Ischemic Acute Kidney Injury Perturbs Homeostasis of Serine Enantiomers in the Body Fluid in Mice: Early Detection of Renal Dysfunction Using the Ratio of Serine Enantiomers

<div><p>The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D−/L-serine from 2.82±0.18 to 1.10±0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D−/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D−/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.</p></div

IRI inverts D−/L-serine ratio in urine.

<p>Urinary serine enantiomers were analyzed using 2D-HPLC. (A) Typical chromatograms showing urinary D−/L-serine in C57BL/6J wild-type mice with or without renal IRI. (B-G) Concentrations of D-serine (B), L-serine (C), creatinine (D), KIM-1 (F), and NGAL (G), and ratios of D−/L-serine (E) in the urine of the wild-type mice were determined (Sham, n = 7; IRI 4, n = 5; IRI 8, n = 5; IRI 20, n = 5; and IRI 40, n = 5). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). NS means ‘not significant’. (H-J) Concentrations of D-serine (H) and L-serine (I), and D−/L-serine ratios (J) in the urine of DAO-null mice were determined. *<i>P</i><0.05, ***<i>P</i><0.001 (two-tailed Student’s <i>t</i> test). Data are plotted as the mean ± SEM.</p

Renal IRI increases D-serine and reduces L-serine in mouse serum.

<p>(A) Shown are typical chromatograms of serum D−/L-serine obtained by 2D-HPLC. [Mice with sham-op (Sham); and those at 4, 8, 20, and 40 h after renal IRI (IRI 4, IRI 8, IRI 20, and IRI 40)] (B-F) Concentrations of serum D-serine (B), L-serine (C), creatinine (E), and cystatin C (F) in the C57BL/6J mice were determined, and ratios of D-serine to L-serine concentrations were calculated (D) (Sham, n = 8; IRI 4, n = 5; IRI 8, n = 9; IRI 20, n = 6; and IRI 40, n = 7). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). NS means ‘not significant’. Data are plotted as the mean ± SEM.</p

Knockout of SR does not affect alterations of serine enantiomers after IRI.

<p>Concentrations of D-serine (A), L-serine (B), and D−/L-serine ratio (C) in the sera of SR-KO mice were determined using 2D-HPLC (Sham, n = 4; IRI 20, n = 4; and IRI 40, n = 4). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). Data are plotted as the mean ± SEM.</p

Lack of DAO activity exacerbates loss of intact proximal tubules and renal dysfunction induced by IRI.

<p>(A and C) Renal cortices in C57BL/6J wild-type (DAO^+/+) and DAO-null mice (DAO^−/−) after sham-op (Sham) or IRI (at 40 h after reperfusion) were stained with H & E (A) or LTL (C). (B) Damaged tubules were evaluated and ATN score was calculated in slices stained with H & E (n = 5, each group). (D) Number of intact proximal tubules was counted in slices stained with LTL (n = 5, each group). (E and F) Serum Cr (E) and BUN (F) in DAO^+/+ [Sham n = 5; IRI 20, n = 5; and IRI 40, n = 7] and DAO^−/− mice [Sham, n = 6; IRI 20, n = 5; and IRI 40, n = 6] were measured. *<i>P</i><0.05, **<i>P</i><0.01, and NS is ‘not significant’ (one-way ANOVA followed by Tukey’s multiple comparison test). Data are plotted as the mean ± SEM.</p

Lack of DAO activity suppresses IRI-induced accumulation of D-serine in serum.

<p>(A-C) The serine enantiomers in the sera of DAO-null mice were determined using 2D-HPLC, and their concentrations (D-serine, A; L-serine, B) and ratio (C) are shown (Sham, n = 6; IRI 20, n = 6; and IRI 40, n = 5). *<i>P</i><0.05, **<i>P</i><0.01 (one-way ANOVA followed by Tukey’s multiple comparison test). NS is ‘not significant’. Data are plotted as the mean ± SEM.</p

IRI reduces the number of proximal epithelial cells with DAO activity.

<p>(A and B) A horizontally sliced section of the kidney in a C57BL/6J wild-type mouse (A) and high magnifications of renal cortex in the mice (Sham or IRI 40) (B) were stained with DAO enzyme histochemistry, a proximal tubular marker (LTL), and a nuclear marker (DAPI). Scale bars, 200 µm. (C and D) DAO activity in the total kidneys of C57BL/6J wild-type mice [Sham, n = 4; IRI 20, n = 5; and IRI 40, n = 6] (C) and SR-KO mice [Sham, n = 3; IRI 20, n = 4; and IRI 40, n = 3] (D) was determined in a quantitative assay. *<i>P</i><0.05, **<i>P</i><0.01 (one-way ANOVA followed by Tukey’s multiple comparison test). Data are plotted as the mean ± SEM.</p