Anandamide Decreases Glomerular Filtration Rate through Predominant Vasodilation of Efferent Arterioles in Rat Kidneys
Yukako Koura*,
Atsuhiro Ichihara*,
Yuko Tada*,
Yuki Kaneshiro*,
Hirokazu Okada,
Constance J. Temm,
Matsuhiko Hayashi* and
Takao Saruta*
*Internal Medicine, Keio University School of Medicine, Tokyo, Japan; Internal Medicine, Saitama Medical College, Saitama, Japan; and Medicine, Division of Nephrology, Indiana University, Indianapolis, Indiana
Correspondence to Dr. Atsuhiro Ichihara, Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: +81-3-5363-3796; Fax: +81-3-3359-2745; E-mail: atzichi{at}sc.itc.keio.ac.jp
ABSTRACT. For determining the effects of anandamide (ANA) onrenal hemodynamics and microcirculation, a clearance study wasperformed in Sprague-Dawley rats that received injections ofANA in doses of 15, 150, and 1500 pmol/kg. At doses up to 150pmol/g, ANA significantly decreased GFR and increased renalblood flow (RBF) without affecting mean arterial pressure (MAP).In the presence of the cannabinoid type 1 (CB1) receptor antagonistAM251, only the 15-pmol/kg dose significantly increased GFRand RBF without altering MAP, with higher doses having no effecton GFR, RBF, or MAP. By contrast, AM281, which antagonizes cannabinoidreceptors nonselectively, inhibited the GFR, RBF, and MAP responsesto ANA. The arteriolar responses to ANA were also assessed invitro by the blood-perfused juxtamedullary nephron technique.Higher doses of ANA significantly increased the diameter ofboth afferent and efferent arterioles, whereas lower doses elicitedpredominant efferent arteriolar dilation. AM251 attenuated theafferent arteriolar response to ANA and inhibited the efferentarteriolar response to ANA, whereas AM281 inhibited the responsesin both arterioles. The CB1 receptor mRNA was expressed in afferentarterioles, and immunohistochemical staining demonstrated thepresence of CB1 receptors in both afferent and efferent arterioles.These results suggest that ANA causes afferent arteriolar dilationvia both CB1 and non-CB1 receptors and greater efferent arteriolardilation via CB1 receptors, resulting in a decreased GFR andan increased RBF without affecting MAP.
Septic shock is principally characterized by severe hemodynamicchanges, including hypotension, decreased systemic vascularresistance, and compromised renal function. Although systemichypotension seems to be involved in the decrease in GFR, somestudies have demonstrated that decreased GFR during the earlystage of sepsis is independent of systemic hemodynamics (1).Accordingly, some factors generated during sepsis may contributeto the compromised renal function independent of systemic hypotension.
Endogenous cannabinoid levels increase during lipopolysaccharide-inducedseptic shock and cause systemic hypotension through the cannabinoidtype 1 (CB1) receptors (2). A recent study suggested that thepolymyxin B–immobilized beads column used clinically totreat endotoxic shock may abolish the hypotensive, immunosuppressive,and cytotoxic effects during endotoxin shock by adsorbing anandamide(ANA), an endogenous cannabinoid generated by activated macrophages(3). Moreover, molecular studies have confirmed the intrarenalpresence of an ANA signaling system that includes ANA, its putativeprecursor, the enzyme catalyzing the breakdown of ANA, and CB1receptors (4–6). However, no studies of the direct effectof ANA on renal function have ever been reported.
The present study was designed to assess the direct effect ofexogenous ANA on renal hemodynamics. Periglomerular microcirculatoryresponses to ANA were also assessed by using an in vitro blood-perfusedjuxtamedullary nephron technique, and CB1 receptors in the kidneywere localized by immunohistochemical techniques and a reversetranscriptase–PCR (RT-PCR) method.
The present study was performed in accordance with the guidelinesand practices established by the Keio University Animal Careand Use Committee. Normal male Sprague-Dawley rats (CharlesRiver Japan, Kanagawa, Japan) that weighed 250 to 300 g werehoused in wire cages and maintained on a 12:12-h light-darkcycle in a temperature-controlled room. Before the experiments,the rats had free access to water and standard rat diet (OrientalYeast, Tokyo, Japan) that contains 110 µmol/g sodium.
Renal Clearance Study
Under anesthesia with pentobarbital sodium (50 mg/kg), tracheostomyand positive pressure ventilation were performed with an artificialrespirator. The right jugular vein, right femoral artery, andright carotid artery were respectively cannulated to infusesolutions, to monitor arterial pressure, and to inject bolusdoses of ANA (Sigma, St. Louis, MO) and CB receptor antagonists.The bladder was catheterized via a suprapubic incision for urinecollection. After surgery, the intravenous infusion was switchedto isotonic saline solution containing 1% albumin, 1.5% p-aminohippuratesodium (PAH; Merck, Sharp & Dohme, West Point, PA), and7.5% inulin (Inutest; Laevosan, Linz, Austria). A 60-min equilibrationperiod was provided after the completion of surgery.
The experimental protocol consisted of three 30-min clearanceperiods to assess control renal function. Between the secondand third periods, the rats received an intra-arterial injectionof saline, the selective CB1 receptor antagonist AM251 (1500pmol/kg; Tocris Cookson, Ballwin, MO), or the nonselective CBreceptors antagonist AM281 (1500 pmol/kg; Tocris Cookson). Therats were then given three intra-arterial bolus injections ofincreasing doses of ANA (15, 150, and 1500 pmol/kg) at 40-minintervals. After a 10-min delay, a 30-min experimental clearanceperiod was provided for each dose of ANA, and an arterial bloodsample was collected at the midpoint of each 30-min clearanceperiod to calculate inulin clearance (Cinulin) and PAH clearance(CPAH). The microhematocrit of all arterial blood samples wasmeasured and remained constant throughout the experiment. Cinulinwas used as an index of GFR, and CPAH was used as an index ofrenal blood flow (RBF) because of the constant hematocrit. Alldrugs were given in total volume of 100 µl.
Additional experiments were performed to investigate the roleof renal sympathetic nerves in renal hemodynamic responses toANA. Bilateral kidney denervation was performed, as describedpreviously (7). The intrarenal norepinephrine content was confirmedto be significantly lower in the renal-denervated rats (20.0± 8.9 ng/g tissue; n = 5) than in the control rats (165.8± 13.5 ng/g tissue; n = 4).
Measurement of Afferent and Efferent Arteriolar Diameter
Afferent and efferent arteriolar diameters were measured invitro by the blood-perfused juxtamedullary nephron techniquecombined with videomicroscopy, as described previously (8).Afferent arteriolar diameters were measured at sites 90 to 150µm upstream from the glomerulus, and efferent arteriolardiameters were measured at sites within 100 µm of theglomerulus, before the first branch. A 10-min equilibrationperiod was allowed before initiating each experimental protocol.The average diameter during the final 2 min of each 5-min periodwas used to statistically analyze steady-state responses.
Afferent and efferent arteriolar diameters were measured beforeand during superfusion with increasing doses of ANA (0.1, 1,and 10 µM). For investigating the involvement of CB1 andCB2 receptors in ANA-mediated afferent and efferent arteriolarrelaxation, the ANA responses were assessed again in the presenceof 1 nM AM251 or 1 nM AM281.
Immunohistochemical Localization of CB1 Receptors
For detecting CB1 receptors, kidneys were perfused in situ withsaline and fixed in Bouin’s solution. Sections were immersedin 3% H2O2 in methanol to inhibit endogenous peroxidase andthen flooded with 5% BSA in PBS to inhibit nonspecific reactions.Polyclonal goat anti-CB1 receptor antibodies (sc-10066; SantaCruz Biotechnology, Santa Cruz, CA; 1:200 dilution) were usedas the primary antibodies, and a biotinylated polyclonal donkeyanti-goat antibody (1:500 dilution) was applied as the secondaryantibody. Immunoreaction was performed using a Vectastain ABC-Elitekit (Vector Laboratories, Burlingame, CA) and visualized with3-amino-9-ethylcarbazole (DAKO, Carpinteria, CA) as a substrate,followed by light counterstaining with hematoxylin. We discriminateefferent arterioles from afferent arterioles on the basis ofthe criteria that the efferent arterioles have a significantintraglomerular segment that runs through the glomerular stalk,whereas the afferent arterioles divide into several primarycapillary branches strictly at the entrance of the glomerulus.
Detection of CB1 Receptor mRNA in Afferent Arterioles
Afferent arterioles were prepared as described previously (9).In brief, the kidneys were cleared of blood by perfusion insitu with ice-cold low-calcium physiologic salt solution (PSS;pH 7.35) followed by an identical solution that contained 1%Evans blue. The renal cortex was sieved with a 180-µmnylon mesh, and the retentate was washed with ice-cold low-calciumPSS. The vascular tissue that remained on the sieve was transferredto the solution that contained 0.075% collagenase (Calbiochem,La Jolla, CA), 0.02% dithiothreitol (Sigma), 0.2% soybean trypsininhibitor (type 1-S; Sigma), and 0.1% BSA dissolved in low-calciumPSS and incubated for 30 min at 37°C. The vascular tissuewas transferred to a 70-µm nylon mesh and washed withice-cold low-calcium PSS. The retained vascular tissue was transferredto a dish that contained ice-cold low-calcium PSS, and afferentarterioles were collected on the stage of a Nikon microscope(model SMZ800) and stored at –80°C. Frozen afferentarterioles were homogenized in Trizol reagent (Life Technologies,Grand Island, NY) and extracted following the manufacturer’sinstructions. RNA was resuspended in DEPC-H2O and stored at–20°C until use. DNAse-I–treated total RNA wassubjected to RT-PCR using SuperScript II Reverse Transcriptase(Invitrogen, Carlsbad, CA) and GeneAmp RNA PCR kit (AppliedBiosystems, Foster City, CA). Primer sequences used were asfollows: forward primer: 5'-ATTTCAAGCAAGGAGCACCCA-3'; reverseprimer: 5'-CATTCGAGCCCACGTAGAGGA-3'.
Quantitative Analysis of CB1 Receptors in Afferent and Efferent Arterioles
The right kidneys from a Sprague-Dawley rat was perfusion fixedand removed. It was further fixed in 3.7% paraformaldehyde for15 min at room temperature, then vibratome-sectioned into 100-µmsections, followed by 0.2% Triton X100 (5 min) for permeabilization.For fluorescence staining, the sections were washed severaltimes in PBS with 0.1%BSA (PBS-BSA) and incubated with primaryantibodies (goat polyclonal CB1) in PBS-BSA for 1 h at roomtemperature followed by overnight at 4°C and washed againin PBS-BSA, and fluorescence-labeled secondary antibody (donkeyanti-goat–Texas-Red; Jackson Laboratory, Bar Harbor, ME)was applied, followed by 1 h of incubation at room temperatureand washing. A control of only secondary antibody was done simultaneously.They were mounted in Antifade (Molecular Probes, Eugene, OR).Microscopy was performed on a BioRad two-photon microscope.The results were analyzed using Voxx software (10) and Metaporph(Fryer, Huntley, IL).
Statistical Analyses
Statistical comparisons within a group were made by one-wayANOVA for repeated measures followed by the Newman-Keuls posthoc test. Differences between two groups were evaluated by two-wayANOVA for repeated measures combined with the Newman-Keuls posthoc test. P < 0.05 was considered significant. Data are reportedas means ± SEM.
Effect of ANA on Arterial Pressure and Renal Hemodynamics Figure 1 shows the changes in mean arterial pressure (MAP) duringintra-arterial injection of increasing doses of ANA in the controlrats (n = 14) and bilaterally renal denervated rats (n = 7).Before ANA administration, the MAP in the control rats and bilaterallyrenal denervated rats averaged 92 ± 2 and 93 ±2 mmHg, respectively, and it was unaltered by ANA up to 150pmol/kg. The 1500-pmol/kg dose, however, elicited a significantdecrease in MAP in both groups, to 84 ± 3 and 73 ±6 mmHg, respectively.
Figure 1. Effect of anandamide (ANA) on mean arterial pressure (MAP) in control rats (; n = 14) and bilaterally renal denervated rats (•; n = 7). *P < 0.05 versus 0 pmol/kg ANA.
Figure 2 shows the responses of Cinulin and CPAH to intra-arterialinjection of ANA in the control rats (n = 14) and bilaterallyrenal denervated rats (n = 7). In the control rats, the 15-,150-, and 1500-pmol/kg doses of ANA significantly decreasedCinulin from 1.49 ± 0.28 to 0.98 ± 0.13, 0.76± 0.12, and 0.78 ± 0.20 ml/min per g kidney weight,respectively, and significantly increased CPAH from 6.1 ±0.7 to 9.9 ± 1.6, 9.3 ± 1.8, and 7.4 ±1.7 ml/min per g kidney weight, respectively. In the bilaterallyrenal denervated rats, the ANA injections also decreased Cinulin,from 1.26 ± 0.22 to 1.02 ± 0.25, 1.00 ±0.24, and 0.59 ± 0.06 ml/min per g kidney weight, respectively,and increased CPAH, from 4.8 ± 1.0 to 8.6 ± 1.6,9.6 ± 1.4, and 8.5 ± 1.3 ml/min per g kidney weight,respectively. Urinary sodium excretion (UNaV) was significantlyhigher in the bilaterally renal denervated rats (19.8 ±2.6 mmol/d) than in the control rats (9.3 ± 1.3 mmol/d),but the ANA injections had no significant effect on UNaV ineither group.
Figure 2. Effect of ANA on inulin clearance (Cinulin) and p-aminohippurate clearance (CPAH) in control rats (; n = 14) and bilaterally renal denervated rats (; n = 7). *P < 0.05 versus 0 pmol/kg ANA.
Renal Hemodynamic Responses to ANA during Inhibition of CB Receptors Figure 3 shows the MAP responses to the ANA injections in ratsthat received an intra-arterially injection of AM251 (n = 13)or AM281 (n = 15) at the 1500-pmol/kg dose. The MAP was unaffectedby the ANA injections in either group.
Figure 3. Effect of ANA on MAP in rats that were treated with AM251 (; n = 13) and AM281 (•; n = 15). *P < 0.05 versus 0 pmol/kg ANA.
Figure 4 shows the responses of Cinulin and CPAH to the intra-arterialANA injections in the rats that were treated with AM251 (n =13) or AM281 (n = 15). In the presence of AM251, the 15-pmol/kgdose of ANA significantly increased Cinulin from 2.57 ±0.54 to 8.35 ± 2.35 ml/min per g kidney weight and CPAHfrom 5.4 ± 1.4 to 8.9 ± 2.3 ml/min per g kidneyweight, but the higher ANA doses (150 and 1500 pmol/kg) didnot affect either Cinulin (4.59 ± 2.84 and 2.23 ±0.82 ml/min per g kidney weight, respectively) or CPAH (8.0± 2.1 and 3.7 ± 0.3 ml/min per g kidney weight,respectively). In the presence of AM281, neither Cinulin norCPAH was influenced by any doses of ANA. Before and after theinjections of 15, 150, and 1500 pmol/kg ANA, Cinulin averaged2.10 ± 0.42, 2.28 ± 0.52, 1.60 ± 0.32,and 1.72 ± 0.39 ml/min per g kidney weight, respectively,and CPAH averaged 11.7 ± 3.7, 12.8 ± 3.6, 11.8± 3.8, and 10.4 ± 5.4 ml/min per g kidney weight,respectively. The ANA injections had no effect on UNaV in thepresence of either AM251 or AM281.
Figure 4. Effect of ANA on Cinulin and CPAH in rats that were treated with AM251 (; n = 13) and AM281 (; n = 15). *P < 0.05 versus 0 pmol/kg ANA.
Arteriolar Responses to ANA before and during CB1 Receptor Inhibition Figure 5 shows the afferent (n = 9) and efferent (n = 6) arteriolarresponses to graded concentrations of ANA of 0.1, 1, and 10µM before and during superfusion with the CB1 receptorantagonist AM251 (1 nM). Basal afferent arteriolar diametersaveraged 12.1 ± 1.2 µm and were unaffected by ANAat the 0.1- and 1-µM concentrations. Afferent arteriolardiameter during superfusion with 0.1 and 1 µM ANA averaged12.8 ± 1.2 and 13.8 ± 1.3 µm, respectively,but the 10-µM concentration significantly increased afferentarteriolar diameters to 15.7 ± 1.2 µm. The selectiveCB1 receptor blockade with AM251 had no effect on afferent arteriolardiameter, but it significantly attenuated the dilator responsesof afferent arterioles to 10 µM ANA. In the presence ofAM251, superfusion with 10 µM ANA increased afferent arteriolardiameter from 12.3 ± 1.2 to 13.1 ± 1.2 µm,but the increase was significantly smaller than that observedbefore administration of AM251.
Figure 5. Afferent (n = 9) and efferent (n = 6) arteriolar responses to ANA before () and during superfusion with AM251 (•). *P < 0.05 versus 0 µM ANA; P < 0.05 versus the response before the treatment with AM251.
Basal efferent arteriolar diameter averaged 13.8 ± 1.2µm, and in response to 0.1, 1, and 10 µM ANA, itsignificantly increased to 16.0 ± 1.1, 19.2 ±1.0, and 20.9 ± 1.5 µm, respectively. At each concentrationof ANA, the increase in efferent arteriolar diameter was significantlygreater than the increase in afferent arteriolar diameter. AM251did not alter efferent arteriolar diameter, and it completelyinhibited the efferent arteriolar response to ANA. In the presenceof AM251, efferent arteriolar diameter before and during superfusionwith 0.1, 1, and 10 µM ANA averaged 12.9 ± 0.7,13.0 ± 1.2, 12.8 ± 0.8, and 12.7 ± 1.2µm, respectively.
Arteriolar Responses to ANA before and during CB1 and 2 Receptors Inhibition Figure 6 shows afferent (n = 7) and efferent (n = 5) arteriolarresponses to graded ANA concentrations of 0.1, 1, and 10 µMbefore and during superfusion with 1 nM AM281, which antagonizesCB receptors nonselectively. Basal afferent arteriolar diameteraveraged 12.2 ± 1.1 µm and was unaffected by 0.1µM ANA. Afferent arteriolar diameter during superfusionwith 0.1 µM ANA averaged 11.9 ± 0.9 µm, butin response to 1 and 10 µM ANA, it significantly increasedto 12.9 ± 0.9 and 15.3 ± 0.8 µm. The nonselectiveblockade of CB receptors with AM281 had no effect on afferentarteriolar diameter but completely inhibited the afferent arteriolarresponses to ANA. In the presence of AM281, afferent arteriolardiameter before and during superfusion with 0.1, 1, and 10 µMANA averaged 12.0 ± 1.0, 12.1 ± 0.8, 12.2 ±1.0, and 12.1 ± 0.9 µm, respectively.
Figure 6. Afferent (n = 7) and efferent (n = 5) arteriolar responses to ANA before () and during superfusion with AM281 (•). *P < 0.05 versus 0 µM ANA; P < 0.05 versus the response before the treatment with AM281.
Basal efferent arteriolar diameters averaged 12.0 ± 2.0µm, and 0.1, 1, and 10 µM ANA significantly increasedefferent arteriolar diameter to 13.4 ± 1.9, 16.0 ±1.5, and 17.2 ± 1.3 µm, respectively. The dilatorresponses to all concentrations of ANA in efferent arterioleswere significantly greater than in afferent arterioles. AM281did not alter efferent arteriolar diameter, but it completelyinhibited the efferent arteriolar responses to ANA. In the presenceof AM281, efferent arteriolar diameter before and during superfusionwith 0.1, 1, and 10 µM ANA averaged 12.0 ± 2.1,12.5 ± 2.2, 12.2 ± 1.9, and 11.4 ± 1.7µm, respectively.
Immunohistochemical Localization of CB1 Receptors
The CB1 receptors were found exclusively in the intrarenal vascularsystems, and there was no staining in the glomeruli, tubules,or interstitium. Figure 7, A and B, shows the similar stainingfor CB1 receptors in both afferent and efferent arterioles.These stainings were not seen when the antibodies were preabsorbedwith competing peptide for CB1 receptors (sc-10066P; Santa CruzBiotechnology; data not shown). There was no staining of CB1receptor in the glomeruli, tubules, or interstitium.
Figure 7. Immunohistochemical localization and mRNA expression of CB1 receptors in control rat kidneys. (A and B) Afferent (Af) and efferent (Ef) arterioles adjacent to the glomerulus (Glo) exhibit immunoreactivity of CB1 receptor (3-amino-9-ethylcarbazole). (C) The CB1 receptor mRNA is expressed in afferent arterioles of rat kidneys as well as the rat brain (B). Magnification, x200 in A and B.
Detection of CB1 Receptor mRNA in Afferent Arterioles
The RT-PCR analysis verified the mRNA expression of CB1 receptorin afferent arterioles. Figure 7C shows the 169-bp band (arrow),which is close to the predicted RT-PCR product size for CB1receptor, in the kidneys and brain. These bands disappearedin the absence of RT.
Quantitative Analysis of CB1 Receptors in Afferent and Efferent Arterioles Figure 8 shows immunofluorescent staining of CB1 receptors inboth afferent and efferent arterioles. Quantitative analysisusing the two-photon microscopy (n = 4) revealed a slightlybut significantly greater immunostaining for CB1 receptors inafferent arterioles (122.9 ± 3.9 densitometric units)than in efferent arterioles (100.4 ± 7.2 densitometricunits).
Figure 8. Immunofluorescent quantitative analysis for CB1 receptors in afferent and efferent arterioles. (A) A control kidney showing the autofluorescence seen with two-photon microscopy. Note that there is no vessel visible at all; only tubules possess autofluorescence. One angle of rotation is shown of two glomeruli (B and C) stained with anti-CB1. The vessels have been outlined for easy visualization. The afferent arteriole is the larger of the two vessels and can be observed connecting to an interlobular artery as the three-dimensional projection is rotated (not shown). Immunofluorescent staining for CB1 receptors is significantly greater in afferent arterioles than in efferent arterioles (D).
Increased ANA levels in the blood of sepsis patients cause systemicvasodilation that results in hypotensive shock. The resultsof the present study consistently demonstrated a significantdecrease in MAP in response to administration of 1500 pmol/kgANA. In addition, at doses of 150 pmol/kg and lower, ANA administrationsignificantly increased CPAH and decreased Cinulin without affectingMAP or hematocrit, and similar results were observed in therats that were subjected to bilateral renal denervation. Theseresults suggest that ANA increases RBF and decreases GFR independentof its effects on BP and renal nerves and that intrarenal vasculatureis more sensitive to ANA than systemic resistance arteries.
ANA significantly increased RBF, and the nonselective CB receptorsantagonist AM281 inhibited the increases in RBF at all dosesof ANA. The present in vitro studies demonstrated that ANA elicitssignificant vasodilation in afferent arterioles, which accountfor 90% of the preglomerular vascular resistance. In addition,the ANA-induced afferent arteriolar dilation was attenuatedby the blockade of CB1 receptors with AM251 and inhibited bythe nonselective blockade of CB receptors with AM281. Theseresults suggest that the RBF responses to ANA are wholly dependenton the CB receptors-mediated afferent arteriolar dilation.
Intra-arterial ANA injections caused a significant decreasein GFR, represented by Cinulin, but did not alter UNaV, suggestingthat the renal tubular system may regulate urinary sodium excretionnormally even in the presence of ANA. This concept was supportedby our immunohistochemical studies that showed staining of CB1receptors in the renal vasculature system alone with no stainingin the glomeruli, tubules, or interstitium. It therefore islikely that ANA has no luminal effects and that the changesin GFR are due to the effect of ANA on the renal vasculature.Lower doses of ANA induced a vasodilation only in efferent arterioles,and higher doses of ANA increased afferent arteriolar diameterand dilated efferent arterioles to a greater extent. Becausepredominant efferent arteriolar vasodilation decreases intraglomerularpressure, the differential effects of ANA on afferent and efferentarterioles seem to account for the decreased GFR.
Why does ANA dilate efferent arterioles so much more than afferentarterioles? To answer this question, we performed pharmacologicexperiments and immunohistochemical staining of CB1 receptorsand assessed the role of CB receptors in the mechanism by whichANA exerts different effects on afferent and efferent arterioles.The pharmacologic studies revealed that CB1 receptors contributeto the afferent and efferent arteriolar dilator responses toANA, whereas non-CB1 receptors contribute to the dilator responseto ANA in afferent arterioles alone. These findings were consistentwith the results of immunohistochemical staining demonstratingthat both afferent and efferent arterioles have CB1 receptorimmunoreactivities. In addition, RT-PCR analysis showed theCB1 receptor mRNA expression in afferent arterioles, and thetwo-photon microscopy showed a greater immunostaining for CB1receptors in afferent arterioles compared with efferent arterioles.In afferent arterioles, therefore, non-CB1 receptors may antagonizethe function of CB1 receptors.
Recent studies demonstrated the absence of CB2 receptor in ratkidneys (11) and suggested the existence of a nonclassical CBreceptor that is neither a CB1 receptor nor a CB2 receptor butis also blocked by an ordinary CB receptor antagonist (12).Because the CB receptor antagonist SR141716A also inhibits Ca2+-activatedK+ channels and voltage-dependent K+ channels (12), endothelium-derivedhyperpolarizing factors may contribute to the function of theANA-specific non-CB1, non-CB2 receptors. Because endothelium-derivedhyperpolarizing factors predominantly modulate afferent, notefferent, arterioles (13,14), it is possible that endothelium-derivedhyperpolarizing factors mediate the function of non-CB1 receptors,which is blocked by AM281.
The great increase in Cinulin after 15 pmol/kg ANA was observedin the presence of the CB1 receptor antagonist AM251 and disappearedin the presence of the nonselective CB receptor antagonist AM281.These results suggest that low doses of ANA may increase GFRthrough the non-CB1 receptor–mediated afferent arteriolardilation. However, higher doses of ANA did not affect Cinulinin the presence of AM251. Excessive activation of afferent arteriolarnon-CB1 receptors may cause excessive hypoperfusion in the kidneys,and the alterations of GFR and RBF may resume as a result. Furtherstudies will be needed to clarify the function of non-CB receptorsin the kidneys.
In conclusion, the present study demonstrated for the firsttime that ANA significantly decreases GFR and increases RBFindependent of its effects on BP and renal nerves and that ANAelicits vasodilation in afferent and efferent arterioles throughCB receptors with a greater effect on efferent arterioles. Becausethe CB receptors were present exclusively in the renal vascularsystem, the ANA-induced predominant vasodilation in efferentarterioles accounts for the decrease in GFR.
Acknowledgments
This work was supported in part by a grant from the Ministryof Education, Science and Culture of Japan (14571073) and agrant to Atsuhiro Ichihara from the Keio Health Counseling CenterFund. We thank the skillful secretarial work of Ms. Rika Wakita.
Fink MP, Fiallo V, Stein KL, Gardiner WM: Systemic and regional hemodynamic changes after intraperitoneal endotoxin in rabbits: Development of a new model of the clinical syndrome of hyperdynamic sepsis. Circ Shock 22: 73–81, 1987[Medline]
Varga K, Wagner JA, Bridgen DT, Kunos G: Platelet- and macrophage-derived endogenous cannabinoids are involved in endotoxin-induced hypotension. FASEB J 12: 1035–1044, 1998[Abstract/Free Full Text]
Wang Y, Liu Y, Sarker KP, Nakashima M, Serizawa T, Kishida A, Akashi M, Nakata M, Kitajima I, Maruyama I: Polymyxin B binds to anandamide and inhibits its cytotoxic effect. FEBS Lett 470: 151–155, 2000[CrossRef][Medline]
Deutsch DG, Chin SA: Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem Pharmacol 46: 791–796, 1993[CrossRef][Medline]
Shire D, Carillon C, Kaghad M, Calandra B, Rinaldi-Carmona M, LeFur G, Caput D, Ferrara P: An amino-terminal variant of the central cannabinoid receptor resulting from alternative splicing. J Biol Chem 270: 3726–3731, 1995[Abstract/Free Full Text]
Deutsch DG, Goligorsky MS, Schmid PC, Krebsbach RJ, Schmid HHO, Das SK, Dey SK, Arreaza G, Thorup C, Stefano G, Moore LC: Production and physiological actions of anandamide in the vasculature of the rat kidney. J Clin Invest 100: 1538–1546, 1997[Medline]
Ichihara A, Inscho EW, Imig JD, Michel RE, Navar LG: Role of renal nerves in afferent arteriolar reactivity in angiotensin-induced hypertension. Hypertension 29: 442–449, 1997[Abstract/Free Full Text]
Ichihara A, Hayashi M, Navar LG, Saruta T: Inducible nitric oxide synthase attenuates endothelium-dependent renal microvascular vasodilation. J Am Soc Nephrol 11: 1807–1812, 2000[Abstract/Free Full Text]
White SM, Imig JD, Kim T-T, Hauschild BC, Inscho EW: Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC. Am J Physiol Renal Physiol 280: F1054–F1061, 2001[Abstract/Free Full Text]
Clendenon JL, Phillips CL, Sandoval RM, Fang S, Dunn KW: Voxx: A PC-based, near real-time volume rendering system for biological microscopy. Am J Physiol 282: C213–C218, 2002
Brown SM, Wager-Miller J, Mackie K: Cloning and molecular characterization of the rat CB2 cannabinoid receptor. Biochim Biophys Acta 1576: 255–264, 2002[Medline]
Bukoski RD, Bátkai S, Járai Z, Wang Y, Offertaler L, Jackson WF, Kunos G: CB1 receptor antagonist SR141716A inhibits Ca2+-induced relaxation in CB1 receptor-deficient mice. Hypertension 39: 251–257, 2002[Abstract/Free Full Text]
Wang X, Loutzenhiser R: Determinants of renal microvascular response to ACh: Afferent and efferent arteriolar actions of EDHF. Am J Physiol Renal Physiol 282: F124–F132, 2002[Abstract/Free Full Text]
Arima S, Endo Y, Yaoita H, Omata K, Ogawa S, Tsunoda K, Abe M, Takeuchi K, Abe K, Ito S: Possible role of P-450 metabolite of arachidonic acid vasodilator mechanism of angiotensin II type 2 receptor in the isolated microperfused rabbit afferent arteriole. J Clin Invest 100: 2816–2823, 1997[Medline]
Received for publication January 16, 2003.
Accepted for publication March 11, 2004.
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Abstract
Introduction
90% of the preglomerular vascular resistance. In addition, the ANA-induced afferent arteriolar dilation was attenuated by the blockade of CB1 receptors with AM251 and inhibited by the nonselective blockade of CB receptors with AM281. These results suggest that the RBF responses to ANA are wholly dependent on the CB receptors-mediated afferent arteriolar dilation. 
; n = 14) and bilaterally renal denervated rats (•; n = 7). *P < 0.05 versus 0 pmol/kg ANA.
; n = 14) and bilaterally renal denervated rats (
; n = 7). *P < 0.05 versus 0 pmol/kg ANA.
