Sphingosine-1-phosphate receptors: Biology and therapeutic potential in kidney disease

Abstract

The major sphingolipid metabolite, sphingosine-1-phosphate (S1P), has important biological functions. S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis, vascular maturation, immunity, chemotaxis, and other important biological pathways. Recently, clinical trials have targeted S1P receptors (S1PRs) for autoimmune diseases and transplantation and have generated considerable interest in developing additional, more selective compounds. This review summarizes current knowledge on the biology of S1P and S1PRs that forms the basis for future drug development and the treatment of kidney disease.

Sphingolipid metabolites are emerging as important lipid signaling molecules in health and disease.^1-3 Among them, sphingosine-1-phosphate (S1P) is known to play important roles in cellular processes. S1P, found at concentrations of 0.1–1 μm in human serum, is produced by phosphorylation of sphingosine by sphingosine kinases (SPHKs) in response to a variety of stimuli. S1P regulates diverse biological processes and functions, including cell growth, cell proliferation, and angiogenesis, and can inhibit apoptosis and lymphocyte trafficking.^4-9 S1P was once considered simply as a breakdown product of ceramide. However, the evolutionally conserved function of S1P from plants to higher organisms has highlighted its importance as a pivotal signaling molecule in normal function and pathophysiology. In addition, the discovery that S1P is a ligand for specific receptors (S1P_1–5, formerly endothelial differentiation gene (EDG)1, -5, -3, -6, -8) has expanded its role as a mediator of diverse physiologic and pathologic conditions.

SPHINGOLIPID RHEOSTAT AND CELL FATE

Although S1P induces cell growth and proliferation and/or inhibits apoptosis, two S1P precursors, ceramide and sphingosine, induce cell cycle arrest and cell death.^10-13 Proapoptotic ceramide is formed by activation of sphingomyelinase or through the de novo sphingolipid biosynthetic pathway starting with serine palmitoyl transferase^14-16 (Figure 1). Different apoptotic stimuli such as endotoxin, interleukin-1 (IL-1), or tumor necrosis factor-α can activate these enzymes,¹⁸ whereas other stimuli such as growth factors, lipopolysaccharide, hypoxia, or IL-1β can activate SPHK.^19,20 Activation of SPHK results in an increase in S1P levels and inhibition of ceramide-induced apoptosis. However, S1P is also converted into sphingosine by sphingosine-1-phosphate phosphatase (SPP), which may induce apoptosis by itself or by increasing proapoptotic ceramide synthesis.^21-24 Because these sphingolipid metabolites have opposite actions on cell fate, the concept of a ‘sphingolipid rheostat’ has been developed that addresses the importance of balance of these mediators and not the absolute amount of metabolites in determining the ultimate cell fate.²⁵

Figure 1. Sphingosine-1-phosphate (S1P) biosynthetic pathway.

The production of S1P can lead to intracellular or extracellular actions. Extracellular actions are mediated by S1P_1–5 receptors in an autocrine or paracrine manner. Adapted from Alvarez et al.¹⁷

S1P SYNTHESIS AND DEGRADATION

Intracellular S1P level is tightly regulated by a balance between its synthesis by SPHK and degradation by endoplasmic reticulum SPP (reversible) or S1P lyase (irreversible).²⁵ By downstream phosphorylation, growth factors and cytokines can activate SPHK, which mediates phosphorylation of sphingosine, producing S1P. Two isoforms of SPHK, known as SPHK1 and SPHK2, have been characterized.^26,27 Despite similar amino-acid sequences, they have different kinetics of expression during development²⁸ as well as different subcellular localization,^29,30 suggesting that these two isoforms serve different functions. Overexpression of SPHK1 improves cell survival from apoptosis by inhibiting the activation of caspases and stress-activated protein kinases.^29,31 SPHK1 activation promotes cell proliferation^29,32 and regulates cell transformation mediated by Ras activation.³¹ In contrast, much less is known about the role of SPHK2, although several reports suggest that it has proapoptotic functions.^33,34 However, a possibility that the function of the two isoenzymes in vivo is complementary has been suggested by the finding that SPHK1/SPHK2 double knockout mice are embryonic lethal, because of a severe defect in angiogenesis and neural tube formation, with almost no detectable level of S1P. In contrast, mice with a single knockout of either isoenzyme do not display an abnormal phenotype.⁶ SPHK2 also phosphorylates and activates FTY720, a strong immune modulatory drug currently in clinical trials,³⁵ which, in its phosphorylated form, is an agonist at S1P₁ receptors.

S1P can be degraded reversibly by an S1P-selective phosphatase (SPP) to produce sphingosine or by S1P lyase to form hexadecenal and phosphoethanolamine.^21,22,36 Two mammalian SPPs have been recently described.^21,22 SPPs regulate the dynamic balance between various sphingolipid mediators and ultimately cell fate by mediating dephosphorylation of S1P to sphingosine. The irreversible pathway of S1P degradation depends on S1P lyase, a pyridoxal-dependent enzyme that cleaves the C2–3 bond of S1P.^36,37 S1P lyase can potentiate apoptosis in response to DNA damage by the p53 and mitogen-activated protein kinase pathway in HEK-293 cells.³⁸

S1P RECEPTORS

The first hint that the biological functions of S1P were receptor mediated came with the discovery of five orphan G-protein-coupled receptors, originally named the endothelial differentiation gene (EDG).^39-43 Five high-affinity S1P receptors (S1PRs), EDG1/S1P₁, EDG5/S1P₂, EDG3/S1P₃, EDG6/S1P₄, and EDG8/S1P₅, have been characterized (Figure 2). Each of these S1PRs couples to heterodimeric G-proteins (α_i, α_q, or α_12/13) with subsequent activation of downstream signaling molecules, such as phospholipase C/D,^44-46 small GTPases,^47,48 mitogen-activated protein kinase,^49-51 and Akt.^52,53 The diversity of responses mediated by S1PRs, including immunity, cell migration, angiogenesis, and heart development, depends on the pattern of receptor expression and the associated downstream effectors. For example, activation of S1P₁ or S1P₃ increases random or chemotactic migration of endothelial cells (ECs), whereas binding to S1P₂ has an opposite effect.^54-56 The S1PRs can differentially regulate small GTPases of the Rho family, particularly Rho and Rac, which are important in cytoskeletal rearrangement, and produce effects on maintenance/disruption of cell barrier integrity or cell migration.^57,58 S1P activation of S1P₁ mediates cortical actin reassembly via Rac activation and leads to endothelial barrier enhancement, whereas S1P₃ activation in pulmonary epithelial cells leads to disruption of tight junctions, possibly by activating Rho, resulting in increased lung vascular permeability.^59-61 The role of S1P₁ signaling in vascular maturation is demonstrated by findings in S1P₁ receptor null mice, which despite normal angiogenesis die from intracranial hemorrhage at embryonic day 12.5; deletion of the receptor prevents activation of the small GTPase, Rac, which leads to failure in migration of vascular smooth muscle cells and pericytes, a defect in vascular maturation, and ultimately to hemorrhage.⁶¹

Figure 2. Sphingosine-1-phosphate (S1P) receptors.

Intracellular or extracellular sphingosine is phosphorylated by SPHK. S1P or phosphorylated FTY720 in serum binds to specific members of the S1PR family, which are coupled to different G proteins: EDG1/S1P₁, EDG5/S1P₂, EDG3/S1P₃, EDG6/S1P₄, and EDG8/S1P₅. SPH: sphingosine, SPHK, sphingosine kinase, S1PP, sphingosine-1-phosphate phosphatase, FTY720-P, phosphorylated FTY720.

S1P AND IMMUNE CELLS

In circulating blood and lymph, S1P is generated predominantly by mononuclear phagocytes, activated mast cells, and platelets.^62,63 Intracellularly produced S1P is secreted and then binds to albumin and other plasma proteins yielding high concentrations of up to 5 μm in extracellular compartments compared to relatively lower concentrations in tissues. A high level of S1P produced by SPHK elicits a variety of functional responses in immune systems in a paracrine or autocrine manner, depending on the expression pattern of S1PR subtypes in immune cells (Table 1). Until recently, S1P–S1PR signaling in immune cells has been examined mainly in lymphocytes, mast cells, and dendritic cells (DCs).

Table 1.

S1P receptors in immune cells

Immune cell	S1PR	Functions
T cell	S1P1	Decreased chemotaxis at low concentration (0.1–100 nm) of S1P, increased chemotaxis at high concentration (0.3–3 μm) of S1P, decreased proliferation, decreased IFN-γ and IL-4 secretion
	S1P4	Decreased proliferation, decreased IFN-γ and IL-2 secretion, enhanced IL-10 secretion
B cell	S1P1	Decreased chemotaxis at low concentration (0.1–100 nm), increased chemotaxis at high concentration (0.3–3 μm)
	S1P4	Decreased proliferation
Mast cell	S1P1	Increased chemotaxis, no effect on degranulation
	S1P2	Increased degranulation
Dendritic cell	S1P1	Increased chemotaxis
	S1P2	Decreased chemotaxis
	S1P3	Migration to S1P
Macrophage	S1P1	Increased chemotaxis

IFN-γ, interferon-γ; IL, interleukin; S1P, sphingosine-1-phosphate; S1PR, S1P receptor.

In human and mouse CD4 and CD8 T cells, S1P₁ and S1P₄ are the predominant S1PRs mediating the functional responses to S1P.⁶⁴ The effect of S1P on T-cell function is concentration dependent. At low concentrations (0.1–100 nm), S1P inhibits apoptosis and enhances chemotaxis to a variety of chemokines; at higher concentrations (0.3–3 μm), S1P inhibits T-cell chemotaxis,⁶⁵ an effect that is mediated through S1P₁ but not S1P₄ receptors.⁶⁵ In addition, S1P at a concentration range of 1 nm to 1 μm can inhibit CD4 T-cell proliferation and production of interferon-γ and IL-4 without affecting IL-2.⁶⁶ These multiple immune modulatory functions of S1P are mediated through S1P₁ activation.⁶⁶ S1P₄ receptor activation also produces immunosuppression by inhibiting T-cell proliferation and secretion of effector cytokines, such as IL-2 or interferon-γ, while enhancing IL-10 secretion.⁶⁷

Mast cells play an important role in innate and acquired immunity and in allergic diseases by producing a variety of vasoactive substances that increase vascular permeability and leukocyte recruitment.^68,69 Mast cells are also known to be an important source of plasma S1P. Antigen-induced IgE aggregation leads to activation of SPHK in mast cells, and subsequent release of S1P into the extracellular space is important in mast cell migration to the site of inflammation and in the degranulation process.^70-72 SPHK2 intrinsically regulates mast cell calcium influx and responses, whereas SPHK1 is important in regulating the levels of circulating S1P and thus extrinsically affecting mast cell responsiveness.⁷³ Elevated S1P levels have been detected in bronchoalveolar lavage fluid of asthmatic patients.⁷⁴ Mast cell-released S1P can transactivate S1P₁ and S1P₂ receptors in an autocrine manner. S1P₁ activation mediates mast cell migration through cytoskeletal rearrangement, whereas S1P₂ activation inhibits chemotaxis primarily by downregulation of Rac activity and activation of Rho.^75,76 However, defects in degranulation have been observed in bone marrow-derived mast cells from S1P₂-deficient mice or immortalized rat basophilic leukemia cells transfected with antisense to S1P₂, whereas rat basophilic leukemia cells transfected with antisense to S1P₁ showed normal granule content despite decreased chemotaxis, thus suggesting that S1P₂ activation mediates the degranulation process in mast cells.

DCs are antigen-presenting cells that link innate and adaptive immune responses. In normal steady state, immature DCs are present in virtually all tissues, participating in immune surveillance. On antigenic stimuli, DCs undergo maturational processing and migrate to draining lymph nodes to activate the adaptive immune response. The expression profile of S1PRs in mouse DCs varies with the DC maturation status;⁷⁷ mature DCs express higher levels of S1P₃ mRNA compared with immature DCs.⁷⁸ S1P binding to S1PRs activates the small GTPase family, inducing migration of DCs without affecting the maturation process.⁷⁹ FTY720, a nonspecific S1PR analog, can inhibit the migratory response of mature DCs to S1P.⁷⁹ On DC maturation, S1P increases cytokine production from DCs leading to a Th2-dependent response.⁸⁰ Further evidence for the effects of S1P–SPHK on DCs comes from the observation that S1P in a dose-dependent manner (0.01–10 μm) significantly reduces the percentage of apoptotic mature DCs and increases accumulation of peripheral immature DCs at the location of antigen, thereby facilitating subsequent antigen uptake.⁸¹ Furthermore, S1P regulates migration and endocytosis of mature murine DCs via S1P₃ but not S1P₁.⁷⁸ These data suggest that S1P signaling can be important in immune modulation through effects on S1PRs expressed on DCs. Although some aspects of immune cell regulation by S1P–S1PR signaling have been clarified, the precise role of S1P in individual immune cells or in the complex immune system still needs to be investigated.

S1P EFFECTS ON LYMPHOCYTE TRAFFICKING

Naive T cells continuously circulate between blood and lymphatic tissues as part of the immune surveillance system. The lymphocyte homing process from blood into secondary lymphoid organs via high endothelial venules depends on various chemokines and signaling through their respective receptors, such as CCL19 and CCL21 with CCR7, CXCL12 with CXCR4, and CXCL13 with CXCR5.^82-84

Recent in vivo studies using FTY720, a novel immunomodulator, have clarified the mechanism of lymphocyte egress from secondary lymphoid organs. FTY720, a sphingosine analog, is activated in vivo by SPHK2 to form the active principle, FTY720-P. FTY720-P, a structural analog of S1P, binds to and activates four different S1PRs.^85,86 Activation of S1P₁ receptors, for example, by FTY720-P, induces peripheral lymphopenia by sequestering lymphocytes from blood and spleen into secondary lymphoid organs,^86-88 thereby suggesting a role for S1P–S1P₁ signaling in normal lymphocyte trafficking. Infusion of S1P into the rat bloodstream also evokes lymphopenia.⁸⁶ In mice whose hematopoietic cells lack S1P₁ receptors, T cells are absent from peripheral blood because of the inability of T cells to egress from the thymus.⁸⁹ The observations that S1PR activation by S1P and FTY720-P and the absence of S1PRs in lymphocytes derived from S1P₁-knockout mice both lead to blockade of lymphocyte egress from secondary lymph organs seem incongruous at first glance. This has been a controversial area in which two hypotheses have been proposed. The first model is the stromal-gate control model. In this model, S1P acts on ECs to reduce the permeability of the endothelium in blood vessels and lymphoid vascular sinuses, thereby preventing the exodus of lymphocytes. The second model is referred to as the S1P–S1P₁ control-of-lymphocytes model. In this model, FTY720 or S1P acts as ‘functional antagonist’ leading to the downregulation of S1P₁ receptors expressed on lymphocytes rendering them unresponsive to a gradient of S1P level (blood>lymphatic tissue).⁹⁰ Recently, however, we and others have demonstrated that selective S1P₁ antagonists including VPC44116 increase endothelial permeability and do not induce lymphopenia,^91,113 supporting the stromal-gate control model. It may be that both models may contribute variably to the lymphopenia observed with FTY720. This finding establishes the intrinsic requirement of lymphocyte S1P₁ receptor in lymphocyte egress from secondary lymphoid organs. S1P–S1P₁ signaling in lymphocytes appears to be important in the adaptive immune response because antigen-specific T cells in lymph nodes lose their S1P responsiveness with transient downregulation of S1P₁ receptors shortly after antigenic challenge, thereby permitting clonal expansion in lymph nodes. However, several days later, antigen-specific T cells recover from transient S1P unresponsiveness with S1P₁ receptor expression and re-entry into the peripheral circulation to produce immune responsiveness. These findings support the association of loss of S1P responsiveness with initial retention of antigen-specific T cells and also the association of acquisition of S1P responsiveness with their egress from lymph node, confirming the role of lymphocyte S1P–S1P₁ signaling in lymphocyte trafficking.^89,92 Failure to respond to S1P has also been demonstrated in lymphocytes from FTY720-treated animals. Lymphocytes from these FTY720-treated animals showed almost complete downregulation of S1P₁ receptors.⁸⁹

S1P EFFECTS ON CELL SURVIVAL

It is now clear that S1P is an extracellular ligand for specific G-protein-coupled receptors that are implicated in a variety of biological and pathological events. However, the following lines of evidence suggest that S1P can also function as an intracellular second messenger, although the intracellular target of S1P has not yet been identified: (1) yeast does not express S1PRs but still responds to S1P;^93,94 (2) dihydro-S1P (sphinganine), a compound identical to S1P except that it does not have a 4,5-trans double bond, can bind to and activate all S1PRs, but cannot reproduce all the effects of S1P, especially those associated with cell survival;^7,95 (3) microinjection of S1P increases the level of intracellular S1P and mobilizes calcium in a pertussis toxin-insensitive manner, whereas extracellular S1P has no effect on calcium mobilization.^7,96,97

Although S1P enhances cell proliferation and survival, ceramide and sphingosine, two precursors of S1P are associated with cell growth arrest and apoptosis.^10-13 An increase in S1P level or decrease in ceramide can prevent oocytes from radiation-induced apoptosis in wild-type mice.⁷ Similarly, pretreatment of human ECs with S1P protects against early apoptosis, which was associated with an increase in ceramide generation induced by radiation exposure; however, no protection was noted with late apoptosis, which was predominantly dependent on DNA damage.⁹⁸ The balance between sphingosine and S1P is decisive in mast cell activation following FcεR I activation.^70,99 interconvertible, these results suggest that the balance of proapoptotic ceramide and sphingosine vs antiapoptotic S1P determines cell fate. More importantly, these effects were neither blocked by pertussis toxin nor reproduced by dimethyl sphingosine, indicating that the cytoprotective effect of S1P is not S1PR-dependent.^{29,31,100-102} Decreasing SPHK1 expression, the principal enzyme in the generation of S1P, by small interfering RNA induced cell cycle arrest and apoptosis in MCF-7 breast cancer cell lines, suggesting an important role of endogenous SPHK in cell survival.¹⁰³ In addition, overexpression of SPHK reduced apoptosis by inhibiting Jun N-terminal kinase caspase activation.³¹ SPHK meditates ischemic preconditioning in murine heart.¹⁰⁴ The protective effect of intracellular S1P on cell survival is mediated in part by activation of extracellular signal-regulated kinase 1/2, a stress-activated protein kinase known to be associated with cell survival and proliferation, and also via activation of the Akt–phosphatidyl inositol-3-kinase pathway.^105,106 Although these latter studies did not explicitly demonstrate a receptor-independent mechanism.^104-106

Although many findings support the role of S1P as an intracellular second messenger to promote cell survival and proliferation, there are several recent reports demonstrating that the prosurvival effect of S1P can also be mediated by S1PRs. Extracellular S1P in nanomolar concentrations inhibited cytokine-induced rat islet cell apoptosis, and this effect was mimicked by dihydro-S1P and, more importantly, was inhibited by the S1PR antagonist.²⁰ In ECs, antisense oligonucleotide to S1P₁ or S1P₃ receptor diminished the protective effect of S1P on cell survival.¹⁰⁷ Like the intracellular S1P effects on cell survival, downstream signaling events following these ligand–receptor interactions also seem to involve extracellular signal-regulated kinase 1/2 activation or Ca-dependent pathways. Indeed, intracellular S1P may be delivered extracellularly to activate G-protein-coupled receptor-mediated cell survival pathways. Although the mechanism of S1P’s export or inside-out signaling is not known, ATP binding cassette (ABC) transporters in mast cells may mediate S1P export.¹⁰⁸ Inhibition of an ABC family member (ABCC1) by either MK571, a selective inhibitor of ABCC1, or antisense to ABCC1 mRNA blocked S1P export.¹⁰⁸ In contrast, inhibition of ABCB1 (P-glycoprotein) did not block S1P export.¹⁰⁸ These studies underscore the complexity of S1P effects on cell survival. Evidence supports both an intracellular and extracellular role of S1P in mediating cell survival.

S1P EFFECTS ON ENDOTHELIAL AND EPITHELIAL BARRIERS

Cell barriers between different compartments are important in normal physiology. The EC barrier separates circulating vascular components from the interstitium, and disruption of the EC barrier results in increased vascular permeability with subsequent inflammation and organ dysfunction; in neoplastic disease, this is accompanied by neoangiogenesis and subsequent tumor metastasis. S1P maintains EC barrier integrity primarily by binding to S1P₁.^109,110 S1P₁ is necessary for maintaining the EC barrier; despite normal angiogenesis, pericyte deficiency-induced incomplete vascular maturation and intracranial hemorrhage result in embryonic lethality in S1P₁-knockout mice.⁶¹ On the other hand, S1P₁, S1P₂, and S1P₃ receptors have a redundant or cooperative function for development of a stable and mature vascular system during embryonic development.¹¹¹ S1P can induce a rapid increase in transmonolayer electrical resistance in the pulmonary microvascular endothelium, and this effect was markedly reduced by S1P₁ antisense oligonucleotide treatment.¹¹² Downstream signaling events following interaction of S1P–S1P₁ involve phosphatidyl inositol-3-kinase and Rac-1 activation with cortical actin rearrangement—events that may be critical to maintain cellular architecture.^59,112 Plasma S1P acting on S1P₁ receptors is important in maintaining skin and lung capillary integrity. Vascular administration of S1P₁ antagonist reversed the protection from vascular endothelial growth factor-induced leakage by selective S1P agonist in skin. S1P₁ antagonism also enhanced basal pulmonary capillary leakage, and this was prevented by the coadministration of S1P₁ agonist.¹¹³ Involvement of S1P₂ in endothelial permeability has also been demonstrated.

Activation of S1P₁ receptors expressed on EC is important in reducing permeability, hence maintaining the integrity of vascular EC barrier function, whereas S1P₂ receptor activation has the opposite effect. Activation of S1P₂ in ECs resulted in Rho-ROCK- and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability.¹¹⁴ JTE013, a selective S1P₂ antagonist, improved endothelial barrier integrity. These data provide evidence that the balance of S1P₁ and S1P₂ receptor activities regulates vascular permeability by S1P.

Human ECs also express S1P₃ in addition to S1P₁ receptors; however, the role of S1P₃ in endothelial barrier function has not been fully demonstrated. S1P–S1P₁ signaling couples to the G_i pathway with subsequent Rac activation, whereas S1P–S1P₃ signaling has been known to couple to G_i, G_q/11, and G_12/13 pathways and activate Rho GTPase instead of Rac GTPase. Rho GTPases mediate endothelial dysfunction and abnormal smooth muscle cell proliferation and are implicated in several diseases such as hypertension, atherosclerosis, vascular spasm, and bronchial asthma.^115,116 Consistent with the known function of Rho GTPase, silencing S1P₃ by small interfering RNA enhanced the transmonolayer electrical resistance and cortical actin, suggesting that S1P₃ functions as a negative regulator of S1P-induced endothelial barrier enhancement.¹¹⁷

Polarized epithelial cells form barriers that regulate the vectorial transport of various ions and solutes. In contrast to the barrier-enhancing effect of S1P–S1P₁ signaling in endothelium, less is known about the role of S1P in epithelial barrier function. However, a recent study suggests the importance of S1P₃ receptors in lung epithelial barrier disruption.⁶⁰ Intratracheal but not intravenous delivery of S1P induced acute pulmonary edema with disruption of tight junctions in wild-type but not in S1P₃ null mice. However, the role of S1P–S1PR signaling in other epithelial cell types or subsequent downstream signaling processes that mediate epithelial barrier disruption has not been demonstrated and needs to be elucidated.

S1P DRUG DEVELOPMENT

Before the realization that FTY720 is a prodrug and that the active form, phospho-FTY720, is an S1PR agonist, predictions of a useful S1PR drug centered on antagonists and, more recently, on SPHK inhibitors—the reasoning being that interdiction of the prosurvival, proangiogenesis functions of S1P could be valuable in the setting of neoplastic disease.¹¹⁸ The paucity of useful S1PR antagonists has heretofore prevented this idea from being tested in vivo. However, the recent demonstration that administration of an S1P₁ antagonist resulted in increased vascular permeability,^91,113 presumably by interfering with endogenous S1P tone, suggests that S1P₁ antagonists as a class might have a toxic liability. The utility, if any, of S1PR antagonists selective for other S1PR types awaits the development of such agents.

As mentioned previously, phospho-FTY720, acting as an agonist at lymphocyte S1P₁ receptors, evokes a long-lasting lymphopenia that is the result of disruption of normal lymphocyte trafficking from the secondary lymphoid tissue. (Curiously, S1P₁ receptor antagonists do not affect circulating lymphocyte levels (see above),^91,113 which suggests a lack of endogenous S1P tone on lymphocyte trafficking.) An explanation commonly offered to explain the efficacy of FTY720 in transplantation and autoimmune disease models is that by trapping lymphocytes in lymphoid tissues, effector T lymphocytes are directed away from sites of inflammation and allografts. However, FTY720 also causes a transient bradycardia in experimental animals and humans. In rodents, the bradycardia results from agonist activity at the S1P₃ receptor. These twin observations have prompted a search for S1P₁ receptor-selective agonists, and the screening of complex chemical libraries has apparently yielded a rich vein of such compounds. Although FTY720-dependent decrease in atherosclerosis was demonstrated,¹¹⁹ a fundamental, but as yet unanswered, question is whether efficacy of the non-selective FTY720 in a variety of disease models such as atherosclerosis can be duplicated by more selective agents. An early example of such an S1P₁-selective compound is SEW2871, which is structurally not related to sphingosine or FTY720. The scrutiny that the pharmaceutical industry is giving to S1P signaling will address whether more selective novel compounds have a similar efficacy to FTY720. Table 2 lists currently available S1P compounds and their sources.

Table 2.

S1P compounds

Compound	Activity	Reference	Commercial source
FTY720a	S1P1,3,4,5 agonist	Kiuchi et al.120	Cayman Chemical
AAL149, AAL151b	S1P1,3,4,5 agonist	Brinkmann et al.85	None
SEW2817c	S1P1 agonist	Sanna et al.121	Sigma-Aldrich
Compound 26d	S1P1 agonist	Li et al.122	None
VPC01091e	S1P1,4,5 agonist, S1P3 antagonist	Zhu et al.123	None
JTE013	S1P2 antagonist	Ohmori et al.124	Tocris
VPC23019	S1P1,3 antagonistf	Davis et al.125	Avanti Polar Lipids
VPC44116	S1P1,3 antagonistg	Foss et al.91	None
S1P (R isomer)h	S1P1 antagonist	Sanna et al.113	Avanti Polar Lipids
N,N-dimethylsphingosinei	SPHK inhibitorj	Yatomi et al.126	Avanti, others
Compounds II, IVk	SPHK inhibitor	French et al.127	Sigma-Aldrich
2-acetyl-4-tetrahydroxybutylimidazole (THI)	S1P lyase inhibitor	Schwab et al.128	None

S1P, sphingosine-1-phosphate; SPHK, sphingosine kinase.

^aOrally available prodrug, receptor active form is phospho-FTY720, K_d at S1P₁>1 nm.

^bEnantiomers of a FTY720 analog, only AAL151 is activated by SPHK2.

^cOrally available, ca. 2 log orders less potent than FTY720.

^dOrally available, more potent than SEW2871.

^eOrally available prodrug, FTY720 analog, very long lasting in vivo, selective SPHK2 substrate.

^fTen-fold more potent at S1P₁ receptor (K_i 30 nm) than S1P₃ receptor, very short lived in vivo.

^gPhosphonate analog of VPC23019, T_1/2 in rats 3.1 h.

^hPhosphonate analog of VPC23019.

ⁱVery low potency at some sphingosine kinase types.

^jNote that, unlike S1P receptors, sphingosine kinases differ widely among mammalian species, thus claims for inhibition should not be extrapolated to other species.

^kWe have been unable to verify claims for inhibitory activity of compound II in recombinant mouse or human sphingosine kinases.

S1P AND KIDNEY DISEASE

The biological functions of S1P have been explored in diverse cell types and tissues. However, the exact role of S1P in kidney physiology and pathophysiology remains unclear. S1P₁ receptors are expressed in the kidney¹²⁹ and play an important role in maintaining EC integrity^57,130 and in trafficking of lymphocytes.³ Recently, we demonstrated expression of S1P_1–4 receptors in whole mouse kidneys with a rank order of S1P₁>S1P₃>S1P₂>S1P₄, whereas S1P₅ mRNA was not found.¹³¹ Cultured mesangial cells also express S1P_1–5 receptors.¹³² However, whether the corresponding receptor subtypes are expressed at the protein level has not been investigated because of lack of antibodies.

The therapeutic relevance of S1P as a target in kidney disease has been explored with FTY720. FTY720 prolonged allograft survival in animal solid organ transplantation models,^133,134 mainly by eliciting lymphopenia resulting from a reversible redistribution of lymphocytes from the circulation to secondary lymphatic tissue. However, a recent phase III clinical trial using 2.5 or 5 mg of FTY720 failed to demonstrate an improvement in efficacy for the prevention of renal allograft rejection when combined with cyclosporine and mycophenylate. FTY720 (5 mg) combined with a reduced dose of cyclosporine was discontinued because of increased incidence of acute rejection and, more importantly, because FTY720 was associated with significantly lower creatinine clearance and several unexpected side effects that need to be further investigated.¹³⁵

S1P IN RENAL ISCHEMIA–REPERFUSION INJURY

The mechanisms involved in renal ischemia–reperfusion injury (IRI) are complex,^136,137 and both innate and adaptive immunity play a prominent role.^138,139 Following IRI, ECs and tissue-resident leukocytes are activated, leading to leukocyte infiltration.¹³⁹ Bone marrow-derived cells are important in mediating injury associated with IRI.¹⁴⁰ Neutrophil infiltration of kidneys subjected to IR is associated with injury.^{136,139,141-144} More recent studies support the role of macrophages^145,146 and T cells in the early antigen-independent inflammatory response following reperfusion.^143,147,148

Troncoso et al¹⁴⁹ demonstrated that FTY720 is protective following kidney IRI. We confirmed these observations and provided data on the optimal dose of FTY720 that confers renal tissue protection after IRI. The tissue-protective effect of FTY720 was associated with a decrease in lymphocyte, macrophage, and neutrophil infiltration into postischemic kidney, a result consistent with the prevailing view that inflammatory cell infiltration appears to play an important role in the pathogenesis of IRI.^139,142,150 Loss of significant protection from IRI with the highest doses of FTY720 suggested the possibility that S1PR subtypes may have opposing and/or different functional effects.

Using selective agonists and antagonists, we further demonstrated that the protective effect of FTY720 is due primarily to activation of S1P₁ receptors. Administration of VPC44116, a selective S1P₁ receptor antagonist, reversed FTY720-induced protective effects. Interestingly, the effect of VPC44116 in blocking FTY720 was not associated with reversal of lymphopenia induced by FTY720 treatment. The finding that VPC44116 blocked the protective effect of FTY720 suggests that FTY720-mediated protection is not consistent with the concept of FTY720-induced ‘functional antagonism.’ The possibility that FTY720 may have additional effects independent of lymphocytes cannot be excluded. Furthermore, the protective effect of FTY720 is mimicked by SEW2871, a selective S1P₁ agonist. SEW2871 induces renal tissue protection after IRI by inhibiting lymphocyte egress and reducing levels of the proinflamma-tory molecules tumor necrosis factor-α, P-selectin, and intercellular adhesion molecule-1.¹⁵¹ Whether the protective effect of FTY720 is due to the action on immune cells and/or kidney cells is not clear. However, a pronounced increase in S1P₁ mRNA expression occurring far before the peak of kidney leukocyte infiltration suggests a possible role of kidney tissue S1P₁ receptor activation that could be important in renal tissue protection. Activation of S1P₁ receptors on DCs reduces IL-12 and IL-10 production.¹⁵² IL-12 activates natural killer T cells,¹⁵³ which contribute importantly to the innate immune system. IL-10 is a potent anti-inflammatory cytokine and has been shown to protect kidneys from acute injury.¹⁵⁴ S1P₁ receptor activation in vascular ECs is implicated in angiogenesis, cell proliferation, stimulation of EC nitric oxide synthase enzyme,⁵⁷ and reduction of monocyte adhesion.¹⁵⁵ These effects on ECs may lead to maintenance of endothelial barrier function.¹⁵⁶ Our data confirm this hypothesis and show a beneficial effect of FTY720 to reduce vascular permeability following renal IRI.¹³¹ Whether the effect of FTY720 to preserve vascular permeability is due to a direct effect on ECs or due to an indirect effect through immune cells is not certain. The possible involvement of other S1PR subtypes by high doses of FTY720 is not clear in this study and needs to be explored. FTY720 also reduced cyclosporine A nephrotoxicity, and this protective effect was accompanied by reduced lymphocyte and macrophage infiltration and fibrosis.¹⁵⁷ The protective effect of FTY720 in a chronic kidney disease model, such as diabetic nephropathy, needs to be further explored, and it might provide a new therapeutic tool in retarding the progression of kidney disease.

S1P IN KIDNEY MESANGIAL CELLS

Mesangial cells play a key role in the pathogenesis of many kidney diseases such as glomerulonephritis or diabetic nephropathy by producing profibrotic cytokines and/or inducing extracellular matrix protein accumulation. S1P stimulates the proliferation of mesangial cells by activating extracellular signal-regulated kinase 1/2 pathways. The suppression of endogenous S1P₂ and S1P₃ by antisense oligonucleotides reduced the antiapoptotic effects of S1P, indicating that the antiapoptotic effect of S1P is mediated by S1P₂ and S1P₃ receptors in mesangial cells.¹⁵⁸ Mesangial cell S1P3 activation also mediates transforming growth factor-β and Smad protein activation by FTY720 with upregulation of connective tissue growth factor and collagen.¹⁵⁹ This study suggests that S1PR signaling in mesangial cells can be associated with fibrotic processes and progression of chronic kidney disease and needs to be further clarified in future studies.

CONCLUSION

S1P, through intracellular and extracellular effects, governs a number of cellular processes that determine cell fate. S1P acts as an intracellular mediator that counterbalances the effects of sphingosine and ceramide. The balance between these signaling mediators ultimately determines cell fate. In addition, S1P is a classic ligand for five membrane-bound G-protein-coupled receptor subtypes. The unique tissue distribution of the receptor subtypes and the differing signaling pathways and downstream cellular effects resulting from S1PR subtype activation underscore the need for discovery and testing of novel subtype-specific S1PR compounds for the treatment of a variety of disorders. Currently, S1PR compounds are being used in clinical trials for multiple sclerosis and in preclinical studies for a number of different disorders. Given the importance of the immune system in kidney disease, S1PR compounds hold great promise for the treatment of various kidney disorders. The treatment of acute kidney injury is an important priority given the high morbidity and mortality associated with this disorder and its less-recognized contribution to the prevalence of end-stage renal disease. S1PR analogs appear to be particularly attractive for the treatment of acute kidney injury.