Fucoidan from Laminaria japonica Inhibits Expression of GLUT9 and URAT1 via PI3K/Akt, JNK and NF-κB Pathways in Uric Acid-Exposed HK-2 Cells

Abstract

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.

Keywords: fucoidan; glucose transporter 9; urate transporter 1; uric acid.

Figure 1

UA-promoted expression of URAT1 and GLUT9 in HK-2 cells. Cells of 80% confluency were exposed to different concentrations of UA (0, 50,100, 200, 400 and 600 μg/mL, Sigma) for 24 h, and were then lysed in radio immunoprecipitation assay (RIPA) lysis buffer supplemented with phenylmethanesulfonyl fluoride (PMSF). Western blotting analysis was used to determine protein contents of URAT1 and GLUT9, β-actin was used to normalize them. Data were expressed as folds of vehicle. ^ABCD p < 0.01, ^abcd p < 0.05.

Figure 2

FPS repressed the UA-induced expression of URAT1 and GLUT9 in HK-2 cells. Cells were grown to 80% confluence, and were then treated with combinations of 200 μg/mL UA and different concentrations of FPS (0, 25, 50, 100, 200 and 400 μg/mL) for 24 h. Cells were lysed in RIPA buffer supplemented with PMSF. URAT1 and GLUT9 expression levels were assessed using Western blotting. Data were normalized with β-actin content, and were expressed as folds of vehicle. ^ABCD p < 0.01, ^abcd p < 0.05.

Figure 3

FPS repressed the UA-induced expression of URAT1 and GLUT9 via NF-κB signal pathway in HK-2 cells. Cells (80% confluence) were exposed to NF-κB pathway inhibitor QNZ (300 nmol/L) for 2 h and then to UA (200 μg/mL) and FPS (25 μg/mL) for another 24 h. Cell lysate in RIPA buffer containing PMSF were used to determine URAT1 and GLUT9 contents normalized with β-actin, and p-p65 content normalized with p65. Data were expressed as folds of vehicle. ^ABCDE p < 0.01, ^abcde p < 0.05.

Figure 4

FPS repressed the UA-induced expression of URAT1 and GLUT9 via the JNK signal pathway in HK-2 cells. Cells at 80% confluence were treated with JNK pathway inhibitor SP600125 (SP, 20 μmol/L) for 2 h, and were then exposed to UA (200 μg/mL) and FPS (25 μg/mL) for another 24 h. Cell lysate in RIPA buffer containing PMSF were conducted to determine relative contents of URAT1, GLUT9 and p-JNK. Data were expressed as folds of vehicle. ^ABC p < 0.01, ^abcd p < 0.05.

Figure 4

FPS repressed the UA-induced expression of URAT1 and GLUT9 via the JNK signal pathway in HK-2 cells. Cells at 80% confluence were treated with JNK pathway inhibitor SP600125 (SP, 20 μmol/L) for 2 h, and were then exposed to UA (200 μg/mL) and FPS (25 μg/mL) for another 24 h. Cell lysate in RIPA buffer containing PMSF were conducted to determine relative contents of URAT1, GLUT9 and p-JNK. Data were expressed as folds of vehicle. ^ABC p < 0.01, ^abcd p < 0.05.

Figure 5

FPS-repressed UA-induced expression of URAT1 and GLUT9 via PI3K/Akt signal pathway in HK-2 cells. Cells were grown to 80% confluence, treated with PI3K/Akt pathway inhibitor wortmanin (WM, 1 μmol/L) for 2 h, and were then exposed to UA (200 μg/mL) and FPS (25 μg/mL) for 24 h. Cell lysate in RIPA buffer containing PMSF were used to determine relative contents of URAT1, GLUT9 and p-AKT proteins. Data were expressed as folds of vehicle. ^ABC p < 0.01, ^abc p < 0.05.