < 0.05 versus healthy children. Table 3 Creatinine and studied uraemic toxin concentrations (in z-score) in the overall paediatric CKD Stages 1–5 group and according to the different CKD stages [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  [AuthorQuery id="AQ9" rid="9"]?>Uraemic toxins  Overall  Stages 1–2 (≥ 60 mL /min/ 1.73 m2)  Stage 3  (30–59 mL /min/ 1.73 m2)  Stage 4  (15–29 mL /min/ 1.73 m2)  Stage 5 (<15 mL /min/ 1.73 m2)  Number (%)  57 (100)  19 (33.3)  21 (36.8)  9 (15.8)  8 (14.0)  Water-soluble molecules             Creatinine, z-score  8.9 (4.1–21.7)•  2.8 (1.3–5.4)•  8.9 (7.2–12.1)*,•  24.4 (20.5–31.5)*,+,•  47.9 (41.1–67.2)*,+,•   SDMA, z-score  11.1 (5.2–20.7)•  4.8 (3.5–7.0)•  10.7 (6.5–13.6)*,•  25.7 (18.3–29.6)*,+,•  33.2 (23.4–50.1)*,+,•   ADMA, z-score  −0.2 (−0.7–1.2)  −0.3 (−0.9–1.1)  −0.5 (−0.9–0.7)  1.6 (−0.3–2.3)  2.6 (−0.1–3.7)*,•  Middle molecules             CfD, z-score  9.5 (3.9–21.2)•  2.8 (1.8–6.2)•  9.5 (5.7–11.9)*,•  22.8 (16.2–28.4)*,+,•  36.1 (28.3–42.0)*,+,•   B2M, z-score  8.6 (4.7–18.1)•  4.5 (2.4, 5.9)•  9.5 (5.3–12.9)*,•  18.7 (16.7–23.8)*,+,•  48.1 (32.3–65.1)*,+,•  Protein-bound toxins             pCG, z-score  0.5 (−0.8–3.3)  0.1 (−1.0–1.3)  −0.5 (−1.0–1.0)  3.8 (1.8–9.8)*,+,•  6.6 (1.0–49.2)+,•   HA, z-score  1.7 (−0.1–5.3)•  0.3 (−1.0–1.0)  2.0 (0.0–4.6)•  2.0 (0.3–13.7)  20.2 (14.3–21.6)*,+,•   IAA, z-score  1.7 (0.3–3.1)•  0.4 (−0.6–2.2)  1.7 (0.4–2.7)•  1.4 (0.6–2.8)•  5.2 (3.5–16.9)*,+,•   IxS, z-score  5.0 (1.7–16.3)•  1.9 (0.0–4.6)•  2.4 (1.7–7.6)•  17.8 (13.5–24.4)*,+,•  32.2 (21.1–52.9)*,+,•   pCS, z-score  2.7 (0.5–5.5)•  0.7 (−0.4–3.2)  2.1 (0.5–3.8)•  7.5 (4.7–10.0)*,+,•  7.8 (1.9–19.8)•   CMPF, z-score  2.1 (0.3–5.3)•  1.6 (0.0–3.7)•  2.9 (0.2–5.3)•  2.0 (0.9–3.6)•  5.7 (4.0–13.6)•  The uraemic toxin concentrations were expressed for each participant as a z-score relative to our control population, using age categories as appropriate. Data are median (25th–75th percentiles) or number (percentage). Bonferroni correction was applied to address multiple testing. * P < 0.05 versus Stages 1–2. + P < 0.05 versus stage 3. • P < 0.05 versus healthy children. FIGURE 1 View largeDownload slide Creatinine and water-soluble uraemic toxin concentrations in a non-dialysis paediatric CKD population according to CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). Significantly elevated ADMA concentrations were only found for CKD Stage 5 (P = 0.04). All box plots of SDMA were significantly elevated compared with the healthy controls (all P < 0.001). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 1 View largeDownload slide Creatinine and water-soluble uraemic toxin concentrations in a non-dialysis paediatric CKD population according to CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). Significantly elevated ADMA concentrations were only found for CKD Stage 5 (P = 0.04). All box plots of SDMA were significantly elevated compared with the healthy controls (all P < 0.001). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 2 View largeDownload slide Middle molecule concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group. *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 2 View largeDownload slide Middle molecule concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group. *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 3 View largeDownload slide Protein-bound uraemic toxin concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. FIGURE 3 View largeDownload slide Protein-bound uraemic toxin concentrations in a non-dialysis paediatric CKD population according to the different CKD stages. The concentrations are presented as z-scores compared with the control group (z = 0). *P < 0.05 compared with CKD Stages 1–2; +P < 0.05 compared with CKD Stage 3. The degree of accumulation of the evaluated uraemic toxins according to CKD stages was very different for each solute. SDMA, CfD, β2M, IxS and CMPF accumulated in the very early stages of CKD: median concentrations in children with CKD Stages 1–2 were 4.8 (25th–75th percentile 3.5–7.0), 2.8 (1.8–6.2), 4.5 (2.4–5.9), 1.9 (0.0–4.6), and 1.6 (0.0–3.7) SD higher than in healthy children, respectively. The median of the normalized SDMA [z = 11.1 (5.2–20.7)] and CfD [z = 9.5 (3.9–21.2)] concentrations in children with CKD Stages 1–2 were even higher than those of creatinine [z = 8.9 (25th–75th percentile 4.1–21.7)]. In the cohort with different CKD stages, the median concentrations of SDMA, CfD, β2M and IxS further increased to 33.2 (25th–75th percentile 23.4–50.1), 36.1 (28.3–42.0), 48.1 (32.3–65.1) and 32.2 (21.1–52.9) SD relative to the controls, respectively. In contrast, CMPF concentrations were equally elevated in the different stages of CKD (P > 0.05 when comparing concentrations with Stages 1–2 and 3). In contrast, pCS, pCG and IAA concentrations were only higher than the control group in CKD Stages 3, 4 and 5. Additionally, ADMA and HA concentrations were only increased in the cohort with end-stage renal disease (CKD Stage 5; z=2.6 (25th–75th percentile −0.1–3.7) and 20.2 (14.3–21.6), respectively. DISCUSSION The main contribution of the present study is the reporting of reference values of a representative set of uraemic toxins in a healthy paediatric population stratified by age and sex. In addition to these reference values, we report concentrations of uraemic toxins in a cohort of paediatric patients with non-dialysis CKD Stages 1–5. First, our report presents reference values for uraemic toxins in a large group of healthy children covering all age categories. In the paediatric setting, it is of utmost importance to include a sufficient subset of children of all age categories, because there might be an association between normal values and age, as is the case, for example, for creatinine. Our results indicate that such an association is present for CfD and HA but not for the other evaluated uraemic toxins. The age dependency of HA may reflect changes in fruits and vegetables consumption with age, as HA is produced by the liver from aromatic compounds (phenolic acids and hydroxycinnates) derived from dietary sources of polyphenol [41, 42]. The age independency of ADMA diverges from the observations by Lücke et al. [25], who included both children and adults and found a decrease of ADMA from 1.0 µmol/L in infancy to ∼0.4 µmol/L in adolescence. The results of Lücke et al., correspond with those of other reports in young healthy adolescents and young healthy adults [26, 27]. We hypothesize that the lack of association between age and ADMA concentrations is because of the predominance of younger children and the absence of adolescents >18 years of age in our healthy paediatric population. Compared with healthy adults, we observed similar concentrations for SDMA, IxS, pCS, IAA and β2M in our control group [22]. In contrast, lower concentrations compared with adults were found for CfD, CMPF, HA and pCG [22]. For CfD, this correlates with the increase in CfD concentrations according to age, as we observed in the current study, and the lower concentrations demonstrated in previous paediatric studies [22, 28, 29]. As mentioned earlier, the lower concentrations of CMPF and HA may be attributable to differences in dietary intake in different age groups, as, for example, strong correlations of CMPF with intake of fatty fish were described [43]. Our study is the first to present reference values for pCG, IAA, CMPF and HA in healthy children. Therefore, no comparison for these substances to other reports can be made. Although the number of publications for the paediatric population are scant, the concentrations of ADMA, SDMA and β2M concentrations observed in our control group are consistent with previously published mean/median concentrations in healthy children [25–27, 30–39]. In contrast, we observed higher CfD concentrations than two previous studies in a paediatric population [28, 29]. The higher CfD concentrations in the current study may reflect a difference in age distributions among studies, as especially younger children (all <5–10 years) were included in the previously reported studies, which fits with the increased CfD concentrations according to age demonstrated in the current study [28, 29]. We also found higher concentrations of IxS and lower concentrations of pCS compared with one small study evaluating 16 healthy children [40]. Second, our report describes concentrations of uraemic toxins in children with mild to severe CKD. To the best of our knowledge, this is the first study determining pCG, HA, IAA, IxS, pCS and CMPF concentrations in children with CKD Stages 1–5. We demonstrate that serum concentrations of IxS and CMPF were increased already in patients with early stages of CKD, and this is in line with observations in adults [13, 22, 44]. CMPF concentrations were equally elevated in CKD Stages 1–4, with a sharp further increase in the CKD Stage 5 cohort. In contrast, pCS, pCG, IAA and HA concentrations appeared to be elevated only in patients with more advanced CKD. This pattern was comparable to previously published adult studies for IAA, but we found that pCS and pCG were increased already in children with more limited CKD, whereas in adults, pCS concentrations were only elevated in patients with CKD Stage 5–5D and pCG concentrations even only in haemodialysis patients [14, 22, 45]. The increase of pCS and pCG in patients with earlier stages of CKD in this paediatric population may be attributable to differences in diet, microbiome or metabolism. Additional studies will be necessary to explore the underlying mechanisms. In contrast, few paediatric studies have been published for ADMA, SDMA, CfD and β2M. For ADMA, we did not observe elevated concentrations in the overall CKD group compared with the healthy group, which is in accordance with other paediatric studies but in contrast with two recent studies reporting elevated concentrations [26, 32, 34, 39]. A similar discrepancy in ADMA concentrations between studies is present in reports of adult CKD populations; these differences were explained by the participants, e.g. kidney function and underlying primary kidney disease [46–49]. We observed an elevation in ADMA concentrations in more advanced CKD stages, in line with other observations and several studies in adults [32, 39, 50]. Moreover, differences in ADMA accumulation patterns according to underlying kidney disease are plausible, since impaired ADMA degradation by the renal dimethylarginine dimethylamino hydrolase (DDAH) enzyme (abundant in endothelial and renal tubular cells) rather than reduced renal filtration plays a central role in the accumulation of ADMA in CKD [46–49]. For SDMA, β2M and CfD, we found an increase in concentrations in patients with minor renal insufficiency, with a further exponential increase according to CKD stage, which dovetails nicely with previously published studies in adults and children [27, 30, 51–57]. It is worthwhile noting that the normalized difference in SDMA and CfD concentrations between healthy children and those with CKD Stages 1–2 was even greater than that for creatinine. These molecules might thus be more sensitive predictors of an early decrease in GFR rather than creatinine [27, 52]. Without doubt, clinical uraemic toxicity is even more complex, as only a small selection of uraemic toxins are presented here and a host of retained compounds remain unidentified [5]. Nevertheless, the uraemic toxins described in this article are the state of the art to represent uraemic toxicity and can be used to model their removal during dialysis. However, further studies are needed to correlate serum concentrations of these toxins with hard clinical outcomes to determine which ones do really contribute to the toxicity of the uraemic syndrome. The paediatric setting is ideally suited for this goal, as in these patients several hard outcomes related to uraemic toxicity, such as growth and cognitive functioning, can be easily followed over time. In conclusion, this study reported reference values for a set of representative uraemic toxins of different classes in a healthy population. The complexity of uraemic retention in childhood is clearly illustrated by the variability in the accumulation patterns of the different uraemic toxins in this study [5]. Understanding the complexity of uraemic retention, especially in the paediatric population, is necessary since childhood CKD is known to differ from adult CKD in terms of aetiology, comorbidities and organ maturation [3]. Consequently, this article is a first step towards constructing a paediatric reference frame of uraemic toxins in healthy and CKD children to ensure the use of biologically relevant uraemic toxin concentrations in experimental settings in the future. ACKNOWLEDGEMENTS The authors are indebted to our laboratory staff—Sophie Lobbestael, Tom Mertens and Maria Van Landschoot—for their technical support. FUNDING This study was funded by the Agency for Innovation by Science and Technology (IWT) from the ‘Applied Biomedical Research with a Primary Societal Goal’ (TBM) programme in Flanders (Belgium) (UToPaed project, grant number IWT-TBM 150195). AUTHORS’ CONTRIBUTIONS E.S. provided substantial contributions to the conception and design; the acquisition, analysis and interpretation of data; drafted the work and approved the final version and agreed to be accountable for all aspects of the work. W.V.B. provided substantial contributions to the conception and design; interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. A.R., J.V.W and S.E. provided substantial contributions to the conception and design, interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. G.G. provided substantial contributions to the conception and design; analysis and interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. V.V.B., K.V.H. and M.C. provided substantial contributions to the acquisition of data; critically revised the intellectual content of the work and approved the final version and agreed to be accountable for all aspects of the work. S.R. provided substantial contributions to the analysis and interpretation of data; drafted and critically revised the intellectual content and approved the final version and agreed to be accountable for all aspects of the work. CONFLICT OF INTEREST STATEMENT W.V.B. received lecture fees, travel and grant support from Fresenius Medical Care, Baxter Gambro, Leo Pharma and Astellas. A.R. received lecture fees and travel support from Ferring Pharmaceuticals. G.G. received lecture fees from Fresenius Medical Care and Baxter and travel support from Baxter. J.V.W. served on paid advisory boards for Alexion, Astellas and Ferring Pharmaceuticals for the last 2 years and received lecture fees from Alexion, Astellas and Ferring Pharmaceuticals. S.E. received lecture fees and travel support from Fresenius Medical Care. The other authors have no conflicts of interest to declare. REFERENCES 1 Harambat J, van Stralen KJ, Kim JJ et al.  . Epidemiology of chronic kidney disease in children. Pediatr Nephrol  2012; 27: 363– 373 Google Scholar CrossRef Search ADS PubMed  2 McDonald SP, Craig JC, Australian et al.  . Long-term survival of children with end-stage renal disease. N Engl J Med  2004; 350: 2654– 2662 Google Scholar CrossRef Search ADS PubMed  3 Kaspar CD, Bholah R, Bunchman TE. A review of pediatric chronic kidney disease. Blood Purif  2016; 41: 211– 217 Google Scholar CrossRef Search ADS PubMed  4 Shroff R, Ledermann S. 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Nephrology Dialysis TransplantationOxford University Press

Published: Jul 24, 2017

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