Am J Physiol Renal Physiol 308: F1178–F1187, 2015. First published October 29, 2014; doi:10.1152/ajprenal.00079.2014.

Involvement of neutral sphingomyelinase in the angiotensin II signaling pathway Rocio Bautista-Pérez,1,2 Leonardo del Valle-Mondragón,3 Agustina Cano-Martínez,4 Oscar Pérez-Méndez,1 Bruno Escalante,5 and Martha Franco2 1

Department of Molecular Biology, Instituto Nacional de Cardiología I. Ch., Mexico City, Mexico; 2Department of Nephrology, Instituto Nacional de Cardiología I. Ch., Mexico City, Mexico; 3Department of Pharmacology, Instituto Nacional de Cardiología I. Ch., Mexico City, Mexico; 4Department of Physiology, Instituto Nacional de Cardiología I. Ch., Mexico City, Mexico; and 5CINVESTAV-Monterrey. Mexico City, Mexico Submitted 5 February 2014; accepted in final form 27 October 2014

Bautista-Pérez R, del Valle-Mondragón L, Cano-Martínez A, Pérez-Méndez O, Escalante B, Franco M. Involvement of neutral sphingomyelinase in the angiotensin II signaling pathway. Am J Physiol Renal Physiol 308: F1178 –F1187, 2015. First published October 29, 2014; doi:10.1152/ajprenal.00079.2014.—The possibility that angiotensin II (ANG II) exerts its effects through the activation of neutral sphingomyelinase (nSMase) has not been tested in kidneys. The results of the present study provide evidence for the activity and expression of nSMase in rat kidneys. In isolated perfused rat kidney, ANG II-induced renal vasoconstriction was inhibited by GW4869, an inhibitor of nSMase. We used nSMase for investigating the signal transduction downstream of ceramide. nSMase constricted the renal vasculature. An inhibitor of ceramidase (CDase), N-oleoylethanolamine (OEA), enhanced either ANG II- or nSMase-induced renal vasoconstriction. To demonstrate the interaction between the nSMase and cytosolic phospholipase A2 (cPLA2) signal transduction pathways, we evaluated the response to nSMase in the presence and absence of inhibitors of arachidonic acid (AA) metabolism: arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2; 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of all AA pathways; indomethacin, an inhibitor of cyclooxygenase (COX); furegrelate, a thromboxane A2 (TxA2)-synthase inhibitor; and SQ29548, a TxA2-receptor antagonist. In these experiments, the nSMase-induced renal vasoconstriction decreased. ANG II or nSMase was associated with an increase in the release of thromboxane B2 (TxB2) in the renal perfusate of isolated perfused rat kidney. In addition, the coexpression of the ceramide with cPLA2, was found in the smooth muscle layer of intrarenal vessels. Our results suggest that ANG II stimulates ceramide formation via the activation of nSMase; thus ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 and the release of TxA2. renal vasoconstriction; angiotensin II; sphingomyelinase; phospholipase A2; cyclooxygenase; thromboxane A2

of lipids that play essential roles as structural cell membrane components and also serve as substrates for enzymes that generate second messengers involved in cell signaling. Thus sphingolipids can be hydrolyzed by sphingomyelinases (SMases) (43). SMases are divided into three major classes, alkaline, acid, and neutral, according to the optimal pH for their activity, primary structure, and localization (15). Acid and alkaline SMases contribute to the hydrolysis of sphingomyelin in the luminal border of cells, in the extracellular space, or in the endosomal system, whereas neu-

SPHINGOLIPIDS ARE A FAMILY

Address for reprint requests and other correspondence: R. Bautista-Pérez, Juan Badiano No. 1, Sección XVI, Tlalpan, 14080, Mexico City, Mexico (e-mail: [email protected], [email protected]). F1178

tral SMase (nSMase) functions in the inner leaflet of the plasma membrane (32). SMases cleave the phosphodiester linkage of sphingomyelin to produce phosphocholine and ceramide. A recent study reported that increased levels of vascular ceramide lead to vasoconstriction due to increased thromboxane A2 (TxA2) release in the vessels of spontaneously hypertensive rats (SHR) (45). Along the same lines, several studies indicated that the abnormal production of TxA2 is linked to the pathophysiology of renal and ANG II-dependent hypertension (7, 26). Interestingly, treatment with the ANG II type 1 (AT1) receptor antagonist losartan has been shown to reduce vascular ceramide levels and prevent the SMase-induced contraction in isolated carotid arteries from SHR (45). In addition, several studies have reported that an accumulation of a sphingolipid, ceramide, its metabolites, or a combination contributes to the pathogenesis of renal injury (4, 5, 53, 54). Therefore, it is possible that the interaction among ANG II, ceramide, and TxA2 plays important roles in maintaining renal function and systemic blood pressure. In this regard, in blood vessels ANG II has also been reported to induce the activation of cPLA2, which is responsible for the release of arachidonic acid (AA) from cell membrane phospholipids. The released AA is metabolized by cyclooxygenase (COX) into prostaglandins and TxA2; these products modulate the vasoconstrictor response to ANG II in both renal and peripheral circulation to promote the maintenance of systemic pressure (6, 20). Interestingly, Huang et al. (16) suggested that cross talk may occur between the SMase and phospholipase A2 (PLA2) signal transduction pathways. Another study demonstrated that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates the cPLA2␣ action in renal mesangial cells (17). Therefore, one could speculate that ANG II stimulates ceramide formation via the activation of SMases; thus ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 in the kidney, and this mechanism may be important for the regulation of blood pressure, most likely by modulating vasoconstrictor responses. To test this hypothesis, we developed the following specific aims: 1) to verify the activity and expression of nSMase in the kidney, 2) to determine whether ANG IIinduced renal vasoconstriction depends on nSMase, 3) to evaluate the renal vascular response to SMase, and 4) to determine whether the SMase and cPLA2 signal transduction pathways interact in isolated perfused rat kidneys.

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MATERIALS AND METHODS

Rats were anesthetized using pentobarbital sodium (45 mg/kg ip), and the right renal artery was cannulated using a 19-gauge needle. The kidney was removed and placed in a water-jacketed chamber maintained at 37°C (Langendorff preparation) (2, 3). The kidney was then perfused with Krebs-Henseleit solution under constant flow (6 ml/ min) without recirculation using an infusion pump (SC-Q 403U/VM4, Watson-Marlow, Wilmington, MA) to obtain a basal perfusion pressure of 100 mmHg. The Krebs-Henseleit solution (pH 7.4, 37°C) contained the following components: 118.5 mmol/l NaCl, 4.8 mmol/l KCI, 2.5 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 25 mmol/l NaHCO3, 5 mmol/l glucose, and 0.25% BSA. The perfusion pressure was continuously monitored using a pressure transducer (model PT300, Grass Instruments, West Warwick, RI) and recorded on a polygraph (model 7D, Grass Instruments, Quincy, MA). Because the flow was maintained at a constant rate, changes in the perfusion pressure were used as an index of changes in the resistance of the renal vasculature. A decrease in perfusion pressure indicated vasodilatation, whereas an increase in perfusion pressure indicated vasoconstriction. The responses are expressed as absolute changes (⌬) in perfusion pressure (in mmHg) from basal pressure.

Materials

Experimental Protocol

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Fig. 1. Enzymatic activity of neutral sphingomyelinase (nSMase) in the medulla and cortex of the rat kidney. Each bar represents the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.01 medulla vs. cortex.

Amplex red, an nSMase antibody (sc-26215), ␤-actin, cPLA2 antibody (sc-376618), anti-␣-smooth muscle actin (␣-SM actin; sc130616), donkey anti-goat IgG-TR, donkey anti-goat IgG-FITC, goat anti-mouse IgG-FITC, and goat anti-mouse IgG-TR were used (Santa Cruz Biotechnology, Santa Cruz, CA). Enhanced chemiluminescence detection solution was used (GE Healthcare, Pittsburgh, PA; Vectashield, Vector Laboratories). Horseradish peroxidase (HRP), choline oxidase, alkaline phosphatase, sphingomyelin, ANG II, GW4869, dithiothreitol (DTT), SMase (from B cereus), N-oleoylethanolamine (OEA), arachidonyl trifluoromethyl ketone (AACOCF3), 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, and a ceramide antibody were purchased from Sigma (St. Louis, MO). Dibromo-dodecenyl-methylsulfimide (DDMS), furegrelate, and SQ29548 were purchased from Cayman Chemical (Ann Arbor, MI). Male Wistar rats (250 –320 g) were housed in institutional animal facilities with free access to food and water. All studies were performed in accordance with the Mexican Federal Regulation for Animal Experimentation and Care (NOM-062- ZOO-1999, published in 2001) and approved by the Bioethics and Investigation Committees of Instituto Nacional de Cardiologia I. Ch.

The following seven series of experiments was performed in the isolated perfused rat kidney. Protocol 1. To evaluate whether ANG II induces SMase activity, experiments were conducted in the presence and absence of GW4869 (10 mM), an inhibitor of nSMase, or DTT (1 mM), an inhibitor of aSMase. The inhibitors were added to the perfusing Krebs-Henseleit solution 30 min before the administration of ANG II (28, 31). ANG II

β-actin nSMase

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nSMase activity was estimated as described previously by Walters and Wrenn (48). The reaction mixture contained the following components in a total volume of 200 ␮l: 25 ␮g renal homogenate proteins in 100 mM Tris·HCl, pH 7.4, 10 mM MgCl2, 100 ␮M Amplex red, 2 U/ml HRP, 0.2 U/ml choline oxidase, 8 U/ml alkaline phosphatase, and 500 ␮M sphingomyelin. The reaction mixture was incubated at 37°C for 30 min. Fluorescence was measured using a microplate reader (Laurier Research Instrumentation, Otego, NY) with excitation at 545 nm and emission at 590 nm.

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Western Blotting to Detect nSMase Forty micrograms of protein was separated by SDS-PAGE on a 12% polyacrylamide gel and transferred onto a nitrocellulose membrane. Then, the membrane was washed and probed using a polyclonal antibody against nSMase (1:1,000) and donkey anti-goat IgGHRP (1:1,000). Finally, enhanced chemiluminescence detection solution was added, and the membrane was exposed to Kodak Omat film.

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Fig. 2. nSMase protein expression level in the medulla and cortex of the rat kidney. Each bar represents the mean ⫾ SE; n ⫽ 5. *P ⬍ 0.01 medulla vs. cortex.

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ANG II Fig. 3. Effect of either GW4869 (10 mmol/l), an inhibitor of nSMase, or DTT (1 mmol/l), an inhibitor of aSMase, on ANG II-induced renal vasoconstriction. Each bar represents the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.01 ANG II⫹GW4869 vs. ANG II and ANG II⫹DTT.

was administered as random bolus doses (1, 2, 4, 8, and 16 ng) that were added to the perfusate line just proximal to the kidney. Protocol 2. We used exogenous nSMase as a tool for investigating the signal transduction that is downstream of ceramide. nSMase (0.4 U) was administered as a bolus injection into the perfusate line of the isolated rat kidneys (36). To further examine whether the enzymatic action of nSMase is essential for the regulation of renal vascular tone,

we also examined the effects of heat-inactivated nSMase (56°C for 30 min) on isolated perfused rat kidneys. Protocol 3. To examine the possibility that ceramide may cause vasoconstriction through intracellular conversion into sphingosine, we evaluated the response to either ANG II (4 ng) or nSMase (0.4 U) in the presence and absence of OEA, an inhibitor of ceramidase (10⫺4 mM). OEA was added to the perfusing Krebs-Henseleit solution 30 min before the evaluation of the response to either ANG II or nSMase (22). Protocol 4. To demonstrate that nSMase catalyzes the hydrolysis of sphingolipids to release ceramide, which can interact with cPLA2␣ to generate AA and eicosanoids, we evaluated the response to nSMase (0.4 U) in the presence and absence of AACOCF3 (1 ␮M), an inhibitor of cPLA2␣; ETYA (1 ␮M), an inhibitor of all AA pathways; DDMS (10 ␮M), a selective inhibitor of the formation of 20-hydroxyeicosatrienoic acid (20-HETE); indomethacin, an inhibitor of COX (10⫺5 M); furegrelate, a TxA2-synthase inhibitor (10⫺5 M); and SQ29548, a TxA2 receptor antagonist (10⫺6 M). The inhibitors were added to the perfusing KrebsHenseleit solution 30 min before the administration of nSMase. Additionally, we evaluated the response to ANG II (4 ng) in the presence and absence of AACOCF3 or SQ29548 (1, 24, 40). Protocol 5. To evaluate the response to a small dose of ANG II in the presence of GW4869 (10 mM), AACOCF3, ETYA, indomethacin, and furegrelate, the inhibitors were added to the Krebs-Henseleit solution 30 min before the administration of ANG II (10⫺10 M). ANG II was added to the Krebs-Henseleit solution and was infused for 30 min, and the response was measured for time. Protocol 6. To evaluate whether other agonists produce vasoconstriction via the nSMase/ceramide/TxA2 pathway, we evaluated the vascular reactivity to a continuous infusion of either ET-1 or NE in isolated perfused rat kidneys in presence of GW4869, this was added

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Fig. 4. A: typical tracing of either nSMase- or ANG II-induced renal vasoconstriction (increased perfusion pressure) in isolated perfused rat kidneys. SMase (0.4 U) and ANG II (4 ng) were administered as a bolus in the absence and presence of N-oleoylethanolamine (OEA). B and C: effect of OEA, an inhibitor of ceramidase, on either the nSMase- or ANG II-induced renal vasoconstriction. Each bar shows the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.01 SMase⫹OEA vs. SMase, ANG II⫹OEA vs. ANG II.

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to the Krebs-Henseleit solution for 30 min before and 30 min after the beginning of an infusion of either norepinephrine (NE) or endothelin (ET-1; 10⫺10 M). Protocol 7: release of TxA2 in isolated perfused rat kidney. The short half-life of TxA2 impedes its measurement at physiological concentrations. Consequently, the measurement of TxB2, which has a half-life of 20 –30 min, is accepted as an indicator of TxA2 production. Thus, after a bolus injection of either ANG II (4 ng) or nSMase (0.4 U), the renal perfusate was collected over 5 min; during this time, the response to either ANG II or nSMase (0.4 U) was increasing and reaching the maximum renal vasoconstriction. The samples were stored at ⫺20°C, and TxB2 was measured using a specific enzymatic immunoassay kit (Cayman Chemical) according to the manufacturer’s instructions. The levels of TxB2 are expressed in picograms per milliliter (pg/ml) (33). Immunofluorescence and Confocal Microscopy After the stimulation with ANG II, the kidneys were used for immunofluorescence. The kidney sections were incubated with 10% blocking serum in PBS for 1 h at room temperature. After blocking, the tissue slices were incubated at 4°C overnight with the following primary antibodies (1:500): nSMase, neutral ceramidase (CDase), ceramide, or cPLA2. After incubation, the sections were washed and incubated with the secondary antibody (donkey anti-goat IgG-FITC, goat anti-mouse IgG-FITC) for 4 h at room temperature. The tissues were costained with anti-␣-SM mouse antibody to confirm that the nSMase, nCDase, ceramide, or cPLA2 was expressed in the vascular smooth muscle cells. Negative controls were run using an identical protocol but excluding the primary antibody. For the dual immunostaining of the ceramide with cPLA2, the sections was processed as described above. After incubation, the sections were washed and incubated with the secondary antibody (donkey anti-goat IgGFITC, donkey anti-goat IgG-TR, goat anti-mouse IgG-FITC, and goat anti-mouse IgG-TR) for 4 h at room temperature. The tissues were SMase

A

mounted on microscope coverslips using Vectashield. Signals were examined under an FV1000 Confocal Laser Scanning Biological Microscope. Image analysis was performed using ImageJ software (Image J 1.36b, National Institutes of Health). Statistical Analysis Data are presented as means ⫾ SE. Student’s t-test or one-way ANOVA with Bonferroni’s posttest for multiple comparisons was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The null hypothesis was rejected when P ⬍ 0.05. RESULTS

SMase Activity Our aim was to first verify the activity and expression of nSMase in the kidney and then to evaluate whether nSMase can be stimulated by ANG II in an isolated perfused rat kidney. The enzymatic activity of nSMase was measured in the medulla and cortex of the rat kidney. The enzymatic activity in the medulla was higher than that in the cortex (Fig. 1). SMase Expression Western blot analysis revealed that the expression of nSMase was significantly higher in the renal medulla than in the cortex (Fig. 2). Vascular Response to ANG II in Isolated Perfused Rat Kidney When ANG II was administered as random bolus doses (1, 2, 4, 8, and 16 ng), the multiple doses of ANG II increased the renal perfusion pressure in a dose-dependent manner in the ANG II (4 ng)

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Fig. 5. A: effect of inhibitors of arachidonic acid metabolism on nSMase-induced renal vasoconstriction. B: effect of AACOCF3, an inhibitor of cPLA2, and SQ29548, a thromboxane A2 (TxA2) receptor antagonist, on ANG II-induced renal vasoconstriction. Each bar shows the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.001 with vs. without AACOCF, EYTA, DDMS, indomethacin, furegrelate, or SQ29548. AJP-Renal Physiol • doi:10.1152/ajprenal.00079.2014 • www.ajprenal.org

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In this study, we used exogenous nSMase as a tool for investigating the signal transduction that is downstream of ceramide. A bolus injection of nSMase (0.4 U) increased the renal perfusion pressure by 30 ⫾ 1.9 mmHg in the isolated perfused rat kidneys; the responses to the bolus injections were transient and not sustained (Fig. 4). To further examine whether the enzymatic action of nSMase is essential for inducing renal vasoconstriction, we also examined the effects of heat-inactivated nSMase on isolated perfused rat kidney. Heatinactivated nSMase had no effect on rat vascular tone, indicating that the intact enzymatic action of nSMase is required for renal vasoconstriction.

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To examine the possibility that ceramide may cause vasoconstriction through an intracellular conversion into sphingosine, we evaluated the response to either nSMase (0.4 U) or ANG II (4 ng) in the presence and absence of OEA, an inhibitor of ceramidase. The renal perfusion pressure from basal was not affected by OEA alone, but the inhibition of the ceramidase with OEA enhanced the nSMase-induced renal vasoconstriction (Fig. 4A) and the ANG II-induced renal vasoconstriction (Fig. 4B). Accordingly, we believe that in the isolated perfused rat kidney, the ceramide levels increase in response to nSMase and ANG II.

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Fig. 6. A: typical tracing of ANG II-induced renal vasoconstriction in isolated perfused rat kidneys. The inhibitors were added to the Krebs-Henseleit solution 30 min before the administration of ANG II (10⫺10 M). B: effect of inhibitors of either GW4869 or arachidonic acid metabolism on ANG II-induced renal vasoconstriction. Each bar shows the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.001 with vs. without GW4869, AACOCF, EYTA, indomethacin, or furegrelate.

Interaction Between the SMase and cPLA2 Signal Transduction Pathways in Isolated Perfused Rat Kidneys To demonstrate that nSMase catalyzes the hydrolysis of sphingolipids to release ceramide, which interacts with cPLA2␣ to generate AA and eicosanoids, we evaluated the response to nSMase in the presence and absence of AACOCF3, ETYA, DDMS, indomethacin, furegrelate, or SQ29548. The pretreatment with either AACOCF3 or ETYA inhibited the nSMaseinduced renal vasoconstriction. DDMS did not affect the nSMase-induced renal vasoconstriction. These results suggest that the 20-HETE pathway cannot explain the nSMase-induced

isolated perfused rat kidneys, and for every dose, the responses were transient and not sustained; therefore, the response time was different. Interestingly, the ANG II-induced renal vasoconstriction was inhibited by GW4869. Furthermore, the use of DTT did not alter the effect of ANG II on isolated perfused rat kidneys (Fig. 3). In addition, GW4869 alone did not change the basal perfusion pressure remained at a steady level (100 –110 mmHg).

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Fig. 7. Production of thromboxane B2 measured after the administration of either SMase (0.4 U; A) or ANG II (4 ng; B) in isolated perfused rat kidneys in the presence and absence of furegrelate, a thromboxane A2-synthase inhibitor. Each bar shows the mean ⫾ SE; n ⫽ 10. *P ⬍ 0.01 SMase⫹ furegrelate and SMase vs basal, ANG II⫹furegrelate and ANG II vs. basal. #P ⬍ 0.01 SMase⫹furegrelate vs. SMase, ANG II⫹furegrelate vs. ANG II.

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vasoconstriction in the isolated perfused rat kidney. The pretreatment with indomethacin decreased the nSMase-induced renal vasoconstriction; these results suggest that renal vasoconstriction depends on the COX pathway (Fig. 5A). In light of the observation concerning indomethacin, we decided to investigate the role of TxA2 as a potential mediator of the vasoconstriction. When we applied either furegrelate (a TxA2-synthase inhibitor) or SQ-29,548 (a TxA2 receptor antagonist), both diminished the nSMase-induced renal vasoconstriction (Fig. 5A). In addition, when we evaluated the response to ANG II in the presence and absence of AACOCF3, ETYA, indomethacin, and furegrelate, all diminished the ANG II-induced renal vasoconstriction (Figs. 5B). In addition, infusion of ANG II (10⫺10 M) increased perfusion pressure by 29 ⫾ 1.7 mmHg and was sustained. The presence of GW4869, AACOCF3, ETYA, indomethacin, or furegrelate in the perfusion solution reduced the maximal ANG II response by 60% (Fig. 6). Our results suggest that either nSMase or ANG II stimulates TxA2 synthesis in renal tissues. To test whether other agonists produce vasoconstriction via the nSMase/ceramide/TxA2 pathway, we evaluated the vascular reactivity to a continuous infusion of either ET-1 or NE in isolated perfused rat kidneys in the presence of GW4869. ET-1 and NE increased perfusion pressure by 38 ⫾ 3 mmHg (P ⬍

0.01) and 31 ⫾ 4 mmHg (P ⬍ 0.01), respectively. The renal vascular reactivity to either ET-1 or NE was not attenuated by GW4869. These results showed that ET-1 and NE are not associated with the activation of nSMase in the renal vasculature. TxB2 Production in Isolated Perfused Rat Kidneys in Response to Either ANG II or nSMase To demonstrate that either ANG II or nSMase stimulates TxA2 synthesis in kidneys, we measured TxB2 production in isolated perfused rat kidneys in response to either ANG II or nSMase. The bolus injection of either nSMase (0.4 U) or ANG II (4 ng) was also associated with an increase in the release of TxB2, the TxA2 metabolite; TxB2 production was inhibited by furegrelate (Fig. 7). Immunofluorescence Using Confocal Microscopy After the stimulation with ANG II, the kidneys were used for immunofluorescence. Representative images show the immunofluorescence staining for nSMase, nCDase, ceramide, cPLA2 (green), and vascular SM-␣-actin (red) and the colocalization of nSMase, nCDase, ceramide, or cPLA2 with vascular smooth muscle cell

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Fig. 8. Immunofluorescence staining of nSMase in the renal cortex (A), nSMase in the renal medulla (B), and ceramidase (CDase) in the renal cortex (C). Representative images show immunofluorescence staining for the either nSMase or CDase enzymes (green), and vascular smooth muscle ␣-actin (red), as well as the colocalization of either nSMase or CDase enzymes and vascular smooth muscle cell (yellow or orange) expression. AJP-Renal Physiol • doi:10.1152/ajprenal.00079.2014 • www.ajprenal.org

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(yellow or orange) expression Figure 9C also shows a section from a kidney doubly labeled with ceramide and cPLA2; coexpression also can be observed in the intrarenal vessels. (Figs. 8 and 9). DISCUSSION

In the present study, our results provide evidence of the activity and expression of nSMase in rat kidneys. Interestingly, it has been reported that treatment with the AT1 receptor antagonist losartan reduces vascular ceramide levels and prevents SMase-induced contraction in isolated carotid arteries from SHR (47). However, the possibility that ANG II exerts some of its effects through the activation of nSMase has not been determined in the vasculature. When we evaluated the response to ANG II in isolated perfused rat kidneys, we found that the ANG II-induced renal vasoconstriction was inhibited by GW4869. These results suggest that ANG II stimulates ceramide formation via the activation of nSMase. In addition, when we evaluated the response to exogenous nSMase in isolated perfused rat kidneys, we found that it induced renal

vasoconstriction. Therefore, it is possible that ceramide may regulate renal vasoconstriction. This observation is consistent with studies that demonstrated that treatment with ceramide and/or SMase leads to contraction in isolated vessels (28, 36, 55, 56). In contrast, other studies have shown that ceramide induces vasorelaxation (endothelium dependent and endothelium independent) (10, 21–23, 32). The reason for this discrepancy is unknown. In this study, it was not possible to evaluate the renal vascular response to exogenous ceramide because ceramide is not soluble in Krebs-Henseleit solution. However, it is also important to consider whether ceramide can be converted to sphingosine by CDase (47a). In this regard, several studies have demonstrated that sphingosine elicited vasoconstriction in coronary arteries and caused relaxation in aortic rings as well as in pulmonary artery rings (17, 36). In addition, it has been reported that OEA is a potent inhibitor of CDase in the kidney (34, 47a). In this study, we also demonstrate the expression of CDase in intrarenal vessels. Therefore, to investigate the role of sphingolipid metabolites, especially

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C Ceramide

Fig. 9. Immunofluorescence staining of ceramide (green; A) and cortical (c) PLA2 (green; B). Representative images show immunofluorescence staining for either ceramide or cPLA2 and vascular smooth muscle ␣-actin (red), as well as the colocalization of either ceramide or cPLA2 and vascular smooth muscle cell (yellow or orange) expression. C: ceramide (green), cPLA2 (red), as well as the colocalization of ceramide and cPLA2 (yellow or orange) expression. The arrow indicates the colocalization of ceramide and cPLA2 on the renal vasculature. AJP-Renal Physiol • doi:10.1152/ajprenal.00079.2014 • www.ajprenal.org

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sphingosine, in mediating vasoconstriction, we utilized an inhibitor of CDase, OEA. In these experiments, we observed that either ANG II-induced renal vasoconstriction or nSMaseinduced renal vasoconstriction was enhanced in the presence of OEA; these results suggest that the inhibition of CDase produces an accumulation of ceramide in isolated perfused rat kidneys. Therefore, sphingosine is not involved in the renal vasoconstriction stimulated by either ANG II or nSMase. However, it has been reported that OEA causes vasorelaxation partly via an endothelium-dependent mechanism (14). More recently, attention has turned to a possible role of COX metabolites in the OEA actions in the vasculature (49). Interestingly, a recent study reported that the SMase-induced ceramide increase in the carotid artery of SHR leads to vasoconstriction via a PLA2, COX-1, and thromboxane synthasedependent mechanism (46). On the other hand, it has been reported that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates the cPLA2␣ action in renal mesangial cells (19). To clarify the possible involvement of cPLA2 activation in SMase-induced renal vasoconstriction, we tested the effect of AACOCF3, an inhibitor of cPLA2␣. Our data show that the presence of AACOCF3 diminished either the SMaseinduced renal vasoconstriction or the ANG II-induced renal vasoconstriction by ⬃50%. Interestingly, Leis et al. (27) reported that AACOCF3 is also a potent inhibitor of COXs. This observation and our results suggest that ceramide can regulate the production of eicosanoids. To determine whether the SMase-induced renal vasoconstriction depends on AA metabolism, we tested the effects of ETYA, an inhibitor of all AA pathways, DDMS, a selective inhibitor of the formation of 20-HETE, and indomethacin, an inhibitor of COX. We observed that ETYA and indomethacin diminished the SMaseinduced renal vasoconstriction. Accordingly, we believe that the ANG II/nSMase/ceramide-PLA2 pathway liberates AA, and free AA serves as a substrate for the COX pathway. COX converts AA to prostaglandin endoperoxide H2 (PGH2), the precursor of the prostaglandins (PGE2, PGI2) and the prostanoids (TxA2). In this regard, it has been reported that the prostaglandins PGE2 and PGI2 play a critical role in buffering ANG II-mediated constriction (41). In addition, it has been reported that COX inhibition usually potentiates the vasoconstrictor actions of pressor hormones by removing the modulatory influence of PGE2 and/or PGI2 (11). In the present study, the indomethacin treatment did not potentiate the nSMaseinduced renal vasoconstriction. Therefore, indomethacin decreases nSMase-induced renal vasoconstriction by abolition of the release of vasoconstrictor prostanoids, TxA2 and/or PGH2. However, a recent study reported that indomethacin inhibits endothelial nitric oxide synthase (eNOS) activity (37). On the other hand, it has been reported that TxA2 is a major product of AA metabolism via the COX pathway and is produced in small quantities by the kidney. Functionally, TxA2 acts on the TP receptor (TxA2 receptor) to induce smoothmuscle contraction (40). Therefore, to determine whether TxA2 is involved in SMase-induced renal vasoconstriction, we tested the effects of either furegrelate (an inhibitor of thromboxane synthase) or SQ29548 (a TxA2 receptor antagonist). Both furegrelate and SQ29548 pretreatment decreased the SMaseinduced renal vasoconstriction. When we evaluated the response to ANG II, the presence of either furegrelate or

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SQ29548 diminished the ANG II-induced renal vasoconstriction. In addition, furegrelate decreased the production of TxA2 in isolated perfused rat kidneys in response to either ANG II or nSMase. These results showed that either the SMase-induced renal vasoconstriction or the ANG II-induced renal vasoconstriction involves the production of TxA2. In this regard, other studies have also reported that TxA2 is synthesized in the kidney in response to ANG II (12, 39, 50, 51). Nevertheless, it is important to note that the ceramide/TxA2 pathway contributes 50% of the renal vasoconstriction produced by either nSMase or ANG II; therefore, our results suggest that other mechanisms can explain the renal vasoconstriction produced by either nSMase or ANG II/AT1/nSMase. With respect to this possibility, it has been reported that the activation of nSMase leads to the generation of ceramide, which, in turn, may activate other proteins, such as PKC␨, KV channels, Rho-kinase, voltage-independent Ca2⫹ channels, protein phosphatase 2A (PP2A), or NADPH oxidase (9, 28, 45, 56 –58). On the other hand, it is well known that the activation of the AT1 receptor in smooth muscle cells results in phospholipase C (PLC) activation, leading to the generation of the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates intracellular Ca2⫹ release from the sarcoplasmic reticulum, and DAG causes PKC activation. Additionally, different Ca2⫹ entry channels, such as voltageoperated (VOC), receptor-operated (ROC), and store-operated (SOC) Ca2⫹ channels, as well as Ca2⫹-permeable nonselective cation channels (NSCC), are involved in the elevation of intracellular Ca2⫹ concentration. The elevation in intracellular Ca2⫹ is transient and initiates contractile activity via a Ca2⫹calmodulin interaction, stimulating myosin light chain (MLC) phosphorylation. When the Ca2⫹ concentration begins to decline, the RhoA/Rho-kinase (ROCK) pathway induces Ca2⫹ sensitization in the contractile proteins, thus inhibiting the dephosphorylation of MLC phosphatase (MLCP) and maintaining force generation. In cases in which NO production is

Fig. 10. Diagram illustrating the proposed signaling cascade involved in renal vasoconstriction. COX, cyclooxygenase; AA, arachidonic acid; AT1R, ANG II type 1 receptor. ANG II stimulates ceramide formation via the activation of nSMases. Thus ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 and release thromboxane A2.

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significantly decreased and/or reactive oxygen species (ROS) levels are increased, the activation of RhoA/Rho-kinase may contribute to increased vascular tone (29, 30, 52). Furthermore, Src-dependent pathways and nicotinic acid adenine dinucleotide phosphate (NAADP), another second messenger generated by the enzyme ADP-ribosyl (ADPR) cyclase, may contribute to the Ca2⫹ signaling of ANG II (8, 13, 14, 29, 30). Furthermore, to support the hypothesis that the ANG II/AT1 receptor/nSMase/ceramide-PLA2/TxA2 pathway contributes to the regulation of renal vasoconstriction, we demonstrated the expression of nSMase, ceramide, and cPLA2 in the smooth muscle layer of intrarenal vessels. Accordingly, we believe that AT1 receptor and nSMase, as well as ceramide and cPLA2, may interact in the kidney. Recent studies have indicated that other vasoconstrictor peptides, such as ET-1, and vasodilator peptides, such as bradykinin (BK), activate nSMase. In this regard, it has been reported that ET-1 stimulates nSMase activity through a p38MAPK-dependent pathway, leading to vascular cell adhesion molecule (VCAM-1) expression (38). The activation of the BK-B1 receptor leads to the activation of SMase, and the generated ceramide induces vasodilatation in rat mesenteric small arteries (25). In this regard, we also found that the activation of the BK-B1 receptor produced vasodilation in isolated perfused kidneys (3). Interestingly, the pathophysiological actions of ANG II are mediated by G protein-coupled receptors; therefore, it is possible that other constrictor agonists (e.g., ET-1, NE) could induce renal vasoconstriction through the nSMase/ceramide/ TxA2 pathway. In this study, to test whether other agonists produce vasoconstriction via the nSMase/ceramide/TxA2 pathway, we evaluated the vascular reactivity to a continuous infusion of either ET-1 or NE in isolated perfused rat kidneys. Our results indicate that the renal vascular reactivity to either ET-1 or NE was not attenuated by GW4869. These results showed that ET-1 and NE are not associated with the activation of nSMase in the renal vasculature. Additional studies are required to determine whether other agonists produce vasoconstriction via the nSMase/ceramide/TxA2 pathway. In summary, this study shows that ANG II stimulates ceramide formation via the activation of nSMase, and thus the study shows that ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 and release TxA2 in isolated perfused rat kidneys (Fig. 10). Thus the activation of SMase by ANG II may contribute to the development of vascular dysfunction; therefore, nSMase inhibitors could be tools for the treatment of renal diseases. ACKNOWLEDGMENTS Part of this work was presented at Cell Signaling Networks 2011 in Mérida, México, and at Experimental Biology 2014 in San Diego, CA. GRANTS This study was supported in part by the Instituto Nacional de Cardiología I. Ch (registered project No 12–766) to R. Bautista-Pérez and by financial support from CONACYT Grant 169736, Mexico to A. Cano-Martínez. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: R.B.-P. provided conception and design of research; R.B.-P., L.d.V.-M., and A.C.-M. performed experiments; R.B.-P., O.P.-M.,

B.E., and M.F. analyzed data; R.B.-P., L.d.V.-M., O.P.-M., and B.E. interpreted results of experiments; R.B.-P. prepared figures; R.B.-P. drafted manuscript; R.B.-P. and B.E. edited and revised manuscript; R.B.-P., L.d.V.-M., A.C.-M., O.P.-M., B.E., and M.F. approved final version of manuscript. REFERENCES 1. Awumey EM, Hill SK, Diz DI, Bukoski RD. Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2⫹-induced relaxation of rat mesenteric arteries. Am J Physiol Heart Circ Physiol 294: H2363– H2370, 2008. 2. Bautista R, Sánchez A, Hernández J, Oyekan A, Escalante B. Angiotensin II type AT2 receptor mRNA expression and renal vasodilatation are increased in renal failure. Hypertension 38: 669 –673, 2001. 3. Bautista-Pérez R, Arellano A, Franco M, Osorio H, Coronel I. Enalaprilat-mediated activation of kinin B1 receptors and vasodilation in the rat isolated perfused kidney. Pharmacology 87: 195–203, 2011. 4. Boini KM, Xia M, Li C, Zhang C, Payne LP, Abais JM, Poklis JL, Hylemon PB, Li PL. Acid sphingomyelinase gene deficiency ameliorates the hyperhomocysteinemia-induced glomerular injury in mice. Am J Pathol 179: 2210 –2219, 2011. 5. Boini KM, Zhang C, Xia M, Poklis JL, Li PL. Role of sphingolipid mediator ceramide in obesity and renal injury in mice fed a high-fat diet. J Pharmacol Exp Ther 334: 839 –846, 2010. 6. Brash AR. Arachidonic acid as a bioactive molecule. J Clin Invest 107: 1339 –1345, 2001. 7. Chatziantoniou C, Arendshorst WJ. Angiotensin and thromboxane in genetically hypertensive rats: renal blood flow and receptor studies. Am J Physiol Renal Fluid Electrolyte Physiol 261: F238 –F247, 1991. 8. Che Q, Carmines PK. Src family kinase involvement in rat preglomerular microvascular contractile and [Ca2⫹]i responses to ANG II. Am J Physiol Renal Physiol 288: F658 –F664, 2005. 9. Cogolludo A, Moreno L, Frazziano G, Moral-Sanz J, Menendez C, Castañeda J, González C, Villamor E, Perez-Vizcaino F. Activation of neutral sphingomyelinase is involved in acute hypoxic pulmonary vasoconstriction. Cardiovasc Res 82: 296 –302, 2009. 10. Czyborra P, Saxe M, Fetscher C, Meyer Zu Heringdorf D, Herzig S, Jakobs KH, Michel MC, Bischoff A. Transient relaxation of rat mesenteric microvessels by ceramides. Br J Pharmacol 135: 417–426, 2002. 11. Dunn M. The role of arachidonic acid metabolites in renal homeostasis. Non-steroidal anti-inflammatory drugs renal function and biochemical, histological and clinical effects and drug interactions. Drugs 33, Suppl 1: 56 –66, 1987. 12. Ercan ZS, Sindel S, Türker R. Possible thromboxane A2 mediated effect of angiotensin II in the rabbit isolated perfused kidney. Arch Int Physiol Biochim Biophys 99: 397–400, 1991. 13. Fellner SK, Arendshorst WJ. Angiotensin II Ca2⫹ signaling in rat afferent arterioles: stimulation of cyclic ADP ribose and IP3 pathways. Am J Physiol Renal Physiol 288: F785–F791, 2005. 14. Fellner SK, Arendshorst WJ. Angiotensin II, reactive oxygen species, and Ca2⫹ signaling in afferent arterioles. Am J Physiol Renal Physiol 289: F1012–F1019, 2005. 15. Goni FM, Alonso A. Sphingomyelinases: enzymology and membrane activity. FEBS Lett 531: 38 –46, 2002. 16. Ho WS, Barrett DA, Randall MD. ‘Entourage’ effects of N-palmitoylethanolamide and N-oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors. Br J Pharmacol 155: 837–846, 2008. 17. Hsiao SH, Constable PD, Smith GW, Haschek WM. Effects of exogenous sphinganine, sphingosine, and sphingosine-1-phosphate on relaxation and contraction of porcine thoracic aortic and pulmonary arterial rings. Toxicol Sci 86: 194 –199, 2005. 18. Huang HW, Goldberg EM, Zidovetzki R. Ceramide induces structural defects into phosphatidylcholine bilayers and activates phospholipase A2. Biochem Biophys Res Commun 220: 834 –838, 1996. 19. Huwiler A, Johansen B, Skarstad A, Pfeilschifter J. Ceramide binds to the CaLB domain of cytosolic phospholipase A2, and facilitates its membrane docking and arachidonic acid release. FASEB J 15: 7–9, 2001. 20. Imig JD. Eicosanoids and renal vascular function in diseases. Clin Sci (Lond) 111: 21–34, 2006. 21. Jang GJ, Ahn DS, Cho YE, Morgan KG, Lee YH. C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: role of RhoA/Rho-kinase and intracellular Ca2⫹ concentration. Naunyn Schmiedebergs Arch Pharmacol 372: 242–250, 2005.

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