Figure 1

D-serine suppresses cellular proliferation and induces apoptosis in human tubular cells. (A) D-serine-containing medium significantly impaired proliferation at 24 h and 48 h when compared with the control medium. The cell numbers were expressed as relative numbers compared with the number on day −1 measured by the Muse Count & Viability Kit, which contains fluorescent dyes that distinguish live from dead cells. Each bar represents the average of control, 20 mM D-serine-treated, or 20 mM L-serine-treated cultures (n = 6). On the left graph, **P < 0.01, ***P < 0.001 compared to untreated control cells. ^†††P < 0.001 compared to D-serine-treated cells. (B–D) At 10 and 20 mM, D-serine significantly decreased cell viability (B). At D-serine 20 mM, the cell viability of both HK-2 cells and normal human renal epithelial cells (NHREC) was decreased (C,D). HK-2 cells (B,C) or NHREC (D) were treated with L- or D-serine at 1–20 mM (B) or 20 mM (C,D). Viability was assessed by using MTS (n = 4 cultures for each treatment group). (E) HK-2 cells treated with 20 mM D-serine for 48 h showed a significant increase in Annexin V staining compared with that of control cells and cells treated with 20 mM L-serine. Apoptosis was induced by D-serine in HK-2 cells as shown by Annexin-V binding and 7-Amino actinomycin D (7-AAD) staining (n = 3 cultures per treatment group). (F) Increased activation of caspase 3/7 by D-serine, but not by L-serine, was confirmed (n = 3 cultures per treatment group). (G,H) Increased expression of pro-apoptotic BAX and PUMA in HK-2 cells (G) and NHREC (H), and decreased expression of anti-apoptotic BCL-2 in NHREC (H) treated with D-serine for 48 h (n = 6 cultures per treatment group). (I) HK-2 cells were treated with D-alanine or D-proline at 1–20 mM for 48 h and viability was assessed by using MTS, with no significant changes with either amino acid added (n = 4 cultures for each treatment group). *P < 0.05, **P < 0.01, ***P < 0.001. All data are expressed as mean ± SEM.