Plasma D-asparagine and the D/L-serine ratio reflect chronic kidney diseases in children regardless of physique

Abstract

Biomarkers that accurately reflect renal function are essential in management of chronic kidney diseases (CKD). However, in children, age/physique and medication often alter established renal biomarkers. We studied whether amino acid enantiomers in body fluids correlate with renal function and whether they are influenced by physique or steroid medication during development. We conducted a prospective study of children 2 to 18 years old with and without CKD. We analyzed associations of serine/asparagine enantiomers in body fluids with major biochemical parameters as well as physique. To study consequences of kidney dysfunction and steroids on serine/asparagine enantiomers, we generated juvenile mice with uninephrectomy, ischemic reperfusion injury, or dexamethasone treatment. We obtained samples from 27 children, of which 12 had CKD due to congenital (n = 7) and perinatal (n = 5) causes. Plasma D-asparagine and the D/L-serine ratio had robust, positive linear associations with serum creatinine and cystatin C, and detected CKD with high sensitivity and specificity, uninfluenced by body size or biochemical parameters. In the animal study, kidney dysfunction increased plasma D-asparagine and the D/L-serine ratio, but dexamethasone treatment did not. Thus, plasma D-asparagine and the D/L-serine ratio can be useful markers for renal function in children.

Keywords: d-amino acids; Body size; Clinical laboratory test; Juvenile kidney function.

Fig. 1

Correlations of plasma amino acid enantiomers with biochemical parameters in children with CKD and controls. (a, b) Heatmaps indicate Spearman’s correlation coefficient between amino acid enantiomers and major biochemical parameters (a), or between amino acid enantiomers (b) in plasma of children with CKD and controls. WBC, white blood cells; Hb, hemoglobin; Plt, platelets; Alb, albumin; BUN, blood urea nitrogen; Cre, creatinine; UA, uric acid; Glu, glucose. (c, d) Linear regressions between d-asparagine and d-serine (c) or between l-asparagine and l-serine (d) in the plasma are shown. Closed circles, CKD (n = 12); open, Control (n = 15)

Fig. 2

Plasma d-amino acids and d/l-amino acid ratios correlate with existing renal markers and can detect pediatric CKD. (a-d) Simple linear regression shows associations of plasma d-asparagine (a), d-serine (b), d/l-asparagine (c), and d/l-serine (d) with serum Cre and CysC in children with CKD (closed circles, n = 12) and controls (open circles, n = 15). (e) The true/false positive rates to detect pediatric CKD at each threshold setting for d-asparagine (left), d/l-serine ratio (left), creatinine (right), and CysC (right) are plotted. Values in the plots show cut offs (specificity, sensitivity). (f) FEs of amino acids based on Cre clearance are shown. The dotted line is the FE of inulin, based on Cre in adults

Fig. 3

Correlations of urinary amino acid enantiomers with kidney markers in children with CKD, and controls. (a, b) Heatmaps indicate Spearman’s correlation coefficient between amino acid enantiomers and major kidney parameters (a), or between amino acid enantiomers (b) in urine of children with CKD and in controls. Renal parameters are urinary, unless noted as ‘serum.’ NAG, N-acetyl-beta-d-glucosaminidse. (c, d) Simple linear regression shows associations of urinary d-asparagine (c) or d-serine (d) with eGFR_Cre and eGFR_CysC in children with CKD (closed circles, n = 12) and in controls (open circles, n = 15)

Fig. 4

Correlations of plasma/urinary amino acid enantiomers with age or body size in non-CKD children. (a) Heatmap shows Spearman’s correlation coefficient between plasma/urinary amino acid enantiomers or existing kidney parameters and age / body size in control children (n = 15). BW, body weight; BSA, body surface area; BMI, body mass index. (b-e) Simple linear regression shows associations of plasma/urinary d-asparagine (b, d) or d-serine (c, e) with heights of control children (n = 15)

Fig. 5

In mice, kidney dysfunction elevates plasma d-asparagine and the d/l-serine ratio, but dexamethasone does not. (a) Sham-operation (SO) twice for the SO group, UN/SO for the UN group, and UN/IRI for the IRI group was performed on C57BL6 mice. (b-e) Plasma Cre (b), CysC (c), d-asparagine (d), and d/l-serine ratio (e) in SO (n = 7), UN (n = 11), IRI mice (n = 8) were plotted. (f) C57BL6 mice were treated with Dex for three consecutive days. (g-j) Plasma Cre (g), CysC (h), d-asparagine (i), and d/l-serine ratio (j) in mice with vehicle (Veh, n = 10) and Dex (n = 10) treatment were plotted. Error bars, mean ± s.e.m. ***P < 0.001, analyzed with one-way ANOVA followed by Dunnett’s multiple comparisons test (b-e) or Mann Whitney U test (g-j). ‘ns’, not significant