Exaggerated response to adenosine in kidneys from high salt-fed rats: role of epoxyeicosatrienoic acids
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《美国生理学杂志》
Department of Pharmacology, New York Medical College, Valhalla, New York
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas
ABSTRACT
Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine2A receptors (A2AR) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 μM) and L-NAME (200 μM) and preconstricted to 150 mmHg with infusion of phenylephrine (10–7 M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1–10 μg) resulted in dose-dependent dilation; at 10 μg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (–60 ± 4 vs. –31 ± 8 mmHg; P < 0.05) and the area of response was increased (27 ± 6 vs. 9 ± 4 mm2; P < 0.05), as was EET release (132 ± 23 vs. 38 ± 18 ng; P < 0.05). HS treatment increased A2AR and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 μM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.
adenosine receptors; cytochrome P-450
ADENOSINE, A PRODUCT of ATP metabolism, plays a critical role in the regulation of renal vascular tone and tubular function, thus modulating renal blood flow and glomerular filtration rate (GFR) (30, 40). Adenosine also inhibits renin release, sympathetic neurotransmission, platelet aggregation, and lipolysis (40). The actions of adenosine are mediated by binding to P1 receptors (AR), of which there are four subtypes: A1, A2A, A2B, and A3 (3). In the renal microcirculation, activation of the A1R and A2AR subtypes has opposing effects on microvascular reactivity: vasoconstriction in response to A1R activation is associated with a decrease in adenylyl cyclase activity, whereas endothelium-dependent relaxation via A2AR is related to stimulation of adenylyl cyclase (29). Thus, while A1R enhances proximal tubular NaCl reabsorption (2), A2AR promotes natriuresis (49). As production of adenosine is increased under stressful conditions such as hypoxia, ischemia, and inflammation, adenosine has traditionally been implicated in the renal functional responses to pathological events (28, 33). However, there is now evidence supporting the contribution of adenosine to renal mechanisms that respond to nonpathological challenges to renal function. Adenosine levels have been shown to correlate with salt intake; switching rats from a normal-salt (NS) to a high-salt (HS) or low-salt diet results in parallel changes in renal interstitial and urinary adenosine levels (37). The increase in adenosine concentration during high salt intake may contribute to a reduction of macula densa-mediated renin secretion (22) and enhance sodium excretion.
Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are important modulators of cardiovascular function and have been recognized for their vasodilator, anti-inflammatory, antiproliferative, and profibrinolytic properties (4, 14, 31, 32, 35). Furthermore, the contribution of EETs to blood pressure regulation has been established in several different animal models (16, 25, 43). It is well documented that the activity of renal epoxygenase is increased with dietary salt loading (5, 34). EETs are thought to be natriuretic by virtue of their ability to dilate the renal vasculature (7, 13, 15) as well as regulate Na+ transport in proximal and distal tubules (27, 36, 41). Thus an increase in the production of natriuretic EETs is one of the significant components of the kidney's adaptive response to prevent elevation of blood pressure in response to HS intake.
Increased salt intake results in increased renal salt excretion. This adaptive process prevents progressive salt retention and volume expansion, with elevation of blood pressure. Indeed, salt sensitivity is an important feature of essential hypertension. Although there is much evidence showing an increase in epoxygenase activity and subsequent increased production of antihypertensive EETs in response to salt loading, the stimulus for this increased epoxygenase activity has not been identified. Recently, we linked A2AR activation to EET production in rat arcuate arteries (8). As adenosine levels are increased by dietary salt intake, we propose that adenosine is the stimulus for increased renal epoxygenase activity in response to salt loading. More specifically, we hypothesize that HS intake increases the renal response to adenosine, resulting in increased epoxygenase activity and EET levels.
MATERIALS AND METHODS
Isolated perfused kidney. Male Sprague-Dawley rats weighing 200–300 g (8–10 wk, Charles River) were used in accordance with National Institutes of Health guidelines. The New York Medical College Institutional Animal Care and Use Committee approved all experimental protocols. Rats were placed on either HS (4.0% NaCl) or NS (0.4% NaCl) diet (Harlan Teklad, Madison, WI) for 7 days and on day 8, they were anesthetized with pentobarbital sodium (60 mg/kg). The right kidney was prepared for perfusion as described. Briefly, following a midline laparotomy, the right renal artery was cannulated via the mesenteric artery; the kidney was then removed from the rat and perfused with warmed (37°C), oxygenated (95% O2-5% CO2) Krebs-Henseleit buffer at a flow rate of 7 ml/min. The composition of the Krebs-Henseleit buffer was (in mmol/l) 118 NaCl, 4.7 KCl, 1.19 KH2PO4, 1.19 MgSO4, 1.9 CaCl2, 25 NaHCO3, and 5.5 glucose. NG-nitro-L-arginine methyl ester (200 μM), a nitric oxide synthase (NOS) inhibitor, and indomethacin (10 μM), a nonselective cyclooxygenase (COX) inhibitor, were included in the Krebs-Henseleit buffer to eliminate any potential interaction of either nitric oxide (NO) (34) or COX (9) with CYP-derived AA metabolite levels. In preliminary experiments, we found that the renal response to adenosine was independent of NO release or COX metabolites and that neither the responses to 2-chloroadenosine (2-CA) nor sodium nitroprusside (SNP) changed over the experimental time period of 140 min. Once a stable perfusion pressure (PP) was obtained, pressure was further increased by 50 mmHg by infusing phenylephrine (10–7 M) to amplify vasodilator responses. A representative tracing of this experimental protocol is depicted in Fig. 1. Renal effluents were collected during responses to bolus injections of the stable adenosine analog 2-CA (0.1–10 μg) and SNP (25 ng), a NO donor, in the absence and presence of a selective epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH; 12 μM; synthesized by us (42)] and a selective A2AR antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl} phenol (ZM-241385; 10 μM; Tocris Cookson, Ellisville, MO).
Release of 20-HETE and EETs. Ten-milliliter aliquots of collected perfusates were processed for CYP metabolite measurements. Samples were acidified to pH 4.0 with 9% formic acid. After addition of internal standards, 1 ng of deuterated (D2) 20-HETE (synthesized by us), and 1 ng each of D8 8,9-EET, 11,12-EET, and 14,15-EET (Biomol, Plymouth Meeting, PA), the samples were extracted twice with 2x vol ethyl acetate and evaporated to dryness.
The samples were purified by RP-HPLC on a C18 μBondapak column (4.6 x 24 mm) by using a linear gradient from acetonitrile:water:acetic acid (62.5:37.5:0.05%) to acetonitrile (100%) for 20 min at a flow rate of 1 ml/min. Fractions containing HETEs and dihydroxyeicosatrienoic acids (DHTs), and those containing 8,9-/11, 12-EETs, and 14,15-EET, were collected on the basis of the elution profile of standards monitored by ultraviolet absorbance (205 nm). The fractions were evaporated to dryness and resuspended in 100 μl of acetonitrile.
HPLC fractions containing HETEs, EETs, and DHTs were derivatized to pentafluorobenzyl esters, and HETEs and DHTs were further derivatized to trimethylsilyl ethers (10). Samples were dried with nitrogen and resuspended in 50 μl of isooctane until gas chromatography-mass spectrometry (GC-MS) analyses. A 1-μl aliquot of derivatized CYP-derived AA metabolites, dissolved in isooctane, was injected into a GC (Hewlett Parkard 5890) column (DB-1; 10.0 m, 0.25-mm inner diameter, 0.25-μm film thickness; Agilent Technologies). We used temperature programs ranging from 180 to 300°C (HETEs and DHTs) and 150 to 300°C (EETs) at rates of 25°C and 30°C/min, respectively (24). These temperature gradients separate individual HETEs; however, they could not resolve EET regioisomers. Methane was used as a reagent gas at a flow resulting in a source pressure of 1.3 Torr and the MS (Hewlett-Packard 5989A) was operated in electron capture chemical ionization mode. The endogenous HETEs (ion m/z 391), EETs (ion m/z 319), and DHTs (ion m/z 481) were identified by comparison of GC retention times with authentic HETE and EET standards. Quantitation of 20-HETE was performed by calculating the ratio of abundance with D2 20-HETE (m/z 393). The endogenous EETs were identified by comparison of GC retention times with authentic D8 8,9-, 11,12-, and 14,15-EET (m/z 327) standards. The highly labile 5,6-EET was not measured. The 5,6-, 8,9-, and 11,12-DHTs (m/z 489) exhibited the same retention time and were quantitated together, whereas 14,15-DHT (m/z 489), which had a different retention time, was quantitated separately. Samples were quantitated by reference to a standard curve.
Western immunoblot analysis. After removal of the right kidney, the left kidney of each rat was immediately excised and hemisected, cortex and medulla were separated, and snap-frozen in liquid nitrogen. Tissues were placed in a tube with 0.5 ml of SDS buffer (50 mM Tris, pH 7.0, 2% SDS, 10% glycerol) containing 10 μl/ml of a protease inhibitor cocktail (Sigma, St. Louis, MO) and 1 mg/ml phenylmethylsulfonyl fluoride and homogenized in a tight glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernate was recovered. Protein concentrations were determined with a detergent-compatible protein assay (DC protein assay kit, Bio-Rad, Hercules, CA) according to the procedures described by the manufacturer and samples were stored at –20°C. Samples (30 μg/well) were mixed with an appropriate volume of 5x SDS-PAGE loading buffer (100 mM Tris·HCl, pH 6.8, 200 mM mercaptoethanol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiled for 3 min. Proteins were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were placed in blocking solution containing 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature (RT) for 1 h and incubated for 1 h at RT or overnight at 4°C with the first antibody [anti-A1 receptor antibody (diluted 1:500 in 1% milk, Sigma); anti-A2AR antibody (diluted 1:500 in 1% milk, Chemicon, Temecula, CA); anti-CYP2C23 antibody (diluted 1:1,000 in 1% milk, gift from Dr. J. Capdevila, Vanderbilt University); anti-CYP2C11 antibody (diluted 1:2,500 in 1% milk, Oxford Biomedical Research, Oxford, MI)]. The membranes were then washed three times in TBST and incubated at RT for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (Amersham, Arlington Heights, IL) at 1:5,000 dilution. Membranes were washed, and enhanced chemiluminescence was used to evaluate protein expression. Each membrane was stripped of bound antibodies and reprobed with an anti--actin antibody. The intensity (densitometric units) ratio of adenosine receptors or CYP2C isozymes to -actin on the same membrane was calculated and used for quantitative comparison.
Analysis of data. All data are expressed as means ± SE. Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls test when multiple comparisons were made (i.e., dose-response curves to 2-CA in NS- vs. HS-fed rats, before and after inhibitor) or by t-tests. Paired analysis (paired t-test) was used when comparisons were made of data obtained from the same experimental preparation (i.e., total EET and DHT release in response to 2-CA before and after inhibitor in the same kidney). Unpaired analysis (unpaired t-test) was used when comparisons were made of data obtained from different experimental preparations (i.e., kidneys of HS vs. NS groups). A P value of <0.05 was considered statistically significant.
RESULTS
The responsiveness of isolated, perfused rat kidneys to 2-CA was determined by the magnitude of the response (measured as decrease in PP and area of response). Bolus injections of 2-CA (0.1–10 μg) elicited biphasic responses: a transient vasoconstriction followed by a prolonged dilation (see Figs. 1 and 3). The dilator responses to 2-CA were dose dependent (Fig. 2). In kidneys from HS-fed rats, the responses to 2-CA were greater than in the NS group. At the 10-μg dose of 2-CA, PP was lowered by –60 ± 4 mmHg in kidneys from HS-fed rats compared with –31 ± 8 mmHg in kidneys from NS-fed rats, and the area of response was increased to 27 ± 6 mm2 compared with 9 ± 4 mm2 from kidneys of HS- vs. NS-fed rats (Fig. 3). The selective epoxygenase inhibitor, MS-PPOH (12 μM), decreased the response to 2-CA under both conditions of NS and HS intake, but the reduction was much more pronounced in kidneys from HS rats (Figs. 2 and 3). In kidneys from the HS group, MS-PPOH significantly reduced the response to 2-CA; PP and area of response decreased by 40 and 70%, respectively, whereas lesser changes were evident in NS kidneys. Under NS conditions, renal vascular responses to SNP (25 ng) were unaffected by MS-PPOH, as we previously reported in pressurized rat arcuate arteries (8). This indicates that inhibition of 2-CA-induced dilation was not related to nonselective actions of MS-PPOH or a decrease in renal vascular sensitivity over the time course of the experiment. Vascular responses to SNP in HS-fed rats were also unaffected by MS-PPOH.
As salt loading increases renal epoxygenase activity (5, 34), we assessed the role of EETs in the enhanced dilator response to 2-CA seen in kidneys from HS-fed rats (Fig. 4). Epoxygenase activity was determined as a measurement of total EET and DHT release before and after the addition of MS-PPOH. HS intake significantly increased total EET and DHT release compared with NS in response to 10 μg of 2-CA (132 ± 23 vs. 38 ± 18 ng, respectively), without a relative change in the EET to DHT ratio (1:9 vs. 2:8, in HS vs. NS groups, respectively). This increase was attenuated in the presence of MS-PPOH, suggesting that the exaggerated renal response to 2-CA, with HS intake, is at least partly mediated by de novo synthesis of EETs. Total EET and DHT release in response to SNP in kidneys from HS-fed rats was not significantly affected by MS-PPOH. In contrast to total EET and DHT release, levels of 20-HETE were neither affected by salt intake nor MS-PPOH (Fig. 5).
Previously, we showed that the A2AR-selective agonist, CGS-21680, -induced dilation of rat renal preglomerular microvessels (PGMV), is mediated by stimulation of EET synthesis (8). Because 2-CA is a nonselective agonist of all four adenosine receptors, we wanted to confirm that 2-CA-induced vasodilation in the isolated, perfused rat kidney was also mediated via stimulation of A2AR and subsequent EET synthesis. As seen in Fig. 6A, the responsiveness of isolated, perfused kidneys to 2-CA in the absence and presence of a selective A2AR antagonist, ZM-241385 (10 μM), was determined in NS-fed rats. Administration of 10 μg of 2-CA resulted in vasodilation under control conditions. In the presence of ZM-241385, the dilator response to 2-CA was abolished; in fact, only a transient vasoconstriction was observed. This suggests that 2-CA-induced dilation in the isolated, perfused rat kidney is solely mediated by A2AR activation. The abrogated dilator response was accompanied by a significant reduction in total EET and DHT release (40 ± 11 vs. 7 ± 2 ng before and after ZM-241385, respectively; Fig. 6B).
Figures 7 and 8 present representative Western blots and densitometric analysis of A1R, A2AR, CYP2C23, and CYP2C11 protein expression in renal cortical and/or medullary homogenates. Western blot analysis of cortical and medullary homogenates showed the presence of A1R protein and a weak band corresponding to the A2AR protein, respectively, results that are in agreement with the studies of Jackson et al. and Zou et al. (18, 50). In cortical homogenates from HS-treated rats compared with NS cortical homogenates, expression of the A1R protein was decreased by 20%, whereas in medullary homogenates, A2AR expression was increased. Medullary A1R protein level was similar between NS- and HS-fed rats, whereas cortical A2AR protein levels (data not shown) were undetectable (Fig. 7). HS treatment increased medullary CYP2C23 expression, whereas cortical CYP2C23 expression was unchanged. Both cortical and medullary protein levels of CYP2C11 were unaffected by HS intake (Fig. 8). Thus the enhanced dilator response to 2-CA seen in kidneys obtained from HS-fed rats is mediated by increased EET and DHT release and is associated with a downregulation of cortical A1R protein expression and increased medullary A2AR and CYP2C23 protein expression.
DISCUSSION
In the present study, we examined the renal response to adenosine in isolated, perfused kidneys obtained from rats fed a 4% HS diet or NS diet for 7 days. To define the vascular responses to adenosine, we used a stable adenosine analog, 2-CA, that is not subject to inactivation by either adenosine deaminase/kinase or rapid removal by nucleoside carriers.
Bolus injections of 2-CA resulted in prolonged dose-dependent dilations, the responses to 2-CA being exaggerated in kidneys from rats fed a HS diet compared with those receiving a NS diet for 7 days. At 10 μg, 2-CA caused a dilation under both conditions of NS and HS, but the magnitude of the response was enhanced in kidneys obtained from rats fed HS for 7 days. As seen in Fig. 3, the inhibitory effect of MS-PPOH was greater under HS conditions. However, MS-PPOH did not completely abolish the response; thus EETs may not be the sole mediators of adenosine-induced dilation in isolated, perfused rat kidneys. Our study was performed in the presence of NOS and COX inhibition, which eliminated the contribution of NO and prostaglandins (i.e., PGI2 and PGE2) to the 2-CA response. In a previous study, we showed that the renal responses to 2-CA in the perfused kidney were not dependent on NO or PG synthesis (8). A possible candidate mediator of the epoxygenase-independent dilation in response to adenosine is carbon monoxide (CO), a product of heme degradation by heme oxygenase. Recently, an interaction between CO and adenosine in the nucleus of the solitary tract of rats has been reported: adenosine receptor antagonism attenuated the vasodepressor effect of hemin, while heme oxygenase inhibition attenuated the vasodepressor effect of adenosine (21).
Although EETs can be metabolized via a number of different pathways (39, 44), the rapid hydrolysis to their corresponding DHTs by soluble epoxide hydrolase seems to be the dominant pathway in the kidney (46, 47). Thus total EET and DHT release was measured, to account for total epoxygenase activity. Compared with NS intake, salt loading significantly augmented epoxygenase activity, as reflected by an increase in EET and DHT release in response to 10 μg of 2-CA. Epoxygenase inhibition abolished this increase, suggesting that de novo synthesis of EETs, rather than release of preformed EETs from storage in phospholipids (6), is responsible for this enhanced renal response. The inability of MS-PPOH to significantly alter 20-HETE levels under either NS or HS conditions reflects the selectivity of MS-PPOH as an inhibitor of CYP epoxygenases vs. CYP hydroxylases (1).
Because all four adenosine receptor subtypes are expressed within the kidney (18) and 2-CA is a nonselective agonist of the adenosine receptors, it was necessary to elucidate which receptor isoform mediated the 2-CA-induced vasodilation and EET stimulation. Adenosine-dependent vasodilation is mediated by increased cAMP levels via stimulation of A2R subtypes (A2AR and/or A2BR). Our data revealed that as with the pressurized arcuate artery preparation (8), 2-CA-induced vasodilation of the isolated, perfused rat kidney is also mediated through activation of A2AR, a finding confirmed by experiments with ZM-241385, a selective A2AR antagonist. As activation of A2BR dilates renal arteries in a NO-dependent manner (26), we eliminated this pathway as a mediator of 2-CA-induced dilation by inhibition of NOS.
Most renal EET biosynthesis has been attributed to the CYP2C and 2J epoxygenase families (11, 23, 45). In the rat kidney, CYP2C23 has been identified as the major 2C arachidonate epoxygenase and the isoform of the 2C family that is subject to regulation by dietary salt (11). In agreement with other studies (11, 48), renal CYP2C23 expression was upregulated with increased dietary salt intake. We showed that HS increased medullary CYP2C23 protein expression, without affecting levels of cortical CYP2C23. In contrast to Zhao et al. (48), who reported that an 8% NaCl diet for 14 days increased CYP2C11 cortical protein expression, we did not detect changes in either cortical or medullary homogenates under our experimental conditions of 4% NaCl intake for 7 days. Changes in AR protein expression in response to dietary salt intake have also been observed (50). Under conditions of low salt, the renal expression of A1R is increased, whereas HS diet downregulated A1R expression (38, 50). Similarly, in the present study, we show that the expression of cortical A1R protein is decreased in kidneys from HS-fed rats compared with NS-fed rats. A novel finding of our study is the upregulation of medullary A2AR under conditions of salt loading. In the kidney, activation of A1R and A2AR participates in the regulation of renal vascular resistance (17). Stimulation of A1R constricts the renal vasculature (PGMV, efferent arterioles, and vasa recta) (12); on the other hand, activation of A2AR increases adenylyl cyclase activity, ultimately leading to vasodilation and natriuresis (20, 49). We showed that 2-CA-mediated activation of A2A receptors dilated a PGMV, the arcuate artery, by stimulating EET synthesis (8). As adenosine agonists dilate both pre- and postglomerluar microvessels (12), the question arises whether a similar EET-dependent mechanism evoked by A2AR activation obtains in postglomerular microvessels. Our findings regarding upregulation of the A2AR and increased CYP2C23 isoform expression in the renal medulla suggest that a similar mechanism, involving A2AR-epoxygenase interactions, operates in postglomerular microvessels.
We conclude that stimulation of adenosine with salt loading and downregulation of A1R, with increased adenylyl cyclase activity via A2AR stimulation, may play an important role in the adaptation of the kidney to enhance salt excretion. Indeed, there is evidence linking changes in A2AR-coupled activity to blood pressure regulation: 1) blood pressure is elevated in transgenic A2AR knockout mice and 2) A2AR wild-type mice exhibit a decrease in blood pressure in response to an A2AR agonist (19). The central finding of this study is that the renal response to adenosine is exaggerated in rats fed a HS diet, presumably via A2AR activation and subsequent increased production of EETs. The evidence to date indicates that stimulation of EET synthesis by PGMV in response to adenosine activation of A2AR represents a vascular mechanism that participates in the regulation of PGMV tone and reactivity and, thereby, GFR and salt and water excretion. Because the tone and reactivity of PGMV are key components in renal autoregulation and tubuloglomerular feedback, A2AR activation may be a key link in a renal mechanism that contributes to the regulation of blood pressure.
GRANTS
This research was supported in part by National Institutes of Health Grants HL-34300, HL-25394, and GM-31278.
ACKNOWLEDGMENTS
We thank B. Eng for technical assistance with Western blot analysis and M. Steinberg for editorial assistance in preparing this manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Zou AP, Wu F, Li PL, and Cowley AW Jr. Effect of chronic salt loading on adenosine metabolism and receptor expression in renal cortex and medulla in rats. Hypertension 33: 511–516, 1999.(Elvira L. Liclican, John )
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas
ABSTRACT
Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine2A receptors (A2AR) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 μM) and L-NAME (200 μM) and preconstricted to 150 mmHg with infusion of phenylephrine (10–7 M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1–10 μg) resulted in dose-dependent dilation; at 10 μg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (–60 ± 4 vs. –31 ± 8 mmHg; P < 0.05) and the area of response was increased (27 ± 6 vs. 9 ± 4 mm2; P < 0.05), as was EET release (132 ± 23 vs. 38 ± 18 ng; P < 0.05). HS treatment increased A2AR and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 μM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.
adenosine receptors; cytochrome P-450
ADENOSINE, A PRODUCT of ATP metabolism, plays a critical role in the regulation of renal vascular tone and tubular function, thus modulating renal blood flow and glomerular filtration rate (GFR) (30, 40). Adenosine also inhibits renin release, sympathetic neurotransmission, platelet aggregation, and lipolysis (40). The actions of adenosine are mediated by binding to P1 receptors (AR), of which there are four subtypes: A1, A2A, A2B, and A3 (3). In the renal microcirculation, activation of the A1R and A2AR subtypes has opposing effects on microvascular reactivity: vasoconstriction in response to A1R activation is associated with a decrease in adenylyl cyclase activity, whereas endothelium-dependent relaxation via A2AR is related to stimulation of adenylyl cyclase (29). Thus, while A1R enhances proximal tubular NaCl reabsorption (2), A2AR promotes natriuresis (49). As production of adenosine is increased under stressful conditions such as hypoxia, ischemia, and inflammation, adenosine has traditionally been implicated in the renal functional responses to pathological events (28, 33). However, there is now evidence supporting the contribution of adenosine to renal mechanisms that respond to nonpathological challenges to renal function. Adenosine levels have been shown to correlate with salt intake; switching rats from a normal-salt (NS) to a high-salt (HS) or low-salt diet results in parallel changes in renal interstitial and urinary adenosine levels (37). The increase in adenosine concentration during high salt intake may contribute to a reduction of macula densa-mediated renin secretion (22) and enhance sodium excretion.
Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are important modulators of cardiovascular function and have been recognized for their vasodilator, anti-inflammatory, antiproliferative, and profibrinolytic properties (4, 14, 31, 32, 35). Furthermore, the contribution of EETs to blood pressure regulation has been established in several different animal models (16, 25, 43). It is well documented that the activity of renal epoxygenase is increased with dietary salt loading (5, 34). EETs are thought to be natriuretic by virtue of their ability to dilate the renal vasculature (7, 13, 15) as well as regulate Na+ transport in proximal and distal tubules (27, 36, 41). Thus an increase in the production of natriuretic EETs is one of the significant components of the kidney's adaptive response to prevent elevation of blood pressure in response to HS intake.
Increased salt intake results in increased renal salt excretion. This adaptive process prevents progressive salt retention and volume expansion, with elevation of blood pressure. Indeed, salt sensitivity is an important feature of essential hypertension. Although there is much evidence showing an increase in epoxygenase activity and subsequent increased production of antihypertensive EETs in response to salt loading, the stimulus for this increased epoxygenase activity has not been identified. Recently, we linked A2AR activation to EET production in rat arcuate arteries (8). As adenosine levels are increased by dietary salt intake, we propose that adenosine is the stimulus for increased renal epoxygenase activity in response to salt loading. More specifically, we hypothesize that HS intake increases the renal response to adenosine, resulting in increased epoxygenase activity and EET levels.
MATERIALS AND METHODS
Isolated perfused kidney. Male Sprague-Dawley rats weighing 200–300 g (8–10 wk, Charles River) were used in accordance with National Institutes of Health guidelines. The New York Medical College Institutional Animal Care and Use Committee approved all experimental protocols. Rats were placed on either HS (4.0% NaCl) or NS (0.4% NaCl) diet (Harlan Teklad, Madison, WI) for 7 days and on day 8, they were anesthetized with pentobarbital sodium (60 mg/kg). The right kidney was prepared for perfusion as described. Briefly, following a midline laparotomy, the right renal artery was cannulated via the mesenteric artery; the kidney was then removed from the rat and perfused with warmed (37°C), oxygenated (95% O2-5% CO2) Krebs-Henseleit buffer at a flow rate of 7 ml/min. The composition of the Krebs-Henseleit buffer was (in mmol/l) 118 NaCl, 4.7 KCl, 1.19 KH2PO4, 1.19 MgSO4, 1.9 CaCl2, 25 NaHCO3, and 5.5 glucose. NG-nitro-L-arginine methyl ester (200 μM), a nitric oxide synthase (NOS) inhibitor, and indomethacin (10 μM), a nonselective cyclooxygenase (COX) inhibitor, were included in the Krebs-Henseleit buffer to eliminate any potential interaction of either nitric oxide (NO) (34) or COX (9) with CYP-derived AA metabolite levels. In preliminary experiments, we found that the renal response to adenosine was independent of NO release or COX metabolites and that neither the responses to 2-chloroadenosine (2-CA) nor sodium nitroprusside (SNP) changed over the experimental time period of 140 min. Once a stable perfusion pressure (PP) was obtained, pressure was further increased by 50 mmHg by infusing phenylephrine (10–7 M) to amplify vasodilator responses. A representative tracing of this experimental protocol is depicted in Fig. 1. Renal effluents were collected during responses to bolus injections of the stable adenosine analog 2-CA (0.1–10 μg) and SNP (25 ng), a NO donor, in the absence and presence of a selective epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH; 12 μM; synthesized by us (42)] and a selective A2AR antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl} phenol (ZM-241385; 10 μM; Tocris Cookson, Ellisville, MO).
Release of 20-HETE and EETs. Ten-milliliter aliquots of collected perfusates were processed for CYP metabolite measurements. Samples were acidified to pH 4.0 with 9% formic acid. After addition of internal standards, 1 ng of deuterated (D2) 20-HETE (synthesized by us), and 1 ng each of D8 8,9-EET, 11,12-EET, and 14,15-EET (Biomol, Plymouth Meeting, PA), the samples were extracted twice with 2x vol ethyl acetate and evaporated to dryness.
The samples were purified by RP-HPLC on a C18 μBondapak column (4.6 x 24 mm) by using a linear gradient from acetonitrile:water:acetic acid (62.5:37.5:0.05%) to acetonitrile (100%) for 20 min at a flow rate of 1 ml/min. Fractions containing HETEs and dihydroxyeicosatrienoic acids (DHTs), and those containing 8,9-/11, 12-EETs, and 14,15-EET, were collected on the basis of the elution profile of standards monitored by ultraviolet absorbance (205 nm). The fractions were evaporated to dryness and resuspended in 100 μl of acetonitrile.
HPLC fractions containing HETEs, EETs, and DHTs were derivatized to pentafluorobenzyl esters, and HETEs and DHTs were further derivatized to trimethylsilyl ethers (10). Samples were dried with nitrogen and resuspended in 50 μl of isooctane until gas chromatography-mass spectrometry (GC-MS) analyses. A 1-μl aliquot of derivatized CYP-derived AA metabolites, dissolved in isooctane, was injected into a GC (Hewlett Parkard 5890) column (DB-1; 10.0 m, 0.25-mm inner diameter, 0.25-μm film thickness; Agilent Technologies). We used temperature programs ranging from 180 to 300°C (HETEs and DHTs) and 150 to 300°C (EETs) at rates of 25°C and 30°C/min, respectively (24). These temperature gradients separate individual HETEs; however, they could not resolve EET regioisomers. Methane was used as a reagent gas at a flow resulting in a source pressure of 1.3 Torr and the MS (Hewlett-Packard 5989A) was operated in electron capture chemical ionization mode. The endogenous HETEs (ion m/z 391), EETs (ion m/z 319), and DHTs (ion m/z 481) were identified by comparison of GC retention times with authentic HETE and EET standards. Quantitation of 20-HETE was performed by calculating the ratio of abundance with D2 20-HETE (m/z 393). The endogenous EETs were identified by comparison of GC retention times with authentic D8 8,9-, 11,12-, and 14,15-EET (m/z 327) standards. The highly labile 5,6-EET was not measured. The 5,6-, 8,9-, and 11,12-DHTs (m/z 489) exhibited the same retention time and were quantitated together, whereas 14,15-DHT (m/z 489), which had a different retention time, was quantitated separately. Samples were quantitated by reference to a standard curve.
Western immunoblot analysis. After removal of the right kidney, the left kidney of each rat was immediately excised and hemisected, cortex and medulla were separated, and snap-frozen in liquid nitrogen. Tissues were placed in a tube with 0.5 ml of SDS buffer (50 mM Tris, pH 7.0, 2% SDS, 10% glycerol) containing 10 μl/ml of a protease inhibitor cocktail (Sigma, St. Louis, MO) and 1 mg/ml phenylmethylsulfonyl fluoride and homogenized in a tight glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernate was recovered. Protein concentrations were determined with a detergent-compatible protein assay (DC protein assay kit, Bio-Rad, Hercules, CA) according to the procedures described by the manufacturer and samples were stored at –20°C. Samples (30 μg/well) were mixed with an appropriate volume of 5x SDS-PAGE loading buffer (100 mM Tris·HCl, pH 6.8, 200 mM mercaptoethanol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiled for 3 min. Proteins were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were placed in blocking solution containing 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature (RT) for 1 h and incubated for 1 h at RT or overnight at 4°C with the first antibody [anti-A1 receptor antibody (diluted 1:500 in 1% milk, Sigma); anti-A2AR antibody (diluted 1:500 in 1% milk, Chemicon, Temecula, CA); anti-CYP2C23 antibody (diluted 1:1,000 in 1% milk, gift from Dr. J. Capdevila, Vanderbilt University); anti-CYP2C11 antibody (diluted 1:2,500 in 1% milk, Oxford Biomedical Research, Oxford, MI)]. The membranes were then washed three times in TBST and incubated at RT for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (Amersham, Arlington Heights, IL) at 1:5,000 dilution. Membranes were washed, and enhanced chemiluminescence was used to evaluate protein expression. Each membrane was stripped of bound antibodies and reprobed with an anti--actin antibody. The intensity (densitometric units) ratio of adenosine receptors or CYP2C isozymes to -actin on the same membrane was calculated and used for quantitative comparison.
Analysis of data. All data are expressed as means ± SE. Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls test when multiple comparisons were made (i.e., dose-response curves to 2-CA in NS- vs. HS-fed rats, before and after inhibitor) or by t-tests. Paired analysis (paired t-test) was used when comparisons were made of data obtained from the same experimental preparation (i.e., total EET and DHT release in response to 2-CA before and after inhibitor in the same kidney). Unpaired analysis (unpaired t-test) was used when comparisons were made of data obtained from different experimental preparations (i.e., kidneys of HS vs. NS groups). A P value of <0.05 was considered statistically significant.
RESULTS
The responsiveness of isolated, perfused rat kidneys to 2-CA was determined by the magnitude of the response (measured as decrease in PP and area of response). Bolus injections of 2-CA (0.1–10 μg) elicited biphasic responses: a transient vasoconstriction followed by a prolonged dilation (see Figs. 1 and 3). The dilator responses to 2-CA were dose dependent (Fig. 2). In kidneys from HS-fed rats, the responses to 2-CA were greater than in the NS group. At the 10-μg dose of 2-CA, PP was lowered by –60 ± 4 mmHg in kidneys from HS-fed rats compared with –31 ± 8 mmHg in kidneys from NS-fed rats, and the area of response was increased to 27 ± 6 mm2 compared with 9 ± 4 mm2 from kidneys of HS- vs. NS-fed rats (Fig. 3). The selective epoxygenase inhibitor, MS-PPOH (12 μM), decreased the response to 2-CA under both conditions of NS and HS intake, but the reduction was much more pronounced in kidneys from HS rats (Figs. 2 and 3). In kidneys from the HS group, MS-PPOH significantly reduced the response to 2-CA; PP and area of response decreased by 40 and 70%, respectively, whereas lesser changes were evident in NS kidneys. Under NS conditions, renal vascular responses to SNP (25 ng) were unaffected by MS-PPOH, as we previously reported in pressurized rat arcuate arteries (8). This indicates that inhibition of 2-CA-induced dilation was not related to nonselective actions of MS-PPOH or a decrease in renal vascular sensitivity over the time course of the experiment. Vascular responses to SNP in HS-fed rats were also unaffected by MS-PPOH.
As salt loading increases renal epoxygenase activity (5, 34), we assessed the role of EETs in the enhanced dilator response to 2-CA seen in kidneys from HS-fed rats (Fig. 4). Epoxygenase activity was determined as a measurement of total EET and DHT release before and after the addition of MS-PPOH. HS intake significantly increased total EET and DHT release compared with NS in response to 10 μg of 2-CA (132 ± 23 vs. 38 ± 18 ng, respectively), without a relative change in the EET to DHT ratio (1:9 vs. 2:8, in HS vs. NS groups, respectively). This increase was attenuated in the presence of MS-PPOH, suggesting that the exaggerated renal response to 2-CA, with HS intake, is at least partly mediated by de novo synthesis of EETs. Total EET and DHT release in response to SNP in kidneys from HS-fed rats was not significantly affected by MS-PPOH. In contrast to total EET and DHT release, levels of 20-HETE were neither affected by salt intake nor MS-PPOH (Fig. 5).
Previously, we showed that the A2AR-selective agonist, CGS-21680, -induced dilation of rat renal preglomerular microvessels (PGMV), is mediated by stimulation of EET synthesis (8). Because 2-CA is a nonselective agonist of all four adenosine receptors, we wanted to confirm that 2-CA-induced vasodilation in the isolated, perfused rat kidney was also mediated via stimulation of A2AR and subsequent EET synthesis. As seen in Fig. 6A, the responsiveness of isolated, perfused kidneys to 2-CA in the absence and presence of a selective A2AR antagonist, ZM-241385 (10 μM), was determined in NS-fed rats. Administration of 10 μg of 2-CA resulted in vasodilation under control conditions. In the presence of ZM-241385, the dilator response to 2-CA was abolished; in fact, only a transient vasoconstriction was observed. This suggests that 2-CA-induced dilation in the isolated, perfused rat kidney is solely mediated by A2AR activation. The abrogated dilator response was accompanied by a significant reduction in total EET and DHT release (40 ± 11 vs. 7 ± 2 ng before and after ZM-241385, respectively; Fig. 6B).
Figures 7 and 8 present representative Western blots and densitometric analysis of A1R, A2AR, CYP2C23, and CYP2C11 protein expression in renal cortical and/or medullary homogenates. Western blot analysis of cortical and medullary homogenates showed the presence of A1R protein and a weak band corresponding to the A2AR protein, respectively, results that are in agreement with the studies of Jackson et al. and Zou et al. (18, 50). In cortical homogenates from HS-treated rats compared with NS cortical homogenates, expression of the A1R protein was decreased by 20%, whereas in medullary homogenates, A2AR expression was increased. Medullary A1R protein level was similar between NS- and HS-fed rats, whereas cortical A2AR protein levels (data not shown) were undetectable (Fig. 7). HS treatment increased medullary CYP2C23 expression, whereas cortical CYP2C23 expression was unchanged. Both cortical and medullary protein levels of CYP2C11 were unaffected by HS intake (Fig. 8). Thus the enhanced dilator response to 2-CA seen in kidneys obtained from HS-fed rats is mediated by increased EET and DHT release and is associated with a downregulation of cortical A1R protein expression and increased medullary A2AR and CYP2C23 protein expression.
DISCUSSION
In the present study, we examined the renal response to adenosine in isolated, perfused kidneys obtained from rats fed a 4% HS diet or NS diet for 7 days. To define the vascular responses to adenosine, we used a stable adenosine analog, 2-CA, that is not subject to inactivation by either adenosine deaminase/kinase or rapid removal by nucleoside carriers.
Bolus injections of 2-CA resulted in prolonged dose-dependent dilations, the responses to 2-CA being exaggerated in kidneys from rats fed a HS diet compared with those receiving a NS diet for 7 days. At 10 μg, 2-CA caused a dilation under both conditions of NS and HS, but the magnitude of the response was enhanced in kidneys obtained from rats fed HS for 7 days. As seen in Fig. 3, the inhibitory effect of MS-PPOH was greater under HS conditions. However, MS-PPOH did not completely abolish the response; thus EETs may not be the sole mediators of adenosine-induced dilation in isolated, perfused rat kidneys. Our study was performed in the presence of NOS and COX inhibition, which eliminated the contribution of NO and prostaglandins (i.e., PGI2 and PGE2) to the 2-CA response. In a previous study, we showed that the renal responses to 2-CA in the perfused kidney were not dependent on NO or PG synthesis (8). A possible candidate mediator of the epoxygenase-independent dilation in response to adenosine is carbon monoxide (CO), a product of heme degradation by heme oxygenase. Recently, an interaction between CO and adenosine in the nucleus of the solitary tract of rats has been reported: adenosine receptor antagonism attenuated the vasodepressor effect of hemin, while heme oxygenase inhibition attenuated the vasodepressor effect of adenosine (21).
Although EETs can be metabolized via a number of different pathways (39, 44), the rapid hydrolysis to their corresponding DHTs by soluble epoxide hydrolase seems to be the dominant pathway in the kidney (46, 47). Thus total EET and DHT release was measured, to account for total epoxygenase activity. Compared with NS intake, salt loading significantly augmented epoxygenase activity, as reflected by an increase in EET and DHT release in response to 10 μg of 2-CA. Epoxygenase inhibition abolished this increase, suggesting that de novo synthesis of EETs, rather than release of preformed EETs from storage in phospholipids (6), is responsible for this enhanced renal response. The inability of MS-PPOH to significantly alter 20-HETE levels under either NS or HS conditions reflects the selectivity of MS-PPOH as an inhibitor of CYP epoxygenases vs. CYP hydroxylases (1).
Because all four adenosine receptor subtypes are expressed within the kidney (18) and 2-CA is a nonselective agonist of the adenosine receptors, it was necessary to elucidate which receptor isoform mediated the 2-CA-induced vasodilation and EET stimulation. Adenosine-dependent vasodilation is mediated by increased cAMP levels via stimulation of A2R subtypes (A2AR and/or A2BR). Our data revealed that as with the pressurized arcuate artery preparation (8), 2-CA-induced vasodilation of the isolated, perfused rat kidney is also mediated through activation of A2AR, a finding confirmed by experiments with ZM-241385, a selective A2AR antagonist. As activation of A2BR dilates renal arteries in a NO-dependent manner (26), we eliminated this pathway as a mediator of 2-CA-induced dilation by inhibition of NOS.
Most renal EET biosynthesis has been attributed to the CYP2C and 2J epoxygenase families (11, 23, 45). In the rat kidney, CYP2C23 has been identified as the major 2C arachidonate epoxygenase and the isoform of the 2C family that is subject to regulation by dietary salt (11). In agreement with other studies (11, 48), renal CYP2C23 expression was upregulated with increased dietary salt intake. We showed that HS increased medullary CYP2C23 protein expression, without affecting levels of cortical CYP2C23. In contrast to Zhao et al. (48), who reported that an 8% NaCl diet for 14 days increased CYP2C11 cortical protein expression, we did not detect changes in either cortical or medullary homogenates under our experimental conditions of 4% NaCl intake for 7 days. Changes in AR protein expression in response to dietary salt intake have also been observed (50). Under conditions of low salt, the renal expression of A1R is increased, whereas HS diet downregulated A1R expression (38, 50). Similarly, in the present study, we show that the expression of cortical A1R protein is decreased in kidneys from HS-fed rats compared with NS-fed rats. A novel finding of our study is the upregulation of medullary A2AR under conditions of salt loading. In the kidney, activation of A1R and A2AR participates in the regulation of renal vascular resistance (17). Stimulation of A1R constricts the renal vasculature (PGMV, efferent arterioles, and vasa recta) (12); on the other hand, activation of A2AR increases adenylyl cyclase activity, ultimately leading to vasodilation and natriuresis (20, 49). We showed that 2-CA-mediated activation of A2A receptors dilated a PGMV, the arcuate artery, by stimulating EET synthesis (8). As adenosine agonists dilate both pre- and postglomerluar microvessels (12), the question arises whether a similar EET-dependent mechanism evoked by A2AR activation obtains in postglomerular microvessels. Our findings regarding upregulation of the A2AR and increased CYP2C23 isoform expression in the renal medulla suggest that a similar mechanism, involving A2AR-epoxygenase interactions, operates in postglomerular microvessels.
We conclude that stimulation of adenosine with salt loading and downregulation of A1R, with increased adenylyl cyclase activity via A2AR stimulation, may play an important role in the adaptation of the kidney to enhance salt excretion. Indeed, there is evidence linking changes in A2AR-coupled activity to blood pressure regulation: 1) blood pressure is elevated in transgenic A2AR knockout mice and 2) A2AR wild-type mice exhibit a decrease in blood pressure in response to an A2AR agonist (19). The central finding of this study is that the renal response to adenosine is exaggerated in rats fed a HS diet, presumably via A2AR activation and subsequent increased production of EETs. The evidence to date indicates that stimulation of EET synthesis by PGMV in response to adenosine activation of A2AR represents a vascular mechanism that participates in the regulation of PGMV tone and reactivity and, thereby, GFR and salt and water excretion. Because the tone and reactivity of PGMV are key components in renal autoregulation and tubuloglomerular feedback, A2AR activation may be a key link in a renal mechanism that contributes to the regulation of blood pressure.
GRANTS
This research was supported in part by National Institutes of Health Grants HL-34300, HL-25394, and GM-31278.
ACKNOWLEDGMENTS
We thank B. Eng for technical assistance with Western blot analysis and M. Steinberg for editorial assistance in preparing this manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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