Sphingosine kinase-1 pathway mediates high glucose-induced fibronectin expression in glomerular mesangial cells

Abstract

Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy.

Fig. 1.

Glomerular injury and activation of SphK1 pathway in STZ-induced diabetic kidney. A, Glomerular histopathology analysis by Periodic Acid-Schiff (PAS) staining. The pictures display representative glomeruli of PAS-stained sections in control and diabetic group at an original magnification of ×200. B, Glomerular size (tuft area) was measured by tracing the tuft. *, P < 0.01 vs. control. C, Mesangial matrix area was defined as the PAS-positive area (red area). *, P < 0.01 vs. control. D, The mesangial matrix index represented the ratio of mesangial matrix area divided by tuft area. *, P < 0.01 vs. control. E, Relative levels of SphK1 and SphK2 mRNA in control and diabetic kidney were analyzed by real-time PCR. *, P < 0.01 vs. control. F, The protein expression levels of SphK1 and FN in the rat kidneys were detected by Western blot analysis. SphK activity (G) and S1P levels (H) were measured by LC-MS/MS assays. *, P < 0.01 vs. control.

Fig. 2.

Time- and concentration-dependent effects of HG on SphK1 and FN expression in GMC. A, GMC were cultured by HG (22 mm glucose) for 0–48 h. B, GMC were cultured by 5.5–30 mm glucose for 48 h. At the end of the incubation period, cells were lysed, and protein expression was determined by Western blot analysis. *, P < 0.05; **, P < 0.01 vs. control for SphK1; #, P < 0.05; ##, P < 0.01 vs. control for FN.

Fig. 3.

HG activates SphK1 pathway in GMC. GMC were serum starved for 24 h, then exposed to media containing 5.5 mm glucose (NG), 22 mm glucose (HG), or NG plus 16.5 mm Mtol for 48 h. A, Real-time PCR was conducted to determine relative mRNA expression levels of SphK1 and SphK2 in GMC. *, P < 0.01 vs. NG. B, SphK1 protein expression was evaluated by Western blot analysis. *, P < 0.01 vs. NG. C, SphK activity was determined by LC-MS/MS. *, P < 0.01 vs. NG. D, Levels of S1P were measured by LC-MS/MS. *, P < 0.01 vs. NG.

Fig. 4.

Roles of ROS and AGE in HG-induced SphK1 activation. GMC were cultured in NG and HG in the presence or absence of NAC (5 mm) or pyridoxamine (PM) (5 mm) for 48 h. A, LC-MS/MS analysis of SphK1 activity. B, Western blot analysis of SphK1 expression. *, P < 0.01 vs. NG; #, P < 0.01 vs. HG.

Fig. 5.

DMS attenuates HG-induced FN expression in GMC. GMC were treated with HG in the presence or absence of DMS (2.5 μm) for 48 h. A, SphK activity was measured by LC-MS/MS after 48 h of exposure to NG and HG. *, P < 0.01 vs. NG; #, P < 0.01 vs. HG. B, FN expression in GMC was evaluated by confocal microscopic analysis. C, Representative Western blotting for FN expression in GMC. *, P < 0.01 vs. NG; #, P < 0.01 vs. HG.

Fig. 6.

SphK1-siRNA blocks HG-induced FN expression in GMC. A, Real-time PCR shows relative mRNA expression of SphK1 and SphK2 in GMC transfected with SphK1 or control siRNA. *, P < 0.01 vs. control. B, The protein expression of SphK1 in GMC transfected with SphK1 or control siRNA was evaluated by Western blot analysis. *, P < 0.01 vs. control. LC-MS/MS assay was used to analyze SphK activity (C) and S1P levels (D) in GMC transfected with SphK1 or control siRNA, followed by incubation with NG or HG for 48 h. *, P < 0.01. E, Western blotting was performed to evaluate expression of SphK1 (upper panel) and FN (lower panel) in GMC transfected SphK1 or control siRNA, followed by incubation with NG or HG for 48 h. *, P < 0.01.

Fig. 7.

Effects of SphK1 overexpression on FN expression in GMC. GMC overexpressing empty vector (vector), SphK^WT, or dominant-negative SphK1 (SphK^G82D) were exposed to NG or HG for 48 h. A, SphK activity was analyzed by LC-MS/MS. B, S1P levels were analyzed by LC-MS/MS. C, The expression of SphK^WT, SphK^G82D (upper panel), and FN (lower panel) in the transfected GMC was determined by Western blotting. *, P < 0.01.

Fig. 8.

Time- and concentration-dependent effects of S1P on FN expression in GMC. FN expression levels were measured in (A) GMC treated with S1P (1 μm) for the indicated time periods or (B) treated for 60 min with S1P at the indicated concentrations. *, P < 0.05; **, P < 0.01 vs. control.

Fig. 9.

Effects of S1P₂ receptor antagonist JTE-013 on FN induction by S1P and HG in GMC. Cells treated with 10 μm JTE-013 for 24 h in the presence of S1P. Data are represented as mean ± sd. *, P < 0.05 vs. control; #, P < 0.05 vs. S1P alone.

Fig. 10.

Effects of exogenous S1P on AP-1 activity and FN expression in GMC. A, GMC were incubated with or without curcumin (20 μm) for 3 h, followed by stimulation with S1P for 1 h. Nuclear proteins (5 μg) were subjected to EMSA. The arrow shows the specific binding of AP-1. *, P < 0.01. B, FN mRNA levels were measured by real-time PCR in GMC preincubated with or without curcumin (20 μm) for 3 h, followed by stimulation with S1P for 1 h. *, P < 0.01. C, FN expression was evaluated in GMC preincubated with or without curcumin (20 μm) for 3 h, followed by stimulation with S1P for 1 h. *, P < 0.01.

Fig. 11.

SphK1 pathway mediates HG-induced activation of AP-1 in GMC. AP-1 DNA-binding activity was assayed by EMSA in GMC (A) exposed to HG for the indicated time periods. B, The specific binding of consensus AP-1 was verified by competition assays and the subunit components for AP-1 dimers identified by supershift assays. C, GMC were treated with HG in the presence or absence of the DMS (2.5 μmol/liter) for 6 h. *, P < 0.01 vs. NG; #, P < 0.01 vs. HG. D, GMC were transfected with SphK^G82D or empty vector followed by incubation with HG for 6 h. The arrows indicate the specific binding of AP-1. *, P < 0.01 vs. vector + NG; #, P < 0.01 vs. vector + HG.