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Original article 143
Caveolin-1 and Hsp70 interact
ion in microdissected proximaltubules from spontaneously hypertensive rats as an effectof LosartanVictoria Bocanegrab, Walter Manuchaa,b, Marcelo Rodrıguez Penab,Valeria Cacciamania and Patricia G. Vallesa,bBackground Caveolin is required to traffic the AT1 receptor
through the exocytic pathway. The chaperone Hsp70
regulates a diverse set of signaling pathways via their
interactions with proteins.
Method Here we examined the AT1 receptor antagonist
Losartan effect on caveolin-1 and Hsp70 protein association
in spontaneously hypertensive rat (SHR) proximal tubules.
Hsp70 involvement in Losartan oxidative stress regulation
was also studied. Five-week-old SHRs were randomized for
receiving Losartan (40 mg/kg per day) (SHRLos) or no
treatment (SHRH2O) during 6 weeks. Wistar–Kyoto rats
(WKY) were normotensive controls.
Results By western blotting, the relative abundance of
caveolin-1 was two-fold higher in microdissected proximal
tubule membrane fractions from treated SHRs vs. WKYH2O.
Hsp70 membrane translocation was demonstrated in
SHRLos through out the up-regulation of Hsp70 expression
in microdissected proximal tubule membrane fractions
when compared with WKYH2O (P < 0.001). Conversely,
decreased Hsp70 protein levels were shown in
microdissected proximal tubule cytosol fraction from
SHRLos (P < 0.01). Interaction between caveolin-1 and
Hsp70 was further determined by coimmunoprecipitation
and by immunofluorescence co-localization in SHRLos
proximal tubule membranes. After membrane translocation
of Hsp70, the decreased NADPH oxidase activity (RFU/
mprot per min incubation) near controls demonstrated on
microdissected proximal tubule membranes from SHRLos
vs. SHRH2O (P < 0.01) was reversed by the preincubation
with anti-Hsp70 antibody. In addition, interaction between
opyright © Lippincott Williams & Wilkins. Unauth
Portions of this study were presented as an abstract form at the World Congressof Nephrology in Rio de Janeiro, Brasil, April 21–25, 2007.
0263-6352 � 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
Hsp70 and Nox4 was determined by the
coimmunoprecipitation strategy showing that membrane
overexpression of Hsp70 was associated with decreased
Nox4 after Losartan treatment in SHRs.
Conclusion After Losartan administration interaction of
caveolin-1 and Hsp70 was shown in SHR proximal tubules.
Translocation of Hsp70 to proximal tubule membranes in
SHRLos might exert a cytoprotective effect by down-
regulation of NADPH subunits Nox4. J Hypertens 28:143–
155 Q 2010 Wolters Kluwer Health | Lippincott Williams &
Wilkins.
Journal of Hypertension 2010, 28:143–155
Keywords: angiotensin II AT1 receptor, caveolin-1, Hsp70, NADPH subunitsNox4, oxidative stress
Abbreviations: AngII, angiotensin II; angiotensin II type 1 receptor, AT1R;RAS, renin–angiotensin system; ROS, reactive oxygen species; SBP,systolic blood pressure; SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rats
aArea de Fisiopatologıa, Departamento de Patologıa, Facultad de CienciasMedicas, Universidad Nacional de Cuyo, Mendoza and bIMBECU-CONICET(National Council of Scientific and Technical Research of Argentina), Argentina
Correspondence to Dr Patricia G. Valles, Area de Fisiologıa Patologica,Departamento de Patologıa Facultad de Ciencias Medicas Universidad Nacionalde Cuyo Centro Universitario, CP: 5500 Mendoza, ArgentinaTel: +54 0261 4135000- Int 2624; fax: +54 0261 4287370;e-mail: [email protected]
Received 4 March 2009 Revised 13 August 2009Accepted 4 September 2009
See editorial commentary on page 9
IntroductionCaveolae are small invaginations, located at or near the
plasma membrane, that are characterized by the presence
of caveolin, a 21–24-kDa cytoskeletal protein that exists
as several subunits (caveolin-1, 2 and 3) [1]. Caveolins are
crucial structural components of caveolae. Specialized
motifs in the caveolin proteins function to recruit lipids
and proteins to caveolae for participation in intracellular
trafficking of cellular components and operation in signal
transduction [2]. Apart from its structural role, caveolins
have been implicated in interactions with signaling
proteins through its scaffolding domain [3–5]. The angio-
tensin II (AngII) type 1 receptor (AT1R) is a nonpalmi-
toylated G-protein-coupled receptor (GPCR) that in
smooth muscle cells co-immunoprecipitates with caveo-
lin [6]. The AT1R is found, similar to caveolin, in many
cell types including smooth and cardiac muscle cells as
well as endothelial and epithelial cells [7]. AT1 caveolin
complex in caveolae may coordinate AngII-induced sig-
naling. Caveolin is required for normal renal AT1R
expression [6]. Acting as a molecular chaperone, this
protein is necessary for correct transport of the AT1R
to the plasma membrane [8]. Mislocalized caveolin
mutant expression, absence of caveolin, or the disruption
of the formation of a caveolin–AT1R complex have a
orized reproduction of this article is prohibited.
DOI:10.1097/HJH.0b013e328332b778
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144 Journal of Hypertension 2010, Vol 28 No 1
profound effect on the trafficking of AT1R to the plasma
membrane. Moreover, caveolin-1 null mice have 55%
reduction in renal AT1R levels compared with controls
[9–11]. Caveolins have also been directly implicated in
interactions with signaling proteins. In the genetic model
of rat hypertension, spontaneously hypertensive rats
(SHRs), a role for the renin–angiotensin system (RAS)
in the development and/or maintenance of hypertension
has been demonstrated [12]. AngII is not only a powerful
vasoactive peptide, but also a potent proinflammatory
cytokine and growth factor in renal tissue [13]. Many
effects of AngII are dependent on the AT1R stimulation
of reactive oxygen species (ROS) production by NADPH
oxidase. AngII up-regulation stimulates oxidative stress
in proximal tubules from SHR [14]. AngII has been
shown to promote production of ROS which, in turn,
mediate several effects of AngII, notably protein syn-
thesis of sodium transporters and growth factors, contri-
buting to the development of hypertension-induced
tubulointerstitial renal injury [15]. Induction of the stress
response includes synthesis of heat shock protein Hsp70,
molecular chaperone that has a critical role in the recov-
ery of cells from stress and in cytoprotection, guarding
cells from subsequent insults. Hsp70 protects stressed
cells by its ability to recognize nascent polypeptides,
unstructured regions of proteins and exposed hydro-
phobic stretches of amino acids. In doing so, chaperones
hold, translocated or refold stress denatured proteins and
prevent their irreversible aggregation with other proteins
in the cell [16]. In addition, chaperones of the Hsp70
family and their co-chaperones interact with a growing
number of signaling molecules [17]. In the present study,
we test the hypothesis of AngII AT1 blockade effect on
caveolin-1 and Hsp70 interaction and the translocated
molecular chaperone involvement in Losartan-induced
Nox4 down-regulation and NADPH oxidase inactivation
in SHR-microdissected proximal tubules.
MethodsAnimal preparationFive-week-old SHRs were randomized for receiving the
AngII type 1 antagonist, Losartan (40 mg/kg per day)
(SHRLos) or water (SHRH2O) by gastric gavage during
6 weeks. Wistar–Kyoto rats (WKYLos) and (WKYH2O)
were controls. The animals were individually housed in a
temperature and humidity-controlled room provided with
a 12 : 12-h dark–light cycle. All rats were given standard
chow with unlimited access to tap water. Weekly systolic
blood pressure (SBP) in conscious animals was measured
by using a tail-cuff sphygmomanometer. Three indepen-
dent SBP measurements per animal were recorded and
averaged. All procedures were conducted in accordance
with conventional animal care guidelines.
Tissue preparationAfter 6 weeks of treatment, kidneys from anesthetized
rats (pentobarbital 50 mg/kg) were perfused via a retro-
opyright © Lippincott Williams & Wilkins. Unautho
grade cannula with ice-cold phosphate-buffered saline
(PBS) solution to rinse all the blood and rapidly removed.
The kidneys were cut in half longitudinally, and the
cortex and medulla were divided with a fine stainless
steel blade. The isolated cortexes were placed in ice-cold
isolation buffer containing 300 mmol/l sucrose, 18 mmol/l
Tris–HCl, 5 mmol/l sodium ethylene-glycol-tetraacetic
acid (Naþ EGTA), 4 mg/ml aprotinin, 4 mg/ml leupeptin,
2 mg/ml chymostatin, 2 mg/ml pepstatin, and 100 mg/ml
4-2 aminoethyl-benzenesulfonyl fluoride (AEBSF) (pH
7.4) and were homogenized by using a Dounce style
tissue homogenizer. The homogenate was centrifuged
at 7000 r.p.m. for 15 min at 58C to remove incompletely
homogenized fragments and nuclei. The supernatant was
re-centrifugated at 19 500 r.p.m. for 45 min at 58C to
produce a pellet containing enriched membrane fractions
and the supernatant as cytosol. The pellets were resus-
pended in isolation buffer and the aliquots were saved
at �708C.
Microdissection of proximal tubule segmentsThe rats were anesthetized with an intraperitoneal injec-
tion of pentobarbital sodium (50 mg/kg). After perfusing
the kidneys through the aorta with a cold, wash solution
consisting of (in mmol/l) 135 NaCl, 3 KCl, 1.5 CaCl2, 1
MgCl2, 2 KH2PO4, 5.5 glucose, 5 L-alanine, and 10
HEPES (pH 7.4), the kidneys were quickly removed
and placed in ice-cold dissection solution consisting of
wash solution with 1 mg/ml hyaluronidase (359 U/mg;
Sigma), and 1 mg/ml soybean trypsin inhibitor (Sigma).
Thin coronal slices (�1 mm) were cut from cortex and
transferred to the dissection chamber. The single prox-
imal tubule segments were manually dissected (tubular
segment length 0.4–1.1 mm) from the outer cortex with
the aid of fine stainless steel needles under an Olympus
stereomicroscope (10–40�) with dark-field illumination,
at 48C. Tubules were stored on ice-cold HEPES solution
until dissection for a maximum of 60 min. Around 50
proximal tubule segments were dissected from each
cortex rat kidney. This pool was homogenized with a
Duonce style tissue homogenizer and the membrane
fractions, obtained from centrifugation were stored at
�708C.
Reverse transcription–polymerase chain reactionanalysisTotal RNA was obtained by using Trizol reagent (Gibco
BRL). Two micrograms of total RNA were denatured in
the presence of 0.5 mg/50 ml Oligo (dT)15 primer and 40
U recombinant ribonuclease inhibitor RNasin (Promega,
Madison, Wisconsin, USA). Reverse transcription was
performed in the presence of a mixture by using 200
units of Reverse Transcriptase M-MLV RT in reaction
buffer, 0.5 mmol/l dNTPs each, and incubated for 60 min
at 428C. The cDNA (10 ml) was amplified by polymerase
chain reaction (PCR) by standard conditions. Each cDNA
aliquot was amplified (35 cycles) for Nox4, p22 and p47
rized reproduction of this article is prohibited.
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Losartan on caveolin-1 and Hsp70 interaction in SHR Bocanegra et al. 145
Table 1 Primer sequences and PCR product lengths for Nox4, p22and p47 NADPH oxidase subunits and b-actin
Primer SequenceAnnealingC8
Predictedproduct
p22 50-GTTTGTGTGCCTGCTGGAGT-30 56 31650-TGGGCGGCTGCTTGATGGT-30
p47 50-TCACCGAGATCTACGAGTTC-30 56 18050-ATCCCATGAGGCTGTTGAAGT-30
Nox4 50-TCAACTGCAGCCTGATCCTTT-30 56 6650-TCTGTGATCCGCGAAGGTAAG-30
b-actin 50-TGGAGAAGAGCTATGAGCTGCCTG-30 56 20150-GTGCCACCAGACAGCACTGTGTTG-30
NADPH oxidase subunits and b-actin (primers designed,
Table 1). Densitometric analysis was performed by using
National Institutes of Health Image 1.6 software (Ras-
band Wayne et al. Division of computer Research and
Technology NIH, Bethesda). The Nox4, p22 and p47
signals were standardized against b-actin signal for each
sample and results were expressed as a ratio.
Protein determination and western blot analysisProtein concentrations from cortex were quantified by
Lowry assay by using BSA as a standard. A total of 20 mg
of proteins from membrane fractions from cortex hom-
ogenates and microdissected proximal tubules were elec-
trophoresed in polyacrylamide minigels (Bio-Rad Mini
Protean II). For each gel, an identical gel was run in
parallel and subjected to Coomassie staining. The Coo-
massie-stained gel was used to ascertain identical loading
or to allow potential correction for minor differences in
loading after scanning and densitometry of major bands.
The other gel was subjected to blotting. After transferring
to nitrocellulose membranes by electroelution, specific
binding sites were blocked with 5% milk in 80 mmol/l
Na2HPO4, 20 mmol/l NaH2PO4, 100 mmol/l NaCl, and
0.1% Tween 20, pH 7.5, for 1 h at room temperature,
washed and then incubated with primary antibodies
directed to Hsp70 dilution 1 : 2500 (Sigma Chemical
Co.), caveolin-1 1 : 4000, Nox4 1 : 1000 or p22 1 : 1000
from Santa Cruz Biotechnology and also to p47 1 : 1000
(Stress Gen, San Diego, California, USA). The labeling
was visualized with secondary biotinilated antibodies and
then with horseradish peroxidase-conjugated streptavi-
din (DAKO). The signal was detected with an enhanced
chemiluminescence system and quantified by exposure
to hyperfilm (Amersham PharmaciaBiotech). For quanti-
fication of Hsp70, caveolin-1, Nox4, p22 and p47 protein
levels, the photographs were digitalized using a scanner
(LACIE Silver Scanner for Macintosh) and the Desk
Scan software (Adobe Photo Shop) on a desktop compu-
ter. Densitometric analysis was performed by using the
US National Institute of Health Image 1.66 software
(Rasband Wayner et al. Division of Computer Research
and Technology NIH, Bethesda, Maryland, USA). The
magnitude of the immunosignal was given as a percen-
tage of control renal tissue.
opyright © Lippincott Williams & Wilkins. Unauth
Caveolin-1 immunoprecipitation: Hsp70 coprecipitationCoimmunoprecipitation was carried out by using Dyna-
beads M-280 Tosylactivated (Dynal, Biotech), following
the specification of the Dynabeads protocol. Anti-caveo-
lin-1 antibody was dissolved in a 0.1 mol/l borate buffer
(pH 9.5), added to the Dynabeads and then vortexed for
1 min. Following 48 h incubation, rotating at 48C, samples
were placed in the magnet and the supernatants were
removed and discarded. The coated beads were washed
with a buffer containing PBS (pH 7.4) with 0.1% BSA and
then with 0.2 mol/l Tris (pH 8.5) with 0.1% BSA. Sub-
sequently, equal volumes of membrane and cytosol
samples were adjusted to contain equal quantities of
protein and added to the coated beads. Following 1 h
incubation at 2–88C, the samples were placed in the
magnet and the supernatants were removed and dis-
carded. The beads were washed using a 0.1 mol/l Na-
phosphate (pH 7.4) and were suspended in an equal
volume of 2� sample buffer, and boiled for 3 min. The
supernatant was removed and stored at �708C. The
western blot analysis of the immunoprecipitated samples
was carried out the same as described before in western
blot analysis. Immunoprecipitation was performed using
Protein A/G Plus-Agarose and either anti-caveolin-1 anti-
body or preimmune rabbit IgG. After extensive washing
with buffer A, samples were separated by SDS-PAGE,
transferred to nitrocellulose membranes and probed with
an mAb against Hsp70. The Hsp70 level was standar-
dized against caveolin-1 level for each experimental
condition and the results were expressed as a ratio.
Nox4 immunoprecipitation (Hsp70 coprecipitation)Immnoprecipitation and western blot analysis were car-
ried out by using the protocol above described. Mem-
brane fractions underwent immunoprecipitation using an
antibody against Nox4. Antibody against Nox-4 was dis-
solved in a 0.1 mol/l borate buffer (pH 9.5), added to the
Dynabeads and then vortexed for 1 min. Following 48 h
incubation, rotating at 48C, samples were placed in the
magnet and the supernatants were removed and dis-
carded. Subsequently, equal volumes of membrane
samples were adjusted to contain equal quantities of
protein and added to the coated beads. Immunoblotting
of the immunoprecipitated samples was performed using
Protein A/G Plus-Agarose and either anti-Nox4 antibody
or preimmune rabbit IgG as outlined earlier. After exten-
sive washing with buffer A, samples were separated by
SDS-PAGE, transferred to nitrocellulose membranes,
and probed with mAb against Hsp70. The Hsp70 level
was standardized against Nox4 level for each experimen-
tal condition, and results were expressed as a ratio.
Immunofluorescence confocal microscopyAfter rinsing the kidneys with PBS, they were perfused
with 50 ml of 4% paraformaldehyde in tetra-borate buffer
(OR 9.4 mmol/l sodium borate, 0.34 mmol/l sodium
sulfite, 0.16 mmol/l boric acid, pH 7.4.), removed and
orized reproduction of this article is prohibited.
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146 Journal of Hypertension 2010, Vol 28 No 1
Fig. 1
Systolic arterial blood pressure (BP) during the course of theexperiment. BP in 40 WKY rats administered with either vehicle(WKYH2O) or Ang II AT1 receptor antagonist (Losartan 40 mg/kg)(WKYLos), and in 40 SHRs administered with either vehicle (SHRH2O)or AngII AT1 receptor antagonist 40 mg/kg (SHRLos). SHRH2O vs.WKY with vehicle, ���P<0.001 SHRLos vs. SHR with vehicle,P<0.001. AngII, angiotensin II; SHR, spontaneously hypertensive rat;WKY, Wistar–Kyoto rat.
placed in paraformaldehyde buffer overnight at 48C.
After fixation procedure they were cryoprotected in a
PBS solution supplemented with 0.9 mol/l sucrose over-
night, at 48C. Then the kidneys were washed twice with
PBS, placed in isopentane and stored at �708C. Tissue
sections (5 um) were obtained by using a cryostat. After
neutralization with NH4Cl buffer, the sections were
permeabilized for 1 h with BSA 1%, Triton X-100 1%
before incubation overnight with primary antibodies,
polyclonal anti-caveolin-1,1 : 100 (Santa Cruz Biotechnol-
ogy) and monoclonal anti-Hsp70, 1 : 100 (Sigma Aldrich,
in 0.1% BSA and 1% Triton X-100 in PBS). Secondary
antibodies were fluorescein isothiocyanate-conjugated
secondary antibody (goat antirabbit diluted 1 : 100) and
antimouse Alexa in PBS/BSA/Triton X-100, for 1 h at
room temperature. After being washed, the tissues were
stained with Streptavidin-FITC (DAKO), 1 : 100 for 1 h.
The coverslips were mounted in PBS-Glicerol for con-
focal microscopy. Confocal fluorescence images were
taken by using a Zeiss LSM 510 microscope.
NADPH oxidase activity assayNADPH oxidase activity was measured by luminol tech-
nique. Luminol (5-amino-2,3-dihydro-1,4-phthalazine
SIGMA). Samples were homogenized and centrifuged
at 6000 r.p.m. for 30 min. The supernatant was separated
and again centrifuged to 19 500 r.p.m. and the protein
concentration of the membrane fraction lysate was quan-
tified by Lowry assay using BSA as a standard. Sample
(40 ml) of the membrane fraction re-suspended in lysis
buffer was rapidly read in the spectrofluorometer (Fluoro
Count TM; AF10001, Cambers Company, USA) in order
to establish the basal value of each sample. Then, 2 ml
of b-NADH (b-nicotinamide adenine dinucleotide,
reduced form SIGMA) 0.1 mmol/l and 2 ml of Luminol
5 mmol/l in DMSO were incorporated and we proceeded
to read for a 10 min space (360 excitation and 460 emis-
sion). The values were expressed as relative fluorescence
units by micrograms of protein and per minute of
incubation.
Statistical analysisThe results were assessed by one-way analysis of variance
for comparisons among groups. Significance of differ-
ences was estimated by the Bonferroni test. A P< 0.05
was considered to be significant. Student’s test was
performed to compare the means when the experimental
design consisted of two samples. Statistical significance
was assessed by Student’s impaired t test. A P< 0.05 was
considered to be significant. Results are given as mean
�SEM. Statistical tests were performed by using Graph-
Pad In Sat version 5.00 for Windows XP (Graph Pad
Software, Inc, San Diego, California, USA).
ResultsThroughout the experiments, SBP in the SHRH2O group
was significantly higher than that in the WKY group.
opyright © Lippincott Williams & Wilkins. Unautho
Losartan administration induced a significant reduction in
SBP compared to the levels in the SHRH2O group. After
14 days, SHRLos showed similar SBP levels than did
WKYH2O (Fig. 1). Body weight was greater in both
WKY groups than in the two SHR groups during the whole
course of the experiments. After Losartan administration,
no difference in body weight between SHRLos and
SHRH2O was shown (152.36� 4.98 vs. 155.78� 2.2 g,
respectively).
Effect of the angiotensin II AT1 receptor antagonistLosartan on caveolin-1 levelsTo examine if caveolin-1 protein levels were decreased
due to degradation subsequent to AT1 receptor intern-
alization in cortex membranes from SHR, immunoblot
analysis was performed. Antibody against caveolin-1
protein recognized a band at 22 kDa. As shown in
Fig. 2, caveolin-1 protein was significantly decreased in
cortex membrane fraction from SHR without treatment
compared to control (60.16� 4.12%, n¼ 8, P< 0.05)
(Fig. 2a), whereas higher caveolin-1 protein levels were
detected in SHR after AngII AT1 receptor antagonist
treatment (254.45� 11.19%, n¼ 8, P< 0.001) (Fig. 2a).
To further confirm the caveolin-1 down-regulation in
proximal tubules, western blot analysis was performed
in isolated membranes from microdissected proximal
tubules. Compared with control, decreased caveolin-1
protein levels were shown in proximal tubule membrane
fraction from SHR without treatment (25.89� 8.40%,
n¼ 4, P< 0.01) in contrast after AngII AT1 receptor
inhibition, SHR proximal tubule membrane fraction
increased the relative amount of caveolin-1 protein
(196.49� 12.6%, n¼ 4, P< 0.001) (Fig. 2b). These find-
rized reproduction of this article is prohibited.
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Losartan on caveolin-1 and Hsp70 interaction in SHR Bocanegra et al. 147
Fig. 2
Effect of Losartan on caveolin-1 expression in isolated cell membranes from renal cortex and cell membranes from microdissected proximal tubulesegments in SHR. (a) Representative western blot and data depicting caveolin-1 protein abundance in renal membrane cortex of WKY and SHRrandomized for receiving the angiotensin II type 1 antagonist, Losartan (WKYLos and SHRlos) or water (WKYH2O and SHRH2O) by gastric gavageduring 6 weeks. Densitometric analysis revealed higher caveolin-1 abundance in SHRLos ���P<0.001 vs. WKYH20 group, yyyP<0.001 vs.SHRH2O group. Decreased caveolin-1 protein levels was observed in SHRH2O vs. control �P<0.05. Protein levels have been normalized in termsof change from WKYH2O. Data are representative of eight experiments that have similar results. (b) Semiquantitative immunoblotting ofmicrodissected proximal tubule membrane fractions from WKY and SHR with and without Losartan treatment. Immunoblots reacted with anti-Cav-1antibody and revealed a single 22 kDa band. The intensity of the bands was quantified by densitometric analysis and was expressed as arbitrary units.Protein levels have been normalized in terms of change from WKYH2O. Densitometric analysis revealed higher caveolin-1 abundance in SHR withLosartan ���P<0.001 vs. control, yyyP<0.001 vs. SHRH2O. Decreased caveolin-1 protein levels was observed in SHRH2O vs. control ��P<0.01.Data are expressed as mean�SEM; n¼4. SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat.
ings let us suggest that in SHR, intrarenal AngII may have
a direct effect by enhancing caveolin-1 degradation.
Angiotensin II AT1 receptor antagonist treatmentinduced expression of Hsp70 protein levels at theplasma membrane in spontaneously hypertensive ratsAs well as its structural role, caveolin-1 has been directly
implicated in interactions with signaling proteins. To gain
insight into the mechanisms involved in AT1 receptor
opyright © Lippincott Williams & Wilkins. Unauth
Fig. 3
AT1 receptor antagonism mediated Hsp70 translocation to the membranemediated Hsp70 translocation to the plasma membrane in membrane cortedensitometry of Hsp70 in renal cortex of WKY and SHR rats randomized foSHRLos) or water (WKYH2O and SHRH2O) by gastric gavage during 6 weused for western blot analysis. Antibody against Hsp70 protein recognizedchange from WKYH2O. Densitometric analysis revealed Hsp70 higher abucontrol, yyyP<0.001 vs. SHRH2O. In contrast, in cytosol cortex fraction (b),yyP<0.01. Decreased expression Hsp70 was shown in membrane fractionSHRLos yyyP<0.001. Data represent the mean�SEM; n¼9. SHR, spont
inhibition in SHR, the expression of chaperone Hsp70
at the protein levels was performed. We hypothesized that
AngII type I antagonist treatment would mediate Hsp70
translocation to the plasma membrane. Membrane and
cytosol preparations from renal cortex were used for wes-
tern blot analysis. Antibody against Hsp70 protein recog-
nized a band at �70 kDa. Figure 3 shows that AngII AT1
antagonist significantly increased Hsp70 protein levels in
cortex membrane from SHR compared with control
orized reproduction of this article is prohibited.
in renal cortex homogenates from SHR rats. AT1 receptor inhibitionx homogenates from SHR rats. Representative western blot andr receiving the angiotensin II type 1 antagonist, Losartan (WKYLos andeks. Membrane (a) and cytosol (b) preparations from renal cortex werea band at �70 kDa. Protein levels have been normalized in terms of
ndance in membrane cortex fraction (a) from SHRLos ���P<0.001 vs.decreased Hsp70 expression was shown in the SHRLos vs. SHRH2O,s (a) from Losartan-treated WKY vs. WKYH2O, ��P<0.01 and vs.aneously hypertensive rat; WKY, Wistar–Kyoto rat.
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148 Journal of Hypertension 2010, Vol 28 No 1
Fig. 4
AT1 receptor antagonist Losartan, mediated Hsp70 translocation to the membrane in microdissected proximal tubule segments in SHR.Semiquantitative immunoblotting of membrane (a) and cytosol (b) fractions of the microdissected proximal tubule segments from WKY and SHR withand without Losartan treatment. Immunoblots reacted with anti-Hsp70 antibody and revealed a single 70 kDa band. The intensity of the bands wasquantified by densitometry and was expressed as arbitrary units. Increased expression Hsp70 was shown in proximal tubule membrane fractions (a)from Losartan-treated SHR ���P<0.001 vs. control and yyP<0.01 vs. SHR without treatment. Decreased expression Hsp70 was shown in proximaltubule membrane fractions (a) from Losartan-treated WKY vs. WKYH2O ���P<0.001 and yyyP<0.001 vs. SHRLos. Data represent themean�SEM; n¼5. Hsp70 protein levels in proximal tubule cytosol fraction (b) from isolated proximal tubules were lower in SHRLos compared withcontrol, ��P<0.01 and compared with SHRH20, yyP<0.01, n¼5. SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat.
(164.5� 14.8%, n¼ 9, P< 0.001), whereas decreased
Hsp70 protein levels were shown in cytosol fraction. More-
over, up-regulation of Hsp70 expression was confirmed
in microdissected proximal tubule membranes from
Losartan-treated SHR when compared with control
(204� 7.8%, n¼ 5, P< 0.001) and also vs. SHR without
treatment (204� 7.8% vs. 131� 7.6%, n¼ 5, P< 0.01)
(Fig. 4a). As expected, in contrast, in cytosol fraction
from microdissected proximal tubules, Hsp70 protein
levels were lower in SHRLos compared with control
(50.34� 7.7%, n¼ 5, P< 0.01) and SHRH2O (112.32�9.6% vs. 50.34� 7.7%, n¼ 5, P< 0.01) (Fig. 4b).
Losartan showed a different response in cortex mem-
brane from WKY rats, decreased Hsp70 expression was
shown when compared with WKRH2O (49.08� 13.5%,
n¼ 5, P< 0.01) and with SHRLos (49.08� 13.5% vs.
164.5� 14.8%, n¼ 5, P< 0.001) (Fig. 3a). These results
were confirm in microdissected proximal tubule mem-
branes from Losartan-treated WKY when compared with
control (38.26� 13.12%, n¼ 5, P< 0.001) and with
SHRLos (38.26� 13.12% vs. 101.27� 14.48%, n¼ 5,
P< 0.001) (Fig. 4a).
Interaction of caveolin-1 and Hsp70 as an effect ofLosartan: coimmunoprecipitation andimmunofluorescenceThe tonic-phase AngII signaling is generated in the
caveolae domain. The caveolae may function as unique
cell surface signal transduction domain. We next hypo-
thesized that caveolin-1 was associated with the chaper-
one Hsp70. To evaluate this proposal, interaction of
caveolin-1 and Hsp70 was studied under experimental
conditions.
opyright © Lippincott Williams & Wilkins. Unautho
To determine whether the angiotensin AT1 receptor
blockade could be involved in the interaction of both
proteins, membrane and cytosolic fractions from SHR
cortex with and without Losartan treatment and their
controls were immunoprecipitated with anti-caveolin-1
antibody, then they were analyzed for the presence of
coprecipitating protein Hsp70. As shown in Fig. 5, Hsp70
was present in caveolin-1 immunoprecipitates prepared
from cortical cytosol and membrane fractions. In mem-
brane fractions from SHR with Losartan treatment, the
amount of Hsp70 coprecipitated with caveolin, expressed
as a ratio, rose above two-fold of control (3.56 vs. 1.62)
Conversely, decreased Hsp70 protein levels in cytosolic
fraction from SHR after Losartan treatment compared
with control was shown. The levels of Hsp70 that copre-
cipitated with caveolin-1 in membrane fractions from
SHR without treatment were similar to those seen in
controls (Fig. 5). Due to these results we can infer Hsp70
translocation after AT1 receptor antagonist treatment in
SHR. Coprecipitation was not observed in membrane
samples from cortex incubated without caveolin-1 anti-
body.
Furthermore in order to explore a potentially direct
relationship between caveolin-1 and the relocated
Hsp70 after AT1 receptor blockade, immunocytochemi-
cal colocalization was used. In WKY proximal tubule
epithelial cells minimal caveolin-1 was identified on
the apical and basolateral surfaces as would be expected
(Fig. 6a); moderate cytosolic expression of Hsp70 occurs
(Fig. 6b); the overlap between these proteins was neg-
ligible (Fig. 6c, caveolin-1 green and Hsp70 red). In SHR
without treatment, caveolin-1 appears at the apical and
basolateral membranes (Fig. 6d); Hsp70 becomes more
rized reproduction of this article is prohibited.
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Losartan on caveolin-1 and Hsp70 interaction in SHR Bocanegra et al. 149
Fig. 5
Interaction of caveolin-1 and Hsp70 after angiotensin AT1 receptor blockade in SHR cortex membrane fractions. Representative immunoprecipitationof caveolin-1. Membrane fractions from rat cortex were immunoprecipitated with caveolin-1 antibody and coprecipitated and analyzed for Hsp70. Theamount of Hsp70 coprecipitating with caveolin-1 was expressed as a ratio. Higher ratio between both proteins was shown in membrane cortex fromSHR after AT1 receptor inhibition, whereas decreased Hsp70 protein levels in cytosolic fraction of SHR after Losartan treatment related to controlwas shown. The Hsp70 that coprecipitated with caveolin-1 in SHR without Losartan was similar to control; n¼5. SHR, spontaneously hypertensiverat; WKY, Wistar–Kyoto rat.
prominent in cytosol, as well (Fig. 6e); and the merged
image (Fig. 6f) showed no colocalization. On the contrary,
after Losartan administration to SHR, antibody against
caveolin-1 protein brightly stained the basolateral and
apical membranes of tubular epithelial cells of proximal
opyright © Lippincott Williams & Wilkins. Unauth
Fig. 6
Immunofluorence/cytochemical localization of caveolin-1 and Hsp70 in proxLosartan treatment were double-labeled with an antibody against caveolin-1isothiocyanate-conjugated and antimouse Rhodamine Red X-conjugated seexperiments. In WKY proximal tubule epithelial cells slight caveolin-1 and Hcytosol, respectively (Fig. 6a–b); the overlap between these proteins wastreatment, caveolin-1 appears at the apical and basolateral membranes (Fig.the merged image (Fig. 6f) showed no colocalization. In contrast, after Losa(green) and Hsp70(red) in apical and basolateral plasma membrane of epiMagnification 600�. SHR, spontaneously hypertensive rat; WKY, Wistar–K
tubules, and resulted in Hsp70 relocation to the apical
membrane of tubular cells of proximal tubules, demon-
strating colocalization in the merged image. These find-
ings are consistent with the immunoprecipitation and
provide confirmation of interactions between these two
orized reproduction of this article is prohibited.
imal tubules. Renal cortex tissues from WKY and SHR without and withand with an antibody against Hsp70 followed by antirabbit fluoresceincondary antibodies. Images are representative of two differentsp70 staining was identified on the apical and basolateral surfaces andnegligible (Fig. 6c, caveolin-1 green and HSP70 red). In SHR without6d); HSP70 becomes more prominent in cytosol, as well (Fig. 6e); andrtan administration to SHR, colocalization of immunoreactive caveolin-1thelial proximal tubule cells was shown in the merged image (6I).
yoto rat.
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150 Journal of Hypertension 2010, Vol 28 No 1
Fig. 7
NADPH oxidase activity after angiotensin II AT1 receptor antagonist, Losartan in SHR proximal tubule membranes. Hsp70 involvement. Proximaltubule membrane fractions from SHR and WKY rats administered either with Losartan or vehicle were incubated in parallel with 25 mg of anti-Hsp70antibody (Sigma) or in the absence of the antibody against Hsp70. This mixture was resuspended and incubated for 20 min at room temperature.Differential centrifugation was then repeated at 35000 g for 15 min at 48C. Top panel: representative western blot of Hsp70 demonstrating theeffects of anti-Hsp70 antibody (anti-Hsp70 Ab) in experimental and control groups. Lower panel: the decreased NADPH activity shown in SHRLoswithout anti-Hsp70 Ab was counteracted in the presence of the antibody against Hsp70: SHRLos anti-Hsp70 Ab vs. SHRLos, ��P<0.01; n¼5.SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat.
proteins in proximal tubule epithelial cells following AT1
receptor inhibition in SHR.
Decreased NADPH oxidase activity after angiotensin IIAT1 receptor blockade in spontaneously hypertensiverats proximal tubule membranes: involvement of Hsp70We next investigated whether Hsp70 translocation to
plasma membrane could be involved in the mechanism
responsible for the effect of Losartan on ROS. NADPH
oxidase represents a key ROS-producing system, there-
fore we examined NADPH activity. Proximal tubule
membranes from SHR and WKY rats were incubated
in the presence and in the absence of anti-Hsp70 anti-
body. As seen in Fig. 7, the decreased NADPH oxidase
activity induced by Losartan in proximal tubule mem-
branes from SHR was abolished by the addition of anti-
Hsp70 antibody (SHRLos vs. SHRLos Anti Hsp70Ab;
214� 10 vs. 306� 14, n¼ 6, P< 0.01). On the contrary,
increased NADPH activity in SHR without Losartan
showed no differences after proximal tubule membranes
incubation with anti-Hsp70 antibody.
Losartan decreased Nox4 NADPH oxidase protein levelsin cell membranes isolated from renal cortex and frommicrodissected proximal tubules in spontaneouslyhypertensive ratsIn the subsequent studies, we explored which one of the
NADPH oxidase subunits were involved in SHR Losar-
tan effect. Expression of NADPH oxidase subunits p47,
p22 and Nox4 at mRNA expression and protein levels
were studied. No significant differences were observed in
opyright © Lippincott Williams & Wilkins. Unautho
mRNA abundance for p47, p22 and Nox4 in proximal
tubule membrane fractions of SHR and WKY with and
without Losartan treatment (Fig. 8). However, increased
expression of Nox4 protein abundance was counteracted
after Losartan administration, in SHR cortex membrane
fractions (Fig. 9a). To further confirm the Nox4 down-
regulation in proximal tubules, western blot analysis was
performed in isolated membranes from microdissected
proximal tubules. Lower Nox4 protein levels were shown
after Losartan treatment on isolated membranes from
SHR microdissected proximal tubule segments when
compared to SHR without treatment (Fig. 9b). Conver-
sely, no differences were demonstrated in the protein
expression pattern of subunit p47, nor in the p22 protein
level expression in SHR microdissected proximal tubules
with and without angiotensin AT1 receptor blockade
(Fig. 10).
Interaction between Hsp70 and Nox4:coimmunoprecipitationWe further studied whether after AngII AT1 receptor
inhibition, the membrane translocated Hsp70 interacts
with Nox4 or not. To confirm this association the coim-
munoprecipitation strategy was used. Immunoprecipi-
tated Nox4 was western blotted for Hsp70. In control
cortex membranes, baseline levels of immunoprecipi-
tated Nox4 were observed. The increased level of
Hsp70 contrasts with the decreased immunoprecipitation
of Nox4 occurring in cortex membrane fractions from
SHR after Losartan administration. In membrane frac-
tions from SHRLos the amount of Hsp70 coprecipitated
rized reproduction of this article is prohibited.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Losartan on caveolin-1 and Hsp70 interaction in SHR Bocanegra et al. 151
Fig. 8
Effect of Losartan on mRNA expression of p22, p47 and Nox4 NADHPH oxidase subunits in microdissected proximal tubule membranes from SHRand WKY rats. Representative gels of p22 (a), p47 (b) and Nox4 (c) mRNA from WKY and SHR microdissected proximal tubule with and withoutLosartan treatment are shown. The corresponding housekeeping b-actin is included in below. Histograms show the relative concentration of mRNAsfor p22, p47 and Nox4 to b-actin mRNA. Data represent the means�SEM of four independent experiments. SHR, spontaneously hypertensive rat;WKY, Wistar–Kyoto rat.
Fig. 9
Nox4 NADPH oxidase subunit expression in isolated cell membranes from renal cortex and from microdissectedproximal tubule membranes in SHR andWKY rats. Effect of Losartan. (a) Representative western blot and densitometry of Nox4 in membrane fractions from renal cortex obtained from WKY andSHR rats administered either with Losartan or vehicle. The intensity of the bands was quantified by densitometric analysis and was expressed as arbitraryunits. Protein levels have been normalized in terms of change from WKYH2O. SHRLos yyyP<0.001 vs. SHRH2O Data are expressed as mean�SEM;n¼5. (b) Semiquantitative immunoblotting of microdissected proximal tubule membrane fractions from WKY and SHR with and without Losartantreatment. Immunoblots reacted with anti-Nox4 antibody and revealed a single 70 kDa band. The intensity of the bands was quantified by densitometricanalysis and was expressed as arbitrary units. Protein levels have been normalized in terms of change from WKYH2O. Decreased SHRLos vs. SHRH2OyyP<0.01. Data are expressed as mean�SEM; n¼4. SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat.
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152 Journal of Hypertension 2010, Vol 28 No 1
Fig. 10
Effect of Losartan on protein expression of p22 and p47 NADPH oxidase subunits expression in isolated cell membranes from microdissectedproximal tubules in SHR and WKY rats. Representative western blot and densitometry of p22 and p47 in membrane fractions from microdissectedproximal tubules obtained from WKY and SHR rats administered either with Losartan or vehicle. The intensity of the bands was quantified bydensitometric analysis and was expressed as arbitrary units. Protein levels have been normalized in terms of change from WKYH2O Data areexpressed as mean�SEM; n¼4. SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto rat.
with Nox4 expressed as a ratio rose to 59% of control (1.44
vs. 0.85, P< 0.05) (Fig. 11). The levels of Hsp70 that
coprecipitated with Nox4 in membrane fractions from
nontreated SHR compared to controls showed no differ-
ences. To ensure specificity of immunoprecipitation
using anti-Nox4 antibody, membrane fractions were
incubated with normal mouse immunoglobulin. Nox4
was not immunoprecipitated by normal mouse immuno-
globuin under any of the experimental conditions.
DiscussionSpontaneously hypertensive rat provides a useful model
for studies on the individual and interacting signaling
mechanisms involving AngII AT1 receptor. Increased
kidney tissue angiotensin has been detected in young
SHR, suggesting that a local intrarenal RAS may be
activated. Moreover, increased expression of type 1 AngII
receptor mRNA and immunoreactive protein have been
demonstrated in proximal tubules at 4 weeks compared
opyright © Lippincott Williams & Wilkins. Unautho
Fig. 11
Nox4 inmunoprecipitation, Hsp70 coprecipitation. Representativecoimmunoprecipitation of Nox4 and Hsp70. Membrane cortex fractionsfrom WKY and SHR kidney rats pretreated with an AT1 receptorantagonist. Cortex membrane tissues were immunoprecipitated withNox4 antibody and were coprecipitated and analyzed for Hsp70. Theamount of Hsp70 coprecipitating with Nox4 was expressed as a ratio.Higher ratio between both proteins was shown in SHR after Losartanadministration, related to homogenate fractions from WKY rats andnontreated SHR; n¼5. SHR, spontaneously hypertensive rat; WKY,Wistar–Kyoto rat.
with those age-matched WKY rats [18]. Evidence for
AngII AT1 inhibition was demonstrated in our study
by a significant reduction in SBP in SHR compared to
the levels in the SHRH2O group. In this study we
demonstrated an interaction between caveolin-1 and
Hsp70 in SHR proximal tubule membranes after Losar-
tan administration. AngII AT1 blockade stimulates move-
ment/translocation of Hsp70 to plasma membrane and
increased caveolin-1 levels. Moreover, caveolin-1 coim-
munoprecipitated and colocalized with Hsp70. These
data also provide evidence of the association between
the translocated Hsp70 and the decrease of NADPH
oxidase activity and Nox4 down-regulation in proximal
tubule cell membranes from SHR, after AngII AT1
receptor inhibition. To our knowledge, this is the first
study showing caveolin-1 and Hsp70 interaction on prox-
imal tubule cell membranes regulated by AngII AT1
receptor blockade and involving Hsp70 in the mechanism
responsible for the effect of Losartan on ROS. Previous
data have provided firm evidence relating the AT1 recep-
tor to the caveolae, both structurally and functionally.
Finding that AT1 receptors coprecipitate with caveolin-1,
like the coprecipitation of G protein subunits with the
caveolin-1 fusion protein supports the notion that caveo-
lin may act as an organizing molecule for signal transduc-
tion of AT1 receptors [6]. A direct interaction with
caveolin is required to traffic the AT1R through the
exocytic pathway, but this does not result in AT1R
sequestration in caveolae. Caveolin-1 therefore acts as
a molecular chaperone rather than a plasma membrane
scaffold for AT1 receptor [8]. Our results showed inten-
sive decrease on caveolin-1 protein expression in the
SHR group as well as a significant increase in caveolin-
1 protein expression in isolated membranes from SHR
proximal tubules after AT1 receptor blockade. It is
possible that in SHR proximal tubule epithelial cell
membranes AngII stimulation results in internalization
rized reproduction of this article is prohibited.
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Losartan on caveolin-1 and Hsp70 interaction in SHR Bocanegra et al. 153
of caveolae and translocation to intracellular compart-
ments in which the caveolin protein is degraded. This
hypothesis may be substantiated by the fact that caveolae
mediate the endocytosis of conformationally modified
albumin for delivery to endosomes and lysosomes for
degradation [19]. As expected from an interaction
restricted to the exocytic pathway, only 2% of caveolin
can be recovered from the VMC cell lysates bound to AT1
receptor [8]. There are several signal-transducing mol-
ecules and receptors that directly bind caveolin [5].
Caveolin itself may play an active role in controlling
signal generation. We next examined if caveolin-1 was
associated with the chaperone Hsp70 after AT1 receptor
inhibition, and we demonstrated that Hsp70 is found in
the proximal tubule cytosolic fraction from SHR and that
AngII AT1 receptor inhibition promotes Hsp70 trans-
location to proximal tubule plasma membrane. After
recruitment of Hsp70 to proximal tubule membrane
fraction, interaction between this protein and caveolin-
1 was shown. The specificity of the observed interaction
was studied by coinmunoprecipitation in which the anti-
body directed against caveolin-1 was used to precipitate
native caveolin-1, and then it was analyzed for the pre-
sence of coprecipitating Hsp70 protein. Binding of Hsp70
and caveolin-1 was increased in proximal tubule mem-
branes after AngII AT1 receptor inhibition, whereas no
interaction between these two proteins was shown in
proximal tubule membrane from SHR without Losartan
administration. Moreover in the present study, relocation
of Hsp70 at the proximal tubule apical domain and
increased expression of Hsp70 and caveolin-1 after angio-
tensin AT1 receptor blockade was shown. Substantial
colocalization of these proteins occurred at this site,
which was not observed in proximal tubules from non-
treated SHR. In a previous study, a mutagenesis approach
was used to study the site specificity of N-glycosylation
sites by inserting nonnatural consensus sequences into
AT1 receptor cDNA. N-glycosylation of ECL2 (extra-
cellular loop 2) was crucial for cell surface expression of
the hAT1 receptor (human AngII receptor subtype 1).
Lanctot et al. [20] hypothesized that a misfolded receptor
mediated greater protein–protein interactions with vari-
ous quality control chaperones. Indeed, Hsp70, which is a
cytoplasmic chaperone that binds to hydrophobic patches
on newly synthesized proteins [20], coprecipitated to a
greater degree with the misfolded AT1 receptor than
with AT1-WT. Hsp70 interacted with the cytoplasmic
side of the receptor, thus allowing the interaction to occur
throughout its biosynthesis, even at the plasma mem-
brane [21].
Absence of membrane Hsp70 translocation was observed
in proximal tubules from Losartan-treated WKY rats. The
different response between proximal tubule cells from
SHR and WKY rats could be explained by a clear distinct
distribution of AT1 receptors in lipid and nonlipid rafts.
SHR proximal tubule cells have been found to be
opyright © Lippincott Williams & Wilkins. Unauth
endowed with more AT1 receptors in both microdomains
than WKY proximal tubule cells, with particular emphasis
in lipid rafts [22]. A series of signaling cascades are
activated after AngII binding to the AT1 receptor, the
peptide being an important mediator of oxidative stress.
Full expression of AngII signaling in VSMCs is depen-
dent on the ROS derived from nicotinamide-adenine
dinucleotide phosphate NADPH oxidase and the
dynamic association of the AngII type I receptor
(AT1R) with caveolae/lipid rafts [23,24]. Consistent with
these data, caveolin-1 siRNA inhibits AngII-stimulated
increase in H2O2 production, indicating the specific
involvement of caveolin-1 in the pathways linking
AT1R signal to the NAD(P)H oxidase [2]. An enhanced
oxidative stress condition coupled with NADPH oxidase
has been demonstrated in proximal tubule cells from
SHRs. In this condition, H2O2 influences renal ion trans-
port, contributing to sodium retention in hypertension
[22]. In addition, AngII-stimulated activation of mem-
brane NAD(P)H oxidase resulting in O2� generation
plays a pivotal role in p27Kip 1 and induction of cellular
hypertrophy in proximal tubular cells [25]. Recently,
Zhuo et al. [26] have shown that AngII activates nuclear
transcription factor NF-kB in proximal tubules, leading
to long-lasting inflammatory and growth-promoting
effects; these responses blocked by inhibition of AT1
receptor-mediated endocytosis of extracellular AngII
with Losartan. Induction of the stress response includes
synthesis of heat shock proteins (HSPs) that have been
well characterized in cells injured from a variety of renal
insults [27,28]. The cellular functions of intracellular
Hsp72 have been thoroughly studied and include limit-
ing protein aggregation, facilitating protein refolding and
chaperoning proteins leading to improve cell survival
[27,29]. Certain HSPs (including Hsp70) provide cellular
protection by decreased oxidative stress linked to up-
regulation of Hsp70 expression [27,30]. We then tested
the hypothesis that plasma membrane translocated
Hsp70 could be involved in the mechanism responsible
for Losartan effect on NADPH oxidase expression and
activity in SHR proximal tubules. From the NADPH
oxidase subunits, after Losartan, Nox4 expression was
down-regulated at the protein level in isolated mem-
branes from SHR microdissected proximal tubules; in
contrast, no changes in gene expression of Nox4 were
shown. These results allow us to suggest that Hsp70
mediated decrease in the expression of Nox4 in isolated
membranes of Losartan SHR proximal tubule, probably
inducing the movement of Nox4 into small intracellular
vesicles (early or late endosomes), in route to their
degradation. Previously a segregated compartmentaliza-
tion of Nox and subunits that impair their assembly, has
been described for Apocynin, resulting in decreased
NADPH oxidase activity and ROS production [31].
However, it remains to be determined how Hsp70
regulate Nox4 and NADPH oxidase subunit degradation
and trafficking in Losartan SHR proximal tubules.
orized reproduction of this article is prohibited.
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154 Journal of Hypertension 2010, Vol 28 No 1
NADPH oxidase subunits p47 and p22 at mRNA expres-
sion and protein levels showed no difference in cortex
membranes derivated from SHR before and after AT1
receptor inhibition.
Nox4, a 578-amino acid protein with 39% sequence
identity to gp91phox/Nox2 was originally described as a
renal oxidase (ReNox), because its expression is limited
to the kidney, highly expressed in the proximal convo-
luted tubules [32]. When overexpressed in NIH-3T3
fibroblasts, Nox4 increased superoxide production and
induced a cellular senescence phenotype, an effect that is
likely mediated by increased production of ROS [32].
Expression of antisense Nox4 mRNA in HEK293 cells,
which contain endogenous Nox4, resulted in a decreased
NADH and NADPH-dependent superoxide production
in vitro [33].
In the current study, the decreased NADPH oxidase
activity induced by Losartan in SHR microdissected
proximal tubule membranes was abolished by the
addition of the anti-Hsp70 antibody, suggesting that
the relocated Hsp70 could be involved in the mechanism
responsible for the effect of Losartan on ROS. In
addition, our investigation of the interaction between
Hsp70 and Nox4 expressed in SHR proximal tubule
membrane fractions, revealed that AngII AT1 inhibition
induced this association. A physical association and func-
tional interaction between the higher amount of Hsp70
coprecipitated with lower NADPH subunits Nox4, was
revealed in this study.
Giving support to our results, a functional interaction of
Hsp70 with NQO1, a potent antioxidant that catalyzes
two-electron reduction of various quinones, with NADH
or NADPH as an electron donor, was previously found in
coimmunoprecipitation; interaction of Hsp70 could only
be observed with wild-type NQO11 but not with mutant
NQO12 protein [34]. Additional evidence linking Hsp70
with NQO1 has come from site-directed mutagenesis
studies of a proposed Hsp70-binding motif located
near the N terminus of the NQO1 protein [35].
Hsp70-catalyzed protein folding requires ATP hydroly-
sis. In contrast, uncoupling of oxidative phosphorylation
and electron transport results in efficient generation of
adenosine 50-triphosphate (ATP) by mitochondria and
increased transfer of electrons to molecular oxygen, with
increased production of superoxide radicals [36].
In conclusion, due to the interaction of caveolin-1 and
Hsp70 after AngII AT1 receptor blockade, a role of both
proteins in AT1 regulation is demonstrated in cell mem-
branes from SHR proximal tubules. Plasma membrane
translocated Hsp70 could be involved in the mechanism
responsible for Losartan cytoprotective effect on prox-
imal tubules by decreasing oxidative stress through the
down-regulation of NADPH subunits Nox4. Such an
opyright © Lippincott Williams & Wilkins. Unautho
action may be responsible, at least in part, for Losartan
proximal tubule cell protection against oxidative stress in
hypertension induced-tubular injury. Future studies are
required to further study the intracellular signalling
mechanisms involved in the (AT1) receptor/caveolin-1
(Cav-1)/Hsp70 chaperone/Nox4 (NADPH oxidase
subunit) pathway.
AcknowledgementsThe work was performed with financial support from
CONICET, PICT/2005 N 33827 and from the Research
and Technology Council of Cuyo University (CIUNC)
Mendoza, Argentina/N: 1143/04 to P.G.V.
There are no conflicts of interest.
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