THP-1 cell adhesion to HUVECs (Non-static rotational assay) and expression of PKCα in THP-1 cells.

<p>(A) SRPO significantly reduced PMA-triggered THP-1 cell adhesion to the HUVECs. THP-1 cells were pretreated with SRPO (10 μM) for 1 h and stimulated with PMA (10 nM) for 10 min before the assay. Preliminary experiments with trypan blue staining demonstrated that THP-1 cells were not damaged by SRPO (10 μM, 1h) treatment (data not shown). Adhesion assays were performed as described in Materials and Methods. Data are the mean ± SD of three independent experiments in each group. (B) SRPO attenuated PMA-induced PKCα activation in THP-1 cells. THP-1 cells were incubated in the presence or absence of SRPO (10 μM) for 1 h and stimulated with PMA (10 nM) for 10 min before the assay, and membrane proteins and total PKCα protein were detected by immunoblotting. Data are representative of three independent experiments.</p

Effects of SRPO on HFFD-induced obesity.

<p>(A) Body weight (n = 17, 28, 29). (B) Epididymal fat weight (n = 13, 18, 18). (C) Liver weight (n = 17, 23, 25). Values are the mean ± SE in each group. (Abbreviations: NC = normal chow; HFFD = high-fat high-fructose diet; VEH = vehicle; SRPO = Sarpogrelate hydrochloride.)</p

SRPO significantly decreased leukocyte-endothelial interactions and serum MCP-1 level.

<p>(A) Image of leukocyte-endothelial interactions in the femoral arteries of mice (with the margins of vessels indicated by dotted lines). Arrows indicate adhered or rolling leukocytes. (B) The numbers of rolling (left) and adherent cells (right) in all groups were calculated as described in Methods. Values are the mean ± SE (n = 10, 16, 18). (C) Effect of SRPO on serum MCP-1 levels in HFFD-induced obesity. Values are the mean ± SE (n = 13, 13, 11). Serum MCP-1 level of the HFFD + VEH group was higher than in the NC group, and SRPO prevented the increase in serum MCP-1 level on the HFFD + VEH group.</p

THP-1 cell adhesion to HUVECs (Flow chamber assay) and expression of E-selectin in HUVECs.

<p>(A) SRPO significantly reduced the number of PRP-induced THP-1 cell that adhered to HUVECs. HUVEC monolayers were stimulated with 3 ng/ml TNF-α for 3.5 h and exposed to PPP or PRP for 20 min. PRP was pretreated or not with SRPO (10 μM) just before addition to the HUVECs. THP-1 cells were perfused over activated HUVEC monolayers at a flow rate of 1.0 dyne/cm². Adhesion assays were performed as described in Materials and Methods. Data are the mean ± SD of three independent experiments in each group. (B) SRPO significantly reduced the expression of PRP-induced E-selectin in HUVECs. E-selectin expression was determined by FIA as described in Materials and Methods. Data are the mean ± SD of 6 independent experiments in each group.</p

Fibroblast growth factor 23 is upregulated in the kidney in a chronic kidney disease rat model

<div><p>The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)₂D₃ or transforming growth factor-β1 (TGFβ1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)₂D₃ level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of <i>αKlotho</i>, <i>NaPi-IIa</i> and <i>NaPi-IIc</i> was decreased, and the mRNA expression of <i>TGFβ1</i>, <i>collagen1a1</i>, <i>osteopontin</i> and <i>FGF23</i> was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. <i>FGF23</i> and <i>osteopontin</i> mRNAs were expressed in HK2 cells incubated with TGFβ1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)₂D₃ and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFβ1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.</p></div

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p

Immunohistochemistry and in situ hybridization in the kidney of hemi-nephrectomized rats fed a high-P diet.

<p>FGF23: FGF23 immunohistochemistry (brown). Osteopontin: osteopontin immunohistochemistry (brown). ×200: high magnification. <i>FGF23</i> in situ hybridization (red spots. arrow: positive cells). ×400: high magnification.</p

Immunohistochemistry and in situ hybridization in the kidney of partial nephrectomy rat model.

<p>FGF23: FGF23 immunohistochemistry (brown). Osteopontin: osteopontin immunohistochemistry (brown). ×200: high magnification. <i>FGF23</i> in situ hybridization (red spots; arrow: positive cells). ×400: high magnification.</p

Immunofluorescence in the kidney of hemi-nephrectomized rats fed a high-P diet.

<p>FGF23 immunofluorescence (green). αSMA immunofluorescence (red). Merge: FGF23 (green), αSMA (red), and DAPI (blue). ×400: high magnification.</p

FGF23 expression in the partial nephrectomy rat model.

<p>(a) Serum FGF23 concentration. (b) FGF23 mRNA expression in the kidney. Sham group was used as a normalization control. (c) Western blot of FGF23 in the kidney. (d) Histology in the kidney. HE: hematoxylin-eosin staining. MT: Masson’s trichrome staining to evaluate fibrosis. VK: Von Kossa staining to evaluate calcification. *P<0.05, **P<0.01, ***P<0.001; sham group (n = 6), partial nephrectomy mild group (PN mild) (n = 6), partial nephrectomy severe group (PN severe) (n = 6).</p