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作者：H. ThomasLee, AyukoOta-Setlik, HuaXu, Vivette D.D‘Agati, Marlene A.Jacobson, Charles W.Emala作者单位：1 Department of Anesthesiology and Department of Pathology, College of Physicians andSurgeons of Columbia University, New York, New York 10032; and Department of Pharmacology, Merck ResearchLaboratories, West Point, Pennsylvania 19486 5 c* c$ j1 r/ G
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& ]& b7 @6 a. J( p. {! n1 k" k$ ^- [4 b 【摘要】
1 H9 M! k0 c6 U A 3 adenosine receptor(AR) activation and inhibition worsen and improve, respectively, renalfunction after ischemia-reperfusion (I/R) injury in rats. Wesought to further characterize the role of A 3 ARs inmodulating renal function after either I/R or myoglobinuric renalinjury. A 3 knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R ormyoglobinuric renal injury. C57 control mice pretreated with theA 3 AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N 6 -(3-iodobenzyl)- N -methyl-5'-carbamoyladenosine(IB-MECA)] demonstrated improved or worsened renal function,respectively, after I/R or myoglobinuric renal injury. Higher doses ofIB-MECA were lethal in C57 mice subjected to renal ischemia.H 1 but not H 2 histamine receptor antagonistprevented death in mice pretreated with IB-MECA before renalischemia. Improvement in renal function was associated withsignificantly improved renal histology. In conclusion,preischemic A 3 AR activation (0.125 mg/kg IB-MECA)exacerbated renal I/R injury in mice. Mice lacking A 3 ARsor blocking A 3 ARs in wild-type mice resulted insignificant renal protection from ischemic or myoglobinuric renal failure.
! l. f9 k" Q+ s: H& l% _ 【关键词】 acute renal failure histamine ischemicreperfusion injury; `0 t4 O9 h$ V, m% v! H2 _4 R
INTRODUCTION5 \8 {& K5 e* r+ s- Q) v. t4 L
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ACUTE RENAL FAILURE (ARF) is a major contributor of perioperative mortalityand morbidity ( 7, 20 ). Ischemic-reperfusion (I/R)injury, toxic nephropathy, and myoglobinuria all can lead to ARF, whichis frequently complicated by many other life-threatening complicationsincluding sepsis and multiorgan failure ( 1, 20, 21 ). Theprognosis for patients with ARF is poor (with mortality ~50%) andhas changed little over the past 40 years ( 1, 7 ).' j7 o8 q$ f8 J9 }, E; U3 p0 X! Q5 X
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We demonstrated previously that adenosine receptor (AR) modulationssignificantly affect renal function after I/R injury in rats ( 14, 16, 17 ). All four known AR subtypes (A 1,A 2a, A 2b, and A 3 ) are expressed inthe kidney ( 18, 28 ), but the function of A 3 ARis unknown. We recently showed that preischemic activation ofA 3 ARs exacerbates renal failure after I/R injury ( 14 ). Conversely, preischemic administration of anA 3 AR antagonist protected the rat kidney against I/R injury.- r; F. R: G2 X$ @ u3 d* Q
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In this study, we aimed to extend our previous findings of modulationof A 3 ARs on renal function after I/R injury utilizing micedeletionally lacking A 3 ARs [A 3 AR knockout(A 3 KO)] ( 23, 26 ). We hypothesized thatA 3 KO mice would be endogenously protected against I/Rinjury. We also hypothesized that preischemic activation orinhibition of A 3 ARs would worsen and protect,respectively, renal failure after I/R- or myoglobinuria-induced renalfailure in mice. A 3 AR activation degranulates mast cellsand increases plasma histamine in rodents ( 4, 6, 22, 26 ).Therefore, we investigated the mechanism of A 3 ARmodulation of renal failure by using selective blockers of histaminereceptors and an effector (compound 48/80) that increases endogenoushistamine release. n }6 _6 x9 I6 }+ E! L" H
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METHODS T5 j# O+ s& V, |: t
1 A8 E9 d& ~/ }2 g# D4 A! @* SA 3 KO mice. Homozygous A 3 KO (A 3 / ) micebreeding pairs were provided by Merck Research Laboratories (WestPoint, PA). Generation and initial characterization of theA 3 KO mice have been described in detail previously( 23, 26 ). The A 3 KO mice were obtained afterthey were backcrossed against C57BL/6 mice for 12 generations to obtain a congenic line. C57BL/6 mice (Harlan Laboratories, Indianapolis, IN)served as background A 3 / controls (C57).
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Renal injury protocol. A 3 KO and C57 mice (25-30 g body wt) were anesthetizedwith pentobarbital sodium (50 mg/kg or to effect ip) and placed supine on a heating pad under a warming light to maintain body temperature between 36 and 38°C. Additional pentobarbital sodium was given asneeded on the basis of response to tail pinch. Bilateral flank incisions were made, and the left kidney was subjected to 30 min ofischemia with microaneurysm clips after right nephrectomy. Theduration of ischemia was chosen to maximize reproducibility ofrenal injury and to minimize mortality in these mice. Separate groupsof C57 and A 3 KO mice were injected with 8.5 mg/kg glycerol intramuscularly after 16 h of water deprivation to inducemyoglobinuric renal injury. Some C57 mice were pretreated either with3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5 dicarboxylate (MRS-1191; 1 mg/kg ip), a selective A 3 ARantagonist, or with 0.125 mg/kg N 6 -(3-iodobenzyl)- N -methyl-5'-carbamoyladenosine(IB-MECA; 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/kg ip), a selectiveA 3 AR agonist, 15 min before renal ischemia orglycerol injection.
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To determine whether histamine release by mast cell degranulationinduced by IB-MECA plays any role in lethality and exacerbation ofrenal injury by A 3 AR activation, some C57 mice werepretreated with 10 mg/kg diphenylhydramine or 10 mg/kg ranitidine ip 30 min before 1 mg/kg IB-MECA. Some A 3 KO and C57 mice werepretreated with 5 mg/kg of compound 48/80 (to degranulate mast cells)15 min before being subjected to renal I/R injury., ]. j! O2 O( p/ J& f4 {, P" M
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Assessment of renal injury. Renal function was assessed by measuring plasma creatinine 24 hafter renal ischemia or glycerol injection by using acommercially available colorimetric method (Sigma). For histologicalpreparations, explanted kidneys were bisected along the long axis andwere fixed in 10% formalin solution overnight. After automateddehydration through a graded alcohol series, transverse kidney sliceswere embedded in paraffin, sectioned at 5 µm, and stained withhematoxylin-eosin. Morphological assessment was performed by anexperienced renal pathologist (V. D. D'Agati) who was blinded tothe treatment for each animal. A grading scale of 0-4, as outlinedby Jablonski et al. ( 10 ), was used for thehistopathological assessment of I/R- or myoblobinuria-induced damage ofthe proximal tubules.
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* q9 U; m1 b; K& s- AMeasurement of plasma histamine levels. Plasma histamine levels were measured in A 3 KO and C57 mice20 min after IB-MECA or compound 48/80 treatment (5 min afterinitiation of renal ischemia) by using an ELISA specific formice (RDI, Flanders, NJ) according to the manufacturer's instructions.
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PCR for A 3 ARs. We analyzed the presence of the wild-type or mutantA 3 AR DNA in C57 and A 3 KO mice by PCR. Todifferentially confirm the genotype of A 3 KO and C57 mice,primers were designed to recognize the 5'- and 3'-end of thefull-length wild-type A 3 AR (sense:5'-GACTGGCTGAACATCACCTACAT-3' and antisense:5'-ATAGAAGTGCATCTTGACTTGCAG-3') and the PGKneo insert (sense:5'-CTATGACTGGGCACAACAGACAAT-3' and antisense:5'-ATCAGCCATGATGGATACTTTCTC-3') in A 3 KOs( 23 ). All primers were purchased from SigmaGenoSys (The Woodlands, TX). Genomic DNA was isolated from mouse tails by using the Wizard Genomic DNA extraction kit (Promega, Madison, WI).DNA concentrations were determined by using spectrophotometric absorbance at 260/280. PCR was performed with Taq polymerase(Promega). Genomic DNA (0.2 µg) was used as a template and amplifiedfor 30 cycles with a PTC-200 thermal cycler (MJ Research, Waltham, MA)using a primer annealing temperature of 60°C. The products wereresolved in a 6% polyacrylmide gel and stained with Syber green(Roche, Indianapolis, IN) for analysis with a FluorS Multi Imager(Bio-Rad, Hercules, CA).
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Chemicals. Unless otherwise specified, all reagents were purchased from Sigma (St.Louis, MO).3 x* O* `* W5 I* V. G8 I: U
q: U A$ E! D& g5 s4 ~3 @4 PStatistics and data analysis. The data were analyzed with Student's t -test when comparingmeans between two groups or with one-way analysis of variance plusDunnett post hoc multiple comparison test to compare mean values acrossmultiple treatment groups.) h# T3 h n& t6 n# P
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Genotyping of wild-type and A 3 KO C57 mice. To confirm the genotypes of mice used in these studies, genomic DNAextracted from the tail was analyzed by PCR for the expression of thefull-length A 3 AR (wild-type or C57 mice) or the plasmid insert (PGKneo) originally used to disrupt the A 3 AR gene(A 3 KO mice) ( 23 ). The full-lengthA 3 AR (~220 bp) was only detected in C57 wild-type mice,whereas the PGKneo insert (~290 bp) was only detected in theA 3 KO mice (Fig. 1 ).1 O% X" z9 b! |
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Fig. 1. Representative PCR products using specific primers of mouseA 3 adenosine receptor (AR; C57 mice) and PKGneo[A 3 AR knockout (A 3 KO) mice]. Total DNA (0.2 µg, extracted from tail) was used in each PCR reaction. DNA encodingA 3 AR was only detected in C57 control mice( n = 2), whereas the PKGneo insert was only detected inA 3 KO mice ( n = 4).$ s* W! ?% F/ {! b: P
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Renal function assessment. C57 control and A 3 KO mice that underwent sham operationshad similar baseline hemodynamic values (heart rate and blood pressure) and renal function [creatinine (Cr) = 0.3 ± 0.1 mg/dl, n = 3, for C57, and 0.3 ± 0.1 mg/dl, n = 3, for A 3 KO]. However, 24 h after renal I/R injury, A 3 KO mice had significantly lowerplasma creatinines (Cr = 0.7 ± 0.1 mg/dl, n = 8) compared with C57 controls (Cr = 3.0 ± 0.3 mg/dl, n = 8, P 2 ). A 3 KO mice were alsoprotected against myoglobinuric renal injury compared with control miceas demonstrated by reduced creatinines at 24 h (Cr = 1.8 ± 0.3 mg/dl, n = 8 for A 3 KO, vs. 4.1 ± 0.2 mg/dl, n = 4, for C57, P 2 ).
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Fig. 2. Comparison of mean creatinine measured from sham-operated C57 mice(C57 sham; n = 3), sham-operated A 3 KO mice(A 3 KO sham; n = 3), C57 mice subjected toischemia and reperfusion (C57 IR; n = 8),A 3 KO mice subjected to ischemia and reperfusion(A 3 KO IR; n = 8), C57 mice pretreated withIB-MECA before ischemia and reperfusion (IB-MECA IR; n = 6), C57 mice pretreated with MRS-1191 beforeischemia and reperfusion (MRS IR; n = 9), C57mice subjected to glycerol injury (C57 Glyc; n = 4),A 3 KO mice subjected to glycerol injury (A 3 KOGlyc; n = 8), C57 mice pretreated with IB-MECA beforeglycerol injury (IB-MECA Glyc; n = 4), and C57 micepretreated with MRS-1191 before glycerol injury (MRS Glyc; n = 6). * P P P
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$ n) P& d' C6 u& V. W- v3 GTo further illustrate the role of the A 3 AR in modulatingrenal function after injury, control C57 mice were pretreated with anA 3 AR antagonist or agonist before I/R or glycerol-induced myoglobinuric injury. C57 mice pretreated with MRS-1191 (a selective A 3 AR antagonist) demonstrated improved renal functionafter either I/R-induced or glycerol-induced renal injury (Cr = 1.8 ± 0.2 mg/dl, n = 9, for MRS-1191 I/R, or3.1 ± 0.2 mg/dl, n = 6, for MRS-1191 glycerol, respectively, P 2 ) compared with I/R orglycerol injury alone. In contrast, C57 mice pretreated with IB-MECA, a selective A 3 AR agonist, failed to survive theischemic period at doses of 2 ( n = 6), 1 ( n = 6), 0.5 ( n = 6), or 0.25 mg/kg( n = 6). At 0.125 mg/kg IB-MECA, C57 mice survivedrenal I/R injury and demonstrated significantly worsened renal function(Cr = 4.5 ± 0.2 mg/dl, n = 6, P Fig. 2 ). C57mice pretreated with 0.0625 mg/kg IB-MECA before renal I/R did notexhibit significantly worsened renal function (Cr = 3.4 ± 0.2 mg/dl, n = 4). Selective A 3 AR agonist(Cr = 0.6 ± 0.1 mg/dl, n = 5, after 0.125 mg/kg IB-MECA) or antagonist (Cr = 0.6 ± 0.1 mg/dl, n = 4, after 1 mg/kg MRS-1191) has no impact on renalprotection observed in A 3 KO mice subjected to I/R injury.5 A+ F. H& _6 U; ^
% o2 h/ ~1 u) L+ s8 z. JIB-MECA (0.5 mg/kg) also exacerbated renal function in C57 mice aftermyoglobinuric renal injury (Cr = 4.8 ± 0.1 mg/dl, n = 4, P mice injected with IB-MECA (2 mg/kg)alone without I/R or myoglobinuric injury survived and showed normalrenal function. When C57 ( n = 4) and A 3 KO( n = 4) mice were pretreated with compound 48/80 (5 mg/kg) and subjected to renal I/R injury, all the mice died duringrenal ischemia.
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: b5 B; t3 D4 W8 FIncrease in plasma histamine by IB-MECA or compound 48/80 is lethalwhen associated with renal ischemia. A 3 AR activation causes mast cell degranulation andhistamine release in rodents ( 6, 22, 26 ). We questionedwhether the systemic elevations in histamine played a role in thelethality and impaired renal function of A 3 AR activationcoupled with renal ischemia and reperfusion. We measured plasmahistamine levels in A 3 KO and C57 mice after IB-MECA orcompound 48/80 (induces mast cell degranulation) pretreatment by usinga mouse-specific ELISA. A 3 KO and control C57 mice thatunderwent sham operations had similar baseline plasma histamine levels(4.6 ± 1.0 and 5.0 ± 1.6 ng/ml, respectively, n = 3). IB-MECA (1 mg/kg) increased plasma histamineconcentration to 226 ± 24.8 ng/ml in C57 mice ( n = 6) and to 31.0 ± 4.3 ng/ml in A 3 KO mice( n = 4, P vs. C57) 20 min afterinjection (5 min after initiation of renal ischemia). Plasmahistamine concentrations increased in C57 and A 3 KO micepretreated with compound 48/80 (320 ± 8.8 ng/ml, n = 4, and 249 ± 29.3 ng/ml, n = 5, respectively).$ q% T) Y4 V+ X1 Q3 g& W3 y
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Because all control C57 mice pretreated with compound 48/80 or0.25-2.0 mg/kg IB-MECA and all A 3 KO mice pretreatedwith compound 48/80 died during ischemia, we questioned whetherinhibition of histamine receptor subtypes would alter lethality orrenal function. Thirty minutes of pretreatment with H 1 (diphenylhydramine, 10 mg/kg ip) but not with H 2 (ranitidine, 10 mg/kg ip) histamine receptor antagonist prevented deathin C57 mice treated with IB-MECA and in C57 or A 3 KO micetreated with compound 48/80. However, pretreatment withdiphenylhydramine did not protect renal function after I/R injury inC57 mice treated with IB-MECA (Cr = 4.0 ± 0.2 mg/dl, n = 4). Similarly, when C57 mice were pretreated with diphenyhydramine before compound 48/80 and subjected to renal I/R, theysurvived renal ischemia but failed to show improved renalfunction compared with the I/R injury alone group (Cr = 2.9 ± 0.2 mg/dl, n = 3). Therefore, although elevatedplasma histamine and activation of the H 1 histaminereceptor coupled with renal ischemia are lethal in mice,H 1 receptor activation alone cannot account forA 3 AR activation-mediated exacerbation of renal function.+ t4 h5 W3 ?6 t
) ` ]& f2 v9 p' s# W! fInjured A 3 KO mice maintained improved renal histology. In Fig. 3, enhancedrenal protection of A 3 KO mice against I/R- andglycerol-mediated injury are further supported by representative histological slides. C57 control mice subjected to 30 min ofrenal ischemia (Fig. 3 B ) or glycerol injection (Fig. 3 C ) followed by 24 h of reperfusion resulted insignificant renal injury, as evidenced by severe tubular necrosis,medullary congestion and hemorrhage, and the development ofproteinaceous casts. A 3 KO mice (Fig. 3, D and E ) or C57 mice (Fig. 3, F and G )pretreated with the A 3 AR antagonist MRS-1191 before I/R-or glycerol-induced injury showed improved renal morphology. TheJablonski ( 10 ) scale histology grading scores are shown inFig. 4. Twenty-four hours after 30 min ofrenal ischemia or glycerol treatment in C57 control mice resulted in severe acute tubular necrosis (grade: 3.0 ± 0.3, n = 6, and 3.7 ± 0.4, n = 4, respectively). A 3 KO mice subjected to I/R or glycerolinjury showed significant improvements in histological scoring comparedwith C57 mice subjected to I/R (grade: 0.7 ± 0.5, n = 4, and 1.7 ± 0.3, n = 4, respectively). Additionally, C57 mice pretreated with MRS-1191 beforeI/R or glycerol injury showed significant improvements in histologicalscoring compared with C57 mice subjected to I/R injury (grade: 2.1 ± 0.2, n = 5, and 2.3 ± 0.3, n = 4, respectively). IB-MECA-pretreated C57 mice subjected to I/R orglycerol injury failed to show statistically significant worseninghistological injury compared with C57 mice (grade: 3.7 ± 0.4, n = 4, and 3.8 ± 0.4, n = 3, respectively).
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2 F7 h I& V, k( [' y) ?8 D9 l- v+ PFig. 3. Representative light microscopic photographs ofouter medulla of the kidney. A : sham-operated C57 mice. B : C57 mice subjected to ischemia and reperfusion. C : C57 mice subjected to glycerol injury. D :A 3 KO mice subjected to ischemia and reperfusion. E : A 3 KO mice subjected to glycerol injury. F : C57 mice pretreated with MRS-1191 before ischemiaand reperfusion. G : C57 mice pretreated with MRS-1191 beforeglycerol injury. Magnification, ×200.
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Fig. 4. Jablonski grading scale scores for histological appearance of acutetubular necrosis from sham-operated C57 mice ( n = 3),sham-operated A 3 KO mice ( n = 3), C57 micesubjected to ischemia and reperfusion ( n = 6),A 3 KO mice subjected to ischemia and reperfusion( n = 4), C57 mice pretreated with IB-MECA beforeischemia and reperfusion ( n = 4), C57 micepretreated with MRS-1191 before ischemia and reperfusion( n = 5), C57 mice subjected to glycerol injury( n = 4), A 3 KO mice subjected to glycerolinjury ( n = 4), C57 mice pretreated with IB-MECA beforeglycerol injury ( n = 3), and C57 mice pretreated withMRS-1191 before glycerol injury ( n = 4).* P P P
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DISCUSSION; V: k/ Y$ ~) [- W" V7 s
, s ~& q: u7 K- |' M9 KThe major findings of our study are the following: 1 )mice deletionally lacking A 3 ARs are protected against bothischemic and myoglobinuric forms of renal injury, 2 )blocking A 3 ARs protected and activating A 3 ARsworsened renal failure after I/R- and myoglobinuria-induced injury inC57 control mice, 3 ) high doses of the A 3 ARagonist (IB-MECA) coupled with renal ischemia are lethal in C57mice presumably by degranulating resident mast cells and releasinghistamine, and 4 ) blocking H 1 histaminereceptors prevented A 3 AR agonist- or mast celldegranulation-induced mortality during renal ischemia in C57control or A 3 KO mice.
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In previous studies by our laboratory, we showed thatpreischemic A 1 or postischemicA 2a AR activation protects against renal I/R injury in rats( 14, 16, 17 ). In addition, we demonstrated in rats thatA 3 AR activation with IB-MECA and inhibition with MRS-1191worsened and protected, respectively, against I/R-induced renal failure( 14 ). In the present study, we utilized mice that lackA 3 ARs to further probe the role of A 3 ARs inrenal I/R and myoglobinuric injury.
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The A 3 AR subtype is the most recently characterized memberof the AR family ( 5, 11 ). Although the expression of the A 3 AR subtype in the kidney has been demonstrated( 18, 28 ), its function in the kidney is unknown. Thepresent studies in mice agree with our previous studies in rats in thatpretreatment with the highly selective A 3 AR agonistIB-MECA worsened renal I/R injury, whereas pretreatment with the highlyselective A 3 adenosine antagonist MRS-1191 protected renalfunction after I/R injury ( 14 ). Although MRS-1191 is apotent and selective antagonist of human A 3 receptors, ithas only limited potency and selectivity at the mouse A 3 ARreceptor ( K i ~ 1.42 µM) ( 12, 13 ). For this reason, we cannot rule out the possibility thatMRS-1191 might act in part by blocking other AR subtypes. For thisreason, the data derived from the use of A 3 KO mice areimportant to bolster the conclusion on the basis of the use ofMRS-1191.
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* Q _& p& o- pIB-MECA also exacerbated glycerol-induced myoglobinuric renal failure.Furthermore, mice lacking the A 3 AR exhibited endogenous protection against both I/R-induced and myoglobinuric-induced renalinjury. Because the A 3 AR agonist alone had no effect on renal function in both rats ( 14 ) and mice, it can beconcluded that A 3 AR activation must be coupled with an I/Ror myoglobinuric renal insult for the receptor to have a detrimentaleffect on renal function. The detrimental effects ofpreischemic A 3 AR agonist treatment on the kidneydiffers from the heart, where significant cardiac protection withA 3 AR agonist pretreatment has been reported ( 19 ).1 o, Y6 V; y( A) n7 s
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The mechanism(s) by which A 3 AR activation or inhibitionexacerbates or protects against, respectively, renal injury remains tobe determined. A 3 adenosine agonists causeapoptosis in multiple cell lines, including cardiomyocytes,human leukemia cell lines, and human proximal tubule (HK-2) cells, viaincompletely characterized mechanisms ( 11, 15, 24 ).Chronic A 3 AR activation or overexpression is detrimentalto cell survival ( 11 ). Moreover, overexpression ofA 3 AR is embryologically lethal in mice with prominentfragmentation of DNA ( 27 ). Additionally, mice deletionallylacking A 3 ARs are protected against cardiac I/R injury byunclear mechanisms ( 3, 8, 9 ).
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; C& [2 b( K( E8 O, w, j: ?The A 3 AR activation degranulates resident mast cells,which results in the release of stored inflammatory mediators,including histamine and proteolytic enzymes, as well as otherproinflammatory mediators (e.g., IL- and TNF- ) ( 6, 22 ). We examined the role of mast cell degranulation andhistamine release after A 3 AR activation utilizinghistamine receptor blockers and by measuring plasma histamineconcentrations. We demonstrate that the A 3 AR agonistIB-MECA profoundly increases plasma histamine levels in C57 mice(~45-fold increase). In contrast, compound 48/80 increased histaminelevels in both C57 and A 3 KO mice. It is interesting to notethat IB-MECA increased plasma histamine levels even in A 3 KOmice by approximately sevenfold, although this increase is significantly less than that observed in C57 mice. It may be that IB-MECA has some additional mechanism of action to increase plasma histamine levels independently of A 3 AR activation.9 G4 V3 O* P, j! h" m' c. p) w
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We show that 0.125 mg/kg IB-MECA, a selective A3 AR agonist, worsenedrenal function after I/R renal injury. Moreover 0.5 mg/kg IB-MECAexacerbated renal injury after glycerol-mediated myoglobinuric renalinjury. Surprisingly, C57 mice were very sensitive to A 3 ARagonist (IB-MECA) injection when subjected to renal ischemia, because doses 0.25 mg/kg were lethal. Moreover, degranulation of mastcells with compound 48/80 coupled with renal ischemia was alsolethal in both C57 and A 3 KO mice. IB-MECA and compound 48/80 injections without renal ischemia were well tolerated in both A 3 KO and C57 mice. The mechanism of mortality afterhigher doses of IB-MECA (0.25-2 mg/kg) coupled with renalischemia is likely related to H 1 histamine receptoractivation. Systemic effects of histamine release on the cardiac andvascular systems likely account for mortality after high doses ofIB-MECA. Death associated with elevated systemic histamine complicatesthe interpretation of A 3 AR-mediated exacerbation of renalfunction after renal ischemia and is the major limitation ofthe present, in vivo approach.
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Pretreatment with an H 1 histamine receptor blocker(diphenylhydramine) prevented death during renal ischemia inC57 mice treated with 1 mg/kg IB-MECA, whereas pretreatment with anH 2 histamine receptor blocker (ranitidine) failed to rescueC57 mice from death. Although C57 mice survived renal ischemiaafter H 1 histamine receptor antagonist and IB-MECA, theirrenal function was not improved. This indicates that an increase inplasma histamine concurrent with renal ischemia is lethal inmice because of H 1 histamine receptor activation. However,H 1 histamine receptor activation cannot account forexacerbation of renal function after ischemia- orglycerol-induced renal injury, because pretreatment with H 1 histamine blocker failed to improve plasma creatinine or histology. Itremains to be determined whether other histamine receptor subtype blockades (H 2, H 3, or H 4 ) with orwithout concomitant H 1 histamine receptor blockade arerequired to protect against renal I/R injury.
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We recognize that the background strain of mice is an importantdeterminant of renal injury ( 2 ). In this study, weutilized commercially available C57BL/6 mice as controls because thehomozygous A 3 KO mice were backcrossed against C57BL/6 micefor 12 generations to obtain a congenic line. Backcrossing to thisextent makes a congenic line that is 99.99% genetically C57BL/6.
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4 d# O6 t0 t g) m* z* N: `% i* iIn conclusion, we demonstrate that mice lacking A 3 ARexpression are protected against both I/R-induced andmyoglobinuria-induced renal failure. Moreover, A 3 ARagonist and antagonist worsened and protected, respectively, I/R- andmyoglobinuric-mediated renal injury in C57 control mice. Themechanism(s) of A 3 AR-mediated exacerbation of renalfunction after I/R or myoglobinuric injury and the signaling pathway(s)responsible for potent renal protection in A 3 KO mice remainto be determined. These findings further support the potential role ofa selective A 3 AR antagonist for protection againstperioperative renal failure.
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0 z0 k0 x$ n' M7 V" _$ b- B. cACKNOWLEDGEMENTS5 x4 F+ E4 Z2 o8 c5 O% a* |" Q
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This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases Grant RO1-DK-58547.- V6 _# t' b# _, W
【参考文献】. Z- n c# r7 [: F) K7 Z; y
1. Aronson, S,andBlumenthal R. Perioperative renal dysfunction and cardiovascular anesthesia: concerns and controversies. J Cardiothorac Vasc Anesth 17:117-130,1998.
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% X2 s) X- h2 p7 t0 p2 @, u" c2. Burne, MJ,Haq M,Matsuse H,Mohapatra S,andRabb H. Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model. Transplantation 69:1023-1025,2000 .
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