Fig 1.
Gemigliptin attenuates vascular calcification in the abdominal aorta.
(A) Representative photomicrographs of the abdominal aorta stained by Von Kossa staining (original magnification, × 17 and × 100). (B) The percentage of calcified area was calculated as the ratio of the Von Kossa positive area versus the total tissue area. (C) Representative photomicrographs of immunohistochemistry for RUNX2 (original magnification, × 200) are shown. (D) Representative immunoblot images and the quantification of RUNX2 expression are shown. GAPDH was used as the loading control. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 5 for LP, low protein group; n = 5 for adenine group; n = 6 for AG10, adenine-gemigliptin [10mg/kg] group; n = 6 for AG20; adenine-gemigliptin [20mg/kg] group). RUNX2, runt-related transcription factor-2. *P<0.5, **P<0.01, and ***P<0.001 compared with LP group, and #P<0.5, ###P<0.001 compared with adenine group.
Table 1.
Body weight, food intake, and biochemical parameters in an animal model of chronic kidney disease.
Fig 2.
Gemigliptin attenuates high phosphate-induced vascular calcification in VSMCs.
(A) Visualization of calcium deposition on VSMCs was assessed by alizarin red staining. Representative pictures were shown. (B) After HCl decalcification, calcium content of the cells was assayed. The value was normalized by the total protein and expressed as percentage of control. (C) Cell viability was determined using MTT assay. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 6–7 in each group). Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3 mM phosphate and 50 μM gemigliptin. ***P<0.001 compared with control and ###P<0.001 compared with phosphate group.
Fig 3.
Gemigliptin attenuates the expression of sodium phosphate co-transporter PiT-1.
The expression of PiT-1 was evaluated by qRT-PCR and western blot after treatment with 3 mM phosphate and/or 50 μM gemigliptin for 2 days (A) or 4 days (B). GAPDH was used as the loading control. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 5–6 in each group). Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.
Fig 4.
Gemigliptin modulates ROS generation and the expression of NADPH oxidase NOX4 and NADPH oxidase subunit p22phox.
(A) Intracellular ROS generation was visualized by fluorescence microscopy, and (B) quantified in the cellular lysate after staining with 10 μM H2DCF-DA. (C) The level of H2O2 generation was measured using the Amplex red hydrogen peroxide assay kit. The mRNA expression of NOX4 and p22phox was analyzed by qRT-PCR after treating VSMCs with phosphate and/or gemigliptin for (D) 7 days and (E) 14 days. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 6–7 in each group). Con, control; Pi, 3 mM phosphate; PG50 –PG500, 3 mM phosphate and 50 μM– 500 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.
Fig 5.
Gemigliptin attenuates phosphate-induced phospho-PI3K-AKT signaling pathway.
(A) VSMCs were treated with 3 mM phosphate and/or 50 μM gemigliptin for 24 h. Representative immunoblot images are shown. Graph represents quantitative data as (B) ratio of phospho-AKT/total-AKT or (C) phospho-PI3K/total PI3K. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 4–5 in each group). Con, control; Gemi50, 50 μM gemigliptin; Pi, 3 mM phosphate; PG50, 3 mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control, #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.
Fig 6.
Gemigliptin attenuates high phosphate-induced Wnt signaling.
The expression of FDZ3 and DKK-1 was evaluated by qRT-PCR (A and B) and western blot (C and D) in VSMCs after incubating with 3 mM phosphate and/or 50 μM gemigliptin for 7 days (A and C) and 14 days (B and D). The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 5–6 in each group). FDZ3, frizzled-3; DKK-1, dickkopf-1; Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.
Fig 7.
Gemigliptin attenuates high phosphate-induced osteogenic differentiation and restores VSMC markers.
The mRNA expression of VSMC markers (α-SMA and SM22α) and osteogenic markers (CBFA1, OSX, E11, DMP-1, and SOST) were evaluated by qRT-PCR in cultured VSMCs after incubating with 3 mM high phosphate and/or 50 μM gemigliptin for 7 days (A and C) and 14 days (B and D). The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 6–7 in each group). CBFA1, core-binding factor alpha a; OSX, osterix; OC, osteocalcin; SOST, sclerostin, Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3 mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate.
Fig 8.
A flow diagram of the effect of gemigliptin against high-phosphate-induced vascular calcification.
Gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in VSMCs via multiple steps including downregulation of PiT-l expression, and suppression of ROS generation, phosphor-PI3K/AKT, and Wnt signaling pathway.