Chronic sphingosine 1-phosphate 1 receptor activation attenuates early-stage diabetic nephropathy independent of lymphocytes

Abstract

Sphingosine 1-phosphate (S1P), a pleiotropic lipid mediator, binds to five related G-protein-coupled receptors to exert its effects. As S1P1 receptor (S1P1R) activation blocks kidney inflammation in acute renal injury, we tested whether activation of S1P1Rs ameliorates renal injury in early-stage diabetic nephropathy (DN) in rats. Urinary albumin excretion increased in vehicle-treated diabetic rats (single injection of streptozotocin), compared with controls, and was associated with tubule injury and increased urinary tumor necrosis factor-α (TNF-α) at 9 weeks. These effects were significantly reduced by FTY720, a non-selective, or SEW2871, a selective S1P1R agonist. Interestingly, only FTY720 was associated with reduced total lymphocyte levels. Albuminuria was reduced by SEW2871 in both Rag-1 (T- and B-cell deficient) and wild-type diabetic mice after 6 weeks, suggesting that the effect was independent of lymphocytes. Another receptor, S1P3R, did not contribute to the FTY720-mediated protection, as albuminuria was also reduced in diabetic S1P3R knockout mice. Further, both agonists restored WT-1 staining along with podocin and nephrin mRNA expression, suggesting podocyte protection. This was corroborated in vitro, as SEW2871 reduced TNF-α and vascular endothelial growth factor mRNA expression in immortalized podocytes grown in media containing high glucose. Whether targeting kidney S1P1Rs will be a useful therapeutic measure in DN will need direct testing.

Figure 1. FTY720 and SEW2871 reduce urinary albumin excretion in diabetic rats

To determine the effect of FTY720 and SEW2871 in diabetic rats, 24-h urine collections were obtained for measurement of urine albumin at baseline (W0) and at weeks (W) 3, 6, and 9 of the study. Open bars, control group; black bars, vehicle-treated diabetes group; dark gray bars, diabetes group treated with FTY720 (0.3 mg/kg, oral gavage); and light gray bars, diabetes group treated with SEW2871 (0.1 mg/kg, i.p.). Data are means ± s.e.m. *P < 0.05, **P < 0.005 and ***P < 0.0005 compared with control; ⁺P < 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.005 compared with vehicle-treated diabetes group.

Figure 2. FTY720 and SEW2871 improve renal histology in diabetic nephropathy

Masson's trichrome staining of kidney sections from normal control rat (a, e), diabetic rat treated with vehicle (b, f), and a diabetic rat treated with FTY720 (c, g) or SEW2871 (d, h) at 9 weeks of the study. Representative photographs show extensive tubular damage with vacuolization (arrows), interstitial fibrosis (blue staining of interstitial collagen), and increased intertubular space (arrowhead) in vehicle-treated diabetic rats and minimal changes in treated animals. Bar = 50 μm.

Figure 3. FTY720 and SEW2871 reduce urinary tumor necrosis factor-α (TNF-α) level

Urine collections (24-h) were obtained for measurement of TNF-α in rats after 9 weeks of streptozotocin (STZ)-induced diabetes. Open bar, control group; filled bar, vehicle-treated diabetes group; hatched bar, diabetes group treated with FTY720 (0.3 mg/kg, oral gavage); and gray bar, diabetes group treated with SEW2871 (0.1 mg/kg, i.p.). Values are means ± s.e.m. *P < 0.05, **P < 0.01, and ***P < 0.005 compared with control; ⁺P < 0.05 and ⁺⁺P < 0.01 compared with vehicle-treated diabetes group.

Figure 4. SEW2871 reduces urinary albumin excretion (UAE) in Rag-1 diabetic mice

Urine collections were obtained for measurement of UAE at week 6 in wild-type (WT) and Rag-1 mice after streptozotocin (STZ)-induced diabetes. Open bars, control group; black bars, vehicle-treated diabetes group; and gray bars, diabetes group treated with SEW2871 (1 mg/kg, i.p.). Data are means ± s.e.m. *P < 0.05 and **P < 0.01 compared with control; ⁺P < 0.05 compared with vehicle-treated diabetes group. Blood glucose (BG) values are shown below the bars for each group.

Figure 5. FTY720 reduces albuminuria independent of sphingosine 1-phosphate 3 receptor (S1P3R) effects

Urine collections were obtained for measurement of urinary albumin excretion (UAE) at week 4 in S1P3^+/+ (wild type, WT) and S1P3^–/– mice after streptozotocin (STZ)-induced diabetes. Open bars, control group; black bars, vehicle-treated diabetes group; and gray bar, diabetes group treated with FTY720 (0.3 mg/kg, oral gavage). Data are means ± s.e.m. *P < 0.05 compared with control; ⁺P < 0.05 compared with vehicle-treated diabetes group. Blood glucose (BG) values are shown below the bars for each group.

Figure 6. Sphingosine 1-phosphate 1 receptor (S1P1R) agonists regulate podocyte-specific proteins

(a, b) Reverse transcriptase-PCR (RT-PCR) was performed on whole rat kidney total RNA isolated after 9 weeks of diabetes from control, vehicle-treated diabetic (D) rats, and diabetic rats treated with FTY720 or SEW2871. (a) Gel analysis of PCR products. (b) Expression of nephrin and podocin mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and data were calculated as expression relative to control. Black bars, vehicle-treated diabetic rats; dark gray bars, diabetic rats treated with FTY720 (0.3 mg/kg, oral gavage); and light gray bars, diabetic rats treated with SEW2871 (0.1 mg/kg, i.p.). Data are means ± s.e.m. *P < 0.05, **P < 0.005 compared with control (dashed line); ⁺P < 0.05, ⁺⁺P < 0.01 compared with vehicle-treated diabetes group. (c–f) WT-1 staining of rat kidney after 9 weeks of diabetes from control (c), vehicle-treated diabetic (d) rats, and diabetic rats treated with FTY720 (e) or SEW2871 (f) (magnification × 400).

Figure 7. Role of sphingosine 1-phosphate 1 receptor (S1P1R) agonists in immortalized podocytes in vitro

(a, b) Total RNA from immortalized podocytes was extracted and subjected to analysis by reverse transcriptase-PCR (RT-PCR) for S1P receptor mRNA expression (a) and sphingosine kinase 1 and 2 (SphK1 and SphK2) mRNA expression (b). (c, d) Total RNA from immortalized podocytes subjected to normal glucose (NG) or high glucose (HG) media for 14 days with or without SEW2871 (n = 5–6 each group) was extracted and subjected to analysis by RT-PCR for tumor necrosis factor-α (TNF-α) mRNA expression (c) and vascular endothelial growth factor (VEGF) mRNA expression (d). Expression of TNF-α and VEGF mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Open bars, NG; black bars, HG treated with vehicle; and gray bars, HG treated with SEW2871 (1 μmol/l). Data are means ± s.e.m. *P < 0.05, **P < 0.01 compared with NG; ⁺P < 0.05 compared with HG + vehicle.