European Journal of Pharmacology 603 (2009) 108–113

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European Journal of Pharmacology

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Pulmonary, Gastrointestinal and Urogenital Pharmacology

Preventive mechanisms of agmatine against ischemic acute kidney injury in rats

Takahiro Sugiura a, Shuhei Kobuchi a, Hidenobu Tsutsui a,b, Masanori Takaoka a, Toshihide Fujii a,Kentaro Hayashi a, Yasuo Matsumura a,⁎a Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japanb Laboratory of Clinical Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tondabayashi, Osaka 584-8540, Japan

⁎ Corresponding author. Department of Pharmacologceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 56690 1051.

E-mail address: matumrh@gly.oups.ac.jp (Y. Matsum

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.11.062

a b s t r a c t

a r t i c l e i n f o

Article history:

The excitation of renal symp Received 30 August 2008Received in revised form 12 November 2008Accepted 24 November 2008Available online 10 December 2008

Keywords:AgmatineAdrenaline α2 receptorImidazoline I1 receptorIschemic acute kidney injuryRenal sympathetic nerve activityNorepinephrine

athetic nervous system plays an important role in the development of ischemicacute kidney injury in rats. Recently, we found that agmatine, an adrenaline α2/imidazoline I1-receptoragonist, has preventive effects on ischemic acute kidney injury by suppressing the enhanced renalsympathetic nerve activity during renal ischemia and by decreasing the renal venous norepinephrineoverflow after reperfusion. In the present study, we investigated preventive mechanisms of agmatine againstischemic acute kidney injury in rats. Ischemic acute kidney injury was induced by clamping the left renalartery and vein for 45 min followed by reperfusion, 2 weeks after the contralateral nephrectomy.Pretreatment with efaroxan (30 μmol/kg, i.v.), an α2/I1-receptor antagonist, abolished the suppressive effectsof agmatine on the enhanced renal sympathetic nerve activity during renal ischemia and on the elevatednorepinephrine overflow after reperfusion, and eliminated the preventing effects of agmatine on theischemia/reperfusion-induced renal dysfunction and histological damage. On the other hand, pretreatmentwith yohimbine (6 μmol/kg, i.v.), an α2-receptor antagonist, eliminated the preventing effects of agmatine onthe ischemia/reperfusion-induced renal injury and norepinephrine overflow, without affecting the loweringeffect of agmatine on renal sympathetic nerve activity. These results indicate that agmatine prevents theischemic renal injury by sympathoinhibitory effect probably via I1 receptors in central nervous system and bysuppressing the norepinephrine overflow through α2 or I1 receptors on sympathetic nerve endings.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Agmatine is a polycationic amine synthesized from L-arginine, bythe enzyme arginine decarboxylase, and has been identified as anendogenous clonidine-displacing substance in the mammalian brain(Li et al., 1994). Moreover, agmatine may serve as a neurotransmitterin the central nervous system (Reis and Regunathan, 2000). Agmatinehas several biologic effects including neuroprotective (Kim et al., 2004,2006), antidepressant and anxiolytic effects (Zomkowski et al., 2002;Aricioglu and Altunbas, 2003). Most recently, we noted that agmatineovercame ischemia/reperfusion-induced acute kidney injury in rats(Sugiura et al., 2008).

Acute kidney injury is a frequent clinical syndrome with highmorbidity and mortality (Ympa et al., 2005). The precise mechanismsunderlying the ischemia/reperfusion-induced acute kidney injury arenot fully understood, but it has been reported that several causalfactors (e.g., ATP depletion, reactive oxygen species, phospholipaseactivation, neutrophil infiltration, and vasoactive peptides) arecontributive to the pathogenesis of this renal damage (Edelstein

y, Osaka University of Pharma-9-1094, Japan. Tel./fax: +81 72

ura).

l rights reserved.

et al., 1997). In addition, enhancement of renal sympathetic nerveactivity and its consequent effect on norepinephrine overflow fromnerve endings are considered to be involved in the development of theischemia/reperfusion-induced acute kidney injury (Ogawa et al.,2002; Kurata et al., 2006). We have found that renal sympatheticnerve activity is significantly augmented during renal ischemia. Inaddition, we noted that ischemic acute kidney injury is ameliorated byrenal denervation or ganglionic blockade and that the effect isaccompanied by suppression of elevated renal venous norepinephrinelevels after reperfusion (Fujii et al., 2003). In our recent study, thepreischemic treatment with agmatine exerted the suppressive effecton the enhancement of renal sympathetic nerve activity andconsequent elevation of renal venous norepinephrine levels observedin ischemic acute kidney injury rats (Sugiura et al., 2008).

Agmatine was firstly isolated as an endogenous agonist forimidazoline I receptors (Li et al., 1994). There is accumulating evidencethat agmatine affects imidazoline I and adrenalineα2 receptors (Piletzet al., 1995; Pinthong et al., 1995; González et al., 1996; Li et al., 2001;Li and He, 2001). Imidazoline I receptors have been subclassified into 3major groups, based largely upon ligand selectivities and subcellulardistribution (Eglen et al., 1998), and I1 subtype is known to existprincipally and to play a role in controlling sympathetic nerve activityin the rostral ventrolateral medulla (Ernsberger et al., 1990; Mayorovet al., 2001). Agmatine administered intravenously was reported to

109T. Sugiura et al. / European Journal of Pharmacology 603 (2009) 108–113

suppress the sympathetic nerve activity of anesthetized rats (Sunet al., 1995) and to reduce norepinephrine release provoked bypreganglionic electrical stimulation in pithed spontaneously hyper-tensive rats (Raasch et al., 2003). In the current study, in order toinvestigate the receptor types and relatedmechanisms involved in theprotective effect of agmatine against ischemic acute kidney injury, weevaluated effects of efaroxan, an α2/I1-receptor antagonist, andyohimbine, an α2-receptor antagonist, on the renoprotective effectof agmatine.

2. Materials and methods

2.1. Animals and experimental design

Male Sprague-Dawley rats (10 weeks of age; Japan SLC, Shizuoka,Japan) were used. The animals were housed in a light-controlled roomwith a 12-h light/dark cycle and were allowed ad libitum access tofood and water. Experimental protocols and animal care methods inthe experiments were approved by the Experimental AnimalCommittee at Osaka University of Pharmaceutical Sciences (Osaka,Japan). Two weeks before the study (at 8 weeks of age), the rightkidney was removed through a small flank incision under pentobar-bital anesthesia (40 mg/kg, i.p.). After a 2-week recovery period,uninephrectomized rats were divided into sham-operated control,vehicle-treated ischemic acute kidney injury, and drug-treatedischemic acute kidney injury groups. To induce ischemic acute kidneyinjury, the rats were anesthetized with pentobarbital (50 mg/kg, i.p.),and the left kidney was exposed through a small flank incision. Theleft renal artery and vein were occluded with a nontraumatic clampfor 45 min. At the end of the ischemic period, the clamp was releasedto allow reperfusion. Each drug used in this study or vehicle (0.9%saline) was administered into the left external jugular vein (1 ml/kg).Agmatine or vehicle was injected 5 min before the start of ischemia,and efaroxan or yohimbine was given 10 min before the ischemia. Insham-operated control rats, the left kidney was treated identically,with the exception of the clamping. The animals exposed to 45-minischemiawere housed in metabolic cages at 24 h after reperfusion and5-h urine samples were collected. At the end of urine collection, bloodsamples were drawn from the thoracic aorta, and then the left kidneyswere excised under pentobarbital anesthesia (50 mg/kg, i.p.). Theplasma was separated by centrifugation and used for measurement ofrenal function parameters. The kidneys were used for light micro-scopic observation.

In separate experiments, we examined the effect of efaroxan oryohimbine on the suppression of norepinephrine overflow byagmatine. Under pentobarbital (50 mg/kg, i.p.) anesthesia, anabdominal midline incision of uninephrectomized rats was madeand the left kidney was exposed. A 26-gauge needle was inserted intothe left renal vein for venous blood sampling. Each blood sample wastaken baseline and 24 h after reperfusion following 45-min ischemia.The sampling period (only one sample from each animal) was 2min induration. Plasma was immediately separated by centrifugation. Thesesamples were stored at −80 °C until the assay for norepinephrineconcentration.

As described below, in another set of experiments, electricalsignals of renal neural activity were directly recorded for evaluation ofchanges in renal sympathetic nerve activity during 45-min ischemicperiod.

2.2. Renal nerve recording

For the measurement of renal sympathetic nerve activity,uninephrectomized rats were anesthetized with pentobarbital(50 mg/kg, i.p.) and given additional doses as required. Depth ofanesthesia was assessed as stability of heart rate and blood pressure,which were continuously monitored with a pressure transducer

connected to a polygraph system (RM6000G, Nihon Kohden, Osaka,Japan). Data collectionwas donewhen the hemodynamic parametersmaintained stable conditions. Surgical preparation of the animalsand basic experimental techniques were identical to those describedpreviously (Shokoji et al., 2003). Renal sympathetic nerve activitywas recorded from the left renal nerve branch before and duringischemia. The nerve was isolated near the aortic-renal arterialjunction through a left flank incision and placed on a Teflon-coatedstainless steel bipolar electrode. The renal nerve and electrode werecovered with silicone rubber. The renal nerve discharge wasamplified using a differential amplifier (AVB-11A; Nihon Kohden)with a band-pass filter (low frequency, 50 Hz; high frequency, 1 kHz).The amplified and filtered signal was visualized on a dual-beamoscilloscope (VC-11; Nihon Kohden) and monitored by an audiospeaker. The output from the amplifier was integrated by anintegrator (EI601G; Nihon Kohden) with 1-s resetting. The outputfrom the integrator was recorded and analyzed with PowerLab(ML750; ADInstruments Pty Ltd., Castle Hill, Australia). For thequantification of renal sympathetic nerve activity, the height ofintegrated nerve discharge was measured for 20 s in each experi-ment. Changes in nerve activity were expressed as percentages ofcontrol resting spontaneous nerve activity.

2.3. Analytical procedures

Blood urea nitrogen and creatinine levels in plasma or urine weredetermined using a commercial assay kit, the BUN-test-Wako, andCreatinine-test-Wako (Wako Pure Chemical Industries, Osaka,Japan), respectively. Creatinine clearance (Ccr, ml/min/kg) wascalculated from the formula: Ccr=Ucr×UF/Pcr, where Ucr and Pcrare creatinine concentration in urine and plasma, respectively, andUF is urine flow.

Norepinephrine concentration in renal venous plasma wasmeasured by high-performance liquid chromatography with anamperometric detector (HTEC-500; Eicom, Kyoto, Japan), as pre-viously reported (Hayashi et al., 1991).

2.4. Histological studies

Excised left kidneys were processed for light microscopicobservation, according to standard procedures. The kidneys werethen preserved in phosphate-buffer 10% formalin, after which thekidneys were chopped into small pieces, embedded in paraffin wax,cut at 4 μm, and stained with hematoxylin and eosin. Histopatho-logical changes were analyzed for tubular necrosis, proteinaceouscasts, and medullary congestion, as described by Caramelo et al.(1996). Tubular necrosis and proteinaceous casts were graded asfollows: no damage (0), mild (1; unicellular, patchy isolateddamage), moderate (2; damage less than 25%), severe (3; damagebetween 25 and 50%), and very severe (4; more than 50% damage).The degree of medullary congestion was defined as no congestion(0), mild (1; vascular congestion with identification of erythrocytesby ×400 magnification), moderate (2; vascular congestion withidentification of erythrocytes by ×200 magnification), severe(3; vascular congestion with identification of erythrocytes by ×100magnification), and very severe (4; vascular congestion withidentification of erythrocytes by ×40 magnification). The scoring ofthe histological data was performed by independent observers in adouble blind manner.

2.5. Drugs

Agmatine, efaroxan and yohimbine were purchased from SigmaChemical (St. Louis, MO, USA). These drugs were dissolved in saline(0.9%). Other chemicals were obtained from Nacalai Tesque (Kyoto,Japan) and Wako Pure Chemical Industries (Osaka, Japan).

Fig. 1. Typical responses of RSNA and integrated RSNA to injection of vehicle (0.9% saline) (A), agmatine (300 μmol/kg) (B), agmatine (300 μmol/kg)+efaroxan (30 μmol/kg) (C) andagmatine (300 μmol/kg)+yohimbine (6 μmol/kg) (D) during the 45-min ischemic period in anesthetized rats. Percent changes in integrated RSNA in responses to injection of vehicle,agmatine (300 μmol/kg), agmatine (300 μmol/kg)+efaroxan (10 μmol/kg), agmatine (300 μmol/kg)+efaroxan (30 μmol/kg), agmatine (300 μmol/kg)+yohimbine (3 μmol/kg) andagmatine (300 μmol/kg)+yohimbine (6 μmol/kg) (E). After efaroxan or yohimbine was given 10 min before the ischemia, vehicle or agmatine was given 5 min before the ischemia.Each point and bar represents the mean±S.E.M. (n=6). ##Pb0.01, compared with basal level. ⁎⁎Pb0.01, compared with vehicle-treated AKI rats. †Pb0.05; ††Pb0.01, compared withagmatine (300 μmol/kg)-treated AKI rats. RSNA, renal sympathetic nerve activity. AKI, acute kidney injury.

110 T. Sugiura et al. / European Journal of Pharmacology 603 (2009) 108–113

2.6. Statistical analysis

All values were expressed as means±S.E.M. Relevant data wereprocessed by Instat (Graph-PAD Software for Science). Nerve record-ing studies were analyzed by one-way repeated measures ANOVA

Fig. 2. Effects of efaroxan or yohimbine on renal protection by treatment with agmatine. Ren(B) at 24 h after ischemia/reperfusion. After efaroxan or yohimbine was given 10 min before tbar represents the mean±S.E.M. (n=6). ##Pb0.01, compared with sham-operated rats. ⁎Pbwith agmatine (300 μmol/kg)-treated AKI rats. AKI, acute kidney injury.

followed by Dunnett's multiple range test for within-group data. Foramong-group data, we used one-way ANOVA followed by Bonferroni'smultiple comparison test in nerve recording, renal function ornorepinephrine concentration studies. Correlation between norepi-nephrine concentration in renal venous plasma and renal functionwas

al function parameters are blood urea nitrogen (BUN) (A), and creatinine clearance (Ccr)he ischemia, vehicle or agmatine was given 5min before the ischemia. Each column and0.05; ⁎⁎Pb0.01, compared with vehicle-treated AKI rats. †Pb0.05; ††Pb0.01, compared

Fig. 3. Light microscopy of the inner zone (A–E), the outer zone inner stripe (F–J), and the outer zone outer stripe (K–O) of medulla of the kidney of AKI rats treated with injection ofvehicle (B, G, and L), agmatine (300 μmol/kg) (C, H, and M), agmatine (300 μmol/kg)+efaroxan (30 μmol/kg) (D, I, and N) and agmatine (300 μmol/kg)+yohimbine (6 μmol/kg) (E, J,and O) at 24 h after ischemia/reperfusion, and sham-operated rats (A, F, and K). After efaroxan or yohimbine was given 10 min before the ischemia, vehicle or agmatine was given5 min before the ischemia. Severe proteinaceous casts in tubuli (B), congestion and hemorrhage (G), and tubular necrosis (L) are observed in vehicle-treated AKI rats. Magnification×200. AKI, acute kidney injury.

Table 1Effects of efaroxan or yohimbine on renal tissue protection by treatment with agmatine

Experimental group Proteinaceous castsin tubuli

Medullarycongestion

Tubularnecrosis

AKI+vehicle 3.50±0.22 3.67±0.21 3.83±0.17AKI+agmatine 300 μmol/kg 1.67±0.33a 1.83±0.31b 1.50±0.22b

AKI+agmatine 300 μmol/kg+efaroxan 30 μmol/kg

2.83±0.48 2.83±0.48 3.17±0.31

AKI+agmatine 300 μmol/kg+yohimbine 6 μmol/kg

3.55±0.22 3.00±0.37 3.33±0.42

Values represent the mean±S.E.M. (n=6) of histopathological grade. Grade: no change(0), mild (1), moderate (2), severe (3), very severe (4). AKI, acute kidney injury.

a Pb0.05, compared with AKI+vehicle.b Pb0.01, compared with AKI+vehicle.

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analyzed by Pearson correlation test. Histological data were analyzedusing Kruskal–Wallis nonparametric test combined with a Steel-typemultiple comparison test. For all comparisons, differences wereconsidered significant at Pb0.05.

3. Results

3.1. Effects of agmatine on the enhanced renal sympathetic nerve activityduring ischemic period in the absence or presence of treatment withefaroxan or yohimbine

As reported previously (Sugiura et al., 2008), the preischemictreatment with agmatine (300 μmol/kg, i.v.) markedly suppressedthe enhanced renal sympathetic nerve activity during the ischemicperiod (Fig. 1A, B and E). This suppressive effect was partially butsignificantly attenuated by efaroxan (10 μmol/kg, i.v.), an α2/I1receptor antagonist, and the higher dose of efaroxan (30 μmol/kg, i.v.)completely abolished the agmatine's effect (Fig. 1C and E). On theother hand, the suppressive effect of agmatine on the renalsympathetic nerve activity was not affected by the treatment withyohimbine (3, 6 μmol/kg, i.v.), an α2 receptor antagonist (Fig. 1Dand E).

3.2. Effects of agmatine on ischemia/reperfusion-induced renaldysfunction and histological damage in the absence or presence oftreatment with efaroxan or yohimbine

As shown in Fig. 2, the renal function of rats subjected to 45-minischemia showed a marked deteriorationwhenmeasured at 24 h afterthe reperfusion. As compared with sham-operated rats, vehicle-treated acute kidney injury rats showed significant increase in BUNand decrease in Ccr. Intravenous injection of agmatine (300 μmol/kg)to ischemic acute kidney injury rats markedly attenuated theischemia/reperfusion-induced renal dysfunction, as demonstratedpreviously (Sugiura et al., 2008). This agmatine-induced improvementwas almost completely inhibited by efaroxan treatment at the lowerdose (10 μmol/kg) as well as the higher dose (30 μmol/kg). Yohimbine

at the higher dose (6 μmol/kg) but not at the lower dose (3 μmol/kg),also inhibited significantly the renoprotective effect of agmatine.

Histopathological examination revealed severe lesions in thekidney of vehicle-treated acute kidney injury rats after the ischemia/reperfusion. These changes were characterized by proteinaceous castsin tubuli in the inner zone of medulla (Fig. 3B), medullary congestion,and hemorrhage in the outer zone inner stripe of medulla (Fig. 3G),and tubular necrosis in the outer zone outer stripe of medulla (Fig. 3L).Intravenous injection of agmatine significantly attenuated the devel-opment of all these lesions (Fig. 3C, H, M and Table 1). Treatment withefaroxan (Fig. 3D, I and N) or yohimbine (Fig. 3E, J and O) at the higherdose negated the improvement of all these lesions induced byagmatine (Table 1).

3.3. Effects of agmatine on ischemia/reperfusion-induced elevation ofnorepinephrine concentration in renal venous plasma in the absence orpresence of treatment with efaroxan or yohimbine

As shown in Fig. 4, norepinephrine concentrations in renal venousplasma (24 h after reperfusion) of vehicle-treated acute kidney injuryrats were remarkably increased, compared with sham-operated rats,and these increases were significantly suppressed by the treatmentwith agmatine, as demonstrated previously (Sugiura et al., 2008).

Fig. 4. Effects of efaroxan or yohimbine on NE overflow suppressed by treatment withagmatine. After efaroxan or yohimbinewas given 10min before the ischemia, vehicle oragmatine was given 5 min before the ischemia. Blood samples were taken during 2 minat 24 h after reperfusion. Each column and bar represents the mean±S.E.M. (n=6).##Pb0.01, compared with sham-operated rats. ⁎Pb0.05, compared with vehicle-treated AKI rats. †Pb0.05; ††Pb0.01, compared with agmatine (300 μmol/kg)-treatedAKI rats. NE, norepinephrine. AKI, acute kidney injury.

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Agmatine-induced decreasing effect was almost completely inhibitedby efaroxan treatment at both the lower and higher doses. Yohimbineat the higher dose but not at the lower dose, also abolished theagmatine's effect.

Yohimbine treatment (6 μmol/kg) without agmatine tended tofurther elevate the increased norepinephrine concentration afterischemia/reperfusion (379±67 pg/ml), but these changes were notstatistically significant.

Norepinephrine concentrations in renal venous plasma after theischemia/reperfusion of each group corresponded well with thechange in renal functional parameters (Fig. 2). Thus, norepinephrine

Fig. 5. Correlation between renal venous norepinephrine concentrations and blood urea nefaroxan or yohimbine was given 10 min before the ischemia, vehicle or agmatine was give

concentrations in renal venous plasma 24 h after reperfusionpositively correlated with levels of BUN and Ccr (Fig. 5).

4. Discussion

Renal sympathetic nervous system and circulating catecholaminesare considered to be involved in the pathogenesis of acute kidneyinjury since pharmacological blockade of sympathetic nerve exerts anefficient protective effect on this failure (Ogawa et al., 2002; Kurataet al., 2006). We also noted that a ganglion blocking agent or renaldenervation efficiently attenuated renal dysfunction and degenerationinduced by ischemia/reperfusion (Fujii et al., 2003). Most recently, wefound that the intravenous treatment (100–300 μmol/kg) withagmatine to the ischemic acute kidney injury rats efficientlysuppressed the enhancement of renal sympathetic nerve activity inthe ischemic period and the increased norepinephrine overflow afterreperfusion. Moreover, similar suppressive effects were observed alsoby the intracerebroventricular injection of agmatine at a small dose(600 nmol/kg), which produced no significant influence by theintravenous injection, thereby suggesting that the inhibitory effectof agmatine on the enhanced renal sympathetic nerve activity seemsto occur at least partly at the central nervous system (Sugiura et al.,2008).

Agmatinebinds to adrenalineα2 and imidazoline I receptors (Li et al.,1994, 2001; Piletz et al.,1995; Pinthonget al.,1995;González et al.,1996;Li and He, 2001). Previous studies support the concept that centrallyacting antihypertensive drugs such as clonidine and related imidazolinederivatives mediate sympathoinhibition not only via activation ofcentral nervous α2 receptors but also via I1 receptors (Ernsbergeret al., 1990, 1993; Tolentino-Silva et al., 2000). In addition, clonidine isknown to lessen post-ischemic acute kidney injury (Solez et al.,1980). Inthe present study, we therefore evaluated the receptors involved in thesuppressive effects of agmatine on renal sympathetic nerve activity.Results clearly indicated that the suppressive effects of agmatine onischemia-enhanced renal sympathetic nerve activity were not affectedby injection of yohimbine, an α2 receptor antagonist, but efaroxan, anα2/I1 receptor antagonist, dose-dependently and efficiently attenuatedthe above agmatine's action. Thus, it is reasonable to consider thatagmatine suppresses the ischemia-enhanced renal sympathetic nerveactivity through the activation of I1 receptors. In contrast, the

itrogen (BUN) or creatinine clearance (Ccr) at 24 h after ischemia/reperfusion. Aftern 5 min before the ischemia. AKI, acute kidney injury.

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renoprotective effects of agmatine on ischemia/reperfusion-inducedrenal dysfunction were completely abolished even by the treatmentwith the lower dose of efaroxan or with yohimbine. In general,enhancement of renal sympathetic nerve activity during ischemia iswell related to renal dysfunction at 24 h after reperfusion (Fujii et al.,2003; Kurata et al., 2006). However, findings obtained by α2 and/or I1receptor blockade on the agmatine-induced actions exhibited thatchanges in renal sympathetic nerve activities did not always correlatewith the extent of the postischemic renal dysfunction.

Earlier studies have indicated that norepinephrine overflow intothe renal vein could be useful to assess the activity of renalsympathetic nervous system, since there was a significant linearrelationship between the frequency of stimulation and norepinephr-ine concentrations in renal venous plasma, when renal nerves wereelectrically stimulated in anesthetized dogs (Oliver et al., 1980). Usingthe ischemic acute kidney injury model, we have demonstrated thatrenal sympathetic nerve activity is enhanced by the ischemia and theenhancement well correlates with norepinephrine concentrations inrenal venous plasma after reperfusion (Fujii et al., 2003). However, inthe present study, the lower dose of efaroxan completely abolishedthe agmatine-induced decreasing effect on the elevation of renalvenous norepinephrine level after reperfusion, in spite of only partialinhibition against the agmatine's action on the ischemia-enhancedrenal sympathetic nerve activity. In addition, yohimbine treatment atthe higher dose abolished the effect of agmatine on the renal venousnorepinephrine level, without affecting the renal sympathetic nerveactivity. The most important observation is that norepinephrineconcentrations in renal venous plasma, but not renal sympatheticnerve activity, correlated well with the changes in renal dysfunctionafter the ischemia/reperfusion (Fig. 5). Taken together, it is reasonableto consider that agmatine suppresses not only the enhanced renalsympathetic nerve activity but also norepinephrine overflow inperipheral renal sympathetic nerve endings. It seems likely thatagmatine suppresses increased renal sympathetic nerve activitythrough the activation of I1 receptors and decreases elevated renalvenous norepinephrine overflow through the activation of α2 or I1receptors. These effects would eventually improve the ischemia/reperfusion-induced renal injury.

In separate pilot study, we obtained evidence that the intracer-ebroventricular injection of efaroxan (100–300 nmol/kg), but notyohimbine (30–90 nmol/kg), attenuated the suppressive effect ofagmatine (300 μmol/kg, i.v.) on the ischemia-enhanced renalsympathetic nerve activity (unpublished observations). In conclusion,our results indicate that agmatine can suppress the enhanced renalsympathetic nerve activity during renal ischemia via the activation ofimidazoline I1 receptors in central nervous system and the renalnorepinephrine overflow after the ischemia/reperfusion through theactivation of α2 or I1 receptors on peripheral renal sympathetic nerveendings. These suppressive effects are probably responsible for therenoprotection against ischemia/reperfusion-induced renal injury.

Acknowledgments

This study was supported in part by a “High Technology ResearchCenter” Project for Private Universities: matching fund subsidy fromthe Ministry of Education, Culture, Sports, Science and Technology(2002–2006 and 2007–2009) and Nihon Bioresearch Inc (6-104Majima, Fukuju-cho, Hashima, Gifu 501-6251, Japan).

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