Exogenous adenosine triphosphate (ATP)
preserves proximal tubule microfilament
structure and function in vivo in a maleic acid
model of ATP depletion.
P S Kellerman
J Clin Invest.
1993;
92(4)
:1940-1949.
https://doi.org/10.1172/JCI116787
.
The hallmark of ischemic acute renal failure is a rapid and early decline in proximal tubule
ATP. Since we have previously shown that over half of apical microfilament losses occur
within the first 5 min of experimental ischemic injury, we postulated that microfilament
(F-actin) structure and cellular location are dependent on cellular ATP levels. To test this
hypothesis, we used maleic acid to selectively inhibit renal cortical ATP production in vivo.
Maleic acid significantly decreased tissue ATP and apical F-actin in a dose-dependent
manner relative to equimolar sodium chloride controls, yet higher doses of maleic acid
quantitatively resulted in net actin polymerization, primarily in the cytoplasm. Functionally,
maleic acid decreased glomerular filtration rate (GFR) and tubular reabsorption of sodium
(TRNa) in a dose-dependent manner relative to sodium chloride controls. Administration of
exogenous ATP resulted in significant increases in tissue ATP, net actin depolymerization,
and relocation of F-actin from the cytoplasm back to the apical surface coinciding with
increases in GFR and TRNa. Thus, ATP depletion induced by maleic acid resulted in
significant cytoskeletal and functional alterations that were ameliorated by exogenous ATP.
We therefore conclude that the structure and cellular location of F-actin necessary for
normal functioning of proximal tubule cells in vivo is dependent on tissue ATP levels.
Research ArticleFind the latest version:
Exogenous
Adenosine
Triphosphate (ATP)
Preserves
Proximal
Tubule
Microfilament Structure and
Function
In
Vivo in
a
Maleic Acid Model of
ATP
Depletion
PaulS. Kellerman,
withthe technical assistance of Shannon Norenberg and Naomi Guse
DivisionofNephrology, Department ofInternal Medicine, University ofCalifornia, Davis,MedicalCenter,Sacramento, California 95817
Abstract
The hallmark ofischemic acute renal failure is a
rapid
and earlydecline
inproximal
tubule ATP.Since
wehavepreviously
shownthatoverhalfof
apical
microfilament lossesoccurwithin the first 5 min ofexperimental
ischemicinjury,
wepostulated
that microfilament
(F-actin)
structureand cellular locationaredependentoncellular ATP levels. Totestthis
hypothesis,
weused
maleic acid
toselectively
inhibit renal cortical ATP pro-duction in vivo. Maleic acidsignificantly
decreasedtissue ATP andapical
F-actin inadose-dependent
mannerrelative
toequi-molarsodium chloride
controls,
yethigher
dosesofmaleic
acidquantitatively
resulted innetactin
polymerization, primarily
in thecytoplasm.
Functionally, maleic acid
decreasedglomerular
filtration rate
(GFR)
and tubularreabsorption
of sodium(TRN.)
in a dose-dependent mannerrelativetosodium
chloride controls.Administration of
exogenous ATP resulted insignifi-cant
increases
intissue ATP,
netactin depolymerization,
and relocation ofF-actin from
the cytoplasm back to theapical
surface coinciding
with increases in GFR andTRN,.
Thus,
ATPdepletion induced by maleic
acid resulted insignificant
cytoskel-etaland
functional alterations
thatwereameliorated by
exoge-nousATP. Wetherefore
conclude that thestructureand cellu-larlocation
of F-actin necessary for normalfunctioning
of prox-imal tubule cells in vivois dependent
ontissue ATP levels.(J.
Clin. Invest. 1993.
92:1940-1949.) Key
words: maleic acid-microfilaments
* adenosine triphosphate - proximal tubule .actin
Introduction
Avery
early
eventduring
invivo experimental ischemic
acute renalfailure is
arapid
decrease inATPto<10-
15% of
control values ( 1-3). Structurally,ischemic
injury
is characterized byextensive
disruption
of the
apical surface of proximal
tubule cells (4-7). Functionally and biochemically, there is loss ofThis work waspresentedinpartat boththe American Society of Clini-cal ResearchmeetinginMay 1991 inSeattle, WA, and the American Society of Nephrology meeting in Baltimore, MD,inNovember 1992. Address reprint requests to Paul Kellerman, M.D., Division of Ne-phrology, University of California, Davis, Medical Center, 4301 X Street,Sacramento, CA 95817.
Receivedfor publication 4 August 1992 and in revisedform 18 May 1993.
unidirectional transport of solute and epithelial polarity as de-finedby surface membrane characteristics (8, 9). Using spec-trofluorometry of rhodamine-phalloidin-stained sections of outercortical proximal tubules, we have recently demonstrated that the majority of apical filamentous (F-) actin lossoccurs
during the first 5 min of experimental ischemic injury (10). Since both ATP depletion and apical microfilament disruption arevery early events during ischemia, and the binding of ATP
toF-actin has been postulatedtobe necessary for
optimal
fila-mentous actin growth, we hypothesize that microfilamentstructureandcellular location in vivoaredependent on cellu-lar ATPlevels. To test this hypothesis, we utilized maleic acid,
aninhibitor of the Krebs cycle that is selectively taken up by renaltubule cells, to decrementally decrease renal ATP levels invivo. Torestoretissue ATP levels in the
kidney,
weinfused exogenous ATP-MgCl2 as previouslydescribed
bySiegel et al. (3).Thus,
this model of in vivo ATPdepletion
and repletion enabledus toobservedATP-dependent alterations
in proximal tubuleactin structure and renal tubular and glomerular func-tion.Methods
MaleSprague-Dawleyrats (Harlan Teklad, Madison, WI) weighing 200-300 gwerefed standardratchow.Animalswereinjected intraperi-toneally with 250, 400, and 1000 mg/kg of disodium maleic acid
(Sigma Immunochemicals,St.Louis,MO),orequimolaramountsof sodium chloride. Animalswereanesthetized with sodium pentobarbi-tal(6.25 mg/ 100 gratwt), and killed for renal cortical F-actin staining and renaladenine nucleotide assays 30 and 60 min following injection of maleic acid.Asecondsetofrats wasinfused with 25 Mmol of ATP-MgCl2 during the last 20 min before death 60 min after maleic acid for F-actinstaining and adenine nucleotide assays. A third set of rats un-derwent 30-min urine collections bracketed by arterial blood samples fordetermination of inulin clearance and tubular handling of sodium 60 minfollowing maleic acid injection without and with ATP-MgCl2 infusions. Last, for quantitative actin determinations, a fourth set of ratswaskilled 60 min after injection with maleic acid without and with exogenousATP-MgC12.
Rhodamine-phalloidin staining ofF-actin. F-actin in outer cortical renaltissue was stained as previously described ( 10). Outer cortical tissue sliceswerequicklyfrozen in liquid nitrogen, embedded in the
cryofixativeTissue-Tek OCT (Miles, Inc., Elkhart, IN), and 5-,um sec-tions cut on acryotome (2800 Frigocut; Reichert Scientific Instru-ments, Buffalo, NY). Following fixation in 3.7% formaldehyde, the sectionswerestained with 1:10 dilution of rhodamine-phalloidin (Mo-lecular Probes, Inc., Eugene, OR). Following mounting in 1:1 PBS/ glycerol, the samples were viewed with a Leitz Orthoplan universal largefield microscope (Orthoplan; E. Leitz, Inc., Rockleigh, NJ)
equippedwithaploemopak 2.1fluorescencevertical illuminator con-taininganexcitation/barrier filter specific for rhodamine. Photographs weretakenwith a Vario Orthomat 2 microscopic camera and the Ploe-mopak (E.Leitz, Inc.).Theexposure time was variable and automati-J.Clin. Invest.
©TheAmerican Society for Clinical Investigation, Inc.
0021-9738/93/10/1940/10 $2.00
callyadjusted by thecameraunit dependentontheamountof fluores-cencequenching.
Spectrofluorometry.
The technique ofspectrofluorometry is well established ( 1 1, 12) and has been previously performed with this fluor by us and others(10, 13). Apical fluorescence of rhodamine-phalloi-din, which is linearly relatedtoF-actin concentration ( 14),was mea-suredwitha MPV compactmicroscope photometer (E. Leitz, Inc.) mounted on thefluorescence microscope. The microspectrophotome-ter wasinterfaced withanIBMpersonal computer througha custom-designed card and software that allowed the computertocontrol the shutter sequencers,toaverage fluorescence ina250-mswindow,andto compile data. Becauseof the distinct qualitative differences between experimental groups, itwas notpossibletoblind the observertothe experimental protocol. Thus, thefollowingcriteriawereutilizedto stan-dardize the observations: (a) All sectionswere5/m
thick.(b) Saturat-ingquantities ofrhodamine-phalloidin were used. There was no differ-enceinquantitative fluorescence found between 1:20 and 1:10 dilu-tions. (c) Thespectrofluorometryfield was less than 1 x 1 mandwas placedentirelywithin the apicalfluorescencefor each reading.(d)All observations were madewithin 1 s of field placement to minimize fluorescence quenching. To minimizereadingfluorescence outside thespectrofluorometry field,the microscopefielddiaphragmwas maxi-mally closed. (e) To eliminateinterassayvariability influorescencedue tostaining,spectrofluorometryfieldsize,ordecline in photon emission withincreasing lamp time, each experiment included a new control kidney, with allspecimens undergoing sectioning, staining, and quanti-fication thesameday.(f)Foreachslide,meanfluorescence expressed inmVwasobtained from 50 observations of tubules, with each obser-vationconsisting ofa separate tubule. Although mean absolute fluores-cence wasmeasured within eachexperiment, summation datawere standardized byexpressingthe percentchange inmeanfluorescence relative to the controls within the same experiment. (g) The validity of the technique using independent observations by two individuals has previously demonstrated a correlation coefficient of 0.96 (P<0.001) (10).
DNAaseinhibition assay. Actin wasquantified asoriginally de-scribed by Blikstadetal.(15)andmodified byCheungetal(16).60 minafter maleic acidinjection, theoutercorticeswerehomogenized
with a polytron (PT-3000; Brinkmann Instruments, Inc., Westbury, NY), and thelysatesmixed 1:1 with buffercontaining 10 mM Tris-HC1 (pH=7.5), 150 mMNaCI,3 mMMgCl2,0.1% Triton X-100, 0.2 mM ATP, and 0.1 mMdithioerythritol. Beef pancrease DNAase I
(SigmaImmunochemicals)wasmixed with 3 ml ofcalfthymusDNA typeI(SigmaImmunochemicals)toobtainamaximalrateof0.25-0.3 ODU/15s asmeasuredat260nmat25°C.Followingconstructionof astandardinhibitioncurveusingsonicated monomeric actin from rab-bit muscle(SigmaImmunochemicals), equalamountsof
lysate
sam-ples were addedtotheDNAase,followed 2-4slaterbyDNAsubstrate,
with thepercent inhibition read at 260nm.To assay totalactin,F-actin wasdepolymerized bymixingthelysate withanequal volume ofa1.5 Mguanidinehydrochloridesolutioncontaining 1 mMCaCI2,2 mM Tris-HCl (pH=7.5), and 1 mMATP,andtheabsorbancewasagain
measuredat 260 nm. Protein determination via the Lowry method ( 17) allowedstandardization ofactin per mg ofprotein between experi-mentalgroups. Only DNAase inhibition experiments with standard curvesshowingrvaluesof>0.99wereconsidered valid, and experi-mental valuesfalling between 25-75% inhibition were used in all calcu-lations.
Adenine nucleotide assays. Adenine nucleotides were measured fol-lowing extraction withperchlorateas perWilliamson and Corkey ( 18). Whole rat kidneys were quickly frozen between stainless steel tongs cooled inliquidnitrogen and pulverized with a liquid nitrogen-cooled stainless steelmortarandpestle inpreparation for extraction. After a smallamountof the powdered tissue was placed in a drying oven at 105°C for 24 h, the remainder was placed in a centrifuge tube on crusheddryice and extracted with cold 8%perchlorate(3.5:1 wt/wt). Following homogenization at 7500 rpm on a polytron (PT-3000;
Brinkmann Instruments, Inc.) and centrifugation at 12,000 rpm for 10
min,
the supernatant was saved. The remaining pellet was again ex-tracted with 6% perchlorate (2.5:1 wt/wt), homogenized, and centri-fugedasabove. The supernatantswerethenpooled, 2NKOH/0.5M triethanolamine addedtoneutralize the pHto6-7, the sample centri-fugedat9,000 rpm for 10min,and the resultant supernatant saved for adenine nucleotide assays.Adenine nucleotides were measured enzymatically ( 19) on a spec-trophotometer(UVI6OU; Shimadzu Scientific Instruments, Inc., Co-lumbia, MD). ATP was measured by mixing Tris base (0.5 M), glu-cose(0.2 M), NADP (2 mg/ml), and the extracted supernatant and obtaining a baseline absorbance reading at 340 nm. Hexokinase/
G6PDH (200 U/ml)wasthenadded, the absorbance read at 60
min,
and theconcentration of ATP in
gmol/ml
calculated by dividing the difference in readings by 6.22, the coefficient of extinction. ADP and AMP were measured by mixing extracted supernatant with triethano-lamine (50 mM), MgCI2 (10 mM), phosphoenolpyruvate (10 mg/ ml), ATP(6 mM), NADH (2 mg/ml) and lactate dehydrogenase (5 mg/ml). Following thebaseline reading, 10,ul
ofpyruvate kinase (2.5 mg/ml) was added, the second reading obtained, and ADP concentra-tioncalculated from the difference in the readings divided by the coeffi-cient of extinction. Myokinase (5,d
of 2 mg/ml) was then added, the final reading obtained, and AMP concentration calculated from the difference of the second and third readings divided by two times the coefficient of extinction.Functional studies. 60 min following injection of maleic acid, inu-lin clearances and sodium handinu-ling were measured during a 30-min collectionperiod. Following general anesthesia with sodium pentobar-bital(6.25 mg/ 100 gratwt), the femoral vein and artery were cannu-lated with Intramedic polyethylene PE-50 tubing (Clay Adams, Parsip-pany,NJ) and the inulin, dissolved in normal saline, infused at 3.78 ml/h. Theanimals were then injected intraperitoneally with either disodiummaleic acidorequimolar sodium chloride, and subsequently underwent bladder cannulation and tracheostomy. Following 60 min of inulin infusiontoattain steady plasma levels, urinewascollected for 30min,with arterial blood samples collected at thestartandfinish of theurine collection.
Inulinwasmeasured using astandard anthrone assay. Briefly, a standardcurvefrom stock inulin and dilutions of plasma (1:10) and urine (1:1000or 1:2000)wereprepared.Following addition of 70% sulfuric acid and anthrone reagent (0.2 g in 100mlof 70% sulfuric acid), the sampleswereincubatedat50°C for 30min,cooledtoroom temperature, and readat630nm.
Plasma and urinesodium values were determined by flame photom-etry(Beckman Instruments, Fullerton, CA).
Statistics. DatawereanalyzedforsignificanceusingANOVA, with simultaneous multiple comparisonsbetween groupsusingDuncan's test.Resultswereconsideredstatisticallydifferent if thePvaluewas at least<0.05, and reportedas P<0.05orNS.Variancewasexpressedas standarderrorof themean.
RESULTS
Effect
of maleic acid on renal adenine nucleotides, proximal
tubule
apical
F-actin, tubular
handling of
sodium,
and
glomer-ular
filtration
rate. Maleicacid causedsignificant
dose-depen-dent decreases in tissueATP, total adenine
nucleotides,
andapicalF-actin.
Asshown in Table I, 250 mg/kg, 400 mg/kg, and 1000
mg/kg of maleic acid
significantly
decreased ATP levels in adose-dependentmanner to82.9%, 52.5%,and 37.3% of control
values,
respectively.
Tissue ADP decreased and tissue AMPincreased with maleic
acid,
but thesechanges
occurred in anon-dose-dependent
manner.Primarily
because of theTableI.Renal Adenine NucleotidesfollowingMaleic Acid without
and withExogenous ATP-MgC12
Treatment ATP ADP AMP TAN
Control 1.58±0.04 0.303±0.01 0.127±0.003 2.03±0.04
250 mg/kg 1.31±0.03* 0.286±0.01 0.129±0.008 1.79±0.06*
400mg/kg 0.83±0.09* 0.287±0.01 0.163±0.014* 1.28±0.09*
1000 mg/kg 0.59±0.03* 0.264±0.01* 0.149±0.004* 1.11±0.10*
400mg/kg
+ATP 1.12±0.05$ 0.35±0.02t 0.161±0.01 1.68±0.02*
Allvaluesareexpressed in
limol/g
wetkidneywt.* P<0.05vs.con-trols,*P<0.05 vs.400mg/kg (n= 17for
controls,
n =6 for 250and 400mg/kg,n = 12for1000 mg/kg,andn =5for400
mg/kg
plus
exogenousATP).
mental ATP
changes,
totaladenine nucleotides
decreased inadose-dependent
manner.Fig.
1 A demonstratesrhodamine-phalloidin
staining of
control
proximal
tubules,
with smoothhomogenous
apical
flu-orescence
representing
apical
brush border andterminal
webmicrofilaments,
andlittle F-actin
fluorescence
inthe
cyto-plasm. Treatmentwith
250 mg/kgof maleic acid (Fig.
1 B) causedsignificant
inhomogeneity
and losses(arrows) of
theapical fluorescence of F-actin relative
tocontrols,
yet therewas nochangein
thescantcytoplasmic F-actin.
400mg/kg
(Fig.
1C)
and 1000 mg/kg(Fig.
1D) of maleic acid
causedprogres-sive
significant
lossesof
apical F-actin
fluorescence, with
fluo-rescence
appearing
in thecytoplasm.
Apical
fluorescence
wasquantified using
spectrofluorometry
(Fig. 2). 250, 400,
and 1000mg/kgof disodium maleic acid progressively
andsignifi-cantly
decreasedapical F-actin by
24.1±1.9%,
34.5+6.5%,
and54.6±2.2%,
respectively, relative
tocontrols.Fig.
3 representstwo
focal planes of
the same tubules 60 minafter
treatmentwith
400 mg/kgof maleic
acid. While
higher
dosesof maleic
acid
resulted in severeapical F-actin degradation (A),
exten-sive
newappearanceof F-actin
occurredthroughout
thecyto-plasm
(B).The
specificity of maleic acid
was demonstrated by twodifferent
means. Whencompared
withnoninjected
controls,animals
injected with sodium
equimolar
(3.13 M)
to1000 mg/ kgof disodium maleic acid
showed nodifferences in
proximal tubuleF-actin
staining
orrenal ATP(1.61±0.04
vs.1.58±0.04
,Omol/g
wetwtkidney).
Furthermore, theinjection
of 400 mg/ kg of the maleicacid isomer, sodium
fumarate, showed nodifferences
inapical F-actin staining
orrenal ATP (1.61±0.06vs. 1.58±0.04
,umol/g
wet wtkidney).Since maleic acid caused dose-dependent apical structural changes, the effect of maleic acid on renal function was also evaluated. There were significant dose-dependent decreases in glomerular filtration rate
(GFR)'
asmeasured by inulinclear-ances
relative
toequimolar sodium chloride controls (Fig. 4).250,
400, and 1000 mg/kg decreased GFR to 56.2%, 34.8%, and12.4%,
respectively, of control values as measured in ml/ min per g kidney (1.3±0.13 vs. 0.73±0.17, 1.41±0.17 vs.0.49±0.08,
1.29±0.15vs.0.16±0.03;
P <0.05for
all groups vs.1.Abbreviation used in this paper: GFR, glomerular filtration rate.
controls). There were no significant differences in GFR be-tween noninjected controls and control animals injected with increasing equimolar doses of sodium chloride (1.52±0.12 vs.
1.3+0.13,
1.41+0.17, and 1.29±0.15 ml/min per g kidney). Fig. 5 demonstrates alterations in tubular reabsorption of so-diumfollowing maleic acid relative to equimolar sodium chlo-ride controls. Although there was a slight downward trendasexpected due to thekidneys' normal response to the progres-sive sodiumloads, therewere nodifferences between250, 400, and 1000mg/kg relative to noninjected controls (99.5±0.18% vs. 98.5±0.17%,
97.8±0.17%,
and96.3+0.66%, respectively). When comparedwith
equimolar saline controls,
250mg/kg,
400 mg/kg, and 1000 mg/kg of maleic acid significantly de-creased tubular reabsorption of sodium ina dose-dependent manner(92.5±3%, 81.2±3%, and33.7±6.1%, respectively).
Thus,maleic acid induceddose-dependent decreases in tis-sue ATP and ADP, apical microfilaments, glomerular
filtra-tion
rate, andproximal
tubulereabsorptive
function.Effect of exogenous ATP on renal adenine nucleotides,
proximal tubule apical
microfilaments, actin polymerization,
tubularfunction, andglomerularfiltration
rate. To test thespec-ificity of
theassociation
oftissue
ATPandmicrofilament
struc-tureand cellular
location,
we infused 25 imol of exogenous ATP-MgCl2into
ratsfollowing injection of high-dose
maleicacid.
ExogenousATP-MgCl2
increased tissue ATP andre-versed theproximal tubule cytoskeletal and
functional
alter-ations
seenwith
ATPdepletion induced by
maleicacid.
30 min
after
injection of
400mg/kg
of maleicacid,
ATP hadsignificantly
decreased to 57.9%of
controls(1.59±0.04
vs.0.92±0.06
,mol/g
wet wtkidney) (Fig. 6).
Without additionof
exogenousATP-MgCl2,
at60 minfollowing
injection,
ATP was52.2%of
controlvalues (1.59±0.04 vs.0.83±0.09ttmol/g
wet wt kidney, P < 0.05).Following infusion of
exogenousATP-MgC12
during the last 20min,
ATP levelssignificantly
increased to 70.4%
of
control values (1.12±0.05 ,imol/g wet wtkidney)
when comparedwith either
30 or 60 minof maleic
acid
alone(Fig.
6, TableI).
ADPlevels alsosignificantly
in-creased, whereas
AMPlevelsremained unchanged (Table I).
Fig.
7,Aand B, are twofocal
planesofthe
sametubulesdemon-strating
thealterations
inmicrofilament staining
seen30
minfollowing injection with
400 mg/kgof maleic acid
andbefore
exogenous
ATP-MgCl2 infusion.
Fig.
7Ademonstratesproxi-mal tubule(p)apical
F-actin disruption,
andFig. 7 B showsF-actin
appearing in the cytoplasmof
kidneys,identical to that seen inFig.
3 60 minfollowing maleic acid injection.
In con-trasttotheproximal
tubules, there was no effect of ATPdeple-tion
onthetypical
basolateralF-actin staining of
distal tubules(d). Fig.
7,Cand
Ddemonstrate the
changes seen inthe
proxi-mal tubules60 minafter treatment with 400 mg/kg of maleic
acid followed
by exogenousATP-MgCl2.
A low powerview
(Fig.
7C) demonstrates that theF-actin has moved from thecytoplasm
backtotheapical
portions of
alltheproximal
tubule cells. Thedistal
tubule (D) F-actin remains unchanged withATP
repletion.
A high-power view further details the move-ment of F-actin out of the cytoplasm and back toward theapical surface
(arrow) inproximal
tubules. Additionally, abright
yellow line
(arrowhead)
has
appeared atthe
apexof
thecells,
likely representing
addition of new F-actin to the distal endsof
microfilaments
in themicrovilli. Quantification
withlev-Figure1.Rhodamine-phalloidin fluorescence of F-actin in proximal tubules treated with maleic acid.
Formaldehyde(
3.7%)-fixedsections(5Am)
ofsuperficial renalcortex werestained witha1:10 dilution ofrhodamine-phalloidin. (A)Cross-sections of controlproximaltubules with ho-mogenousapical fluorescenceof F-actin. (B) Following 250 mg/kg of maleicacid, apicalfluorescence decreased and becameinhomogeneouswith patchy areasof microfilament losses (arrows). (C) Following 400 mg/kg of maleicacid,therewereextensive lossesofapicalF-actin,with increases in cytoplasmicfluorescence. (D) Following 1000 mg/kg of maleicacid,therewasalmostcompletelossofapicalF-actin,with cyto-plasmicopacification by F-actinstaining. Magnificationwas984xfor A, B, C, andD.
n=7 n=6
0
-10
-20
-30
-40
-50
-60
-70
n=7
250 400 1000
MAleic
Acid Dose(mg/kg)
Figure 2. Maleic acid-induced quantitative changes of proximal tu-buleapical F-actin fluorescence relative to controls. Apical F-actin
significantlydecreased inadose-dependentmannerfollowing maleic acid. See Methods for detailed description of spectrofluorometry. *P <0.05.
els seen with250 mg/kg of maleic acid, approaching butnot
reachingstatistical significance when compared with 400mg/
kg of maleicacid alone.
Toquantify changes in cellular actin polymerization, G-ac-tin andtotal actinweremeasured with the DNAase inhibition assay (Fig. 8). 60 min following 400 mg/kg of maleic acid, both percent
(top
graph)
andquantity (bottom
graph)
ofG-ac-tinsignificantly decreased from 56.7±1.2% to 44.7±1.8%, and 0.345±0.026 to 0.264±0.11 mg/mg of protein, respectively.
Administration of
exogenous ATP-MgCl2 following maleic acidsignificantly increased the percentage of G-actin back to-ward controllevelsrelative to maleic acid alone (50.8± 1.1 % vs. 44.7±1.8%), while G-actin quantity increased relativetoma-leic acid alone, approaching but not reaching statistical signifi-cance(0.318±0.023 vs. 0.264±0.11 mg/mg of protein). Total actin remained unchanged with ATP depletion and repletion (datanotshown). Thus,ATPdepletionresulted in net F-actin polymerization, while partial restoration of cellular ATPwith
exogenousATP-MgCl2 resulted in net actin depolymerization back toward control values.
Functionally,
GFRsignificantly
improvedin those animalsFigure3.Effect of high dose maleic acidon outercorticalproximaltubules stained with
rhodamine-phalloidin.
(A)Following400 mg/kg of maleicacid, therewereextensive apicallossesofproximaltubule F-actin.(B)Adifferent focal plane of thesametubulesdemonstratingthe appearance of F-actinthroughout thecytoplasm. Magnificationwasx787 forAandB.treated with 400 mg/kg of maleic acid with
exogenousATP-MgCl2
vs.those
whoreceived 400
mg/kg of maleic acid alone
(0.79±0.08
vs.0.49±0.08
ml/min
per gkidney) (Fig.
9
A).
Similarly, tubular sodium reabsorption improved
significantly
with maleic acid followed by exogenous ATP vs.
maleic
acidalone
(90.1±2.7%
vs.81.2±3%)
(Fig.
9
B).
Discussion
In
polarized
epithelial cells, distinct
membranedomains
en-able functional
specialization. Proximal tubule cells
unidirec-tionally
transportsolute from lumen
toblood and have
aspe-cialized
apical
membrane domain both with microvilli for
in-creased
resorptive surface
area as well aslipid
andintegral
membrane
proteins distinct from the basolateral membrane
(20). Theactin
cytoskeletonis
adynamic
organelle innonmus-"N
1.50
1.00
0.50
0.00
T
AT
T\"~~~~T
I16
I
I
I
- \ ~~*
\T
K*
I~~~~~~
\ *CON 250 400 1000
100
90
80 70 60
50
40 30 20
10 0
Maleic
AcidDose(mg/kg)
Figure4. Dose-dependent decreases in glomerular filtration rate (GFR),asmeasured by inulin clearance, following maleic acid. Inu-lin clearances were measured by anthrone assay 60 min following maleic acid injection. Open triangles, noninjected controls (n=6), oranimalsinjectedwithequimolar NaCl (n= 10, 6, and 1 1,
respec-tively); filled triangles, maleic acid-treated animals (n=6, 10, and
6,respectively). *P<0.05.
cle
cells
such
asproximal
tubule cells.G-actin,
ormonomericactin, is soluble in
thecytoplasm
and
ispolymerized
toF-actin,
or
microfilaments
(21, 22).
Themajor
structural componentof the microvilli of
proximal
tubule cells is
F-actin, arranged
inbundles
of
30-50 microfilaments attached via vinculin
tothe
distal
tips
of the microvilli and attached
proximally
to asub-membranous
band
of actin
known asthe
terminal
web.Link-ing
microfilaments
tothe surface membrane and
toother
fila-ments are
multiple
actin-associated proteins,
some whoseac-tions
arecalcium dependent. The terminal web in
turnattaches
to
the cell membrane
atboth
thetight
and intermediate
junc-tions
(23, 24).
During experimental ischemic injury
toproximal tubules
in
vivo,
ATPdecreases
to25%
of control values within 30
s, andbelow 10-15% of control
valueswithin
5min
(
1-3).
De-creasesin
ATPhave been shown
to causesignificant disruption
of
microfilament bundles
infibroblasts (25), endothelial (26),
100 A
90 A
. ~~~* -r
N 80
70*
60 50
40T
30
CON 250 400 1000
Mleic
AcidDose(mg/kg)
Figure5. Dose-dependent decreases in tubular reabsorption of so-dium(TRNa)following maleic acid.
TRNa
was measured 60min after maleic acid injection. Open triangles, noninjected control animals (n=5)oranimalsinjected with equimolar NaCl (n= 10, 5, and 1 1,respectively); filled triangles, maleic acid-treated animals (n=6, 1 1,
2.00 1.50 1.00 0.50 ,&+ATP A -ATP .100 90 z 80 7 * -60 50 40 1Infusi-on 30 0 10 20 30 40 50 60
TIME
(mio)W
Figure6. Effect of exogenousATP-MgCI2 ontissue ATPfollowing highdose maleic acid. Solidtriangles, control animals(n = 17)and animals 30min(n 5)and 60mmn(n 6)aftertreatmentwith 400 mg/kgof maleic acid without exogenousATP-MgCl2;opentriangle,
animals treated with 400mg/kgof maleic acid followedby25'smol
of exogenousATP-MgCl2(n=6).Renal ATPsignificantlydecreased
to57.9%of control valuesby30mmnand52.2% of control valuesby 60mmnfollowingmaleicacid(*JP<0.05).Administration of
exoge-nousATP-MgCl2 significantlyincreased tissue ATPto70.4% of
con-trol values 60mmnfollowingmaleic acid(§P<0.05vs.both 30 and 60minafter maleic acid without exogenousATP-MgCl2).
andepithelial cells(27). We haverecentlydemonstrated that the majorityofproximaltubule apical F-actindisruption oc-cursduringthefirst 5minof in vivo ischemia(1I0),concurrent
withrapidly decliningATPlevels. Experimental ischemic
in-juryinvivo also results inpartiallossofproximaltubule polar-ity,with resultant decreases in tubularreabsorptionof sodium
(2).A recentstudyshowed thatchemicallyinduced ATP
de-pletioninLLC-PK,cells resulted in decreases in G-actin indica-tive of actin polymerization, alongwithdissociation of Na'-K'-ATPase both from the cytoskeleton and the basolateral
membrane, consistent with loss ofpolarity (28). Usingan iso-lated perfused kidney system, we have previously demon-strated that cytochalasinD, aselectivedisrupterof microfila-ments, causessignificant proximal tubulardysfunction (29).
Thus,theactincytoskeleton playsarole in the maintenance of
epithelial cellstructure andpolarity required for normal cell function.
Maleic acid has been used as an experimental model of
proximal renal tubular acidosis because it causes decreased
reabsorption ofsodium, bicarbonate, chloride, protein,
glu-cose,andphosphorus (30-36).Because it istransportedbythe
sameorganic transporterthat also transports para-aminohip-puric acid,maleic acid isselectivelytakenupand concentrated in theproximaltubulecells ofthekidney.Duetospecific inhibi-tionof the Krebscyclein the mitochondria ofproximaltubule
cells, maleic acidcausesdecreases inATP(37, 38).Although
maleate is the trans-isomer offumarate,rather thaninterfering
withfumarase,itmostlikelyformsmaleylcoA,whichacts to
deplete the Krebs cycle ofcoenzyme A (39, 40). Although early studies demonstrated inhibition of membrane Na',
K'-ATPase in vivo(37), morerecentinvitro vesicle studies have demonstrated that maleic acid does not directly alter
membrane
transporters
(41, 42). Thus,
it has beenproposed
that decreases in cellular ATP induced
by
maleicacid starvethe ionic pumps
of
energy,resulting
in lossofunidirectional
transport
of solute.Areview of
previous
studies
demonstrates that maleic acid doesnot actas anappropriate
modelof
proximal
renaltubular acidosis. In contrast toclinical renal tubularacidosis,
maleic acid results insignificant apical
membranedisruption
andvac-uolization
atbothlight
andelectronmicroscopic
levels(35, 38,
43-45).
Additionally,
in the few instances where GFR has beenmeasured,
prior
studies
haveshownsignificant
decreases in GFRwith maleic acid(30, 31, 33-35),
anotherfeature
in-consistent withproximal
renaltubular acidosis. The decrease inGER
hasbeenpostulated
tobedueeither
toincreasedpro-duction of adenosine
(46)
ortobackleakof inulin
(47).
Before thisstudy,
asystematic
dose response has never been per-formed for tissueATP,
renalfunction,
andmostsignificantly,
F-actin location andnetcellularactin
polymerization.
We
postulate
that maleic acidactsas amodelof selectivecortical
ATPdepletion
invivo,
thusmimicking
ischemicin-jury. Although
thereare noother viable in vivo models of selec-tive renal ATPdepletion, Shanley
andcolleagues
demon-stratedthatinfusion of
multiple
metabolic inhibitorsintoanisolated
perfused
kidney
system
resulted inproximal
tubuleapical
membranestructural alterations and"clubbing"
charac-teristicof
experimental
ischemia andhypoxic
injury
(48).
Us-ing
maleicacid,
we have demonstrated asignificant
dosere-sponse
for
renal ATPdepletion,
withconcurrentdose-depen-dent alterations in
proximal
tubuleapical F-actin, GFR,
and tubularsodium
reabsorption
similarto thatseenduring
isch-emicinjury. Raising
tissue ATPwith exogenous ATP in this in vivo maleic acid model resulted insignificant
reversal of this process, bothstructurally
andfunctionally. Using
time-courseexperiments,
we demonstrated that exogenous ATP did notjust
prevent
maleic acid-inducedapical
lossesof
F-actin,
butactually
resulted in relocation of F-actin from thecytoplasm
backtothe
apical
surface ofproximal
tubule cells. This is the firstdemonstration
that microfilament structure and cellular location invivo canbemodified
by
exogenous ATP.Concur-rentwith this reversal
of microfilament
structureand cellular locationby
exogenousATP,
both GFR and tubularreabsorp-tion of sodium
significantly improved.
These interventional datastrongly
support
thehypothesis
that microfilament struc-ture and cellular location necessary for normal function ofproximal
tubules in vivoaredependent
oncellular ATP levels. Similartoexperimental
ischemicinjury
in vivo(10O),
distal tubules did not altertheir F-actin structure with renal ATPdepletion
orrepletion
in this model.Very
little is knownregarding
thedynamics
of
actinpoly-merization in vivo. In
vitro,
G-actinpolymerizes
toF-actin viairreversible
hydrolysis
of ATP(49).
Only
very smallamountsof
G-actin arerequired
for filamentgrowth
invitro,
so theoverwhelming majority
ofactin ispresent
in thepolymerized
form. In contrast,
approximately
half of actin in cells exists in the monomeric form(50),
andmicroinjection
of G-actin into normal ratkidney
cells does not result in furthergrowth
ofexisting
microfilaments(51).
Themaintenance of thisdepoly-merizedstateismost
likely
duetoinhibition ofpolymerization
by
actin-associated
proteins (51). Additionally, LLC-PK1
cells treated with metabolic inhibitors have demonstrated decreasedamounts of cellular
G-actin,
indicating
netpolymerization
Cellular AdenosineTriphosphateand Proximal TubuleMicrofilaments 1945
1.50
1.00
0.50
0.00
A
with decreased cellularenergylevels(28). Basedonthesedata, investigators have postulated that cellularenergyisrequiredto
maintainadepolymerized rather thanapolymerizedstateof actin(50).
Our datasuggestthatenergy mayberequired for both poly-merization and depolypoly-merization of different actin pools in
vivo, allowing for rapid interconversion between
monomeric
and filamentous forms of actin. As ATP levels decreased in proximal tubule cells, cytoplasmicF-actinappearedwhile api-cal F-actin waseither lost into the lumenor depolymerized. Restoration of cellularATP levelsstructurallyresulted in
relo-cation of F-actin from thecytoplasm backtothe apical surface. Ifthis change in F-actin location was due to movement of
microfilaments rather than alterations inpolymerization, then
wewouldexpectnochangeinnetcellularactinpolymerization with ATP depletion and repletion. Netcellular actin polymer-ization, whichrepresentsthesumof actinpools,increasedwith
maleic acid-induced ATP depletion in vivo, similarto that
seenduringATPdepletion in the
LLC-PKI
proximal tubule cell model(28). Alterations in the cytoskeleton in vivooc-curred with smaller decrements in measured ATP than that
100
70 r
60
50
40
30
-0.40
0.30
0.20
CON 400 400+ATP
CON 400
TREA TMENT
400+ATP
Figure8. Effect ofexogenousATPfollowing maleic acidoncellular
actinpolymerization. Actinwasquantifiedwith the DNAase
inhibi-tionassay60min following400 mg/kg of maleic acid withoutand
withexogenousATP-MgCl2.Totalactinremained unchanged inall
treatmentgroups(datanotshown). Following maleic acidalone,
G-actinsignificantlydecreasedfrom56.7±1.2%to44.7±1.8% (top)
and from0.345±0.026to0.264±0.011 mg/mgprotein (bottom). Relativetomaleic acidalone,maleicacid plusexogenousATP sig-nificantly increased thepercentof G-actinto50.8±1.1,andincreased G-actinquantityto0.318±0.023mg/mgprotein,nearing butnot
reaching significance.n=6in eachgroup,*P<0.05.
CON 400 400+ATP
7REA7MENT
B
90
70
CON 400 400+ATP
TREA7TMABNV7
Figure9. Effect ofexogenousATP-MgCl2onglomerularfiltrationrate
(GFR)and tubularreabsorptionof sodium(TRN.)followingmaleic acid. 60minafter 400mg/kgofmaleicacid,both GFRandTRN.
decreased relativetoequimolarNaCl controls.(A) Followinginfusion with 25
gmol
of ATP-MgCl2, GFRsignificantlyincreasedto0.79±0.08 ml/minper gfrom 0.49±0.08 ml/minper gwith 400
mg/kgofmaleic acid alone.(B) FollowingexogenousATPinfusion,
TRNa significantlyincreasedto90.1±2.7% from 81.2±3% with 400 mg/kgof maleic acid alone.*P<0.05.
seen in cell culture (28), perhaps due to contribution from other unaffectedcortical cells. With ATP repletion, netactin polymerization decreased back toward control values,yetnew
F-actin formedattheapical surface. The differential polymer-ization ofapical and cytoplasmic F-actin withATPdepletion andrepletionsuggeststhat theremaybetwopoolsof actin in epithelial cells that respond in contrastingfashionstothesame
cellularATPlevels.Thishypothesis isfurther supported byour
recentstudies of F-actinstructureandcellularlocation during reperfusion following ischemia (Kellerman,P.S., manuscript
inpreparation;seealso reference 52). When renal ATP
recov-eredtolevelssimilartothatseenwithhigher dose maleic acid,
apical F-actin losses continued while cytoplasmic polymeriza-tion ofF-actinidenticaltothatseenwithmaleic acidwas
ob-served.Thus, theremaybe thresholdcellular ATP levels
neces-saryforpolymerization of different actin pools. The
differen-tial response to adenine nucleotides by the apical and
CellularAdenosineTriphosphateand ProximalTubuleMicrofilaments 1947
r. I ~~~~... . _
cytoplasmic pools of actin may be maintained via differences intheiractin-associated proteins, since thereareunlikely to be structural barriers within the cytosol. This concept has some
support in a recent study
demonstrating
actin populations within the same cell with different sensitivities to depolymeriza-tionbyprofilin,anactin-associated protein (53). This hypoth-esis of intracellular compartmentalizationofactinwillrequire furtherstudy for confirmation.Acknowledgments
The author extends hisgratefulappreciationtoJudyLundand Kather-ine Sanders for their technical assistance in
sectioning,
Gunilla Thulin andDr. KarenGaudio for their technical adviceonATPinfusions,and Drs.GeorgeKaysen and Stuart Linas for their overallsuggestions
andguidance.
This workwassupportedby aGrant-In-Aid from the American HeartAssociation, California affiliate, a YoungInvestigator Award from the National Kidney Foundation, andgeneral research funds from theUniversityofCalifornia,Davis School of Medicine.
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