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LABORATORY STUDY
OXYGEN METABOLITES AND HYPOXIC RENAL
INJURY: EFFECT OF MITOCHONDRIAL
ELECTRON TRANSPORT BLOCKADE
Paul F. Shanley, M.D.1,* and Ginger C. Johnson, M.S.
2
1Department of Pathology, College of Medicine, State University ofNew York, Upstate, 750 Adams St., Syracuse, NY 13210, USA
2Department of Pathology (B216), University of Colorado HealthSciences Center, 4200 East Ave., Denver, CO 80262, USA
ABSTRACT
Isolated perfusion of the rat kidney causes hypoxic damage in the cells ofthe thick ascending limb of the loop of Henle. The cell damage is drivenby active solute transport, which generates an imbalance of oxygensupply and demand. This injury is paradoxically prevented by addingthe mitochondrial electron transport inhibitors rotenone or antimycinto the perfusion media. The present study shows that rotenone and anti-mycin decrease production of hydrogen peroxide in the thick ascendinglimb during perfusion. The findings support the hypothesis that the injuryin this model is dependent on mitochondrial electron flow and suggestthat mitochondrial electron flow, driven by the work of active solutetransport in the presence of limited oxygen availability, may result inthe generation of toxic oxygen metabolites.
Key Words: Hypoxia; Mitochondria; Kidney; Free radicals; Reactiveoxygen species; Electron transport
249
Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com
*Corresponding author. SUNY, Upstate, Department of Pathology, College of Medicine,
State University of New York, Upstate, 750 Adams St., Syracuse, NY 13210. Fax: 315-464-4675; E-mail: [email protected]
RENAL FAILURE, 24(3), 249–258 (2002)
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INTRODUCTION
The isolated perfused rat kidney has been extensively studied as amodel of hypoxic injury. Perfusion of the kidney in vitro with a cell freebuffered salt solution consistently causes epithelial cell damage in the thickascending limb of the loop of Henle (TAL).[1,2] The isolated perfusedkidney (IPK) model has received some attention for its potential implica-tions for the pathophysiology of hypoperfusion mediated acute renal fail-ure[3] and also for its more general implications for understanding cellulardefense strategies against hypoxia.[4,5] The injury is known to be caused bythe combination of a low oxygen tension in the renal medulla and theoxygen demand imposed by active ion transport in the TAL. Improvingthe oxygen delivery in the IPK by adding red blood cells or hemoglobin tothe perfusate prevents the TAL damage[2] as does decreasing solute trans-port work by inhibition of the sodium pump with ouabain.[6,7]
One paradoxical finding in this model is that TAL damage is not wors-ened or reproduced by blockade of mitochondrial electron transport. In fact,electron transport blockade with agents such as rotenone (inhibitor at complexI of the electron transport chain) or antimycin (a blocker at complex III)actually prevents the hypoxic damage usually seen in this model.[8] Thisprotection from injury occurs despite the marked ATP depletion that resultsfrom exposure of kidneys to these metabolic toxins. Consideration of thisremarkable feature of the hypoxic damage in the TAL led to the proposalthat the injury may be dependent on mitochondrial electron transportactivity itself during relative oxygen deprivation.[9,10] The mechanism ofsuch a ‘‘mitochondrial electron flow dependent’’ cell injury is not known.One hypothesis involves the possibility that potentially toxic oxygen metabo-lites might be generated by mitochondrial activity during relative oxygendeprivation. More specifically, mitochondrial electron flow, in the presenceof inadequate terminal oxygenation of the electron transport chain, results inreduction of the electron transport intermediates. The reduced electron trans-porters might then interact with the limited oxygen available resulting in par-tial reduction of the oxygen to superoxide, hydrogen peroxide andhydroxyl radical.[11,12]
The present study examines this hypothesis by determining the effects ofrotenone and antimycin on hydrogen peroxide production in the TAL duringisolated perfusion of the kidney.
DESIGN AND METHODS
Catalase is irreversibly inhibited by 3-amino-1,2,4-triazole (AMTZ) inthe presence of hydrogen peroxide.[13] Since hydrogen peroxide is requiredfor catalase inactivation by AMTZ, exposure of cells to AMTZ provides
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a sensitive method for detecting cellular production of H2O2. In the presentexperiments, AMTZ was added to the perfusion medium in the isolatedperfused kidney and catalase activity was measured in the inner stripe ofthe outer medulla after short perfusions under various experimentalconditions. The inner stripe of the outer medulla is the zone of the kidneywhere the TAL tubules are concentrated and constitute the bulk of epithelialcell mass. Experimental groups for which outer medullary catalase activitywas measured were as follows:
1) In vivo, untreated and flushed with KPO4 buffer for baselinecatalase activity (n¼ 9)
2) Standard perfusion for 10min without aminotriazole (n¼ 7)3) Standard perfusion for 10min with AMTZ (n¼ 8)4) Rotenone perfusion for 10min with AMTZ (n¼ 5)5) Antimycin perfusion for 10min with AMTZ (n¼ 3)6) Standard perfusion for 20min with AMTZ added at 10min (n¼ 4)7) Rotenone perfusion for 20min with AMTZ added at 10min (n¼ 3)
Perfusion of rat kidneys was as previously described.[14] The recirculatingperfusionmediumwasKrebs-Henseleit-albumin solution at pH 7.4 with 5mMglucose as substrate and no amino acids added. Temperature was maintainedat 37�C and perfusion pressure was 100mmHg at the cannula tip. The bovineserum albumin (BSA) was at 6.7 g%. Rotenone and antimycin were at 10�5Min the standard perfusate from the start of the experiments where they wereused. Aminotriazole was added to the perfusate and perfusions were carriedout for a total of 10min after addition of the AMTZ in all cases. The dose ofaminotriazole used was determined in a preliminary dose-response analysiswhere from 50 to 1000mg AMTZ were added to exactly 125 cc of perfusionmedium. It was found that 200mg AMTZ (19mM) was adequate for maximalcatalase inactivation in the outer medulla of the isolated perfused kidneyunder standard conditions (data not shown). Following the 10min ofperfusion in the presence of AMTZ, the reaction was stopped and perfusatewas flushed out of the kidney by introduction of 15 cc KPO4 buffer (5mM, pH7.8) into the arterial line via 3-way stopcock. The kidney was then removedfrom the perfusion circuit and the inner stripe of the outer medulla wasdissected from the remaining tissue and snap frozen in liquid nitrogen. Thefrozen tissue was weighed and then homogenized in 50mMKPO4 buffer(5mL/mg tissue). The homogenate was centrifuged at 20,000RPM for20min at 4�C and catalase activity was determined on the supernatant asdescribed in detail by Brown et al.[15] In brief, the change in absorbance permin at 240 nm was measured spectrophotometrically after addition of super-natant to aH2O2/KPO4 solution and the enzyme activity was determined froma standard curve.
In selected experiments, perfusions under standard conditions or withrotenone were for 60min and aminotriazole was omitted. Following these
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perfusions the kidneys were fixed by perfusion with glutaraldehyde and pre-pared for examination by transmission electron microscopy as previouslydescribed.[16] The ultrastructure shown represents typical TAL epithelialcell morphology in the various experimental groups. Quantitative aspectsof the TAL lesions under standard perfusion conditions and with mitochon-drial electron transport inhibitors have been described elsewhere.[8]
RESULTS
Perfusion of the isolated rat kidney for 60min under standard condi-tions results in extensive fragmentation of epithelial cells in the thick ascend-ing limb of the loop of Henle and addition of rotenone to the perfusateprevents this damage (Fig. 1). Compared to the standard perfusion, theTAL in the rotenone perfused kidneys have an intact smooth luminalplasma membrane and do not exhibit nuclear pyknosis. There is some per-ipheral nuclear chromatin clumping in the rotenone perfused TAL and someof the mitochondria appear to be in a condensed state.
When aminotriazole is added to the standard perfusion media, 10min ofkidney perfusion results in a marked inhibition of catalase activity in theouter medulla. Addition of rotenone or antimycin to the perfusion media
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Figure 1. Electron micrographs of thick ascending limb of loop of Henle after perfusionfixation in vivo (A), following 60min of standard perfusion (B) and after 60min of perfusionwith addition of rotenone to the standard perfusate (C). Standard perfusion causes hypoxic
cell death in the tubules. Nuclei are pyknotic and cells are fragmented. These lesions areprevented by the addition of rotenone.
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containing aminotriazole prevents the inactivation of catalase. The data inFigure 2 show this for perfusions lasting a total of 10min where aminotria-zole is present from the onset (groups 2–5). In a second series of experiments,aminotriazole was added after a 10min equilibration period and theperfusions carried out for a total of 20min (groups 6 and 7). Again, the
MITOCHONDRIAL BLOCKADE PREVENTS DAMAGE 253
Figure 1. Continued.
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addition of rotenone prevented catalase inactivation by AMTZ. In thissecond series of experiments catalase activity under standard conditionswas 2.07� 0.36U/mg tissue and with rotenone added catalase activity was6.97� 1.56U/mg tissue ( p<0.02 by t-test).
DISCUSSION
Mitochondrial electron transport blockade with rotenone or antimycinprevents hypoxic damage in the thick ascending limb of the loop of Henle inthe isolated perfused kidney. This unexpected finding suggested that the
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Figure 2. Catalase activity in the outer medulla after 10min of isolated perfusion of the
kidney under various conditions is shown. The dotted lines represent the range covered bythe mean� SEM for catalase activity in the outer medulla in vivo in untreated rats and areprovided for reference. Standard kidney perfusion without aminotriazole (AMTZ) causes nostatistically significant decrease in medullary tissue catalase activity from the in vivo level. The
presence of AMTZ in the medium during otherwise standard perfusion results in a decrease intissue catalase activity indicating H2O2 production during the perfusion. Addition of eitherrotenone or antimycin prevents the loss of catalase activity caused by the presence of AMTZ
indicating inhibition of H2O2 production. ANOVA indicates p<0.001 for differences amongthe groups. Standard perfusions with aminotriazole (**) show statistically significant differ-ence from all other groups by Bonferroni’s method of post-test analysis. None of the other
groups differ significantly from each other or from the in vivo level.
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injury in this system is not simply an effect of homeostasis failure from ATPdepletion. The pace of injury generation in this model (with complete cellfragmentation within one hour) along with evidence that the damage wasdriven by cell work[6,7] suggested a more active mechanism of injury and theconcept of a mitochondrial electron flow dependent mechanism was pro-posed by Brezis.[9,10] In the time since that proposal was made, more hasbeen learned about the mechanism of this injury. Dissociation of the injuryfrom the degree of ATP depletion in the TAL has been confirmed,[17] calciumand pH dependence has been established[16,18,19] and the possibility that acti-vation of programmed cell death plays a role has been suggested.[20]
However, the possible role of mitochondrial activity in the mechanism ofdamage remains essentially unexplored.
One hypothesis possibly linking mitochondrial activity to cell injuryduring hypoxia involves the generation of partially reduced, toxic oxygenmetabolites. Continued electron flow in the face of inadequate terminal oxy-genation could result in relative reduction of electron chain intermediates.These reduced intermediates might then interact directly with availableoxygen to produce potentially toxic oxygen metabolites such as superoxide,hydrogen peroxide and hydroxyl radical.[11,12]
There is evidence that mitochondrial electron transport chain intermedi-ates are relatively reduced in the thick ascending limb of Henle duringisolated kidney perfusion. Epstein has shown incomplete oxidation of cyto-chrome oxidase in the medulla of the isolated perfused kidney by opticalspectroscopy.[21] The reduction of this cytochrome is attenuated by additionof furosemide to the perfusate, which suggests that ion transport in the TALaccounts for the partial reduction. Furthermore, mitochondrial electrontransport blockade with rotenone or antimycin restores a high oxidationstate to the cytochrome aa3. The latter finding indicates that adequateoxygen is available in this system for maintenance of fully oxidized cyto-chrome aa3 if electron flow is inhibited.
The present study examined the effects of inhibition of mitochondrialelectron transport activity on the production of hydrogen peroxide in theinner stripe of the outer medulla in the isolated perfused kidney.The electron transport blockade was accomplished with rotenone, aninhibitor at complex I of the mitochondrial electron transport chain, orantimycin, an inhibitor in complex III. As is the case with isolatedmitochondria, these agents cause a rapid decrease in oxygen utilizationin the isolated perfused kidney and ATP depletion can be demonstratedin cortex and medulla.[8]
Hydrogen peroxide production was measured as a representative of thepotentially toxic oxygen metabolites. A very sensitive assay developed formeasuring H2O2 production in bacteria[13] was adapted for kidney tissuemeasurement. The method is based on the inactivation of catalase by amino-triazole in the presence of small amounts of hydrogen peroxide. Thus
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a decrease in tissue catalase activity in the presence of added aminotriazoleindicates production of H2O2 in the tissue.
The kidney was dissected after perfusions so that only the inner stripeof the outer medulla was studied since this is the region where the thickascending limb of the loop of Henle is concentrated and where the TALmake up most of the parenchymal mass. The outer stripe tissue level ofcatalase activity after a perfusion in the presence of AMTZ was assumed,therefore, to reasonably estimate thick ascending limb of Henle H2O2
production.The study showed that there was hydrogen peroxide production in
the thick ascending limb of Henle during standard isolated perfusion ofthe kidney and that the H2O2 production was eliminated by mito-chondrial electron transport blockade with addition of either rotenoneor antimycin. Thus, the findings are consistent with the possibility thattoxic oxygen products play a role in the cell fragmentation injury andmay explain the paradoxical effects of these metabolic poisons in thismodel.
The effectiveness of antimycin in preventing hydrogen peroxide pro-duction in the TAL may be surprising since many studies have concludedthat addition of antimycin is, in fact, an effective means of generatingoxygen free radicals in isolated mitochondria.[11] The protective effect ofantimycin in this model has been consistent and impressive, however, andthe discrepancy with other systems is unexplained. The findings here suggestthat maintenance of the oxygenation of the terminal portion of the electrontransport chain is critical to preventing partial reduction of oxygen at leastin the IPK model.
The mechanism of hypoxic damage in the thick ascending limb ofHenle in the isolated perfused kidney model appears to involve calcium,solute transport activity and mitochondrial electron flow in the face ofinadequate terminal oxygenation. It is rapid in onset, is preventableby acidosis and perhaps involves a cell death program. Recent informationconcerning the mitochondrial permeability transition and its potentialimportance as a trigger for either necrosis or apoptosis[22,23] may providea framework for future work on this model. The known synergism betweencalcium mechanisms and free radical production in producing thepermeability transition[22] makes the hypothesis attractive. One mightsuggest that relative hypoxia disrupts calcium homeostasis and, at thesame time, sets up conditions for mitochondrial electron flow to causereduction of electron transport chain intermediates. Cell work, such astubular solute transport, might drive the mitochondrial electron flow.Reduced cytochromes could interact directly with the limited oxygen avail-able and produce toxic oxygen products. These, together with abnormalcalcium availability, trigger a mitochondrial permeability transition andrapid cell destruction.
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ACKNOWLEDGMENT
This work was supported by Grant DK38516-03 from the NationalInstitutes of Health. We thank Alan S. Jones for expert assistance with theelectron microscopy.
REFERENCES
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22. Lemasters, J.J.; Nieminen, A.L.; Qian, T.; Trost, L.C.; Elmore, S.P.;Nishimura, Y.; Crowe, R.A.; Cascio, W.E.; Bradham, C.A.; Brenner, D.A.;Herman, B. The Mitochondrial Permeability Transition in Cell Death: ACommon Mechanism in Necrosis, Apoptosis and Autophagy. Biochimica etBiophysica Acta 1998, 1366(1–2), 177–196.
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