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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,b

Background 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: pvalles@fcm.uncu.edu.ar

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

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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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