d-Serine Mediates Cellular Proliferation for Kidney Remodeling

Abstract

Background: d-serine, a long-term undetected enantiomer of serine, is a biomarker that reflects kidney function and disease activity. The physiologic functions of d-serine are unclear.

Methods: The dynamics of d-serine were assessed by measuring d-serine in human samples of living kidney donors using two-dimensional high-performance liquid chromatography, and by autoradiographic studies in mice. The effects of d-serine on the kidney were examined by gene expression profiling and metabolic studies using unilateral nephrectomy mice, and genetically modified cells.

Results: Unilateral nephrectomy in human living kidney donors decreases urinary excretion and thus increases the blood level of d-serine. d-serine is quickly and dominantly distributed to the kidney on injection in mice, suggesting the kidney is a main target organ. Treatment of d-serine at a low dose promotes the enlargement of remnant kidney in mouse model. Mechanistically, d-serine activates the cell cycle for tissue remodeling through an mTOR-related pathway.

Conclusions: d-serine is a physiologic molecule that promotes kidney remodeling. Besides its function as a biomarker, d-serine has a physiologic activity that influences kidney function.

Keywords: basic science; biomarker; cell cycle; chronic kidney disease; d-serine; glomerular filtration rate; kidney remodeling; kidney transplantation; mTOR; physiological activity; renal-physiology; urinary excretion.

Graphical abstract

Figure 1.

Blood level of d-serine increases in living kidney donors after nephrectomy. (A–D) Inulin clearance (A), plasma levels of d- (B) and l-serine (C), and plasma ratio of d-serine to total serine (D) of living kidney donors were measured before (pre) and after nephrectomies (post). n=10; statistics, paired two-tailed Student’s t test. (E) Blood levels of d-serine were plotted with inulin clearance. n=20; statistics, Pearson’s correlation. LN, log-natural transformed. (F–H) Clearance of d- (F) and l-serine (G), and clearance ratio of d- over l-serine (H) of living kidney donors. n=10; statistics, paired two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001. d-Ser, d-serine; l-Ser, l-serine.

Figure 2.

d-serine accumulates in the kidney. (A) Standardized uptake values (SUV), corrected for body weight, were measured in each organ of 12-week-old Balb/c male mice at the indicated time points after the intravenous injection with ³H-labeled d- or l-serine. (B) Time course of SUV of each organ with ³H-labeled either d- or l-serine injections. n=4–5; statistics, two-way ANOVA (P<0.001 for interaction effect in kidney, blood, heart, pancreas, liver, and spleen, *P<0.05, **P<0.01, ***P<0.001; ***P<0.001 for main effect of chirality in lung and P<0.001 for main effect of time in lung). Data, mean±SEM.

Figure 3.

Blood level of d-serine increase in mice after unilateral nephrectomy. (A–D) 10-week-old mice were fed with a serine-free diet for 1 week, subjected to either unilateral nephrectomy (UNX) or sham operation, and then sacrificed 2 days after operation. Kidney per body weight (A), plasma ratio of d-serine to total serine (B), plasma levels of d- (C) and l-serine (D). n=6 (A) and 8 (B–D); statistics, unpaired two-tailed Student’s t test. **P<0.01. Data, mean±SEM.

Figure 4.

d-serine promotes kidney enlargement after unilateral nephrectomy. (A–E) Mice were fed with a serine-free diet and water with or without 0.1% d-serine for 1 week, subjected to either UNX or sham operation, and then sacrificed 2 days after operation. Kidney weight per body weight (A), plasma levels of d- (B), and l-serine (C), and plasma levels of urea nitrogen (D) and creatinine (E). n=6–7; statistics, two-way ANOVA, (A)*P<0.05 for main effect of d-serine; ***P<0.001 for main effect of operation; (B) P<0.05 for interaction effect; (C) *P<0.05 for main effect of operation; (D) *P<0.05 for main effect of operation. *P<0.05, **P<0.01, ***P<0.001. Data, mean±SEM.

Figure 5.

d-serine activates cell cycle for tissue remodeling. Mice were fed with a serine-free diet and water with or without 0.1% d-serine for 1 week, subjected to either UNX or sham operation, and then sacrificed 2 days after operation. (A and B) Analyses of Reactome pathway enrichment (A) and gene ontology terms (B) using RNA sequencing data of kidney cortexes from UNX mice treated with d-serine over vehicle-treated UNX mice (n=3). False discovery rate (FDR) was calculated using the Benjamini and Hochberg procedure. (C and D) Heatmaps of cyclins (C) and cell-division cycle (Cdc) (D) related genes. (E) Relative mRNA expressions of cell cycle–related genes. n=6–7; statistics, unpaired two-tailed Student’s t test. *P<0.05. Data, mean±SEM.

Figure 6.

d-serine promotes cellular proliferation in kidney. Mice were fed with a serine-free diet and water with or without 0.1% d-serine for 1 week, subjected to either UNX or sham operation, and then sacrificed 2 days after operation. (A) Representative images of kidney cortex stained with anti-Ki67 antibody. Bars, 50 μm. (B) Numbers of Ki67-positive nuclei in proximal tubules per field. n=6–7; statistics, two-way ANOVA (**P<0.01 for main effect of treatment and operation). (C) Representative images of immunofluorescence staining for Ki67 (red) in the lectin-positive proximal tubules (green) in kidney cortex sections. DAPI (blue) were stained for nuclei. Bars, 50 μm. Data, mean±SEM.

Figure 7.

Contents of d- and l-serine in reagents. (A) Concentration of amino acids in FCS (left) and dialyzed FCS (right). (B and C) Purity of d- (B) and l-serine (C). Levels of d- and l-serine were measured.

Figure 8.

d-serine activates cellular proliferation via an mTOR-related pathway. (A and B) Time course of relative growth of normal human RPTEC cells (A) and HK-2 cells (B) treated with or without 10 μM of d-serine in a serine-free medium. n=18; statistics, two-way ANOVA, (A) P<0.01 and (B) P<0.001 for interaction effect. (C) Western blots of phospho-S6K (p-S6K) at Thr389 from HK-2 cells that were treated with 5 μM of either d- or l-serine for 10 minutes in a serine-free medium. Representative images of five independent experiments and their quantification. RI, relative index; a.a., amino acids. Statistics, one-way ANOVA with Dunnett’s post-hoc test. (D) Immunoblots for phospho-S6 ribosomal protein (p-S6RP) at Ser235/236 from the kidney cortexes of 10-week-old C57 BL/6 male mice that had been fed with a serine-free diet and water with or without 0.1% d-serine for 1 week, subjected to unilateral nephrectomy (UNX) or not, and then sacrificed 2 days after operation. Representative images of three independent experiments and their quantification. RI, relative index. Statistics, one-way ANOVA with Dunnett’s post-hoc test. (E–G) Time course of relative growth of p18-revertant (Rev; Rheb wild-type) mouse embryonic fibroblasts (MEF (E), p18-deficient MEF (F), and Rheb-deficient MEF (G). Cells were treated with or without 5 μM of d-serine upon replacement to a serine-free medium. n=15; statistics, two-way ANOVA ((E) *P<0.05 for main effect of d-serine; (E–G) P<0.001 for main effect of time). (H) High content microscopy quantification of mTOR and LAMP2 colocalization in HK-2 cells that were incubated in a culture medium, then starved for amino acids in the presence of 5 μM of either d- or l-serine for 10 minutes. Mask overlay, primary objects (algorithm-defined cellular boundaries on the basis of CellMask, pseudo-color in red, and internal secondary objects, computationally defined colocalization between mTOR, green, and LAMP2, yellow). Scale bars, 10 μm. n=30; statistics, one-way ANOVA with Bonferroni’s post-hoc test. (I) Schematic summary of this study. d-serine promotes cellular proliferation via the mTOR/p18/Rheb pathway. As a result, d-serine mediates kidney remodeling after unilateral nephrectomy. *P<0.05, **P<0.01, ***P<0.001. Data, mean±SEM.