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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：WeiWang, SuparoekJittikanont, Sandor A.Falk, PingLi, LiliFeng, Patricia E.Gengaro, Brian D.Poole, Russell P.Bowler, Brian J.Day, James D.Crapo,  Robert W.Schrier作者单位：1 Department of Medicine, University of Colorado HealthSciences Center, Denver 80262; Department of Medicine, BaylorCollege of Medicine, Houston, Texas 77030; and Department ofMedicine, National Jewish Medical and Research Center, Denver, Colorado80206

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      【摘要】
   Acute renal failure(ARF) during sepsis is associated with increased nitric oxide (NO) andoxygen radicals, including superoxide (O 2 − ). BecauseO 2 − reacts with NO in a rapid manner, it plays animportant role in modulating NO levels. Therefore, scavenging ofO 2 − by superoxide dismutase (SOD) may be critical forpreserving NO bioavailability. In mice, substantial renal extracellularSOD (EC-SOD) expression implies its important role in scavengingO 2 − in the kidney. We hypothesized that duringendotoxemic ARF, EC-SOD is decreased in the kidney, resulting inincreased O 2 − and thus decreased vascular NObioavailability with resultant renal vasoconstriction and ARF. In thepresent study, normotensive endotoxemic ARF was induced in mice usinglipopolysaccharide (LPS; 5 mg/kg ip). Sixteen hours after LPS,glomerular filtration rate (GFR; 50 ± 16 vs. 229 ± 21 µl/min, n = 8, P blood flow (RBF; 0.61 ± 0.10 vs. 0.86 ± 0.05 ml/min, n = 8, P in endotoxemic kidneyswere decreased at 16 h compared with controls. A catalyticantioxidant, metalloporphyrin, reversed the deleterious effects ofendotoxemia on renal function as GFR (182 ± 40 vs. 50 ± 16 µl/min, n = 6, P ± 0.10 ml/min, n = 6, P wereobtained with tempol, a chemically dissimilar antioxidant. Specificinhibition of inducible nitric oxide synthase (iNOS), L - N 6 -(1-iminoethyl)-lysine, reversedthe renal protective effect on GFR and RBF observed with antioxidanttreatment during endotoxemia. In summary, renal EC-SOD expression isdecreased during endotoxemia. Antioxidant therapy preserved GFR and RBFduring endotoxemia. The reversal of this protective effect byinhibition of iNOS suggests the importance of the bioavailability of NOfor preservation of renal function during early endotoxemia. 7 @5 C$ h* W2 e& f) }% w, e3 o
      【关键词】 sepsis extracellular superoxide dismutase lipopolysaccharide kidney
   INTRODUCTION: s  h5 `( }" O1 _
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DURING ENDOTOXEMIA, reactive oxygenspecies (ROS) have been shown to be increased in several species( 5, 16, 27 ). The injurious effects of ROS are attenuated,at least in part, by antioxidants. Superoxide dismutase (SOD) is anendogenous antioxidant which can scavenge superoxide(O 2 − ) during circumstances of stress such asendotoxemia. There are three isoforms of endogenous SOD; however,extracellular SOD (EC-SOD) is the most abundant in the vasculature aswell as in the kidney in mice ( 17 ). To date it is notknown whether EC-SOD is altered in the kidney during endotoxemia. Thisis an important question because endotoxemia-related sepsis isfrequently associated with acute renal injury and ROS may play a rolein this renal failure.

In addition to increased generation of ROS, endotoxemia also results inthe induction of nitric oxide synthase (iNOS) and increased circulatingnitric oxide (NO) ( 9, 29 ). We hypothesized that renalEC-SOD may be downregulated during endotoxemia, resulting in increasedO 2 −. This increase in O 2 − may lead toincreased scavenging of NO ( 22, 23 ). The reduction inbioavailable NO in the renal vasculature may lead to renal vasoconstriction and ultimately to acute renal failure (ARF). To testthis hypothesis, the present study was undertaken in a mouse model ofendotoxemic ARF. We determined 1 ) renal EC-SOD mRNA andprotein expression during endotoxemia, 2 ) the effect ofantioxidant administration on renal function during endotoxemia, and 3 ) whether specific iNOS inhibition reverses any renalprotective effect of antioxidant treatment during endotoxemia.
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MATERIALS AND METHODS+ |$ i; ^' S5 i! \: D; \: i* o
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Animals. The experimental protocol was approved by the Animal Ethics ReviewCommittee at the University of Colorado Health Sciences Center. C57BL/6mice were purchased from Jackson Laboratories (Bar Harbor, ME). Malemice aged 8-10 wk were used throughout the study. Mice weremaintained on a standard rodent chow and had free access to water.

Materials. Chemicals were purchased from Sigma (St. Louis, MO) unless otherwisespecified. Manganese (III)mesotetrakis( N -ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP)was a kind gift of Incara Pharmaceuticals (Research Triangle Park, NC).
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Measurement of renal blood flow, glomerular filtration rate, andmean arterial pressure. The animals were anesthetized with pentobarbital sodium (60 mg/kg) andplaced on a thermostatically controlled surgical table. A tracheotomywas performed, at which time a steady steam of 100% oxygen was blownover the tracheal tube throughout the experiment. Catheters (custompulled from PE-250) were placed in the jugular vein for maintenanceinfusion and the carotid artery for blood pressure determinations. Thekidney was exposed by a left subcostal incision and was dissected freefrom perirenal tissue, and renal arteries were isolated for thedetermination of renal blood flow (RBF) using a blood flowmeter andprobe (0.5v; Transonic Systems, Ithaca, NY) as described by Traynor( 26 ). Mean arterial pressure (MAP) was measured via acarotid artery catheter connected to a Transpac IV transducer andmonitored continuously using Windaq Waveform recording software (DataqInstruments). An intravenous maintenance infusion of 2.25% BSA innormal saline (NS) at a rate of 0.25 µl · g bodywt 1 · min 1 was started 1 hbefore experimentation. FITC-inulin (0.75%) was added to the infusionsolution for the determination of glomerular filtration rate (GFR) asdescribed by Lorenz et al. ( 13 ). A bladder catheter(PE-10) was used to collect urine. Two 30-min collections of urine wereobtained under oil and weighed for volume determination. Blood forplasma inulin determination was drawn between urine collections. FITCin plasma and urine samples was measured using a CytoFluor plate reader(PerSeptive Biosystems, Foster City, CA).

Western blot analysis. Whole kidney lysate was mixed with sample buffer containing 50 mM Tris,0.5% glycerol, 0.01% bromophenol blue, and 0.75% SDS (pH 6.8).Identical amounts of protein were fractionated by a 4-15%Tris/glycine polyacrylamide gradient gel (EC-SOD determination) or 15%polyacrylamide separating gel (MnSOD and Cu/ZnSOD determinations) andtransferred to a nitrocellulose membrane (Millipore, Bedford, MA).Membranes were blocked using 5% milk in TTBS [50 mM Tris, 150 mMNaCl, 0.1% Tween 20 (pH 7.5)] at room temperature for 60 min and weresubsequently incubated at 4°C overnight with rabbit anti-EC-SODantibody (1:5,000) or 1 µg/ml rabbit anti-MnSOD antibody (UpstateBiotechnology, Lake Placid, NY) and 1 µg/ml sheep anti-Cu/ZnSOD (Upstate Biotechnology). An additional 1-h incubation was performed with a secondary antibody, goat anti-rabbit IgG or donkey anti-sheep IgG, coupled to horseradish peroxidase (Amersham, Piscataway, NJ) at1:5,000 dilution in TTBS. Detection of the protein bands was carriedout using enhanced chemiluminescence (Amersham). Membranes were thenstripped and blotted with rabbit anti-mouse actin (Sigma) to examinethe actual loading of the proteins. Relative densitometry was measuredas the ratio of the densitometry of a specific protein to that of actin.. M5 |# y5 R' v; H
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RNA extract and RNase protection assay. Total RNA was prepared by using TRIzol reagent (Invitrogen, Carlsbad,CA). An RNase protection assay was performed on 2-4 µg of RNAwith the RNase Protection Assay Kit I (Torrey Pines Biolabs, Houston,TX) according to the manufacturer's instructions. Full-length mouseEC-SOD cDNA was obtained from ATCC (GenBank accession no. BF300486 ). A268-bp Sac I- Pst I fragment of EC-SOD was isolatedfrom the full-length cDNA and inserted in the pBluescript KS II vector.The clone was verified by DNA sequencing and linearized withappropriate restriction enzymes. [ - 32 P]UTP (3,000 Ci/mmol, ICN)-labeled antisense RNA probes were synthesized by an invitro transcription system (Promega, Madison, WI). Antisense RNA probeswere hybridized with the RNA samples at 90°C for 25 min. Unhybridizedsingle-strand RNA was digested by ribonuclease A/T 1 (Sigma)for 30 min. Double-strand RNA was precipitated by stop solution at 80°C for 15-30 min and centrifuged at maximum speed for 30 min. The samples were resolved by a 6% sequencing gel. The gel wasdried and exposed to X-ray film." T5 W# T; l0 I. t1 V9 x8 O( l
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Measurement of plasma NO levels. Plasma NO levels were determined by measuring plasmaNO 2 /NO 3 levels using a nitrate/nitritecolorimetric assay kit (Cayman Chemical, Ann Arbor, MI).- L% l2 [3 R& U  W
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Statistical analysis. Values are expressed as means ± SE. Multiple comparisons wereassessed by ANOVA using a post hoc Newman-Keuls test. Survival analysiswas analyzed by the Kaplan-Meier method. P considered statistically significant.
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RESULTS- {9 V5 a' v) M: b

Normotensive endotoxemic ARF model in mice. Mice were injected intraperitoneally with 5 mg/kg LPS( Escherichia coli 026:B6, Sigma), a relatively low andnonlethal dose of LPS that permitted surgery and physiologicalmeasurements without excessive mortality. With this dose of LPS, therewas no significant change in MAP (82 ± 0.8 vs. 82 ± 2.2 mmHg, n = 6, P = not significant), thusallowing measurement of renal function in the absence of hypotension.7 e; \8 f: N9 [' r
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Effect of LPS on SOD expression in the kidney. We s tern blot analysis was used to examine all three isoformsof SOD protein expression in the mice kidneys. In control mice, therewas high expression of EC-SOD in the control kidney, but the expressionwas significantly decreased in mice treated with LPS at 16 h (Fig. 1 A ). The relative densitometrywas 2.6 ± 0.13 vs. 1.2 ± 0.05 ( n = 6, P 1 B ). Two bands at ~34 and 32 kDa can be observed in both groups. These represent intact andproteolytically processed forms of EC-SOD, respectively ( 4, 6 ). The top band (the intact form) is much strongerthan the bottom band (proteolytic form) in vehicle-treatedmice, whereas the two bands are similar in density in the kidney ofLPS-treated mice. The ratio between the two bands are 2.97 ± 0.12 in the control group vs. 1.27 ± 0.07 in the LPS-treated group( n = 6/group, P were examined, there isno difference between the control and LPS-treated group (Fig. 2, A and B ).
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Fig. 1. Extracellular (EC) superoxide dismutase (EC-SOD) proteinexpression in the kidney. Western blot analysis was used to examineEC-SOD protein expression in the kidney in control (CON) andendotoxemic mice ( A ). Kidneys were harvested at 16 hafter LPS (5 mg/kg) administration. A rabbit polyclonal anti-EC-SODantibody was used as the primary antibody, and protein bands weredetected using the enhanced chemiluminesence (ECL) method. Relativedensitometry of EC-SOD was calculated as the ratio of EC-SOD (the sumof the top and bottom bands) to actin( B, n = 6) in each group.

Fig. 2. MnSOD and Cu/ZnSOD protein expressions in the kidney.Western blot analysis was used to examine MnSOD ( A ) andCu/ZnSOD ( B ) protein expressions in the kidney in control(CON) and endotoxemic mice. Kidneys were harvested at 16 h afterLPS (5 mg/kg) administration. A rabbit polyclonal anti-MnSOD antibodyand a sheep polyclonal anti-Cu/ZnSOD antibody were used as the primaryantibodies, respectively, and protein bands were detected using the ECLmethod.
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Effect of LPS on EC-SOD mRNA expression in the kidney in mice. To examine whether the decreased EC-SOD protein expression wasregulated at the transcriptional level, EC-SOD mRNA in the kidney wasmeasured using an RNase protection assay. As shown in Fig. 3, EC-SOD mRNA was decreased in thekidney in endotoxemic mice, thus corresponding to EC-SOD proteinexpression.7 i& \! o8 t( ~; g- u

Fig. 3. RPA was used to examine EC-SOD mRNA in the kidney incontrol (CON) and endotoxemic mice. Kidney cortex was harvested at16 h after LPS (5 mg/kg) administration. Total RNA (2-4 µg)was loaded and [ - 32 P]UTP-labeled antisense EC-SOD andL32 probes were used.1 G  P& v  g8 ^1 w  `3 w% R, O
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Effect of MnTE-2-PyP on GFR, RBF, and MAP in endotoxemic mice. MnTE-2-PyP (10 mg/kg) or vehicle (NS) was injectedintraperitoneally 30 min before LPS (5 mg/kg) (Fig. 4 ). At 16 h after LPS injection,GFR, MAP, and RBF were examined in control mice and MnTE-2-PyP-treatedmice. There was no difference in MAP between vehicle- andMnTE-2-PyP-treated endotoxemic mice. Sixteen hours after LPS injection,there were significant decreases in GFR (50 ± 16 vs. 229 ± 21 µl/min, n = 8, P vs. 0.86 ± 0.05 ml/min, n = 8, P controls (NS).The decreased GFR and RBF were dramatically reversed by MnTE-2-PyP,which brought GFR and RBF up to 182 ± 40 µl/min ( n = 6, P ml/min ( n = 6, P The above experiments were performed when MnTE-2-PyP was injected before LPS. Further experiments were undertaken, in which MnTE-2-PyP was administered 6 h after LPS administration. In theseexperiments, GFR was partially protected (85 ± 10 vs. 37 ± 12 µl/min of vehicle-treated controls, n = 8, P+ e) p6 _, n3 n# S+ p$ l; p

Fig. 4. Effects of manganese (III) mesotetrakis( N -ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) on renalfunction during endotoxemia. MnTE-2-PyP (10 mg/kg) or vehicle [normalsaline (NS)] was injected 30 min before LPS injection (5 mg/kg ip). Inthe control group, vehicle (NS) alone was injected. Sixteen hours afterLPS injection, glomerular filtration rate (GFR; Fig. 6 A ) andrenal blood flow (RBF; Fig. 6 B ) were measured by FITC-inulinclearance and blood flowmeter, respectively. Values are means ± SE.3 E$ R: n) ~: }' F% K9 ]/ i+ y

Effect of MnTE-2-PyP on mortality in endotoxemia. A large dose of LPS (30 mg/kg) was injected intraperitoneally, and themortality of MnTE-2-PyP (10 mg/kg ip 30 min before LPS)- vs.vehicle-treated mice was examined for 48 h. The 24-h survival ratewas 0% in vehicle-treated mice ( n = 18), whereas itwas 50% in MnTE-2-PyP-treated mice ( n = 8, P 5 ).
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Fig. 5. MnTE-2-PyP improved survival in mice with a lethal doseof LPS. Mortality was checked for 48 h after LPS administration.MnTE-2-PyP (10 mg/kg) or vehicle was injected 30 min before LPS (30 mg/kg ip).
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Effect of iNOS inhibition with L -N 6 -(1-iminoethyl)-lysine on the protectiveeffect of MnTE-2-PyP during endotoxemia. L - N 6 -(1-iminoethyl)-lysine (L -NIL; 10 mg/kg ip, Alexis Biochemicals, Carlsbad, CA), aselective inhibitor of iNOS ( 14 ), was administered 30 minbefore LPS (5 mg/kg ip) either alone or with MnTE-2-PyP (10 mg/kg ip).As shown in our previous study ( 9, 29 ), plasma NO levelswere significantly higher in endotoxemic mice compared with controlmice (227 ± 16 vs. 2.5 ± 0.4 µM, n = 6; P after the treatment with L -NIL (51 ± 4 vs. 227 ± 16 µM, n = 6, P L -NIL was administered with MnTE-2-PyP, itabolished the renal protective effect of MnTE-2-PyP during endotoxemiabecause GFR decreased from 138 ± 8 to 49 ± 9 µl/min( n = 4, P Fig. 6 A ) and RBF decreased from1.05 ± 0.09 to 0.55 ± 0.08 ml/min ( n = 4, P 6 B ), respectively.
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Fig. 6. Effect of L - N 6 -(1-iminoethyl)-lysine (L -NIL) on the protective effect of MnTE-2-PyP on renal functionduring endotoxemia. Vehicle (NS) or L -NIL (10 mg/kg ip) andor MnTE-2-PyP (10 mg/kg ip) was injected 30 min before LPS (5 mg/kgip). GFR ( A ) and RBF ( B ) were measured byFITC-inulin clearance and blood flowmeter, respectively. Values aremeans ± SE.

Effect of tempol on renal function in endotoxemic mice. To examine whether the protective effect of MnTE-2-PyP was specific tosuperoxide scavenging, the effect of another superoxide dismutase,tempol (Calbiochem Bioscience, La Jolla, CA), was also examined duringendotoxemia. LPS ( E. coli 0111:B4, 2.0 mg/kg, LISTBiological Laboratories, Campbell, CA) was used in this study. This LPScompound is purer and more potent than the LPS from Sigma. The 2 (LIST)and 5 mg/kg (Sigma) dosages resulted in comparable effects on GFR andRBF. Similar to what was observed with MnTE-2-PyP, tempol significantlyimproved both RBF (1.21 ± 0.05 vs. 0.67 ± 0.04 ml/min, n = 4, P n = 7, P by L -NIL because RBF (1.21 ± 0.05 vs. 0.85 ± 0.05 ml/min, n = 4, P n = 4, P decreased significantly with theadministration of L -NIL (10 mg/kg) with tempol comparedwith tempol alone during endotoxemia.* f& J" [( {% Y6 F* A% r8 K

DISCUSSION
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In several studies, sepsis has been identified as the most commoncause of ARF ( 1, 11 ). Moreover, the combination of sepsisand ARF is associated with mortality as high as 60-80%. However,there is no consensus either about the pathophysiology ofsepsis-related ARF or the appropriate treatment.

The present study was therefore undertaken to examine thepathophysiology of endotoxemia-related ARF in a normotensive mouse model. In this model, the endotoxemia was associated with a progressive increase in NO ( 9, 25, 29, 30 ), an effect that does not occur in iNOS knockout mice ( 10 ). Sepsis is also known toincrease O 2 −, a known scavenger of NO ( 22, 23 ).% T+ q% T6 ^2 ^7 O: w

To focus on the potential effect of ROS in endotoxemia-related ARF, therole of endogenous and exogenous ROS scavengers was examined. EC-SOD, asecreted endogenous antioxidant enzyme, is the predominant form of SODin the vasculature and is highly expressed in mouse kidneys ( 8, 17, 19 ). In the present study, endotoxemia was associated with adecrease in renal EC-SOD, an effect that could lead to increasedO 2 − and enhanced scavenging of NO during endotoxemia( 18 ). The decrease in renal EC-SOD protein was associatedwith diminished EC-SOD mRNA as assessed by an RNase protection assay,thus indicating an effect mediated by either decreased transcription ordecreased mRNA stability. In contrast to EC-SOD, neither mitochondrial(MnSOD) nor cytosolic (Cu/ZnSOD) antioxidants were affected in ourmodel. There was also evidence for a posttranslational effect onEC-SOD, because EC-SOD cleavage products were observed during endotoxemia.

Further study of the role of ROS during endotoxemia was undertaken byexamining the effect of a potent exogenous antioxidant, MnTE-2-PyP, inendotoxemia-related ARF. The antioxidant properties of this agentinclude scavenging O 2 −, H 2 O 2,and ONOO ( 2, 3, 20 ). The administration ofthis antioxidant before LPS was associated with a highly significantimprovement in both GFR and RBF during endotoxemia. Renal protectioncould also be demonstrated when the antioxidant was administered 6 h after LPS. A significant decrease in mortality at 24 h was alsoobserved when the antioxidant was administered with an otherwiseuniformly fatal dose of LPS (30 mg/kg).- g: m; ]& c7 l7 u$ n
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An increase in NO, secondary to decreased scavenging by ROS, couldcontribute to the beneficial effect of the antioxidant against theendotoxin-induced renal vasoconstriction and ARF. To examine whethersome of the protective effect of the antioxidant was due to increasedbioavailability of NO, the potent antioxidant was administered incombination with the specific inhibitor of iNOS. The administration of L -NIL decreased plasma NO and reversed the renal protectiveeffect of the antioxidant on GFR and RBF, thus supporting a vascularprotective effect of NO. Similar results were also observed whentempol, a chemically dissimilar superoxide dismutase, was studied.$ w. r1 D- I8 `5 P9 |: c

This role of endogenous and exogenous antioxidants in modulatingendotoxemia-related renal vasoconstriction by enhancing the bioavailability of NO has implications for the early phase of endotoxemia ( 24 ). The early phase of this normotensivemouse model of endotoxin-induced ARF has been shown to be associated with activation of the sympathetic and renin-angiotensin systems ( 29 ). The systemic pressor effects of these eventscounterbalance the systemic vasodilatory effects of NO duringendotoxemia and thereby support MAP. Nevertheless, these events occurat the expense of renal vasoconstriction. This sequence of events hasbeen supported by demonstrating a renal protective effect of acuterenal denervation in this normotensive endotoxemic model of ARF( 29 ). The present results provide further understanding ofthe early events that occur during endotoxin-related ARF.

The increase in NO, which results from the endotoxin-relatedinduction of NOS, is scavenged by the increased O 2 −. Although this decrease in NO may attenuate the systemic vasodilation, the present results suggest that NO bioavailability is important incounteracting renal vasoconstriction during early endotoxemia. This invivo effect of iNOS-related NO may override any downregulation ofconstitutive nitric oxide synthase in the kidney, as suggested by invitro studies in the rat ( 25 ). However, at a later stage, an injurious effect of iNOS-induced NO on tubules may be observed ( 12, 15, 21, 28 ). The role of the cytokine TNF- hasalso been implicated in this early phase of endotoxemia-related ARF ( 10 ) and therefore would be expected to contribute notonly to the induction of NOS but also to enhanced ROS activity( 7 ).
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In summary, the early ARF in endotoxemia involves a complex sequence ofevents leading to renal vasoconstriction. The present resultsdemonstrate that the predisposition to renal vasoconstriction duringendotoxemia involves a downregulation of renal EC-SOD with resultantscavenging of bioavailable NO by ROS. Antioxidant treatment bychemically dissimilar compounds exhibited an impressive amelioration ofthe endotoxin-mediated decrease in GFR and RBF. Increased NO bioavailability appears to be involved because the beneficial effect ofantioxidants was reversed by the specific inhibition of iNOS.Scavenging of renal iNOS-related NO by ROS thus appears to be animportant factor in the renal vasoconstriction associated with early(16 h) endotoxemia in mice.

ACKNOWLEDGEMENTS
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This work was supported by National Institutes of Health GrantsDK-52599 and P01-HL-31992.5 {; }/ _2 A* G, C5 L, h
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