Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has been implicated in regulating vascular tone and participating in chronic and acute kidney injury. However, little is known about the role of S1P in the renal microcirculation. Here, we directly assessed the vasoresponsiveness of preglomerular and postglomerular microvascular segments to exogenous S1P using the in vitro blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (0.001-10 mM) evoked concentration-dependent vasoconstriction in preglomerular microvessels, predominantly afferent arterioles. After administration of 10 mM S1P, the diameter of afferent arterioles decreased to 35%65% of the control diameter, whereas the diameters of interlobular and arcuate arteries declined to 50%612% and 68%66% of the control diameter, respectively. Notably, efferent arterioles did not respond to S1P. The S1P receptor agonists FTY720 and FTY720-phosphate and the specific S1P1 receptor agonist SEW2871 each evoked modest afferent arteriolar vasoconstriction. Conversely, S1P2 receptor inhibition with JTE-013 significantly attenuated S1P-mediated afferent arteriolar vasoconstriction. Moreover, blockade of L-type voltage-dependent calcium channels with diltiazem or nifedipine attenuated S1P-mediated vasoconstriction. Intravenous injection of S1P in anesthetized rats reduced renal blood flow dose dependently. Western blotting and immunofluorescence revealed S1P1 and S1P2 receptor expression in isolated preglomerular microvessels and microvascular smooth muscle cells. These data demonstrate that S1P evokes segmentally distinct preglomerular vasoconstriction via activation of S1P1 and/or S1P2 receptors, partially via L-type voltagedependent calcium channels. Accordingly, S1P may have a novel function in regulating afferent arteriolar resistance under physiologic conditions. 25: 177425: -178525: , 201425: . doi: 10.1681 Sphingosine 1-phosphate (S1P) is recognized as an important signaling molecule in diverse biologic processes. 1,2 Growing evidence indicates that S1P plays an important role in regulating vascular reactivity. 3-5 S1P is a bioactive sphingolipid metabolite and is released from erythrocytes, platelets, and endothelial cells. 6,7 The majority of S1P effects are mediated via five distinct receptors (S1P1-S1P5 receptors), which represent a family of small G proteincoupled receptors (GPCRs) 5 ; however, S1P can also exist in the cytoplasm as a second messenger involved in Ca 2+ mobilization or cell survival and proliferation. 8,9 S1P1-S1P3 receptors are expressed by a wide variety of tissues, whereas S1P4 and S1P5 receptors are mainly expressed in cells of the immune and nervous systems. 4,10 In the vasculature, endothelial cells mainly express S1P1 and S1P3 with variable expression of S1P2, whereas vascular smooth muscle cells express S1P2 and S1P3 with variable expression of S1P1. [3][4][5] Studies in animals show that application of exogenous S1P J Am Soc Nephrol

Inflammation contributes to ANG II-associated impairment of renal autoregulation and microvascular P2X1 receptor signaling, but its role in renal autoregulation in mineralocorticoid-induced hypertension is unknown. Autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. Hypertension was induced in uninephrectomized control rats (UNx) by subcutaneous implantation of a DOCA pellet plus administration of 1% NaCl in the drinking water (DOCA-salt) for 3 wk. DOCA-salt rats developed hypertension that was unaltered by anti-inflammatory treatment with pentosan polysulfate (DOCA-salt+PPS) but was suppressed with "triple therapy" (hydrochlorothiazide, hydralazine, and reserpine; DOCA-salt+TTx). Baseline arteriolar diameters were similar across all groups. UNx rats exhibited pressure-dependent vasoconstriction with diameters declining to 69 ± 2% of control at 170 mmHg, indicating intact autoregulation. DOCA-salt treatment significantly blunted this pressure-mediated vasoconstriction. Diameters remained between 91 ± 4 and 98 ± 3% of control over 65-170 mmHg, indicating impaired autoregulation. In contrast, pressure-mediated vasoconstriction was preserved in DOCA-salt+PPS and DOCA-salt+TTx rats, reaching 77 ± 7 and 75 ± 3% of control at 170 mmHg, respectively. ATP is required for autoregulation via P2X1 receptor activation. ATP- and β,γ-methylene ATP (P2X1 receptor agonist)-mediated vasoconstriction were markedly attenuated in DOCA-salt rats compared with UNx (P < 0.05), but significantly improved by PPS or TTx (P < 0.05 vs. DOCA-salt) treatment. Arteriolar responses to adenosine and UTP (P2Y2 receptor agonist) were unaffected by DOCA-salt treatment. PPS and TTx significantly reduced MCP-1 and protein excretion in DOCA-salt rats. These results support the hypothesis that hypertension triggers inflammatory cascades but anti-inflammatory treatment preserves renal autoregulation in DOCA-salt rats, most likely by normalizing renal microvascular reactivity to P2X1 receptor activation.

Sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of the preglomerular microvasculature. Our recent studies indicate that S1P-mediated afferent arteriolar vasoconstriction is enhanced in ischemia-reperfusion (IR) induced renal injury and S1P2 receptor protein expression is increased in renal microvessels. Reactive oxygen species (ROS) play a critical role in the development of IR renal injury. We hypothesized that inhibition of S1P2 receptor activation protects against renal IR injury via ROS reduction. Renal IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. In some rats, IR was induced during treatment with the selective S1P2 receptor antagonist, JTE-013 (0.1mg/kg BW, IP) prior to ischemia and repeated at the time of reperfusion. After reperfusion, plasma was collected and kidneys were harvested for histological analysis and ROS detection. Plasma creatinine (Cr) concentration was markedly increased in IR rats (4.2±0.1 mg/dl, n=6) compared to sham controls (1.1±0.1 mg/dl, n=5, p<0.05). JTE-013 treatment (n=4) significantly reduced plasma Cr in IR rats (2.0±0.3 mg/dl, p<0.05). Histological analysis revealed no significant cell damage in control kidney sections stained with hematoxylin and eosin whereas severe cell damage was detected in IR kidneys, including cell necrosis, disruption of the plasma membrane, nuclear pyknosis, cellular vacuolization, and the presence of luminal casts and sloughed cells in proximal tubules, particularly in the outer stripe of the outer medulla. Additionally, there was significant trapping of erythrocytes in the medulla. In contrast, JTE-013 treated IR rats showed less necrosis and cell damage in the proximal tubules. Renal ROS levels were assessed in frozen kidney sections using the fluorogenic probe, H 2 DCFDA. Fluorescence intensity was markedly increased in IR rats (18.7±2.4 in cortex and 19.0±2.5 in medulla) compared to control (10.9±0.7 and 9.6±0.7, p<0.05, respectively). JTE-013 treatment reduced ROS levels to values not different from control (13.5±1.1 and 15.4±1.5, p>0.05 vs. control, respectively). These data suggest that blockade of S1P2 receptors protects against renal IR injury by reducing tissue ROS accumulation.

Sphingosine-1-phosphate (S1P) is an important regulator of resistance vessels and is implicated in pathological processes including diabetic nephropathy and acute kidney injury. Previous studies show that S1P causes marked vasoconstriction in preglomerular but not postglomerular microvessels, however, the intracellular signaling pathways underlying S1P-mediated vasoconstriction are undefined. We postulated that S1P-mediated afferent arteriolar (AA) vasoconstriction involves activation of voltage-dependent L-type Ca 2+ channels (L-VDCC) and the rho kinase pathway. Studies were conducted in vitro using the blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (n=6) evoked concentration-dependent AA vasoconstriction. Control diameter averaged 13.8±0.9 μm and declined to 95±2, 85±4, 75±6, 60±5 and 46±5% of control diameter in response to S1P (from 10 -9 to 10 -5 M), respectively. Superfusion with nifedipine (10 -6 M, n=6), a L-VDCC inhibitor, increased basal diameter by 39±18% (p<0.05) and significantly attenuated the S1P-induced vasoconstriction. S1P reduced AA diameter to 99±1, 98±1, 90±3, 79±3 and 52±5% of control (p<0.05 vs. S1P alone at 10 -8 to 10 -6 M) during nifedipine treatment. Superfusion with the rho kinase inhibitor, Y27632 (10 -5 M, n=6), increased diameter by 60±12% and inhibited S1P-induced vasoconstriction. AA diameter averaged 103±1, 101±1, 93±3, 72±9 and 56±10% of control in response to increasing concentrations of S1P (p<0.05 vs. S1P alone at 10 -9 to 10 -7 M). In contrast, S1P-induced vasoconstriction was unaltered by Ca 2+ store depletion using the Ca 2+ -ATPase inhibitors, thapsigargin (Tg) and cyclopiazonic acid (CPA). Although Tg (10 -6 M, n=4) increased diameter by 38±16%, the S1P-induced vasoconstrictor profile was not different from that without Tg. AA declined to 99±3, 95±5, 85±10, 60±13 and 32±8% (p>0.05), respectively. Similar results were obtained with CPA (10 -5 M, n=6). S1P reduced diameter to 97±2, 93±2, 75±5, 53±6 and 36±5% of the control (p>0.05), respectively, suggesting that mobilization of intracellular Ca 2+ store is not required for S1P-induced vasoconstriction. Overall, these data indicate that both L-VDCC and rho kinase pathways contribute to S1P-mediated AA vasoconstriction.

Sphingosine‐1‐phosphate (S1P) potently vasoconstricts renal microvessels and participates in ischemia‐reperfusion (IR) induced acute kidney injury (AKI). We postulated that IR enhances renal vascular reactivity to S1P and contributes to AKI. Reactivity of juxtamedullary afferent arterioles (AA) to S1P was assessed in vitro in rats receiving 60 min bilateral ischemia followed by 24 hr reperfusion (B60IR) using the blood‐perfused juxtamedullary nephron technique (n=5–7/group). Plasma creatinine increased in B60IR (3.7±0.2 vs. 1.2±0.1 mg/dl in sham, p<0.05). Baseline AA diameter decreased in B60IR (11.8±0.7 vs. 14.6±0.6 μm in shams, p<0.05). S1P evoked concentration‐dependent AA vasoconstriction in both groups, however, the response was markedly enhanced in B60IR. S1P (10−10–10−5 M) reduced AA diameter to 93±1, 88±1, 76±1, 64±5, 46±5 and 35±4% of control in B60IR vs. 100±1, 94±2, 91±2, 81±4, 59±5 and 32±4%, respectively in shams. Conversely, AA responses to norepinephrine (NE) or KCl were unchanged. NE (10−8–10−6 M) reduced AA diameter to 92±2, 81±5 and 42±8% of control in B60IR compared to 92±2, 78±5 and 34±6% in shams. KCl (30, 60 and 90 mM) decreased AA diameter to 89±4, 51±6 and 40±7% in B60IR vs. 80±4, 38±4 and 32±4% in shams (p>;0.05). These data reveal that IR enhances AA reactivity to S1P and may contribute importantly to IR‐AKI.Funding sources: AHA10SDG3770010, DK44628 and HL074167

Afferent arterioles (AA) are highly responsive to purinoceptor stimulation. ATP contributes importantly to renal autoregulation by activating P2 receptors. Pressure‐mediated, and ATP and β,γ‐methylene ATP (P2X1 receptor agonist)‐induced AA vasoconstriction is impaired in DOCA‐salt hypertensive rats (DOCA rats) while the function of other purinoceptors is unclear. We postulated that P2Y and A1 receptor reactivity is intact in AA from DOCA rats. AA responses to UTP (a P2Y2 receptor agonist) and adenosine were studied in juxtamedullary nephrons. Systolic blood pressure averaged 125±5 and 180±7 mmHg in uninephrectomized (UNx) and DOCA rats, respectively. UTP (10−8 to 10−4 mol/L, n=5) evoked concentration‐dependent vasoconstriction of AA in UNx rats. Diameter declined by 2±1, 6±2, 12±2, 23±5 and 49±9%, respectively. UTP‐induced vasoconstriction was unchanged in DOCA rats (n=5, p>0.05) leading to declines of 3±2, 10±2, 14±3, 29±5 and 43±9%, respectively. The AA response to adenosine was also indistinguishable between two groups (n=6 each, p>0.05). Preglomerular microvascular P2X1 receptor protein expression was unaltered in DOCA rats. These data demonstrate that AA P2Y2 and A1 receptor signaling is preserved in DOCA rats. This study suggests that impairment of AA autoregulatory behavior in DOCA‐salt hypertension cannot be attributed to impaired P2Y2 and A1 receptor signaling.