Activation of sphingosine-1-phosphate 1 receptor in the proximal tubule protects against ischemia-reperfusion injury

Abstract

Agonists of the sphingosine-1-phosphate receptor (S1PR) attenuate kidney ischemia-reperfusion injury (IRI). Previous studies suggested that S1P1R-induced lymphopenia mediates this protective effect, but lymphocyte-independent mechanisms could also contribute. Here, we investigated the effects of S1PR agonists on kidney IRI in mice that lack T and B lymphocytes (Rag-1 knockout mice). Administration of the nonselective S1PR agonist FTY720 or the selective S1P1R agonist SEW2871 reduced injury in both Rag-1 knockout and wild-type mice. In vitro, SEW2871 significantly attenuated LPS- or hypoxia/reoxygenation-induced apoptosis in cultured mouse proximal tubule epithelial cells, supporting a direct protective effect of S1P1R agonists via mitogen-activated protein kinase and/or Akt pathways. S1P1Rs in the proximal tubule mediated IRI in vivo as well: Mice deficient in proximal tubule S1P1Rs experienced a greater decline in renal function after IRI than control mice and their kidneys were no longer protected by SEW2871 administration. In summary, S1PRs in the proximal tubule are necessary for stress-induced cell survival, and S1P1R agonists are renoprotective via direct effects on the tubule cells. Selective agonists of S1P1Rs may hold therapeutic potential for the prevention and treatment of acute kidney injury.

Figure 1.

FTY720 reduces kidney IRI in Rag-1 KO mice. (A) Effects of FTY720 on plasma creatinine in WT and Rag-1 KO mice exposed to kidney IRI. Mice were treated with FTY720 24 hours and 1 hour before ischemia. There were no differences between baseline plasma creatinine levels in sham-operated WT (0.10 ± 0.02; n = 2) and Rag-1 KO (0.09 ± 0.03; n = 2) mice. Data are means ± SEM; n = 2 to 8 for each group (n = 2 for sham-operated mice, n = 8 for WT-vehicle, n = 8 for WT-FTY720, n = 6 for Rag-1 KO-vehicle, and n = 6 for Rag-1 KO-FTY720). ††P < 0.01 versus WT-vehicle; *P < 0.05 versus each respective vehicle treatment. (B through G) Hematoxylin and eosin (H&E) staining of kidney sections from WT and Rag-1 KO IRI; insets show a ×2.5 magnified image. (B) WT-sham. (C) Rag-1 KO sham. (D) WT vehicle. (E) Rag-1 KO vehicle. (F) WT FTY720. (G) Rag-1 KO FTY720. Bar = 100 μm. (H) Semiquantitative scoring of tubular injury in H&E-stained kidney sections using a scale of 0 to 5 as described in the Concise Methods section. Data are means ± SEM; n = 2 to 5 for each group. ***P < 0.001, *P < 0.05 versus respective vehicle treatment; ††P < 0.001 versus WT-vehicle.

Figure 2.

Selective activation of S1P₁R mediates tissue protection from kidney IRI in Rag-1 KO mice. (A) Effects of SEW2871 and VPC44116 administered to Rag-1 KO mice 1 hour before kidney IRI. Data are means ± SEM; n = 3 to 7 per group. *P < 0.05 versus vehicle; ##P < 0.01 versus SEW2871. (B through D) H&E staining of mouse kidney sections from Rag-1 KO mice pretreated with SEW2871 (B), VPC44116 (C), SEW2871+VPC44116 (D) before IRI; insets show a ×2.5 magnified image. Bar = 100 μm. (E) Tubular injury score. Data are means ± SEM; n = 5 to 7 for treated groups, and n = 3 for sham and SEW2871+VPC44116. **P < 0.01, *P < 0.05 versus vehicle.

Figure 3.

Selective activation of S1P₁Rs attenuates leukocyte infiltration in kidney after IRI in Rag-1 KO mice. The effects of SEW2871 and VPC44116 on leukocyte infiltration in Rag-1 KO mouse kidneys 24 hours after IRI were examined. FACS analysis of total live (7AAD⁻) leukocytes (CD45⁺) and subsets of CD45⁺ cells expressed as percentage of total CD45⁺ cells as determined by cell count per gram of kidney weight. Fold changes relative to cell numbers of each population in vehicle-treated mice are shown; n = 4 to 5. ***P < 0.001, **P < 0.01, *P < 0.05 versus vehicle.

Figure 4.

LPS induces expression of S1P₁R in cultured kidney epithelial cells. (A) S1P₁R expression in TKPTS cells 24 hours after LPS doses of 0.1 to 25.0 μg/ml; n = 3 to 4 replicates. **P < 0.01, ***P < 0.001 versus 0 LPS. (B) mRNA expression of S1PR subtypes in TKPTS cells in response to 1 μg/ml LPS treatment for 24 hours. Inset is a representative gel of S1PRs PCR products from vehicle- and LPS-treated cells. S1P₄R and S1P₅R expression levels were too low to detect and therefore were not included in gel analysis. **P < 0.01 versus vehicle-treated cells. Results in A and B are from representative experiments that were repeated three times.

Figure 5.

S1P₁R agonists attenuate LPS-induced injury in cultured kidney epithelial cells through MAPK and Akt pathways and knockdown of S1P₁R expression abolishes protection by S1P₁R agonist. Necrotic and/or apoptotic cell death measured by FACS analysis of Annexin V and 7-AAD labeling 24 hours after treatment of TKPTS cells. (A) TKPTS cells were treated with SEW2871 (1 μM) and/or with VPC44116 (10 μM) 1 hour before treatment with or without LPS (1 μg/ml); n = 3 to 4. †††P < 0.001, †P < 0.05 relative to vehicle; ***P < 0.001 relative to LPS. (B) Cells were pretreated with the ERK inhibitor PD098059 (5 μM), Akt inhibitor LY294002 (5 μM), or pertussis toxin (PTX; 100 ng/ml) 1 hour before SEW2871 and LPS. †††P < 0.001, ††P < 0.01, and †P < 0.05 relative to vehicle; *P < 0.05 relative to LPS; ##P < 0.01, #P < 0.05 relative to LPS+SEW2871. Results are from a representative experiment that was performed three times with n = 3 to 4 replicates of each treatment. (C) Relative mRNA levels of S1P₁R (reduced by approximately 50%) and S1P₃R (unchanged) after transfection of TKPTS cells with 25 nM scrambled sequence or S1P₁R siRNA; n = 3. Inset shows RT-PCR products for S1P₁R, S1P₃R, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a representative gel. **P < 0.01 relative to siRNA-scramble transfected cells. Experiment was performed three times. (D) Apoptotic cell death in siRNA-transfected cells after LPS treatment (1 μg/ml) for 24 hours with and without 1 hour of pretreatment with SEW2871 (1 μM). ††P < 0.01, †P < 0.05 relative to vehicle; **P < 0.01 relative to LPS; ±P < 0.05 relative to LPS in scramble.

Figure 6.

Generation of PEPCK-Cre-S1P₁ floxed mice results in reduced copy number of S1P₁Rs in kidney cortex. (A) Genotyping of PEPCK-Cre and S1P₁ floxed mice. Agarose gel analysis of PCR products amplified from tail DNA. Lane 1, molecular weight markers; lane 2, PEPCK-Cre; lane 3, S1P₁^fl/wt; lane 4, PEPCK-CreS1P₁^fl/wt; lane 5, S1P₁^fl/fl; lane 6, PEPCK-CreS1P₁^fl/fl. Size of predicted PCR products (number of bp) is shown at right side of gel. (B and C) Immunofluorescent localization of YFP reporter (GFP immunoreactivity, green) indicative of Cre recombinase expression in kidney (B) and liver (C) sections of PEPCK-CreRosa26^YFP/wt mice. Nuclei were labeled with DAPI, blue. Bar = 40 μm. (D) Representative gel of S1P₁R mRNA levels in different tissues from PEPCK-Cre, PEPCK-CreS1P₁^fl/wt, and PEPCK-CreS1P₁^fl/fl mice (lanes from left to right). Regions of cortex (CTX) and inner medulla/papilla (IM) were dissected from kidney. (E) Expression of S1P₁R (mRNA levels relative to GAPDH, determined by real-time RT-PCR) in kidney cortex (Kid-CTX) and medulla (Kid-IM), liver, and lung from PEPCK-Cre, PEPCK-CreS1P₁^fl/wt, and PEPCK-CreS1P₁^fl/fl mice.

Figure 7.

S1P₁Rs are necessary for PTEC survival during kidney IRI. (A) Plasma creatinine levels of PEPCK-Cre, PEPCK-CreS1P₁^fl/wt, and PEPCK-CreS1P₁^fl/fl mice pretreated with vehicle or SEW2871 1 hour before mild kidney ischemia (26 minutes) and 24 hours of reperfusion. †††P < 0.001 versus respective sham; ***P < 0.001, *P < 0.05 versus respective vehicle; ### P < 0.001 versus vehicle-treated PEPCK-Cre mice. (B through J) Representative H&E staining of mouse kidney sections after sham surgery or IRI (treatments as in A) from PEPCK-Cre sham (B), PEPCK-Cre vehicle (C), PEPCK-Cre SEW2871 (D), PEPCK-CreS1P₁^fl/wt sham (E), PEPCK-CreS1P₁^fl/wt vehicle (F) PEPCK-CreS1P₁^fl/wt SEW2871, (G), PEPCK-CreS1P₁^fl/fl sham (H), PEPCK-CreS1P₁^fl/fl vehicle (I), and PEPCK-CreS1P₁^fl/fl SEW2871 mice (J). (K) Semiquantitative tubular injury score of kidneys from sham-, vehicle-, and SEW2871-treated PEPCK-Cre, PEPCK-CreS1P₁^fl/wt, and PEPCK-CreS1P₁^fl/fl mice. Data are means ± SEM; n = 3 to 7. †††P < 0.001 versus respective sham; ***P < 0.001; ###P < 0.001 versus vehicle-treated control mice.